Research And Development Of A New Process Technology For Loading Of Vitamin A Palmitate By CD-MOF

Objective:Vitamin A（VA）is an important and easily deficient fat-soluble vitamin that is often present in the form of vitamin A esters and beta-carotene.Vitamin A has attracted a great deal of attention as a dietary supplement or medicine,but it degrades with increasing temperature,UV exposure,or the presence of oxygen,and improving its stability has been a challenge.Vitamin A Palmitate（VAP）is a widely used vitamin A ester,and its structure is similar to that of vitamin A.The structural skeleton contains multiple conjugated double bonds and ester bonds.In order to prevent degradation of VAP during preparation and storage,the goal of this study was to develop a green and efficient drug loading process and find a suitable carrier to solidify the liquid oil drug for stability enhancement.Methods:In this study,different types of CD-MOF were used as carriers for loading liquid,unstable drugs by a solvent-free method.Taking VAP as the model drug,in view of the porous structure and drug loading potential of CD-MOF,the oil drug VAP was adsorbed and diffused into theγ-CD-MOF cavity by a new solvent-free method.Based on the consideration of availability and cost,the extended study ofβ-CD-based MOF carrier and optimization.1.Study onγ-CD-MOF loaded VAP by solvent-free method:The solvent-free drug loading method rapidly combined oil VAP withγ-CD-MOF and adsorbed on the porous surface.VAP progressively entered the cavity as the loading time increased,and VAP entering the interior was protected by the carrier.The optimal conditions ofγ-CD-MOF-loaded VAP were investigated.The contents ofγ-CD-MOF loaded VAP and the remaining contents ofγ-CD-MOF treated at high temperature were used as indexes to determine the contents of VAP.The single factor method was used to investigate and determine the carrier type and the level range of key factors（time,material ratio,and ratio of MOF to zirconia beads）.Then,through the extraction experiment with free VAP and the long-term stability experiment at high temperature,the existence of unprotected VAP was preliminary investigated.In order to reduce the influence of possible free VAP,washing and heating pretreatment were used.2.Study on new excipients ofβ-CD based CD-MOF:This study attempted to design a new CD-MOF（β-CD-POF（I）,POF:Potassium Organic Framework）to implement stable loading of VAP.By exploring and optimizing the synthesis process,the single crystal culture of the carrier was successfully realized,and its spatial structure was analyzed by single crystal X-ray diffraction.The new-type carrier with a unique spatial structure was used to load the unstable molecule VAP to improve drug loading efficiency.3.Study onβ-CD-POF（I）loaded VAP:The solvent-free process ofβ-CD-POF（I）loaded VAP was preliminary determined by investigating the material ratio percentage and washing treatment after loading.Using KOH-β-CD-MOF andβ-CD as controls,the thermal stability tests were conducted at different temperature conditions to determine the function ofβ-CD-POF（I）.In order to further improve the protective efficacy,nitrogen protection was adopted in the thermal stability experiment to reduce instability factors.On the other hand,the degradation of the raw material and the loaded compound under strong light was investigated.4.Study of loading mechanism:In order to further explore the reason ofβ-CD-POF（I）loading VAP efficiently and enhancing its stability,molecular simulation was used to dock VAP withβ-CD-POF（I）,and to predict the possible distribution of VAP inβ-CD-POF（I）.In addition,the Synchrotron radiation-Fourier transform infrared spectroscopy（SR-FTIR）,powder X-ray diffraction（PXRD）,differential scanning calorimetry（DSC）,powder X-ray diffraction（PXRD）,thermogravimetric analysis（TGA）and nitrogen adsorption isotherm were conducted to verify the successful encapsulation of VAP intoβ-CD-POF（I）,molecular simulations was conducted to explore the possible encapsulation mechanism between VAP and CD-MOF and Differential scanning calorimeter（DSC）was employed under nitrogen purge.The possible encapsulation mechanism between VAP andβ-CD-POF（I）was explored by combining the properties of drugs and the binding mode ofβ-CD-POF（I）.5.Technology and optimization of supercritical fluid assisted drug loading:Vitamin A palmitate was loaded with KOH-CD-MOF andβ-CD-POF（Ⅰ）in supercritical CO₂ fluid.The horizontal range of three key factors（reactor temperature,reactor pressure,and reaction time）in supercritical CO₂ fluid-assisted CD-MOF loading of VAP was investigated by single factor method.Taking the stability experiment data of VAP as the index,the differences of drug loading and stability under various key parameters were investigated,and the optimal drug loading process was optimized.Results:1.γ-CD-MOF solvent-free method loading VAP,a green and efficient method to improve the stability of VAPThe final optimized solvent-free drug loading conditions were as follows:The neutral KOH-CD-MOF was used as the carrier,the loading ratio was 2%,the ratio of MOF to zirconia beads was 1:3,mixed for 1 h at room temperature,and the drug loading range was 1.8%-2.0%.The stability experiment results showed that the VAP protection of micron-scale VAP@KOH-CD-MOF was better than that of nano-scale VAP@KOH-CD-MOF.Heat treatment and pre-washing treatment could improve the stability of VAP in the VAP@KOH-CD-MOF complexes.Proper washing could remove the free VAP that was adsorbed on the surface,promoting the redistribution of VAP inγ-CD-MOF and improving the overall stability of VAP@KOH-CD-MOF.In addition,the protective effect of CD-MOF on VAP increased slightly with the increase in treatment temperature,but the drug loss rate increased after pretreatment.2.Exploring a new excipient（β-CD-POF（I）,POF:Potassium Organic Framework）β-CD-POF（I）was successfully prepared by using the more economicalβ-CD as raw material in the synthesis process.The molar ratio ofβ-CD to potassium iodide was 1:8,and the crystal was arranged in order by using anhydrous ethanol as the conversion solvent.