LITERATURE begins to spread (Rudramurthy et al.





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About 17 million of lives, mostly of children and of
elderly, are taken by bacterial infections. Although there have been
significant achievements in the process of discovering and developing
antibiotics, infectious diseases still remain as the second main cause of death
globally (De
Lima Procópio et al. 2012). This can be attributed to the
increasing antimicrobial resistance that occurs due to the misuse of
antibiotics. Antibiotic resistance is becoming a global threat as the emergence
of multi-drug resistant organism increases and begins to spread (Rudramurthy
et al. 2016).


            The discovery of antibiotics derived
from Streptomyces started in 1942 and 80% of antibiotic used now are from Streptomyces (De Lima Procópio et al. 2012). The Gram-positive bacteria has the
nature of producing a class of bioactive secondary metabolites, called
polyketides metabolites. Some of these metabolites which are bioactive are potentially
used as antibiotics and immunosuppressant (Khan et al. 2011; ?mura
et al. 2001; Patzer & Braun


            The occurrence of antimicrobial drug
resistance has led to the development of nanoparticles as drug delivery system (Rudramurthy et al. 2016). Nanotechnology provides a better
drug delivery system because the drug does not interact with the carrier and is
delivered directly to the right site of action as its protected  against enzymatic degradation (Asadi 2014).


            Nanoparticles as antimicrobial
delivery system produce the desired therapeutic effect because of their large
surface area to volume ratio (Ravishankar Rai & Jamuna Bai 2011), hence their interaction with the
microorganism increases and leads to

therapeutic outcome (Hajipour et al. 2012; Whitesides
2005). As nanoparticles can enter the host
cell, antimicrobial agent can be released from the nanoparticles to treat
intracellular infections(Zhang et al. 2010).


            Extensive research had been done on
poly lactic-co-glycolic acid (PLGA) to be used as nanoparticles. PLGA is a
synthetic polymer which has been approved by US Food and Drug Administration
and European Medicines Agency (Prokop & Davidson 2008; Vert
et al. 1994). The
biodegradable nature (Dinarvand et al. 2011), nano-size, solubility in most
solvents, enabling of controlled and sustained release of drugs (Anderson & Shive 2012; Baldwin
& Saltzman 1998; Panyam &
Labhasetwar 2003), bio-compatibility and no
significant toxicity (Athanasiou et al. 1996) make PLGA nanoparticles to be an
effective drug delivery system.


            Alginate, a co-polymer of guluronic
acid and mannuronic acid, has also been approved by the US Food and Drug
Administration (Tønnesen & Karlsen 2002). Owing to the negative charge of alginate
(Mariappan et al. 2005) , it is muco-adhesive which is an
important characteristic of a drug delivery system to improve the
bioavailability. Alginate nanoparticles are also cytocompatible and
biocompatible to be administered to the body(Lee & Mooney 2012; Sarei
et al. 2013).


            This study is done to evaluate the
therapeutic enhancement of the biologically active polyketides metabolites of Streptomyces when encapsulated into the alginate-coated
PLGA nanoparticles as delivery system, as compared to the free polyketides












there have been advancements in the development of antimicrobials, treating
infectious diseases is becoming a great challenge. This is due to the
difficulty of antimicrobials to cross the cell membranes to reach the site of action
especially in intracellular infection, and instead acting on normal cells that
leads to toxicity and the emergence of antimicrobial resistance (Zhang et al. 2010). Drawbacks and limitations of
conventional medicines such as poor bioavailability and lack of selectivity led
to the development of nanotechnology (Nevozhay et al. 2006) . Nanotechnology
provides a better drug delivery system because the drug does not interact with
the carrier and is delivered directly to the right site of action as its
protected  against enzymatic degradation (Asadi 2014). One of the extensively explored
nanotechnology fields is nanoparticles.


            Nanoparticles helps in drug delivery
to the microorganism through various mechanisms as shown in the Figure 1 below.

