Grazing incidence x-ray diffraction studies of lipid-peptide mixed monolayers during shear flow

Grazing Incidence X-ray Diffraction (GIXD) studies of monolayers of biomolecules at the air-water interface give quantitative information of in-plane packing, coherence lengths of the ordered diffracting crystalline domains and the orientation of hydrocarbon chains. Rheo-GIXD measurements revel quantitative changes in the monolayer under shear. Here we report GIXD studies of monolayers of Alamethicin peptide, DPPC lipid and their mixtures at the air-water interface under the application of steady shear stresses. The Alamethicin monolayer and the mixed monolayer show flow jamming transition. On the other hand, pure DPPC monolayer under the constant stress flows steadily with a notable enhancement of area/molecule, coherence length, and the tilt angle with increasing stress, suggesting fusion of nanocrystallites during flow. The DPPC-Alamethicin mixed monolayer shows no significant change in the area/DPPC molecule or in the DPPC chain tilt but the coherence length of both phases (DPPC and Alamethicin) increases suggesting that the crystallites of individual phases are merging to bigger size promoting more separation of phases in the system during flow. Our results show that Rheo-GIXD has the potential to explore in-situ molecular structural changes under rheological conditions for a diverse range of confined biomolecules at the interfaces.Comment: 21 pages, 11 figures, 2 table

Anomalous phase transitions in soft colloid-polymer binary mixtures

We have shown earlier [1] that these PGNPs resemble star polymers or spherical brushes in terms of their morphology in the melt. However, these particles show dynamics in melt which is quite different from other soft colloidal particles. Since most of the work on soft colloidal particles have been performed in solutions we have now explored the phase behavior of the PGNPs in good solvent using microscopic structural and dynamical measurements on binary mixtures of homopolymers and soft colloids consisting of polymer grafted nanoparticles. We observe anomalous structural and dynamical phase transitions of these binary mixtures, including appearance of spontaneous orientational alignment and logarithmic structural relaxations, as a function of added homopolymers of different molecular weights. Our experiments points to the possibility of exploiting the phase space in density and homopolymer size, of such hybrid systems, to create new materials with unique properties

Patterning Self-Organizing Microvascular Networks within Engineered Matrices

Introduction Hierarchically arranged self-organizing vasculature is a long-sought goal of tissue engineering. In native tissues, various factors such as fluid flow, biomechanical cues and biochemical cues (growth factors & cytokines) work in synergy to achieve high precision over vasculature. To this end, by employing spatiotemporally controlled growth factor’s availability within engineered tissues could help in guiding the developing vasculature. However, the conventional approaches for growth factor delivery often focuses on their immobilization or coupling within the engineered matrices (hydrogel), via various linker proteins or peptides. Even though it provides stable release rates, but imparts limitations upon upscaling with high specificity of multiple growth factors delivery. To overcome this limitation, the present study employed oligonucleotides based aptamers, that are affinity ligands designed to recognize proteins with high affinity and specificity.1 The developed aptamer-functionalized biomaterials were systematically studied for achieving patterned self-organizing microvascular networks in 3D microenvironments. Materials & Methods The aptamer-functionalized hydrogels were prepared via photo-polymerization of gelatin methacryloyl (GelMA) and acrydite functionalized aptamers having DNA sequence specific for binding to vascular endothelial growth factor (VEGF165). Visible light photoinitiator, tris(2,2′-bipyridyl)dichloro-ruthenium(II) hexahydrate with sodium persulfate was used. For patterning, aptamer-functionalized hydrogels 3D printing technique was employed. The human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs) were used. The construct was 3D bioprinted as lines of aptamer-functionalized bio-ink next to plain GelMA lines (blue beads), making an interface. After 3D printing, the constructs were crosslinked and loaded with VEGF for 1 hr. It was expected that the 3D bioprinted aptamer lines would be able to sequester VEGF from the culture medium, compared to GelMA lines. To study the programmable/triggered growth factor (VEGF) release efficiency, the complementary sequences (CSs) were also added at specific time-points and their effect of microvascular network formation was studied. Results & Discussion The results obtained from physicochemical analysis of the aptamer-functionalized hydrogels confirmed the higher aptamer retention capacity of acrydite functionalized aptamers within the hydrogels, in comparison with the control aptamers for as long as 10 days at 37 °C. The VEGF ELISA experiments confirmed triggered release of VEGF from the aptamer functionalized hydrogels in response to CS addition. Without CS addition, these hydrogels could sustain a controlled release for until 10 days. Furthermore, in co-culture experiments, the developed patterned aptamer-functionalized hydrogels showed high cellular viability and ability to guide microvascular network formation (by HUVECs and MSCs) only within the aptamer-functionalized regions of the pattern, and not in GelMA regions, after 10 days of culture (Figure 1). However, differences in the microvascular organization was observed in the samples with triggered VEGF release on on day 5, compared to the samples without the VEGF release. These observations altogether confirmed the ability of patterned aptamer functionalized hydrogels in controlling self-organizing microvascular networks. Conclusions The present study confirms the potential of patterned aptamer-functionalized hydrogels in guiding self-organizing microvascular networks within 3D microenvironment, by spatiotemporally controlling VEGF bioavailability. Acknowledgements: This work is supported by an ERC Consolidator Grant under grant agreement no 724469. References 1. D. Rana, A. Kandar, N. Salehi-Nik, I. Inci, B. Koopman, J. Rouwkema. BioRxiv (2020) doi: https://doi.org/10.1101/2020.09.22.308619

