Shawn C. Owen

Design of three-dimensional biomimetic scaffolds.

A detailed understanding of the biophysical features that affect cell growth and development is important in guiding the design of biomimetic scaffolds. The cellular microenvironment is a network of structural and functional components that provide mechanical and chemical stimuli, which influence cell function and fate. Important developmental signals are conveyed to cells through interactions with neighboring cells, the extracellular matrix (ECM), and growth factors. Currently, there are number of approaches to create 3D tissue models in vitro that allow for control over cell adhesion, the physical properties of the surrogate matrix, and the spatial distribution of growth factors. This review describes some of the most significant biological features of the ECM, and several engineering methods currently being implemented to design and tune synthetic scaffolds to mimic these features.

Colloidal aggregation affects the efficacy of anticancer drugs in cell culture

Many small molecules, including bioactive molecules and approved drugs, spontaneously form colloidal aggregates in aqueous solution at micromolar concentrations. Though it is widely accepted that aggregation leads to artifacts in screens for ligands of soluble proteins, the effects of colloid formation in cell-based assays have not been studied. Here, seven anticancer drugs and one diagnostic reagent were found to form colloids in both biochemical buffer and in cell culture media. In cell-based assays, the antiproliferative activities of three of the drugs were substantially reduced when in colloidal form as compared to monomeric form; a new formulation method ensured the presence of drug colloids versus drug monomers in solution. We also found that Evans Blue, a dye classically used to measure vascular permeability and to demonstrate the “enhanced permeability and retention (EPR) effect” in solid tumors, forms colloids that adsorb albumin, as opposed to older literature that suggested the reverse.

Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry

Antibody drug conjugates (ADCs) have emerged as an important pharmaceutical class of drugs designed to harness the specificity of antibodies with the potency of small molecule therapeutics. The three main components of ADCs are the antibody, the linker, and the payload; the majority of early work focused intensely on improving the functionality of these pieces. Recently, considerable attention has been focused on developing methods to control the site and number of linker/drug conjugated to the antibody, with the aim of producing more homogenous ADCs. In this article, we review popular conjugation methods and highlight recent approaches including “click” conjugation and enzymatic ligation. We discuss current linker technology, contrasting the characteristics of cleavable and non-cleavable linkers, and summarize the essential properties of ADC payload, centering on chemotherapeutics. In addition, we report on the progress in characterizing to determine physicochemical properties and on advances in purifying to obtain homogenous products. Establishing a set of selection and analytical criteria will facilitate the translation of novel ADCs and ensure the production of effective biosimilars.

Hyaluronic acid click hydrogels emulate the extracellular matrix.

Hydrogels are used to create 3D microenvironments with properties that direct cell function. The current study demonstrates the versatility of hyaluronic acid (HA)-based hydrogels with independent control over hydrogel properties such as mechanics, architecture, and the spatial distribution of biological factors. Hydrogels were prepared by reacting furan-modified HA with bis-maleimide-poly(ethylene glycol) in a Diels-Alder click reaction. Biomolecules were photopatterned into the hydrogel by two-photon laser processing, resulting in spatially defined growth factor gradients. The Youngs modulus was controlled by either changing the hydrogel concentration or the furan substitution on the HA backbone, thereby decoupling the hydrogel concentration from mechanical properties. Porosity was controlled by cryogelation, and the pore size distribution, by the thaw temperature. The addition of galactose further influenced the porosity, pore size, and Youngs modulus of the cryogels. These HA-based hydrogels offer a tunable platform with a diversity of properties for directing cell function, with applications in tissue engineering and regenerative medicine.

Colloidal drug formulations can explain "bell-shaped" concentration-response curves

Drug efficacy does not always increase sigmoidally with concentration, which has puzzled the community for decades. Unlike standard sigmoidal curves, bell-shaped concentration–response curves suggest more complex biological effects, such as multiple-binding sites or multiple targets. Here, we investigate a physical property-based mechanism for bell-shaped curves. Beginning with the observation that some drugs form colloidal aggregates at relevant concentrations, we determined concentration–response curves for three aggregating anticancer drugs, formulated both as colloids and as free monomer. Colloidal formulations exhibited bell-shaped curves, losing activity at higher concentrations, while monomeric formulations gave typical sigmoidal curves, sustaining a plateau of maximum activity. Inverting the question, we next asked if molecules with bell-shaped curves, reported in the literature, form colloidal aggregates at relevant concentrations. We selected 12 molecules reported to have bell-shaped concentration–response curves and found that five of these formed colloids. To understand the mechanism behind the loss of activity at concentrations where colloids are present, we investigated the diffusion of colloid-forming dye Evans blue into cells. We found that colloidal species are excluded from cells, which may explain the mechanism behind toxicological screens that use Evans blue, Trypan blue, and related dyes.

