Correspondence: Ashish Ranjan, Department of Radiology and Imaging Sciences, National Institutes of Health, Bethesda, MD 20892, USA. Tel.: +1 301 496 3593; fax: +1 540 231 3426; e-mail: email@example.com
Intracellular pathogens like Salmonella evade host phagocytic killing by various mechanisms. Classical antimicrobial therapy requires multiple dosages and frequent administration of drugs for a long duration. Intracellular delivery of antimicrobials using nanoparticle may effectively devise therapies for bacterial infections. This review will address the mechanisms used by Salmonella to avoid host pathogenic killing, reasons for therapeutic failure and advances in nanoparticle drug delivery technology for efficient intracellular bacterial clearance.
In the last few decades, development of chronic carriers of bacterial organisms like Salmonella is increasingly becoming a global health concern (Gunn et al., 2011). Salmonellae are rod-shaped, gram-negative, facultative anaerobes in the family Enterobacteriacea (Malik-Kale et al., 2011). Clinically, Salmonella spp. are classified as enteric (typhoid form) and gastroenteritis types (nontyphoidal form) (Perrett & Jepson, 2007). Enteric forms are seen exclusively in human beings and are caused by Salmonella Typhi and Salmonella Paratyphi (Connor & Schwartz, 2005). In contrast, gastroenteritis is a self-limiting disease condition seen in both human and various animal species including birds, cattle, and pigs and is caused mainly by Salmonella enteric spp. Typhimurium (Alvarez-Ordonez et al., 2011). Based on population-based active surveillance for culture-confirmed Salmonella in the United States by the Foodborne Diseases Active Surveillance Network (FoodNet), an estimated 1.4 million cases of nontyphoidal salmonellosis were observed between 1996 and 1999 (Voetsch et al., 2004). Furthermore, risk assessment studies in the USA and the world for salmonellosis indicate high mortality and morbidity in human and animal populations with economic losses in billions of dollars (Hope et al., 2002; Crump et al., 2004; Behravesh et al., 2011).
Salmonellosis can occur in either an acute or chronic form. Acute salmonellosis can be treated with aminoglycoside and quinolone classes of antimicrobials (Asperilla et al., 1990). Treatment of chronic salmonellosis is difficult owing to drug resistance, poor management practices and the presence of a significant percentage of carriers without clinical signs (Feglo et al., 2004; Solnik-Isaac et al., 2007). Development of a chronic state is mainly by the evasion of host phagocytic killing mechanisms and establishment of specialized intracellular niches sequestered from the host immune system (Monack et al., 2004). The intracellular localization of Salmonella spp. presents unique therapeutic challenges (Pasmans et al., 2008). At a cellular level, many therapeutically active polar drugs are not able to traverse the mammalian cell membrane efficiently. Consequently, necessary intracellular drug levels for bacterial clearance are not met. This may result in antimicrobial treatment failure and high relapse rates.
To reduce treatment failure and relapse, nanotechnology-based approaches may be helpful. Nanotechnology can be used to fabricate the nanoparticles and cross-link them to a variety of antimicrobials (Gamazo et al., 2007). This review will give insights into the potential of nanomedicine for the therapy of intracellular infections.
Salmonella and intracellular survival
The interaction of Salmonella spp. with mammalian phagocytic and nonphagocytic cells is a complex interplay of numerous genes and protein products that is triggered by the bacterium in response to killing by the host (Haraga et al., 2008). Salmonellae possess two types of genes encoding type III secretion systems (TTSS). Their encoded proteins play an important role in extracellular and intracellular survival (Prost et al., 2007). Upon phagocytosis, Salmonellae are found in membrane-bound vacuoles, also referred to as Salmonella-containing vacuoles (Catron et al., 2002; Bakowski et al., 2008; Garcia-del Portillo et al., 2008). The biogenesis of Salmonella-containing vacuoles is normally by the activation of invasion-associated TTSS encoded by a Salmonella pathogenicity island 2 (SPI-2). The SPI-2 upon induction inside the Salmonella-containing vacuoles secretes more than 19 effector proteins across the vacuolar membrane. These effector proteins play an important role in Salmonella-containing vacuoles membrane integrity, promote subcellular localization, avoid lysosomal killing, prevent the action of intracellular antimicrobial factors and reorganize the host cytoskeleton (Rajashekar et al., 2008). Thus, formation of Salmonella-containing vacuoles results in the prevention of direct fusion with late endosomes or lysosomes and evasion of bacterial killing by the host phagocytic cell (Abrahams & Hensel, 2006). In contrast, Salmonella pathogenicity island 1 assists in extracellular survival, invasion of epithelial cells, and infection mainly in the intestinal lumen (Miki et al., 2004). Alternative mechanisms of intracellular survival may be mediated by the Salmonellae phoP–phoQ genetic components activating the transcription of genes within Salmonella-containing vacuoles providing resistance against antimicrobial peptides (Ernst et al., 1999). The phoP–phoQ proteins in Salmonellae produce a remodeling of the lipid A domain of the lipopolysaccharide resulting in an outer membrane that serves as an effective permeability barrier to divalent cations or cationic peptides like antimicrobial peptides (Rosenberger et al., 2004; Murata et al., 2007).
