Intracellular delivery crossing the endomembrane barrier is the "last mile to target" for nano delivery systems carrying biomacromolecules, including genetic medicines. Nevertheless, a mass of nanomedicines is currently restricted by their equivocal safety and delivery efficiency. Here, we establish a universal strategy independent of nanomaterials. Such a policy broadly facilitates the intracellular delivery of all kinds of tested nanomedicines, subtly by inducing ARF6 GTPases to their overactivated GTP-bound state. ARF6, one member of ARF subfamily in small GTPases, is verified to regulate intracellular vesicle transport and lipid metabolism through GTP/GDP conversion. ARF6 biased to GTP-bound state causes the increased endocytosis and reduced exocytosis of eleven types of nanoparticles. This universal effect is derived from the formation of a hybrid type of endosomes triggered by overactivated ARF6 via regulating cholesterol-associated vesicles and lipid raft/caveolae pathways. Due to the mild microenvironment in hybrid endosomes, the internalized protein and nanoparticles are steadily delivered to the cytoplasm, avoiding the intensive degradation in lysosomes. Based on these findings, we identify QS11, a safe small molecule inhibitor of ARF GTPase-activating proteins, significantly enhances the antitumor efficacy of siEGFR-loaded nanoparticles by inducing ARF6 overactivation. In sum, these findings reveal that the tactics of tuning ARF6 GTPases to GTP-bound form will widely benefit cellular nano delivery.

Abstract Intracellular delivery crossing the endomembrane barrier is the “last mile to target” for nano delivery systems carrying biomacromolecules, including genetic medicines. Nevertheless, a mass of nanomedicines is currently restricted by their equivocal safety and delivery efficiency. Here, we trimmed nano delivery at the cell perspective and established a general strategy independent of nanomaterials. Such a policy broadly facilitates the intracellular delivery of all kinds of tested nanomedicines, subtly by inducing ARF6 GTPases to their overactivated GTP-bound state. We discovered that ARF6, one member of ARF subfamily in small GTPases, regulated intracellular vesicle transport and lipid metabolism through GTP/GDP conversion. More importantly, we demonstrated that ARF6 biased to GTP-bound form induced GTPase overactivation, promoting endocytosis and reducing exocytosis of cargoes, including transferrin proteins and eleven types of nanogranules. This universal effect was mechanistically derived from forming a particular category of hybrid endosomes triggered by overactivated ARF6 via regulating cholesterol-associated vesicles and lipid raft/caveolae pathways. Cargoes were steadily and slowly delivered to the cytoplasm due to the mild microenvironment in hybrid endosomes. Based on these findings, we identified that QS11, a safe small molecule inhibitor of ARF GTPase-activating proteins, enhanced the antitumor efficacy of siEGFR-loaded nanoparticles by inducing ARF6 overactivation. In sum, it demonstrates that the tactics of tuning ARF6 GTPases to GTP-bound form will widely benefit cellular nano delivery.

Nano-tumor interactions are fundamental for cancer nanotherapy, and the cross-talk of nanomedicines with the extracellular matrix (ECM) is increasingly considered essential. Here, we specifically investigate the nano-ECM interactivity using drug-free nanoparticulates (NPs) and highly metastatic cancer cells as models. We discover with surprise that NPs closely bind to specific types of ECM components, namely, retraction fibers (RFs) and migrasomes, which are located at the rear of tumor cells during their migration. This interaction is observed to alter cell morphology, limit cell motion range and change cell adhesion. Importantly, NPs are demonstrated to inhibit tumor cell removal in vitro, and their anti-metastasis potential is preliminarily confirmed in vivo. Mechanically, the NPs are found to coat and form a rigid shell on the surface of migrasomes and retraction fibers via interaction with lipid raft/caveolae substructures. In this way, NPs block the recognition, endocytosis and elimination of migrasomes by their surrounding tumor cells. Thereby, NPs interfere with the cell-ECM interaction and reduce the promotion effect of migrasomes on cell movement. Additionally, NPs trigger alteration of the expression of proteins related to cell-cell adhesion and cytoskeleton organization, which also restricts cell migration. In summary, all the findings here provide a potential target for anti-tumor metastasis nanomedicines.

