Synthesis and in-vivo evaluation of antibiotic loaded silver nanoparticles-

chitosan composite spheres for methicillin-resistant Staphylococcus aureus

Amir Zeb, Mariya Javed, Sana Mushtaq, Haroon Iqbal, Nargis Aman, Muhammad Samie,

Muhammad Arfat Yameen

Abstract

Using of antibiotics incoherently and improperly made the microorganisms resistant to

conventional antibiotics. One of the best examples is methicillin-resistant S. aureus (MRSA)

which has high frequency of infection in community and hospital associated settings. In current

research, an attempt was made to enhance the antibacterial activity of amoxicillin with the help

of silver nanoparticles (Ag NPs) and biodegradable chitosan (CS) polymer against MRSA.

The Ag NPs-CS composite spheres’ optimal formulation (C03) was carefully chosen on the

bases of evaluation studies. FTIR outcomes exhibited no substantial interfaces among drug and

polymer while XRD results showed the constancy and integrity of formulated drug. SEM

micrographs exhibited that the spheres were uneven, varied, porous and had irregular surface.

The mean diameter of the composite spheres was 02 mm while the mean diameter of

nanoparticles was 264 nm.

All MRSA strains were 100% resistant with all tested antibiotics except vancomycin and

amoxicillin MIC was ranging from 4-128 mg/L. In in-vivo study, infection was produced by

inoculation of MRSA on skin abrasion and different formulations were applied, tissue was

removed and was homogenized and no of colonies/ml was counted. The amoxicillin loaded Ag

NPs-CS composite spheres and the blank composite spheres showed considerable result.
The in-vivo and in-vitro study evaluated that the blank silver nanoparticles-chitosan composite

spheres showed its own antibacterial activity so the activity of amoxicillin was enhanced after its

loading in the blank silver nanoparticles-chitosan composite spheres. Additional extensive in-

vitro and in-vivo studies are needed for evaluating the enhanced and effective use of these

formulations against microorganisms.

Introduction

Methicillin is semisynthetic penicillin which was first brought into use clinically in 1960. Just a

year later, S. aureus strains showing resistance to methicillin were reported (Chongtrakool et al.,

2006). From that time, MRSA strains have made feel their presence around the world and they

are still one of the most common hospital pathogens (Ayliffe, 1997) . Emerged in 1960s, MRSA

specifically target those patients who are vulnerable to risk factors that come with health care

(Baba et al., 2002). The infections caused by MRSA have been reported in general population.

They prevail within the community and thus people who lack traditional risk factors like recent

admittance, surgery, or long term residence in care facilities fall prey to these infections (CDC,

2003).

MRSA infections have broken out as epidemical in hospitals across the world over the past four

decades (Diekema et al., 2001), exceptionally, only patients having established risk factors

(Brumfitt & Miller, 1989; Lowy, 1998). Most presently, however, patients living in community

without established risk factors have been reported with MRSA infections. (CDC, 2003; Sattler,

2002).

MRSA are known as nosocomial pathogens around the world (Diekema et al., 2001). Recent

hospitalization, surgery, indwelling percutaneous medical devices, residence in a long-term-care

facility, dialysis and catheters are established risk factors for MRSA infection. Lately,

nevertheless, MRSA symptoms have been reported in persons having no established factors for

MRSA attainment, who reside in healthy society. For the reason that they are evidently got into

the community; these infections are identified as community-acquired (CA)-MRSA (Naimi et

al., 2001). CA-MRSA infections may transcend grave and even deadly infections in

contradictory healthy hosts (Dakota, 1999; Naimi et al., 2001). CA-MRSA specifically targets

children and youth and it is the main cause of infections same to those caused by community-

acquired methicillin susceptible S aureus (MSSA) (Groom et al., 2001)

To combat this resistance problem, new drugs and delivery systems were introduced. The present

time has seen enormous growth in research and applications in the field of nanotechnology and

great attempt has been made to advance it for drug delivery system. (Wilczewska et al., 2012).

The focus of nanotechnology is on developing therapeutic agents in biocompatible moieties like

nanoparticles, nanocapsules, micellar system and conjugates. Generally these systems can be

applied to supply customary and riskless delivery of drugs, to enhance the standard of oral

bioavailability, to keep up drug effect on spotted tissues, to prepare drug for intravascular use

and to increase the indelibility of therapeutic agents opposed to enzymatic degradation

specifically resulting from peptides, proteins, and nucleic acid drugs (Panyam & Labhasetwar,

2003).

