In this study, an injectable, photocurable gelatin system, consisting of acrylated gelatin and thiolated gelatin, with tunable mechanical, biodegradation, and biological properties was used as a potential cell-supportive scaffold for the repair of focal corneal wounds. The mechanical property of hydrogels can be readily modified (postcure shear modulus of between 0.3 and 22 kPa) by varying the ratio of acrylate to thiol groups, photointensity, and solid content, and the biodegradation times also varied with the change of solid content. More importantly, the generated hydrogels exhibited excellent cell viability in both cell seeding and cell encapsulation experiments. Furthermore, the hydrogels were found to be biocompatible with rabbit cornea and aided the regeneration of a new tissue under a focal corneal wound (exhibiting epithelial wound coverage in <3d), and ultraviolet irradiation did not have any obvious harmful effect on the cornea and posterior eye segment tissues. Along with their injectability and tunable mechanical properties, the photocurable thiol-acrylate hydrogels showed promise as corneal substitutes or substrates to construct a new corneal tissue.

Biophysical studies were undertaken to investigate the binding and release of short interfering ribonucleic acid (siRNA) from lyotropic liquid crystalline lipid nanoparticles (LNPs) by using a quartz crystal microbalance (QCM). These carriers are based on phytantriol (Phy) and the cationic lipid DOTAP (1,2-dioleoyloxy-3-(trimethylammonium)propane). The nonlamellar phase LNPs were tethered to the surface of the QCM chip for analysis based on biotin-neutravidin binding, which enabled the controlled deposition of siRNA-LNP complexes with different lipid/siRNA charge ratios on a QCM-D crystal sensor. The binding and release of biomolecules such as siRNA from LNPs was demonstrated to be reliably characterised by this technique. Essential physicochemical parameters of the cationic LNP/siRNA lipoplexessuch as particle size, lyotropic phase behaviour, cytotoxicity, gene silencing and uptake efficiencywere also assessed. The SAXS data show that when the pH was lowered to 5.5 the structure of the lipoplexes did not change, thus indicating that the acidic conditions of the endosome were not a significant factor in the release of siRNA from the cationic lipidic carriers.

Metformin is an oral hypoglycemic drug that has been shown to inhibit cancer cell proliferation via up-regulation of AMPK (AMP-activated protein kinase), and possibly inhibition of mTOR (mammalian target of rapamycin). The purpose of this study was to evaluate the effects of metformin on a feline injection site sarcoma cell line. Cells from a feline injection site sarcoma cell line were treated with metformin at varied concentrations. A dose-dependent decrease in cell viability following metformin treatment was observed, with an IC50 of 8.0mM. Using flow cytometry, the mechanism of cell death was determined to be apoptosis or necrosis. To evaluate the role of mTOR inhibition in metformin-induced cell death, Western blot was performed. No inhibition of mTOR or phosphorylated mTOR was found. Although metformin treatment leads to apoptotic or necrotic cell death in feline injection site sarcoma cells, the mechanism does not appear to be mediated by mTOR inhibition.

The aim of this study was to develop a 2-N, 6-O-sulfated chitosan (26SCS) modified electrospun fibrous PCL scaffold for bone morphogenetic protein-2 (BMP-2) delivery to improve osteoinduction. The PCL scaffold was modified by an aminolysis reaction using ethylenediamine (ED) and 26SCS was immobilized via electrostatic interactions (PCL-N-S). Scaffolds were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and contact angle measurements. In vitro BMP-2 adsorption and release kinetics indicated that modified PCL-N-S scaffolds showed higher levels of binding of BMP-2 (about 30-100 times), moderative burst release (about one third), and prolonged releasing time compared to the unmodified PCL scaffold. The bioactivity of released BMP-2 determined by alkaline phosphatase (ALP) activity assay was maintained and improved 8-12 times with increasing concentration of immobilized 26SCS on the scaffolds. In vitro studies demonstrated that bone marrow mesenchymal stem cells (BMSCs) attached more readily to the PCL-N-S scaffolds with increased spreading. In conclusion, 26SCS modified PCL scaffolds can be a potent system for the sustained and bioactive delivery of BMP-2.