As an important part of tumor microenvironment, tumor associated macrophages (TAMs) play a vital role in the occurrence, development, invasion, and metastasis of many malignant tumors and can significantly promote the formation of tumor blood vessels and lymphatic vessels, hence TAMs are greatly associated with poor prognosis. The research on nanomedicine has achieved huge progress, and nano-drugs have been widely utilized to treat various diseases through different mechanisms. Therefore, developing nano-drugs that are based on TAMs-associated anti-tumor mechanisms to effectively suppress tumor growth is expected to be a promising research filed. This paper introduces relevant information about TAMs in terms of their origin, and their roles in tumor genesis, development and metastasis. Furthermore, TAMs-related anti-tumor nano-drugs are summarized. Specifically, a wide range of nano-drugs targeting at TAMs are introduced, and categorized according to their therapeutic mechanisms toward tumors. Additionally, various nano delivery platforms using TAMs as cell carriers which aim at inhibiting tumor growth are reviewed. These two parts elucidate that the exploration of nanomedicine is essential to the study on TAMs-related anti-tumor strategies. This review is also intended to provide novel ideas for in-depth investigation on anti-tumor molecular mechanisms and nano-drug delivery systems based on TAMs.

Mucus, which is secreted by the goblet cells of enterocytes, constitutes the first obstacle encountered for the intestinal absorption of nanomedicines. For decades, mucus has simply been regarded as a physical barrier that hinders the permeation and absorption of drugs, because of its high viscosity and reticular structure, whereas the interaction of mucus ingredients with nanomedicines is usually neglected. It is unclear whether glycoproteins, as the main components of mucus, interact with nanomedicines. We also do not know how the potential interaction affects the subsequent transportation of nanomedicines through the intestinal epithelium. In this study, mucin as the key element of mucus was investigated to characterize the interaction of nanomedicines with mucus. PEG-modified gold nanoparticles (PGNPs) were fabricated as model nanoparticles. Mucin was found to adhere to the nanoparticle surface to form a corona structure and induce the clustering of PGNPs by joining particles together, demonstrating the interaction between mucin and PGNPs. In addition, two intestinal epithelia, Caco-2 (non-mucus secretion) and HT-29 (high mucus secretion), were compared to evaluate the influence of mucin on the cellular interaction of PGNPs. Amazingly, mucin altered the trafficking characteristic of PGNPs in intestinal epithelium. Both in vitro and in vivo investigations demonstrated more nanoparticles being internalized by cells due to the mucin coverage. However, mucin induced a significant reduction in the transcytosis of PGNPs across epithelial monolayers. The mechanism exploration further revealed that the "more endocytosis but less transcytosis (MELT)" effect was mainly attributed to the strengthened retrograde pathway in which more PGNPs were transported to Golgi apparatus and exocytosed back to the apical but not the basolateral side of the epithelial monolayers. The "MELT" effect endowed mucin with duality in the nanoparticle transportation. Therefore, the rational regulation based on the "MELT" effect will provide new insight into overcoming the mucus obstacle as a barrier and enhancing the oral absorption rate of nanomedicines.

