An analysis of the microencapsulation of ceftiofur in chitosan particles using the spray drying technology

Carbohydrate Polymers 234 (2020) 115922

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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

An analysis of the microencapsulation of ceftiofur in chitosan particles using the spray drying technology

T

Ignacio M. Helblinga,*, Carlos A. Busattoa, Federico Karpa, Germán A. Islanb, Diana A. Estenoza, Julio A. Lunaa a b

INTEC (Universidad Nacional del Litoral – CONICET), 3450 Güemes, 3000, Santa Fe, Argentina CINDEFI (Universidad Nacional de La Plata – CONICET), 50 and 115, 1900, La Plata, Argentina

A R T I C LE I N FO

A B S T R A C T

Keywords: Ceftiofur Spray drying Microencapsulation Chitosan Drug delivery Mathematical modeling

Ceftiofur is a third-generation cephalosporin approved to treat numerous infections in production animals. Its commercial formulations are administered daily due to the mean life time, leading to several inconveniences, like operative challenges and non-uniform plasma levels. The objective of this work was to microencapsulate ceftiofur in chitosan particles using spray drying technology to extend the delivery and consequently reduce the dosage frequency. The effect of formulation factors on particle features was studied using a multilevel factorial design. In addition, ceftiofur thermal stability was assayed by differential scanning calorimetry and microbiological assays. Finally, a pharmacokinetic model was developed to predict theoretical plasma concentration in goats. Results showed that ceftiofur thermal stability increased after microencapsulation, indicating a protective effect of chitosan particles. Besides, MIC, IC50 and inhibition halos against E. coli and S. aureus were similar than those of the commercial product. In addition, suitable plasma levels can be theoretically maintained in goats during 48 h with a single injection. These findings suggest that chitosan microparticles could be a good vehicle for ceftiofur administration.

1. Introduction Infectious diseases are one of the most serious problems in animal breeding. They can cause a reduction in production yields and might lead to the animal’s death in the most severe cases, generating large economic losses. Hence, sanitation programs using antibiotics have been developed to treat and prevent these diseases. Treatments usually require dosage forms that generate an adequate drug concentration in the focus of infection during a sufficient period of time to inhibit the microorganism growth. This concentration is known as minimum inhibitory concentration (MIC) and the interrelation between MIC and the spatial and time variables is crucial to eliminate the pathogens and to achieve the success of the treatment. Ceftiofur (CTF) is a third-generation cephalosporin approved by the US Food and Drug Administration (FDA) to treat several of these infectious diseases. For example, this antibiotic has been successfully used to treat respiratory diseases in cattle, pigs and chickens (FDA, 1992), and in horses (Folz et al., 1992). In addition, CTF has been used for the

treatment of mastitis in dairy cattle (Cortinhas, Tomazi, Zoni, Moro, & Veiga Dos Santos, 2016; Oliver et al., 2004). Besides, it has been reported its administration in cattle, pigs, sheep, horses, and goats with different infections (Brown et al., 2000; Drillich et al., 2001, 2006; Galvao, Greco, Vilela, Sa Filho, & Santos, 2009; Hurtgen, 2006; Salmon et al., 1995; Schukken et al., 2011; Scott, Schouten, Gaiser, Belschner, & Jordan, 2005; Tang et al., 2010; Witte, Bergwerff, Scherpenisse, Drillich, & Heuwieser, 2010). The commercial formulations of CTF typically contain the antibiotic dispersed in an oily vehicle and they are administered by injection. The mechanism of action is based on the inhibition of the synthesis of the bacterial cell wall. Briefly, the antibiotic binds to the penicillin-binding proteins inhibiting the synthesis of peptidoglycans that constitute the cell wall, and hence promotes the subsequent bacterial lysis (Hornish & Kotarski, 2002). In general, CTF is considered a broad-spectrum antibiotic exhibiting high activity against Gram positive and Gram-negative bacteria (Drillich et al., 2006; Galvao et al., 2009; Guglick et al., 1998; Prescott, 2002; Witte et al., 2010). Moreover, aminothiazole and metoximin groups of ceftiofur confer it a

Abbreviations: CTF, ceftiofur; CHT, chitosan; DSC, differential scanning calorimetry; FDA, US Food and Drug Administration; FTIR, fourier-transform infrared spectroscopy; IC50, half maximal inhibitory concentration; MIC, minimum inhibitory concentration; MPs, microparticles; PHBV, poly(3-hydroxybutyrate-co-3-hydroxyvalerate); PLGA, poly lactic-co-glycolic acid; TPP, sodium tripolyphosphate; 2FN, two-fluid nozzle; 3FN, three-fluid nozzle ⁎ Corresponding author. E-mail address: [email protected] (I.M. Helbling). https://doi.org/10.1016/j.carbpol.2020.115922 Received 19 September 2019; Received in revised form 24 January 2020; Accepted 26 January 2020 Available online 31 January 2020 0144-8617/ © 2020 Elsevier Ltd. All rights reserved.

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objective of the present work was to investigate the microencapsulation of ceftiofur using the spray drying technology and chitosan as polymer to prepare the MPs. The spray drying technology involves a single step minimizing the consumed time. In addition, it can be easily scale up using bigger dryer apparatus or different equipment operated in parallel. This process has been widely used in the pharmaceutical industry to transform liquid feed into dry solid powder in a one-step continuous process (Harsha et al., 2017). By other hand, chitosan was selected due to its interesting properties, like biocompatibility, biodegradability, non-toxicity, antimicrobial activity, and its relatively low cost. In the present contribution, MPs were prepared by the spray drying method and optimal operative conditions were determined. The resulting particles were characterized in terms of particle size, morphology, water content, encapsulation efficiency and in vitro drug release behavior. The effect of formulation factors on particle features was studied using a multilevel factorial design. In addition, ceftiofur thermal stability after microencapsulation was assayed by differential scanning calorimetry (DSC) and microbiological tests with E. coli and S. aureus. Finally, a pharmacokinetic model was developed to predict theoretical plasma concentration in goats.

