Search

Naturally occurring oligoamines, such as spermine, spermidine, and putrescine, are well-known regulators of gene expression. These oligoamines frequently have short alkyl spacers with varying lengths between the amines. Linear polyethylenimine (PEI) is a polyamine that has been widely applied as a gene vector, with various formulations currently in clinical trials. In order to emulate natural oligoamine gene regulators, linear random copolymers containing both PEI and polypropylenimine (PPI) repeat units were designed as novel gene delivery agents. In general, statistical copolymerization of 2-oxazolines and 2-oxazines leads to the formation of gradient copolymers. In this study, however, we describe for the first time the synthesis of near-ideal random 2-oxazoline/2-oxazine copolymers through careful tuning of the monomer structures and reactivity as well as polymerization conditions. These copolymers were then transformed into near-random PEI-PPI copolymers by controlled side-chain hydrolysis. The prepared PEI-PPI copolymers formed stable polyplexes with GFP-encoding plasmid DNA, as validated by dynamic light scattering. Furthermore, the cytotoxicity and transfection efficiency of polyplexes were evaluated in C2C12 mouse myoblasts. While the polymer chain length did not significantly increase the toxicity, a higher PPI content was associated with increased toxicity and also lowered the amount of polymers needed to achieve efficient transfection. The transfection efficiency was significantly influenced by the degree of polymerization of PEI-PPI, whereby longer polymers resulted in more transfected cells. Copolymers with 60% or lower PPI content exhibited a good balance between high plasmid-DNA transfection efficiency and low toxicity. Interestingly, these novel PEI-PPI copolymers revealed exceptional serum tolerance, whereby transfection efficiencies of up to 53% of transfected cells were achieved even under 50% serum conditions. These copolymers, especially PEI-PPI with DP500 and a 1:1 PEI/PPI ratio, were identified as promising transfection agents for plasmid DNA.

19F magnetic resonance imaging (MRI) using fluoropolymer tracers has recently emerged as a promising, noninvasive diagnostic tool in modern medicine. However, despite its potential, 19F MRI remains overlooked and underused due to the limited availability or unfavorable properties of fluorinated tracers. Herein, we report a straightforward synthetic route to highly fluorinated 19F MRI nanotracers via aqueous dispersion polymerization- induced self-assembly of a water-soluble fluorinated monomer. A polyethylene glycol-based macromolecular chaintransfer agent was extended by RAFT-mediated N-(2,2,2- trifluoroethyl)acrylamide (TFEAM) polymerization in water, providing fluorine-rich self-assembled nanoparticles in a single step. The resulting nanoparticles had different morphologies and sizes ranging from 60 to 220 nm. After optimizing their structure to maximize the magnetic relaxation of the fluorinated core, we obtained a strong 19F NMR/MRI signal in an aqueous environment. Their non-toxicity was confirmed on primary human dermal fibroblasts. Moreover, we visualized the nanoparticles by 19F MRI, both in vitro (in aqueous phantoms) and in vivo (after subcutaneous injection in mice), thus confirming their biomedical potential., O.S. and V.M.P. acknowledge the financial support from Czech Grant Foundation (grant Nr. 22-03102S) and Charles University Prague (grant PRIMUS/21/SCI/007). D.H. acknowledges the financial support from the Czech Grant Foundation (project number 22-02836S). D.J. acknowledges the financial support from the Ministry of Health of the Czech Republic (NU22-08-00286) and from the project National Institute for Research of Metabolic and Cardiovascular Diseases (Programme EXCELES, P r o j e c t no. LX22NPO5104)Funded by the European UnionNext Generation EU. The authors thank Dr. Carlos V. Melo for editing the manuscript.

In biomedicine, polymer blends are frequently applied in wound dressing design or drug delivery. Within these applications, poly(2-alkyl/aryl-2-oxazoline)s (PAOx) are emerging as a popular matrix d...

