The ability of epithelial cells to organize into polarized, three-dimensional (3D) structures correlates closely with their normal or malignant status. In a versatile model of morphogenesis, we have shown in past studies that inhibiting a number of key signaling pathways in human breast cancer cells grown in laminin-rich ECM gels leads to ‘reversion&’ of the malignant phenotype. The resulting growth-arrested polarized structures resemble normal ‘acini&’. These studies have helped us understand how polarity of the normal structures in the mammary gland may be disrupted as breast cancer progresses. We now have used two additional models to study signaling integration in single mammary cells and also mammary organoids, where we have modeled mammary invasion into the stromal collagen during branching morphogenesis and discovered unexpected mechanisms by which MMPs (matrix metalloproteinases) signal and how tumor cells usurp these pathways to invade. Most recently we have succeeded in modeling where breast cancer cells go to become dormant and how and why they may ‘wake up&’. I will discuss data under review to provide additional proof of how single cells from adult breast can make physiologically correct structures (acini) of the mammary gland, and show that all signaling pathways including those of glucose metabolism, must integrate to maintain homeostasis. We show how biochemical and mechanical signaling from the ECM, the ECM receptors and MMPs interconnect with cells and their nuclei to build tissue architecture by reciprocal and reiterating loops to achieve tissue specificity.

Introduction: Glioblastoma (GBM) is the most common and aggressive form of primary brain cancer in adults. The GBM tumor niche consists of biochemical and mechanical cues that act in conjunction to fuel tumor growth. The gold standards for studying cancer biology are 2D monolayer culture, organotypic explant cultures, and animal models. Currently, there is a lack of 3D in vitro models that permit studying cancer cells in a more physiologically relevant and controllable manner. Clinical evidence has shown that GBM tumor development is coupled with a substantial increase in matrix stiffness, with ~26 kPa in GBM vs. ~1 kPa in normal brain tissues. While the important roles of niche cues on GBM progression have been well recognized, little is known about how matrix stiffness regulates GBM cell fates in 3D. Here we report the development of 3D biomimetic hydrogels to study the effect of matrix stiffness on brain cancer cell behavior.
Materials and Methods: Synthetic materials, such as poly(ethylene-glycol) (PEG), allow independent control of various hydrogel properties and present a blank slate for incorporating naturally-derived ECM ligands. To tune the hydrogel stiffness, the concentration of multi-arm PEG molecules was varied. To allow for cell-mediated degradation, MMP-cleavable sequences and nondegradable linear PEG molecules were used to crosslink the hydrogel. To mimic the biochemical properties of brain tissue and allow cell adhesion, hyaluronic acid (HA) and CRGDS peptides were also incorporated. A commercially available brain tumor cell line (U87) was used and cultured in hydrogels mimicking normal brain (1 kPa) or tumor (26 kPa) tissue stiffness. Hydrogels were characterized using mechanical testing and quantification of equilibrium swelling ratio over time. Cellular fates in 3D hydrogels were analyzed by monitoring cell proliferation, cell morphology, and gene expression.
Results and Discussion: Both soft and stiff hydrogels supported substantial cell proliferation of U87 cells in 3D, with formation of tumor spheroids throughout the hydrogels. Within 21 days of culture, cells proliferated 16-fold in hydrogels with brain-like stiffness (1 kPa) and 3-fold in hydrogels with tumor-like stiffness (26 kPa). In addition, increasing hydrogel stiffness limited cell spreading at early time points, but seemed to permit longer cell protrusion at later time points. Furthermore, changes in hydrogel stiffness resulted in differential gene expression for Ras protein HRAS, mechanotransduction proteins RhoA and ROCK1, and ECM remodeling proteins, including HA synthases 1 and 2 and MMP9. Our results highlight the importance of matrix stiffness in influencing brain tumor cell behavior in 3D. Such biomimetic hydrogels could provide a 3D in vitro model for elucidating tumor-niche interactions in a controlled manner.

Glioblastoma multiforme (GBM), the most prevalent primary brain cancer, is characterized by diffuse infiltration of tumor cells into brain tissue, which severely complicates surgical resection and likely gives rise to the almost universal tumor recurrence. This diffuse infiltration is frequently guided by anatomical “tracks” in the brain in the form of blood vessels or white matter tracts, which give rise to the highest migration speeds observed in vivo. Despite this observation, little is known about the biophysical and biochemical mechanisms through which these tissue interfaces promote invasive motility, which in turn may derive from a lack of appropriate culture paradigms. To address this need, we developed a culture system in which tumor cells are sandwiched between a ventral fibronectin-coated dorsal surface representing vascular basement membrane and a dorsal hyaluronic acid (HA) surface representing brain parenchyma. We find that inclusion of the dorsal HA surface induces formation of adhesive complexes and significantly slows cell migration relative to a free fibronectin-coated surface. This retardation is amplified by inclusion of integrin binding peptides in the dorsal layer and expression of CD44, suggesting that it acts through biochemically specific mechanisms rather than simple physical confinement. Moreover, both the reduction in migration speed and assembly of dorsal adhesions depend on myosin activation and the stiffness of the ventral layer, implying that mechanochemical feedback directed by the ventral layer can influence adhesive signaling at the dorsal surface.

Patients with glioblastoma multiforme (GBM), the most aggressive form of primary brain tumor, have a poor prognosis due to a rapid diffuse infiltration of tumor cells into normal parenchyma. The biochemical and biophysical interactions between tumor cells and brain extracellular matrix play an important role in the rapid progression of the tumor. Platforms to replicate the tumor microenvironment are a critical topic in the field of cancer research, and offer unique opportunities to engage next generation genomic tools. These technologies may serve as diagnostic platforms for clinical assessment of therapeutic strategies using patient-specific biopsies. As a result, they will turn into a powerful clinical tool to characterize the tumor microenvironment, and the associated intercellular signaling network in individual patients, enabling personalized therapy. We have developed a versatile gelatin-based biomaterial platform to present combinations of mechanical, structural, and cellular cues inspired by the native glioblastoma microenvironment. Strategies to decorate these biomaterials with biomolecular cues (e.g. hyaluronic acid), common glioma mutations (e.g. EGFR) and chemokines that regulate cell motility, proliferation and survival (e.g. CXCL12) demonstrate an impact in their response to microenvironment. Moreover, spatial and temporal gradients regulate the cell proliferation, migration, and differentiation during cancer. Therefore, we use a microfluidic approach to fabricate patterned biomaterials that have the ability to examine transitions between defined environments (e.g., glioma core, periphery and neural tissue). We developed a series of gelatin and hyaluronic acid (HA) macromers in order to create libraries of composite hydrogel structures. Hydrogels containing brain-mimetic HA show significant impact on GBM malignancy metrics in comparison to 2D culture or through the use of 3D GelMA hydrogels. Glioma cell clusters were observed exclusively in HA containing gels as well as HA-dose dependent gene expression patterns. Using this tool we aim to generate a brain tumor biochip to examine how the heterogeneities within the tumor microenvironment impact glioma malignant transformation, growth, and the formation of immunomodulatory zones that limit therapeutic efficacy. We combined this technology with clinical specimens and data obtained from diagnosis of patients in order to prognosticate the cell dynamics in tumor progression and lead, as a result, to the design of personalized therapy.

To investigate the possibility of treating multidrug-resistant tumors with targeted chemo-photothermal treatment, we conducted in vitro and in vivo studies using a doxorubicin (DOX)-resistant DLD-1 cell line (DLD-1/DOX) and nude mice with human xenograft tumors, respectively. The chemo-photothermal treatment consisted of DOX-loaded-poly(lactic-co-glycolic acid)-Au half-shell nanoparticles with targeting moieties of anti-death receptor-4 monoclonal antibody conjugated to the Au surface. The cells or xenografted tumors were exposed to near infrared light for 10 min, which caused an increase in temperature to 45oC. Chemo-photothermal treatment resulted in a large reduction in the rate of tumor xenograft growth on DLD-1/DOX tumor-bearing mice with a much smaller dose of DOX than conventional DOX chemotherapy. These results demonstrate that targeted chemo-photothermal treatment can provide high therapeutic efficacy and low toxicity in the treatment of multidrug- resistant tumors.

A synthetic extracellular matrix (sECM) from hyaluronic acid (HA) affords highly reproducible, manufacturable, approvable, and affordable biomaterials. These injectable clinical materials are being developed for clinical use to deliver autologous and allogeneic cells to sites in need of repair. Tissue repair targeted by us, our collaborators and partners include heart, kidney, brain, liver, fat, bone, cartilage, and vocal folds. The HA materials offer a cell-friendly niche for cell retention and proliferation, as well as being permissive and conducive to angiogenesis and nutrient flow.

We report increased stimulation of dendritic cells via heterodimers of immunostimulants formed at a discrete molecular distances. Many vaccines present spatially organized agonists to immune cell receptors. These receptors cluster suggesting that signaling is increased by spatial organization and receptor proximity, but this has not been directly tested for multiple, unique receptors. In this study we probe the spatial aspect of immune cell activation using heterodimers of two covalently attached immunostimulants.

As a technology to investigate T cell - antigen presenting cell interactions, we have created spatially structured surfaces that mimic nanoscale features of the Immunological Synapse. We show that biomimetic nanoarrays functionalised with antibody fragments engage with human CD4+ T cells to form an "artificial immune synapse" and demonstrate that the level of T cell signalling can be controlled by varying the spacing of ligand anchoring points on the nanoscale.
Although it is well known that TCR-ligand engagements plays a key role in activation of T Cells, there is some controversy over the minimum stimulation required to induce signalling clusters on the submicron scale, and structurally important features have been described on the micro- and nano- lengthscales. To investigate the effect of such structures in controlling signalling, we have prepared biomimetic surfaces that present antibodies and antibody fragments with nanoscale spacing using nanopatterned gold arrays. Using the technique of diblock copolymer micelle nanolithography, arrays of gold nanoparticles with spacings ranging from 25-104nm have been formed. By controlling the nanoarray structure, and the biological ligands bound to the surface, we investigate how the spatial distribution of selected stimulatory ligands affects cell activation and downstream signalling. The nanoarrays have been succesfully functionalised with biologically complex antibody F(ab')2 fragments, bound through the di-sulphide hinge region to the gold nanoparticle.
Specifically, we assess CD4+ engagement with these biomimetic nanopatterns, showing that the degree of signalling decreases with increasing spacing of anti-CD3 ligands. Parallel studies of NK cell stimulation by CD16 binding nanoarrays showed intriguingly similar results. These results indicate that immune cell activation can be driven by the spatial organisation of receptor-ligand engagement on a sub-100nm scale. The elucidation of these signalling and activation requirements will help mechanistic understanding of the activation process, and direct immunotherapy towards new nanoscale targets.

