Andrew S. Khalil

Serum-dependence of affinity-mediated VEGF release from biomimetic microspheres.

Vascular endothelial growth factor (VEGF) activity is highly regulated via sequestering within the ECM and cell-demanded proteolysis to release the sequestered VEGF. Numerous studies have demonstrated that VEGF activity mediates cellular events leading to angiogenesis and capillary formation in vivo. This has motivated the study of biomaterials to sustain VEGF release, and in many cases, the materials are inspired by the structure and function of the native ECM. However, there remains a need for materials that can bind to VEGF with high specificity, as the in vivo environment is rich in a variety of growth factors (GFs) and GF-binding moieties. Here we describe a strategy to control VEGF release using hydrogel microspheres with tethered peptides derived from VEGF receptor 2 (VEGFR2). Using biomaterials covalently modified with varying concentrations of two distinct VEGFR2-derived peptides with varying serum stability, we analyzed both biomaterial and environmental variables that influence VEGF release and activity. The presence of tethered VEGF-binding peptides (VBPs) resulted in significantly extended VEGF release relative to control conditions, and the resulting released VEGF significantly increased the expansion of human umbilical vein endothelial cells in culture. VEGF release rates were also strongly influenced by the concentration of serum. The presence of Feline McDonough Sarcoma-like tyrosine kinase 1 (sFlt-1), a serum-borne receptor fragment derived from VEGF receptor 1, increased VEGF release rates, although sFlt-1 was not sufficient to recapitulate the release profile of VEGF in serum. Further, the influence of serum on VEGF release was not due to protease activity or nonspecific VEGF interactions in the presence of serum-borne heparin. VEGF release kinetics correlated well with a generalizable mathematical model describing affinity-mediated release of VEGF from hydrogel microspheres in defined conditions. Modeling results suggest a potential mechanism whereby competition between VEGF and multiple VEGF-binding serum proteins including sFlt-1, soluble kinase insert domain receptor (sKDR), and α2-macroglobulin (α2-M) likely influenced VEGF release from microspheres. The materials and mathematical model described in this approach may be useful in a range of applications in which sustained, biologically active GF release of a specific GF is desirable.

Nanostructured Mineral Coatings Stabilize Proteins for Therapeutic Delivery

Proteins tend to lose their biological activity due to their fragile structural conformation during formulation, storage, and delivery. Thus, the inability to stabilize proteins in controlled-release systems represents a major obstacle in drug delivery. Here, a bone mineral inspired protein stabilization strategy is presented, which uses nanostructured mineral coatings on medical devices. Proteins bound within the nanostructured coatings demonstrate enhanced stability against extreme external stressors, including organic solvents, proteases, and ethylene oxide gas sterilization. The protein stabilization effect is attributed to the maintenance of protein conformational structure, which is closely related to the nanoscale feature sizes of the mineral coatings. Basic fibroblast growth factor (bFGF) released from a nanostructured mineral coating maintains its biological activity for weeks during release, while it maintains activity for less than 7 d during release from commonly used polymeric microspheres. Delivery of the growth factors bFGF and vascular endothelial growth factor using a mineral coated surgical suture significantly improves functional Achilles tendon healing in a rabbit model, resulting in increased vascularization, more mature collagen fiber organization, and a two fold improvement in mechanical properties. The findings of this study demonstrate that biomimetic interactions between proteins and nanostructured minerals provide a new, broadly applicable mechanism to stabilize proteins in the context of drug delivery and regenerative medicine.

BMP-2-coated mineral coated microparticles improve bone repair in atrophic non-unions.

Atrophic non-unions are a major clinical problem. Mineral coated microparticles (MCM) are electrolyte-coated hydroxyapatite particles that have been shown in vitro to bind growth factors electrostatically and enable a tuneable sustained release. Herein, we studied whether MCM can be used in vivo to apply Bone Morphogenetic Protein-2 (BMP-2) to improve bone repair of atrophic non-unions. For this purpose, atrophic non-unions were induced in femurs of CD-1 mice (n = 48). Animals either received BMP-2-coated MCM (MCM + BMP; n = 16), uncoated MCM (MCM; n = 16) or no MCM (NONE; n = 16). Bone healing was evaluated 2 and 10 weeks postoperatively by micro-computed tomographic (µCT), biomechanical, histomorphometric and immunohistochemical analyses. µCT revealed more bone volume with more highly mineralised bone in MCM + BMP femurs. Femurs of MCM + BMP animals showed a significantly higher bending stiffness compared to other groups. Histomorphometry further demonstrated that the callus of MCM + BMP femurs was larger and contained more bone and less fibrous tissue. After 10 weeks, 7 of 8 MCM + BMP femurs presented with complete osseous bridging, whereas NONE femurs exhibited a non-union rate of 100 %. Of interest, immunohistochemistry could not detect macrophages within the callus, indicating a good biocompatibility of MCM. In conclusion, the local application of BMP-2-coated MCM improved bone healing in a challenging murine non-union model and, thus, should be of clinical interest in the treatment of non-unions.

