Preparation and Characterization of Tiamulin-Loaded Niosomes for Oral Bioavailability Enhancement in Mycoplasma-Infected Broilers

Abstract

Mycoplasma infections pose significant challenges in the poultry industry, necessitating effective therapeutic interventions. Tiamulin, a veterinary antibiotic, has demonstrated efficacy against Mycoplasma species. However, the emergence of resistant Mycoplasma species could dramatically reduce the therapeutic potential, contributing to economic losses. Optimizing the tiamulin’s pharmacokinetic profile via nanocarrier incorporation could enhance its therapeutic potential and reduce the administration frequency, ultimately reducing the resistant strain emergence. Niosomes, a type of self-assembled non-ionic surfactant-based nanocarrier, have emerged as a promising drug delivery system, offering improved drug stability, sustained release, and enhanced bioavailability. In this study, niosomal nanocarriers encapsulating tiamulin were prepared, characterized and assessed in Mycoplasma-inoculated broilers following oral administration. Differential scanning colorimetry (DSC) confirmed the alterations in the crystalline state following components integration into the self-assembled structures formed during the formulation procedure. Transmission electron microscopy (TEM) showed the spherical nanostructure of the formed niosomes. The formulated nanocarriers exhibited a zeta potential and average hydrodynamic diameter of −10.65 ± 1.37 mV and 339.67 ± 30.88 nm, respectively. Assessment of the pharmacokinetic parameters following oral administration to Mycoplasma gallisepticum-infected broilers revealed the ability of the niosomal nanocarriers to increase the tiamulin’s bioavailability and systemic exposure, marked by significantly higher area under the curve (AUC) (p < 0.01) and prolonged elimination half-life (T_1/2) (p < 0.05). Enhanced bioavailability and prolonged residence time are crucial factors in maintaining therapeutic concentrations at reduced doses and administration frequencies. This approach provides a viable strategy to decrease the risk of subtherapeutic levels, thereby mitigating the development of antibiotic resistance. The findings presented herein offer a sustainable approach for the efficient use of antibiotics in veterinary medicine.

1. Introduction

Mycoplasma gallisepticum stands out as one of the most formidable causative agent of chronic respiratory disease in chickens, significantly affecting the poultry industry. The prevailing strategy for controlling Mycoplasma infections involves the use of antimicrobial agents. Tiamulin is a semisynthetic antibiotic derived from pleuromutilin that originates from the tricyclic diterpenoid pleuromutilin found in the fungus Pleurotus mutilus. As an efficient antimycoplasmal drug extensively utilized in veterinary medicine, tiamulin exerts its activity by hindering bacterial protein synthesis, specifically by binding to the 50S ribosomal subunit. Nevertheless, the swift rise of antibiotic-resistant Mycoplasma strains, as reported in recent studies, undermines the remarkable benefits that can be attained through the use of tiamulin [1,2,3,4]. This is further exacerbated by the emergence of multiple drug resistance and the alarming risk posed by the outbreak of untreatable infections with Mycoplasma [5].

Frequent and prolonged exposure of bacterial populations to antibiotics creates a selective pressure favoring the survival and proliferation of resistant strains [6]. Consequently, the reduced exposure of Mycoplasma populations to tiamulin can impede the development of resistance by minimizing the opportunities for genetic mutations or adaptations that confer antibiotic resistance [7]. Reducing the administration frequency of antibiotics is a crucial approach in combatting resistance development [8]. In addition, augmenting the bioavailability of antibiotics at reduced doses represents another pivotal strategy to mitigate resistance development. The improved bioavailability can facilitate the achievement of therapeutic concentrations with lower overall doses, minimizing the selective pressure on bacteria to evolve resistance [9]

Addressing the pressing issue of antibiotic resistance necessitates innovative approaches to enhance the precision and efficacy of antibiotic delivery. One promising avenue is the utilization of advanced drug delivery mechanisms, with nanoparticles emerging as a focal point in recent research [10,11,12]. Nanoscopic materials have shown the ability to improve the effectiveness of loaded therapeutic cargo [13,14,15,16,17,18,19,20]. Additionally, nanoparticles previously demonstrated their efficacy as carriers for antibiotics such as tobramycin, cephalexin, and ciprofloxacin [21,22,23]. Niosomes are nano-sized carriers consisting of bilayers formed by self-assembled non-ionic surfactants, typically stabilized through the incorporation of cholesterol [24,25,26]. Featured by their biodegradability, biocompatibility, and chemical stability, niosomes have been widely employed to enhance drug bioavailability [24,27,28,29]. The combination of reduced administration frequency and enhanced bioavailability, facilitated by innovative drug delivery systems such as niosomes, holds significant promise for addressing the challenge of antibiotic resistance developed by Mycoplasma infections [30]. This approach not only seeks to preserve the effectiveness of tiamulin but also aligns with the broader goal of responsible antibiotic use in safeguarding the long-term viability of antimicrobial therapies in the poultry industry.

