Optimizing Production, Characterization, and In Vitro Behavior of Silymarin–Eudragit Electrosprayed Fiber for Anti-Inflammatory Effects: A Chemical Study

Abstract

Introduction

Inflammatory bowel disease (IBD) encompasses chronic or recurrent inflammation of the gastrointestinal (GI) tract, primarily including ulcerative colitis (UC), which affects the colon, and Crohn’s disease (CD), which can involve the entire GI tract [1]. IBD predominantly affects individuals aged 20–40 years, imposing a significant socioeconomic burden. The relapse rate for IBD is 50–80%, requiring long-term management and increasing healthcare costs. There have been numerous pharmacological medical therapies which have been proposed for IBD, like Salicylates, glucocorticoids and immunosuppressives [2], but its medical management remains challenging, and investigations into novel treatments such as herbal extracts containing polyphenols as a new strategy for management of IBD are currently in the center of attention [3,4].

Pharmacological studies with experimental animals over the past decade indicate that polyphenols are effective in preventing and alleviating complications of ulcerative colitis (UC) [5]. Among the various botanicals examined, curcumin, the primary component of turmeric, is a highly researched phytochemical for treating inflammatory bowel disease (IBD) and other conditions, due to its non-toxic, antioxidant, anti-inflammatory, and cytoprotective properties. Numerous preclinical studies have shown that curcumin improves survival rates and reduces discomfort caused by chemical ulcerogens, scavenges free radicals, influences multiple signaling pathways, and inhibits enzymes like COX-1, COX-2, and LOX. Clinical studies suggest that curcumin can inhibit clinical relapse in IBD patients [6,7]. Similarly, resveratrol, found in grapes and other berries, offers multiple pharmacological benefits, including preventing colitis, reducing colorectal carcinogenesis, and improving disease prognosis by modulating immune cell numbers and inflammatory markers [7,8]. Quercetin, a flavonoid present in various fruits and vegetables, exhibits free-radical scavenging, antioxidant, and anti-inflammatory properties, effective in early stages of TNBS-induced colitis [7,9]. Kaempferol, found in many edible plants, has antioxidant, anti-inflammatory, antimicrobial, anticancer, and other protective activities, significantly reducing DSS-induced colitis [7,10]. Ellagic acid from berries and pomegranates, rutoside from buckwheat, green tea polyphenols, and grape seed polyphenols have all been shown to reduce the severity of dextran sodium sulfate and 2,4,6-trinitrobenzene sulfonic acid-induced colitis, alleviate oxidative stress, and prevent inflammation-associated colon carcinogenesis, due to their anti-inflammatory and antioxidant properties [11,12]. Collectively, these phytochemicals exhibit significant potential in treating IBD and other inflammatory conditions.

Silymarin, a herbal extract from milk thistle seeds, with a complex composition of four flavonolignans, silybin, isosilybin, silydianin and silychristin, is most known for its hepatoprotective qualities [13]. The drug has also been shown to positively treat the colon, especially in cases involving ulcerative colitis [14]. Additionally, silymarin has antioxidant properties, enhances antioxidant enzyme activity, interacts with cell membranes to prevent lipid deformity, and manages toxic stress. Additionally, silymarin exhibits anti-inflammatory effects by stabilizing mast cells, inhibiting neutrophil infiltration, decreasing adhesion molecules, downregulating leukotriene and prostaglandin synthesis, and inhibiting key cytokines involved in inflammatory responses such as interleukin (IL)-6 and transforming growth factor (TGF)-β, which are key cytokines in the differentiation of regulatory T cells CD4 + CD25-Foxp3- and T helper (Th) 17 cells; it also has inhibitory effects on inducible nitric oxide (NO) synthase activity [15,16,17,18,19]. The therapeutic and biochemical effects of silymarin that have been studied show promising evidence of its positive influence on various types of cells. In cases of Amanita mushroom poisoning, silymarin has been shown to be an active treatment and to reduce patient mortality [5,20]. Additionally, silymarin has demonstrated a strong anti-angiogenesis effect on the colon cancer cell line in vitro [21]. Due to the significant antioxidant, anti-inflammatory, and immunomodulatory properties of silymarin, we investigated its potential protective antioxidant activity through an assay. However, one problem with silymarin is that the drug is poorly soluble in water [22], leading to investigations involving drug encapsulation for the targeted release of silymarin in the body. Studies have accomplished extremely small particle size (461 ± 173 nm) with a combination of polyvinylpyrrolidone and silymarin by homogenization via nanoprecipitation, demonstrating increased solubility and enhanced antioxidant activities.

