SILVER NANOPARTICLES FUNCTIONALIZED GEL: PHYSICO- …

www.wjpps.com Vol 7, Issue 2, 2018.

76

Almeida et al. World Journal of Pharmacy and Pharmaceutical Sciences

SILVER NANOPARTICLES FUNCTIONALIZED GEL: PHYSICO-

CHEMICAL PROPERTIES AND ANTIBACTERIAL ACTIVITY

AGAINST ESCHERICHIA COLI, PSEUDOMONAS AERUGINOSA

AND STAPHYLOCOCCUS AUREUS

Eduardo José Jucá Mallmann1, Samuel Veloso Carneiro

1, Francisco Afrânio Cunha

1,3,

Maria Conceição Dos Santos Oliveira Cunha2, Everardo Albuquerque Menezes

3,

Tamara Gonçalves-Araújo4

and Pierre Basílio Almeida Fechine1*

1Grupo de Química de Materiais Avançados (GQMat)- Departamento de Química Analítica e

Físico-Química, Universidade Federal do Ceará – UFC, Campus do Pici, CP 12100, CEP

60451-970 Fortaleza – CE, Brazil. 2Mestranda da Universidade UNILAB- Universidade da Integração Internacional da Lusofonia

Afro-Brasileira, Fortaleza – CE, Brasil. 3Laboratório de Microbiologia da Faculdade de Farmácia, Odontologia e Enfermagem,

Universidade Federal do Ceará. 4Faculdade de Farmácia, Odontologia e Enfermagem, Universidade Federal do Ceará.

ABSTRACT

This study was performed to verify the stability, physicochemical

properties and antibacterial activity of a carbomer based gel

functionalized with silver nanoparticles (AgNPs) obtained by green

synthesis. Three formulations of Gel-AgNP were done: 0.5, 1.0 and

2.0% of AgNPs. The gel is skin-compatible and had shown interesting

rheological properties, assuming a pseudoplastic profile. In addition,

the stability of the gel was obtained and it was extremely satisfactory

for six months. The antibacterial activity was performed against

Escherichia coli, Pseudomonas aeruginosa and Staphylococcus

aureus, showing satisfactory results for 1.0 and 2.0% AgNPs gels

trough well-diffusion technique. The AgNPs gel could provide an

alternative to conventional antibacterial formulations (topical use) for

veterinary or human applications.

KEYWORDS: Silver nanoparticles; Carbomer gel; Green synthesis;

Antibacterial activity.

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 6.647

Volume 7, Issue 1, 76-93 Research Article ISSN 2278 – 4357

*Corresponding Author

Pierre Basílio Almeida

Fechine

Grupo de Química de

Materiais Avançados

(GQMat)- Departamento de

Química Analítica e Físico-

Química, Universidade

Federal do Ceará – UFC,

Campus do Pici, CP 12100,

CEP 60451-970 Fortaleza –

CE, Brazil.

Article Received on

29 November 2017,

Revised on 19 Dec. 2017,

Accepted on 09 Jan. 2017

DOI: 10.20959/wjpps20182-10686

javascript:abreDetalheGrupo('0089106Z8KT1AM')




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INTRODUCTION

Some of recurrent employment of silver nanoparticles (AgNPs) are in paint with antibacterial

properties[1]

, wound dressing, also with anti-inflammatory and antimicrobial properties[2-4]

,

cosmetic industries[5]

and large use as growth controlling antimicrobial.[6-11]

Due to the

emergency and the raising of multi-resistant microorganisms, many researchers have been

studied a viable alternative in treatment of patients infected by those pathogens. Once the

new antibiotics demand a lot of time and money to be developed, and still there is the risk of

the inefficacy of them, even if they are newly released, because the microorganism may

previously develop resistance to them.[12,13]

Antiseptics silver-based have been largely

employed due to its large spectrum of activity against microorganisms and low resistance-

inducing of them, when compared to antibiotics.[14]

Among metallic nanoparticles, the silver are the ones that show the best bacteriostatic and

bactericide effects.[30]

