Name: Preparation of Bioactive Compound Encapsulated Chitosan Nanoparticles
Uploaded: 2023-04-07T14:35:00
Description: Watch this Scientific Journal Video about Preparation of Bioactive Compound Encapsulated Chitosan Nanoparticles at JoVE.com

The video outlines a procedure for preparing nanoparticles using chitosin. It involves dissolving chitosin in acetic acid, adding Tween 80, incorporating bioactive compounds like carvacrol or thymol, and using pentasodium tripolyphosphate. The final steps include centrifugation, washing to remove impurities, and reconstituting the nanoparticles in deionized water.

preparation of bioactive compound encapsulated chitosan nanoparticles

Antimicrobial Efficacy of Nano-Herbal-Coated Fabrics

The protective white coat is a mandatory personal protective equipment (PPE) item in microbiology laboratories and healthcare facilities, and it protects from direct exposure to pathogens, spills, and burns. These cotton coats promote microbial growth due to many factors-the woven fabric provides attachment sites and aeration, cotton and starch used in the manufacturing process along with exfoliated epithelial cells from the user supply nutrients, and the proximity to the user gives warmth and moisture. The accumulation of microbes on textiles can also cause health problems such as allergies and nosocomial infection, unpleasant odors, and fabric deterioration1.
Unlike regular clothing, protective coats are infrequently washed or disinfected, as found in many surveys2,3. Many studies show evidence of lab coats acting as a vector of microbial transmission and the risk of nosocomial infections in the healthcare setting2,4, particularly resistant strains3 such as methicillin-resistant Staphylococcus aureus (MRSA); thus, they raise health concerns of PPE, which is meant to protect from microbial contamination. There are not enough cross-sectional studies on lab coat-associated infections in the context of Biosafety Level 2 (BSL-2) facilities or microbiology teaching labs, but many regulatory authorities restrict the use of lab coats within the containment level. However, many academic institutions in North America struggle to meet the requirements due to practical constraints, such as laundering and storing inside the facility, the incidents of wearing lab coats in public areas such as cafeterias and libraries are common. One practical solution to these issues is the application of antimicrobial agents in textile finishing.
Antimicrobial fabrics are gaining increasing popularity in sportswear, activewear, and socks, mainly intended to reduce body odor. However, the use of these fabrics is not common in PPE development, except for some silver-coated cotton masks and healthcare garments5. We report the development of an antimicrobial fabric for lab coats, which inhibits common pathogens found in BSL-2 labs and renders effective protection from the cross-contamination of common pathogens.
Currently, a variety of antimicrobial fabrics and finishings are available in the market, but most of these use heavy metal colloidal particles (e.g., silver, copper, zinc), organometallics, or synthetic chemicals such as triclosan and quaternary ammonium compounds, which are not environmentally friendly1 and may lead to health issues such as skin irritation and allergies6. Some synthetic formulations pose concerns due to non-target microbes, such as normal flora or inducing antimicrobial resistance (AMR). The US Food and Drug Administration (FDA) regulates commercial antimicrobial fabrics, which must be non-toxic to the user and free from eco-toxicity. Therefore, antimicrobial fabrics based on natural biocides that inhibit a broad spectrum of microbes are preferable. Essential oils (EOs) are used widely as antimicrobial and therapeutic agents, but their use in antimicrobial finishing is limited due to their durability6,7,8. Based on our knowledge and market research on nano-herbal finishing8, no herbal-based antimicrobial fabric is commercially available. This is because synthetic coatings are easy to manufacture and have long durability. A few nano-herbal-coated textiles reported only for research purposes include neem7, moringa9, and curry leaves9.
The present study uses two bioactive components extracted from oregano EOs, carvacrol and thymol, which are effective against a wide range of bacterial pathogens and viruses but are generally recognized as safe for humans10. However, these bioactive components are volatile, and therefore their antimicrobial potential is short-lived if applied directly to the fabric. Nano-herbal encapsulation is a process in which bioactive components or drugs are loaded inside a polymeric shell that protects the core from environmental degradation, and thus enhances the shelf life. In addition, the small size of the polymeric particles, which generally range from 10 nm to 100 nm, enhances the efficacy of the application and slows the release of the bioactive compounds onto the fabric. These bioactive compounds are used for various purposes, such as food preservation10, but not for textile coating.
Among many polymeric encapsulants, chitosan is an attractive candidate due to many of its attributes, such as nontoxicity, biodegradability, mucoadhesivity, and biocompatibility11. It is a natural polysaccharide, obtained by the deacetylation process from chitin, which is found in seashells and fungal cell walls. It is used in biochemical and food preservation applications such as drug or protein delivery11,12,13, controlled release14, and antimicrobial films10. Chitosan is not readily soluble in water but forms a colloidal suspension in acidic media. Bioactive molecules are loaded into chitosan nanoparticles (NPs) by a simple two-step ionic gelation method14,15,16. In this process, hydrophobic bioactive compounds such as carvacrol and thymol form an oil-in-water emulsion, which is aided by a surfactant, Tween 80. Subsequently, a polyanionic compound, pentasodium tripolyphosphate (TPP), is used to form the cross-linkages between the amino groups along the polycationic polymer molecules and phosphate groups of TPP molecules to stabilize the complex. This complexation process solidifies the bioactive compounds within the matrix of chitosan, which is subsequently purified and coated onto cotton swatches to produce antimicrobial fabric.
The nano-formulations must be tested first for antimicrobial effectiveness in emulsion form before being applied to the fabric. This can be conveniently evaluated by a qualitative method, such as Kirby-Bauer disk diffusion, well diffusion, and the cylinder plate assay. However, the cylinder plate assay17 provides the flexibility to load varying volumes of the formulation and compare the zone of clearance. In this method, the antimicrobial formulations are loaded in stainless steel cylinders and placed on a soft agar layer, which is inoculated with the test microorganism or pathogen. The diameter of the zone of clearance produced against the test organism is proportional to the inhibitory potential of the antimicrobial formulation, and therefore can be used as an alternative to broth dilution methods. However, the size of the clear zones is only a comparative or qualitative measure within a specific plate unless specific standards are maintained. Antimicrobial agents act against the pathogens either by inhibiting their growth (biostatic) or killing the cells (biocidal), which can be quantified by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), respectively. However, the efficacy and behavior of the bioactive chemicals are different in their formulations (liquid state) and when coated on a substrate such as a fabric18. This is because multiple factors play a role in the efficacy, such as the stability of the adherence of the antimicrobial agents to the fabric, moisture content, substrate type, and adherence of the microbes. If the intended purpose is only bacteriostatic activity, a qualitative assay such as the &#34;Parallel Streak Method&#34;19 can provide a relatively quick and easy evaluation of diffusible antimicrobial formulation. However, if the bactericidal effects are to be determined, &#34;Assessment of Antibacterial Finishes on Textile Materials&#34;20 can be employed, which provides the log reduction of the spiked pathogen.

