Semi-synthetic degradable notochordal cell-derived matrix hydrogel for use in degenerated intervertebral discs: Initial in vitro characterization.

Low back pain is the leading cause of disability worldwide, but current therapeutic interventions are palliative or surgical in nature. Loss of notochordal cells (NCs) and degradation of the healthy matrix in the nucleus pulposus (NP), the central tissue of intervertebral discs (IVDs), has been associated with onset of degenerative disc changes. Recently, we established a protocol for decellularization of notochordal cell derived matrix (NCM) and found that it can provide regenerative cues to nucleus pulposus cells of the IVD. Here, we combined the biologically regenerative properties of decellularized NCM with the mechanical tunability of a poly(ethylene glycol) hydrogel to additionally address biomechanics in the degenerate IVD. We further introduced a hydrolysable PEG-diurethane crosslinker for slow degradation of the gels in vivo. The resulting hydrogels were tunable over a broad range of stiffness's (0.2 to 4.5 kPa), matching that of NC-rich and -poor NP tissues, respectively. Gels formed within 30 min, giving ample time for handling, and remained shear-thinning post-polymerization. Gels also slowly released dNCM over 28 days as measured by GAG effusion. Viability of encapsulated bone marrow stromal cells after extrusion through a needle remained high. Although encapsulated NCs stayed viable over two weeks, their metabolic activity decreased, and their phenotype was lost in physiological medium conditions in vitro. Overall, the obtained gels hold promise for application in degenerated IVDs but require further tuning for combined use with NCs.

In Vivo Retention Quantification of Supramolecular Hydrogels Engineered for Cardiac Delivery.

Recent advances in the field of cardiac regeneration show great potential in the use of injectable hydrogels to reduce immediate flush-out of injected factors, thereby increasing the effectiveness of the encapsulated drugs. To establish a relation between cardiac function and retention of the drug-encapsulating hydrogel, a quantitative in vivo imaging method is required. Here, the supramolecular ureido-pyrimidinone modified poly(ethylene glycol) (UPy-PEG) material is developed into a bioactive hydrogel for radioactive imaging in a large animal model. A radioactive label is synthesized, being a ureido-pyrimidinone moiety functionalized with a chelator (UPy-DOTA) complexed with the radioactive isotope indium-111 (UPy-DOTA-111 In) that is mixed with the hydrogel. Additionally, bioactive and adhesive properties of the UPy-PEG hydrogel are increased by supramolecular introduction of a UPy-functionalized recombinant collagen type 1-based material (UPy-PEG-RCPhC1). This method enables in vivo tracking of the nonbioactive and bioactive supramolecular hydrogels and quantification of hydrogel retention in a porcine heart. In a small pilot, cardiac retention values of 8% for UPy-PEG and 16% for UPy-PEG-RCPhC1 hydrogel are observed 4 h postinjection. This work highlights the importance of retention quantification of hydrogels in vivo, where elucidation of hydrogel quantity at the target site is proposed to strongly influence efficacy of the intended therapy.

Synthesis and Self-Assembly of Bay-Substituted Perylene Diimide Gemini-Type Surfactants as Off-On Fluorescent Probes for Lipid Bilayers.

Interest in bay-substituted perylene-3,4:9,10-tetracarboxylic diimides (PDIs) for solution-based applications is growing due to their improved solubility and altered optical and electronic properties compared to unsubstituted PDIs. Synthetic routes to 1,12-bay-substituted PDIs have been very demanding due to issues with steric hindrance and poor regioselectivity. Here we report a simple one-step regioselective and high yielding synthesis of a 1,12-dihydroxylated PDI derivative that can subsequently be alkylated in a straightforward fashion to produce nonplanar 1,12-dialkoxy PDIs. These PDIs show a large Stokes shift, which is specifically useful for bioimaging applications. A particular cationic PDI gemini-type surfactant has been developed that forms nonfluorescent self-assembled particles in water ("off state"), which exerts a high fluorescence upon incorporation into lipophilic bilayers ("on state"). Therefore, this probe is appealing as a highly sensitive fluorescent labelling marker with a low background signal for imaging artificial and cellular membranes.

Controlling and tuning the dynamic nature of supramolecular polymers in aqueous solutions.

Structural and kinetic exchange properties of supramolecular polymers composed of mono- and bivalent ureidopyrimidinone-based monomers are investigated in aqueous solutions. It is shown that exchange dynamics can be controlled by mixing different types of monomers. This tunability widens the scope in their design as biomaterials.

