Novel Honokiol-eluting PLGA-based scaffold effectively restricts the growth of renal cancer cells.

Renal Cell Carcinoma (RCC) often becomes resistant to targeted therapies, and in addition, dose-dependent toxicities limit the effectiveness of therapeutic agents. Therefore, identifying novel drug delivery approaches to achieve optimal dosing of therapeutic agents can be beneficial in managing toxicities and to attain optimal therapeutic effects. Previously, we have demonstrated that Honokiol, a natural compound with potent anti-tumorigenic and anti-inflammatory effects, can induce cancer cell apoptosis and inhibit the growth of renal tumors in vivo. In cancer treatment, implant-based drug delivery systems can be used for gradual and sustained delivery of therapeutic agents like Honokiol to minimize systemic toxicity. Electrospun polymeric fibrous scaffolds are ideal candidates to be used as drug implants due to their favorable morphological properties such as high surface to volume ratio, flexibility and ease of fabrication. In this study, we fabricated Honokiol-loaded Poly(lactide-co-glycolide) (PLGA) electrospun scaffolds; and evaluated their structural characterization and biological activity. Proton nuclear magnetic resonance data proved the existence of Honokiol in the drug loaded polymeric scaffolds. The release kinetics showed that only 24% of the loaded Honokiol were released in 24hr, suggesting that sustained delivery of Honokiol is feasible. We calculated the cumulative concentration of the Honokiol released from the scaffold in 24hr; and the extent of renal cancer cell apoptosis induced with the released Honokiol is similar to an equivalent concentration of direct application of Honokiol. Also, Honokiol-loaded scaffolds placed directly in renal cell culture inhibited renal cancer cell proliferation and migration. Together, we demonstrate that Honokiol delivered through electrospun PLGA-based scaffolds is effective in inhibiting the growth of renal cancer cells; and our data necessitates further in vivo studies to explore the potential of sustained release of therapeutic agents-loaded electrospun scaffolds in the treatment of RCC and other cancer types.

Role of fiber diameter in adhesion and proliferation of NIH 3T3 fibroblast on electrospun polycaprolactone scaffolds.

The goal of the current study was to find the quantitative relationship between electrospun polycaprolactone scaffold fiber diameter and NIH 3T3 fibroblast adhesion and growth kinetics. By varying 3 important process parameters--solution concentration, voltage, and collector screen distance--different average fiber diameters ranging from 117 to 1,647 nm were obtained. Although 117 nm represented the lowest possible fiber diameter obtainable, these fibers had beads in them. An increase in fiber diameter to 428 nm led to uniform fibers without any beads. Fiber distribution pattern was a single mode for all the scaffolds except at the largest-diameter end. The diameter distribution changed from single to bimodal at 1,647 nm, suggesting some instability in the process. It was found that cell adhesion and growth kinetics are significantly affected as a function of fiber diameter. Beaded scaffolds offered the lowest cell adhesion and minimal growth kinetics despite having the lowest average fiber diameter. When uniform fibers were formed and the average fiber was in the nanofiber range (428-1051 nm), cell adhesion and growth kinetics decreased as a function of increasing fiber diameter. Cell adhesion kinetics remained invariant when the average fiber diameter was in the micron range (1,647 nm), whereas cell-growth kinetics were slightly greater than with 900 nm scaffolds. We propose that the uniformness of fibers and the average fiber diameter may play an important role in modulating cellular attachment and proliferation in electrospun tissue engineering scaffolds.

A flexible approach to the preparation of polymer scaffolds for tissue engineering.

Porous polyester thermoset xerogels have been produced via sol-gel chemistry as a first step in the development of sol-gel derived tissue engineering scaffolds templated by replica molding and/or salt leaching. The pore structure of these untemplated thermosets is tunable and can be altered independent of or in tandem with alterations in composition. Cytocompatibility studies on these xerogels imply the effects of both pore size and materials chemistry, with fully aliphatic polyesters with large pore structures allowing the growth of mammalian cells. To the best of our knowledge, this represents the first report examining the preparation and potential of sol-gel derived porous polymer xerogels as tissue engineering scaffolds.

pH-responsive drug release from functionalized electrospun poly(caprolactone) scaffolds under simulated in vivo environment.

The difference in the tumor environment from the normal healthy tissue can be therapeutically exploited to develop new strategies for controlled and site-specific drug delivery. In the present study, a continuous flow system is designed to represent the in vivo environment of a tumor tissue and drug release is studied at different pH that represents normal tissue pH, tumor tissue pH, and stomach pH. The results obtained from these experiments were translated to a human embryonic kidney cell culture system and the effect of drug released from these functionalized PCL scaffolds on cell viability was studied. A significant decrease in cell viability was observed with the doxorubicin hydrochloride concentration that would be released at acidic pH, either present as a result of tumor extracellular environment or could be achieved via fabrication of a composite scaffold with a polyvinyl alcohol hydrogel containing acid. In the end, a study using zebrafish as an animal model is also undertaken in order to study the drug release from the scaffolds in vivo.

