Monday, November 14, 2011

Nanotchnology and Treatment of Bacterial Infections

The new age drugs are nanoparticles of polymers, metals or ceramics, which can combat conditions like cancer [83] and fight human pathogens like bacteria [84-88].
The development of new resistant strains of bacteria to current antibiotics [89] has become a serious problem in public health; therefore, there is a strong incentive to develop new bactericides [86]. Bacteria have different membrane structures which allow a general classification of them as Gram-negative or Gram positive. The structural differences lie in the organization of a key component of the membrane, peptidoglycan. Gram negative bacteria exhibit only a thin peptidoglycan layer (~2–3 nm) between the cytoplasmic membrane and the outer membrane [90]; in contrast, Gram-positive bacteria lack the outer membrane but have a peptidoglycan layer of about 30 nm thick [91].
Silver has long been known to exhibit a strong toxicity to a wide range of micro-organisms [92]; for this reason silver-based compounds have been used extensively in many bactericidal applications [93, 94]. Silver compounds have also been used in the medical field to treat burns and a variety of infections [95]. Several salts of silver and their derivatives are commercially employed as antimicrobial agents [96]. Commendable efforts have been made to explore this property using electron microscopy, which has revealed size dependent interaction of silver nanoparticles with bacteria [87]. Nanoparticles of silver have thus been studied as a medium for antibiotic delivery [97], and to synthesize composites for use as disinfecting filters [98] and coating materials [99]. However, the bactericidal property of these nanoparticles depends on their stability in the growth medium, since this imparts greater retention time for bacterium–nanoparticle interaction. There lies a strong challenge in preparing nanoparticles of silver stable enough to significantly restrict bacterial growth.
Studies were carried out on both antibiotic resistant (ampicillin- resistant) and nonresistant strains of gram-negative (Escherichia coli) and a non-resistant strain of gram-positive bacteria (Staphylococcus aureus). A multi-drug resistant strain of gram-negative (Salmonella typhus, resistant to chloramphenicol, amoxycilin and trimethoprim) bacteria was also subjected to analysis to examine the antibacterial effect of the nanoparticles [100]. Efforts have been made to understand the underlying molecular mechanism of such antimicrobial actions. The effect of the nanoparticles was found to be significantly more pronounced on the gram-negative strains, irrespective of whether the strains were resistant or not, than on the gram-positive organisms. This could be attributed this enhanced antibacterial effect of the nanoparticles to their stability in the medium as a colloid, which modulates the phosphotyrosine profile of the bacterial proteins and arrests bacterial growth.
The bactericidal effect of silver ions on micro-organisms is very well known; however, the bactericidal mechanism is only partially understood. It has been proposed that ionic silver strongly interacts with thiol groups of vital enzymes and inactivates them (101, 102). Experimental evidence suggests that DNA loses its replication ability once the bacteria have been treated with silver ions [95]. Other studies have shown evidence of structural changes in the cell membrane as well as the formation of small electron-dense granules formed by silver and sulfur [95, 103].
Silver ions have been demonstrated to be useful and effective in bactericidal applications, but due to the unique properties of nanoparticles nanotechnology presents a reasonable alternative for development of new bactericides. Metal particles in the nanometer size range exhibit physical properties that are different from both the ion and the bulk material. This makes them exhibit remarkable properties such as increased catalytic activity due to morphologies with highly active facets [104-109]. Several electron microscopy techniques can be applied to study the mechanism by which silver nanoparticles interact with these bacteria. We can use high angle annular dark field (HAADF) scanning transmission electron microscopy (STEM), and developed a novel sample preparation that avoids the use of heavy metal based compounds such as OsO4. High resolutions and more accurate x-ray microanalysis were obtained.
Generally, the encapsulation of antibiotics in liposomes or in nanoparticles increased the maximal tolerated dose and the therapeutic index of the antibiotics compared with the free drug. This can be explained by a modification of the pharmacokinetic profile of the antibiotic when encapsulated as well as by a modification of its bio distribution. For instance, in a liposome formulation of amikacin (Mikasome\, Gilead), which is in clinic evaluation, the antibiotic was found 2- to 6-fold more active than the free drug and the free streptomycin in an acute experimental model of murine tuberculosis in which bacteria were located into macrophages.
In a model of mice infected by Mycobacterium avium, amikacin in liposomes could reduce viable bacterial count in liver, spleen, and, to a lesser extent, lungs by approximately 3-log10 compared with the untreated control.
In another example, the entrapment of ampicillin in poly(isobutylcyanoacrylate) nanoparticles increased by 120-fold the efficacy of the antibiotic in an experimental acute infection of mice by Salmonella typhi murium. In this model, 100% of the infected mice treated with a single dose of the nanoparticles survived, whereas all the untreated animals died after 10 days. Such high activity was explained by a complete sterilization of the organs where the intracellular bacteria were located. Treatment with liposomes was less efficient. The survival of mice did not exceed 60%, and the infected organs were never completely sterilized in mice that survived.
Ampicillin-loaded nanoparticles were also found more efficient than liposomes for the treatment of listeriosis in a model of chronic infection of mice by Listeria monocytogenes [188]. In this case, it was shown that the spleen was not totally sterilized, and a reinfection occurred after several days whatever the treatment was. In this model, reinfection was believed to occur from non dividing bacteria, but even nanoparticles loaded with ciprofloxacine, a fluoroquinolone with antibacterial activity against both dividing and nondividing bacteria, could not totally eradicate the infectious reservoir [110].
