**8. Antibacterial activity of carrier systems for intracellular infection**

Treatment of intracellular bacterial infection remains both a medical and economic challenge. Pathogens thriving or maintaining themselves in cells, or simply taking transient refuge therein, are indeed shielded from many of the humoral and cellular means of defense. They also seem more or less protected against many antibiotics [182]. Various infectious diseases are caused by facultative organisms that are able to survive in phagocytic cells. The intracel‐ lular location of these microorganisms protects them from the host defence systems and from some antibiotics with poor penetration into phagocytic cells. Intracellular infections are especially difficult to eradicate because bacteria fight for their survival using several ingenious mechanisms: inhibition of the phagosome–lysosome fusion, resistance to attack by lysosomal enzymes, oxygenated compounds and defensins of the host macrophages, escape from the phagosome into the cytoplasm [183]. Thus, the need for the development of improved antimicrobial chemotherapeutics and prophylaxis strategies is increasing [4]. In spite of the availability of a wide variety of *in vitro* active antibiotics, therapeutic deficiencies are reported, mainly because of the inability of the drugs to reach the bacteria harboring intracellular compartments or to perform their activity in the intracellular environments [182, 183]. However, the poor cellular penetration limits these use in the treatment of infections caused by intracellular pathogens [183]. One strategy utilized to improve the penetration of antibiotics into phagocytic cells is the use of carrier systems that deliver these drugs directly to the target cells [185]. Several *in vivo* and *in vitro* studies have reported the potential applications of various carrier systems to enhance the selectivity of antibiotics for phagocytic cells and sustain therapeutic efficiency in the treatment of intracellular infections [31].

#### **8.1. Infections due to mycobacteria**

having certain dimensions [166]. Several recent studies showed that the potential of zeolites in medical applications is due to their structural properties and stability in biological envi‐ ronments [167]. Zeolites have also been explored as suitable hosts for the encapsulation of drug molecules, in search for efficient drug delivery sysytems. Both zeolites and drugs have been administrated simultaneously to a patient without loss of the individual pharmacological effect of the drugs [164, 167]. Coating or impregnating zeolite with metallic silver nanoparticles to prepare zeolite composites can enhance the antibacterial ability of materials, and these materials can inhibit bacterial growth effectively [168]. It has been reported that silver embedded zeolite A was found to be antibactrerial against *E. coli*, *Bacillus subtilis* and *staphy‐ lococcus aureus* [165]. Moreover, polymer composites of plasticized poly (vinylchloride) pellets with silver zeolites demonstrated activity against *S. epidermidis* and *E. coli*, while polyurethane composites with silver zeolites showed antimicrobial action against *E. coli* and polylactid acidpolylactide (PLA)/silver zeolite composites also presented activity against *S. aureus* and

Quantum dots (QDs) are nanocrystals formed by semiconductor materials, showing attractive photophysical properties, containing high quantum yield, resistance to photobleaching, and harmonic photoluminescence, making them potentially powerful tools in a range of biomed‐ ical applications [170, 171]. QDs are typically in the size range between 1 nm and 10 nm, composed of groups II–VI (e.g., CdSe) or II–V (e.g., InP) elements of the periodic table. QDs are highly bright, photostable and possess high quantum yield [172]. Due to their very small size, they possess unique properties and behave in different way than crystals in macro scale [173]. Water-soluble QDs may be cross-linked to biomolecules such antibodies, oligonucleo‐ tides, or small molecule ligands to render them specific to biological targets [174]. A variety of techniques have been explored to label cells internally with QDs, using passive uptake, receptor-mediated internalization, chemical transfection, and mechanical delivery. QDs have been loaded passively into cells by exploiting the innate capacity of many cell types to uptake their extracellular space through endocytosis [175, 176]. Krauss group utilized CdSe/ZnS streptavidin-coated QDs to detect solitary pathogenic *E. coli* O157:H7 in phosphate buffer saline solution [177]. Biotinylated anti-*E. coli* O157:H7 distinguished streptavidin-coated QDs via famous avidin–biotin binding. Once treated, QD labeled antibody selectively targeted pathogenic *E. coli* O157:H7 over common lab strain *E. coli* DH5α. This assay represented 2 orders of magnitude more sensitivity than using an organic dye with minimal non-specific binding between the QDs and the bacterial cells [178]. Recently, Luo et al. reported that CDTe QDs coupled to a rocephin antibiotic complex exhibited antibacterial activity against *Escheri‐ chia coli* [179]. The mechanism for the antimicrobial activity of QDs is unclear, but it is possible that QDs can produce singlet O2, a source of free radicals, under irradiation. Heavy metal ion oxides can also form the QDs core and result in antimicrobial activity [180]. A recent and excellent review emphasized the application of bioconjugated quantum dots for the detection of food contaminants such as pathogenic bacterial toxins like botulinum toxin, enterotoxins

*E. coli*, with silver being effectively released from the films [169].

produced by *Staphylococcus aureus* and *Escherichia coli* [181].

