Development of injectable organic/inorganic colloidal composite gels made of self-assembling gelatin nanospheres and calcium phosphate nanocrystals

Colloidal gels are a particularly attractive class of hydrogels for applications in regenerative medicine, and allow for a “bottom-up” fabrication of multi-functional biomaterials by employing micro- or nanoscale particles as building blocks to assemble into shape-specific bulk scaffolds. So far, however, the synthesis of colloidal composite gels composed of both organic and inorganic particles has hardly been investigated. The current study has focused on the development of injectable colloidal organic–inorganic composite gels using calcium phosphate (CaP) nanoparticles and gelatin (Gel) nanospheres as building blocks. These novel Gel–CaP colloidal composite gels exhibited a strongly enhanced gel elasticity, shear-thinning and self-healing behavior, and gel stability at high ionic strengths, while chemical – potentially cytotoxic – functionalizations were not necessary to introduce sufficiently strong cohesive interactions. Moreover, it was shown in vitro that osteoconductive CaP nanoparticles can be used as an additional tool to reduce the degradation rate of otherwise fast-degradable gelatin nanospheres and fine-tune the control over the release of growth factors. Finally, it was shown that these colloidal composite gels support attachment, spreading and proliferation of cultured stem cells. Based on these results, it can be concluded that proof-of-principle has been obtained for the design of novel advanced composite materials made of nanoscale particulate building blocks which exhibit great potential for use in regenerative medicine.     

Structure–function–property–design interplay in biopolymers: Spider silk

Spider silks have been a focus of research for almost two decades due to their outstanding mechanical and biophysical properties. Recent advances in genetic engineering have led to the synthesis of recombinant spider silks, thus helping to unravel a fundamental understanding of structure–function–property relationships. The relationships between molecular composition, secondary structures and mechanical properties found in different types of spider silks are described, along with a discussion of artificial spinning of these proteins and their bioapplications, including the role of silks in biomineralization and fabrication of biomaterials with controlled properties.     

Squalene-PEG: Pyropheophorbide-a nanoconstructs for tumor theranostics

Novel nanoscale drug delivery biomaterials are of great importance for the diagnosis and treatment of different cancers. We have developed a new pegylated squalene (SQ-PEG) derivative with self-assembly properties. Supramolecular assembly with a lipophilic photosensitizer pyropheophorbide-a (Ppa) by nanoprecipitation gave nanoconstructs SQ-PEG:Ppa with an average size of 200 nm in diameter and a drug loading of 18% (w/w). The composite material demonstrates nanoscale optical properties by tight packing of Ppa within Sq-PEG:Ppa resulting in 99.99% fluorescence self-quenching. The biocompatibility of the nanomaterial and cell phototoxicity under light irradiation were investigated on PC3 prostate tumor cells in vitro. SQ-PEG:Ppa showed excellent phototoxic effect at low light dose of 5.0 J/cm² as a consequence of efficient cell internalization of Ppa by the nanodelivery system. The diagnostic potential of SQ-PEG:Ppa nanoconstructs to deliver Ppa to tumors in vivo was demonstrated in chick embryo model implanted with U87MG glioblastoma micro tumors.     

Resilin: Protein-based elastomeric biomaterials

Resilin is an elastomeric protein found in insect cuticles and is remarkable for its high strain, low stiffness, and high resilience. Since the first resilin sequence was identified in Drosophilia melanogaster (fruit fly), researchers have utilized molecular cloning techniques to construct resilin-based proteins for a number of different applications. In addition to exhibiting the superior mechanical properties of resilin, resilin-based proteins are autofluorescent, display self-assembly properties, and undergo phase transitions in response to temperature. These properties have potential application in designing biosensors or environmentally responsive materials for use in tissue engineering or drug delivery. Furthermore, the capability of resilin-based biomaterials has been expanded by designing proteins that include both resilin-based sequences and bioactive domains such as cell-adhesion or matrix metalloproteinase sequences. These new materials maintain the superior mechanical and physical properties of resilin and also have the added benefit of controlling cell response. Because the mechanical and biological properties can be tuned through protein engineering, a wide range of properties can be achieved for tissue engineering applications including muscles, vocal folds, cardiovascular tissues, and cartilage.     

