LITERATUREREVIEW  1.1       INTRODUCTION  About 17 million of lives, mostly of children and ofelderly, are taken by bacterial infections. Although there have beensignificant achievements in the process of discovering and developingantibiotics, infectious diseases still remain as the second main cause of deathglobally (DeLima Procópio et al. 2012). This can be attributed to theincreasing antimicrobial resistance that occurs due to the misuse ofantibiotics.

Antibiotic resistance is becoming a global threat as the emergenceof multi-drug resistant organism increases and begins to spread (Rudramurthyet al. 2016).             The discovery of antibiotics derivedfrom Streptomyces started in 1942 and 80% of antibiotic used now are from Streptomyces (De Lima Procópio et al. 2012).

The Gram-positive bacteria has thenature of producing a class of bioactive secondary metabolites, calledpolyketides metabolites. Some of these metabolites which are bioactive are potentiallyused as antibiotics and immunosuppressant (Khan et al. 2011; ?muraet al. 2001; Patzer & Braun2010).              The occurrence of antimicrobial drugresistance has led to the development of nanoparticles as drug delivery system (Rudramurthy et al. 2016). Nanotechnology provides a betterdrug delivery system because the drug does not interact with the carrier and isdelivered directly to the right site of action as its protected  against enzymatic degradation (Asadi 2014).

             Nanoparticles as antimicrobialdelivery system produce the desired therapeutic effect because of their largesurface area to volume ratio (Ravishankar Rai & Jamuna Bai 2011), hence their interaction with themicroorganism increases and leads to enhancedtherapeutic outcome (Hajipour et al. 2012; Whitesides2005). As nanoparticles can enter the hostcell, antimicrobial agent can be released from the nanoparticles to treatintracellular infections(Zhang et al. 2010).              Extensive research had been done onpoly lactic-co-glycolic acid (PLGA) to be used as nanoparticles.

PLGA is asynthetic polymer which has been approved by US Food and Drug Administrationand European Medicines Agency (Prokop & Davidson 2008; Vertet al. 1994). Thebiodegradable nature (Dinarvand et al. 2011), nano-size, solubility in mostsolvents, enabling of controlled and sustained release of drugs (Anderson & Shive 2012; Baldwin& Saltzman 1998; Panyam &Labhasetwar 2003), bio-compatibility and nosignificant toxicity (Athanasiou et al. 1996) make PLGA nanoparticles to be aneffective drug delivery system.             Alginate, a co-polymer of guluronicacid and mannuronic acid, has also been approved by the US Food and DrugAdministration (Tønnesen & Karlsen 2002). Owing to the negative charge of alginate(Mariappan et al.

2005) , it is muco-adhesive which is animportant characteristic of a drug delivery system to improve thebioavailability. Alginate nanoparticles are also cytocompatible andbiocompatible to be administered to the body(Lee & Mooney 2012; Sareiet al. 2013).             This study is done to evaluate thetherapeutic enhancement of the biologically active polyketides metabolites of Streptomyces when encapsulated into the alginate-coatedPLGA nanoparticles as delivery system, as compared to the free polyketidesmetabolites.         1.2      NANOPARTICULATE-BASED DRUG DELIVERYPLATFORM: PROMISING APPROACH AGAINST BACTERIAL INFECTIONS Althoughthere have been advancements in the development of antimicrobials, treatinginfectious diseases is becoming a great challenge.

This is due to thedifficulty of antimicrobials to cross the cell membranes to reach the site of actionespecially in intracellular infection, and instead acting on normal cells thatleads to toxicity and the emergence of antimicrobial resistance (Zhang et al. 2010). Drawbacks and limitations ofconventional medicines such as poor bioavailability and lack of selectivity ledto the development of nanotechnology (Nevozhay et al. 2006) . Nanotechnologyprovides a better drug delivery system because the drug does not interact withthe carrier and is delivered directly to the right site of action as itsprotected  against enzymatic degradation (Asadi 2014). One of the extensively explorednanotechnology fields is nanoparticles.             Nanoparticles helps in drug deliveryto the microorganism through various mechanisms as shown in the Figure 1 below.