Immune cell stimulation has been shown to be enhanced by co-treatment of multiple agonists. One such pairing, Toll-like receptor (TLR) 4 phospholipid agonists with TLR 7/8 imidazoquinoline agonists, have shown synergistic increased in cell surface markers and cytokines indicative of dendritic cell (DC) activation. We have designed caged agonists of TLR 4 and 7/8 to probe the mechanism of the observed synergy. Protecting agonists with orthogonal photo-labile groups allows for spatially- and temporally-controlled release of agonists within a single cell. Agonist moieties critical for TLR binding and activation, the phosphate of monophosphoryl Lipid A (MPLA) and primary amine of resiquimod, were protected with photo-labile nitrophenyl and coumarin groups, respectively. We show that caged compounds elicit minimal stimulation in a model macrophage cell line, and that upon photo-deprotection, the parent compounds reform and regain activity. For activation of single cells and precise deprotection at the organelle level, two-photon confocal microscopy was employed. Immune stimulation, assessed by direct observation of endocytosis in bone marrow-derived dendritic cells (BMDCs), again was knocked out with the cage and regained following exposure with the respective wavelength. We demonstrate that a spatio-temporal relationship exists between TLR 4 and 7/8 activation pathways and that this synergy can be exploited for creating multi-functional, robust materials for immune cell programming.

Tissue and organ development is regulated by a complex interplay of multiple extracellular cues, and emulating the process with synthetic scaffolds remains challenging. One challenge is the independent control in a single scaffold of soluble signals, bound signals, and mechanical properties. We describe here a two-component core-shell microcapsule construct 150-200 µm in diameter that consists of a polymeric alginate core and a shell comprised of peptide amphiphile (PA) nanofibers. We demonstrate that the release of bone morphogenetic protein 4 (BMP-4) from the polymer core can be tuned by changing the degree of calcium cross-linking of alginate. At the same time the PA nanofiber shell on the capsule surface provides a substrate for cellular attachment without affecting BMP-4 release. Moreover, using C2C12 premyoblasts, we show that cell proliferation and spreading on PA substrates can be modulated by molecularly tuning the bioactivity of nanofibers displaying the fibronectin-derived RGDS epitope. Combining the tailorable PA design and tunable BMP-4 release afforded an independent control over C2C12 cell distribution and osteogenic differentiation on the microcapsule surface. The microcapsules investigated could be easily injected and thus serve as a novel bioactive scaffold for cell therapies in regenerative medicine.

INTRODUCTION
The availability of forming technology able to mass produce porous polymeric microspheres with diameters ranging from 150 to 300 µm is significant for some biomedical applications where tissue augmentation is required1,2. Moreover, appropriate assembly of microspheres into scaffolds is an important challenge to enable direct usage of the as-formed structures in treatments of chronic wound such as fistula3. This specified range of microspheres has the advantage of large enough interstices between the packed spherical particles for migration of cells into scaffold for tissue regeneration.
EXPERIMENTAL METHOD
Poly Lactic-Co-Glylolic Acid (PLGA, 50:50 co-polymer) was used to generate the required size of microspheres. Different concentrations (5, 10 and 15 wt%) of PLGA solutions were prepared by combining appropriate amounts of polymer in Dimethyl Carbonate (DMC). Electrospraying of the polymer solutions was conducted by adopting single-axial cone-jetting to produce the hollow microspheres with different size ranges. The applied voltage was varied according to the flow rate used in order to obtain a stable cone jet and produce monodisperse microspheres. When the required size of microspheres was achieved with the 15% concentration of the polymer solution at the specific flow-rate and range of voltage supply, the single-axial cone-jet mode spraying was performed once again to electrospray the polymer solution for 1800s into a metallic beaker filled with liquid nitrogen. Once the required size of microspheres was collected in liquid nitrogen, the frozen products were immediately placed into freeze-dryer in order to remove the residual solvent from the structure of microspheres (TIPS).
RESUTLS AND DISCUSSION
The required size of microspheres for the development of the desired scaffold was obtained at a flow rate 225µl/min after collection in liquid nitrogen. Due to thermal phase separation, when the frozen microspheres were placed in freeze dryer, the residual solvent was removed from the structure of particles, and this led to porosity on the surface of microspheres.
During the procedure of electrospraying, flow rate was changed, therefore in order to obtain stable cone-jet and monodisperse particles, voltage supply was also varied.
CONCLUSION
Electrospray of PLGA in DMC, a low toxicity solvent with moderate conductivity and low dielectric constant, followed by freeze drying generated porous microspheres within the required diameter range of 150-300 µm suitable for use as minimally invasive, in situ forming scaffolds.
REFERENCES
1. H. Keshaw, N. Thapar, A.J. Burns, N. Mordan, J.C. Knowles, A. Forbes, R.M. Day, Acta Biomaterialia 6 (2010) 1158-1166
2. J.J. Blaker, J.C. Knowles, R.M. Day, ActaBiomater 4 (2008) 364-272
3. J.J. Blaker, J. Pratten, D. Ready, J.C. Knowles, A. Forbes, R.M. Day, Journal of Alimentary Pharmacology & Therapeutics 28 (2008) 614-622

Delivery of biological effectors into cells is a technological challenge of significant importance for diagnosis and treatment of disease and for fundamental biological studies. Vertical nanowire (NW) arrays have shown promise as a potential universal platform for drug delivery. In contrast to conventional techniques which rely on biochemical pathways, vertical NW platforms physically penetrate the cell membrane, enabling biomolecules to be directly introduced to the cytoplasm to avoid endosomal degradation routes. However, the NW arrays are restricted to in vitro applications because the NWs are attached to a planar 2D substrate. In this work, we grow ZnO NWs on suspended nanoparticles to produce "spiky particles", which can be used for biomolecular delivery into cells by utilizing the sharp NWs' cell membrane penetration ability. Naturally occurring particle phagocytosis by cells encourages spiky particle engulfment, and once the cell membrane is penetrated by the NWs, the biomolecules bound on the particle surface can be directly released into the cytosol. The particle surface is protected by a thin layer of alumina to reduce the cytotoxicity of ZnO materials, and is further functionalized with amino groups to allow noncovalent binding of biomolecules. We demonstrate synthesis of spiky particles with tunable particle size, NW length and NW density. The spiky particle-cell interface and interaction studies suggest that cells actively engulf the spiky particles. The cytotoxicity of spiky particles at high doses is mainly due to the ZnO content, and can be reduced by the thin alumina protection layer. Cell viability is found to be similar for treatments with spiky particles compared to plain particles. In addition, spiky particle-mediated DNA plasmid transfection was demonstrated. By combining the advantages of the NW penetration ability and the suspension nature of nanoparticles, the spiky particles present a direct cell membrane penetrant vehicle for drug delivery, with potential applications in vivo.

The use of nanomaterials in medicine is a growing field. Thereby drug delivery with nanoparticles is coming into focus, as it promises the possibility of targeted and controlled delivery of medicine to diseased cells, for example with mesoporous silica nanoparticles (MSN). MSN offer a large surface area and pore volume, a defined and tunable pore size, and various functionalization possibilities of the inner and outer surface. [1] Here we demonstrate a modular toolbox which allows the sequential covalent attachment of different functionalities to the surface of colloidal MSN. In the presented system, poly(2-vinylpyridine) (PVP) was used as a pH-responsive cap system. To solubilize the particles in a neutral environment, polymeric poly(ethylene glycol) (PEG) was anchored to the cap system. To demonstrate the possibility for targeting, folic acid was attached to PEG and the functionality was demonstrated in vitro and in first in vivo studies in mice. After uptake of the MSN by cancer cells, endosomal entrapment represents a further bottle-neck. With the presented system we were able to deliver membrane permeable drugs which can diffuse through the endosomal membrane after opening of the polymer shell. Membrane-impermeable cargos could be delivered with an additionally attached photosensitizer similar to our previous published system. [2,3] Furthermore stability tests in cell culture medium revealed an enhanced stability of the system due to the coating and first in vivo studies in mice revealed a good tolerability of the MSN.
Acknowledgement
The authors are grateful for funding from DFG through the SFB 749, the NIM cluster and the “Fonds der Chemischen Industrie”.
References:
[1] V. Cauda, A. Schlossbauer, J. Kecht, A. Zuerner, T. Bein, J. Am. Chem. Soc. 2009, 131, 11361.
[2] S. A. Mackowiak, A. Schmidt, V. Weiss, C. Argyo, C. von Schirnding, T. Bein, C. Braeuchle, Nano Lett. 2013, 13, 2576.
[3] A. Schlossbauer, A.M. Sauer, V. Cauda, A. Schmidt, H. Engelke, U. Rothbauer, K. Zolghadr, H. Leonhardt, C. Braeuchle, T. Bein, Adv. Healthcare Mater. 2012, 3, 316.

Islet cell transplantation is considered as a promising treatment for type 1 diabetes. To avoid immune response after transplantation, we decided to use hydrogel system because hydrogels are expected to be the most excellent cell carriers. Hydrogel contains a lot of water in its structure, so it can prevent bioactive agents such as drugs, proteins and cells from denaturation. Also, to minimize surgical process, hydrogels which can be injected into body using syringe are required and a lot of researches have been reported. In this study, we designed injectable hydrogel by mixing oppositely charged nanoparticles together. There are many kinds of biopolymers and peptides, so we made charged nanoparticles using this polymers. For example, it is well known that gelatin has a lot of RGD sequences in its contents, so it is expected that the gelatin containing hydrogel can enhance cell adhesion. Among a lot of candidates, we used gelatin, hyaluronic acid, heparin and glycol chitosan. The size of nanoparticles were successfully controlled around 200 nm. Gelatin from porcine skin and glycol chitosan nanoparticles showed positively charges and gelatin from bovine skin, heparin and hyaluronic acid nanoparticles exhibited negatively charges. By blending oppositely charged nanoparticles, we confirmed the gelatin behavior and also found that the hydrogel using this system is injectable by shear thinning property. Finally, we applied this hydrogel into islet cell carrier to control the high glucose level by in vivo experiment.

Engineered nano- and microparticles that co-deliver antigen and immunostimulatory molecules are of interest as next-generation subunit vaccines and so-called ‘smart&’ adjuvants, given their ability to mimic pathogens while avoiding toxicity and anti-vector immune responses. To demonstrate that mesoporous oxide nanoparticles warrant development as particulate vaccines and adjuvants, we co-loaded mesoporous silica nanoparticles (MSNPs) with a model protein antigen, ovalbumin (OVA), and an immunostimulatory RNA (isRNA) known to activate Toll-like receptor (TLR) 7 and TLR8 and then encapsulated cargo-loaded MSNPs in a supported lipid bilayer (SLB) that we further modified with various targeting and phagosomolytic moieties. We found that high-surface-area MSNPs are able to encapsulate 50-60 wt% of OVA or isRNA individually and simultaneously encapsulate ~30 wt% of both OVA and isRNA, capacities that exceed those of state-of the-art liposomes and polymeric nanoparticles by up to 100-fold. We, furthermore, found that a SLB composition of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) modified with 5 wt% of a single-chain antibody fragment (scFv) against DEC-205 triggers efficient uptake of MSNPs by bone marrow-derived dendritic cells (BMDCs) and that the degree of silica condensation in the MSNP core can be controlled to tailor antigen release from burst (100% in 12 hours) to sustained (2-5% per day for several weeks) rates. We employed fluorescently-labeled OVA and isRNA to demonstrate that incorporating phagosomolytic peptides (e.g. ‘H5WYG&’) and/or lipids (e.g. 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, or DOPE) in the SLB enables phagosomal release of isRNA and cytosolic dispersion of OVA, which, in turn, trigger DC maturation (determined by staining for CD40, CD80, and CD86) and cross-presentation of OVA-derived peptides (determined by staining for MHC class I H-2K^b molecules complexed with the OVA-derived peptide, SIINFEKL). Furthermore, DCs pulsed with DEC-205-targeted MSNPs loaded with OVA and isRNA induce vigorous in vitro proliferation of OVA-specific CD8⁺ T cells, whereas DCs pulsed with free OVA or OVA complexed with Imject Alum trigger weak T cell responses. Upon immunization of C57Bl/6 mice, MSNPs loaded with OVA and isRNA and targeted to DEC-205, additionally, induce high-titer (>10⁵), OVA-specific IgG responses that are 100- and 10,000-fold higher than the titers achieved using OVA complexed to Imject Alum and free OVA, respectively. Importantly, these MSNPs also trigger OVA-specific CD8⁺ T cell responses, the magnitude of which is higher for MSNPs with sustained release kinetics than for MSNPs with burst release kinetics. Our results indicate that mesoporous oxide nanoparticles are an important class of antigen delivery vehicles and warrant further development.