The activity of silver salts against bacteria has been reported since the

old ages[15]

and the silver is used, nowadays, to control the growth of bacteria in a large scale

of applications, such as dental works, catheters and burns.[16,17]

Silver ions and compounds

containing silver are, actually, extremely toxic to microorganisms, showing biocides effects

in lot of bacteria.[18]

Aymonier and co-workers[19]

published a work showing functionalized

AgNPs with amphiphilic macromolecules associated to antimicrobials as capping agents. The

silver nanoparticles (AgNPs) show more intensive activity against microorganisms when

compared to its ions and salts of silver.[20-23]

Beyond the activity against bacteria and viruses,

[18,24-26] these nanoparticles had been used also to treat immunologic and inflammatory

disorders. Due to these reasons, the research concerning silver nanoparticles had increased on

the last years.[27,28]

Other metallic nanoparticles (that show a high surface area and a

reasonable amount of atoms on its surface) are also targets of researches due to its unique

physic-chemical properties, such as catalytic activities, optic and electronic properties,

antimicrobial activity and magnetic properties.[29-31]

According the resistance of microorganism to currently available antibiotics, AgNPs show

themselves as a new and effective alternative to combat pathogens, once its use does not

induce the resistance on the germs, even its mechanisms are not elucidated yet. A viable

alternative would be using silver as synergic agent to the antibiotics, however, this alternative

needs more studies and data.[32,33]


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MATERIALS AND METHODS

Obtaining the gel

Carbomer 940 and sodium carboxymethylcellulose were purchased from Chemistry (São

Paulo, Brazil). Trietanolamine (TEA) was purchased from Basf (São Paulo, Brazil) and

propylene glycol from Mapric (São Paulo, Brazil). AgNPs were synthetized starting from

AgNO3 (Dinâmica- São Paulo) 5mM solutions. The reduction of Ag+ ions was performed by

1.0g of ribose (Sigma- Brazil), and 0.5g of sodium citrate (Dinâmica- São Paulo) was used as

stabilizer agent. The temperatures of synthesis were 50 °C, 80 °C and 100 °C during 10

minutes. The AgNPs functionalized gel was produced using the nanoparticles synthetized at

100 °C.

Definite amount of CMC and carbomer 940 were dissolved in deionized water and mixed

using Omni Mixer Homogenizer (Model M50). It was added propylene glycol after 30 min of

stirring. This mixture was agitated for additional 10 min. The dispersion was then allowed to

hydrate and swell for 60 min and the pH of the neutralized sample was measured. The

Carbopol dispersion was neutralized with 98% TEA until the desired pH value of 6.0. During

neutralization, the mixture was stirred gently with a spatula until homogeneous gel was

formed. AgNPs were added in two different concentrations (1.0% and 2.0% w/w). These gel

formulations, were packaged under sterile conditions, labeled with appropriate details and

stored at room temperature for further uses. This last procedure is illustrated at Fig. 1.

Figure 1: Obtaining the gel functionalized with silver nanoparticles: (a) production of

AgNPs; (b, c) purification; (d) concentration, (e) production of gel and (f) gel.


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2.2. Antibacterial activity

Gel silver nanoparticles (Gel-AgNP) in two different concentrations 1.0% and 2.0% w/w

were tested for antimicrobial activity as method suggested by Srinivasulu et al.[34]

using

various Gram-positive and Gram-negative bacteria by the agar well-diffusion method.

Approximately, 20 mL of nutrient agar medium was poured into sterilized petri-dishes. The

bacterial test organisms were grown in nutrient broth for 24 h. A 100 μL nutrient broth

culture of each bacterial organism (1 × 105 CFUmL

−1) was used to prepare bacterial lawns.

Agar wells of 8 mm diameter were prepared with the help of a sterilized stainless steel cork

borer. The wells were loaded with 60 μL of Ag nanoparticles gel, 60 μL of culture with gel

without AgNPs as a negative control, along with 60 μL of 30 μg mL−1

of 1.0% silver

sulfadiazine as a positive control. The plates were incubated at 37 °C for 24 h and then were

examined for the presence of zones of inhibition.

