ABSTRACT metabolite of Streptomyces. The formulation was prepared

 

 

ABSTRACT

 

Antimicrobial drug resistance is becoming one
of the great challenges in the treatment of infectious disease. To address this
matter, alginate-coated PLGA nanoparticles were produced as a carrier for the
novel fraction extracted from polyketide metabolite of Streptomyces. The formulation was prepared to increase the
therapeutic activity of the metabolite. Alginate-coated PLGA nanoparticles were
prepared using double emulsion solvent evaporation method and the particle
size, zeta potential, FTIR, entrapment efficiency and antimicrobial activity
was evaluated. The particle size of the nanoparticles was 352.40 ± 27.87 nm
with PDI and zeta-potential of 0.352 ± 0.026 and -22.70 ± 0.10. This showed
that the nanoparticles are within the acceptable range, monodispersed and
physically stable. FTIR analysis confirmed the presence of alginate on the
nanoparticles which shows that the nanoparticles were successfully coated with
alginate. Alginate-coated PLGA nanoparticles have higher entrapment efficiency
compared to the uncoated nanoparticles. However, alginate-coated PLGA
nanoparticles showed no antimicrobial effect which might be due to degradation
of the polyketide metabolites. In conclusion, the alginate-coated PLGA
nanoparticles were successfully formulated but further studies should be done
to investigate more on this formulation to increase the therapeutic efficacy of
the polyketide metabolites.

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Keywords: PLGA; alginate; nanoparticles; polyketide metabolites;
Streptomyces

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Introduction

 

Despite
the advancement in the discovery of new antibiotics, treatment of infectious
disease, especially intracellular infections are facing so many challenges
because of the  presence of toxicity and drug
delivery to the microorganism (Zhang et al. 2010). Besides that, some antibiotics have
a narrow spectrum, cannot reach the site of action or some undergo degradation
even before reaching the target site of action (Ranghar et al. 2014). The emergence of antimicrobial drug
resistance makes the treatment of infectious disease even more difficult. This
has led to the development of a new drug delivery system which is the
nanotechnology (Rudramurthy et al. 2016), as in this delivery system, the
carrier does not interact with the drug and the drug can be distributed
directly to the site of action due the protection against the enzymatic
degradation (Asadi 2014).

 

            One of the extensively explored
nanotechnology fields is nanoparticles. The size range between 10-1000nm (Soppimath et al. 2001) enables nanoparticle to enhance the
therapeutic efficacy of drug delivery system (Xu et al. 2007) as they have high
surface-to-volume ratio (Ravishankar Rai & Jamuna Bai 2011) and can easily penetrate through the
cells to the site of action (Parveen et al. 2012). Furthermore, nanoparticles provide a
targeted delivery system as the particle size and the surface characteristics
can be manipulated (Singh et al. 2010). Controlled and sustained release of
antimicrobial drug can be achieved by formulating nanoparticles as they
function as depot which minimises the possible side effects and reduces the
need of higher dosage, thus improving patients’ adherence (Ladavière & Gref 2015; Parveen
et al. 2012; Singh & Lillard
2009)

 

            One of the polymer matrix that has been
widely explored for pharmaceutical application as a drug carrier is poly
lactic-co-glycolic acid (PLGA).  PLGA is
approved by both US Food and Drug Administration and European Medical Agency (Prokop & Davidson 2008; Vert
et al. 1994), soluble in most
common solvents (Uhrich et al. 1999; Wu
& Wang 2001), mucoadhesive (Tafaghodi et al. 2004), biodegradable, biocompatible and
has shown no significant toxicity (Athanasiou et al. 1996). Alginate, on the other hand, is
also approved by US Food and Drug Administration (Tønnesen & Karlsen 2002) and been recognised as ” Generally
Referred As Safe” (GRAS) material (Sosnik 2014). Alginate is also mucoadhesive,
cytocompatible and biocompatible. Based on a study by (Zhai et al. 2015), PLGA/alginate composite microsphere
has shown to have higher entrapment efficiency and has a more sustained release
of bovine serum albumin as compared  to PLGA
composite microsphere.  In another study
by (Liu
et al. 2004), alginate coated
tetracycline loaded PLGA microspheres shows a higher encapsulation efficiency,
higher quantity of released tetracycline and a more sustained release compared
to the naked PLGA microsphere.

