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Back to Journal »International Journal of Nanomedicine» Volume 14

Solid lipid nanoparticles with enteric coating can improve the stability, palatability and oral bioavailability of enrofloxacin

Authors: Li C, Zhou KX, Chen D, Xu W, Tao Y, Pan Y, Meng K, Shabbir MAB, Liu Q, Huang L, Xie S 

Published on March 1, 2019, Volume 2019: 14 pages, 1619-1631 pages

DOI https://doi.org/10.2147/IJN.S183479

Single anonymous peer review

Editor who approved for publication: Dr. Mian Wang

Chao Li,1 Kaixiang Zhou,1 Dongmei Chen,1,2 Wei Xu,1 Yanfei Tao,1 Yuanhu Pan,2 Kuiyu Meng,2 Muhammad Abu Bakr Shabbir,2 Qianying Liu,1 Lingli Huang,2 Shuyu Xie1 1 Veterinary drug residues ( HZAU) and MAO Key Laboratory of Veterinary Drug Residue Detection, Wuhan, Hubei; 2 Huazhong Agricultural University, Animal Products Quality and Safety Risk Assessment Laboratory, Wuhan 430070, Hubei Background: Enrofloxacin has poor palatability, variable oral bioavailability, and gastric mucosa Stimulating effect and light instability limit the application of enrofloxacin. ENR). An enteric-coated particle combining solid lipid nanoparticles (SLN) with an enteric coating was explored to overcome these shortcomings. Materials and methods: The sentinel lymph node loaded with ENR was prepared by thermal homogenization and phacoemulsification, and enteric-coated particles with the sentinel lymph node as the inner core were prepared by wet granulation and polyacrylic resin II (PRII) coating. Optimize formulations by using orthogonal or single factor test screening. Results: The best SLNs with loading capacity (LC) and price as inspection indicators consisted of 10 mL 3% polyvinyl alcohol/0.8 g ENR and 2.4 g octadecanoic acid. The size, LC, polydispersity index and zeta potential of SLN are 308.5±6.3 nm, 15.73%±0.31%, 0.352±0.015 and -22.3 mv, respectively. The best enteric-coated granules use 15% PRII as the coating material. The release of enteric-coated particles in simulated intestinal fluid (SIF, pH=8) was significantly faster than simulated gastric juice (SGF, pH=2), and was slower than SLNs and natural ENR. The particles showed good stability in the influencing factor test. The pellets showed similar daily feed intake to the control group and had a higher daily feed intake than ENR powder and single-coated pellets. Compared with ENR soluble powder, the area under the plasma concentration-time curve and the average retention time of enteric particles after intragastric administration increased from 4.26±0.85 µg h/mL and 6.80±2.28 hours to 11.24±3.33 µg h/mL and 17.97± 4.01 hours, respectively. Conclusion: Enteric-coated granules combined with SLNs and enteric coating significantly improve the stability, palatability, sustained-release performance and oral bioavailability of ENR. This new technology will be a potential measure to overcome similar shortcomings of other drugs. Keywords: Enrofloxacin, solid lipid nanoparticles, enteric coating, palatability, bioavailability, photostability

Enrofloxacin (ENR), the full name of ethyl ciprofloxacin, is the first animal-specific fluoroquinolone drug successfully developed by Bayer in Germany1, because of its broad antibacterial spectrum, strong bactericidal activity, and few adverse reactions. 2 However, , ENR has a bitter taste. 3,4 Poor palatability limits its oral administration, because animals with a keen sense of taste, especially pigs and cattle, are more likely to reject bitter drugs. 5 Oral preparations ENR currently on the market cannot conceal the bitter taste of ENR, which limits the oral clinical application of pigs. In addition, the drug has a short elimination half-life (T1/2β) and mean residence time (MRT) in most mammals, and its bioavailability is variable due to its poor water solubility. Therefore, there is a need for a controlled-release preparation with good palatability, less gastric irritation, and good absorption.

Some taste-masking formulas have been prepared to improve the palatability of ENR. Chun et al. prepared ENR carbopol complex to mask the bitter taste, but due to the lack of direct animal experiments, its taste masking effect is uncertain. 4 Double-layered ENR particles may effectively mask the bitter taste of ENR, but the release data of the particles are not provided in simulated gastric juice (SGF). 3 Preparations prepared from hydrogel materials (sodium alginate, chitosan) have low pH-dependent release. 6,7 Therefore, the large release of these hydrogel formulations in SGF may cause gastric irritation due to the irritating effect of enrofloxacin on the gastric mucosa. This may affect the oral compliance of the animal. Reducing the release of ENR in the stomach may be a potential way to achieve excellent release because ENR has maximum solubility at low pH (>100 mg/mL at pH=1). 8 EC In addition, ENR has high photodegradability. 9 It should be stored away from light, which increases storage and transportation costs. Therefore, the development of a new dosage form with good palatability, less gastric irritation and good stability is an urgent need for ENR.