Event Abstract Back to Event Saving vision with light: Photo-modulated ocular drug delivery Johan D. Basuki1*, Xavier D. Mulet1, Chen Hao2*, Hong D. Zhang3, Keith D. Mclean1* and Timothy D. Hughes1* 1 CSIRO, Manufacturing, Australia 2 Wenzhou Medical University, School of Optometry and Ophthalmology and Eye Hospital, China 3 University of Melbourne, Centre for Eye Research Australia, Australia Introduction: Age-related macular degeneration (AMD) is the leading cause of blindness in Australia today[1]. The only way to save vision requires monthly injection to the back of the eye with biomacromolecular anti-angiogenics, such as bevacizumab (Avastin®) and ranibizumab (Lucentis®)[2]. This invasive procedure, with the potential for sight threatening complications, has obvious negative impacts on patient’s quality of life as well as posing a major burden on the global health care system. A drug delivery system that serves as a reservoir to enable sustained release or ‘on-demand’ release of therapeutic protein/antibody would be beneficial, since it will significantly reduce the number of injections into the back of the eye[3]. The release of therapeutic payloads from the drug reservoir can be modulated on demand by light, particularly due to its external spatiotemporal control[4]. Materials/Method: In order to fabricate a photo-responsive drug delivery system a thermo-rensponsive polymer was formulated into a hydrogel containing light sensitive nanoparticles (LSNPs) and therapeutic payloads (Fig. 1). The release of different payloads, ranging from small molecules (doxorubicin, trimacinolone acetonide) to biomacromolecules (lysozyme, bovine serum albumin, and IgG/antibody), can be switched on and off upon exposure to visible light (Fig. 2). This hydrogel can be modified to a dispersion of microparticles or a thermo-reversible polymer solution for ocular injection, while additional surface coating can be applied for long-term sustained release. Results and Discussion: Released lysozyme, bevacizumab, and ranibizumab from this formulation exhibited above 85% biological activities after their exposure to light, in particular bevacizumab due to its binding affinity to human VEGF in the anti-angiogenic AMD therapy (Fig. 3). The microparticulate formulation did not show in vitro toxicity to ocular cells, and was injected subconjunctivally through a 30-gauge needle to the rabbit. In this preliminary animal study the corneal and retinal examinations of the injected eye confirmed the biocompatibility of the formulation. Conclusion: Due to its minimum toxicity and high versatility, this implantable photo-modulated drug delivery system has a potential to improve the treatment of ocular diseases. The release rate of the loaded drug can be tuned on demand by internal (e.g. LSNPs and polymer concentration) and external parameters (e.g. light intensity) for controlled dose. Fengxiang Qie; Randy Suryadinata; Linda Ge; Tianwei Tan; Xiaojuan Hao; Mark GreavesReferences:[1] Hugh R Taylor, Jill E Keeffe, Hien T V Vu, Jie Jin Wang, Elena Rochtchina, M Lynne Pezzullo and Paul Mitchell, "Vision loss in Australia", Medical Journal of Australia, Vol. 182, June 2005[2] Jayakrishna Ambati, John P. Atkinson and Bradley D. Gelfand, "Immunology of age-related macular degeneration", Nature Reviews Immunology, Vol. 13, June 2013[3] Owen A. Anderson, James W.B. Bainbridge and David T. Shima, "Delivery of anti-angiogenic molecular therapies for retinal disease", Drug Discovery Today, Vol. 15, April 2010[4] Salvatore Sortino, "Photoactivated nanomaterials for biomedical release applications", Journal of Materials Chemistry, Vol. 22, Nov. 2012 Keywords: Drug delivery, stimuli-response, Polymeric material, Bioactive molecule Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: General Session Oral Topic: Biomaterials for therapeutic delivery Citation: Basuki JD, Mulet XD, Hao C, Zhang HD, Mclean KD and Hughes TD (2016). Saving vision with light: Photo-modulated ocular drug delivery. