Charge-guided masking of a membrane-destabilizing peptide enables efficient endosomal escape for targeted intracellular delivery of proteins

Intracellular delivery of biologicals such as peptides,proteins,and nucleic acids presents a great opportunity for innovative therapeutics.However,the endosome entrapment remains a major bottleneck in the intracellular delivery of biomacromolecules,largely limiting their therapeutic potential.Here,we converted a cell-penetrating peptide(CPP),low molecular weight protamine(LMWP),to endosomal escape peptides(EEPs)by masking LMWP with a pH-responsive counter-ionic peptide.The resulting masked CPPs(mLMWP and mLMWP2)effectively promoted the escape of peptide/protein cargoes from endosomes into the cytoplasm.Consequential lysosome repair and lysophagy were initiated upon the endolysosomal leakage.Minimal reactive oxygen species(ROS)elevation or cell death was observed.Based on mLMWP2,we constructed an intracellular protein delivery system containing an antibody as a targeting module,mLMWP2 as an endosomal escape module,and the desired protein cargo.With the HER2-targeting delivery system,we efficiently translocated cyclization recombination enzyme(Cre)and BH3-interacting domain death agonist(BID)into the cytosol of HER2^(+)cells to exert their biological activity.Thereby,the modular delivery system shows its potential as a promising tool for scientific studies and therapeutic applications.

siRNA-functionalized lanthanide nanoparticle enables efficient endosomal escape and cancer treatment

Attaching DNA/RNA to nanomaterials is the basis for nucleic acid-based assembly and drug delivery.Herein,we report that small interfering RNA(siRNA)effectively coordinates with ligand-free lanthanide nanoparticles(NaGdF4 NPs),and forms siRNA/NaGdF4 spherical nucleic acids(SNA).The coordination is primarily attributed to the interaction between Gd and phosphate backbone of the siRNA.Surprisingly,an efficient encapsulation and rapid endosomal escape of siRNA from the endosome/lysosome were achieved,due to its flexible ability to bound to phospholipid head of endosomal membrane,thereby disrupting the membrane structure.Resorting to the dual properties of NaGdF4 NPs,siRNA loading,and endosomal escape,siRNA targeting programmed cell death-ligand 1(siPD-L1)/NaGdF4 SNA triggers significant gene silencing in vitro and in vivo,and effectively represses the tumor growth in both CT26 tumor model and 4T1 orthotopic murine model.

Modeling on in vivo disposition and cellular transportation of RNA lipid nanoparticles via quantum mechanics/physiologically-based pharmacokinetic approaches

The lipid nanoparticle(LNP)has been so far proven as a strongly effective delivery system for mRNA and siRNA.However,the mechanisms of LNP's distribution,metabolism,and elimination are complicated,while the transportation and pharmacokinetics(PK)of LNP are just sparsely investigated and simply described.This study aimed to build a model for the transportation of RNA-LNP in Hela cells,rats,mice,and humans by physiologically based pharmacokinetic(PBPK)and quantum mechanics(QM)models with integrated multi-source data.LNPs with different ionizable lipids,particle sizes,and doses were modeled and compared by recognizing their critical parameters dominating PK.Some interesting results were found by the models.For example,the metabolism of ionizable lipids was first limited by the LNP disassembly rate instead of the hydrolyzation of ionizable lipids;the ability of RNA release from endosomes for three ionizable lipids was quantitively derived and can predict the probability of RNA release.Moreover,the biodegradability of three ionizable lipids was estimated by the QM method and the is generally consistent with the result of PBPK result.In summary,the transportation model of RNA LNP among various species for the first time was successfully constructed.Various in vitro and in vivo pieces of evidence were integrated through QM/PBPK multi-level modeling.The resulting new understandings are related to biodegradability,safety,and RNA release ability which are highly concerned issues of the formulation.This would benefit the design and research of RNA-LNP in the future.

Perturbation of autophagy pathways in murine alveolar macrophage by 2D TMDCs is chalcogen-dependent

Increasing risks of incidental and occupational exposures to two-dimensional transition metal dichalcogenides(2D TMDCs)due to their broad application in various areas raised their public health concerns.While the composition-dependent cytotoxicity of 2D TMDCs has been well-recognized,how the outer chalcogenide atoms and inner transition metal atoms differentially contribute to their perturbation on cell homeostasis at non-lethal doses remains to be identified.In the present work,we compared the autophagy induction and related mechanisms in response to WS_(2),NbS_(2),WSe_(2)and Nb Se_(2)nanosheets exposures in MH-S murine alveolar macrophages.All these 2D TMDCs had comparable physicochemical properties,overall cytotoxicity and capability in triggering autophagy in MH-S cells,but showed outer chalcogen-dependent subcellular localization and activation of autophagy pathways.Specifically,WS_(2)and NbS_(2)nanosheets adhered on the cell surface and internalized in the lysosomes,and triggered m TOR-dependent activation of autophagy.Meanwhile,WSe_(2)and Nb Se_(2)nanosheets had extensive distribution in cytoplasm of MH-S cells and induced autophagy in an m TOR-independent manner.Furthermore,the 2D TMDCs-induced perturbation on autophagy aggravated the cytotoxicity of respirable benzo[a]pyrene.These findings provide a deeper insight into the potential health risk of environmental 2D TMDCs from the perspective of homeostasis perturbation.