Author Spotlight: An Antimicrobial Fabric Using Nano-Herbal Encapsulation of Essential Oils  

An Antimicrobial Fabric Using Nano-Herbal Encapsulation of Essential Oils

This research outlines the protocol to develop a safe and eco-friendly antimicrobial fabric which can be used to design lab coats. The study answers many key questions in the field of antimicrobial fabric development. For example, how effectively the treated fabric neutralizers pathogens.The key challenge in antimicrobial fabric development is to choose the right ratios of bioactive compounds without compromising antimicrobial efficacy while maintaining skin friendly finishes. Our antimicrobial fabric provides effective protection against four human pathogens with up to four log reduction, or simply 99.99%effectiveness. It also resists up to 10 wash cycles while retaining an efficacy of up to 90%The major advantage of this method over existing standard techniques is that it provides a simplified and realistic approach to accurately evaluate the efficacy of antimicrobial fabrics.It better represents the typical scenarios of lab coat usage, such as any accidental microbial spills that must be neutralized within a short time. The application of the technique in the manufacturing of protective coats will reduce the biohazard risk in biosafety labs and nosocomial infections in healthcare setting. We are in the process of testing full length antimicrobial lab coats in Biosafety Level 2 labs.Our future research questions would focus on the effectiveness of preventing microbial contamination in the long run and continuous usage.

Watch this Scientific Journal Video about Preparation of Bioactive Compound Encapsulated Chitosan Nanoparticles at JoVE.com

Preparation of Bioactive Compound Encapsulated Chitosan Nanoparticles  

To begin, prepare chitosin solution by dissolving 0.6 grams of chitosin flakes in 50 milliliters of 1%acetic acid. Agitate overnight at room temperature to get a homogenous emulsion. Add 0.5 grams of Tween 80 and stir at 60 degrees Celsius for two hours at 1, 000 RPM to get a homogenous solution.After bringing the solution to room temperature, add 0.75 grams of bioactive compound carvacrol or thymol dropwise or gradually while stirring the mixture for 20 minutes. Add 50 milliliters of pentasodium tripolyphosphate dropwise to the mixture while stirring at room temperature. Continue stirring for 30 minutes to get a homogenous emulsion.For purification, centrifuge the emulsion at 10, 000 G for 30 minutes at four degrees Celsius. Decant the supernatant and collect the formed nanoparticles. Wash the particles using aqueous Tween 80 with double the volume of pellets formed to remove the unbound or free bioactive compounds.Then wash the particles with deionized water twice to get rid of impurities. Reconstitute the nanoparticles by resuspending the pellet in 30 milliliters of deionized water.