great affinity for the penicillin-binding protein and a great stability against beta-lactamase enzymes respectively, enhancing its microbicide activity and effectiveness compared to other antibiotics (Klein & Cunha, 1995; Yancey et al., 1987). Despite its efficacy, several inconveniences are associated to the actual therapies with ceftiofur in production animals. For example, the commercial formulations need to be administered daily during the period of sanitation since CTF has a short mean life time. It has been reported that the mean life time of CTF is about four hours in sheep, horses and goats, and seven to twelve hours in cattle (Cervantes, Brown, Gronwall, & Merritt, 1993; Courtin, Craigmill, Wetzlich, Gustafson, & Arndt, 1997; Craigmill, Brown, Wetzlich, Gustafson, & Arndt, 1997; Whittem, Freeman, Hanlon, & Parton, 1995). Therefore, formulations are injected once a day to maintain plasma concentrations between the therapeutic window during around five and twelve days for acute and severe diseases, respectively. This dairy administration represents a great challenge with operative complications when a large number of animals are involved. Another drawback of the dairy dosage is the resulting non-uniform concentration profile, presenting maximum and minimum values at early and largest times, respectively. Undesired effects have been reported due to the high plasma concentrations achieved shortly after injections (Ahmed & Kasraian, 2002; Sun, Scruggs, Peng, Johnson, & Shukla, 2004). By other hand, concentrations lower than MIC can occur between two consecutive injections at larger times, which could compromise the therapy effectiveness. With the goal of overcoming the aforementioned inconveniences, biodegradable and biocompatible microparticles (MPs) have been evaluated as delivery system for ceftiofur. The use of biodegradable MPs has several advantages. Firstly, it permits the sustained release of the antibiotic at effective levels for longer times allowing to reduce the administration frequency and avoiding the operative complications. Secondly, the controlled release generates a more homogeneous profile after injection minimizing the undesired effects and enhancing the effectiveness of the therapy. By last, a protective effect might occur after microencapsulation increasing the antibiotic stability. Different researches were conducted to evaluate the microencapsulation of ceftiofur in biodegradable particles. For example, Hao et al. encapsulated CTF in gelatin MPs prepared by an w/o emulsion method and studied the effect of formulation variables on particle features (Hao et al., 2010; Hao, Wang et al., 2013; Hao, Wu et al., 2013). These authors further investigated the preparation of poly (lactic-co-glicolic acid) (PLGA) and gelatin MPs and their pharmacokinetics in beagle dogs (Hao, Wang et al., 2013; Hao, Wu et al., 2013). In this study, MPs with an average diameter between 5−35 μm were administered via intravenous injection into the animals and the results showed that longer times with plasma levels between the therapeutic windows were achieved when PLGA MPs were used. By other hand, Vilos et al. prepared poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) MPs with an average diameter of 1−2 μm and a encapsulation efficiency of about 39 % (Vilos et al., 2012). In this work, the in vitro release experiments showed a sustained release profile during approximately 2 days. These authors also studied the pharmacokinetics, toxicity, and therapeutic activity of the PHBV MPs in rats reporting a sustained release with detectable levels above the MIC for at least 17 days (Vilos et al., 2014). In addition, Vilos et al. prepared PLGA MPs by a double emulsion w/o/w technique obtaining particles with an average size of 1.5–2.2 μm and a encapsulation efficiency of about 40 % (Vilos et al., 2015). In this research, authors assayed the MPs in rats reporting a sustained release during 20 days. Clearly, the use of MPs is an interesting alternative to the conventional formulations. However, the research carried out to date is closer to the academic area than to the industrial application. To date, CTF -loaded MPs have been prepared using only batch processes, e.g. emulsion methods. These processes have the disadvantage that they are difficult to scale up for industrial production, and are time-consuming methods with relatively low encapsulation efficiencies. Therefore, the

2. Materials and methods 2.1. Materials Chitosan (CHT, Mw = 63.97 ± 2.31 kDa, 93 % DD, Eastar Holding Group Dong Chen Co. Ltd.), sodium tripolyphosphate (TPP, 85 %, Sigma-Aldrich), sodium hydrogen phosphate (Anedra), potassium dihydrogen phosphate (Anedra), sodium chloride (Ciccarelli) and potassium chloride (Ciccarelli) were used as received. Ceftiofur hydrochloride (98 %) and myritol 318 were kindly provided to us by Zoovet Productos Veterinarios S.A. (Santa Fe, Argentina). All other reagents were of analytical grade, except for acetonitrile which was HPLC grade. All aqueous solutions were prepared with ultrapure water. 2.2. Preparation of chitosan microparticles CHT MPs were prepared by the spray drying method. Briefly, CHT solution (1–2 % w v−1) was prepared by dissolving the polymer in 1 % v v−1 acetic acid solution. Then, ceftiofur hydrochloride (0–20 % w w−1) was dispersed in the polymeric solution. Thereafter, 0.22 % w v−1 TPP solution (0.000-0.150 g per gram of chitosan) was added dropwise and stirred at 300 rpm during 10 min. Alternately, the active principle was first dispersed into propylene glycol or myritol 318 (0−1 g per gram of chitosan) and then dispersed into the polymeric solution. After homogenization, formulations were dried using a Mini Spray Dryer B290 (Buchi, Switzerland) with a standard two-fluid nozzle (2 F N) and an inner nozzle tip of 0.7 mm. Several operational conditions were evaluated varying the inlet temperature (140–160 °C), liquid flow rate (2 - 10 mL min−1), and air flow rate (11.12 - 17.53 L min−1). The compressed air pressure was 6 bar. Dry microparticles were collected in a glass collection vessel and stored at room temperature until further assays. Yields were calculated based on resulting mass. Further information about the spray dryer apparatus (scheme, operation, nozzles) and the description of the drying process can be founded in the operation manual (AG, 2016). 2.3. Preparation of coated chitosan microparticles Coated microparticles were fabricated using a Mini Spray Dryer B290 (Buchi, Switzerland) with a standard three-fluid nozzle (3 F N) with an inner nozzle tip of 0.7 mm. A CHT solution containing ceftiofur hydrochloride, myritol 318 and TPP was pumped through the central channel to form the MP core. Simultaneously, a CHT solution (1–2 % w v−1) containing TPP (0.025 g per gram of chitosan) was pumped through the external channel as coating solution using an external 2

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peristaltic pump. Formulations were dried at 155 °C, using a liquid flow rate of 10 mL min−1, and a spray air flow of 17.5 L min−1. Dry microparticles were collected in a glass collection vessel and stored at room temperature until further assays.

were calculated from the slope of the cumulative amount of CTF released vs the square root of time plots.

2.4. Microparticles characterization

Ceftiofur concentration in samples was measured using a HPLC system (Prominence LC20A, Shimadzu, Japan) equipped with a ZORBAX® Eclipse XDB-C18 column (4.6 × 250 mm, 5 μm particle size) and a UV–vis detector. The mobile phase consisted of a mixture of disodium hydrogen phosphate buffer (pH 6.8)/acetonitrile (70:30 v/v), at a flow rate of 1.0 mL min−1. The column temperature was set to 30 °C and the detection wavelength was 292 nm (Palur et al., 2013). The elution time of ceftiofur was 3.2 ± 0.2 min.

2.5. Quantitative determination

2.4.1. Particle size Dried microparticles were dispersed in isopropyl alcohol to avoid swelling and observed under an optical microscope (DM 2500 M, Leica, Germany) coupled with a camera LEICA DFC 290 HD. The average particle diameter and sphericity degree were determined using an image processing program. Approximately 300 particles per sample were analyzed. The sphericity degree was measured by the roundness factor (R), which can be calculated by the inverse of the aspect ratio (Barreiros, Ferreira, & Figueiredo, 1996; Moura, Martins, & Duarte, 2015):

A R=4 π D2

2.6. Dissolution study in aqueous environment The viscosity of supernatants from microparticles suspensions were measured to qualitatively assess the extent of MPs dissolution in acidic aqueous environment. For each sample, 25 mg of MPs were placed in 3 mL of 0.1 % v v−1 acetic acid solution and the vials were incubated at 37 °C during 24 h under orbital stirring (75 rpm). Then, samples were centrifuged for 7 min at 4000 rpm, and the supernatant was collected. Viscosity of the supernatants (0.5 mL) was measured using a Brookfield DV3TRV (cone/plate) viscometer with a CP-40 configuration. Determinations were made by triplicate at shear rate of 112.5 s-1 and 25 °C.

(1)

where A and D are the area and the diameter of particles, respectively. The roundness factor is equal to 1 when the particle is a perfect sphere. As the value departs from 1, the degree of sphericity decreases. 2.4.2. Water content and absorption The water content of microparticles after the spray drying process was calculated gravimetrically. Briefly, 20 mg of microparticles were dried at 60 °C during 48 h. Then, samples were incubated in a dissecator at room temperature during 1 h and weighed. Absorption assays were performed in order to determine the water content at equilibrium. About 20 mg of microparticles were dried in oven at 60 °C during 48 h and then incubated at 25 °C and 70 % of relative humidity during 3 days. After that, samples were weighed and water content was calculated gravimetrically. Assays were run in triplicate.

