Background
Phagocytic effecter function is the basic to host defense against intracellular pathogens such as
Staphylococcus aureus (
S. aureus), the gram positive coccal bacteria, the etiological agent of local infections (e.g., wound infection, furuncle and cellulitis) to systemic dissemination (bacteremia) and finally to metastatic infections (e.g. endocarditis, osteomyelitis and septic arthritis) [
1]. Microbes upon entrance to host body are destined to engulfment by professional phagocytes such as neutrophils, macrophages and dendritic cells. Among few organisms,
S.aureus possess diverse mechanism to avoid destruction in phagolysosomes [
2]. Recent in-vitro studies revealed greater resistance of
S.aureus to killing by macrophages [
3]. Upon internalization by macrophages,
S. aureus is widely assumed to be confined within phagosome following its maturation and fusion with endosomes and lysosomes, creates an inhospitable environment for invading microorganisms, boosting acidification, and augmentation of ROS, and other charged anti-microbial peptides [
4].
S. aureus has evolved a diversified array of antioxidant tools both enzymatic and non-enzymatic to resist immune mediated oxidative attack [
5].
With the emergence of MRSA (methicillin resistant
S. aureus) strains with reduced susceptibility to vancomycin, and as there are numerous discrepancies for antibiotics action, starting from the degree of plasma membrane permeability, segregation to diverse compartments, to inactivation by intracellular environment [
6]. Thus there is the need of combinational therapy with efficacious antibiotics having intracellular bactericidal activity, being harsher to pathogen and least severe to host cells. Thus, different antibiotics or drugs amalgamation can implement successive anti staphylococcal mission and further not only reduce infection but can also aid in protection of host cell from
Staphylococcus aureus infection induced cell death.
Riboflavin (vitamin B-2) is an essential micronutrient found in a large variety of foods. Vitamin B-2 is necessary for maintaining proper functioning of the nervous, endocrine, cardiovascular and immune systems. Riboflavin is known to elevate immune functions by activation of macrophages, conferring bactericidal action from its spectrum of actions. Riboflavin participates in numerous diverse internal redox reactions as a part of metabolism. An inadequate intake of this vitamin would contribute to difficulties in intermediary metabolism [
7]. Riboflavin kinase (RFK) in cell converts Riboflavin into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are essential cofactors of dehydrogenases, reductases, and oxidases including the phagocytic NADPH oxidase 2 (Nox2) and participate in wide range of redox reactions [
8,
9]. Riboflavin as a proteasome inhibitor quenches inflammation by reduction of proinflammatory cytokines [
10]. Moreover, in-vivo treatment of mice with Riboflavin reduces the mortality of mice with septic shock [
11] and enhances the resistance to bacterial infections [
12].
Imbalance between Reactive oxygen species (ROS) and antioxidant enzymes confers to cytotoxicity, and thus balance between these two ensures prevention from chronic diseases [
13]. ROS produced by NADPH oxidase (NOX) envisage its role as defense and signaling molecules related to innate immunity and various other cellular responses [
14]. In the early innate immune response H
2O
2 kills bacteria through classic ROS respiratory burst. Superoxide anion does not diffuse across the membranes efficiently and is rapidly dismuted to H
2O
2 by superoxide dismutase (SOD). However, H
2O
2 can diffuse more freely and causes direct oxidative damage to many pathogens. NOX-2 derived ROS is the main, but not the only source of oxidative attack on invading organism [
15] and are critical components for host defense against bacterial infection [
16]. As the balance in the levels and rates of production of ROS and NO dictates oxidative versus nitrosative stress, these differences may be crucial in understanding how immune responses are regulated in host cells upon treatment with Riboflavin and antibiotics during
S. aureus infection. Cells contain several anti-oxidant systems to protect themselves from the injury induced by increased intracellular ROS. However the effect of antibiotics along with Riboflavin was not investigated in infection induced oxidative stress and inflammation in macrophages [
17].
Azithromycin (AZM), the macrolide antibiotic has the potential to accumulate inside the cell and therefore have an intracellular responsiveness [
18]. Intracellular azithromycin enhances phagocytic killing of staphylococci. Azithromycin has the ability to concentrate within neutrophils, and dynamically act against intracellular organisms [
19]. Ciprofloxacin (CIP) which belongs to group of fluoroquinolones is potent synthetic agents active against a variety of bacterial species in vitro. Fluroquinolone antibiotics accumulation in mammalian PMNs can be largely found intracellularly than extracellularly. Further, it has also been seen that enhanced trapping of antibiotic is observed in infected phagocytic cell and thus more clearance of bacteria.
