Andrea J. O’Connor

Separation of biological molecules using mesoporous molecular sieves

The selective separation and purification of biological molecules is important in a number of industries, especially those processing food or pharmaceuticals. However, the applications of molecular sieves for these separations have been limited by the available pore sizes (<1.5 nm). With the development of synthetic mesoporous molecular sieves the available pore size range has been extended, providing pores large enough to allow access for a number of biological molecules. These materials have unique properties desirable for adsorption including a highly regular structure, uniform pore sizes and a high surface area, thus offering potential for separations based on size exclusion and targeted surface chemistry. They can also be tailored to suit particular separations and are expected to have advantages over currently used adsorbents, such as bentonite and activated carbon. This work investigates the use of mesoporous molecular sieves for the separation of biological molecules. Adsorption experiments involving proteins (lysozyme and trypsin) and a vitamin (riboflavin), as model biological solutes, have been conducted to assess the potential of MCM-41 and MCM-48 as selective adsorbents. The results demonstrate the potential of these materials for use in size exclusion separations.

An MMP13-Selective Inhibitor Delays Primary Tumor Growth and the Onset of Tumor-Associated Osteolytic Lesions in Experimental Models of Breast Cancer

We investigated the effects of the matrix metalloproteinase 13 (MMP13)-selective inhibitor, 5-(4-{4-[4-(4-fluorophenyl)-1,3-oxazol-2-yl]phenoxy}phenoxy)-5-(2-methoxyethyl) pyrimidine-2,4,6(1H,3H,5H)-trione (Cmpd-1), on the primary tumor growth and breast cancer-associated bone remodeling using xenograft and syngeneic mouse models. We used human breast cancer MDA-MB-231 cells inoculated into the mammary fat pad and left ventricle of BALB/c Nu/Nu mice, respectively, and spontaneously metastasizing 4T1.2-Luc mouse mammary cells inoculated into mammary fat pad of BALB/c mice. In a prevention setting, treatment with Cmpd-1 markedly delayed the growth of primary tumors in both models, and reduced the onset and severity of osteolytic lesions in the MDA-MB-231 intracardiac model. Intervention treatment with Cmpd-1 on established MDA-MB-231 primary tumors also significantly inhibited subsequent growth. In contrast, no effects of Cmpd-1 were observed on soft organ metastatic burden following intracardiac or mammary fat pad inoculations of MDA-MB-231 and 4T1.2-Luc cells respectively. MMP13 immunostaining of clinical primary breast tumors and experimental mice tumors revealed intra-tumoral and stromal expression in most tumors, and vasculature expression in all. MMP13 was also detected in osteoblasts in clinical samples of breast-to-bone metastases. The data suggest that MMP13-selective inhibitors, which lack musculoskeletal side effects, may have therapeutic potential both in primary breast cancer and cancer-induced bone osteolysis.

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.

Hydrogels with smart systems for delivery of hydrophobic drugs

ABSTRACT Introduction: Smart hydrogel systems present opportunities to not only provide hydrophobic molecule encapsulation capability but to also respond to specific delivery routes. Areas covered: An overview of the design principles, preparation methods and applications of hydrogel systems for delivery of hydrophobic drugs is given. It begins with a summary of the advantages of hydrogels as delivery vehicles over other approaches, particularly macromolecular nanocarriers, before proceeding to address the design and preparation strategies and chemistry involved, with a particular focus on the introduction of hydrophobic domains into (naturally) hydrophilic hydrogels. Finally, the applications in different delivery routes are discussed. Expert opinion: Modifications to conventional hydrogels can endow them with the capability to carry hydrophobic drugs but other functions as well, such as the improved mechanical stability, which is important for long-term in vivo residence and/or self-healing properties useful for injectable delivery pathways. These modifications harness hydrophobic-hydrophobic forces, physical interactions and inclusion complexes. The lack of in-depth understanding of these interactions, currently limits more delicate and application-oriented designs. Increased efforts are needed in (i) understanding the interplay of gel formation and simultaneous drug loading; (ii) improving hydrogel systems with respect to their biosafety; and (iii) control over release mechanism and profile.

Cubosomes and other potential ocular drug delivery vehicles for macromolecular therapeutics

Introduction: Many macromolecular therapeutics designed to treat posterior segment eye diseases (PSEDs) are administered through frequent ocular injection, which can further deteriorate eye health. Due to the high frequency of injection and the high cost of the therapeutics, there is a need to develop new ways in which to deliver these therapeutics: ways which are both safer and more cost effective. Areas covered: Using the most common PSED, age-related macular degeneration, as an example of a debilitating ocular disease, this review examines the key barriers limiting the delivery of macromolecular therapeutics to the posterior segment of the eye and defines the key requirements placed on particulate drug delivery vehicles (DDVs) to be suitable for this application. Recent developments in macromolecular drug delivery to treat this disease as well as the remaining shortcomings in its treatment are surveyed. Lastly, an emerging class of DDVs potentially suited to this application, called cubosomes, is introduced. Expert opinion: Based on their excellent colloidal stability and high internal surface area, cubosomes hold great potential for the sustained release of therapeutics. Novel production methods and a better understanding of the mechanisms through which drug release from these particles can be controlled are two major recent developments toward successful application.

Formation and characterisation of a modifiable soft macro-porous hyaluronic acid cryogel platform

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.

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion

Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies.

