W/O/W Microemulsions for Nasal Delivery of Hydrophilic Compounds: A Preliminary Study.

The administration of hydrophilic therapeutics has always been a great challenge because of their low bioavailability after administration. For this purpose, W/O/W microemulsion resulted to be a potential successful strategy for the delivery of hydrophilic compounds, interesting for the nasal mucosal therapy. Herein, an optimized biphasic W/O microemulsion was designed, through a preliminary screening, and it was inverted in a triphasic W/O/W microemulsion, intended for the nasal administration. In order to enhance the mucosal retention, surface modification of the biphasic W/O microemulsion was performed adding didodecyldimethylammonium bromide, and then converting the system into a cationic triphasic W/O/W microemulsion. The developed samples were characterized in terms of droplet size, polydispersity, zeta potential, pH and osmolality. The physical long-term stability was analyzed storing samples at accelerated conditions (40 ± 2 °C and 75 ± 5 % RH) for 6 months in a constant climate chamber, following ICH guidelines Q1A (R2). In order to verify the potential retention on the nasal mucosa, the two triphasic systems were analyzed in terms of mucoadhesive properties, measuring the in vitro interaction with mucin over time. Furthermore, fluorescein sodium salt was selected as a model hydrophilic drug to be encapsulated into the inner core of the two triphasic W/O/W microemulsions, and its release was analyzed compared to the free probe solution. The cytocompatibility of the two platforms was assessed on two cell lines, human fibroblasts HFF1 and Calu-3 cell lines, chosen as pre-clinical models for nasal and bronchial/tracheal airway epithelium.

Drug delivery of 6-bromoindirubin-3'-glycerol-oxime ether employing poly(D,L-lactide-co-glycolide)-based nanoencapsulation techniques with sustainable solvents.

BACKGROUND: Insufficient solubility and stability of bioactive small molecules as well as poor biocompatibility may cause low bioavailability and are common obstacles in drug development. One example of such problematic molecules is 6-bromoindirubin-3'-glycerol-oxime ether (6BIGOE), a hydrophobic indirubin derivative. 6BIGOE potently modulates the release of inflammatory cytokines and lipid mediators from isolated human monocytes through inhibition of glycogen synthase kinase-3 in a favorable fashion. However, 6BIGOE suffers from poor solubility and short half-lives in biological aqueous environment and exerts cytotoxic effects in various mammalian cells. In order to overcome the poor water solubility, instability and cytotoxicity of 6BIGOE, we applied encapsulation into poly(D,L-lactide-co-glycolide) (PLGA)-based nanoparticles by employing formulation methods using the sustainable solvents Cyrene™ or 400 g/mol poly(ethylene glycol) as suitable technology for efficient drug delivery of 6BIGOE. RESULTS: For all preparation techniques the physicochemical characterization of 6BIGOE-loaded nanoparticles revealed comparable crystallinity, sizes of about 230 nm with low polydispersity, negative zeta potentials around - 15 to - 25 mV, and biphasic release profiles over up to 24 h. Nanoparticles with improved cellular uptake and the ability to mask cytotoxic effects of 6BIGOE were obtained as shown in human monocytes over 48 h as well as in a shell-less hen's egg model. Intriguingly, encapsulation into these nanoparticles fully retains the anti-inflammatory properties of 6BIGOE, that is, favorable modulation of the release of inflammation-relevant cytokines and lipid mediators from human monocytes. CONCLUSIONS: Our formulation method of PLGA-based nanoparticles by applying sustainable, non-toxic solvents is a feasible nanotechnology that circumvents the poor bioavailability and biocompatibility of the cargo 6BIGOE. This technology yields favorable drug delivery systems for efficient interference with inflammatory processes, with improved pharmacotherapeutic potential.

Determining the effect of ocular chemical injuries on topical drug delivery.

