Unraveling GLUT-mediated transcytosis pathway of glycosylated nanodisks

Glucose transporter(GLUT)-mediated transcytosis has been validated as an efficient method to cross the blood-brain barrier and enhance brain transport of nanomedicines.However,the transcytosis process remains elusive.Glycopeptide-modified nanodisks(Gly-A7R-NDs),which demonstrated high capacity of brain targeting via GLUT-mediated transcytosis in our previous reports,were utilized to better understand the whole transcytosis process.Gly-A7R-NDs internalized brain capillary endothelial cells mainly via GLUT-mediated/clathrin dependent endocytosis and macropinocytosis.The intracellular Gly-A7R-NDs remained intact,and the main excretion route of Gly-A7R-NDs was lysosomal exocytosis.Glycosylation of nanomedicine was crucial in GLUT-mediated transcytosis,while morphology did not affect the efficiency.This study highlights the pivotal roles of lysosomal exocytosis in the process of GLUT-mediated transcytosis,providing a new impetus to development of brain targeting drug delivery by accelerating lysosomal exocytosis.

Overloading of differentiated Caco-2 cells during lipid transcytosis induces glycosylation mistakes in the Golgi complex

Overloading the intestine enterocytes with lipids induced alteration of the Golgi complex(GC;Sesorova et al.,2020)and could cause glycosylation errors.Here,using differentiated Caco-2 cells with the established 0[I]blood group phenotype(no expression of the blood antigens A and B[AgA,AgB]under normal conditions)as a model of human enterocytes we examined whether the overloading of these cells with lipids could cause errors in the Golgi-dependent glycosylation.We demonstrated that under these conditions,there were alterations of the GC and the appearance of lipid droplets in the cytoplasm.Rare cells produced AgA and AgB.This suggested that after overloading of enterocytes with lipids,AgA were mistakenly synthesized in individual enterocytes by the Golgi glycosyltransferases.These mistakes could explain why in the absence of AgA and AgB antibodies against them exist in the blood.

Alteration of the Expression Levels of Rab3A Affects the Extent of Transcytosis of HRP-Labeled Marker Proteins in Rat CNS Neurons

It has been hypothesized that Rab3A, a small GTPase, may be closely involved in the process of dense core vesicle exocytosis in various cell types. This possibility was investigated by disrupting the expression levels of Rab3A-mRNA using a small interfering RNA of the Rab3A GTPase (Rab3A-siRNA) and examining the effect of this on transcytosis of wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP). Rab3A-siRNA and WGA-HRP were injected into the right vagus nerves of adult rats which were killed 12, 24 or 48 hours later. In some animals, portions of the brain stem containing the nucleus of solitary tract (NST) were prepared for electron microscopy. In other animals, the nodose ganglion of the vagus nerve was used to determine the levels of expression of Rab3A-mRNA using RT-PCR techniques. It was found that the expression of Rab3A-mRNA was markedly depressed in animals at 12 h after the Rab3A-siRNA injection. In the NST, there was an accumulation of HRP-reaction product (RP), recognized as electron dense lysosomal-like structures, in both axons and terminals in the NST 12 h after injection. Some HRP-RP was found in membrane bound vesicles in close proximity to cell membranes and appeared to be in the process of transcytosis. This neuronal transcytosis of HRP-RP appeared to occur at random locations over the axodendritic membranes. These findings indicate that inhibiting the expression of Rab3A-mRNA using Rab3A-siRNA can modulate the level of transcytosis of proteins across neuronal membranes confirming the potentially important role of this GTPase in the process of transcytosis.

Orange-derived extracellular vesicles nanodrugs for efficient treatment of ovarian cancer assisted by transcytosis effect

Extracellular vesicles(EVs)have recently received much attention about the application of drug carriers due to their desirable properties such as nano-size,biocompatibility,and high stability.Herein,we demonstrate orange-derived extracellular vesicles(OEV)nanodrugs(DN@OEV)by modifying cRGD-targeted doxorubicin(DOX)nanoparticles(DN)onto the surface of OEV,enabling significantly enhancing tumor accumulation and penetration,thereby efficiently inhibiting the growth of ovarian cancer.The obtained DN@OEV enabled to inducement of greater transcytosis capability in ovarian cancer cells,which presented the average above 10-fold transcytosis effect compared with individual DN.It was found that DN@OEV could trigger receptor-mediated endocytosis to promote early endosome/recycling endosomes pathway for exocytosis and simultaneously reduce degradation in the early endosomes-late endosomes-lysosome pathway,thereby inducing the enhanced transcytosis.In particular,the zombie mouse model bearing orthotopic ovarian cancer further validated DN@OEV presented high accumulation and penetration in tumor tissue by the transcytosis process.Our study indicated the strategy in enhancing transcytosis has significant implications for improving the therapeutic efficacy of thedrugdelivery system.