Functionalization of microparticles with mineral coatings enhances non-viral transfection of primary human cells

Gene delivery to primary human cells is a technology of critical interest to both life science research and therapeutic applications. However, poor efficiencies in gene transfer and undesirable safety profiles remain key limitations in advancing this technology. Here, we describe a materials-based approach whereby application of a bioresorbable mineral coating improves microparticle-based transfection of plasmid DNA lipoplexes in several primary human cell types. In the presence of these mineral-coated microparticles (MCMs), we observed up to 4-fold increases in transfection efficiency with simultaneous reductions in cytotoxicity. We identified mechanisms by which MCMs improve transfection, as well as coating compositions that improve transfection in three-dimensional cell constructs. The approach afforded efficient transfection in primary human fibroblasts as well as mesenchymal and embryonic stem cells for both two- and three-dimensional transfection strategies. This MCM-based transfection is an advancement in gene delivery technology, as it represents a non-viral approach that enables highly efficient, localized transfection and allows for transfection of three-dimensional cell constructs.

Controlled Self-assembly of Stem Cell Aggregates Instructs Pluripotency and Lineage Bias

Stem cell-derived organoids and other 3D microtissues offer enormous potential as models for drug screening, disease modeling, and regenerative medicine. Formation of stem/progenitor cell aggregates is common in biomanufacturing processes and critical to many organoid approaches. However, reproducibility of current protocols is limited by reliance on poorly controlled processes (e.g., spontaneous aggregation). Little is known about the effects of aggregation parameters on cell behavior, which may have implications for the production of cell aggregates and organoids. Here we introduce a bioengineered platform of labile substrate arrays that enable simple, scalable generation of cell aggregates via a controllable 2D-to-3D “self-assembly”. As a proof-of-concept, we show that labile substrates generate size- and shape-controlled embryoid bodies (EBs) and can be easily modified to control EB self-assembly kinetics. We show that aggregation method instructs EB lineage bias, with faster aggregation promoting pluripotency loss and ectoderm, and slower aggregation favoring mesoderm and endoderm. We also find that aggregation kinetics of EBs markedly influence EB structure, with slower kinetics resulting in increased EB porosity and growth factor signaling. Our findings suggest that controlling internal structure of cell aggregates by modifying aggregation kinetics is a potential strategy for improving 3D microtissue models for research and translational applications.

335. High Content Analysis Platform for Optimization of Lipid Mediated CRISPR-Cas9 Delivery Strategies in Human Cells

Background: Non-viral gene-editing of human cells using the CRISPR-Cas9 system requires optimized delivery of multiple components. Both the Cas9 endonuclease and a single guide RNA, that defines the genomic target, need to be present and co-localized within the nucleus for efficient gene-editing to occur. This work describes a new high-throughput screening platform for the optimization of CRISPR-Cas9 delivery strategies. Methods: By exploiting high content image analysis and microcontact printed plates, multi-parametric gene-editing outcome data from hundreds to thousands of isolated cell populations can be screened simultaneously. Employing this platform, we systematically screened four commercially available cationic lipid transfection materials with a range of RNAs encoding the CRISPR-Cas9 system. Analysis of Cas9 expression and editing of an endogenous and fluorescent mCherry reporter transgene within human embryonic cells was monitored over several days after transfection. Design of experiments analysis enabled rigorous evaluation of delivery materials and RNA concentration conditions. Results: The results of this analysis indicated that the concentration and identity of transfection material have significantly greater effect on gene-editing than ratio or total amount of RNA. Cell subpopulation analysis on microcontact printed plates, further revealed that low cell number and high Cas9 expression 24 hours after CRISPR-Cas9 delivery, were strong predictors of gene-editing outcomes. After purifying the population for high Cas9 expression, 24 hours after CRISPR-Cas9 delivery, greater than 92% of cells were properly edited. Significance: These results suggest design principles for the development of materials and transfection strategies with lipid-based materials. This platform could be applied to rapidly optimize materials for gene-editing in a variety of cell/tissue types in order to advance genomic medicine, regenerative biology and drug discovery.View Large Image | Download PowerPoint Slide