In vitro evaluation of PVA gels loaded with Copaiba Oil and Duotrill

*duarteap@gmail.com Obstract Enrofloxacin can be slowly delivered through polymeric systems and the addition of oil could increase the polymeric gels hydrophobicity and help the continuous release. The present work intended to develop and characterize microstructurally (XRD and FTIR) and in vitro (swelling and antimicrobial tests) the PVA hydrogels loaded with copaiba oil and Duotrill (enrofloxacin) to treat bacterial infections, as pyelonephritis, in the veterinary field. Duotrill  and oil combined diminished the gels degree of crystallinity and it was observed interaction between phases due to a new band found only in PVA hydrogels loaded with copaiba oil and Duotrill (PVA-D-O) FTIR spectrum. The samples with oil swelled less than samples without it, where copaiba oil altered the samples’ hydrophilicity. PVA-D-O presented lower weight loss and higher gel fraction than PVA, indicating the loaded material increased the gels stability. All samples containing oil and Duotrill 


Introduction
Pyelonephritis is a name used to describe an inflammatory process of the pelvis and renal parenchyma originated by bacterial infections all over the lower urinary tract. These bacterial infections are generally caused by aerobic bacteria, e.g. Escherichia coli and Staphylococcus sp., and rarely by species of Proteus, Streptococcus, Klebsiella and Enterobacter [1,2] . There is a wide variety of antibiotics to treat bacterial diseases in animals, specially dogs and cats. Consequently, there is also an increase of intoxication due to the incorrect use (overdose) of this medicine. Some cases, the drugs' collateral effects and toxins could lead to death [3] . One possible alternative to avoid poisoning is the use of drug delivery systems (DDS), e.g. hydrogels [4] . [5] developed polyvinyl alcohol (PVA) hydrogels (3D networks of hydrophilic polymers) with structural integrity by physical crosslinking [5] , using freeze-thawing method [6] . The development of PVA hydrogels by freeze-thawing are based on the polymer's hydroxyl groups, which form crystallites through of intra-and inter-chain hydrogen bonding [7] .

Stauffer and Peppast
The PVA physical hydrogels are biocompatible, stable at room temperature, ease form film by solution casting and suffer natural biodegradation under physiological conditions [7][8][9] Biodegradation or erosion mechanism of PVA physical hydrogels is essential to drug delivery by implantable biomaterials [9]. Among PVA gels used as DDS, there are: Jensen et al. [9] , who developed gels with high potential for drug delivery through spontaneous erosion [9] ; Marques [10] studied hydrogels loaded with ibuprofen and obtained gels with excellent mechanical propriety and an efficient controlled ibuprofen delivery [10] . delay the microbial activity, due to phenolic and terpenoids groups to which is attributed their antimicrobial activities [11] . Brandelero et al. [12] added copaiba and lemongrass oils directly to starch-polyvinyl alcohol-alginate device, which presented improved antimicrobial properties [12,13] . Kavoosi et al. [14] developed gelatin/PVA hydrogel loaded with Zataria essential oil (ZO) for wound-dressing, obtaining increased the antimicrobial activities due to the addition of ZO, which also decrease the films' swelling ability. ZO probably contributed to the gels hydrophobicity due to its characteristics [12,14] . The essential oils have a nature hydrophobic due to substances that stimulate create region non-polar in the polymeric matrix. This efficiency is linked with rate of proportion between hydrophilic and hydrophobic of film, and with characteristics of the compounds added such as polarity or structural chemical [12,15] .
Copaiba oil, obtained from copaiba trees (Copaifera sp., Fabaceae), is a natural antimicrobial agent native from western Africa and South America (specifically from the Amazon, north of Brazil). The Amazon indigenous people use copaiba oil for treatment of various diseases, e.g. stomach ulcers and tonsillitis [16][17][18] . Antimicrobial studies of the copaiba oleoresin found its high potential as medicine [19] . Sachetti et al. [16] observed that the oleoresin did not cause negative effects (toxicity) to rats, but further studies are necessary [16] . Copaiba oil could add the antimicrobial property and hydrophobicity to PVA hydrogels.
Among the hydrophilic drugs that could be added to PVA, there is Duotrill (commercial name), an enrofloxacin based antibiotic [20] . The enrofloxacin is a fluoroquinolone used in the veterinary medicine. According to Vancutsem et al. [21] the fluoroquinolones is efficient in the treatment of bacterial diseases in several animals, including birds, except to juvenile dogs and horses, since it effects their cartilage [21,22] . The enrofloxacin is excellent to treat pyelonephritis, since this antibiotic has a wide spectrum of action against Gram-negative (E. coli, Pseudomonas sp and Enterobacter sp) and some Gram-positive bacteria (Streptococcus sp and Staphylococcus sp) and Mycoplasma and Chlamydia [2,20,22] . Enrofloxacin can be loaded to polymers to be slowly delivered, e.g. enrofloxacin loaded to Poly(3-hydroxybuty rate-co-3-hydroxyvalerate) (PHBV) microspheres showed delivery for 13 days and when inserted intramuscular, it was detected in rats' blood for 3 days. Nonetheless it could be inferred that the therapeutic concentration was maintained for long periods when enrofloxacin was delivered through PHBV microspheres [23] .
The goal of present work was to develop and characterize microstructurally and in vitro the PVA hydrogels loaded with copaiba oil and Duotrill (enrofloxacin) intended to treat bacterial infections in the veterinary field.