Targeting HER2+ breast cancer cells: Lysosomal accumulation of anti-HER2 antibodies is influenced by antibody binding site and conjugation to polymeric nanoparticles

Humanized monoclonal antibodies (mAb) against HER2 are being engineered to treat cancer. We utilized phage-display technology to generate a novel anti-HER2 mAb (named 73JIgG) that binds an epitope of HER2 distinct from that of trastuzumab. Although these mAbs bind to the same cell surface receptor, they have different cell distribution profiles. After 3h of incubation, almost 10% of the total 73JIgG reaches the lysosome compared to less than 3% of trastuzumab. Interestingly, 73JIgG disassociates from HER2 whereas trastuzumab remains bound to the receptor. Importantly, HER2 distribution is not affected by the antibody binding epitope, thus negating this mechanism as the reason for the difference in intracellular trafficking of 73JIgG versus trastuzumab. Each of trastuzumab and 73JIgG was chemically-modified with either a small molecule or polymeric nanoparticle to better understand the influence of conjugation on cellular localization. Relative to antibody alone, antibody-nanoparticle conjugates resulted in a higher concentration of antibodies in the lysosome whereas antibody-small molecule conjugates did not affect cell trafficking to the lysosome. Given the importance of lysosomal targeting, these results demonstrate the importance of understanding the influence of the antibody-conjugate on cell trafficking for ultimate optimization of treatment selection.

Double Click: Dual Functionalized Polymeric Micelles with Antibodies and Peptides

Polymeric nanoparticle micelles provide a possible platform for theranostic delivery, combining the role of therapeutics and diagnostics in one vehicle. To explore dual-functional micelles, the amphiphilic copolymer of poly(d,l-lactide-co-2-methyl-2-carboxytrimethylene carbonate)-graft-poly(ethylene glycol)-X (P(LA-co-TMCC)-g-PEG-X) was self-assembled to form micelles, with X representing either azide or furan. Micelles of P(LA-co-TMCC)-g-PEG-azide and P(LA-co-TMCC)-g-PEG-furan terminal functional groups were used to conjugate dibenzylcyclooctyne and maleimide-modified probes, respectively, taking advantage of orthogonal coupling chemistry. To verify the utility of the dual-functional micelles, trastuzumab-maleimide antibodies and FLAG-dibenzylcyclooctyne peptides were covalently bound by sequential click chemistry reactions. SKOV-3luc cells that were treated with the dual-functionalized micelles showed colocalization of the antibodies and peptides by confocal imaging, demonstrating the promise of this approach.

Blood vessel hyperpermeability and pathophysiology in human tumour xenograft models of breast cancer: a comparison of ectopic and orthotopic tumours

BackgroundHuman tumour xenografts in immune compromised mice are widely used as cancer models because they are easy to reproduce and simple to use in a variety of pre-clinical assessments. Developments in nanomedicine have led to the use of tumour xenografts in testing nanoscale delivery devices, such as nanoparticles and polymer-drug conjugates, for targeting and efficacy via the enhanced permeability and retention (EPR) effect. For these results to be meaningful, the hyperpermeable vasculature and reduced lymphatic drainage associated with tumour pathophysiology must be replicated in the model. In pre-clinical breast cancer xenograft models, cells are commonly introduced via injection either orthotopically (mammary fat pad, MFP) or ectopically (subcutaneous, SC), and the organ environment experienced by the tumour cells has been shown to influence their behaviour.MethodsTo evaluate xenograft models of breast cancer in the context of EPR, both orthotopic MFP and ectopic SC injections of MDA-MB-231-H2N cells were given to NOD scid gamma (NSG) mice. Animals with matched tumours in two size categories were tested by injection of a high molecular weight dextran as a model nanocarrier. Tumours were collected and sectioned to assess dextran accumulation compared to liver tissue as a positive control. To understand the cellular basis of these observations, tumour sections were also immunostained for endothelial cells, basement membranes, pericytes, and lymphatic vessels.ResultsSC tumours required longer development times to become size matched to MFP tumours, and also presented wide size variability and ulcerated skin lesions 6 weeks after cell injection. The 3 week MFP tumour model demonstrated greater dextran accumulation than the size matched 5 week SC tumour model (for P < 0.10). Immunostaining revealed greater vascular density and thinner basement membranes in the MFP tumour model 3 weeks after cell injection. Both the MFP and SC tumours showed evidence of insufficient lymphatic drainage, as many fluid-filled and collagen IV-lined spaces were observed, which likely contain excess interstitial fluid.ConclusionsDextran accumulation and immunostaining results suggest that small MFP tumours best replicate the vascular permeability required to observe the EPR effect in vivo. A more predictable growth profile and the absence of ulcerated skin lesions further point to the MFP model as a strong choice for long term treatment studies that initiate after a target tumour size has been reached.

