Preparation and characterization of CS/ PEO/ cefazolin nanofibers with in vitro and in vivo testing

INTRODUCTION

So far, various methods with different features have been used to cover the wound types. For having wound coatings some features should be considered, such as: biocompatibility, biodegradability, wound healing accelerator and prevention of scarring. Recently, some of the nanoparticles such as silver are receiving considerable attention as an antimicrobial agent. But the implication of nanoparticles on health and environment needs to be assessed completely before their application as antimicrobials. The size of the nanoparticles is small and these can easily access the skin, lungs, and brain and cause adverse effects. Traditional methods have deficiencies and often do not have above characteristics. To achieve this goal, in this study, we prepared the nanofibers of chitosan and PEO polymers with cefazolin additives by electrospinning device. Chitosan is a biocompatible and natural polymer with different properties such as: biodegradability, biocompatibility [1,2], lack of toxicity, the coagulability, the antigenicity, the lack of water solubility and gelation properties, antibacterial, anti-fungal and anti-viral effects, has wide application in wound dressing and injuries [3,4], wound healing [5], drug delivery systems [6] and varied applications in tissue engineering [7,8] . Application of chitosan in bone tissue engineering [9, 10], in vitro [11] and living tissue [12] reviewed and the strengthening effect of chitosan was shown in the bone formation.

Nevertheless, electrospinning of alone chitosan is not possible, because it has poor solubility in water and high viscosity in aqueous solutions and can be performed just in the presence of other polymers. PEO has become a very useful synthetic polymer, because it has the diverse molecular weight and substantially solubility in most solvents, especially water [13, 14].The properties of this polymer containing; biocompatibility [15], low toxicity [16],   as well as the ability for fibers production in aqueous solution [17-18]. The use of this polymer with natural polymers such as chitosan leads to the formation of uniform fibers with high mechanical ability, without decreasing the wound healing potential of chitosan [2, 19]. On the other hand, it was found that using 50% aqueous acetic acid as solvent, chitosan-based nanofibers with the high amount of chitosan can be fabricated from 2 wt% Chitosan and 4wt% of PEO (Chitosan/PEO with the ratio of 90/10) solutions without any other additives. It was also found that the matrix with a chitosan/PEO ratio of 90/10 retained excellent integrity of the fibrous structure in water [20]. In this study, cefazolin antibiotic was used for increasing the antibacterial properties of nanofibers. Cefazolin (first generation of cephalosporin) is a glycopeptide antibiotic that inhibits the formation of bacterial cell wall and causes cell lysis. Every bacteria need to trans-peptidase and carboxypeptidase enzymes for connected precursors and building their cell walls. Cephalosporin antibiotics prevent of trans-peptidase enzymes activity. Each newer generation of cephalosporins has significantly greater Gram-negative antibacterial properties than the preceding generation, in most cases with decreased activity against Gram-positive organisms. Fourth-generation cephalosporins, however, have true broad-spectrum activity. Resistance to cephalosporin antibiotics can involve either reduced affinity of existing penicillin binding protein (PBP) components or the acquisition of a supplementary β-lactam-insensitive PBP. Cefazolin with 450-500 kDa molecular weight, is water-soluble and relatively stable against changes of pH and temperature and is used for skin infections and soft tissues [21]. As a first-generation cephalosporin antibiotic, cefazolin and other first-generation antibiotics are very active against Gram-positive bacteria and some Gram-negative bacteria. Cefazolin is used in a variety of infections for example: skin and skin structure infections (due to S. aureus penicillin-sensitive and penicillin-resistant, group A beta-hemolytic streptococci, and other strains of streptococci); bone and joint infections (due to S. aureus); urinary tract infections (due to E. coli, P. mirabilis and Klebsiella species).

 In other investigation, the CS-PEO-1% F. silica-0.5% cefazolin mat was considered as the drug delivery system for biomedical and wound dressing [22].