2.7. Differential scanning calorimetry (DSC) assay Samples of ceftiofur and MPs were analyzed using a Mettler differential scanning calorimeter (TA 3000 with 30 DSC module, MettlerToledo). Briefly, about 5 mg of samples was placed in an aluminum pan and heated at a rate of 10 °C per min from 0 °C to 250 °C. An empty aluminum pan served as the inert control material. Nitrogen was used as purge gas.

2.4.3. Particle morphology The morphology of microparticles was studied by scanning electron microscopy (SEM). Samples were put over an aluminum stub coated with a carbon adhesive tape, and the non-adhered sample was removed by airflow. The microparticle morphology was examined using an acceleration voltage of 15 kV in a Phenom ProX desktop scanning electron microscope.

2.8. Fourier-transform infrared spectroscopy (FTIR) analysis FTIR spectra of ceftiofur, and MPs samples were recorded using an IR Spectrophotometer (FTIR-8201PC, Shimadzu) in the range of 400–4000 cm−1 with a 4 cm−1 resolution and 40 scans per spectrum. Approximately 2 mg of samples and 100 mg of potassium bromide were dried at 110 °C during 24 h. Then, powders were blended with a mortar and the mixture was compacted using an IR hydraulic press at a pressure of 6 tons for 3 min. The resulting disks were conditioned in a desiccator until measurements.

2.4.4. Encapsulation efficiency Approximately 10 mg of microparticles were dispersed in 10 mL of methanol and stirred at 150 rpm at room temperature during 24 h. Then, samples were sonicated during 1 min and centrifuged at 2000 rpm during 2 min. Ceftiofur concentration in the supernatant was quantified by the HPLC technique described in Section 2.5. Experiments were run in triplicate. Encapsulation efficiency (EEf) was calculated as follows:

EEf = 100

2.9. Application test Formulations M19 and M21 were dispersed in 5 mL of both pH 7.4 saline solution and myritol 318, in a glass vial. Then, vials were vigorously agitated and the macroscopic condition was observed. Finally, a 3 mL syringe with a concentric, luer lock tip (SS + 03L1, Terumo) was filled with the dispersions and thereafter, the fluids were uploaded in an empty glass vial to assess the ease of application. Assays were conducted by triplicate.

Aexp Atheo

(2)

where Aexp and Atheo are the experimental and theoretical CTF loading in the MPs, respectively.

2.10. Microbiological studies

2.4.5. In vitro release assays Approximately 20 mg of CTF-loaded MPs were dispersed in 50 mL of 1 mM phosphate buffer pH 7.4 and incubated at 37 ± 1 °C under constant stirring at 75 rpm. At predetermined times, aliquots of 4 mL were taken and replaced with fresh medium to maintain constant volume. CTF concentration in samples was measured by the HPLC method detailed in Section 2.5. The commercial product Cefafur® (Zoovet Productos Veterinarios S.A., Argentine) was used as control for comparison purposes. Assays were conducted by triplicate. Release rates

2.10.1. Bacterial strains Escherichia coli ATCC 25922 and Staphylococcus aureus (ATCC 6538) were used for susceptibility tests according to the Clinical and Laboratory Standards Institute (CLSI) for the measurement of inhibition zones and broth dilution MICs. In all cases, the inoculum was prepared by suspending five representative colonies obtained from an 18-20-h culture on Mueller-Hinton agar medium in sterile physiological solution 3

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significant when p-value was lower than 0.05. Additionally, in vitro delivery profiles were quantitatively compared using the difference factor (f1) and the similarity factor (f2). The procedure for calculating these factors can be founded in the literature (Costa & Sousa Lobo, 2001; Helbling et al., 2018). Usually, profiles with values of f1 lower than 15 (0–15) and f2 higher than 50 (50–100) can be considered equals.

and adjusting the cell density to the 0.5 McFarland scale. 2.10.2. Broth dilution tests The minimum inhibitory concentration (MIC) and the half maximal inhibitory concentration (IC50) for E. coli and S. aureus was determined by the microdilution plates test. Briefly, 100 μL of Mueller-Hinton medium were placed in a 96-well plate and successive dilutions (1/2) were done for the antibiotic and microparticles samples. MPs were dispersed in sterilized ultra-pure water, and diluted with MuellerHinton medium to obtain a range of concentrations equivalent to free ceftiofur (0.006–12.500 μg mL−1). For comparison purposes, solutions of the antibiotic in DMSO/water mixture were prepared and tested. Finally, 100 μL of a 1/100 diluted inoculum (previously adjusted to 0.5 Mc Farland) were added to each well. Growth controls (positive control) and sterility control (negative control) for medium were also run. The microdilution plates were incubated at 37 °C for 24 h and the absorbance at 600 nm was measured in a microplate reader TECAN Infinite 200 PRO. All experiments were performed in triplicate.

2.12. Mathematical modeling A pharmacokinetic model to predict ceftiofur plasma concentration in animals was developed. The CFT concentration in plasma can be calculated by difference between the amount released from the system and the rate of metabolization in the animal’s body. Hence, a global mass balance can be written:

∂C ∂D ∂M = − ∂t ∂t ∂t

(3)

where dC/dt, dD/dt and dM/dt represent the temporal change of CTF concentration in plasma, the release rate from the delivery system and the metabolism rate in the animal’s body, respectively. CTF metabolism can be described by the Hill equation. Taking into account the total volume of distribution, Eq. (3) can be re-written as:

2.10.3. Agar diffusion tests (cylinder-plate method) Inhibition halos against E. coli and S. aureus were determined by a modified disk diffusion method according to international clinical standards (CLSI/NCCLS), replacing disks for sterile glass cylinders (8 × 6 × 10 mm3 of external and internal diameter, and length respectively). Briefly, the surface of a sterile Mueller-Hinton agar plate was inoculated with the previously prepared inoculums using a sterile cotton swab. The glass cylinders were then placed on the agar plate. MPs were dispersed in sterile ultra-pure water at an equivalent concentration of 10 μg mL−1 of ceftiofur and 50 μL of these samples were placed inside the cylinders. For comparison purposes, cefafur® and solutions of the antibiotic in DMSO/water mixture at the same concentration (10 μg mL−1) were prepared and tested. Agar plates were incubated at 37 °C for 24 h and the sizes of the resulting inhibition zones were appropriately measured. Ultra-pure water and a mixture of DMSO/water were used as negative control in the corresponding tests. Assays were run by triplicate.

∂C M ∂FDL Vm Ch = tot − ∂t Vd ∂t Vd (K h + Ch)

(4)

where C is the CTF concentration in plasma, t is the time, Mtot is the CTF load in the delivery system, Vd is the total volume of distribution, FDL is the fraction of drug released from the delivery system, Vm is the maximum rate of metabolism, K is a constant including for interaction factors, and h is the Hill coefficient. Integration of Eq. (4) over time allows to estimate the concentration of CTF in plasma. A similar approach was developed for progesterone in our previous work (Helbling et al., 2018). Note that Eq. (4) is a general equation valid for any delivery system, for example liquid formulations, microparticles, hydrogels, implants, among others. When the delivery system is a drug dispersion administered by intravenous injection, a particular case can be obtained. In this situation, the total amount of CTF is available in plasma from the beginning of the experiment and hence dFDL/dt is equal to zero, simplifying Eq. (4) to:

2.10.4. Viability assay against bacterial biofilms with the Live/Dead BacLight® kit For the microbiological assays, E. coli (EC) or St. aureus (SA) growing at late exponential phase were inoculated in a soft nutrient agar previously thermostated at 40 °C. A drop of around 20 μL was placed on the surface of a glass slide, followed by incubation for 24 h to allow biofilm formation. MPs were dispersed in sterile ultra-pure water at an equivalent concentration of 297 μg mL−1 of CTF. Then, 20 μL of these samples were added to cover the resulting biofilms and incubated for 1 h. Samples were carefully washed with sterile ultra-pure water and bacterial viability was then monitored using the LIVE/DEAD BacLight® kit (Thermo Fischer Scientific) according to the manufacturer´s instructions. The commercial kit is composed of two fluorescent dyes, SYTO9® (green) and propidium iodide (red). A mixture of both dyes was prepared in equal proportions dissolving 0.75 μl of each one in 0.5 mL of sterile ultra-pure water, and applied onto the entire biofilm and held in darkness for 20 min. Fluorescence from both live and dead bacteria was recorded using a Leica DM 2500 epifluorescence microscope (Leica, Germany) equipped with UV filters (495–505 nm). Living bacteria were observed green-colored by U-MWG2 filters (excitation between 510 and 550 nm and emission at 590 nm) while dead bacteria were observed red-colored with U-MWB2 filters (excitation 460 and emission 490−520 nm) [17].