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02105 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. * Correspondence: Dr. Johan D Basuki, CSIRO, Manufacturing, Clayton, Australia, johan.basuki@csiro.au Dr. Chen Hao, Wenzhou Medical University, School of Optometry and Ophthalmology and Eye Hospital, Wenzhou, China, chenhao@mail.eye.ac.cn Dr. Keith D Mclean, CSIRO, Manufacturing, Clayton, Australia, Keith.McLean@csiro.au Dr. Timothy D Hughes, CSIRO, Manufacturing, Clayton, Australia, tim.hughes@csiro.au Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Johan D Basuki Xavier D Mulet Chen Hao Hong D Zhang Keith D Mclean Timothy D Hughes Google Johan D Basuki Xavier D Mulet Chen Hao Hong D Zhang Keith D Mclean Timothy D Hughes Google Scholar Johan D Basuki Xavier D Mulet Chen Hao Hong D Zhang Keith D Mclean Timothy D Hughes PubMed Johan D Basuki Xavier D Mulet Chen Hao Hong D Zhang Keith D Mclean Timothy D Hughes Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Event Abstract Back to Event Amphiphilic core cross-linked star polymers as key building blocks of the hydrogels with hydrophobic domains for drug delivery Dunyin Gu1, Katharina Ladewig1*, Molly Klimak1*, David Haylock2, Keith M. Mclean2, Andrea J. O'Connor1 and Greg G. Qiao1 1 The University of Melbourne, Department of Chemical and Biomolecular Engineering, Australia 2 Commonwealth Scientific and Industrial Research Organisation, Manufacturing Flagship, Materials Science and Engineering, Australia Introduction: Core cross-linked star (CCS) polymers represent an intriguing platform in the application of drug delivery. CCS polymers as drug carriers are more robust to environmental variations, compared with the self-assembled micelles or vesicles, which tend to disassemble below their critical association concentration or under external stimuli, followed by an undesired burst release of drugs[1],[2]. Also, the ease of designing the size or functionality of arms and cores separately makes CCS polymers more attractive than other types of unimolecular containers[3]. Like many other molecular carriers, however, the limited circulation period of CCS polymers in body would decrease the duration of drug action. In order to overcome this constraint, CCS polymers can be built into hydrogel to form a stable drug-delivery implant. Materials and Methods: In this work, CCS polymers were obtained by using ring-opening polymerization (ROP) in two steps, with firstly poly(ethylene glycol)-poly(ε-caprolactone) (PEG-PCL) copolymers being formed as macroinitiators (MIs) and then these MIs being cross linked by [4, 4'-bioxepane]-7, 7'-dione (BOD) (Fig1)[4]. Various characterization tools, such as NMR, GPC, DLS, TEM, UV-Vis spectroscopy, flow cytometry and CLSM, have been adopted to investigate the structures, drug loading and release properties (pirarubicin and doxorubicin used as model hydrophobic drugs), cytotoxicity and cellular uptake of the resulting CCS polymers. Furthermore, the functionalized PEG arm was used to introduce the reactive end groups in the outer layer of the CCS, allowing for the formation of hydrogels in the presence of certain cross-linkers (Fig2). Results and Discussion: A range of CCS polymers were made with different hydrophilic/hydrophobic balance by tuning the ratio of PEG to CL and BOD blocks. These star polymers are soluble in both organic solvents and water and exist in a unimolecular state, as characterized by DLS and TEM, which provides the opportunity to easily stabilise hydrophobic drugs in aqueous environment without the need for lengthy encapsulation techniques. Their drug loading capacity is heavily influenced by the hydrophobicity and core size. The in vitro drug release study illustrated high stability of CCS-drug complex at neutral pH and a faster release profile under acidic conditions due to the degradation of pH-sensitive PCL segments. The CCS polymers also demonstrated very low toxicity, with cell viability remaining above 80% even up to high polymer concentrations. CLSM and flow cytometry analyses indicated highly efficient cellular uptake. The hydrogels based on the PEG-PCL-BOD CCS polymers feature the uniform distribution of hydrophobic domains as the hydrophobic drug depots. The swelling, mechanical and drug loading properties of the hydrogels are largely determined by the variations in hydrophobic content of the embedded CCS polymers. Conclusions: A library of well-defined amphiphilic PEG-PCL-BOD CCS polymers was prepared through facile ROP. They have large loading capacities for hydrophobic drugs, with the CCS-drug complex displaying high stability at neutral pH and a faster release under acidic condition. The unimolecular drug containers are bio-compatible and can be internalised by cells with high efficiency. The functionalized CCS polymers could become key building blocks of the hydrogels with hydrophobic domains as drug-delivery implants for cancer therapy. The authors acknowledge the Australian Research Council under the Future and Super Science Fellowship Schemes (FT110100411, G.G.Q.; FS110200025, G.G.Q. & K.L.).; D.G. thanks The University of Melbourne for the provision of MIRS.References:[1] H. Wei, X. Zhang, C. Cheng, S. X. Cheng and R. X. Zhuo, Biomaterials, 2007, 28, 99-107.[2] S. Peng, K. Wang, D. S. Guo and Y. Liu, Soft Matter, 2014, 11, 290-296.