Reaching the Tumor: Mobility of Polymeric Micelles Inside an In Vitro Tumor-on-a-Chip Model with Dual ECM.

Degradable polymeric micelles are promising drug delivery systems due to their hydrophobic core and responsive design. When applying micellar nanocarriers for tumor delivery, one of the bottlenecks encountered in vivo is the tumor tissue barrier: crossing the dense mesh of cells and the extracellular matrix (ECM). Sometimes overlooked, the extracellular matrix can trap nanoformulations based on charge, size, and hydrophobicity. Here, we used a simple design of a microfluidic chip with two types of ECM and MCF7 spheroids to allow "high-throughput" screening of the interactions between biological interfaces and polymeric micelles. To demonstrate the applicability of the chip, a small library of fluorescently labeled polymeric micelles varying in their hydrophilic shell and hydrophobic core forming blocks was studied. Three widely used hydrophilic shells were tested and compared, namely, poly(ethylene glycol), poly(2-ethyl-2-oxazoline), and poly(acrylic acid), along with two enzymatically degradable dendritic hydrophobic cores (based on hexyl or nonyl end groups). Using ratiometric imaging of unimer:micelle fluorescence and FRAP inside the chip model, we obtained the local assembly state and dynamics inside the chip. Notably, we observed different micelle behaviors in the basal lamina ECM, from avoidance of the ECM structure to binding of the poly(acrylic acid) formulations. Binding to the basal lamina correlated with higher uptake into MCF7 spheroids. Overall, we proposed a simple microfluidic chip containing dual ECM and spheroids for the assessment of the interactions of polymeric nanocarriers with biological interfaces and evaluating nanoformulations' capacity to cross the tumor tissue barrier.

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.

Understanding the temperature induced aggregation of silica nanoparticles decorated with temperature-responsive polymers: Can a small step in the chemical structure make a giant leap for a phase transition?

Temperature-responsive nanomaterials have gained increasing interest over the past decade due their ability to undergo conformational changes in situ, in response to a change in temperature. One class of temperature-responsive polymers are those with lower critical solution temperature, which phase separate in aqueous solution above a critical temperature. When these temperature-responsive polymers are grafted to a solid nanoparticle, a change in their surface properties occurs above this critical temperature, from hydrophilic to more hydrophobic, giving them a propensity to aggregate. This study explores the temperature induced aggregation of silica nanoparticles functionalised with two isomeric temperature-responsive polymers with lower critical solution temperature (LCST) behavior, namely poly(N-isopropyl acrylamide) (PNIPAM), and poly(2-n-propyl-2-oxazoline) (PNPOZ) with similar molecular weights (5000 Da) and grafting density. These nanoparticles exhibited striking differences in the temperature of aggregation, which is consistent with LCST of each polymer. Using a combination of small-angle neutron scattering (SANS) and dynamic light scattering (DLS), we probed subtle differences in the aggregation mechanism for PNIPAM- and PNPOZ-decorated silica nanoparticles. The nanoparticles decorated with PNIPAM and PNPOZ show similar aggregation mechanism that was independent of polymer structure, whereby aggregation starts by the formation of small aggregates. A further increase in temperature leads to interaction between these aggregates and results in full-scale aggregation and subsequent phase separation.

Judging Enzyme-Responsive Micelles by Their Covers: Direct Comparison of Dendritic Amphiphiles with Different Hydrophilic Blocks.

