Halloysites are naturally occurring nanotubes (118). The halloysite nanotube can be loaded with drugs and can be used for sustained release. This lengthens the effective life of the drug as the drug migrates out of the tubes slowly. Drug loaded halloysite tubes can be encapsulated to significantly influence the rate of drug release. This helps in extending the effectiveness of drugs without increasing strength. Compared with carbon nanotubes (CNTs), halloysite nanotubes are cheaper and have a larger surface area. As the halloysite nanotube has a larger surface area, it allows for more control of drug loading and drug release. Activation of drug loaded halloysite nanotubes can be done by coating the nanotubes with nanomagnetic material that can be then heated at a specific electromagnetic energy. Thus, heating can provide release of a drug on demand.
The benefits of using halloysite in drug delivery applications are longer delivery times, more control of the drug release profile, and improved safety profiles. Drug loaded halloysite technology can be used for drug loaded wound care products. There are a number of potential benefits of this technology, including the elimination of the high initial delivery rate and a better safety profile. Using drug loaded halloysites, we can deliver drugs more uniformly, which increases the effectiveness of a clinical dose. Due to this, less drug loading is required per patch.
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There are many different wound care products, ranging from a simple bandage to long term treatment, to promote healing and reduce the chances of infection and scarring. The drug loaded halloysite delivery system is designed to offer superior clinical benefits over current wound care systems, especially in the area of burn care. Drugs are loaded into halloysite tubes and embedded into the bandage and are released over an extended time period. This increases the duration of drug effectiveness and reduces the rate at which a bandage needs to be changed. In the current study, we are making halloysite-PCL scaffolds that can be used as a drug loaded bandage in the future.
Materials and Methods
Drug Loading in Halloysites
Figure 6. Drug loading in halloysite (119)
Dissolve any desired 100mg drug in 5ml of water or alcohol. Sonicate it until the drug dissolves (usually 5 to 30 minutes). Once the solution becomes transparent add 50mg of halloysite powder. Sonicate again for 30 minutes. Then keep the solution in a vacuum machine and apply the vacuum for 20 minutes. After 20 minutes, stop the vacuum, remove the tube containing halloysites from the vacuum machine. Keep it in room atmosphere for 20 minutes. Repeat steps 5 &6 three times. Figure 6.1 shows the drug loading process in halloysites (119).
Drug Release from Halloysites
Take the drug loaded hallyosite and add 1ml of water. Incubate on magnetic stirrer for 10 minutes. Centrifuge at 7000 rpm for 2 minutes. Take the supernatant in the fresh tube and mark it as the #1 tube. To the pallet add 1 ml of water. Incubate again on the magnetic stirrer for 10 minutes. Centrifuge at 7000 rpm for 2 minutes. Take the supernatant in the fresh tube and mark it as the #2 tube. To the pallet add 1 ml of water. Repeat this process for 10 minutes six times, 30 minutes 2 times and 1.30 hours 3 times. Take out the magnetic stirrer, then measure the concentration of the drug released with the UV Spectrometer.
A bacterial experiment was performed to find out the effect of the drug loaded halloysite PCL scaffold on the bacteria. 1 liter of LB broth was prepared by mixing 10 grams of NaCl, 10 grams of tryptone, 5 grams of yeast extract, 15 grams of agar, and enough distilled water to make a final volume of 1 liter. This LB broth was sterilized by autoclaving at 1210C for 15 minutes. After autoclaving, LB broth was poured into different plates, which were allowed to solidify over night. Next day, a loop full culture of E-coli bacteria was spread on the LB broth plates. Bacteria were allowed to grow over night. Next day, the drug loaded halloysite-PCL scaffolds were kept on the bacterial culture in the LB broth plate. The effect of the drug was observed the next day.
Loading of the halloysite nanotubes with drugs was based on the vacuum cycling of a halloysite suspension in a saturated solution containing a drug, as was described earlier. The air located in the cores of the tubes was replaced by the drug solution during this process. This cycle was repeated three times in order to get the highest loading. Brilliant green, chlorhexidine, iodin, curcuma longa, povidine iodine, amoxicillin and neosporin drugs were used for loading in the halloysites.
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Figures 6.2, 6.3, 6.4, 6.5 and 6.6 show drug release profiles form the halloysites for brilliant green, chlorhexidine, iodine, curcuma long and povidone iodine respectivly. A drug release study was performed in water at room temperature. The suspension of the halloysite nanotubes was constantly stirred with the magnetic stirrer during the entire release process in order to establish an equilibrium condition. Samples for analysis were taken from the suspension by centrifugation. Concentration of the drug was determined by UV spectrophotometer. 98.9% of Brilliant green was released in the first 2.5 hours (Figure 6.2). 94.2% of Chlorhexidine was released in the first 5 hours (Figure 6.3). 99.8% of Iodine was released in the first 5 hours (Figure 6.4). 93.4% of Curcuma longa was released in the first 8 hours (Figure 6.5). 76% of Povidone iodine was released in the first 6.5 hours (Figure 6.6).
