Bioactive Polyurethane-Poly(ethylene Glycol) Diacrylate Hydrogels for Applications in Tissue Engineering.

Polyurethanes (PUs) are a highly adaptable class of biomaterials that are among some of the most researched materials for various biomedical applications. However, engineered tissue scaffolds composed of PU have not found their way into clinical application, mainly due to the difficulty of balancing the control of material properties with the desired cellular response. A simple method for the synthesis of tunable bioactive poly(ethylene glycol) diacrylate (PEGDA) hydrogels containing photocurable PU is described. These hydrogels may be modified with PEGylated peptides or proteins to impart variable biological functions, and the mechanical properties of the hydrogels can be tuned based on the ratios of PU and PEGDA. Studies with human cells revealed that PU-PEG blended hydrogels support cell adhesion and viability when cell adhesion peptides are crosslinked within the hydrogel matrix. These hydrogels represent a unique and highly tailorable system for synthesizing PU-based synthetic extracellular matrices for tissue engineering applications.

Pooling of Samples for SARS-CoV-2 Detection Using a Rapid Antigen Test.

While molecular assays, such as reverse-transcription polymerase chain reaction (RT-PCR), have been widely used throughout the coronavirus disease 2019 (COVID-19) pandemic, the technique is costly and resource intensive. As a means to reduce costs and increase diagnostic efficiency, pooled testing using RT-PCR has been implemented. However, pooling samples for antigen testing has not been evaluated. Here, we propose a proof-of-concept pooling strategy for antigen testing that would significantly expand SARS-CoV-2 surveillance, especially for low-to-middle income countries, schools, and workplaces. Our laboratory-based testing demonstrates that combining of up to 20 nasal swab specimens per pool can expand surveillance with antigen tests, even if a pool contains only one positive sample.

Considerations for the Use of Polysorbates in Biopharmaceuticals.

PURPOSE: Polysorbates are commonly added to protein formulations and serve an important function as stabilizers. This paper reviews recent literature detailing some of the issues seen with the use of polysorbate 80 and polysorbate 20 in protein formulations. Based on this knowledge, a development strategy is proposed that leads to a control strategy for polysorbates in protein formulations. METHODS: A consortium of Biopharmaceutical scientists working in the area of protein formulations, shared experiences with polysorbates as stabilizers in their formulations. RESULTS: Based on the authors experiences and recent published literature, a recommendation is put forth for a development strategy which will lead into the appropriate control strategy for these excipients. CONCLUSIONS: An appropriate control strategy may comprise one or more elements of raw material, in-process and manufacturing controls. Additionally, understanding the role, if any, polysorbates play during stability will require knowledge of the criticality of the excipient, based upon its impact on CQAs due to variations in concentration and degradation level.

Effects of fibrin pad hemostat on the wound healing process in vivo and in vitro.

Fibrin Pad is a hemostatic pad designed to control surgical-related bleeding. It consists of a fully absorbable composite matrix scaffold coated with human-derived active biologics that immediately form a fibrin clot upon contact with targeted bleeding surfaces. Studies were conducted to investigate the effect of Fibrin Pad and its biologics-free composite matrix component (Matrix) on the wound healing process in in vitro and in vivo models. Fibrin Pad was evaluated in solid organ, soft tissue defects, and subcutaneous tissues. Immunocompromised rodents were used to avoid xeno-mediated responses. Extracts created from both materials were evaluated for biological activity using in vitro cell culture assays. Neither Fibrin Pad nor Matrix alone showed any inhibition of the wound healing of treated defect sites. An apparent accelerated healing was noted in the soft tissue and subcutaneous tissue defects with Fibrin Pad as compared to Matrix. Both materials showed desirable properties associated with tissue scaffolds. The in vitro study results show that Fibrin Pad extract can induce dose-dependent increases in fibroblast proliferation and migration. These studies confirm that the biologic components of Fibrin Pad can enhance wound healing processes in in vitro assays and fully support wound healing at the site of in vivo application.