**7.11. Quantum dots**

168 Application of Nanotechnology in Drug Delivery

Tuberculosis, caused by *Mycobacterium tuberculosis*, is a ordinary lung infection that is even endemic to specified regions. Its prevalence has increased recently because it is often associated with AIDS. The *Mycobacterium avium* complex (MAC) complex is the main cause of hardships in immunodepressed patients [186]. There are drugs that are efficient against tuberculosis, but these are used in extended treatment, increasing the risk of side effects [187]. Moreover, tuberculosis has emerged as an occupational disease in the health care set-up. Although an effective therapeutic regimen is available, patient non-compliance (because of the need of taking antitubercular drugs daily or several times a week) results in treatment failure as well as the emergence of drug resistance [188]. The use of delivery systems facilitates the selective shuttling of antibiotic to the site of infection and such systems provide slow and prolonged drug release, which permits administration over longer intervals of time [189]. The encapsu‐ lation of antitubercular drugs in polymeric particles is another strategy to improve the current therapeutic regimen of tuberculosis. In the last few years several antitubercular drugscontaining PLGA and PLA microparticles and mainly nanoparticles have been comprehen‐ sively studied [190]. Fawaz et al*.* encapsulated the synthetic drug ciprofloxacin in polyisobutylcyanoacrylate (PIBCA) nanoparticles. When testing these nanoparticles against a *M. avium* infection in a human macrophage culture, it was found that though nanoparticle associated ciprofloxacin was more effective than unbound ciprofloxacin, it was much less so than anticipated [191]. Rifampicin-loaded polybutylcyanoacrylate nanoparticles have shown enhanced antibacterial activity both *in vitro* and *in vivo* against *S. aureus* and *M. avium* due to an effective delivery of drugs to macrophages [192]. The encapsulation of different antibiotics in liposomes has shown good antibacterial efficacy in both macrophage cell lines and in animal models of MAC-due disease [193]. Ciprofloxacin efficiently inhibits the growth of *M. avium in vitro* in a murine macrophage-like cell line using negatively charged liposomes and *in vivo* using specific stealth liposomes in a mouse model of tuberculosis infection [194]. Similar results have been obtained using stealth liposomes of isoniazid and rifampicin, which show controlled release and reduce toxicity *in vivo* in mice infected with *M. tuberculosis* [195].

ticles were also effective against non-dividing bacteria, Page-Clisson *et al.* studied the effec‐ tiveness of these carriers in a model of persistent *Salmonella typhimurium* infection [206]. They found that although at early stages of the infection, when bacteria are actively dividing, there was an antibacterial effect, neither free nor nanoencapsulated ciprofloxacin or ampicillin could significantly reduce infection in the liver or the spleen at later stages [206]. Liposomal cipro‐ floxacin, administered intravenously and intraperitoneally to mice infected with intracellular *S. typhimurium*, has increased habitation time in plasma and the concentration of drug in the liver, spleen, lungs and kidneys is also increased, while when administered intratracheally its pulmonary retention is increased. Compared with free ciprofloxacin, it extends survival and reduces the number of bacteria in the liver and spleen [207]. Therefore, alternative methods such as DDS which achieve high protective and bactericidal activity should be taken into

Nanoparticle based Drug Delivery Systems for Treatment of Infectious Diseases

http://dx.doi.org/10.5772/58423

171

account in the future as suitable treatments for *Salmonella*-induced infections [203].

*Lysteria monocytogenes* is a facultative intracellular parasite able to cause meningitis and septicaemia. The encapsulation of ampicillin in liposomes decreases the survival of *L. mono‐ cytogenes* in mouse peritoneal macrophages to different extents, depending on the composition of the liposomes [208]. Chitosan-coated plastic films, alone or loaded with antimicrobial agents, were evaluated for their effect against *L. monocytogenes*. These chitosan-coated films inhibited this pathogen growth in a concentration-dependent manner whereas chitosancoated films impregnated with antibiotics were significantly more effective against *L. monocytogenes* [209]. Formulation of gentamicin in liposomes containing DOPE (dioleylphosphatidylethanolamine) and sensitive to pH has been reported to increase the concentration of drug in mouse macro‐ phages infected with *L. monocytogenes*, increasing its bactericidal activity. This formulation is more effective against *L. monocytogenes* than against other bacteria owing to its location in the cytosol [210]. Furthermore, the efficacy of liposomes and free antibiotic were distinguished in *Listeria-*infected mice. Seven days after the treatment, ampicillin-loaded liposomes had reduced the infection by 3.2 logs in the liver and 2.8 logs in the spleen, while free ampicillin was ineffective [208]. In another example, ampicillin-encapsulated polyisohexylcyanoacrylate nanoparticles have been investigated against *L. monocytogenes* in mouse peritoneal macro‐