Solid lipid nanoparticles (SLNs) are a new type of nano-drug delivery system with high melting point natural or synthetic solid lipids as the backbone material, because they have good physiological compatibility and outstanding physical and chemical properties (nano size and large ratio). Surface area effect). 10 It is often used to improve permeability and palatability, 11 to achieve controlled release, 12, 13 and to increase oral bioavailability. 14 However, due to the maximum solubility of ENR in gastric juice, the release of ENR from SLN in the stomach is very rapid. In addition, the sentinel lymph node cannot completely encapsulate the drug. Therefore, simple nanoparticles may not be able to effectively reduce the release of ENR in the stomach, achieve the best sustained-release effect, and completely mask the bitter taste.

Coating technology, including enteric coating, is a production process in which polymer materials are coated on drugs by chemical or physical methods. 15 After coating, the chance of contact between the drug and animal taste buds is reduced, 16 the drug can be directly irradiated from natural light. The coating of functional materials (ie, water-insoluble, enteric-coated materials) can give the drug specific target release characteristics in the gastrointestinal tract. In recent years, coating technology has been widely used to increase palatability and delivery of drugs to specific targets. Choi and Kim used Eudragit acrylic resin to coat peony, which masked the bitter taste of the drug. 17 There are also reports that when coated with a mixture of gelatin, partially hydrogenated soybean oil and glyceryl monostearate, the taste of gabapentin is significantly improved. 18 Alsulays et al. reported that the cumulative release of lansoprazole enteric coating was less than 10% under acidic conditions, and as high as 80% during the alkaline buffer phase. 19 The stability of fluidized bed coating to all-trans retinoic acid under high concentration conditions is reported to be enhanced by light (4,500±500 lx). 20 However, due to ENR's maximum bitterness, a simple coating may not significantly improve palatability. It happens that coating tablets or granules with enteric materials may reduce the dissolution of the drug in the stomach, but cannot obtain sufficient taste masking.

In order to overcome the shortcomings of simple coating and insufficient taste-masking and controlled-release properties of SLNs, this study creatively combined SLNs with coating to prepare ENR taste-masking enteric granules. Optimize the formula by using orthogonal experiments or single factor screening. The properties, stability, palatability and sustained release in vitro and in vivo were further evaluated.

ENR standard (98% ENR content) was purchased from Dr Ehrenstorfer. ENR (≥96.0% ENR content) was purchased from Wuhan Konglong Century Technology Development Co., Ltd. (Wuhan, China). ENR soluble powder was purchased from Guangdong Wen's Dahuanong Biotechnology Co., Ltd. (Guangdong, China). Stearyl acid and behenic acid were purchased from Aladdin (Shanghai, China). Polyvinyl alcohol (PVA) and Poloxamer 188 were purchased from Sigma (St. Louis, MO, USA). Polyvinylpyrrolidone (PVPK30) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Polypropylene resin II (PRII) was purchased from Shanghai Yuan Biotechnology Co., Ltd. (Shanghai, China). Ethyl cellulose (EC) and sodium carboxymethyl cellulose were purchased from Sinopharm Chemical Reagent Co., Ltd.

27 clinically healthy three-way hybrid pigs (15-20 kg) were obtained from the Animal Husbandry Engineering Center of Huazhong Agricultural University (HAZU) (Wuhan, China). The pigs were domesticated in the laboratory animal room of the National Reference Laboratory for Veterinary Drug Residues (HZAU), and they were freely fed non-toxic feed and fasted for 1 week. The relative humidity and temperature of the breeding environment are maintained at 45%-65% and 18°C-25°C, respectively. All experimental protocols are authorized by the HAZU Institutional Animal Care and Use Committee (approval number: HZAUSW-2018-009) and follow the guidelines of the Hubei Provincial Department of Science and Technology.

Preparation of ENR enteric-coated granules

The production process of ENR enteric-coated granules is shown in Figure 1.

Figure 1 Production process of enrofloxacin enteric-coated granules containing SLNs core. Abbreviations: ENR-SLNs, SLNs loaded with enrofloxacin; RT, room temperature; SLNs, solid lipid nanoparticles.

SLNs are produced by hot melt homogenization and ultrasonic emulsification methods. In short, a certain amount of ENR is dissolved in the molten solid lipid under the stirring of a magnetic stirrer (9001, Shanghai Huxi Analytical Instrument Factory Co., Ltd., Shanghai, China). After being completely dissolved, a preheated emulsifier solution of a certain volume and concentration is quickly poured into the molten lipid solution under stirring to form colostrum. A 6 mm microprobe (VCX 130 Vibra-CellTM, Sonics & Materials, Inc., Newtown, CT, USA) with 95% amplitude was used to sonicate the primary emulsion for 4 minutes to form a hot O/W emulsion. In the preparation process of O/W emulsion, the drug loading (LC) of SLNs was used as the evaluation index, and the emulsifier was optimized by orthogonal test. The type, concentration and volume of the emulsifier are also selected as variables. The corresponding levels are PVA, PVP, Poloxamer 188; 1%, 2%, 3%; 10 mL, 15 mL, 20 mL, respectively. After determining the variables and their levels, SPSS software (version 20, IBM) was used to design the orthogonal experiment (Table 1). In orthogonal experiments, one to nine SLNs consist of 15 mL 3% PVA, 20 mL 3% poloxamer 188, 20 mL 2% PVA, 10 mL 1% PVA, 15 mL 1% poloxamer 188, 20 mL 1% PVP K30, 10 mL 3% PVP K30, 15 mL 2% PVP K30, and 10 mL 2% Poloxamer 188 are 0.8 g ENR and 2.4 g octadecanoic acid, respectively. The hot O/W nanoemulsion was cooled at room temperature to form SLN.