preparation-of-bioactive-compound-encapsulated-chitosan-nanoparticles

Research

JoVE Journal

Biology

Lab coats are widely used in biohazard laboratories and healthcare facilities as protective garments to prevent direct exposure to pathogens, spills, and burns. These cotton-based protective coats provide ideal conditions for microbial growth and attachment sites due to their porous nature, moisture-holding capacity, and retention of warmth from the user&#39;s body. Several studies have demonstrated the survival of pathogenic bacteria on hospital garments and lab coats, acting as vectors of microbial transmission.
A common approach to fix these problems is the application of antimicrobial agents in textile finishing, but concerns have been raised due to the toxicity and environmental effects of many synthetic chemicals. The ongoing pandemic has also opened a window for the investigation of effective antimicrobials and eco-friendly and toxic-free formulations. This study uses two natural bioactive compounds, carvacrol and thymol, encapsulated in chitosan nanoparticles, which guarantee effective protection against four human pathogens with up to a 4-log reduction (99.99%). These pathogens are frequently detected in lab coats used in biohazard laboratories.
The treated fabrics also resisted up to 10 wash cycles with 90% microbial reduction, which is sufficient for the intended use. We made modifications to the existing standard fabric tests to better represent the typical scenarios of lab coat usage. These refinements allow for a more accurate evaluation of the effectiveness of antimicrobial lab coats and for the simulation of the fate of any accidental microbial spills that must be neutralized within a short time. Further studies are recommended to investigate the accumulation of pathogens over time on antimicrobial lab coats compared to regular protective coats.

Antimicrobial lab coats prevent the cross-contamination of pathogen accumulation and accidental bio-spills. Here, we describe the protocol for developing a skin-friendly antimicrobial fabric using nano-herbal encapsulation and modified standard tests to precisely evaluate the efficacy and suitability for typical usage of the lab coat.

Lab coats are widely used in biohazard laboratories and healthcare facilities as protective garments to prevent direct exposure to pathogens, spills, and ...

Qualitative Screening of Nanoparticle-Treated Fabric: The Parallel Streak Method

Take a nanoparticles treated fabric and cut it into 50 millimeters by 25 millimeter swatches. Inoculate the microbial cultures from fresh purity plates to Trypticase soy broth at 35 degrees Celsius overnight with moderate shaking. Dilute the overnight cultures to 150 million CFU per milliliter, or corresponding to the turbidity of 0.5 McFarland standard.To inoculate the diluted culture with a four millimeter sterile loop, load a loop full of broth culture and transfer it to the surface of the Mueller-Hinton agar or MHA agar plate by making five parallel streaks of six centimeters in length and one centimeter apart from each other. Gently press the fabric swatch using a sterile spatula across the five streaks so that the fabric is in the center and touches all five streak lines. Incubate at 35 degrees Celsius for 24 hours.For the qualitative evaluation of antimicrobial efficacy, examine the incubated plate for the interruption of growth along the streaks beyond the edges of the fabric. Calculate the average width of a zone of inhibition along the streak line W on either side of the fabric swatch using the equation shown on the screen. The results of the parallel streak method are presented in this figure, the untreated fabric and the thymol nanoparticles coated fabric against S-aureus and the untreated fabric and the thymol nanoparticles coated fabric against C-Albicans are shown here.These results show clear zones for the treated fabric swatches.

Quantitative Screening of Nanoparticle-Treated Fabric: The Log Reduction Method

Take a nanoparticles-treated fabric, and cut it into 50 millimeters by 50 millimeters square swatches. Prepare the overnight cultures of microbes of interest by inoculating the isolated colonies from the purity plates to sterile Trypticase soy broth. Incubate at 35 degrees Celsius for 18 to 24 hours.Dilute the overnight cultures to 150 million CFU per milliliter or corresponding to the turbidity of 0.5 McFarland standard. Next, to determine the appropriate volume of cultures for spiking, add a series of volumes of the diluted broth culture onto the fabric swatches, and choose the volume such that the fabric swatch absorbs the water fully and leaves no residual or free liquid. Spike the volume of each culture onto the swatches placed in sterile Petri plates.Similarly, perform the negative control with the same type of untreated fabric swatches for each corresponding test. To recover microbes by viable plate counts, air dry the inoculated swatches inside the Petri plates at room temperature for the required contact periods to be tested. Transfer the swatches aseptically to separate sterile centrifuge tubes, and screw the caps tightly.Add Letheen Broth or any relevant neutralizing buffers to make a 1:100 dilution. After closing centrifuge tubes, vortex for one minute at a medium speed. Serially dilute the suspension with the sterile PBS in subsequent 1:10 dilutions, such that the colony counts of the untreated group becomes too low to count.Plant the dilutions on appropriate media plates which support the growth of the microorganism or any media that optimize the growth and provide good contrast to make the colony counting accurate. Incubate the bacterial plates at 35 degrees Celsius for two days and the fungal plates at room temperature for five days. Count the viable colony-forming units directly using a colony counter or imaging software.Calculate the microbial log reduction R due to the antimicrobial fabric using the equation shown on the screen. Here, A is the log value of the number of colony-forming units recovered from untreated fabric, and B is the same for treated fabric. The log reduction results of antimicrobial fabric coated with thymol-encapsulated chitosan nanoparticles tested against S.aureus on blood agar, E.coli on purple lactose agar, P.aeruginosa on cetrimide agar, and C.albicans on Sabouraud dextrose agar are shown here.The pathogens were spiked on untreated and treated swatches for 30 minutes and recovered upon neutralization and dilution plating. The dilution ratios are shown between the treated and untreated series. The log reduction of three bacteria and one fungus due to the contact of antimicrobial fabrics impregnated with two bioactive compounds are shown here.The antimicrobial efficacy is weakened after wash cycles for both carvacrol and thymol-coated fabrics.