Influence of Hydrophobic Side-Chain Length in Amphiphilic Gradient Copoly(2-oxazoline)s on the Therapeutics Loading, Stability, Cellular Uptake and Pharmacokinetics of Nano-Formulation with Curcumin.

Due to the simple one-step preparation method and a promising application in biomedical research, amphiphilic gradient copoly(2-oxazoline)s are gaining more and more interest compared to their analogous block copolymers. In this work, the curcumin solubilization ability was tested for a series of amphiphilic gradient copoly(2-oxazoline)s with different lengths of hydrophobic side-chains, consisting of 2-ethyl-2-oxazoline as a hydrophilic monomer and 2-(4-alkyloxyphenyl)-2-oxazoline as a hydrophobic monomer. It is shown that the length of the hydrophobic side-chain in the copolymers plays a crucial role in the loading of curcumin onto the self-assembled nanoparticles. The kinetic stability of self-assembled nanoparticles studied using FRET shows a link between their integrity and cellular uptake in human glioblastoma cells. The present study demonstrates how minor changes in the molecular structure of gradient copoly(2-oxazoline)s can lead to significant differences in the loading, stability, cytotoxicity, cellular uptake, and pharmacokinetics of nano-formulations containing curcumin. The obtained results on the behavior of the complex of gradient copoly(2-oxazoline)s and curcumin may contribute to the development of effective next-generation polymeric nanostructures for biomedical applications.

Vibrational characterization of the pesticide molecule Tebuconazole.

Pesticide use worldwide exhibits a positive effect on agricultural production while it may negatively affect organisms living in soil, water or the air. Importantly, numerous negative health effects also occur in humans exposed to (accumulated) pesticides or their metabolites over a long period of time. To prevent both environmental catastrophes and adverse human health impacts, initial studies of the selected pesticides need to be performed together with the constant post-approval control; risk assessment analysis and on site monitoring have to be continuously carried out. Given this, Raman spectroscopy, especially surface-enhanced Raman spectroscopy (SERS), during the last decade has become a powerful analytical technique since it can offer quick, selective, and in situ detection of selected pollutants found in analyzed samples at very low concentrations. Moreover, the structural changes caused by the pollutant-biomacromolecule interaction can also be recognized in the molecule-specific Raman spectral signatures of biomolecules. In this study, we report a vibrational characterization of the fungicide molecule Tebuconazole (TB) which is listed to be a possible carcinogen. Even though its international and common use there is no evidence about the use of Raman/SERS spectroscopy to detect it sensitively and selectively as well as to analyse its impacts on biological systems. Therefore, we have recorded and calculated Raman and infrared spectra of TB. Furthermore, SERS spectra of TB were also registered and comprehensively analysed in view of the employed SERS substrates, dependence on the excitation wavelengths and pH of the analysed molecular systems. The molecule of TB interacts preferentially through the triazole moiety with the colloidal metal nanoparticles (NPs) whereas the silver NPs prepared by reduction of silver nitrate with hydroxylamine hydrochloride resulted to be the most effective ones. Consequently, the limit of detection was determined to be 1.4 µM≈430 ppb. The present paper thus could serve significantly for further investigations focused on both conducting vibrational analyses of structurally related molecules as well as providing a more precise explanation of the mechanism of action of TB and its influence on biological macromolecules.

Chromosomal Aberrations in Cattle.

Chromosomal aberrations and their mechanisms have been studied for many years in livestock. In cattle, chromosomal abnormalities are often associated with serious reproduction-related problems, such as infertility of carriers and early mortality of embryos. In the present work, we review the mechanisms and consequences of the most important bovine chromosomal aberrations: Robertsonian translocations and reciprocal translocations. We also discuss the application of bovine cell cultures in genotoxicity studies.

Alkyl Chain Length in Poly(2-oxazoline)-Based Amphiphilic Gradient Copolymers Regulates the Delivery of Hydrophobic Molecules: A Case of the Biodistribution and the Photodynamic Activity of the Photosensitizer Hypericin.