Ocular chemical injuries (OCIs) commonly cause ocular damage and visual loss and treatment uses topical therapies to facilitate healing and limit complications. However, the impact of chemical injury on corneal barrier function and treatment penetration is unknown. Therefore, the aim of this study was to determine the effect of OCI on drug penetration and absorption. Porcine corneal explants were used to assess histological damage, electrical resistance, and the trans-corneal penetration/corneal adsorption of reference compounds (sodium fluorescein and rhodamine B) and dexamethasone. Corneal explants were injured with either 1 M sulfuric acid, or 1 M sodium hydroxide. Dexamethasone penetration was measured using high-performance liquid chromatography (HPLC) and that of fluorescein and rhodamine using fluorescence. Dexamethasone corneal adsorption was measured using enzyme-linked immunoabsorbant assay (ELISA). Both acid and alkaline injuries reduced trans-corneal electrical resistance. NaOH injury increased hydrophilic fluorescein penetration (NaOH 8.59 ± 1.50E-05 cm.min-1 vs. Hanks' Balanced Salt Solution (HBSS) 1.64 ± 1.01E-06 cm.min-1) with little impact on hydrophobic rhodamine B (1 M NaOH 6.55 ± 2.45E-04 cm.min-1 vs. HBSS 4.60 ± 0.972E-04 cm.min-1) and dexamethasone penetration (1 M NaOH 3.00 ± 0.853E-04 cm.min-1 vs. HBSS 2.69 ± 0.439E-04 cm.min-1). By contrast, H2SO4 decreased trans-corneal penetration of hydrophilic fluorescein (H2SO4 1.16 ± 14.2E-07 cm.min-1) and of hydrophobic dexamethasone (H2SO4 1.88 ± 0.646E-04 cm.min-1) and rhodamine B (H2SO4 4.60 ± 1.42E-05 cm.min-1). Acid and alkaline OCI differentially disrupted the corneal epithelial barrier function. Acid injury reduced penetration of hydrophobic dexamethasone and rhodamine B as well as hydrophilic fluorescein, which may translate clinically into reduced drug penetration after OCI, while alkaline injury increased fluorescein penetration, with minimal effect on dexamethasone and rhodamine B penetration.

Reliability and efficacy of maximum fluorescein tear break-up time in diagnosing dry eye disease.

This study aims to investigate the reliability and efficacy of maximum fluorescein tear break-up time (FTBUTmax) in diagnosing dry eye disease (DED). 147 participants were enrolled in this study. Ocular symptoms were assessed by Ocular Surface Disease Index (OSDI). The fluorescein tear break-up time (FTBUT) examination, corneal fluorescein staining (CFS), and Schirmer I test were performed on both eyes. Each participant underwent 3 consecutive FTBUT tests, and five types of FTBUT values including FTBUTmax, the minimum FTBUT (FTBUTmin), the first FTBUT (FTBUT1), the average of three FTBUTs (FTBUT123) and the average of the first and second FTBUT (FTBUT12) were recorded. FTBUTmax was larger than the other FTBUT values, but no differences were found among the values of FTBUT1, FTBUT123, FTBUT12 and FTBUTmin. In the ROC analysis, FTBUTmax had the largest or the second largest area under the ROC (AUROC) in all three DED diagnostic criteria, while FTBUTmin had the least AUROC of them. ROC efficacy of FTBUTmax was significantly higher than that of FTBUT123, FTBUT12, FTBUT1 and FTBUTmin in the OSDI criteria and higher than that of FTBUT1 and FTBUTmin in Schirmer I test and CFS tests. FTBUTmax has a close correlation with OSDI, Schirmer I test and CFS, and is an effective tool for the DED diagnosis.

Enhancement of Skin Permeation of a Hydrophilic Drug from Acryl-Based Pressure-Sensitive Adhesive Tape.

PURPOSE: Penetration enhancers are necessary to overcome a formidable barrier function of the stratum corneum in the development of topical formulations. Recently, non-lamella liquid crystal (NLLC)-forming lipids such as glycerol monooleate and phytantriol (PHY) are gaining increasing attention as a novel skin permeation enhancer. In the present study, fluorescein sodium (FL-Na) was used as a model hydrophilic drug, and acryl-base pressure-sensitive adhesive (PSA) tape containing NLLC forming lipids, mono-O-(5,9,13-trimethyl-4-tetradecenyl) glycerol ester (MGE) or PHY, was prepared to enhance drug permeation through the skin. METHODS: A PSA patch containing FL-Na was prepared by mixing FL-Na entrapped in NLLC and acrylic polymer. FL permeation through excised hairless rat skin, and also human skin, was investigated. Changes in lipid structure, folding/unfolding state of keratin in the stratum corneum, and penetration of MGE into the stratum corneum were investigated using confocal Raman microscopy. RESULTS: Enhanced FL permeation was observed by the application of a PSA patch containing MGE and PHY. Especially, dramatically enhancement effect was confirmed by 15% of MGE contained formulation. Penetration of MGE provided diminished orthorhombic crystal structure and a peak shift of the aliphatic CH3 vibration of keratin chains toward lower wavenumbers. CONCLUSION: The present results suggested that the formulation development by adding MGE may be useful for improving the skin permeation of mal-permeable drugs such as hydrophilic drugs.