Challenges in intracellular therapy
Persistent intracellular infection can reduce susceptibility to antimicrobials leading to higher incidences of treatment failure (Kanungo et al., 2008). For example, persistent salmonellosis may reduce susceptibility to antimicrobials like nalidixic acid and ciprofloxacin (Crump et al., 2008; Parry et al., 2011). Such cases requires accurate epidemiological assessment for antibiotic resistance and prolonged therapy (Ong et al., 2007). However, prolonged therapy is often associated with patient noncompliance (Tanaka et al., 1998). Salmonellae have also evolved sophisticated multidrug efflux system to reduce the cellular accumulation of drugs (Wasaznik et al., 2009). This is facilitated by the use of pumps belonging to the resistance-nodulation-division (RND) gene family (Piddock, 2006). These drug efflux systems helps in avoidance of bactericidal action of bile salts in the intestinal lumen and of antimicrobial peptide intracellularly.
Cellular barriers in optimizing antimicrobial therapy
Therapeutic success against intracellular pathogens depends on the ability of drug molecules to traverse the eukaryotic cell membrane (Vakulenko & Mobashery, 2003). Intracellular penetration of a drug molecule is dependent on its polarity. Polar drugs are poorly permeable across the nonpolar, lipophilic cell membrane. For example, aminoglycosides like gentamicin are polar and cationic with a net charge of approximately +3.5 at pH 7.4 (Ristuccia & Cunha, 1982). Hence, their permeability across cell membranes is very low (Abraham & Walubo, 2005; Lecaroz et al., 2006). Drugs entrapped in the endosome inside cells can affect their biological activity. Late endosomal pH of 5 can inactivate or increase the minimum inhibitory concentration of the drug molecule. For example, gentamicin shows a 64-fold increase in minimum inhibitory concentration at pH 5 (Gamazo et al., 2006). Thus, active drug molecules should also be protected from endosomal pH. Finally, for complete clearance, drug molecules should target the subcellular niche where the intracellular bacterium resides which is extremely difficult to achieve.
Nanomedicine-based salmonellosis chemotherapy
Goals of antibacterial nanomedicine
Nanotechnology is a multidisciplinary scientific field focused on materials whose physical and chemical properties can be controlled at the nanoscale range (1–100 nm) by incorporating chemistry, engineering, and manufacturing principles (Kim et al., 2010). The convergence of nanotechnology and medicine, suitably called nanomedicine, can potentially advance the fight against a range of diseases (Sanhai et al., 2008). In particular, the application of nanomedicine for antibacterial therapy can sustain drug release over time, increase solubility and bioavailability, decrease aggregation and improve efficacy (Swenson et al., 1990; Gelperina et al., 2005; Dillen et al., 2006). The improved biodistribution profile of drugs encapsulated in a nanocarrier in the target organ of infection (for example, liver and spleen) is because of phagocytosis by the blood monocytes and macrophages of the liver, spleen, and bone marrow (Prior et al., 2000). This is evidenced by enhanced gentamicin accumulation in Salmonella infected liver and spleen in mouse models (Fierer et al., 1990). Additionally, this approach can reduce dosage, frequency of drug administration, as well as healthy organ toxicity observed with conventional therapy (Mingeot-Leclercq et al., 1995; Ahmad et al., 2007). For example, liposomal encapsulation of gentamicin allows a significant reduction (50%) in the total treatment duration in disseminated Mycobacterium avium infections in mice relative to usual antimicrobial therapy (de Steenwinkel et al., 2007). Similarly, reduced build-up of gentamicin in the kidneys upon parenteral administration in rats has been reported (Abrahams & Hensel, 2006). Therefore, nanomedicine approach can limit the distribution of drugs to target organs of infection (Lecaroz et al., 2006).