Self-assembled nanostructures of amphiphilic gradient copoly(2-oxazoline)s have recently attracted attention as promising delivery systems for the effective delivery of hydrophobic anticancer drugs. In this study, we have investigated the effects of increasing hydrophobic side chain length on the self-assembly of gradient copolymers composed of 2-ethyl-2-oxazoline as the hydrophilic comonomer and various 2-(4-alkyloxyphenyl)-2-oxazolines as hydrophobic comonomers. We show that the size of the formed polymeric nanoparticles depends on the structure of the copolymers. Moreover, the stability and properties of the polymeric assembly can be affected by the loading of hypericin, a promising compound for photodiagnostics and photodynamic therapy (PDT). We have found the limitation that allows rapid or late release of hypericin from polymeric nanoparticles. The nanoparticles entering the cells by endocytosis decreased the hypericin-induced PDT, and the contribution of the passive process (diffusion) increased the probability of a stronger photoeffect. A study of fluorescence pharmacokinetics and biodistribution revealed differences in the release of hypericin from nanoparticles toward the quail chorioallantoic membrane, a preclinical model for in vivo studies, depending on the composition of polymeric nanoparticles. Photodamage induced by PDT in vivo well correlated with the in vitro results. All formulations studied succeeded in targeting hypericin at cancer cells. In conclusion, we demonstrated the promising potential of poly(2-oxazoline)-based gradient copolymers for effective drug delivery and sequential drug release needed for successful photodiagnostics and PDT in cancer therapy.

Encapsulation of anticancer drug curcumin and co-loading with photosensitizer hypericin into lipoproteins investigated by fluorescence resonance energy transfer.

Low-density lipoproteins (LDL) and high-density lipoproteins (HDL) are natural occurring vehicles attractive for drug delivery and targeting tumor cells. Here we have investigated the encapsulation and interaction of a well-known anticancer agent curcumin with LDL and HDL. LDL particles have been found to accumulate more curcumin molecules inside their structure than HDL. The chemical stability of curcumin is enhanced and its photo-physical properties are altered due to encapsulation inside both lipoproteins. Combining photodynamic therapy with chemotherapy can improve anticancer treatment by overcoming drug resistance in cancer therapy. Therefore, we have also investigated a co-loading of curcumin with a natural potent photosensitizer hypericin into molecules of LDL using fluorescence resonance energy transfer. The loading patterns of curcumin and hypericin into LDL particles were found to be different as revealed by the fluorescence resonance energy transfer experiments. Present study illustrates the potential of LDL nanoparticles in combination therapy because of simultaneous loading of more than one type of drugs into these nanoparticles with high level of efficiency.

Phosphorescence Kinetics of Singlet Oxygen Produced by Photosensitization in Spherical Nanoparticles. Part II. The Case of Hypericin-Loaded Low-Density Lipoprotein Particles.

The phosphorescence kinetics of singlet oxygen produced by photosensitized hypericin (Hyp) molecules inside low-density lipoprotein (LDL) particles was studied experimentally and by means of numerical and analytical modeling. The phosphorescence signal was measured after short laser pulse irradiation of aqueous Hyp/LDL solutions. The Hyp triplet state lifetime determined by a laser flash-photolysis measurement was 5.3 × 10-6 s. The numerical and the analytical model described in part I of the present work (DOI: 10.1021/acs.jpcb.8b00658) were used to analyze the observed phosphorescence kinetics of singlet oxygen. It was shown that singlet oxygen diffuses out of LDL particles on a time scale shorter than 0.1 µs. The total (integrated) concentration of singlet oxygen inside LDL is more than an order of magnitude smaller than the total singlet oxygen concentration in the solvent. The time course of singlet oxygen concentrations inside and outside the particles was calculated using simplified representations of the LDL internal structure. The experimental phosphorescence data were fitted by a linear combination of these concentrations using the emission factor E (the ratio of the radiative singlet oxygen depopulation rate constants inside and outside LDL) as a fitting parameter. The emission factor was determined to be E = 6.7 ± 2.5. Control measurements were carried out by adding sodium azide, a strong singlet oxygen quencher, to the solution.

Unravelling the Excellent Chemical Stability and Bioavailability of Solvent Responsive Curcumin-Loaded 2-Ethyl-2-oxazoline-grad-2-(4-dodecyloxyphenyl)-2-oxazoline Copolymer Nanoparticles for Drug Delivery.

A new gradient copolymer has been synthesized by the living cationic ring-opening polymerization of hydrophilic 2-ethyl-2-oxazoline with lipophilic 2-(4-dodecyloxyphenyl)-2-oxazoline (EtOx-grad-DPOx). The prepared copolymer is capable of assembling in water to yield polymeric nanoparticles that are successfully loaded with an anticancer agent, curcumin. Self-assembly of the copolymer was found to be tuned by the polarity as well as the hydrogen bonding ability of solvents. Solvent took distinctive role in the preparation of unloaded and curcumin-loaded nanoparticles. The stability of the nanoparticles was increased by curcumin loading promoted by curcumin-polymer interactions. Further, the chemical stability of curcumin in water is largely enhanced inside the polymeric nanoparticles. Curcumin-loaded (EtOx-grad-DPOx) copolymer nanoparticles showed excellent stability in the biological medium, low cytotoxicity, and concentration dependent uptake by U87 MG and HeLa cells, which indicate the possibility of their efficient application in drug delivery.