Ag NPs are rising as one of the quickest developing item classifications in the nanotechnology

business with spotlight on antimicrobial action. This has prompted expanding number of

medicinal utilizations of Ag NPs. A portion of the items which are as of now accessible in the

business sector incorporate injury dressings, prophylactic gadgets, surgical instruments and bone

prostheses (Arora et al., 2009). Embeds, for example, heart valves, focal venous catheters,
neurosurgical catheters, bone concrete (Hackenberg et al., 2011). Poly-methyl methacrylate

stacked with nano Ag is being considered as bone concrete as the nano Ag can impel

antimicrobial movement (Bechert et al., 2004). Ultrahigh atomic weight polyethylene has been

the favoured decision for creating embeds for aggregate joint substitution parts, however it is

vulnerable to wear and tear which is a noteworthy disadvantage. To beat this, Ag NPs were

included, and the nearness of silver nanoparticles radically lessened the wear and tear of the

polymer (Morley et al., 2007). The available utilized strategies to counteract surgical

contamination incorporate anti-infection agents and cleaning agents. Surgical cross sections are

utilized to scaffold vast injuries and for tissue repairs. In spite of the fact that these lattices are

successful, they are defenceless to microbial diseases. Ag NPs covered polypropylene cross

section is said to have great antimicrobial action and can be viewed as a perfect possibility for

surgical lattices (Cohen et al., 2007).

Another moiety the chitosan (CS), -(1-4) linked 2-amino-2-deoxy-D-glucose, is an innate

biopolymer on this planet after cellulose (Chung et al., 2004). CS is a natural un-inebriated

biopolymer inferred by deacetylation of chitin, a leading constituent of the cuticle of crustacean,

for instance, crab, shrimp, and crawfish. Great care has been taken to its mercenary usage in

biomedical, diet, and chemical factories (No et al., 2002).

Because of its astounding biocompatibility, biodegradability and nontoxicity (Kean & Thanou,

2010; Kumar et al., 2004), CS has been effectively utilized as a part of nanomedicine for

conveying helpful medications (De Campos et al., 2004), proteins and genes (Roy et al., 1999).

Till this time, CS has been accounted for to help in the combination of metal nanoparticles, for

the most part gold (Huang & Yang, 2004; Potara et al., 2009), and Ag NPs (Potara et al., 2011;

Wei & Qian, 2008).

In the present research, an attempt was made to enhance the antibacterial activity of amoxicillin

with the help of silver nanoparticles (Ag NPs) and biodegradable chitosan (CS) polymer against

MRSA.

Material and methods

Sample Collection

Biochemical and molecular identified strains of MRSA were taken from the previous study. The

strains were refreshed by inoculating on mueller hinton agar (MHA) at 37 ℃.

Antimicrobial Susceptibility Test

Disc Diffusion

Kirby-Bauer disc diffusion method was carried out for antimicrobial susceptibility testing for all

the isolates. For the inoculum preparation, overnight grown colonies in saline suspension was

used. Turbidity of the bacterial suspension was compared with 0.5 McFarland standard. Bacterial

lawn was made on MHA plates by using socked sterile cotton swab in suspensions.

Antimicrobial susceptibility by disc diffusion was accomplished by using ampicillin,

amoxicillin/clavulanic acid, cefoxitin, methicillin, oxacillin and vancomycin antibiotic discs.

Minimum Inhibitory Concentrations (MIC)

Antibiotic stock solution of, levofloxacin, penicillin G, tetracycline and vancomycin were

prepared according to the manufacturer’s guidelines. Dilutions were prepared from zero up to

512 mg/L and MHA plates were prepared by adding adequate amount of molten media and

antibiotic solution. The bacterial suspension’s turbidity was compared with 0.5 McFarland

standard and 02-03 µl of inoculum suspension was dispensed on MHA plates and spread with the
help of cotton swab. Then suspension was allowed to absorb properly on the surface and

incubation at 37°C for overnight.

Synthesis of the CS, Ag NPs-CS and amoxicillin (AMX) loaded Ag NPs-CS Composite

Spheres

CS, Ag NPs-CS and AMX loaded Ag NPs-CS composite spheres were prepared by the method

of Wang et al., 2015 with slight modifications (Wang et al., 2015). Ag NPs-CS composite

spheres are prepared by adding 200 mg CS in 10 mL of 2% acetic acid solution. After that 10 ml

of 5 mM AgNO3 solution was added and mixed by constant stirring for 30 min. With the help of

syringe pump, AgNO3–CS mixture solution was added drop wise to 25 ml 25% NaOH solution.

