Think Beyond the Core: Impact of the Hydrophilic Corona on Drug Solubilization Using Polymer Micelles.

Polymeric micelles are typically characterized as core-shell structures. The hydrophobic core is considered as a depot for hydrophobic molecules, and the corona-forming block acts as a stabilizing and solubilizing interface between the core and aqueous milieu. Tremendous efforts have been made to tune the hydrophobic block to increase the drug loading and stability of micelles, whereas the role of hydrophilic blocks is rarely investigated in this context, with poly(ethylene glycol) (PEG) being the gold standard of hydrophilic polymers. To better understand the role of the hydrophilic corona, a small library of structurally similar A-B-A-type amphiphiles based on poly(2-oxazoline)s and poly(2-oxazine)s is investigated by varying the hydrophilic block A utilizing poly(2-methyl-2-oxazoline) (pMeOx; A) or poly(2-ethyl-2-oxazoline) (pEtOx; A*). In terms of hydrophilicity, both polymers closely resemble PEG. The more hydrophobic block B bears either a poly(2-oxazoline) and poly(2-oxazine) backbone with C3 (propyl) and C4 (butyl) side chains. Surprisingly, major differences in loading capacities from A-B-A > A*-B-A > A*-B-A* is observed for the formulation with two poorly water-soluble compounds, curcumin and paclitaxel, highlighting the importance of the hydrophilic corona of polymer micelles used for drug formulation. The formulations are also characterized by various nuclear magnetic resonance spectroscopy methods, dynamic light scattering, cryogenic transmission electron microscopy, and (micro) differential scanning calorimetry. Our findings suggest that the interaction between the hydrophilic block and the guest molecule should be considered an important, but previously largely ignored, factor for the rational design of polymeric micelles.

In Vitro Blood-Brain Barrier Permeability and Cytotoxicity of an Atorvastatin-Loaded Nanoformulation Against Glioblastoma in 2D and 3D Models.

Inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of the family of statins have been suggested as therapeutic options in various tumors. Atorvastatin is a statin with the potential to cross the blood-brain barrier; however, the concentrations necessary for a cytotoxic effect against cancer cells exceed the concentrations achievable via oral administration, which made the development of a novel atorvastatin formulation necessary. We characterized the drug loading and basic physicochemical characteristics of micellar atorvastatin formulations and tested their cytotoxicity against a panel of different glioblastoma cell lines. In addition, activity against tumor spheroids formed from mouse glioma and mouse cancer stem cells, respectively, was evaluated. Our results show good activity of atorvastatin against all tested cell lines. Interestingly, in the three-dimensional (3D) models, growth inhibition was more pronounced for the micellar formulation compared to free atorvastatin. Finally, atorvastatin penetration across a blood-brain barrier model obtained from human induced-pluripotent stem cells was evaluated. Our results suggest that the presented micelles may enable much higher serum concentrations than possible by oral administration; however, if transport across the blood-brain barrier is sufficient to reach the therapeutic atorvastatin concentration for the treatment of glioblastoma via intravenous administration remains unclear.

Probing the Complex Loading-Dependent Structural Changes in Ultrahigh Drug-Loaded Polymer Micelles by Small-Angle Neutron Scattering.

Drug-loaded polymer micelles or nanoparticles are being continuously explored in the fields of drug delivery and nanomedicine. Commonly, a simple core-shell structure is assumed, in which the core incorporates the drug and the corona provides steric shielding, colloidal stability, and prevents protein adsorption. Recently, the interactions of the dissolved drug with the micellar corona have received increasing attention. Here, using small-angle neutron scattering, we provide an in-depth study of the differences in polymer micelle morphology of a small selection of structurally closely related polymer micelles at different loadings with the model compound curcumin. This work supports a previous study using solid-state nuclear magnetic resonance spectroscopy and we confirm that the drug resides predominantly in the core of the micelle at low drug loading. As the drug loading increases, neutron scattering data suggests that an inner shell is formed, which we interpret as the corona also starting to incorporate the drug, whereas the outer shell mainly contains water and the polymer. The presented data clearly shows that a better understanding of the inner morphology and the impact of the hydrophilic block can be important parameters for improved drug loading in polymer micelles as well as provide insights into the structure-property relationship.

Loading-Dependent Structural Model of Polymeric Micelles Encapsulating Curcumin by Solid-State NMR Spectroscopy.

