Abstract
Microbial infections are considered as one of the most important
concerns of the world community. Developing drug delivery systems based
on formulation of nanoparticles with antimicrobial agents have shown
beneficial effectiveness against microbial infections and related
antimicrobial resistance. In this study we prepared and characterized a
chitosan based hydrogel loaded with zinc oxide nanoparticles for
controlling the release of vancomycin and also improving its
antibacterial effect. Characterization studies demonstrated that the
developed biopolymeric hydrogel was able to sustained and controlled the
release of vancomycin in response to acidic media for 96 hours.
Furthermore, antimicrobial studies showed siginificant and efficient
antibacterial activity of prepared hydrogel against S. aureus andP. aeruginosa. Based on obtained results, it can be concluded
that the prepared chitosan hydrogel containing ZnO nanoparticles has a
desirable activity for controlling the release of vancomycin and
improving its antibacterial properties.
Keywords: Hydrogel, Chitosan, Nanoparticle, Zinc oxide,
Vancomycin, Antibacterial activity
Introduction
In the early twentieth century, the discovery of antimicrobial or
antibiotic agents was a milestone in the field of pharmacy that led to a
remarkable decline in mortality and morbidity (1). However, long time
therapy or treating infections with high doses leads to drug resistance
(2, 3). Today, microbial resistance is considered as one of the most
important concerns of the world. The World Health Organization has
declared that microbial resistance is one of the top 10 global public
health threats for humanity (4). Vancomycin as a complex tricyclic
glycopeptide antibacterial agent is extensively used to treat
gram-positive infections like meticillin-resistant Staphylococcus Aureus
(MRSA) (5, 6). High dose administartion and prolonged therapy with
vancomycin, enhancing the risks of toxicity and aggravation of
deletrious impacts (7-10). Due to the undeniable beneficial effects of
antibiotics, development of new drug delivery systems to maintain the
beneficial effects of antibiotics and reduce their side effects is
essential.
Inorganic nanoparticles (NPs) have shown antibacterial effects due to
their distinctive physical and chemical properties and can interact with
bacterial cells, modifying cell membrane penetration and impeding with
molecular pathways (11-13). Formulattion of NPs with antibiotics exert
synergistic effects against bacteria, inhibit biofilm formation and have
been used to prevent multidrug-resistant organisms and combination of
NPs and antimicrobial agents may be useful in fighting the current
crisis of antimicrobial resistance (14, 15). Recently, application of
zinc oixde (ZnO) nanoparticles in infection disease have been considered
due to their potential biocompatibility over other metal oxides and also
their remarcable antibacterial activities over a wide spectrum of
bacterial species (16-19).
For treatment of microbial infections, it is crucial that antimicrobial
agents can be released in a sustained manner to efficiently treatment
and prevent biofilm formation (20). Hydrogels have been used as carriers
for antimicrobial agents and also instruments for co-delivery of
antmicrobial agents to achieve synergistic effects. This codelivery
approach significantly reduce antibiotic toxicity by decreasing the
required doses and administration intervals
(21-24).
Chitosan is a biodegradable and biocompatible polymeric material from a
natural source with high efficiency for preparing hydrogel carriers (25,
26). Chemically crosslinked chitosan hydrogels can be achieved by using
genipin as a natural crosslinkers which its cytotoxicity is
approximately 10,000 times less than of glutaraldehyde (27, 28).
According to the above mentioned facts, the main aim of present study is
preparation and characterization of chitosan hydrogl loaded with ZnO NPs
for controlling release of vancomycin and also improving its
antibacterial activity.
Materials and Methods
Materials
Chitosan (MW: 127kD, deacetylation degree: 97%) was purchased from
Primex Co. (Iceland). Genipin and triton-100 were obtained from
Sigma-Aldrich (USA). Zinc acetate was purchased from Samchun pure
chemical (South Korea). Vancomycin was obtained from Afa chemi company
(Iran). All the other analytical grade reagents were obtained from Merck
(Darmstadt, Germany).
Bacterial strains: Standard strains of Staphylococcus
aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 15442 have been
used from iranian biological resource center.