∂C Vm Ch =− ∂t Vd (K h + Ch)

(5)

Eq. (5) can be used to calculate the metabolic parameters of Hill equation. Eqs. (4) and (5) were implemented in a MatLab® routine and solved using the ode23 MatLab® function. Typical computing time was less than 1 min per simulation. 3. Results and discussion 3.1. MPs preparation by the spray drying method CFT-loaded CHT MPs were prepared by the spray drying technology using the apparatus presented in Fig. 1.a. First of all, preliminary studies were conducted to determine the optimal operational conditions. Optimal values of 155 °C ± 2 °C, 10 ± 1 mL min−1, and 17.5 ± 0.5 L min−1 were observed for inlet temperature, liquid flow rate, and spray air flow, respectively. It is known that operational conditions affect the drying process. For example, if the flow rate of the polymer solution is too high compared with the gas flow, then the solution cannot be completely dried and a wet powder is obtained. On the other hand, low inlet temperatures usually require slow solution flow rates to achieve a successful drying. Therefore, optimal conditions determination is useful to optimize the fabrication process. Overall, the fabrication process was simple and fast, and involved only one step. MPs were collected as a dry powder ready to use or store.

2.11. Statistical analysis Media comparisons were made by t-tests in the commercially available Statgraphics software package (v16.1, Statgraphics Technologies Inc., USA). Results were considered to be statistically 4

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Fig. 1. CFT-loaded CHT MPs fabrication. (a) Mini Spray Dryer B-290. (b) 2 F N used to prepare uncoated MPs. (c) 3 F N used to prepare coated MPs. (d) SEM image of uncoated MPs. (e) SEM image of coated MPs.

aqueous media. More details about the ionic bonds formation in CHT structures can be consulted in the literature (Berger et al., 2004). For this study, uncoated particles were used with a CTF content of 8 % w w1 and without additives. From Table 1, it can be observed that CTF loading efficiency decreased with the increment of crosslinker agent (ttest, p-value < 0.05). The particle size and roundness also decreased as the TPP content raised (t-test, p-value < 0.05). These results could be due to the formation of the ionic bounds during the drying process which results in particles with less swelling degree, possibly reducing their average size and the amount of antibiotic that can be loaded. At this respect, it was reported that an increase in crosslinking density induces a decrease in the swelling of CHT MPs, improving the stability of the network and decreasing the drug delivery (Berger et al., 2004). In addition, the use of TPP led to the formation of CHT MPs with more irregular shapes due to the ionic interaction (Shu & Zhu, 2000). A similar result was observed in our previous work, where CHT MPs crosslinked with TPP were prepared by the spray drying method to encapsulate progesterone (Helbling et al., 2018). In that work, we observed that more irregular particles were obtained as crosslinking density increases, possible due to the ionic bonds formation that acts as fixed points deforming the shape of the MPs. Finally, no clear trend was observed for the yield (t-test, p-value > 0.05). The next step was to evaluate the incorporation of additives. For this study, uncoated particles were used with a CTF content of 8 % w w−1 and a TPP content of 0.05 g g-1 CHT. When no additive was used, numerous agglomerates of CTF were observed dispersed in the chitosan solutions. These agglomerates occasionally clogged the dryer nozzle generating inconveniences during MPs fabrication. In addition, a low loading efficiency was obtained with an average value of 50.55 %. Moreover, these agglomerates might rise the heterogeneity in the drug distribution within particles. On the contrary, the dispersion of CTF in the CHT solutions enhanced when propylene glycol or myritol 318 was added. No agglomerates of antibiotic were observed when these additives were employed. Besides, the loading efficiency of CTF improved up to more than 80 % (t-test, p-value < 0.05) and the additives did not affect the particles size, roundness, nor yield in the range of concentrations studied (t-test, p-value > 0.05). These findings suggest that propylene glycol or myritol 318 are useful to appropriately disperse the antibiotic in the polymeric solution before the drying step. A suitable

Their average water content after fabrication was 15.17 ± 0.96 % and the water absorption at equilibrium was 16.93 ± 1.26 %. Using the optimal conditions previously determined, production ranged between 94−247 mg min−1. By other hand, uncoated and coated MPs were prepared using two different nozzle type identified as 2 F N and 3 F N, respectively. Photographs of these nozzles are presented in Fig. 1.b and. c. An external pump was required when the 3 F N was used. In addition, examples of SEM images of uncoated and coated MPs are presented as an illustration in Fig. 1.d and. e, respectively. As it can be observed, both type of particles presented spherical morphology and their surfaces were smooth without appreciable roughness or pores. Besides, their dimensions did not differ significantly. Furthermore, CTF encapsulation efficiency up to 100 % were achieved. These results support the idea of employing the spray drying technology to prepare MPs containing the antibiotic. Clearly, this method is simpler and faster than the emulsion technique, and it can be scale up easily changing the apparatus or operating several equipment in parallel mode. 3.2. Multilevel factorial design A multilevel factorial design was conducted to identify possible effects of formulation factors on particle features. Uncoated particles were used when analyzing the effect of ceftiofur load, TPP content, type of additive, and myritol content. Also, when a formulation parameter was studied, all other parameters remained constant during MPs fabrication. Results are presented in Table 1. First of all, the CTF content was studied varying the theoretical loading from 0 to 20 % w w−1. For this study, uncoated particles were used with a TPP content of 0.05 g g−1 CHT and without additive. Results showed that loading efficiency improved as the initial amount of antibiotic was increased, but the yield decreased (t-test, p-value < 0.05). No clear trend was observed for the particle size and the degree of sphericity, suggesting that the average diameter and roundness do not vary significantly with the initial content of antibiotic (t-test, pvalue > 0.05). Secondly, the content of TPP was analyzed varying its load from 0.00 to 0.15 g per gram of CHT. This reagent was used as crosslinking agent since it is a non-toxic polyanion that interacts with the amino groups of the CHT chains via electrostatic forces. The interaction forms ionic bonds that avoid the dissolution of the MPs in 5

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Table 1 Multilevel factorial design to study the effect of formulation factors on particle features. Values are expressed as Mean ± SEM. Formulation parameters

Levels

Actual Ceftiofur loading (%) (n = 3)

Loading efficiency (%) (n = 3)

Average size (μm) (n = 300)

Roundness (dimensionless) (n = 300)

Yield (%) (n = 3)

Identification number

Ceftiofur content (% w w−1)

0 2 4 8 10 15 20 0.000 0.025 0.050 0.100 0.150 Without Propylene Glycol Myritol 318 0.00 0.30 1.00 0.00 1.00 2.00