Self-assembled magnetic fluorescent polymeric micelles for magnetic resonance and optical imaging

Stable and cytocompatible hybrid PEGylated micelles with multimodal imaging capabilities are described. The F₃O₄-encapsulated polymeric micelles composed of cores containing magnetic nanoparticles and polyethylene glycol (PEG) shells are synthesized by self-assembly of amphiphilic poly(HFMA-co-VBK)-g-PEG copolymers and oleic acid stabilized Fe₃O₄ nanoparticles. The Fe₃O₄ magnetic nanoparticles in the core produce T₂-weighted MR imaging functionalities, whereas the small fluorescent monomer carbazole in the polymer shell introduces good fluorescent properties. The multifunctional micelles exhibit excellent paramagnetic properties with the maximum saturation magnetization of 9.61 emu/g and transverse relaxivity rate of 157.44 mm⁻¹ S⁻¹. In vivo magnetic resonance imaging (MRI) studies reveal enhanced contrast between the liver and spleen. Fluorescence spectra show characteristic emission peaks from carbazole at 350 nm and 365 nm and vivid blue fluorescence can be observed by 2-photon confocal scanning laser microscopy (CLSM). In vivo optical imaging demonstrates the unique fluorescent characteristics of the Fe₃O₄-encapsulated polymeric micelles in the liver and spleen and the excellent multifunctional properties suggest potential clinical use as nanocarriers in magnetic resonance imaging and optical imaging.     

Coassembly of amphiphilic peptide EAK16-II with histidinylated analogues and implications for functionalization of β-sheet fibrils in vivo

EAK16-II (AEAEAKAKAEAEAKAK) is one of the first building blocks of environmentally responsive materials. This self-assembling peptide undergoes solution-to-gel transition when transferred from a low to high ionic strength environment. Previously we have demonstrated the histidinylated analogue EAKIIH6 (AEAEAKAKAEAEAKAKHHHHHH) coassembles with the parent peptide to render His-tags as a functionalization mechanism in vitro and in vivo. The present study aimed to understand the pathways by which the analogue coassembles with EAK16-II. The results presented herein suggested two competing but not mutually exclusive events in the coassembly. Atomic force microscopic and gel electrophoretic data showed that EAKIIH6 self-sorted to high molecular weight species without EAK16-II. Self-sorting of EAKIIH6 was inhibited by the parent peptide in a concentration dependent manner. Injecting mixtures containing EAKIIH6 subcutaneously rendered His-tags detectable in live mice for at least 312 h, despite diluting the histidinylated analogue by 10–50 folds compared to a previous formulation. The study provided a formulation by which in vivo display of His-tags was attained without excess amphiphilic peptides. By increasing coassembling efficiency, the likelihood of generating immunogenic aggregates outside the main fibrils could be minimized. These findings provide insights for rational functionalization of in situ self-gelling materials.     

Reducible polyamidoamine-magnetic iron oxide self-assembled nanoparticles for doxorubicin delivery

We report a reducible copolymer self-assembled with superparamagnetic iron oxide nanoparticles (SPIONs) to deliver doxorubicin (DOX) for cancer therapy. The copolymer of reducible polyamidoamine (rPAA) with poly(ethylene glycol)(PEG)/dodecyl amine graft was synthesized by Michael addition. rPAA@SPIONs were formed by the alkyl grafts of reducible copolymers intercalated with the oleic acid layer capped on the surface of magnetite nanocrystals. The intercalating area formed a reservoir for hydrophobic anti-cancer drug (DOX), whilst the PEG moiety in the copolymers helped the nanoparticle well-dispersible in aqueous solution. We employed two-photon excited fluorescence (TPEF) and coherent anti-Stokes Raman (CARS) to investigate drug delivery in intra-cellular structures of live cells, and used Vivaview^® technique to show real-time inhibition efficacy of nanoparticles in live cells. rPAA@SPIONs present efficiently drug loading with reducible responsibility in vitro tests. Finally, rPAA@SPIONs were tested in mice with xenograft MDA-MB-231 breast tumor though i.v. injection and inhibited tumor growth efficiently. MRI was used to monitor nanoparticles aggregation in tumor site. Histology and Prussian blue on kidney, liver, and heart in mice indicated that DOX/rPAA@SPIONs showed no significant toxicity for mice organs after 24 days treatment.     