ZnO nanoparticles preparation
25 ml of 0.2 N zinc acetate solution was prepared and poured into a
flask and placed on a magnetic stirrer. 4.5 ml triton x-100 was added to
the solution and incubated at room temperature for 24 hours. Then NaOH
0.1M was added to the prepared solution drop wise until the pH of
solution reach to 9 by color change and precipitation. The solution was
stirred for 24 hours to complete sedimentation. The solution was
centriguged with rate of 8000 rpm for 30 min. The sediment was washed
twice with deionised water and absolute ethanol, respectively and dried
in 60°C in the oven. Then dried precipitate was calcined in a furnace at
400°C for 1 hour which the color was changed from white to grey.
Nanoparticles characterization
Field emission scanning electron microscope (Hitachi S-4160, Germany)
was used to investigate the size and surface morphology of prepared ZnO
nanoparticles. Furthermore, crystallinity of ZnO was studied using an
X-ray diffractometer (D4-BRUKER) fitted with a Cu-Ka source.
Hydrogel synthesis
Hydrogel were synthetized with genipin at different concentations.
Firstly, a 2% (w/v) chitosan solution was prepared by dissolving
chitosan powder into acetic acid 1% and let it to dissolve for 24 hours
under stirring. 100 mg of vancomycin was added into the prepared
solution. After that, 10 mg of ZnO nanoparticles were added and let to
disperse completely for 24 hours. Solution of genipin was then prepared
by dissolving genipin powder in ethanol with different concentrations of
2 and 4 mg/mL. This soluion was added to the chitosan solution and mixed
for 30 minutes to form the hydrogel precursor solution. The precursor
solution was sonicated for 30 min in the ultrasonic bath (Backer vClean)
and then was dried in oven for 24 hours at 50 °C. The
same method was used For preparations of hydrogels without ZnO
nanoparticles, except adding ZnO nanoparticles. Table 1 summerized
details of hydogel formulations.
Hydrogel characterization
The conjugation between amine group of chitosan polymer and C-OH group
of genipin was confirmed by FTIR. The samples of chitosan polymer and
chitosan hydrogel were mixed with dried potassium bromide (KBr)
separately and FTIR was carried out in the spectral range of 400 to 4000
cm−1 for each one.
Field-emission scanning electron microscopy (FESEM) (Tescan Mira) was
used to evaluate the morphology of chitosan hydrogel prepared with 2 mg
genipin (as a selected formulation). Sample were mounted to the sample
stub using double-sided carbon tape, and images were obtained quickly to
prevent sample shrinkage from drying.
Hydrogel swelling analysis
The swelling profiles of different formulation of hydrgels were
investigated in PBS (pH 7.4) and Citrate buffer (pH 5.8) at 37°C during
24 hours. At each time interval (30 minutes,1, 2, 3, 4, 24 hours)
hydrogel sample mass was recorded. Swelling behavior of prepared
hydrogels was calculated as a percentage using Equation (1), where Wf is
the weight of the hydrogel at each time point and Wi is the initial dry
weight of the hydrogel.
Equation 1: Swelling (%) = ((Wf − Wi)/Wi) × 100
In vitro release study
In vitro release of vancomycin from different hydrogels was
investigated at 37 °C in two different pHs (PBS with pH 7.4 and citrate
buffer with pH 5.8) under stirring. An appropriate amount of hydrogel
(50mg) was dispersed in 2 ml of buffer solution then the obtained
suspension was poured in a dialysis bag (molecular weight cutoff 12 kDa)
and then it was plunged in 50 ml buffer solution. At specified time
intervals (0.5, 1, 2, 3, 4, 24, 48, 72 and 96 hours), 2ml of media was
withdraw and replaced by 2 ml of fresh media. The concentration of
released vancomycin was measured by reading amount of UV absorption at
280 nm.
Furthermore, the released quantity of ZnO nanoparticles from the
hydrogel formulations was investigated in two different pHs (PBS with pH
7.4 and citrate buffer with pH 5.8). For this, 50 mg of hydrogel was
dispersed in 2 ml of buffer solution then the obtained suspension was
poured in a dialysis bag (molecular weight cutoff 12 kDa) and then it
was plunged in 50 ml buffer solution. At specified time intervals (2,
12, 24 and 48 hours), 2ml of media was withdraw and replaced by 2 ml of
fresh media. The concentration of released ZnO nanoparticles was
measured by inductively coupled plasma optical emission spectrometry
(ICP-OES) (Spectro Arcos, Germany) (29). All experiments were carried
out in triplicates.