Figure 1.1 Mechanism of drug deliveryto microorganisms by nanoparticles. (a) by fusing with the cell membrane and exposingthe drug to the cell and (b) binding to the cell wall and functions as drugdepot causing a continuous release of drugs which will diffuse into themicroorganismSource : (Zhanget al. 2010) Dueto the size range from 10 -1000 nm (Soppimath et al. 2001), nanoparticles exhibit uniquecharacteristics (Wilczewska et al.

2012) . The small size of the nanoparticleenhances the therapeutic efficacy of the drug delivery system (Xu et al. 2007). The nano-sizealso causes nanoparticles to have a high surface-to-volume ratio (Ravishankar Rai & Jamuna Bai 2011) which enhance the antimicrobialactivity of the drug by increasing the exposure towards microorganism (Hajipour et al. 2012; Whitesides 2005).

They also can easily enter the cell due to their small size leading toincreased efficacy of drug delivery to a specific site of action. This is dueto their ability to pass through the smallest capillaries and avoid from beingtaken up by phagocytes so they can remain in the blood for a longer period oftime (Parveen et al. 2012).

  Figure 1.2             The importance of size and shape ofnanoparticles as drug carrierSource : (Farokhzad & Langer 2009)                        Targeted drug delivery systemtowards microorganisms also can be achieved by using the nanoparticles(Gao et al. 2014). This can be doneby manipulating the particle size and surface characteristic which can lead topassive and active drug targeting (Singh et al. 2010). Active drug targeting meansconjugation of a nanoparticle to a tissue or cell-specific ligand (Lamprecht et al. 2001) while passive drug targeting is the incorporationof the drug into the nanoparticles which passively targets the specific site ofaction (Maeda 2001; Sahooet al. 2002) as shown inFigure 1.

3. Due to the increased vascular permeability in bacterial infectionas bacterial components trigger inflammatory mediators, passive targeting ofnanoparticles can be easily achieved. As macrophage possesses scavengingproperty, they can easily take up nanoparticles. This feature helps in thetreatment of bacteria that survives the ingesting of macrophages.

(Gao et al. 2014) Targeted drugdelivery system is also important to prevent toxicity of antimicrobial agents.For an example, aminoglycoside causes ototoxicity and nephrotoxicity that leadsto the need for controlled dosage.(Zhang et al. 2010).  Figure 1.3 Passiveand active drug targetingSource :(Singh & Lillard 2009)             In addition to that, nanoparticlesdo provide controlled and sustained release of antimicrobial drug (Zaidi et al.

2017). Controlled and sustained release isimportant in drug delivery as it increases the drug therapeutic efficacy and minimisesthe side effects (Parveen et al. 2012). In designing an optimum deliverysystem, controlled and sustained release of drugs at optimum therapeutic rateand dosage becomes a major goal (Farrugia & Grover 1999). As the drug reaches the target siteor organ, the nanoparticles can function as a depot to supply the drugcontinuously to the site of action (Singh & Lillard 2009).

Based on a study done by (Pandey et al. 2003), after oral administration ofanti-tubercular drugs-loaded nanoparticles, the plasma concentration of theanti-tubercular drugs could be maintained at a minimum inhibitory concentrationup to 9 days, while free drugs could only last up to a day only. The drugconcentration at the target organ was at therapeutic level till day 11, whilefree drug could only be found at the target organ for not more than 2 days.

Controlled and sustained release of antimicrobial drugs can improve patientadherence as frequency and dose of the required drug will be reduced.(Ladavière & Gref 2015)             In addition, nanoparticles couldalso overcome some limitation of other free drugs, especially in oral drugdelivery system. As most of the free drugs are susceptible for degradation inthe digestive tract, nanoparticle could overcome these limitations as theyprotect the drug (Des Rieux et al. 2006).

Encapsulation of the drug allowsprotection against enzymatic or hydrolytic degradation (Damge et al. 1990) and prevent interaction with otherdrugs.(Zaidi et al. 2017) As bioadhesive properties ofnanoparticles can easily be modified, nanoparticles can adhere to the mucosamembrane and increase the absorption of the drugs, hence increasing the bioavailability(Gabor et al.