Other than antimicrobial activity, antibiotics are also potent immuno-modulators [
20]. They are capable of reducing the production of pro-inflammatory cytokines like IL-1β, IL-6, IL-8, TNF-α, IFN-γ and also regulate anti-inflammatory IL-10 in the cytokine milieu during acute phase inflammatory processes [
21,
22]. Furthermore, antibiotics diminish the release of various oxidizing species like superoxide anion and nitric oxide that take part in the innate immunity of the host phagocytic cells, their prominent modulatory effect on several nuclear transcription factors such as NF-κB and activator protein-1 (AP-1) in the cell cytosol has been documented. [
23].
But as already mentioned, although advancement antibiotic dosing regimen, the increasing prevalence of infections caused by multidrug-resistant bacteria is a global health problem that has been exacerbated by the dearth of novel classes of antibiotics. Herein, we tried combination therapies for the treatment of multidrug-resistant bacterial infections using an in vitro infection model. These efforts include antibiotic–antibiotic combinations, such as AZM and CIP along with the use of certain other agents like Riboflavin, which could delineate S. aureus burden by activation of macrophage weaponry against the pathogen by enhanced production of ROS and cytokines, the therapeutic approach of which will provide adequate coverage for potential pathogens causing infections.
The present study is a contribution to the current knowledge about oxidative stress caused by S. aureus infection in macrophages, suggesting that an investigation of ROS, NO and antioxidant enzymes should be performed in order to detect the contribution of infection induced oxidative stress and inflammation and its amelioration by treatment of host cells with Riboflavin and antibiotics including CIP and AZM. In response to the regulated production of NO and pro inflammatory cytokines, there was a rise in the activity of SOD and enzymes of the Glutathione system and decrease in the expression of COX-2, the enzyme which drives acute inflammation from its onset to its resolution was investigated upon combinational treatment. Production of pro-inflammatory cytokines was regulated by combined treatment with Riboflavin and antibiotics. Thus, this combinational therapy could bestow protection to the host cell and ultimately restrict S. aureus infection induced cell death.
Discussion
The treatment of
S. aureus infections has been increasingly problematic due to the high prevalence of multi-antibiotic-resistant strains, such as methicillin-resistant
S. aureus, [
29] and the emergence of vancomycin-resistant
S. aureus strains. As an alternative to traditional antibiotics in an era of increasing bacterial resistance, new attention has focused upon development of agents that can effectively disarm the pathogen burden and allow clearance by the host. The aim of this study was to determine the intracellular anti-staphylococcal activities of antibiotics CIP and AZM along with the presence of Riboflavin an established modulator of macrophage function to boost pathogen clearance and enhance longevity of the host cell by reducing cell death. It has been reported that Riboflavin at a concentration of more than 25 μg/ml could enhance macrophage function in-vitro [
30]. Our result of recovered or uptaken Riboflavin concentration after treatment of macrophage with 100 μg/ml depicts that intake of Riboflavin by macrophages is more than 40 μg/ml (Table
1) which according to previous reports is more than the threshold value required for macrophage activation in-vitro. Riboflavin uptake is enhanced when macrophages were exposed to
S. aureus and this uptake is incubation time dependent, i.e., 90mins incubation resulted in more Riboflavin intake than 60mins of incubation time. From our experiment of Riboflavin concentration inside the cell we found that the use of antibiotics AZM and CIP further enhanced Riboflavin concentration (Table
1). It has been reported that formation of intra mitochondrial FAD requires transport of Riboflavin from the cytosol into mitochondria via mitochondrial flavokinase. Because FAD itself does not traverse membranes, FAD requires degradation back to Riboflavin [
31]. Therefore trafficking of Riboflavin inside the macrophages could be expected.