2.3. Stability Evaluation

The experimental protocol was based on the guideline “Stability testing: of existing active

substances and related finished products” (CPMP/QWP/122/02). Three batches of the

formulation were produced under similar conditions and were then stored at room

temperature (realtime, 25 ± 2°C / 60% ± 5% humidity ) for 6 months and submitted to

accelerated aging for 60 days (oven at 40±2°C / 75% ± 5% humidity). The stability of the

formulation was defined by samples analysis without significant changes in parameters

physico-chemical. The stability samples (n=3) were taken for analysis at the end of the

following time periods: 30, 60, 90 and 180 days.

2.4. Characterization

2.4.1. pH

The pH was controlled using a potentiometric method (pH meter Metrohm ® pH Meter 744,

glass electrode).

2.4.2. Rheology

The apparent viscosity and rheological profile were evaluated using a Brookfield R/S –CC+

rotational viscometer ® equipped with V3 40/20 spindle. In these rheological tests were

evaluated three different gels (0.5%, 1.0% and 2.0%), with only comparative purposes.


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2.4.3 – UV-Vis Spectroscopy

The silver nanoparticles were identified by UV-VIS spectroscopy through a GENESYS®

10S (Malvern Instruments Ltd- England), UV-Vis spectrophotometer. The absorption band

near 400nm is a characteristic pattern of silver nanoparticles.

2.4.4. XRD

A Rigaku (Tokyo, Japan) X-ray powder diffractometer operating on 40 kV/30 mA with a Cu-

KαI tube (λ= 1.54056 Å) was used to obtain the X-ray diffraction pattern of the silver

nanoparticles. The diffraction patterns were carried out using Bragg-Brentano geometry in

continuous mode with speed of 1°/min and step size of 0.02° (2θ) in the angular range 20–80°

(2θ).

2.4.5 – Scanning Electron Microscopy

The scanning electron microscopy (SEM) was performed at an INSPECT 50 SEM with

EDS/EBSD and lithography.

RESULTS

The all lots of gel were transparent, light brown, uniform in appearance and without smell.

The pH value of SNG was 5.8 ± 0.05 (n=6), which is a physiologically acceptable pH. Two

Tables (1 and 2) show the variation of pH along 6 months and 2 months, when the gels were

stored at 25°C/60°C and 40°C/75°C respectively.

Table 1: Physicochemical characteristics of 1.0% and 2.0% AgNP gel stored at 25ºC

/60%RH.

AgNP GEL 25ºC /60%RH

Months Color

Smell/Odor

Appearance

pH a,b

1% 2%

1% 2%

1% 2%

1% 2%

0 NC NC

NC NC

NC NC

5.8±0.4 5.8±0.6

1 NC NC

NC NC

NC NC

5.9±0.25 5.8±0.4

2 NC NC

NC NC

NC NC

5.7±0.1 5.8±0.4

3 NC NC

NC NC

NC NC

5.8±0.08 6.1±0.5

6 NC NC

NC NC

NC NC

6.0±0.3 6.1±0.8

a= Mean ± SD, n =3; b= statistically significant difference vs. time zero= p = 0.003; NC= Not

Change.


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Table 2: Physicochemical characteristics of 1.0% and 2.0% AgNP gel stored at 40ºC

/75%RH.

AgNP GEL 40ºC/75% RH

Months Color Smell/Odor Appearance pH a,b

0 NC NC NC 5.8±0.4

1 Dark Brown NC PPT 6.4±1.5

2 Dark Brown NC PPT 7.2±1.8

a= Mean ± SD, n =3; b= statistically significant difference vs. time zero, p = 0.003; NC= Not

Change.

The UV-Vis spectroscopy had shown as an excellent technique to characterize AgNPs. The

absorption bands of the three different methods of synthesis of AgNPs are shown in Fig. 2.

Figure 2. UV–VIS absorption spectra of AgNPs. AgRC01-50 °C, AgRC02-80 °C and

AgRC03-100 °C.

