Non-destructive Raman Method Development for Quantifying Active Pharmaceutical Ingredient in an Oral Suspension Through Plastic Dosing Syringes.

For liquid drug products, e.g., solutions or suspensions for oral or parenteral dosing, stability needs to be demonstrated in primary packaging during storage and in dosing devices during in-use periods per quality guidelines from the International Conference on Harmonisation (ICH) and the European Agency for the Evaluation of Medicinal Products (EMEA). One aspect of stability testing for liquid drug products is in-use stability, which typically includes transferring the liquid samples into another container for further sample preparation with extraction diluent and necessary agitation. Samples are then analyzed with traditional chromatography methods, which are laborious, prone to human errors, and time-consuming, especially when this process needs to be repeated multiple times during storage and in-use periods. Being able to analyze the liquid samples non-destructively would significantly improve testing efficiency. We investigated different Raman techniques, including transmission Raman (TRS) and back scatter Raman with a non-contact optic (NCO) probe, as alternative non-destructive tools to the UHPLC method for API quantitation in in-use liquid samples pulled into plastic dosing syringes. The linearity of the chemometric methods for these two techniques was demonstrated by cross-validation sample sets at three levels over an API concentration range of 60 to 80 mg/mL. The accuracy of the chemometric models was demonstrated by the accurate prediction of the API concentrations in independent samples from four different pilot plant batches manufactured at different sites. Both techniques were successful in measuring a signal through a plastic oral dosing syringe, and predicting the suspension API concentration to within 4% of the UHPLC-measured value. For future work, there are opportunities to improve the methodology by exploring additional probes or to expand the range of applications by using different sample presentations (such as prefilled syringes) or formulation matrices for solutions and suspensions.

Protease-Sensitive Hydrogel Biomaterials with Tunable Modulus and Adhesion Ligand Gradients for 3D Vascular Sprouting.

Biomaterial strategies focused on designing scaffolds with physiologically relevant gradients provide a promising means for elucidating 3D vascular cell responses to spatial and temporal variations in matrix properties. In this study, we present a photopolymerization approach, ascending photofrontal free-radical polymerization, to generate proteolytically degradable hydrogel scaffolds of poly(ethylene) glycol with tunable continuous gradients of (1) elastic modulus (slope of 80 Pa/mm) and uniform immobilized RGD concentration (2.06 ± 0.12 mM) and (2) immobilized concentration of the RGD cell-adhesion peptide ligand (slope of 58.8 µM/mm) and uniform elastic modulus (597 ± 22 Pa). Using a coculture model of vascular sprouting, scaffolds embedded with gradients of elastic modulus induced increases in the number of vascular sprouts in the opposing gradient direction, whereas RGD gradient scaffolds promoted increases in the length of vascular sprouts toward the gradient. Furthermore, increases in vascular sprout length were found to be prominent in regions containing higher immobilized RGD concentration.

Design and characterization of hydrogel nanoparticles with tunable network characteristics for sustained release of a VEGF-mimetic peptide.

Peptides that mimic the bioactivity of growth factors are rapidly emerging as therapeutics for a variety of drug delivery applications including therapeutic neovascularization. Neovascularization requires controlled and sustained delivery of proangiogenic factors to stimulate reperfusion of ischemic tissues. To this end, hydrogel nanoparticles were designed to provide sustained and tunable diffusion-based release of a pro-angiogenic peptide, QK. Inverse phase mini-emulsion polymerization (IPMP) was used to generate crosslinked poly(ethylene) glycol (PEG) hydrogel nanoparticles entrapped with the QK peptide. Peptide release kinetics were tuned through adjustments in nanoparticle crosslink density. This was achieved by altering the mole fraction of the crosslinking agent which allowed for the synthesis of low crosslink density (0.754 mmol cm-3) and high crosslink density (0.810 mmol cm-3) nanoparticles. Nanoparticle tracking analysis revealed narrow particle size distributions and similar particle sizes regardless of crosslink density (225 ± 75 nm and 233 ± 73 nm, for low and high crosslink density nanoparticles, respectively). The zeta potential was found to be -26 mV for blank nanoparticles and +4 mV in the case of QK-loaded nanoparticles. The resulting nanoparticle crosslink density impacted both peptide loading as well as release kinetics. In terms of cumulative fractional release and weight of peptide released per mass of nanoparticle, higher crosslink density nanoparticles resulted in slower peptide release kinetics. The IPMP process preserved the QK secondary structure and its bioactivity as confirmed using circular dichroism spectroscopy and a Matrigel tubulogenesis assay, respectively, with released peptide. The presented nanoparticles hold great potential for use as drug delivery carriers for stimulation of therapeutic neovascularization of ischemic tissues.