From the bacterial CFU count of
S. aureus recovered after phagocytosis by Riboflavin pre-treated murine peritoneal macrophages, where, significantly lower number of bacteria has been found in comparison to Riboflavin untreated
S. aureus infected macrophages, indicating the immunomodulatory role of Riboflavin and the presence of antimicrobial property in azithromycin and ciprofloxacin further aided in the clearance of the pathogen. Confocal imaging also depicted more engulfment in presence of Riboflavin alone or with AZM or CIP. Previous results indicated that the phagocytic ability of the macrophages may be significantly limited in macrophages with Riboflavin deficiency [
8]. Therefore, a lower amount of viable intracellular bacteria directly correlated with increased engulfment and subsequent intracellular clearance in the given experimental set up. Taken together Riboflavin pretreated cells were active enough to kill the bacteria in presence of antibiotics. Since treatment with antibiotics and Riboflavin did not exert any toxic effect on macrophage and thus has no effect on the viability of macrophages therefore, reduced amount of viable intracellular bacteria could not be expected from decreased amount of extracellular bacteria or from an impaired phagocytic activity of macrophages due to the treatment with antibiotics and Riboflavin. However, we have not tested whether the treatment of antibiotics used and Riboflavin has any effect directly on the growth of
S. aureus.
Undoubtedly with the elicitation of macrophages function in presence of Riboflavin (Table
1), reactive oxygen species like H
2O
2 and superoxide anion elevation has been found which give the ability to macrophages to destroy ingested cells. Riboflavin deficient macrophages may exhibit an impaired ability of phagocytosis and killing of ingested particles [
32]. Decreased levels in ROS were observed when infected macrophages are treated with antibiotic alone. It has been shown that macrolides like AZM are able to inhibit the production of ROS from neutrophils and this is incubation time dependent [
33], which has been suggested to be partly because of the stabilization of cell membrane. Macrolides attenuate the membrane-destabilizing effect of bioactive phospholipids, such as lysophosphatidylcholine, platelet-activating factor (PAF), and lyso-PAF, and this is accompanied by a dose-related inhibition of superoxide production. Interference with phospholipase/phosphatidic acid phosphohydrolase may also decrease superoxide generation by phagocytes [
34]. The kinetics study of O
2
− and H
2O
2, production in the lysate of macrophage cultures has revealed higher concentration of these anions in CIP and Riboflavin treated infected macrophages but lesser in the RIBO and AZM-treated infected macrophages after incubation. The phagocytosis of microorganisms activates oxidase dependent on adenine dinucleotide phosphatase (NADPH), which induces the production of high levels of superoxide, H
2O
2 a process commonly denominated as respiratory burst.
The enzyme Riboflavin Kinase converts Riboflavin to FMN and FAD. The crucial enzyme for ROS production i.e. NADPH oxidase enzyme complex depends on FAD which is one of the NADPH oxidase component [
14,
35], therefore, elevated H
2O
2 and O
2
− production in Riboflavin pretreated macrophages obtained in this study is also relevant and supported by earlier studies Fig.
3a, b]. Superoxide anion does not diffuse across membrane efficiently and is rapidly dismuted to H
2O
2 by SOD. However, H
2O
2 can diffuse more freely and causes direct oxidative damage to many pathogens. In the early innate immune responses, H
2O
2 kills bacteria through classic ROS respiratory burst.
Further elaboration of ROS, the effecter molecules is documented in the addition of Ciprofloxacin along with Riboflavin as it stimulates the oxidative burst in monocyte and macrophages. Azithromycin when combined with Riboflavin elicits the function of macrophage but lower in comparison to Riboflavin when combined to Fluroquinolone signaling [
36]. In our study pre-treatment of macrophages with antibiotics does not lead to residual toxic effects on macrophages, and the effects are due to changes in response to live cells because in our results by FACS analysis of ROS a significant increase in ROS production on addition of antibiotic CIP was documented which cannot be attributed by dead cells.
Our data suggest that Riboflavin treatment diminishes the nitric oxide production by mouse peritoneal macrophages when exposed to
Staphylococcus aureus. Suppression of nitric oxide induction and pro-inflammatory cytokines by novel proteasome inhibitors has been studied in various experimental models as in LPS stimulated RAW 264.7 macrophage [
10]. Role of Riboflavin as a naturally occurring proteasome inhibitor has been extracted from literature according to which Riboflavin is potent inhibitors of chymotrypsin-like activity of 20S subunit of rabbit muscle proteasomes and mouse immune proteasome and its activity depends on protease site LMP7 and other signaling pathways [
10].