Thermodynamic and physical interactions between novel polymeric surfactants and lipids: toward designing stable polymer-lipid complexes.

Surfactant amphiphilic macromolecules (AMs) were complexed with a 1:1 ratio of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), either by a coevaporation (CE) or postaddition (PA) method, to form AM-lipid complexes with enhanced drug delivery applications. By characterizing the surfactant-lipid interactions, these heterogeneous drug delivery systems can be better controlled and engineered for optimal therapeutic outcomes. In this study, the physical interactions between DOPE:DOTAP liposomes and AM surfactants were investigated. Langmuir film balance and isothermal calorimetry studies showed cooperative intermolecular interactions between pure lipids and AM in monolayers and high thermostability of structure formed by the addition of AM micelles to DOTAP:DOPE vesicles in buffer solution respectively. Increasing the AM weight ratio in the complexes via the CE method led to complete vesicle solubilization--from lamellar aggregates, to a mixture of coexisting vesicles and micelles, to mixed micelles. Isothermal calorimetry evaluation of AM-lipid complexes shows that, at higher AM weight ratios, PA-produced complexes exhibit greater stability than complexes at lower AM weight ratios. Similar studies show that AM-lipid complexes produced by the CE methods display stronger interactions between AM-lipid components than complexes produced by the PA method. The results suggest that the PA method produces vesicles with AM molecules associated with its outer leaflet only (i.e., an AM-coated vesicle), while the CE method produces complexes ranging from mixed vesicles to mixed micelle in which the AM-lipid components are more intimately associated. These results will be helpful in the design of AM-lipid complexes as structurally defined, stable, and effective drug delivery systems.

Efficient intracellular siRNA delivery by ethyleneimine-modified amphiphilic macromolecules.

New materials that can bind and deliver oligonucleotides such as short interfering RNA (siRNA) without toxicity are greatly needed to fulfill the promise of therapeutic gene silencing. Amphiphilic macromolecules (AMs) were functionalized with linear ethyleneimines to create cationic AMs capable of complexing with siRNA. Structurally, the parent AM is formed from a mucic acid backbone whose tetra-hydroxy groups are alkylated with 12-carbon aliphatic chains to form the hydrophobic component of the macromolecule. This alkylated mucic acid is then mono-functionalized with poly(ethylene glycol) (PEG) as a hydrophilic component. The resulting AM contains a free carboxylic acid within the hydrophobic domain. In this work, linear ethyleneimines were conjugated to the free carboxylic acid to produce an AM with one primary amine (1N) or one primary amine and four secondary amines (5N). Further, an AM with amine substitution both to the free carboxylic acid in the hydrophobic domain and also to the adjacent PEG was synthesized to produce a polymer with one primary amine and eight secondary amines (9N), four located on each side of the AM hydrophobic domain. All amine-functionalized AMs formed nanoscale micelles but only the 5N and 9N AMs had cationic zeta potentials, which increased with increasing number of amines. All AMs exhibited less inherent cytotoxicity than linear polyethyleneimine (L-PEI) at concentrations of 10 µM and above. By increasing the length of the cationic ethyleneimine chain and the total number of amines, successful siRNA complexation and cellular siRNA delivery was achieved in a malignant glioma cell line. In addition, siRNA-induced silencing of firefly luciferase was observed using complexes of siRNA with the 9N AM and comparable to L-PEI, yet showed better cell viability at higher concentrations (above 10 µM). This work highlights the promise of cationic AMs as safe and efficient synthetic vectors for siRNA delivery. Specifically, a novel polymer (9N) was identified for efficient siRNA delivery to cancer cells and will be further evaluated.

Preferential cellular uptake of amphiphilic macromolecule-lipid complexes with enhanced stability and biocompatibility.