Materials
Polyvinyl alcohol -PVA, M w 85000-124000 Da and degree of hydrolysis 99%, was purchased from Sigma Aldrich. The copaiba oil, natural product, was obtained commercially from Ashram Aquarius. Ethyl alcohol, 95% purity, was purchased from Vetec. The Duotril  (drug) was obtained commercially from Laboratory Duprat in Brazil. All reagents described were used without further purification.

Preparation of the samples
The method employed to preparation of four samples distinct was based on Oliveira et al. [4] The PVA pristine and PVA hydrogels containing duotril, oil and both oil and duotril were labelled PVA, PVA-D, PVA-O, PVA-D-O, respectively. PVA aqueous solution (10% w/v) was prepared by dissolution in 90°C for 4h, under mechanical stirring and it was named 'PVA'. The duotril was dissolved in distilled H 2 O at room temperature, under magnetic stirring and mixed to PVA solution. This sample was named 'PVA-Duotril' (PVA-D). The copaiba oil was associated to ethyl alcohol (molar ratio 1:1) at room temperature under magnetic stirring and after mixed to PVA solution. This sample was named 'PVA-Oil' (PVA-O). The samples composition is displayed in Table 1.
The duotril and the copaiba oil were mixed to PVA solution when it reached room temperature under stirring and it was named 'PVA-Duotril-Oil' (PVA-D-O). 10 mL of each final solution were poured in petri dishes (diameter of 90 mm), and the samples were freeze-thawed (1 cycle of 16 h at -16°C and 30 min at 25°C followed by 4 cycles of 1h at -16°C and 30 min at 25°C). The samples were dried in room temperature afterwards.

Microstructural analysis
Microstructural characterization of dry samples was performed using Fourier Transform Infrared Spectroscopy (FTIR, PerkinElmer equipment, Spectrum 100 (COPPE/UFRJ), in the ATR mode, wavenumber range of 4000 cm -1 and 600 cm -1 , 32 scans per samples and a spectral resolution of 4 cm -1 ) and X-ray diffraction analysis (XRD, Bruker-AXS D8 Advance Eco diffractometer (CETEM/UFRJ), Cu kα radiation (40 kV/25 mA), in the 2Ɵ angle range of 10º -60º, with a 0.01º step size and a position-sensitive Lynx Eye XE detector with energy discrimination). The degree of crystallinity (Xc) of the samples evaluated by XRD was based on the area of crystalline peaks per area of crystalline and amorphous phases [24] .
The W S is the samples' weight at each interval time. Whereas, W D is the dry weight prior to swelling test and W DS is the dry weight after swelling [25][26][27].
Antimicrobial activity of hydrogels was evaluated according to standard ASTM E2180-07 with some changes, using Staphylococcus aureus. In the initial step, a cell suspension of S. aureus (ATCC 6538) was prepared, adjusting the turbidity on the MacFarland scale to 5, that is equivalent to 10 8 colony forming units per mL (CFU/mL). Afterwards, one (1) mL of this suspension was diluted in 100 mL of agar paste to obtain concentration of 10 6 CFU/mL. The samples were placed on 24-well plates and each sample was added of 200 microliters of agar inoculated paste. The plates were incubated at 30°C for 24h. Thereafter, incubated samples were moved to Falcon tubes and it was added 1,8 mL of buffer solution. Subsequent decimal dilutions were prepared up to 10 -4 and S. aureus survivability was evaluated on PCA agar using the micro-drop plate technique.