We previously demonstrated niosomes’ ability to improve the bioavailability of florfenicol, a veterinary antibiotic, through the oral administration route [31]. Employing a non-invasive administration approach for delivering niosomes to chickens presents substantial advantages over more invasive methods, particularly parenteral administration. This approach prioritizes animal welfare by minimizing stress and discomfort, crucial for maintaining the overall health of the birds. Furthermore, the practicality and ease of non-invasive methods, such as oral administration, make them well suited for large-scale implementation in the poultry industry, streamlining processes and reducing labor intensity. The reduced risk of infection and tissue damage associated with non-invasive routes adds another layer of benefit, contributing to the birds’ health and productivity. The reduced complexity and enhanced compliance associated with non-invasive approaches render them an attractive choice for the seamless incorporation of cutting-edge drug delivery systems into poultry health management practices.

As such, exploring the potential of niosomes as a tiamulin delivery nanocarrier holds promise for overcoming the emerging antibiotic-resistant Mycoplasma strains. Accordingly, we proposed that the niosomes’ formulation capacity to improve tiamulin’s bioavailability and reduce administration frequency could address the growing challenge of antibiotic resistance in Mycoplasma strains, thereby contributing to the sustainability of antimicrobial therapies in the poultry industry. To this end, we developed and characterized a tiamulin-loaded niosomal formulation. Additionally, we assessed pharmacokinetics following oral administration to Mycoplasma-infected chickens. Furthermore, we evaluated oral bioavailability in healthy broilers to deepen our understanding of the nano-formulation’s pharmacokinetic profile in the presence of Mycoplasma infection.

2. Materials and Methods

2.1. Materials

Tiamulin hydrogen fumarate powder with purity ≥98.0% was obtained from Pharma-Swede (Cairo, Egypt). Cholesterol, DDP, Tween 60, and Span 60 were purchased from Sigma Aldrich (Burlington, MA, USA). Methanol and chloroform were purchased from Corner-Lab Company (Cairo, Egypt). Mycoplasma gallisepticum (MG) S 6 strain (ATCC 19610) was obtained from the Microbiological Resources Centre (Mircen, Cairo, Egypt). Mycoplasma gallisepticum field isolate strain (EIS-C3-09) was obtained from the Mycoplasma department of the Animal Health Research Institute (Giza, Egypt). Nylon filter (0.45 µm) was purchased from Millipore (Burlington, MA, USA).

2.2. Preparation of Tiamulin-Loaded Niosomes (TLN)

Tiamulin-loaded niosomes were formulated using the thin film hydration method [32]. In a round flask, tiamulin (1 mg), along with 30 mg of Span 60 and cholesterol in a molar ratio of 1:1, was dissolved in a chloroform and methanol mixture. The combined solution was subsequently rotary-evaporated under reduced pressure at 40 °C until a thin film was formed. The resulting dried thin layer was then hydrated with 10 mL of phosphate buffer of pH 7.4 at 60 °C for 2 h. The formulated mixture underwent sonication for 30 min and was subsequently centrifuged using a cooling centrifuge at a speed of 15,000 rpm and 4 °C for 1 h, facilitating the separation of the entrapped drug from the unentrapped one. The collected niosomal pellets were re-suspended in phosphate buffer, and the prepared formulation was stored at 4 °C for subsequent studies.

2.3. Characterization of TLN

2.3.1. Transmission Electron Microscopy (TEM)

The morphological features of the TLN formulation were determined using TEM (Carl Zeiss, Oberkochen, Germany) [33]. The freshly prepared sample was deposited onto the surface of a carbon-coated copper grid and stained with a drop of a 1% aqueous solution of phosphotungstic acid dye to enhance visualization during TEM analysis.

2.3.2. Particle Size, Size Distribution, and Zeta Potential

The prepared niosome formulation’s particle size, poly dispersity index (PDI), and zeta potential were determined using the Malvern PCS4700 Instrument (Malvern Instruments, Malvern, UK) [33,34]. TLN samples were appropriately diluted with de-ionized water prior to measurements. Three independent measurements were conducted at room temperature.