A long-term and huge challenge in nanomedicine is the substantial uptake and rapid clearance mediated by the mononuclear phagocyte system (MPS), which enormously hinders the development of nanodrugs. Inspired by the natural merits of extracellular vesicles, we therefore developed a combined "eat me/don't eat me" strategy in an effort to achieve MPS escape and efficient drug delivery. Methodologically, cationized mannan-modified extracellular vesicles derived from DC2.4 cells were administered to saturate the MPS (eat me strategy). Then, nanocarriers fused to CD47-enriched exosomes originated from human serum were administered to evade phagocytosis by MPS (don't eat me strategy). The nanocarriers were also loaded with antitumor drugs and functionalized with a novel homing peptide to promote the tumour tissue accumulation and cancer cell uptake (eat me strategy). The concept was proven in vitro as evidenced by the reduced endocytosis of macrophages and enhanced uptake by tumour cells, whereas prolonged circulation time and increased tumour accumulation were demonstrated in vivo. Specially, the strategy induced a 123.53% increase in tumour distribution compared to conventional nanocarrier. The study both shed light on the challenge overcoming of phagocytic evasion and provided a strategy for significantly improving therapeutic outcomes, potentially permitting active drug delivery via targeted nanomedicines.

The merits of nanomedicines are significantly impacted by the surrounding biological environment. Similar to the protein corona generated on the surface of nanoparticles in the circulation system, the intracellular protein corona (IPC) might be formed on nanoparticles when transported inside the cells. However, little is known currently about the formation of IPC and its possible biological influence. Methods: Caco-2 cells, a classical epithelial cell line, were cultured in Transwell plates to form a monolayer. Gold nanoparticles (AuNPs) were prepared as the model nanomedicine due to their excellent stability. Here we focused on identifying IPC formed on the surface of AuNPs during cell transport. The nanoparticles in the basolateral side of the Caco-2 monolayer were collected and analyzed by multiple techniques to verify IPC formation. High-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics was utilized to analyze the composition of IPC proteins. In particular, we established a dual-filtration strategy to exclude various interference in IPC identification. Based on the subcellular localization of specific IPC proteins, we elicited the nano-trafficking network of AuNPs. The transport pathways of AuNPs identified by proteomic analysis were also verified by various conventional technologies. Finally, we explored the influence of IPC on the uptake and stress response of endothelium. Results: The existence of IPC was demonstrated on the surface of AuNPs, in which 227 proteins were identified. Among them, 40 proteins were finally ascertained as the specific IPC proteins. The subcellular location analysis indicated that these "specific" IPC proteins could back-track the transport pathways of nanoparticles in the epithelial cell monolayer. According to the subcellular distribution of IPC proteins and co-localization, we discovered a new pathway of nanoparticles from endosomes to secretory vesicles which was dominant during the transcytosis. After employing conventional imageology and pharmacology strategies to verify the result of proteomic analysis, we mapped a comprehensive intracellular transport network. Our study also revealed the merits of IPC analysis, which could readily elucidate the molecular mechanisms of transcytosis. Besides, the IPC proteins increased the uptake and stress response of endothelium, which was likely mediated by extracellular matrix and mitochondrion-related IPC proteins. Conclusion: The comprehensive proteomic analysis of IPC enabled tracing of transport pathways in epithelial cells as well as revealing the biological impact of nanoparticles on endothelium.

Small GTPase is a kind of GTP-binding protein commonly found in eukaryotic cells. It plays an important role in cytoskeletal reorganization, cell polarity, cell cycle progression, gene expression and many other significant events in cells, such as the interaction with foreign particles. Therefore, it is of great scientific significance to understand the biological properties of small GTPases as well as the GTPase-nano interplay, since more and more nanomedicine are supposed to be used in biomedical field. However, there is no review in this aspect. This review summarizes the small GTPases in terms of the structure, biological function and its interaction with nanoparticles. We briefly introduced the various nanoparticles such as gold/silver nanoparticles, SWCNT, polymeric micelles and other nano delivery systems that interacted with different GTPases. These current nanoparticles exhibited different pharmacological effect modes and various target design concepts in the small GTPases study. This will help to elucidate the conclusion that the therapeutic strategy targeting small GTPases might be a new research direction. It is believed that the in-depth study on the functional mechanism of GTPases can provide insights for the design and study of nanomedicines.