Figure 1.1 Mechanism of drug delivery
to microorganisms by nanoparticles. (a) by fusing with the cell membrane and exposing
the drug to the cell and (b) binding to the cell wall and functions as drug
depot causing a continuous release of drugs which will diffuse into the

Source : (Zhang
et al. 2010)


to the size range from 10 -1000 nm (Soppimath et al. 2001), nanoparticles exhibit unique
characteristics (Wilczewska et al. 2012) . The small size of the nanoparticle
enhances the therapeutic efficacy of the drug delivery system (Xu et al. 2007). The nano-size
also causes nanoparticles to have a high surface-to-volume ratio (Ravishankar Rai & Jamuna Bai 2011) which enhance the antimicrobial
activity 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 to
increased efficacy of drug delivery to a specific site of action. This is due
to their ability to pass through the smallest capillaries and avoid from being
taken up by phagocytes so they can remain in the blood for a longer period of
time (Parveen et al. 2012).



Figure 1.2             The importance of size and shape of
nanoparticles as drug carrier

Source : (Farokhzad & Langer 2009)


            Targeted drug delivery system
towards microorganisms also can be achieved by using the nanoparticles(Gao et al. 2014). This can be done
by manipulating the particle size and surface characteristic which can lead to
passive and active drug targeting (Singh et al. 2010). Active drug targeting means
conjugation of a nanoparticle to a tissue or cell-specific ligand (Lamprecht et al. 2001) while passive drug targeting is the incorporation
of the drug into the nanoparticles which passively targets the specific site of
action (Maeda 2001; Sahoo
et al. 2002) as shown in
Figure 1.3. Due to the increased vascular permeability in bacterial infection
as bacterial components trigger inflammatory mediators, passive targeting of
nanoparticles can be easily achieved. As macrophage possesses scavenging
property, they can easily take up nanoparticles. This feature helps in the
treatment of bacteria that survives the ingesting of macrophages. (Gao et al. 2014) Targeted drug
delivery system is also important to prevent toxicity of antimicrobial agents.
For an example, aminoglycoside causes ototoxicity and nephrotoxicity that leads
to the need for controlled dosage.(Zhang et al. 2010).



Figure 1.3 Passive
and active drug targeting

Source :(Singh & Lillard 2009)


            In addition to that, nanoparticles
do provide controlled and sustained release of antimicrobial drug (Zaidi et al. 2017). Controlled and sustained release is
important in drug delivery as it increases the drug therapeutic efficacy and minimises
the side effects (Parveen et al. 2012). In designing an optimum delivery
system, controlled and sustained release of drugs at optimum therapeutic rate
and dosage becomes a major goal (Farrugia & Grover 1999). As the drug reaches the target site
or organ, the nanoparticles can function as a depot to supply the drug
continuously to the site of action (Singh & Lillard 2009). Based on a study done by (Pandey et al. 2003), after oral administration of
anti-tubercular drugs-loaded nanoparticles, the plasma concentration of the
anti-tubercular drugs could be maintained at a minimum inhibitory concentration
up to 9 days, while free drugs could only last up to a day only. The drug
concentration at the target organ was at therapeutic level till day 11, while
free drug could only be found at the target organ for not more than 2 days.
Controlled and sustained release of antimicrobial drugs can improve patient
adherence as frequency and dose of the required drug will be reduced.(Ladavière & Gref 2015)


            In addition, nanoparticles could
also overcome some limitation of other free drugs, especially in oral drug
delivery system. As most of the free drugs are susceptible for degradation in
the digestive tract, nanoparticle could overcome these limitations as they
protect the drug (Des Rieux et al. 2006). Encapsulation of the drug allows
protection against enzymatic or hydrolytic degradation (Damge et al. 1990) and prevent interaction with other
drugs.(Zaidi et al. 2017) As bioadhesive properties of
nanoparticles can easily be modified, nanoparticles can adhere to the mucosa
membrane and increase the absorption of the drugs, hence increasing the bioavailability
(Gabor et al. 2004).