In vivo evaluation of the delivery and efficacy of a sirolimus-laden polymer gel for inhibition of hyperplasia in a porcine model of arteriovenous hemodialysis graft stenosis

Synthetic arteriovenous (AV) hemodialysis grafts are plagued by hyperplasia resulting in occlusion and graft failure yet there are no clinically available preventative treatments. Here the delivery and degradation of a sirolimus-laden polymer gel were monitored in vivo by magnetic resonance imaging (MRI) and its efficacy for inhibiting hyperplasia was evaluated in a porcine model of AV graft stenosis. Synthetic grafts were placed between the carotid artery and ipsilateral jugular vein of swine. A biodegradable polymer gel loaded with sirolimus (2.5mg/mL) was immediately applied perivascularly to the venous anastomosis, and reapplied by ultrasound-guided injections at one, two and three weeks. Control grafts received neither sirolimus nor polymer. The lumen cross-sectional area at the graft-vein anastomosis was assessed in vivo by non-invasive MRI. The explanted tissues also underwent histological analysis. A specifically developed MRI pulse sequence provided a high contrast-to-noise ratio (CNR) between the polymer and surrounding tissue that allowed confirmation of gel location after injection. Polymer signal decreased up to 80% at three to four weeks after injection, slightly faster than its degradation kinetics in vitro. The MR image of the polymer was confirmed by visual assessment at necropsy. On histological assessment, the mean hyperplasia surface area of the treated graft was 52% lower than that of the control grafts (0.43mm(2) vs. 0.89mm(2); p<0.003), while the minimum cross-sectional lumen area, as measured on MRI, was doubled (5.3mm(2) vs 2.5mm(2); p<0.05). In conclusion, customized MRI allowed non-invasive monitoring of the location and degradation of drug delivery polymer gels in vivo. Perivascular application of sirolimus-laden polymer yielded a significant decrease in hyperplasia development and an increase in lumen area at the venous anastomosis of AV grafts.

Correlation of tissue drug concentrations with in vivo magnetic resonance images of polymer drug depot around arteriovenous graft

Sustained delivery of anti-proliferative drugs to the perivascular area using an injectable polymeric platform is a strategy to inhibit vascular hyperplasia and stenosis. In this study, the concentrations of sirolimus in vascular tissues were evaluated after delivery using an injectable platform made of poly(lactic-co-glycolic acid)-polyethylene glycol-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA). In order to optimize the drug release profile, the effect of two solvents or solid loading of the sirolimus into the polymer gel was first examined in vitro. The early release was slower with loading of dry drug into the polymer, compared to drug dissolution in solvents. Dry sirolimus was therefore used to load the polymer and applied to the perivascular surface of the graft-venous anastomosis at the time of surgical placement of a carotid-jugular synthetic hemodialysis graft in a porcine model. This was replenished by ultrasound-guided injection of additional drug-laden polymer at one, two and three weeks post-operatively. Magnetic resonance imaging (MRI) using pulse sequences specifically designed for optimal detection of the polymeric gel showed that the polymer injected post-operatively remained at the juxta-anastomotic perivascular site at two weeks. Sirolimus was extracted from various segments of the juxta-anastomotic tissues and the drug concentrations were determined using HPLC MS/MS. Tissue sirolimus concentrations at one and two weeks were highest near the venous anastomosis, which were approximately 100- to 500-fold greater than the concentrations necessary to inhibit vascular smooth muscle cell proliferation in vitro. Drug concentrations remained above the inhibitory concentrations for at least six weeks post-operatively. Thus, serial injections of sustained-delivery polymer gel loaded with sirolimus can provide high localized concentrations at target vascular tissues and thus may be useful for the prevention and treatment of vascular proliferative disorders such as hemodialysis graft stenosis. In addition, MRI is useful for the monitoring of the location of the drug depot.