Recent studies demonstrate that pluripotent cells recognize topographical cues in their local environment through mechanotransduction, allowing them identify and adapt to their niche. Much effort has focused on fabricating bioscaffolds that can provide similar cues to control stem cell fate. Not surprisingly, scaffolds with nanoscale topography, mimicking the extracellular matrices naturally encountered by cells, provide the greatest success this regard.
One intense area of research is on the topographical control of neural stem cell (NSC) fate. Recent studies show that scaffolds with flat, isotropic surfaces promote astrocyte differentiation, while anisotropic scaffolds with a high degree nanotopography promote neuron differentiation. This ability to control NSC fate without chemical treatment is a very useful tool for the study of NSCs and their differentiated cell types. However, most surfaces developed to drive NSC differentiation are designed to promote differentiation to only one cell type.
A scaffold system capable of promoting the differentiation of NSCs into multiple cell types would be a very useful in vitro model, especially if the cell types could be individually patterned into custom arrangements to recreate neural structures of multiple cell types. Such a model would allow for the study of complex relationships between neural cells types in a controlled in vitro setting. To accomplish this, a scaffold would require a combination of micron-scale features, for cell patterning, and nano-scale topography, for control of NSC fate. With this in mind, we have developed a novel technique, termed micropatterned nanotemplating, to creating such a scaffold. We are developing this new scaffold to create custom patterns of neurons and astrocytes as a tool to study their interactions in a highly controlled in vitro environment.

Keloids are the type of scar formed by an overgrown fibrous tissue (mostly collagen type I or III) at the site of the healed skin. Since the scar usually extends beyond the borders of the original wound and tends to recur after excision, it is not easy to remove the keloids. In this work, we suggest the plasma treatment as a new way to remove (or reduce) the keloids in the cell levels. Recently, it was reported that the plasma treatment can selectively activate the apoptosis of the cancer cells without any serious damage to the normal cells [C.-H. Kim et al., Appl Phys Lett. 96, 243701 (2010)]. We hypothesized that the reactive oxygen species (ROS) are increased in the keloid-derived stem cells by the plasma treatment as in the cancer cells because of the similar biological properties. The ROS will increase the cell death of the keloid-derived stem cells. Two different cells of the keloid-derived stem cells and the hypertrophic scars, which is a control, were treated by the non-thermal dielectric barrier discharge plasma to avoid the heat effects. Both treated cells were characterized by Fourier-transform infrared (FT-IR) spectroscopy. In the keloid-derived stem cells, it was clearly observed increasing in the stretching mode of the O-H which indicates the O-H defect is strongly affected by the plasma treatment. To confirm the plasma effects on the keloid-derived stem cells, additional results including MTT assay, Raman spectroscopy, and atomic force microscopes will be reported.

Recent studies have shown that the interactions between chondrocytes and mesenchymal stem cells (MSCs) can be harnessed for cartilage repair, with enhanced MSC chondrogenesis, increased chondrocyte proliferation and cartilage matrix formation. To better predict the clinical outcomes of utilizing such cell-cell interactions for cartilage repair, it is important to understand the effects of microenvironmental factors on the ability of these cells to form neocartilage tissues. Culture conditions, such as soluble factors and oxygen tension, play important roles in modulating chondrogenesis. Due to its avascular nature, the local oxygen tension in native cartilage is low (1-7%). As such, the goal of this study is to examine the effects of soluble factors and oxygen tension on chondrocyte-stem cell interactions. In particular, neonatal chondrocytes (NCs) were chosen in this study due to their immunoprivileged property, highly proliferative nature, and the ability to produce abundant cartilage matrix. Adipose-derived stem cells (ADSCs) were used given their relative abundance and potential to differentiate into chondrogenic lineage. NCs and ADSCs were mixed co-cultured in 3D biomimetic hydrogels for 14 days in vitro under two oxygen tension (2 and 20% O2) and two culture medium conditions (GM: serum supplemented vs. CM: serum-free supplemented with TGF-β3). The extent of cell proliferation and cartilage matrix production (sGAG and collagen) were evaluated. Interaction synergy was quantified with interaction index, which is defined as the measured biochemical content in the mixed co-culture normalized by the expected value based the measured content in the controls and the initial cell ratio. Although varying oxygen tension affected cell proliferation and cartilage matrix synthesis of NCs, it did not affect the overall interaction synergy, as shown by the interaction index analyses. Under both oxygen tension, mixed co-culture in CM led to enhanced cell proliferation and cartilage matrix synthesis, as indicated by interaction index analyses (up to 2 folds increase). Culture medium composition was found to be a critical modulator of interaction synergy, and with positive synergy only observed in CM containing TGF-β3 alone, whereas negative synergy was observed in serum containing GM. The presence of TGF-β3 in CM changes the differentiation state of ADSCs, which will likely change the release of paracrine factors and affect their role as a trophic mediator, resulting in differences in interaction synergy between CM and GM groups. Our results showed that soluble factors, but not oxygen tension, play an important role in regulating the interactions between NCs and ADSCs, as well as the resulting cartilage formation. TGF-β3 appeared to be critical in permitting synergistic interaction and enhanced cartilage matrix production and should be taken into account when applying co-culture as a strategy for cartilage tissue regeneration.

Bladder cancer is the second most common genitourinary malignancy and ranks ninth in worldwide cancer incidence. Most bladder cancers are superficial or non-muscle invasive cancers and the tumors are confined in the urothelial lining, and the conventional treatment is surgical transurethral resection of the tumor, along with adjuvant treatment using intravesical immunotherapy and chemotherapy. However, tumor recurrence and progression to more aggressive muscle-invasive tumors is a major problem. One of the shortcomings associated with current intravesical chemotherapy is the short residence time of the drug in the bladder since much of it is lost upon the first voiding of urine. Our work focuses on the development of mesoporous silica nanoparticles as a potential drug delivery system for superficial bladder cancer therapy. A series of β-cyclodextrin modified mesoporous silica nanoparticles with hydroxyl, amino and thiol groups was prepared and their suitability as a mucoadhesive and sustained drug delivery system was assessed using the mucin-particle method and porcine bladder. The thiol-functionalized nanoparticles exhibit significantly higher mucoadhesivity on the urothelium as compared to the hydroxyl- and amino-functionalized nanoparticles due to their ability to form covalent bonds between the thiol groups and the glycoproteins in mucin. Sustained release of doxorubicin loaded into the mesopores of the thiol-functionalized nanoparticles was triggered by the acidic pH of artificial urine, and cytotoxicity against bladder cancer cells was achieved.

Ultrasound (US) triggered drug release is a promising drug delivery method that allows ex-vivo modulation of treatment intensity and duration. The method relies on the synergistic interaction between rupture of sonosensitive particles and enhanced plasma membrane permeability.
Nearly all studies regarding US involve microcapsules containing air. However, the control of the physicochemical properties of the carrier such as size and surface chemistry is extremely important for efficient tumor accumulation. Liposomes, in this respect, can be a great alternative because they are easier to tailor with respect to size and composition.
Conventional liposomal systems show virtually no response to acoustic energy. We will demonstrate how sonosensitivity can be achieved in these particulate materials by the inclusion of instabilities in the lipid membrane that promote local transient lamellar to non lamellar phase transitions. In this work we used X-ray scattering characterization of very concentrated systems and fluorescence microscopy of dilute systems in a highly viscous media to slow down the rate of the transient phase transitions and we were able to clearly detect a lamellar-to 2D hexagonal transformation as a response to ultrasound radiation that is concomitant with more efficient release.

Polymeric nanocomplexes are promising non-viral carriers for anti-cancer compounds due to their self-assembly behavior, versatile functionalization and ability to preferentially accumulate at the tumor site through the enhanced permeability and retention effect. Recently, smart biomaterials that respond to various stimuli have been developed and proposed for a more effective tumor-specific drug delivery. For example, the slightly acidic conditions in the cancer microenvironment can help the selective adsorption of the carrier, trigger macromolecular switches and lead to localized release of the encapsulated cargo. Amongst the materials studied for this purpose, chitosan seems to meet the requirements, combining temperature and pH-sensitive features with biodegradability and low immunogenicity. To overcome the poor water solubility of chitosan and expand its applications in the biomedical field, several chemical modifications have been investigated, either by grafting small molecules or by engineering amphiphilic chitosan derivatives.
Although chemically modified chitosan-based drug delivery systems have recently been demonstrated both in vitro and in vivo, the role of hydrophobic substitution on the pH responsiveness and delivery efficiency is not very well understood. In our study, the pH responsiveness of hydrophobically modified glycol chitosan (HGC) nanoparticles was investigated. Nanoparticles were first obtained by grafting 5-β-cholanic acid at various degrees of substitution and were characterized in terms of size, surface charge and morphology. Delivery of Cy5.5-labelled HGC nanoparticles to a human osteosarcoma MG-63 cell line resulted in rapid internalization and minimal cytotoxicity up to a concentration of 0.5 mg/ml. Encapsulation with a model anti-tumor drug as well as the release behavior under various pH conditions is currently under study.
Accurate characterization of this easy tailorable nanosystem is crucial in order to predict its mechanisms of action and to develop a versatile platform for an effective intracellular drug delivery.

