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Antimicrobial photodynamic therapy is a relatively new option for the treatment of microbial pathogens. It uses photosensitive molecules and visible light to induce oxidative damage to the pathogens. The use of photosensitive molecules could someday be used as a replacement to antibiotics or as an assist to them. This would therefore reduce the amount of bacteria growing a resistance to systemic drugs. Infections on the skin would be a perfect target for Antimicrobial photodynamic therapy.
The leading cause of death to patients who are admitted to hospital with a burn is a skin infection with 75% being caused by S. Aureus and 25% being caused by P. Aeruginosa. (S. Banfi Et al. 2006) The technique of using photosensitizers has already shown to be effective in the treatment of bacteria in vitro that are resistant to drugs. (E. Alves Et al. 2009)
Different structured photosensitizers are known to show different bactericidal effects and efficiency depending on if the bacteria contains a cell wall. Gram-positive bacteria are less resistant to photoinactivation than Gram-negative bacteria due to the presence of the outer membrane layer or cell wall on the Gram-negative bacteria. (S. Banfi Et al. 2006) In a type 1 photochemical reaction the photosensitizer reacts with the biomolecule and produces free radicals. In a type 2 mechanism singlet oxygen is produced and is the main component of cell inactivation. Both type 1 and type 2 mechanism occur simultaneously and the sensitizer, substrate and nature of the medium determine the ratio between the two mechanisms. (D. Caminos Et al. 2005) The singlet oxygen of a type 2 reaction is available to react with proteins, lipids and nucleic acid which causes structural and functional damage eventually leading to cell death. Type 1 reactions produce Hydroxyl and superoxide radicals which are directly linked to cell death however it is thought that type 2 is the more important reaction of photosensitizers. (C. Moore Et al. 2005)
Studies have shown that Porphyrin derivatives can photosensitize the inactivation of pathogens; these studies showed that Gram-positive bacteria were more likely to be photoinactivated by photosensitization than Gram-negative bacteria. The highly organised outer membrane structure of the Gram-negative bacteria intercepts the photogenerated reactive oxygen species and prevents the interaction of photosensitizer with the cytoplasmic membrane. (D. Caminos Et al. 2005) Photosensitizers are typically porphyrin or a phthalocanine derivative.
Photoinactivation of bacteria under solar radiation has been possible in drinking water. This means that the potential use of photosensitizers could be expanded to water treatment systems. Neutral photosensitizers are those which cannot photoinactivate Gram-negative bacteria. Porphyrin is a neutral photosensitizer. Neutral photosensitizers however have been shown to be effective against Gram-negative bacteria upon administration alongside outer membrane disrupting agents. (E. Alves Et al. 2009)
The porphyrins used in this study are Tetraphenylporphyrin and 6 derivatives of porphyrin.
Figure 1.This study used Tetraphenylporphyrin in all graphs and tables it is marked up as 'TPP'. The chemical formula for the Tetraphenylporphyrin used is C44H30N4 (Figure 1.) with the chemical weight being 614.74 g/mol
2.3.2 Porphyrin Derivative 1C:\Users\Matt\Desktop\Porphyrin.bmp
Figure 2.The porphyrin used for porphyrin derivate '1' is marked up in all tables and graphs as '1'. The molecular formula for porphyrin derivative 1 used in this study is C44H26Br4N4 (Figure 2) with the weight of that formula being 930.32004g/mol. Figure 1 shows the location of the bromine atoms.
2.3.3 Porphyrin Derivative 2
Figure 3.The porphyrin used for porphyrin derivate '2' is marked up in all tables and graphs as '2'. The molecular formula for porphyrin derivative 2 used in this study is C44H26Br4N4 (Figure 3) with the weight of that formula being 930.32004g/mol. This porphyrin has a slightly different chemical layout to derivative number 1 as shown by figure 2 that the position of the bromine is on a different carbon atom of each of the outer rings.
