Improving Oralabsorption Of Veterinary Antibacterial Drugs And Its Mechanism Of Composite Nanosystem

Oral administration（mixed drinking or mixed feeding）is the most economical,convenient and safe way in large-scale livestock farming,and is also the mainstream way in the current breeding process.However,FDA statistics found that most of the drugs were poorly absorbed orally,which affected their clinical efficacy.The low absorption efficiency of oral drugs is mainly attributed to two main reasons:first,the physical and chemical properties of drugs,such as low solubility and low permeability,limit their oral absorption;Second,physiological barriers such as the destruction of strong stomach acid,mucus clearance and epithelial barrier efflux hinder drug absorption.These factors are the key to the drug molecules entering the bloodstream inefficiently or at all.However,traditional oral formulations such as granules,tablets,premixes,soluble powders and suspensions are not effective in addressing these challenges of oral delivery.Therefore,how to promote the oral application or improve the oral absorption of drugs has been the majority of researchers committed to solve the scientific problem.Some big pharmaceutical companies,such as Johnson&Johnson Pharmaceuticals and Pfizer-Bio NTech,are trying to fix these problems by reinventing compounds.However,time-consuming and laborious investment does not necessarily yield good results,and potentially increases the cost of trial and error.And most compounds cannot be used to modify,for example,large molecules of protein or peptides,which is also urgent to propose a new solution.In recent years,the development of new preparation technologies has provided opportunities for improving oral drug absorption,such as solid dispersion technology,microspheres and microcapsules technology and liposome technology.These techniques have improved the oral absorption of drugs to varying degrees.Therefore,novel techniques of preparation may be an effective strategy to promote oral absorption of drugs.In this paper,two oral delivery systems based on the new preparation technology were designed and constructed to improve the oral absorption of low solubility and low permeability veterinary drugs.1.Enteric-coated granules based on solid lipid nanoparticles improved the oral absorption of poorly soluble drug enrofloxacinThe low solubility veterinary drug enrofloxacin（ENR）was used as a model to improve the oral absorption of ENR through solid lipid nanotechnology combined with coating technology.According to the physicochemical properties of ENR,ENR solid lipid nanoparticles（ENR-SLNs）were prepared by hot melt emulsification method with octadecanoic acid（OA）as the lipid substrate and polyvinyl alcohol（PVA）as the emulsifier.The optimal prescription for ENR-SLNs was 10 m L 3%PVA containing 0.8 g ENR and 2.4 g OA.The ENR-SLNs were spherical with smooth surface and relatively uniform distribution.Particle size（PS）,zeta potential（ZP）and polydispersity index（PDI）were 308.5±6.3 nm,-22.3±0.36 m V and 0.352±0.013,respectively.Then,ENR enteric-coated granules were prepared with ENR-SLNs as core,polyacrylic resin II（PRII）as coating material,starch as filler and sucrose as sweetener.The quality evaluation of ENR enteric-coated granules showed that the content,granularity,drying weight loss and stability of ENR enteric-coated granules were in accordance with the requirements of the veterinary pharmacopoeia.The release results showed that 15%PRII coated granules showed a relatively slow release rate in SGF,only about 30%of which were released at 4h.Palatability tests revealed that ENR enteric-coated granules with 10%sucrose increased daily feed intake by 11 times,which was similar to that of the untreated group,indicating that they completely masked the bitterness of ENR.The pharmacokinetic results further demonstrated that the area under concentration-time curve(AUC_0-last),peak concentration(C_max)and peak time(T_max)in porcine plasma were 11.24±3.33μg h/m L,0.52±0.05μg/m L and 3.33±1.03 h,respectively.The oral bioavailability of ENR enteric-coated granules was 2.64 times that of ENR soluble powder.2.Core-shell nanosystems improved the oral absorption of low-permeability drug colistinThe innovative core-shell structure nanoparticles（CA-NPs）,employed the low-permeability veterinary drug colistin sulphate（COS）as a model,were used to overcome the three major physiological barriers of the gastrointestinal tract（gastric acid,mucus and epithelial barriers）to achieve efficient oral absorption of COS.The core nanoparticles（HA-NPs）were prepared by self-assembly method,which were spherical in uniform size,with the average PS of 231.06±5.85 nm,ZP of-3.87±0.09 m V and PDI of0.372±0.02.The formation mechanism of HA-NPs was mainly the self-assembly of the carboxyl group of hyaluronic acid（HA）and the amino group of mannosamine（DMH）through electrostatic interaction.Then,shell structure（CA-CMC）was prepared by solvothermal method.CA-CMC was formed from citric acid（CA）and carboxymethyl cellulose（CMC）by lipid and hydrogen bonds.Core-shell structure nanoparticles（CA-NPs）were constructed.The PS,ZP,PDI,loading capacity（LC）and encapsulation efficiency（EE）of CA-NPs were 382.79±9.33,-18.44±0.58,0.238±0.03,19.61±0.94and 88.12±1.53,respectively.The fluorescence resonance energy transfer（FRET）results showed that the distance between the shell and the core of CA-NPs was 4.5 nm.Fluorescence imaging demonstrated that CA-NPs significantly extended the intestinal