Opening Of Tight Junctions Research Articles

Ultrasound-Responsive Hydrogel Incorporated with TGF-β Mimetic Peptides for Endometrium Recovery to Restore Fertility.

Unavoidable damage to the basal layer of the endometrium has a huge negative impact on a woman's reproductive health and menstrual cycle. However, it is difficult for medicine to penetrate a series of biological barriers toward the basal layer in the deeper area of the endometrium. To meet this challenge, we developed an ultrasound-responsive hydrogel incorporated with a transforming growth factor-beta (TGF-β) mimetic peptide to enhance pregnancy outcomes by restoring the function of a wounded endometrium due to its deep-tissue-penetration capability. In vitro studies revealed that the TGF-β-mimetic-peptide-loaded hydrogel could achieve 64.35% of cell migration under ultrasound stimulation even in phosphate-buffered saline of pH 6.0. Upon in situ sonication at the uterus, carboxymethyl chitosan can be released from degraded hydrogel to open tight junctions with reduced interstitial pressure by ultrasound to promote deep penetration. Rat studies revealed that the penetration capability of TGF-β-mimetic-peptide-loaded hydrogel with sonication was 1.6 times higher than that of the control group. Besides the rat uterine model, ex vivo human uterine tissue was also collected and imaged, demonstrating up to ∼700 μm of tissue penetration depth. In addition, compared to control groups, effective uterus recovery without intrauterine stenosis and endometrial cavity fluid was observed from rats with severe uterine injury treated by TGF-β-mimetic-peptide-loaded hydrogel. In addition, fertility restoration in the endometrial injury model was observed after treatment with such an ultrasound-responsive hydrogel incorporated with TGF-β mimetic peptides. Overall, this work provides an effective approach to treating endometrial injury for enhanced pregnancy outcomes.

Mechanism and performance of choline-based ionic liquids in enhancing nasal delivery of glucagon

Proteins and peptides have been increasingly developed as pharmaceuticals owing to their high potency and low side effects. However, their administration routes are confined to injections, such as intra-muscular and intra-venous injections, making patient compliance a challenge. Hence, non-injectable delivery systems are crucial to expanding the clinical use of proteins and peptides. In this context, two choline-based ionic liquids (ILs), namely, choline geranic acid ([Ch][Ger]) and choline citric acid ([Ch][Cit]), have been identified as promising agents for enhancing the permeation and prolonging the retention time of glucagon (GC) after intra-nasal administration. Notably, intra-nasal delivery of GC via ILs (GC/ILs) elicited rapid and smooth reversal of acute hypoglycaemia without leading to rebound hyperglycaemia in type 1 diabetic rats subjected to insulin induction. In addition, ILs could improve the transcellular transport of GC through electrostatic interaction. ILs could also transiently open inter-cellular tight junctions transiently to facilitate the paracellular transport of GC. Safety tests indicated that continuous intra-nasal delivery of ILs led to reversible changes, such as epithelial cell inflammation, goblet cell overgrowth, and impacts on the distribution of nasal cilia. However, these changes could be alleviated by the innate self-repair ability of mucosal epithelial cells. This study highlights the considerable potential of ILs for long-term nasal delivery of biomacromolecules.

Mind the Particle Rigidity: Blooms the Bioavailability via Rapidly Crossing the Mucus Layer and Alters the Intracellular Fate of Curcumin.

