Anna Bogomolova

Coiled Coil Peptides and Polymer–Peptide Conjugates: Synthesis, Self-Assembly, Characterization and Potential in Drug Delivery Systems

Coiled coils are a common structural motif in many natural proteins that can also be utilized in the design and preparation of drug delivery systems for the noncovalent connection of two macromolecules. In this work, two different pairs of peptides forming coiled coil hetero-oligomers were designed, synthesized, and characterized. While the peptide sequences (VAALEKE)4 and (VAALKEK)4 predominantly form coiled coil heterodimers with randomly orientated peptide chains, (IAALESE)2-IAALESKIAALESE and IAALKSKIAALKSE-(IAALKSK)2 tend to form higher hetero-oligomers with an antiparallel orientation of their peptide chains. The associative behavior of these peptides was studied in aqueous solutions using circular dichroism spectroscopy, size-exclusion chromatography, isothermal titration calorimetry and sedimentation analyses. The orientation of the peptide chains in the coiled coil heterodimers was assessed using fluorescence spectroscopy with fluorescence resonance energy transfer labels attached to the ends of the peptides. The formation of the heterodimer can be used as a general method for the selective noncovalent conjugation of a specific targeting moiety with various drug carrier systems; this process involves simple self-assembly in a physiological solution before drug administration. The preparation of targeted macromolecular therapeutics consisting of a synthetic polymer drug carrier and a recombinant protein targeting ligand is discussed.

CTAB/water/chloroform reverse micelles: a closed or open association model?

The micellization of cetyltrimethylammonium bromide (CTAB) in chloroform in the presence of water was examined. Three scenarios of the reverse micelle formation, the closed, open and Eickes association models, were considered in the interpretation of the experimental data. The growth of the aggregates was observed through the changes of NMR signals of associated water, probing the microenvironment of the premicellar aggregates and the interior of reverse micelles. This technique if combined with isothermal titration calorimetry (ITC) revealed that hydrated surfactant premicellar aggregates are already present at ∼6 mM CTAB. NMR, ITC and conductometry were used to determine the critical micelle concentration (cmc) to be ∼40 mM CTAB. It is suggested that the variation of the cmc values reflects the fact that the NMR analysis indicated the beginning of the reverse micelle formation, whereas conductometry and ITC measurements provided the upper limit and an average value of a so-called apparent cmc, respectively. The cmc values were found to be unaffected by the water content. The presence of reverse micelles, the existence of multiple equilibria, and high polydispersity of the samples were evidenced by DOSY NMR spectroscopy. As a result, we validated Eickes association model, according to which cyclic inverse micelles are formed by a structural reorganization of linear associates within a narrow concentration range, called the apparent cmc. New experimental results have also been gained for micellization of cetyltrimethylammonium chloride (CTAC) in chloroform in the presence of water; a similar mechanism of reverse micelle formation has been suggested.

Hydrolytically Degradable Polymer Micelles for Drug Delivery: A SAXS/SANS Kinetic Study

We report kinetic studies of therapeutically highly potent polymer-drug conjugates consisting of amphiphilic N-(2-hydroxypropyl) methacrylamide (HPMA)-based copolymers bearing the anticancer drug doxorubicin (Dox). Highly hydrophobic cholesterol moieties as well as the drug were attached to the polymer backbone by a pH-sensitive hydrazone bond. Moreover, the structure of the spacer between the polymer carrier and the cholesterol moiety differed in order to influence the release rate of the hydrophobic moiety, and thus the disintegration of the high-molecular-weight micellar nanoparticle structure. We performed time-dependent SAXS/SANS measurements after changing pH from a typical blood value (pH 7.2) to that of tumor cells (pH 5.0) to characterize the drug release and changes in particle size and shape. Nanoparticles composed of the conjugates containing Dox were generally larger than the drug-free ones. For most conjugates, nanoparticle growth or decay was observed in the time range of several hours. It was established that the growth/decay rate and the steady-state size of nanoparticles depend on the spacer structure. From analytical fitting, we conclude that the most probable structure of the nanoparticles was a core-shell or a core with attached Gaussian chains. We concluded that the spacer structure determined the fate of a cholesterol derivative after the pH jump. Fitting results for 5α-cholestan-3-onecholestan-3-one and cholesteryl-4-oxopentanoate (Lev-chol) implied that cholesterol moieties continuously escape from the core of the nanoparticle core and concentrate in the hydrophilic shell. In contrast, cholest-4-en-3-one spacer prevent cholesterol escaping. Dox moiety release was only observed after a change in pH. Such findings justify the model proposed in our previous paper. Lastly, the cholesteryl 4-(2-oxopropyl)benzoate (Opb-Chol) was a different case where after the release of hydrophobic Opb-Chol moieties, the core becomes more compact. The physicochemical mechanisms responsible for the scenarios of the different spacers are discussed.