 

            Due to the increasing
antibiotic-resistant microbial pathogens, the need for discovery of novel
antibiotic increases, especially one from the Streptomyces genus. The discovery of antibiotics from this genus
goes long way back to 1942, and up to 80% of antibiotics used now are from Streptomyces. This is because they have
a unique characteristic 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 2010). These metabolites are released as
self-defense and also as a part of symbiosis process where the antibiotic
protects the plant that the microorganism is on. (Bosso et al. 2010).

 

In
this study, alginate coated-PLGA nanoparticles loaded with polyketide
metabolites of Streptomyces were
formulated and the antimicrobial activity were compared to free polyketide
metabolites of Streptomyces and PLGA
nanoparticles loaded with polyketide metabolites of Streptomyces. The prepared nanoparticles were characterised by
particle size, zeta-potential, FTIR and entrapment efficiency.

 

 

 

 

 

 

 

 

 

Materials
and Method

 

Materials

 

The polyketide metabolites of Streptomyces were provided by a novel
antibiotic research group from the Faculty of Health Science, University Kebangsaan
Malaysia. Poly (D, L –lactide-co-glycolide) with a ratio of lactide: glycolide being
75:25 and a molecular weight of 66, 000 – 107, 000 was obtained from Sigma
Aldrich (USA). The polyvinyl alcohol (PVA) was obtained from R & M
Chemicals. Methanol of molecular weight 32.04 g/mol was obtained from Fine
Chemicals Limited (New Dehli). Dichloromethane of molecular weight 84.93g/mol
was purchased from Friendemann Schmidt Chemical.  Sodium alginate was obtained from Nacalai
Tesque, Inc. (Kyoto, Japan). D-mannitol was purchased from Sigma Aldrich
(France). Deionised water was produced in the laboratory using Millipore
Milli-Q. The bacteria strains used for the antimicrobial test were Escherichia coli, Methicillin-resistant Staphylococcus aureus, Enterobacter
aerogenes, Staphylococcus aureus, Bacillus subtilis
and ABR and they were obtained from Novel
Antibiotic Laboratory collection of Faculty of Health Science, Universiti
Kebangsaan Malaysia. All chemicals
used were of analytical grade and used as received.

 

Methods

 

Preparation of alginate coated-PLGA nanoparticles
loaded with polyketide metabolites of Streptomyces

 

The
nanoparticles were synthesized by using double emulsion solvent evaporation method
as described in (Marasini, Khalil, et al. 2016) but with slight modification. Firstly,
1mL of polyketide metabolites of Streptomyces
in methanol (1mg/mL) was added to 3 mL of dichloromethane (DCM). Then, 0.0087g
of PLGA was dissolved in the methanol/DCM mixture using a magnetic stirrer. 1
mL of 0.75% of PVA solution was added dropwise to the methanol/DCM/PLGA using a
dropper. The solution was sonicated using Branson B3510R-MTH Ultrasonic Cleaner
(H030056) for 30 minutes and added dropwise to 6.75 mL of 0.1% Alginate 0.75%
PVA solution. The solution was sonicated again and was left for overnight
stirring for 24 hours. After that, the solution was centrifuged at 3000 RCF for
10 minutes at 4 0C using AllegraTM 64R Beckman Coulter
Bench Top High Speed Refrigerated Centrifuge. The pellets and supernatants were
separated and the pellets were discarded while the supernatants were
centrifuged again at 15 000 RCF for 10 minutes at 4 0C. The formed
pellets were washed with deionised water and centrifuge was repeated for
another two times. Then, cryoprotectant was added to the pellets. The
cryoprotectant used for this formulation is 10mL of 5% mannitol. The samples
were frozen for 3 days and 18 hours, then freeze-dried for 3 days and 14 hours
using SCANVAC CoolsafeTM 110-4. PLGA nanoparticles loaded with
polyketide metabolites of Streptomyces were prepared with the same method,
except after the first sonication, the solution was added to 6.75mL of 0.75%
PVA solution without the alginate.

 

Particle size, polydispersity index
and zeta-potential characterisation

 

10mg
of the freeze dried PLGA nanoparticles and alginate coated-PLGA nanoparticles
were dissolved separately in 3 mL of deionised water. 1 mL of the samples were placed
in a separate disposable cuvette using a micropipette. The particle size
(z-average), polydispersity index (PDI) and zeta potential of both samples were
measured in triplicate using Zetasizer® Nano ZS (Malvern Instrument, UK) by
dynamic light scattering method done at 250C.