[3] J. T. Wiltshire and G. G. Qiao, Aust J Chem, 2007, 60, 699-705.[4] D. Gu, K. Ladewig, M. Klimak, D. Haylock, K. M. McLean, A. J. O'Connor and G. G. Qiao, Polymer Chemistry, 2015, 6, 6475-6487. Keywords: Drug delivery, polymer, amphiphile, Biodegradable material Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: General Session Oral Topic: Biodegradable polymers Citation: Gu D, Ladewig K, Klimak M, Haylock D, Mclean KM, O'Connor AJ and Qiao GG (2016). Amphiphilic core cross-linked star polymers as key building blocks of the hydrogels with hydrophobic domains for drug delivery. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.01074 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. * Correspondence: Dr. Katharina Ladewig, The University of Melbourne, Department of Chemical and Biomolecular Engineering, Melbourne, Australia, Email1 Dr. Molly Klimak, The University of Melbourne, Department of Chemical and Biomolecular Engineering, Melbourne, Australia, Email2 Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Dunyin Gu Katharina Ladewig Molly Klimak David Haylock Keith M Mclean Andrea J O'Connor Greg G Qiao Google Dunyin Gu Katharina Ladewig Molly Klimak David Haylock Keith M Mclean Andrea J O'Connor Greg G Qiao Google Scholar Dunyin Gu Katharina Ladewig Molly Klimak David Haylock Keith M Mclean Andrea J O'Connor Greg G Qiao PubMed Dunyin Gu Katharina Ladewig Molly Klimak David Haylock Keith M Mclean Andrea J O'Connor Greg G Qiao Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Event Abstract Back to Event Photocurable gelatin hydrogels for cell encapsulation Timothy Hughes1*, Lingli Li1, 2*, Conglie Lu2*, Mei Chen2*, Huixiang Ma2*, Lei Wang3*, Xiaojuan Hao1*, Keith Mclean1* and Chen Hao2, 3* 1 CSIRO, Manufacturing, Australia 2 Wenzhou Medical University, Wenzhou Eye Hospital, China 3 Wenzhou Institute of Biomaterials and Engineering, China Introduction: Tissue engineering requires the implantation of cells along with a supportive scaffold. As such, degradable support scaffolds that are amenable to minimally invasive implantation are attractive. With this in mind, we have explored the use of phototriggered thiol-ene reactions as a method to produce injectable and photocurable cell supportive scaffolds that can be cured in the presence of cells (Figure 1). Figure 1: Conceptual diagram of in situ cured scaffold in the presence of cells Materials and Methods: Type A Porcine skin (Bloom 100) Gelatin (Sigma Aldrich) was used for experiments. Vinyl functionalised gelatines were produced from gelatine and vinyl anhydrides (eg: acrylate and pentanoic anhydride)[1]. While thiolylated gelatin was prepared from gelatin and cysteine with EDC and NHS[1]. The products were purified by dialysis (1 kDa MWCO) and freeze dried before being reconstituted with saline (0.9 %). Light triggered thiol-ene reactions were used to crosslink the vinyl functionalised gelatins with multifunctional thiols included thiolated gelatin. The rheological properties of the precursor formulations as well as the final gels and their cure profiles were measured using photorheology. The effect of solids content, ratio of components and light intensity were investigated. The water content, optical transparency and mechanical properties and microscopic morphologies of the gels were also measured. The ability of bovine corneal endothelial cells (bCECs) and fibroblast cells (L929) to grow on the gels was investigated. The in situ curing of gels in the presence of living cells was also explored. Finally a gelatin hydrogel was cured in situ to fill corneal defects in rabbits(trephine, 3 mm diameter by 200 μm deep) to assess the corneal biocompatibility of the hydrogels. Results and Discussion: Thiol-ene crosslinked gelatins were able to be cured within 2 min. of exposure to 365 nm light resulting in optically transparent gels with water contents about 80-85 %. The resulting soft gels possessed high viability of both L929 and bCECs (>80 %). The mechanical properties of the gels could be tuned by altering the feed ratio of the components and the light intensity used during the cure. Live/Dead staining confirmed that both bCEC and L929 retained high cell viability in culture following in situ cure of the gelatin hydrogels containing the cells (Figure 2). Moreover, laser confocal imaging indicated that the cells were evenly distributed within the hydrogel. Figure 2: 3D Laser confocal fluorescent imaging of L929 cells pre stained with DilC(3) after 7 days of cell culture in a hydrogel matrix prepared from a formulation of 150 mg/ml 2:1 Gel-LA to Gel-SH + Irgacure 2959 (0.5 %) cured with 100 mW/cm2 for 1min Corneal defects in rabbit corneas were filled with