Non-invasive and controlled release of bioactive compounds is an important goal in the development of drug delivery systems and novel biomaterials for tissue engineering. This project aims to temporally and spatially control the release of bioactive compounds from phospholipid vesicle carriers by crosslinking them with superparamagnetic iron oxide nanoparticles. The magnetic properties of the nanoparticles allow release to be triggered by an alternating magnetic field (AMF), which induces localised heating and “melts” the vesicle membranes.¹ The aggregates can also be manipulated by a static magnetic field.² Incorporation of these aggregates into hydrogels has created novel responsive biomaterials. Controlled release of ascorbic acid-2-phosphate has been used to induce collagen production by chondrocytes, demonstrating an AMF triggered cellular response in vitro.³ This system has recently been improved after assessment of the performance of each component. Replacement of calcium alginate with hyaluronic acid-based hydrogels reduces gel-induced leakage of vesicle contents and improves compatibility with different cell types. An improved nanoparticle coating has improved release properties and the new system has been shown to be capable of releasing some large biomolecules, such as proteins.
Key references:
[1] R. J. Mart, K. P. Liem, S. J. Webb, Chem. Commun. 2009, 2287-2289
[2] F. de Cogan, A. Booth, J. E. Gough, S. J. Webb, Soft Matter 2013, 9, 2245-2253.
[3] F. de Cogan, A. Booth, J. E. Gough, S. J. Webb, Angew. Chem. Int. Ed. Engl. 2011, 50, 12290-12293.

Semi-flexible polymer networks generate a diverse family of structures. The network generating behaviors of specific semi-flexible biological filaments are well known (i.e. F-actin, microtubules, DNA etc.), however recent developments in tunable synthetic filaments extend this range of accessible structures. A similarly tunable model was developed using the molecular dynamics platform NAMD to provide a guide for generating synthetic filament networks. Structural characteristics of simulated networks may be quantitatively examined using connectivity analysis, radial pair distribution functions and a scaling analysis. These methods provide a basis to calculate morphological properties, including mesh size, packing order, network connectivity, avg. cluster size, filaments per bundle, and space-filling dimensionality. An analytic toolset for describing the structure of filament networks is thus provided by detailing these methods.

Mesenchymal stem cells (MSCs) have emerged as a promising therapeutic modality for tissue regeneration and repair. In particular, the immunomodulatory and angiogenic cytokines secreted by MSCs have been found to play a key role in suppressing inflammation and rendering reparative process in vivo. Despite our understanding of the regenerative mechanisms associated with MSCs, the paracrine function of MSCs under the influence of scaffold materials remains unexplored. Here, rat adipose derived stem cells (rADSCs) were cultured on random or micro-patterned fibrous scaffolds. The anti-inflammatory and angiogenic paracrine characteristics of ADSCs were investigated to understand whether scaffold materials could provide the niche to modulate the stem cell function.
The nano-fibrous scaffold was prepared by electrospinning technic. The random electrospun fibers (REF) were collected on a flat plate and the lattice-patterned, mesh-like electrospun fibers (MEF) were collected on a copper mesh. After amination, the scaffolds were covalently conjugated with hyaluronic acid (HA) to create HA modified scaffolds. Thereafter, rADSCs were cultured on 48-microwell plates and nano-fibrous for 24 h. The total RNA was then extracted for each sample and the relevant gene expression was measured by qPCR assay. The result showed that the expression levels of VEGF, COX2 and TGF-β were all up-regulated in rADSCs cultured on nano-fibrous scaffolds compared to those on microwell plates. The rADSC-derived conditioned medium was also collected and used to culture human umbilical vein endothelial cells (HUVECs) and RAW 264.7 macrophages. When the LPS-induced macrophages were exposed to the rADSC-conditioned medium, the inflammatory cytokine, TNF-α, was down-regulated and the anti-inflammatory cytokine, IL-10 up-regulated; in particular, the MEF group showed the most significant effects to promote the anti-inflammatory function. HUVECs proliferation and tube formation was also promoted by the conditioned medium derived from rADSCs cultured on scaffolds. The HA surface modification did not further improve the paracrine secretion of rADSCs on eitheir REF or MEF scaffolds.
The anti-inflammatory and angiogenic paracrine function of rADSCs were significantly improved through the cellular interaction with nano-fibrous scaffolds and the extracellular environment generated by the oriented fibers could further enhance these improvements. Compared to surface modification, the topographical characteristics of nano-fibers may be the essential factor that determines the cell behavior. The study provides new insights regarding how the interaction between materials and cells could be manipulated to improve the therapeutic function of ADSCs for tissue regeneration and repair.

Elucidating the mechanisms that govern stem cell self-renewal and differentiation is critical for understanding the roles these cells play in organismal development and function as well as for harnessing stem cells to repair tissues damaged by disease or injury. It has become increasingly clear that stem cells are regulated not only by biochemical signals in the niche, but also by biophysical features in the way these signals are presented, though investigating the latter is challenged by experimental complexities in investigating and mimicking the complexity of the extracellular matrix (ECM), cell-cell interactions, and other niche components. Recent work has demonstrated that bioactive, synthetic materials can be harnessed to emulate and thereby study the effects of solid phase, biophysical cues on cell function. For example, activation of many cellular receptors involves the formation of oligomeric protein signaling complexes with ligands presented from the matrix, the surface of neighboring cells, and in some cases even from solution. We have developed multivalent ligands - polymers conjugated to signaling proteins to yield biomimetic signals with nanoscale spatial organization - which potently induce the differentiation of human pluripotent stem cells in vitro and neural stem cells in vitro and in vivo. In addition, these materials combined with optogenetics and super-resolution microscopy have yielded insights into signaling mechanisms that regulate the fate decisions of these cells. Finally, such biomimetic materials can be integrated into safe, scaleable, and robust bioprocesses for pluripotent stem cell expansion and differentiation.

Immunoisolation devices provide diffusional barriers that shield the implanted cells (e.g., islets) from the attack of the host immune system while allow the transport of nutrients, oxygen and therapeutic products (e.g., insulin). Following the implantation of immunoisolation devices, the foreign-body responses (FBRs) are inevitably invoked at the tissue-device interface and lead to the formation of avascular fibrotic capsules that can severely impair the viability and function of implanted cells. Adipose-derived stem cells (ADSCs) are multipotent stem cells that can be conveniently harvested and hold promise to treat various types of diseases. Interestingly, the therapeutic outcomes in many ADSC therapies were mainly attributed to the ADSC paracrine factors capable of promoting angiogenesis and modulating inflammation. It is therefore our interest here to investigate how the encapsulated ADSCs or the paracrine factors of ADSCs would modulate the FBRs and whether encapsulation of stem cells could provide a new approach to enhance the biocompatibility of medical devices.
To investigate the question, ADSCs were aggregated and formed spheroids of 80 mu;m in diameter on ultra-low attachment plates. ADSCs were then loaded into a model device fabricated by two layers of polytetrafluoroethylene filtration membranes with the backing polyester mesh on the outer surface. The conditioned media outside the encapsulation devices were applied to the culture of endothelial cells or endotoxin-stimulated macrophages (sMPhi;). It is found that the secreted factors that diffused across the device membrane had trophic and angiogenic effects on endothelial cells. In addition, sMPhi; changed towards an anti-inflammatory phenotype manifested in the down- or up-regulation of pro- or anti-inflammatory factors, respectively.
Devices with or without ADSC spheroids were implanted subcutaneously in rats. Histological analysis following 4 week implantation revealed abundant formation of blood vessels adjacent to the outer surface of the devices containing ADSC spheroids, and the number of vessels at the interface was significantly higher in comparison to the blank devices without the stem cell encapsulation. In addition, the presence of ADSCs also mitigated the formation of fibrotic capsules, as only loosely collagen fibers were observable surrounding the devices, in contrast to the dense and thick capsule layers surrounding blank devices.
Our study suggests that ADSC spheroids can modulate the tissue FBR to implanted devices. The secreted cytokine repertoire including angiogenic and anti-inflammatory factors from ADSCs may be responsible for improved neovascularization and biocompatibility. The results shed lights on new ways to improve the performance of immunoisolation devices for prolonging the viability and function of cellular implantations.

Neonatal chondrocytes (NChons) are an attractive cell source for cartilage repair given their immunoprivileged nature as well as their potential to proliferate and synthesize abundant cartilage matrix. However, its clinical application is hindered by scarce donor availability. Mesenchymal stem cells (MSCs) have the potential to differentiate into chondrogenic lineage and are an alternative cell source for cartilage repair. Recent studies have also suggested that co-culturing MSCs with chondrocytes led to enhanced neo-cartilage formation. However, most of these studies focused on the interaction between adult chondrocytes and bone-marrow derived MSCs, and how adipose-derived stem cells (ADSCs), an easily accessible and relatively abundant cell source, influence cartilage formation by NChons remains largely unknown. As such, the objectives of this study are to (1) determine the minimum ratio of NChons needed in mixed co-culture with ADSCs to achieve robust cartilage formation using 3D biomimetic hydrogels, and b) examine the potential for cartilage formation in vivo by transplanting a mixed population of NChons and ADSCs in a subcutaneous mouse model. In vitro, mixed co-culture of NChons and ADSCs in 3D hydrogels resulted in robust synergistic interaction, with enhanced cell proliferation and cartilage matrix production. Interaction index, defined as the measured biochemical content in the mixed co-culture normalized by the expected value based the measured content in the controls and the initial cell ratio, revealed that interaction synergy in mixed co-culture peaked at 5-25% NChons. Based on their high cartilage matrix content in vitro, mixed co-culture groups with 10% (10C:90A) and 25% NChons (25C:75A) were further evaluated for their potential to produce cartilage matrix in vivo. After 3 weeks in vivo, the two mixed co-culture groups achieved ~4-9 folds higher cell number and cartilage matrix production than pre-implantation, which is comparable to 100% NChon alone. While total cell number and sGAG content seemed to reach its maximum at 3 weeks, collagen content continued to increase up to 8 weeks. Compressive moduli of mixed co-culture groups also increased 2-3 folds after 8 weeks of in vivo implantation, achieving comparable values as 100% NChon control. Immunostaining showed extensive and homogeneous type II collagen deposition throughout the scaffold in both mixed co-culture groups by week 12, which were comparable to NChon control. In contrast, ADSCs alone resulted in minimal cartilage matrix content and collagen II deposition in vivo. Taken together, our results highlighted the feasibility to catalyze robust cartilage formation in vivo using minimal number of NChons, by harnessing the trophic effects of ADSCs and 3D hydrogel co-culture models. Such strategies may offer a novel therapy for repairing large articular cartilage defects and accelerate the translation of NChons for cartilage repair by alleviating donor scarcity limitation.

