Modeling Diffusivity Through Alginate-Based Microfibers: A Comparison of Numerical and Analytical Models Based on Empirical Spectrophotometric Data

Abstract: The study of mass transport across hollow and solid 3D microfibers to study metabolic profiles is a key aspect of tissue engineering approach. A new modified numerical mathematical model based on Fickian equations in cylindrical coordinates has been proposed for determining the membrane diffusivity of 2% (w/v) alginate-based stents cross-linked with 10% CaCl2. Based on the economical and direct spectrophotometric measurements, using this model, inward diffusivities ranging from 5.2x10-14 m2/s 2.93x10-12m2/s were computed for solutes with Stokes radii ranging between 0.36 to 3.5 nm, diffusing through bare alginate and alginate-chitosan-alginate microfibers. In parallel an analytical solution to the cylindrical Fickian equation was derived to validate the numerical solution using experimental diffusion data from a solid stent. Excellent agreement was found between the numerical and analytical models with a maximum calculated residual value of 4%. Using these models, a flexible computational platform is proposed to conduct custom diffusion and MW cut-off characterization across micro-porous microfibers not limited to alginate in composition

Mechanical Properties of Hydrated Acoustically Sensitive Alginate-Based Microcapsules Confined in a Microfluidic Device as a Function of Size and Composition

Understanding the mechanical properties of alginate-based microcapsules according to size and chemical composition allows researchers to zero in on the treatment and methods required to engineer optimized implantable alginate-based artificial cells for chemotherapy. Cross-linked medium viscosity alginate capsules ranging from 1.1% (w/v)-1.8% (w/v) in composition and 200 μm-1200 μm in size, encapsulating ultrasound contrast agents and blue dextran were compressed within a 40 μm high polydimethylsiloxane microfluidic device and subsequently examined using 2D microscopy for strain deformation aimed at the calculation of poisson ratios and volume loss postcompression. Results indicate a decrease in Poisson ratio as a function of alginate concentration, with statistically significant increases in Poisson ratios and percent volume loss as a function of size and composition. For an average of 120 s observation time post compression, in light of the volume loss correlated to the number of cross-links as a function of capsule size and alginate concentration, a strong case for the dominance of poroelasticity vs. viscoelasticity can be made. While there was a decrease in mean Poisson ratio as a function of concentration, at 1.8% (w/v) the mean strain value converged to 0.5, the theoretical ideal isotropic value associated with soft biological tissue

Three-Dimensional (3D) Printed Microneedles for Microencapsulated Cell Extrusion

Cell-hydrogel based therapies offer great promise for wound healing. The specific aim of this study was to assess the viability of human hepatocellular carcinoma (HepG2) cells immobilized in atomized alginate capsules (3.5% (w/v) alginate, d = 225 µm ± 24.5 µm) post-extrusion through a three-dimensional (3D) printed methacrylate-based custom hollow microneedle assembly (circular array of 13 conical frusta) fabricated using stereolithography. With a jetting reliability of 80%, the solvent-sterilized device with a root mean square roughness of 158 nm at the extrusion nozzle tip (d = 325 μm) was operated at a flowrate of 12 mL/min. There was no significant difference between the viability of the sheared and control samples for extrusion times of 2 h (p = 0.14, α = 0.05) and 24 h (p = 0.5, α = 0.05) post-atomization. Factoring the increase in extrusion yield from 21.2% to 56.4% attributed to hydrogel bioerosion quantifiable by a loss in resilience from 5470 (J/m3) to 3250 (J/m3), there was no significant difference in percentage relative payload (p = 0.2628, α = 0.05) when extrusion occurred 24 h (12.2 ± 4.9%) when compared to 2 h (9.9 ± 2.8%) post-atomization. Results from this paper highlight the feasibility of encapsulated cell extrusion, specifically protection from shear, through a hollow microneedle assembly reported for the first time in literature

Machine learning provides predictive analysis into silver nanoparticle protein corona formation from physicochemical properties

Proteins encountered in biological and environmental systems bind to engineered nanomaterials (ENMs) to form a protein corona (PC) that alters the surface chemistry, reactivity, and fate of the ENMs. Complexities such as the diversity of the PC and variation with ENM properties and reaction conditions make the PC population difficult to predict. Here, we support the development of predictive models for PC populations by relating biophysicochemical characteristics of proteins, ENMs, and solution conditions to PC formation using random forest classification. The resulting model offers a predictive analysis into the population of PC proteins in Ag ENM systems of various ENM size and surface coatings. With an area under the receiver operating characteristic curve of 0.83 and F1-score of 0.81, a model with strong performance has been constructed based upon experimental data. The weighted contribution of each variable provides recommendations for mechanistic models based upon protein enrichment classification results. Protein biophysical properties such as pI and weight are weighted heavily. Yet, ENM size, surface charge, and solution ionic strength also proved essential to an accurate model. The model can be readily modified and applied to other ENM PC populations. The model presented here represents the first step toward robust predictions of PC fingerprints