 

FTIR spectroscopic analysis

 

Alginate,
PLGA nanoparticles and alginate coated-PLGA nanoparticles were characterised by
using FTIR Spectrophotometer (Pelkin-Elmer Spectrum 100, Waltham, USA) by
taking 32 scans at the resolution of 4 cm-1 in the range of 4000 to
650 cm-1.

 

Encapsulation efficiency

 

10
mg of the freeze dried PLGA nanoparticles and alginate coated-PLGA
nanoparticles were dissolved separately in 10 mL of methanol to dissolve the
PLGA. The solutions were analysed by HPLC method (1515 Isocratic HPLC Pump C-18
column) using the Breeze tm software.  The mobile phase used was methanol: deionised
water (50:50, v/v). The injection volume was 20µL and the temperature was
maintained at 250C. The flow rate used for the analysis was
1.5ml/min and UV detection was at 214nm. The concentration of the polyketide
metabolites in nanoparticles formulation was done based on the calibration
curve done with the serial dilution of the polyketide metabolites. The
encapsulation efficiency was calculated using the following formula:

 

Antimicrobial property evaluation

 

Before
the formulation of nanoparticles was done, the susceptibility of the fraction
of polyketides metabolites used were tested against Escherichia coli. Two days before the test was done, the bacteria
strains were streaked on agar petri dish. The streaked agar plates were
incubated for 24 hours in a 370C incubator. The next day, the
bacteria strains were suspended in nutrient broth and incubated for 24 hours at
370C. The suspension later was diluted with Mueller Hinton broth to an
absorbance of OD of 0.08 at 625nm for adequate bacteria concentration. Later,
the bacteria strains were swabbed on a petri dish containing Mueller Hinton
agar than have been prepared earlier. The metabolites with the concentration of
0.5 mg/mL were prepared by dissolving it in methanol. 10 µL of the metabolites
was placed on the agar. The plates were incubated for 24 hours at 370C
and the growth inhibition zone was measured. After preparing both formulation
of nanoparticles, the sensitivity against Escherichia
coli, Methicillin-resistant Staphylococcus
aureus, Staphylococcus aureus, Enterobacter aerogenes,
Bacillus subtilis and ABR
strains were tested. Same procedures were done to prepare the bacteria streaked
Mueller Hinton agar plate.10 µL of 10mg/ml of PLGA nanoparticles and alginate
coated-PLGA nanoparticles which were dissolved with deionised water were placed
on the agar using a micropipette. Free polyketide metabolites of Streptomyces in methanol (0.5 mg/mL) was also placed on the agar. The
positive control were also placed on the agar which were vancomycin for MRSA and
gentamicin for the rest of the bacteria strains. The plates were incubated for
24 hours at 370C and the growth inhibition zone were assessed.

Results and discussion

 

Preparation of alginate coated-PLGA nanoparticles loaded with
polyketide metabolites of Streptomyces

 

In
the beginning of the study, the double emulsion solvent evaporation technique
used was adapted from (Khanal et al. 2016) to formulate PLGA nanoparticle, with
slight modification done on their method. The size of the nanoparticles was 1500
nm and has PDI of 0.73. This method was further modified by increasing the
concentration to 1% but the particle size obtained was higher than the previous
formulation. For the third formulation, 0.1% of PVA was used, but the particle
size was unable to be tested immediately because Zetasizer® Nano ZS instrument
broke down. The particle size
of this formulation when tested after one week was very high, showing particle
aggregation. This is because to evaluate the size of nanoparticles, the
formulation must be freshly prepared. Nanoparticles, if left for a long time,
can cause particles aggregation which affects the particle size and the
polydispersity index. As the Zeta Sizer machine is unavailable, there was a
delay in testing the completed formulation of nanoparticles. The delay was
caused by the need for booking the Zetasizer® Nano ZS
instrument in the Department
of Chemistry, Faculty of Science, University of Malaya. Department of Chemistry
only opens during weekdays and office hours and the preparation of the
nanoparticles took about two days. This made limited time available to prepare new
formulation. Formulation with a reduction in PLGA concentration also showed
larger particle size which were not in nanometre range. The next two formulations
were adapted from (Marasini, Giddam, et al. 2016) and (Marasini, Khalil, et al. 2016) but this time they were both coated with
alginate.  The formulations adapted from (Marasini, Khalil, et al. 2016) showed particle size analysis was within acceptable
range.