important factor in designing a drug delivery system is the choosing of materials
used for that drug delivery system. Choosing the right polymer for designing
nanoparticles is necessary to achieve the desired target (Bala et al. 2004). One of the polymer matrix that has
been widely researched upon is poly lactic-co-glycolic acid (PLGA). PLGA is a
polymer that has been approved by both US Food and Drug Administration and
European Medical Agency (Prokop & Davidson 2008; Vert
et 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 be
formulated into various forms and sizes according to the desired application (Anderson & Shive 2012) and can also encapsulate molecules
of various size. PLGA is soluble in most common solvents (Uhrich et al. 1999; Wu
& Wang 2001). PLGA’s
mucoadhesive property also enables various routes of administration of the
nanoparticles (Tafaghodi et al. 2004).




x: Number of
lactic acid

y: Number of
glycolic acid


Figure 1.4
Chemical structure of PLGA

Source: (Mahapatro & Singh 2011)


            A well-known advantage of using PLGA
is attributed to its biodegradable property. PLGA nanoparticle can degrade in
two ways, mainly by bulk erosion or by surface erosion (Dinarvand et al. 2011). The biodegradable property can
clearly be seen when PLGA breaks down to form metabolites which are lactic acid
and glycolic acid as shown in Figure 1.5. These metabolites are commonly found
in our body and can be metabolized further by the Krebs cycle (Kumari et al. 2010). The end products are carbon dioxide
and water (Jain 2000; Panyam
et al. 2002) which leads to
minimal systemic toxicity of PLGA (Wickline et al. 2007). PLGA polymer enables sustained
release of drugs by diffusion and also degradation of the PLGA matrix (Anderson & Shive 2012; Baldwin
& Saltzman 1998; Panyam &
Labhasetwar 2003). The rate of degradation actually
depends 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 are
present in higher ratio.



Figure 1.5 Hydrolysis of PLGA

Source : (Kumari
et al. 2010)


            Based on in vivo studies and in
vitro studies  by(Athanasiou et al. 1996), PLGA nanoparticles are
biocompatible and there is no significant toxicity found. As for the safety
profile, PLGA have been studied extensively by administering to humans and
already being used in some formulations(Hanafusa et al. 1995; Katz
2001). Administration to pigs and rats
also exhibits long-term biocompatibility (Guzman et al. 1996; Panyam
& Labhasetwar 2003). In another
study, PLGA nanoparticles have shown improved permeability and retention effect,
leading to accumulation of therapeutic agents (Saxena et al. 2006).


on a research done on azithromycin, PLGA nanoparticles loaded with antimicrobial
drugs have lower MIC compared to free antimicrobial drug, where the MIC is
8-fold lower in azithromycin-loaded PLGA nanoparticles. This shows PLGA
nanoparticles as drug delivery system requires a lesser dosage of drugs to
exhibit optimum efficacy, hence the side effect of the drug also reduces. (Mohammadi et al. 2010)






PLGA nanoparticles exhibit a great amount of advantages as a drug delivery
system, there are still some limitations in this system. Optimum drug delivery
must possess both efficient drug loading and drug release profile. PLGA
nanoparticles have poor drug loading profile. Although they have high
encapsulation efficiencies, their drug loading efficiency is about 1% only.
This might be caused by the absorption of the drugs onto the surface of the
PLGA nanoparticles. (Danhier et al. 2012).


            Furthermore, they also cause high
burst release of drug from nanoparticles. This leads to reduced efficacy of the
drug delivery system because the drugs will be unable to be delivered at the target
site of action (Danhier et al. 2012).