After 30 min, the residual alkali was removed by washing. The collected spheres were also

washed with double distilled H2O and dried in freeze dryer for 3 hours.

For AXM loaded Ag NPs-CS composite sphere, amoxicillin (1.32 mg/ml) was added to 0.1%

TPP solution and stirred for 30 minutes. After forming of clear solution, TPP-amoxicillin

solution was added drop wise to AgNO 3-CS solution (same as prepared for blank composites)

and stirred for 30 min. Mixture was added dropwise in 25ml of 25% NaOH solution. Alkali was

removed and washing was done as for blank Ag NPs-CS composite sphere

Formation of Ag NPs

Ten Ag NPs-CS composites spheres were dissolved in 20 µl acetic acid and 01 ml distilled water

with continuous stirring until complete dissolution. The pH was adjusted to 5 and then the

mixture of solution was left for stirring for 30 minutes. The solution was centrifuged at 10,000

rpm for 20 minutes at 04°C. After discarding the supernatant, amount of 5 ml cryoprotectant

(0.5% mannitol or 1% sucrose solution) was added to prevent the adhesion of NPs. The solution
was thoroughly mixed by using the vortex mixer. After complete dispersion of Ag-CS NPs for

the second time at 10,000 rpm for 20 minutes at 04°C the solution was centrifuged. The

supernatant was again discarded and sediment obtained was dispersed in sterile distilled water.

Characterization of Loaded and Blank Ag NPs-CS Composite Spheres

Percentage Yield

Percentage yield of loaded Ag NPs-CS composite spheres was calculated by the following

formula

Total weight of dried composite spheres

Percentage yeild = × 100
Total weight of matrials used ∈internal phase

Loading Capacity (LC) and Entrapment Efficiency (EE)

LC and EE of AMX loaded Ag NPs-CS composite spheres were determined by the help UV-

Spectrophotometer at 273 nm using following formulas

Total AMX Trihydrate – Free AMX Trihydrate

LC= ×100
Dried composite spheres weight

Total AMX Trihydrate−Free AMX Trihydrate

EE= × 100
Total AMX Trihydrate

X-ray Diffraction (XRD)

Encapsulation studies cause the crystallinity of the drug and this effect was examined by XRD

studies of crumble samples of drug, polymer and drug loaded composite spheres. The

diffractometer range that used was among 10° to 80° and the angle of diffraction was 2θ with
step angle of 0.05°/min, under specific measurement conditions scanning of samples was carried

out that was. Cu-Kα radiations, 40 kV voltages, and 20 mA current.

Fourier Transform Infrared Spectroscopy (FTIR)

FTIR was conducted to find out the possible interaction between drug and polymer. An infrared

spectrum of drug, polymer and composite spheres with and without drug was recorded in the

range of 600 to 4000 cm-1 using potassium bromide pellet press technique.

Scanning Electron Microscopy

The morphology and surface characteristics of drug loaded composite sphere and Ag-CS NPs

were determined with scanning electron microscope using gold sputter technique at the voltage

of 10 and 15 kV respectively.

Determination of Particle Size, Poly Dispersion Index (PDI) and Zeta Potential

Particle size, PDI and zeta potential of Ag-CS NPs and AMX loaded Ag-CS NPs were

determined by using zeta-sizer that was based on photon correlation spectroscopy.

Antimicrobial susceptibility studies of Blank and AMX Loaded Ag NPs-CS Composite

Spheres

Agar Well Diffusion assay

Agar well diffusion method is widely used to evaluate the antibacterial activity of formulations

(Magaldi et al., 2004; Valgas et al, 2007). First dilution was made from fresh sample of MRSA

and turbidity was compared with 0.5 McFarland standard. Volume of inoculum of MRSA was

spread through sterile cotton swab on entire plate of nutrient agar. Four wells of size 6-8 mm in

diameter were made on each plate by using sterile cork borer. The wells formed were sealed
properly and were added volume of 50-100 µl of blank, loaded, drug solution and sterile distilled

water to wells on each plate. The prepared plates were then kept for incubation at 37°C overnight

and then zone of inhibition was calculated by scale.