Antimicrobial studies
The agar disc diffusion method was employed to test the antibacterial
activity of chitosan hydrogel with 2 mg genipin. Briefly, From an
overnight culture of S. aureus ATCC 6538 and P. aeruginosaATCC 27853, a suspension of the bacteria was prepared, diluted in
nutrient broth (NB) (about 107 CFU.mL-1), and then
equally dispersed onto Müller-Hinton agar. 5 mm side squares of the
hydrogel samples inclduing chitosan hydrogel (CH), chitosan hydrogel
contained ZnO NPs (CNH) and chitosan hydrogel loaded by vancomycin and
ZnO NPs (CNH loaded vancomycin) were cut out and carefully arranged on
agar petri dishes. The agar plates were incubated at 37°C and the
diameters of the inhibition zones were measured after 24 h according to
the Kirby–Bauer method (30). Vancomycin was used as control and its
concentartion in all experiments was 6.5 and 12.5 µg/ml for antibacteril
study against of S. aureus and P. aeruginosa respectively.
Furthernore, bacterial kiling assay (CFU assay) was used to assess the
bacteria absolute load reduction values of prepared hydrogels. The
hydrogel samples at concentrations of 0.5 mg/mL and 0.7 mg/mL were
prepared in NB to further explore the antimicrobial ability on S.
aureus ATCC 6538 and P. aeruginosa ATCC 27853, respectively. The
prepared tubes were inoculated with bacteria stock suspensions
(107 CFU mL−1) and incubated for 24 h at 37 ℃ in 150
rpm. At the end of 24 h incubation, a series of ten-fold dilutions of
the bacteria from each tube were made in phosphate-buffered saline (PBS)
and equally dispersed onto nutrient agar plates in order to acquire
viable counts. After overnight incubation, the CFU was finally
calculated and expressed in logarithmic scale (31). All tests were
perfomred in triplicate and reported by mean average values.
Statistical analysis
The statistical analysis was performed using GraphPad Prism version 8.
Multiple comparison tests were performed by ANOVA test. Data are
presented as the mean ± standard deviation (SD). The levels of P
<0.05 was defined to be statistically significant difference.
Results and discussion
ZnO nanoparticle characetrization
According to the Figure 1, the synthesized ZnO nanoparticles were
spherical and homogenous with the average size of 33 nm. Furthermore,
crystallinity of ZnO was studied using an X-ray diffractometer (XRD) and
results showed the hexagonal structure which peaks were indexed
according to JCPDS card No. 96-900-4180. The crystallite size ZnO
nanoparticles determined 41.59 nm by Scherrer equation.
Hydrogel characetrization
The conjugation between amine group of chitosan polymer and C-OH group
of genipin was confirmed by FTIR. An amine group of chitosan polymer
undergoes nucleophilic attack at the C–OH group of genipin and
resulting formation of an amid bond which is indicated by absorption
band at 1630 cm−1. This result is in good agreement
with other studies (32, 33). Furthermore, obtained image from FESEM
showed a highly porous hydrogel network with approximately 200-400 nm
hydrogel pore size. (Figure 2)
Hydrogel swelling analysis
The swelling ratio of different hydrogels was studied by using media
with different pH values (Figure 3). According to the results the
swelling ratio of CH4 formulation which prepraed by 4 mg genipin and
without ZnO nanopartilces in compared with CNH4 formulation which loaded
by ZnO nanopartilces showed higher swelling ratio that could be related
to the presence of ZnO nanoparticles in CNH4 formulation. Kumar et
al . also observed the presence of ZnO nanopartilces could decrease the
swelling ratio of prepared chitosan hydrogel (34). However, chitosan
hydrogel formulation prepared with 2 mg genipin did not show significant
difference between swelling ratio in the presence or abesence of ZnO
nanopartilces. This observation could be related to the lower amount of
crosslinker in formultion that cause to increase the pore size of
hydrogel and therefore ZnO nanopartilces could easily pass from the
pores and not making hindrance. According to the obtained results, the
pore size of prepared chitosan hydrogels with 2 mg genipin was 200 nm
and the synthtized ZnO nanoparticles also showed 33 nm size.
Furthermore, highest swelling ratio was observed in CNH2 formulation in
acidic citrate buffer medium which could be related to the electrostatic
repulsion between amine groups of chitosan in the acidic medium (35).