in situ cured gelatine hydrogel and the wounded cornea re-epithelialized in 3 to 7 days with minimal inflammation (Figure 3). Figure 3: Slit lamp microscope images of corneal defects at day 1 (left) and 7 (right) stained with fluorescein filled with gelatin hydrogel matrix prepared from a formulation of 150 mg/ml 2:1 Gel-LA to Gel-SH + Irgacure 2959 (0.5 %) cured with 100 mW/cm2 for 1min Conclusions: Phototriggered thiol-ene reactions are rapid and facile methods of forming crosslinked gelatin hydrogels. They resulted in transparent gels with mechanical properties suitable for soft tissue replacements. In addition, they could be cured in the presence of living cells which maintained high viability for over 7 days post crosslinking. Moreover, the in situ cured gels were able to heal rabbit corneal defects in vivo with low inflammation and rapid re-epithelialization. Therefore, the resulting materials are promising candidates for corneal tissue engineering substrates. This study is supported by Chinese Scholarship Council, the International Scientific and Technological Cooperation Project (No. 2012DFB30020) and High-end Foreign Experts Recruitment Program (China, No.GDW20133300101).References:[1] Li, Lingli, et al., ‘Tissue engineering materials‘, CN104548196A. Keywords: Hydrogel, Tissue Engineering, 3D scaffold, Biodegradable material Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: New Frontier Oral Topic: Biomaterials for ophthalmic applications Citation: Hughes T, Li L, Lu C, Chen M, Ma H, Wang L, Hao X, Mclean K and Hao C (2016). Photocurable gelatin hydrogels for cell encapsulation. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.01497 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. * Correspondence: Dr. Timothy Hughes, CSIRO, Manufacturing, Clayton, Australia, Email1 Dr. Lingli Li, CSIRO, Manufacturing, Clayton, Australia, lingli.li@wibe.ac.cn Dr. Conglie Lu, Wenzhou Medical University, Wenzhou Eye Hospital, Wenzhou, China, zjcilcl@163.com Dr. Mei Chen, Wenzhou Medical University, Wenzhou Eye Hospital, Wenzhou, China, cm-8988@163.com Dr. Huixiang Ma, Wenzhou Medical University, Wenzhou Eye Hospital, Wenzhou, China, 13777770073@163.com Dr. Lei Wang, Wenzhou Institute of Biomaterials and Engineering, Wenzhou, China, Email2 Dr. Xiaojuan Hao, CSIRO, Manufacturing, Clayton, Australia, Xiaojuan.Hao@csiro.au Dr. Keith Mclean, CSIRO, Manufacturing, Clayton, Australia, keith.mclean@csiro.au Dr. Chen Hao, Wenzhou Medical University, Wenzhou Eye Hospital, Wenzhou, China, chenhao@mail.eye.ac.cn Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Timothy Hughes Lingli Li Conglie Lu Mei Chen Huixiang Ma Lei Wang Xiaojuan Hao Keith Mclean Chen Hao Google Timothy Hughes Lingli Li Conglie Lu Mei Chen Huixiang Ma Lei Wang Xiaojuan Hao Keith Mclean Chen Hao Google Scholar Timothy Hughes Lingli Li Conglie Lu Mei Chen Huixiang Ma Lei Wang Xiaojuan Hao Keith Mclean Chen Hao PubMed Timothy Hughes Lingli Li Conglie Lu Mei Chen Huixiang Ma Lei Wang Xiaojuan Hao Keith Mclean Chen Hao Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

A facile method for the synthesis of cell supportive, highly macro-porous hyaluronic acid (HA) hydrogels via cryogelation is presented. Unmodified HA was chemically cross-linked via EDC/NHS zero-length cross-linking at sub-zero temperatures to yield cryogels with high porosity and high pore interconnectivity. The physical properties of the HA cryogels including porosity, average pore size, elasticity and swelling properties were characterised as a function of cryogelation conditions and composition of the precursor solution. The HA cryogels swell extensively in water, with the average porosities observed being ~90% under all conditions explored. The morphology of the cryogels can be controlled, allowing scaffolds with an average pore size ranging from 18 ± 2 to 87 ± 5 μm to be formed. By varying the cross-linking degree and HA concentration, a wide range of bulk elastic properties can be achieved, ranging from ~1 kPa to above 10 kPa. Preliminary cell culture experiments, with NIH 3T3 and HEK 293 cell lines, performed on biochemically modified and unmodified gels show the cryogels support cell proliferation and cell interactions, illustrating the biomedical potential of the platform.

This article demonstrates the importance of stabiliser selection and temperature control when producing cubosomes using the ‘salt-induced’ production technique.

We report a series of amphiphilic, unimolecular, biocompatible, biodegradable and readily functionalisable PEG-PCL-based CCS polymers formed in a well-controlled manner and their application as a carrier of hydrophobic anthracycline drugs.