Introduction Endocytosis of nanomaterials is the first step of nano-bio interaction and current regulation is mostly by nanomaterials but seldom by intracellular signaling proteins. Materials and methods Herein, we synthesized tubular nanocarbon (oxMWCNT) and lamellar-like nanocarbon (oxGRAPHENE) and formulated their aqueous dispersion. A549 and Caco-2 cells were selected as the models of tumor and intestinal epithelial cells, respectively. After knocking down three members of Rho GTPases (Cdc42, Rac1, RhoA) in these two cell lines, their silencing effects on the uptake pathways of nanomaterials with different morphologies were investigated. Results An unexpected finding was that the knock-down led to opposite uptake trends in different types of cells. The endocytosis of carbon nanomaterials increased in Caco-2 cells when Rho GTPases were inactivated, while that in A549 cells decreased. For nanomaterials with different shapes, the involved GTPase member of Rho family, or regulating protein molecule, was different. Concretely, Cdc42 and Rac1 were involved in oxMWCNT endocytosis, while all three GTPases participated in oxGRAPHENE internalization. More interestingly, such difference induced different uptake pathways, namely, the cellular uptake of oxMWCNT was clathrin-mediated and oxGRAPHENE was caveolin-modulated, both with the involvement of dynamin. Conclusion In conclusion, this study provides new insights for the potential intervention in nano-bio interplay.

Drug nanocrystals (NCs) appear to be favorable to improving oral bioavailability of poorly water-soluble drugs as evidenced by the great success they have had in the market. However, the pathway and mechanism of drug NCs through epithelial membrane are still unclear. In an attempt to clarify their transport features, paclitaxel nanocrystals (PTX-NCs), and paclitaxel hybrid NCs with lipophilic carbocyanine dyes, were prepared and characterized as the models. The endocytosis, intracellular trafficking, paracellular transport, and transcytosis of PTX-NCs were carefully investigated with Forster resonance energy transfer (FRET) analysis, as well as a colocalization assay, flow cytometry, gene silencing, Western-blot, transepithelial electrical resistance (TEER) study and other approaches on MDCK cells. It was found that rod-like PTX-NCs could transport through the monolayer intact, and the process of endocytosis proved to be time and energy dependent. Endoplasmic reticulum (ER) and Golgi complexes were colocalized with PTX-NCs in cells, so the ER Golgi complexes/Golgi complexes basolateral membrane pathway may be involved in,the intracellular trafficking and transcytosis of PTX-NCs. It was demonstrated here that cav-1, dynamin, and actin filament modulated the endocytosis process, and Cdc 42 regulated the transcytosis process. In addition, no paracellular transport of PTX-NCs was observed. These findings demonstrated that the rod-like nanocrystals not only enhanced the transcytosis of PTX compared with micropartides of raw drug materials but also changed the pathways of drug delivery. This study certainly provides insight for the oral absorption mechanism of nanocrystals of poorly soluble drugs.

Rho GTPases play an important role on the regulation of cytoskeleton,which can affect the cell morphogenesis,cell migration,endocytosis and vesicle transport by controlling the growth and maintenance of microfilaments and microtubules.It has been known that regulation of cell cytoskeleton is inseparable from the cell uptake of nano-medicine or nano-drug delivery systems.However,only few studies have focused on the impacts ofRho GTPases on cell uptake of nano-medicine or nano-drug delivery systems.This study selected single-walled carbon nanohoms (SWCNHs),which have emerged as promising drug delivery systems,to explore the impacts of Rho GTPases on cell uptake of nano-drug delivery systems.SWCNHs were oxidized with concentrated nitric acid and prepared into nano dispersion by ultrasonic dispersion.Confocal laser scanning microscope (CLSM)and transmission electron microscopy (TEM) were used to observe the cell uptake and intracellular distribution of nanoparticles after incubated A549 cells with the dispersion mentioned above.Mechanism of cell uptake was assessed using various inhibitors.The results showed that the cell uptake of oxSWCNHs was significantly reduced when RhoA was inhibited.The oxSWCNHs were internalized through clathrin-mediated endocytosis and mainly positioned in lysosomes of A549 cells.