Enzymatically degradable polymeric micelles have great potential as drug delivery systems, allowing the selective release of their active cargo at the site of disease. Furthermore, enzymatic degradation of the polymeric nanocarriers facilitates clearance of the delivery system after it has completed its task. While extensive research is dedicated toward the design and study of the enzymatically degradable hydrophobic block, there is limited understanding on how the hydrophilic shell of the micelle can affect the properties of such enzymatically degradable micelles. In this work, we report a systematic head-to-head comparison of well-defined polymeric micelles with different polymeric shells and two types of enzymatically degradable hydrophobic cores. To carry out this direct comparison, we developed a highly modular approach for preparing clickable, spectrally active enzyme-responsive dendrons with adjustable degree of hydrophobicity. The dendrons were linked with three different widely used hydrophilic polymers-poly(ethylene glycol), poly(2-ethyl-2-oxazoline), and poly(acrylic acid) using the CuAAC click reaction. The high modularity and molecular precision of the synthetic methodology enabled us to easily prepare well-defined amphiphiles that differ either in their hydrophilic block composition or in their hydrophobic dendron. The micelles of the different amphiphiles were thoroughly characterized and their sizes, critical micelle concentrations, drug loading, stability, and cell internalization were compared. We found that the micelle diameter was almost solely dependent on the hydrophobicity of the dendritic hydrophobic block, whereas the enzymatic degradation rate was strongly dependent on the composition of both blocks. Drug encapsulation capacity was very sensitive to the type of the hydrophilic block, indicating that, in addition to the hydrophobic core, the micellar shell also has a significant role in drug encapsulation. Incubation of the spectrally active micelles in the presence of cells showed that the hydrophilic shell significantly affects the micellar stability, localization, cell internalization kinetics, and the cargo release mechanism. Overall, the high molecular precision and the ability of these amphiphiles to report their disassembly, even in complex biological media, allowed us to directly compare the different types of micelles, providing striking insights into how the composition of the micelle shells and cores can affect their properties and potential to serve as nanocarriers.

Using Ion Mobility-Mass Spectrometry to Extract Physicochemical Enthalpic and Entropic Contributions from Synthetic Polymers.

Ion mobility-mass spectrometry (IM-MS) experiments are mostly used hand in hand with computational chemistry to correlate mobility measurements to the shape of the ions. Recently, we developed an automatable method to fit IM data obtained with synthetic homopolymers (i.e., collision cross sections; CCS) without resorting to computational chemistry. Here, we further develop the experimental IM data interpretation to explore physicochemical properties of a series of nine polymers and their monomer units by monitoring the relationship between the CCS and the degree of polymerization (DP). Several remarkable points of the CCS evolutions as a function of the DP were found: the first observed DP of each charge state (ΔDPfirst DP), the DPs constituting the structural rearrangements (ΔDPrearr), and the DPs at the half-rearrangement (DPhalf-rearr). Given that these remarkable points do not rely on absolute CCS values, but on their relative evolution, they can be extracted from CCS or raw IM data without accurate IM calibration. Properties such as coordination numbers of the cations, steric hindrance, or side chain flexibility can be compared. This leads to fit parameter predictions based on the nature of the monomer unit. The interpretation of the fit parameters, extracted using solely experimental data, allows a rapid screening of the properties of the polymers.

Unravelling the Miscibility of Poly(2-oxazoline)s: A Novel Polymer Class for the Formulation of Amorphous Solid Dispersions.

Water-soluble polymers are still the most popular carrier for the preparation of amorphous solid dispersions (ASDs). The advantage of this type of carrier is the fast drug release upon dissolution of the water-soluble polymer and thus the initial high degree of supersaturation of the poorly soluble drug. Nevertheless, the risk for precipitation due to fast drug release is a phenomenon that is frequently observed. In this work, we present an alternative carrier system for ASDs where a water-soluble and water-insoluble carrier are combined to delay the drug release and thus prevent this onset of precipitation. Poly(2-alkyl-2-oxazoline)s were selected as a polymer platform since the solution properties of this polymer class depend on the length of the alkyl sidechain. Poly(2-ethyl-2-oxazoline) (PEtOx) behaves as a water-soluble polymer at body temperature, while poly(2-n-propyl-2-oxazoline) (PPrOx) and poly(2-sec-butyl-2-oxazoline) (PsecBuOx) are insoluble at body temperature. Since little was known about the polymer's miscibility behaviour and especially on how the presence of a poorly-water soluble drug impacted their miscibility, a preformulation study was performed. Formulations were investigated with X-ray powder diffraction, differential scanning calorimetry (DSC) and solid-state nuclear magnetic resonance spectroscopy. PEtOx/PPrOx appeared to form an immiscible blend based on DSC and this was even more pronounced after heating. The six drugs that were tested in this work did not show any preference for one of the two phases. PEtOx/PsecBuOx on the other hand appeared to be miscible forming a homogeneous blend between the two polymers and the drugs.