2004).   1.3       POLY LACTIC-CO-GLYCOLIC ACID (PLGA)NANOPARTICLES IN DRUG DELIVERY SYSTEM Animportant factor in designing a drug delivery system is the choosing of materialsused for that drug delivery system.

Choosing the right polymer for designingnanoparticles is necessary to achieve the desired target (Bala et al. 2004). One of the polymer matrix that hasbeen widely researched upon is poly lactic-co-glycolic acid (PLGA). PLGA is apolymer that has been approved by both US Food and Drug Administration andEuropean Medical Agency (Prokop & Davidson 2008; Vertet al. 1994). As nanoparticles,it has proven to be an excellent vector in the drug delivery system (Sharma et al. 2016). PLGA nanoparticles can also beformulated into various forms and sizes according to the desired application (Anderson & Shive 2012) and can also encapsulate moleculesof various size.

PLGA is soluble in most common solvents (Uhrich et al. 1999; Wu& Wang 2001). PLGA’smucoadhesive property also enables various routes of administration of thenanoparticles (Tafaghodi et al. 2004).

   x: Number oflactic acidy: Number ofglycolic acid Figure 1.4Chemical structure of PLGASource: (Mahapatro & Singh 2011)             A well-known advantage of using PLGAis attributed to its biodegradable property. PLGA nanoparticle can degrade intwo ways, mainly by bulk erosion or by surface erosion (Dinarvand et al. 2011). The biodegradable property canclearly be seen when PLGA breaks down to form metabolites which are lactic acidand glycolic acid as shown in Figure 1.

5. These metabolites are commonly foundin our body and can be metabolized further by the Krebs cycle (Kumari et al. 2010). The end products are carbon dioxideand water (Jain 2000; Panyamet al. 2002) which leads tominimal systemic toxicity of PLGA (Wickline et al. 2007).

PLGA polymer enables sustainedrelease of drugs by diffusion and also degradation of the PLGA matrix (Anderson & Shive 2012; Baldwin& Saltzman 1998; Panyam 2003). The rate of degradation actuallydepends on the monomer ratio used to produce PLGA. PLGA 50:50 which has 50 %lactic acid and 50% glycolic acid degrades faster than any other monomers that arepresent in higher ratio.   Figure 1.5 Hydrolysis of PLGASource : (Kumariet al. 2010)             Based on in vivo studies and invitro studies  by(Athanasiou et al. 1996), PLGA nanoparticles arebiocompatible and there is no significant toxicity found.

As for the safetyprofile, PLGA have been studied extensively by administering to humans andalready being used in some formulations(Hanafusa et al. 1995; Katz2001). Administration to pigs and ratsalso exhibits long-term biocompatibility (Guzman et al. 1996; Panyam& Labhasetwar 2003). In anotherstudy, PLGA nanoparticles have shown improved permeability and retention effect,leading to accumulation of therapeutic agents (Saxena et al. 2006).  Basedon a research done on azithromycin, PLGA nanoparticles loaded with antimicrobialdrugs have lower MIC compared to free antimicrobial drug, where the MIC is8-fold lower in azithromycin-loaded PLGA nanoparticles.

This shows PLGAnanoparticles as drug delivery system requires a lesser dosage of drugs toexhibit optimum efficacy, hence the side effect of the drug also reduces. (Mohammadi et al. 2010)  1.3.1    LIMITATIONSOF PLGA NANOPARTICLES  ThoughPLGA nanoparticles exhibit a great amount of advantages as a drug deliverysystem, there are still some limitations in this system. Optimum drug deliverymust possess both efficient drug loading and drug release profile. PLGAnanoparticles have poor drug loading profile.

Although they have highencapsulation efficiencies, their drug loading efficiency is about 1% only.This might be caused by the absorption of the drugs onto the surface of thePLGA nanoparticles. (Danhier et al. 2012).             Furthermore, they also cause highburst release of drug from nanoparticles. This leads to reduced efficacy of thedrug delivery system because the drugs will be unable to be delivered at the targetsite of action (Danhier et al.

2012).   1.4       ALGINATENANOPARTICLES Indesigning drug delivery system, the goal would be to produce a sustainedrelease of drug.