The samples were prepared to X-ray diffraction according shown at Fig. 3, where 1.0mL of

the silver nanoparticle solution was dried at plates of glass under a temperature of 60.


82


°C.

Figure 3. XRD patterns of the AgNPs and diffraction peaks from JCPDS (04-0783) used

for identification and comparison. AgRC01-50 °C, AgRC02-80 °C and AgRC03-100 °C.

To verify the morphology of silver nanoparicles acquired, it was performed the scanning

electron microscopy technique. The obtained results for the chosen sample, AgRC03, are

shown at Figs. 4a, 4b and 4c respectively.

(a)


83


(b)

(c)

Figure 4. SEM image of the AgRC-03at an amplification of (a) 100,000X; (b) 238,000X;

(c) size distribution histogram.

The evaluation of the stability of the gel containing silver nanoparticles ( 1 % and 2 % )

subjected to accelerated stability conditions exhibited respectable stability with respect to

concentration of silver nanoparticles , except 40 ºC / 75 % RH (Table 3). The other results are

plotted in Figs. 5a and 5b, respectively. The results are plotted at Figs. 5a and 5b,

respectively.


84


Table 3: Chemical stability of AgNP gel (1.0 and 2.0%) during stability testing AgNP

content (%)a.

1.0% AgNP GEL 2.0% AgNP GEL

Months 25ºC /60%RH 40ºC/75% RH 25ºC /60%RH 40ºC/75% RH

0 100.2% 100.2% 100.3 100.3

1 99.8±0,2 96 ±0,8 99.2±0.08 93 ±1,5

2 99.5±0,5 94.2± 1.5 98.7±0.2 90.1± 1.1

3 98.9±0.1 96.4± 0.9 97.5±0.1 87.8± 0.5

4 98.1±0.2

97.1±0.8

5 98.9±1.5

96.5±1.6

6 98.6±0,7 87.2± 1.2 95.6±0.5 82.7± 0.5b

a= Mean ± SD, n =3 b= statistically significant difference vs. time zero= p = 0.003.

(a)

(b)

Figure 5. Chemical stability of AgNP during the stability testing of gel (1 and 2%) at (a)

25°C; (b) 40°C.


85


The results of rheological analyses are shown at Figs. 6a, 6b and 6c.The three samples (1, 2

and 3) had shown apparently mean viscosity as 1.8357 Pas, 3.6840 Pas and 3.3636 Pas

respectively.

(a)

(b)

(c)

Figure 6: Results rheological analysis: (a) Shear stress; (b) viscosity x deformation; (c)

viscosity x time.


86


The Microbiological tests were as carried out in Petri dishes, checking the growth of colonies

of Escherichia coli in various gels as shown in Fig. 7.

Figure 7. (A) Antibacterial activity against Escherichia coli for gel without AgNP (1),

1.0% AgNP gel (2), 2.0% AgNP gel (3) and 1.0% silver sulfadiazine (4). (B) For gel

samples without AgNP (a), 1% AgNP gel (b) and 2% AgNP gel (c).

DISCUSSION

A large number of recent works demonstrates the antimicrobial activity of aqueous

suspensions of silver. From this perspective, one can develop topical gels with silver

nanoparticles, taking into consideration the properties of the product, which must conform to

the physiological features of human skin. It was observed that the formulations had

developed a pH between 5.7 and 6.1, which is ideal for use on human skin.

By UV-Vis spectroscopy, there was a band characteristic with wavelength of 420 nm called

plasmon band, which acts as fingerprints, for this type of product.[35-38]

The XRD

spectroscopy technique is complementary to UV -Vis, showing the formed phases of silver.

As verified at literature,[38-41]

the diffractogram is characteristic of silver (JCPDS-04-0793).

According the diffratogram shown in Fig. 3, it can be concluded the preferential growth of

the crystal is assigned to the plan (1 1 1). The geometry is face centered cubic (fcc).