Overcoming intestinal epithelial barriers to enhance bioavailability is a major challenge for oral delivery systems. Desirable nanocarriers should simultaneously exhibit rapid mucus penetration and efficient epithelial uptake; however, they two generally require contradictory structural properties. Herein, we proposed a strategy to construct multiperformance nanoparticles by modifying the rigidity of amphiphilic nanostructures originating from soy polypeptides (SPNPs), where its ability to overcome multibarriers was examined from both in vitro and in vivo, using curcumin (CUR) as a model cargo. Low-rigidity SPNPs showed higher affinity to mucin and were prone to getting stuck in the mucus layer. When they reached epithelial cells, they tended to be endocytosed through the clathrin and macropinocytosis pathways and further transferred to lysosomes, showing severe degradation and lower transport of CUR. Increased particle rigidity generally improved the absorption of CUR, with medium-rigidity SPNPs bloomed maximum plasma concentration of CUR by 80.62-fold and showed the highest oral bioavailability. Results from monocultured and cocultured cell models demonstrated that medium-rigidity SPNPs were least influenced by the mucus layer and changes in rigidity significantly influenced the endocytosis and intracellular fate of SPNPs. Those with higher rigidity preferred to be endocytosed via a caveolae-mediated pathway and trafficked to the ER and Golgi, facilitating their whole transcytosis, and avoiding intracellular metabolism. Moreover, rigidity modulation efficiently induces the reversible opening of intercellular tight junctions, which synergistically improves the transport of CUR into blood circulation. This study suggested that rigidity regulation on food originated amphiphilic peptides could overcome multiple physiological barriers, showing great potential as natural building block toward oral delivery.

Glucoraphanin and sulforaphane mitigate TNFα-induced Caco-2 monolayers permeabilization and inflammation

Intestinal permeabilization is central to the pathophysiology of chronic gut inflammation. This study investigated the efficacy of glucoraphanin (GR), prevalent in cruciferous vegetables, particularly broccoli, and its derivative sulforaphane (SF), in inhibiting tumor necrosis factor alpha (TNFα)-induced Caco-2 cell monolayers inflammation and permeabilization through the regulation of redox-sensitive events. TNFα binding to its receptor led to a rapid increase in oxidant production and subsequent elevation in the mRNA levels of NOX1, NOX4, and Duox2. GR and SF dose-dependently mitigated both these short- and long-term alterations in redox homeostasis. Downstream, GR and SF inhibited the activation of the redox-sensitive signaling cascades NF-κB (p65 and IKK) and MAPK ERK1/2, which contribute to inflammation and barrier permeabilization. GR (1 μM) and SF (0.5–1 μM) prevented TNFα-induced monolayer permeabilization and the associated reduction in the levels of the tight junction (TJ) proteins occludin and ZO-1. Both GR and SF also mitigated TNFα-induced increased mRNA levels of the myosin light chain kinase, which promotes TJ opening. Molecular docking suggests that although GR is mostly not absorbed, it could interact with extracellular and membrane sites in NOX1. Inhibition of NOX1 activity by GR would mitigate TNFα receptor downstream signaling and associated events. These findings support the concept that not only SF, but also GR, could exert systemic health benefits by protecting the intestinal barrier against inflammation-induced permeabilization, in part by regulating redox-sensitive pathways. GR has heretofore not been viewed as a biologically active molecule, but rather, the benign precursor of highly active SF. The consumption of GR and/or SF-rich vegetables or supplements in the diet may offer a means to mitigate the detrimental consequences of intestinal permeabilization, not only in disease states but also in conditions characterized by chronic inflammation of dietary and lifestyle origin.

Inflammation-oriented montmorillonite adjuvant enhanced oral delivery of anti-TNF-α nanobody against inflammatory bowel disease

Oral delivery of proteins faces challenges due to the harsh conditions of the gastrointestinal (GI) tract, including gastric acid and intestinal enzyme degradation. Permeation enhancers are limited in their ability to deliver proteins with high molecular weight and can potentially cause toxicity by opening tight junctions. To overcome these challenges, we propose the use of montmorillonite (MMT) as an adjuvant that possesses both inflammation-oriented abilities and the ability to regulate gut microbiota. This adjuvant can be used as a universal protein oral delivery technology by fusing with advantageous binding amino acid sequences. We demonstrated that anti-TNF-α nanobody (VII) can be intercalated into the MMT interlayer space. The carboxylate groups (-COOH) of aspartic acid (D) and glutamic acid (E) interact with the MMT surface through electrostatic interactions with sodium ions (Na+). The amino groups (NH2) of asparagine (N) and glutamine (Q) are primarily attracted to the MMT layers through hydrogen bonding with oxygen atoms on the surface. This binding mechanism protects VII from degradation and ensures its release in the intestinal tract, as well as retaining biological activity, leading to significantly enhanced therapeutic effects on colitis. Furthermore, VII@MMT increases the abundance of short-chain fatty acids (SCFAs)-producing strains, including Clostridia, Prevotellaceae, Alloprevotella, Oscillospiraceae, Clostridia_vadinBB60_group, and Ruminococcaceae, therefore enhance the production of SCFAs and butyrate, inducing regulatory T cells (Tregs) production to modulate local and systemic immune homeostasis. Overall, the MMT adjuvant provides a promising universal strategy for protein oral delivery by rational designed protein.

