Role of Micelle Size in Cell Transcytosis-Based Tumor Extravasation, Infiltration, and Treatment Efficacy.

Transcytosis-based active transport of cancer nanomedicine has shown great promise for enhancing its tumor extravasation and infiltration and antitumor activity, but how the key nanoproperties of nanomedicine, particularly particle size, influence the transcytosis remains unknown. Herein, we used a transcytosis-inducing polymer, poly[2-(N-oxide-N,N-diethylamino)ethyl methacrylate] (OPDEA), and fabricated stable OPDEA-based micelles with different sizes (30, 70, and 140 nm in diameter) from its amphiphilic block copolymer, OPDEA-block-polystyrene (OPDEA-PS). The study of the micelle size effects on cell transcytosis, tumor extravasation, and infiltration showed that the smallest micelles (30 nm) had the fastest transcytosis and, thus, the most efficient tumor extravasation and infiltration. So, the 7-ethyl-10-hydroxyl camptothecin (SN38)-conjugated OPDEA micelles of 30 nm had much enhanced antitumor activity compared with the 140 nm micelles. These results are instructive for the design of active cancer nanomedicine.

Design Principles for Smart Linear Polymer Ligand Carriers with Efficient Transcellular Transport Capabilities.

The surface functionalization of polymer-mediated drug/gene delivery holds immense potential for disease therapy. However, the design principles underlying the surface functionalization of polymers remain elusive. In this study, we employed computer simulations to demonstrate how the stiffness, length, density, and distribution of polymer ligands influence their penetration ability across the cell membrane. Our simulations revealed that the stiffness of polymer ligands affects their ability to transport cargo across the membrane. Increasing the stiffness of polymer ligands can promote their delivery across the membrane, particularly for larger cargoes. Furthermore, appropriately increasing the length of polymer ligands can be more conducive to assisting cargo to enter the lower layer of the membrane. Additionally, the distribution of polymer ligands on the surface of the cargo also plays a crucial role in its transport. Specifically, the one-fourth mode and stripy mode distributions of polymer ligands exhibited higher penetration ability, assisting cargoes in penetrating the membrane. These findings provide biomimetic inspiration for designing high-efficiency functionalization polymer ligands for drug/gene delivery.

Transcellular Transport Behavior of the Intact Polymeric Mixed Micelles with Different Polymeric Ratios.

In order to better promote the application of the polymeric mixed micelles (PMMs) in oral delivery, in addition to focusing on the improvement of micellar structural stability, it is necessary to obtain the absorption characteristics of the intact micellar particles. In this work, the transport behavior across Caco-2 cells of FS/PMMs composed of Pluronic F127 and Solutol HS15 was tracked by encapsulating an environment-responsive probe into the particles. The specific property of the probe is the water-initiated aggregation-caused quenching (ACQ) ability, by which integral particles can be identified accurately. The influence of polymeric ratios (FS) on the transcellular behavior of FS/PMMs was explored and the single pass intestinal perfusion experiment was used to further illustrate it. Moreover, pharmacokinetics parameters were detected to analyze the relationship among FS ratios, transport behavior, and pharmacokinetic parameters. FS ratios were found to hardly affect the endocytosis pathways and intracellular itinerary of FS/PMMs, but do affect the proportion of each path. FS/PMMs with high HS15 content, namely System-I, were found to primarily undergo receptor-mediated endocytosis pathway and be less susceptible to lysosomal degradation, which would lead to more absorption and higher Cmax and AUC than drug suspension. In contrast, despite System-II with high F127 content cannot contribute to drug plasma concentration, it can prolong the in vivo retention time. These findings provided evidence for the role of polymeric ratios in modulating the transcellular absorption and pharmacokinetic parameters of the drug-loaded PMMs, and would be a step forward in helping PMMs' design to enhance oral drug delivery.

Hydrophobicity Effects of γ-Glutamyl Transpeptidase-Responsive Polymers on the Catalytic Activity and Transcytosis Efficacy.

