Polyelectrolyte-protein synergism: pH-responsive polyelectrolyte/insulin complexes as versatile carriers for targeted protein and drug delivery.

The co-assembly of polyelectrolytes (PE) with proteins offers a promising approach for designing complex structures with customizable morphologies, charge distribution, and stability for targeted cargo delivery. However, the complexity of protein structure limits our ability to predict the properties of the formed nanoparticles, and our goal is to identify the key triggers of the morphological transition in protein/PE complexes and evaluate their ability to encapsulate multivalent ionic drugs. A positively charged PE can assemble with a protein at pH above isoelectric point due to the electrostatic attraction and disassemble at pH below isoelectric point due to the repulsion. The additional hydrophilic block of the polymer should stabilize the particles in solution and enable them to encapsulate a negatively charged drug in the presence of PE excess. We demonstrated that diblock copolymers, poly(ethylene oxide)-block-poly(N,N-dimethylaminoethyl methacrylate) and poly(ethylene oxide)-block-poly(N,N,N-trimethylammonioethyl methacrylate), consisting of a polycation block and a neutral hydrophilic block, reversibly co-assemble with insulin in pH range between 5 and 8. Using small-angle neutron and X-ray scattering (SANS, SAXS), we showed that insulin arrangement within formed particles is controlled by intermolecular electrostatic forces between protein molecules, and can be tuned by varying ionic strength. For the first time, we observed by fluorescence that formed protein/PE complexes with excess of positive charges exhibited potential for encapsulating and controlled release of negatively charged bivalent drugs, protoporphyrin-IX and zinc(II) protoporphyrin-IX, enabling the development of nanocarriers for combination therapies with adjustable charge, stability, internal structure, and size.

Dynamic light scattering and transmission electron microscopy in drug delivery: a roadmap for correct characterization of nanoparticles and interpretation of results.

In this focus article, we provide a scrutinizing analysis of transmission electron microscopy (TEM) and dynamic light scattering (DLS) as the two common methods to study the sizes of nanoparticles with focus on the application in pharmaceutics and drug delivery. Control over the size and shape of nanoparticles is one of the key factors for many biomedical systems. Particle size will substantially affect their permeation through biological membranes. For example, an enhanced permeation and retention effect requires a very narrow range of sizes of nanoparticles (50-200 nm) and even a minor deviation from these values will substantially affect the delivery of drug nanocarriers to the tumour. However, amazingly a great number of research papers in pharmaceutics and drug delivery report a striking difference in nanoparticle size measured by the two most popular experimental techniques (TEM and DLS). In some cases, this difference was reported to be 200-300%, raising the question of which size measurement result is more trustworthy. In this focus article, we primarily focus on the physical aspects that are responsible for the routinely observed mismatch between TEM and DLS results. Some of these factors such as concentration and angle dependencies are commonly underestimated and misinterpreted. We convincingly show that correctly used experimental procedures and a thorough analysis of results generated using both methods can eliminate the DLS and TEM data mismatch completely or will make the results much closer to each other. Also, we provide a clear roadmap for drug delivery and pharmaceutical researchers to conduct reliable DLS measurements.

Fluorine-Containing Block and Gradient Copoly(2-oxazoline)s Based on 2-(3,3,3-Trifluoropropyl)-2-oxazoline: A Quest for the Optimal Self-Assembled Structure for 19F Imaging.