2.3.3. Differential Scanning Calorimetry (DSC)

The thermal behavior of TLN and its contained ingredients, tiamulin, Span 60, and cholesterol, was determined using a DSC analyzer (60F3, NETZSCH-Geratebau GmbH, Maia, Germany) equipped with a liquid nitrogen cooling system [35]. The analysis was performed with a heating rate of 5 °C/min under a nitrogen gas flow of 25 mL/min, and the temperature ranged from room temperature to 200 °C. An empty aluminum pan was used as a reference.

2.3.4. Entrapment Efficiency (EE%)

The TLN suspension was centrifuged at 15,000 rpm for 2 h at a temperature of 4 °C to separate the entrapped tiamulin. Methanol was used to dissolve the entrapped tiamulin. Tiamulin quantification was carried out utilizing high-performance liquid chromatography (HPLC). Tiamulin was separated isocratically using a C18 analytical column (150 mm × 4.6 mm) at a maximum wavelength of 212 nm. The mobile phase consisted of a 40:60 v/v of 1% ammonium carbonate and acetonitrile, delivered at a flow rate of 1 mL per minute with injection volumes of 20 µL [36]. The EE% was determined using the following equation [37].:

$$\mathrm{\%}\mathrm{E}\mathrm{E}={\displaystyle \frac{{\mathrm{E}}_{\mathrm{t}}}{{\mathrm{E}}_{\mathrm{i}}}}\times 100$$

(1)

where E_t is the amount of entrapped tiamulin and E_i is the initial tiamulin amount.

2.3.5. In Vitro Release

The release profile of tiamulin in its solution and niosome-loaded forms was evaluated utilizing a Hanson dissolution apparatus [38]. To a dialysis bag, 2 mg of free or niosome-loaded tiamulin were transferred. The dialysis bag was subsequently submerged in 25 mL of phosphate buffer (pH 7.4) that was used as the release medium. The Hanson dissolution apparatus (USA) was set to operate at 100 rpm and maintained at a temperature of 37 ± 0.5 °C. At specified intervals, 2 mL samples were withdrawn from each receptor compartment and replaced with an equivalent volume of fresh medium. The released tiamulin was quantified using HPLC and the percentage release was determined using the following equation in triplicate:

$$\mathrm{\%}\mathrm{R}\mathrm{e}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{e}={\displaystyle \frac{{\mathrm{R}}_{\mathrm{t}}}{{\mathrm{R}}_{\mathrm{i}}}}\times 100$$

(2)

where R_t is the amount of tiamulin released at time t and R_i is the initial amount of entrapped tiamulin

2.4. Determination of Mycoplasma gallisepticum Strain Sensitivity

2.4.1. Mycoplasma gallisepticum Strain

Mycoplasma gallisepticum field isolate strain (EIS-C3-09), sourced from the Mycoplasma Department at the Animal Health Research Institute in Dokki, Giza, Egypt, was utilized in experimental infection. To enhance the virulence of this strain, it underwent a passage through the yolk sacs of specific pyrogen-free eggs. Subsequently, the yolk and allantoic fluid were collected and utilized for the inoculation process [39]. Following the enhancement in virulence, an in vitro assessment was carried out to determine the strain’s sensitivity to the antimycoplasmal drug, utilizing the minimum inhibitory concentration (MIC) method.

2.4.2. The Growth Media

The growth medium employed for the sensitivity test was fortified with 12% inactivated horse serum [40]. The broth utilized for culture propagation underwent sterilization through filtration using a Millipore filter equipped with a 0.2 µm cellulose membrane. Subsequently, the agar medium was prepared by subjecting the medium to autoclaving at 121 °C for 20 min, with the addition of 1.5% agar. Following autoclaving, cysteine, penicillin, thallium acetate, and swine serum were aseptically introduced into the medium and stored at 4 °C.

2.4.3. Determination of Minimum Inhibitory Concentration (MIC) Against Mycoplasma

The determination of the minimum inhibitory concentration (MIC) of tiamulin against both the Mycoplasma gallisepticum (MG) S 6 strain (ATCC 19610) and the field isolate strain (EIS-C3-09) was conducted through a modified MIC assay method [41]. A Mycoplasma gallisepticum titer of 10⁷ CFU/mL in the Mycoplasma medium was prepared. Serial two-fold dilutions of tiamulin were then prepared within the concentration range of 0.0156–32 µg/mL, utilizing MG culture medium as the diluent (at a 1:1 dilution). All experimental procedures were meticulously performed in triplicate. The MIC was defined as the lowest concentration of tiamulin at which no discernible change in the color of the culture medium was observed.