Optimal properties of nanocarrier for efficient drug delivery
The goal of antibacterial nanomedicine is to achieve intracellular drug delivery especially in the subcellular organelles (Fig. 1). An important component of such goals is to avoid pH-dependent loss of bioactivity in the endosome inside the cell (Gamazo et al., 2006). Rapid escape of drugs from endosome and release at the cytoplasmic pH can be facilitated by incorporating cell-penetrating peptides, fusogenic lipids, or listeriolysin-O onto the nanocarriers (Lee et al., 1996; Reddy & Low, 2000; Moon et al., 2007; Delehanty et al., 2010). The mechanism of endosomal destabilization by these biomolecules is an interplay of endosomal pH and its membrane composition (Wasungu & Hoekstra, 2006). For example, fusogenic lipids such as dioleoylphosphatidylethanolamine do not form bilayers in aqueous media. However, addition of different lipids may favor a bilayer structure. The presence of a negatively charged head group in a stabilizing lipid in acidified endosomes can neutralize the lipid charge and reduces the bilayer stability. This mechanism has been shown to improve cytoplasmic delivery of gentamicin from the endosomes (Lutwyche et al., 1998; Zuhorn et al., 2005). Alternatively, pores on the endosomal membrane can be created by purified listeriolysin-O secreted by the bacterial pathogen Listeria monocytogenes (Vazquez-Boland et al., 2001; Kullberg et al., 2010). Listeriolysin-O activity demonstrates increased biological activity and pore forming ability at low endosomal pH's (Geoffroy et al., 1987; Vazquez-Boland et al., 2001). This property has been employed for the cytosolic delivery of macromolecular therapeutics like peptide antigens, nonviral gene delivery and plasmid DNA (Mandal & Lee, 2002; Saito et al., 2003; Choi & Lee, 2008). However, incorporation of listeriolysin-O in a nanocarrier can potentially induce host immune responses. Therefore, further research is required before clinical use.
Another approach for cytoplasmic delivery, especially for polycationic drugs, is their incorporation into amphiphilic polyanionic carriers. Using this approach, stable dispersions of hydrophilic core–shell nanostructures were obtained by incorporating block copolymers of poly(ethylene oxide-b-sodium acrylate) blended with Poly(acrylic acid sodium salt) homopolymers and complexed with polycationic gentamicin (Ranjan et al., 2010a,b). However, hydrophilic core–shell nanostructures were phagocytosed by endosomal route. Therefore, we modified the hydrophilic core–shell nanostructures by incorporating amphiphilic copolymers into the shells to render them more hydrophobic. Gentamicin encapsulation in core–shell nanostructures that contained some poly(propylene oxide) with an average block length of 68 repeat units in the shells in addition to the hydrophilic polyethylene oxide block enhanced the rate and modulated the route of cell uptake by augmenting nonendosomal uptake (Ranjan et al., 2009a,b). The stabilities of those nanostructures in the presence of phosphate salts, however, were relatively poor. Thus, to improve the stabilities of the core–shell nanostructures at the physiological pH of 7.4, 37 °C, and 0.1 M NaCl, we incorporated a higher molecular weight hydrophobic poly(propylene oxide) with an average of 85 repeat units in the shells, and also more poly(propylene oxide) relative to the hydrophilic polyethylene oxide (Fig. 2). This enhanced hydrophobic interactions contributed to nanostructure stabilities in physiological media in addition to its nonendosomal uptake.
It is also critical that physicochemical characteristics of the nanocarriers like size, zeta potential, pH sensitivity, and surface chemistry are controlled carefully. For example, nanocarriers with a low-positive zeta potential and diameter > 80 nm are rapidly taken up by reticuloendothelial cells (Rudt, 1993). Uptake by macrophages of quantum dot containing anionic carboxylates is more rapid compared with amino-functional polyethylene oxide (Clift et al., 2008). Likewise, the phagocytosis of hydrophilic core–shell nanostructures modified with polyethylene glycol is less efficient by the polymorphonuclear cells (Zahr et al., 2006). In general, preliminary results from our and other studies show that the presence of hydrophobic functional groups on the polymeric surface has a stimulatory effect both in adhesion and internalization by the cells (Mainardes et al., Ranjan et al., 2009; 2010a,b). Thus, we hypothesize that nanocarrier uptake is correlated with particle surface chemistry and should be a subject of further investigation.