This shows the extreme difficulty to eradicate all bacteria from the body even when they are a priori located in the MPS. Another difficulty is to reach infections, which develop outside the MPS and outside macrophages. Indeed, it was suggested that part of the clinical trials, which aimed to treat patients infected by tuberculosis with Mikasome, failed because the antibiotic was released in macrophages that were too far from the extracellular bacilli clustered in cavity caseum in the human infection [111]. In case of tuberculosis, it is now known that targeting the non replicated persistent bacilli still remains a challenge to be addressed [112]. Further improvements of drug delivery systems are still needed to enhance the targeting of the extracellular infectious sites.
So far, most of the very promising data were obtained by treating experimental animal infections with antibiotics associated with nanodevices in comparison with the free drug. However, in front of the somewhat disappointing results obtained with Mikasome\ during clinical trials, questions about the relevance of the animal experimental models (with intact host defense and with highly susceptible bacteria to the antibiotics) were raised.
Indeed, in clinical practice, treatments are often given to patients with impaired host defenses and who may be infected with bacteria of low antibiotic susceptibility. Only a few studies considered experimental models on animals with impaired host defenses [110].
In vitro models must also be handled cautiously because they were not always predictive of the in vivo activity. Indeed, the activity measured in vitro may be found dramatically reduced or significantly promoted because of synergies with lymphocytes when tested in vivo in animal models [113]. Nevertheless, for a systemic treatment of bacterial infection in which the target cells are the MPS macrophages of the liver and the spleen, conventional liposomes and nanoparticles can be suggested as the most relevant delivery systems for antibiotics. The efficacy of liposomes was found very dependent on their physicochemical characteristics. For instance, specific composition may affect the bactericidal activity by interaction with the infected organism [114].
In contrast, such formulations seemed of limited value to treat infections in which bacteria are located outside the main MPS organs (i.e., liver, spleen, and bone marrow), and more efforts are still required to address this goal [115].
Indeed, targeted systems to extracellular bacteria and to other reservoir organs may contribute to make progress in the battle against bacterial infections. It is also needed to develop appropriate strategies to eliminate persistent bacteria, which are either in inaccessible sites or in a state of dormancy within macrophages. Some attempts were made using targeted liposomes with mannose to promote recognition by human phagocytic cells [116]. However, the design of a targeted device seemed very delicate to find the right length of spacer between the targeting moiety and the surface of the device and to balance between the numbers of mannose residues on the lipid surface.
Finally, only a few investigations have considered comparative experiments performed with liposomes and other nanosystems. The nanoparticles seemed more efficient than niosomes, which were, in turn, more efficient than liposomes [117,118]. This superiority of nanoparticles may be explained by a higher stability in biological media. In the future, the problem of stability of delivery systems in biological fluid may become even more important in view of the systemic delivery of targeted antibiotics by the oral route. This is another challenge that emerged and is still poorly documented at the moment [119].
Liposome formulations of antibiotics were also evaluated for the local delivery of antibiotics to be used as controlled release system at the site of the infection. They have proven to be of interest for readily accessible infected tissues such as the eye, wound, and lungs [120]. This strategy was suggested in surgical wound prophylaxis [121,122], in the treatment of keratitis using liposomal formulation of tobramycin in eye drops (123), in the treatment of endophthalmitis by intravitreal injection of amikacin-loaded liposomes, and in lung infections by aerosol delivery of the liposomal formulation of antibiotics [124,125]. Recently, a bioresorbable composite pellet of calcium sulfate and hydroxyapatite nanoparticles was studied as a material for local and sustained delivery of antibiotics in bone infections [126]. In this system, the nanoparticles of hydroxyapatite changed the properties of the material by increasing the specific surface of the device and by allowing a higher loading of antibiotics. The nanoparticles incorporated in the material could also advantageously modify the released profile of antibiotics permitting the release of the total dose of the antibiotic incorporated in the material at the end of the process. This was actually not the case with the material devoid of nanoparticles, which retained up to 25% of the dose of the antibiotic after 10 days.
Finally, the tolerance of the material modified by the nanoparticles was improved because the quantity of acid produced by the dissolution of calcium sulfate and responsible for an inflammatory response was reduced. This example illustrates advantages brought when nanotechnology is associated with other technologies to improve the pharmacological properties of a material used as an implant.
Beside what could be considered as Bartificial^ nanotechnologies including liposomes and nanoparticles, some authors considered the use of Bnatural^ nanotechnologies to fight against resistant bacteria by using bacteriophages. This approach was used once in human with an unexpected success in combination with ciprofloxacin for local treatment of patients with wounds infected by multidrug-resistant Staphylococcus aureus [127].
Data obtained on infected animal models suggested that bacterial infection can be circumvented only with functional phage specific to the bacterial strain [128]. The formidable activity observed was suggested to result from the functional capability of the phage only and not due to a nonspecific immune effect of the host defense [129]. At the moment, no side effect was reported about the use of phages, but the number of studies remained very limited. The level of antibodies against phages found in the rescued animals was not substantially elevated.

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