Natural extracellular matrix (ECM) is a spatially heterogeneous network of proteins, growth factors, and polysaccharides that provides biochemical and biophysical features to the cellular microenvironment. Photopatterning, a technique where light is used to spatially modulate a particular reaction, has shown promise towards introducing heterogeneity to control cellular response, but its utility is largely unexplored within hydrogels with the nanoscale fibrous structure that is characteristic of natural ECM.
To create a scaffold that synthetically replicates the spatial heterogeneity and topographical features of natural ECM, we modified approximately 20% of the repeat units of hyaluronic acid (HA, a linear polysaccharide found throughout the ECM that is known to influence cell behavior) with norbornene, a chemical group with high selectivity toward reaction with thio-radicals. The efficiency and selectivity of this addition click reaction permits initial crosslinking of the electrospun fibers with a dithiol, and then photopatterning of any thiolated molecule in a two-step process. Specifically, solutions of 3 wt% NorHA, 3% polyethylene oxide as a carrier polymer, 1% bovine serum albumin, and dithiothreitol (DTT) were electrospun (fiber diameter: 220 ± 50nm) and first crosslinked in the presence of UV light and a radical initiator (I2959). By limiting the amount of DTT within the network, only a fraction (0.25-0.5) of the available norbornene groups were consumed in this first crosslinking step. Second, the scaffolds were then swollen with 1mM thiolated RGD (an amino acid sequence derived from fibronectin that is known to induce cellular integrin binding) and exposed to UV light through a photomask. To visualize the pattern, fluorescent peptides (GCDD-Rhodamine) were synthesized and included at 0.25 mM during photopatterning. After repeated washes with PBS over 72 hours to remove any unattached RGD and fluorescent peptides, human mesenchymal stem cells (hMSCs) or human umbilical vein endothelial cells (HUVECs) were seeded onto the scaffolds at a range of densities between 5,000-15,000 cells/cm2. Staining for actin (FITC-phalloidin) and nuclei (DAPI) revealed adherent cells with high aspect ratios on scaffold surfaces containing photopatterned RGD.
This work extends photopatterning into electrospun nanofibrous hydrogels to further the ability to synthetically recreate the spatial heterogeneity and structural characteristics of natural ECM. While RGD patterning and cellular response is demonstrated, this technique can further be explored to photopattern any thiolated ligand of interest or dithiol crosslinkers to alter mechanical properties within a nanofibrous hydrogel. Ongoing and future work will investigate patterned cellular adhesion in response to biophysically relevant changes in nanofibrous topography.

Reconstructing organs in vitro requires understanding how cells self-organize in the context of other cells and their supporting 3D scaffold. Although intrinsic signals such as the differential expression of cell-cell adhesion molecules on the surface of lineage-specific cells are known to be key players orchestrating the in vitro self-organization of dissociated tissues, the effect of extrinsic signals such as the physicochemical properties of the reconstituted extracellular matrix (ECM) are poorly defined. We study the delicate balance of these intrinsic and extrinsic signals in order to reconstruct the human mammary and human prostate glands in vitro. Using epithelial cells derived from human breast reduction mammoplasties or prostectomy surgeries, we sort cells into their luminal and basal lineages and reassemble them in well-defined 3D co-cultures using a combination of photolithographic and chemical strategies. By tuning the adhesivity and elasticity of the biomimetic hydrogels used to accommodate these heterotypic cell-populations we can control the 3D positioning of the different cell-types and promote the formation of bilayered acini that resemble the in vivo architecture. Our methods allow us to identify and physically characterize cell-cell/cell-ECM adhesion molecules and downstream effector proteins responsible for the self-organization of epithelial cells as well as to computationally model the sorting processes of these cells. Current efforts are aimed towards using our findings to design environmental context that can direct the reconstruction of functional human glands in vitro.

Stem cells (e.g., mesenchymal stem cells, MSCs) respond to many cues from their microenvironment, which may include chemical signals, mechanics, and topography. Importantly, these cues may be incorporated into scaffolding to control stem cell differentiation and optimize their ability to produce tissues in regenerative medicine. Despite the significant amount of work in this area, the materials have been primarily static and uniform. To this end, we have developed a sequential crosslinking process that relies on our ability to crosslink functional biopolymers (e.g., methacrylated hyaluronic acid, HA) in two steps, namely a Michael-type addition reaction to partially consume reactive groups and then a light-initiated free-radical polymerization to further crosslink the material. With light exposure during the second step comes control over the material in space (via masks and lasers) and time (via intermittent light exposure). We are applying this technique for numerous applications. For example, when the HA hydrogels are crosslinked with MMP degradable peptides with thiol termini during the first step, a material that can be degraded by cells is obtained. However, cell-mediated degradation is obstructed with the introduction of kinetic chains during the second step, leading to spatially controlled cell degradability. Due to the influence of cellular spreading on MSC differentiation, we have controlled cell fates by controlling their spreading, for instance towards osteoblasts in spread areas and adipocytes when cell remained rounded. Towards applications in cartilage repair, we are engineering HA hydrogels to provide an environment for MSC chondrogenesis, including investigating the influence of receptor interactions, the surrounding culture environment, and the degradation behavior on matrix production and distribution within these gels. As one example, we have introduce cadherin signaling into 3D hydrogels to mimic the interactions that cells see during the condensation process, to enhance MSC chondrogenesis. This design is evolving to incorporate degradable cadherin signals to mimic the temporal profiles of this signal during development. Overall, these advanced HA hydrogels provide us the opportunity to investigate diverse and controlled material properties for a range of biomedical applications.

The chemical and mechanical properties of substrate are critical in regulating stem cell fate because it provides niche signals that control proliferation and differentiation. A challenge in culturing hematopoietic cells is to maintain pluripotency through as many expansions as possible. The current culturing method for stem cells is usually accomplished either in vivo, or with the presence of feeder cells. Here we investigated whether they can be directly cultured on polymeric and hydrogel substrates, in the absence of additional factors.
We found that even though the hematopoietic stem cells (HSCs) are considered not adherent, they are very sensitive to the material properties of the substrates. We experimented with mTG cross linked gelatin where the gel moduli were varied from 500Pa to 5000Pa. Cells were plated on these substrates with/without Notch. Cell counts were performed after 5 days and cells were labeled with CD34 and CD38 to determine stemness. The results showed that a 5 fold increase in cell proliferation was observed for cells grown on substrate with the softest modulus. Cells on the hard substrates were comparable to the control. Optical imaging indicated that the cells on the gels were coordinated with a high degree of order, with that on the soft matrix having the highest order. Cells on the control culture dish (hard surface) were not ordered, indicating that the cells were able to sense surface deformation and migrate accordingly. Measurements of the cell moduli showed that the cells were able to conform to the modulus of the substrate. Furthermore, the moduli of single cells in each case was harder than that of cells in the organized clusters, indicating that formation of the cluster may be a factor in preserving stemness. These results show conclusively that these cells interact with the substrates and respond to adhesive properties, even though adhesion is weak.

Introduction: Urinary incontinence (UI) is a chronic and debilitating condition that is attributed to the loss of smooth muscle cells (SMC) and function of the urethral sphincter. Pluripotent stem cells (PSCs) are promising cell sources for smooth muscle tissue repair. Diverse protocols have been developed to direct SMC differentiation of human PSCs. Most methods rely on lengthy and poorly defined processes that result in very low differentiation efficiency, and efficient directed differentiation of PSCs into SMCs remains a major challenge. We hypothesize that hydrogel substrates that mimic the matrix stiffness of the native smooth muscle extracellular matrix would promote the differentiation of hPSCs towards SMC lineages. The goal of this work is to examine the effects of matrix stiffness on PSC differentiation towards SMC lineages using biomimetic hydrogels as substrates.
Materials and Methods: A human embryonic stem cell line (H9) was used in this study. To fabricate hydrogel substrates with varying stiffness, we varied the monomer to crosslinker ratio of poly(acryl amide) (PAA) hydrogels (100:1 to 17:1). To allow H9 attachment, Matrigel was chemically conjugated to PAA surface using a hetero bifunctional crosslinker, Sulfo-SANPAH. To evaluate the effects of biophysical cue on hESC fate decision, H9s were passaged on Matrigel-coated PAA gels with varying stiffness. Cells were also culture on standard 2D tissue culture plastic (TCP) as a control. All samples were cultured using an in-house optimized chemically-defined differentiation medium for 8 days before analyses. Differentiation efficiency was evaluated at day 8 by FACS for smooth muscle progenitor markers (CD31+/CD34+).
Results and Discussion: Mechanical testing confirmed successful generation of hydrogel substrates with varying stiffness (1 kPa, 15 kPa and 36kPa). Matrigel-coated hydrogels with all stiffness supported H9 cell attachment and proliferation. Flow cytometry data showed that culturing H9 cells on hydrogels resulted in up to 40-fold increase in differentiation efficiency towards SMC lineage compared to cells cultured on TCP control, and increasing hydrogel stiffness from 1 kPa to 15 kPa resulted in a great increase in differentiation efficiency. Our results suggest that matrix stiffness plays an important role in modulating PSC differentiation towards SMC lineage. And the platform reported here may be broadly useful for elucidating the effects of matrix stiffness on PSC fate towards other lineages.

Multivalent interaction on cellular membrane involves multiple and clustered ligation between ligands and receptors. Multivalency can lead to enhanced ligand-receptor affinity as well as activation of signaling pathways. Although multivalency is a fundamental mechanism underlying numerous biological events, few studies have designed and investigated multivalent materials for regenerative medicine. Particularly, adhesive oligopeptides have been widely used in material surface modification for promoting cellular attachments; it yet remains unexplored how to take advantage of multivalent binding involving adhesion ligands to regulate cell function. In this study, we intend to investigate the interaction of dendrimer-based multivalent ligands with adipose-derived stem cells (ADSCs) to understand: i) whether multivalent nano-conjugates could promote the viability, and the angiogenic/anti-inflammatory paracrine function of ADSCs; ii) the structure-property relationship that controls the bioactive properties of dendrimeric nano-conjugates.
Using polyamidoamine (PAMAM), a library of peptide-dendrimer nano-conjugates were created to present three types of peptide ligands, RGD, IKVAV and FHRRIKA in different valencies. These nano-conjugates were also complexed with hyaluronic acid to generate particles in the scale of hundreds of nanometers to incorporate the extracellular glycosaminoglycan component to enhance cell-material interactions. The multivalent nanomaterials were found to effectively bind with the membrane of rat ADSCs. The nanomaterial-coated ADSCs were mixed with uncoated ADSCs in a spheroidal culture model. The metabolic activity and proliferation level of ADSCs were either significantly inhibited or promoted depending on the valency of the specific peptide ligands. By qPCR analysis, the bioactive membrane-binding nano-conjugates were found capable of upregulating the mRNA expression of angiogenic and anti-inflammatory paracrine factors, including VEGF, bFGF and COX-2. When the conditioned media harvested from the stem cell culture were studied, it is demonstrated that the paracrine factors secreted by nano-conjugate-treated ADSCs were more effective to promote the angiogenic capability of endothelial cells and down-regulate the inflammatory response of macrophages, in comparison to non-treated stem cells.
Our study suggests that multivalent peptide-dendrimer conjugates or particles can act as unique artificial factors to modulate the stem cell niche in 3D culture. The properties of multivalent materials were determined by the biophysical/biochemical characteristics of nanomaterials, including the valency, physical size and ligand types etc. Adhesive peptide-dendrimer conjugates may be further applied to stem cell transplantation for promoting the therapeutic stem cell function in vivo.
References
[1]Jiang et al. Biomaterials, 2013; 34: 2665-2673;
[2]Conway et al. Nat Nanotechnol, 2013; doi: 10.1038/nnano.2013.205.