Active transcytosis has recently sparked great interest in drug delivery as a novel route for tumor extravasation and infiltration. However, the rational design of transcytosis-inducing nanomedicines remains challenging. We recently demonstrated that the γ-glutamyl transpeptidase (GGT)-responsive polymer cationization induced efficient adsorption-mediated transcytosis (AMT). However, it remains unclear how the nanomedicines' physicochemical properties influence the GGT-responsive cationization and induced transcytosis behaviors. Herein, through a combination of experimental techniques and molecular dynamics (MD) simulations, we find that the random copolymers with high hydrophobic monomers tend to form compact structures accessible to the catalytic site of GGT, leading to a fast cationization and thus high transcytosis efficiency, while the homopolymers of the hydrophilic GGT-sensitive monomers have elongated structures unable to enter the active site and thus exhibit poor GGT sensitivity. As a result, the more hydrophobic polymer-drug conjugates with high camptothecin contents exhibit higher GGT-responsive activity, which in turn leads to faster cationization and cellular internalization, enhanced tumor infiltration, and more potent antitumor activity. These findings indicate the hydrophobicity is a main parameter determining the GGT catalytic activity and transcytosis efficiency of the GGT-activatable co(homo)polymers, providing guidelines for the rational design of GGT-induced charge reversal carriers for transcytotic nanomedicines.

Albumin-Embellished Arsenic Trioxide-Loaded Polymeric Nanoparticles Enhance Tumor Accumulation and Anticancer Efficacy via Transcytosis for Hepatocellular Carcinoma Therapy.

Arsenic trioxide (ATO) has efficient anticancer effect on hepatocellular carcinoma (HCC) in clinical trials, but its off-target distribution and side effects have limited its use. Here, we demonstrate an albumin-embellished ATO-loaded polyethylene glycol-polycaprolactone-polyethyleneimine (PEG-PCL-PEI) nanoparticle (AATONP) to enhance the tumor distribution and intratumor drug release of ATO for HCC therapy. AATONP is prepared by surface embellishment with albumin on the cationic ATO-loaded PEG-PCL-PEI nanoparticles (CATONP). Albumin embellishment can reduce the cationic material's hemolytic toxicity in blood cells while maintaining the rapid internalization and lysosome escape abilities of the positively charged CATONP. AATONP provides sustained and low pH-responsive drug release, facilitating the targeted drug release in the intratumor acidic microenvironment. Moreover, AATONP can significantly improve the circulation time and tumor distribution of ATO via albumin-mediated transcytosis in HCC tumor-bearing mice. Compared with free ATO and the clinically used nanomedicine Genexol/PM, AATONP shows potent antitumor activity against a human HCC xenograft mouse model, leading to a higher tumor inhibition rate of 89.4% in HCC therapy. In conclusion, this work presents an efficient strategy to achieve tumor accumulation and the intratumor drug release of ATO for HCC therapy. An albumin-embellished arsenic trioxide (ATO)-loaded polyethylene glycol-polycaprolactone-polyethyleneimine nanoparticle (AATONP) is designed to enhance tumor distribution and intratumor drug release of ATO for hepatocellular carcinoma therapy. AATONP can achieve enhanced tumor distribution via albumin-mediated transcytosis and exhibit intratumor drug release of ATO via tumor acidic microenvironment-response, leading to potent antitumor activity.

Effect of Cationic Charge Density on Transcytosis of Polyethylenimine.

The adsorption-mediated transcytosis (AMT) induced by the electrostatic interaction between the positively charged surface of carriers and negatively charged cell membrane is a new paradigm enabling nanomedicine's tumor extravasation and infiltration. However, little is known about the correlation between the carrier's charge density and its AMT-induced tumor infiltration efficiency. Herein, we investigate the effect of the cationic polymer's charge on the AMT-induced tumor penetration ability using in vitro multilayer tumor spheroids (MTSs). A cationic polymer, polyethylenimine (PEI), is amidized with acetic anhydride to obtain acetylated PEIs (AcPEIs) with different cationic charge densities. As the amidization ratio increases, the AcPEIs' cytotoxicity, zeta potential, and cell-binding affinity significantly decrease. Notably, not only does the weak cell binding (AcPEIs with high acetylation degrees) lead to slow endocytosis and inefficient transcytosis, so does the strong cell-binding PEI. The PEI with 24% acetylation (AcPEI24%) is found to have the highest transcytosis efficiency because its balanced cell-binding affinity triggers fast adsorption-mediated endocytosis. The subsequent Golgi apparatus/endoplasmic reticulum-mediated exocytosis via extracellular vesicles leads to highly effective transcellular delivery and tumor penetration in MTSs. Therefore, the drug carrier's surface cationic charge density critically influences its AMT-induced tumor penetration efficiency. This study provides mechanistic insights into the design of drug-delivery systems with active transcytosis for improved tumor penetration and enhanced therapeutic efficiency.

