Thermosensitive bioartificial hydrogels as smart injectable and biocompatible systems allowing post-injection chemical crosslinking

Introduction Injectable hydrogels as carriers for drugs and/or cells have gained increasing interest in the last years due to the possibility to vehicle their payload in the desired loco through mini-invasive procedures [1, 2]. In this work, a new library of bioartificial hydrogels was designed combining the chemical versatility and reproducible physicochemical properties of a synthetic polymer with the enhanced cell adhesiveness of a natural polymer. Specifically, an amphiphilic polyurethane (PEU) bearing amino groups was first synthesised and then blended with hyaluronic acid (HA) to obtain thermosensitive bioartificial formulations. The influence of HA molecular weight on polymers miscibility was investigated as well as the thermosensitivity and the injectability of the newly designed bioartificial systems. Modification of the PEU and the HA may allow post-injection chemical crosslinking enhancing the chemical stability of the hydrogel. Experimental Methods The amphiphilic PEU was synthesised in a two step procedure under nitrogen by reacting a commercial triblock copolymer (Poloxamer 407, Poly(ethylene oxide)-Poly(propylene oxide)-Poly(ethylene oxide)) with 1,6-hexamethylene diisocyanate. Then, the prepolymer was chain extended with a diol (N-Boc diethanolamine) containing protected secondary amino groups. Infrared (IR) spectroscopy and Size Exclusion Chromatography (SEC) were then performed to assess the success of the synthesis and to evaluate PEU molecular weight, respectively. Subsequently, the synthesised PEU was subjected to an acidic treatment in chloroform/trifluoroacetic acid 90/10 V/V to remove BOC-protecting groups and the exposed secondary amino groups were quantified through a colorimetric assay (Orange Sodium salt). The synthetic component (D-DHP407) was then blended with a high (HA_400kDa) and low (HA_82kDa) molecular weight HA, reaching different weight ratios. Lastly, formulations were prepared by dissolving both polymers in physiological saline solution and then characterized in terms of thermosensitivity by means of tube investing test and gelation time test at 37 °C; injectability in the sol state through needles of different diameters (G18, G21 and G22) and cytocompatibility according to ISO10993-5. Secondary amino groups in PEU and carboxyl groups in HA may be exploited to graft functional molecules for in situ post-injection crosslinking. Results and Discussion The successful PEU synthesis was proved through IR spectroscopy by the appearance of new bands ascribed to urethane bonds, while SEC analysis gave a molecular weight in the range 30000 – 35000 Da with 1.4 polydispersity index. Secondary amino groups were quantified to be 4.5x1020 groups/g of polymer by means of Orange II Sodium Salt assay. For what concerns hydrogel preparation, D-DHP407 and HA_400kDa were mixed at 98/2, 95/5, 92/8, 88/12 and 83/17 wt. ratios. All tested formulations formed compatible blends, but, due to the high molecular weight of HA_400kDa, further increase in the natural component content highly increased system viscosity affecting injectability. Hence, D-DHP407 was blended with a low molecular weight HA (HA_82kDa) obtaining compatible and injectable blends even at 50/50 wt. ratio. Subsequently, hydrogel temperature-driven gelation was tested and all considered formulations turned out to gel within few minutes at 37 °C, thus suggesting that HA introduction did not affect PEU thermosensitivity. Regarding hydrogel injectability, all blends could not be extruded through G22 needle, while they could be injected through larger needle diameters (G21 and G18). Finally, by increasing HA content, thus decreasing the synthetic component, an increase of hydrogel biocompatibility was observed. Conclusion A new platform of thermosensitive bioartificial hydrogels was developed by blending a custom-made amphiphilic polyurethane, which ensures hydrogel thermo-responsiveness, with hyaluronic acid, responsible for an improved cytocompatibility. Furthermore, the presence of exposed secondary amino groups along PEU chains and carboxylic groups in HA chains opens the possibility to graft functional moieties to both molecules for post-injection crosslinking. Such injectable system is under development as a matrix for in situ treatment of myocardial tissue, by releasing agents promoting direct reprogramming of cardiac fibroblasts into cardiomyocytes. References [1]Zhang Z., Expert. Opin. Biol. Ther., 17:1, 49-62, 2017. [2] Boffito M. et al., J. Biomed. Mater. Res. A, 103(3):1276-90, 2015. Acknowledgement The activity has been carried within the research project BIORECAR. This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 772168)

Thiol-Ene Photo-Click Hydrogels with Tunable Mechanical Properties Resulting from the Exposure of Different -Ene Moieties through a Green Chemistry

Temperature and light responsiveness are widely exploited stimuli to tune the physico-chemical properties of double network hydrogels. In this work, new amphiphilic poly(ether urethane)s bearing photo-sensitive moieties (i.e., thiol, acrylate and norbornene functionalities) were engineered by exploiting the versatility of poly(urethane) chemistry and carbodiimide-mediated green functionalization procedures. Polymers were synthesized according to optimized protocols maximizing photo-sensitive group grafting while preserving their functionality (approx. 1.0 × 10E19, 2.6 × 10E19 and 8.1 × 10E17 thiol, acrylate and norbornene groups/gpolymer), and exploited to prepare thermo- and Vis-light-responsive thiol-ene photo-click hydrogels (18% w/v, 1:1 thiol:ene molar ratio). Green light-induced photo-curing allowed the achievement of a much more developed gel state with improved resistance to deformation (~60% increase in critical deformation, γL). Triethanolamine addition as co-initiator to thiol-acrylate hydrogels improved the photo-click reaction (i.e., achievement of a better-developed gel state). Differently, L-tyrosine addition to thiol-norbornene solutions slightly hindered cross-linking, resulting in less developed gels with worse mechanical performances (~62% γL decrease). In their optimized composition, thiol-norbornene formulations resulted in prevalent elastic behavior at lower frequency compared to thiol-acrylate gels due to the formation of purely bio-orthogonal instead of heterogeneous gel networks. Our findings highlight that exploiting the same thiol-ene photo-click chemistry, a fine tuning of the gel properties is possible by reacting specific functional groups