Detailed insight into the internal structure of drug-loaded polymeric micelles is scarce, but important for developing optimized delivery systems. We observed that an increase in the curcumin loading of triblock copolymers based on poly(2-oxazolines) and poly(2-oxazines) results in poorer dissolution properties. Using solid-state NMR spectroscopy and complementary tools we propose a loading-dependent structural model on the molecular level that provides an explanation for these pronounced differences. Changes in the chemical shifts and cross-peaks in 2D NMR experiments give evidence for the involvement of the hydrophobic polymer block in the curcumin coordination at low loadings, while at higher loadings an increase in the interaction with the hydrophilic polymer blocks is observed. The involvement of the hydrophilic compartment may be critical for ultrahigh-loaded polymer micelles and can help to rationalize specific polymer modifications to improve the performance of similar drug delivery systems.

Collagenase Nanoparticles Enhance the Penetration of Drugs into Pancreatic Tumors.

Overexpressed extracellular matrix (ECM) in pancreatic ductal adenocarcinoma (PDAC) limits drug penetration into the tumor and is associated with poor prognosis. Here, we demonstrate that a pretreatment based on a proteolytic-enzyme nanoparticle system disassembles the dense PDAC collagen stroma and increases drug penetration into the pancreatic tumor. More specifically, the collagozome, a 100 nm liposome encapsulating collagenase, was rationally designed to protect the collagenase from premature deactivation and prolonged its release rate at the target site. Collagen is the main component of the PDAC stroma, reaching 12.8 ± 2.3% vol in diseased mice pancreases, compared to 1.4 ± 0.4% in healthy mice. Upon intravenous injection of the collagozome, ∼1% of the injected dose reached the pancreas over 8 h, reducing the level of fibrotic tissue to 5.6 ± 0.8%. The collagozome pretreatment allowed increased drug penetration into the pancreas and improved PDAC treatment. PDAC tumors, pretreated with the collagozome followed by paclitaxel micelles, were 87% smaller than tumors pretreated with empty liposomes followed by paclitaxel micelles. Interestingly, degrading the ECM did not increase the number of circulating tumor cells or metastasis. This strategy holds promise for degrading the extracellular stroma in other diseases as well, such as liver fibrosis, enhancing tissue permeability before drug administration.

Temperature-Dependent Rheological and Viscoelastic Investigation of a Poly(2-methyl-2-oxazoline)-b-poly(2-iso-butyl-2-oxazoline)-b-poly(2-methyl-2-oxazoline)-Based Thermogelling Hydrogel.

The synthesis and characterization of an ABA triblock copolymer based on hydrophilic poly(2-methyl-2-oxazoline) (pMeOx) blocks A and a modestly hydrophobic poly(2-iso-butyl-2-oxazoline) (piBuOx) block B is described. Aqueous polymer solutions were prepared at different concentrations (1-20 wt %) and their thermogelling capability using visual observation was investigated at different temperatures ranging from 5 to 80 °C. As only a 20 wt % solution was found to undergo thermogelation, this concentration was investigated in more detail regarding its temperature-dependent viscoelastic profile utilizing various modes (strain or temperature sweep). The prepared hydrogels from this particular ABA triblock copolymer have interesting rheological and viscoelastic properties, such as reversible thermogelling and shear thinning, and may be used as bioink, which was supported by its very low cytotoxicity and initial printing experiments using the hydrogels. However, the soft character and low yield stress of the gels do not allow real 3D printing at this point.

Ultra-High to Ultra-Low Drug-Loaded Micelles: Probing Host-Guest Interactions by Fluorescence Spectroscopy.

Polymer micelles are an attractive means to solubilize water insoluble compounds such as drugs. Drug loading, formulations stability and control over drug release are crucial factors for drug-loaded polymer micelles. The interactions between the polymeric host and the guest molecules are considered critical to control these factors but typically barely understood. Here, we compare two isomeric polymer micelles, one of which enables ultra-high curcumin loading exceeding 50 wt.%, while the other allows a drug loading of only 25 wt.%. In the low capacity micelles, steady-state fluorescence revealed a very unusual feature of curcumin fluorescence, a high energy emission at 510 nm. Time-resolved fluorescence upconversion showed that the fluorescence life time of the corresponding species is too short in the high-capacity micelles, preventing an observable emission in steady-state. Therefore, contrary to common perception, stronger interactions between host and guest can be detrimental to the drug loading in polymer micelles.

Like Dissolves Like? A Comprehensive Evaluation of Partial Solubility Parameters to Predict Polymer-Drug Compatibility in Ultrahigh Drug-Loaded Polymer Micelles.