2.5. In Vivo Studies

2.5.1. Experimental Birds

The Institutional Animal Care and Use Committee (IACUC) at the Faculty of Veterinary Medicine, Cairo University, Egypt approved the animal use protocol (Vet CU 09092023749). The experiments involving animal use were conducted in accordance with the guidelines and regulations of the IACUC at the Faculty of Veterinary Medicine, Cairo University, Egypt.

Clinically healthy, one-week-old, Hubbard chickens, verified as Mycoplasma gallisepticum-free, were procured from Elarabia Poultry Breeding Farm. These chicks were nourished with antibiotic- and anticoccidial-free poultry ration (ad libitum), accompanied by unrestricted access to water for a period of two weeks. This dietary regimen was implemented to facilitate the full elimination of antibacterial agents from the body. Chickens were housed in an environment characterized by a constant temperature, relative humidity ranging from 45% to 65%, and a 12 h lighting cycle. Prior to drug administration, the birds underwent an overnight fasting period.

2.5.2. Induction of Mycoplasma gallisepticum Intratracheal Infection

At three weeks of age, chickens free from Mycoplasma gallisepticum underwent thorough screening by collecting tracheal swabs for molecular detection using polymerase chain reaction (PCR) to confirm their negative status for Mycoplasma gallisepticum. Subsequently, these chickens were subjected to inoculation via the intraocular/intranasal route with 0.1 mL containing 107 colony-forming units (CFU) of MG culture. An additional inoculum of 0.1 mL was administered via the intratracheal route [42]. The chickens were observed for 6–21 days till the appearance of the clinical signs [43]. The confirmation of infection was established through the observation of clinical manifestations and the serum plate agglutination test (SPA). The experiments were conducted under conditions adhering to the institutional biosafety guidelines.

2.5.3. Serum Plate Agglutination

A single volume, approximately 0.02 mL, of serum was dispensed onto a pristine white glass plate, followed by an equal volume of stained MG antigen. Subsequently, a stirring rod was employed to evenly distribute the mixture over a circular area of approximately 1.5 cm in diameter. The plate was gently rocked for a duration of 2 min. The occurrence of agglutination was evidenced by the flocculation of the antigen within this 2 min timeframe [44]. The test encompassed both known positive and negative controls, and retests involved serial dilutions of any sera exhibiting agglutination after undergoing heating at 56 °C for 30 min. Sera that maintained strong reactivity even after dilution (1/4 or more) were considered positive.

2.5.4. Serum Plate Agglutination (SPA) Test

On a glass plate, 0.03 mL of antigen was dispensed along with an equivalent volume of fresh serum using a pipette [45]. The contents underwent thorough mixing by stirring with a glass rod, followed by gentle rocking. The interpretation of results took place within a 2 min timeframe. Positive cases revealed a gradual formation of granules, discernible during the rocking motion. Conversely, negative cases displayed the absence of such granular formations. A meticulous record was maintained for all serum plate agglutination (SPA) outcomes. It is noteworthy that in our study, the SPA test employed a crystal violet-stained Mycoplasma gallisepticum commercial antigen procured from Intervet Company Ltd. (Boxmeer, The Netherlands).

2.5.5. Oral Administration of Free Tiamulin or TLN to Mycoplasma gallisepticum-Infected Broilers

Broiler chickens that were experimentally infected with avian pathogenic Mycoplasma gallisepticum (MG) were randomly segregated into two subgroups, each comprising six individuals (n = 6). A single dose of 30 mg/kg body weight of free or noisome-contained tiamulin was administered through the oral route to the first or second subgroups, respectively [46,47].

2.5.6. Oral Administration of Free Tiamulin or TLN to Clinically Healthy Broilers

Clinically healthy chickens were randomly distributed into two subgroups; each subgroup contained six individuals (n = 6). A single dose of free or noisome-contained tiamulin was orally administered at 30 mg/kg body weight to the first or second subgroups in this healthy cohort, respectively.