Polymeric nanoparticles have found application in varied fields including drug delivery and medical imaging. Particle&’s properties have a significant impact on their therapeutic performance including circulation half-life, drug release rates and toxicity. My talk will focus on discussing some of the key outcomes of nanoparticles that can be controlled through engineering particle morphologies. We have devised methods to generate particles of several distinct morphologies and studied their impact on key processes in drug delivery including phagocytosis, circulation, adhesion of vascular walls, and targeting. Based on this understanding, we have designed novel particles that demonstrate enhanced targeting. Our studies demonstrate that particle shape provides a new dimension in engineering of polymeric carriers and opens up new opportunities in drug delivery. In addition to shape, we demonstrate that controlling mechanical properties of carriers also offers unique opportunities. Specifically, we have synthesized flexible particles made from proteins that mimic the physical and functional properties of body&’s own circulating cells such as red blood cells and platelets. Particles that mimic the size, shape and flexibility of natural circulating cells offer unique advantages.

Local heat delivered by magnetic nanoparticles (MNPs) selectively attached to their target proteins can be used to manipulate and break up toxic or obstructive aggregates. We applied this magnetic hyperthermia treatment to the amyloid beta (Aβ) peptide, which unnaturally folds and self-assembles forming amyloid fibrils and insoluble plaques characteristic of amyloidgenic diseases such as Alzheimer&’s disease. We demonstrate remote disaggregation of Aβ plaques using heat dissipated by ferrite MNPs in the presence of an alternating magnetic field. Specific targeting was achieved by MNP functionalization with a targeting peptide sequence that binds a hydrophobic domain of Aβ. Field parameters and nanoparticle composition and size were tailored to maximize hysteretic losses. Transmission electron microscopy image analysis and size exclusion chromatography were used to determine the morphology and size distribution of aggregates before and after field stimulus. We found that the field stimulus is effective at destabilizing the plaque and causing a reduction in fiber length. This targeting scheme has potential as a therapy for amyloidosis and is a noninvasive tool for analyzing and controlling protein aggregation.

The epithelium compartmentalizes the body, and therefore is a significant obstacle to drug delivery. The epithelial tight junctions (TJ) limit paracellular diffusion of molecules, and thus present a major barrier to drug delivery of high molecular weight, protein based therapeutics. We have previously shown that a polypropylene nanostructured thin film in contact with epithelial cells in vitro can modulate the barrier function of the epithelium, allowing for significantly greater delivery of high molecular weight therapeutics (150 kDa) across a CACO2 monolayer by topographical cues alone. Polypropylene thin films with nanocolumns 300 nm high and 200 nm wide are made by nanoimprint lithography whereby a thermoplastic is heated above its glass transition temperature and pressed into a silicon mold. Molds are fabricated by electron beam lithography, allowing for precise design of submicron structures. This method has allowed the fabrication of thin films with varying nanotopographies. Further investigation has shown a relationship between the aspect ratio of the nanostructure and increased permeability. The lower the aspect ratio, the greater the permeability of an epithelial monolayer to high molecular weight therapeutics. Factors connected to aspect ratio - height, width, and shear modulus - are currently being investigated to determine which factors are required for this transport phenotype and to model which nanostructure characteristics are optimal for drug delivery.
The mechanism by which nanotopography can modulate transport of molecules across the epithelium was also investigated. Alterations in localization, expression, and protein level of proteins involved in paracellular transport were found. We have previously shown the morphology of the TJs was altered in cells in contact with the nanostructured film shown by a snaking staining pattern of zonula occuldin-1 (ZO-1) between cells, instead of a linear border. This staining pattern has been observed by other groups in association with increased tight junction permeability. Decreased paracellular localization of claudins 1 and 4, TJ proteins that confer paracellular barrier function, has also been observed. It is hypothesized that active cell engagement with the nanotopography occurs via integrin engagement and signaling by cellular mechanosensing leading to alterations in the permeability of epithelial TJs. Increased phosphorylation of both myosin light chain and FAK are consistent with this hypothesis, as are changes in cellular localization of RhoA. Inhibition of myosin light chain kinase, which causes the contraction of the actin cytoskeleton and creates paracellular gap formation, abolished the snaking pattern of ZO-1 staining. Our studies demonstrate that low aspect ratio nanotopographical cues alone can increase delivery of high molecular weight therapeutics across the epithelium by altering cellular tight junctions.

We identified novel peptide sequences that specifically interact with cancerous collagen matrices. Collagen is the most abundant protein of the extracellular matrix (ECM) in human body, and excessive collagen remodeling occurrs in various diseases. Especially, several types of collagen are not only over-expressed in various human cancers, but large portions of the collagens are degraded and denatured by proteolytic enzymes such as matrix metalloproteinases (MMPs). Thus, collagen is emerging as a new biomarker for cancer diagnosis and prediction of cancer prognosis as well as a novel target for therapeutic purpose. Structural change of collagen for tumor progress is well investigated in the breast cancer, named a tumor-associated collagen signature (TACS), and TACS-3 is reverse correlated with the long-term survival rate of human patients. Concerning the fact that delivering imaging or therapeutic agents to specific cancer tissues is a critical factor for the development of nanomedicine, the development of bio-nanomaterials that specifically interact with tumor-associated collagen is of importance. Here, we report novel peptides that specifically bind cancerous collagen matrices using a phage display. We first constructed collagen-like peptide libraries inspired by collagen molecules that carrying (Gly-Pro-Xaa) hepta- and (Gly-Xaa-Yaa) deca-repeats on the pIII minor coat protein of M13 bacteriophages (phages; Xaa and Yaa are random twenty amino acids). Selected peptides from the constructed libraries exhibited selective and specific binding capability for the cancerous collagen matrices. We utilized engineered M13 phages as a novel bio-nanomaterial for clinical applications for the cancer targeting, due to their manageability and safety to mammalian cells. The engineered phages that display collagen binding peptides will provide a sensitive and selective binding for collagen in cancer cells, a cancer biomarker, and our engineered phages might be useful for cancer imaging and therapy during cancer progression in the future.

Although nanotechnology promises to revolutionize the treatment of infectious disease, existing state-of-the-art nanoparticle delivery vehicles, including many liposomal and polymeric nanoparticle formulations, suffer from limited capacities, uncontrollable release profiles, and complex, specialized synthesis procedures that must be re-adapted for each new cargo molecule, leading to drug- and disease-specific ‘one-off&’ approaches. To address these limitations, we have developed mesoporous silica nanoparticle-supported lipid bilayers (‘protocells&’ - see Nature Materials (2011), 10: 389-397) for high capacity, cell-specific delivery of various therapeutic molecules, including antibiotics. Protocells are composed of a mesoporous silica nanoparticle (MSNP) core encased within a supported lipid bilayer (SLB). MSNPs have high surface areas and can, therefore, be loaded with 20-55 wt% of acidic, basic, and hydrophobic antibiotics, capacities that are 100 to 1000-fold higher than similarly-sized liposomes and polymeric nanoparticles. Furthermore, by controlling the degree of silica condensation in the MSNP core, release rates can be precisely tailored from 100% release within 12 hours to 2% release for nearly two months. Fusion of liposomes to antibiotic-loaded MSNPs creates a coherent SLB that enhances the colloidal stability of protocells in blood and helps retain encapsulated drugs within the MSNP core until protocells reach their target organ or cell. Furthermore, the SLB provides a biocompatible interface for display of targeting and endosomolytic moieties, which we have shown trigger efficient, cell-specific uptake of protocells, followed by cytosolic dispersion of encapsulated antibiotics. Specifically, protocells loaded with the antibiotic, levofloxacin, and targeted to cells infected by Francisella tularensis live vaccine strain (LVS) are internalized by target cells 10,000-times more efficiently than by non-target cells and kill intracellular LVS more effectively than free levofloxacin and levofloxacin-loaded liposomes at just 2 wt% loading (one-twentieth the protocell&’s maximum loading capacity). In summary, protocells combine high cargo capacities, long-term stability in blood, high targeting specificity, and controllable release kinetics and are, therefore, a promising nanoparticle-based delivery platform. We are currently adapting protocells for oral administration and testing the pharmacokinetics, efficacy, and safety of levofloxacin-loaded protocells in rodents and non-human primates infected with Francisella tularensis.

Human pluripotent stem cells (human embryonic stem cells and induced pluripotent stem cells) have the ability to self-renew indefinitely and differentiate into specialized types. Soluble signals (e.g., growth factors or small molecules) have been identified that promote human pluripotent stem (hPS) cell differentiation. Standard differentiation protocols employ Matrigel, an undefined and complex mixture of over 1800 proteins. The role of the insoluble signals in directing differentiation is largely unexplored. To mine this molecular space, we devised arrays of chemically defined surfaces composed of self-assembled monolayers of peptides. Using the results from our array screen, we generated peptide-modified surfaces and used them to elucidate the molecular mechanisms by which insoluble signals guide hPS cell differentiation to each of the primary germ layers. We identified surfaces with surprising assets: They not only permit human pluripotent stem cells to differentiate to specific lineages but even instruct them to do so. Our findings highlight the dramatic effects of insoluble cues on stem cell pluripotency and differentiation. They also underscore the utility of defined surfaces to dissect critical signaling pathways.

The obtaining of particulate nanostructured molecular materials at large scale is currently playing a crucial role in drug delivery and clinical diagnostics. It has been observed that polymeric nanoparticles and nanovesicles are efficient drug carriers that can significantly help to develop new drug delivery routes, and more selective and efficient drugs with a higher permeability to biological membranes and with controlled released profiles, as well as to enhance their targeting towards particular tissues or cells [1-2].
The potential of laquo;bottom-upraquo; strategies, based on molecular self-assembling, is much larger than that of laquo;top-downraquo; approaches for the preparation of such nanostructures. For instance, by precipitation procedures it should be possible to control particle size and size distribution, morphology and particle supramolecular structure. However, conventional methods from liquid solutions have serious limitations and are not adequate for producing such nanoparticulate materials at large scale with the narrow structural variability, high reproducibility, purity and cost needed to satisfy the high-performance requirements and regulatory demands dictated by the US FDA agency.
The solvent power of compressed fluids (CFs) can be tuned by pressure changes. Therefore, using compressed solvent media, it is possible to obtain supramolecular materials with unique physicochemical characteristics (size, porosity, polymorphic nature morphology, molecular self-assembling, etc.) unachievable with classical liquid media. The most widely used CF is CO2, which has gained considerable attention, during the past few years as a laquo;green substituteraquo; to organic solvents. During the past few years, CFs based technologies are attracting increasing interest for the preparation of nanovesicles with application in the field of nanomedicine.
In this presentation a simple one-step and scale-up methodology for preparing multifunctional nanovesicle-drug conjugates will be presented. This method is readily amenable to the integration/encapsulation of multiple bioactive components, like peptides, proteins, enzymes, into the vesicles in a single-step yielding sufficient quantities for clinical research becoming, thereby, nanocarriers to be used in nanomedicine. A couple of examples of novel nanomedicines prepared by this methodology will be presented and their advantages discussed [3-4].
References
[1] M. E. Davis, Z. Chen, D. M. Shin, Nature Reviews-Drug Discovery 2008, 7, 771-782.
[2] J. A. Hubbell, R. Langer, Nature Materials, 2013, 12, 963-966.
[3] N. Ventosa, L. Ferrer-Tasies, E. Moreno-Calvo, M. Cano, M. Aguilella, A. Angelova, S. Lesieur, S. Ricart, J. Faraudo, J. Veciana. Langmuir, 2013, 29, 6519-6528.
[4] I. Cabrera, E. Elizondo, E. Olga; J. Corchero, M. Mergarejo, D. Pulido, A. Cordoba, E. Moreno-Calvo, U. Unzueta, E. Vazquez, I. Abasolo, S. Schwartz, A. Villaverde, F. Albericio, M. Royo, M. Garcia, N. Ventosa, J. Veciana. Nano Letters, 2013, 13, 3766-3774.