Hybrid transcytosis nanopomegranates for sensitizing breast cancer radiotherapy in deep tumor tissue

As a standard cancer treatment method,radiotherapy(RT)has cured or alleviated over half cancer bearing patients worldwide more than 100 years.However,the therapeutic outcome is seriously hindered by the resistant tumor microenvironment(TME).Hypoxia is a critical factor of vicious TME that causes radiation resistance owing to the insufficiency of oxygen for DNA damage maintenance.Moreover,severe vascular dysfunction and pyknomorphic extracellular matrix(ECM)in deep tumor tissues substantially limit radiosensitizer penetration and oxygen diffusion from vessels into tightly packed tumor core.In this study,we develop a hybrid transcytosis nanopomegranate(HTP)with high transcytosis potential in response to TME condition.HTP is architected by self-assembly of small CuS and Au nanoparticles(NPs)at normal physiological condition.HTP can rapidly collapse to transcytosis NPs(CuS and Au NPs)in TME with cationized surface,which enables excellent transcytosis potential and effectively elevates the penetration of CuS and Au into deep tumor tissues.Following the second near-infrared(NIR(II))biowindow laser irradiation,CuS heats the tumor and enhances blood perfusion,eliciting tumor hypoxia alleviation and DNA damage aggravation.Moreover,Au NPs enriched in deep tumor tissues effectively sensitize radio-therapeutic response.Our study provides a new and potential nano-platform to ameliorate tumor hypoxia and sensitize deep tumor tissue radiotherapy.

Transcytosis of Junonia coenia densovirus VP4 across the gut epithelium of Spodoptera frugiperda(Lepidoptera:Noctuidae)

The Junonia coenia densovirus rapidly traverses the gut epithelium of the host lepidopteran without replicating in the gut cells.The ability of this virus to transcytose across the gut epithelium is of interest for the potential use of virus structural proteins as delivery vehicles for insecticidal peptides that act within the insect hemocoel,rather than in the gut.In this study,we used fall armyworm,Spodoptera frugiperda to examine the binding of the virus to brush border membrane vesicle proteins by two-dimensional ligand blot analysis.We also assessed the rate of flux of the primary viral structural protein,VP4 fused to eGFP with a proline-rich linker(VP4-P-eGFP)through the gut epithelium ex vivo in an Ussing chamber.The mechanisms involved with transcytosis of VP4-P-eGFP were assessed by use of inhibitors.Bovine serum albumin(BSA)and eGFP were used as positive and negative control proteins,respectively.In contrast to BSA,which binds to multiple proteins on the brush border membrane,VP4-P-eGFP binding was specific to a protein of high molecular mass.Protein flux was significantly higher for VP4-P-eGFP after 2 h than for albumin or eGFP,with rapid transcytosis of VP4-P-eGFP within the first 30 min.In contrast to BSA which transcytosed following clathrin-mediated endocytosis,the movement of VP4-P-eGFP was vesicle-mediated but clathrin-independent.The specificity of binding combined with the efficiency of transport across the gut epithelium suggest that VP4 will provide a useful carrier for insecticidal peptides active within the hemocoel of key lepidopteran pests including S.frugiperda.

The relationship between amyloid-beta and brain capillary endothelial cells in Alzheimer's disease