Amphiphilic gradient copolymers represent a promising alternative to extensively used block copolymers due to their facile one-step synthesis by statistical copolymerization of monomers of different reactivity. Herein, an in-depth analysis is provided of micelles based on amphiphilic gradient poly(2-oxazoline)s with different chain lengths to evaluate their potential for micellar drug delivery systems and compare them to the analogous diblock copolymer micelles. Size, morphology, and stability of self-assembled nanoparticles, loading of hydrophobic drug curcumin, as well as cytotoxicities of the prepared nanoformulations are examined using copoly(2-oxazoline)s with varying chain lengths and comonomer ratios. In addition to several interesting differences between the two copolymer architecture classes, such as more compact self-assembled structures with faster exchange dynamics for the gradient copolymers, it is concluded that gradient copolymers provide stable curcumin nanoformulations with comparable drug loadings to block copolymer systems and benefit from more straightforward copolymer synthesis. The study demonstrates the potential of amphiphilic gradient copolymers as a versatile platform for the synthesis of new polymer therapeutics. acceptedVersion

In this work, we report our results on the hydrodynamic behavior of poly(2-methyl-2-oxazoline) (PMeOx). PMeOx is gaining significant attention for use as hydrophilic polymer in pharmaceutical carriers as an alternative for the commonly used poly(ethylene glycol) (PEG), for which antibodies are found in a significant fraction of the human population. The main focus of the current study is to determine the hydrodynamic characteristics of PMeOx under physiological conditions, which serves as basis for better understanding of the use of PMeOx in pharmaceutical applications. This goal was achieved by studying PMeOx solutions in phosphate-buffered saline (PBS) as a solvent at 37 °C. This study was performed based on two series of PMeOx samples; one series is synthesized by conventional living cationic ring-opening polymerization, which is limited by the maximum chain length that can be achieved, and a second series is obtained by an alternative synthesis strategy based on acetylation of well-defined linear poly(ethylene imine) (PEI) prepared by controlled side-chain hydrolysis of a defined high molar mass of poly(2-ethyl-2-oxazoline). The combination of these two series of PMeOx allowed the determination of the Kuhn–Mark–Houwink–Sakurada equations in a broad molar mass range. For intrinsic viscosity, sedimentation and diffusion coefficients, the following expressions were obtained: η=0.015M0.77, s0=0.019M0.42 and D0=2600M−0.58, respectively. As a result, it can be concluded that the phosphate-buffered saline buffer at 37 °C represents a thermodynamically good solvent for PMeOx, based on the scaling indices of the equations. The conformational parameters for PMeOx chains were also determined, revealing an equilibrium rigidity or Kuhn segment length, (A) of 1.7 nm and a polymer chain diameter (d) of 0.4 nm. The obtained value for the equilibrium rigidity is very similar to the reported values for other hydrophilic polymers, such as PEG, poly(vinylpyrrolidone) and poly(2-ethyl-2-oxazoline), making PMeOx a relevant alternative to PEG.

Poly(2-alkyl-2-oxazoline)s (PAOx) are regaining interest for biomedical applications. However, their full potential is hampered by the inability to synthesise uniform high-molar mass PAOx. In this work, we proposed alternative intrinsic chain transfer mechanisms based on 2-oxazoline and oxazolinium chain-end tautomerisation and derived improved polymerization conditions to suppress chain transfer, allowing the synthesis of highly defined poly(2-ethyl-2-oxazoline) s up to ca. 50 kDa (dispersity (D)

We describe a new approach to depot drug delivery in which a copolymer poly[N-isopropyl acrylamide-co-N-(3-imidazolylpropyl)methacrylamide] (PNIPAM-co-ImPM) is considered as the main object and matrix for a new formulation strategy that provides the controlled and sustained release of an incorporated drug. The relatively low content of ImPM groups (1.6 mol%) was determined to be sufficient to introduce pH-sensitive behavior to the polymer. Together with NIPAM units, which possess a thermo-sensitive behavior, a dual sensitivity was imparted to the polymer that was investigated by means of turbidimetry and dynamic light scattering. A change in pH from 9 down to 4 was observed to result in the increase of the polymer transition temperature from 32 to 70 °C. The separation process is also accompanied with the formation of ca. 150–300 nm particles while above the transition temperature. The pH value of approximately 6.5 was defined as a boundary value, where certain properties of the system significantly change. This observation assumes a potential attractiveness of the system for biological applications in which injection is possible using a liquid form at pH ca. 5 without the risk of injection needle obstruction. In this way, a depot is formed at the application site upon simultaneously heating to body temperature and increasing the pH to the physiological value of 7.4. An in vivo experiment using the polymer in PBS (pH = 5.0) with paliperidone as a model drug showed excellent results regarding the release of the drug from a depot. The putative mechanism of action for our depot system is thoroughly described in the article.

