Katharina Ladewig

Cyclodextrin-based supramolecular assemblies and hydrogels: recent advances and future perspectives.

The application of cyclodextrin (CD)-based host-guest interactions towards the fabrication of functional supramolecular assemblies and hydrogels is of particular interest in the field of biomedicine. However, as of late they have found new applications as advanced functional materials (e.g., actuators and self-healing materials), which have renewed interest across a wide range of fields. Advanced supramolecular materials synthesized using this noncovalent interaction, exhibit specificity and reversibility, which can be used to impart reversible cross-linking, specific binding sites, and functionality. In this review, various functional CD-based supramolecular assemblies and hydrogels will be outlined with the focus on recent advances. In addition, an outlook will be provided on the direction of this rapidly developing field.

Drug delivery in soft tissue engineering.

Introduction: Tissue defects, sustained through disease or trauma, present enormous challenges in regenerative medicine. Modern tissue engineering (TE) aims at replacing or repairing these defects through a combined approach of biodegradable scaffolds, suitable cell sources and appropriate environmental cues, such as biomolecules presented on scaffold surfaces or sustainably released from within. Areas covered: This review provides a brief overview of the various drugs and bioactive molecules of interest to TE, as well as a selection of materials that have been proposed for TE scaffolds and matrices in the past. It then proceeds to discuss encapsulation, immobilization and controlled release strategies for bioactive proteins, before discussing recent advances in this area with a special focus on soft TE. Expert opinion: Overall, minimal clinical success has been achieved so far in using growth factor, morphogen, or adhesion factor modified scaffolds and matrices; only one growth factor delivery system (Regranex Gel), has been approved by the FDA for clinical use, with only a handful of other growth factors being approved for human use so far. However, many more growth factors are currently in clinical Phase I – II or preclinical trials and many delivery systems utilize materials already approved by the FDA for other purposes. With respect to drug delivery in soft TE, a combination of increased research efforts in hydrogel and support material development as well as growth factor development is needed before clinical success is realized.

Biodegradable and biocompatible poly(ethylene glycol)-based hydrogel films for the regeneration of corneal endothelium.

Corneal endothelial cells (CECs) are responsible for maintaining the transparency of the human cornea. Loss of CECs results in blindness, requiring corneal transplantation. In this study, fabrication of biocompatible and biodegradable poly(ethylene glycol) (PEG)-based hydrogel films (PHFs) for the regeneration and transplantation of CECs is described. The 50-μm thin hydrogel films have similar or greater tensile strengths to human corneal tissue. Light transmission studies reveal that the films are >98% optically transparent, while in vitro degradation studies demonstrate their biodegradation characteristics. Cell culture studies demonstrate the regeneration of sheep corneal endothelium on the PHFs. Although sheep CECs do not regenerate in vivo, these cells proliferate on the films with natural morphology and become 100% confluent within 7 d. Implantation of the PHFs into live sheep corneas demonstrates the robustness of the films for surgical purposes. Regular slit lamp examinations and histology of the cornea after 28 d following surgery reveal minimal inflammatory responses and no toxicity, indicating that the films are benign. The results of this study suggest that PHFs are excellent candidates as platforms for the regeneration and transplantation of CECs as a result of their favorable biocompatibility, degradability, mechanical, and optical properties.

Highly porous and mechanically robust polyester poly(ethylene glycol) sponges as implantable scaffolds

The development of suitable scaffolds plays a significant role in tissue engineering research. Although scaffolds with promising features have been produced via a variety of innovative methods, there are no fully synthetic tissue engineering scaffolds that possess all the desired properties in one three-dimensional construct. Herein, we report the development of novel polyester poly(ethylene glycol) (PEG) sponges that display many of the desirable scaffold characteristics. Our novel synthetic approach utilizes acidchloride/alcohol chemistry, whereby the reaction between a hydroxyl end-functionalized 4-arm PEG and sebacoyl chloride resulted in cross-linking and simultaneous hydrogen chloride gas production, which was exploited for the in situ formation of highly interconnected pores. Variation of the fabrication conditions, including the precursor volume and concentration, allowed the pore size and structure as well as the compressive properties to be tailored. The sponges were found to possess excellent elastic properties, preserving their shape even after 80% compressive strain without failure. The benign properties of the sponges were demonstrated in an in vivo subcutaneous rat model, which also revealed uniform infiltration of vascularized tissue by 8 weeks and complete degradation of the sponges by 16 weeks, with only a minimal inflammatory response being observed over the course of the experiments.