2.5.7. Determination of Tiamulin Concentration in Plasma

Blood samples (0.75–1 mL) were collected from all avian subjects through the median tarsometatarsal vein into heparinized tubes at specific timepoints post-administration. Subsequently, all collected samples underwent centrifugation at 1800× g for 10 min. The resulting clear plasma was carefully transferred to Eppendorf tubes and promptly frozen at –80 °C until the time of assay. The quantification of tiamulin in plasma was conducted using LC-MS, following established protocols with slight adjustments [48]. Chromatographic separation of tiamulin was achieved utilizing a C18 column (Luna, 150 mm × 2 mm, I.D, 5 µm) maintained at a temperature of 30 °C. A gradient elution program was employed with eluent A (water containing 0.1% formic acid) and eluent B (methanol containing 0.1% formic acid) at a flow rate of 0.4 mL/min. The elution commenced at 90% eluent A for 7.0 min, followed by a linear decrease to 10% eluent A over 10 min. Subsequently, the system returned to the initial conditions in 0.1 min and was conditioned with 10% solvent A for 0.9 min. The total run time for a sample was 20.0 min, and the injection volume was set at 20 μL. The mass spectrometer was operated in positive ion and multiple reaction monitoring (MRM) mode with precursor to product qualifier transitions m/z 494.500/192.200 and m/z 494.500/119.200. All parameters of the liquid chromatography (LC) and mass spectrometry (MS) were managed through Analyst_1.5 software. The spray voltage was optimized at 4000 V, source temperature at 550 °C, and curtain gas, and auxiliary gas (nitrogen) pressures were set at 25 and 10 psi, respectively. Nitrogen served as the collision gas at a pressure of 40 psi; collision energy (CE) was adjusted to 29 and 55 V, and collision extraction potential (CXP) to 12 V, for transitions 494.500/192.200 and m/z 494.500/119.200, respectively. The dwell time was set at 100 ms. Standard curves were established by spiking tiamulin standards in chicken plasma and tissues within the concentration range of 0.01–10 μg/mL. Chicken plasma samples were allowed to thaw at room temperature. Subsequently, 0.25 mL of plasma samples were subjected to extraction with 0.75 mL of acetonitrile. The resulting mixture was vortexed for 1 min and then centrifuged at 16,000 g for 10 min. The upper organic layer was carefully transferred and filtrated through a 0.22 mm syringe filter, and 20 µL of the filtered solution was injected into the LC-MS/MS system for analysis. The pharmacokinetic parameters were analyzed using Kinetica 2000 (Version 3.0, InnaPhase Corporation, Philadelphia, PA, USA).

2.6. Statistical Analysis

The pharmacokinetic parameters obtained were analyzed with a non-parametric Mann–Whitney test using GraphPad Prism version 7.00 (USA) to determine the differences between the groups.

3. Results and Discussion

3.1. In Vitro Characterization of TLN

Niosomes can be described as self-assembled spherical vesicles formed from non-ionic surfactants, often stabilized with cholesterol to enhance rigidity and prevent leakage of encapsulated ingredients (Figure 1A) [29]. The synthesis of the noisome formulation was achieved using the thin film hydration method, a widely used technique for the preparation of lipid-based delivery systems due to its simplicity and efficiency. This method involves the formation of a surfactant thin film by evaporating an organic solvent mixture, followed by hydration with an aqueous phase to form the self-assembled vesicles [31]. The transmission electron microphotograph of the TLN formulation showed that the nanoparticles exhibited an approximately spherical shape, appearing as discernible black dots without evident aggregation (Figure 1B). The evaluation of vesicle size is crucial in determining the niosomal effectiveness, as a nanocarrier. Smaller vesicles offer a larger surface area that could enhance drug absorption [49,50,51]. The formulated TLN exhibited a small particle size of 339.67 ± 30.88 nm, suggesting the formation of nano-sized vesicles (Figure 1C). To ascertain the size distribution, and homogeneity of vesicle size within a sample, the PDI was employed [33]. The analysis of the prepared TLN’s PDI unveiled a low value of 0.149 ± 0.07, indicating homogeneity and a narrow distribution of particle size. As illustrated in Figure 1D, the formulated TLN exhibited a net mean negative surface charge of −10.65 ± 1.37 mV, indicating electrostatic stabilization of the colloidal dispersion [49].

The EE% was determined to evaluate the loading efficiency of the niosomal formulation. The TLN formulation exhibited an EE% of 71.05 ± 0.58%. The intrinsic features of Span 60, encompassing characteristics such as the length of the C-H alkyl chains, a low HLB value, and the presence of a hydrophobic moiety, in conjunction with a high gel-liquid phase transition temperature and low surface free energy, can facilitate the generation of niosomes with high entrapment efficiency and a small particle size [37,52]. Additionally, niosome integrity, physical stability, and rigidity could be enhanced by the incorporation of cholesterol within the vesicular structure [53,54,55]. Nevertheless, the increase in the amount of incorporated cholesterol can be accompanied by an increase in the size of the prepared niosomes [35]. Moreover, higher amounts of cholesterol may compete with the drug incorporation within the bilayers leading to a decrease in the entrapment efficiency [56,57]. In our study, the TLN formulation was prepared using 30 mg of Span 60 and cholesterol with a 1:1 molar ratio [37,56,57]. Previous studies reported that increasing the amount of non-ionic surfactants and cholesterol may lead to an increase in the hydrophobic domain volume and the number of niosomes formed [37,58]. This, in turn, could result in an increase in entrapment efficiency and particle size. However, the further increase in the amount of incorporated non-ionic surfactants and cholesterol above 30 mg may lead to a reduction in the entrapment efficiency and larger particle sizes [37,58]. This may be due to the formation of mixed micelles along with the niosomal vesicles. Accordingly, the careful selection of the Span 60 and cholesterol in a 1:1 molar ratio in our study contributed to favorable niosomal characteristics, including a small particle size of 339.67 ± 30.88 nm, homogeneity with a low PDI of 0.149 ± 0.07, and high entrapment efficiency.