Therefore, in this study polymer/ cefazolin composite mats were prepared by the electrospinning method for the efficiently controlled delivery of the cefazolin drug.

The combination of the antibacterial properties of chitosan and adding antibacterial properties with an antibiotic drug is a promising strategy for the preparation of nanofibrous materials suitable for wound dressing applications. In this study, a one- step method–electrospinning in common solvent – has been used for the preparation of nanofibers based on chitosan, PEO and cefazolin antibiotic drug. As the spin ability of pure chitosan is challenging, chitosan/PEO blend was used as matrix phase and cefazolin as reinforcing phase in order to improve the wound healing properties of chitosan nanofiber.

The nanofibers with 1%wt cefazolin have suitable antibacterial properties. The new work in this research was the adding the 1%wt cefazolin antibiotic for increasing the antibacterial activity and in vivo test was showing the good performance of nanofibers. We can use this nanofibers as a new gauze pads in skin burn and the other application in medicine.

MATERIALS AND METHODS

Acetic acid, tween 80 and nutrient agar cultivation environment were prepared from Merck. Cefazolin sodium salt was purchased from Tehran medication. Escherichia coli (E.coli) (ETEC ATCC 35401), Staphylococcus aureus (S.aureus) (ATCC 6538), a medium molecular weight of chitosan and PEO with molecular weight of 900000 g.mol^-1 were purchased from Sigma-Aldrich. Electrospinning (model ES100, Fanavaran nano-meghyas Co. of Iran), scanning electron microscope (model MV2300, Camscan SEM Co.,of Czech Republic), FT-IR (model Spectrum GX, Perkin Elmer Co., of USA), UV-Vis spectrophotometer (model U-3010, Hitachi Co., of Japan), incubator (model JTSL 40,  Jal Tajhiz Co. of Iran) were used.

Preparation of solutions and nanofibers

Preparation of chitosan/PEO solution (90 to10) with and without antibiotic

The chitosan/PEO mixture was prepared by slowly adding 0.27 g of chitosan powder with medium molecular weight to 0.04 g PEO in the appropriate volume of 50% acetic acid. Then, 0.25mL tween 80 was added to this solution as an emulsifier. The prepared solution was stirred using a magnetic stirrer at 200 rpm for 12 h at room temperature, to yield a homogeneous solution.

In order to prepare chitosan/PEO/cefazolin solution, cefazolin with percentages of 0.5, 1, 2 and 3wt% was added to polymeric solutions of chitosan/PEO and mixed. The antibiotic agent is firstly dissolved in the same solvent and slowly added to the polymer solution while stirring to produce a homogeneous solution prior to electrospinning. This method can accommodate a large amount of the antibiotic to be loaded in the nanofibers by adjusting 1wt% concentration of the drug in the electrospinning solution.

Preparation of nanofibers from polymeric solutions

In this stage, for producing electrospun nanofibers, the prepared solutions were supplied into a 5 mL syringe with a stainless steel capillary needle (inner diameter =0.9 mm). The electrospinning was performed at room temperature and the solutions were injected from the syringe pump with a feed rate of 0.3-0.5 mL/h at 17-20 kV voltage. The electrospun nanofibers were assembled on a collector which was placed at 10 cm from the needle tip and rotate at 400 rpm. In order to obtain nanofiber with the appropriate thickness, electrospinning was performed for 10 h. For the complete evaporation of solvent and water from the nanofibers, they were drying at room temperature.

Biodegradation and swelling of nanofibers

A buffer at pH 5.5 and 37^oC temperature was used as a simulation of human skin in order to examine the degradation and swelling of prepared nanofibers.

The degradation and water absorption capacity of scaffolds of the nanofibers were studied by immersing segments of chitosan/PEO/cefazolin nanofibers mats in 500 mL of buffer at 37^oC for 24 h. Afterward, the wet nanofibers were placed between two filters to remove the excess amount of buffer solution.

The morphology of the nanofibers structures and also their degradation and swelling were studied by scanning electron microscope.