– 2.31 ± 0.11 4.27 ± 0.45 8.39 ± 0.94 10.68 ± 1.03 17.28 ± 2.56 23.15 ± 3.98 21.30 ± 3.25 19.14 ± 2.58 14.60 ± 2.21 15.63 ± 2.02 13.23 ± 1.13 5.37 ± 0.75 8.33 ± 0.80

– 22.54 ± 1.07 42.14 ± 4.44 78.51 ± .8.80 95.89 ± 9.25 117.16 ± 17.36 119.89 ± 20.61 105.21 ± 16.05 95.70 ± 12.90 73.00 ± 11.05 78.15 ± 11.00 66.15 ± 5.65 53.70 ± 7.50 83.30 ± 8.00

5.87 5.21 4.56 5.53 4.38 5.86 4.54 5.93 5.42 5.12 4.82 4.51 5.28 5.73

± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.51 1.50 1.39 1.69 0.90 2.25 1.31 2.45 1.56 1.50 1.75 1.73 1.49 1.66

0.88 0.84 0.80 0.84 0.80 0.80 0.81 0.94 0.87 0.84 0.82 0.80 0.84 0.84

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.07 0.09 0.10 0.10 0.13 0.11 0.13 0.11 0.05 0.09 0.13 0.10 0.08 0.10

60.40 51.44 46.44 49.03 37.49 20.19 32.02 23.14 38.09 47.33 36.71 43.24 49.27 40.53

± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.78 2.12 1.15 1.98 1.84 2.33 4.31 1.77 3.34 3.09 2.98 3.25 2.86 4.17

M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14

8.45 ± 1.31 10.11 ± 2.52 12.59 ± 1.88 16.99 ± 1.92 22.13 ± 2.44 28.63 ± 2.03 26.21 ± 2.49

84.53 50.55 62.57 84.95 73.77 95.43 87.37

4.48 5.31 4.21 5.38 4.38 5.04 5.14

± ± ± ± ± ± ±

1.44 1.54 1.20 0.90 1.44 1.86 1.91

0.81 0.84 0.80 0.85 0.81 0.79 0.80

± ± ± ± ± ± ±

0.15 0.17 0.15 0.13 0.12 0.13 0.13

43.28 47.41 34.50 41.87 45.65 40.46 44.30

± ± ± ± ± ± ±

3.94 4.19 4.03 4.57 2.87 3.78 4.52

M15 M16 M17 M18 M19 M20 M21

TPP content (g g−1 CHT)

Additives

Myritol content (g g−1 CHT) CHT content in coating solution

± ± ± ± ± ± ±

13.10 12.60 9.34 9.60 8.13 6.77 8.30

Fig. 2. Analysis of the effect of formulation parameters on in vitro CTF delivery in aqueous medium: (a) Effect of CTF initial content. (b) Effect of TPP initial content. (c) Effect of additives incorporation. (d) Effect of myritol 318 initial content. (e) Effect of MPs coating. Error bars represent the standard deviation.

The use of polymeric coating is a common strategy in matrix-type delivery systems to reduce burst release and delay overall rate. Thus, coated microparticles were fabricated with the 3 F N with the aim of extend the ceftiofur delivery. Chitosan concentration in the coating solution was varied from 0 to 2 % w w−1. For this study, MPs were prepared with a CTF content of 20 % w w−1, a TPP content of 0.025 g g−1 CHT, and a myritol content of 1 g g−1 CHT. Results showed that loading efficiency was higher in coated particles than in uncoated particles (t-test, p-value < 0.05). Besides, coated particles presented a slightly larger mean diameter probably due to the additional polymer layer deposited on the MPs core. However, this increment was not

dispersion is desirable because it prevents the formation of drug agglomerates and results in a homogeneous distribution of the antibiotic inside MPs. Complementary, myritol content was analyzed in more detail since CTF release rate decreased during in vitro tests when this additive was employed, enabling to extend the delivery time (see Fig. 2.c). Hence, its content was varied from 0 to 1 g per g of chitosan. It was found that CTF loading efficiency improved as myritol content increased probably due to the preferential partition of the antibiotic into the oil phase (t-test, p-value < 0.05). In addition, no statistically significant effect was observed on particle size, roundness and yields (ttest, p-value > 0.05).

6

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Table 2 Supernatant viscosities of microparticle suspensions in acidic aqueous solutions. Values are expressed as Mean ± SEM. Viscosity (cP) (n = 3) Phosphate content (g g−1)

Empty MPs

CTF-loaded MPs

Phosphate content (g g−1)

M19

M21

0.00 0.05 0.15

82.85 ± 1.79 40.01 ± 1.08 6.98 ± 0.02

23.49 ± 0.10 14.61 ± 0.30 3.71 ± 0.01

0.025

17.52 ± 0.15

6.10 ± 0.03

administered to production animals, e.g. cattle, goats, and pigs. Hence, it is expected that chitosan-based MPs will allow to maintain CTF plasma levels above the threshold for longer times since they presented an extended in vitro drug release profile. This assumption must be corroborated testing the particles in animals, but it is a good start that opens the possibility of using CHT MPs as an alternative to current formulations. Based on in vitro results, formulation M21 (MPs coated with CHT 2 %) seems to be the best candidate for further studies. Complementary, formulation M19 (non-coated MPs) could be studied for comparison purposes.

statistically significant (t-test, p-value > 0.05). In addition, no clear tendency was observed for roundness and yield suggesting that coating step did not significantly affect these features (t-test, p-value > 0.05). Summarizing, the following strategies can be pointed out from the multilevel factorial design: (a) Ceftiofur dispersion can be enhanced using myritol or propylene glycol; (b) The loading efficiency can be improved increasing the content of antibiotic, myritol, or coating the MP core; (c) Particle size can be reduced by increasing the TPP content, although this has a negative effect on loading efficiency and roundness. 3.3. In vitro release tests

3.4. MPs and CTF stability evaluation Simultaneously to the multilevel factorial design, the in vitro delivery of CTF from MPs was studied in a relevant aqueous medium and results are presented in Fig. 2. As expected, delivery profiles showed that MPs with higher content of CTF released greater amount of antibiotic at same period of time (see Fig. 2.a). However, the release rates of these MPs were similar suggesting that they were not affected by the initial amount of active principle. In addition, delivery rate was slower in crosslinked particles compared to non-crosslinked ones, and it diminished with the increment of TPP content (see Fig. 2.b). This result could be attributed to the reduction of matrix swelling and the increment of the tortuosity, resulting in a small effective diffusion coefficient of the drug. Regarding the incorporation of additives, the release kinetic was not affected when propylene glycol was added to the formulation (see Fig. 2.c). In contrast, the delivery rate was slower when myritol was used suggesting a larger retention of the antibiotic inside the microparticles due to the presence of the oily phase. Moreover, the higher the content of myritol, the slower the release of CTF (see Fig. 2.d). Finally, the effect of particle coating was studied (see Fig. 2.e). The analysis showed that the commercial product Cefafur® delivered the totality of the antibiotic in approximately 1 h while uncoated MPs presented a slower release rate (t-test, p-value < 0.05). Particles coated with CHT 1 % delivered the antibiotic with a similar rate than uncoated particles (t-test, p-value > 0.05), while 2 % CHT coated MPs presented the smallest delivery (t-test, p-value < 0.05). Complementary, profile comparisons resulted in values of f1 > 15 and f2 < 50 for all formulations, taking the commercial product as the reference profile, confirming that the encapsulation of CTF in CHT MPs significantly delay the delivery in aqueous environments in comparison with the actual commercially available products. From the study, it can be concluded that delivery rate can be slowed down increasing the content of TPP and/or myritol, and coating the MP core with an appropriate layer. It is important to note that particles coated with CHT 1 % (M20) presented a similar profile than uncoated particles (M19). A complete drug release was observed at approximately 6 h, suggesting that this coat was not effective and no additional barrier effect was added. On the contrary, the drug delivery was extended to 48 h when CHT 2 % was employed to coat the MPs core (M21), resulting in an appropriate barrier effect. In addition, an important assumption could be made on the in vivo delivery of CTF from the MPs. Cefafur® releases the total amount of antibiotic in 1 h in the in vitro aqueous environment while it maintains CTF plasma levels above the therapeutic threshold of 1 μg ml−1 during 24 h when it is