Functionalization of electrospun poly(caprolactone) fibers for pH-controlled delivery of doxorubicin hydrochloride.

Functionalized electrospun polymer fibers are a promising candidate for controlled delivery of chemotherapeutic drugs to improve the therapeutic efficacy and to reduce the potential toxic effects by delivering the drug at a rate governed by the physiological need of the site of action. In this study, poly(caprolactone) (PCL) fibers were fabricated by electrospinning, followed by hydrolyzation to introduce functional groups on the fiber surface. Characterization studies were performed on these functionalized fibers using X-ray photoelectron spectroscopy, scanning electron microscopy, and Toluidine Blue O dye assay. The pH-sensitivity of the functional groups on the fiber surface and doxorubicin hydrochloride was utilized to bind the drug electrostatically to these functionalized PCL fibers. The effect of pH on drug loading and release kinetics was investigated. Results indicate successful electrostatic binding of the drug to functionalized electrospun fibers and a high drug payload. The drug delivery response can be modulated by introduction of suitable stimuli (pH).

Fibronectin adsorption on functionalized electrospun polycaprolactone scaffolds: experimental and molecular dynamics studies.

Designing scaffolds to modulate protein adsorption is a key to building advanced scaffolds for tissue regeneration. Protein adsorption to tissue engineering scaffolds is critical in early cell attachment, survival, and eventual proliferation. The goal of this study is to examine the effect of functionalization on fibronectin adsorption to electrospun polycaprolactone (PCL) scaffolds through experimentation using fluorescently labeled fibronectin and to couple this experimental data with analysis of interaction energies obtained through molecular dynamics (MD) simulations to develop a better understanding of the adsorption process. This study is the first to analyze and compare experimental and MD simulation results of fibronectin adsorption on functionalized electrospun PCL scaffolds. Electrospun nanofiber PCL scaffolds were treated with either 1 N NaOH (hydrolyzed) or 46% hexamethylenediamine (HMD) (aminated) solution to be compared with untreated (control) scaffolds. We found that aminated PCL scaffolds experimentally adsorbed more fibronectin than control scaffolds, whereas hydrolyzed scaffolds showed decreased adsorption. MD simulations carried out with NVT ensemble at a temperature of 310 K indicated a higher work of adhesion for both functionalized scaffolds over control. Also, the simulations revealed different conformations of fibronectin on each scaffold type after adsorption, with the arginine-glycine-aspartic acid sequence appearing most accessible on the aminated scaffolds. This suggests that functionalization affects not only the quantity of protein that will adsorb on a scaffold but how it attaches as well, which could affect subsequent cell attachment.

Altering surface characteristics of polypropylene mesh via sodium hydroxide treatment.

Incisional hernias represent a serious and common complication following laparotomy. The use of synthetic (e.g. polypropylene) meshes to aid repair of these hernias has considerably reduced recurrence rates. While polypropylene is biocompatible and has a long successful clinical history in treating hernias and preventing reherniation, this material may suffer some limitations, particularly in challenging patients at risk of wound failure due to, for example, an exaggerated inflammation reaction, delayed wound healing, and infection. Surface modification of the polypropylene mesh without sacrificing its mechanical properties, critical for hernia repair, represents one way to begin to address these clinical complications. Our hypothesis is treatment of a proprietary polypropylene mesh with sodium hydroxide (NaOH) will increase in vitro NIH/3T3 cell attachment, predictive of earlier and improved cell colonization and tissue integration of polypropylene materials. Our goal is to achieve this altered surface functionality via enhanced removal of chemicals/oils used during material synthesis without compromising the mechanical properties of the mesh. We found that NaOH treatment does not appear to compromise the mechanical strength of the material, despite roughly a 10% decrease in fiber diameter. The treatment increases in vitro NIH/3T3 cell attachment within the first 72 h and this effect is sustained up to 7 days in vitro. This research demonstrates that sodium hydroxide treatment is an efficient way to modify the surface of polypropylene hernia meshes without losing the mechanical integrity of the material. This simple procedure could also allow the attachment of a variety of biomolecules to the polypropylene mesh that may aid in reducing the complications associated with polypropylene meshes today.

Controlled vacuum seeding as a means of generating uniform cellular distribution in electrospun polycaprolactone (PCL) scaffolds.