Size and Phase Control of Cubic Lyotropic Liquid Crystal Nanoparticles

The effective use of lyotropic liquid crystalline dispersions, such as cubosomes, as drug delivery vehicles requires that they have tailored physical characteristics that suit specific therapeutics and external conditions. Here, we have developed phytantriol-based cubosomes from a dispersion of unilamellar vesicles and show that we can control their size as well as the critical packing parameter (CPP) of the amphiphilic bilayer through regulation of temperature and salt concentration, respectively. Using the anionic biological lipid 1,2-dipalmi-toylphosphatidylserine (DPPS) to prevent the cubic phase from forming, we show that the addition of phosphate buffered saline (PBS) results in a transition from small unilamellar vesicles to the cubic phase due to charge-shielding of the anionic lipid. Using dynamic light scattering, we show that the cubosomes formed following the addition of PBS are as small as 30 nm; however, we can increase the average size of the cubsosomes to create an almost monodisperse dispersion of cubosomes through cooling. We propose that this phenomenon is brought about through the phase separation of the Pluronic F-127 used to stabilize the cubosomes. To complement previous work using the salt-induced method of cubosome production, we show, using synchrotron small-angle X-ray scattering (SAXS), that we can control the CPP of the amphiphile bilayer which grants us phase and lattice parameter control of the cubosomes.

Cyclodextrin-based supramolecular polymeric nanoparticles for next generation gas separation membranes

Cyclodextrin-based supramolecular assemblies derived from poly(dimethylsiloxane) (PDMS) functionalized polyrotaxanes (PRXs) were self-assembled into core–shell morphologies and used as soft nanoparticle (SNP) additives in the selective layer of thin film composite (TFC) membranes for the first time. Various weight percentages (wt%) of the PRX SNP additives were combined with Pebax® 2533 to form the selective layer, and the gas transport properties of the TFC membranes were studied in detail. Increasing the amount of PRX SNP additives led to a significant increase in CO2 permeance of the membranes, with only a slight decrease in the CO2/N2 selectivity, which was attributed to the dynamic nature (i.e., translational and rotational freedom) of the conjugated PDMS chains on the PRXs. In comparison, the performance of membranes prepared using a conventional analogue with fixed PDMS chains was inferior. The excellent gas transport properties observed for membranes are attributed to the novel self-assembly process of the dynamic PRX SNP additives; the sliding nature of the conjugated PDMS chains allow for increased exposure of the CO2-philic PEG backbone and increased size of the hydrophobic core leading to improved membrane selectivity and permeability. The effect of varying operating conditions (feed pressure and temperature) was also investigated and compared between the dynamic and fixed additive systems. Interesting trends were observed with the dynamic PRX system which diverges from conventional systems. This study opens up new avenues for CD-based supramolecular chemistry in the field of membrane technologies for gas separation.

Amphiphilic core cross-linked star polymers as water-soluble, biocompatible and biodegradable unimolecular carriers for hydrophobic drugs

Unimolecular polymeric architectures are ideal candidates for drug encapsulation. In this study we report the facile yet well-controlled formation of a series of biocompatible and biodegradable core cross-linked star (CCS) polymers via the easily scalable, metal free, one- or two- step ring-opening polymerization (ROP) of e-caprolactone, using poly(ethylene glycol) (PEG) as initiator and [4,4′-bioxepane]-7,7′-dione (BOD) as cross-linker. The resulting CCS polymers, which exhibit hydrophilic PEG blocks in their outer shell and hydrophobic poly(e-caprolactone) (PCL) and BOD segments in their inner core, are water-soluble and amphiphilic and exist in a unimolecular state, both in organic solvents and in water. These properties provide the opportunity to easily stabilise water-insoluble, hydrophobic drugs in aqueous environments without the need for conjugation of the drug to the carrier and/or complex encapsulation techniques. The impact of hydrophilic/hydrophobic block length and core size on polymer properties was investigated via gel permeation chromatography (GPC) and dynamic light scattering (DLS). In addition, the change in drug encapsulation properties with varying hydrophilic/hydrophobic balance was studied using pirarubicin – a potent anthracycline – as a model hydrophobic drug. Formation of a drug–CCS polymer conjugate purely based on hydrophobic–hydrophobic interaction of the drug and the hydrophobic component of the CCS was verified by 1H NMR and UV-Vis measurements, and the size change confirmed by DLS and transmission electron microscopy (TEM). The in vitro study of drug–CCS conjugate demonstrated significantly faster release of anthracycline from the CCS polymer under acidic conditions (pH = 5.5) compared with normal physiological pH level (7.4). Furthermore, cytotoxicity and cellular uptake tests performed using Hela cells, demonstrated extremely low toxicity of the macroinitiators and CCS polymers even at high concentrations, while anthracycline-loaded CCS polymers exhibited similar IC50 values to the free drug. Confocal laser scanning microscopy and flow cytometry confirmed high uptake efficiency and intracellular localisation of the CCS polymers upon uptake, respectively.