Involvement of Caveolin-1-mediated transcytosis in the intratumoral accumulation of liposomes.

For achieving efficient cancer treatment, it is important to elucidate the mechanism responsible for the accumulation of nanoparticles in tumor tissue. Recent studies suggest that nanoparticles are not delivered merely through gaps between tumor endothelial cells. We previously reported that the maturation of the vascular structure by the vascular endothelial cell growth factor receptor 2 (VEGFR2) using a previously developed siRNA delivery technology (RGD-MEND) significantly enhanced the accumulation of nanoparticles in types of cancers that area vessel-rich (renal cell carcinoma). This result was completely inconsistent with the generally accepted theory of the enhanced permeability and retention (EPR) effect. We hypothesized that a caveolin-1 (Cav1)-mediated transcellular route would be involved with the penetration of nanoparticles into tumor vasculature. To reveal the exact mechanism responsible for this enhancement, we observed the delivery of long-circulating liposomes (LPs) after Cav1 was co-suppressed by RGD-MEND with VEGFR2. The enhanced delivery of LPs by siRNA against VEGFR2 (siVEGFR2) was accompanied by the elevated expression of the Cav1 protein. In addition, Cav1 knockdown by siRNA against Cav1 (siCav1) canceled the enhanced delivery of LPs by siVEGFR2. The injection of siCav1 had no effect on the formation of alpha smooth muscle actin or vascular endothelial cell adhesion molecules. These results suggest that a Cav1-induced transcellular route and not a paracellular route, at least partially, contributes to the accumulation of nanoparticles in tumors.

Immunization with HIV-1 gp41 subunit virosomes induces mucosal antibodies protecting nonhuman primates against vaginal SHIV challenges.

Human immunodeficiency virus (HIV)-1 is mainly transmitted mucosally during sexual intercourse. We therefore evaluated the protective efficacy of a vaccine active at mucosal sites. Macaca mulatta monkeys were immunized via both the intramuscular and intranasal routes with an HIV-1 vaccine made of gp41-subunit antigens grafted on virosomes, a safe delivery carrier approved in humans with self-adjuvant properties. Six months after 13 vaginal challenges with simian-HIV (SHIV)-SF162P3, four out of five vaccinated animals remained virus-negative, and the fifth was only transiently infected. None of the five animals seroconverted to p27gag-SIV. In contrast, all 6 placebo-vaccinated animals became infected and seroconverted. All protected animals showed gp41-specific vaginal IgAs with HIV-1 transcytosis-blocking properties and vaginal IgGs with neutralizing and/or antibody-dependent cellular-cytotoxicity activities. In contrast, plasma IgGs totally lacked virus-neutralizing activity. The protection observed challenges the paradigm whereby circulating antiviral antibodies are required for protection against HIV-1 infection and may serve in designing a human vaccine against HIV-1-AIDS.

Role of Endocytosis in Nanoparticle Penetration of 3D Pancreatic Cancer Spheroids.

Robust deposition of extracellular matrix is a significant barrier for delivery of nanotherapeutics and small-molecule anticancer drugs to different tumors including pancreatic ductal adenocarcinoma. Here, we investigated permeation and total uptake of polystyrene nanoparticles of different diameters in 3D multicellular spheroid models of pancreatic tumors. Special attention was given to analysis of the impact of endocytic processes on nanoparticle accumulation and distribution in spheroids. We generated spheroids of BxPC3 or PANC-1 cells that were able to internalize 20, 100, and 500 nm fluorescent polystyrene beads with different efficacies, resulting in 20 ≫100 > 500 nm and 100 > 500 > 20 nm trends, respectively. It was found that endocytosis and transcytosis increased overall nanoparticle uptake and facilitated permeation of 20 nm beads in BxPC3 spheroids, whereas 100 and 500 nm particles did not penetrate. In PANC-1 spheroids, penetration of nanoparticles also decreased with the increase of size but was not significantly affected by endocytic processes. Thus, our study showed that passive diffusion and endocytic processes may have a different contribution to nanoparticle accumulation and distribution in spheroid models of pancreatic cancer.

Length of surface PEG modulates nanocarrier transcytosis across brain vascular endothelial cells.