Herein, we describe a new method for the synthesis of superhydrophilic poly(2-alkyl-2-oxazoline)s (PAOx) from poly(2-ethyl-2-oxazoline) (PEtOx). A well-defined linear polyethylenimine was prepared from PEtOx by controlled acidic hydrolysis of its side-chains followed by reacylation with different carboxylic acids. Using this protocol, we obtained a series of new hydrophilic PAOx containing side-chain ether groups with potential in biomaterials science. The relative hydrophilicity of the polymers was assessed, revealing that poly(2-methoxymethyl-2-oxazoline) (PMeO-MeOx) is the most hydrophilic PAOx reported to date. Additionally, the amorphous poly(2-methoxy-ethoxy-ethoxymethyl-2-oxazoline) (PDEGOx) shows the lowest reported glass transition temperature (-25 degrees C) within the PAOx family to date. The biomedical potential of the prepared polymers was further fortified by an in vitro cytotoxicity study, where all polymers appeared to be noncytotoxic. The described synthetic protocol is universal and can be extremely versatile, especially for PAOx that are difficult to prepare by conventional cationic ring-opening polymerization due to the monomer interference and/or degradation.

Sperm morphology is highly diversified across the animal kingdom and recent comparative evidence from passerine birds suggests that postcopulatory sexual selection is a significant driver of sperm evolution. In the present study, we describe sperm size variation among 20 species of African greenbuls and one bulbul (Passeriformes: Pycnonotidae) and analyze the evolutionary differentiation of sperm size within a phylogenetic framework. We found significant interspecific variation in sperm size; with some genera exhibiting relatively long sperm (e.g. Eurillas) and others exhibiting short sperm head lengths (e.g. Phyllastrephus). However, our results suggest that contemporary levels of sperm competition are unlikely to explain sperm diversification within this clade: the coefficients of inter-male variation (CVbm) in sperm length were generally high, suggesting relatively low and homogeneous rates of extra-pair paternity. Finally, in a comparison of six evolutionary or tree transformation models, we found support for both the Kappa (evolutionary change primarily at nodes) and Lambda (lineage-specific evolutionary rates along branches) models in the evolutionary trajectories of sperm size among species. We therefore conclude that African greenbuls have more variable rates of sperm size evolution than expected from a neutral model of genetic drift. Understanding the evolutionary dynamics of sperm diversification remains a future challenge. The final version of this research has been published in Biological Journal of the Linnean Society. © 2016 Oxford University Press

Inspired by the increasing popularity of poly(2-ethyl-2-oxazoline) (PEtOx) for biomedical applications, this study reports the complete and thorough solution analysis of the homologous series of biocompatible PEtOx samples in a very broad range of molecular weights ranging from 11.2 x 10(3) g mol(-1) up to 260 x 10(3) g mol(-1). The main focus of the research was on the determination of the conformational properties of PEtOx macromolecules at a temperature of 37 degrees C in phosphate buffered saline (PBS) simulating the parameters of physiological media. The polymers were studied in PBS solutions by analytical ultracentrifugation, dynamic light scattering (DLS), translational diffusion, and intrinsic viscosity measurements in a temperature range from 15 degrees C up to 72 degrees C. The complete set of Kuhn-Mark-Houwink-Sakurada scaling relationships revealed linear trends over the whole range of the studied molar masses, while the determined scaling indices at 37 degrees C correspond to the coil conformation in a thermodynamically good solvent ([eta] = 0.045 x M-0.62, s(0) = 0.010 x M-0.46 and D-0 = 1750 x M-0.54). Based on the intrinsic viscosity values (most sensitive characteristic to the size variations of polymer coils, [eta] similar to r(3)), it was demonstrated that PEtOx macromolecules in PBS solutions undergo a transition from swollen polymer coils with gradual deterioration of thermodynamic quality of solutions within the temperature range of 15-45 degrees C, reaching theta-conditions at 55 degrees C with further precipitation at 62-72 degrees C. Also, to the best of our knowledge, the conformational parameters (equilibrium rigidity/the Kuhn segment length and the diameter of the polymer chain) of PEtOx macromolecules were evaluated under physiological conditions for the first time and constitute A = 1.8 +/- 0.3 nm and d = 0.7 +/- 0.4 nm. These equilibrium rigidity values classify PEtOx as a flexible macromolecule with rigidity similar to that of poly(ethylene glycol). For the first time, we were able to demonstrate a direct influence of thermosensitivity on the rigidity of the biocompatible polymer: PEtOx. The Kuhn segment length is undoubtedly decreasing when approaching the LCST.