A novel one-pot approach towards dynamically cross-linked hydrogels

Herein, we report the synthesis of sliding-ring (SR) hydrogel networks in a one-pot click-mediated approach using α,ω-dialkyne poly(ethylene glycol) (PEG) and azido-functionalised cyclodextrin, which acts as both sliding cross-link and end-capping agent. This novel approach resulted in polymeric networks that possess a combination of both SR and covalent (CV) cross-link points. The extent of inclusion complexation and the ratio of SR to CV cross-links in the hydrogels was found to be dependent on both the concentration of the precursors and the curing temperature. Based upon model studies where rotaxanes were synthesised from the same precursors, it was observed that an increase in the precursor concentration led to an increase in click efficiency and inclusion ratio, which in turn affects the overall hydrogel rigidity and elasticity. Hydrogels synthesised at higher curing temperatures led to more homogeneous networks that were significantly tougher as a result of the overall increase in cross-linking density and the extent of CV cross-links. We therefore present a facile one-pot method for the synthesis of SR networks with tunable physicochemical properties. Additionally, the resultant hydrogel networks are potentially capable of supporting post-modification with various (bio)molecules or therapeutics utilizing the remaining azide groups on the cyclodextrin cross-links. Preliminary cytotoxicity studies revealed that the hydrogels did not impede cell growth and demonstrate negligible toxicity. Thus, these networks may have potential for soft-tissue engineering or biomedical applications, including sustained release and drug-delivery systems.

Peptide-Based Star Polymers as Potential siRNA Carriers

16- and 32-arm star polymers were synthesised using poly(amido amine) (PAMAM) dendrimers as multifunctional initiators for the ring-opening polymerisation (ROP) of ϵ-Z-l-lysine N-carboxyanhydride (Lys NCA) via the core-first approach. The resulting star polymers were subsequently post-functionalised with poly(ethylene glycol) (PEG) via carbodiimide coupling, potentially improving the biodistribution of the stars in vivo. De-protection of the carboxybenzyl (Cbz)-protected star arms yielded water-soluble cationic poly(l-lysine) (PLL) star polymers with hydrodynamic radii ranging from 2.0 to 3.3 nm. Successful complexation of the PLL star polymers with double-stranded oligodeoxynucleotides (ODNs)—a mimic for small interfering RNA (siRNA)—was achieved at a nitrogen-to-phosphate (N/P) ratio of 5. Cell viability studies using HEK293T cells indicated the ‘safe’ concentration for these polymers is within a suitable window for the delivery of siRNA therapeutics.

Physicochemical and cytotoxicity analysis of glycerol monoolein-based nanoparticles

Lyotropic liquid crystalline dispersions, such as cubosomes, have been proposed as potential drug delivery vehicles. A recently described ‘salt induced’ method of cubosome production may be suitable for the encapsulation of macromolecular bioactive therapeutics, such as proteins, within the cubic phase. Here, we develop and characterise glycerol-monoolein (GMO)-based cubosomes using this novel method of cubosome production. Using the anionic biological lipid 1,2-dipalmitoyl phosphatidylserine (DPPS) to prevent GMO from forming its natural cubic-phase, we validate that addition of phosphate buffered saline (PBS) can be used to reverse the effects of DPPS. However, this transition is dependent on the type of Pluronic® block copolymer stabiliser used to prevent re-flocculation of the cubosome dispersions. Using small angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy, we show that the ‘salt induced’ phase transition, from small unilamellar vesicles to cubosomes, is inhibited when using Pluronic® F127. In contrast, using the larger, more hydrophilic stabiliser Pluronic® F108, cubosomes can be formed, although further analysis using SAXS suggests these GMO-based cubosomes are less thermally stable than those comprising GMO alone. In addition, we find no significant difference in the in vitro cytotoxicity of cubosome dispersions formed using either of these stabilisers, or between those containing DPPS and those without. The ability to control cubic phase transitions may present an opportunity for the incorporation of therapeutically relevant proteins in these nanoparticles.