1.4       ALGINATE


designing drug delivery system, the goal would be to produce a sustained
release of drug. Many drug delivery systems face the problem of desorption of
the outer layer of nanoparticle and that is not the desired outcome. To solve
this, the nanoparticles itself was prepared using sodium alginate, a hydrophilic
polymer (Rajaonarivony et al. 1993). Alginate, the co-polymer of
guluronic acid and mannuronic acid, has also been approved by US Food and Drug
Administration (Tønnesen & Karlsen 2002) and recognised as ” Generally
Referred As Safe” (GRAS) material (Sosnik 2014).



a: Monomers of alginates

b: Chain conformation of alginate

c: Block distribution of alginate

Figure 1.6: Structural characteristic
of alginate

Source: (Draget
& Taylor 2011)


mucoadhesive property, cytocompatibility and biocompatibility of alginate was
the main reason alginate was researched upon (Lee & Mooney 2012; Sarei
et al. 2013). Alginate
nanoparticles protect the therapeutic materials in them and release them at the
target site of action (Aynie et al. 1999). Highly aqueous property of alginate
nanoparticle is attributed to its negative charge (Mariappan et al. 2005).


when alginate is being administered orally, it forms a solid-like-structure
because they form alginic acid that protects the content of the nanoparticle (Draget & Taylor 2011). Alginate is also bioadhesive, which
causes the nanoparticles to adhere longer to the intestinal mucosa, increasing
the absorption of drugs (Tønnesen & Karlsen 2002) It is also endocytosed intact from
the gastrointestinal tract, increasing the bioavailability of the drugs (Florence & Hussain 2001; Yi
et al. 1999) . The bioadhesive
property is mainly due to alginate’s negative charge that makes microfold cells
and enterocytes absorb them (Reis et al. 2006). The pH-sensitive property, in
addition to the negative charge, enable itself to interact with positively
charged drug or molecules by simple electrostatic interaction. (Sun & Tan 2013)


bioavailability can also be seen with alginate nanoparticles as drugs
encapsulated in the alginate nanoparticles remains within therapeutic plasma
concentration and therapeutic concentration at the organ for much longer
compared to free drugs. A study was done by incorporating antituberculosis
drugs into alginate nanoparticles, where after administration, the drugs can be
found in the plasma for about 9 to 11 days while free drugs last up to 12 hours
only. This exhibits the controlled and sustained release nature of the alginate
nanoparticles.  (Ahmad et al. 2006).


safety profile of alginate can be seen during administration of alginate. When
alginate was administered repeatedly into the body, there was no immunoglobulin
G (IgG), immunoglobulin M (IgM) humoral response and accumulation of alginates
at any major organ. In addition to that, alginate nanoparticles have high drug
loading profile because of their higher drug to polymer ratio and high gel
porosity of alginate. High drug to polymer ratio indirectly reduces the cost of
production and dose size of the formulation. 
(Rajaonarivony et al. 1993).










Secondary metabolites, unlike primary
metabolites which are important for its daily activities, are metabolites
produced during production phase and are not essential for cell survival (Martin & Demain 1980). The ability to produce secondary
metabolites is due to the presence of biosynthetic gene clusters that are able
to encode the enzyme to produce the secondary metabolites(Nett et al. 2009). Streptomyces
is a Gram-positive bacteria that produce two large groups of secondary
metabolites which are polyketides and non-ribosomal peptides (Hwang et al. 2014) and some of these metabolites can
potentially function as antibiotics. (Khan et al. 2011; ?mura
et al. 2001; Patzer & Braun