Radiation combined injury models to study the effects of interventions and wound biomechanics.

In the event of a nuclear detonation, a considerable number of projected casualties will suffer from combined radiation exposure and burn and/or wound injury. Countermeasure assessment in the setting of radiation exposure combined with dermal injury is hampered by a lack of animal models in which the effects of interventions have been characterized. To address this need, we used two separate models to characterize wound closure. The first was an open wound model in mice to study the effect of wound size in combination with whole-body 6 Gy irradiation on the rate of wound closure, animal weight and survival (morbidity). In this model the addition of interventions, wound closure, subcutaneous vehicle injection, topical antiseptic and topical antibiotics were studied to measure their effect on healing and survival. The second was a rat closed wound model to study the biomechanical properties of a healed wound at 10 days postirradiation (irradiated with 6 or 7.5 Gy). In addition, complete blood counts were performed and wound pathology by staining with hematoxylin and eosin, trichrome, CD68 and Ki67. In the mouse open wound model, we found that wound size and morbidity were positively correlated, while wound size and survival were negatively correlated. Regardless of the wound size, the addition of radiation exposure delayed the healing of the wound by approximately 5-6 days. The addition of interventions caused, at a minimum, a 30% increase in survival and improved mean survival by ∼9 days. In the rat closed wound model we found that radiation exposure significantly decreased all wound biomechanical measurements as well as white blood cell, platelet and red blood cell counts at 10 days post wounding. Also, pathological changes showed a loss of dermal structure, thickening of dermis, loss of collagen/epithelial hyperplasia and an increased density of macrophages. In conclusion, we have characterized the effect of a changing wound size in combination with radiation exposure. We also demonstrated that the most effective interventions mitigated insensible fluid loss, which could help to define the most appropriate requirements of a successful countermeasure.

MMP-sensitive PEG diacrylate hydrogels with spatial variations in matrix properties stimulate directional vascular sprout formation.

The spatial presentation of immobilized extracellular matrix (ECM) cues and matrix mechanical properties play an important role in directed and guided cell behavior and neovascularization. The goal of this work was to explore whether gradients of elastic modulus, immobilized matrix metalloproteinase (MMP)-sensitivity, and YRGDS cell adhesion ligands are capable of directing 3D vascular sprout formation in tissue engineered scaffolds. PEGDA hydrogels were engineered with mechanical and biofunctional gradients using perfusion-based frontal photopolymerization (PBFP). Bulk photopolymerized hydrogels with uniform mechanical properties, degradation, and immobilized biofunctionality served as controls. Gradient hydrogels exhibited an 80.4% decrease in elastic modulus and a 56.2% decrease in immobilized YRGDS. PBFP hydrogels also demonstrated gradients in hydrogel degradation with degradation times ranging from 10-12 hours in the more crosslinked regions to 4-6 hours in less crosslinked regions. An in vitro model of neovascularization, composed of co-culture aggregates of endothelial and smooth muscle cells, was used to evaluate the effect of these gradients on vascular sprout formation. Aggregate invasion in gradient hydrogels occurred bi-directionally with sprout alignment observed in the direction parallel to the gradient while control hydrogels with homogeneous properties resulted in uniform invasion. In PBFP gradient hydrogels, aggregate sprout length was found to be twice as long in the direction parallel to the gradient as compared to the perpendicular direction after three weeks in culture. This directionality was found to be more prominent in gradient regions of increased stiffness, crosslinked MMP-sensitive peptide presentation, and immobilized YRGDS concentration.

Describing congenital heart disease by using three-part segmental notation.