Thioacetate-Based Initiators for the Synthesis of Thiol-End-Functionalized Poly(2-oxazoline)s.

New functional initiators for the cationic ring-opening polymerization of 2-alkyl-2-oxazolines are described to introduce a thiol moiety at the α terminus. Both tosylate and nosylate initiators carrying a thioacetate group are obtained in multigram scale, from commercial reagents in two steps, including a phototriggered thiol-ene radical addition. The nosylate derivative gives access to a satisfying control over the cationic ring-opening polymerization of 2-ethyl-2-oxazoline, with dispersity values lower than 1.1 during the entire course of the polymerization, until full conversion. Cleavage of the thioacetate end group is rapidly achieved using triazabicyclodecene, thereby leading to a mercapto terminus. The latter gives access to a new subgeneration of α-functional poly(2-oxazoline)s (butyl ester, N-hydroxysuccinimidyl ester, furan) by Michael addition with commercial (meth)acrylates. The amenability of the mercapto-poly(2-ethyl-2-oxazoline) for covalent surface patterning onto acrylated surfaces is demonstrated in a microchannel cantilever spotting (µCS) experiment, characterized by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary-ion mass spectrometry (ToF-SIMS).

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.

Fundamental Studies on Poly(2-oxazoline) Side Chain Isomers Using Tandem Mass Spectrometry and Ion Mobility-Mass Spectrometry.

When polymer mixtures become increasingly complex, the conventional analysis techniques become insufficient for complete characterization. Mass spectrometric techniques can satisfy this increasing demand for detailed sample characterization. Even though isobaric polymers are indistinguishable using simple mass spectrometry (MS) analyses, more advanced techniques such as tandem MS (MS/MS) or ion mobility (IM) can be used. Here, we report proof of concept for characterizing isomeric polymers, namely poly(2-n-propyl-2-oxazoline) (Pn-PrOx) and poly(2-isopropyl-2-oxazoline) (Pi-PrOx), using MS/MS and IM-MS. Pi-PrOx ions lose in intensity at higher accelerating voltages than Pn-PrOx ions during collision-induced dissociation (CID) MS/MS experiments. A Pn/i-PrOx mixture could also be titrated using survival yield calculations of either precursor ions or cation ejection species. IM-MS yielded shape differences in the degree of polymerization (DP) regions showing the structural rearrangements. Combined MS techniques are thus able to identify and deconvolute the molar mass distributions of the two isomers in a mixture. Finally, the MS/MS and IM-MS behaviors are compared for interpretation. Graphical Abstract .

Gas-Phase Dynamics of Collision Induced Unfolding, Collision Induced Dissociation, and Electron Transfer Dissociation-Activated Polymer Ions.

Polymer characterizations are often performed using mass spectrometry (MS). Aside from MS and different tandem MS (MS/MS) techniques, ion mobility-mass spectrometry (IM-MS) has been recently added to the inventory of characterization technique. However, only few studies have focused on the reproducibility and robustness of polymer IM-MS analyses. Here, we perform collisional and electron-mediated activation of polymer ions before measuring IM drift times, collision cross-sections (CCS), or reduced ion mobilities (K0). The resulting IM behavior of different activated product ions is then compared to non-activated native intact polymer ions. First, we analyzed collision induced unfolding (CIU) of precursor ions to test the robustness of polymer ion shapes. Then, we focused on fragmentation product ions to test for shape retentions from the precursor ions: cation ejection species (CES) and product ions with m/z and charge state values identical to native intact polymer ions. The CES species are formed using both collision induced dissociation (CID) and electron transfer dissociation (ETD, formally ETnoD) experiments. Only small drift time, CCS, or K0 deviations between the activated/formed ions are observed compared to the native intact polymer ions. The polymer ion shapes seem to depend solely on their mass and charge state. The experiments were performed on three synthetic homopolymers: poly(ethoxy phosphate) (PEtP), poly(2-n-propyl-2-oxazoline) (Pn-PrOx), and poly(ethylene oxide) (PEO). These results confirm the robustness of polymer ion CCSs for IM calibration, especially singly charged polymer ions. The results are also discussed in the context of polymer analyses, CCS predictions, and probing ion-drift gas interaction potentials. Graphical Abstract.

