Ofloxacin was obtained as a free gift sample from Caside pharmaceuticals, India. Poly Hydroxyl Butyrate co Valerate (PHBV) 12% was purchased from Goodfellow Chemicals, USA. Steel plates were obtained as gift sample from KRR pvt limited, which were cut down into small plates with equal dimensions (1 inch - 1 inch). Other chemicals such as Dichoromethane, Sodium formate were purchased from Sigma Aldrich, India.
3.2 Polymer coating:
The steel plates were coated by dip coating method. Prior to coating, the plates were undergone surface treatment by using hydrogen peroxide and sodium hydroxide . The polymer solution was prepared by using 15% of PHBV (w/v) in Dichloromethane and to which the drug was added at a ratio of 5:1 (Polymer: Drug) respectively. Implants were kept immersed in the solution for a period of 60 min. Later they were withdrawn manually and were kept outside at room temperature for the solvent to get vaporized.
After 24 hours, by following spray coating protocol, 3% of sodium formate solution was prepared and coated over the primary polymer coating. The second coating was done three times for each implants.
3.3 Surface characterization:
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The surface morphology of coated implants was investigated by using scanning electron microscope (SEM). The implants were cut down into small fragments and were mounted onto metal stubs using double-sided adhesive tape, sputter-coated with a thin platinum layer using an Auto-sputter fine coater (JFC 1600, JEOL, Japan) and directly analyzed by cold field emission SEM (JEOL, JSM-6701F, Japan).
Fig 3.1: Cold field emission Scanning Electron Microscope at CeNTAB, SASTRA
3.4 Determination of ofloxacin drug loading:
A small amount of free drug was dissolved in 0.1M HCl. The ofloxacin content was assayed by means of UV spectrophotometer (UV - 1601 Spectrophotometer, Shimadzu). Followed by, the standard concentration profiles were done and the drug loading efficiency was calculated.
3.5 In vitro drug release:
In vitro release patterns were studied using conventional dialysis technique. Ofloxacin coated stainless steel implants were placed in a dialysis bag and dialyzed against 200 mL of phosphate saline buffer (PBS).The PBS was prepared initially and the pH was adjusted to 7.4 with sodium hydroxide. Followed by 0.1M of HCl was added at the ratio of 9:1 (v/v) of PBS and HCl respectively. pH of the final solvent was 6.2. During the dissolution process the temperature was stabilized at 37±1 °C, and the sink conditions were maintained through out the course of study. The in vitro release was aided by continuous stirring using a magnetic stirrer.
3.6 Thickness of Film:
The thickness of the coated film was done by screw gauge apparatus. Prior to any coating, the thickness of implant is measured and after the coating again, the thickness of the plate is measured. The difference between these values indicates the thickness of film.
3.7 Zone of Inhibition:
Zone of inhibition was determined by the Kirby-Bauer antibiotic testing method. Known quantities of bacteria are grown on agar plates in the presence of coated implants. If the bacteria are susceptible to a particular antibiotic, an area of clearing surrounds the wafer where bacteria are not capable of growing (called a zone of inhibition).The size of the zone and the rate of antibiotic diffusion are used to estimate the bacteria's sensitivity to that particular antibiotic. In general, larger zones correlate with smaller minimum inhibitory concentration (MIC) of antibiotic for those bacteria. This information can be used to choose appropriate antibiotics to combat a particular infection.
The bacterial strains that were used are Staphylococcus aureus (ATC 6538) and Clostridium sporogenes (ATC 11437). All the bacteria were cultures in Nutrient Agar. The mediums that were used for inoculating the S.aureus and C.sporogenes were Muller hinton agar and Brucella blood agar respectively. Under aseptic conditions, first the plates were cultured with standardized inoculums of each bacterial strain respectively using sterile cotton swabs. Followed by which the antibiotic coated implants were carefully placed on the plate. The plates were incubated under their respective environments for a period of 24 hrs and following which the zone of inhibition was observed.
4.1 Nature of films:
Based upon various factors, the film properties got varied. In case of plates which were undergone surface treatment initially prior to dip coating process, had a uniform coating of the antibiotic over it, with a fine thickness of few microns were obtained [Figure 2]. Also the adhesive property of these films were found to be good, where only under external pressure they get peeled away from the surfaces of the plates, otherwise they remain intact with the surface of the plates. In case of stainless steel plates which were not undergone any surface treatment initially, had poor adhesive properties and the thickness were not uniform in nature and were larger in thickness.
