Cross-Linkable Liposomes Stabilize a Magnetic Resonance Contrast-Enhancing Polymeric Fastener

Liposomes are commonly used to deliver drugs and contrast agents to their target site in a controlled manner. One of the greatest obstacles in the performance of such delivery vehicles is their stability in the presence of serum. Here, we demonstrate a method to stabilize a class of liposomes that load gadolinium, a magnetic resonance (MR) contrast agent, as a model cargo on their surfaces. We hypothesized that the sequential adsorption of a gadolinium-binding chitosan fastener on the liposome surface followed by covalent cross-linking of the lipid bilayer would provide enhanced stability and improved MR signal in the presence of human serum. To investigate this hypothesis, liposomes composed of diyne-containing lipids were assembled and functionalized via chitosan conjugated with a hydrophobic anchor and diethylenetriaminepentaacetic acid (DTPA). This postadsorption cross-linking strategy served to stabilize the thermodynamically favorable association between liposome and polymeric fastener. Furthermore, the chitosan-coated, cross-linked liposomes proved more effective as delivery vehicles of gadolinium than uncross-linked liposomes due to the reduced liposome degradation and chitosan desorption. Overall, this study demonstrates a useful method to stabilize a broad class of particles used for systemic delivery of various molecular payloads

Matrix Rigidity-Modulated Cardiovascular Organoid Formation from Embryoid Bodies

<div><p>Stem cell clusters, such as embryoid bodies (EBs) derived from embryonic stem cells, are extensively studied for creation of multicellular clusters and complex functional tissues. It is common to control phenotypes of ES cells with varying molecular compounds; however, there is still a need to improve the controllability of cell differentiation, and thus, the quality of created tissue. This study demonstrates a simple but effective strategy to promote formation of vascularized cardiac muscle - like tissue in EBs and form contracting cardiovascular organoids by modulating the stiffness of a cell adherent hydrogel. Using collagen-conjugated polyacrylamide hydrogels with controlled elastic moduli, we discovered that cellular organization in a form of vascularized cardiac muscle sheet was maximal on the gel with the stiffness similar to cardiac muscle. We envisage that the results of this study will greatly contribute to better understanding of emergent behavior of stem cells in developmental and regeneration process and will also expedite translation of EB studies to drug-screening device assembly and clinical treatments.</p></div

Immunohistochemical analysis of cardiomyogenic and endothelial differentiation within EBs.

<p>(A) Fluorescent images of EBs stained for sarcomeric α-actinin (red) and CD31 (green). (A-1, 5, & 9) EBs cultured in suspended state for 23 days. (A-2, 6 & 10) EBs cultured on the pure collagen gel with <i>E</i> of 0.2 kPa. (A-3, 7, & 11) EBs cultured on the CCP gel with <i>E</i> of 6 kPa. (A-4 & 8) EBs cultured on the CCP gel with <i>E</i> of 40 kPa. Scale bar represents 200 µm. Images on the second row are magnified views of those on the first row. Images on the third row are three-dimensional confocal images of EBs. (B) Percentage of the EB area positively stained by antibodies to sarcomeric α-actinin. The difference of the values between EBs cultured on the gel with <i>E</i> of 6 kPa and other three conditions is statistically significant (*p<0.05). (C) Percentage of EB area positively stained with an antibody to CD31. The difference of the values between EBs cultured on the gel with <i>E</i> of 6 and 40 kPa is statistically not significant (*p>0.1). Values and error bars represent the mean and the standard error of at least 20 EBs, respectively.</p

Cell cycle analysis of cardiomyoblasts using EdU incorporation.

<p>(A) Fluorescent images of sarcomeric α-actinin positive cells (red) incorporating EdU (green). Blue color represents cell nuclei stained by Hoechst 33342. Images represent EBs cultured in suspended state (A-1 and A-5) and on hydrogels with <i>E</i> of 0.2 (A-2 and A-5), 6 (A-3 and A-5), and 40 kPa (A-4 and A-5). Scale bar represents 20 µm. Images on the first row were taken on Day 15, and those on the second row were taken on Day 23. (B) Quantified percentage of cardiomyoblasts incorporating EdU on Day 15 (B-1) and 23 (B-2). In (B-1), the difference of values between EBs cultured on the gel with <i>E</i> of 6 kPa and 40 kPa was statistically significant (*p<0.05). In (B-2), the difference of values between EBs cultured on the gel with <i>E</i> of 6 kPa and EBs on the gels with <i>E</i> of 0.2 and 40 kPa was statistically significant (**p<0.05). Symbols ‡, ◊, †, •, *, ** and brackets indicate statistically significant groups (p<0.05). (C) The degree of decrease in the percentage of cardiomyoblasts incorporating EdU between Day 15 and Day 23.</p

Additional file 1: of Proangiogenic alginate-g-pyrrole hydrogel with decoupled control of mechanical rigidity and electrically conductivity

Supporting Information. (DOCX 618 kb

Histological analysis of EBs cultured in suspended state or on hydrogels with controlled elastic moduli.

