Melissa D. Krebs

Controlled delivery of antibodies from injectable hydrogels.

Therapeutic antibodies are currently used for the treatment of various diseases, but large doses delivered systemically are typically required. Localized controlled delivery techniques would afford major benefits such as decreasing side effects and required doses. Injectable biopolymer systems are an attractive solution due to their minimally invasive potential for controlled release in a localized area. Here, alginate-chitosan hydrogels are demonstrated to provide controlled delivery of IgG model antibodies and also of Fab antibody fragments. Also, an alternate delivery system comprised of poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with antibodies and encapsulated in alginate was shown to successfully provide another level of control over release. These biopolymer systems that offer controlled delivery for antibodies and antibody fragments will be promising for many applications in drug delivery and regenerative medicine.

Collagen and collagen-chondroitin sulfate scaffolds with uniaxially aligned pores for the biomimetic, three dimensional culture of trabecular meshwork cells.

Glaucoma is a disease in which damage to the optic nerve leads to progressive, irreversible vision loss. The intraocular pressure (IOP) is the only modifiable risk factor for glaucoma and its lowering is considered a useful strategy for preventing or slowing down the progression of glaucomatous neuropathy. Elevated intraocular pressure associated with glaucoma is due to increased aqueous humor outflow resistance, primarily through the trabecular meshwork (TM) of the eye. Current in vitro models of the trabecular meshwork are oversimplified and do not capture the organized and complex three‐dimensional nature of this tissue that consists primarily of collagen and glycoasaminoglycans. In this work, collagen and collagen‐chondroitin sulfate (CS) scaffolds were fabricated via unidirectional freezing and lyophilization to induce the formation of aligned pores. Scaffolds were characterized by scanning electron microscopy, dynamic mechanical analysis, and a chondroitin sulfate quantification assay. Scaffold characterization confirmed the formation of aligned pores, and also that the CS was leaching out of the scaffolds over time. Primary porcine trabecular meshwork (TM) cells were seeded onto the surface of scaffolds and their gene expression, proliferation, viability, migration into the scaffolds, and morphology were examined. The TM cells were viable and proliferated 2 weeks after seeding. The cells migrated down into the internal scaffold structure and their morphology reflected the topography and alignment of the scaffold structure. This work is a promising step toward the development of a three dimensional in vitro model of the TM that can be used for testing of glaucoma pharmacological agents in future experimentation and to better our understanding of the trabecular meshwork and its complex physiology. Biotechnol. Bioeng. 2017;114: 915–923.

Injectable and microporous scaffold of densely-packed, growth factor-encapsulating chitosan microgels.

In this work, an emulsion crosslinking method was developed to produce chitosan-genipin microgels which acted as an injectable and microporous scaffold. Chitosan was characterized with respect to pH by light scattering and aqueous titration. Microgels were characterized with swelling, light scattering, and rheometry of densely-packed microgel solutions. The results suggest that as chitosan becomes increasingly deprotonated above the pKa, repulsive forces diminish and intermolecular attractions cause pH-responsive chain aggregation; leading to microgel-microgel aggregation as well. The microgels with the most chitosan and least cross-linker showed the highest yield stress and a storage modulus of 16kPa when condensed as a microgel paste at pH 7.4. Two oppositely-charged growth factors could be encapsulated into the microgels and endothelial cells were able to proliferate into the 3D microgel scaffold. This work motivates further research on the applications of the chitosan microgel scaffold as an injectable and microporous scaffold in regenerative medicine.

Cell-interactive alginate-chitosan biopolymer systems with tunable mechanics and antibody release rates

This work investigates techniques to produce biocompatible hydrogels with tunable stiffness without the addition of crosslinking agents or altering cell binding sites. Alginate and water-soluble chitosan salts were used to form polyelectrolyte complexes (PECs), where the storage and loss moduli could be increased by raising gelation temperatures. The largest change, a 6.5-fold increase in storage modulus, occurred when the crosslinking temperature was increased from 37 to 50°C while using chitosan with chlorine counterions. Osteogenic MC3T3 cells were shown to have significantly higher proliferation on the stiffer PECs prepared at 50°C compared to 37°C. Gelation temperature showed minimal effect on antibody release, but the inclusion of CaSO4 provided a longer overall release where the rate was nearly linear for several weeks. However, CaSO4 inhibited the strengthening effect of increased gelation temperature. PECs containing glutamate counterions demonstrated an increase in stiffness with decreased chitosan content.

Zwitterionic cryogels for sustained release of proteins

This work reports the preparation of zwitterionic cryogels for the sustained release of proteins for up to 4 months. In addition, high protein loading efficiency of up to 80% was achieved. Control studies with different hydrogels (such as non-ionic cryogels and non-ionic and zwitterionic gels prepared at room temperature) were performed to understand the mechanism behind the excellent protein loading and release properties of the zwitterionic cryogels.

Sustained delivery of anti-VEGF from injectable hydrogel systems provides a prolonged decrease of endothelial cell proliferation and angiogenesis in vitro

Therapeutic antibodies are attractive treatment options for numerous diseases based on their ability to target and bind to specific proteins or antigens. Bevacizumab, an antiangiogenic antibody, has shown promise for multiple diseases, including various cancers and macular degeneration, where excessive VEGF secretion induces aberrant angiogenesis. In many cases local, sustained delivery of a therapeutic antibody would be preferable to maximize the therapeutic at the disease site, eliminate the need for repeated doses, and reduce systemic side effects. The biodegradable polysaccharides alginate and chitosan can electrostatically interact to form a polyelectrolyte complex (PEC), and have proved effective as a carrier for controlled release of antibodies. In this work, an alginate–chitosan PEC system was designed to produce targeted 30-day delivery of non-specific IgG and anti-VEGF antibodies. The release of anti-VEGF was slow relative to IgG release, suggesting that release rate is antibody specific and is based on the interactions of the PEC with charges present on the antibody surface. The anti-VEGF released from the PEC was shown to successfully inhibit VEGF-induced proliferation and angiogenesis in vitro throughout the 30-day test period.

Quantification of cellular distribution as Poisson process in 3D matrix using a multiview light-sheet microscope

The absence of quantitative in vitro cell-extracellular matrix models represents an important bottleneck for basic research and human health. Randomness of cellular distributions provides an opportunity for the development of a quantitative in vitro model. However, quantification of the randomness of random cell distributions is still lacking. In this paper, we have imaged cellular distributions in an alginate matrix using a multiview light-sheet microscope and developed quantification metrics of randomness by modeling it as a Poisson process, a process that has constant probability of occurring in space or time. Our light-sheet microscope can image more than 5 mm thick optically clear samples with 2.9 ±0.4 μm depth-resolution. We applied our method to image fluorescently labeled human mesenchymal stem cells (hMSCs) embedded in an alginate matrix. Simulated randomness agrees well with the experiments. Quantification of distributions and validation by simulations will enable quantitative study of cell-matrix interactions in tissue models.