Table 1 The factors and levels of L9 (34) orthogonal design are referred to as: PVA, polyvinyl alcohol; PVPK30, polyvinylpyrrolidone K30.

Preparation of coated granules with SLNs as the core

After preparing ENR-loaded SLNs (ENR-SLNs), a certain amount of sucrose and starch were added to the nanosuspension during the stirring process to prepare preliminary soft materials. Put the preliminary soft material into a drying oven (DGX-9243B-1, Fuma Testing Equipment Co., Ltd., Shanghai, China) (50°C, 4 hours) to prepare a soft material suitable for granulation. After granulating with a granulator (YK-60, Zhongcheng Pharmaceutical Machinery Co., Ltd., Changsha, China), the prepared granules were dried again (50°C, 1 hour) to remove moisture. Use the coating pan (BY-400, Zhongcheng Pharmaceutical Machinery Co., Ltd.) to use 10% (W/W) ethyl cellulose or 5%, 10% and 15% (W/W) polyacrylic resin ( PRII) Coating.) ENR taste-masking enteric-coated granules are obtained after coating. The optimal coating content of PRII was evaluated by in vitro release performance.

Preparation of simple coated enteric granules

Except for the production process of ENR-SLNs, the preparation process of the simple coated enteric-coated granules is the same as the preparation process of the above-mentioned ENR double taste-masking enteric-coated granules. In short, 60% starch, 15% sucrose and 10% ENR are completely mixed. Then, 5% starch used as a binder was made into a starch paste by boiling with 50 mL of water. The prepared starch paste is mixed with the above mixture and granulated to form granules. The granules are coated with 10% PRII (750 mL of 2% alcohol solution).

Determination of particle size, polydispersity index (PDI) and zeta potential

The morphology of SLN was observed by scanning electron microscope (JSM-6390LV, NTC, Co., Ltd., Tokyo, Japan). The size, PDI and zeta potential were measured at 25°C by using Zetasizer ZX3600 (Malvern Instruments, Worcestershire, UK) and laser particle size analyzer BT-9300S (Better, Liaoning, China). The sample was diluted in distilled water to ensure that the concentration used for size and PDI testing was 2.7 mg/mL, and the concentration used for zeta potential measurement was 0.3 mg/mL to obtain the best thousand counts of 20-400 per second. All determinations were repeated in triplicate by using separate formulations.

Load capacity (LC) and package efficiency (EE) of SLN

The determination of LC and EE of SLN was described in our previous work. 21 In short, the nanosuspension was collected by centrifugation at 14,000 rpm (Hitachi Centrifugation CR21GIII; Hitachi Koki Co., Ltd., Japan) for 60 minutes at 4°C. The free ENR in the supernatant was measured by a Waters 2695 series HPLC equipped with a Waters 2587 UV detector (Waters Corp., Milford, MA, USA) to determine EE. The precipitated SLN was resuspended in distilled water and lyophilized for 48 hours (freeze drying system; Labconco, Missouri, USA) to determine the LC. After freeze-drying, 10 mg of the dried nanoparticles were added to a 15 mL tube containing 10 mL of acetonitrile/water solution (V/V; 1:1) and placed in a boiling water bath to destroy the nanoparticles. Add the heated SLN to a 10 mL volume and centrifuge at 8,000 rpm for 10 minutes. The filtered supernatant was injected into HPLC for analysis. This assay was repeated in triplicate using different samples from related formulations. EE and LC are defined as follows:

ENR particles were released in vitro in SGF (pH=2, 100 mL containing 2.0 g NaCl and 3.2 g pepsin, and then adjusted to pH 2 with HCl) and simulated intestinal fluid (SIF; pH=8, 1,000 mL SIF containing 6.8 g KH2PO3 And 10 g trypsin, and then use NaOH solution to adjust the pH of the pig to 8 through the dissolution tester RC806 (Tianjin Tianda Tianfa Co., Ltd., Tianjin, China). The maximum solubility of ENR in SGF and SIF is 1.99 and respectively 0.31 mg/mL. According to tank conditions, 1 g enteric-coated granules (containing 100 mg ENR) or 155 mg enteric-coated granules (containing 15.5 mg ENR) (n=3) are placed under stirring with a rotating propeller at 38°C. 200 mL of buffer solution in a dissolution cup at 100 revolutions per minute. 1 mL samples are collected from the dissolution cup at a fixed time to determine the released drug. After each sample, an equal volume of fresh SGF or SIF is added to keep the volume constant. Measured by HPLC The drug concentration in the release medium. The cumulative release rate and time are used as the ordinate and abscissa to draw the cumulative release curve. ENR particles with different types and contents of coating materials are released in vitro to select the best formulation. The same process will be used to draw the cumulative release curve. The in vitro release of natural ENR, ENR-SLN and simply coated particles was used as a control. The particle size and loss on drying of the various particles meet the requirements of the Chinese Veterinary Pharmacopoeia (2015). 22