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Figure 4.1: Antibiotic coated plates. (a)Plates undergone
surface treatment prior to coating and (b) plate not undergone
any surface treatment.
4.2 Thickness of film:
The thickness of coated film was around 9 µm. The particles were distributed uniformly over the plate surface. In the case of plates, which did not undergo any surface treatment, the thickness of film was larger in size.
4.3 Zone of inhibition:
In all bacterial strains, S.aureus, C sporogens, P aeruginosa, the zone of inhibition was noticed. In S aureus, the diameter of the inhibited area was 64mm. In the case of C sporogenes, the bacterial strain had good viability and the area of inhibition was 19mm [Figure 3]. Also in P aeruginosa the zone of inhibition was noted and was of significant distance. Rest while in the control groups, there was no growth of any contaminants.
Figure 4.2: Ofloxacin coated plates exhibiting antibiotic sensitivity against
(a) Clostridium sporogenes; (b) Staphylococcus aureus
4.4 Scanning Electron Microscopy Image:
The scanning electron microscopy image of the antibiotic coated implants shows that the antibiotic particles are incorporated uniformly over the film. The drug - ofloxacin - particle size was of few microns.
Figure 4.3: SEM image of ofloxacin coated film
4.5 In vitro drug release:
The maximum absorbance peak of ofloxacin content was obtained it 294nm by spectrometry. 98.5% of encapsulation efficiency was achieved. The best fit straight line equation of the calibration curve was found to be A = 2.456 + 0.0056. The invitro release of the ofloxacin coated plates is shown in the figure 5. Initially for the first one hour, controlled release was achieved, were at least 16 % of the encapsulated drug gets released into the medium. Followed by which the drug is released at a constant rate where the total amount of drug gets released into the medium over a period of 6 hours. This might be due to incorporation of drug in the film rather than by micro spheres. Thereby the drug must have been released directly into the medium at a faster rate.
Figure 4.4 : Drug release pattern of antibiotic loaded plates.
The hypothesis of this study is that the encapsulated drug gets released during the presence of aerobic organisms in the environment. Niekus et al. demonstrated the formation of hydrogen peroxide from superoxide dismutase in Campylobacter sputorum subspecies bubulus. They demonstrated the ability to produce superoxide anion radicals (O2-) during oxidation of formate and lactate. Furthermore they showed that C.sputorum was found to produce H2O2 while oxidizing formate. Based upon this principle, the work was designed so that, it could be applied for clinical applications especially in combating infections.
Mechanism of release:
The principle of the study depends upon the production of enzymes, super oxide dismutase, and catalase, by the microorganisms respectively. Thereby the superior formate coating gets degraded and the antibiotic is released to the target site.
According to our hypothesis, in the presence of oxygen in the medium, the formate gets oxidized thereby leading to production of super oxide anions. Since these radicals are lethal to the microorganisms, they tend to stimulate to release superoxide dismutase such a way to breakdown the radicals into hydrogen peroxide (H2O2). By nature as such hydrogen peroxide has bactericidal effects thereby they can also inhibit the growth of microorganisms. Hence this is lead to release of enzyme catalase, to breakdown the hydrogen peroxide to water and oxygen. Thereby the drug comes in contact with the medium and hence it gets released.
To confirm this overall mechanism, the drug release was tested with C.sporogenes, S.aureus and P.aeruginosa. C. sporogenes are anaerobic organisms, which are catalase and superoxide dismutase negative, while the later organisms are aerobic organism, are catalase and superoxide dismutase positive. Prior to procedure it was found that all organisms are sensitive to ofloxacin. When the antibiotic loaded plates were kept, the area of the inhibited zone was more in the S.aureus and P.aeruginosa than C.sporogenes. Being a catalase and superoxide dismutase negative, there should not be any zone of inhibition. But in our study, an inhibited zone area of 19mm was found. This might be due to high concentration of loaded drug in the implant and due to presence of any free drug without proper coating of formate. Comparing to C.sporogenes, S.aureus had a significant area of inhibition even though both are sensitive to the drug.
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By comparing the minimum inhibitory concentration (MIC) value of ofloxacin against all the organisms, C sporogenes is highly sensitive to the drug than other organisms. In our study we can see that, even at such a high concentration level of the drug, the C. sporogenes are able to survive in the medium while the S.aureus growth has been totally inhibited in the other case. This indicates that the drug release from the coated plates should be following the above mechanism.