CONTRIBUTION OF CHOLESTEROL MOIETIES ATTACHED ON MPEG-b-PCL-b-PLL TO THE CELL UPTAKE, ENDOSOMAL ESCAPE AND GENE KNOCKDOWN OF THE MICELLEPLEXES OF siRNA

To further enhance the transfection efficiency of a micelleplex system based on monomethoxy poly（ethylene glycol）-block-poly（e-caprolactone）-block-poly（L-lysine） （MPEG-b-PCL-b-PLL）, cholesterol （Chol） moieties are attached to the e-termini of PLL segments to obtain MPEG-b-PCL-b-PLL/Chol. The structure and morphology of the copolymer are studied by IH-NMR, TEM and DLS （dynamic light scattering）. The cytotoxicity, cell uptake, endosomal release and mRNA knockdown are studied by MTT （3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide） assay, flow cytometry, CLSM （confocal laser scanning microscopy） and RT-PCR （real-time polymerase chain reaction）. The results show that compared to their precursor MPEG-b-PCL-b-PLL, the cholesterol-grafted copolymer shows significantly lower toxicity, more rapid cellular endocytosis and endosome escape, and consequently displays enhanced siRNA transfection efficiency even at a lower N/P ratio. These improvements are ascribed to enhanced interaction of the cholesterol moieties with both cellular membrane and endosomal membrane. Moreover, effect of the PLL block length is examined. The final conclusion is that long enough PLL segments and incorporation of proper fraction of cholesterol onto the PLL segments benefit the enhancement of siRNA transfection efficiency.

Enhanced endosomal escape of dendrigraft poly-L-lysine polymers for the efficient gene therapy of breast cancer

Dendrimer,such as dendrigraft poly-L-lysine(DGL)polymers,with high surface charge density,well-defined structure,and narrow poly-dispersity is often employed as a gene vector,but its transfection efficiency is still partially inhibited due to poor endosomal escape ability.Herein,we used a surface modification strategy to enhance the endosomal escape ability of DGL polymers,and thus improved its gene transfection efficiency.A library of phenylboronic acid(PBA)modified DGL polymers(PBA-DGLs)was designed to screen efficient small interfering RNA(siRNA)vectors.The lead candidate screened from the library shown a capability of inducing nearly 90% gene silencing in MDA-MB-231 cells.The study of the transfection mechanism revealed that PBA modification not only improves siRNA cellular uptake,but,more importantly,endows DGL polymers the ability of endosomal escape.One of the top candidates from polyplexes was further shielded with hyaluronic acid to construct targeted nanoparticles,and the yielding nanoparticles significantly suppressed the tumor growth in a breast cancer model by effective siRNA delivery.This research provides a general and effective strategy to enhance the endosomal escape and transfection efficiency of dendrimer.

Endosomal escape of protein nanoparticles engineered through humanized histidine-rich peptides

Poly-histidine peptides such as H6(HHHHHH)are used in protein biotechnologies as purification tags,protein-assembling agents and endosomal-escape entities.The pleiotropic properties of such peptides make them appealing to design protein-based smart materials or nanoparticles for imaging or drug delivery to be produced in form of recombinant proteins.However,the clinical applicability of H6-tagged proteins is restricted by the potential immunogenicity of these segments.In this study,we have explored several humanized histidine-rich peptides in tumor-targeted modular proteins,which can specifically bind and be internalized by the target cells through the tumoral marker CXCR4.We were particularly interested in exploring how protein purification,self-assembling and endosomal escape perform in proteins containing the variant histidine-rich tags.Among the tested candidates,the peptide H5 E(HEHEHEHEH)is promising as a good promoter of endosomal escape of the associated fulllength protein upon endosomal internalization.The numerical modelling of cell penetration and endosomal escape of the tested proteins has revealed a negative relationship between the amount of protein internalized into target cells and the efficiency of cytoplasmic release.This fact demonstrates that the His-mediated,proton sponge-based endosomal escape saturates at moderate amounts of internalized protein,a fact that might be critical for the design of protein materials for cytosolic molecular delivery.

Convenient preparation of charge-adaptive chitosan nanomedicines for extended blood circulation and accelerated endosomal escape

A major impediment in the development of chitosan nanoparticles （CTS NPs） as effective drug delivery vesicles is their rapid clearance from blood and endosome entrapment. To overcome these problems, a convenient and promising template system was developed by decorating poly（methacrylic acid） （PMAA） to the surface of 10-hydroxy camptothecin （HCPT）-loaded CTS NPs （HCPT-CTS/ PMAA NPs）. The results show that the presence of negatively charged PMAA significantly elongated the blood circulation time of HCPT-CTS NPs from 12 to 24 h, and reduced the blood clearance （C1） from 30.57 to 6.72 mL/h in vivo. The calculated area under curve （AUC0-24h） and terminal elimination half-life （tl/2） of HCPT-CTS/PMAA NPs were 4.37-fold and 2.48-fold compared with those of HCPT-CTS NPs. Furthermore, the positively charged HCPT-CTS/PMAA NPs triggered by tumor acidic microenvironment （pH 6.5） result in a 453-fold higher cellular uptake than the negatively charged counterparts at pH 7.4. Additionally, HCPT-CTS/PMAA NPs have the ability to escape endosomal entrapment via ＂proton sponge effect＂ after incubation with HepG2 cells for 3 h at pH 6.5. Taken together, these findings open up a convenient, low-cost, but effective way to prepare HCPT-CTS/PMAA NPs as a candidate for developing vectors with enhanced long blood circulation and endosomal escape ability in future clinical experiments.