The palatability of ENR enteric-coated granules was studied through pig feeding and drinking experiments. In short, 15 pigs were randomly divided into 5 groups with 3 pigs in each group. The 5 groups included blank group, ENR powder group, simple enteric-coated granules, 10% sucrose ENR enteric-coated granules, 15% sucrose ENR enteric-coated granules group. Before the experiment, the average daily feed intake of each group was measured for 3 consecutive days to ensure that the selected pig herd was healthy and had a normal appetite, and to eliminate individual possible errors. During the experiment, the pigs were freely fed medicated feed with different ENR formulas for 5 consecutive days. At 9 am every day, enough feed without or mixed ENR formula is added to the tank, and the remaining feed is weighed at 9 am the next day. The daily feed intake of each group is equal to the feed added on the first day minus the remaining feed on the second day. After the 7-day cleaning period of the daily feed intake experiment, 6 pigs were randomly selected to study the taste of ENR-SLN through 5 days of daily water consumption. In short, 6 pigs were randomly divided into control group and ENR-SLNs group (n=3). At 9 am every day, add excess drinking water (corresponding to 25 mg ENR/L) without or mixed with ENR-SLN, and the rest is weighed at 9 am the next day. The daily water consumption of each group is equal to the water added on the first day minus the remaining water on the second day.

The particle size is determined by the double sieve method of "Chinese Veterinary Pharmacopoeia" (2015). In short, 8.0 g of the drug is placed in a horizontal sieve and sieved for 3 minutes from left to right. Weigh the particles that cannot pass the 2mm aperture No. 1 sieve and the powder that can pass the 0.2mm aperture No. 5 sieve, and calculate their proportion to the total particles.

The ENR enteric coated granules (8.0 g) were spread flat in an open tray and placed in a 40°C dry box. The pellets are weighed at fixed time points until the weight of the pellets remains constant. The weight loss of the sample during drying is calculated by subtracting the remainder from the added amount. This assay was repeated in triplicate using different batches of formulations.

The stability of ENR enteric-coated granules is evaluated by high temperature, high humidity, strong light and other influencing factors tests. Put the enteric-coated granules in a container, and then place them at 60°C and 40°C (high temperature test), 25°C and 90%±5% (humidity test) or 4,500±500 l× (light) for 10 days, respectively . Samples were taken on the fifth and tenth days to evaluate the changes in appearance, drug content, particle size and in vitro release properties.

Before the experiment, 12 healthy pigs were randomly divided into two groups (ENR soluble powder group and enteric-coated granule group), each with 6 pigs. ENR enteric-coated granules (content: 10%) and ENR soluble powder (Guangdong Wen's, content: 5%) were resuspended or dissolved in 20mL CMCC-Na, and 2.5 mg/kg body weight was administered to pigs by gavage. Blood was collected from the anterior vena cava of the pig, and blank plasma was collected before administration. After administration, blood was collected at 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12, 24, 36, 48, and 72 hours. 3 ml of blood was collected at each time point, placed in a centrifuge tube containing heparin sodium, and centrifuged at 4,000 r/min to separate plasma. After pretreatment, the plasma drug concentration was detected by high performance liquid chromatography.

The pharmacokinetic parameters were calculated by WinNonlin software (version 6.4; Pharsight Corporation, Mountain View, CA, USA). By observing the plasma concentration-time curve, the maximum plasma concentration (Cmax) and the time to reach Cmax (Tmax) are obtained. The area under the plasma concentration-time curve (AUC0-last) is determined using the log-linear trapezoidal rule.

To quantitatively measure ENR, 0.8 mL of plasma was measured into a 10 mL centrifuge tube containing 1.2 mL of acetonitrile. The plasma and acetonitrile were mixed thoroughly under vortex for 2 minutes to precipitate the protein, and then centrifuged at 12,000 r/min for 20 minutes to obtain the supernatant. The supernatant was dried in nitrogen and reconstituted with 800 μL of mobile phase. The liquid was filtered through a 0.22 μm filter and subjected to HPLC (Waters Corp.) for detection. The chromatographic conditions are as follows: Column: Agilent SB C18 (250 × 4.6 mm × 5 μm); detection wavelength: 278 nm; column temperature: 25°C; mobile phase: 0.1% formic acid (phase A) and acetonitrile (phase B) 86:14; Flow rate: 1.00 mL/min; Injection volume: 40 μL. Use the standard curve to determine the content of ENR in solution and plasma. The HPLC method has been validated in terms of linearity, accuracy, precision, and detection and quantification limits. The linear range of ENR is 0.1 to 1.8 μg/mL (R2=0.9985). The detection limit and quantification limit were 0.025 and 0.08 μg/mL, respectively. The relative standard deviation of precision is less than 2%, and the recovery rate of three different additive concentrations is 96.8%~103.3%.