Hydrophobically-modified gelatin hydrogel as a carrier for charged hydrophilic drugs and hydrophobic drugs.

Gelatin molecules have been chemically crosslinked using potentially cytotoxic reagents to prepare stable hydrogels. Hydrophobic interaction is a means of forming physical crosslinks that is a good candidate for enhancing the stability of gelatin hydrogels without using cytotoxic chemicals. In this study, we proposed a new method to fabricate hydrogels from hydrophobically-modified gelatin (HMG) with high content of hydrophobic segments. HMG was first dissolved in dimethyl sulfoxide and poured into a vial with the desired shape. After the solution was freeze-dried, the solid construct was hydrated. The HMG hydrogel containing basic fibroblast growth factor promoted angiogenesis in vivo, indicating that the positively charged hydrophilic growth factor formed an electrostatic complex with negatively charged HMG hydrogel and was gradually released in vivo with the degradation of the hydrogel. In addition, we showed that the hydrophobic segments of HMG enhanced the adsorption of fluorescein sodium, a model for hydrophobic therapeutic agents, to the hydrogel through hydrophobic interaction. Furthermore, in vitro experiments indicated that the hydrophobic agents would be released from the hydrogel in a controlled manner in vivo. These results show that the HMG hydrogel has significant potential as a carrier for both charged hydrophilic drugs and hydrophobic drugs.

The Relationship between Fluorescein Angiography Leakage after Infliximab Therapy and Relapse of Ocular Inflammatory Attacks in Ocular Behçet's Disease Patients.

Purpose: To clarify the relationship between fluorescein angiography (FA) leakage after infliximab therapy and ocular attack relapse in patients with ocular Behçet's disease (BD). Methods: Patients with ocular BD were divided into two groups based on the presence (Group 1) or absence (Group 2) of ocular attacks after IFX therapy. FA leakage was evaluated by FA score in each of the optic discs, macula, large retinal vessels, and capillary vessels. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the relationship between FA score after IFX therapy and ocular attack relapse. Results: The areas under the curves obtained from the ROC curve of optic disc score and capillary vessels score after IFX therapy were 0.867 (95% confidence interval [CI]: 0.788-0.946) and 0.788 (95% CI: 0.649-0.927), respectively. Conclusions: FA leakage in the optic disc and capillary vessels after IFX therapy was strongly related to ocular attack relapse.

Tunable Decoupling of Dual Drug Release of Oppositely Charged, Stimuli-Responsive Anisotropic Nanoparticles.

Multicompartmentalized nanostructures are of interest because they can provide unique physicochemical properties and multifunctionalities in each compartment. Furthermore, stimuli-responsive anisotropic nanostructures (ANPs) with distinct opposite charges would be useful for drug delivery systems because different drug release kinetics could be achieved from each compartment in response to both charge and stimuli. In this study, stimuli-responsive ANPs were formed via electrohydrodynamic cojetting of poly(N-isopropylacrylamide)-based copolymers with opposite charges. The positively charged compartment consisted of poly(N-isopropylacylamide-co-stearyl acrylate-co-allylamine) (poly(NIPAM-co-SA-co-AAm)) (i.e., PNSAAm) and poly(N-isopropylacylamide-co-stearyl acrylate-co-acrylic acid) (poly(NIPAM-co-SA-co-AAc)) (i.e., PNSAAc). The two distinct compartments of ANPs were physically cross-linked through hydrophobic interactions within the copolymers. Oppositely charged, small-molecule model drugs (fluorescein sodium salt and rhodamine 6G) were separately encapsulated within each compartment and released based on changes in noncovalent interactions and temperature. Furthermore, two different biomacromolecule drugs with opposite charges, bovine serum albumin and lysozyme (which were complexed with polysaccharides by hydrophobic ion pairing), were loaded within the ANPs. Electrostatic interactions between the encapsulated drugs and each ANP compartment controlled the rate of drug release from the ANPs. In addition, these ANPs showed a thermally induced actuation, leading to drug release at different rates due to the collapse of poly(NIPAM)-based copolymers under aqueous conditions. This work may be useful for decoupled drug release kinetics.

