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.

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.

Searching for combination therapy by clustering methods: Stimulation of PKC in Golgi apparatus combined with hypericin induced PDT.

Cancer cell metabolism is a very attractive target for anticancer treatments. This work focuses on protein kinase C (PKC) signaling in the U87 MG glioma. By means of western blot, fluorescence and time-resolved fluorescence microscopy the correlation between the Golgi apparatus (GA), lysosomes and mitochondria were evaluated. The known regulators of PKC were applied to cancer cells. Phorbol myristate acetate (PMA) was chosen as the activator of PKC. Gö6976, hypericin and rottlerin, the inhibitors of PKCα and PKCδ were selected as well. Stabilization, destabilization processes occurring in cells allow classification of observations into several groups. Multiple versions of hierarchical cluster analysis have been applied and similarities have been found between organelles and PKC regulators. The method identified GA as an extraordinary organelle whose functionality is significantly influenced by PKC regulators as well as oxidative stress. Therefore, combination therapy has been designed according to the results of the cluster analysis. Furthermore, the efficacy of photodynamic therapy mediated by hypericin, and the consequent apoptosis, was significantly increased during the treatment. To our knowledge, this is the first demonstration of the effectiveness of the clustering in the given area.

Benefits of hypericin transport and delivery by low- and high-density lipoproteins to cancer cells: From in vitro to ex ovo.

Lipoproteins are very attractive natural-based transport systems suitable for applications in diagnostics and cancer therapy. Low- and high-density lipoproteins (LDL, HDL) were selected for hypericin (hyp) delivery in cancer cells. Hyp was used, as it is a well-known model for hydrophobic molecules, in order to estimate the LDL and HDL transport efficacy. We applied fluorescence techniques, absorption and Raman spectroscopy to characterize the state and alteration of LDL and HDL in the absence and presence of hyp. The fluorescence intensity of hyp loaded in lipoproteins was two times weaker in HDL than LDL. We demonstrated that there are faster redistribution kinetics of hyp from HDL than from LDL. As a consequence, hyp uptake by glioma and breast cancer cells was driven more via endocytosis when hyp was delivered by LDL than by HDL. Hyp induced photodynamic action was stronger when hyp was delivered by HDL than LDL. Ex ovo hyp fluorescence pharmacokinetics demonstrated differences in biodistributions of hyp in lipoproteins topical applications. However, hyp was successfully delivered to cancer cells grafted on quail's chorioallantoic membrane. The results presented in this paper could provide strategies to develop adequate and targeted anticancer therapy.

Advances in the Study of Cerium Oxide Nanoparticles: New Insights into Antiamyloidogenic Activity.

There seems to be general agreement that oxidative stress is involved in many pathological conditions including Parkinson's, Alzheimer's, and other neurodegenerative diseases, and overall aging. Cerium oxide nanoparticles, also known as nanoceria (CeO2-NPs), have shown promise as catalytic antioxidants, based on their ability to switch between Ce3+ and Ce4+ valence states. In the present work we have synthesized and characterized CeO2-NPs, examined the effect of CeO2-NPs on amyloidogenesis of insulin, and analyzed the impact of CeO2-NPs on oxidative stress and biocompatibility in vitro in three types of invasive cancer cells, and in vivo in the preclinical model of the chorioallantoic membrane (CAM) of quail embryos. The different experimental techniques revealed a high stability and homogeneity of the "naked" CeO2-NPs synthesized by precipitation from a reversal microemulsion. The CeO2-NPs were 5-6 nm in diameter (TEM) and monodispersed and have a ζ +46.9 mV ζ potential in Milli-Q water. We demonstrated for the first time that CeO2-NPs affect insulin fibrillation in a dose-dependent manner. The inhibiting, IC50, and disassembling, DC50, concentrations were calculated to be ∼100 ± 3.5 and ∼200 ± 5.5 µg/mL, respectively. Furthermore, CeO2-NPs demonstrated reliable biocompatibility and sufficient uptake by glioma and breast cancer cells. The presence of a high concentration of CeO2-NPs within the cells resulted only in local changes in metabolic activity and generation of oxidative stress at a low level. Moreover, high biocompatibility with CeO2-NPs was shown in vivo in the CAM.

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.

In vitro identification of mitochondrial oxidative stress production by time-resolved fluorescence imaging of glioma cells.

Oxidative phosphorylation and glycolysis are important features, by which cells could bypass oxidative stress. The level of oxidative stress, and the ability of cells to promote oxidative phosphorylation or glycolysis, significantly determined proliferation or cell demise. In the present work, we have employed selective mitochondrial probe MitoTracker™ Orange CMTM/Ros (MTO) to estimate the level of oxidative stress in cancer cells at different stressed conditions. MTO is partially sensitive to decrease of mitochondrial membrane potential and to reactive oxygen species (ROS) generated in mitochondria. We have demonstrated, that fluorescence lifetime of MTO is much more sensitive to oxidative stress than intensity-based approaches. This method was validated in different cancer cell lines. Our approach revealed, at relatively low ROS levels, that Gö 6976, a protein kinase C (PKC) α inhibitor, and rottlerin, an indirect PKCδ inhibitor, increased mitochondrial ROS level in glioma cell. Their involvement in oxidative phosphorylation and apoptosis was investigated with oxygen consumption rate estimation, western blot and flow-cytometric analysis. Our study brings new insight to identify feeble differences in ROS production in living cells.