Many drug delivery systems face the problem of desorption ofthe outer layer of nanoparticle and that is not the desired outcome. To solvethis, the nanoparticles itself was prepared using sodium alginate, a hydrophilicpolymer (Rajaonarivony et al. 1993).

Alginate, the co-polymer ofguluronic acid and mannuronic acid, has also been approved by US Food and DrugAdministration (Tønnesen & Karlsen 2002) and recognised as ” GenerallyReferred As Safe” (GRAS) material (Sosnik 2014).   a: Monomers of alginatesb: Chain conformation of alginatec: Block distribution of alginateFigure 1.6: Structural characteristicof alginateSource: (Draget& Taylor 2011) Themucoadhesive property, cytocompatibility and biocompatibility of alginate wasthe main reason alginate was researched upon (Lee & Mooney 2012; Sareiet al. 2013). Alginatenanoparticles protect the therapeutic materials in them and release them at thetarget site of action (Aynie et al. 1999).

Highly aqueous property of alginatenanoparticle is attributed to its negative charge (Mariappan et al. 2005).  Furthermore,when alginate is being administered orally, it forms a solid-like-structurebecause they form alginic acid that protects the content of the nanoparticle (Draget & Taylor 2011). Alginate is also bioadhesive, whichcauses the nanoparticles to adhere longer to the intestinal mucosa, increasingthe absorption of drugs (Tønnesen & Karlsen 2002) It is also endocytosed intact fromthe gastrointestinal tract, increasing the bioavailability of the drugs (Florence & Hussain 2001; Yiet al. 1999) . The bioadhesiveproperty is mainly due to alginate’s negative charge that makes microfold cellsand enterocytes absorb them (Reis et al. 2006).

The pH-sensitive property, inaddition to the negative charge, enable itself to interact with positivelycharged drug or molecules by simple electrostatic interaction. (Sun & Tan 2013) Improvedbioavailability can also be seen with alginate nanoparticles as drugsencapsulated in the alginate nanoparticles remains within therapeutic plasmaconcentration and therapeutic concentration at the organ for much longercompared to free drugs. A study was done by incorporating antituberculosisdrugs into alginate nanoparticles, where after administration, the drugs can befound in the plasma for about 9 to 11 days while free drugs last up to 12 hoursonly. This exhibits the controlled and sustained release nature of the alginatenanoparticles.  (Ahmad et al.

2006).  Thesafety profile of alginate can be seen during administration of alginate. Whenalginate was administered repeatedly into the body, there was no immunoglobulinG (IgG), immunoglobulin M (IgM) humoral response and accumulation of alginatesat any major organ. In addition to that, alginate nanoparticles have high drugloading profile because of their higher drug to polymer ratio and high gelporosity of alginate. High drug to polymer ratio indirectly reduces the cost ofproduction and dose size of the formulation. (Rajaonarivony et al.

1993).      1.5       POLYKETIDE METABOLITES OF STREPTOMYCES  Secondary metabolites, unlike primarymetabolites which are important for its daily activities, are metabolitesproduced during production phase and are not essential for cell survival (Martin & Demain 1980). The ability to produce secondarymetabolites is due to the presence of biosynthetic gene clusters that are ableto encode the enzyme to produce the secondary metabolites(Nett et al. 2009). Streptomycesis a Gram-positive bacteria that produce two large groups of secondarymetabolites which are polyketides and non-ribosomal peptides (Hwang et al. 2014) and some of these metabolites canpotentially function as antibiotics.

(Khan et al. 2011; ?muraet al. 2001; Patzer & Braun2010).  Production of antibioticsfrom Streptomyces are in a smallamount and is produced during the transition phase in colonial development.During this phase, the growth of mycelium slows down due to nutrient exhaustionand the development of aerial mycelium depends on the release of nutrients fromthe breakdown of the vegetative hyphae (Miguélez et al. 2000; Parish1979).

The functions of the antibioticthat is produced by Streptomyces areto compete with other microorganisms that it encounters with and acts as a partof symbiosis process where the antibiotic can protect the plant that it is on. (Bosso et al. 2010)             Thefirst antibiotic discovered from the genus Streptomycesis streptothricin in 1942.