Neurovascular dysfunction,as an integral part of Alzheimer's disease,may have an important influence on the onset and progression of chronic neurodegenerative processes.The bloodbrain barrier(BBB)pathway is one of the main pathways that mediates the clearance of amyloidbeta(Aβ)in the brain parenchyma.A large number of studies have shown that receptors and ATPbinding cassette transporte rs expressed on endothelial cells play an important role in Aβtransport across the BBB,but the specific mechanism is not clear.In this review,we summarize the possible mechanisms of Aβproduction and clearance,and in particular the relationship between Aβand brain capillary endothelial cells.Aβis produced by abnormal cleavage of the amyloid precursor protein via amyloidogenic processing under pathological conditions.Dys regulation of Aβclearance is considered to be the main reason for the massive accumulation of Aβin the brain parenchyma.Several pathways mediating Aβclearance from the brain into the periphery have been identified,including the BBB pathway,the blood-cerebros pinal fluid barrier and arachnoid granule pathway,and the lymphoidrelated pathway.Brain ca pilla ry endothelial cells are the key components of Aβclearance mediated by BBB.Receptors(such as LRP1,RAGE,and FcRn)and ATP-binding cassette transporters(such as P-gp,ABCA1,and ABCC1)expressed on endothelial cells play a critical role in Aβtranscytosis across the BBB.The toxic effects of Aβcan induce dysregulation of receptor and transpo rter expression on endothelial cells.Excessive Aβexerts potent detrimental cerebrovascular effects by promoting oxidative stress,inducing chronic inflammation,and impairing endothelial structure and functions.All of these are main causes for the reduction in Aβclearance across the BBB and the accumulation of Aβin the brain parenchyma.Therefo re,studies on the intera ctions between Aβand brain capillary endothelial cells,including their receptors and transporters,studies on inhibition of the toxic effects of Aβon endothelial cells,and studies on promoting the ability of endothelial cells to mediate Aβclearance may provide new therapeutic strategies for Aβclearance in Alzheimer's disease.

The endocytosis and intracellular fate of nanomedicines: Implication for rational design

Nanomedicines employ multiple endocytic pathways to enter cells.Their following fate is interesting,but it is not sufficient understood currently.This review introduces the endocytic pathways,presents new technologies to confirm the specific endocytic pathways and discusses factors for pathway selection.In addition,some intriguing implication about nanomedicine design based on endocytosis will also be discussed at the end.This review may provide new thoughts for the design of novel multifunctional nanomedicines.

Light-activated arginine-rich peptide-modified nanoparticles for deep-penetrating chemo-photo-immunotherapy of solid tumor

Poor permeation of drugs and“immune-cold”tumor microenvironment in solid tumors are the two major challenges which lead to the inefficient therapeutic efficacy for cancer treatment.Here,light-activated penetrable nanoparticles(PEGVAL&DOX&ICG@RNPs)for co-delivery of the chemotherapeutic drug doxorubicin(DOX),the photosensitizer agent indocyanine green(ICG),and the angiotensin II receptor blockers valsartan(VAL)were developed to achieve deep drug penetration and synergistic photo-chemo-immunotherapy of solid tumor.Studies showed that under the first-wave of laser irradiation,the polyethylene glycol(PEG)hydrophilic layer as an“inert”surface could detach from the nanoparticles,release VAL and expose the arginine-rich peptide modified-cores that can facilitate deep drug penetration via a transcytosis pathway.When exposed to the second-wave of laser irradiation,the synergistic chemo-photo-immunotherapy can be achieved.As expected,in 4T1 tumorbearing mice,PEG-VAL&DOX&ICG@RNPs treatment could effectively inhibit the growth of tumors,down-regulateα-smooth muscle actin expression level of cancer-associated fibroblasts cells in tumors,induce dendritic cells(DCs)maturation,and promote intratumoral infiltration of cytotoxic T lymphocytes.Moreover,combination therapy by PEG-VAL&DOX&ICG@RNPs and anti-PD-1 monoclonal antibody can elicit memory T cell response for preventing tumor recurrence and metastasis in vivo.This work provides a promising delivery strategy to overcome the current limitations of nanomedicine for achieving more effective therapeutic index of“immune-cold”solid tumor treatment.

“One-for-All”approach:a black technology for nanomedicine development?

Cancer nanomedicines require different,even opposite,properties to voyage the cascade drug delivery process involving a series of biological barriers.Currentlyapproved nanomedicines can only alleviate adverse effects but cannot improve patient survival because they fail to meet all the requirements.Therefore,nanocarriers with synchronized functions are highly requisite to capacitate efficient drug delivery and enhanced therapeutic efficacies.This perspective article summarizes recent advances in the two main strategies for nanomedicine design,the All-in-One approach(integration of all the functions in one system)and the One-for-All approach(one functional group with proper affinity enables all the functions),and presents our views on future nanomedicine development.