Polystyrene Microsphere and 5-Fluorouracil Release from Custom Designed Wound Dressing Films

Custom-designed wound dressing films of chitosan and alginate have been prepared by a casting/solvent evaporation method for hydrophobic therapeutic agent encapsulation. In this parametric study, the propylene glycol (PG) and calcium chloride (CaCl2) concentrations were varied for chitosan and alginate films, respectively. Mechanical and chemical inter-related responses under observations included thickness (th), elasticity (E), tensile strength (TS), sorption ability (S%) and kinetics of in-vitro drug release, specifically in terms of membrane time to burst (tB) and duration of release (tR). As shown by results of a one tailed t-test significance testing at the 95% confidence interval (α = 0.05), alginate films were significantly more elastic (p = 0.003), thinner (p = 0.004) and more susceptible to osmotic burst (p = 0.011) and characterized by a longer duration of release (p = 0.03). Meanwhile chitosan films exhibited superior moisture permeability (p = 0.006) and sorption characteristics (p = 0.001), indicative of higher hydrophilicity. There were no significant differences in tensile strength (p = 0.324) for alginate and chitosan-based formulations. Preliminary testing was conducted using 0.71 μm in diameter microspheres for modeling film dissolution into Lactated Ringer’s solution. Experimental release profiles were modeled for each film from which the average release from alginate films (MAGCa = 81%) was estimated to be twice the percentage associated with chitosan films (MCD = 42%). The film comprised of 2.5% (w/v) medium MW chitosan/dextran 70 kDa (5:1) was selected for studying the release of 5-Fluorouracil (5-FU) as a model hydrophobic drug. Diffusion coupled with film disintegration is immediate (tB = 0) in case of encapsulated 5-FU as compared to the control film encapsulating microspheres characterized by tB = 70 min ± 7 min. This shift in release profile and the ability to modulate the timing of membrane burst can be attributed to the approximate ratio (1: 505) in molecular size between drug and microsphere. This hypothesis has been validated by the film pore size measured to be 430 nm ± 88 nm using atomic force microscopy

Sustainable, Alginate-Based Sensor for Detection of Escherichia coli in Human Breast Milk

There are no existing affordable diagnostics for sensitive, rapid, and on-site detection of pathogens in milk. To this end, an on-site colorimetric-based sustainable assay has been developed and optimized using an L16 (54) Taguchi design to obtain results in hours without PCR amplification. To determine the level of Escherichia coli (E. coli) contamination, after induction with 150 µL of breast milk, the B-Per bacterial protein extraction kit was added to a solution containing an alginate-based microcapsule assay. Within this 3 mm spherical novel sensor design, X-Gal (5-Bromo-4-Chloro-3-Indolyl β-D-Galactopyranoside) was entrapped at a concentration of 2 mg/mL. The outward diffusing X-Gal was cleaved by β-galactosidase from E. coli and dimerized in the solution to yield a blue color after incubation at 40 °C. Color intensity was correlated with the level of E. coli contamination using a categorical scale. After an 8 h incubation period, a continuous imaging scale based on intensity normalization was used to determine a binary lower limit of detection (LOD), which corresponded to 102 colony forming unit per mL (CFU/mL) and above. The cost of the overall assay was estimated to be $0.81 per sample, well under the$3 benchmark for state-of-the-art immune-based test kits for pathogen detection in biofluids. Considering the reported binary LOD cutoff of 102 CFU/mL and above, this proposed hydrogel-based assay is suited to meet global requirements for screening breast milk or milk for pathogenic organisms of 104 CFU/mL, with a percentage of false positives to be determined in future efforts

Cross-Linked Alginate Film Pore Size Determination Using Atomic Force Microscopy and Validation Using Diffusivity Determinations

The deficit of organ donors has fueled the need for advances in tissue engineering and regenerative medicine. Microencapsulation in alginate immuno-isolation membranes has been used to treat many disabling metabolic disorders, namely, phenylketonuria, kidney failure and diabetes mellitus. Systematic nutrient flux determinations are hindered by the lack of experimental data on alginate-based membrane topography and the pore size thus preventing the full therapeutic potential of the bio-membranes to be reached. In this study, samples of cross-linked alginate membranes were subjected to the following analytical characterization: 1) pore size characterization using atomic force microscopy operated in contact mode to detect and measure pore size; 2) differential scanning calorimetry to confirm biopolymer cross-linking; and 3) diffusivity measurements using spectrophotometry and fluorescence microscopy to confirm the presence of through pores and to calculate reflection coefficients. The pore sizes for the pre-clinical standard formulation of 1.5% (w/v) medium viscosity alginate cross-linked with 1.5% CaCl2 and 0.5% (w/v) alginate and chitosan cross-linked with 20% CaCl2 are 5.2 nm ± 0.9 nm and 7.0 nm ± 3.1 nm, respectively. An increase in the glass transition temperatures as a function of cross-linker concentration was observed. Diffusivity values obtained from the inward diffusivity of creatinine into macrocapsules (d = 1000 μm ± 75 μm) and the outward diffusivity of FITC dextrans from macrocapsules (d = 1000 μm ± 75 μm) and microcapsules (d = 40 μm ± 5 μm) were shown to correlate strongly (R2 = 0.9835) with the ratio of solute to pore sizes, confirming the presence of through pores. Reflection coefficients approaching and exceeding unity correlate with the lack of permeability of the membranes to MW markers that are 70 kDa and greater