SUMMARY Cubosomes are considered to be promising drug delivery vehicles due to their high internal surface area which allows for the encapsulation and release of therapeutics over sustained periods of time. 1 However, to date the use of cubosomes as drug delivery vehicles for protein therapeutics has been inhibited by their energy intensive production methods. Recently, a new method of cubosome production was proposed that uses chargeshielding of an ionic lipid within a phytantriol-based dispersion to induce a lamellar to cubic phase transition following the addition of phosphate buffered saline (PBS). 2 Here, we demonstrate the versatility of this method of cubosome production using the cubic phase forming amphiphile glycerol monooleate (GMO). GMO displays a significantly lower cytotoxicity than phytantriol and is therefore a superior cubic phase forming amphiphile for drug delivery applications. In addition, we show that an established method of protein encapsulation frequently used with liposomes can be transferred to the GMO based liposomes and GMO based cubosomes can subsequently be formed following the addition of PBS. INTRODUCTION Traditionally, cubosomes are formed through sonication or homogenization of a bulk surfactant phase in excess water to produce a dispersion of cubic phase nanoparticles. They have a large internal surface area and are therefore regarded as having great potential for the sustained delivery of therapeutics. 1 This large internal surface area of the cubic phase is a result of the complex curvature of the bicontinuous amphiphilic bilayer. The bilayer arranges to form non-intersecting water channels that traverse the nanoparticle, creating an environment that permits the encapsulation of hydrophilic, hydrophobic and amphiphilic molecules. Until recently, one limitation to the use of cubosomes for the delivery of bioactive therapeutics was their energy intensive production methods, mentioned above. These techniques are likely to denature many protein therapeutics, such as Avastin ® and Lucentis ® that are used in the treatment of macular degeneration, thus rendering them inactive. Recently, a new method of cubosome production that uses charge-shielding was established in which a lamellar to cubic phase transition occurs following the addition of PBS to phytantriol-based dispersions. 2 We show here that the other commonly used cubic phase forming amphiphile, GMO, can be used to form cubosomes using the same salt-induced production method. Substituting phytantriol with GMO maximizes the therapeutic doses that can be delivered with cubosomes, as it displays lower cytotoxicity in vitro. We show that by using GMO as the cubic phase forming amphiphile, the viability of cells exposed to cubosomes is higher than those treated with phytantriol-based cubosomes. In addition, we propose a method for the encapsulation of protein-based therapeutics in the cubosomes. EXPERIMENTAL METHODS Cubosomes were prepared as described previously. 2 In brief, GMO (Nu-Check Prep, Inc., U.S.A) was dissolved in chloroform with 1,2-dipalmitoyl phosphatidyl serine (DPPS) (4% w/w) (Corden Pharmaceuticals, Switzerland) and the triblock copolymer, Pluronic® F-108 (8% w/w) (Sigma Aldrich) to stabilise the dispersion. The chloroform was removed by rotary evaporation followed by 24 hours drying under vacuum. The resulting mixture was heated to 70 C for 10 minutes. Milli-Q water was added to give a final GMO concentration of 2.5% w/v and the solution was sonicated for 3 minutes using a 3 seconds on, 2 seconds off pattern at 30% of maximum power (Misonix XL2000, Misonix Incorporated). To induce the lamellar to cubic phase transition PBS (Sigma Aldrich) was added to give a final salt concentration of 150 mM (1x PBS). Dispersions were analysed through dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) to determine their size and phase. A freeze-thaw method was used to evaluate the suitability of this method for the encapsulation of protein therapeutics into liposomes prior to the cubic phase transition. In short, 200 μL of liposome dispersion was sterilised using a 0.22 μm syringe filter and placed into a 1.5 mL centrifuge tube. The dispersions were then cycled 1–3 times through a 5 minute freeze-thaw (-196°C – 45°C) cycle. Size and polydispersity of the liposomes were recorded using a Zetasizer-Nano instrument (Malvern, U.K). PBS was then added to the dispersions as described above and the size and the polydispersity of the dispersions remeasured. In our future work we will use fluorescently labelled model proteins, such as Ovalbumin and Bovine Serum Albumin, to determine the encapsulation efficiency and cellular uptake of liposomes and cubosomes produced through the freeze-thaw method. RESULTS AND DISCUSSION The introduction of 4% (w/w) DPPS to the cubic phase-forming amphiphile GMO leads to the formation of the lamellar phase following sonication. We have used Pluronic® F-108 as the surfactant to stabilise the dispersions as it has been demonstrated that, unlike the more commonly used F-127, this surfactant does not result in changes to the phase of GMO cubosomes. 