In vivo Activity of Blank and AMX Loaded Ag NPs-CS Composite Spheres

Ethical committee of CIIT approved guidelines are in agreement with scientific practice act 1986

for the animal instruction and guideline and it was followed for performance of research work.

Female (Balb/c) mice having age limit range 06-08 weeks and weight limit range of within 20-30

g were purchased from National Institute of Health, Islamabad, Pakistan. Five animals in each

group were kept under typical condition of environment at a temperature range from 25 ± 2°C.

The circadian rhythm (12 h light/dark cycles) was maintained for twelve hours, as lights were

turned on at 8.00 am and turned off at 8.00 pm.

The groups were arranged naming Group A, Group B, Group C and Group D.

Table: Grouping of animals

Groups No. of animals Type of treatment

A 5 Negative control (No treatment no infection)
B 5 Positive control (Infection and treatment with plan
ointment)
C 5 AMX loaded composite spheres ointment
D 5 Blank composite spheres ointment

Cyclophosphamide used was obtained from Korea united pharma Inc. Two doses of

cyclophosphamide were given to each animal prior to infection. At day first, one day before the
infection first dose given was of 150 mg/kg per body weight was given intraperitoneally (I.P) to

each animal. While dose no second was of 100 mg/kg per body weight was given at day four

after the infection to each mice. The treatment of cyclophosphamide caused reduction of

peripheral blood neutrophils to <100/ml blood which provide a more susceptible atmosphere in

the mice to cause infection.

Density of bacterial cells was compared with 3.0 McFarland standard. Approximately the density

of the cells of was 9.0 × 10 8. After dilutions were made wounds were made on each mice skin.

Before the creation of wounds, mice were sedated with I.P injections of ketamine/xylazine

(ketamine 75 mg/kg and xylazine 5 mg/kg) cocktail per body weight and then shaved on the back

surfaces. By using 28-guage needle, skin abrasion wounds were made on the back or dorsal area

of the mice by making 6×6 cross scratches lines within defined area of 1×1 cm. he dermis

remains safe during making the scratches and only stratum corneum and upper layer of epidermis

was damaged. Five minutes after wounding, 50 µl suspension containing 9.0×10 8 CFU of MRSA

in Phosphate buffer saline (PBS) was inoculated by using pipette tip on the area having cross

scratches. Immediately after the inoculation of bacteria and on a daily basis images were taken

thereafter.

Figure: Mouse model of skin abrasion

Cholesterol hydrophilic petrolatum an absorption ointment was made according to the

specifications of U.S.P (1). Bees wax (665 mg) was melted at 50°C and stearyl alcohol (250 mg)

was added and melted at same temperature, afterward white petrolatum (08 g) was added and

melted. At last cholesterol (250 mg) was added and mixed through magnetic stirrer and then

allowed to cool. The cold ointment was then autoclaved in order to obtained sterile ointment.

Fifty composite spheres were then dissolved in 100 µl acetic acid and 05 ml sterile water and

then added to sterile ointment drop wise by continuous mixing through sterile spatula.

Ointment was applied on day second after inoculation of MRSA up to next three days. Ointment

was applied three times in each day. While for each application up to 30 mg of ointment was

applied this was estimated by weighing the pellet of ointment on a spatula. The concentration of

composite spheres was 1.636 mg per gram of ointment.

Animals were euthanized after four days treatment with ointment by cervical dislocation.

Immediately after the mice were euthanized, the wounds, approximately 2 cm 2, were cut out and

make uniform together with 1 ml of phosphate-buffered saline by using Dounce homogenizer.

The sample of homogenized tissue of each mouse in each group was serially diluted for proper

bacterial count. For the purpose four sterile test tubes were taken for each sample and the tubes

were marked as tube A, tube B, tube C and tube D respectively. Then 0.1 ml homogenized tissue

was added to tube A that contained 9.9 ml of sterile distilled water. This was the first dilution

and was 100 times diluted. From tube A, 0.5 ml of diluent was added to tube B which contained

4.5 ml sterile distilled water. Similarly from tube B, 0.01 ml of diluent was added to tube C that

contained 9.99 ml of sterile distilled water. From tube C, 1.0 ml of diluent was added to tube D

that contained 9.0 ml of sterile distilled water.

From tube D, 0.1 ml of diluent was added to nutrient agar plate by using micropipette and was

spread on the entire plate by using sterile spreader. Plate was then kept for incubation at 37 °C

for 16-18 hours. Colonies was then counted by using colony counter. The dilution factor for

these serial dilutions was 100×10×1000×100.