Striking Effect of Polymer End-Group on C60 Nanoparticle Formation by High Shear Vibrational Milling with Alkyne-Functionalized Poly(2-oxazoline)s.

Buckminsterfullerene (C60) has a large potential for biomedical applications. However, the main challenge for the realization of its biomedical application potential is to overcome its extremely low water solubility. One approach is the coformulation with biocompatible water-soluble polymers, such as poly(2-oxazoline)s (PAOx), to form water-soluble C60 nanoparticles (NPs). However, uniform and defined NPs have only been obtained via a thin film hydration method or using cyclodextrin-functionalized PAOx. Here, we report the mechanochemical preparation of defined and stable C60:PAOx NPs by the introduction of a simple alkyne group as a polymer end-group. The presence of this alkyne bond is proven to be crucial in the mechanochemical synthesis of stable, defined sub-100 nm C60:PAOx NPs, with high C60 content up to 8.9 wt %.

Mechanochemical Preparation of Stable Sub-100†nm γ-Cyclodextrin:Buckminsterfullerene (C60) Nanoparticles by Electrostatic or Steric Stabilization.

Buckminster fullerene (C60 )'s main hurdle to enter the field of biomedicine is its low bioavailability, which results from its extremely low water solubility. A well-known approach to increase the water solubility of C60 is by complexation with γ-cyclodextrins. However, the formed complexes are not stable in time as they rapidly aggregate and eventually precipitate due to attractive intermolecular forces, a common problem in inclusion complexes of cyclodextrins. In this study we attempt to overcome the attractive intermolecular forces between the complexes by designing custom γ-cyclodextrin (γCD)-based supramolecular hosts for C60 that inhibit the aggregation found in native γCD-C60 complexes. The approach entails the introduction of either repulsive electrostatic forces or increased steric hindrance to prevent aggregation, thus enhancing the biomedical application potential of C60 . These modifications have led to new sub-100 nm nanostructures that show long-term stability in solution.

Recognition-Mediated Hydrogel Swelling Controlled by Interaction with a Negative Thermoresponsive LCST Polymer.

Most polymeric thermoresponsive hydrogels contract upon heating beyond the lower critical solution temperature (LCST) of the polymers used. Herein, we report a supramolecular hydrogel system that shows the opposite temperature dependence. When the non-thermosesponsive hydrogel NaphtGel, containing dialkoxynaphthalene guest molecules, becomes complexed with the tetra cationic macrocyclic host CBPQT4+ , swelling occurred as a result of host-guest complex formation leading to charge repulsion between the host units, as well as an osmotic contribution of chloride counter-ions embedded in the network. The immersion of NaphtGel in a solution of poly(N-isopropylacrylamide) with tetrathiafulvalene (TTF) end groups complexed with CBPQT4+ induced positive thermoresponsive behaviour. The LCST-induced dethreading of the polymer-based pseudorotaxane upon heating led to transfer of the CBPQT4+ host and a concomitant swelling of NaphtGel. Subsequent cooling led to reformation of the TTF-based host-guest complexes in solution and contraction of the hydrogel.

Side chain variations radically alter the diffusion of poly(2-alkyl-2-oxazoline) functionalised nanoparticles through a mucosal barrier.