Analysis of surface morphology and measurement of nanofibers diameter

   For analyzing the morphology of surface and nanofibers diameter of the samples, scanning electron microscopy was used. For this purpose, the nanofibers had been collected on the aluminum foil on the collector and were cut. Ionic coater E5200 model applying 1kV voltage on prepared samples and they were covered by gold for 5 minutes. Also, ''Image J'' software was used to determine the average nanofibers diameter.

Drug releasing template from nanofibers by UV-Vis spectrometery

Releasing template of cefazolin from nanofibers scaffold was studied by UV-Vis spectrometery. 5×5 cm segments of chitosan/PEO/cefazolin nanofibers were placed in 5 mL of buffer at 37^oCin order to investigate the drug release into the buffer solution. Sampling from this solution was performed at specific times and the UV-Vis spectrum of released active substance was recorded from 200-800 nm wavelengths. The maximum wavelength (λ _max) for cefazolin is 273 nm.

FT-IR analysis of prepared nanofibers

     FT-IR is one of the commonly used spectroscopic methods for studying polymers. We used this technique for investigation of chemical changes during the nanofibers preparation and confirming the chemical attachment. The FT-IR spectra of the polymeric nanofibers were measured by placing the samples between two KBr windows spacer with a 0.25 mm thickness. For this purpose, FT-IR spectrum of chitosan/PEO and chitosan/PEO/cefazolin nanofibers were recorded.

In vitro and in vivo testing of nanofibers

For investigating of in vitro antibacterial property of nanofibers, we tested them by using agar plate method. The antibacterial activity of electrospun CS/PEO and CS/PEO/Cefazolin nanofibers, against the Gram positive bacteria S. aureus ATCC 6538 and Gram negative E. coli ATCC 35401 which is commonly found on burn wounds, was assessed by a semi-quantitative way based on diffusion which known as antibiogram on bacterial culture medium, Mueller-Hinton agar, was prepared and used for antibacterial testing. The bacterial species Staphylococcus aureus and Escherichia coli are recommended in most test methods. These two species are potentially pathogenic and therefore require proper physical containment facility in their handling. Many studies have used them as the test microorganism which can be cultured and handled in a standard laboratory with minimal health risk.

Nanofibers were first sterilized by using ethanol 75% treatment. Then these nanofibers mats caught into 5 mm disks and were dropped in the culture on a Mueller-Hinton agar plate. The plates were incubated at 37^oC for 24 h, and the inhibition zone of bacteria was measured to determine minimum inhibitory concentration (MIC).

Furthermore, the wound healing activity was evaluated by burn wound model in an adult rat. Healthy male rats weighing 150– 200g were selected for the study. Animals were divided into 4 groups, consisting 2 animals in each group.

The wound healing activity was conducted with the protocol as shown in table 2. The animal was unconscious with chloroform and 1.5 ×1.5 cm segment on the back side of rats was burned by a pipe with a diameter 1.5 cm of boiling water for several minutes and skin wounds has been sterilized with 70% alcohol.

After, the wound was blotted with sterile gauze in control group, the respective nanofibers on the wound of animals in treatment groups (Group- III, IV) and were investigated on days 3, 5, 7, 10 after the injury and wound size was measured (with three repeat tests).

 RESULTS AND DISCUSSION

Electrospinning of chitosan /PEO with a ratio of 90 to 10

SEM images of samples that have been collected on aluminum foil, showed all nanofibers are electrospun regularly and without beads (Fig. 1).

Electrospinning of chitosan/PEO/cefazolin solution

SEM images showed the morphology of electrospun nanofibers of chitosan/PEO containing the different concentration of cefazolin (Fig.2). In 0.5 and 1 wt% concentration of cefazolin, the uniform nanofibers were formed on the surface of the collector. The addition of 2 and 3wt% of the drug caused formation a significant bead between the nanofibers, because there is no perfect dissolution of antibiotic in solutions and distribution of antibiotic could not produce homogenous solutions throughout the mats that contained 2 and 3wt% antibiotic. The SEM image of these nanofibers showed white beads and nanofibers diameter and the number of beads in the nanofibers structure increased in 3wt% and more of drug. We have chosen 1wt% of antibiotic for using in next tests.