Figure 6.:Brilliant Green release profile from halloysite
Figure 6. Chlorhexidine release profile from halloysite
Figure 6. Iodine release profile from halloysite
Figure 6. Curcuma longa release profile from halloysite
Figure 6. Povidone iodine release profile from halloysite
Single-celled organisms were the first life on the planet. Bacteria have adapted and evolved over millions of years. Today there are numerous varieties of bacteria, can be divided into two classes, gram-negative and gram-positive, based on a differential staining process called the Gram stain. The Gram stain separates bacteria based on the ability of the cell wall to retain crystal violet stain when decolorized by an organic solvent like ethanol. The differences in the cell wall also play an important role in the types of antibiotics that will be effective against them.
The drug loaded halloysite-PCL scaffolds were used for the disc diffusion assay to examine the effectiveness of the drug released from the halloysite-PCL scaffold on Escherichia coli (E. coli). E.coli is a gram-negative bacteria. A bacterial culture of E. coli was spread on an agar plate and lines were drawn, on the outside of the plate to create "sectors". A halloysite-PCL scaffold with a particular drug was placed in each sector, and the plates were allowed to grow overnight at 37°C. After incubation, an even growth of bacteria, or lawn, should cover the plate. The only place where the bacteria did not grow was in a region around the drugloaded halloysite-PCL scaffold. This clear region is called the zone of clearance. Figure 6.7 shows the zone of clearance formed by amoxicillin in the upper part and povidone-iodine in the lower part. Figure 6.8 shows a zone of clearance by a Chlorhexidine. Figure 6.9 shows a zone of clearance by a Neosporin. The zone of clearance formed by amoxicillin was the largest, and the zone of clearance formed by Neosporin was the smallest.
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Figure 6. Zone of clearance formed by Amoxicillin in upper part and povidone-iodine in lower part
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Figure 6. zone of clearance formed by Chlorhexidine
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Figure 6. zone of clearance formed by Neosporin
Halloysite minerals have relatively high specific surface areas (SSA) which vary from 50 tol40 m2g-1. For halloysite nanotubes with an inner radius of 7nm, the capillary force in terms of height of the water column is in terms of atmosphere h - 200 atm. Hence, it is understandable that this high capillary force helps halloysites in the quick adsorption of several materials. Therefore, they can absorb a relatively big class of compounds from inorganic salts to organic molecules, as well as polymers and biologically active agents. Absorption of salts and small organic molecules mainly takes place by their intercalation into interlayer space (120; 121; 122; 123), whereas big polymer molecules, proteins and drugs, are bound to the halloysite outer and inner faces. Intercalation does not occur with polymers, proteins, and other macromolecules due to their large molecular size. The adsorptive and ion exchange properties of halloysite nanotubes are highly affected by their surface charges, which are pH-dependent. The surface charge pH dependence of kaolinite and halloysite were studied by Tari et al.. However, the halloysite has a higher negative surface charge compared to kaolinite, showing the predominance of silica properties at the outer surface. According to the literature, a portion of the drug might be irreversibly bound onto the sites of halloysites, while the majority of the drug should enter the lumen of halloysite and would be available to release (124; 125; 126; 127). Brilliant green, chlorhexidine, iodine, curcuma longa, povidine iodine, amoxicillin and neosporin drugs were successfully loaded in halloysite. A drug release study was performed in water at room temperature. The suspension of halloysite nanotubes was constantly stirred with a magnetic stirrer during the entire release process in order to establish an equilibrium condition. Samples for analysis were taken from the suspension by centrifugation. The concentration of the drug was determined by UV spectrophotometer. Each drug had a different release profile. This indicated that the halloysite can be used for drug delivery.
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The drug loaded halloysite-PCL scaffolds were used for disc diffusion assay to examine the effectiveness of the drug released from the halloysite-PCL scaffold on Escherichia coli. The drug loaded halloysite-PCL scaffold was placed in each sector and allowed to grow overnight at 37°C. After incubation, an even growth of bacteria covered the plate. The only place where the bacteria did not grow was in a region around the drug loaded halloysite-PCL scaffold. This clear region is called the zone of clearance, which was present for chlorhexidine, povidine iodine, amoxicillin and neosporin. The zone of clearance was not present for drugs including clindamycin phosphate, brilliant green, iodin, curcuma longa. Thus, the results of the current study indicate that the halloysite PCL scaffold can be used as a drug loaded nano band aid.