In our study of thioglycolate elicited murine peritoneal macrophage which were infected by
S. aureus the pathway by which Riboflavin is suppressing the NO production is through increasing cellular levels of PI-κB and decrease nuclear translocation of NF-κB, further decreasing degradation of ubiquinated PI-κB by the proteasome, and this inhibition by Riboflavin results in decreased translocation of NF-κB to the nucleus [
10]. It has been reported that low levels of H
2O
2 can enhance NF-kB activity and higher H
2O
2 inhibited its activity [
37]. The overall effect of NO and ROS on NF-kB activity follows a biphasic pattern and many of these observations are cell and mechanism dependent [
38]. In addition, lower NO produced by
S. aureus stimulated macrophages (Fig.
5) can combine lesser with superoxide to diminish the generation of additional product with enhanced toxicity, such as peroxynitrite, suggesting protection from more harmful ROS in presence of Riboflavin at 60 and 90 mins post incubation [
39,
40].
It has been suggested that bacteria that has survived avoiding the host immune response must have to scavenge the host derived O
2
− and H
2O
2 by enhancing bacterial SOD and catalase enzyme activity. Higher amount of SOD enzyme activity might have counteracted with the increased superoxide radical and H
2O
2, as evidenced by the increased SOD activity in lysate of macrophages exposed to
S. aureus from Riboflavin treated macrophages. The increased SOD activity (Fig.
6b) in lysate, therefore, neutralizes the bactericidal activity of host macrophage derived O
2
− by converting it into H
2O
2, another oxygen metabolite having potential antimicrobial activities whereas, decreased catalase activity (Fig.
6a) in the lysate of macrophages exposed to recovered
S. aureus from Riboflavin treated macrophages suggests enhanced phagocytic capacity of the macrophages. Lesser Catalase and more SOD enzyme activity has also been documented when in addition to Riboflavin, cells were further co-treated with Ciprofloxacin at 60 and 90 mins of incubation. Macrolide antibiotics like AZM are also known to alter the physiological redox homeostasis leading to oxidative stress and lipid peroxidation [
41]. This induction of oxidative stress may further reduce the catalase activity in 60 and 90 mins post incubation in presence of Riboflavin (Fig.
3a). Reports also suggests that in acute infection i.e., for 60 and 90 mins Ciprofloxacin increases the SOD activity leading to more production of H
2O
2 from more generated superoxide anion [
42]. A study investigated the effect of Riboflavin therapy on diabetic cardiomyopathy documented that Riboflavin can increase SOD activity in the heart tissue [
43]. So, increase in SOD activity in all Riboflavin treated groups can be attributed to antioxidant property of Riboflavin. Earlier it was demonstrated that TNF-α mediated activation of NF-κB Pathway may be inhibited by transient over expression of cytosolic SOD1 or mitochondrial SOD 2 but not catalase [
44].
Glutathione is the master component of the antioxidant defenses in the cell. Both cellular activities of glutathione reductase and concentration of reduced glutathione are markers of flavin status and FMN and FAD participate in a range of redox reactions including the Glutathione system Our results demonstrate an increase in both Glutathione and Glutathione reductase activity when macrophages were stimulated in presence of Riboflavin at 60 and 90 mins of incubation. Presence of Riboflavin at a dose of 100 μg/ml converted it to FAD which may acted as a co-enzyme to increase glutathione reductase activity at 60 and 90 mins of incubation in the cell lysate [Table
3]. Further activation of glutathione reductase might have increased the reduced glutathione content in the cell as evident from our study at 60 and 90 mins of incubation (Fig.
6c) [
45]. FAD transports hydrogen from NADPH to oxidised glutathione to convert it into the reduced form [
46]. Reduced glutathione acts as an endogenous antioxidant in different cell types function of which can be enhanced by the activity of Riboflavin.
Increase of GSH and decrease of LPO in Riboflavin treated groups can be indicators of reduction of oxidative stress, as lipid peroxidation is a potent agent for cellular damage which acts by inhibiting membrane enzymes and receptors, depreciation of LPO and augmentation of GSH is more prominent when Riboflavin is combined with AZM/CIP (Fig.
6c and
d).
The defense of an organism depends on the recognition of harmful stimuli followed by appropriate activation of innate and adaptive immune responses, leading to cytokine production. Several cytokines have pro-inflammatory actions that drive the innate immune response, cause inflammation and activate adaptive immune responses. During infection and sepsis elevated levels of pro-inflammatory cytokines, e.g. TNF-α and IFN- γ have been reported [
47]. Pro-inflammatory cytokines like TNF-α, IL-1β, and IL-6 are inducer of systemic inflammation [
20] and can be detrimental to host cell inducing hemodynamic shock and cell death. Our research has focused primarily on macrophages because they are highly sensitive to Riboflavin stimulation and respond by reducing production of TNF-α, IL-1β, IL-6 and IFN-γ.