Antioxidant activities were demonstrated by the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) method [10]. Biopolymer-based and lipid-based systems have also been investigated [23], as new procedures involving nano-encapsulation of silymarin can help the drug bypass factors, limiting its poor oral consumptive properties for more effective drug loading and drug release. While silymarin nanoparticles have also been prepared using emulsion solvent evaporation and freeze-drying methods to improve solubility [24], an electrospray method for improved silymarin solubility and pH-dependent targeted drug release in the colon has not been developed. The hepatoprotective activity of silymarin nanoformulations has been confirmed with a mixture of soy lecithin, with an average diameter between 138.9 nm and 1155 nm, indicating improved therapeutic efficacy of silymarin with a nano approach [25]. Silymarin nanoencapsulations even been shown to have antimicrobial applications when formulated in combination with chitosan (WCS) and poly-γ-glutamic acid (γ-PGA) for improved water solubility in food additives and food packaging, compared to unencapsulated silymarin [26]. The therapeutic and biochemical effects of silymarin that have been investigated show promising evidence of its positive influence on various types of cells. For example, silymarin has been shown to increase superoxide dismutase activity in humans, which plays a key role in defending cells from free radicals. While various delivery methods for silymarin such as biopolymer-based and lipid-based systems have been investigated [7], new procedures involving nano-encapsulation and micro-encapsulation of silymarin can help the drug bypass factors limiting its poor oral consumptive properties for more effective drug loading and drug release.

Electrospraying (electro-hydrodynamic spraying) is a process for generating droplets by applying an electric field. In this process, a drug/polymer solution is subjected to an electric field flowing out from a capillary nozzle maintained at high potential [27]. When the electric field attains a critical value, a jet is formed. The electric field then causes deformation and distribution of the jet into droplets. Indeed, electrospraying and electrospinning are based on the same principles, except that the jet formed in electrospinning does not break into droplets but produces a micro- or nanofiber [28]. Generally, both nanofibers and nanoparticles function to increase bioavailability, due to their unique physicochemical properties. The main difference in, and reason for, the use of nanofibers with silymarin is that they typically offer a larger surface area-to-volume ratio compared to nanoparticles; this can enhance bioavailability and provide controlled release, better stability, and improved interaction with biological tissues, which is necessary for pH-dependent target release in the colon surface area and a greater sustained-release profile of nanofibers. The major advantage of electrospun fibers is that the system can be set up for release and delivery of multiple drugs, with multi polymer and drug systems [29]. Conversely, nanoparticles provide a more uniform and potentially faster release profile due to their smaller size, which can be advantageous for rapid-onset applications. The electrospraying procedure has many advantages when compared to nanoprecipitation, such as producing smaller droplet sizes with narrow distribution, the absence of droplet agglomeration and coagulation, since charged droplets are self-dispersing in the space, and easy control of motion and deposition efficiency of charged droplets. Essentially, highly charged potential forces can result in dividing charged droplets into smaller droplets. This is defined as Coulomb fission of the droplets, which causes original dispersed droplets to form many smaller, more stable droplets. The bulk forces include electro-dynamic forces, inertia, gravity, and drag forces, which are the physics governing electrospraying. When the induced droplet flows and deforms, (as a Taylor cone-jet), surface stresses act against surface tension including electro-dynamic stress (proportional to the charge density on the surface of the jet, and on the local electric field), pressure differential across the jet–air interface, and stresses due to liquid dynamic viscosity and inertia [30]. The literature reviews on the process and its application in the pharmaceutical field include the following: nano/micro particles were produced for drug delivery such as PLGA nanoparticles with Paclitaxel [31], electrosprayed coenzyme Q10 in copovidone (Kollidon® VA64) [32], streptokinase-loaded PLGA nanoparticles [33], encapsulating drugs such as paclitaxel and topotecan in PLGA-chitosan [34], resveterol in Hyaluronic acid-ceramide and soluplus [35], Doxorubicin in PVA-silk fibroin [36], Oridonin in PLGA [37], encapsulating DNA, and enzymes such as PEG and trehalose were added to PLGA to prepare vascular endothelial growth factor (VEGF)- or bone morphogenetic protein 7 (BMP-7)-loaded carriers via electrospraying [38], polymeric coatings on medical implants [39], and biomolecule carriers for tissue and bone regeneration. These are applications explored for electrospraying techniques [40,41]. Polyvinylpyrrolidone and Sodium dodecyl sulphate loaded with silymarin-laden nanocontainers with a particle size of <1000 nm have been developed successfully for improved aqueous solubility y (26,432.76 ± 1749.00 μg/mL) and dissolution (~92% in 20 min), compared to plain drug powder [42].Cellulose acetate (CA) fibers with silymarin were made into fibers using the electrospinning method with 608 ± 133 nm diameter with 12.5 kV voltage application when the needle is 15 cm away from the spinning drum, and researched the stable drug release 120 min 1/1 phosphate buffer/methanol medium pH 7.4 at 37 °C [43]. In another study, polycaprolactone (PCL) loaded with silymarin with different concentrations (mainly 5, 7.5 and 10 wt%) were produced by electrospinning to develop a functional wound dressing. In vitro drug-release studies were conducted using phosphate-buffered solution (PBS) at pH 7.4 and 36 °C, and time-dependent release values were examined [44]. Eudragit®-based electrospray/spin optimization was conducted by employing Eudragit® E PO and Chlorpheniramine Maleate, where 35% Eudragit E PO at a gap distance of 175 mm and a flow rate of 1 mL/h were identified as optimum conditions for fiber production [45] and for ketoprofen (KET)-loaded Eudragit® L and Eudragit® S nanofibers. The electrospinning technique for buccal administration to treat oral mucositis was used, where optimum conditions were a lower flow rate (0.5 mL/h), as higher flow rates led to thicker fibers and structural deformations, due to insufficient drying. A voltage of 15 kV and 20% w/v Eudragit® S100 with 10% w/v KET demonstrated high drug loading efficiency [46]. In another study, Eudragit® L 100 was electrospun with diclofenac sodium, where the Eudragit® L 100 was set to 20% (w/v) in ethanol and DMAc in a 5:1 ratio, with an applied voltage of 10 kV, and a flow rate 1.0 mL/h. Different drug concentrations were tested: 9.1%, 16.7%, and 33.3% (w/w); higher drug concentrations were observed to increase the fiber diameter and potentially affect the surface morphology [47]. In another study, Eudragit® S100 Nanofibers were prepared with Aspirin and a Eudragit® S100 concentration of 15% w/v, with a polymer: drug ratio of 5:1, an applied voltage of 15 kV and an average flow rate of 1 mL/h, resulting in average diameter of 800 ± 110 nm [48]. In another study, Eudragit® L100 fibers 25% (w/v) were mixed in N, N-Dimethylacetamide (DMAc), ethanol, and methanol, with an applied voltage of 12 kV and flow rate maintained at 2.0 mL/h [49]. Finally, another study employed 13 % w/v Eudragit® S100 with 5-Fluorouracil, a mixture of ethanol and N,N-dimethylformamide (DMF) (8:2 v/v), along with other ingredients, with an applied voltage of 14.5 kV–16 kV and flow rate of 1.5 mL/h, with drug concentration maintained at 10% w/v [50].

