16 research outputs found

    Multicompartment Lipid Cubic Nanoparticles with High Protein Upload: Millisecond Dynamics of Formation

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    Membrane shapes, produced by dynamically assembled lipid/protein architectures, are crucial for both physiological functions and the design of therapeutic nanotechnologies. Here we investigate the dynamics of lipid membrane–neurotrophic BDNF protein complexes formation and ordering in nanoparticles, with the purpose of innovation in nanostructure-based neuroprotection and biomimetic nanoarchitectonics. The kinetic pathway of membrane states associated with rapidly occurring nonequilibrium self-assembled lipid/protein nanoarchitectures was determined by millisecond time-resolved small-angle X-ray scattering (SAXS) at high resolution. The neurotrophin binding and millisecond trafficking along the flexible membranes induced an unusual overlay of channel-network architectures including two coexisting cubic lattices epitaxially connected to lamellar membrane stacks. These time-resolved membrane processes, involving intercalation of discrete stiff proteins in continuous soft membranes, evidence stepwise curvature control mechanisms. The obtained three-phase liquid-crystalline nanoparticles of neurotrophic composition put forward important advancements in multicompartment soft-matter nanostructure design

    Synthesis and Solution Properties of PCL‑<i>b</i>‑PHPMA Diblock Copolymers Containing Stable Nitroxyl Radicals

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    This work focuses on the synthesis and the aqueous solution properties of novel amphiphilic PCL-<i>b</i>-PHPMA diblock copolymers possessing 2,2,6,6-tetramethyl­piperidine-1-oxyl (TEMPO) stable radicals covalently conjugated to the hydrophobic poly­(ε-caprolactone) (PCL) block. A new synthetic approach (a four-step pathway) combining ring-opening polymerization (ROP), carbodiimide chemistry (DCC method), a reversible addition–fragmentation chain transfer (RAFT) polymerization technique, and finally chemical oxidation was employed to successfully produce a series of TEMPO-containing PCL-<i>b</i>-PHPMA diblock copolymers for the first time. EPR spectroscopy was applied to verify successful oxidation of the synthesized diblock copolymers and to investigate the dynamics of the polymer chains before and after micellization. The diblock copolymers self-assembled in PBS solution into spherical radical-containing nanoparticles (RNPs), which were characterized by <sup>1</sup>H NMR spectroscopy, dynamic (DLS), static (SLS) light scattering, and cryo-transmission electron microscopy (cryo-TEM). These novel RNPs could find applications, e.g., as drug delivery systems and for the treatment of oxidative stress injuries

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

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    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

    Fluorinated 2‑Alkyl-2-oxazolines of High Reactivity: Spacer-Length-Induced Acceleration for Cationic Ring-Opening Polymerization As a Basis for Triphilic Block Copolymer Synthesis

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    The synthesis of defined triphilic terpolymers with hydrophilic, lyophilic, and fluorophilic blocks is an important challenge as a basis for the development of multicompartment self-assembled structures with potential for, e.g., cascade catalysis and multidrug loading. The synthesis of fluorophilic poly­(2-oxazoline)­s generally suffers from a very low reactivity of fluorinated 2-oxazoline monomers in cationic ring-opening polymerization (CROP). We report a systematic study on overcoming the extremely low reactivity of 2-perfluoroalkyl-2-oxazolines in CROP by the insertion of methyl and ethyl hydrocarbon spacers between the 2-oxazoline ring and the trifluoromethyl group. The kinetic studies showed the gradual increase of the rate of polymerization with increasing of the hydrocarbon spacer length. The monomer with an ethyl spacer was found to have similar reactivity as 2-alkyl-2-oxazolines and allowed the synthesis of defined triphilic triblock copolymers

    DNA/Fusogenic Lipid Nanocarrier Assembly: Millisecond Structural Dynamics

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    Structural changes occurring on a millisecond time scale during uptake of DNA by cationic lipid nanocarriers are monitored by time-resolved small-angle X-ray scattering (SAXS) coupled to a rapid-mixing stopped-flow technique. Nanoparticles (NPs) of nanochannel organization are formed by PEGylation, hydration, and dispersion of a lipid film of the fusogenic lipid monoolein in a mixture with positively charged (DOMA) and PEGylated (DOPE-PEG<sub>2000</sub>) amphiphiles and are characterized by the inner cubic structure of very large nanochannels favorable for DNA upload. Ultrafast structural dynamics of complexation and assembly of these cubosome particles with neurotrophic plasmid DNA (pDNA) is revealed thanks to the high brightness of the employed synchrotron X-ray beam. The rate constant of the pDNA/lipid NP complexation is estimated from dynamic roentgenograms recorded at 4 ms time resolution. pDNA upload into the vastly hydrated channels of the cubosome carriers leads to a fast nanoparticle–nanoparticle structural transition and lipoplex formation involving tightly packed pDNA

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

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    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