High-Throughput Methods for Miniaturization of Implantable Artificial Cells

Using three different fabrication methods, namely Electrostatic Spraying, On-Chip Synthesis and Inkjet Bio-Printing, miniaturization of alginatebased microcapsules ranging from 20–100 μm has been achieved. With a limited amount of published cell and drug-delivery data, the projected therapeutic applications differ by miniaturization technique, dependent in turn on the starting alginate concentration. The capsules miniaturized using the On-Chip Synthesis method would be ideal for implantation in tumors in non-invasive drug delivery applications. As for the other two methods with documented cell encapsulation feasibility, the benefits would be in the areas of programmed cell delivery, metabolic disorders and wider bio-membrane porosity studies. The effect of miniaturization on the strength, porosity and permeability of alginate capsules using Inkjet Bio-Printing with a throughput of 1.8 × 106 microcapsules/hr are the following: (1) Strength has been enhanced by a higher degree of cross-linking, thickening the cross-linking solution or subsequent coating steps as shown by results of spectrophotometric measurements; (2) Neither porosity nor the reflection coefficients have been affected for blue dextran (MW = 2000 kDa) and FITC dextran (MW = 4 kDa), as shown by results of Atomic Force Microscopy measurements of pore sizes; and (3) Permeability has been enhanced as a result of miniaturization when comparing standard 1000 μm macrocapsules (1.5% MV alginate cross-linked with 1.5% CaCl2) to 30 μm minicapsules (0.5% LV alginate cross-linked with 15% CaCl2 and coated with 0.5% chitosan) as shown by results of the Fluorescence Microscopy measurements. In light of these preliminary findings, the hypothesis of diffusion across the membrane being inversely correlatedto the surface to volume ratio (S/V) has been confirmed for artificial cell miniaturization, and the membrane strength and porosity can be tuned for a wide array of promising applications in cutting-edge areas of nanomedicine

Immobilization of R. erythropolis in alginate-based artificial cells for simulated plaque degradation in aqueous media

Cholesterol degradation rates of free and immobilized Rhodococcus erythropolis (ATCC # 25544) were studied utilizing the bacterium’s cholesterol oxidase enzyme pathway to degrade cholesterol in an aqueous simulated non-calcified plaque solution. An L16 (45) Taguchi design was used to minimize the glycolipid bio-surfactant by-product in the growth medium, to improve bacterial viability in the immobilized state. As an expected outcome of miniaturization, there is a significant difference between the atomized (d = 850 ± 50 μm) and inkjet-bioprinted (d = 32 ± 5 μm) lumped kinetic degradation rates after 48 h (p = 0.029, α = 0.05) per ml of jetted alginate. Based on a biphasic cholesterol degradation model, at an initial bacterial cell density of Nlow = 4.53 × 108/ml, for an initial cholesterol concentration of 3 mg/ml, the percentage mass of metabolite degraded is 37.0% ± 0.42%, 57.8% ± 0.04% and 65.1% ± 0.01% for the free, atomized and inkjet immobilized bacteria, respectively

Adsorption of chitin derivatives onto liposomes: Optimization of adsorption conditions

A 2′ factorial design is used to optimize the adsorption conditions of the hydrophilic anionic polyelectrolytes, Carboxymethylchitin (CMC) and Carboxymethyl/Glycolchitin (CO) onto liposomes at physiological ionic strength (I) and pH using phosphate buffered saline (PBS, I= 154mM, pH = 7.4). Positive ([+]) or high surface affinity liposomes (DSPC:CHOL:DMTAP, 5:4:1), and Neutral ([N]) or low surface affinity liposomes (DSPC:CHOL, 1:1) were used as adsorption surfaces. Results of the calculations of the main effects indicate that polymer molecular weight (mwt), Surface Affinity (S), Number of Adsorption Shots (Sh), Temperature (T), and the combinations mwt ×S, mwt ×Sh are the most important process parameters. Results of a study conducted at T = 37 °C show that no loss occurs from the positive surface at the highest particle concentratiqn, Np = 4.043 × 1011. Finally, the extent of polymer-induced particle aggregation PTIS decreased when the diameter of the uncoated liposomes is doubled from 0.22 to 0.45m. These results are as expected, given the stiffness and the dimensions of the macromolecules