Thermoresponsive Nanoparticles Based on Poly(2-alkyl-2-Oxazolines) and Pluronic F127

We synthesized statistical poly(2-isopropyl-2-oxazoline-co-2-butyl-2-oxazolines) (POXs) that are molecularly dissolved below their cloud point temperature in aqueous milieu and are incorporated into micellar nanoparticles of biocompatible Pluronic F127 (F127) after heating their solution above transition temperature, T(tr). A functional comonomer 2-(but-3-enyl)-2-oxazoline copolymerized into one of the POXs (polymer E) allows introduction of fenolic moieties and subsequent radionuclide labeling with iodine-125. Self-assembly of the polymer E with F127 leads to formation of radioactive nanoparticles with hydrodynamic diameter 20 nm in aqueous solution by heating to 37 °C. The nanoparticles are intended to be used as radioimaging tool in solid tumor diagnostics.

Study of Complex Thermosensitive Amphiphilic Polyoxazolines and Their Interaction with Ionic Surfactants. Are Hydrophobic, Thermosensitive, and Hydrophilic Moieties Equally Important?

The temperature-driven self-assembly of nonionic amphiphilic tailor-made triblock copolymers has been studied by DLS, NMR, ITC, and SAXS. The composition of these triblock copolymers is more complex than that of the vast majority of poly(2-alkyl-2-oxazoline)s: a statistical thermoresponsive (iPrOx) and hydrophobic (BuOx) central block with terminal hydrophilic blocks (MeOx). In general, as temperature increases, nanoparticles form in a process starting with single molecules that become loose aggregates and ends with the formation of compact nanoparticles. Here, we first attempt to resolve the effects of each block on nanoparticle formation. It has been proven that the iPrOx/MeOx ratio determines the value of the cloud point temperature, whereas the different BuOx-iPrOx blocks determine the character of the process. Finally, we complete our investigation by presenting the thermodynamic and structural profiles of the complexation between these triblock poly(2-alkyl-2-oxazoline)s and two ionic surfactants. The addition of an ionic surfactant promotes a rearrangement of the polymer molecules and the formation of complexes followed by the appearance of polymer-surfactant hybrid micelles. Analysis of the interaction shows a strong and nonspecific reaction between the polymers and the anionic surfactant sodium dodecyl sulfate and weak but polymer-state-sensitive interactions between the polymer and the cationic surfactant hexadecyltrimethylammonium bromide.

Small‐angle X‐ray scattering and light scattering study of hybrid nanoparticles composed of thermoresponsive triblock copolymer F127 and thermoresponsive statistical polyoxazolines with hydrophobic moieties

A combination of new thermoresponsive statistical polyoxazolines, poly[(2-butyl-2-oxazoline)-stat-(2-isopropyl-2-oxazoline)] [pBuOx-co-piPrOx], with different hydrophobic moieties and F127 surfactant as a template system for the creation of thermosensitive nanoparticles for radionuclide delivery has recently been tested [Panek, Filippov, Hrubý, Rabyk, Bogomolova, Kucka & Stěpanek (2012). Macromol. Rapid Commun. 33, 1683–1689]. It was shown that the presence of the thermosensitive F127 triblock copolymer in solution reduces nanoparticle size and polydispersity. This article focuses on a determination of the internal structure and solution properties of the nanoparticles in the temperature range from 288 to 312 K. Here, it is demonstrated that below the cloud point temperature (CPT) the polyoxazolines and F127 form complexes that co-exist in solution with single F127 molecules and large aggregates. When the temperature is raised above the CPT, nanoparticles composed of polyoxazolines and F127 are predominant in solution. These nanoparticles could be described by a spherical shell model. It was found that the molar weight and hydrophobicity of the polymer do not influence the size of the outer radius and only slightly change the inner radius of the nanoparticles. At the same time, molar weight and hydrophobicity did affect the process of nanoparticle formation. In conclusion, poly(2-oxazoline) molecules are fully incorporated inside of F127 micelles, and this result is very promising for the successful application of such systems in radionuclide delivery.