Amphiphilic macromolecules (AM) were electrostatically complexed with a 1:1 ratio of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) to form AM-lipid complexes with drug delivery applications. The complexes exist as AM-coated liposomes and their drug delivery properties can be tuned by altering the AM-lipid weight ratio. The complexation and tuning are achieved in a simple, efficient, and scalable manner. The gradual increase in lipid ratios concurrently increased the zeta potential of the complexes, which directly correlates to increased cell uptake of the complexes in vitro with preferential uptake noted in BT-20 carcinoma cells versus normal fibroblasts. Increasing AM content increased complex steric stability in the presence of serum proteins and reduced the inherent cytotoxicity towards fibroblasts in vitro. AM-lipid complexes solubilized paclitaxel and showed drug-mediated, dose-dependent cytotoxicity towards target BT-20 cells in vitro. AM-lipid complexes make good candidates as drug delivery systems due to their tunable zeta potential, steric stability, inherently low cytotoxicity, and ability to load and deliver insoluble chemotherapeutic agents. Significantly, their preferential uptake in a carcinoma cell line over normal cells in vitro demonstrates a unique, passive targeting approach to delivery anti-cancer therapeutics.

Micellar nanocarriers assembled from doxorubicin-conjugated amphiphilic macromolecules (DOX-AM).

Amphiphilic macromolecules (AMs) have unique branched hydrophobic domains attached to linear PEG chains. AMs self-assemble in aqueous solution to form micelles that are hydrolytically stable in physiological conditions (37 degrees C, pH 7.4) over 4 weeks. Evidence of AM biodegradability was demonstrated by complete AM degradation after 6 d in the presence of lipase. Doxorubicin (DOX) was chemically conjugated to AMs via a hydrazone linker to form DOX-AM conjugates that self-assembled into micelles in aqueous solution. The conjugates were compared with DOX-loaded AM micelles (i.e., physically loaded DOX) on DOX content, micellar sizes and in vitro cytotoxicity. Physically encapsulated DOX loading was higher (12 wt.-%) than chemically bound DOX (6 wt.-%), and micellar sizes of DOX-loaded AMs (approximately 16 nm) were smaller than DOX-AMs (approximately 30 nm). In vitro DOX release from DOX-AM conjugates was faster at pH 5.0 (100%) compared to pH 7.4 (78%) after 48 h, 37 degrees C. Compared to free DOX and physically encapsulated DOX, chemically bound DOX had significantly higher cytotoxicity at 10(-7) M DOX dose against human hepatocellular carcinoma cells after 72 h. Overall, DOX-AM micelles showed promising characteristics as stable, biodegradable DOX nanocarriers.

Cells isolated from umbilical cord tissue rescue photoreceptors and visual functions in a rodent model of retinal disease.

Progressive photoreceptor degeneration resulting from genetic and other factors is a leading and largely untreatable cause of blindness worldwide. The object of this study was to find a cell type that is effective in slowing the progress of such degeneration in an animal model of human retinal disease, is safe, and could be generated in sufficient numbers for clinical application. We have compared efficacy of four human-derived cell types in preserving photoreceptor integrity and visual functions after injection into the subretinal space of the Royal College of Surgeons rat early in the progress of degeneration. Umbilical tissue-derived cells, placenta-derived cells, and mesenchymal stem cells were studied; dermal fibroblasts served as cell controls. At various ages up to 100 days, electroretinogram responses, spatial acuity, and luminance threshold were measured. Both umbilical-derived and mesenchymal cells significantly reduced the degree of functional deterioration in each test. The effect of placental cells was not much better than controls. Umbilical tissue-derived cells gave large areas of photoreceptor rescue; mesenchymal stem cells gave only localized rescue. Fibroblasts gave sham levels of rescue. Donor cells were confined to the subretinal space. There was no evidence of cell differentiation into neurons, of tumor formation or other untoward pathology. Since the umbilical tissue-derived cells demonstrated the best photoreceptor rescue and, unlike mesenchymal stem cells, were capable of sustained population doublings without karyotypic changes, it is proposed that they may provide utility as a cell source for the treatment of retinal degenerative diseases such as retinitis pigmentosa.