residence time to 8 h.The whole-body imaging of mice showed that CA-NPs was significantly higher than the control group at different time points,and still maintained strong whole-body fluorescence imaging at 6 h,revealing its efficient and sustained absorption into blood.The fluorescence distribution of CA-NPs in the heart,liver,spleen,lung and kidney showed that the fluorescence intensity of CA-NPs at different times was strongest in the liver,followed by the kidney and lung,indicating that CA-NPs may be mainly metabolized by the liver.The AUC_0-last,C_maxand T_maxof CA-NPs in rat plasma were 4.97±0.95,0.86±0.08 and 0.85±0.33,respectively,which significantly improved the 303%oral bioavailability of COS.The in vitro MIC of COS against E.coli and Klebsiella pneumoniae was 0.25μg/m L.The therapeutic effect of bacteremia model showed that CA-NPs doubled the survival rate,was the first to regain weight and to clear bacteria from the blood,and had significantly lower levels of proinflammatory cytokines on day 7 compared with the same dose of COS.The results of the treatment of lung infection model showed that the clinical symptom score of CA-NPs group was 1-2,the weight was the first to recover 100%,and the survival rate was improved.To investigate the oral safety of CA-NPs,mice were evaluated after 14 consecutive days of administration.The results of oral FITC-labeled 40 k Da dextran（FITC-DX40）showed that CA-NPs reversible opening of tight junctions（TJs）did not lead to harmful entry of bacteria into the bloodstream.Organ coefficient,histopathological observation,physiological and biochemical indexes,and inflammatory and immune factor levels revealed that the oral administration of CA-NPs was safe.3.Oral absorption mechanism of core-shell nanosystemsIt is further explored the mechanism of efficient oral absorption of CA-NPs.The result demonstrated that CA-NPs was stable under physiological environment,due to the shell structure of CA-CMC had protective and acid-resistant properties.CA-NPs significantly reduced the release of CA-NPs under acidic conditions,and the cumulative release was about 30%in 4 h,indicating that the acidic environment had little effect on the release of CA-NPs.Fluorescence intensity,morphology,PS,ZP and structure indicated that the shell structure of CA-NPs gradually shed with the increase of p H value,which was mainly attributed to the fracture of CA-CMC lipid bond under alkaline conditions.The results of ELISA and isothermal titration calorimeter（ITC）showed that the affinity between HA-NPs and mucin was low,so as to avoid mucin capture.Compared with PEG-NPs,HA-NPs first reached the bottom of 40μm and reached the maximum number of particles at the depth of 100μm,and its mean square displacement（MSD）were increased by 7.03 times.The rapid diffusion of HA-NPs in mucus may be attributed to the synergistic effect of surface density,near electricity neutral and hydrophilic functional groups.Subsequently,the transmembrane transport properties and mechanism of CA-NPs were investigated.The apparent permeability coefficient（Papp）of CA-NPs and HA-NPs was increased by 4.02 and 2.54 times,respectively,compared with COS.Confocal laser scanning microscope（CLSM）visualization and flow cytometry（FCM）quantitative analysis showed that CA-NPs and HA-NPs were highly efficient taken up by Caco-2 cells.CA-NPs and HA-NPs achieved efficient transcellular transport via GLUT transporter mediated and clathrin mediated as well as intracellular transport via the Golgi pathway.TEM imaging of mice intestinal tissue also directly demonstrated the transcellular transport pathway of CA-NPs.The CA concentration of CA-NPs in the process of formulation optimization was 5 mg/m L,cell TJs were reversibly opened.Immunofluorescence labeling and western blot analysis of ZO-1 protein showed that CA-NPs reversibly opened TJs at 1 h and recovered within 24 h.TEM imaging of mice intestinal tissue further demonstrated CA-NPs-mediated TJs opening and recovery.Subsequently,the paracellular transport efficiency of reversible opening of its TJs was evaluated,and it was found that the permeability of two membrane impermeable substances,calcein and FITC-DX4,increased by 2.27 or 2.14 times,respectively.This revealed that CA-NPs-mediated TJs opening contributed to its transmembrane transport.The mechanism of reversible opening of cellular TJs by CA-NPs was further investigated.The results showed that CA-NPs activated p KC,p KA,and p KG protein kinases by reducing extracellular Ca²⁺concentration,thus reversibly opening TJs.These results suggested that the efficient transmembrane transport of CA-NPs was achieved through the combination of transcellular and paracellular pathways.In the study,ENR enteric-coated granules and core-shell structure CA-NPs were designed and constructed in order to address the key problems of low solubility or low permeability and physiological barrier of the body faced by oral drugs.Both of them overcomed the corresponding scientific difficulties of low solubility or low permeability drugs and significantly improved the oral absorption of loaded drugs as well as established the relevant design theory.On the one hand,this paper provided a new idea for rational design of low cost and high efficiency oral delivery carriers;On the other hand,it also provided a theoretical basis for other researchers to develop oral delivery systems to overcome the deficiencies of physical and chemical properties of drugs and gastrointestinal physiological barriers.