Nanovehicles capable of releasing carried cargo in response to external stimuli promise to enable spatiotemporal control over drugs and signaling pathways. Among various kinds of stimuli, light is a promising “trigger” because it has excellent spatial and temporal resolution. We have previously reported o-nitrobenzyl-containing polymeric nanocapsules (~200 nm in diameter) as delivery vehicles that degrade into small molecules upon ultraviolet (UV) irradiation. However, UV is detrimental to living tissues and the penetration depth of UV light is extremely low, which greatly limits the applicability of these UV-responsive polymers in vivo. Lanthanide-doped upconverting nanoparticles (UCNPs) (~20 nm in diameter) with ladder-like energy levels can readily convert bio-benign low-energy near-infrared (NIR) light to high-energy visible and UV light. We co-loaded polymeric nanocapsules with cargo and UCNPs by a facile electrospray process. Under 980 nm irradiation, UCNPs emit 350 nm UV light, triggering polymer degradation and cargo release. This system holds great promise as a biomedical research tool and candidate for clinical applications.

The abnormal assembly of beta-amyloid (Aβ) peptides into neurotoxic, beta-sheet rich amyloid aggregates is one of the critical pathological events of Alzheimer&’s disease (AD). In this context, the suppression of Aβ aggregation is considered to be an attractive therapeutic strategy. Here, we propose a novel strategy for the inhibition of Aβ self-assembly, by delivering photo-excited porphyrin. Light-induced treatment is regarded as a promising therapy for various local diseases due to its temporal and spatial controllability. Photodynamic therapy (PDT), for example, utilizes visible or near-infra red light and light-sensitive molecules to induce a localized cell death of tumor cells by generating reactive oxygen species. In this work, we examined the inhibition of Aβ aggregation and decrease in cell cytotoxicity by meso-tetra(4-sulfonatophenyl)porphyrin (TPPS) under the blue LED light whose emission wavelength corresponds to absorbance of TPPS, using circular dichroism (CD), dot blot, atomic force microscopy (AFM), native gel electrophoresis, and cell viability assay (MTT). CD and dot blot assay results show that TPPS alters the secondary structure of the protein aggregates in concentration dependent manner under the light condition. AFM and gel electrophoresis results further confirm that conversion of Aβ peptide into aggregates of fibrous form was blocked by photo-excited TPPS, leaving small monomeric or oligomeric peptides. Furthermore, MTT assay performed with PC 12 cell line clearly verifies that the photo-induced suppression significantly lowers the cytotoxicity. Based on the photochemical property of the porphyrin, we speculate that photo-induced damage of Aβ peptides in the presence of TPPS under light illumination would preclude the self-assembly of the peptides into aggregates. Our findings suggest the potential of utilizing photo-excited dye molecules as a therapeutic agent for photodynamic therapy of AD.

Polymeric micelles have drawn increasing interest for delivering anti-cancer agents due to their small size and versatility for functionalization. A current challenge in the use of polymeric micelles is the relatively short blood circulation. Zwitterionic materials have shown great potential in drug delivery because of their excellent anti-biofouling property. In this study, star-branched poly(2-methacryloyloxyethyl phosphorylcholine) grafted poly-lactide (PLA-PMPC) was synthesized by the combination of ring opening polymerization, atom transfer radical polymerization and click chemistry. The chemical structure of PLA-PMPC was verified by 1H nuclear magnetic resonance (1H-NMR). The self-assembled micelles of PLA-PMPC with diameter of 40~80 nm showed low cytotoxicity and good hydrophilicity. These PC-containing star-branched copolymer micelles could escape from spleen filtration. The blood circulation time was investigated by using DiI fluorescent pigment as a model drug. The PLA-PMPC micelles considerably enhanced the blood circulation of DiI and the plasma half-life reached 19.3h, which was 1.48-fold higher than that of PLA-mPEG. The prolonged circulation time attributed to the covalently reinforced core-shell structure of amphiphilic copolymer, since the PMPC layer inhibited serum protein adsorption. Furthermore, an in vivo murine tumor model evaluation showed that the distribution of DiI delivered through PLA-PMPC micelles in liver and lung was similar to that of PLA-mPEG, while the concentration of DiI in spleen was much lower. Moreover, PLA-PMPC micelles delivery significantly enhanced the accumulation of DiI at the tumor site. The relative accumulation concentration of DiI delivered by PLA-PMPC was 2.37-fold higher that that of PLA-mPEG. In summary, star branched PLA-PMPC micelles can efficiently prolong the circulation time of hydrophobic drugs and successfully mediate the tumor-target accumulation, which demonstrated that PLA-PMPC might be a promising vehicle with high efficacy and low side effects for future cancer therapy.

Nanomaterials engineered in novel multi-modular systems in which every component works in a synergistic way with others have the potential to lead to a completely new class of tools for nanomedicine. The development of nanostructures able to release drugs directly within the target after a stimulus can drastically improve the therapeutics efficiency by reducing side effects. Gold nanoparticles offer one of the most suitable platforms for the development of modular nano-devices. On one hand their surface properties enable effective coating by peptides yielding stable and non-cytotoxic systems. On the other, their intriguing photophysics, characterized by the surface plasmon resonance, can be exploited for novel excitation schemes.
Doxorubicin is a widely used but toxic cancer chemotherapeutic agent. In order to localize its therapeutic action while minimizing its side effects, it was covalently conjugated to 30 nm peptide-encapsulated gold nanospheres by click-chemistry and then photo-released in a controlled fashion through the cleavage of the 1,2,3-triazolic ring by a multiphoton process with 561 nm irradiation at microW power. Selective apoptosis of human osteosarcoma (U2OS) cells was observed only in the irradiated 100x100 micron area in less than 6 minutes after the stimulus. Notably, the apoptotic effect of doxorubicin was completely inhibited for at least 8 hours until its release “on demand” was externally light-triggered.
The therapeutical efficiency of these gold-based nanosystems, engineered for the exogenous-controlled intracellular release of drugs by exploiting multiphoton stimulation is discussed together with the promising applications of nanocarriers that can ensure high spatial and temporal controlled drug delivery.

We have demonstrated the production of non-spherical collagen microparticles with the encapsulation of MDA231 cancer cells by soft robotic PDMS/Ecoflex micro-molding method and investigated cell viability in these microparticles. Collagen microspheres have been commonly produced by microfluidic flow focusing system which yields mono-dispersed microspheres in microchannels. These mono-dispersed microspberes have been attractive to many biomedical applications such as tissue engineering and cellular microenvironment study, because it has biodegradable nature and a highly porous structure that allows adequate transport of nutrients and oxygen to cells encapsulated within the microspheres. Non-spherical collagen particles are also in demanded for similar types of applications. However, the production of these particles has not been performed well because of the difficulty of non-spherical collagen particle production in dispersed media. Additionally, the traditional micro-molding approach also did not allow the production of non-spherical collagen particles due to the surface absorption of the collagen. Therefore, we invented a new soft robotic micro-mold that has more than 100 individual non-spherical particle template. These templates are deformed by pneumatic pressure which pushes for microparticles out from the templates after collagen solution is gelled. The soft robotic mico-mold is made of the top PDMS/ Ecoflex substrate and the bottom PDMS substrate, which are patterned from polylactic acid (PLA) original hard plastic molds formed by 3D printer. The top substrate and the bottom PDMS substrate with a pneumatic channel are bonded together via oxygen plasma process. When air is pumped to the pneumatic channel, the top substrate is deformed and cross-linked collagen in the template is pushed out without being damaged. With this method, non-spherical collagen microparticles such as square, triangular and cross shapes with dimension of 100-500 um can be generated. In addition, the viability of MDA231 cell encasplated in non-spherical microspheres has been confirmed. These new non-spherical collagen microparticles which have never been investigated before will provide fresh opportunities for soft biomaterial research.

Introduction: Poly(ethylene glycol) (PEG) hydrogels have been widely used for delivery of biomolecules due to their tissue-like water content and tunable physicochemical properties. To create chemically stable PEG hydrogel network, two crosslinking mechanisms have been commonly used including chain-growth (CG) or step-growth (SG), which result in differential gel crosslinking density and subsequent material property. However, how different crosslinking mechanism and crosslinking density of PEG hydrogel change protein release from such hydrogels remains poorly understood. To address this limitation, here we aimed to evaluate the effects of PEG hydrogel crosslinking mechanism and density on protein release.

Materials and Methods: BSA protein was encapsulated in PEG hydrogels formed by either CG or SG polymerization. Gel crosslinking density was further tuned by varying PEG molecular weight (MW), concentration (Conc.), or crosslink functionality. To form CG hydrogels, PEG-diacrylate (PEGDA) with different MW(2k, 4k, 5kDa) and conc. (10, 15, 20 (w/v)%) was dissolved in PBS solution with BSA (2 (w/v)%) and photoinitiator (Irgacure 2959, 0.05 (w/v)%). For SG hydrogels, norbornene-terminated PEG (4- or 8-arm PEG, 5k or 10k) and thiol-terminated PEG (2-, 4- or 8-arm, 1.5k, 5k or 10k) were mixed in PBS solution (2% BSA, 0.05% Irgacure 2959) to be reacted in stoichiometric balanced ratio. To quantify protein release, hydrogels were immersed in PBS and supernatant was taken for up to 42 days. Total releasable BSA was calculated as percentage of total accumulated BSA release to initially encapsulated BSA. To quantify release kinetics, BSA fractional release curve was fitted into 3D Fickian diffusion model.

Results and Discussion: For CG gels, both total releasable BSA and BSA release kinetics were mostly dependent upon PEG conc. Decreasing PEG conc. from 20% to 10% resulted in a ~3-fold increase in total releasable protein. In contrast, varying PEG MW only had mild effect on protein release. For SG gels, BSA release was sensitive to both varying PEG MW and PEG conc. Moreover, SG gels further delayed the BSA release by using higher crosslink functionality (from 4arm to 8arm). Our findings suggest that SG gels are capable of producing fewer structural defects during network formation, giving more tunability for controlled release of biomolecules.
Conclusions: Our results showed that PEG hydrogel crosslinking mechanism (CG vs. SG) can differentially impact protein release from the resulting hydrogels. Furthermore, varying PEG MW did not change protein release from CG gels, but can be used be further tune protein release from SG gels. In sum, this study provides valuable insight on the understanding the effects of PEG hydrogel crosslinking mechanism and density on protein release, and may be used to guide rational design of PEG hydrogels to achieve desirable protein release kinetics.