Colonies were counted by using colony counter and multiplied by dilution factor. Formulas are

given below.

no of colonies
CFU ml plated
=
ml Total dilution factor

While, Total Dilution Factor (TDF) = Multiplication of all Individual dilution factor (IDF) OR

TDF = (IDFA) (IDFB) (IDFC) (IDFD)

Amount transfered
IDF=
amount transfered+ Amount already ∈tub e

Statistical Analysis

Graph pad prism 6 was used for statistical analysis and data interpretation and one way analysis

of variance (ANOVA) was used for statistical analysis. The value of p ≤ 0.05 was considered as

“statistically significant”.

Results

Samples Collection

Six strains of MRSA were used in the current study, which were taken from a previous study of

Mariya Javed that were collected from Holy Family Hospital Rawalpindi, Pakistan
Antimicrobial Susceptibility Testing

Disc Diffusion Test

All MRSA strains were 100% resistant with, amoxicillin, cefotaxitin, methicillin except

vincomycin Table 3.1.

Table Error! No text of specified style in document.-1 Resistance profile of MRSA

Serial No Antibiotic Disc Resistance

1 Ampicillin 06 (100)
2 Amoxicillin/ Clavulanic Acid 06 (100)
3 Cefoxitin 06 (100)
4 Methicillin 06 (100)
5 Oxacillin 06 (100)
6 Vancomycin 0

MIC

All the strains of MRSA were resistant to amoxicillin MIC ranging from 4-128 mg/L, while all

strains were susceptible to vincomycin MIC ranging from 1-8 mg/L. Table 3.2 shows the MICs.

Table Error! No text of specified style in document.-2 MIC with antibiotics

MIC with Amoxicillin MIC with Vancomycin

Antibiotic Dilutions (mg/L) N (%) N (%)

0.25 0 (0) 0 (0)

0.5 0 (0) 0 (0)

1 0 (0) 01 (16.7)
2 0 (0) 03 (50)

4 01 (16.7) † 01 (16.7)

8 0 (0) 01 (16.7) †

32 02 (33.3) 0 (0)

128 03 (50) 0 (0)

t= 2.982 t= 4.620
T-test P <0.05 P >0.05
†
Breakpoint concentration

Preparation of CS, Ag NPs-CS and AMX loaded Ag NPs-CS Composite Spheres

About 30 different trails were conducted for the preparation of different formulations. For the

preparation of CS spheres different concentrations of CS, acetic acid and NaOH were used. The

most stable was found to be the one containing 200mg dissolved in 2% acetic acid and added in

25ml of 25%NaOH After stirring for 30 min, composite spheres having dark colour were yielded

as shown in Figure 2.2

Similarly for the preparation of Ag NPs-CS composite spheres different concentrations of CS,

acetic acid. NaOH and AgNO3 were used Table 2.3. While for the preparation of AMX loaded
Ag NPs-CS composite sphere same method was followed and different concentrations was

selected Table 2.4.

Figure: Ag NPs-CS composite spheres

Characterization

Percentage Yield, LC and EE

For the purpose, composite spheres were dissolved in 1% acetic acid and absorbance was taken

of the whole solution ant then centrifuged at 10,000 rpm for 20 minutes at 4°C and absorbance of

the supernatant was taken. Average percentage yield of the AMX loaded Ag NPs-CS composite

spheres was 65-70%. Similarly % LC was calculated for different trials by using UV

spectrophotometer and average % LC was 45-50 % while, % EE as in the range of 45-70%.

UV-Vis absorbance spectra of synthesized AMX loaded Ag NPs-CS composite spheres.

Prominent peaks were observed: the peak at 273 nm is characteristic peak of AMX, while the

peak at 410 nm is characteristic peak of nano size Ag particles.

UV-Vis absorbance spectra of synthesized AMX loaded Ag NPs-CS composite spheres.

Prominent peaks were observed: the peak at 273 nm is characteristic peak of AMX, while the

peak at 410 nm is characteristic peak of nano size Ag particles.