Despite decades of research, our understanding of the molecular interactions between drugs and polymers in drug-loaded polymer micelles does not extend much beyond concepts such as "like-dissolves-like" or hydrophilic/hydrophobic. However, polymer-drug compatibility strongly affects formulation properties and therefore the translation of a formulation into the clinics. Specific interactions such as hydrogen-bonding, π-π stacking, or coordination interactions can be utilized to increase drug loading. This is commonly based on trial and error and eventually leads to an optimized drug carrier. Unfortunately, due to the unique characteristics of each drug, the deduction of advanced general concepts remains challenging. Furthermore, the introduction of complex moieties or specifically modified polymers hampers systematic investigations regarding polymer-drug compatibility as well as clinical translation. In this study, we reduced the complexity to isolate the crucial factors determining drug loading. Therefore, the compatibility of 18 different amphiphilic polymers for five different hydrophobic drugs was determined empirically. Subsequently, the obtained specificities were compared to theoretical compatibilities derived from either the Flory-Huggins interaction parameters or the Hansen solubility parameters. In general, the Flory-Huggins interaction parameters were less suited to correctly estimate the experimental drug solubilization compared to the Hansen solubility parameters. The latter were able to correctly predict some trend regarding good and poor solubilizers, yet the overall predictive strength of Hansen solubility parameters is clearly unsatisfactory.

Drug induced micellization into ultra-high capacity and stable curcumin nanoformulations: Physico-chemical characterization and evaluation in 2D and 3D in vitro models.

Curcumin (CUR) is a natural extract from the plant Curcuma longa and part of turmeric, a spice and herbal remedy in traditional medicine. Thousands of papers claim a plethora of health benefits by CUR, but a growing number of reports and contributions caution that many experimental data may be artifacts or outright deny any suitability of CUR due to its problematic physicochemical properties. Two major issues often encountered with CUR are its extraordinarily low solubility in water and its limited chemical stability. Here, we report on a novel nanoformulation of CUR that enables CUR concentrations in water of at least 50 g/L with relative drug loadings of >50 wt% and high dose efficacy testing in 3D tumor models. Despite this high loading and concentration, the CUR nanoformulation comprises polymer-drug aggregates with a size <50 nm. Most interestingly, this is achieved using an amphiphilic block copolymer, that by itself does not form micelles due to its limited hydrophilic/lipophilic contrast. The ultra-high loaded nanoformulations exhibit a very good stability, reproducibility and redispersibility. In order to test effects of CUR in conditions closer to an in vivo situation, we utilized a 3D tumor test system based on a biological decellularized tissue matrix that better correlates to clinical results concerning drug testing. We found that in comparison to 2D culture, the invasively growing breast cancer cell line MDA-MB-231 requires high concentrations of CUR for tumor cell eradication in 3D. In addition, we supplemented a 3D colorectal cancer model of the malignant cell line SW480 with fibroblasts and observed also in this invasive tumor model with stroma components a decreased tumor cell growth after CUR application accompanied by a loss of cell-cell contacts within tumor cell clusters. In a flow bioreactor simulating cancer cell dissemination, nanoformulated CUR prevented SW480 cells from adhering to a collagen scaffold, suggesting an anti-metastatic potential of CUR. This offers a rationale that the presented ultra-high CUR-loaded nanoformulation may be considered a tool to harness the full therapeutic potential of CUR.

More Is Sometimes Less: Curcumin and Paclitaxel Formulations Using Poly(2-oxazoline) and Poly(2-oxazine)-Based Amphiphiles Bearing Linear and Branched C9 Side Chains.

A known limitation of polymer micelles for the formulation of hydrophobic drugs is their low loading capacity (LC), which rarely exceeds 20 wt%. One general strategy to overcome this limitation is to increase the amphiphilic contrast, that is, to make the hydrophobic core of the micelles more hydrophobic. However, in the case of poly(2-oxazoline) (POx)-based amphiphilic triblock copolymers, a minimal amphiphilic contrast was reported to be beneficial. Here, this subject is revisited in more detail using long hydrophobic side chains that are either linear (nonyl) or branched (3-ethylheptyl). Two different backbones within the hydrophobic block are investigated, in particular POx and poly(2-oxazine) (POzi), for the solubilization and co-solubilization of the two highly water insoluble compounds, curcumin and paclitaxel. Even though high loading capacities can be achieved for curcumin using POzi-based triblock copolymers, the solubilization capacity of all investigated polymers with longer side chains is significantly lower compared to POx and poly(2-oxazine)s with shorter side chains. Although the even lower LC for paclitaxel can be somehow improved by co-formulating curcumin, this study corroborates that in the case of POx and POzi-based polymer micelles, an increased amphiphilic contrast leads to less drug solubilization.

Poly(2-oxazoline)s based biomaterials: A comprehensive and critical update.