Nanomedicine for the treatment of salmonellosis
Antimicrobials encapsulated nanocarriers have been tested in vitro and in vivo against salmonellosis. In vitro treatment using ampicillin-loaded polycyanoacrylate nanocarriers shows marked destruction of the intracellular Salmonella in peritoneal cells and J774A.1 murine macrophage cells (Pinto-Alphandary et al., 1994; Balland et al., 1996). The killing action of the ampicillin nanocarriers was attributed to cell wall destruction of the Salmonella, shown by the presence of numerous spherical bodies in the cell cytoplasm. Also, the actions of these nanocarriers were time dependent. For example, intracellular Salmonella clearance upon a 12-h treatment produced significant differences compared with free ampicillin. In contrast, shorter incubation with the nanocarrier showed higher cell uptake, but did not translate into significant clearance (Balland et al., 1994). This suggests that evaluation of nanocarriers in an in vitro infection models be performed for longer durations.
Along with polymeric carriers, liposomes have also been investigated for cytoplasmic delivery of anitmicrobials (Lutwyche et al., 1998; Cordeiro et al., 2000). Liposomes are efficient nanocarriers, but their stability in the blood plasma is a concern. Break-up of the liposome in blood plasma often tends to release any encapsulated drugs prematurely. To address this, cholesterol has been incorporated into the lipid bilayer to increase stiffness of the liposome walls (Vitas et al., 1996; Mugabe et al., 2005). However, such stable modifications can also compromise the liposomal uptake by the macrophage cells. Therefore, it is critical that the lipid components are appropriately balanced for greater drug delivery.
Treatment for salmonellosis is also dependent on the physiological state, antimicrobial class, and duration of infection (Page-Clisson et al., 1998a,b). For example, acute Salmonella infection is more efficiently cleared by polymeric ampicillin nanocarriers, gentamicin and ciprofloxacin containing liposomes, and gentamicin loaded into core–shell nanostructures (Fierer et al., 1990; Magallanes et al., 1993; Webb et al., 1998; Ranjan et al., 2009a,b). However, polymeric ampicillin nanocarriers are ineffective in treating chronic murine salmonellosis. This is because ampicillin is more effective against replicating pathogens. Chronic infection is generally characterized by changes in the intracellular microenvironment and successful adaptation of dormant bacteria in specialized vacuoles in the lymph nodes, spleen, and liver. This is evidenced by in vitro treatment using liposomes and our core–shell nanostructure encapsulating gentamicin. These nanocarriers show highly efficient intracellular clearance of cytoplasm-resident Listeria (3.16 log reduction in CFU). This is better than clearance of vacuolar-resident Salmonella (0.53 log reduction in CFU) (Lutwyche et al., 1998; Ranjan et al., 2010a,b). It is clear that the vacuolar-resident Salmonella may not have been exposed to a high dose of the antimicrobial owing to membrane barriers around the Salmonella within the cells. In contrast, cytoplasm-resident Listeria directly interacts with gentamicin, favoring efficient clearance. Thus, the stage of infection, i.e. acute or chronic, and subcellular location of the bacterium is a limiting factor in instituting a nanoparticle-based therapy. It is important that the choice of antimicrobial encapsulated nanocarrier should take into consideration these clinical situations. For example, ciprofloxacin encapsulated polycyanoacrylate nanoparticles are relatively better in mice chronically infected with Salmonella compared with ampicillin carriers (Page-Clisson et al., 1998a,b).
As described previously, several nanocarriers are realistic candidates for intracellular treatment. However, they should have high encapsulation efficiency with sustained and prolonged intracellular antibiotic release. For example, our core–shell nanostructures can incorporate up to 25% by weight of gentamicin, and about 25–30% of the gentamicin is released over 24 h in phosphate buffer saline at pH 7.4 (Ranjan et al., 2010a,b). But, as the gentamicin begins to leave the complex, the net anionic character of the complexes increases. As this occurs, greater electrostatic attraction between the polymer and gentamicin slows or completely prevent further release. Therefore, nanocarrier needs to be modified such that they degrade slowly to release 100% of the encapsulate drug. We recently reported biodegradable silica xerogel nanocarrier for complete drug release (Seleem et al., 2009a,b). Xerogel nanostructures are prepared by a sol–gel process. This involves formation of a colloidal suspension (sol) that acts as a precursor for globally connected integrated solid matrix (gel) that can be dried to form xerogel (Quintanar-Guerrero et al., 2009). The xerogels can be fabricated and tuned at low temperatures to carry biologically active agents like gentamicin (Xue et al., 2006). Silica xerogels nanostructures prepared by our technique can incorporate 17% gentamicin by weight and releases 90% of gentamicin in 30 h in vitro. Gentamicin release from these nanostructures is biphasic. A total of 20–25% of drug is initially released at a burst rate followed by a slower and steady state. Biphasic release may be problematic in vivo because burst release can result in encapsulated drug acting similar to its free form. This is reflected in an incomplete in vivo clearance of intracellular Salmonella in the livers (1.15 log reduction in CFU) and spleen (0.41 log reduction in CFU). Therefore, although the results are encouraging, careful engineering and chemical principles are required in particle synthesis to address these issues before further clinical application.
