8.7.1. Test-1
The susceptibility test was performed to determine if the immobilised drug was releasing and to investigate activity of the drug after hydrolysing from the monolayers. Figure 8-13 shows the photographic images of the zone of
Chapter 8: Immobilisation of Ciprofloxacin®
inhibition for the control discs and discs coated with Ciprofloxacin®. Release of the drug from the standard filter paper disc coated with the drug was observed to inhibit the growth of S. aureus whereas the metal disc coated with drug did not show any drug elution and inhibition. In contrast, the metal discs coated with Ciprofloxacin® were observed to inhibit the growth of E.coli. A similar trend was followed in all five sets for both bacteria. It may be argued that the metal discs placed on S. aureus were not coated with the drug, but this may not be the case. All metal discs used for both S. aureus and E. coli were from the same batch. Also, the XPS studies on the drug coated metal discs confirmed the functionalisation of Ciprofloxacin® (Figure 8-14).
Figure 8-13 Antibacterial susceptibility test against S. aureus (a) and E. coli(b). Label description: A - control standard filter paper disc without drug, B - standard filter paper disc
with drug; C - Control Ti6Al4V metal disc without drug; D - metal disc coated with drug.
Ciprofloxacin®, being a wide spectrum antibiotic, should inhibit the growth of S. aureus and previous studies have proved this [175]. Whereas, the results obtained in this study for S. aureus are unclear. One possible reason for not inhibiting the growth may be due to the temperature used to incubate the culture plates after placing the discs. Since the S. aureus inoculated plates were maintained at 30 °C, this might have affected the hydrolysis of the drug from the monolayer. Since the drugs on the standard filter paper discs were physisorbed, it has released the drug and inhibited the growth. The temperature used to incubate E. coli (37.5 °C) was observed to hydrolyse the drug from the metal disc and inhibit the bacterial growth. Thus the temperature might have been the factor that affected the drug release.
After the experimentation time, the samples were removed from the culture plate and rinsed with ethanol for 2 minutes before examining the presence of
Chapter 8: Immobilisation of Ciprofloxacin®
Ciprofloxacin® using XPS on these discs. Figure 8-14 shows the XPS spectra obtained for a control, drug coated, drug coated metal disc from S. aureus culture and drug coated metal disc from on E. coli culture.
Figure 8-14 XPS spectra of F 1s region for control, drug coated and drug coated discs placed on S. aureus and E. coli bacteria.
The spectra clearly show the existence of the drug on the surface of the Ti6Al4V discs placed on the culture medium; however, their intensities varied. The variation in the intensities might be due to the fact that after placing on the medium, adherence of the agar medium to the metal discs are possible. Since XPS probes typically 1-10 nm of the surface, the adhered medium might have reduced the penetration depth of the photoelectrons. Although the results showed the drug released from the metal substrate is active for E. coli, no activity was observed for S. aureus and the reason for this should be further explored. In addition to the drug release profile, the release of drug in this bacterial culture (again a mild condition) further shows the possibility for the formation of anhydrides rather than amide during the reaction between the acyl chloride and Ciprofloxacin®.
682 684 686 688 690 692 694 696 Rela tiv e Int ensit y ( A rb it rar y Un it s)
Binding Energy (eV) Control (MC)
Drug coated
After placement on the S. aureus culture
After the placement on the E. coli culture
Chapter 8: Immobilisation of Ciprofloxacin®
8.7.2. Test-2
Since the drug eluted from the metal discs did not show any activity against the gram positive bacteria S. aureus, this test was mainly performed to investigate antibacterial susceptibility of Ciprofloxacin® released from the metal samples in Tris-HCl buffer. This test was also performed to qualitatively observe if there was an increase in the inhibition zone diameter with respect to immersion time intervals in the Tris-HCl buffer solution. Theoretically, as the immersion time of the drug coated sample in Tris-HCl is increased, the amount of drug eluted from the metal surface should increase. As the concentration of the drug in Tris-HCl buffer increases, there should be an increase in the diameter of the inhibition zone. Figure 8-15 shows the photographic images of the zone of inhibition for the discs coated with Ciprofloxacin® eluted at different time intervals.
Figure 8-15 Antibacterial susceptibility test against a) S. aureus and b) E. coli. Label description: C- control disc with no drug; CD – control with drug on; TC: discs coated with Tris-HCl buffer; 1, 2, 4, 6 – immersion time intervals (in weeks) of the samples in Tris-HCl
buffer solution.
It can be observed from the graph that the discs coated with the drug showed anti-bacterial activity confirming the drug was active upon their release. Furthermore, the discs coated with drugs eluted at different time intervals showed an increased zone of inhibition diameter (Figure 8-16) for both S. aureus and E.coli. As expected this may be due to the fact that as the sample immersion time was increased, more Ciprofloxacin® molecules eluted from the surface to the buffer solution thereby increasing the drug concentration. This study qualitatively showed the release of the drug and its antibacterial
Chapter 8: Immobilisation of Ciprofloxacin®
activity. Since the present study did not use standard discs coated with Ciprofloxacin®, the MIC for S. aureus and E. coli was not determined.
Figure 8-16 Inhibited zone diameters for S. aureus and E. coli.
8.8. Summary
The use of 16-PhDA monolayers to modify SLM fabricated structures to deliver therapeutics has been demonstrated. Experimental results showed a covalent attachment of the Ciprofloxacin® molecules to SAMs and sustained release in Tris-HCl buffer solution. The total drug immobilised to the monolayers was estimated to be approximately 1.2 ± 0.1 µg/cm2. The antibacterial susceptibility study revealed that the drug was active upon its release. Thus the attachment, stability and the delivery of Ciprofloxacin® from SLM fabricated surface shows the potential to use this approach to deliver therapeutics directly from customised implant surfaces.
0 5 10 15 20 Control Control with Tris- HCl buffer Control with drug
Week 1 Week 2 Week 4 Week 6
Inh ibit ed z o n e w idt h ( mm ) Gram positive Gram Negative