Functionalised nanomaterials are gaining popularity for use as drug delivery vehicles and, in particular, mucus penetrating nanoparticles may improve drug bioavailability via the oral route. To date, few polymers have been investigated for their muco-penetration, and the effects of systematic structural changes to polymer architectures on the penetration and diffusion of functionalised nanomaterials through mucosal tissue have not been reported. We investigated the influence of poly(2-oxazoline) alkyl side chain length on nanoparticle diffusion; poly(2-methyl-2-oxazoline), poly(2-ethyl-2-oxazoline), and poly(2-n-propyl-2-oxazoline) were grafted onto the surface of thiolated silica nanoparticles and characterised by FT-IR, Raman and NMR spectroscopy, thermogravimetric analysis, and small angle neutron scattering. Diffusion coefficients were determined in water and in a mucin dispersion (using Nanoparticle Tracking Analysis), and penetration through a mucosal barrier was assessed using an ex vivo fluorescence technique. The addition of a single methylene group in the side chain significantly altered the penetration and diffusion of the materials in both mucin dispersions and mucosal tissue. Nanoparticles functionalised with poly(2-methyl-2-oxazoline) were significantly more diffusive than particles with poly(2-ethyl-2-oxazoline) while particles with poly(2-n-propyl-2-oxazoline) showed no significant increase compared to the unfunctionalised particles. These data show that variations in the polymer structure can radically alter their diffusive properties with clear implications for the future design of mucus penetrating systems.

Reversible Calcium(II)-Ion Binding through an Apparent pKa Shift of Thermosensitive Block-Copolymer Micelles.

There is an increasing need for smart materials capable of removing multivalent ions from aqueous streams without the inconvenience of brine regeneration as in ion-exchange processes. Herein, we present a thermoresponsive micellar system consisting of polystyrene-poly(methoxy diethyleneglycol acrylate) block copolymer surfactants modified with carboxylic acid end groups (PS-PMDEGA-COOH) that can be used to switch between the adsorption and desorption of divalent calcium(II) cations by a mild temperature trigger, thus providing a new type of thermoregenerable ion-adsorbing materials. The switch of calcium(II)-binding capacity is demonstrated to result from a shift in the pKa value of the carboxylic acid groups by the collapse and redissolution of the PMDEGA block and the associated change in local polarity.

Thermoresponsive interplay of water insoluble poly(2-alkyl-2-oxazoline)s composition and supramolecular host-guest interactions.

A series of water insoluble poly[(2-ethyl-2-oxazoline)-ran-(2-nonyl-2-oxazoline)] amphiphilic copolymers was synthesized and their solubility properties in the presence of different supramolecular host molecules were investigated. The resulting polymer-cavitand assemblies exhibited a thermoresponsive behavior that could be modulated by variation of the copolymer composition and length. Interestingly, the large number of hydrophobic nonyl units across the polymer chain induced the formation of kinetically-trapped nanoparticles in solution. These nanoparticles further agglomerate into larger aggregates at a temperature that is dependent on the polymer composition and the cavitand type and concentration. The present research expands the understanding on the supramolecular interactions between water insoluble copolymers and supramolecular host molecules.

Tuning temperature responsive poly(2-alkyl-2-oxazoline)s by supramolecular host-guest interactions.

A poly[(2-ethyl-2-oxazoline)-ran-(2-nonyl-2-oxazoline)] random copolymer was synthesized and its thermoresponsive behavior in aqueous solution modulated by the addition of different supramolecular host molecules. The macrocycles formed inclusion complexes with the nonyl aliphatic side-chains present in the copolymer, increasing its cloud point temperature. The extent of this temperature shift was found to depend on the cavitand concentration and on the strength of the host-guest complexation. The cloud point temperature could be tuned in an unprecedented wide range of 30 K by supramolecular interactions. Since the temperature-induced breakage of the inclusion complexes constitutes the driving force for the copolymer phase transition, the shift in cloud point temperature could be utilized to estimate the association constant of the nonyl side chains with the cavitands.

Solution polymeric optical temperature sensors with long-term memory function powered by supramolecular chemistry.

A poly[(2-ethyl-2-oxazoline)-ran-(2-nonyl-2-oxazoline)] copolymer in combination with hydroxypropylated cyclodextrins has been demonstrated to lead to a supramolecular self-assembly process that results in the formation of kinetically trapped thermoresponsive nanoparticles. Selection of the cyclodextrin type provides control over the nanoparticle phase-transition thermodynamics, thus affording optical temperature sensors with an unprecedented, long-term thermal memory function, which is reversible or irreversible. This research also sheds light onto kinetic and dynamic supramolecular assemblies, thus providing important insight because similar supramolecular processes are at the foundation of living matter.