    Thermoresponsive Polymer Micelles as Potential Nanosized Cancerostatics

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    An effective chemotherapy for neoplastic diseases requires the use of drugs that can reach the site of action at a therapeutically efficacious concentration and maintain it at a constant level over a sufficient period of time with minimal side effects. Currently, conjugates of high-molecular-weight hydrophilic polymers or biocompatible nanoparticles with stimuli-releasable anticancer drugs are considered to be some of the most promising systems capable of fulfilling these criteria. In this work, conjugates of thermoresponsive diblock copolymers with the covalently bound cancerostatic drug pirarubicin (PIR) were synthesized as a reversible micelle-forming drug delivery system combining the benefits of the above-mentioned carriers. The diblock copolymer carriers were composed of hydrophilic poly­[<i>N</i>-(2-hydroxypropyl)­methacrylamide]-based block containing a small amount (∼5 mol %) of comonomer units with reactive hydrazide groups and a thermoresponsive poly­[2-(2-methoxyethoxy)­ethyl methacrylate] block. PIR was attached to the hydrophilic block of the copolymer through the pH-sensitive hydrazone bond designed to be stable in the bloodstream at pH 7.4 but to be degraded in an intratumoral/intracellular environment at pH 5–6. The temperature-induced conformation change of the thermoresponsive block (coil–globule transition), followed by self-assembly of the copolymer into a micellar structure, was controlled by the thermoresponsive block length and PIR content. The cytotoxicity and intracellular transport of the conjugates as well as the release of PIR from the conjugates inside the cells, followed by its accumulation in the cell nuclei, were evaluated in vitro using human colon adenocarcinoma (DLD-1) cell lines. It was demonstrated that the studied conjugates have a great potential to become efficacious in vivo pharmaceuticals

    Binding of HSA to Macromolecular <i>p</i>HPMA Based Nanoparticles for Drug Delivery: An Investigation Using Fluorescence Methods

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    Amphiphilic poly­(<i>N</i>-(2-hydroxypropyl)­methacrylamide) copolymers (<i>p</i>HPMA) bearing cholesterol side groups in phosphate buffer saline self-assemble into nanoparticles (NPs) which can be used as tumor-targeted drug carriers. It was previously shown by us that human serum albumin (HSA) interacts weakly with the NPs. However, the mechanism of this binding could not be resolved due to overlapping of signals from the complex system. Here, we use fluorescence labeling to distinguish the components and to characterize the binding: On the one hand, a fluorescent dye was attached to <i>p</i>HPMA, so that the diffusion behavior of the NPs could be studied in the presence of HSA using fluorescence lifetime correlation spectroscopy. On the other hand, quenching of the intrinsic fluorescence of HSA revealed the origin of the binding, which is mainly the complexation between HSA and cholesterol side groups. Furthermore, a binding constant was obtained

    Fluorophilic–Lipophilic–Hydrophilic Poly(2-oxazoline) Block Copolymers as MRI Contrast Agents: From Synthesis to Self-Assembly

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    This work focuses on the synthesis and self-assembly of triphilic poly­(2-oxazoline) triblock copolymers with high fluorine content toward our future aim of developing poly­(2-oxazoline) magnetic resonance imaging (MRI) contrast agents. A highly fluorinated 2-substituted-2-oxazoline monomer, namely 2-(1<i>H</i>,1<i>H</i>,2<i>H</i>,2<i>H</i>-perfluorooctyl)-2-oxazoline, was synthesized using the Grignard reaction. The polymerization kinetics of the synthesized monomer was studied, and it was used for the preparation of triblock copolymers with hydrophilic 2-methyl-2-oxazoline, hydrophobic 2-octyl-2-oxazoline, and fluorophilic blocks by cationic ring-opening polymerization yielding polymers with low relatively dispersity (1.2–1.4). The presence of the blocks with the different nature in one copolymer structure facilitated self-assembly of the copolymers in water and dimethyl sulfoxide as observed by dynamic light scattering, cryo-transmission electron microscopy, and small-angle neutron scattering. The nanoparticle morphology is strongly influenced by the order and length of each block and the nature of solvent, leading to nanoparticles with core–shell structure as confirmed by small-angle neutron scattering. The reported poly­(2-oxazoline) block copolymers with high fluorine content have high potential for future development of MRI contrast agents

    Self-Assembly Thermodynamics of pH-Responsive Amino-Acid-Based Polymers with a Nonionic Surfactant

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    The behavior of pH-responsive polymers poly­(<i>N</i>-methacryloyl-l-valine) (P1), poly­(<i>N</i>-methacryloyl-l-phenylalanine) (P2), and poly­(<i>N</i>-methacryloylglycyne-l-leucine) (P3) has been studied in the presence of the nonionic surfactant Brij98. The pure polymers phase-separate in an acidic medium with critical pH<sub>tr</sub> values of 3.7, 5.5, and 3.4, respectively. The addition of the surfactant prevents phase separation and promotes reorganization of polymer molecules. The nature of the interaction between polymer and surfactant depends on the amino acid structure in the side chain of the polymer. This effect was investigated by dynamic light scattering, isothermal titration calorimetry, electrophoretic measurements, small-angle neutron scattering, and infrared spectroscopy. Thermodynamic analysis revealed an endothermic association reaction in P1/Brij98 mixture, whereas a strong exothermic effect was observed for P2/Brij98 and P3/Brij98. Application of regular solution theory for the analysis of experimental enthalpograms indicated dominant hydrophobic interactions between P1 and Brij98 and specific interactions for the P2/Brij98 system. Electrophoretic and dynamic light scattering measurements support the applicability of the theory to these cases. The specific interactions can be ascribed to hydrogen bonds formed between the carboxylic groups of the polymer and the oligo­(ethylene oxide) head groups of the surfactant. Thus, differences in polymer–surfactant interactions between P1 and P2 polymers result in different structures of polymer–surfactant complexes. Specifically, small-angle neutron scattering revealed pearl-necklace complexes and “core–shell” structures for P1/Brij98 and P2/Brij98 systems, respectively. These results may help in the design of new pH-responsive site-specific micellar drug delivery systems or pH-responsive membrane-disrupting agents
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