Formulation Optimization And Quality Evaluation Of Tilmicosin-Loaded Nanostructured Lipid Carriers And Its Absorption Mechanism In Intestinal Epithelial Cells

Tilmicosin(TMS)is the first-line drug among macrolide antibiotics for preventing and treating the respiratory infection in livestock and poultry due to its strong antimicrobial effect.In addition,It has been exhibited the potential antiviral and anti-inflammatory activities.Oral administration is the most widely used for tilmicosin in veterinary clinic,due to its parenteral administration can easily lead to serious cardiac and renal toxicity.However,its low aqueous solubility,poor palatability and instability in gastric acid affect its clinical efficacy.Nanostructured lipid carriers(NLCs)are the efficient drug oral delivery system with high loading capacity and good stability,which can avoid drug leakage,increase drug permeability and improve oral bioavailability.Therefore,in the present study timicocin-loaded nanostructured lipid carriers(TMS-NLCs)was optimized by orthogonal design.Then the physicochemical and pharmaceutical properties of TMS-NLCs prepared by high shear-ultrasound method were characterized,and its absorption effect and mechanism in intestinal epithelial cells were investigated.Firstly,single factor method and orthogonal experimental method(L9,34)was used to find the optimized formulation and preparation process of TMS-NLCs.The optimum conditions acquired were determined as 1:9 of solid-liquid lipid ratio,3:10 of emulsion-lipid ratio,3:10 of drug-lipid ratio and 1:1 of cold-water dispersion dilution ratio.Additionally,Tween 80 was dissolved in oil phase,oil phase was added into water phase when oil-water was mixed,and pre-emulsion was dispersed into cold water within 60 sec after ultrasonication to form TMS-NLCs.The characterization about the optimum TMS-NLCs was 283.03 ± 6.64 nm of hydrodynamic diameter,0.219±0.027 of polydispersity index(PDI),-30.04±1.36 mV of zeta potential,(93.46±0.50)%of encapsulation efficiency(EE),(9.23±0.08)%of drug loading(DL).Under transmission electron microscopy,the surface of TMS-NLCs was smooth and spherical with relatively uniform size distribution and no agglomeration.Then,the quality evaluation of the optimal TMS-NLCs formulation were carried out.The TMS-NLCs suspension was lyophilized to form the freeze-dried powder.The interaction between drug and lipid matrix was then investigated by X-ray diffractometry(XRD),differential scanning calorimetry(DSC)and fourier transform infrared(FT-IR).The physicochemical stability of TMS-NLCs was comprehensively evaluated via its storage,high-low temperature and high-speed centrifugation,dilution and simulated gastrointestinal fluids stability.TMS-NLCs and free TMS were firstly separated by ultrafiltration centrifugation(UC),and then determined by HPLC.Its in vitro release behavior was studied by dialysis bag method.The results showed that 10%sucrose was the best cryoprotectant for lyophilization.The results of XRD,DSC and FT-IR were consistently revealed that TMS was dissolved in the lipid matrix of TMS-NLCs in the form of molecule,suggesting that the amorphous structure of TMS completely encapsulated in NLCs.Comprehensive evaluation about physicochemical properties revealed the TMS-NLCs with good stability.A UC-HPLC method with good repeatability,high accuracy,and simple operation was successfully established to determine entrapment efficiency and drug loading of TMS-NLCs.The standard curve for HPLC analysis of TMS was a good linear ranging from 1 to 20 μg/mL as indicated by R2=0.9999.The precision of the method as expressed by RSD%of inter-day and intra-day assays was less than 5%.The adsorption rate of Millipore membrane,the recovery rate of sample addition and the recovery rate of ultrafiltration can meet the methodological requirements.In vitro release studies showed that TMS-NLCs exhibited the sustained release behaviors in simulated gastric and intestinal fluids.All the results indicated that NLCs could be used as a new delivery carrier for oral administration of TMS.Ultimately,the permeability and absorption mechanism of TMS-NLCs were investigated in Caco-2 and MDCK-chAbcg2/Abcb1 cell monolayer.The integrity,permeability and morphology of cell monolayer were firstly evaluated to confirm the successful establishment of cell monolayer.MTT method was then used to screen the appropriate concentration range for transport and evaluate the toxicity of tracers and inhibitors.Finally,a series of single and bidirectional transport experiments were conducted to confirm the permeability of TMS-NLCs and elucidate the absorption mechanism in cell monolayer.The results showed that transepithelial electrical resistance(TEER)value of MDCK-chAbcg2/Abcb1 cell monolayer reached over 1700 Ω·cm2 6-days after culture,while its TEER values of Caco-2 kept above 450 Ω·cm 21 days after culture.Furthermore,there was no significant changes in TEER values after transport;both cells differentiated into intestinal epithelial-like structures under FE-TEM;the Lucifer Yellow Papp in both cells was less than 0.50 ×10-6 cm/s;and the Papp values of propranolol were equal to or greater than 10×10-6 cm/s.All the above results confirmed that the monolayer models of both cell lines were successfully established.MTT analysis showed that all the drug concentrations selected in the transport experiment had no obvious cytotoxicity,which could ensure the reliability of the transport results.TMS was found to be the substrate of P-gp using the MDCK-chAbcg2/Abcbl and Caco-2 cells,NLCs enabled TMS to escape the efflux effect of P-gp similar to verapamil.The results of bidirectional transport revealed that NLCs could not only enhance the uptake of TMS in Caco-2 or MDCK-chAbcg2/Abcb1 cell manolayer,but also reduce the efflux of P-gp,eventually enhancing the permeability of TMS.Moreover,the transcellular transport of TMS-NLCs in cell monolayers was an energy-dependent process,and the cellular internalization was mainly mediated by the caveolae/lipid raft-mediated endocytosis pathway revealed by pretreating Caco-2 or MDCK-chAbcg2/Abcb1 cell monolayers with different endocytosis inhibitors.In addition,the particles could be found in the basolateral medium.Taken together,TMS-NLCs has the potential to eacape the efflux of P-gp and avoid lysosome degradation of intestinal barrier to transport across cells in an intact form.