The application of DSC allows for the assessment of ingredients’ crystallinity [59]. The DSC thermogram of pure tiamulin revealed a clear endothermic peak at 147.79 °C, corresponding to its melting point and indicating its crystalline nature (Figure 1E). In the thermal analysis of the Span 60 surfactant, an endothermic peak was observed at its melting point of 52.09 °C, while cholesterol exhibited a similar endothermic peak at 149 °C. The introduction of tiamulin into the niosomes, as evident in the TLN thermogram, led to a reduction in tiamulin’s crystallinity, likely attributed to the incorporation of tiamulin within the surfactant bilayer of the niosomal structure in an amorphous form. The absence of peaks corresponding to Span 60 and cholesterol in the TLN thermogram indicated the loss of their crystalline pattern that could be attributed to their amorphous assembly as a bilayer. Collectively, the DSC analysis highlighted the changes in the key ingredients’ crystalline state following their incorporation in the vesicular structures generated during the formulation process.

The release profile of tiamulin from the solution form and niosomal formulation was investigated over an 8 h period using a dialysis bag system (Figure 1F). The tiamulin solution exhibited rapid release, with nearly 30% of the drug released within the first hour, reaching almost 100% by the end of this study. In contrast, TLN demonstrated a significantly slower release, with approximately 10% released in the first hour and around 40% after 8 h. This sustained release profile of TLN was likely attributable to the structural integrity of the niosomes, which could act as a diffusion barrier, effectively modulating the release of the encapsulated tiamulin [29].

3.2. In Vitro Antimicoplasmal Activity

The MIC values of free tiamulin and its niosome-contained form against MG S 6 strain (ATCC 19610) and the field isolate strain (EIS-C3-09) were determined (Supplementary Figure S1). The MIC of free tiamulin and its niosome-loaded form against MG S 6 strain (ATCC 19610) were both 0.0312 µg/mL ± 0.0 (

Table 1. MIC of tiamulin (μg/mL) against Mycoplasma Gallisepticum.

	MIC of Tiamulin 1 (µg/mL)
Bacterial Strain	Free Tiamulin	TLN
MG S 6 strain (ATCC 1961)	0.0312 ± 0.0	0.0312 ± 0.0
MG field isolate strain (EIS-C3-09)	0.0624 ± 0.0	0.0624 ± 0.0

¹ Results represent the mean value ± SD (n = 3).

). Similarly, the observed MIC values for free tiamulin and its niosome-encapsulated form against the MG field isolate strain (EIS-C3-09) were both 0.0624 µg/mL ± 0.0. These findings suggest that niosomal nanocarriers did not influence the internalization of loaded tiamulin or its interaction with Mycoplasma gallisepticum. The comparable MIC values indicated that the efficacy remained consistent, whether tiamulin was used in its free or niosome-incorporated forms.

3.3. Pharmacokinetic Profile of Orally Administered Niosomal Tiamulin in Mycoplasma-Infected Broilers

To evaluate the niosomal potential to improve the oral bioavailability of the encapsulated drug, we assessed the tiamulin’s pharmacokinetic properties after the administration of its free or niosomes-loaded form to Mycoplasma-infected broilers via the oral route. The results revealed significant differences in key pharmacokinetic parameters between the administered free and noisome-contained tiamulin (Figure 2). Chicken-derived plasma samples were analyzed by LCMS to determine the levels of tiamulin after oral administration. Using standard tiamulin concentrations that ranged from 0.1 to 40 µg/mL in chicken plasma, the calibration curve created showed linear correlation coefficient higher than 0.997 (Supplementary Figure S2).