Synthesis of ultra small nanoparticles (< 50 nm) of mesoporous MCM-48 for bio-adsorption

Mono-dispersed spherical MCM-48 mesoporous silica nanoparticles (MSN) with cubic Ia3d mesostructure were synthesized under different stirring conditions using hexadecyltrimethyl ammonium bromide as structure directing agent and EO20PO70EO20 copolymer as particle size control. Results indicated that the EO20PO70EO20 works well in the reduction of nanoparticle size. In addition, increasing the stirring rate transformed mesoporosity from 2-D hexagonal P6mm to 3-D cubic Ia3d. The biomolecule adsorption properties of prepared ultra small nanoparticles of MCM-48 were compared with that of MCM-48 with ten times larger particle sizes. MCM-48 with smaller particle sizes adsorbed much more vitamin B12 compared with MCM-48 with larger particles. In addition, diffusion of vitamin in the intraparticle spaces occurred only for MCM-48 with ultra-small nanoparticles.

Biofabrication of human articular cartilage: a path towards the development of a clinical treatment

Cartilage injuries cause pain and loss of function, and if severe may result in osteoarthritis (OA). 3D bioprinting is now a tangible option for the delivery of bioscaffolds capable of regenerating the deficient cartilage tissue. Our team has developed a handheld device, the Biopen, to allow in situ additive manufacturing during surgery. Given its ability to extrude in a core/shell manner, the Biopen can preserve cell viability during the biofabrication process, and it is currently the only biofabrication tool tested as a surgical instrument in a sheep model using homologous stem cells. As a necessary step toward the development of a clinically relevant protocol, we aimed to demonstrate that our handheld extrusion device can successfully be used for the biofabrication of human cartilage. Therefore, this study is a required step for the development of a surgical treatment in human patients. In this work we specifically used human adipose derived mesenchymal stem cells (hADSCs), harvested from the infra-patellar fat pad of donor patients affected by OA, to also prove that they can be utilized as the source of cells for the future clinical application. With the Biopen, we generated bioscaffolds made of hADSCs laden in gelatin methacrylate, hyaluronic acid methacrylate and cultured in the presence of chondrogenic stimuli for eight weeks in vitro. A comprehensive characterisation including gene and protein expression analyses, immunohistology, confocal microscopy, second harmonic generation, light sheet imaging, atomic force mycroscopy and mechanical unconfined compression demonstrated that our strategy resulted in human hyaline-like cartilage formation. Our in situ biofabrication approach represents an innovation with important implications for customizing cartilage repair in patients with cartilage injuries and OA.

Facile In Situ Synthesis and Impregnation of Silver Nanoparticles in a Hydrophobic Polymer for Antimicrobial Biomaterials

Device-associated infection (DAI) remains a challenge to modern medicine as more patients are being implanted with medical devices that provide surfaces and microenvironments for bacterial colonization. In addition, there is an urgent need for alternatives to antibiotics in preventing and treating these infections as a result of increases in drug resistance. Silver nanoparticles (Ag NPs) have emerged as a promising non-antibiotic antimicrobial agent against a wide range of bacteria. However, for them to be clinically useful, they must be properly incorporated into devices which often possess wetting properties detrimental to not only the incorporation but also the release of such nanoparticles. This study takes advantage of polyethylene glycol to form and stabilize hydrophilic Ag NPs in situ within a hydrophobic polycaprolactone (PCL) matrix. Results showed that Ag NPs were formed in situ and uniformly dispersed in the matrix. Interestingly, the lower concentration of Ag appeared to have more prolonged Ag release. This new method has potential to significantly improve the antimicrobial efficacy and ease of fabrication of Ag-containing materials for medical devices. Introduction There is an alarming increase in the number and occurrence of multi-antibiotic resistant bacterial strains due to the misuse and overuse of antibiotics worldwide. Such resistance makes eradication very difficult. Therefore, there has been increasing interest in using non-drug antimicrobial agents to prevent as well as treat microbial infections. The use of inorganic, non-antibiotic antimicrobial agents is a promising treatment in the fight against infection because these agents have been shown to act upon multiple bacterial pathways that make it difficult for bacteria to develop resistance [1]. Silver (Ag) and silver nanoparticles in particular have attracted great attention because of their antimicrobial activities against a broad range of pathogens. Ag nanoparticles were shown to produce radical oxygen species that caused oxidative stress to bacteria [2, 3] or disrupt the bacterial cell wall [4] leading to increased permeability and eventually cell death. Ag ions and Ag nanoparticles have a potent antimicrobial effect at concentrations as low as parts per billion [5, 6]. Recently, there has been an increased interest in Ag nanoparticles which have similar antimicrobial activities to that of Ag ions but have much lower dissolution rates leading to extended efficacy. However, to be useful in clinical settings, Ag nanoparticles need to be properly incorporated into the devices to be implanted to allow for effective release of Ag to eradicate or prevent infection. In this study, we present a facile method to incorporate antimicrobial Ag nanoparticles into a hydrophobic polymer in situ and, in addition, improve the release of Ag from the polymer matrix. PCL was chosen as a model hydrophobic polymer because it is a FDA-approved, biodegradable polymers commonly used in various biomaterial applications. Advances in Science and Technology Online: 2014-10-31 ISSN: 1662-0356, Vol. 96, pp 9-14 doi:10.4028/www.scientific.net/AST.96.9