Effects Of Cyclodextrin Complexation And Macromolecular Modification On Its Encapsulation And Release Behavior

With its unique hydrophilic and hydrophobic cavity chemical structure,cyclodextrin（CD）can be used as a drug carrier,forming an inclusion compound with a variety of small molecules,significantly increasing the solubility of hydrophobic drugs in water and protecting and releasing sensitive natural drugs.Due to the solubility,rigidity of cavity and the adaptability of inclusion,the application of CD was limited.Compared with CD and its derivatives,cyclodextrin polymers have good water-solubility,good biocompatibility,strong tolerance to the spatial configuration and size of guest molecules,and can encapsulate guest molecules larger than cyclodextrin cavities,which has obvious advantages in the ability of inclusion.In this paper,cyclodextrins were taken as the subject,click chemistry was introduced to the macromolecular modification of cyclodextrins,study the eencapsulaton and release behaviors of cyclodextrins and their macromolecular modified polymers for hydrophobic drugs.Alamethicin（ALM）inhibits the growth of bacteria by disrupting their cell membrane through permeabilization/pore formation.Because of its high hydrophobicity,ALM exhibits poor solubility in aqueous medium thus limiting its application.The first part of this study proposed a method for encapsulation of ALM withγ-cyclodextrin（γ-CD）by forming a complex in water.By adjusting the molar ratios ofγ-CD and ALM to 1:1,5:1 and 10:1 respectively,three correspondingγ-CD/ALM complexes were prepared:γ-CD/ALM 1,γ-CD/ALM₅ andγ-CD/ALM₁0.The formation ofγ-CD/ALM complex was confirmed by FT-IR.Particle size distribution indicated that aggregation of the complex was inhibited at higherγ-CD/ALM molar ratio.Moreover,γ-CD/ALM complex exhibited significant antimicrobial activity against L.monocytogenes with the minimum inhibitory concentration being dependent onγ-CD/ALM molar ratio（>0.429 mg/mL for 1:1;>1.094 mg/mL for 5:1;>2.078 mg/mL for 10:1）;Optimumγ-CD/ALM molar ratio for maximum antimicrobial activity is the result of competing effects of higher solubility and shielding of hydrophobic sites of ALM byγ-CD.Molecular dynamics simulation suggested a stable configuration ofγ-CD/ALM complex when hydrophobic residues are inserted into the hydrophobic cavity ofγ-CD;ALM have multiple hydrophobic points,larger cyclodextrin cavities and higher cyclodextrin concentration are likely to be combined with more hydrophobic points,improved the inclusion effect between CD and guest molecules.Furthermore,explore the effect of CD on the antibacterial mechanism of ALM.In this part,transmission electron microscopy of L.monocytogenes treated withγ-CD/ALM complex indicated cell lysis due to pore formation.This was further corroborated by fluorescent dye leakage from DMPC/cholesterol liposomes when exposed toγ-CD/ALM complex for molar ratios of 1,5 and 10.It indicated thatγ-CD only served as a carrier to facilitate the solubility and bioavailability of ALM in water solution,rather than induced dye leakage through membrane penetration.The extent of dye leakage increased with ALM-lipid ratio in the range of 0.00015 to 0.16.Dye leakage ofγ-CD/ALM complex was found to be the highest for molar ratio of 5,consistent with our earlier results of antimicrobial activity of the complex against L.monocytogenes.All atom molecular dynamics（MD）simulation showed thatγ-CD/ALM complex can effectively bind to the 3:1 POPE/POPG bilayer,a mimic of bacterial cell membrane.Transmembrane MD simulation of sixγ-CD/ALM complex aggregates inα-helical conformation showed water channel with barrel stave like pore structure.In addition,circular dichroism spectrum indicated that ALM in the complex has a helical conformation in solution as well as in the presence of liposome.These results proved thatγ-CD can change the conformation of ALM in aqueous solution through complex inclusion,and ALM changes from an irregular structure to a helical structure,so that it has antibacterial activity in aqueous solution.The previous study found that higher cyclodextrin concentration are likely to improve the inclusion effect between CD and guest molecule（ALM）,thus the cyclodextrin monomer polymerization modification for cyclodextrin polymers to improve the local concentration of cyclodextrins.Here water-solubleβ-cyclodextrin polymers（β-CDPs）employing thiolatedβ-cyclodextrin（β-CD-（SH）₇）and maleimide-functionalized hydroxypropyl-β-cyclodextrin（HPCD-AMI）were successfully synthesized in aqueous solution via thiol-ene‘click’chemistry.The intermediate and final synthesizedβ-CDPs were characterized by Fourier transform infrared spectroscopy（FT-IR）,¹H nuclear magnetic resonance（¹H NMR）,Matrix-assisted laser desorption ionization-time of flight mass spectroscopy（MALDI-TOF）,size exclusion chromatography（SEC）,X-ray Powder Diffractometer（XRD）,and thermalgravimetric analysis（TGA）.It was found that the Mw ofβ-CDPs increased with both feed ratio of HPCD-AMI/β-CD-（SH）₇ and reaction temperature.Moreover,the ¹H NMR indicated thatβ-CDPs prepared at elevated temperature have higher degree of thiol-ene reaction compared to that prepared at room temperature,thereby leading to more compact star-like branched structure.Phase solubility study showed that the affinity of curcumin withβ-CDPs was significantly higher than that with the nativeβ-CD and HPβ-CD.Increasing the Mw resulted in an increased or decreased complex-forming capacity,depending on the microstructure ofβ-CDPs.Besides,it was found thatβ-CDPs exhibited better dissolution abilities.Cyclodextrin was modified to chitosan via Diels-Alder reaction between furfural functionalized chitosan（CF）and N-maleoyl alanine functionalized hydroxypropylβ-cyclodextrin（HPCD-AMI）,formed macromolecular hydrogel in aqueous media without any catalyst or initiator.The CF and HPCD-AMI were confirmed by Fourier transform infrared spectroscopy and ¹H Nuclear magnetic resonance spectroscopy.The resultant CFCD hydrogel was characterized in terms of thermal peripteries,microstructure,rheology behavior,and swelling capacity.The rheology analysis found that the storage modulus G’ranged from 1 pa to1200 pa as the degree of furfural substitute on chitosan increased from 2.6%to 28.3%,indicating the hydrogel strength can be tuned readily by reaction stoichiometry.The swelling behaviors proved that CFCD hydrogel was pH-responsive with low swelling capacity,which would be preferable for drug delivery.Drug adsorption analysis showed the introduction of cyclodextrin into CFCD hydrogels promoted drug adsorption capacity.In addition,methyl orange cumulative release in PBS buffer was only 48.85%after 24 h,suggesting CFCD hydrogel had good sustained release capacity on the loaded drug.