β-CD-POF（I）single crystal was prepared by solvent diffusion method and its structure was analyzed by X-ray diffractometer.3.Loading efficiency and mechanism of VAP@β-CD-POF（I）was determined（1）The process ofβ-CD-POF（I）solvent-free loading VAP was determined,and the material ratio was set at 2%for the follow-up stability test.Stability experiments showed thatβ-CD-POF（I）was superior to KOH-β-CD-MOF andβ-CD to protect VAP at 40℃,60℃or 70℃.The stability of the complexes ofβ-CD-POF（I）@VAP under nitrogen protection was better than that of the samples directly exposed to the environment at 70°C.High-light conditions have less effect on the stability of VAP.The conclusion was that VAP@KOH-β-CD-MOF could not exist stably in the drug stability test chamber.The degradation of VAP by intense light treatment was lower than that by high temperature treatment,and the protective effect of KOH-β-CD-MOF on VAP was still weaker than that ofβ-CD-POF（I）.（2）Molecular modeling and characterizations of the synchrotron radiation Fourier transform infrared spectroscopy（SR-FTIR）,differential scanning calorimeter（DSC）,powder X-ray diffraction（PXRD）,thermogravimetric analysis（TGA）,and nitrogen adsorption isotherm were performed to confirm that the VAP was successfully encapsulated into the channel formed by dicyclodextrin pairs.VAP is prone to oxidation,pyrolysis,decarboxylation,and so on due to the abundance of conjugated double bonds and an ester bond in the structure.The cavities formed by the dicyclodextrin pairs ofβ-CD-POF（I）provide space for the VAP molecules.It was determined from the results of the molecular docking that VAP molecules were more probably to stay inβ-CD bimolecular pairs,and the docking free energy was measured to be-6.108 kcal/mol.In light of the spatial structure and VAP instability ofβ-CD-POF（I）,the following hypothesis on the mechanism of action between VAP andβ-CD-POF（I）was put forward:VAP was adsorbed throughout the cavity ofβ-CD-POF（I）,on the one hand,long-parallel tubular channels could separate VAP molecules and prevent mutual oxidation of molecules;on the other hand,flexible VAP molecules were restrained in narrow dicyclodextrin cavities,which could not realize self-oxidation.In summary,the mechanism of the stability enhancement to VAP was determined to be the constraint and separation effect ofβ-CD pairs on VAP.Their combined impact resulted in well-encapsulated multiple conjugated double bonds in VAP molecules and further enhanced stability.4.Supercritical CO₂ fluid-assisted CD-MOF loaded with vitamin A palmitateIn order to better optimize the solvent-free drug loading,after the pre-mixing of CD-MOF and VAP,the supercritical fluid was used for follow-up processing,and the mixing effect could be clearly seen.The supercritical fluid-assisted loading of KOH-CD-MOF was better than the solvent-free method,but the stability protection effect decreased with the increase of the material ratio proportion.The initial loading conditions of KOH-CD-MOF were as follows:5%material ratio（which could be increased within a certain range）,reaction at 40°C and 20 MPa for 1 h.The initial loading conditions ofβ-CD-POF（I）were as follows:2%material ratio（could be increased within a certain range）,reaction at 40°C,20 MPa for 0.5 or 2 h.By improving the loading equipment and changing the loading strategy,the experimental conditions were effectively adjusted.By virtue of supercritical CO₂,which had dual characteristics of both gas and liquid and a large solubility to certain drugs,liquid VAP was diffused and adsorbed in the porous structure of MOF.Conclusions:The solvent incubation method has some drawbacks,including a poor drug utilization rate,a large amount of organic solvent,and a lengthy reaction time.Based on this,a solvent-free method with a comparatively mild and green drug loading process was created to achieve the solid state of liquid drugs and improve the stability of some unstable liquid drugs.Furthermore,the extensive study and optimization of theβ-CD-POF（I）carrier improved the availability and significantly reduced the preparation cost.β-CD-POF（I）had dicyclodextrin units and long-parallel tubular channels,with cavities formed in the structure to provide space for VAP molecules.Molecular simulation and a series of characterization experiments confirmed that VAP was successfully encapsulated into the channel formed by dicyclodextrin pairs.The results of molecular docking showed that the free energy of the docking was-6.108 kcal/mol.The binding and separation effects ofβ-CD-POF（I）on the chain of VAP molecules jointly promoted the well-encapsulation of multiple conjugated double bonds in VAP molecules.In order to better optimize the drug loading mode without introducing organic solvent,the supercritical fluid was used for follow-up treatment after the pre-mixing of CD-MOF and VAP.By virtue of the dual properties of supercritical CO₂ as both gas and liquid,the loading of CD-MOF assisted by supercritical fluid on VAP was realized,which also improved the stability of VAP.In conclusion,the development and optimization of solvent-free drug loading technology,as well as the exploration and optimization of new carriers,were beneficial in addressing the problem of the poor stability of vitamin A esters and provided new ideas for the development of unstable drug preparations on CD-MOF carriers.