The inclusion of antibiotics in the polymer solution can have some effect on the electrospinnability of the polymer and the morphology of the nanofibers, due to the changes in viscosity, surface tension, and conductivity of the solution. The determination of nanofibers diameters was achieved with “Image J” software regardless of the addition of higher amounts of antibiotics. By using this software, the diameter of at least 50 nanofibers are determined and an average diameter is calculated. The average diameter of the nanofibers containing 1wt% of the drug is 92.5 nm. This diameter is a suitable diameter for nanofibers and is lower than the other research of electrospinning of CS /PEO/1wt% antibiotic nanofibers that is a good result [22]. The morphology of nanofibers with 0.5 wt% of cefazolin was suitable and without bead but in 2 and 3 wt% of cefazolin the nanofibers cannot appear very suitable. The low diameter of  nanofiber has a good advantage such as more surface area to volume ratio, the better interaction of nanofibers with cells, more control of the humidity wound and more ability for loading the drugs in pores and finally increasing the antibacterial properties.

Antibiotic releasing

In order to investigate the releasing of nanofibers containing antibiotic, nanofibers were immersed in phosphate buffer with pH 5.5 and 37°C. In the period of time, the releasing of the drug was measured. First, it is necessary to draw the calibration curve and then, the calibration curve of cefazolin was determined by taking the absorbance of cefazolin concentrations between 0 and 2 mg/mL as parameters as well as the calibration curve was fitted to the Lambert–Beer law. A linear relationship between the cefazolin concentration (y) and the optical absorbance (x) was obtained (y =0.925x - 0.187 and R²= 0.855). All experiments were repeated three or more times and the experimental data are expressed as means ± standard error deviation. Fig.3 shows the relationship between absorption and antibiotic concentration.

To evaluate the amount of drug loaded by nanofibers, a UV-Vis spectrometer was used for analysis.

Fig.4 shows the release profile of cefazolin in time. Cefazolin wasn’t released rapidly, its delivery happened moderately during the two days. In the early times, most of the drug releasing from nanofibers containing 1wt% antibiotic, was approximately 26% and increased in the next times. This release continued until around two days. Probably the connection between drug and chitosan is hydrogen bonds and maximum releasing of nanofibers are about 65% and then remain constant for the next time.

This releasing template can be attributed to the porous structure of prepared nanofibers that cause to penetration of buffer to the nanofibers pores thus the releasing process occurs slowly. Moderate delivery of active substance during some days results in better healing and curing of the wound.

The release of the agent in an aqueous environment was found to follow a biphasic profile: an initial burst release followed by a much slower process thereafter. The high burst release can be ascribed to two reasons. First, the very small diameter and the high surface area in the nanofibers provide short diffusion pathway and are conducive to mass transfer of the drug. Second, during electrospinning, the majority of cationic drugs is likely to be localized on the surface of the nanofibers due to their ionic strength. In such a spatial arrangement, the drug can easily be dissolved and released into a solution.

FT-IR spectroscopy of nanofibers

FT-IR spectroscopy of electrospun nanofibers in a range from 400 to 4000cm^-1 at room temperature were shown in Fig. 5. Fig. 5a shows the band between 3600-3200cm⁻¹ assigned to the stretching mode of the O-H and N-H bonds of chitosan and O-H bond of PEO. N-H bending vibration of the NH₂ groups appears at about 1569cm^-1. Moreover, the bands located between 2960-2874 cm^-1are ascribed to the symmetrical stretching mode of the C-H bond and band in the 1243 cm^-1 is assigned to the symmetrical bending vibration of the methylene groups. The band at 1736 cm⁻¹ correspond to C=O stretching vibration of the carbonyl group of chitosan. Also, the band of 1342 cm^-1 is assigned –C–O stretching of the primary alcoholic group in chitosan. As shown in Fig. 5b, the broad band at around 3359 cm⁻¹ is assigned to the stretching mode of the O-H and N-H bonds in the chitosan and cefazolin and O-H bond in the PEO backbone. Moreover, the bands located in 1630 cm^-1 ascribed to the stretching mode of the carboxyl group in the cefazolin.