Phosphonium Polyelectrolyte Complexes for the Encapsulation and Slow Release of Ionic Cargo.

Polyelectrolyte complexation, the combination of anionically and cationically charged polymers through ionic interactions, can be used to form hydrogel networks. These networks can be used to encapsulate and release cargo, but the release of cargo is typically rapid, occurring over a period of hours to a few days and they often exhibit weak, fluid-like mechanical properties. Here we report the preparation and study of polyelectrolyte complexes (PECs) from sodium hyaluronate (HA) and poly[tris(hydroxypropyl)(4-vinylbenzyl)phosphonium chloride], poly[triphenyl(4-vinylbenzyl)phosphonium chloride], poly[tri(n-butyl)(4-vinylbenzyl)phosphonium chloride], or poly[triethyl(4-vinylbenzyl)phosphonium chloride]. The networks were compacted by ultracentrifugation, then their composition, swelling, rheological, and self-healing properties were studied. Their properties depended on the structure of the phosphonium polymer and the salt concentration, but in general, they exhibited predominantly gel-like behavior with relaxation times greater than 40 s and self-healing over 2-18 h. Anionic molecules, including fluorescein, diclofenac, and adenosine-5'-triphosphate, were encapsulated into the PECs with high loading capacities of up to 16 wt %. Fluorescein and diclofenac were slowly released over 60 days, which was attributed to a combination of hydrophobic and ionic interactions with the dense PEC network. The cytotoxicities of the polymers and their corresponding networks with HA to C2C12 mouse myoblast cells was investigated and found to depend on the structure of the polymer and the properties of the network. Overall, this work demonstrates the utility of polyphosphonium-HA networks for the loading and slow release of ionic drugs and that their physical and biological properties can be readily tuned according to the structure of the phosphonium polymer.

Topical application of highly concentrated water-in-oil emulsions: Physiological skin parameters and skin penetration in vivo - A pilot study.

Important aspects in the development of new dermal drug delivery systems are the formulations' physicochemical properties and stability. Moreover, their influence on skin physiology and their penetration performance in vivo are of crucial interest. We have recently developed novel concentrated water-in-oil emulsions based on a non-ionic silicone surfactant; the present study deals with the effect of these formulations on physiological skin parameters of healthy volunteers after repeated application. Variations in skin condition and barrier integrity were investigated using classical biophysical and spectroscopic techniques. After four weeks of continuous treatment, no signs of skin irritation could be observed. Both tested emulsions had a positive effect on skin properties despite their relatively high water content and low lipid content. In vivo tape stripping studies revealed penetrated amounts of the incorporated model drug fluorescein sodium of almost 50% of the applied dose, with a superior performance of emulsions with isopropyl myristate when compared to liquid paraffin. In summary, our study confirmed the suitability of the developed W/O emulsions for pharmaceutic and cosmetic applications.

Noninvasive characterization of in situ forming implant diffusivity using diffusion-weighted MRI.

In situ forming implants (ISFIs) form a solid drug-eluting depot, releasing drug for an extended period of time after a minimally-invasive injection. Clinical use of ISFIs has been limited because many factors affect drug release kinetics. The aim of this study was to use diffusion-weighted MRI (DWI) to noninvasively quantify spatial-temporal changes in implant diffusivity in situ. ISFIs were formed using poly(lactic-co-glycolic) acid, with a molecular weight of either 15 kDa or 52 kDa, and fluorescein as the mock drug. Drug release, polymer erosion, polymer degradation, and implant diffusivity were analyzed in vitro over 21 days. DWI was also performed in vivo over 5 days. Spatial diffusivity maps of the implant were generated using DWI data. Results showed constant diffusivity at the implant shell ((1.17 ±â€¯0.13) × 10-3 mm2/s) and increasing diffusivity within the interior over time (from (0.268 ±â€¯0.081) × 10-3 mm2/s during day 1 to (1.88 ±â€¯0.04) × 10-3 mm2/s at 14 d), which correlated with increasing porosity of the implant microstructure. Implants formed in vivo followed the same diffusivity trend as those in vitro. This study validates the use of DWI to provide novel functional information about implant behavior through its ability to noninvasively characterize transport properties within the implant both in vitro and in vivo.