2.3.4 Porphyrin Derivative 3C:\Users\Matt\Desktop\Porphyrin.bmp
Figure 4.The porphyrin used for porphyrin derivate '3' is marked up in all tables and graphs as '3'. The molecular formula for porphyrin derivative 3 used in this study is C44H26Br4N4 (Figure 4) with the weight of that formula being 930.32004g/mol. Similar to derivative 1 and 2 this porphyrin has bromine on a different carbon atom to porphyrins 1 and 2.
2.3.5 Porphyrin Derivative 4C:\Users\Matt\Desktop\Porphyrin.bmp
Figure 5.The porphyrin used for porphyrin derivate '4' is marked up in all tables and graphs as '4'. The molecular formula for porphyrin derivative 4 used in this study is C48H38N4O4 (Figure 5) with the weight of that formula being 734.83972g/mol.
2.3.6 Porphyrin Derivative 5
The porphyrin used for porphyrin derivate '5' is marked up in all tables and graphs as '5'. The molecular formula for porphyrin derivative 5 used in this study is C44H26CL4N4 (Figure 6) with the weight of that formula being 752.51604g/mol.
2.3.7 Porphyrin Derivative 6
Figure 7.The porphyrin used for porphyrin derivate '6' is marked up in all tables and graphs as '6'. The molecular formula for porphyrin derivative 6 used in this study is C48H38N4O8 (Figure 7) with the weight of that formula being 798.83732g/mol.
2.3.8 Porphyrins as photosensitizers
Synthetic meso-arylsubstituted porphyrins are a good place to start when designing a porphyrin as a photosensitizer as they are versatile. Ionic or non ionic chemicals can be positioned equally along the tetrapyrrole ring which changes the polarity of the photosensitizer. Cationic porphyrins are said to be more active than anionic or non ionic against both Gram-positive and Gram-negative. Cationic porphyrins have also shown the ability to be able to inactivate bacteria without any additional help from membrane disrupting agents. (S. Banfi 2006) Derivatives of porphyrin can photosensitize and inactivate many different pathogens. Cationic porphyrins are known to be able to inactivate gram negative bacteria without a permeabilization agent. An electrostatic interaction is thought to occur due to the positive charges of the photosensitizer and the negatively charged sites of the outer area of Gram-negative bacteria. (D. Caminos Et al. 2005)
2.4 Reactive Oxygen Species
Originally Reactive oxygen species were thought to only be released by phagocytes during a host defence role. They are now recognised to come from the NADPH oxidase complex as the primary source. Further research has suggested they are used in cellular signals. Reactive Oxygen Species are in a more reactive state than molecular oxygen; with the oxygen being reduced. One of the primary Reactive Oxygen Species is superoxide; this is formed by a one electron reduction of molecular oxygen. Hydrogen Peroxide is produced by a further reduction of Oxygen and may occur spontaneously at a low pH. Hydrogen Peroxide can also be catalysed by some enzymes which are the superoxide dismutase therefore Hydrogen Peroxide is the most likely follow on from Superoxide formation. Hydroxyl radicals may also be produced; these radicals are highly reactive and have a short half-life and are expected to react with the first molecule that is encountered. Superoxide may react with nitric oxide within the body which is another reactive molecule and forms peroxynitrite. Some key signalling molecules are Reactive Oxygen species for example Hydrogen Peroxide whilst others are highly damaging to the biological world. The higher the concentration of the damaging reactive oxygen species the greater the damage that is caused to cells. If reactive oxygen species are to be seen as a signalling molecule they must be produced by a cell and stimulated to be a signalling molecule and have an action in a cell; whether it be the one that produced it or a neighbouring cell. They also need to be removed so that you can turn off or reverse the signal. The reactive oxygen species that matches the above are hydrogen peroxide and superoxide. (J. Hancock Et al. 2001)