In this study, the polymer Eudragit® S100 was combined with silymarin to create a colon-targeted drug-delivery system using vertical electrospray technology. The resulting complex, termed the SILS100-Electrofiber complex, was formulated with an 11.2% ratio of silymarin to Eudragit® S100. This complex represents the first successful micro-encapsulation of silymarin into fibers. The paper details the electrospray process and its optimization, characterizes the complex, and examines its pH-dependent release properties. The findings demonstrate that this formulation enhances aqueous solubility and improves the dissolution rate in the colon’s specific pH environment, indicating its potential for effective colon-targeted drug delivery.

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Materials

The experimental materials consisted of silymarin, an Antioxidant Colorimetric Assay Kit, and Spin-X^® Centrifuge Tubes purchased from Sigma Aldrich, St. Louis, MO, USA, Eudragit^® S100 donated from Evonik Industries, Essen, Germany, Spectrum Spectra/Por Float-A-Lyzer G2 Dialysis Devices (3.5–5 kD), Simulated Gastric Fluid (SGF) without pepsin and Simulated Intestinal Fluid (SIF) without pepsin purchased from Fisher Scientific Inc. located in Pittsburg, PA, USA.

Madiyar, F.; Suskavcevic, L.; Daugherty, K.; Weldon, A.; Ghate, S.; O’Brien, T.; Melendez, I.; Morgan, K.; Boetcher, S.; Namilae, L. Optimizing Production, Characterization, and In Vitro Behavior of Silymarin–Eudragit Electrosprayed Fiber for Anti-Inflammatory Effects: A Chemical Study. Bioengineering 2024, 11, 864. https://doi.org/10.3390/bioengineering11090864

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