Diblock PLA-PEG nanoparticles were produced to establish the role of PEG chain length on brain vascular endothelial cell transcytosis. 100-nm nanoparticles tagged with fluorescent pyrene butanol and coated with PEG chains (Mw: 1-10 kDa), at similar PEG surface density, were used to study endocytosis and transcytosis phenomena on mouse vascular endothelial cell monolayers. The transport mechanisms were then investigated through inhibitory processes. Our results show that there is an evident correlation between PEG chain length and nanoparticle translocation. The highest transcytosis rates were obtained with PEG5000 and PEG10000 and macropinocytosis appeared to play a central role in cell uptake. This study constitutes the first systematic exploration of the role of PEG chain length on nanoparticle endocytosis and transcytosis in an in vitro model of the blood-brain barrier.

Controversies in Management of Hyperkalemia.

BACKGROUND: Hyperkalemia is a common electrolyte disorder that can result in morbidity and mortality if not managed appropriately. OBJECTIVES: This review evaluates the classic treatments of hyperkalemia and discusses controversies and new medications for management. DISCUSSION: Potassium (K+) plays a key role in determining the transmembrane potentials of "excitable membranes" present in nerve and muscle cells. K+ is the predominant intracellular cation, and clinical deterioration typically ensues when patients develop sufficiently marked elevation in extracellular fluid concentrations of K+ (hyperkalemia). Hyperkalemia is usually detected via serum clinical laboratory measurement. The most severe effect of hyperkalemia includes various cardiac dysrhythmias, which may result in cardiac arrest and death. Treatment includes measures to "stabilize" cardiac membranes, to shift K+ from extracellular to intracellular stores, and to promote K+ excretion. Calcium gluconate 10% dosed 10 mL intravenously should be provided for membrane stabilization, unless the patient is in cardiac arrest, in which case 10 mL calcium chloride is warranted. Beta-agonists and intravenous insulin should be given, and some experts recommend the use of synthetic short-acting insulins rather than regular insulin. Dextrose should also be administered, as indicated by initial and serial serum glucose measurements. Dialysis is the most efficient means to enable removal of excess K+. Loop and thiazide diuretics can also be useful. Sodium polystyrene sulfonate is not efficacious. New medications to promote gastrointestinal K+ excretion, which include patiromer and sodium zirconium cyclosilicate, hold promise. CONCLUSIONS: Hyperkalemia can be deadly, and treatment requires specific measures including membrane stabilization, cellular shift, and excretion.

Transcytosis-Targeting Peptide: A Conductor of Liposomal Nanoparticles through the Endothelial Cell Barrier.

The ultimate goal in the area of drug-delivery systems is the development of a nanoparticle that can penetrate the endothelial cell monolayer for the targeting of tissue parenchyma. In the present study, we identify a transcytosis-targeting peptide (TTP) that permits polyethyleneglycol (PEG)-modified liposomes (PEG-LPs) to penetrate through monolayers of brain-derived endothelial cells. These endothelial cells were layered on a gelatin nanofiber sheet, a nanofiber meshwork that allows the evaluation of transcellular transport of nanosized particles (ca. 100 nm). Systematic modification of the sequences results in the identification of the consensus sequence of TTP as L(R/K)QZZZL, where Z denotes hydrophilic amino acids (R/K/S and partially D). The TTP-modified liposomes are bound on the heparin sulfate proteoglycan, and are then taken up via lipid raft-mediated endocytosis. Subsequent intracellular imaging of the particles reveals a unique intracellular sorting of TTP-modified PEG liposomes (TTP-PEG-LPs); namely the TTP-LPs are not localized with the lysosomes, whereas this co-localization is dominant in the unmodified PEG liposomes (PEG-LPs). The in vivo endothelial penetration of liposomes in adipose tissue is conferred by the dual modification of the particles with TTP and tissue-targeting ligands. This technology promises innovations in intravenously available delivery system to tissue parenchyma.

Topical delivery of Avastin to the posterior segment of the eye in vivo using annexin A5-associated liposomes.