Improving treatment for Parkinson's disease: Harnessing photothermal and phagocytosis-driven delivery of levodopa nanocarriers across the blood-brain barrier

Parkinson's disease (PD) poses a significant therapeutic challenge, mainly due to the limited ability of drugs to cross the blood-brain barrier (BBB) without undergoing metabolic transformations. Levodopa, a key component of dopamine replacement therapy, effectively enhances dopaminergic activity. However, it encounters obstacles from peripheral decarboxylase, hindering its passage through the BBB. Furthermore, levodopa metabolism generates reactive oxygen species (ROS), exacerbating neuronal damage. Systemic pulsatile dosing further disrupts natural physiological buffering mechanisms. In this investigation, we devised a ROS-responsive levodopa prodrug system capable of releasing the drug and reducing ROS levels in the central nervous system. The prodrug was incorporated within second near-infrared region (NIR-II) gold nanorods (AuNRs) and utilized angiopep-2 (ANG) for targeted delivery across the BBB. The processes of tight junction opening and endocytosis facilitated improved levodopa transport. ROS scavenging helped alleviate neuronal oxidative stress, leading to enhanced behavioral outcomes and reduced oxidative stress levels in a mouse model of PD. Following treatment, the PD mouse model exhibited enhanced flexibility, balance, and spontaneous exploratory activity. This approach successfully alleviated the motor impairments associated with the disease model. Consequently, our strategy, utilizing NIR-Ⅱ AuNRs and ANG-mediated BBB penetration, coupled with the responsive release of levodopa, offers a promising approach for dopamine supplementation and microenvironmental regulation. This system holds substantial potential as an efficient platform for delivering neuroprotective drugs and advancing PD therapy.

Impact of milking interval and time on milk spontaneous lipolysis and composition in dairy cows

Milk lipolysis is defined as the hydrolysis of triglycerides, the major component of milk fat, resulting in the release of short-chain fatty acids (FA) responsible for rancid flavor and partial glycerides that impair functional properties such as foaming and creaming abilities. Milk lipolysis is a complex phenomenon that depends on both animal parameters and breeding factors. Milk spontaneous lipolysis is known to be higher in milk from evening milkings than from morning milkings. This may be related to the longer length of overnight milking intervals or to the nycthemeral cycle. In this experiment, our objective was thus to study the impact of both milking intervals and time of day on milk spontaneous lipolysis in twice-daily-milking systems with one of 3 milking intervals: Short Day - Long Night (SD-LN, 6.30 a.m. and 4.30 p.m.,); Long Day - Short Night (LD-SN, 6:30 a.m. and 8:30 p.m.,); and Balanced Day and Night (BDN, 6:30 a.m. and 6:30 p.m.,). To achieve this goal, 21 multiparous dairy cows in mid-lactation were used in a 3 × 3 Latin square design over 3 periods. The experiment lasted 5 weeks, corresponding to 3 experimental periods of 6 d alternating with 8 d of milking with conventional hours (morning-evening gap of 10 h). We confirmed that milk spontaneous lipolysis was influenced by milking interval, but not the milking time. Indeed, we observed more lipolysis in SD-LN evening milk (+0.20 mEq/100 g fat) and LD-SN morning milk (+0.22 mEq/100 g fat), both of which corresponded to a 10 h interval between successive milkings. High lipolysis milk came from cows that produced less milk with a higher milk fat content. No significant difference between milkings was observed for BDN. Milk protein, total P and citrate contents increased according to the duration of mammary gland storage of milk (from 10 to 14 h). There was no effect of milking intervals on milk fat globule diameter. The milk Na+/K+ ratio, indicating an opening of tight junctions in the mammary gland, increased only in evening milkings with BDN and LD-SN. In conclusion, we found that the effect of milking intervals on lipolysis is stronger than that of the nycthemeral cycle.