Attractive features, such as increased dissolution velocity, increased saturation solubility, improved bioadhesivity, versatility in surface modification and ease of post-production processing, have widened the applications of nanosuspensions for various routes. One major problem with the intravenous administration of colloidal particles is their interaction with the reticulo-endothelial system [212]. The applications of nanosuspensions in parenteral and oral routes have been very well investigated and applications in pulmonary and ocular delivery

**8.4. Lysteriosis**

phages [211].

**9. Specific applications of biodegradable NPs**

#### **8.2. Brucellosis**

Brucellosis is an infectious disease caused by *Brucella* spp. Four species, *Brucella abortus*, *Brucella melitensis*, *Brucella suis* and *Brucella canis*, have been recognized as human pathogens each associated with a different natural host animal [196]. These small coccobacilli are mainly localized intracellularly within phagocytic cells making treatment difficult, since most antibiotics, although highly active *in vitro*, do not actively pass through cellular membranes [197]. However in the last two decades many experiments have provided good evidence criteria for its antibiotic treatment, the most suitable antimicrobial therapy for human brucel‐ losis continues to be a controversial subject [198]. Because of its intracellular location, long treatments with several antibiotics are required. Relapses are frequent owing to the low efficacy of many drugs and the lack of patient agreement [199]. Thus, alternative methods such as drug delivery systems to achieve high intracellular bactericidal activity should be consid‐ ered [198]. Gentamicin, encapsulated in different types of liposomes, has been evaluated against murine monocytes infected with *B. abortus*. All such liposomes reduced the number of bacteria, the most effective being SPLVs (stable plurilamellar vesicles) [200]. Rifampicinloaded mannosylated dendrimers have indicated specific pH-dependent delivery of this antibiotic to rat alveolar macrophages [201]. Recently, gentamicin loaded poly (D, L-lactideco-glycolide) (PLGA) have been obtained by the several emulsion solvent evaporation method for the treatment of brucellosis [202]. Thus, alternative methods such as DDS to achieve high intracellular bactericidal activity seem promising. The possible use of drug delivery systems containing aminoglycosides may be one of the most appropriate therapeutic advances in human brucellosis treatment in the recent years [203].

#### **8.3. Salmonellosis**

Salmonellosis is one of the most serious food-borne diseases affecting humans. It may be considered the most important pandemic zoonosis under natural conditions [204]. Bacteria of the genus *salmonella* are facultative intracellular parasites that cause salmonellosis and typhoid fever. Antibiotics effective against this type of bacteria have limitations owing to the problems of formulation, low penetration, or the appearance of side effects; these can be solved using carrier systems [205]. Several studies using antibiotic-loaded nanoparticles have been per‐ formed in order to recognize the suitability and efficacy of these carriers in experimental models of salmonellosis [204]. In order to recognize whether polyalkycyanoacrylate nanopar‐ ticles were also effective against non-dividing bacteria, Page-Clisson *et al.* studied the effec‐ tiveness of these carriers in a model of persistent *Salmonella typhimurium* infection [206]. They found that although at early stages of the infection, when bacteria are actively dividing, there was an antibacterial effect, neither free nor nanoencapsulated ciprofloxacin or ampicillin could significantly reduce infection in the liver or the spleen at later stages [206]. Liposomal cipro‐ floxacin, administered intravenously and intraperitoneally to mice infected with intracellular *S. typhimurium*, has increased habitation time in plasma and the concentration of drug in the liver, spleen, lungs and kidneys is also increased, while when administered intratracheally its pulmonary retention is increased. Compared with free ciprofloxacin, it extends survival and reduces the number of bacteria in the liver and spleen [207]. Therefore, alternative methods such as DDS which achieve high protective and bactericidal activity should be taken into account in the future as suitable treatments for *Salmonella*-induced infections [203].