It is known that crosslinked CHT chains are stable in aqueous medium, but their stability in acidic solution depends on crosslinking degree (Berger et al., 2004). Therefore, microparticle stability was assayed in acidic aqueous environment by measuring the supernatants viscosities of particle suspensions as an indicative of the polymer dissolution. A similar approach was utilized by Santa María et al. to determine the stability of alginate microparticles prepared by spray drying (Santa-Maria, Scher, & Jeoh, 2012). The results of the study are presented in Table 2. The 0.1 % v v−1 acetic acid solution used as medium had a viscosity of 1.53 ± 0.01 cP while non-crosslinked empty MPs presented the highest value indicating large polymer dissolution. As expected, supernatant viscosity decreased with the increment of phosphate content in the MPs. This could be associated to the ionic interactions between CHT chains and TPP, which act as fixed points avoiding polymer chains dissolution and thus resulting in more stable particles. In addition, this tendency was observed in both empty and CTF-loaded MPs. However, the latter showed smaller viscosity values probably related to the hydrophobic nature of the antibiotic that reduces the water intake, resulting in less particle swelling and polymer dissolution. Summarizing, this study indicates that the stability of CHT MPs can be improved increasing the content of TPP and CTF. By other hand, the viscosity of formulations M19 and M21 were measured to analyze the effect of the coating and are also presented in Table 2. Formulation M21 had a smaller viscosity than M19. Evidently, the coating delays or reduces the polymer dissolution, enhancing the stability of the MPs. Indeed, the value of 6.10 ± 0.03 cP is significantly lower than the expected viscosity of a non-coated particles with the same phosphate content (t-test, p-value < 0.05). This result suggest that the additional external polymer layer effectively improve the particle stability. Therefore, it can be concluded that microparticle stability in aqueous environment can also be improved by coating the core with an external layer of CHT. The stability of CTF after the microencapsulation process was first investigated by DSC and results are presented in Fig. 3a. The pure CTF sample exhibited two endothermic peaks corresponding to crystal melting at approximately 138 °C and 168 °C, suggesting the presence of multiple crystalline forms. In addition, this spectrum showed an exothermic peak starting at around 205 °C, which corresponds to the thermal degradation of the antibiotic. It is known that CTF is sensitive to light and temperature. When analyzing the microparticle samples, two endothermic peaks were also observed at around 139−141 °C and 153−161 °C for M19 and M21 respectively, corresponding to crystals 7

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Fig. 3. Stability assays: (a) CTF stability studied by DSC. (b) Interaction studied by FTIR. (c) Microparticle stability in aqueous saline solution. (d) Microparticle stability in myritol.

needle generating several operative inconveniences. Dispersion assays were carried out in saline and oily vehicle in order to evaluate the stability of MPs in these two mediums to determine the best condition for the dosage. Results showed that both formulations, M19 and M21, were better dispersed in myritol than in the aqueous saline solution. In the aqueous solution, numerous aggregates were formed and needle clogged several times during the fluid dispensing, possibly due to the large size of aggregates. In contrast, particles were successfully dispersed in myritol and no aggregates nor precipitates were observed, suggesting a better stability of the MPs in this vehicle. Moreover, about 250 mg of particles per mL of oil were successfully dispersed and uploaded with the syringe without needle clogging. Photographs of the dispersion of formulation M21 in the saline solution and in myritol are presented in Fig. 3c and d respectively as illustration. From the dispersion test, it can be concluded that CTF-loaded CHT MPs are better dispersed in myritol than in the saline aqueous solution and therefore, myritol is recommended as a better vehicle for the administration of the particles.

melting. This result indicates that CTF is in a crystalline form inside the MPs. With regard to degradation, formulation M19 presented the exothermic peak started at approximately 225 °C. This delay in the beginning of degradation postulate a protective effect of the CHT MPs on the antibiotic. This effect was improved when the particle core was coated with CHT 2 % as can be observed in the spectrum of the M21 sample, where the degradation disappeared almost completely in that range of temperature. These findings report a protective effect of CHT MPs on CTF, minimizing the thermal degradation of the antibiotic and extending its stability after microencapsulation, e.g. during product storage. Complementary, FTIR analysis was performed to investigate possible interactions between the drug and the polymer. Results are presented in Fig. 3b. The spectra of pure CTF showed the characteristic signals of this molecule, similar to those reported by Solar et al. (Solar et al., 2015). In addition, M19 and M21 presented very similar spectrums showing the peaks at approximately 1750, 1700, 1650 and 1550 cm−1 corresponding to the signals of carbonyl, thioester, amide, and amine groups of CTF respectively. This analyze indicates that no significant interactions occur between the polymer and the antibiotic. Microparticle-based formulations are stored as dry powder and need to be re-suspended in an adequate sterile liquid vehicle before its administration to animals. Sterilization is commonly performed by gamma irradiation (Desai & Park, 2006; Sakar et al., 2017) while dispersion is usually made in aqueous or oily vehicle. The stability of dispersions is a key factor for proper administration. Formulations need to be stable after dispersion until the time of injection into the animal’s body, since particle agglomeration or precipitation can clog the syringe

3.5. Microbiological assays The stability of CTF after the microencapsulation process was also investigated by both microdilution plates test and inhibition growth in agar plates test using E. coli and S. aureus as representative of gramnegative and gram-positive bacteria, respectively. Results are presented in Table 3. For comparison purposes, non-encapsulated antibiotic (pure CTF) and the commercial product were also tested. The absorbance values, measured at 600 nm for the sterility control and the growth

Table 3 Stability of CTF after the microencapsulation process measured by microbiological assays. Values are expressed as Mean ± SEM. Formulations

Pure CTF M19 M21 Commercial product

Escherichia coli

Staphylococcus aureus

MIC (μg mL−1) (n = 3)

IC50 (μg mL−1) (n = 3)

Inhibition halos (mm) (n = 3)

MIC (μg mL−1) (n = 3)

IC50 (μg mL−1) (n = 3)

Inhibition halos (mm) (n = 3)

0.48 ± 0.03 0.29 ± 0.04 0.78 ± 0.01 n.d.

0.21 ± 0.03 0.14 ± 0.04 0.47 ± 0.01 n.d.

14.98 20.47 14.49 11.85

0.78 ± 0.02 0.78 ± 0.03 0.78 ± 0.01 n.d.

0.25 ± 0.02 0.25 ± 0.03 0.47 ± 0.01 n.d.