Structure-dependent capacity of procyanidin dimers to inhibit inflammation-induced barrier dysfunction in a cell model of intestinal epithelium

Diet is of major importance in modulating intestinal inflammation, as the gastrointestinal tract is directly exposed to high concentrations of dietary components. Procyanidins are flavan-3-ol oligomers abundant in fruits and vegetables. Although with limited or no intestinal absorption, they can have GI health benefits which can promote overall health. We previously observed that epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) dimers inhibit in vitro colorectal cancer cell proliferation and invasiveness. Inflammation-mediated intestinal barrier permeabilization can result in a chronic inflammatory condition and promote colorectal cancer onset/progression. Thus, this study investigated the structure-dependent capacity of ECG, EGCG and (−)-epicatechin (EC) dimers to inhibit tumor necrosis factor alpha (TNFα)-induced inflammation, oxidative stress, and loss of barrier integrity in Caco-2 cells differentiated into an intestinal epithelial cell monolayer. Cells were incubated with TNFα (10 ng/ml), in the absence/presence of ECG, EGCG and EC dimers. The three dimers inhibited TNFα-mediated Caco-2 cell monolayer permeabilization, modulating events involved in the loss of barrier function and inflammation, i.e. decreased tight junction protein levels; increased matrix metalloproteinases expression and activity; increased NADPH oxidase expression and oxidant production; activation of the NF-κB and ERK1/2 pathways and downstream events leading to tight junction opening. For some of these mechanisms, the galloylated ECG and EGCG dimers had stronger protective potency than the non-galloylated EC dimer. These differences could be due to differential membrane interactions as pointed out by molecular dynamics simulation of procyanidin dimers-cell membrane interactions and/or by differential interactions with NOX1. Results show that dimeric procyanidins, although poorly absorbed, can promote health by alleviating intestinal inflammation, oxidative stress and barrier permeabilization.

Liposome‐Based Permeable Eyedrops for Effective Posterior Segment Drug Delivery

AbstractTopical eyedrop administration is identified as an ideal non‐invasive strategy for ocular drug delivery. However, multiple complex ocular barriers greatly restrict their effectiveness in the treatment of posterior ocular disease. Herein, a liposome‐based permeable eyedrops (pDrops) capable of overcoming multiple ocular barriers and achieving efficient posterior drug delivery is presented. pDrops have a core‐shell structure in which drugs are encapsulated inside the liposome core with a chitosan shell. This chitosan coating significantly enhances the pDrops’ binding to mucin in tears and facilitates the temporary opening of tight junctions in cornea/conjunctive epithelial cells, thereby achieving prolonged preocular retention and enhanced posterior segment drug delivery. In this study, hydrophilic ganciclovir (GCV) and hydrophobic curcumin (CUR) are employed as model drugs. Upon topical instillation, pDrops effectively overcome ocular barriers and delivered GCV to the posterior segment in both rat and rabbit eyes. Notably, CUR delivery by pDrops demonstrates significantly enhanced therapeutic efficacy in light‐damaged retina of mice. Considering that pDrops can deliver both hydrophobic and hydrophilic drugs to the posterior segment of the eye, it can potentially become a feasible platform for the non‐invasive delivery of various drug molecules and improve the treatment efficiency of posterior ocular diseases.

Enhancing paracellular and transcellular permeability using nanotechnological approaches for the treatment of brain and retinal diseases.

