Novel Photosensitizers As Antimicrobial Agents For Photodynamic Antimicrobial Chemotherapy

In recent years, the overuse of antibiotics has led to the emergence and spread of superbug. The so-called superbugs refer to the bacteria which are multidrug-resistant to present antibiotics. This has added to a lot of difficulties for the treatment of wound infection in clinic. Although there is only a potential likelihood for the explosive prevalence of multiantibiotics-resistant superbugs, what terror it will cause has brought great panics in many countries around the world. Therefore, the development of new anti-infection strategy is urgent. Photodynamic antimicrobial chemotherapy (PACT) is one of the most promising new antimicrobial therapies in treating infections caused by bacteria, Fungi and viruses, especially in treating infections by multidrug-resistant bacteria Photodynamic antimicrobial therapy is an oxidative damage mechanism relying on a combined action of three factors----light, photosensitizer and oxygen, which will not bring some drug-resistant problems related with such factors as single-drug administration, the concentration of photosensitizer, inadequate exposure time etc.Additionally, superbugs are most likely to be found in the infection site of the wound, in the infection of the lungs, the blood and the urinary tract. Except for blood infection, PACT is relatively easy to implement in the other parts’ infections. Hence, in terms of drug-resistance, bactericidal effect and convenience, PACT has its great advantages. Currently, a lot of research in foreign countries has been done into the photodynamic inactivation of different bacteria with different photosensitizers, which aims at discovering a desired photosensitizer. A desired antimicrobial photosensitizer should have the following characteristics-----high efficacy yet low toxicity, good selectivity, strong permeability of the cell wall and high effectiveness in microorganism inactivation while doing little harm to the normal tissues. In previous reports, photosensitive drugs as antibacterial agents have no highlighted selectivity, with some effective to Gram-positive bacteria and some effective to Gram-negative bacteria We expect that the desired photosensitive drugs can be effective in treating the above two bacteria and the potential bacteria Therefore, it is of great practical significance to achieve the promotion of PACT. Based on the above objectives, we were screened antibacterial sensitive drugs in vitro and in vivo, the major contents of paper includes the following two parts:1. The design and screening of high efficacy antimicrobial photosensitizer1.1The photodynamic antibacterial effect of a-D-galactopyranosyl Zinc PhthalocyaninesPhthalocyanines photosensitizers have its own advantages but there is limitation to the use of Phthalocyanines because of its poor water solubility and easily gathering. Considering the rapid metabolism and reproduction of bacteria, galactose is used as modification to increase its water solubility and its biocompatibility. So,drugs that bacteria selectively take in are well designed so that photosensitive drugs are preferentially concentrated in bacteria rather than in the surrounding tissues or cells so as to achieve selective sterilization. The novel a-D-galactopyranosyl zinc phthalocyanines (T1, T2, T3, T4) have been synthesized to study the biological properties and antibacterial properties. The study showed that the bacterial uptake to four phthalocyanine compounds (T1-T4) reached maximum in30minutes. With increased irradiation time, the number of colonies decreased and when the energy density reached6J/cm2, the inactivation rate of bacteria reached maximum, which no longer increased with increased laser energy density. Of the four a-D-galactopyranosyl zinc phthalocyanines compounds (T1-T4), the irradiation of T1had effective inactivation to Gram-positive drug-resistant bacteria MRSA with low dark toxicity, but the inactivation effect to Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) was very poor. The laser confocal showed that MRSA had maximum uptake of T1and T1had the strongest fluorescent.1.2The antibacterial effect of Porphyrin compoundsPhthalocyanines photosensitizers are effective in fighting against Gram-positive bacteria but ineffective to Gram-negative bacteria, which greatly limit the use of such photosensitizers. Porphyrins is another kind of natural photosensitizer, But its parent nucleus of photosensitive efficiency and selectivity are not ideal. Considering the reproduction of bacteria need huge amounts of carbon and nitrogen sources, double ethylenediamine is used as the modification of the original porphyrin compounds, and obtained a new type of antibacterial photosensitizer. Amino heme was taken as an example and a study of PACT was conducted against Gram-negative and Gram-positive bacteria, showing that it had good antibacterial effect in vitro. Experiments on rats with acute trauma infection in vivo indicated that PACT with Amino heme could obviously kill P. aeruginosa and promote the wound healing. Amino heme photosensitizer has its unique antibacterial advantages, but its limitation is unstability and easy decomposition to light, which greatly limits its use. To further study the effect of the amino-substituted porphyrins on the stability and the biological activity of photosensitizers, Amino heme was further designed and five amino porphyrin compounds were obtained. The experiments in vitro showed that the five compounds PA1-PA5were all slower in photobleaching and a strong ability to generate singlet oxygen, of which PA1had the highest rate of generating singlet oxygen (0.69), higher than that of the tetraphenyl porphyrin (0.64), and the singlet oxygen yield of PA2-PA4was0.15-0.37. The