Antioxidants play an important role by protecting organisms from reactive oxygen species produced by different cells and cellular compartments. They are found throughout the organism and in different cells in different places. They can be obtained through diet. They catalyze the reaction of Oxygen radicals into hydrogen peroxide and oxygen. They come from the family of metalloenzymes. (L. Del Rio Et al. (May 2002) Ascorbic acid is the reduced form of the antioxidant Vitamin C with Dehydroascorbic acid being the oxidized form of ascorbic acid. (P. David Et al. (2002)
2.5 Staphylococcus aureus
Staphylococcus aureus is a bacterium that is often found within the skin flora. Around 20% of the population of humans carry staphylococcus aureus on their skin. (J. Kluytmans Et al. 1997) 60% are intermittent carriers and the remaining 20% never carry the bacteria. (T. Foster 2004) Staphylococcus aureus is a Gram-positive bacteria that is non-flagellated that can cause meningitis and sepsis. Recently Staphylococcus aureus has become resistant to many strains of antibiotics and this strain is known as methicillin-resistant Stapylococcus aureus. Staphylococcus aureus forms a biofilm on medical equipment and it is unknown whether it can spread across a solid object. On soft agar it is known to spread at 100 µM/min. (C. Kaito and K. Sekimizu 2006) The strain of Stapylococcus aureus used in this study is Stapylococcus aureus (NCTC 6571)
2.6 Pseudomonas aeruginosa
Pseudomonas aeruginosa is a Gram-negative rod that is motile and belonging to the family Pseudomonadaceae. Among the critically ill and those admitted to intensive care is it the main cause of nosocomial infections. It is spread widely throughout the natural world but is highly concentrated in the hospital environment this is due to the wards promotion of bacterial growth. Along with antibiotic resistance pseudomonas aeruginosa can resist temperatures, high salt concentrations and antiseptics. The antimicrobial pattern of pseudomonas aeruginosa varies between different geographical locations. (K. Okon Et al. 2010)
The strain of Pseudomonas aeruginosa used in this study is Pseudomonas aeruginosa (NCIMB 12649)
DBPF is also known as 1,3-dipenylisobenzofuran is the most frequently used method for assessing the amount of Reactive oxygen species produced. It is an Oxygen scavenging agent. (M.Krieg 1993) Singlet Oxygen goes under a 1,4-addition to DBPF which produces endo-peroxide which is degraded at an ambient temperature to give o-dibenzoylbenzene. It is this chemical change which can be measured using a spectrophotometer. (J. Howard and G. Mendenhall 1975)
2.8 Previous experimental processes
2.9 Experimental aims
This study has two main aims these are:
Testing porphyrins for their ability to release reactive oxygen species and measuring how much is produced.
Testing porphyrins for their ability to kill bacteria once they have been photoactivated.
Null Hypothesis: As the amount of reactive oxygen species produced by the porphyrins increases the antibacterial effect will not change.
Alternative Hypothesis: As the amount of reactive oxygen species produced by the porphyrins increases the antibacterial effect will also increase.
All methods were completed using aseptic technique
3.1 Bacterial growth
1. Overnight cultures of Staphylococcus aureus and Pseudomonas aeruginosa were grown in nutrient broth and incubated for 24 hours at 37oC.
2. 1ml of each broth was transferred into 9ml of bacterial peptone water and a ten-fold serial dilution was performed until 10-5 was achieved. This was done by transferring 1ml of the previously added to peptone water into 9ml of a fresh peptone water 4 times after the broth transfer.
3. 0.1ml of each dilution was transferred to an appropriately labeled nutrient agar plate and spread evenly on the surface then incubated for 24 hours at 37oC.
4. Results were gathered using a plate counter.
3.2 Fixed porphyrin on the universal
Each of the 7 porphyrins was prepared based on their molecular weight to 1mM using the formula: W(g) = M(w) x Vol(l) x Conc.(M)
10ml of chloroform was used
Tetraphenylporphyrin has a molecular weight of 614.74 g/mol
Therefore the weight required to make this 1mM was 0.0061g
2. Once all the derivatives had been prepared at 1mM concentration 0.1ml of each was pipetted into a universal and left to evaporate the chloroform leaving behind the porphyrin derivatives.