XRD

Figure 3.4 shows the XRD pattern of simple CS composite spheres (A) and AMX loaded (C) and

blank (B) Ag NPs-CS composite spheres. We observed four distinctive peaks (B and C) for Ag

NPs that are (1110), (200), (220) and (311). Whereas for AMX trihydrate showed characteristic

peaks at about 12.2, 15.18, 18.08, 18.12, 19.38 and 26.74 (2 theta). Where’s the obtained some

peaks have location and intensity with different values. This change in B and C does not

ascertain the alteration in the crystallinity of the drug but that might be a reason of alteration in

sample size and preparation as composite spheres and polymer was crumbled before analysis,
sample synthesis and size can alter the XRD pattern. During the procedure the change in

crystallinity of the drug was not confirmed. The mentioned peaks observed were consistent with

those found in the Joint Committee on Powder Diffraction Standards (JCPDS) database (PDF

No. 65-3107).

700

600

500

400

300

200

ABC
100

0
3 111 219 327 435 543 651 759 867 975 083 191 299 407
1 1 1 1

Figure Error! No text of specified style in document.-1 XRD graphs of (A) fabricated CS

composite spheres and (B) blank and (C) AMX loaded Ag NPs-CS composite spheres

FTIR

Figure 3.5 shows the Ag NPs-CS composite spheres FTIR spectra. The bands among 3,462 cm− 1

and 3,441 cm−1 were related to the stretching vibrations of amino groups from NH-amine. The

bands between 2,925 cm−1 and 2,891 cm−1 related to the alkane C-H-stretching lipids. The bands

between 1,632 cm−1 and 1,597 cm−1 related to the amino groups of amide. The bands between

1,385 cm−1 and 1,387 cm−1 were associated with the C=C stretching of aromatic amine groups.

The bands between 1,077 cm−1 and 1,075 cm−1 were related to the carbonyl stretch in proteins.
The Ag NPs were bonded by protein, which served as a stabilizing agent, either through free

amine groups or cysteine residues. These proteins were present as enzymes that could reduce

AgNO3 ions to form Ag nanoparticles (Ali, Sasikala, Gunasekaran, & Thajuddin, 2011).

Where’s FTIR spectrum of pure AMX showed characteristic peaks at 1249.87 cm 1 (C-C

stretching), 1313.52 cm 1 (C-N stretching), 1573.91 cm 1 (C-H stretching), 1,614 cm 1 (C=O

stretching), 1687 cm 1 (–CONH2 stretching), 1776 cm 1 (-COOH stretching), 3325 cm 1 (sec. –

NH or –OH) and some prominent bands like 846–567 cm 1 (–CH aromatic ring bending and

heteroaromatics). Characteristic peaks of drug were also present in the FTIR spectrum of

composite spheres (A) with some expansion and decrease in intensity, representing the

nonappearance of chemical relations between drug and polymer after production of composite

sphere (Thombre & Gide, 2016).

Figure Error! No text of specified style in document.-2 FTIR spectra of Blank Ag NPs-CS

composite spheres
Figure Error! No text of specified style in document.-3 FTIR spectra of Loaded Ag NPs-CS

composite spheres

Scanning Electron Microscopy (SEM)

Figure 3.6 shows the SEM photographs of the contrive Ag NPs-CS composite spheres. It shows

the SEM graphs of the surface and the “zoom-in” of their whole sphere counter parts. It also

signify the SEM displays of the morphology, and the “zoom-in” of their cross sectional sphere

counter parts. The outcomes display that the spheres contains numerous uneven macro apertures,

predominantly in their interior. The morphology of the CS spheres, that constitute comparatively

even arrangements matched with the Ag NPs-CS composite spheres.

SEM of Ag-CS NPs were performed at 15 kV with different magnifications as shown in Figure

3.7. The presence of NPs was further confirmed by particle size, poly dispersion index and zeta

potential.
Figure Error! No text of specified style in document.-4 SEM photographs of synthesized Ag

NPs-CS composite spheres

Note: a: SEM of surface of blank Ag NPs-CS spheres, b: Cross sectional SEM of blank Ag

NPs-CS spheres, c: SEM of surface of loaded Ag NPs-CS spheres, d: Cross sectional SEM

of loaded Ag NPs-CS spheres

Figure: SEM photographs of synthesized loaded Ag-CS NPs at different magnifications

Determination of Particle Size, PDI and Zeta Potential

For the purpose, AMX loaded Ag NPs-CS composite spheres were dissolved in 1% acetic acid

and pH was adjusted to 5. The solution was then subjected to centrifugation at 10,000 rpm for 20

minutes and the supernatant was then discarded and to obtain NPs 5 ml cryprotectant (0.5 %

mannitol) was added and then again centrifuged at 10,000 rpm for 20 minutes and the obtained

NPs were then dispersed in sterile distilled water. The NPs solution was then subjected to

particle size, PDI and zeta potential.