Manipulation of immune-vascular crosstalk:new strategies towards cancer treatment

Tumor vasculature is characterized by aberrant structure and function,resulting in immune suppressive profiles of tumor microenvironment through limiting immune cell infiltration into tumors,endogenous immune surveillance and immune cell function.Vascular normalization as a novel therapeutic strategy tends to prune some of the immature blood vessels and fortify the structure and function of the remaining vessels,thus improving immune stimulation and the efficacy of immunotherapy.Interestingly,the presence of"immune-vascular crosstalk"enables the formation of a positive feedback loop between vascular normalization and immune reprogramming,providing the possibility to develop new cancer therapeutic strategies.The applications of nanomedicine in vascular-targeting therapy in cancer have gained increasing attention due to its specific physical and chemical properties.Here,we reviewed the recent advances of effective routes,especially nanomedicine,for normalizing tumor vasculature.We also summarized the development of enhancing nanoparticle-based anticancer drug delivery via the employment of transcytosis and mimicking immune cell extravasation.This review explores the potential to optimize nanomedicine-based therapeutic strategies as an alternative option for cancer treatment.

A nanocleaner specifically penetrates the blood-brain barrier at lesions to clean toxic proteins and regulate inflammation in Alzheimer's disease

Insurmountable blood-brain barrier(BBB) and complex pathological features are the key factors affecting the treatment of Alzheimer's disease(AD).Poor accumulation of drugs in lesion sites and undesired effectiveness of simply reducing Aβ deposition or TAU protein need to be resolved urgently.Herein,a nanocleaner is designed with a rapamycin-loaded ROS-responsive PLGA core and surface modification with KLVFF peptide and acid-cleavable DAG peptide [R@(ox-PLGA)-KcD].DAG can enhance the targeting and internalization effect of nanocleaner towards neurovascular unit endothelial cells in AD lesions,and subsequently detach from nanocleaner in response to acidic microenvironment of endosomes to promote the transcytosis of nanocleaner from endothelial cells into brain parenchyma.Then exposed KLVFF can capture and carry Aβ to microglia,attenuating Aβ-induced neurotoxicity.Strikingly,rapamycin,an autophagy promoter,is rapidly liberated from nanocleaner in the high ROS level of lesions to improve Aβ degradation and normalize inflammatory condition.This design altogether accelerates Aβ degradation and alleviates oxidative stress and excessive inflammatory response.Collectively,our finding offers a strategy to target the AD lesions precisely and multi-pronged therapies for clearing the toxic proteins and modulating lesion microenvironment,to achieve efficient AD therapy.

Intelligent lesion blood–brain barrier targeting nano-missiles for Alzheimer's disease treatment by anti-neuroinflammation and neuroprotection

The treatment of Alzheimer's disease(AD)is one of the most difficult challenges in neurodegenerative diseases due to the insufficient blood‒brain barrier(BBB)permeability and unsatisfactory intra-brain distribution of drugs.Therefore,we established an ibuprofen and FK506 encapsulated drug co-delivery system(Ibu&FK@RNPs),which can target the receptor of advanced glycation endproducts(RAGE)and response to the high level of reactive oxygen species(ROS)in AD.RAGE is highly and specifically expressed on the lesion neurovascular unit of AD,this property helps to improve targeting specificity of the system and reduce unselective distribution in normal brain.Meanwhile,these two drugs can be specifically released in astrocytes of AD lesion in response to high levels of ROS.As a result,the cognition of AD mice was significantly improved and the quantity of Aβplaques was decreased.Neurotoxicity was also alleviated with structural regeneration and functional recovery of neurons.Besides,the neuroinflammation dominated by NF-κB pathway was significantly inhibited with decreased NF-κB and IL-1βin the brain.Overall,Ibu&FK@RNPs can efficiently and successively target diseased BBB and astrocytes in AD lesion.Thus it significantly enhances intracephalic accumulation of drugs and efficiently treats AD by anti-neuroinflammation and neuroprotection.