As it can be seen, the nanoparticles show a spheric morphology as predominant geometry,

even that others types of particles are present, such as prismatic and as rod particles (Fig. 4b).

This results corroborates the literature[42-46]

that shows the predominance of spheric silver

nanoparticles when the plasmon bands are assigned to the region of 400 nm.


87


About evaluation of the stability of the gels, it was found that AgNP test values for the

sample gel AgNP 1.0% stored at 25 °C / 60% RH was found to vary between 100.2 and 98.6

% after 12,3,4,5 and 6 months storage compared with the initial value of 100.9 % the

observed differences in assay values not statistically significant. The assay values were still

not significantly different. However, the assay values for the GSN samples stored at

40ºC/75% RH were 96 ±0,8, 94.2± 1.5, 96.4± 0.9 and 87.2± 1.5 after 1,2,3 and 6 moths,

respectively, indicating loss of SN after six months. For the 2.0% AgNP gel sample stored at

25ºC /60%RH were found to range between 100.0% and 95.6 after 1, 2, 3, 4, 5 and 6 months

of storage and compared to the initial value 100.3% the observed differences in assay values

not statistically significant. The assay values were still not significantly different. However,

the assay values for the Gel-AgNP samples stored at 40ºC/75% RH were 93 ±1,5, 90.1± 1.1,

87.8± 0.5 and 82.7± 0.5 after 1,2,3 and 6 months, respectively, indicating loss of AgNP after

six months.

The profiles observed in the rheological behavior can be understood by morphology and the

interaction between the gel and AgNP. Due to its high surface area per volume (AgNPs),

which show a high tendency to aggregate to minimize the total energy system, which

involves both attractive and repulsive forces.[47]

The surface charge may provide physical

stability to the system by preventing agglomeration of the nanoparticles through electrostatic

repulsion.[48]

Furthermore, these charges were exploited to improve the interaction between

nanoparticles and skin.[49,50]

According to the data obtained, these forces cause the change in

viscosity of the gel samples. All samples are characterized as pseudoplastic profile.

Pseudoplastic materials have an apparent viscosity decreased according as the shear rate

increases. It cannot be expressed by the number an isolated.[51]

The strain rate is also shear

function (shown in Fig. 6a).[52]

Observing the graphs, it appears that all gels showed a

distinction between the ascending and descending curves. This effect indicates that the fluid

is independent of time and hysteresis between the curves is indicative of a thixotropic

behavior.[53]

This profile is viable for high gels, because they deform during application,

which facilitates the spreading, but the viscosity returns to its original value when the process

is over. Thus, the product can not drain.[54]

The gel obtained in our study showed high activity against the bacteria E. coli (Figure 7). In a

previous study it was observed that the gel containing AgNPs may destroy the structure of

bacterial cell membranes in order to enter the bacterial cell. The AgNPs then condensed DNA


88


and combined and coagulated with the cytoplasm of the damaged bacteria, resulting in the

leakage of the cytoplasmic component and the eventual death of the bacteria.[57]

Our gel was

activated in the minimum concentration of 1% AgNPs that proves the high antimicrobial

activity of AgNPs present in the cream and may represent a valuable ally in the treatment of

wounds.[58]

CONCLUSIONS

The gel functionalized with silver nanoparticles (1.0 and 2.0%) had shown effective action

against the strains of related bacteria on this work. These results sign for a further study,

investigating its activity at higher concentrations, for example. The physicochemical

properties had shown the skin compatibility (pH) of the formulations, nicer odor and color,

such as the rheological behavior is satisfactory. About the stability, the formulations had

shown a good profile for 25ºC/60ºC for six months.

ACKNOWLEDGMENTS

We gratefully acknowledge the financial support of Brazilian Agencies for Scientific and

Technological Development CNPq, CAPES and Funcap. Also, Projeto Central Analítica and

Laboratório de Raios-X from Universidade Federal do Ceará.

Conflicts of Interest

The authors declare no conflict of interest.

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SILVER NANOPARTICLES FUNCTIONALIZED GEL: PHYSICO- …