 

When the right formulation was
obtained, alginate coated-PLGA nanoparticles loaded with polyketide
metabolites of Streptomyces were formulated. The polyketide metabolites provided
were said to dissolve in organic solvent. Polyketide metabolites were dissolved
in dichloromethane to be incorporated into the nanoparticles but they were not
fully soluble. Dichloromethane was evaporated and the polyketides were
dissolved in methanol later. The methanol containing polyketide metabolites
were used in the formulation to prepare the nanoparticles.

 

Particle
size, polydispersity index and zeta-potential characterisation

 

The
result of characterisation of particle size, polydispersity index and
zeta-potential of alginate coated-PLGA nanoparticles loaded with polyketide
metabolites of Streptomyces and PLGA
nanoparticles loaded with polyketide metabolites of Streptomyces are shown in Table 1. The particle size of uncoated
PLGA nanoparticles was twice bigger than the PLGA nanoparticles coated with
alginate. The particle size of alginate coated-PLGA nanoparticles loaded with
polyketide metabolites of Streptomyces
and PLGA nanoparticles loaded with polyketide metabolites of Streptomyces are in the acceptable range
of 10 – 1000nm (Soppimath
et al. 2001). The result was unexpected as alginate-coated PLGA
nanoparticles were expected to be larger than the uncoated nanoparticles. This
is because based on the study done by (Liu et al. 2004), the presence of alginate coating increases the
particle size where the particle size of alginate coated PLGA microspheres were
much larger than the uncoated PLGA microspheres. Despite that, there was
another study by (Oliveira
et al. 2012) reported that alginate coating shown a significant
reduction in the size of the chitosan-SmRho nanoparticles. This result might be
due to particles aggregation. Furthermore, the polyketide metabolites received from
the Faculty of Health Science was limited
which made only one formulation only could be done from the received
metabolites and limits the study to analyse more about the effect of alginate
coating on particle size.

 

The
PDI of the alginate coated-PLGA nanoparticles were smaller than the uncoated
PLGA nanoparticles. PDI shows the size distribution of the nanoparticles and
smaller or closer value to zero indicates the sample is more homogenous. PDI of
PLGA nanoparticles indicates a slight broader distribution of the particle size
while PDI of alginate coated-PLGA nanoparticles indicates a more homogenous
sample. These results do correspond to the particle size of both samples. Alginate
coated-PLGA nanoparticles has much more negative zeta-potential as compared to
the uncoated nanoparticles.

Zeta-potential
is used to indicate the surface charge of the nanoparticles and the stability
of the formulation. High value of zeta-potential of the formulations, either
positive or negative, indicates the formulation is electrically stable, while
formulation with low zeta-potential tends to aggregate leading to poor physical
stability. Zeta potential of higher value than 20 mV indicates the formation of
stable dispersion (Cosgrove 2010). The zeta-potential of the alginate
coated-PLGA nanoparticles show more negative value, which shows the presence of
alginate as alginate has a negative charge. This also proves that alginate
coated-PLGA nanoparticles formulation is more stable than the uncoated
nanoparticles. These results correlate with the PDI of the PLGA nanoparticles
which is much higher than alginate coated-PLGA nanoparticles indicating
significant aggregation has occurred.

 

Particle size(nm) ± SD

Polydispersity index ± SD

Zeta-potential(mV) ± SD

PLGA nanoparticles loaded with polyketide
metabolites of Streptomyces
 
Alginate coated-PLGA nanoparticles loaded with
polyketide metabolites of Streptomyces

 
749.00 ± 84.53
 
 
 
352.40
± 27.87

 
0.570
± 0.016
 
 
 
0.352
± 0.026

 
-19.60
± 1.16
 
 
 
-22.70
± 0.10

 

Table
1 Particle size, polydispersity index and zeta-potential characterization of alginate
coated-PLGA nanoparticles loaded with polyketide metabolites of Streptomyces and PLGA nanoparticles
loaded with polyketide metabolites of Streptomyces

 

FTIR spectroscopic analysis

 

FTIR
spectroscopic was done to analyse the chemical structure of the nanoparticles
and compare the chemical structure of PLGA nanoparticles and alginate
coated-PLGA nanoparticles. There is a weak peak that can be seen in the alginate
coated-PLGA nanoparticles formulation to differentiate both formulations at
1742.0 cm-1 which can also be seen in the FTIR spectra of alginate which
represents the C=O stretching. This indicates that the nanoparticles were
successfully coated with alginate.