13.86 15.91 14.79 11.97

8

± ± ± ±

0.53 0.92 0.12 0.63

± ± ± ±

1.17 1.76 0.29 0.54

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inhibition compared to S. aureus which is in agreement with those observed previously for MIC, IC50 and inhibition halos. Also, inhibition seems to be higher for the M19 in comparison with the M21. From microbiological tests, it can be concluded that the spray drying process does not alter ceftiofur properties, remaining its microbicide power similar to that of non-encapsulated drug. A potential application in biofilm inhibition could be attractive taking into account the capacity of these MPs to inhibit bacteria growing in situ.

control of the microdilution plates test, were 0.052 ± 0.002 and 0.678 ± 0.013 respectively for E. coli, and 0.052 ± 0.001 and 0.390 ± 0.003 respectively for S. aureus. The samples absorbance ranged between these limits. For E. coli, calculated MIC and IC50 values were smaller for M19 than for pure CTF. This means that a lesser amount of formulation was required to achieve the same effect that the non-encapsulated antibiotic. This enhanced microbicide power could be due to the antimicrobial activity of CHT. Contrary, MIC and IC50 values of M21 were bigger than those of pure CTF. This result might be related to the additional external layer that coat the particle core. This layer effectively delays the delivery of CTF as was observed before, and hence, a larger time or a more amount of MPs in the same time are required to achieve a similar effect that pure CTF. Despite this fact, these values were similar to the referential value of 0.50 μg mL−1 reported in the literature (Steeve, Prescott, & Dowling, 2013). For S. aureus, MIC values of M19 and M21 were equal than that of pure CTF, and similar to the referential value of 1 μg mL−1 reported in the literature (Steeve et al., 2013). In addition, IC50 was also similar to the referential value. These findings suggest that the spray drying process does not affect ceftiofur structure, remaining the antimicrobial activity similar to that of non-encapsulated antibiotic (t-test, p-value > 0.05). Complementary, the inhibition halos of M19, M21, pure CTF, and the commercial product are presented in Table 3 and photographs of the tests are showed in Fig. 4.a–.d as illustration. As can be observed for E. coli, the halo of M19 was bigger than the corresponding of pure CTF. This result is in concordance to the MIC and IC50 values observed previously, associated to the bacteriostatic property of chitosan that results in a synergic effect. A similar phenomena has been reported previously (Azadi, Seward, Larsen, Shapley, & Tripathi, 2012; Fei Liu, Lin Guan, Zhi Yang, Li, & De Yao, 2001; Kong, Chen, Xing, & Park, 2010). On the other hand, the halo of M21 was very close to the observed value for pure CTF and greater than that of the commercial product. For S. aureus, the halos of both MPs were almost equal to the corresponding of pure CTF and higher than the observed value for the commercial product. No inhibition halos were observed when ultrapure water and a mixture of DMSO/water were used (negative control). This study confirms the stability of CTF during the spray drying process, remaining unchanged its antimicrobial activity after the microencapsulation. Fig. 4 also shows the results obtained for the Live/Dead BacLight® kit (see Fig. 4.e–.h). As it can be seen, E. coli presented an enhanced

3.6. Theoretical study in goats A mathematical model was developed with the aim of predict the CTF plasma concentration in animals after a formulation dosage. First of all, the model was validated using reported values of CTF administration. At this respect, Cárceles García and Varón studied the administration of CTF in goats (Cárceles García & Varón, 2016). In this work, the authors administered the antibiotic dispersed in saline aqueous solution by intravenous injection and quantified the corresponding plasma concentrations. Their experimental results are shown in Fig. 5.a. These data were fitted using Eq. (5), and the values of metabolic parameters were calculated, resulting in: Vd = 3.96 L, Vm =283.22 mg min−1 kg−1, K =0.04 mg ml−1 kg−1, and h = 1.93. The model prediction is also presented in Fig. 5.a, showing a good agreement between experimental and simulated results (f1 < 15, f2 > 50). In the same work, the authors studied the administration of two ceftiofur formulations via subcutaneous injection and quantified the corresponding plasma concentrations (Cárceles García & Varón, 2016). The formulations consisted in a dispersion of the antibiotic in aqueous saline solution and in a poloxamer 407 solution that gelled in situ at body temperature. From these experimental data and using the values of metabolic parameters previously calculated, Eq. (4) was employed to calculate the delivery rate (dFDL/dt) for both formulations. Results are presented in Fig. 5.b and referred as "aqueous formulation" and "P407 formulation", respectively. The corresponding plasma concentrations and the theoretical predictions are showed in Fig. 5.c. It can be observed that aqueous formulation delivered the antibiotic during less than 12 h while the gel controlled the release over a more extended period of time. The model predicted the experimental data with good agreement (f1 < 15, f2 > 50). These results confirm the validity of the model and its accuracy to predict plasma concentration of CTF after the administration of diverse formulations.

Fig. 4. Antimicrobial activity of CFT-loaded CHT MPs: (a) Inhibition of MP19 against E. coli. (b) Inhibition of MP19 against S. aureus. (c) Inhibition of MP21 against E. coli. (d) Inhibition of MP21 against S. aureus. (e) Live/Dead BacLight® kit for MP19 against E. coli. (f) Live/Dead BacLight® kit for MP19 against S. aureus. (g) Live/ Dead BacLight® kit for MP21 against E. coli. (h) Live/Dead BacLight® kit for MP21 against S. aureus. 9

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Fig. 5. Ceftiofur administration in goats: (a) Intravenous injection (experimental data were reproduced from (Cárceles García & Varón, 2016)). (b) Release rate (experimental data identified as “Aqueous formulation” and “P407 formulation” were reproduced from (Cárceles García & Varón, 2016)). (c) Plasma concentration (experimental data identified as “Aqueous formulation” and “P407 formulation” were reproduced from (Cárceles García & Varón, 2016)).

use or store. In addition, minimal requirements were needed like the apparatus and a clean supply of compressed air. Clearly, the spray drying technology is a good alternative to the emulsion technique, and it can be scale up easily for higher volume production using a bigger apparatus or operating several equipment in a parallel mode. Once optimized the fabrication process, a multilevel factorial design was conducted to study how formulation factors affect the features of the MPs. From this study, it was observed that the use of an oily phase, like myritol or propylene glycol, enhanced the dispersion of CTF in the CHT solutions. This better dispersion increased the loading efficiency and avoided the formation of agglomerates that can potentially hinder the dosage through a syringe. Besides, the loading efficiency was improved increasing the content of antibiotic, and coating the particle core with an external layer of CHT. In addition, the in vitro delivery of CTF was significantly delayed in aqueous medium when the antibiotic was microencapsulated inside CHT MPs in comparison with the commercial products. Moreover, the delivery rate can be slowed down increasing the content of TPP and/or myritol, and coating the MP core with an appropriate external layer of CHT. On the other hand, the stability of the MPs in aqueous environment was improved increasing the content of TPP and CTF, and coating the core with an external layer of CHT. The next step was to evaluate the stability of CTF after the microencapsulation process to determine if the spray drying method alters the structure of the molecule. The microbiological tests showed that the antimicrobial activity did not change nor decrease after microencapsulation, presenting values of CIM, IC50, and inhibition halos similar to those observed for the non-encapsulated active principle. Moreover, the DSC analysis showed a decrease in the thermal degradation of CTF when it is encapsulated inside the CHT MPs, suggesting a protective effect which is enhanced when the particle core is coated with a 2 % CHT solution. These findings indicate that the stability of CTF can be extended microencapsulating the antibiotic inside CHT MPs. Finally, a mathematical model was developed to predict the CTF plasma concentrations in production animals after the administration of different formulations. The model was first validated using reported values of CTF administration in goats and the plasma concentrations obtained after the dosage of different formulations (aqueous and in situ gelling) were predicted with good agreement. Then, the behavior of the CHT MPs was studied and simulation results showed that a 2 % CHT coated MPs might maintain plasma levels above 1 μg ml−1 during 48 h with a single dose. This research opens the possibility of developing a novel product for ceftiofur therapies in animals, improving the antibiotic stability, minimizing the dosage frequency, saving time and effort, and simplifying operative steps. Future assays must be carried out to evaluate the in vivo performance of the CTF-loaded CHT MPs.