3 This is important because by changing the DPPS concentration in the dispersion we can obtain greater control over the phase and water channel diameter of the cubosomes formed using F-108. Following the addition of PBS to the lamellar GMODPPS-F-108 dispersion we observe an immediate change in the turbidity of the GMO based dispersions. The transition from a translucent lamellar phase to a ‘milky’ dispersion suggests the formation of a cubic phase. Synchrotron SAXS confirmed the presence of cubosomes in the primitive phase (Figure 1). Figure 1 SAXS scatterplot of a GMO-DPPS-F-108 dispersion following the addition of PBS. * Indicates primitive phase peaks. This new method of cubosome production uses processes that may not denature protein therapeutics; however, it is yet to be shown whether these therapeutics can be encapsulated into the cubic phase. As the precursors for the formation of cubosomes are liposomes, we subsequently investigated methods used to encapsulate proteins within liposomes. One such method is the freezethaw method of encapsulation of proteins into pre-formed liposomes. 5 To evaluate whether this method of encapsulation may be suitable for the encapsulation of bioactive therapeutics within GMO and phytantriol-based cubosomes the lamellar phase was cycled through 5minute freeze-thaw cycles (Figure 2). We observe that following one freeze-thaw cycle there is a reorganisation of the amphiphile bilayer which results in significantly larger liposomes being formed, however, the size and polydispersity does not significantly change as we repeat the freeze-thaw cycle. Following the addition of PBS to liposomes that were subjected to the freeze-thaw procedure we observed a reduction in the size and polydispersity of the nanoparticles (Figure 2). We believe that this change may be due to the difference in amphiphile density within the liposome compared to that of the cubosome. Using the salt-induced cubosome production method spherical liposomes are converted to cubosomes, which, due to their highly structured internal network have a greater density of amphiphiles within the nanoparticle compared to the liposome precursor. Figure 2 Size (bars) and polydispersity (triangles) of GMO-based dispersions before (lamellar) and after the addition of PBS (cubosomes). The lamellar phase was exposed to up to 3 freeze-thaw cycles in liquid nitrogen. CONCLUSION We have shown here that the salt-induced cubosome production method can be used to form GMO-based cubosomes from a lamellar dispersion. This may be important for the use of cubosomes in the field of drug delivery as GMO-based cubosomes are significantly less toxic than those based on phytantriol and therefore a higher dose of therapeutic may be delivered to the target site. Although it has been suggested that the salt-induced method of cubosome production is a promising technique for the encapsulation of protein therapeutics, their successful encapsulation has yet to be demonstrated using this method of cubosome production. Using the freezethaw cycle to encapsulate proteins into the lamellar phase we believe we can maximize the encapsulation potential within cubosomes following the addition of PBS. With their ability to release therapeutics over a sustained period of time, the encapsulation of proteins into cubosomes is expected to extend the duration of action for these proteins and increase patient comfort through the reduced frequency of treatment. REFERENCES 1. Mulet X., et al., J Colloid Interf. Sci., (2013), 393, 1. 2. Muir, B., et al., J. Phys. Chem. B (2012), 116, 3551 3. Chong, J., et al., Soft Matter (2011), 7, 4768 4. Shen, H. H., et al., Biomaterials (2010), 31, 9473 5. Xu, X., et al., Pharm. Res. (2012), 29, 1919 ACKNOWLEDGMENTS The authors acknowledge infrastructure support by the PFPC and CMSE, the Australian Synchrotron for beamtime and scholarships provided by The University of Melbourne, The Melbourne Materials Institute and CSIRO (TH). KL acknowledges funding from the Australian Research Council (ARC) through their Super Science Fellowship scheme. 20 30 40 50 60 0 0.1 0.2 0.3 In te n si ty ( a .u .) q (Å)-1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 100 200 300 400 500 600

Cold atmospheric pressure plasma (APP) is a recent, cutting-edge antimicrobial treatment. It has the potential to be used as an alternative to traditional treatments such as antibiotics and as a promoter of wound healing, making it a promising tool in a range of biomedical applications with particular importance for combating infections. A number of studies show very promising results for APP-mediated killing of bacteria, including removal of biofilms of pathogenic bacteria such as Pseudomonas aeruginosa. However, the mode of action of APP and the resulting bacterial response are not fully understood. Use of a variety of different plasma-generating devices, different types of plasma gases and different treatment modes makes it challenging to show reproducibility and transferability of results. This review considers some important studies in which APP was used as an antibacterial agent, and specifically those that elucidate its mode of action, with the aim of identifying common bacterial responses to APP exposure. The review has a particular emphasis on mechanisms of interactions of bacterial biofilms with APP.