Production of antibiotics
from Streptomyces are in a small
amount and is produced during the transition phase in colonial development.
During this phase, the growth of mycelium slows down due to nutrient exhaustion
and the development of aerial mycelium depends on the release of nutrients from
the breakdown of the vegetative hyphae (Miguélez et al. 2000; Parish
1979). The functions of the antibiotic
that is produced by Streptomyces are
to compete with other microorganisms that it encounters with and acts as a part
of symbiosis process where the antibiotic can protect the plant that it is on. (Bosso et al. 2010)


first antibiotic discovered from the genus Streptomyces
is streptothricin in 1942. The discovery of streptomycin later on in 1944 led
to more screening of antibiotics from Streptomyces
genus. Figure 1.6 shows the production of secondary metabolites from the genus Streptomyces. The increase in
antibiotic-resistant microbial pathogens present causes the need for discovery
of novel antibiotics especially from the Streptomyces species (Bush et al. 2011; Fischbach
& Walsh 2009) because of their
ability in producing more secondary metabolites apart from the ones that have
been isolated (Baltz 2008, 2011; Craney
et al. 2013).



1.7 Secondary metabolites from Streptomyces with the molecular and
computational tools in the middle.

: (Hwang
et al. 2014)




Antimicrobial drug delivery has many
challenges despite advancement in the development of the antibiotics (Zhang et al. 2010). Some of the limitations are due to
the narrow spectrum of certain antimicrobial agents (Ranghar et al. 2014), the presence of toxic effect
towards healthy cells and difficulty in the transportation across the cell
membrane of the microorganism (Zhang et al. 2010). Furthermore, the available dosage
form such as oral and topical formulation causes the non-target distribution of
the antimicrobial agents, poor uptake into the cells and the degradation of the
antimicrobial agents before reaching the target (Ranghar et al. 2014). The major problem in the
antimicrobial treatment is the acquired resistance towards the antimicrobial


occurrence of antimicrobial drug resistance has led to the development of
nanoparticles as drug delivery system because of its high surface to volume
ratio and the modifiable physicochemical and biological property according to
the desired application (Rudramurthy et al. 2016). The high surface to volume ratio is
due to the nano-size of the particles enhancing the therapeutic efficacy of the
drug delivery system (Xu et al. 2007). The small size
enhances the antimicrobial activity of the drug by increasing the exposure
towards microorganism (Hajipour et al. 2012; Whitesides 2005).


done on PLGA nanoparticles, have shown improved bioavailability of the drugs.
Based on a study by (Toti et al. 2011), when PLGA nanoparticles are loaded
with azithromycin and rifampin, they directly target the C trachomatis
infections and exhibit controlled and sustained release of the drugs. There are
some limitations of using PLGA nanoparticles, such as having poor drug loading
property and high burst release of drugs. Alginate nanoparticles, on the other
hand, shows high drug encapsulation efficiency and controlled and sustained
release of the drugs (Ahmad et al. 2006).


overcome the limitation of these delivery systems, both PLGA and alginate are
incorporated together to form nanoparticles. Combination
of PLGA polymer which is hydrophobic, and alginate which is hydrophilic, gives
advantages to both hydrophobic and hydrophilic nanoparticulate system (George & Abraham 2006; Makadia
& Siegel 2011). The combination creates synergy
effects leading to optimization of its delivery efficiency of the active
component. Polyketide, metabolites of Streptomyces
are 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 antibiotics
from this genus is increasing due to the development of antimicrobial
resistance (Bush et al. 2011; Fischbach
& Walsh 2009) and the
therapeutic effect of the drug can be enhanced through this formulation.

research is done to compare the antimicrobial actions of free polyketide with alginate-coated
PLGA nanoparticles loaded with polyketide and to evaluate whether nanoparticles
as drug delivery system could enhance the antimicrobial activities of the



1.7       OBJECTIVES




formulate and characterize PLGA- alginate nanoparticles loaded with polyketide
metabolites of Streptomyces




To synthesize PLGA- alginate nanoparticles
loaded with polyketide metabolites of Streptomyces using double emulsion solvent
evaporation method

To characterize the PLGA- alginate
nanoparticles loaded with polyketide metabolites of Streptomyces

To evaluate the antimicrobial activity
of  PLGA- alginate nanoparticles loaded
with polyketide metabolites of Streptomyces