Once validated, the model was used to predict the delivery rate and the plasma concentrations in goats after a theoretical administration of the formulations M19 and M21. The in vitro release kinetics presented in Fig. 2.e were used as an approach of the in vivo delivery rates since similar sink conditions are presented in both situations. In the animal’s body, the antibiotic is continuously consumed maintaining its tissue concentration close to zero. It is considered that the diffusion of the antibiotic molecules from tissues to the systemic circulation is very fast, and the time involved in this step is negligible. Similarly, ceftiofur concentration in the release medium during the in vitro assays is negligible compared to its solubility due to the large volume of aqueous medium. Therefore, similar concentration gradients exist in both situations. Since the concentration gradient is the main factor that determines the release rate, it is assumed that the delivery rate is similar in both situations and hence the in vitro kinetics can be used as an approximation of the in vivo delivery. In addition, the in vitro assays were performed at 37 °C and in PBS to mimic the corporal temperature and saline environment. From these release data and using Eq. (4), the delivery rate was calculated and the results are presented in Fig. 5.b. The corresponding plasma concentration are included in the Fig. 5.c. Both microparticles have similar initial release rates, however M21 showed a more sustained delivery along time. This behavior was corroborated in the corresponding plasma levels. Compared to aqueous and poloxamer formulations presented in the Cárceles García and Varón work, formulation M21 could have a better control over delivery. This formulation presented a lower burst release and maintained suitable CTF concentrations above the threshold value of 1 μg ml−1 for a longer period of time. This extended delivery could reduce the frequency of dosage from 24 h to 48 h, simplifying the sanitation therapy. The results observed in this study support the idea of using the CHT MPs as an alternative formulation for CTF administration in production animals. Besides, the use of a mathematical model is a very useful tool that allows to perform numerous simulations in short times with the goal of analyze and compare different delivery systems. This saves time and money, and avoids excessive experimentation with animals which not only has a high economic cost but also ethical concerns.

4. Conclusions The objective of the present work was to encapsulate ceftiofur in chitosan microparticles using the spray drying technology and evaluate the formulation as an alternative to the actual commercially available products. First of all, the fabrication process was optimized determining the best operating conditions, e.g. optimal values for inlet temperature, liquid flow rate, and spray air flow. Microencapsulation of ceftiofur was successfully achieved using these conditions, and a good encapsulation efficiency was obtained. The fabrication process was simple and fast, and involved only one step, collecting the MPs as a dry powder ready to 10

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CRediT authorship contribution statement

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Ignacio M. Helbling: Conceptualization, Methodology, Software, Investigation, Writing - original draft, Writing - review & editing. Carlos A. Busatto: Methodology, Software, Formal analysis, Investigation, Writing - review & editing. Federico Karp: Investigation. Germán A. Islan: Investigation, Resources. Diana A. Estenoz: Resources, Supervision, Funding acquisition. Julio A. Luna: Conceptualization, Resources, Supervision, Project administration, Funding acquisition. Declaration of Competing Interest The authors declare that there is no conflict of interest. Acknowledgments Authors would like to acknowledge to Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), to Universidad Nacional del Litoral (UNL) of Argentine, and to ZOOVET Productos Veterinarios S.A. for the financial support granted to this contribution. References AG, B. L. (2016). Operation manual of B-290 mini spray dryer. www.buchi.com. Ahmed, I., & Kasraian, K. (2002). Pharmaceutical challenges in veterinary product development. Advanced Drug Delivery Reviews, 54(6), 871–882. Azadi, G., Seward, M., Larsen, M. U., Shapley, N. C., & Tripathi, A. (2012). Improved antimicrobial potency through synergistic action of chitosan microparticles and low electric field. Applied Biochemistry and Biotechnology, 168(3), 531–541. Barreiros, F. M., Ferreira, P. J., & Figueiredo, M. M. (1996). Calculating shape factors from particle sizing data. Particle & Particle Systems Characterization, 13(6), 368–373. Berger, J., Reist, M., Mayer, J. M., Felt, O., Peppas, N. A., & Gurny, R. (2004). Structure and interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications. European Journal of Pharmaceutics and Biopharmaceutics, 57(1), 19–34. Brown, S. A., Chester, S. T., Speedy, A. K., Hubbard, V. L., Callahan, J. K., Hamlow, P. J., ... Robb, E. J. (2000). Comparison of plasma pharmacokinetics and bioequivalence of ceftiofur sodium in cattle after a single intramuscular or subcutaneous injection. Journal of Veterinary Pharmacology and Therapeutics, 23(5), 273–280. Cárceles García, C., & Varón, E. F. (2016). Estudio farmacocinético de formulaciones poliméricas de liberación controlada y excreción en leche de ceftiofur en caprino. Facultad de medicina. España: Universidad de Murcia. Cervantes, C. C., Brown, M. P., Gronwall, R., & Merritt, K. (1993). Pharmacokinetics and concentrations of ceftiofur sodium in body fluids and endometrium after repeated intramuscular injections in mares. American Journal of Veterinary Research, 54(4), 573–575. Cortinhas, C. S., Tomazi, T., Zoni, M. S. F., Moro, E., & Veiga Dos Santos, M. (2016). Randomized clinical trial comparing ceftiofur hydrochloride with a positive control protocol for intramammary treatment of nonsevere clinical mastitis in dairy cows. Journal of Dairy Science, 99(7), 5619–5628. Costa, P., & Sousa Lobo, J. M. (2001). Modeling and comparison of dissolution profiles. European Journal of Pharmaceutical Sciences, 13(2), 123–133. Courtin, F., Craigmill, A. L., Wetzlich, S. E., Gustafson, C. R., & Arndt, T. S. (1997). Pharmacokinetics of ceftiofur and metabolites after single intravenous and intramuscular administration and multiple intramuscular administrations of ceftiofur sodium to dairy goats. Journal of Veterinary Pharmacology and Therapeutics, 20(5), 368–373. Craigmill, A. L., Brown, S. A., Wetzlich, S. E., Gustafson, C. R., & Arndt, T. S. (1997). Pharmacokinetics of ceftiofur and metabolites after single intravenous and intramuscular administration and multiple intramuscular administrations of ceftiofur sodium to sheep. Journal of Veterinary Pharmacology and Therapeutics, 20(2), 139–144. Desai, K. G., & Park, H. J. (2006). Study of gamma-irradiation effects on chitosan microparticles. Drug Delivery, 13(1), 39–50. Drillich, M., Arlt, S., Kersting, S., Bergwerff, A. A., Scherpenisse, P., & Heuwieser, W. (2006). Ceftiofur derivatives in serum, uterine tissues, cotyledons, and lochia after fetal membrane retention. Journal of Dairy Science, 89(9), 3431–3438. Drillich, M., Beetz, O., Pfutzner, A., Sabin, M., Sabin, H. J., Kutzer, P., ... Heuwieser, W. (2001). Evaluation of a systemic antibiotic treatment of toxic puerperal metritis in dairy cows. Journal of Dairy Science, 84(9), 2010–2017. FDA (1992). Implantation injectable dosage form; New animal drugs–ceftiofur sterile powder for injection. Federal register. Washington, D.C: US Food and Drug Administration418–462. Fei Liu, X., Lin Guan, Y., Zhi Yang, D., Li, Z., & De Yao, K. (2001). Antibacterial action of chitosan and carboxymethylated chitosan. Journal of Applied Polymer Science, 79(7), 1324–1335. Folz, S. D., Hanson, B. J., Griffin, A. K., Dinvald, L. L., Swerczek, T. W., Walker, R. D., &

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