3. Broth was transferred into the universals containing the porphyrin
4. Staphylococcus aureus and Pseudomonas was transferred into 2 universals for each derivative.
5. One universal was incubated on the stirring incubator for 24 hours at 37oC in the dark and the other was incubated at the same temperature and time under UV light.
6. A serial dilution was completed as mentioned in the Bacterial growth and each dilution was plated and incubated corresponding to the stirring incubator in light or dark for 24 hours at 37oC
7. Results were counting using the plate counter
8. A sample of each derivative was centrifuged and placed in the spectrophotometer at 5nm intervals between 200-650nm to see if any of the porphyrin had dissolved into the broth
3.3 Porphyrin mixed in the broth
1. The above porphyrin concentration was used.
2. 100ml of each derivative was added to 20ml of broth
3. The 2 bacteria were transferred into the broth and incubated in the stirring incubator in UV light and darkness for 24 hours at 37oC
3.4 Porphyrin directly on the plate
1. Using the above samples of chloroform and porphyrin dilutions of 0.1mM (1 tenth) and 0.45mM (1 fifth) this was done by adding 10ml of chloroform and 5ml of chloroform respectively to the initial dilution
2. 0.01ml of all 3 dilution strengths of each derivative was pipetted onto nutrient agar 3 times per plate and allowed to evaporate the chloroform off. One was completed for light and one for dark
3. 0.1ml of Staphylococcus aureus was added to each plate and spread evenly
4. 0.1ml of Pseudomonas aeruginosa was added to a repeat batch of plates method points 1- 2 and spread evenly
4. The plates to be kept in the dark of both bacteria were incubated in darkness the other half which were to be exposed to light had white light shone on them for 1 hour before being incubated in darkness at 37oC for 24 hours
3.5 Testing ROS production
1. A light box was created using a GG475 cut-off filter. This blocks the wavelength of light that degrades DPBF but still allows the correct wavelength that is required to activate the porphyrins.
2. 0.3ml DPBF (50µM) and 1.7ml DMF were added to a cuvette and placed within the light box for 20 minutes with a lamp 30cm away. The cuvette was removed and then replaced before and after a reading with the spectrophotometer.
3. 0.3ml DBPF, 1.7ml DMF and 10µl of each porphyrin were added to a cuvette. This was repeated twice. One time being left the spectrophotometer and being kept in darkness. The other was placed in the light box as mentioned above.
4. DMF solvent was used to blank the spectrophotometer. Absorbance's were read at 410nm every 5 minutes.
3.6 Retesting porphyrin directly on the plate with DMF instead of Chloroform
1. 10µl of 10µM of each porphyrin derivative diluted in DMF instead of chloroform was added to the agar plates 3 times per plate and allowed to evaporate the DMF off. One plate was completed for light and one for dark.
2. 0.1ml of Staphylococcus aureus was added to each plate and spread evenly
3. The plates to be kept in the dark were incubated in darkness the other half which were to be exposed to light had white light shone on them for 1 hour before being incubated in darkness at 37oC for 24 hours
4.1 Bacterial growth
Growth of bacteria in Light (Per 20ml broth)
Growth of bacteria in Dark (Per 20ml broth)
4.2 Bacterial growth with porphyrin fixed on universal
All per 20ml of broth
* meaning that these were the only plates to show results in the dilution series
4.3 Bacterial growth with porphyrin added directly to broth
No growth present
4.4 DBPF absorbance
4.5 Porphyrins in the dark
Figure 13. Figure 14.
Figure 15. Figure 16.
Figure 17. Figure 18.
4.6 Porphyrins in the light
Figure 20. Figure 21.
Figure 22. Figure 23.
Figure 24. Figure 25.
4.7 Bacterial growth after porphyrin photoactivation on the plates Staphylococcus aureus and Pseudomonas aeruginosa
Staphylococcus aureus light
Staphylococcus aureus dark
Pseudomonas aeruginosa light
Pseudomonas aeruginosa dark