Lipid carriers for mRNA delivery

Messenger RNA(mRNA)is the template for protein biosynthesis and is emerging as an essential active molecule to combat various diseases,including viral infection and cancer.Especially,mRNA-based vaccines,as a new type of vaccine,have played a leading role in fighting against the current global pandemic of COVID-19.However,the inherent drawbacks,including large size,negative charge,and instability,hinder its use as a therapeutic agent.Lipid carriers are distinguishable and promising vehicles for mRNA delivery,owning the capacity to encapsulate and deliver negatively charged drugs to the targeted tissues and release cargoes at the desired time.Here,we first summarized the structure and properties of different lipid carriers,such as liposomes,liposome-like nanoparticles,solid lipid nanoparticles,lipid-polymer hybrid nanoparticles,nanoemulsions,exosomes and lipoprotein particles,and their applications in delivering mRNA.Then,the development of lipid-based formulations as vaccine delivery systems was discussed and highlighted.Recent advancements in the mRNA vaccine of COVID-19 were emphasized.Finally,we described our future vision and perspectives in this field.

Pulmonary delivery of siRNA against acute lung injury/acute respiratory distress syndrome

The use of small interfering RNAs(si RNAs)has been under investigation for the treatment of several unmet medical needs,including acute lung injury/acute respiratory distress syndrome(ALI/ARDS)wherein si RNA may be implemented to modify the expression of pro-inflammatory cytokines and chemokines at the m RNA level.The properties such as clear anatomy,accessibility,and relatively low enzyme activity make the lung a good target for local si RNA therapy.However,the translation of si RNA is restricted by the inefficient delivery of si RNA therapeutics to the target cells due to the properties of naked si RNA.Thus,this review will focus on the various delivery systems that can be used and the different barriers that need to be surmounted for the development of stable inhalable si RNA formulations for human use before si RNA therapeutics for ALI/ARDS become available in the clinic.

Achieving efficient RNAi therapy: progress and challenges

RNA interference(RNAi)has been harnessed to produce a new class of drugs for treatment of various diseases.This review summarizes the most important parameters that govern the silencing efficiency and duration of the RNAi effect such as small interfering RNA(siRNA)stability and modification,the type of delivery system and particle sizing methods.It also discusses the predominant barriers for siRNA delivery,such as off-target effects and introduces internalization,endosomal escape and mathematical modeling in RNAi therapy and combinatorial RNAi.At present,effective delivery of RNAi therapeutics in vivo remains a challenge although significant progress has been made in this field.

Synchronous conjugation of i-motif DNA and therapeutic siRNA on the vertexes of tetrahedral DNA nanocages for efficient gene silence

The functionality of DNA biomacromolecules has been widely excavated,as therapeutic drugs,carriers,and functionalized modification derivatives.In this study,we developed a series of DNA tetrahedron nanocages(Td),via synchronous conjugating different numbers of i-(X)and therapeutic siRNA on four vertexes of tetrahedral DNA nanocage(aX-Td@bsiRNA,a+b=4).This i-motif-conjugated Td exhibited good endosomal escape behaviours in A549 tumor cells,and the escape efficiency was affected by the number of i-motif.Furthermore,the downregulating mRNA and protein expression level of epidermal growth factor receptor(EGFR)caused by this siRNA embedded Td were verified in A549 cells.The tumor growth inhibition efficiency of the 2X-Td@2siRNA treated group in tumorbearing mice was significantly higher than that of non-i-motif-conjugated Td@2siRNA(3.14-fold)and free siRNA(3.63-fold).These results demonstrate a general strategy for endowing DNA nanostructures with endosomal escape behaviours to achieve effective in vivo gene delivery and therapy.

Evaluation of the intracellular trafficking of siRNAs in A375 cells by confocal laser scanning microscopy

Investigation intracellular trafficking of siRNAs following their delivery to cells is of great interest to elucidate dynamics of siRNA in cytoplasm. In this study, we present a novel confocal laser scanning microscopy （CLSM） method to evaluate a novel delivery system of 3＇-peptide-siRNA therapeutic, which was named 3＇-pAs-siRNA/CLD. This method could not only calculate the content of the intracellular 3＇-peptide-siRNA, but also quantify its co-localization with cellular substructure. We observed that 3＇-pAs-siRNA/CLD, which provided the better antitumor capability, also had a better cell uptake, endosome escape and a longer retention time in A375. This novel strategy was proved to be efficient, quantified and visualized, thus making the dynamics research of siRNA in cytoplasm clear and simplified.