Development Of Curcumin Supersaturated Self-Nanoemulsion Drug Delivery System:Characterization,Stabilization,and Mechanisms Of Enhanced Absorption

Objective:Supersaturated self-nano-emulsion drug delivery systems are mostly thermodynamically unstable during the process of dispersion and digestion because of the generation of precipitates,which restricts the development and utilization of this dosage form.In this study,the water-insoluble curcumin was used as a model drug to develop a supersaturated self-nanoemulsion system by incorporating hydrophilic polymers to improve drug absorption and bioavailability.Furthermore,the mechanism of drug supersaturation using hydrophilic polymer was discussed by comprehensively characterizing the nano-emulsion solution and formation of precipitates in the process of self-emulsion emulsification.In addition,the molecular dynamic simulation was also used to describe the mechanism of precipitates formed during the self-emulsion system.In conclusion,the mechanism of enhanced absorption of supersaturated self-nanoemulsion was elucidated on three levels:in vitro intestinal absorption,cellular uptake,and in vivo pharmacokinetics.Methods:(i)Preparation and in vitro dispersion evaluation of curcumin self-nanoemulsion:The composition of the nano-emulsion system was screened through solubility tests,compatibility of the oil phase and surfactants,emulsification ability of surfactants,and construction of pseudo-triple phase diagram.Then CUR-SNEDDS prescriptions were prepared and optimized based on the hierarchical analysis(AHP)method combined with the star point design-effect surface method.The morphology,particle size and dispersion,Zeta potential,and emulsifying time of emulsion droplets after CUR-SNEDDS emulsification were characterized by transmission electron microscopy,laser nano-size meter,and stopwatch.The artificial gastrointestinal fluid was used as a dispersion system to study the in vitro supersaturation stability of CUR-SNEDDS.(ii)Construction of curcumin self-nanomil supersaturated drug delivery system based on hydrophilic polymer crystallization inhibition behavior:CUR-SNEDDS was used as a supersaturated production tool,based on the in vitro dispersion experiments by taking the crystal nucleation time and crystal growth rate as the index,the best precipitation inhibitor was optimized.Moreover,the hydrophilic polymers dosage and prescription drug loading were also optimized by maintaining the supersaturated concentration and the area under the time curve(AUC)of CUR.In summary,Curcumin supersaturated self-nanoemulsions were prepared,and the quality of curcumin supersaturated self-nanoemulsions was evaluated.(iii)Study on the mechanism of polymer stabilization of curcumin supersaturation:The solution conductivity,viscosity,nanoemulsions quality,stability,and CUR equilibrium solubility were systematically evaluated during the in vitro dispersion studies of SNEDDS and its two supersaturated SSNEDDS,Such as using X-ray powder diffraction(XRD)and differential scanning calorimeter(DSC),infrared spectroscopy(FT-IR),Raman spectroscopy(Roman)and proton nuclear magnetic resonance spectroscopy(¹H-NMR)to characterize the precipitates generated during the dispersion process,and combined with the Gromacs2018 program to analyze the CUR and polymerization process.Molecular dynamics simulation was performed to clarify the mechanism of CUR supersaturation maintained by the polymer.(iv)Curcumin supersaturated self-nanoemulsion in vitro and in vivo evaluation:HPLC and laser nanoparticle size analyzer was used to investigate the curcumin stability in CUR-SNEDDS,CUR-SSNEDDS-I,and CUR-SSNEDDS-II at different p H environments for 24 h,and also the stability of CUR content and the quality of nano-emulsion stored at 25?for three months.The differences in dissolution and digestion behaviors of CUR-SNEDDS,CUR-SSNEDDS-I,and CUR-SSNEDDS-II were compared via in vitro dissolution and digestion experiments.Furthermore,the absorption and bioavailability of CUR-SNEDDS and two types of CUR-SSNEDDS were evaluated by isolated rat intestinal tract and in vivo pharmacokinetic study.(v)Study on absorption mechanism of curcumin supersaturated self-nanometer emulsion absorption:Caco-2 cells were used as a cellular uptake model,with different endocytosis and intracellular transport inhibitors,and analyzed via flow cytometry(quantitative)and fluorescence microscopy(qualitative)to study CUR-SNEDDS,CUR-SNEDDS-I,and CUR-SNEDDS-II cell uptake pathways.Results:(i)The prepared CUR-SNEDDS system consisted of capryol90-kolliphor RH40-transcutol HP(7.93?66.71?25.36),with a maximum drug loading of(65.12±1.25)mg·g^-1.The appearance of CUR-SNEDDS remained yellow transparent,and the nanoemulsion droplets appeared spherical with uniform distribution.The emulsification time was(19.64±0.11)s,the average particle size was(15.06±1.12)nm,the polydispersity index(PDI)was(0.18±0.01),and the Zeta potential was(-7.41±0.52)m V.In vitro dispersion experiments showed that the supersaturation of CUR decreased significantly,and with the increase of drug loading,the nucleation and growth rate of crystals increased,and the nanoemulsion droplets'size increased,adhered,and distributed unevenly.