Tunable Growth Factor Delivery from Injectable Hydrogels for Tissue Engineering

Current sustained delivery strategies of protein therapeutics are limited by the fragility of the protein, resulting in minimal quantities of bioactive protein delivered. In order to achieve prolonged release of bioactive protein, an affinity-based approach was designed which exploits the specific binding of the Src homology 3 (SH3) domain with short proline-rich peptides. Specifically, methyl cellulose was modified with SH3-binding peptides (MC-peptide) with either a weak affinity or strong affinity for SH3. The release profile of SH3-rhFGF2 fusion protein from hyaluronan MC-SH3 peptide (HAMC-peptide) hydrogels was investigated and compared to unmodified controls. SH3-rhFGF2 release from HAMC-peptide was extended to 10 days using peptides with different binding affinities compared to the 48 h release from unmodified HAMC. This system is capable of delivering additional proteins with tunable rates of release, while maintaining bioactivity, and thus is broadly applicable

Hydrogel for Simultaneous Tunable Growth Factor Delivery and Enhanced Viability of Encapsulated Cells <i>in Vitro</i>

Poor cell survival <i>in vitro</i> and <i>in vivo</i> is one of the key challenges in tissue engineering. Prosurvival therapeutic proteins, such as insulin-like growth factor-1 (IGF-1), can promote cell viability but require controlled delivery systems due to their short half-lives and rapid clearance. Biocompatible materials are commonly used for drug delivery platforms or to encapsulate cells for increased viability, but few materials have been used for both applications simultaneously. In this work, we present a dual-use platform. A blend of hyaluronan and methylcellulose, known to promote cell survival, was covalently modified with Src homology 3 (SH3)-binding peptides and demonstrated tunable, affinity-based release of the prosurvival fusion protein SH3–IGF-1. The material also significantly increased the viability of retinal pigment epithelium cells under anchorage-independent conditions. This novel platform is applicable to a broad range of cells and protein therapeutics and is a promising drug delivery/cell transplantation strategy to increase the viability of both exogenous and endogenous cells in tissue engineering applications

Stability of Self-Assembled Polymeric Micelles in Serum

The stability of polymeric nanoparticles in serum is critical to their use in drug delivery where dilution after intravenous injection often results in nanoparticle disassembly and drug unloading; however, few investigate this in biologically relevant media. To gain greater insight into nanoparticle stability in blood, the stability of self-assembled polymeric micelles of poly(d,l-lactide-<i>co</i>-2-methyl-2-carboxytrimethylene carbonate)-<i>g</i>-poly(ethylene glycol), P(LA-<i>co</i>-TMCC)-<i>g</i>-PEG, were tested in both serum and individual serum protein solutions. By encapsulating Förster resonance energy transfer pairs and following their release by fluorescence, these micelles demonstrated excellent thermodynamic and kinetic stability in the presence of serum. Further analyses by fast protein liquid chromatography and dynamic light scattering confirmed these data. Moreover, these micelles are compatible with red blood cells, as shown by a hemolysis assay. The stability and compatibility demonstrated in blood suggest that these micelles may be stable <i>in vivo</i>, which is critical for intravenous drug delivery applications. This comprehensive approach to understanding micelle stability and compatibility is broadly applicable

Independently Tuning the Biochemical and Mechanical Properties of 3D Hyaluronan-Based Hydrogels with Oxime and Diels–Alder Chemistry to Culture Breast Cancer Spheroids

For native breast cancer cell growth to be mimicked in vitro as spheroids, a well-defined matrix that mimics the tumor microenvironment is required. Finding a biomimetic material for 3D cell culture other than Matrigel has challenged the field. Because hyaluronan is naturally abundant in the tumor microenvironment and can be chemically modified, we synthesized a hyaluronan (HA) hydrogel with independently tunable mechanical and chemical properties for 3D culture of breast cancer cells. By modifying HA with distinct bioorthogonal functional groups, its mechanical properties are controlled by chemical cross-linking via oxime ligation, and its biochemical properties are controlled by grafting bioactive peptides via Diels–Alder chemistry. A series of hydrogels were screened in terms of stiffness and peptide composition for cancer spheroid formation. In the optimal hydrogel formulation, the 3D breast cancer spheroids showed decreased drug diffusion into their core and upregulation of cellular multidrug-resistant efflux pumps similar to what is observed in drug-resistant tumors. Our results highlight the potential of these tunable and well-defined gels in drug screening assays

Microsphere-loaded channels effectively release dbcAMP <i>in vitro</i>.

<p><b>A</b>) Cumulative release profiles of dbcAMP from free-floating microspheres and microsphere-loaded channels. The process of embedding microspheres into channel walls is likely responsible for early degradation of PLGA and faster drug release from channels. <b>B</b>) Schematic of the entubulation strategy. NSPCs are seeded on fibrin scaffold within a chitosan channel. Drug-loaded PLGA microspheres release the differentiation factor dibutyryl cyclic-AMP in a local and sustained manner, influencing NSPCs to preferentially differentiate into neurons. <b>C</b>) Viability of NSPCs in a three-dimensional fibrin scaffold. Simultaneous staining of CalceinAM (green) and Ethidium homodimer (red) for live and dead cells respectively show good cell viability of NSPCs in fibrin scaffolds at 1 week. Scale bar represents 100 µm. <b>D–G</b>) Immunostaining of NSPCs for DAPI-nuclear stain and betaIII-tubulin with various dbcAMP treatments. Scale bar represents 100 µm. <b>H</b>) Quantification of betaIII-tubulin immunostained NSPCs with various dbcAMP treatments. <b>I,J</b>) Quantitative RT-PCR data for (I) betaIII tubulin and (J) nestin mRNA expression with various dbcAMP treatments, normalized to housekeeping gene HPRT. Data represented as mean ± standard (n = 3 to 6). Statistical differences denoted by *, p<0.05.</p

The regenerated bridge tissue contains host axons, blood vessels, and fibroblasts.

