Albumin Hydrogels Formed by Electrostatically Triggered Self-Assembly and Their Drug Delivery Capability

Biological hydrogels are fundamentally biocompatible and have intrinsic similarities to extracellular matrices in medical applications and drug delivery systems. Herein we demonstrate the ability to form drug-eluting protein hydrogels using a novel mechanism that involves the electrostatically triggered partial denaturation and self-assembly of the protein via changes in pH. Partial denaturation increases the protein’s solvent exposed hydrophobic surface area, which then drives self-assembly of the protein into a hydrogel within 10 min at 37 °C. We describe the properties of an albumin hydrogel formed by this mechanism. Intrinsic drug binding properties of albumin to all-trans retinoic acid (atRA) are conserved through the partial denaturation process, as confirmed by fluorescence quenching. atRA released from the hydrogel inhibited smooth muscle cell migration as per an in vitro scratch wound assay. Atomistic molecular dynamics and potential of mean force calculations show the preservation and potential creation of new atRA binding sites with a binding energy of −41 kJ/mol. The resulting hydrogel is also biocompatible and exhibits rapid postgelation degradation after its implantation in vivo. This interdisciplinary work provides a new tool for the development of biocompatible protein hydrogel drug delivery systems

Albumin Hydrogels Formed by Electrostatically Triggered Self-Assembly and Their Drug Delivery Capability

Biological hydrogels are fundamentally biocompatible and have intrinsic similarities to extracellular matrices in medical applications and drug delivery systems. Herein we demonstrate the ability to form drug-eluting protein hydrogels using a novel mechanism that involves the electrostatically triggered partial denaturation and self-assembly of the protein via changes in pH. Partial denaturation increases the protein’s solvent exposed hydrophobic surface area, which then drives self-assembly of the protein into a hydrogel within 10 min at 37 °C. We describe the properties of an albumin hydrogel formed by this mechanism. Intrinsic drug binding properties of albumin to all-trans retinoic acid (atRA) are conserved through the partial denaturation process, as confirmed by fluorescence quenching. atRA released from the hydrogel inhibited smooth muscle cell migration as per an in vitro scratch wound assay. Atomistic molecular dynamics and potential of mean force calculations show the preservation and potential creation of new atRA binding sites with a binding energy of −41 kJ/mol. The resulting hydrogel is also biocompatible and exhibits rapid postgelation degradation after its implantation in vivo. This interdisciplinary work provides a new tool for the development of biocompatible protein hydrogel drug delivery systems

Targeting Heparin to Collagen within Extracellular Matrix Significantly Reduces Thrombogenicity and Improves Endothelialization of Decellularized Tissues

Thrombosis within small-diameter vascular grafts limits the development of bioartificial, engineered vascular conduits, especially those derived from extracellular matrix (ECM). Here we describe an easy-to-implement strategy to chemically modify vascular ECM by covalently linking a collagen binding peptide (CBP) to heparin to form a heparin derivative (CBP–heparin) that selectively binds a subset of collagens. Modification of ECM with CBP–heparin leads to increased deposition of functional heparin (by ∼7.2-fold measured by glycosaminoglycan composition) and a corresponding reduction in platelet binding (>70%) and whole blood clotting (>80%) onto the ECM. Furthermore, addition of CBP–heparin to the ECM stabilizes long-term endothelial cell attachment to the lumen of ECM-derived vascular conduits, potentially through recruitment of heparin-binding growth factors that ultimately improve the durability of endothelialization in vitro. Overall, our findings provide a simple yet effective method to increase deposition of functional heparin on the surface of ECM-based vascular grafts and thereby minimize thrombogenicity of decellularized tissue, overcoming a significant challenge in tissue engineering of bioartificial vessels and vascularized organs

A Thermoresponsive Biodegradable Polymer with Intrinsic Antioxidant Properties

Oxidative stress in tissue can contribute to chronic inflammation that impairs wound healing and the efficacy of cell-based therapies and medical devices. We describe the synthesis and characterization of a biodegradable, thermoresponsive gel with intrinsic antioxidant properties suitable for the delivery of therapeutics. Citric acid, poly­(ethylene glycol) (PEG), and poly-<i>N</i>-isopropylacrylamide (PNIPAAm) were copolymerized by sequential polycondensation and radical polymerization to produce poly­(polyethylene glycol citrate-<i>co</i>-<i>N</i>-isopropylacrylamide) (PPCN). PPCN was chemically characterized, and the thermoresponsive behavior, antioxidant properties, morphology, potential for protein and cell delivery, and tissue compatibility in vivo were evaluated. The PPCN gel has a lower critical solution temperature (LCST) of 26 °C and exhibits intrinsic antioxidant properties based on its ability to scavenge free radicals, chelate metal ions, and inhibit lipid peroxidation. PPCN displays a hierarchical architecture of micropores and nanofibers, and contrary to typical thermoresponsive polymers, such as PNIPAAm, PPCN gel maintains its volume upon formation. PPCN efficiently entrapped and slowly released the chemokine SDF-1α and supported the viability and proliferation of vascular cells. Subcutaneous injections in rats showed that PPCN gels are resorbed over time and new connective tissue formation takes place without signs of significant inflammation. Ultimately, this intrinsically antioxidant, biodegradable, thermoresponsive gel could potentially be used as an injectable biomaterial for applications where oxidative stress in tissue is a concern