We designed and synthesized a new delivery system for the anticancer drug doxorubicin based on a biocompatible hydrophilic poly(2-ethyl-2-oxazoline) (PEtOx) carrier with linear architecture and narrow molar mass distribution. The drug is connected to the polymer backbone via an acid-sensitive hydrazone linker, which allows its triggered release in the tumor. The in vitro studies demonstrate successful cellular uptake of conjugates followed by release of the cytostatic cargo. In vivo experiments in EL4 lymphoma bearing mice revealed prolonged blood circulation, increased tumor accumulation and enhanced antitumor efficacy of the PEtOx conjugate having higher molecular weight (40 kDa) compared to the lower molecular weight (20 kDa) polymer. Finally, the in vitro and in vivo anti-cancer properties of the prepared PEtOx conjugates were critically compared with those of the analogous system based on the well-established PHPMA carrier. Despite the relatively slower intracellular uptake of PEtOx conjugates, resulting also in their lower cytotoxicity, there are no substantial differences in in vivo biodistribution and anti-cancer efficacy of both classes of polymer-Dox conjugates. Considering the synthetic advantages of poly(2-alkyl-2-oxazoline)s, the presented study demonstrates their potential as a versatile alternative to well-known PEO- or PHPMA-based materials for construction of drug delivery systems.

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.

Auger electrons-emitting radioisotopes (such as iodine-125) are a potentially effective cancer treatment. They are extremely biologically effective, but only within a short range (nanometers). Their use as an effective cancer therapy requires that they will be transported within close proximity of DNA by an intercalator, where they induce double-strand breaks leading to cell death. This type of therapy may be even more beneficial when associated with drug delivery systems. In this report, we describe an optimized triple-targeted polymer delivery system for the intercalator ellipticine, which contains radioisotope iodine-125 with high specific radioactivity (63.2 GBq/mg). This compound is linked to an N-(2-hydroxypropyl)methacrylamide copolymer via an optimized acid-sensitive hydrazone linker. The system is stable at pH 7.4 (representing the pH of blood plasma), and the radioiodine-containing biologically active intercalator is released upon a decrease in pH (44% of the intercalator is released after 24 h of incubation in pH 5.0 buffer, which mimics the pH in late endosomes). The active compound is a potent intercalator, as shown with direct titration with a DNA solution, and readily penetrates into cell nuclei, as observed by confocal microscopy. Its polymer conjugate is internalized into endosomes and releases the radioactive intercalator, which accumulates in the cell nuclei. In vivo experiments on mice with 4T1 murine breast cancer resulted in a statistically significant increase in the survival of mice treated with the polymer radioconjugate. The free radiolabeled intercalator was also shown to be effective, but it was less potent than the polymer conjugate.

Poly(2-oxazoline)s (in literature abbreviated as PAOx, POx, or POZ, herein referred to as PAOx) represent an emerging class of biocompatible polymers outperforming polyethylene glycol (PEG) in many aspects, including their high synthetic versatility and structural modularity. In this review, we provide a brief introduction to PAOx chemistry and biology to sketch their potential in biomaterials science. Further, we provide a detailed comprehensive overview of the literature on PAOx-protein conjugates with emphasis on their critical evaluation and comparison with analogous systems based on PEG. Based on this literature overview, PAOx seem to be an excellent alternative to PEG in the construction of therapeutic polymer-protein conjugates.

Tropical forests store 40–50 per cent of terrestrial vegetation carbon 1. However, spatial variations in aboveground live tree biomass carbon (AGC) stocks remain poorly understood, in particular in tropical montane forests 2. Owing to climatic and soil changes with increasing elevation 3, AGC stocks are lower in tropical montane forests compared with lowland forests 2. Here we assemble and analyse a dataset of structurally intact old-growth forests (AfriMont) spanning 44 montane sites in 12 African countries. We find that montane sites in the AfriMont plot network have a mean AGC stock of 149.4 megagrams of carbon per hectare (95% confidence interval 137.1–164.2), which is comparable to lowland forests in the African Tropical Rainforest Observation Network 4 and about 70 per cent and 32 per cent higher than averages from plot networks in montane 2,5,6 and lowland 7 forests in the Neotropics, respectively. Notably, our results are two-thirds higher than the Intergovernmental Panel on Climate Change default values for these forests in Africa 8. We find that the low stem density and high abundance of large trees of African lowland forests 4 is mirrored in the montane forests sampled. This carbon store is endangered: we estimate that 0.8 million hectares of old-growth African montane forest have been lost since 2000. We provide country-specific montane forest AGC stock estimates modelled from our plot network to help to guide forest conservation and reforestation interventions. Our findings highlight the need for conserving these biodiverse 9,10 and carbon-rich ecosystems.