Glycogen-graft-poly(2-alkyl-2-oxazolines) – the new versatile biopolymer-based thermoresponsive macromolecular toolbox

This study is focused on thermoresponsive glycogen-graft-poly(2-alkyl-2-oxazolines), a new group of nanostructured hybrid dendrimeric stimuli-responsive polymers connecting the body’s own biodegradable polysaccharidic dendrimer glycogen with the widely tuneable thermoresponsive behavior of polypeptide-analogic poly(2-alkyl-2-oxazolines), which are known to be biocompatible. Glycogen-graft-poly(2-alkyl-2-oxazolines) were prepared by a simple one-pot two-step procedure involving cationic ring-opening polymerization of 2-alkyl-2-oxazolines followed by termination of the living cationic ends with sodium glycogenate. As confirmed by light and X-ray scattering, as well as cryo-transmission electron microscopy, the grafted dendrimer structure allows easy adjustment of the cloud point temperature, the concentration dependence and nanostructure of the self-assembled phase separated polymer by crosstalk during graft composition, the graft length and the grafting density, in a very wide range.

Novel thermosensitive telechelic PEGs with antioxidant activity: synthesis, molecular properties and conformational behaviour

We report on the synthesis and solution properties of novel tailor-made polymer conjugates, which are highly compelling for biomedical applications due to their antioxidant activity and the potential to fine-tune their thermosensitive properties. These conjugates consist of polyethylene glycol (PEG) polymers containing antioxidant moieties, namely 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate or 2-benzamido-3-(3,5-di-tert-butyl-4-hydroxyphenyl)acrylate, as end groups that differ in activity and hydrophobicity. It was shown that all of the synthesized conjugates have low critical solution temperatures (LCSTs) characteristic of type II polymers on a phase diagram. By simply varying the PEG molecular weight, the solution properties, including the LCST value, could be easily tuned across a broad temperature range of 20–90 °C, providing an ideal method for the creation of thermosensitive polymers. It was also established that the LCST value and the polymer conjugate conformation depend on the antioxidant structure. From dynamic light scattering and small-angle X-ray scattering data, we were able to construct a complete sequence diagram of the conformational phase behaviour of the polymers with increasing temperature. It was observed that the conjugate conformation changes are the result of water shifting from a thermodynamically favourable solvent to an unfavourable one. This process then leads to compaction of the conjugate, followed by its aggregation.

Polyelectrolyte pH-Responsive Protein-Containing Nanoparticles: The Physicochemical Supramolecular Approach

We report on the physicochemical properties and self-assembly behavior of novel efficient pH-sensitive nanocontainers based on the Food and Drug Administration-approved anionic polymer Eudragit L100-55 (poly(methacrylic acid-co-ethyl acrylate) 1:1) and nonionic surfactant Brij98. The features of the interaction between Eudragit L100-55 and Brij98 at different pH values and their optimal ratio for nanoparticle formation were studied using isothermal titration calorimetry. The influence of the polymer-to-surfactant ratio on the size and structure of particles was studied at different pH values using dynamic light scattering and small-angle X-ray scattering methods. It was shown that stable nanoparticles are formed at acidic pH at polymer-to-surfactant molar ratios from 1:43 to 1:139. Trypsin was successfully encapsulated into Eudragit-Brij98 nanoparticles as a model bioactive component. The loading efficiency was determined by labeling trypsin with radioactive iodine-125. Eudragit-Brij98 nanoparticles effectively protected trypsin against pepsin digestion. The results showed that trypsin encapsulated into novel pH-sensitive nanocontainers retained more than 50% of its activity after treatment with pepsin compared with nonencapsulated trypsin. The described concept will contribute both to understanding the principles of and designing next-generation nanocontainers.

Thermodynamic and kinetic analysis of phase separation of temperature-sensitive poly(vinyl methyl ether) in the presence of hydrophobic tert-butyl alcohol

NMR and isothermal titration calorimetry (ITC) techniques were chosen to examine interactions in a poly(vinyl methyl ether) (PVME)/tert-butyl alcohol (t-BuOH)/water ternary system. The effects of PVME and t-BuOH concentrations on phase separation temperature were examined. Molecules of t-BuOH additive hydrophobically associate with PVME and decrease the phase separation temperature. Thermodynamic parameters connected with phase separation were calculated from NMR results using an approach based on the van’t Hoff equation. Presence of t-BuOH increases the number of PVME monomeric units in one cooperative domain (where the units undergo phase separation as whole—‘all-or-none’). NMR time-resolved relaxation measurements show very different dynamics of the solvent releasing process for low and high PVME concentrations above phase separation temperature. ITC data show that the presence of t-BuOH restricts water solvation of PVME globules. Presented results on the PVME/t-BuOH/D2O system show that the PVME solution properties are not constant in time. The analysis of measurements (and resulting properties) should always be done while considering strong time-dependent behaviour of PVME solutions.