The use of fluorinated contrast agents in magnetic resonance imaging (MRI) facilitates improved image quality due to the negligible amount of endogenous fluorine atoms in the body. In this work, we present a comprehensive study of the influence of the amphiphilic polymer structure and composition on its applicability as contrast agents in 19F MRI. Three series of novel fluorine-containing poly(2-oxazoline) copolymers and terpolymers, hydrophilic-fluorophilic, hydrophilic-lipophilic-fluorophilic, and hydrophilic-thermoresponsive-fluorophilic, with block and gradient distributions of the fluorinated units, were synthesized. It was discovered that the CF3 in the 2-(3,3,3-trifluoropropyl)-2-oxazoline (CF3EtOx) group activated the cationic chain end, leading to faster copolymerization kinetics, whereby spontaneous monomer gradients were formed with accelerated incorporation of 2-methyl-2-oxazoline or 2-n-propyl-2-oxazoline with a gradual change to the less-nucleophilic CF3EtOx monomer. The obtained amphiphilic copolymers and terpolymers form spherical or wormlike micelles in water, which was confirmed using transmission electron microscopy (TEM), while small-angle X-ray scattering (SAXS) revealed the core-shell or core-double-shell morphologies of these nanoparticles. The core and shell sizes obey the scaling laws for starlike micelles predicted by the scaling theory. Biocompatibility studies confirm that all copolymers obtained are noncytotoxic and, at the same time, exhibit high sensitivity during in vitro 19F MRI studies. The gradient copolymers provide the best 19F MRI signal-to-noise ratio in comparison with the analogue block copolymer structures, making them most promising as 19F MRI contrast agents.

Reversible multilayered vesicle-like structures with fluid hydrophobic and interpolyelectrolyte layers.

Hydrophobic blocks of amphiphilic block copolymers often form glassy micellar cores at room temperature with a rigid structure that limits their applications as nanocapsules for targeted delivery. Nevertheless, we prepared and analyzed core/shell micelles with a soft core, formed by a self-assembled block copolymer consisting of a hydrophobic block and a polycation block, poly(lauryl acrylate)-block-poly(trimethyl-aminoethyl acrylate) (PLA-QPDMAEA), in aqueous solution. By light and small-angle neutron scattering, by transmission electron microscopy and by fluorescence spectroscopy, we showed that these core/shell micelles are spherical and cylindrical with a fluid-like PLA core and a positively charged outer shell and that they can encapsulate and release hydrophobic solutes. Moreover, after mixing these PLA-QPDMAEA core/shell micelles with another diblock copolymer, consisting of a hydrophilic block and a polyanion block, namely poly(ethylene oxide)-block-poly(methacrylic acid) (PEO-PMAA), we observed the formation of novel vesicle-like multicompartment structures containing both soft hydrophobic and interpolyelectrolyte (IPEC) layers. By combining small-angle neutron scattering with self-consistent field modeling, we confirmed the formation of these complex vesicle-like structures with a swollen PEO core, an IPEC inner layer, a PLA soft layer, an IPEC outer layer and a loose PEO corona. Thus, these multicompartment micelles with fluid and IPEC layers and a hydrophilic corona may be used as nanocapsules with several tunable properties, including the ability to control the thickness of each layer, the charge of the IPEC layers and the stability of the micelles, to deliver both hydrophobic and multivalent solutes.

Role of pKA in Charge Regulation and Conformation of Various Peptide Sequences.

Peptides containing amino acids with ionisable side chains represent a typical example of weak ampholytes, that is, molecules with multiple titratable acid and base groups, which generally exhibit charge regulating properties upon changes in pH. Charged groups on an ampholyte interact electrostatically with each other, and their interaction is coupled to conformation of the (macro)molecule, resulting in a complex feedback loop. Their charge-regulating properties are primarily determined by the pKA of individual ionisable side-chains, modulated by electrostatic interactions between the charged groups. The latter is determined by the amino acid sequence in the peptide chain. In our previous work we introduced a simple coarse-grained model of a flexible peptide. We validated it against experiments, demonstrating its ability to quantitatively predict charge on various peptides in a broad range of pH. In the current work, we investigated two types of peptide sequences: diblock and alternating, each of them consisting of an equal number of amino acids with acid and base side-chains. We showed that changing the sequence while keeping the same overall composition has a profound effect on the conformation, whereas it practically does not affect total charge on the peptide. Nevertheless, the sequence significantly affects the charge state of individual groups, showing that the zero net effect on the total charge is a consequence of unexpected cancellation of effects. Furthermore, we investigated how the difference between the pKA of acid and base side chains affects the charge and conformation of the peptide, showing that it is possible to tune the charge-regulating properties by following simple guiding principles based on the pKA and on the amino acid sequence. Our current results provide a theoretical basis for understanding of the complex coupling between the ionisation and conformation in flexible polyampholytes, including synthetic polymers, biomimetic materials and biological molecules, such as intrinsically disordered proteins, whose function can be regulated by changes in the pH.