The discovery of streptomycin later on in 1944 ledto more screening of antibiotics from Streptomycesgenus. Figure 1.6 shows the production of secondary metabolites from the genus Streptomyces. The increase inantibiotic-resistant microbial pathogens present causes the need for discoveryof novel antibiotics especially from the Streptomyces species (Bush et al. 2011; Fischbach& Walsh 2009) because of theirability in producing more secondary metabolites apart from the ones that havebeen isolated (Baltz 2008, 2011; Craneyet al. 2013).   Figure1.

7 Secondary metabolites from Streptomyces with the molecular andcomputational tools in the middle.Source: (Hwanget al. 2014) 1.

6       JUSTIFICATION Antimicrobial drug delivery has manychallenges despite advancement in the development of the antibiotics (Zhang et al. 2010). Some of the limitations are due tothe narrow spectrum of certain antimicrobial agents (Ranghar et al.

2014), the presence of toxic effecttowards healthy cells and difficulty in the transportation across the cellmembrane of the microorganism (Zhang et al. 2010). Furthermore, the available dosageform such as oral and topical formulation causes the non-target distribution ofthe antimicrobial agents, poor uptake into the cells and the degradation of theantimicrobial agents before reaching the target (Ranghar et al. 2014). The major problem in theantimicrobial treatment is the acquired resistance towards the antimicrobialagents.               Theoccurrence of antimicrobial drug resistance has led to the development ofnanoparticles as drug delivery system because of its high surface to volumeratio and the modifiable physicochemical and biological property according tothe desired application (Rudramurthy et al. 2016).

The high surface to volume ratio isdue to the nano-size of the particles enhancing the therapeutic efficacy of thedrug delivery system (Xu et al. 2007). The small sizeenhances the antimicrobial activity of the drug by increasing the exposuretowards microorganism (Hajipour et al. 2012; Whitesides 2005).             Studiesdone on PLGA nanoparticles, have shown improved bioavailability of the drugs.Based on a study by (Toti et al.

2011), when PLGA nanoparticles are loadedwith azithromycin and rifampin, they directly target the C trachomatisinfections and exhibit controlled and sustained release of the drugs. There aresome limitations of using PLGA nanoparticles, such as having poor drug loadingproperty and high burst release of drugs. Alginate nanoparticles, on the otherhand, shows high drug encapsulation efficiency and controlled and sustainedrelease of the drugs (Ahmad et al. 2006).  Toovercome the limitation of these delivery systems, both PLGA and alginate areincorporated together to form nanoparticles. Combinationof PLGA polymer which is hydrophobic, and alginate which is hydrophilic, givesadvantages to both hydrophobic and hydrophilic nanoparticulate system (George & Abraham 2006; Makadia& Siegel 2011).

The combination creates synergyeffects leading to optimization of its delivery efficiency of the activecomponent. Polyketide, metabolites of Streptomycesare chosen as the encapsulated drug in this research as Streptomyces has the most abundant source of antibiotics(Liu et al. 2013), the discovery of novel antibioticsfrom this genus is increasing due to the development of antimicrobialresistance (Bush et al. 2011; Fischbach& Walsh 2009) and thetherapeutic effect of the drug can be enhanced through this formulation.Thisresearch is done to compare the antimicrobial actions of free polyketide with alginate-coatedPLGA nanoparticles loaded with polyketide and to evaluate whether nanoparticlesas drug delivery system could enhance the antimicrobial activities of thecompound.  1.7       OBJECTIVES 1.7.1    GENERAL OBJECTIVE Toformulate and characterize PLGA- alginate nanoparticles loaded with polyketidemetabolites of Streptomyces 1.7.2    SPECIFIC OBJECTIVES i)                   To synthesize PLGA- alginate nanoparticlesloaded with polyketide metabolites of Streptomyces using double emulsion solventevaporation methodii)                 To characterize the PLGA- alginatenanoparticles loaded with polyketide metabolites of Streptomycesiii)               To evaluate the antimicrobial activityof  PLGA- alginate nanoparticles loadedwith polyketide metabolites of Streptomyces

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