The role of caveolin-1 in the biofate and efficacy of anti-tumor drugs and their nano-drug delivery systems

As one of the most important components of caveolae,caveolin-1 is involved in caveolaemediated endocytosis and transcytosis pathways,and also plays a role in regulating the cell membrane cholesterol homeostasis and mediating signal transduction.In recent years,the relationship between the expression level of caveolin-1 in the tumor microenvironment and the prognostic effect of tumor treatment and drug treatment resistance has also been widely explored.In addition,the interplay between caveolin-1 and nano-drugs is bidirectional.Caveolin-1 could determine the intracellular biofate of specifc nano-drugs,preventing from lysosomal degradation,and facilitate them penetrate into deeper site of tumors by transcytosis;while some nanocarriers could also affect caveolin-1 levels in tumor cells,thereby changing certain biophysical function of cells.This article reviews the role of caveolin-1 in tumor prognosis,chemotherapeutic drug resistance,antibody drug sensitivity,and nano-drug delivery,providing a reference for the further application of caveolin-1 in nano-drug delivery systems.

Localization of Banana bunchy top virus and cellular compartments in gut and salivary gland tissues of the aphid vector Pentalonia nigronervosa

Banana bunchy top virus （BBTV） （Nanoviridae： Babuvirus） is transmitted by aphids of the genus Pentalonia in a circulative manner. The cellular mechanisms by which BBTV translocates from the anterior midgut to the salivary gland epithelial tissues are not understood. Here, we used multiple fluorescent markers to study the distribution and the cellular localization of early and late endosomes, macropinosomes, lysosomes, microtubules, actin filaments, and lipid raft subdomains in the gut and principal salivary glands ofPentalonia nigronervosa. We applied colabeling assays, to colocalize BBTV viral particles with these cellular compartments and structures. Our results suggest that multiple potential cellular processes, including clathrin- and caveolae-mediated endocytosis and lipid rafts, may not be involved in BBTV internalization.

Milk-derived exosomes exhibit versatile effects for improved oral drug delivery

As endogenous courier vesicles,exosomes play crucial roles in macromolecule transmission and intercellular communication.Therefore,exosomes have drawn increasing attention as biomimetic drug-delivery vehicles over the past few years.However,few studies have investigated the encapsulation of peptide/protein drugs into exosomes for oral administration.Additionally,the mechanisms underlying their biomimetic properties as oral delivery vehicles remain unknown.Herein,insulin-loaded milk-derived exosomes(EXO@INS)were fabricated and the in vivo hypoglycemic effect was investigated on type I diabetic rats.Surprisingly,EXO@INS(50 and 30 IU/kg)elicited a more superior and more sustained hypoglycemic effect compared with that obtained with subcutaneously injected insulin.Further mechanism studies indicated that the origin of excellent oral-performance of milk-derived exosomes combined active multi-targeting uptake,pH adaptation during gastrointestinal transit,nutrient assimilation related ERK1/2 and p38 MAPK signal pathway activation and intestinal mucus penetration.This study provides the first demonstration that multifunctional milk-derived exosomes offer solutions to many of the challenges arising from oral drug delivery and thus provide new insights into developing naturally-equipped nanovehicles for oral drug administration.

Antibody-activated trans-endothelial delivery of mesoporous organosilica nanomedicine augments tumor extravasation and anti-cancer immunotherapy

Tumor vasculature constitutes a formidable hurdle for the efficient delivery of cancer nanomedicine into tumors.The leverage of passive pathway through inter-endothelial gaps in tumor blood vessels might account for limited extravasation of nanomedicine into tumor microenvironment(TME).Herein,Annexin A1 antibody-installed mesoporous organosilica nanoplatforms carrying immunotherapeutics of anti-PD-L1 antibody(aPD-L1)and Indoximod are developed to target at caveolar Annexin-A1 protein of luminal endothelial cells and to trigger the active trans-endothelial transcytosis of nanomedicine mediated by caveolae.Such strategy enables rapid nanomedicine extravasation across tumor endothelium and relatively extensive accumulation in tumor interstitium.aPD-L1 and Indoximod release from aPD/IND@MON-aANN in a reduction-responsive manner and synergistically facilitate the intratumoral infiltration of cytotoxic T lymphocytes and reverse the immunosuppressive TME,thus demonstrating substantial anti-tumor efficacy in subcutaneous 4T1 breast tumors and remarkable anti-metastatic capacity to extend the survival of 4T1 tumor metastasis model.Moreover,aPD/IND@MON-aANN nanomedicine also exhibits distinct superiority over the combination therapy of free drugs to potently attenuate the progression of urethane-induced orthotopic lung cancers.Collectively,aPD/IND@MON-aANN nanoplatforms with boosted delivery efficiency via antibody-activated trans-endothelial pathway and enhanced immunotherapeutic efficacy provides perspectives for the development of cancer nanomedicines.