Spatiotemporally controlled, aptamers-mediated growth factor release locally manipulates microvasculature formation within engineered tissues

Spatiotemporally controlled growth factor (GF) delivery is crucial for achieving functional vasculature within engineered tissues. However, conventional GF delivery systems show inability to recapitulate the dynamic and heterogeneous nature of developing tissue's biochemical microenvironment. Herein, an aptamer-based programmable GF delivery platform is described that harnesses dynamic affinity interactions for facilitating spatiotemporal control over vascular endothelial GF (VEGF165) bioavailability within gelatin methacryloyl matrices. The platform showcases localized VEGF165 sequestration from the culture medium (offering spatial-control) and leverages aptamer-complementary sequence (CS) hybridization for triggering VEGF165 release (offering temporal-control), without non-specific leakage. Furthermore, extensive 3D co-culture studies (human umbilical vein-derived endothelial cells & mesenchymal stromal cells), in bi-phasic hydrogel systems revealed its fundamentally novel capability to selectively guide cell responses and manipulate lumen-like microvascular networks via spatiotemporally controlling VEGF165 bioavailability within 3D microenvironment. This platform utilizes CS as an external biochemical trigger for guiding vascular morphogenesis which is suitable for creating dynamically controlled engineered tissues

Grazing incidence x-ray diffraction studies of lipid-peptide mixed monolayers during shear flow

Grazing Incidence X-ray Diffraction (GIXD) studies of monolayers of biomolecules at the air-water interface give quantitative information of in-plane packing, coherence lengths of the ordered (diffracting) domains and orientation of hydrocarbon chains of the model mem branes. Rheo-GIXD measurements revel quantitative changes in the monolayer under shear. Here we report GIXD studies of monolayers of Alamethicin peptide, DPPC lipid and their mixtures at the air-water interface under the application of steady shear stresses. The Alame thicin monolayer and the mixed monolayer show flow jamming transition. On the other hand, pure DPPC monolayer under the constant stress flows steadily with a notable enhancement of 1 arXiv:1908.00228v1 [cond-mat.soft] 1 Aug 2019 area/molecule, coherence length, and the tilt angle with increasing stress, suggesting fusion of domains during flow. The DPPC-Alamethicin mixed monolayer shows no significant change in the area/DPPC molecule or in the DPPC chain tilt angle but the coherence length of both phases (DPPC and Alamethicin) increases suggesting that the domains of individual phases aremergingtobiggersizepromotingmoreseparationofphasesinthesystemduringflow. Our results show that Rheo-GIXD has the potential to explore in-situ molecular structural changes under rheological conditions for a diverse range of confined biomolecules at the interfaces