Data are expressed as mean ± SD. Statistical significance is defined by SPSS software (version 20, IBM) as P values ​​of 0.05 and 0.01.

The maximum solubility of ENR in molten octadecanoic acid and behenic acid is 33.33%. Due to its low price, octadecanoic acid was chosen as the lipid matrix. The best ratio of drug to lipid is 1:3. According to the analysis of orthogonal experiment, when PVA is used as emulsifier, the size of ENR-SLNs is in the nanometer range. The PDI of samples 1 and 3 were 0.229±0.007 and 0.155±0.030, respectively, while the other samples exceeded 0.5 (Table 2). The zeta potentials of all nine samples are negative. Taking into account the practicality of enteric-coated particles, LC is the most important factor in choosing a dosage form. The factors affecting LC are: emulsifier type> concentration> volume (Table 2). The final optimal aqueous phase is 10 mL 3% PVA per 0.8 g ENR and 2.4 g octadecanoic acid.

Table 2 Optimization of emulsifier by orthogonal test Note: K1, K2, K3 are the average of the three levels of each factor; R is the maximum value of K1, K2, and K3 in each level and the mixed value Difference. Abbreviations: LC, load capacity; PDI, polydispersity index.

As shown in Figure 2, the scanning electron microscope observation shows that ENR-SLN is spherical and has a relatively uniform volume distribution. The average size, PDI and zeta potential of the best SLN are 308.5±6.3 nm, 0.352±0.015 and -22.3 mv, respectively. The LC and EE of the best SLN are 15.73%±0.31% and 68.78%±1.35%, respectively. Then the ENR-SLNs with these physicochemical properties will be used in future research.

Figure 2 Scanning electron micrograph of the sentinel lymph node with enrofloxacin. Abbreviation: SLNs, solid lipid nanoparticles.

The effect of coating material on the release of granules in vitro

The in vitro release of ENR-SLNs and enteric-coated particles in SIF and SGF is shown in Figure 3 and Figure 4. In SGF (pH=2), ENR-SLNs and simple coated particles showed a closed release profile, but the release rate was slightly slower than that of the local ENR. Please note that 96.56% of natural ENR is rapidly dissolved in simulated SGF within 1 hour, while approximately 95.62% is released from SLNs within 4 hours, and 95.17% is released from simple coated granules within 6 hours. It shows that the sustained-release performance of ENR-SLNs and simply coated granules is not obvious. The in vitro release of particles with different coating materials was significantly slower than that of natural ENR, ENR-SLN and simple coated particles (Figure 3). As the PRII content increases from 5% to 15%, the release rate of enteric-coated particles decreases. In order to achieve the slowest release in SGF, 15% PRII was selected as the best coating material. When 10% ethyl cellulose is used, the release rate of the particles is the slowest compared with other particles. Within 36 hours, only half of the ENR was released from the particles.

Figure 3 The cumulative release curve of SLNs and particles in simulated SGF (pH=2) (n=3). Abbreviations: EC, ethyl cellulose; ENR, enrofloxacin; ENR-SLNs, enrofloxacin-loaded SLNs; PR, polyacrylic resin; SGF, simulated gastric juice; SLNs, solid lipid nanoparticles.

Figure 4 The cumulative release curve of SLNs and particles in simulated SIF (pH=8) (n=3). Abbreviations: ENR, enrofloxacin; ENR-SLNs, enrofloxacin-loaded SLNs; PR, polyacrylic resin; SIF, simulated intestinal fluid; SLNs, solid lipid nanoparticles.

In SIF (pH = 8), ENR-SLN shows a closed release profile, but compared with natural ENR, the release rate is slower (Figure 4). Note that 97.15% of natural ENR dissolves in SIF within 4 hours, while SLNs releases about 94.27% within 8 hours, indicating that ENR-SLNs has a certain slow-release performance than natural ENR. Enteric-coated particles with 15% PRII as the coating material are slower than natural ENR and ENR-SLN enteric-coated particles, but significantly faster than those in SGF (pH=2). These results indicate that the combination of enteric-coated particles and SLN with 15% PR coating has a localized release in the intestine.