TNF-α is a multifunctional pro-inflammatory cytokine that belongs to the tumor necrosis factor (TNF) super family [
48]. Increased TNF-α from the Riboflavin plus CIP or Riboflavin plus AZM treated macrophages at early infection (at 30 min) indicating bacterial killing and better efficacy of both this antibiotics in presence Riboflavin (Fig.
7a). However, suppression of TNF-α level at late stage of infection by both the antibiotics in presence of Riboflavin indicates the combination to be effective in down regulating inflammation with increasing time which was beneficial to the host cell. Suppression of IL-1β, IFN-γ as well as IL-6 (Fig.
7d,
b and
c) by the Riboflavin and CIP or AZM treated macrophages at late stage of infection further confirms their anti-inflammatory nature. Macrolide antibiotics reportedly repress the production of pro-inflammatory cytokines in macrophages [
49]. Apart from the microbicidal activity of fluroquinolone Ciprofloxacin, reports also pictures CIP also a potent immunomodulatory agent [
50]. As already discussed Riboflavin is a potent protease inhibitor and this inhibitory effect governs the TNF-α signaling pathway through NF-κβ. Classical NF-κβ consist of heterodimers of p65 and p50 and these are the potent activators of plethora of general proinflammatory cytokines such as IL-6, TNF-α etc. In an inactive cell p50/65, is maintained in the inactive state in the cytoplasm of cells when it is bound to I-κB. Riboflavin has the capacity to inhibit proteasome activity in conjunction with their capacity to increase cellular levels of PI-κB and decrease nuclear translocation of NF-κB, suggests that the mechanism by which these agents suppress production of TNF-α and NO, involves decreased degradation of ubiquinated P-IκB by the proteasome, resulting in depressed translocation of NF-κB to the nucleus. Thus, Riboflavin exerts is anti-inflammatory effects by inhibiting NF-κB activation [
10]. Based on these reports, the capacity to increase cellular levels of P-IκB and subsequently decrease nuclear translocation of NF-κB, we hypothesized that Riboflavin possibly down-regulate the NF-κB pathway. Given the important role of TNF-α in sepsis such suppression by combination therapy could be a therapeutic effect [
51]. It is also known that exposure of staphylococci to beta-lactam antibiotics greatly enhances the release of bacterial cell wall fragments that have strong proinflammatory properties, such as peptidoglycan and lipoteichoic acid [
52,
53], resulting in the induction of higher TNF-α levels from monocytes [
54]. Previous reports demonstrated the clinical benefits of antibiotic and TNF-α inhibitor combination therapies relating to both arthritis severity and bone destruction in addition to reductions in the mortality rate by a combination therapy in experimental
S. aureus–induced septic arthritis [
55]. Recently we have observed that certain interaction exists between TNF-α and MMP-2 during the course of septic arthritis in
S. aureus infected mice, hence further experiments are warranted to figure out the impact of TNF-α on MMP-2 activation via receptors for TNF-α (TNFR-I and TNFR-II) during
S. aureus induced septic arthritis [
56].
Among the pro-inflammatory cytokine milieu, anti-inflammatory cytokine IL-10 was also evaluated and it was found that early infection in presence of Riboflavin did not effect this anti-inflammatory cytokine, but at 60 and 90 mins of post incubation IL-10 was found to be increasing in presence of Riboflavin [
57] and this effect was more prominent in acquaintances of Riboflavin with AZM and CIP (Fig.
7f).
Riboflavin is also known to modulate MCP-1 function which is a potent chemoattractant and a regulatory mediator involved in a variety of inflammatory diseases [
58]. Evidences further support the action of fluroquinolones and AZM on suppression of MCP1 [
59]. Our report shows a reduction in the value of this chemo-attractant in Riboflavin treated groups. Further in comparison to only Riboflavin treated group, combination with AZM or CIP further down regulated production of chemokine MCP 1 (Fig.
7e). Riboflavin action as an inflammation regulator has not only been justified but its role as an anti-inflammatory cytokine enhancer was also established from this study.