Dual stimuli-responsive polyurethane-based hydrogels as smart drug delivery carriers for the advanced treatment of chronic skin wounds

The design of multi-stimuli-responsive vehicles for the controlled and localized release of drugs is a challenging issue increasingly catching the attention of many research groups working on the advanced treatment of hard-to-close wounds. In this work, a thermo- and pH-responsive hydrogel (P-CHP407) was prepared from an ad hoc synthesized amphiphilic poly(ether urethane) (CHP407) exposing a significant amount of –COOH groups (8.8 ± 0.9 nmol/gpolymer). The exposure of acid moieties in P-CHP407 hydrogel led to slightly lower initial gelation temperature (12.1 °C vs. 14.6 °C, respectively) and gelation rate than CHP407 hydrogel, as rheologically assessed. Nanoscale hydrogel characterization by Low Field NMR (LF-NMR) spectroscopy suggested that the presence of carboxylic groups in P-CHP407 caused the formation of bigger micelles with a thicker hydrated shell than CHP407 hydrogels, as further proved by Dynamic Light Scattering analyses. In addition, P-CHP407 hydrogel showed improved capability to change its internal pH compared to CHP407 one when incubated with an alkaline buffer (pH 8) (e.g., pHchange_5min = 3.76 and 1.32, respectively). Moreover, LF-NMR characterization suggested a stronger alkaline-pH-induced interaction of water molecules with micelles exposing –COOH groups. Lastly, the hydrogels were found biocompatible according to ISO 10993 and able to load and release Ibuprofen: delivery kinetics of Ibuprofen was enhanced by P-CHP407 hydrogels at alkaline pH, suggesting their potential use as smart delivery systems in the treatment of chronic infected wounds

Injectable thermosensitive formulation based on polyurethane hydrogel/mesoporous glasses for sustained co-delivery of functional ions and drugs

Mini-invasively injectable hydrogels are widely attracting interest as smart tools for the co-delivery of therapeutic agents targeting different aspects of tissue/organ healing (e.g., neo-angiogenesis, inflammation). In this work, copper-substituted bioactive mesoporous glasses (Cu-MBGs) were prepared as nano- and micro-particles and successfully loaded with ibuprofen through an incipient wetness method (loaded ibuprofen approx. 10% w/w). Injectable hybrid formulations were then developed by dispersing ibuprofen-loaded Cu-MBGs within thermosensitive hydrogels based on a custom-made amphiphilic polyurethane. This procedure showed almost no effects on the gelation potential (gelation at 37 °C within 3–5 min). Cu2+ and ibuprofen were co-released over time in a sustained manner with a significantly lower burst release compared to MBG particles alone (burst release reduction approx. 85% and 65% for ibuprofen and Cu2+, respectively). Additionally, released Cu2+ species triggered polyurethane chemical degradation, thus enabling a possible tuning of gel residence time at the pathological site. The overall results suggest that hybrid injectable thermosensitive gels could be successfully designed for the simultaneous localized co-delivery of multiple therapeutics

Embedding Ordered Mesoporous Carbons into Thermosensitive Hydrogels: A Cutting-Edge Strategy to Vehiculate a Cargo and Control Its Release Profile

9siThe high drug loading capacity, cytocompatibility and easy functionalization of ordered mesoporous carbons (OMCs) make them attractive nanocarriers to treat several pathologies. OMCs’ efficiency could be further increased by embedding them into a hydrogel phase for an in loco prolonged drug release. In this work, OMCs were embedded into injectable thermosensitive hydrogels. In detail, rod-like (diameter ca. 250 nm, length ca. 700 nm) and spherical (diameter approximately 120 nm) OMCs were synthesized by nanocasting selected templates and loaded with ibuprofen through a melt infiltration method to achieve complete filling of their pores (100% loading yield). In parallel, an amphiphilic Poloxamer®407-based poly(ether urethane) was synthesized (Mn 72 kDa) and solubilized at 15 and 20% w/v concentration in saline solution to design thermosensitive hydrogels. OMC incorporation into the hydrogels (10 mg/mL concentration) did not negatively affect their gelation potential. Hybrid systems successfully released ibuprofen at a slower rate compared to control gels (gels embedding ibuprofen as such), but with no significant differences between rod-like and spherical OMC-loaded gels. OMCs can thus work as effective drug reservoirs that progressively release their payload over time and also upon encapsulation in a hydrogel phase, thus opening the way to their application to treat many different pathological states (e.g., as topical medications).openopenMonica Boffito; Rossella Laurano; Dimitra Giasafaki; Theodore Steriotis; Athanasios Papadopoulos; Chiara Tonda-Turo; Claudio Cassino; Georgia Charalambopoulou; Gianluca CiardelliBoffito, Monica; Laurano, Rossella; Giasafaki, Dimitra; Steriotis, Theodore; Papadopoulos, Athanasios; TONDA TURO, Chiara; Cassino, Claudio; Charalambopoulou, Georgia; Ciardelli, Gianluc