An [18F]-Positron Emitting Fluorophore Allows Safe Evaluation of Small Molecule Distribution in the CSF, CSF Fistulas, and CNS Device Placement.

The small molecule fluorescein is commonly used to guide the repair of cerebral spinal fluid leaks (CSFLs) in the clinic. We modified fluorescein so that it is also visible by positron emission tomography (PET). This probe was used to quantitatively track the fast distribution of small molecules in the CSF of rats. We tested this probe in models relevant to the clinical diagnosis and treatment of central nervous system (CNS) diseases that affect CSF flow. In this study, fluorescein was radiolabeled with fluorine-18 to produce Fc-AMBF3. [18/19F]-Fc-AMBF3 was introduced at trace quantities (13.2 nmols, 100 µCi) intrathecally (between L5 and L6) in rats to observe the dynamic distribution and clearance of small molecules in the CSF by both [18F]-PET and fluorescence (FL) imaging. Murine models were used to demonstrate the following utilities of Fc-AMBF3: (1) utility in monitoring the spontaneous CSFL repair of a compression fracture of the cribriform plate and (2) utility in quantifying CSF flow velocity during neurosurgical lumboperitoneal shunt placement. Fc-AMBF3 clearly delineated CSF-containing volumes based on noninvasive PET imaging and in ex vivo FL histology. In vivo morbidity (n = 16 rats, <2.7 mg/kg, 77 times the PET dose) was not observed. The clearance of the contrast agent from the CNS was rapid and quantitative (t1/2 = 33.8 ± 0.6 min by FL and t1/2 = 26.0 ± 0.5 min by PET). Fc-AMBF3 was cleared from the CSF through the vasculature and/or lymphatic system that supplies the cribriform plate and the temporal bone. Fc-AMBF3 can be used to diagnose CSFLs, image CSFL repair, and determine the CSF flow velocity in the CNS or through lumboperitoneal shunts by PET/FL imaging. In conclusion, Fc-AMBF3 PET imaging has been demonstrated to safely and dynamically quantitate CSF flow, diagnose fistulas associated with the CSF space, and approximate the clearance of small molecules in the CSF.

In vivo demonstration of blood-brain barrier impairment in Moyamoya disease.

BACKGROUND: Moyamoya disease (MMD) is a cerebrovascular disorder characterized by fragile vascular system. Previous studies suggested that the blood-brain barrier (BBB) destabilizing cytokine angiopoietin-2 plays a critical role in increasing vascular plasticity and endothelial disintegration in MMD. The aim of this study was to assess cerebrovascular integrity in vivo in patients affected by MMD. METHODS: We retrospectively analyzed 11 patients that underwent bypass for MMD (MMD group), 11 patients that underwent bypass for atherosclerotic cerebrovascular disease (ACVD-control group I), and 5 patients that underwent clipping for unruptured aneurysms (non-ischemic-control group II). Sodium fluorescein (NaFL) extravasation was evaluated during videoangiography when checking for bypass patency. A grading system (0, +, ++, +++) was used to define the extent of extravasation. Frequency and intensity of leakage was compared among different groups. RESULTS: NaFL extravasation appeared in 10/11 (91%) patients with MMD and in 8/11 (73%) patients with ACVD during bypass procedures. Extravasation was observed in none of the patients undergoing clipping for unruptured aneurysms. Although both chronic ischemic patient groups showed a comparably high incidence of NaFL extravasation, the MMD group was characterized by a much greater intensity of NaFL extravasation (grade +++ in 82%) than the ACVD group (grade +++ in 27%, p < 0.05). CONCLUSIONS: We demonstrate blood-brain barrier impairment in MMD patients for the first time in vivo. This may be due to mechanisms intrinsic to the unique pathology of MMD, probably explaining the higher association with hemorrhage and post-operative hyperperfusion.