Physicochemical Evaluation of Insulin Complexes with QPDMAEMA-b-PLMA-b-POEGMA Cationic Amphiphlic Triblock Terpolymer Micelles.

Herein, poly[quaternized 2-(dimethylamino)ethyl methacrylate-b-lauryl methacrylate-b-(oligo ethylene glycol)methacrylate] (QPDMAEMA-b-PLMA-b-POEGMA) cationic amphiphilic triblock terpolymers were used as vehicles for the complexation/encapsulation of insulin (INS). The terpolymers self-assemble in spherical micelles with PLMA cores and mixed QPDMAEMA/POEGMA coronas in aqueous solutions. The cationic micelles were complexed via electrostatic interactions with INS, which contains anionic charges at pH 7. The solutions were colloidally stable in all INS ratios used. Light-scattering techniques were used for investigation of the complexation ability and the size and surface charge of the terpolymer/INS complexes. The results showed that the size of the complexes increases as INS ratio increases, while at the same time the surface charge remains positive, indicating the formation of clusters of micelles/INS complexes in the solution. Fluorescence spectroscopy measurements revealed that the conformation of the protein is not affected after the complexation with the terpolymer micellar aggregates. It was observed that as the solution ionic strength increases, the size of the QPDMAEMA-b-PLMA-b-POEGMA/INS complexes initially decreases and then remains practically constant at higher ionic strength, indicating further aggregation of the complexes. atomic force microscopy (AFM) measurements showed the existence of both clusters and isolated nanoparticulate terpolymer/protein complexes.

Formation of core/corona nanoparticles with interpolyelectrolyte complex cores in aqueous solution: insight into chain dynamics in the complex from fluorescence quenching.

Formation of interpolyelectrolyte complexes (IPECs) of poly(methacrylic acid) (PMAA) bearing a fluorescent label (umbelliferone) at the chain end and poly[3,5-bis(trimethyl ammoniummethyl)-4-hydroxystyrene iodide]-block-poly(ethylene oxide) (QNPHOS-PEO) acting as a fluorescence quencher, was followed using a combination of scattering, calorimetry, microscopy and fluorescence spectroscopy techniques. While scattering and microscopy measurements indicated formation of spherical core/corona nanoparticles with the core of the QNPHOS/PMAA complex and the PEO corona, fluorescence measurements showed that both static and dynamic quenching efficiency were increased in the nanoparticle stability region. As the dynamic quenching rate constant remained unchanged, the quenching enhancement was caused by the increase in the local concentration of QNPHOS segments in the microenvironment of the label. This finding implies that the local dynamics of PMAA end chains affecting the interaction of the label with QNPHOS segments was independent of both PMAA and QNPHOS chain conformations.

Local pH and Effective pK of a Polyelectrolyte Chain: Two Names for One Quantity?

In recent experiments, the "local pH" near polyelectrolyte chains was determined from the shift in the effective acidity constant of fluorescent pH indicators attached to the macromolecules. This indirect determination raises the question if the analyzed quantity was indeed the "local pH" and what this term actually means. In this study, we combined experiments and simulations to demonstrate that the shift in ionization constant is slightly lower than the difference between the pH and the "local pH". This offset is caused by correlations between fluctuations in chain conformation, small-ion distribution, and fluorophore ionization.