The maximum plasma concentration (C_max) of tiamulin was markedly higher in Mycoplasma-infected broilers receiving niosomal tiamulin than those administered with free tiamulin (Figure 2). This observation suggests that the nanoparticle formulation facilitated more efficient drug absorption. Enhanced drug absorption could be attributed to the nano-formulation’s capacity to improve the solubility and permeability of the incorporated tiamulin across biological barriers, such as the intestinal epithelium. Previous studies reported the niosomal ability to increase the loaded drug absorption after oral intake [60,61]. Furthermore, it was previously demonstrated that the surfactant constituents within the niosomal formulations could also potentially inhibit the efflux facilitated by P-glycoprotein (P-gp), a transmembrane efflux protein pump found in the gastrointestinal tract that pumps the drug out of the enterocytes back into the intestinal lumen [61,62]. It could be suggested that the suppressive impact of non-ionic surfactants on P-gp are thought to stem from their capacity to alter membrane fluidity [63]. Consequently, a reduction in P-gp activity could result in increased drug absorption and higher concentrations in the bloodstream, leading to improved bioavailability.

The mean residence time (MRT) of tiamulin was significantly prolonged in Mycoplasma-infected broilers that were treated with TLN (Figure 3). This prolonged exposure could be advantageous in preventing infections or maintaining therapeutic levels over an extended period, potentially reducing the frequency of drug administration. This finding could suggest the niosomes’ ability to sustain the incorporated tiamulin release and extend its retention within the body [31]. The elimination T_1/2 of tiamulin was substantially longer in the oral TLN-administered group, indicating a slower drug elimination process. The prolonged T_1/2 could indicate that the formulated niosomes may have protected the loaded tiamulin from rapid metabolism or degradation [62,64]. Niosomes could act as drug reservoirs that release the contained tiamulin gradually over time, ultimately leading to prolonged presence of the administered drug. Additionally, niosomal encapsulation could protect tiamulin from enzymatic degradation in the gut, which could allow for reduced drug metabolism and enhanced systemic exposure [65]. The AUC_(0–∞) was significantly higher in the Mycoplasma-infected broilers that received oral TLN (Figure 3). This observation reflects an overall increase in the drug exposure, potentially leading to higher bioavailability and a more pronounced therapeutic effect [66].

These findings highlight the potential of niosomal nanoparticle-based drug delivery systems for optimizing the therapeutic effectiveness of tiamulin in Mycoplasma infections and may have broader implications for drug delivery strategies in various medical contexts. Further investigations are warranted to elucidate the precise mechanisms behind these observed enhancements and to validate their applicability in clinical settings.

3.4. Effect of Mycoplasma Infection on the Absorption and Elimination of Orally Administered Niosomal Tiamulin

To address the effect of Mycoplasma infection on the pharmacokinetic profiles of noisome-delivered tiamulin after oral administration, the pharmacokinetic parameters were assessed in healthy chickens after oral intake of the free tiamulin or in its noisome-loaded form (Figure 4).

The C_max of tiamulin in healthy broilers orally administered with tiamulin-loaded niosomal nanoparticles was found to be similar to that in broilers receiving free tiamulin, suggesting comparable initial drug exposure (Figure 5). The comparable C_max values between the two formulations in healthy broilers suggest that niosomal nanoparticles did not significantly affect the initial drug absorption or peak plasma concentration of tiamulin in healthy broilers. This implies that in healthy conditions, both forms of the drug can initially achieve similar levels in the bloodstream upon oral administration. Conversely, the plasma AUC_0-∞ of tiamulin was significantly higher (p < 0.05) in healthy broilers treated with niosomal nanoparticles, indicating a more extensive overall drug exposure (Figure 5). Interestingly, a four-fold increase in C_max of tiamulin was detected in Mycoplasma-infected broilers compared to healthy broilers following oral administration of TLN. This could be attributed to the presence of Mycoplasma infections that might alter gastrointestinal physiology, permeability and drug absorption, resulting in different C_max values [67].

On the other hand, the MRT and elimination T_1/2 of tiamulin were significantly longer (p < 0.01) in healthy broilers receiving free tiamulin than those administered tiamulin-loaded niosomal nanoparticles. The MRT and elimination T_1/2 of tiamulin were significantly longer (1.7-fold) in Mycoplasma-infected broilers than healthy broilers after oral administration of free tiamulin. In poultry, as in other species, the primary route of metabolism for orally taken tiamulin is hepatic metabolism [68]. Due to the first-pass effect, orally administered tiamulin may undergo hepatic metabolism, which reduces the intact drug fraction reaching the systemic circulation [69]. The liver capacity to metabolize orally administered drugs can be adversely affected by the inflammation or dysfunction caused by Mycoplasma infection [70,71]. Thus, the longer MRT and elimination T_1/2 detected in Mycoplasma-infected broilers than healthy ones could be attributed to Mycoplasma-induced liver impairment. The fact that oral administration of tiamulin in the form of a niosomal formulation to healthy broilers markedly shortened the tiamulin MRT and elimination T_1/2, which were not observed in Mycoplasma-infected broilers, could highlight the significant influence of liver metabolism on the niosomal clearance [61]. Similar to the free drug, orally administered nanoparticles that enter the portal circulation following absorption suffer from the first-pass metabolism [61,72]. The results presented herein suggest the dramatic influence of liver metabolism on the nano-formulation compared to the free drug in healthy broilers that may justify the detected longer free tiamulin persistence in healthy broilers. Future studies should also consider tracking tiamulin metabolites following administration in both free and niosome-incorporated forms to better understand the influence of liver metabolism on the pharmacokinetic profile of the nano-formulation.