Ultrashort peptide nanofibrous hydrogels for the acceleration of healing of burn wounds

There is an unmet clinical need for wound dressings to treat partial thickness burns that damage the epidermis and dermis. An ideal dressing needs to prevent infection, maintain skin hydration to facilitate debridement of the necrotic tissue, and provide cues to enhance tissue regeneration. We developed a class of ‘smart’ peptide hydrogels, which fulfill these criteria. Our ultrashort aliphatic peptides have an innate tendency to self-assemble into helical fibers, forming biomimetic hydrogel scaffolds which are non-immunogenic and non-cytotoxic. These nanofibrous hydrogels accelerated wound closure in a rat model for partial thickness burns. Two peptide hydrogel candidates demonstrate earlier onset and completion of autolytic debridement, compared to Mepitel^®, a silicone-coated polyamide net used as standard-of-care. They also promote epithelial and dermal regeneration in the absence of exogenous growth factors, achieving 86.2% and 92.9% wound closure respectively, after 14 days. In comparison, only 62.8% of the burnt area is healed for wounds dressed with Mepitel^®. Since the rate of wound closure is inversely correlated with hypertrophic scar formation and infection risks, our peptide hydrogel technology fills a niche neglected by current treatment options. The regenerative properties can be further enhanced by incorporation of bioactive moieties such as growth factors and cytokines.     

Active targeting of early and mid-stage atherosclerotic plaques using self-assembled peptide amphiphile micelles

Inflammatory cell adhesion molecules expressed by endothelial cells on the luminal surface of atherosclerotic plaques, such as vascular cell adhesion molecule-1 (VCAM-1), provide a rational target for diagnostic and therapeutic delivery vehicles. Therefore, the potential of using spherical, self-assembled micelles synthesized from VCAM-1 targeted peptide amphiphile molecules was examined for the ability to specifically bind to both early and mid-stage atherosclerotic plaques. In vitro, cells incubated with VCAM-1 targeted and dye-labeled micelles show enhanced fluorescence signal as compared to cells incubated with a PEG micelle control. In vivo, VCAM-1 targeted and Cy7-labeled peptide amphiphile micelles were shown to specifically accumulate at atherosclerotic plaques in both early and mid-stage ApoE −/− mice through co-localization of Cy7 signal with anti-VCAM-1 antibody staining in fixed tissue. No specific accumulation was observed with a PEG micelle control. Histological analysis of excised tissue provided evidence for the in vivo biocompatibility of these micelle formulations as no tissue damage was observed. These results demonstrate that VCAM-1 targeted micelles have potential as a platform for targeted drug delivery to multiple stages of atherosclerotic plaque formation due to their established specificity and safety.     

Fibrin-binding,peptide amphiphile micelles for targeting glioblastoma

Glioblastoma-targeted drug delivery systems facilitate efficient delivery of chemotherapeutic agents to malignant gliomas, while minimizing systemic toxicity and side effects. Taking advantage of the fibrin deposition that is characteristic of tumors, we constructed spherical, Cy7-labeled, targeting micelles to glioblastoma through the addition of the fibrin-binding pentapeptide, cysteine–arginine–glutamic acid–lysine–alanine, or CREKA. Conjugation of the CREKA peptide to Cy7-micelles increased the average particle size and zeta potential. Upon intravenous administration to GL261 glioma bearing mice, Cy7-micelles passively accumulated at the brain tumor site via the enhanced permeability and retention (EPR) effect, and Cy7-CREKA-micelles displayed enhanced tumor homing via active targeting as early as 1 h after administration, as confirmed via in vivo and ex vivo imaging and immunohistochemistry. Biodistribution of micelles showed an accumulation within the liver and kidneys, leading to micelle elimination via renal clearance and the reticuloendothelial system (RES). Histological evaluation showed no signs of cytotoxicity or tissue damage, confirming the safety and utility of this nanoparticle system for delivery to glioblastoma. Our findings offer strong evidence for the glioblastoma-targeting potential of CREKA-micelles and provide the foundation for CREKA-mediated, targeted therapy of glioma.