Paracellular permeability across epithelial and endothelial cells is, in large part, regulated by apical intercellular junctions also referred to as tight junctions (TJs). These junctions contribute to the spatial definition of different tissue compartments within organisms, separating them from the outside world as well as from inner compartments, with their primary physiological role of maintaining tissue homeostasis. TJs restrict the free, passive diffusion of ions and hydrophilic small molecules through paracellular clefts and are important for appropriate cell polarization and transporter protein localisation, supporting the controlled transcellular diffusion of smaller and larger hydrophilic as well as hydrophobic substances. This traditional diffusion barrier concept of TJs has been challenged lately, owing to a better understanding of the components that are associated with TJs. It is now well-established that mutations in TJ proteins are associated with a range of human diseases and that a change in the membrane fluidity of neighbouring cells can open possibilities for therapeutics to cross intercellular junctions. Nanotechnological approaches, exploiting ultrasound or hyperosmotic agents and permeation enhancers, are the paradigm for achieving enhanced paracellular diffusion. The other widely used transport route of drugs is via transcellular transport, allowing the passage of a variety of pro-drugs and nanoparticle-encapsulated drugs via different mechanisms based on receptors and others. For a long time, there was an expectation that lipidic nanocarriers and polymeric nanostructures could revolutionize the field for the delivery of RNA and protein-based therapeutics across different biological barriers equipped with TJs (e.g., blood-brain barrier (BBB), retina-blood barrier (RBB), corneal TJs, etc.). However, only a limited increase in therapeutic efficiency has been reported for most systems until now. The purpose of this review is to explore the reasons behind the current failures and to examine the emergence of synthetic and cell-derived nanomaterials and nanotechnological approaches as potential game-changers in enhancing drug delivery to target locations both at and across TJs using innovative concepts. Specifically, we will focus on recent advancements in various nanotechnological strategies enabling the bypassing or temporally opening of TJs to the brain and to the retina, and discuss their advantages and limitations.

Electromagnetic pulse induced blood-brain barrier breakdown through tight junction opening in rats.

The blood-brain barrier (BBB) is the main obstacle to hydrophilic and large molecules to enter the brain, maintaining the stability of the central nervous system (CNS). But many environmental factors may affect the permeability and structure of the BBB. Electromagnetic pulses (EMP) irradiation has been proven to enhance the permeability of the BBB, but the specific mechanism is still unclear. To explore the potential mechanism of EMP-induced BBB opening, this study investigated the permeability, fine structure and the proteins expression of the tight junction (TJ) of the BBB in the rats exposed to EMP. Using the leakage of fluorescein isothiocyanate-labeled dextran with different molecular mass under different field intensity of EMP exposure, we found that the tracer passing through the BBB is size-dependent in the rat exposed to EMP as field intensity increased. Transmission electron microscopy showed TJ of the endothelial cells in the EMP-exposed group was open, compared with the sham-irradiated group. But the levels of TJ proteins including ZO-1, claudin-5, or occludin were not changed as indicated by western blot. These data suggest that EMP induce BBB opening in a field intensity-dependent manner and probably through dysfunction of TJ proteins instead of their expression. Our findings increase the understanding of the mechanism for EMP working on the brain and are helpful for CNS protection against EMP.

Dendritic Oligoethylenimine Decorated Liposome with Augmented Corneal Retention and Permeation for Efficient Topical Delivery of Antiglaucoma Drugs.

The topically administered glaucoma medications usually encounter serious precorneal drug loss and low corneal penetration, leading to a low bioavailability. In addition, due to the complexity of glaucoma etiology, a single medication is often insufficient. In this work, we report a novel dendritic oligoethylenimine decorated liposome for codelivery of two antiglaucoma drugs, latanoprost and timolol. The liposome showed a uniform nanoscopic particle size, positive surface charge, and excellent dual-drug loading capacity. A prolonged precorneal retention is observed by using this liposomal delivery system. This liposomal delivery system presents increased cellular uptake and tight junctions opening capacity, contributing respectively to the transcellular and paracellular permeation, thereby enhancing the trans-corneal transportation. Following topical administration of one eye drop in brown Norway rats, the dual-drug-loaded liposome formulation resulted in a sustained and effective intraocular pressure reduction as long as 5 days, without inducing ocular inflammation, discomfort, and tissue damage.