The ablative fractional coagulation zone influences skin fluorescence intensities of topically applied test molecules-An in vitro study with fluorescence microscopy and fluorescence confocal microscopy.

BACKGROUND: Ablative fractional laser (AFL) increases uptake of topically applied skin agents. The coagulation zone (CZ) surrounding vertically ablated channels may influence uptake of drugs. OBJECTIVES: To investigate impact of CZ thickness on skin fluorescence intensities (FI) of a hydrophilic molecule by means of fluorescence microscopy (FM) and fluorescence confocal microscopy (FCM). Second, to compare FI of hydrophilic and lipophilic test molecules by FCM. STUDY DESIGN/METHODS AND MATERIALS: Microchannels with CZ thicknesses of 0, 20, and 80 µm were generated by microneedles or AFL (10,600 nm). Channels were 700 µm deep and number of channels kept constant per skin area. After 4 hours of incubation, FI induced by sodium fluorescein (NAF, hydrophilic, logarithmic partition-coefficient (logP) = -1.52, MW = 376.26) were quantified in both CZ and surrounding skin by FM (0-1,500 µm) and FCM (0-90 µm). FI of NAF and carboxyfluorescein (CAF, lipophilic, logP = 2.9, MW = 376.32) were compared by FCM. RESULTS: By FM, NAF-induced FI were higher in CZ than in surrounding skin (P ≤ 0.001). Highest NAF-FI were induced in skin pretreated with a thin CZ (CZ-20 µm), assessed by both FM and FCM and in particular, FI were higher than in skin pretreated with no CZ (CZ-0 µm) (FM P ≤ 0.041, FCM P < 0.012). Skin FI remained constant to a depth of 500 µm, which corresponded to approximate depth of microchannels (CZ-0 µm, CZ-20 µm, CZ-80 µm: 0-500 µm P ≥ 0.107). In accordance with FM data, FCM showed higher FI within CZ than in surrounding skin, but gradually decreased to zero at a depth of 90 µm. NAF-FI were higher than CAF-FI (P ≤ 0.036), and highest CAF-FI were induced by CZ-0 µm and CZ-20 µm compared to CZ-80 µm (P ≤ 0.009). CONCLUSIONS: The influence of the CZ thickness on skin FI differs between small hydrophilic and lipophilic test molecules. Results may have clinical relevance for laser-assisted drug delivery. Lasers Surg. Med. 51:68-78, 2019. © 2018 Wiley Periodicals, Inc.

Three-Dimensional Transport Model for Intravitreal and Suprachoroidal Drug Injection.

Purpose: Quantitative understanding of the transport of therapeutic macromolecules following intraocular injections is critical for the design of efficient strategies in treating eye diseases, such as neovascular (wet) age-related macular degeneration (AMD) and macular edema (ME). Antiangiogenic treatments, such as neutralizing antibodies against VEGF or recently characterized antiangiogenic peptides, have shown promise in slowing disease progression. Methods: We developed a comprehensive three-dimensional (3D) transport model for intraocular injections using published data on drug distribution in rabbit eyes following intravitreal and suprachoroidal (SC) injection of sodium fluorescein (SF), bevacizumab, and ranibizumab. The model then was applied to evaluate the distribution of small molecules and antiangiogenic proteins following intravitreal and SC injections in human eyes. Results: The model predicts that intravitreally administered molecules are substantially mixed within the vitreous following injection, and that the long-term behavior of the injected drug does not depend on the initial mixing. Ocular pharmacokinetics of different drugs is sensitive to different clearance mechanisms. Effective retinal drug delivery is impacted by RPE permeability. For VEGF antibody, intravitreal injection provides sustained delivery to the retina, whereas SC injection provides more efficient, but short-lived, retinal delivery for smaller-sized molecules. Long-term suppression of neovascularization through SC administration of antiangiogenic drugs necessitates frequent injection or sustained delivery, such as microparticle-based delivery of antiangiogenic peptides. Conclusions: A comprehensive 3D model for intravitreal and SC drug injection is developed to provide a framework and platform for testing drug delivery routes and sustained delivery devices for new and existing drugs.

Efflux Pump Substrates Shuttled to Cytosolic or Vesicular Compartments Exhibit Different Permeability in a Quantitative Human Blood-Brain Barrier Model.