The segmental classification of congenital heart disease, first conceptualized nearly 50 years ago, is now well established. The Van Praagh classification system, in particular, is commonly used throughout North America to facilitate communication between physicians from various specialties who are involved in diagnosing and managing congenital cardiovascular abnormalities. In the Van Praagh system, a three-part notation consisting of letters separated by commas and encompassed by a set of braces is used to succinctly describe the visceroatrial situs, the orientation of the ventricular loop, and the position and relation of the great vessels. For example, the notation "{S, D, S}" describes the normal anatomic configuration, in which the morphologic right atrium and largest hepatic lobe are on the patient's right side and the morphologic left atrium, stomach, and spleen are on the left side (situs solitus); the ventricular loop is curved rightward (dextro- or d-loop); and the aorta is posterior to and rightward of the main pulmonary artery (situs solitus). Because the Van Praagh notation imposes on its users a systematic approach to anatomic description, it is a helpful device for structuring the interpretation of imaging studies as well as the reporting of results: First, the morphologic right and left atria and ventricles must be identified; next, the visceroatrial situs and ventricular loop orientation may be determined from the positions of the cardiac chambers; and last, the position and relation of the great vessels (normal, inverted, transposed, or malpositioned) can be established. The article provides concise, step-by-step guidance for applying the Van Praagh system in the radiology reading room.

Enhanced mechanical performance and biological evaluation of a PLGA coated ß-TCP composite scaffold for load-bearing applications.

Porous ß-tricalcium phosphate (ß-TCP) has been used for bone repair and replacement in clinics due to its excellent biocompatibility, osteoconductivity, and biodegradability. However, the application of ß-TCP has been limited by its brittleness. Here, we demonstrated that an interconnected porous ß-TCP scaffold infiltrated with a thin layer of poly (lactic-co-glycolic acid) (PLGA) polymer showed improved mechanical performance compared to an uncoated ß-TCP scaffold while retaining its excellent interconnectivity and biocompatibility. The infiltration of PLGA significantly increased the compressive strength of ß-TCP scaffolds from 2.90 MPa to 4.19 MPa, bending strength from 1.46 MPa to 2.41 MPa, and toughness from 0.17 MPa to 1.44 MPa, while retaining an interconnected porous structure with a porosity of 80.65%. These remarkable improvements in the mechanical properties of PLGA-coated ß-TCP scaffolds are due to the combination of the systematic coating of struts, interpenetrating structural characteristics, and crack bridging. The in vitro biological evaluation demonstrated that rat bone marrow stromal cells (rBMSCs) adhered well, proliferated, and expressed alkaline phosphatase (ALP) activity on both the PLGA-coated ß-TCP and the ß-TCP. These results suggest a new strategy for fabricating interconnected macroporous scaffolds with significantly enhanced mechanical strength for potential load-bearing bone tissue regeneration.

In vitro evaluation of an injectable chitosan gel for sustained local delivery of BMP-2 for osteoblastic differentiation.

We investigated the effect of sustained release of bone morphogenetic protein-2 (BMP-2) from an injectable chitosan gel on osteoblastic differentiation in vitro. We first characterized the release profile of BMP-2 from the gels, and then examined the cellular responses of preosteoblast mouse stromal cells (W-20-17) and human embryonic palatal mesenchymal (HEPM) cells to BMP-2. The release profiles of different concentrations of BMP-2 exhibited sustained releases (41% for 2 ng/mL and 48% for 20 ng/mL, respectively) from the chitosan gels over a three-week period. Both cell types cultured in the chitosan gels were viable and significantly proliferated for 3 days (p < 0.05). Chitosan gels loaded with BMP-2 enhanced ALP activity of W-20-17 by 3.6-fold, and increased calcium mineral deposition of HEPM by 2.8-fold at 14 days of incubation, compared to control groups initially containing the same amount of BMP-2. In addition, schitosan gels loaded with BMP-2 exhibited significantly greater osteocalcin synthesis of W-20-17 at seven days, and of HEPM at both 7 and 14 days compared with the control groups (p<0.05). This study suggests that the enhanced effects of BMP-2 released from chitosan gels on cell differentiation and mineralization are species and cell type dependent.