Poly(2-oxazoline)s have been investigated for decades as biomaterials. Pioneering early work suggested that hydrophilic poly(2-oxazoline)s are comparable to poly(ethylene glycol) regarding their potential as biomaterials, but the ready commercial availability of the latter has led to its meteoric rise to become the gold standard of hydrophilic synthetic biomaterials. In contrast, poly(2-oxazoline)s almost fell into oblivion. However, in the last decade, this family of polymers has gained much more interest in general and as biomaterials in particular. The rich chemistry and comparably straightforward synthesis of poly(2-oxazoline)s gives many opportunities for tailoring the properties of the resulting biomaterials, allowing the chemist to explore new conjugation chemistry, and to fine-tune the molar mass, hydrophilic-lipophilic balance as well as architecture. Thus, the wide range of demands for various applications of biomaterials can be suitably addressed. This review aims to give a comprehensive and critical update of the development of poly(2-oxazoline) based biomaterials, focusing on the last 5 years, which have seen an explosive increase of interest. We believe that the research regarding this diverse family of polymers will remain strong and will keep growing, in particular after the promising first-in-human studies of a poly(2-oxazoline) drug conjugate. This review aims at researchers and students new to this polymer family and seasoned poly(2-oxazoline) experts alike and attempts to showcase how the chemical diversity of poly(2-oxazoline)s allows a relatively facile and broad access to biomaterials of all kinds.

Investigating the Influence of Aromatic Moieties on the Formulation of Hydrophobic Natural Products and Drugs in Poly(2-oxazoline)-Based Amphiphiles.

Many natural compounds with interesting biomedical properties share one physicochemical property, namely, low water solubility. Polymer micelles are, among others, a popular means to solubilize hydrophobic compounds. The specific molecular interactions between the polymers and the hydrophobic drugs are diverse, and recently it has been discussed that macromolecular engineering can be used to optimize drug-loaded micelles. Specifically, π-π stacking between small molecules and polymers has been discussed as an important interaction that can be employed to increase drug loading and formulation stability. Here, we test this hypothesis using four different polymer amphiphiles with varying aromatic content and various natural products that also contain different relative amounts of aromatic moieties. In the case of paclitaxel, having the lowest relative content of aromatic moieties, the drug loading decreases with increasing relative aromatic amount in the polymer, whereas the drug loading of curcumin, having a much higher relative aromatic content, is increased. Interestingly, the loading using schizandrin A, a dibenzo[ a, c]cyclooctadiene lignan with intermediate relative aromatic content is not influenced significantly by the aromatic content of the polymers employed. The very high drug loading, long-term stability, ability to form stable highly loaded binary coformulations in different drug combinations, small-sized formulations, and amorphous structures in all cases corroborate earlier reports that poly(2-oxazoline)-based micelles exhibit an extraordinarily high drug loading and are promising candidates for further biomedical applications. The presented results underline that the interaction between the polymers and the incorporated small molecules may be more complex and are significantly influenced by both sides, the used carrier and drug, and must be investigated in each specific case.

Drug Specificity, Synergy and Antagonism in Ultrahigh Capacity Poly(2-oxazoline)/Poly(2-oxazine) based Formulations.

Polymer micelles offer the possibility to create a nanoscopic environment that is distinct from the bulk phase. They find applications in catalysis, drug delivery, cleaning, etc. Often, one simply distinguishes between hydrophilic and hydrophobic, but fine-tuning of the microenvironment is possible by adjusting the structure of the polymer amphiphile. Here, we investigated a small library of structurally similar amphiphiles based on poly(2-oxazoline)s and poly(2-oxazine)s with respect to their solubilization capacity for two extremely water insoluble drugs, curcumin and paclitaxel. We found very significant and orthogonal specificities even if only one methylene group is exchanged between the polymer backbone and side chain. More strikingly, we observed profound synergistic and antagonistic solubilization patterns for the coformulation of the two drugs. Our findings shed new light on host-guest interaction in polymer micelles and such pronounced host-guest specificities in polymer micelles may not only be interesting in drug delivery but also for applications such as micellar catalysis.

A Thermogelling Supramolecular Hydrogel with Sponge-Like Morphology as a Cytocompatible Bioink.

Biocompatible polymers that form thermoreversible supramolecular hydrogels have gained great interest in biomaterials research and tissue engineering. When favorable rheological properties are achieved at the same time, they are particularly promising candidates as material that allow for the printing of cells, so-called bioinks. We synthesized a novel thermogelling block copolymer and investigated the rheological properties of its aqueous solution by viscosimetry and rheology. The polymers undergo thermogelation between room temperature and body temperature, form transparent hydrogels of surprisingly high strength (G' > 1000 Pa) and show rapid and complete shear recovery after stress. Small angle neutron scattering suggests an unusual bicontinuous sponge-like gel network. Excellent cytocompatibility was demonstrated with NIH 3T3 fibroblasts, which were incorporated and bioplotted into predefined 3D hydrogel structures without significant loss of viability. The developed materials fulfill all criteria for future use as bioink for biofabrication.