This review summarized the recent findings on targeting of intracellular pathogens especially Salmonella. As discussed, incorporation of antimicrobials in a nanocarrier provides a novel method for intracellular drug delivery and enhancing their killing effect. However, complete eradication of intracellular pathogens using this methodology is yet to be realized. Targeted drug delivery and their intracellular bioactivity are two separate issues. In our opinion, antibacterial nanomedicine in its true sense is the delivery of targeted drug to the subcellular niche where a bacterium resides. Currently available technologies deliver drugs to the cell endosome or cytoplasm and hence may not be fully targeted. Endosomal or cytoplasmic delivery exposes drug initially to the cellular microenvironment prior to their interaction with the bacteria. Phenotypic changes in the cellular microenvironment can increase tolerance against the antimicrobials, alter drug targets and modify their bioactivity (Murillo et al., 2009). Intracellular bioactivity in such scenarios can, however, be improved by modifying the cellular environment. For example, alkalinization of endosomal pH by agents like chloroquine can decrease sequestration of drug and improve their cytotoxicity (Lee & Tannock, 2006). Further, drugs that are environmentally sensitive in their action can be combined with other therapies to enhance their efficacy. For example, macrolide antibiotic Bafilomycin A1 when used alone is not effective against intracellular Diplorickettsia massiliensis infection (Subramanian et al., 2011). But, when combined with chlaramphenicol, they are active at lower concentrations. Similarly, the incorporation of streptomycin and doxycycline into macromolecular polymeric complexes simultaneously is more effective in treating murine brucellosis relative to free drugs (Seleem et al., 2009a,b). Thus, environmentally sensitive therapies may be an elegant treatment approach for improving the intracellular bioactivity of drugs in many clinical situations.
While the goals of improving antimicrobial levels in the infected cells cannot be overstated, an effective interventional strategy directly against the bacteria also needs to be pursued simultaneously. Examples of such an approach includes blocking access to micronutrients like iron or targeting of specific bacterial genes involved in intracellular bacterial growth. To obtain iron, a bacterium produces strong iron chelators called siderophores (Jain et al., 2011). Deletion of genes (for example, entF) responsible for siderophore production has been shown to affect bacterial multiplication in iron minimal media. Therefore, incorporation of micronutrient chelators in a drug delivery system is highly recommended. Similarly, modulations of bacterial genes by synthetic oligonucleotide has been shown to inhibit intracellular bacterial growth (Mitev et al., 2009). For example, phosphorodiamidate morpholino oligomers (PMO) are high molecular weights antisense oligomer. But, owing to their polarity, they are poorly cell membrane permeable. Conjugation of these oligomers to cell-penetrating peptide can result in better intracellular accumulation and clearance of Salmonella from macrophage cells. Most importantly, these conjugated oligomers can enter macrophages and even enter Salmonella-containing vacuoles. The major drawbacks of PMO are their lack of in vivo delivery to the desired organ. Therefore, combining such agents with a nanocarrier is a potentially exciting next step for cell-based therapy. Finally, the arsenals of nanomaterials continue to expand. It is important that the nanostructures are characterized and designed carefully. For example, core–shell nanostructure confers higher gentamicin encapsulation, but incomplete release. Thus, the effects of molecular parameters on size and charge and correlating these aspects with uptake into macrophages, long-term release, and therapeutic efficacy require further investigation.
In conclusion, nanotechnology is a highly promising technology that can enhance the safety and therapeutic efficacy of antimicrobials against many intracellular infections. It is critical that the physicochemical properties like particle size, composition, charge, and surface area be appropriately controlled to direct them to specific locations in the body. In addition, biocompatibility and subcellular delivery of nanostructure may ease the clinical translation.