Also, as shown in Fig. 5b and 6, the absorptions bands of thiodiazole part of cefazolin are stretching vibrations of C=N (1399cm⁻¹), C–S (561 cm⁻¹) and in-plane vibrational mode involving NCS moiety (1034cm⁻¹).The other band at 920 cm⁻¹ may be related to the stretching vibration of C-C group. The band at 839 cm⁻¹ correspond to N-H stretching and bending vibration of the tetrazole ring.

Biodegradation and swelling of nanofibers

            The order of degradation of nanofibers is the loss of their structure into a polymeric mass without a specific form. Fig.7 shows SEM images of CS/PEO containing the different concentration of cefazolin nanofibers after immersion in the buffer. Neither chitosan, nor PEO can degrade in the buffer for 24 h. Electrospun nanofibers of chitosan/PEO/ 3 wt% cefazolin have been completely resolved in the buffer. This result is probably due to insertion of cefazolin into nanofibers. Cefazolin is water-soluble and could possibility increase the solubility of nanofibers within the buffer and water. So that by increasing the percentage of cefazolin disturbing of the structure can be seen more.

Also, CS and PEO are both hydrophilic polymers. The great tendency to interact with water is probably due to the increased H-bond interactions between the functional groups of the CS, PEO and cefazolin with the water molecules.

It is notable that since CS and PEO are both hydrophilic polymers which are soluble in an acidic aqueous environment, in order to apply the CS-PEO nanofibrous mats for biomedical applications, an appropriate mat with desirable wettability must be prepared.

Finally, swelling and degradation test showed that the most of degradation and swelling related to CS/PEO containing 3wt% cefazolin nanofibers, and least of degradation and swelling related to CS/PEO containing 0.5wt% cefazolin nanofibers. 

The water uptake or swelling percent of samples was calculated by using following formula, where W₀ is initial weight of the dry sample and Ws is a swollen weight of the sample at equilibrium.

Swelling percent = (Ws−W₀) / W₀100

The plot of swelling percents is presented in Fig. 8.

In vitro and in vivo testing of nanofibers

In the microbial test, the zone diameter formed around of nanofibers is considered as an index of antibacterial activity. Quantitative methods are used to determine antimicrobial minimum inhibitory concentrations (MICs). These MICs provide estimates of the susceptibility of bacteria to antimicrobial compounds. Quantitative methods that require measurement of zone diameters provide reproducible estimates of the susceptibility of bacteria to antimicrobial compounds. One such standardized procedure requires the use of standardized inoculum concentrations.  This procedure uses paper disks impregnated with 30-mg cefazolin to test the susceptibility of microorganisms to cefazolin.

The size of the zone is indicative of the level of antibacterial activity in the nanofibers and is affected by the potency of the antibacterial agent, the amount that has been leached into the agar and the rate of release. However, the zone of inhibition should not be expected if the antibacterial agent in the sample cannot diffuse into the agar, such as the inherently antibacterial chitosan nano membranes, or textiles on which the antibacterial agents have been durably attached. This zone of inhibition method has been used to examine the antibacterial activity of electrospun nanofibers loaded with an antibiotic.

The antibacterial study of chitosan/PEO/1wt%cefazolin nanofibers (Fig.9) shows that this scaffold has the antibacterial effect and inhibition zone was created in the presence of both of Gram-negative and Gram-positive bacteria. This means that cefazolin destroyed both type bacteria membranes but cefazolin inhibitory effect on the growth of Gram-positive bacteria is more than that of Gram-negative bacteria.