Echogenic Gold Nanorod Incorporated Hybrid Poly(2-oxazoline) Nanocapsules for Real-Time Ultrasound/Fluorescent Imaging and Targeted Cancer Theranostics.

In recent years, various nanocarrier systems have been explored to enhance the targeting of cancer cells by improving the ligand-receptor interactions between the nanocarrier and cancer cells for selective cancer cell imaging and targeted delivery of anticancer drugs. Herein, we report multifunctional hydrogen-bonded multilayer nanocapsules functionalized with both folic acid-derived quantum dots (FAQDs) and gold nanorods (AuNRs) for targeted cancer therapy and cancer cell imaging using fluorescence microscopy and medical-range ultrasound imaging systems. The encapsulation efficiency of nanocapsules was found to be 49% for 5-fluorouracil (5-FU). The release percentage reached a plateau at 37% after 1 h at pH 7.4 and increased to 57% after 3 h when the release pH was decreased to pH 5.5 (i.e., the pH of the tumor environment). Under ultrasound irradiation, the release was significantly accelerated, with a total release of 52% and 68% after only 6 min at pH 7.4 and pH 5.5, respectively. While the sonoporation process plays an important role in anticancer activity experiments under ultrasound exposure by generating temporary pores, the targeting ability of FAQDs brings the capsules closer to the cell membrane and improves the cellular uptake of the released drug, thereby increasing local drug concentration. In vitro cytotoxicity experiments with HCT-116 and HEp-2 cells demonstrated anticancer activities of 96% and 98%, respectively. The nanocapsules showed enhanced ultrasound scattering signal intensity and bright spots under ultrasound exposure, most likely caused by high scattering ability and internal reflections of preloaded AuNRs in the interior structure of the nanocapsules. Hence, the demonstrated nanocapsule system not only has the potential to be used as an integrated system for early- stage detection and treatment of cancer cells but also has the ability for live tracking and imaging of cancer cells while undergoing treatment with chemotherapy and radiation therapy.

Poly(2-alkyl-2-oxazoline)s: A polymer platform to sustain the release from tablets with a high drug loading.

Sustaining the release of highly dosed APIs from a matrix tablet is challenging. To address this challenge, this study evaluated the performance of thermoplastic poly (2-alkyl-2-oxazoline)s (PAOx) as matrix excipient to produce sustained-release tablets via three processing routes: (a) hot-melt extrusion (HME) combined with injection molding (IM), (b) HME combined with milling and compression and (c) direct compression (DC). Different PAOx (co-)polymers and polymer mixtures were processed with several active pharmaceutical ingredients having different aqueous solubilities and melting temperatures (metoprolol tartrate (MPT), metformin hydrochloride (MTF) and theophylline anhydrous (THA)). Different PAOx grades were synthesized and purified by the Supramolecular Chemistry Group, and the effect of PAOx grade and processing technique on the in vitro release kinetics was evaluated. Using the hydrophobic poly (2-n-propyl-2-oxazoline) (P n PrOx) as a matrix excipient allowed to sustain the release of different APIs, even at a 70% (w/w) drug load. Whereas complete THA release was not achieved from the P n PrOx matrix over 24 â€‹h regardless of the processing technique, adding 7.5% w/w of the hydrophilic poly (2-ethyl-2-oxazoline) to the hydrophobic P n PrOx matrix significantly increased THA release, highlighting the relevance of mixing different PAOx grades. In addition, it was demonstrated that the release of THA was similar from co-polymer and polymer mixtures with the same polymer ratios. On the other hand, as the release of MTF from a P n PrOx matrix was fast, the more hydrophobic poly (2-sec-butyl-2-oxazoline) (P sec BuOx) was used to retard MTF release. In addition, a mixture between the hydrophilic PEtOx and the hydrophobic P sec BuOx allowed accurate tuning of the release of MTF formulations. Finally, it was demonstrated that PAOx also showed a high ability to tune the in vivo release. IM tablets containing 70% MTF and 30% P sec BuOx showed a lower in vivo bioavailability compared to IM tablets containing a low PEtOx concentration (7.5%, w/w) in combination with P sec BuOx (22.5%, w/w). Importantly, the in vivo MTF blood level from the sustained release tablets correlated well with the in vitro release profiles. In general, this work demonstrates that PAOx polymers offer a versatile formulation platform to adjust the release rate of different APIs, enabling sustained release from tablets with up to 70% w/w drug loading.

Stable amorphous solid dispersion of flubendazole with high loading via electrospinning.