The palatability of different ENR formulations

The normal daily feed intake and the daily feed intake of different ENR formula feeds are shown in Table 3. In the first 3 days of the experiment, the daily feed intake of different groups of pigs was similar, indicating that the selected pigs were physiologically consistent. During the experiment, the daily feed intake of pigs in the ENR powder group (5 mg/kg body weight) dropped sharply from about 1.5 kg/day to less than 0.2 kg/day. In contrast, the daily feed intake of pigs in the pellet group (consisting of 10% or 15% sucrose) (5 mg/kg bw) was not significantly different from the control and normal daily feed intake. In simple enteric-coated granules (5 mg/kg bw), the daily feed intake of pigs in the granule group was lower than that of the control and granule groups. The average water intake of the control group was 9.7±1.2 L/group/day, while the average daily water intake with ENR-SLNs (25 mg/L) dropped to 7.5±1.6 L/group/day. These results indicate that SLNs alone cannot completely overcome the palatability of ENR. The combination of enteric-coated particles with SLNs and enteric coating significantly improves the palatability of ENR, which will help ENR's clinical application.

Table 3 Daily feed intake of pigs (mean ± standard deviation, n=3) Note: The control group only feeds; 10% pellets contain 10% sucrose in the pellets; 15% pellets contain 15% sucrose in the pellets. a The statistical significance compared with the control is P<0.01. b Compared with mixed feed ENR, the statistical significance is P<0.01. c Compared with simple coating, the statistical significance is P<0.05. d Compared with simple coating, the statistical significance is P<0.01. Abbreviations: ENR, enrofloxacin.

Properties of ENR enteric-coated granules

The best ENR enteric-coated granule dosage form is used; 15% PRII is the coating material, and 10% sucrose is the flavoring agent. ENR enteric-coated granules are evaluated in accordance with the Chinese Veterinary Pharmacopoeia (2015). The average proportion of granules that pass the No. 5 sieve and those that cannot pass the No. 1 sieve account for 7.60%±0.12% of the total granules, which is lower than the 15% required by the Pharmacopoeia. Therefore, the size of most ENR enteric-coated granules (92.4%) is 0.2-2 mm. The average weight loss on drying is 1.90%±0.10%, which is lower than the 2.0% required by the Pharmacopoeia.

The test results of the influencing factors of high temperature, high humidity and strong light of granules are shown in Table 4. The appearance of enteric-coated granules was white, and there was no change during the whole process of the influencing factor test. Under strong light, only 4.89% and 6.0% of the ENR in the particles degrade after 5 and 10 days, respectively, and up to 15% and 20% of the natural ENR are degraded after 5 and 10 days, respectively. These results indicate that the particles can improve the stability of ENR to strong light. ENR particles are stable at a high temperature of 40°C, but are slightly sensitive to a high temperature of 60°C. Under high humidity, 4.82% and 7.0% of ENR in the pellets degrade after 5 and 10 days, respectively. The particle size of ENR enteric-coated particles did not change significantly. After 10 days of influencing factors test, the dosage and particle size of ENR enteric-coated particles meet the requirements of "Chinese Veterinary Pharmacopoeia" (labeled amount: 90% to 110%, unqualified particle size: <15%). The release rate of ENR enteric-coated particles in 5 days and 10 days under high temperature and strong light conditions did not change significantly, but the release rate was slightly faster under high humidity conditions in 10 days (Figure 5). These results indicate that the enteric-coated particles have good stability.

Table 4 Inspection of particle influencing factors (mean ± standard deviation, n=3)

Figure 5 The influence of the influencing factor experiment on the release ability of enteric-coated particles (n=3). Note: (A) The effect of high temperature on the release ability of enteric-coated particles. (B) The effect of high humidity on the release ability of enteric-coated particles. (C) The effect of strong light on the release ability of enteric-coated particles. HT: high temperature (40°C); HI: high humidity (25°C, 90%±5%); HL: high light (4,500±500 lx).

After intragastric administration of the particles, ENR in plasma quickly reached a peak concentration of 0.52±0.05μg/mL in 3.33±1.03 hours, and then slowly decreased and maintained above 0.03μg/mL for 72 hours. In contrast, the soluble powder reached a peak of 0.60±0.12 μg/mL faster at 1.12±0.44 hours, and then rapidly dropped to 0.03 μg/mL 24 hours after intragastric administration (Figure 6). The pharmacokinetic parameters are shown in Table 5. The AUC0-last, T1/2β, and MRT of the particles are 11.24±3.33 μg h/mL, 12.59±3.53 hours and 17.97±4.01 hours, respectively, and these parameters are 4.26±0.85 μg h/mL, 4.71±1.58 for soluble powder, respectively Hours and 6.80±2.28 hours (Table 5). Compared with the soluble powder, the bioavailability, T1/2β and MRT of the prepared ENR enteric-coated particles are increased by 2.64, 2.67 and 2.64 times, respectively. The results show that the prepared granules have a slow-release effect and improve the oral bioavailability of ENR.

Figure 6 Plasma enrofloxacin concentration curve-time of prepared granules and reference preparation (soluble powder) in pigs (n = 6). Remarks: Granules: 10% Enrofloxacin enteric-coated granules; Powder: 5% Enrofloxacin soluble powder.