For a clear analysis of inflammation, a well known hallmark of acute inflammation COX2 expression was also analysed in our experimental setup [
60]. A decrease in COX2 expression at 90 mins in all Riboflavin treated group was documented. NO has been found to modulate PGE2 synthesis in macrophage cell line and rat islet cells [
61]. Our study shows declined production of NO which might be responsible for reducing COX2 expression as reduction in expression of iNOS tend to reduce expression of COX2 modulated by cytokines. IL-1β stimulated the expression of inducible nitric oxide synthase (iNOS) by β-cells [
62]. So, from our study it can be speculated that lower level of IL-1β might have reduced the iNOS synthesis which by similar mechanism reduced COX2 expression. Reduction of inflammatory mediator like COX2 certainly illustrates Riboflavin as a potent inflammation regulator. In the in-vivo system treatment with azithromycin and Riboflavin completely eradicated the bacteria from blood and spleen. TNF-α, IFN-γ, IL-6, and MCP1 were down regulated by treatment with azithromycin and Riboflavin [
63]. Treatment with Riboflavin also altered the antioxidant status which was further elaborated by using this combinational therapy in mouse peritoneal macrophage in the present study.
Caspase-1, however, seems to be uniquely involved in participating in the inflammatory response by cleaving the precursors of IL-1 beta, IL-18, and IL-33. Indeed, the rate-limiting step in inflammation due to IL-1 beta or IL-18 is the activation of caspase-1 [
64]. Recently, a role for ROS has emerged in the activation of the NLRP3 inflammasome, one pathway for generation of active caspase-1 and secretion of mature IL-1β [
65]. There is a distinct role for ROS in up-regulation of mRNA for inflammatory cytokines such as IL-1β and TNF-α. As, ROS upregulate Caspase 1 as well as IL-1β, So increase in ROS production on Riboflavin treatment might have upregulated Caspase 1 as well as IL-1β in early infections. In late infections reduction in IL-1β might have reduced Caspase 1 expression as there are reports that IL-1β secretion and reactive oxygen species (ROS) down-modulate IL-1β rather than activating it in phagocytes [
66].
The isoalloxazine moiety of Riboflavin chelates metals such as cadmium, cobalt, copper, iron, molybdenum, manganese, nickel, silver, and zinc [
67]. Some bound metals are easily oxidized and reduced and play a role in the formation of free radicals. Our study also evidenced an enhanced free radical production behind which iron chelation by Riboflavin may play a significant role. Iron has important role to play in the disease manifestation of a pathogen in the macrophage [
68].
Pathogens release siderophores which chelate iron present in the macrophage for their own survival, but presence of Iron chelators like Riboflavin in the medium will chelate the iron and make it unavailable to the bacteria secreted siderophores, Thus depletion of Iron will hinder the pathogen’s replication and thus their survival will be at a stake.
It appears desirable to keep the concentration of pro-inflammatory products of pathogens in the tissue low during the whole course of an infection to decline host cell death. In clinical practice, a favourable outcome depends on the antibiotic treatment after hospital admission. Therefore, the reduction of potentially deleterious pathogen derived compounds by rapid initiation of an effective antibiotic therapy along with choosing compounds which synergistically act with antibiotics to boost the killing process and further do not release large amounts of pathogen products is a promising strategy to over -stimulate phagocytic cells and decrease tissue injury. It restricts host cell damage which depicts a protective approach of Riboflavin along with antibiotics like AZM and CIP.
Similar results were also found in case of bone marrow derived macrophages (BMDM) when pre-treated with Riboflavin and exposed to S.aureus. The phagocytic activity increased with the treatment of riboflavin along with increased ROS production leading to augmentation in the killing of pathogen. This comparative analysis of BMDM to peritoneal macrophages showed similar mechanism of action, confirms human relevance, or at least efficacy during initial infection conditions.
The use of these novel mechanisms can be implemented in targeted delivery of drugs through targeted drug carriers like microcapsules, liposomes and micelles etc. in in-vivo system. In arthritis, a diversity of organisms are present in deep infection, the dominant bacteria are Gram positive pathogens with S. aureus. Established treatments rely on the controlled release of antibiotics. Therefore, this targeted drug carrier will selectively and effectively localize pharmacologically active moiety of Riboflavin and CIP at pre selected target in therapeutic concentration while limiting its access to non-targeted cells thereby minimizing toxic effect and maximizing therapeutic index.