(ii)When different polymers were introduced into the prescription of CUR-SNEDDS,the crystal inhibition behavior becomes obvious.HPMC and Soluplus significantly inhibited the nucleation and growth of drug crystals in simulated artificial gastrointestinal fluid,while HPMCAS and PVP only inhibited crystal growth in artificial intestinal fluids.As the degree of polymerization also affects the ability to inhibit crystals;therefore,the greater the degree of polymerization of HPMC,the lower is the ability to inhibit crystals(HPMC55-6>HPMC4000>HPMC15000).The greater the degree of polymerization of HPMC,the stronger the ability to inhibit crystals(LG<MG?HG).Finally,2%HPMC55-60and 5%Soluplus were selected as supersaturated prescriptions containing precipitation inhibitors,CUR-SSNEDDS-I and CUR-SSNEDDS-II,respectively,with a drug loading of 100%.Compared with CUR-SNEDDS,the particle size of CUR-SSNEDDS-II increased to(57.03±0.35)nm.After CUR-SSNEDDS-I and CUR-SSNEDDS-II are emulsified in water,the emulsion droplets are spherical with uniform particle size distribution.The absolute value of the Zeta point increased to(-17.87±1.42)m V and(-18.10±1.30)m V,respectively,indicating that the nanoemulsion formed after the introduction of HPMC55-60 and Soluplus is more stable.(iii)During the emulsification process of the three CUR self-nano-emulsion preparations,as the water content ratio increases,the equilibrium solubility and solution viscosity of the drug in the system decreased significantly.Compared with CUR-SNEDDS,the equilibrium solubility of the two SSNEDDS prescription drugs and the mass stability of the nanoemulsion were significantly improved,and CUR-SNEDDS-I significantly increased the viscosity of the system.Furthermore,neither affected the SNEDDS structural transformation.The precipitates produced during the in vitro emulsification process were significantly reduced,and the precipitation morphology was changed.Both the precipitation characterization and molecular dynamics results showed that CUR interacts with HPMC55-60 and Soluplus molecules,forming hydrogen bonds and having a?-?stacking effect.Therefore,HPMC55-60 and Soluplus inhibit the aggregation of CUR molecules and maintain the stable supersaturation state by changing the physical characteristics such as viscosity,solubility,and droplet stability of the solution during self-nano-emulsification and forming hydrogen bonds and?-?stacking effects with drug molecules.(iv)CUR-SNEDDS,CUR-SSNEDDS-I,and CUR-SSNEDDS-II did not degrade CUR within 24 hours in an alkaline biological medium;the drug loading and the quality of nano-emulsion were stable after 3 months of storage at room temperature.In the in vitro dissolution process,all of them could significantly improve the speed and degree of CUR dissolution.During in vitro digestion,CUR-SSNEDDS-I and CUR-SSNEDDS-II significantly inhibited the formation of precipitates,and the proportion of CUR in the hydrophilic phase increased by 2.63 and 4.18 times,respectively.The quality and stability of the nanoemulsion were improved,and the morphology and particle size of Soluplus droplets remained stable and did not increase significantly.The results of in vitro intestinal absorption showed that compared with CUR-SNEDDS,the absorption of CUR in the duodenum,jejunum,ileum,and colon was significantly enhanced in the CUR-SSNEDDS-I group,and P_appwere increased by 3.12,1.73,1.29,and 1.94 times respectively,while in CUR-SSNEDDS-II group,P_apponly increased in duodenum and jejunum.The in vivo pharmacokinetics results showed that after oral administration to SD rats,compared with CUR-SNEDDS,the Cmax of CUR-SSNEDDS-I and CUR-SSNEDDS-II increased by 2.29 and 1.60 times,and AUC0-t increased by 3.50 and 1.92 times,respectively.Therefore,HPMC55-60 and Soluplus significantly improved the stability,dissolution,and digestion supersaturation of CUR in vitro,thus promoting the in vivo absorption and increased the bioavailability.(v)The cellular uptake of CUR-SNEDDS is affected by time,concentration,temperature,and P-gp.The main uptake pathways include endocytosis and macrocytophagy mediated by grid protein,caveolin,and dynamic protein,and depend on the transmission between the endoplasmic reticulum and Golgi apparatus,Golgi apparatus to the cell membrane,tubulin,and lysosome acidification.Compared with CUR-SNEDDS,CUR-SNEDDS-I and CUR-SSNEDDS-II cell intake rates were significantly higher,which is not regulated by P-glycoprotein,but both rely on the actin-mediated microcytosis pathway,and intracocytotransmission is affected by the lysosomal acidification pathway,where CUR-SSNEDDS-II does not rely on the tubulin transfer pathway.The results showed that HPMC55-60 and Soluplus increased the cellular uptake of CUR by inhibiting the P-glycoproteins,thereby changing the pathway of endocytosis and intracellular transport.Conclusion:CUR-SSNEDDS(I and II)prepared by incorporating HPMC55-60and Soluplus can improve the solubility,stability,dissolution,dispersion,and digestion stability of CUR,and interact with CUR molecules in the process of SNEDDS emulsification to form hydrogen bonds and?-?stacking effect,resulting in changes in the viscosity of the system,and improving the solubilization and mass stability of nano-emulsions by inhibiting the intermolecular aggregation of CUR in order to maintain the supersaturated state of curcumin.At the same time,HPMC55-60 and Soluplus inhibited P-gp on the surface of the cell membrane,changed the pathway of endocytosis and intracellular transport,and increased the uptake rate of CUR in the Caco-2 cells.In summary,CUR-SSNEDDS enhances drug absorption by improving the stability of CUR,maintaining supersaturated stability,inhibiting P-gp,and changing the pathway of endocytosis and intracellular transport.