Representative in vitro blood-brain barrier (BBB) models can support the development of strategies to efficiently deliver therapeutic drugs to the brain by aiding the characterization of their internalization, trafficking, and subsequent transport across the BBB. A collagen type I (COL1) hydrogel-based in vitro BBB model was developed to enable the simultaneous characterization of drug transport and intracellular processing using confocal microscopy, in a way that traditional insert-based in vitro BBB models cannot. Human induced pluripotent stem cells (hiPSCs) were differentiated into cells that exhibited a BBB-like phenotype on COL1 hydrogels, which included the expression of key BBB-specific proteins and low permeability of representative small and large molecule therapeutics. Furthermore, the BBB phenotype observed on the COL1 hydrogel was similar to that previously reported on porous inserts. The intracellular visualization of two small molecule efflux pump substrates within the hiPSC-derived BBB-like cells demonstrated a difference in cytosolic and vesicular accumulation, which complemented permeability measurements demonstrating a difference in transport rate. The easy-to-construct COL1-based hiPSC-derived BBB model presented here is the first in vitro two-dimensional BBB experimental system that enables the simultaneous quantification of cellular permeability and visualization of intracellular processes by utilizing confocal microscopy, which can provide insights regarding the relationship between transport and intracellular trafficking of therapeutic drugs.

Contributions of the glycocalyx, endothelium, and extravascular compartment to the blood-brain barrier.

The endothelial cells that form the blood-brain barrier (BBB) are coated with glycocalyx, on the luminal side, and with the basement membrane and astrocyte endfeet, on the abluminal side. However, it is unclear how exactly the glycocalyx and extravascular structures contribute to BBB properties. We used two-photon microscopy in anesthetized mice to record passive transport of four different-sized molecules-sodium fluorescein (376 Da), Alexa Fluor (643 Da), 40-kDa dextran, and 150-kDa dextran-from blood to brain, at the level of single cortical capillaries. Both fluorescein and Alexa penetrated nearly the entire glycocalyx volume, but the dextrans penetrated less than 60% of the volume. This suggested that the glycocalyx was a barrier for large but not small molecules. The estimated permeability of the endothelium was the same for fluorescein and Alexa but several-fold lower for the larger dextrans. In the extravascular compartment, co-localized with astrocyte endfeet, diffusion coefficients of the dyes were an order of magnitude lower than in the brain parenchyma. This suggested that the astrocyte endfeet and basement membrane also contributed to BBB properties. In conclusion, the passive transport of small and large hydrophilic molecules through the BBB was determined by three separate barriers: the glycocalyx, the endothelium, and the extravascular compartment. All three barriers must be taken into account in drug delivery studies and when considering BBB dysfunction in disease states.

Photography-based method for assessing fluorescein clearance test in dogs.

BACKGROUND: The fluorescein clearance test (FCT) provides insight into the tear film dynamics. The purpose of this study was to describe an inexpensive and practical method for assessing FCT in dogs, using photography and software analysis, and to assess the retention time of 1 vs. 2 eye drops on the canine ocular surface. METHODS: (i) In vivo - Eight healthy German Shepherd dogs were recruited. Following topical anesthesia with 0.5% proxymetacaine, each eye sequentially received (1 week apart) either 1 drop (35 µL) or 2 drops (70 µL) of 0.5% fluorescein. A Schirmer strip was inserted in the ventral conjunctival fornix for 10 s at the following times: each 10 min for 100 min, 24 h, 48 h and 72 h. (ii) In vitro - Schirmer strips were placed for 10 s in contact with microplate wells containing 1 or 2 drops of 0.5% fluorescein. In both experiments, the fluorescein-impregnated Schirmer strips were immediately imaged, and the area and intensity of fluorescein uptake were analyzed with ImageJ software. For the in vitro experiment, images were evaluated by the same examiner (repeatability) or two examiners (reproducibility). RESULTS: Photography-based FCT was easy to perform and showed high repeatability and reproducibility (coefficients of variation ≤2.75%). In vivo, the area and intensity of fluorescein uptake on Schirmer strips were significantly greater at 30 min and 40 min post- fluorescein instillation in the 2 drops vs. 1 drop groups (p ≤ 0.044). Compared to baseline, the residual fluorescein uptake on Schirmer strips was < 5% at 60 min and 90 min in the 1 drop and 2 drops groups, respectively. CONCLUSIONS: Photography-based FCT is a practical and reliable diagnostic tool with various clinical and research applications in veterinary medicine. Instillation of two drops provided greater amount and longer retention on the anesthetized canine ocular surface than a single drop. Fluorescein clearance time of a single drop in dolichocephalic dogs is 60 min.