Copper Metal–Organic Framework Nanoparticles Stabilized with Folic Acid Improve Wound Healing in Diabetes

The successful treatment of chronic nonhealing wounds requires strategies that promote angiogenesis, collagen deposition, and re-epithelialization of the wound. Copper ions have been reported to stimulate angiogenesis; however, several applications of copper salts or oxides to the wound bed are required, leading to variable outcomes and raising toxicity concerns. We hypothesized that copper-based metal–organic framework nanoparticles (Cu-MOF NPs), referred to as HKUST-1, which are rapidly degraded in protein solutions, can be modified to slowly release Cu²⁺, resulting in reduced toxicity and improved wound healing rates. Folic acid was added during HKUST-1 synthesis to generate folic-acid-modified HKUST-1 (F-HKUST-1). The effect of folic acid incorporation on NP stability, size, hydrophobicity, surface area, and copper ion release profile was measured. In addition, cytotoxicity and <i>in vitro</i> cell migration processes due to F-HKUST-1 and HKUST-1 were evaluated. Wound closure rates were assessed using the splinted excisional dermal wound model in diabetic mice. The incorporation of folic acid into HKUST-1 enabled the slow release of copper ions, which reduced cytotoxicity and enhanced cell migration <i>in vitro</i>. <i>In vivo</i>, F-HKUST-1 induced angiogenesis, promoted collagen deposition and re-epithelialization, and increased wound closure rates. These results demonstrate that folic acid incorporation into HKUST-1 NPs is a simple, safe, and promising approach to control Cu²⁺ release, thus enabling the direct application of Cu-MOF NPs to wounds

Repair of critical sized cranial defects with BMP9-transduced calvarial cells delivered in a thermoresponsive scaffold

<div><p>Large skeletal defects caused by trauma, congenital malformations, and post-oncologic resections of the calvarium present major challenges to the reconstructive surgeon. We previously identified BMP-9 as the most osteogenic BMP in vitro and in vivo. Here we sought to investigate the bone regenerative capacity of murine-derived calvarial mesenchymal progenitor cells (iCALs) transduced by BMP-9 in the context of healing critical-sized calvarial defects. To accomplish this, the transduced cells were delivered to the defect site within a thermoresponsive biodegradable scaffold consisting of poly(polyethylene glycol citrate-co-N-isopropylacrylamide mixed with gelatin (PPCN-g). A total of three treatment arms were evaluated: PPCN-g alone, PPCN-g seeded with iCALs expressing GFP, and PPCN-g seeded with iCALs expressing BMP-9. Defects treated only with PPCN-g scaffold did not statistically change in size when evaluated at eight weeks postoperatively (p = 0.72). Conversely, both animal groups treated with iCALs showed significant reductions in defect size after 12 weeks of follow-up (BMP9-treated: p = 0.0025; GFP-treated: p = 0.0042). However, H&E and trichrome staining revealed more complete osseointegration and mature bone formation only in the BMP9-treated group. These results suggest that BMP9-transduced iCALs seeded in a PPCN-g thermoresponsive scaffold is capable of inducing bone formation in vivo and is an effective means of creating tissue engineered bone for critical sized defects.</p></div

Schematic representation of the overall experimental design.

<p>(A) Murine calvarial cells (CALs) were immortalized via retrovirally introduced SV40 large T antigen to produce iCALs. iCALs were then transduced with BMP9 via adenoviral vector and mixed with PPCN scaffolding material. (B) qPCR analysis demonstrating relative expression of BMP9 in iCALs infected with ad-BMP9 (blue bars) compared to control (ad-GFP)-infected cells (red bars). Gene transcript expression was normalized against GAPDH expression. (C) The mixture was subsequently tested in our murine craniofacial defect model. Four millimeter diameter full-thickness calvarial defects were created in the left parietal bone of 8-week-old male athymic (nu/nu) mice. The newly created empty defect reveals the underlying dura mater. PPCN alone or PPCN and adenovirally transduced immortalized calvarial cells were used to fill the defect site.</p

Time-course microCT imaging of the calvarial defects.

<p>At 24–48 hours postoperatively, baseline microCT imaging was performed and analyzed to determine defect volume. Follow-up imaging and analysis was performed at 2, 4, 6, 8, and 12 weeks postoperatively to quantify residual defect volume and new bone ossification. Representative images are shown.</p

Assessment of the Trichrome histology.

<p>Histologic analysis of tissue microsections harvested 12 weeks post-treatment. (A, B) Chondroid matrix and a smaller proportion of mature bone is shown in the defect site of a PPCN + iCAL + AdGFP-treated mouse. (C, D) A visibly higher proportion of mature bone can be appreciated in samples taken from the defect sites treated with PPCN + iCAL + AdBMP9.</p

Histologic evaluation of the BMP9-induced calvarial defect repair.

<p>Histologic analysis of tissue microsections harvested 12 weeks post-treatment. Yellow arrows indicate the original defect borders. (A) The defect site of this AdGFP-treated mouse shows incomplete healing; there is some ingrowth of bone, but fibrous tissue fills part of the defect. (B) Conversely, the defect site of an AdBMP9-treated mouse has been completely bridged with new bone. All sections show no trace of PPCN material, indicating complete resorption.</p