Statistical analysis
The statistical analysis was performed using the one-way ANOVA analysis and Tukey test. The ANOVA one-way analysis, 95% significance level, was used to evaluate the parameter amount of drug and/or oil, with four levels: PVA, PVA-Drug, PVA-Oil and PVA-Oil-Drug. The gels' swelling capacity, weight loss and gel fraction were used as response data. Tukey test, α=0.05, was conducted to determine if the difference between each pair was significant.
Samples PVA-D-O displayed the main bands of PVA and copaiba oil, similar to sample PVA-O, although the bands intensity varied (Figure 1(d)). It was expected to observe high intensity of the bands between 3400-2900 cm -1 and 1800-1100 cm -1 with the addition of enrofloxacin [37] . This effect was not observed, but there is a band at 871 cm -1 in PVA-D-O sample, that was not observed in PVA, PVA-D or PVA-O samples, indicating a possible interaction between of copaiba oil and Duotrill  .
The XRD analysis revealed probable overlapped peaks and they were deconvoluted to distinguish the crystalline amount from the amorphous one, Figure 2. The addition of copaiba oil to PVA altered the XRD spectrum, although the main peak of all spectra is at 2θ ~39°, a wide peak that apparently is the overlap of different peaks (peaks at 2θ = ~32°, ~39° and ~47°). The main PVA peak (2θ ~20°) was not identified in the samples [38] . Nonetheless, the addition of Duotrill  led to the presence of another peak at 2θ ~19°, which could be related to the main enrofloxacin peak at 2θ ~25° [ 39,40] .
The XRD curves deconvolution, Figure 2, revealed the peaks at 2θ = ~32°, ~39° (both probably related to crystalline phase) and a wide peak at 2θ = ~47° (possibly related to the amorphous phase). The addition of copaiba oil or duotril to PVA altered the chains packing (decreasing the Xc of the samples), but the addition of both revealed a considerable synergic effect on the samples Xc.
The presence of enrofloxacin peak in the XRD spectra of samples could indicate incomplete incorporation of the drug in the PVA gel [41] , or even simple physical presence of the loaded material between PVA chains [42] . Although the position of the peaks remained similar after loading, altering the samples Xc could indicate interaction between the materials [43] . Duotrill  and oil combined diminished the gels degree of crystallinity and there is an interaction between phases revealed by a new band found only in PVA-D-O sample's FTIR.

In vitro analysis
The swelling tests revealed the all samples swelled at least 180%. There was a peak of media uptake at the onset of all curves and the equilibrium swelling degree (ESD) was reach after 1 day of immersion, Figure 3. The condition to occur the ESD is when the swelling forces (media entrance stretches the network) and elastic forces of the network (chains relaxation and crosslinking are responsible for a partial network's contraction) reach the equilibrium [44] . PVA gels usually reaches ESD at 37°C of approximately 400% and at least 300% [4] . The samples in this work presented relatively low ESD (evaluated at room temperature). The temperature could have influenced the ESD, increasing it when evaluated at 37°C [4,25] . The ANOVA   analysis on the ESD revealed that samples with oil swelled less than samples without it (p < 0.05). Thereby, copaiba oil alters the samples' hydrophilicity due to its hydrophobic characteristic / substances [45] . The PVA samples weight loss was higher than the PVA-D-O weight loss, as well as PVA presented lower gel fraction than PVA-D-O(p < 0.05), Figure 3 (b). It seems that the PVA chains presented lower mobility and higher inter / intra-connections in PVA-D-O samples. This is an indication that Duotrill  and copaiba oil combined increased the structural stability of the PVA gels [12] . Table 3 shows the results obtained by inhibition of S. aureus. The samples loaded with oil reduced the S. aureus proliferation considerably, but the highest values of inhibition (total inhibition) was observed in samples containing Duotrill  . Classically, copaiba oil [46] , as well as enrofloxacin [47] , inhibits S. aureus, although some organisms could develop resistance to enrofloxacin [48] . The gels of the present work seem to have incorporated 'oil' and Duotrill  presenting activity against S. aureus.

Conclusions
Duotrill  and copaiba oil combined diminished the gels degree of crystallinity and there is an interaction between phases revealed by a new band found only in PVA-D-O sample's FTIR. The samples with oil swelled less than samples without it, where copaiba oil altered the samples' hydrophilicity. PVA-D-O presented lower weight loss and higher gel fraction than PVA, indicating the loaded material increased the gels stability. All hydrogels containing copaiba oil and Duotrill  reduced S. aureus load, but the combination of both did not result in a greater reduction. Gels loaded with copaiba oil or Duotrill  are potential materials to treat bacterial infections in the veterinary field.