A major challenge encountered in using electrospun scaffolds for tissue engineering is the non-uniform cellular distribution in the scaffold with increasing depth under normal passive seeding conditions. Because of the small surface pores, typically few microns in diameter, cells tend to congregate and proliferate on the surface much faster compared to penetrating the scaffold interior. In order to overcome this problem, we used a vacuum seeding technique on polycaprolactone electrospun scaffolds while using NIH 3T3 fibroblasts as the model cell system. This serves as a precursor to the bilayer skin model where the fibroblasts would be residing at an intermediate layer and the keratinocytes would be on the top. Vacuum seeding was used in this study to enhance fibroblasts seeding and proliferation at different depths. Our results show that the kinetics of cell attachment and proliferation were a function of varying vacuum pressure as well as fiber diameter. Cell attachment reached a maxima somewhere between 2-8 in. Hg vacuum pressure and fell for lower vacuum pressures presumably because of cell loss through the filtration process. Cell proliferation and collagen secretion over five days indicated that vacuum pressure did not affect cellular function adversely. We also compared the combined impact of scaffold architecture (400 nm versus 1100 nm average diameter fiber scaffolds) and vacuum pressure. At a given pressure, more cells were retained in the 400 nm scaffolds compared to 1100 nm scaffolds. In addition, the cell intensity profile shows cell intensity peak shift from the top to the inner layers of the scaffold by lowering the vacuum pressure from 0 in. Hg to 20 in. Hg. For a given vacuum pressure the cells were seeded deeper within the 1100 nm scaffold. The results indicate that cells can be seeded in electrospun scaffolds at various depths in a controlled manner using a simple vacuum seeding technique. The depth of seeding is a function of pressure and scaffold fiber diameter.

Role of electrospun fibre diameter and corresponding specific surface area (SSA) on cell attachment.

In order to develop scaffolds for tissue regeneration applications, it is important to develop an understanding of the kinetics of cell attachment as a function of scaffold geometry. In the present study, we investigated how the specific surface area of electrospun scaffolds affected cell attachment and spreading. Number of cells attached to the scaffold was measured by the relative intensity of a metabolic dye (MTS) and cell spreading was analysed for individual cells by measuring the area of projected F-actin cytoskeleton. We varied the fibre diameter to obtain a specific surface area distribution in the range 2.24-18.79 microm(-1). In addition, we had one case where the scaffolds had beads in them and therefore had non-uniform fibres. For each of these different geometries, we varied the cell-seeding density (0.5-1 x 10(5)) and the serum concentration (0-12%) over the first 8 h in an electrospun polycaprolactone NIH 3T3 fibroblast system. Cells on beaded scaffolds showed the lowest attachment and almost no F-actin spreading in all experiments indicating uniform fibre diameter is essential for electrospun scaffolds. For the uniform fibre scaffolds, cell attachment was a function of scaffold specific surface area (SSA) (18.79-2.24 microm(-1)) and followed two distinct trends: when scaffold SSA was < 7.13 microm(-1), cell adhesion rate remained largely unchanged; however, for SSA > 7.13 microm(-1) there was a significant increase in cellular attachment rate with increasing SSA. This indicated that nanofibrous scaffolds increased cellular adhesion compared to microfibrous scaffolds. This phenomenon is true for serum concentrations of 7.5% and higher. For 5% and lower serum concentration, cell attachment is low and higher SSA fails to make a significant improvement in cell attachment. When cell attachment was investigated at a single-cell level by measuring the projected actin area, a similar trend was noted where the effect of higher SSA led to higher projected area for cells at 8 h. These results indicate that uniform electrospun scaffolds with SSA provide a faster cell attachment compared to lower SSA and beaded scaffolds. These results indicate that continuous electrospun nanofibrous scaffolds may be a good substrate for rapid tissue regeneration.

Forced and natural convective drying of trehalose/water thin films: implication in the desiccation preservation of Mammalian cells.

Trehalose is believed to offer desiccation protection to mammalian cells by forming stable glassy matrices. The goal of the current study was to explore the desiccation kinetics of thin films of trehalose-water solution under forced and natural convective conditions and to investigate the thermophysical state of mammalian cells at the bottom of the thin film. We developed a finite difference model based on the mass and energy conservation equations coupled to the water transport model from the cells. The boundary conditions were obtained from correlations or experimental measurements and the Gordon-Taylor equation was used to predict the glass transition temperature at every location. Results indicated that there are three distinct regimes for drying for both forced and natural convection, characterized by the slope of the moisture content plot as a function of time. Our results also indicate that the surface of the solution reached the glassy state in less than 10 min for the Reynolds (forced) numbers explored and approximately 30 min for some Rayleigh (natural convective) numbers; however, significant water was trapped at this instant. Larger drying force hastened quicker glass formation but trapped more water. The numerical model was capable of predicting the drying kinetics for the dilute region accurately, but deviated while predicting the other regimes. Based on these experimental validations of the model, the osmotic response of different cells located at the bottom of the solution with orders of magnitude difference in their membrane permeability (Lp) was predicted. The results suggested that extracellular glass formed around cells at the bottom of a trehalose-water solution by the propagation of glass into the solution; however it takes more than an order of magnitude time (approximately 7 min to >100 min for forced convective drying) to remove sufficient water to form glass around cells from the time when the first surface glass is formed. This is attributed to low diffusivity of water through the glass. In addition, the water transport from the glassy matrix could be either diffusion or Lp limited. For diffusion-limited transport, lowering the film thickness at the beginning of drying by half almost lowers the drying time by an order of magnitude. In summary, the optimal design of convective desiccation protocols requires accounting for the size of the cell, their membrane permeability (Lp) and the starting thickness of the solution.