Influence of the solvent and the enantiomeric purity on the transition between different supramolecular polymers.

The self-assembly of two enantiomerically pure hexa(oligo(p-phenylene vinylene))-substituted benzenes having 24 stereocenters was studied in pure methylcyclohexane (MCH) and in a mixture of MCH/toluene (4:1). Irrespective of the solvent a cooperative supramolecular polymerization mechanism was determined for these star-shaped molecules by using temperature-dependent CD and UV/Vis spectroscopy. Quite remarkably, a transition from one helical supramolecular state (A) to a second more thermodynamically stable supramolecular helical assembly (B) was observed. The rate of the A→B transition was strongly dependent on the nature of the solvent; being faster in the solvent mixture than in pure MCH. By using size exclusion chromatography we could relate the increased rate to a decreased stability of the supramolecular A state in the solvent mixture. Next, we mixed the two enantiomerically pure hexa-substituted benzene derivatives in a so-called majority-rules experiment, which lead to the anitcipated chiral amplification in the A state. More importantly it appeared that the A→B transition was significantly hampered in these mixed systems. Furthermore, the absence of chiral amplification in the B state revealed the formation of separated enantiomerically pure assemblies. Therefore, by using a wide variety of spectroscopic and chromatographic techniques we determined the influence of solvent and enantiomeric purity on the transition between different supramolecular states.

White-light emitting hydrogen-bonded supramolecular copolymers based on pi-conjugated oligomers.

Three different pi-conjugated oligomers (a blue-emitting oligofluorene, a green-emitting oligo(phenylene vinylene), and a red-emitting perylene bisimide) have been functionalized with self-complementary quadruple hydrogen bonding ureidopyrimidinone (UPy) units at both ends. The molecules self-assemble in solution and in the bulk, forming supramolecular polymers. When mixed together in solution, random noncovalent copolymers are formed that contain all three types of chromophores, resulting in energy transfer upon excitation of the oligofluorene energy donor. At a certain mixing ratio, a white emissive supramolecular polymer can be created in solution. In contrast to their unfunctionalized counterparts, bis-UPy-chromophores can easily be deposited as smooth thin films on surfaces by spin coating. No phase separation is observed in these films, and energy transfer is much more efficient than in solution, giving rise to white fluorescence at much lower ratios of energy acceptor to donor. Light emitting diodes based on these supramolecular polymers have been prepared from all three types of pure materials, yielding blue, green, and red devices, respectively. At appropriate mixing ratios of these three compounds, white electroluminescence is observed. This approach yields a toolbox of molecules that can be easily used to construct pi-conjugated supramolecular polymers with a variety of compositions, high solution viscosities, and tuneable emission colors.

Identification of oligo(p-phenylene vinylene)-naphthalene diimide heterocomplexes by scanning tunneling microscopy and spectroscopy at the liquid-solid interface.

Co-assembly of a melamine derivative covalently equipped with two oligo(p-phenylene vinylene) chromophores and a naphthalene diimide dye results in the formation of heterocomplexes at the liquid-solid interface which was shown using bias dependent imaging and scanning tunneling spectroscopy, despite the disordered nature of the assemblies.

Visualization of various supramolecular assemblies of oligo(para-phenylenevinylene)-melamine and perylene bisimide.

A melamine derivative has been covalently equipped with two oligo(para-phenylenevinylene) (OPV) chromophores. This procedure yields a bifunctional molecule with two hydrogen-bonding arrays available for complementary binding to perylene bisimide derivatives. Depending on the solvent, hydrogen-bonded trimers, tetramers, and dimers on a graphite surface are observed for pure OPV-melamine by using scanning tunneling microscopy (STM). Upon the addition of perylene bisimide, linear tapes of perylene bisimide, 12-membered rosettes that consist of alternating hydrogen-bonded OPV-melamine and perylene bisimide moieties are visualized. These results provide direct evidence for the possible modes of hydrogen bonding within a supramolecular co-assembly in solution. Subsequently, the optical properties of pure OPV-melamine and co-assemblies with a perylene bisimide derivative were characterized in solution. In an apolar solvent, OPV-melamine self-assembles into chiral superstructures. Disassembly into molecularly dissolved species is reversibly controlled by concentration and temperature. Complementary hydrogen bonding to a perylene bisimide derivative in an apolar solvent yields multicomponent, pi-stacked dye assemblies of enhanced stability that are characterized by fluorescence quenching of the constituent chromophores. Titration experiments reveal that a mixture of hydrogen-bonded oligomers is present in solution, rather than a single discrete assembly. The solution experiments are consistent with the STM results, which revealed various supramolecular assemblies. Our system is likely not to be optimally programmed to obtain a discrete co-assembled structure in quantitative yield.