Hydrogels formed by ultrashort peptides are emerging as cost-effective materials for cell culture. However, L-peptides are labile to proteases, while their D-isomers are thought to not support cell growth as well. In contrast, the self-assembly behaviour and biological performance of heterochiral peptides (i.e., made of both D and L amino acids) are largely unknown. In this study, we evaluate the effects of amino acid chirality on tripeptide self-assembly and hydrogelation at physiological pH, and cytocompatibility in fibroblast cell culture. A series of uncapped hydrophobic tripeptides with all combinations of D, L amino acids was prepared, tested for self-assembly under physiological conditions, and analysed by circular dichroism, FT-IR, cryo-TEM, AFM, and Thioflavin T fluorescence imaging. Amino acid chirality has a profound effect on the peptides' supramolecular behaviour. Only selected isomers form hydrogels, and of amyloid structure, as confirmed by rheology and XRD. Importantly, they are able to maintain the viability and proliferation of fibroblasts in vitro. This study identifies two heterochiral gels that perform well in cell culture and will assist in the design of innovative and cost-effective peptide gel biomaterials.

The present study has evaluated a commercial pericardial material for its capacity to assist as a natural extracellular matrix (ECM) patch for the delivery and retention of mesenchymal stem cells for cardiac repair. The repair of cardiac tissue with cells delivered by an appropriate bioscaffold is expected to offer a superior, long-lasting treatment strategy. The present material, CardioCel®, is based on acellular pericardium that has been stabilized by treatments, including a low concentration of glutaraldehyde, that eliminate calcification after implantation. In the present study, we have assessed this material using human bone marrow mesenchymal stem cells at various cell densities under standard, static cell culture conditions. The initial seeding densities were monitored to evaluate the extent of cell attachment and cell viability, with subsequent cell proliferation assessed up to 4 weeks using an MTS assay. Cell morphology, infiltration, and spreading were tracked using scanning electron microscopy and phalloidin staining. The efficacy of long-term cell survival was further assessed by examining the extent and type of new tissue formation on seeded scaffolds at 70 days; both type I and type III collagens were present in fibrillar structures on these scaffolds indicating that the seeded stem cells had the capacity to differentiate into collagen-producing cells necessary to repair damaged ECM. These data show that the CardioCel® scaffold is an appropriate substrate for the stem cells and has the potential to both retain seeded stem cells and to act as a template for cell propagation and new tissue formation.

Although rhBMP-2 has excellent ability to accelerate the repair of normal bone defects, limitations of its application exist in the high cost and potential side effects. This study aimed to develop a composite photopolymerisable hydrogel incorporating rhBMP-2 loaded 2-N, 6-O-sulfated chitosan nanoparticles (PH/rhBMP-2/NPs) as the bone substitute to realize segmental bone defect repair at a low growth factor dose. Firstly rhBMP-2 loaded 2-N, 6-O-sulfated chitosan nanoparticles (rhBMP-2/NPs) were prepared and characterized by DLS and TEM. Composite materials, PH/rhBMP-2/NPs were developed and investigated by SEM-EDS as well as a series of physical characterizations. Using hMSCs as an in vitro cell model, composite photopolymerisable hydrogels incorporating NPs (PH/NPs) showed good cell viability, cell adhesion and time dependent cell ingrowth. In vitro release kinetics of rhBMP-2 showed a significantly lower initial burst release from the composite system compared with the growth factor-loaded particles alone or encapsulated directly within the hydrogel, followed by a slow release over time. The bioactivity of released rhBMP-2 was validated by alkaline phosphatase (ALP) activity as well as a mineralization assay. In in vivo studies, the PH/rhBMP-2/NPs induced ectopic bone formation in the mouse thigh. In addition, we further investigated the in vivo effects of rhBMP-2-loaded scaffolds in a rabbit radius critical defect by three dimensional micro-computed tomographic (μCT) imaging, histological analysis, and biomechanical measurements. Animals implanted with the composite hydrogel containing rhBMP-2-loaded nanoparticles underwent gradual resorption with more pronounced replacement by new bone and induced reunion of the bone marrow cavity at 12 weeks, compared with animals implanted with hydrogel encapsulated growth factors alone. These data provided strong evidence that the composite PH/rhBMP-2/NPs are a promising substitute for bone tissue engineering.

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.