In vitro drug release:
The pattern of drug release varies with each technique. The release kinetics can be adjusted by the choice of the coating technique, the film thickness and polymer composition . In our study, by following dip coating method, the drugs are incorporated in the surface of the PHBV film. In our study, we observed that the total encapsulated drug gets eliminated into the medium over a time period of 6 hours. The thickness of film is 9µm. Also the pH of the solvent can play major role, since ofloxacin is highly soluble in acidic solution than basic. In our study, the final pH of the releasing medium was 6.1. Since the medium is too acidic, it would have triggered the ofloxacin that are coated in the plates to get released at a faster rate. Sree Harsha et al showed that by varying the pH of the releasing the medium, the releasing pattern of the ofloxacin loaded drug delivery systems gets altered . They showed that when the pH of the medium is too acidic the drug gets easily dissolved in the medium and gets released at a faster rate comparing with the other case, where the pH of the releasing medium is basic.
In drug-polymer matrix system, the drug is dispersed or dissolved in the polymer, and the release rate depends upon various factors . The diffusion of a drug molecule through the polymer matrix is dependent upon the solubility of the drug in the polymer matrix and the surrounding medium, the diffusion coefficient of the drug molecule, the molecular weight of the drug, its concentration throughout the polymer matrix, and the distance necessary for diffusion . The faster release of drug from the coated drug may be due to the presence of higher concentration of the drug in the film. The obtained release pattern may be due to combination of both surface and bulk erosion mechanism and also the pH of the releasing medium. It can be presumed that when the antibiotic coated plates are exposed to the release media, the drug- polymer coated thin film becomes hydrated in presence of aqueous media, leads to generate more porosity throughout the film, which allows more drug to release from coating. Also the thickness of the film plays a vital role on the drug elution. As the thickness of the film is very less the entire surface degradation process proceeds rapidly .
A local drug delivery system, which targets only the infected tissues, would eliminate some of the complications associated with extended courses of parenteral antibiotic treatment. Generally for treating bone infections, implantable drug delivery carriers must deliver the antibiotic at a steady rate for at least 6 week, remain immobilized at the site of implantation, and be biocompatible . Since the release lasted only for few hours in our study, it indicates that this system may not be suitable for treating bone infections. Rather than dip coating, if we use drug encapsulated microspheres with appropriate polymer, the desired f90duration of drug release can be achieved.
It is well known that the products of oxygen reduction-hydrogen peroxide, superoxide radical, and hydroxyl radical-are highly toxic for cells and bring about damage to cell macromolecules . Also hydrogen peroxide plays a major role in overcoming the formation of biofilm and promotes wound healing [18, 83, 84]. In our study, for the release of the drug from the medium it can be noted that, formation of hydrogen peroxide is essential. The formed hydrogen peroxide can itself act as a antibactericidal agent other than the loaded drug and thereby improving the treatment efficacy.
The rate of enzyme getting released and its properties get varied between organisms. The breakdown of hydrogen peroxide to water and oxygen by catalase is associated with a release of energy - exothermic reaction. Based upon this, temperature based biosensors can be made to detect the organism in the given sample. Sensors are been devised to detect the micro organism based upon microbial redox reactions. The approach to the detection of biological activity described in this study is based on the recording of microbial redox reactions. In two laboratory applications, measurement of microbial activity in a biological wastewater treatment plant and in a biofilm, two sensor systems were investigated which monitor the microbiological activity online and in real time and thus its used for monitoring and control. The findings obtained showed considerable potential for optimizing biological processes on the basis of microbial activity .
Limitation of the study:
At random concentrations of the organisms, the zone of inhibition studies were performed.
The minimum concentrations of organism for stimulating the drug release from the plate should be evaluated.
In studying the invitro drug release; pH of the releasing medium was 6.1.
In vivo release from the coated plates should be evaluated.
The present study, demonstrates the potential of ofloxacin coated plates for treating infections caused by aerobic micro organisms. Plates were coated with antibiotic by dip coating method, followed by spray coating of sodium formate. Antibacterial effects were studied against S.aureus and C.sporogenes. Comparing with S.aureus,a significant zone of inhibition was formed in the case of C.sporogenes. In drug release study, the total amount of encapsulated drug was released over a period of 6 hours. Also the hydrogen peroxide produced during the course of mechanism could play a major role in preventing formation of biofilms. Hence these results indicates that these smart drug delivery systems releases drug during the presence the aerobic micro organisms and thereby improving the treating efficacy.