<p><b>A</b>) Representative image of endogenous axonal regeneration into the tissue bridge based on betaIII tubulin staining. <b>B</b>) Evidence of association between betaIII-positive endogenous axons with surviving GFP-positive NSPCs at six weeks. Synaptophysin staining is observed at the interface (inset). <b>C</b>) RECA1 staining for endothelial cells show blood vessel formation throughout the tissue bridge at 2 weeks. <b>D</b>) Prolyl-4-hydroxylase (rPH) staining of bridge tissue indicates that the majority of cells are collagen producing fibroblasts.</p

Differentiation profiles of NSPCs are impacted by dbcAMP treatment.

<p><b>A–L</b>) Representative images of tissue samples demonstrating NSPC differentiation profile of (A–C) nestin-positive progenitor cells, (D–F) BetaIII-positive neurons, (G–H) CC1-positive oligodendrocytes, and (J–L) GFAP-positive astrocytes. Scale bar represents 50 µm. <b>M</b>) Quantification of NSPC differentiation profile for the various treatment groups. Mean ± standard deviation are plotted, n = 3 to 5; significant differences noted with an asterisks, p<0.05. <b>N</b>) Deconvoluted confocal image of betaIII-positive NSPC-derived neurons (arrows) 6 weeks post-transplantation. Scale bar represents 50 µm.</p

PEG-Graft Density Controls Polymeric Nanoparticle Micelle Stability

Polymeric nanoparticle micelles typically comprise amphiphilic block copolymers, having a hydrophobic core that is useful for chemotherapeutic encapsulation, and a hydrophilic corona for aqueous stability. Formulations often require the use of excipients to overcome poor particle stability, yet these excipients can be cytotoxic. In order to create a stable polymeric nanoparticle micelle without the use of excipients, we investigate a series of amphiphilic polymers where the hydrophobic core composition and molar mass is maintained and the hydrophilic corona is varied. With the graft copolymer, poly­(d,l-lactide-<i>co</i>-2-methyl-2-carboxytrimethylenecarbonate)-<i>g</i>-poly­(ethylene glycol) (P­(LA-<i>co</i>-TMCC)-<i>g</i>-PEG), we demonstrate how PEG density can be tuned to improve the stability of the resulting self-assembled micelle. Increased PEG density leads to micelles that resist aggregation during lyophilization, allowing resuspension in aqueous media with narrow distribution. Furthermore, high PEG density micelles resist dissociation in serum protein containing media, with almost no dissociation seen in serum after 72 h. By changing the number of PEG chains per polymer backbone from 0.5 to 6, we observe increased stability of the nanoparticle micelles. All formulations are cytocompatible, as measured with MDA-MB-231 cells, and show no evidence for hemolysis, as measured with red blood cells. Importantly, PEG density does not impact drug loading within the nanoparticle micelle core, as demonstrated with the potent chemotherapeutic drug, docetaxel, confirming the role of the hydrophobic core for encapsulation. The surface properties of the polymeric nanoparticle micelles can thus be selectively modulated by variation in PEG density, which in turn influences stability, obviates the need for excipients and provides key insights into the design of drug delivery platforms

A New Spin on Antibody–Drug Conjugates: Trastuzumab-Fulvestrant Colloidal Drug Aggregates Target HER2-Positive Cells

While the formation of colloidal aggregates leads to artifacts in early drug discovery, their composition makes them attractive as nanoparticle formulations for targeted drug delivery as the entire nanoparticle is composed of drug. The typical transient stability of colloidal aggregates has inhibited exploiting this property. To overcome this limitation, we investigated a series of proteins to stabilize colloidal aggregates of the chemotherapeutic, fulvestrant, including the following: bovine serum albumin, a generic human immunoglobulin G, and trastuzumab, a therapeutic human epidermal growth factor receptor 2 antibody. Protein coronas reduced colloid size to <300 nm and improved their stability to over 48 h in both buffered saline and media containing serum protein. Unlike colloids stabilized with other proteins, trastuzumab-fulvestrant colloids were taken up by HER2 overexpressing cells and were cytotoxic. This new targeted formulation reimagines antibody–drug conjugates, delivering mM concentrations of drug to a cell

Channel implantation after spinal cord transection facilitates tissue bridging, NSPC survival, and behavioural improvement over time.

<p><b>A</b>) Photograph of the surgical implantation of fibrin-filled chitosan channels. <b>B</b>) Tissue bridges obtained from animals 2 weeks after implantation. <b>C,D</b>) Longitudinal section of tissue bridge demonstrating NSPC survival after 6 weeks in an animal receiving dbcAMP pre-treatment (dbcAMP, 4div). Boxed area in (C) is magnified in (D). <b>E</b>) NSPC survival after 2 and 6 weeks for various treatment groups. <b>F</b>) Assessment of functional recovery using the BBB locomotor scale. After 6 weeks, rats receiving transplants of dbcAMP-pre-treated NSPCs show a statistically significant increase in hindlimb function relative to untreated animals (*, p<0.05). Mean ± standard deviation shown for n = 4 to 6.</p