<p>Cross-sections of EBs were stained with Hematoxylin & Eosin. (A) (A-1 and A-6) EBs formed by culturing ES cells in suspended state for 8 days. (A-2 and A-7) EBs cultured in suspended state for additional 15 days. (A-3 and A-8) EBs cultured on the pure collagen gel with <i>E</i> of 0.2 kPa, (A-4 and A-9) EBs cultured on the CCP gel with <i>E</i> of 6 kPa, and (A-5 and A-10) EBs cultured on the CCP gel with <i>E</i> of 40 kPa. Images on the first and second rows represent EBs cultured in medium supplemented with 10% FBS and that without FBS, respectively. Thin arrows in A-2, 3, and 4 indicate cystic EBs. The thick arrow in A-4 indicates columnar epithelium, and the arrowhead in A-8 indicates a neuroectodermal rosette. Scale bar represents 250 µm. (B) Quantified necrotic area percentage of EBs cultured in suspension and on hydrogels with <i>E</i> of 0.2, 6, and 40 kPa. Black bars represent average diameter of EBs cultured in media supplemented with 10% FBS and grey bars do those cultured without FBS. The difference of values for EBs cultured in the medium supplemented with 10% FBS (black bar) and free of FBS (grey bar) is statistically significant for all four different conditions (*p<0.05). Values and error bars represent the mean and the standard deviation of at least 10 EBs, respectively.</p

Composition and properties of hydrogels.

<p>M: Molar ratio of N,N′-methylenebis(acrylamide) to acrylamide; E: Elastic modulus, Q: Degree of swelling.</p

Analysis of stiffness-modulated contraction in EBs.

<p>(A) Percentage of contracting EBs. The difference of the values between EBs cultured on the gel with <i>E</i> of 6 kPa and other three conditions is statistically significant (*p<0.05). Values and error bars represent the mean and the standard error of at least 100 EBs, respectively. (B) Frequency of EB contractions. Values and error bars represent the mean and the standard deviation of at least 5 EBs, respectively. (C) Effects of <i>E</i> of the hydrogel on the sarcomeric α-actinin (Actn2) mRNA expression. (D) Effects of <i>E</i> of the hydrogel on cardiac troponin T type 2 (Tnnt2) mRNA expression. Values and error bars represent the mean and the standard error. In (C) and (D), * indicate statistical significance of difference between conditions (*p<0.05).</p

Generation of Cell-Instructive Collagen Gels through Thermodynamic Control

Recent studies have demonstrated the usefulness of three-dimensional hydrogel scaffolds for cell instruction. However, the control of gel architectures in cell-friendly conditions remains a challenge. Here, we report a novel method to generate unique three-dimensional collagen gel structures for the modulation of cell phenotypes. This was achieved by directing collagen self-assembly with unreactive hydrophilic polyethylene glycol (PEG) chains. Our approach allowed the fiber sizes and mechanics of three-dimensional collagen gels to be readily controlled. It also enabled the recapitulation of distinctive structures such as large perimysial collagen cables. Through different experiments, we elucidated the underlying mechanism for this PEG-mediated thermodynamic regulation of gel structure. We further used these cell-instructive three-dimensional gels to bring about pronounced morphological changes in encapsulated fibroblasts and their activation to form contractile bundles. Overall, our platform fills a gap in the existing array of collagen scaffolds and can potentially be adapted to a variety of self-assembling systems

Size analysis of EBs cultured in suspended state or on hydrogels with controlled elastic moduli.

<p>(A) Bright field images of EBs. (A-1 and A-6) EBs formed by culturing ES cells in suspended state for 8 days. (A-2 and A-7) EBs cultured in suspended state for additional 15 days. (A-3 and A-8) EBs cultured on the pure collagen gel with <i>E</i> of 0.2 kPa, (A-4 and A-9) EBs cultured on the CCP gel with <i>E</i> of 6 kPa, and (A-5 and A-10) EBs cultured on the CCP gel with <i>E</i> of 40 kPa. Images in the first and second rows represent EBs cultured in medium supplemented with 10% FBS and that without FBS, respectively. Arrows in A-1 to A-3 indicate cystic EBs. The scale bar represents 1 mm. (B) The quantified analysis of average diameters of EBs cultured in suspended state or on collagen-based hydrogels of controlled <i>E</i>. Black bars represent average diameter of EBs cultured in the medium supplemented with 10% FBS, and grey bars represent EBs cultured without FBS. Values and error bars represent the mean and the standard error of at least 50 EBs, respectively. No statistical significance was found between conditions.</p