In this work, an important step is taken towards the bioavailability improvement of poorly water-soluble drugs, such as flubendazole (Flu), posing a challenge in the current development of many novel oral-administrable therapeutics. Solvent electrospinning of a solution of the drug and poly (2-ethyl-2-oxazoline) (PEtOx) is demonstrated to be a viable strategy to produce stable nanofibrous amorphous solid dispersions (ASDs) with ultrahigh drug-loadings (up to 55 wt% Flu) and long-term stability (at least one year). Importantly, at such high drug loadings, the concentration of the polymer in the electrospinning solution has to be lowered below the concentration where it can be spun in absence of the drug as the interactions between the polymer and the drug result in increased solution viscosity. A combination of experimental analysis and molecular dynamics simulations revealed that this formulation strategy provides strong, dominant and highly stable hydrogen bonds between the polymer and the drug, which is crucial to obtain the high drug-loadings and to preserve the long-term amorphous character of the ASDs upon storage. In vitro drug release studies confirm the remarkable potential of this electrospinning formulation strategy by significantly increased drug solubility values and dissolution rates (respectively tripled and quadrupled compared to the crystalline drug), even after storing the formulation for one year.

Tannic Acid-Stabilized Self-Degrading Temperature-Sensitive Poly(2-n-propyl-2-oxazoline)/Gellan Gum Capsules for Lipase Delivery.

In recent years, stable hydrogen-bonded stimuli-responsive polymer capsules have been receiving great interest for the encapsulation and release of sensitive molecules such as lipase enzymes. Compartmental capsules having a liquid gel core stabilized with temperature-responsive hydrogen-bonded multilayers are advantageous over other conventional systems because of their ability to maintain hydrophilic lipase and other hydrophobic compounds in compatible protected molecular vehicle environments and prolong their native properties, e.g., in the body. In this work, we report a methodology to stabilize an aqueous liquid gellan gum (GG) core in a capsule using neutral and nontoxic building blocks, namely, poly(2-n-propyl-2-oxazoline) (PnPrOx) and tannic acid (TA), to fabricate temperature-responsive capsules, comprising both lipase and hydrophobic oil droplets. The capsules were fabricated by adding GG droplets to a PnPrOx suspension at a temperature (T) higher than its cloud point temperature (TCP). Notably, the formed capsules were not stable in water without TA stabilization via hydrogen bonding. Scanning electron microscopy (SEM) investigations of the GG/building block interphase revealed that the collapsed PnPrOx globules that are present above the TCP stabilized the GG interphase as a Pickering emulsion, while undergoing a configurational transformation into its linear form by interacting with TA in the next step of capsule formation resulting in a smooth PnPrOx/TA capsule wall. The encapsulation efficiencies of the capsules for model fluorescent molecules were found to be 52, 54, and 24% for FITC-dextran, rhodamine, and Nile red, respectively. The stability experiments exhibited swelling and shell thinning at certain locations followed by complete rupture of the capsules at 37 °C, while the capsules were stable for several weeks at temperatures below the TCP of PnPrOx. The capsules were found to be stable in stimulated gastric fluid (SGF) for several hours at 37 °C while successfully releasing the encapsulated lipase and Nile red (model hydrophobic compound) in stimulated intestinal fluid (SIF). The released lipase was found to retain almost 100% of its activity. The reported capsules have high potential for use as carriers for encapsulation and release of a variety of payloads ranging from proteins and vitamin supplements to enzymes and probiotics through the oral route of administration.

Hydrogen-Bonded Multilayer Thin Films and Capsules Based on Poly(2-n-propyl-2-oxazoline) and Tannic Acid: Investigation on Intermolecular Forces, Stability, and Permeability.

In recent years, hydrogen-bonded multilayer thin films and capsules based on neutral and nontoxic building blocks have been receiving interest for the design of stimuli-responsive drug delivery systems and for the preparation of thin-film coatings. Capsule systems made of tannic acid (TA), a natural polyphenol, as a hydrogen bonding donor and poly(2-n-propyl-2-oxazoline) (PnPropOx), a polymer with lower critical solution temperature around 25 °C, as a hydrogen bonding acceptor are advantageous over other conventional hydrogen-bonded systems because of their high stability in physiological pH range, biocompatibility, good renal clearance, stealth behavior, and stimuli responsiveness for temperature and pH. In this work, investigations on the interactive forces in TA/PnPropOx capsule formation, film thickness, stability, and permeability are reported. The multilayer thin films were assembled on quartz substrates, and the layer-by-layer film growth was investigated by UV-vis spectroscopy, atomic force microscopy, and profilometry. Hollow capsules were fabricated by sequential coating of TA and PnPropOx onto CaCO3 colloidal particles, followed by template dissolution with a 0.2 M ethylenediaminetetraacetic acid solution. The obtained capsules and multilayer thin films were found to be stable over a wide pH range of 2-9. It is found that both hydrogen bonding and hydrophobic interactions are responsible for the enhanced stability of the capsules at higher pH range. Swelling followed by dissolution of the capsules was observed at a pH value lower than 2, while the capsules undergo shrinking at a pH value higher than 8 and finally transform into a particle-like morphology before dissolution. The TA/PnPropOx capsules reported here could be used as a temperature-responsive drug delivery system in controlled drug delivery applications.