Effective delivery to the retina is presently one of the most challenging areas in drug development in ophthalmology, due to anatomical barriers preventing entry of therapeutic substances. Intraocular injection is presently the only route of administration for large protein therapeutics, including the anti-Vascular Endothelial Growth Factors Lucentis (ranibizumab) and Avastin (bevacizumab). Anti-VEGFs have revolutionised the management of age-related macular degeneration and have increasing indications for use as sight-saving therapies in diabetes and retinal vascular disease. Considerable resources have been allocated to develop non-invasive ocular drug delivery systems. It has been suggested that the anionic phospholipid binding protein annexin A5, may have a role in drug delivery. In the present study we demonstrate, using a combination of in vitro and in vivo assays, that the presence of annexin A5 can significantly enhance uptake and transcytosis of liposomal drug carrier systems across corneal epithelial barriers. This system is employed to deliver physiologically significant concentrations of Avastin to the posterior of the rat eye (127 ng/g) and rabbit retina (18 ng/g) after topical application. Our observations provide evidence to suggest annexin A5 mediated endocytosis can enhance the delivery of associated lipidic drug delivery vehicles across biological barriers, which may have therapeutic implications.

Fine-tuning of liposome integrity for differentiated transcytosis and enhanced antitumor efficacy.

Transcytosis-inducing nanomedicines have been developed to improve tumor extravasation. However, the fate during transcytosis across multicell layers and the structural integrity of the nanomedicines before reaching tumor cells could impact antitumor therapy. Here, a BAY 87-2243 (a hypoxia-inducible factor-1 inhibitor)-loaded liposomal system (HA-P-LBAY) modified by low molecular weight protamine (LMWP) and crosslinked by hyaluronic acid (HA) was constructed. This system could accomplish differentiate cellular transport in endothelial and tumor cells by fine-tuning its structural integrity, i.e. transcytosis across the endothelial cells while preserving structural integrity, facilitating subsequent retention and drug release within tumor cells via degradation-induced aggregation. In vitro cellular uptake and transwell studies demonstrated that HA-P-LBAY were internalized by endothelial cells (bEnd.3) via an active, caveolin and heparin sulfate proteoglycan (HSPG)-mediated endocytosis, and subsequently achieved transcytosis mainly through the ER/Golgi pathway. Moreover, the fluorescence resonance energy transfer (FRET) study showed that HA-crosslinking maintained higher integrity of HA-P-LBAY after transcytosis, more efficiently than electrostatic coating of HA (HA/P-LBAY). In addition, more HA-P-LBAY was retained in tumor cells (4T1) compared to HA/P-LBAY corresponding to its enhanced in vitro cytotoxicity. This may be attributed to better integrity of HA-P-LBAY post endothelial transcytosis and more degradation of HA in tumor cells, leading to more liposome aggregation and inhibition of their transcytosis, which was inferred by both TEM images and the HAase responsiveness assay proved by FRET. In vivo, HA-P-LBAY exhibited more potency in tumor suppression than the other formulations in both low and high permeability tumor models. This highlighted that fine-tuning of structural integrity of nanocarriers played a key role no matter whether the transcytosis of nanocarriers contributed to cellular transport. Collectively, this study provides a promising strategy for antitumor therapies by fine-tuning liposome integrity to achieve active trans-endothelial transport with structural integrity and selective aggregation for prolonged tumor retention.

Electrostatic properties of confluent Caco-2 cell layer correlates to their microvilli growth and determines underlying transcellular flow.

Recently, Rajapaksa et al. (2010) showed that the rate of uptake of potential vaccine delivery nanoparticles in the mucosal layer is a function of the electrostatic properties of the corresponding solvent. This fundamentally implies that the dominant driving forces that may be capitalized on for mucosal vaccine strategies are electrostatic in nature. We hypothesize that the driving force normal to the cell (in the direction from apical to basolateral across the cell) is of particular importance. In addition, it has been theoretically shown that the electrostatic properties of mucosal cells are directly related to their development of brush border. Here we correlate the development of brush border on a human mucosal epithelial model (Caco-2) cultured in DMEM on 3.0 µm pore sized polycarbonate membranes to their corresponding electrostatic properties characterized by measuring their normal zeta potential. Properties of normal streaming potential, hydraulic permeability, and brush border development (as determined by size and number) were monitored for 2, 6, and 16 days (after cells were confluent). Human endothelial cells (HECs), which lack brush border, were used as the control. Our results demonstrate that normal zeta potential of Caco-2 cells significantly changed from -5.7 ± 0.11 mV to -3.4 ± 0.11 mV for a period between 2 and 16 days, respectively. The zeta potential of the control cell line, HECs, stayed constant (statistically not different, P > 0.05) for the duration of the experiments. Our results show that the calculated increase in surface area of the Caco-2 cells with microvilli from 6 to 16 days was directly proportional to the corresponding measured zeta potential difference. These results imply that microvilli alter the electrostatic local environment around Caco-2 cells and, hence, enhance the normal electrostatic selective transport of solute across the mucosal barrier.