The observed differences in pharmacokinetic parameters between TLN and free tiamulin provide valuable insights into the potential advantages of niosomal nanoparticles as a drug delivery system. While the infected broilers benefited from the extended exposure to tiamulin, healthy broilers primarily exhibited advantages in terms of AUC_0-∞ following TLN oral intake. These findings highlight the importance of tailoring drug delivery systems to specific clinical conditions, with niosomal nanoparticles showing promise for optimizing tiamulin therapy in poultry, both in treating infections and potentially in preventive measures for healthy broilers. Further research should delve into the mechanistic aspects of these differences and their clinical implications.

Furthermore, the findings from our study on the pharmacokinetic profile of orally administered niosomal tiamulin in Mycoplasma-infected broilers offer strategic opportunities to mitigate the risk of tiamulin resistance. The prolonged MRT and elimination T_1/2 observed in Mycoplasma-infected broilers suggest sustained drug action, providing a foundation to optimize dosage and administration frequency. By tailoring the dosage regimen based on the sustained release properties of niosomal tiamulin, there is potential to reduce the need for frequent administration, minimizing the risk of subtherapeutic levels that could contribute to resistance. Additionally, the enhanced bioavailability and increased C_max support the possibility of maintaining therapeutic levels at lower doses. Considering these advantages, exploring combination therapy with antibiotics having different mechanisms of action becomes a viable strategy, potentially reducing selective pressure and further safeguarding against resistance development.

Our study extensively explored the pharmacokinetic profile of orally administered niosomal tiamulin in healthy and Mycoplasma-infected broilers. The higher C_max observed in niosomal tiamulin-treated broilers indicated more efficient drug absorption, likely attributed to improved solubility and permeability across biological barriers. The MRT and elimination T_1/2 further suggest sustained drug release and extended retention. Importantly, the elevated AUC_(0–∞) in Mycoplasma-infected broilers receiving oral niosomal tiamulin signifies increased overall drug exposure, enhancing bioavailability and therapeutic efficacy. These findings highlight the potential of niosomal nanoparticles for precise and impactful antibiotic delivery in managing Mycoplasma infections in poultry. Considering the convenience of oral administration in poultry farming, our study supports the suitability of the oral route for administering niosomal tiamulin, offering practical and effective drug delivery in the context of poultry health management.

4. Conclusions

Our study encompassed a comprehensive exploration of the pharmacokinetic profile of nanocarrier-loaded tiamulin administered orally to healthy and Mycoplasma-infected chickens. The characterization techniques confirmed the nano-sized vesicles’ homogeneity, small size, and negative zeta potential, indicative of good stability. DSC further supported the amorphous nature of tiamulin within the niosomal structure. Oral niosomal tiamulin exhibited significant advantages, including higher C_max and AUC_(0–∞) as well as prolonged MRT and elimination T_1/2 in Mycoplasma-infected broilers. These findings signify enhanced drug absorption and extended exposure, ultimately leading to improved bioavailability and precise antibiotic delivery in managing Mycoplasma infections in poultry. The convenience of oral administration in poultry farming further supports the suitability of this route for niosomal tiamulin, providing a practical and effective drug delivery strategy. Moreover, our study opens up strategic possibilities to address the threat of tiamulin resistance. The heightened bioavailability and protracted residence form the basis for refining dosage schedules and sustaining therapeutic concentrations at reduced doses and administration frequencies. This approach presents a viable means of diminishing the likelihood of subtherapeutic levels that promote resistance development. In essence, our study presents a forward-thinking strategy for promoting sustainable and efficient antibiotic utilization in veterinary medicine. Ongoing research and clinical validation are imperative to translate these promising findings into practical strategies for addressing Mycoplasma infections and curtailing antibiotic resistance in poultry.