uptake of PA1-PA5by MRS A, E.coli, P.aeruginosa showed that the uptake reached maximum in30minutes incubation. The laser confocal microscopy revealed that three strains were selective in taking in5compounds. The uptake of PA1-PA2by three strains was higher whereas the uptake of PA3-PA5was relatively low. The irradiation bacterial effect of PA1was better than the other four compounds. The MIC of the three strains was16μM, the MBC for MRS A, E.coli’s was31μM and the MBC for P.aeruginosa was62.5uM. The dark toxicity of PA1was very low (MBC>500μM). The colony counting method was used to detect the concentration curves of PA1-PA5in the light and dark reactions to three strains. The results showed that with increasing concentration, PA1had significant bactericidal effect and the greatest bactericidal effect against MRSA happened in the concentration of15.6uM. When the concentration reached31.25μM, PA1almost completely killed MRSA. However, within this concentration range, PA1had minor inactivation to3T3fibroblasts.These results indicate that PA1has good antibacterial properties against Gram-positive and negative bacteria, and it can be used as a novel broad-spectrum antimicrobial photosensitizers, which will have a broad and bright prospects.2Antibacterial effect evaluation of porphyrin photosensitizers in the skin-infection diseases2.1Effects of Photodynamic antimicrobial therapy of porphyrin photosensitizer on wound infectionSkin trauma is a common disease, and it is more difficult to treat in clinic. The PA1compounds is taken as the study object, mainly focusing on the effect of PA1on the trauma model in rats with mixed wound infection of MRSA, E.coli, P.aeruginosa. The results showed that PACT could greatly promote the skin healing in rats with large areas of wound infections. Its mechanism lay in inducing the high expression of BFGF, TGF-β1and promoting the synthesis and redistribution of fibrolos in the wounds. The expression of BFGF and TGF-β1in the PACT group is the highest while the expression of the control group is the lowest.(p<0.01). PACT could significantly downregulate inflammation condition of the infected tissues in the burns, reducing the amount of TNF-and IL-6and the number of bacteria and promoting the healing speed of the wounds. The concentration change of PA1was positively correlated with PACT effect, showing good dose-dependent relation.Trauma tend to damage the barrier system of the body, which makes it easy for bacteria to penetrate the barrier and cause sepsis. While PACT can be used in vitro, bacteria in vivo can only be removed by immunity in itself. The combined advantage of PACT and antibiotics is that PACT can remove bacteria in vitro while bacteria in vivo can be cleaned through antibiotics, small amount of which can work. Therefore, the combined therapy of PACT and antibiotics has been used to treat trauma models in seriously-infected rats. The results showed that PACT combined with antibiotics could significantly reduce the inflammation expression of the seriously-infected tissues in rats with severe wounds, greatly reducing the number of bacteria in the wound tissues and promoting the wound healing. The result was far better than that of the PACT group and the antibiotics group. The combined therapy of PACT and antibiotics was used to treat rats with a large area of severe infections, with the mortality20%, whereas the mortality of the control group was60%, which indicated the effectiveness of the combined therapy. The explanation to the treatment of a large area of trauma infections with the combined therapy of PACT and antibiotics was as follows:It could significantly improve the amount of BFGF and TGF-β1, promoting the fiber composition and redistribution of the wound and thus accelerating the healing speed. At the same time, it could greatly reduce the secretion of inflammation-related factors TNF-a and IL-6, reducing the inflammation of the infected tissues in trauma, thus contributing to the healing of the skin lesion.2.2Effects of photodynamic antimicrobial chemotherapy on burn infection model.Scalds/Burns is a common form of skin wounds and frequently-occurring disease in clinical. Bacterial infection, the main complications of burn patients, is the dominating factor of death. So, Anti-infection treatment is the important link for burn patients. The results showed that PACT combined with antibiotics could significantly reduce the inflammation expression of the burned tissues in rats with severe burns, greatly reducing the number of bacteria in the wound tissues and promoting the wound healing. The result was far better than that of the PACT group and the antibiotics group. The combined therapy of PACT and antibiotics was used to treat rats with burned severe infections, with the mortality0%, whereas the mortality of the control group was50%, which indicated the effectiveness of the combined therapy. The explanation to the treatment of degree Ⅲ burns infection in rats with the combined therapy of PACT and antibiotics was as follows: It could significantly improve the expression of bFGF and CD31in trauma, facilitating the proliferation of new capillaries and fibroblast cells in the early stage and the growth of wound granulation tissues, and enhancing the wound healing. It could greatly improve the rats’immunity system, regulating the secretion of inflammation-related factors TNF-a and IL-6and reducing the inflammation of burn tissues, thus contributing to the healing of the skin lesion.In summary, PA1is a broad-spectrum photosensitizer with good antimicrobial effect. The experiments in vitro and in vivo reveal that the photosensitizer has the naure of high efficiency and low toxicity. Therefore, it can be used as a novel broad-spectrum photosensitizer against bacteria, with broad prospects.