Comparative study of the potential of poly(2-ethyl-2-oxazoline) as carrier in the formulation of amorphous solid dispersions of poorly soluble drugs.

Despite the fact that solid dispersions are gaining momentum, the number of polymers that have been used as a carrier during the past 50 years is rather limited. Recently, the poly(2-alkyl-2-oxazoline) (PAOx) polymer class profiled itself as a versatile platform for a wide variety of applications in drug delivery, including their use as amorphous solid dispersion (ASD) carrier. The aim of this study was to investigate the potential of poly(2-ethyl-2-oxazoline) (PEtOx) by applying a benchmark approach with well-known, commercially available carriers (i.e. polyvinylpyrrolidone (PVP) K30, poly(vinylpyrrolidone-co-vinyl acetate) (PVP-VA) 64 and hydroxypropylmethylcellulose (HPMC)). For this purpose, itraconazole (ITC) and fenofibrate (FFB) were selected as poorly water-soluble model drugs. The four polymers were compared by establishing their supersaturation maintaining potential and by investigating their capability as carrier for ASDs with high drug loadings. Spray drying, as well as hot melt extrusion and cryo-milling were implemented as ASD manufacturing technologies for comparative evaluation. For each manufacturing technique, the formulations with the highest possible drug loadings were tested with respect to in vitro drug release kinetics. This study indicates that PEtOx is able to maintain supersaturation of the drugs to a similar extent as the commercially available polymers and that ASDs with comparable drug loadings can be manufactured. The results of the in vitro dissolution tests reveal that high drug release can be obtained for PEtOx formulations. Overall, proof-of-concept is provided for the potential of PEtOx for drug formulation purposes.

Influence of the Aliphatic Side Chain on the Near Atmospheric Pressure Plasma Polymerization of 2-Alkyl-2-oxazolines for Biomedical Applications.

Plasma polymerization is gaining popularity as a technique for coating surfaces due to the low cost, ease of operation, and substrate-independent nature. Recently, the plasma polymerization (or deposition) of 2-oxazoline monomers was reported resulting in coatings that have potential applications in regenerative medicine. Despite the structural versatility of 2-oxazolines, only a few monomers have been subjected to plasma polymerization. Within this study, however, we explore the near atmospheric pressure plasma polymerization of a range of 2-oxazoline monomers, focusing on the influence of the aliphatic side-chain length (methyl to butyl) on the plasma polymerization process conditions as well as the properties of the obtained coatings. While side-chain length had only a minor influence on the chemical composition, clear effects on the plasma polymerization conditions were observed, thus gaining valuable insights in the plasma polymerization process as a function of monomer structure. Additionally, cytocompatibility and cell attachment on the coatings obtained by 2-oxazoline plasma polymerization was assessed. The coatings displayed strong cell interactive properties, whereby cytocompatibility increased with increasing aliphatic side-chain length of the monomer, reaching up to 93% cell viability after 1 day of cell culture compared to tissue culture plates. As this is in stark contrast to the antifouling behavior of the parent polymers, we compared the properties and composition of the plasma-polymerized coatings to the parent polymers revealing that a significantly different coating structure was obtained by plasma polymerization.

Poly(2-ethyl-2-oxazoline) conjugates with doxorubicin for cancer therapy: In vitro and in vivo evaluation and direct comparison to poly[N-(2-hydroxypropyl)methacrylamide] analogues.

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.

Hydrogen bonded capsules by layer-by-layer assembly of tannic acid and poly(2-n-propyl-2-oxazoline) for encapsulation and release of macromolecules.