The mean particle size was recorded as 255 nm and the mean zeta potential was observed as 25.8

mV as shown in the Figure.

Figure: (a) Mean particle size and (b) Zeta potential of the Ag-CS NPs

Antimicrobial susceptibility studies of Blank and AMX Loaded Ag NPs-CS Composite

Spheres

Agar Well Diffusion assay

Six strains were used for antimicrobial susceptibility testing. All the strains showed

resistant to AMX trihydrate with different concentrations. While at concentration of 1.132 mg/ml

showed slight ZOI. Where’s AMX loaded and blank Ag NPs-CS composite spheres solution

showed clear zone of inhibition. ZOI was observed on day 1, day 2 and day 3 respectively. The

mean of ZOI for AMX loaded and blank Ag NPs-CS composite spheres solution and drug

solution are given in the table 3.1

Table Error! No text of specified style in document.-3 ZOI of the Formulations on different

strains

Formulations A1 A2 A3 A4 A5 A6

L NPs 26 mm 22 mm 26 mm 28 mm 26 mm 26 mm

BNPs 20 mm 14 mm 22 mm 16 mm 22 mm 16 mm

D.S 0 mm 12 mm 16 mm 10 mm 16 mm 22 mm

DW 0 mm 0 mm 0 mm 0 mm 0 mm 0 mm

In vivo Activity of Blank and AMX Loaded Ag NPs-CS Composite Spheres

In negative control group, bacterial load was zero as no pathogen was administered while the

positive control group showed the highest rate of ~ 9 log10 CFU/ml for MRSA which was

considered as 100% growth. Among the groups treated with blank CNPs, AXM loaded Ag NPs;

the group treated with AMX loaded Ag NPs-CS Composite Spheres showed significant decrease

(P < 0.05) in microbial burden almost 28% MRSA infection whereas blank Ag NPs-CS

Composite Spheres showed 20% decrease. Other group showed very slight change in microbial

burden as compare to positive control which can be seen in Fig. 5

 MRSA
10
9
8
7
6
Log10 CFU

5
4
3
2
1
0
Negative Control Positive Control Blank Loaded

Figure:

Discussion

Current study was designed to develop and evaluate antimicrobial effect of antibiotic loaded Ag

NPs against MRSA. Silver (Ag) has widely been studied for its antimicrobial effects and has

very old history of use. Additionally, it has been well established that at low and therapeutic

concentrations, Ag is non-toxic to normal bodily cells and do not cause any physical or

biochemical damage to cells (Martinez-Castanon et al., 2008). Broad spectrum antibacterial

effects of Ag are associated with bacteriostatic and bactericidal activities of ionic form of silver

containing compounds. These activities are not limited to bacteria only and extend to viruses and

fungi (Li et al., 2010). Nano particles i.e. NPs have recently been employed in medicine and

found to be far better than traditional formulation in terms of lesser adverse effects and

associated complications. Similarly, Ag NPs have widely been studied for its antibacterial,

antifungal and antiviral effects. The ability of NPs to penetrate into the cellular compartment and
cytoplasm of bacteria justifies the antimicrobial effects of NPs but exact mechanism is still

unknown and yet to be discovered. Recent study on E. coli has shown that when treated with Ag

NPs, morphology of plasma membrane structure is altered and it becomes more permeable for

intracellular contents to come out of the cell and hence bacterial death occurs as result of efflux

and non-functional transport system across the cell membrane (Sondi & Salopek-Sondi, 2004).

Apparently, MRSA is resistant to Amoxicillin (AMX). We investigated the role of Ag NPs in

susceptibility of MRSA to AMX in nano particle based formulations. In current study, a natural

bio polymer CS was used, which has reported antimicrobial (antibacterial and antifungal)

activities (Li et al., 2002). Though the precise mechanism of its action is still unknown, there

have been many proposed hypothesized mechanisms show that toxicity is associated with

interaction of polycataionic CS with anionic groups on surface of cell (plasma membrane and

cell wall) and may cause increase the permeability of membrane for intracellular content to

efflux out of cells, membrane disruptions and essential protein leakage (Juneja et al., 2006).