Biofunctionalization of nanoceria with sperminated hyaluronan enhances drug delivery performance for corneal alkali burn therapy

Corneal alkali burn (AB) injury is the most common sight-threatening injury that is highly associated with oxidative stress, inflammation, apoptosis, angiogenesis, and cell deficiency. Most therapeutic options are ineffective in improving and reversing pathological conditions. This study aims to develop a delivery system based on sperminated hyaluronan (sH)-functionalized nanoceria (Ce-sH nanoparticles (NPs)) with enhanced barrier penetration capability and increased ocular drug bioavailability. The functional surface properties and therapeutic bioactivities of Ce-sH NPs were evaluated. We observed a high tight-junction opening capacity with higher spermine content, resulting in excellent permeability and cellular uptake efficiency (fourfold that of bare Ce NPs). The NPs exhibited high drug (insulin-like growth factor 1, IGF-1) entrapment efficiency (10-fold) and hyaluronidase-triggered controlled slow release (up to 168 h) compared with bare Ce NPs. The IGF-1-loaded Ce-hsH NPs (I@Ce-hsH) had the best wound healing rate (>90 %). In contrast to a clinically used eye drop (dexamethasone), the I@Ce-hsH significantly reduced corneal defects to <1 % and restored the tissue transparency to a normal range within 7 d post-instillation in a rat model of AB. In summary, our bioactive nanoformulations successfully reversed corneal injury and has great potential in the design of biomaterials for ocular therapeutics.

Capsaicin indirectly regulates TRPA1 via the arachidonic acid cascade, resulting in TJ opening.

Capsaicin induces the reversible opening of tight junctions (TJs) and enhances the delivery of hydrophilic macromolecules through a paracellular route. We previously revealed that TRPA1 is involved in the capsaicin-induced Ca2+ influx and TJ permeability increase, although there are no reports that capsaicin directly activates TRPA1. In this study, we investigated the upstream factors of TRPA1 using RNA-seq analysis, and found that the cyclooxygenase 2 (COX2) gene was upregulated by capsaicin. Cyclooxygenase 2 converts arachidonic acid (AA), a metabolite by phospholipase A2 (PLA2), to prostaglandins. Prostaglandin E2 (PGE2) production was stimulated by capsaicin, and capsaicin-induced Ca2+ influx was effectively inhibited by PLA2 and COX2 inhibitors. The AA-induced TJ permeability increase was inhibited by a TRPA1 antagonist, but the capsaicin- and AA-induced TJ permeability increases were hardly inhibited by a COX2 inhibitor. These results suggest that capsaicin-induced PLA2 activation and AA production are the important steps for the TJ permeability increase.

Mercaptonicotinic acid activated thiolated chitosan (MNA-TG-chitosan) to enable peptide oral delivery by opening cell tight junctions and enhancing transepithelial transport

Recent advances in peptide delivery and nanotechnology has resulted in emergence of several non-parenteral administration routes that replace subcutaneous injections associated with patient discomfort. Thiolated biopolymers are relatively new materials being explored to enhance mucoadhesivity and permeability in these efforts, yet their pH dependent reactivity remains an obstacle. This work focussed on improving the mucoadhesivity of thiolated chitosans by activating them with mercaptonicotinic acid, in a bid to create a novel thiomerized chitosan that can open cell tight junctions for application in oral delivery. The synthesized mercaptonicotinic acid activated thiolated chistoan (MNA-TG-chitosan), along with thiolated chitosan (TG-chitosan) and unmodified chitosan were then used to create insulin nanoparticles (insNPs) using spray drying encapsulation process. Use of MNA-TG-chitosan in place of chitosan resulted in reduction of particle size of insNPs from 318 to 277 nm with no significant changes in polydispersity index (~ 0.2), encapsulation efficiency (~ 99%), insulin loading content (~ 25%) and morphology. Results from in-vitro cytotoxicity on TR146, CaCo2 and HepG2 cell lines revealed no significant effects on cell viability at 50–1000 μg/mL concentration. insNPs encapsulated with the new material, MNA-TG-chitosan, resulted in a 1.5-fold and 4.4-fold higher cellular uptake by HepG2 liver cells where insulin is metabolized, approximately 40% and 600% greater insulin transport through TR146 buccal cell monolayers, and 40% and 150% greater apparent permeability than insNPs encapsulated with unmodified chitosan and TG-chitosan respectively. The higher permeation achieved on using MNA-TG chitosan was attributed to the greater opening of the cell tight junction evidenced by reduction of transepithelial electrical resistance of TR146 buccal cell monolayers. This study demonstrates MNA-TG-chitosan as a promising material for improved peptide oral delivery.