 

Encapsulation
efficiency

 

The
encapsulation efficiency of alginate coated-PLGA nanoparticles was 33.4% and encapsulation
efficiency of PLGA nanoparticles was lower than of alginate coated-PLGA
nanoparticles. This shows that alginate coating provides more binding site for
the polyketide metabolites that increase the entrapment efficiency. Polyketide
metabolites form covalent bonds with alginate which increases the amount of
drug trapped in the nanoparticles.

 

Antimicrobial property evaluation

 

To
access the antimicrobial property of the fraction of polyketide metabolites, it
was tested first against Escherichia coli.
The zone of inhibition observed after 24-hour incubation was 12mm. The same
fraction was used to be incorporated into both PLGA nanoparticles and
PLGA-alginate nanoparticles. After formulating the nanoparticles, an antimicrobial
screening test was done to test the sensitivity of the microorganism against
both nanoparticles formulation. This test supposedly to be done before
proceeding with broth dilution test on both formulations. Broth dilution test
is done to determine the minimum inhibitory concentration (MIC) of the
antimicrobial substances. Based on the result of the screening test, both the
formulation and the metabolite had no total zone of inhibition as can be seen
in Figure 2. The presence of the zone of inhibition determines the
susceptibility of the metabolites and the formulation towards the
microorganisms. This shows that the metabolite and the nanoparticles have no or
minimum antimicrobial activity towards all the microorganism tested against. As
there was no total inhibitory zone, broth dilution test was not done on both
formulations. The result obtained from this test was unexpected because
earlier, the same fraction with same concentration was tested against E.coli
and has shown sensitivity. This shows that the metabolites might have been
degraded when the second test was carried out. Poor antimicrobial effect might
be due to the incorrect solvent used when formulating the nanoparticles. Dichloromethane
is a polar solvent which increases the probability of possible interaction with
the polyketide metabolites, causing inactivation of a certain functional group.
The physicochemical properties of the polyketide metabolites are unknown
leading to possible chemical reaction occurred cannot be predicted. This might
have caused the polyketide metabolites to lose its antimicrobial activity.
Furthermore, the method of evaporating dichloromethane might have led to
physical degradation of the polyketide metabolites. This is because evaporation
enables the loss of volatile compound in the polyketide metabolites, which
might be essential for the antimicrobial activity. Vaporization of the compound
essential for the antimicrobial activity affects the stability of the
polyketide metabolites, causing degradation. Besides that, the absence of the
antimicrobial effect of both formulations might also be due to the poor
entrapment efficiency. Both formulation had low entrapment efficiency which causes
the formulation not to exhibit similar antibacterial effect as the polyketide
metabolites.

 

Conclusions

 

In
summary, the alginate-coated PLGA nanoparticles were successfully formulated
using the double-emulsion solvent evaporation technique. Additionally, PLGA
nanoparticles were also formulated to see the effect of alginate coating in
improving the effectiveness of the formulation. Both formulations were
characterised by particle size, zeta-potential, FTIR and entrapment efficiency.
Both formulations were successfully produced within the nanometre range and
alginate coating showed a significant reduction in the particle size. FTIR
analysis confirmed that the nanoparticles were successfully coated with
alginate. Alginate coating of the PLGA nanoparticles managed to increase the
entrapment efficiency of the formulation despite the smaller size. However,
there is no antimicrobial activity was seen for both formulations. This is
mainly because, at the end of the studies, the polyketide metabolites also exhibit
minimum antimicrobial activity, which indicates that they have degraded by
then. Further studies can be done on this formulation to investigate more about
the drug loading and drug releases profile. 

 

Acknowledgement

 

I
would like express my sincere gratitude to Dr. Fazren Azmi for his guidance and
expert opinion throughout this research project.

References