| Since a number of therapeutic targets are located intracellularly,drug molecules need to enter the cells to achieve therapeutic effects by interacting with suitable targets.Therapeutic molecules in traditional formulations will be usually distributed to the organs and tissues throughout the body by blood circulatory system.However,it is often difficult for drug molecules to achieve efficient cellular internalization and delivery to the intracellular targets only by their diffusion,leading to insufficient therapy and obvious off-target side effects.Although nanocarriers bring promising future for precise drug delivery to intacellular targets,the complex multiple physiological barriers in the body severely hinder the transport and delivery efficiency of nanocarriers.In the process of transport from diseased tissues,such as tumor tissues,to relevant intracellular targets,the precisely designed nanocarriers must overcome the triplebarriers composed of extracellular matrix(ECM),cell membrane and intracellular matrix.The ECM and intracellular matrix provide net-structural biological gels barriers by numerous protein fibers,with pore sizes ranging from tens to hundreds of nanometers,which seriously hinder the diffusion of nanocarriers.Served as the cell boundary,cell membrane consists of lipid bilayer and forms an important barrier for the entry of external substances.Therefore,the ideal nanocarriers must be able to overcome the ECM barrier effectively by enhanced diffusion to reach the cell,further span the cell membrane,and then cross the intracellular matrix barrier for improved transport to targets,resulting in sufficient drug release and therapeutic effects.However,it is difficult for present traditional nanocarriers to efficiently and sequentially overcome the above triple-barriers for delivering drugs to intracellular targets.Therefore,more reasonable design of the carrier is needed to improve their delivery efficiency.Natural enveloped viruses are mainly composed of two parts: the outer lipid envelopes and the inner nucleocapsids with different sizes.The two components do not completely press each other,forming a special non-adherent core-shell structure,which plays a crucial role in endowing enveloped virus with efficient capacity to overcome biological multi-barriers and deliver their genome into host cells.This phenomenon inspires us to design nanocarriers by mimicking the core-shell structure and invasion function of enveloped virus.We can screen the advantageous core-shell carriers that can efficiently overcome ECM barrier by changing the core sizes.Then the carriers are controlled to cross the cell membrane by membrane fusion,releasing drug-loaded cores into cytosol and promote the trafficking efficiency of cores to targets for drug release in the intracellular matrix by optimizing the modification strategy.Based on the above assumptions,the following research was carried out.This work will be divided into two parts.In the first part of the study,inspired by the fact that the core-shell structure of enveloped virus contributes to its efficient penetration through the extracellular barrier,and the released inner nucleocapsid can further rapidly traffick in the intracellular matrix,we designed a series of nanoparticles(NPs).Firstly,the superior core-shell NPs were fabricated and optimized to overcome the ECM barriers efficiently.Then the transport ability of core particles in the intracellular matrix was improved by optimizing the modification strategy,laying a foundation for sequential overcoming the triple-barriers of ECM,cell membrane and intracellular matrix.A series of core-shell carriers with lipid shells and spherical cores of mesoporous silica nanoparticles(MSNs)with different sizes were prepared by microfluidic device.They had similar sizes(approximately 204.3 nm)and surface charges(approximately-25.4 m V).By using multiple-particle tracking method and super-resolution microscopy,it was confirmed that the non-adherent core-shell structures Lip@S60 and Lip@S100,whose structures were similar with enveloped virus,could effectively overcome the ECM barrier.Their mean square displacement(MSD)values were 2 to 5.6 times higher than that of common adherent core-shell carriers.In order to simulate the intracellular process of the nucleocapsid in enveloped virus and realize enhanced transport of the core particles by efficiently overcoming the intracellular matrix barrier,the S60 and S100 cores were further screened and modified.It was verified that the S60 coated with PEGylated lipid membrane(LM)could absorb less proteins due to its near-neutral surface charge(approximately-6.7 m V)and exhibit effective intracellular trafficking in cytosol with 32.1-fold MSD than naked S60 by intracellular multiple-particle tracking and protein corona evaluation.Thus,the screened LM core was verified that it could diffuse rapidly in the intracellular matrix and was a suitable choice to efficiently overcome the intracellular matrix barrier.In the first part,we filtered out the non-adherent core-shell structure that can effectively overcome the ECM barrier.And its core was optimized to obtain S60 wrapped in PEGylated lipid membrane(LM),which could be rapidly transported in the intracellular matrix.Further,in order to sequentially overcome the triple-barriers of ECM,cell membrane and intracellular matrix to achieve efficient intracellular drug delivery and further improved entry of the enveloped virus-mimetic core-shell structure into cells,we carried out the second part of the study to integrate the above-mentioned advantages of core-shell structure and the respective of core and shell to achieve efficient drug delivery.In the second part,a solid tumor model with obvious ECM-cell membraneintracellular matrix triple-barriers was selected to evaluate the delivery efficiency of nanocarriers.Inspired by the membrane fusion mechanism of enveloped virus into cells,on the basis of the superior core-shell structure and core particles screened in the first part,we further aimed at the cell membrane barrier and replaced the common lipid shell with homologous cancer cell membrane with membrane fusion potential.Finally,the enveloped virus-mimetic core-shell nanocarrier(CCM@LM)that can sequentially overcome the triple-barriers of ECM,cell membrane and intracellular matrix was successfully constructed.The particle size of CCM@LM was about 188.5 nm and the surface charge was about-23.8 m V.The encapsulation efficiencies of antitumor drugs doxorubicin and mefuparib hydrochloride increased to 93.7 ± 1.3% and 76.7 ± 1.7%,respectively.Firstly,CCM@LM exhibited a homologous tumor-targeting effect and immune escape capacity due to the cancer cell membrane shell,and could accumulate in tumor tissue for a long time without being cleared from the body quickly over more than 24 h post-administration.After entering tumor tissue,the non-adherent core-shell structure endowed CCM@LM with the capacity to efficiently overcome the ECM barrier,and its MSD value and tumor penetration efficiency are 44.9 and 23.3 times higher than those of the CCM vesicles,respectively.Combined the fluorescence resonance energy transfer with intracellular single-particle tracking techniques,it was validated that CCM@LM overcame the cell membrane barrier by membrane fusion similar to enveloped viruses and successfully released the LM core into cytosol.Then the LM particle could efficiently cross the intracellular matrix barrier and exhibited facilitated trafficking to perinuclear area to promote the drug delivery into the nucleus.Finally,the envelope virus-mimetic core-shell nanocarrier,CCM@LM,which coloaded with low-dose doxorubicin and mefuparib hydrochloride showed significantly stronger anti-tumor effect than Doxil,a first-line chemotherapeutic drug,yielding a tumor-inhibition rate up to 95.0%.Via mimicking the structure and function of enveloped virus,the drug carriers developed in this work efficiently overcame the triple delivery barriers of ECM,cell membrane and intracellular matrix,significantly improved the efficiency of drug delivery and anti-tumor effect.This work provides novel ideas and methods for the subsequent design of efficient drug delivery systems against multiple physiological barriers. |
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