Table 5 The pharmacokinetic parameters of ENR after swine oral administration of ENR enteric-coated granules and ENR soluble powder (mean ± standard deviation, n=6) Note: a, b, c, d are statistically significant compared with soluble powder P <0.01. Abbreviation: AUC0-last, area under the curve; CL, body clearance; Cmax, maximum plasma concentration; ENR, enrofloxacin; F, relative bioavailability; MRT, average residence time; T1/2β, elimination half-life; Tmax, Time to reach Cmax.

Coating technology and nanoparticles are often used to produce functional formulations with controlled release capabilities23,24 and targeted release capabilities25 and improve the palatability of undesirable drugs. 26 However, single-coated granules, tablets, capsules and other dosage forms will be eaten by animals. In this way, the coated drug will be released and attached to the taste buds of the animal's mouth. The study also showed that the daily feed intake of pigs using single-coated pellets was improved compared to those using ENR powder, but still improved compared to the control group. These results indicate that the single-coated particles did not completely resolve the bitter taste of ENR. According to reports, lipid nanoparticles are also an effective way to improve the palatability of bitter drugs because the drugs can be encapsulated and distributed in lipid materials. 3 At the same time, SLNs are often used to improve the oral bioavailability of encapsulated drugs because of their adhesiveness.27 However, SLNs cannot completely encapsulate drugs and are usually released suddenly, so nanoparticles may not be able to completely mask the bitter taste of encapsulated drugs. Our research also showed that the daily water consumption of pigs in the octadecanoic acid nanosuspension group was slightly lower than that of normal pigs. In order to overcome the problems of poor palatability and variable bioavailability of ENR, enteric coated granules were developed with SLNs as the core. Wet granulation technology is used to prepare granules containing ENR-SLNs, which are then coated with polymer film-forming materials.

In the preparation of sentinel lymph nodes, the choice of lipids is very important in formulation design. Lipids must be selected according to their good ability to dissolve and encapsulate drugs. Our previous work showed that compared with other melted lipids, fatty acids have a higher solubility for ENR because ENR is freely soluble in acid solutions. 28 Considering that short-chain fatty acids with low melting points are not convenient for storage and transportation, palmitic acid, capric acid and lauric acid are not considered. Therefore, the maximum solubility of ENR in molten long-chain fatty acids of octadecanoic acid and behenic acid was determined to select the best encapsulating lipid matrix. Due to its low price and satisfactory solubility, octadecanoic acid was finally chosen. In order to obtain a higher LC to meet clinical practicability, an orthogonal experimental design was used to optimize the emulsifier. Based on LC, size and PDI, the best SLN formulation containing 10 mL of 3% PVA was selected for further characterization. According to previous reports, palatability and cheap price, after ENR-SLNs are formulated, different amounts of sucrose and starch are added to the nanosuspension as flavoring and diluents.

In order to achieve the best palatability and sustained release, different coating materials were selected and the in vitro release was evaluated. In SGF (pH=2), the release of particles with different coating materials was significantly slower than that of natural ENR and ENR-SLN. These results indicate that particles with different PRII contents are relatively stable in gastric juice. The sustained release of particles containing 5% and 10% PRII showed no significant difference, while the particles containing 15% PRII had the slowest release in SGF. Therefore, 15% PRII content was used in the best formula. When 10% ethyl cellulose is used as the coating material, the release rate of the particles is the slowest compared with the particles with PRII as the coating material. However, ethyl cellulose is a water-insoluble substance, and its solubility has nothing to do with pH. 29 According to the solubility of ENR, particles containing 10% ethyl cellulose will be released very slowly in simulated SIF (pH=8). Taking into account the gastrointestinal emptying time of pigs (≤ 50 hours), 30 particles use 15 % PRII but not formulated with 10% ethyl cellulose. In addition, ENR is a concentration-dependent antibiotic; effective antibacterial activity requires a higher Cmax level. In SGF (pH=2), the release of particles with 15% PRII is slower than the release of natural ENR and ENR-SLNs, but in SIF (pH=8) it is faster than itself. ENR may be completely released within the gastrointestinal emptying time of pigs, and can maintain a higher Cmax than the ethylcellulose coating. These results indicate that the particles combining SLN and 15% PRII coating have a localized release in the intestine and reduce gastric irritation. Although we did not use ethyl cellulose as a coating material in our study, we proved that ethyl cellulose can be coated with time-dependent antibiotics (ie, macrolides) to prepare excellent sustained-release preparations. The release of ENR from internal SLN may be due to a combination of diffusion and erosion. 31-33 Because of the highest solubility under acidic conditions, ENR-SLN is released very quickly in SGF (pH=2). Burst release may be the rapid dissolution of free and unembedded drugs and the absorption of ENR on the surface. For enteric-coated particles containing SLNs, due to the insolubility of outer PRII in SGF, the main mechanism of drug release may be diffusion from the semipermeable membrane of PRII coating. Due to double obstruction, the speed of enteric-coated particles is significantly slower than that of sentinel lymph nodes. In SIF, the outer PRII coating of the particles dissolves quickly, causing the particles to disintegrate. The release rate of the particles in SIF mainly depends on the disintegration rate of the particles and the diffusion of the drug from the sentinel lymph node. Therefore, the ENR in the particles is released faster in SIF than in SGF.