In vitro release study
The in vitro release of different formualtions were investigated
for 96 hours. According to the results in Figure 4, the presence of ZnO
nanoparticles affected the release profile of vancomysin in CH4
formulatio which prepared using high amount of genipin as crosslinker
agents. In this formulatiln the presence of nanoparticles decreased the
release rate of vancomycin. However, the CH2 formulation with or without
nanoparticles which prepared by lower amount of genipin did not show any
differnce in vancomycine release profile. The effect of ZnO
nanoparticles in decreasing swelling ratio of chitosan hydrogel with
high amounts of crosslinker and therfore lower drug releas was also
previously observed by PT et al. (34). On the other hand, in CH2
formulation , the lower amount of crosslinker caused to increase the
pore size of hydrogel and therefore ZnO nanopartilces could easily pass
from the pores and didn’t able to make hindrance. Therefore, the study
was continued by CNH2 formulation which showed more desirable swelling
ratio and release profile. This finding was in agreement with the
previous studies. Oustadi et al. indicated that the swelling ratio and
degradation rate of prepared hydrogel was decreased by increasing the
genipin concentration (36).
According to the Figure 4B, the mentioned hyrogel showed a controlled
release behaviour in the response of pH. In fact,in acidic pH the
release of vancomycin was increased due to the electrostatic repulsion
between amine groups of chitosan in the acidic medium. This controlled
release profile is very applicable for antibiotic delivery to the
infection sites which are more acidic and cause to have high
concentration of antibiotic in the infection site against pathogens
(37). Lui et al, also observed a pH responsive release behaviour
for genipin crosslinked chitosan hydrogel. In their study the drug
release decreased by increasing the amount of genipin as a crosslinker
and they showed drug release could increase in acidic media. These
findings are related to the impeding the diffusion of drug from the
hydrogel matrix by increasing the amount of crosslinker agents and also
related to the protonation of amine groups in chitosan chains in the
acidic media (35).
Furthermore, the released quantity of ZnO nanoparticles from the
hydrogel formulations was investigated in two different pHs (pH 7.4 and
pH 5.8). According to the Figure 4C, high amount of genipin as
crosslinker agent in CNH4 formulation caused to decrease relase of ZnO
NPs in comparrison with CNH2 formulation which showed higher ZnO NPs
release. Additionally, in CNH2 formulation observed controlled release
behaviour in response to pH is similar to vancomycin release in acidic
pH which could be related to the electrostatic repulsion between amine
groups of chitosan in the acidic medium.
Antimicrobial studies
The antimicrobial studies were performed on formulation with 2 mg
genipin since better results of swelling ratio and release profile was
observed from this formulation. According to the obtained results,
inhibition zone of prepared hydrogel contained both ZnO NPs and
vancomycin was significantly broader than free vancomycin againstS. aureus and P. aeruginosa (Figure 5).
Bacterial kiling assay was used to assess the bacteria absolute load
reduction values of prepared hydrogel. The results demonstarted that
chitosan hydrogel contained ZnO NPs and vancomycin significantely
reduced the growth of both S. aureus and P. aeruginosa in
comparrison with vancomyicn (Figure 6). The obtained results suggest
that prepared hydrogel (CNH2 formulation) has a valuable potential to
use as an instrument for enhancing antibacterial activity of vancomycin.
This efficiency could be related to both characteristics of this
hydrogel. First, the loaded ZnO NPs that can act as an animicrobial
agent which could help vancomycine to generate higher antimicrobial
activity and the second, controlled release property of this system. The
antmicrobial mechanism of ZnO nanoparticles is not completely understood
however direct contact of ZnO-NPs with cell walls and ROS formation has
been proposed (38). Vasile et al. also observed developing
gentamicin controlled release system using chitosan and ZnO could
efficinetly increase antibacetrial activity againts bacteria (39).
Conclusion
In conclusion, in this study we developed a chitosan based
hydrogelloaded with zinc oxide nanoparticles for controlled release of
vancomycin and analyzed its antimicrobial effectivness. Furthermore, the
effect of genipin concentration on hydrogel properties were evaluated to
optimize the formulation. The results were encouraging since chitosan
hydrogel prepared by 2 mg genipin showed pH responsive controlled
release behaviour and also significantely enhanced antimicrobial
effevtivenss against S. aureus and P. aeruginosa in
comparrison with free vancomyicn. Finally, this prepared hydrogel could
be an attractive and low-cost option for developing an efficient
antibiotic controlled delivery system for different skin, bone or other
tissues microbial complications. .