Elucidating the kinetics of sodium fluorescein for fluorescence-guided surgery of glioma.

OBJECTIVE: The use of the optical contrast agent sodium fluorescein (NaFl) to guide resection of gliomas has been under investigation for decades. Although this imaging strategy assumes the agent remains confined to the vasculature except in regions of blood-brain barrier (BBB) disruption, clinical studies have reported significant NaFl signal in normal brain tissue, limiting tumor-to-normal contrast. A possible explanation arises from earlier studies, which reported that NaFl exists in both pure and protein-bound forms in the blood, the former being small enough to cross the BBB. This study aims to elucidate the kinetic binding behavior of NaFl in circulating blood and its effect on NaFl accumulation in brain tissue and tumor contrast. Additionally, the authors examined the blood and tissue kinetics, as well as tumor uptake, of a pegylated form of fluorescein selected as a potential optical analog of gadolinium-based MRI contrast agents. METHODS: Cohorts of mice were administered one of the following doses/forms of NaFl: 1) high human equivalent dose (HED) of NaFl, 2) low HED of NaFl, or 3) pegylated form of fluorescein. In each cohort, groups of animals were euthanized 15, 30, 60, and 120 minutes after administration for ex vivo analysis of fluorescein fluorescence. Using gel electrophoresis and fluorescence imaging of blood and brain specimens, the authors quantified the temporal kinetics of bound NaFl, unbound NaFl, and pegylated fluorescein in the blood and normal brain tissue. Finally, they compared tumor-to-normal contrast for NaFl and pegylated-fluorescein in U251 glioma xenografts. RESULTS: Administration of NaFl resulted in the presence of unbound and protein-bound NaFl in the circulation, with unbound NaFl constituting up to 70% of the signal. While protein-bound NaFl was undetectable in brain tissue, unbound NaFl was observed throughout the brain. The observed behavior was time and dose dependent. The pegylated form of fluorescein showed minimal uptake in brain tissue and improved tumor-to-normal contrast by 38%. CONCLUSIONS: Unbound NaFl in the blood crosses the BBB, limiting the achievable tumor-to-normal contrast and undermining the inherent advantage of tumor imaging in the brain. Dosing and incubation time should be considered carefully for NaFl-based fluorescence-guided surgery (FGS) of glioma. A pegylated form of fluorescein showed more favorable normal tissue kinetics that translated to higher tumor-to-normal contrast. These results warrant further development of pegylated-fluorescein for FGS of glioma.

Mechanical load increase-induced changes in cytoskeletal structure and cellular barrier function in human cerebral endothelial cells.

Globally, approximately a billion patients are estimated to suffer from neurological disorders. Although there are many therapeutic candidates for the central nervous system, treatment of brain disorders is restricted by the blood-brain barrier (BBB), which is a highly selective membrane that protects the brain from exogenous substances. This study was undertaken to develop a novel strategy to overcome the BBB and improve the efficiency of drug delivery to the brain by mechanical load increase using hypergravity. Human cerebral microvascular endothelial cells were exposed three times to 20 min hypergravity (10g), with a 20-min rest period between each exposure. The applied hypergravity reversibly decreased the cellular metabolic activity and increased the permeation rate of fluorescein sodium salt, fluorescein isothiocyanate-labeled dextran (FD-4), and fluorescein-labeled jacalin. Following the exposure to hypergravity, we also observed structural changes of the cytoskeleton and tight junctions, and an alteration in the expression levels of related genes. These results indicate that increased mechanical load due to the applied hypergravity affects the cytoskeletal arrangement and tight junctions, thereby weakening the cell barrier function and enhancing the permeability of the paracellular pathway. Thus, the mechanical load increase by hypergravity has the potential of being used as a novel strategy to overcome the BBB for brain drug delivery.