Insights into the formulation properties, biocompatibility, and permeability of poorly water-soluble methoxyflavones with PEG400 and propylene glycol.

Herein, thermal and non-thermal techniques were used to elucidate the putative physical and chemical interactions between poorly water-soluble Kaempferia methoxyflavones and PEG400/propylene glycol. Additionally, the biocompatibility of methoxyflavone-glycol solutions was evaluated using Caco-2 cells whereas the absorptive transport was investigated by measuring the apparent permeability coefficient (P app) of the methoxyflavones and transepithelial electrical resistance (TEER) of the Caco-2 cell monolayer. Data from differential scanning calorimetry, Fourier-transform infrared (FTIR), and proton nuclear magnetic resonance (1H NMR) spectroscopic analysis revealed physico-chemical compatibility between the three methoxyflavones and PEG400/propylene glycol. Furthermore, PEG400 and propylene glycol solutions of the methoxyflavones were shown to be compatible with Caco-2 cells at pharmacologically effective concentrations. In vitro transport studies across the Caco-2 cell monolayer revealed high P app values of 24.07 × 10-6 to 19.63 × 10-6 cm s-1 for PEG400 solutions of the methoxyflavones. The TEER values of the Caco-2 cell monolayers indicated that the increased drug transport was partly due to increased tight junction openings, but without compromising the epithelial barrier integrity. The good pharmaceutical and biocompatibility profiles, as well as improved transport of the methoxyflavones in PEG400 and propylene glycol solutions, are suggestive of the worthiness of this approach for further consideration pertaining to the development of these drugs into oral liquid dosage forms.

Borneol-modified PEGylated graphene oxide as a nanocarrier for brain-targeted delivery of ginsenoside Rg1 against depression

Depression is a chronic mental disorder which threatens human health and lives. However, the treatment of depression remains challenging largely due to blood brain barrier (BBB), which restricts drugs from entering the brain, resulting in a poor distribution of antidepressants in the brain. In this work, a novel brain-targeted drug delivery system was developed based on borneol-modified PEGylated graphene oxide (GO-PEG-BO). GO-PEG-BO was characterized and proved to possess excellent biocompatibility. By incorporating borneol, GO-PEG-BO could penetrate BBB efficiently by opening tight junctions and inhibiting the efflux system of BBB. The targeted distribution of GO-PEG-BO in the brain was observed by an in vivo biodistribution study. Moreover, GO-PEG-BO exhibited a neuroprotective effect, which is beneficial to the treatment of depression. Ginsenoside Rg1 (GRg1), which can relieve depressive symptoms but difficult to cross BBB, was loaded to GO-PEG-BO for the therapy of depression. In depressive rats, GRg1/GO-PEG-BO improved stress-induced anhedonia, despair and anxiety, and comprehensively relieved the depressive symptoms. In conclusion, GO-PEG-BO could serve as a promising nanocarrier for brain-targeted drug delivery, and provide a new strategy for the therapy of depression.

Inhibiting PI3K/Akt-Signaling Pathway Improves Neurobehavior Changes in Anti-NMDAR Encephalitis Mice by Ameliorating Blood–Brain Barrier Disruption and Neuronal Damage