#### **8.4. Lysteriosis**

than anticipated [191]. Rifampicin-loaded polybutylcyanoacrylate nanoparticles have shown enhanced antibacterial activity both *in vitro* and *in vivo* against *S. aureus* and *M. avium* due to an effective delivery of drugs to macrophages [192]. The encapsulation of different antibiotics in liposomes has shown good antibacterial efficacy in both macrophage cell lines and in animal models of MAC-due disease [193]. Ciprofloxacin efficiently inhibits the growth of *M. avium in vitro* in a murine macrophage-like cell line using negatively charged liposomes and *in vivo* using specific stealth liposomes in a mouse model of tuberculosis infection [194]. Similar results have been obtained using stealth liposomes of isoniazid and rifampicin, which show controlled

Brucellosis is an infectious disease caused by *Brucella* spp. Four species, *Brucella abortus*, *Brucella melitensis*, *Brucella suis* and *Brucella canis*, have been recognized as human pathogens each associated with a different natural host animal [196]. These small coccobacilli are mainly localized intracellularly within phagocytic cells making treatment difficult, since most antibiotics, although highly active *in vitro*, do not actively pass through cellular membranes [197]. However in the last two decades many experiments have provided good evidence criteria for its antibiotic treatment, the most suitable antimicrobial therapy for human brucel‐ losis continues to be a controversial subject [198]. Because of its intracellular location, long treatments with several antibiotics are required. Relapses are frequent owing to the low efficacy of many drugs and the lack of patient agreement [199]. Thus, alternative methods such as drug delivery systems to achieve high intracellular bactericidal activity should be consid‐ ered [198]. Gentamicin, encapsulated in different types of liposomes, has been evaluated against murine monocytes infected with *B. abortus*. All such liposomes reduced the number of bacteria, the most effective being SPLVs (stable plurilamellar vesicles) [200]. Rifampicinloaded mannosylated dendrimers have indicated specific pH-dependent delivery of this antibiotic to rat alveolar macrophages [201]. Recently, gentamicin loaded poly (D, L-lactideco-glycolide) (PLGA) have been obtained by the several emulsion solvent evaporation method for the treatment of brucellosis [202]. Thus, alternative methods such as DDS to achieve high intracellular bactericidal activity seem promising. The possible use of drug delivery systems containing aminoglycosides may be one of the most appropriate therapeutic advances in

Salmonellosis is one of the most serious food-borne diseases affecting humans. It may be considered the most important pandemic zoonosis under natural conditions [204]. Bacteria of the genus *salmonella* are facultative intracellular parasites that cause salmonellosis and typhoid fever. Antibiotics effective against this type of bacteria have limitations owing to the problems of formulation, low penetration, or the appearance of side effects; these can be solved using carrier systems [205]. Several studies using antibiotic-loaded nanoparticles have been per‐ formed in order to recognize the suitability and efficacy of these carriers in experimental models of salmonellosis [204]. In order to recognize whether polyalkycyanoacrylate nanopar‐

release and reduce toxicity *in vivo* in mice infected with *M. tuberculosis* [195].

human brucellosis treatment in the recent years [203].

**8.2. Brucellosis**

170 Application of Nanotechnology in Drug Delivery

**8.3. Salmonellosis**

*Lysteria monocytogenes* is a facultative intracellular parasite able to cause meningitis and septicaemia. The encapsulation of ampicillin in liposomes decreases the survival of *L. mono‐ cytogenes* in mouse peritoneal macrophages to different extents, depending on the composition of the liposomes [208]. Chitosan-coated plastic films, alone or loaded with antimicrobial agents, were evaluated for their effect against *L. monocytogenes*. These chitosan-coated films inhibited this pathogen growth in a concentration-dependent manner whereas chitosancoated films impregnated with antibiotics were significantly more effective against *L. monocytogenes* [209]. Formulation of gentamicin in liposomes containing DOPE (dioleylphosphatidylethanolamine) and sensitive to pH has been reported to increase the concentration of drug in mouse macro‐ phages infected with *L. monocytogenes*, increasing its bactericidal activity. This formulation is more effective against *L. monocytogenes* than against other bacteria owing to its location in the cytosol [210]. Furthermore, the efficacy of liposomes and free antibiotic were distinguished in *Listeria-*infected mice. Seven days after the treatment, ampicillin-loaded liposomes had reduced the infection by 3.2 logs in the liver and 2.8 logs in the spleen, while free ampicillin was ineffective [208]. In another example, ampicillin-encapsulated polyisohexylcyanoacrylate nanoparticles have been investigated against *L. monocytogenes* in mouse peritoneal macro‐ phages [211].