We report hydrogen bonded capsules with the built-in ability to release loaded bioactive molecules at a physiological temperature of 37 °C. The use of neutral and non-toxic building blocks such as tannic acid (TA) and poly(2-n-propyl-2-oxazoline)s (PnPropOx) as hydrogen bonding donor and acceptor results in stable hollow capsules. The temperature induced morphological changes of the shell were investigated using a scanning electron microscope and an optical microscope and revealed pore formation in the shell when the temperature (T) increases beyond the cloud point temperature (TCP) of PnPropOx. Furthermore, confocal laser scanning microscopic investigation of the hollow capsules loaded with different probes of varying hydrodynamic diameters revealed that the open and closed state of the capsules could be effectively manipulated by varying the incubation time and hydrodynamic radius of the probes. Such hydrogen bonded capsules have high potential for use in temperature responsive sustained drug delivery applications.

Ultra-high performance size-exclusion chromatography in polar solvents.

Size-exclusion chromatography (SEC) is amongst the most widely used polymer characterization methods in both academic and industrial polymer research allowing the determination of molecular weight and distribution parameters, i.e. the dispersity (Ɖ), of unknown polymers. The many advantages, including accuracy, reproducibility and low sample consumption, have contributed to the worldwide success of this analytical technique. The current generation of SEC systems have a stationary phase mostly containing highly porous, styrene-divinylbenzene particles allowing for a size-based separation of various polymers in solution but limiting the flow rate and solvent compatibility. Recently, sub-2µm ethylene-bridged hybrid (BEH) packing materials have become available for SEC analysis. These packing materials can not only withstand much higher pressures up to 15000psi but also show high spatial stability towards different solvents. Combining these BEH columns with the ultra-high performance LC (UHPLC) technology opens up UHP-SEC analysis, showing strongly reduced runtimes and unprecedented solvent compatibility. In this work, this novel characterization technique was compared to conventional SEC using both highly viscous and highly polar solvents as eluent, namely N,N-dimethylacetamide (DMAc), N,N-dimethylformamide (DMF) and methanol, focusing on the suitability of the BEH-columns for analysis of highly functional polymers. The results show a high functional group compatibility comparable with conventional SEC with remarkably short runtimes and enhanced resolution in methanol.

Sulfolane as Common Rate Accelerating Solvent for the Cationic Ring-Opening Polymerization of 2-Oxazolines.

The search for alternative solvents for the cationic ring-opening polymerization (CROP) of 2-methyl-2-oxazoline (MeOx) is driven by the poor solubility of P(MeOx) in polymerization solvents such as acetonitrile (CH3CN) and chlorobenzene as well as in MeOx itself. In this study, solvent screening has revealed that especially sulfolane is a good solvent for PMeOx. Unexpectedly, an increased propagation rate constant (kp) was found for the CROP of MeOx in sulfolane. Further extended kinetic studies at different temperatures (60-180 °C), revealed that the acceleration is due to an increase in frequency factor, while the activation energy (Ea) of the reaction is hardly affected. In order to explore the versatility of sulfolane as polymerization solvent for the CROP of 2-oxazolines in general, also the polymerization kinetics of other 2-oxazoline monomers, such as 2-ethyl-2-oxazoline (EtOx) and 2-phenyl-2-oxazoline (PhOx), have been studied, revealing a common acceleration of the CROP of 2-oxazoline monomers in sulfolane. This also enabled more controlled synthesis of PMeOx-block-PPhOx block copolymers that otherwise suffers from solvent incompatibility.

Hydrogen bonded multilayer films based on poly(2-oxazoline)s and tannic acid.

In recent years, the layer-by-layer (LbL) assembly based on hydrogen bonding interactions is gaining popularity for the preparation of thin film coatings, especially for biomedical purposes, based on the use of neutral, non-toxic building blocks. The use of tannic acid (TA) as hydrogen bonding donor is especially interesting as it results in LbL films that are stable under physiological conditions. In this work, investigations on the LbL thin film preparation of TA with poly(2-oxazoline)s with varying hydrophilicity, namely poly(2-methyl-2-oxazoline) (PMeOx), poly(2-ethyl-2-oxazoline) (PEtOx) and poly(2-n-propyl-2-oxazoline) (PnPropOx), are reported. The LbL assembly process is investigated by quartz crystal microbalance and UV-vis spectroscopy revealing linear growth of the film thickness. Furthermore, isothermal titration calorimetry demonstrates the LbL assembly of TA, and PMeOx is found to be mostly enthalpy driven while the LbL assembly of TA with PEtOx and PnPropOx is mostly entropy driven. Finally, scanning electron microscopy and ellipsometry demonstrate the formation of smooth thin films for LbL assembly of TA with all three polymers. Such poly(2-oxazoline) coatings have high potential for use as anti-biofouling coatings.