Porphyromonas gingivalis bacteremia increases the permeability of the blood-brain barrier via the Mfsd2a/Caveolin-1 mediated transcytosis pathway.

Bacteremia induced by periodontal infection is an important factor for periodontitis to threaten general health. P. gingivalis DNA/virulence factors have been found in the brain tissues from patients with Alzheimer's disease (AD). The blood-brain barrier (BBB) is essential for keeping toxic substances from entering brain tissues. However, the effect of P. gingivalis bacteremia on BBB permeability and its underlying mechanism remains unclear. In the present study, rats were injected by tail vein with P. gingivalis three times a week for eight weeks to induce bacteremia. An in vitro BBB model infected with P. gingivalis was also established. We found that the infiltration of Evans blue dye and Albumin protein deposition in the rat brain tissues were increased in the rat brain tissues with P. gingivalis bacteremia and P. gingivalis could pass through the in vitro BBB model. Caveolae were detected after P. gingivalis infection in BMECs both in vivo and in vitro. Caveolin-1 (Cav-1) expression was enhanced after P. gingivalis infection. Downregulation of Cav-1 rescued P. gingivalis-enhanced BMECs permeability. We further found P. gingivalis-gingipain could be colocalized with Cav-1 and the strong hydrogen bonding between Cav-1 and arg-specific-gingipain (RgpA) were detected. Moreover, P. gingivalis significantly inhibited the major facilitator superfamily domain containing 2a (Mfsd2a) expression. Mfsd2a overexpression reversed P. gingivalis-increased BMECs permeability and Cav-1 expression. These results revealed that Mfsd2a/Cav-1 mediated transcytosis is a key pathway governing BBB BMECs permeability induced by P. gingivalis, which may contribute to P. gingivalis/virulence factors entrance and the subsequent neurological impairments.

Glucose Transporter 1-Mediated Transcytosis of Glucosamine-Labeled Liposomal Ceramide Targets Hypoxia Niches and Cancer Stem Cells to Enhance Therapeutic Efficacy.

Tumour hypoxia plays an important role in modulating tumorigenesis, angiogenesis, invasion, immunosuppression, resistance to treatment, and even maintenance of the stemness of cancer stem cells (CSCs). Moreover, the targeting and treatment of hypoxic cancer cells and CSCs to reduce the influence of tumor hypoxia on cancer therapy remains an imperative clinical problem that needs to be addressed. Since cancer cells upregulate the expression of glucose transporter 1 (GLUT1) through the Warburg effect, we considered the possibility of GLUT1-mediated transcytosis in cancer cells and developed a tumor hypoxia-targeting nanomedicine. Our experimental results indicate that glucosamine-labeled liposomal ceramide can be efficiently transported between cancer cells by GLUT1 transporters and substantially accumulated in the hypoxic area in in vitro CSC spheroids and in vivo tumor xenografts. We also verified the effects of exogenous ceramide on tumor hypoxia, including important bioactivities such as upregulation of p53 and retinoblastoma protein (RB), downregulation of hypoxia-inducible factor-1 alpha (HIF-1α) expression, disruption of the OCT4-SOX2 network of stemness, and inhibition of CD47 and PD-L1 expression. To achieve an ideal therapeutic outcome, we combined treatment of glucosamine-labeled liposomal ceramide with paclitaxel and carboplatin, and we found an excellent synergistic effect, with tumor clearance being noted in three-fourths of the mice. Overall, our findings provide a potential therapeutic strategy for the treatment of cancer.

Strategies for enhancing antibody delivery to the brain.

Antibodies and antibody conjugates have emerged as important tools for cancer therapy. However, a major therapeutic challenge for the use of antibodies is their inability to cross the blood-brain barrier (BBB) to reach tumors localized in the central nervous system (CNS). Multiple methods have been developed to enhance antibody delivery to the CNS, including direct injection, mechanical or biochemical disruption of the BBB, conjugation to a 'molecular Trojan horse', cationization, encapsulation in nanoparticles and liposomes, and more recently, stem cell-mediated antibody delivery. In this review, we discuss each of these approaches, highlighting their successes and the obstacles that remain to be overcome.