The disruption of the blood–brain barrier (BBB) is hypothesized to be involved in the progression of anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis, but its mechanism is still unclear. Recently, the phosphatidylinositol 3-kinase (PI3K)/threonine kinase (Akt) pathway is involved in the regulation of the BBB in various diseases. This study is aimed to investigate the mechanism of BBB damage and neurobehavior changes in anti-NMDAR encephalitis mice. Female C57BL/6J mice were actively immunized to establish an anti-NMDAR encephalitis mouse model and evaluate the neurobehavior changes of mice. To study its potential mechanism, LY294002 (PI3K inhibitor, 8 mg/kg) and Recilisib (PI3K agonist, 10 mg/kg) were treated by intraperitoneal injection, respectively. Anti-NMDAR encephalitis mice showed neurological deficits, increased BBB permeability, open endothelial tight junctions (TJs), and decreased expression of TJ-related proteins zonula occludens (ZO)-1 and Claudin-5. However, administration of PI3K inhibitor significantly reduced the expression of p-PI3K and p-Akt, improved neurobehavior function, decreased BBB permeability, and upregulated the expressions of ZO-1 and Claudin-5. Furthermore, PI3K inhibition reversed the decline of NMDAR NR1 in the membranes of hippocampal neurons, which reduced the loss of neuron-specific nucleoprotein (NeuN) and microtubule-associated protein 2 (MAP2). In contrast, administration of the PI3K agonist Recilisib showed a tendency to exacerbate BBB breakdown and neurological deficits. Our results showed that the activation of PI3K/Akt, along with the changes in TJ-related proteins ZO-1 and Claudin-5, may be closely related to BBB damage and neurobehavior changes in anti-NMDAR encephalitis mice. PI3K inhibition attenuates BBB disruption and neuronal damage in mice, thereby improving neurobehavior.

Unsaturated free fatty acid emulsion infusion into carotid artery enhances drug delivery to the rat brain.

To determine whether the blood-brain barrier (BBB) opens to enhance drug delivery during the acute stage of unsaturated fat embolism. We infused oleic, linoleic, and linolenic acid emulsions through the right common carotid artery of rats, followed by trypan blue for gross and lanthanum for electron microscopic (EM) examination. Doxorubicin and temozolomide were also administered, and then the rats were euthanized at 30min, 1h, and 2h. Trypan blue hue was analyzed to semiquantitatively measure BBB opening. Desorption electrospray ionization-mass spectrometry (DESI-MS) imaging was used to evaluate drug delivery. Trypan blue staining observed in each group 30min after emulsion infusion increased at 1h and decreased after 2h in the oleic acid group. The linoleic and linolenic acid groups showed weak staining over time. The hue and trypan blue analysis results were corroborative. EM showed tight junction opening, whereas DESI-MS imaging showed increased doxorubicin and temozolomide signal intensities in ipsilateral hemispheres of all three groups. We demonstrated that oleic, linoleic, and linolenic acid emulsions opened the BBB, promoting drug delivery to the brain. Hue analysis and DESI-MS imaging are appropriate for analysis of doxorubicin and temozolomide concentrations in brain tissue.

Quaternization of high molecular weight chitosan for increasing intestinal drug absorption using Caco-2 cells as an in vitro intestinal model

Potential use of a quaternized chitosan (MW 600 kDa) with 65% of 3-chloro-2-hydroxypropyltrimethylammonium (600-HPTChC65) as an absorptive enhancer was investigated in Caco-2 monolayers. 600-HPTChC65 (0.005% w/v) quickly reduced transepithelial electrical resistance (TEER) to the maximum level in 40 min with full recovery within 6 h after removal. Its TEER reduction was corresponded to increased FD4 transport across the monolayers and disrupted localization of tight junction proteins ZO-1 and occludin at the cell borders. 600-HPTChC65 was densely localized at the membrane surface and intercellular junctions. This chitosan (0.08–0.32% w/v) reduced the efflux ratio of [3H]-digoxin by 1.7- 2 folds, suggesting an increased [3H]-digoxin transport across the monolayers. Its binding with P-gp on Caco-2 monolayer increased the signal of fluorescence-labeled anti-P-gp (UIC2) reactivity due to conformational change. 600-HPTChC65 (0.32% w/v) had no effect on P-gp expression in the Caco-2 monolayers. These results suggest that 600-HPTChC65 could enhance drug absorption through tight junction opening and decreased P-gp function. Its interaction with the absorptive barrier mainly resulted in disrupting ZO-1 and occludin organization as well as changing in P-gp conformation.