Chiral alignment of OPV chromophores: exploitation of the ureidophthalimide-based foldamer.

The ability of foldamers to adopt a secondary structure in solution has been exploited to organize peripheral functionality. Our previously reported poly(ureidophthalimide) foldamer proved to be an excellent scaffold for the chiral organization of peripherally positioned oligo(p-phenylenevinylene) (OPV) chromophores. Facile high-yielding synthesis gave access to the required OPV-decorated building blocks. A condensation polymerization provided polymers of sufficient length to allow construction of a helical architecture comprising several turns. Short and long chains were separated by chromatography. Circular dichroism studies in THF of the longer chains indicate the presence of helically arranged OPVs. However, such an effect is not observed in CHCl3. Remarkable are the measurements of the OPV foldamers in heptane. A bisignate Cotton effect is observed in heptane of a sample with a THF history. No Cotton effect is observed in heptane of a sample with a CHCl3 history. In this example of supramolecular synthesis, the solvent dictates the expression of supramolecular chirality in a secondary structure. The short-chain oligomeric fractions that are unable to create a full turn revealed on scanning tunneling microscopy analysis the presence of circular architectures at the graphite/1-phenyloctane interface. This is in full agreement with the proposed conformation of the decorated foldamers.

Hydrogen-bonded oligo(p-phenylenevinylene) functionalized with perylene bisimide: self-assembly and energy transfer.

We describe the synthesis, supramolecular ordering on surfaces and in solution, and photophysical characterization of OPV4UT-PERY, an oligo(p-phenylenevinylene) (OPV) with a covalently attached perylene bisimide moiety. In chloroform, the molecule forms dimers through quadruple hydrogen bonding of the ureido-s-triazine array. This is supported by scanning tunneling microscopy (STM) studies, which reveal dimer formation at the liquid (1,2,4-trichlorobenzene)/solid (graphite) interface. Moreover, contrast reversal in bias-dependent STM imaging provides information on the ordering and different electronic properties of the oligo(p-phenylenevinylene) and perylene bisimide moieties. In dodecane, the molecule self-assembles into H-type aggregates that are still soluble as a result of the hydrophobic shell formed by the dodecyloxy wedges. The donor-acceptor molecule is characterized by efficient energy transfer from the photoexcited OPV to the perylene bisimide. Mixed assemblies with analogous OPVs lacking the perylene bisimide unit have been prepared in dodecane solution and energy transfer to the incorporated perylene bisimides has been studied by fluorescence spectroscopy.

Energy transfer in chiral co-assemblies of triple hydrogen-bonded oligo(p-phenylene vinylene)s and porphyrin.

Pi-pi stacking of hydrogen-bonded porphyrin and oligo(p-phenylene vinylene)s (OPVs) yields helical co-assemblies which exhibit energy transfer from OPV to porphyrin.

Synthesis and energy-transfer properties of hydrogen-bonded oligofluorenes.

A set of fluorene oligomers has been synthesized by stepwise palladium-catalyzed (Suzuki) couplings of fluorene monomers. Ureidopyrimidinones (UPy), functional groups that can dimerize via quadruple hydrogen bonds, were attached to both ends of the oligofluorenes. The resulting bis-UPy-terminated oligomers self-assemble into supramolecular chain polymers. For comparison, oligofluorenes of the same oligomer lengths but without terminal hydrogen-bonding groups were synthesized. Chains of hydrogen-bonded fluorenes can be simply endcapped by a variety of chain stoppers, molecules that have one UPy group. In this manner, we have endcapped the hydrogen-bonded fluorene chains with either oligo(p-phenylenevinylene) or perylene bisimide. Energy-transfer experiments in solution and the solid state demonstrate that oligofluorenes can donate energy to a variety of energy acceptors, but that this energy transfer occurs most effectively when the donor fluorene is hydrogen-bonded to the acceptor.