Another proposed mechanism is based on chelates formation of CS with trace elements and/or

essential nutritive components within the cytoplasm which results in inhibition vital enzymatic

activities (Rabea et al., 2003). Additionally, it has also been reported that CS, because of the

presence polycataionic groups, can interfere with metabolic machinery through electrostatic

entangling with surface of bacterial cell (Chung et al., 2004; Je & Kim, 2006). The ability of CS

to load sufficient drug make it potential carrier in development of various pharmaceutical

formulations, such as drug loaded CNPs, which show improved antibacterial activity against S.

aureus and E. coli in some previous studies (Kong et al., 2010).

The purpose of current study was to investigate the potential use of AMX loaded Ag NPs-CS

spheres to overcome the bacterial resistance to conventional antibiotics and make it available for

the treatment of wounds and burns.

Literature reveals no study available on drug loaded Ag NPs-CS composite spheres. We

proposed a simplified approach to prepare Ag NPs-CS composite spheres in a one-step process

and successfully loaded AMX trihydrate by using TPP as cross linker. In vivo and In vitro

evaluation of the composite spheres was carried out.

MRSA is resistant to AMX trihydrate (Miller, 2002), which is a conventional antibiotic, in

current study AMX was loaded to Ag NPs-CS composite spheres and showed significant

synergetic effect against MRSA in skin abrasion model of mouse as depicted in Table 3.2.

Besides this it showed substantial wound healing effect in mouse model as shown in Figure 3.17.

Similarly disc diffusion and MIC showed significant resistance to all antibiotics except

vincomycin as shown in Table 3.1, for the purpose different concentrations of antibiotics were

used. All the used strains of MRSA were resistant to amoxicillin MIC ranging from 4-128 mg/L

while all the strains were susceptible to vincomycin MIC ranging from 1-8 mg/L as shown in

Table 3.2.

While In vitro evaluation also revealed profound effect against MRSA as compared AMX and

blank Ag NPs-CS composite spheres solution as shown in Table 3.1 and showed clear ZOI as

depicted in Figure 3.16.

AMX loaded Ag NPs-CS composites which was derived from composite spheres were

characterized on the bases of various physicochemical parameters, including particle size, size

distribution and zeta potential. AMX loaded Ag NPs-CS spheres showed typical size which was
01-1000 nm as shown in Figure 3.8 and Zeta potential greater than 38 mV and PDI less than 9.0

as shown in Figure 3.9. While PDI was less than 0.9. SEM results showed irregular surface and

the zoom in spheres counter parts showed irregular macro pores, particularly on their interior as

shown in the Figure 3.6. The present findings of SEM were similar to that of Wang et al, 2015

(Wang et al., 2015). Similarly SEM results of Ag-CS NPs are shown in Figure 3.7.

The results of XRD were helpful in revealing loading of AMX in Ag NPs-CS composite spheres.

The original peaks of chitosan were unchanged that illustrate the chitosan and silver binding did

not harm AMX that is no phase change of AMX was observed as depicted in Figure 3.4. XRD

showed similar patterns to the previous study (Wang et al., 2015).

The FTIR was useful technique that easily demonstrated the relationship between Ag NPs-CS

composite spheres and AMX Trihydrate and which revealed the presence of AMX Trihydrate in

Ag NPs-CS composite spheres. The peaks were similar to the work already done by Ali et al,

2011 (Ali et al., 2011). Additional characteristic peaks were observed like, C-C stretching, C-N

stretching, C-H stretching, C=O stretching, –CONH2 stretching, -COOH stretching, sec. –NH or

–OH and some prominent bands like –CH aromatic ring bending and heteroaromatics. These

bandings indicates the presence of AMX trihydrate in composite spheres. Some additional peaks

were observed in some old studies (Thombre & Gide, 2016).

Average % yield of the AMX loaded Ag NPs-CS composite spheres was 65-70%. Similarly %

LC were calculated for different trials by using UV spectrophotometer and average % LC was

45-50% and % EE was in the range of 70%.

Conclusion

Hence it is finally concluded that AMX loaded Ag NPs-CS composite spheres can be effectively

used to synergize the antibacterial effect of AMX against MRSA. Further study in this regard is

required to obtain fruitful results in the MRSA resistant to antibiotics and the same formulation

can be used for antifungal and antiviral effects.

1. Yadav S, Papneja P, Batra S, Sharma M. Formulation and Evaluation of Ointment and

Cream with Their Mathematical Treatment of Absorption Through Skin: A Review.

World Journal of Clinical Pharmacology, Microbiology and Toxicology 2015:1:09-26.