The palatability of granules coated with 15% PRII was evaluated. The daily feed intake of pigs in the pellets and SLNs group (consisting of 10% or 15% sucrose) (5 mg/kg bw) was not statistically different from the control and normal days (P<0.01) higher than ENR powder group and simple enteric granule group. In order to reduce the price of the formula, 10% sucrose is used as a flavoring agent. These results indicate that the enteric-coated particles combining SLNs and enteric coating significantly improve the palatability of ENR, which is helpful for the clinical application of ENR.

The particle size and loss on drying of ENR particles meet the requirements of the Chinese Veterinary Pharmacopoeia (2015). The prepared enteric-coated particles have good stability under high temperature, high humidity and strong light. In addition, the particles can significantly improve the stability of ENR under strong light. This may be due to the light-shielding effect of lipids and coating materials. The appearance, drug content and particle size range of enteric-coated particles did not change significantly under high temperature, high humidity and strong light. This result indicates that the prepared enteric-coated particles have good stability. The core material of enteric-coated particles also includes SLN, sucrose and starch. Therefore, it is impossible to study only the internal SLN changes. The in vitro release changes of the particles under high temperature, high humidity and strong light were compared with the release of the initial formulation to indirectly evaluate the stability of internal SLNs. The in vitro release profile of enteric-coated particles did not change except for 10 days under high humidity conditions. This indicates that the enteric-coated particles have good stability under high temperature and light, but are slightly sensitive to high humidity. Therefore, ENR enteric-coated granules need to take good moisture-proof measures during storage and transportation. The good stability of the enteric-coated particles may be due to the excellent stability of the internal nanoparticles and the PRII coating. According to Wang et al., the octadecanoic acid nanosuspension exhibited excellent stability at room temperature for 9 months, with no change in particle size and zeta potential, and a slight decrease in LC. 34 It is well known that the stability of nanoparticles in solid powder is better than that in liquid. The good stability of ENR-SLN in enteric-coated granules is predictable. Therefore, the stability of ENR-loaded octadecanoic acid SLNs was not studied separately in this study.

The prepared enteric-coated particles have a slow-release effect after intragastric administration, and the bioavailability is improved. Compared with the soluble powder, the bioavailability, T1/2β and MRT of the prepared ENR enteric-coated particles are increased by 2.64, 2.67 and 2.64 times, respectively. The improved bioavailability, controlled release and palatability of enteric-coated particles may be closely related to gastrointestinal transport (Figure 7). After pigs take the pellets, the octadecanoic acid encapsulation and PRII coating isolate ENR and taste buds in the mouth, so the taste is very good. When the particles subsequently reach the stomach, the internal ENR of the enteric-coated particles is less released during the time of gastric emptying (2-6 hours), and there is almost no irritation to the gastric mucosa. The increase in particle bioavailability reduces the amount of ENR metabolized in the gastric juice, resulting in more ENR reaching the intestine. When the particles reach the small intestine, due to the dissolution of the outer layer of PRII, the gastric emptying begins to disintegrate, and the pH of the small intestine is close to neutral. The ENR-SLN space is released after the particles disintegrate. Due to the nano size and large surface area, ENR-SLN has high adhesion and permeability, so the particles show higher oral bioavailability.

Figure 7 The transport process of enrofloxacin particles in the gastrointestinal tract of pigs. Abbreviations: ENR-SLNs, SLNs loaded with enrofloxacin; SLNs, solid lipid nanoparticles.

In our previous work, we demonstrated that ENR-SLNs exhibit good transmembrane transport, resulting in higher intracellular accumulation and stronger antibacterial activity as well as longer in vitro antibacterial time against intracellular Salmonella. 21 Pharmacokinetics showed that enteric-coated particles have similar Cmax compared with ENR soluble powder, and the plasma concentration is maintained for a longer time. The similar enhancement of absorption of Cmax and enteric-coated particles and the enhanced antibacterial activity of internal nanoparticles against intracellular bacteria will not easily produce drug resistance, which will help treat the intestinal tract caused by intracellular bacteria such as Salmonella and Lawsonia intracellularis Infect. The pharmacodynamics of enteric-coated particles and their effects on intestinal pathogens will be studied to evaluate possible future clinical applications.

Select lipid materials, emulsifiers, coating materials and other auxiliary materials through orthogonal test or single factor screening to formulate the best ENR enteric-coated granules. ENR enteric-coated particles combined with SLN and enteric coating significantly improve the stability, palatability, controlled release and oral bioavailability of ENR. The combination of SLNs and enteric coating will be a potential measure to improve the stability, palatability, sustained release and bioavailability of other drugs.

This work was supported by the National Key Research and Development Program (2017YFD0501402) and the National Natural Science Foundation of China (approval number: 31772797).

The authors report no conflicts of interest in this work.

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