Rational Design And Oral Evaluation Of Zwitterionic Nanocarriers Mediated By Amino Acid Transporters

Zwitterionic polymers are a class of electrically neutral polymer materials with a pair of opposing charge groups in their constitutional unit structure.The advantages of zwitterionic nanocarriers are particularly apparent in the field of oral drug delivery.On the one hand,due to hydration,zwitterionic nanocarriers could form a dense hydration layer on the surface of the nanocarriers,giving zwitterionic nanocarriers superior mucus permeability.On the other hand,zwitterionic constitutional units generally have weak cellular affinities（i.e.,transporter-substrate interaction etc.）,endowing zwitterionic nanocarriers with enhanced cellular uptake capacity.Although the oral delivery of zwitterionic nanocarriers has significant advantages,there are still some scientific problems that need to be solved in zwitterionic nanocarriers.Firstly,some complexities and limitations exist in the zwitterion modification of nanocarriers.Secondly,zwitterionic nanocarriers have a"barrel effect",and their ability of cellular uptake is their shortcoming.In the end,intestinal epithelial cells express a large number of amino acid transporters with different categories,and amino acid substrates have a unified zwitterionic structure（i.e.,α-amino andα-carboxylic groups）.Currently,there is a lack of development of zwitterionic nanocarriers based on amino acid structures.In view of the above scientific problems,the main research contents and results of this paper are as follows:1.In view of the limitations of modification method of zwitterionic nanocarriers and the easy modification characteristic of commercial Pluronic,a simple modification method of betaine-based zwitterionic Pluronic analogues for hydrophobic nanocarriers was developed in this paper.Three betaine-based zwitterionic Pluronic analogues(PCB₂₀-PPO2K-PCB₂₀,PCB₂₀-PPO3K-PCB₂₀ and PCB₂₀-PPO4K-PCB₂₀,PPP2K,PPP3K and PPP4K for short)were firstly synthesized.Here,PLGA was used as a hydrophobic NPs model to explore the adsorption modification mechanism of zwitterionic Pluronic analogues.The results showed that the zwitterionic Pluronic analogues with PPO molecular weight more than 2 k Da could be effectively adsorbed to the surface of PLGA NPs forming typical core-shell NPs.The morphological sizes of the NPs were all below 100 nm,which were weakly electrically negative.PLGA@PPP2K was not stable due to the weak hydrophobicity of PPO in PPP2K.When its concentration was higher than 2 mg/m L,the prepared PLGA@PPP3K or PLGA@PPP4K both had good freeze-drying stability.In vitro experimental results showed that the cellular uptake capacity of the PLGA@PPP4K was approximately 2.2 times that of PLGA@F127,and the PAT1 transporter was involved in mediating the cellular internalization of the PLGA@PPP4K.In addition,partial PLGA@PPP4K could bypass the lysosomal degradation pathway and use the intracellular retrograde pathway for transcytosis.The results in vivo showed that PLGA@PPP4K could be efficiently absorbed by the intestinal mucosa,enter the blood circulation system and mainly accumulate in liver tissue due to the First-Pass Effect.INS-TDCS complexes were prepared by hydrophobic ion pairing and loaded into the PLGA core.Using insulin as a model drug,the oral delivery ability of PLGA@PPP4K was comprehensively investigated.The results showed that the relative pharmacological bioavailability of insulin in the PLGA@PPP4K group was approximately11-fold higher than that of the oral insulin group.2.Compared with its superior mucus permeability,the cellular uptake ability of zwitterionic nanocarriers is insufficient.However,the mucus permeation and cellular uptake characteristics of polyguanidine-based cationic nanocarriers are opposite.Therefore,zwitterion-based and polyguanidine-inserted hybrid micelles were developed with good mucus permeability as well as cellular internalization capacity.Firstly,three betaine-based amphiphilic block copolymers（PLA-b-PCB15,PLA-b-PCB30 and PLA-b-PCB45,CB15,CB30 and CB45 for short）and one polyguanidine-based amphiphilic block copolymer（PLA-b-Gua15,Gua15 for short）were synthesized by ATRP.By adjusting the molar ratio of Gua15（10%,20%,30%,40%and 50%）and the types of CB copolymer,a library with betaine-based and polyguanidine-inserted hybrid micelles（Gua15@CB15 series,Gua15@CB30 series,Gua15@CB45 series and Gua15@PEG series）was constructed.TEM showed that the sizes of hybrid micelles were all below 50 nm,and the zeta potential increased gradually with the increasing of Gua15.The experimental results in vitro confirmed that the longer the zwitterionic chain,the better the effect of shielding polyguanidine,mainly reflected in the decrease of zeta potential（from+38.4 m V to+19.8 m V）and the enhancement of mucus permeability.However,the longer the zwitterionic chain,the stronger the effect of inhibiting cellular internalization of polyguanidine.Overall,30%Gua15@CB30 had the best ability to balance mucus penetration and cellular uptake,and had the highest transcytosis delivery efficiency which was about 30 times higher than that of PEG-M（as well as 6 times higher than that of CB30-M）.The in vitro transcytosis efficiency of 40%Gua15@CB45 was basically the same as that of 30%Gua15@CB30 in the presence of a mucus layer and electrostatic interaction（or salt bridge effect）resulted in a significant decrease in the transcytosis efficiency of 20%Gua15@CB15 and 40%Gua15@PEG.Polybetaine played an important role in the intracellular transport and transcytosis of 30%Gua15@CB30 mainly by retrograde pathway.Surprisingly,the intestinal mucosal in situ absorption capacity of40%Gua15@CB45 was better than that of 30%Gua15@CB30.These results suggested that in the complex intestinal mucosal environment,mucus permeability should be taken more importance in the rational design of oral nanocarriers than cellular uptake capacity.3.Based on the high expression of amino acid transporters with different categories in intestinal epithelial cells,the development of broad-spectrum amino acid-based zwitterionic nanocarriers has important application prospects in oral administration.Firstly,two Glu-derivatized amphiphilic block copolymers[PLA-b-P（Glutamine）₂₅ and PLA-b-P（Glutamate）₂₅,PPQ and PPD for short]were synthesized by ATRP.Amino acid-based zwitterionic micelles（PPD-M and PPQ-M）were prepared by simple nanoprecipitation.The sizes measured by DLS and TEM were mainly located at 20-30 nm and zwitterionic micelles both presented weakly negatively charged.Compared with PEG-M,the mucus permeability of amino acid-based zwitterionic micelles was both increased by 2times,and the cellular uptake ability was both increased by 3-4 times.PPQ-M had the highest cellular uptake capacity because its glutamine constitutional unit structure is more close to glutamine substrate.Multiple amino acid transporters,including ASCT2 and PAT1,have been shown to synergistically mediate cellular uptake of zwitterionic micelles.In addition,the intracellular transport of zwitterionic micelles was dominated by retrograde pathways,and some of them were delivered to lysosome for further degradation.Amino acid zwitterionic micelles could gently open the TJs,but their paracellular pathway delivery capacity remained to be studied with model drugs.Compared with PEG-M,amino acid-based zwitterionic micelles showed excellent intestinal mucosal absorption.Some zwitterionic micelles,especially PPQ-M,were successfully delivered to the basolateral side of the epithelial cell layer,and the basolateral micelles will have a high chance of transcytosis into the circulatory or lymphatic system.