The initiation and extension of axon are critical to neuron development and neural circuit formation. The importance of extracellular cues to axon initiation and outgrowth is emerging as one of the major themes in neural development. Nanostructures and nanomaterials serve as promising candidates to provide topographical cues to neuronal adhesion and development. Previous studies showed that neuron cells were able to sense nanoscale structures and responded differently in their neurite extension. In the present work, we use patterned nanopillar structures as controllable topographical cues to culture hippocampal neurons, and found that nanopillars have significant guidance effect on neurite outgrowth and elongation. More interestingly, the axon specification occurs in the first 12 hours after cell plating, which is much earlier than the usual time point for cells growing on normal flat surfaces. It indicated that the topographical cues can indeed accelerating neural development. We further studied this topographical influence on axon initiation and elongation by varying the diameter, height, pitch and shape of the nanopillars, and different effects were observed. This work will provide new insights on the role of topographical cues for neuronal development in vivo, as well as the possibility of using nanoscale topographic features to control neuronal development.

Many cells display electrotaxis meaning directional migration in an electrical field. For instance this comprises various cancer cells, stem cells and epithelial cells. Such effects have practical importance as a therapeutic option, for promoting wound healing, guiding tissue regeneration, or to better understand the progress of metastatic disease. A prerequisite is however that it can be studied in a reliable manner and a consistent theory explaining the behavior can be formed. The underlying mechanisms are currently not well understood.
One reason is lack of experimental reproducibility, as a consequence of that most experiments are performed in setups with insufficient control over electrochemical conditions. Minor alterations in geometry, medium conductivity and pH can substantially influence the response of the cells and the homogeneity and strength of the field. Main components of the standard system are silver:silverchloride (Ag:AgCl) electrodes immersed in saline and connected via agar bridges to either side of a narrow cell culture chamber. Longer bridges ensure that toxic Ag+ does not enter the chamber within the experimental time frame. We here propose the use of microfluidic systems to offer better control over important experimental parameters, improve properties in terms of handling and the possibility to include complex features. A prerequisite for the design of such devices is that the electrochemistry of the experiment is clarified. Only then can the boundary conditions for the miniaturization be understood, the right control parameters be selected and strategies for their control be implemented.
In an effort to outline these influences we performed a set of experiments focusing on the effect of DC stimulation on pH and conductivity fluctuations in a µ-fluidic device. Silicon rubber channels were constructed and connected to standard agar bridges. A pH-sensitive foil imaging system was integrated to investigate pH distribution within the channel. Furthermore, a miniaturized system for conductivity measurements was designed for monitoring conductivity alterations in medium as a consequence of the current flow. In addition, we investigated the influence of DC stimulation on the cell culture medium, using stimulated medium to analyze proliferation and viability of an electotactic cell line (MAT-LyLu). These results were in turn correlated to measurements concerning the migration speed of Ag+ in the agar bridges, the purpose being to clarify if other effects, apart from Ag+, influence viability. Ag+ was quantified using stripping voltammetry.
Only with clear data excluding such indirect effects of stimulation, reliable conclusions regarding the effect of the DC field itself can be drawn. Through the extensive set of measurements outlined here we can for the first time provide detailed and accurate information about the electrochemistry of an electrotactic system, pointing the way for future development towards microfluidic devices.

Natural polyphenols are a class of organic compounds that are commonly found in food. Some natural polyphenols, such as limonene in citrus fruits, Eugenol in cloves, and methyl salycilate in wintergreen give off strong flavors and aromas. While other polyphenols, such as curcumin from turmeric root, Resveratrol from grapes and cinnamaldehyde from cinnamon are known to possess medicinal properties including antioxidant, antibacterial, antifungal and anticancer. Despite the strong flavoring and potential for medicinal uses, these polyphenols have poor bioavailability and bio-absorptivity due to their low water solubility. In addition, the polyphenols are very susceptible to oxidation, light and heat degradation, thus reducing their stability and limiting their shelf life. As such, encapsulation and controlled delivery of these natural polyphenols could be a promising approach to overcome these drawbacks. In this work, an oil-in-water (o/w) starch nanoparticles stabilized Pickering emulsions will be prepared using a food grade starch, CAPSUL TA. Limonene and tea tree oil will be used as model flavor and antibacterial compounds, respectively. Limonene is used widely used as a flavoring and fragrance compound because of its pleasant citrus aroma. On the other hand, tea tree oil is an essential oil extract that has been traditionally used as a topical antiseptic and antifungal treatment. Moreover, it has been shown to be effective against MRSA (methicillin-resistant Staphylococcus aureus), a multi-antibiotic resistant bacteria. The o/w Pickering emulsion was prepared using 4 wt % CAPSUL TA starch nanoparticles, 5 wt % canola oil, 91 wt % water and 0.2 vol % limonene or tea tree oil, and mixed using a hand disperser follow by probe sonication. The physical properties of the Pickering emulsion were evaluated using DLS and optical microscopy. For controlled release of the volatile compounds, various concentrations of alpha-amylase was added to the Pickering emulsion incubated with stirring at 37 degrees Celcius, and the release of limonene or tea tree oil into the headspace was captured using a SPME (solid phase microextraction) fiber and injected into a GC/MS for analysis. The controlled release and delivery of tea tree oil from the o/w Pickering emulsion will be evaluated using a model E. coli bacterial biofilm. The effectiveness of this delivery system will be evaluated based on the disruption of the biofilm and the resultant decrease bacterial viability. This study highlights several potential applications of this Pickering emulsion incluing dental biofilm treatment and the release of flavor compounds in the mouth.

In the past few years, the Desai lab has shown that nanoporous thin-film polymer membranes can achieve zero-order release kinetics of bio-molecules from a reservoir device. Additionally, a method for nano-porous thin-film polycaprolactone fabrication using zinc oxide nano-wires as a template has been described. In order to improve process control, scale-up, reproducibility and control over nano-features, we explore a new fabrication method utilizing on hot-press lamination techniques. This novel technique uses a laminator to create a nano-wire polymer template from an anodized aluminum oxide nano-porous membrane. A polycaprolactone film is then cast over the nano-wire template to create a nano-porous membrane. The pore size and density is controlled by the features of the anodized aluminum oxide membrane, which can be fabricated with precise control over a range of pore sizes in the nano-scale range. In addition to a novel method for fabricating nano-porous membranes, we present a characterization of diffusion as a function of membrane design and molecular properties. We investigate the release of a range of bio-molecules in size from peptides to antibodies to determine at what pore size to molecular weight or radius ratio we observe single-file diffusion and zero order release kinetics.

The highly specific interactions between saccharides and cell surface lectins offer a convenient way to target nanostructures towards different cell types. To this end, a simple, versatile method has been developed to provide a range of saccharide coatings for magnetite nanoparticles and liposomes. These coating agents were formed through hydrazide addition to the reducing end of saccharides¹, forming pyranose adducts with good anomeric purity that hydrolyzed very slowly, with only 25% hydrolysis after 24 days.
Glycolipids, which allow the coating of phospholipid vesicles (800 nm), were formed through this procedure using a hydrazide-terminated PEG-lipid. The formation of hydrazide adducts was successful for a range of saccharides, from simple monosaccharides such as glucose to a more complex trisaccharide, siallyllactose. There is also scope to further modify these coatings using transglycosidases², which was demonstrated using a galactosyl transferase.
Catechol-sugar conjugates were synthesized through the same methodology then used to coat magnetite nanoparticles (30 nm), with aggregation by fluorescein-tagged lectins confirming functionalization. These coated nanoparticles were applied to cell culture, where 3T3 fibroblast cells incubated with glucose- and GlcNAc-coated nanoparticles showed a greater response to a static magnetic field than those incubated with non-functionalized nanoparticles. To visualize the organelles and surface targeted by these saccharide coated particles, a fluorescent coating is also under development. Once completed, it is anticipated that the resulting multi-functional nanoparticles will not only possess superparamagnetic properties,³ allowing hyperthermia and magnetic cell sorting, but will also be capable of visualizing targeted cell components. When combined with saccharide functionalized liposomes, they may also have applications in the targeted delivery of biomolecules.^4
1 K. Godula and C. R. Bertozzi, J. Am. Chem. Soc, 2010, 132, 9963-9965
² G. T. Noble, F. L. Craven, J. Voglmeir, R. Sardzik, S. L. Flitsch and S. J. Webb, J. Am. Chem. Soc., 2012, 134, 13010-13017
³ R. T. Gordon, J. R. Hines and D. Gordon, Med. Hypotheses, 1979, 5, 83-102
⁴ F. De Cogan, A. Booth, J.E. Gough and S.J. Webb, Angew. Chem. Int. Ed., 2011, 50, 12290-12293

Endothelial filopodia play key roles in guiding the tubular sprouting during angiogenesis. However, their dynamic morphological characteristics, with the associated implications in cell motility, have been subjected to limited investigations. In the present work, the interaction between endothelial cells and extracellular matrix fibrils was recapitulated in vitro, where a specific focus was paid to derive the key morphological parameters to define the filopodial guiding process. Based on 1-D gelatin fibrils patterned by near-field electrospinning (NFES), we show that the behavior of endothelial filopodia underpin the cellular contractility. ROCK inhibition resulted in abnormally long filopodia which retained the ability to sense chemical
cues, but reduced the ability for motility guidance. Breakage of trailing filopodia was sometimes observed during cell motility, but this effect might be diminished for filopodia shorter than ~10micron. Adopting these 1-D fibril platforms, facile imaging processing was facilitated using an open source software CellProfiler. The morphological phenotypes of endothelial cells under different treatments were identified.

Lipids are amphiphilic molecules that self-assemble in aqueous media. Depending on the molecular structure and external conditions (e. g. temperature, concentration, and charge density) several self-assembled structures are observed (Liposome, hexasome, cubosome, and so on). This rich phase behavior enables the application of lipid-based materials as carriers for cellular delivery of drugs and genes. There are many biological barriers that a delivery vehicle has to overcome.
In this work we will present strategies to modulate lipid materials that promote membrane fusion. The majority of the lipid materials used currently are presented as flat lipid layer stacks intercalated with DNA or siRNA which have zero or positive Gaussian curvature. We will report our most recently found topologically active lipid materials containing siRNA that has negative (Gaussian) curvature and is therefore active with respect to membrane fusion. A clever selection of lipid identity and composition allows for the fragmentation of this material into a few hundreds nanometer particulates which is imperative for the applications of drug and gene delivery.

A significant body of research has focused recently onto inhalation therapy as a well-accepted treatment for many lung diseases. The aim of this work was to develop and in-vitro evaluate new series of polymeric carrier systems for controlled pulmonary drug delivery. These carriers combine the benefits of nanoparticles (NPs) and respirable hydrogel microparticles while avoiding their shortcomings. The developed carriers are based on poly(ethylene glycol)-chitosan (PEG-CS) copolymer and crosslinked with tripolyphosphate (TPP) and/or sodium hyaluronate (SHA) in form of hydrogel NPs. The drug-loaded hydrogel NPs were then used to develop respirable 2-5 microns size microparticles through controlled spray drying of an aqueous suspension