The role of glutathione conjugation on the transcellular transport process of PEGylated liposomes across the blood brain barrier.

Notwithstanding the growing evidence of improved drug delivery efficiency to the brain by ligand modification of PEGylated liposomes, the comprehensive knowledge of their transport processes and payload across the BBB is yet to be revealed. Herein, this study sought to understand the glutathione (GSH) ligand effect on transcellular transport mechanisms of liposomes through the blood-brain barrier (BBB) by comparing PEGylated liposomes (PEG-L) and GSH PEGylated liposomes (GSH-PEG-L). Endocytosis and exocytosis of liposomes including the role of secreted extracellular vesicles (EVs) of brain endothelial cells (BECs) were assessed. Furthermore, pharmacokinetics and brain distribution analysis of gemcitabine loaded liposomes were carried out in healthy rats to ascertain the in vivo applicability. Our findings suggested that the presence of GSH increased the cellular uptake of liposomes by up to 3-fold in human brain microvascular endothelial cells depending on the dose but not in astrocytes. The cell exposure to liposomes particularly GSH-PEG-L dramatically increased the cell secretion of small and microvesicles with liposomal components, though different liposomes preferred different vesicles for exocytosis. This correlated with GSH-PEG-L transport efficiency of 4 % across the in vitro BBB model in 24 h, 1.7-fold higher than that of PEG-L (p < 0.05). In rats, while PEG-L and GSH-PEG-L showed similar pharmacokinetic profiles and prolonged circulation properties, 3.8 % of the total injected dose (ID) of gemcitabine was found in the brain of the GSH-PEG-L group at 8 h post-injection, compared with 2.8 % ID in the PEG-L group. A brain: blood concentration ratio of 1.27 ± 0.12 indicated that an active transport mechanism to cross the BBB for GSH-PEG-L. Overall, this study revealed that GSH augmented the transcellular transport efficiency of liposomes through BBB to improve targeted brain delivery by enhancing cellular uptake and vesicular exocytosis route of BECs.

Transepithelial delivery of insulin conjugated with phospholipid-mimicking polymers via biomembrane fusion-mediated transcellular pathways.

Epithelial barriers that seal cell gaps by forming tight junctions to prevent the free permeation of nutrients, electrolytes, and drugs, are essential for maintaining homeostasis in multicellular organisms. The development of nanocarriers that can permeate epithelial tissues without compromising barrier function is key for establishing a safe and efficient drug delivery system (DDS). Previously, we have demonstrated that a water-soluble phospholipid-mimicking random copolymer, poly(2-methacryloyloxyethyl phosphorylcholine30-random-n­butyl methacrylate70) (PMB30W), enters the cytoplasm of live cells by passive diffusion manners, without damaging the cell membranes. The internalization mechanism was confirmed to be amphiphilicity-induced membrane fusion. In the present study, we demonstrated energy-independent permeation of PMB30W through the model epithelial barriers of Madin-Darby canine kidney (MDCK) cell monolayers in vitro. The polymer penetrated epithelial MDCK monolayers via transcellular pathways without breaching the barrier functions. This was confirmed by our unique assay that can monitor the leakage of the proton as the smallest indicator across the epithelial barriers. Moreover, energy-independent transepithelial permeation was achieved when insulin was chemically conjugated with the phospholipid-mimicking nanocarrier. The bioactivity of insulin as a growth factor was found to be maintained even after translocation. These fundamental findings may aid the establishment of transepithelial DDS with advanced drug efficiency and safety. STATEMENT OF SIGNIFICANCE: A nanocarrier that can freely permeate epithelial tissues without compromising barrier function is key for successful DDS. Existing strategies mainly rely on paracellular transport associated with tight junction breakdown or transcellular transport via transporter recognition-mediated active uptake. These approaches raise concerns about efficiency and safety. In this study, we performed non-endocytic permeation of phospholipid-mimicking polymers through the model epithelial barriers in vitro. The polymer penetrated via transcytotic pathways without breaching the barriers of biomembrane and tight junction. Moreover, transepithelial permeation occurred when insulin was covalently attached to the nanocarrier. The bioactivity of insulin was maintained even after translocation. The biomimetic design of nanocarrier may realize safe and efficient transepithelial DDS.