Nattokinase Attenuates Retinal Neovascularization Via Modulation of Nrf2/HO-1 and Glial Activation.

Purpose: Nattokinase (NK), an active ingredient extracted from traditional food Natto, has been studied for prevention and treatment of cardiovascular diseases due to various vasoprotective effects, including fibrinolytic, antihypertensive, anti-atherosclerotic, antiplatelet, and anti-inflammatory activities. Here, we reported an antineovascular effect of NK against experimental retinal neovascularization. Methods: The inhibitory effect of NK against retinal neovascularization was evaluated using an oxygen-induced retinopathy murine model. Expressions of Nrf2/HO-1 signaling and glial activation in the NK-treated retinae were measured. We also investigated cell proliferation and migration of human umbilical vein endothelial cells (HUVECs) after NK administration. Results: NK treatment significantly attenuated retinal neovascularization in the OIR retinae. Consistently, NK suppressed VEGF-induced cell proliferation and migration in a concentration-dependent manner in cultured vascular endothelial cells. NK ameliorated ischemic retinopathy partially via activating Nrf2/HO-1. In addition, NK orchestrated reactive gliosis and promoted microglial activation toward a reparative phenotype in ischemic retina. Treatment of NK exhibited no cell toxicity or anti-angiogenic effects in the normal retina. Conclusions: Our results revealed the anti-angiogenic effect of NK against retinal neovascularization via modulating Nrf2/HO-1, glial activation and neuroinflammation, suggesting a promising alternative treatment strategy for retinal neovascularization.

The effect of hypergravity in intestinal permeability of nanoformulations and molecules.

The oral administration of drugs remains a challenge due to rapid enzymatic degradation and minimal absorption in the gastrointestinal tract. Mechanical forces, namely hypergravity, can interfere with cellular integrity and drug absorption, and there is no study describing its influence in the intestinal permeability. In this work, it was studied the effect of hypergravity on intestinal Caco-2 cells and its influence in the intestinal permeability of different nanoformulations and molecules. It was shown that the cellular metabolic activity and integrity were maintained after exposure to different gravity-levels (g-levels). Expression of important drug transporters and tight junctions' proteins was evaluated and, most proteins demonstrated a switch of behavior in their expression. Furthermore, paracellular transport of FITC-Dextran showed to significantly increase with hypergravity, which agrees with the decrease of transepithelial electrical resistance and the increase of claudin-2 at higher g-levels. The diffusion of camptothecin released from polymeric micelles revealed a significant decrease, which agrees with the increased expression of the P-gp observed with the increase in g-levels, responsible for pumping this drug out. The neonatal Fc receptor-mediated transport of albumin-functionalized nanoparticles loaded with insulin showed no significant changes when increasing the g-levels. Thus, this study supports the effect of hypergravity on intestinal permeability is dependent on the molecule studied and the mechanism by which it is absorbed in the intestine.

Transdermal electroosmotic flow generated by a porous microneedle array patch.

A microneedle array is an attractive option for a minimally invasive means to break through the skin barrier for efficient transdermal drug delivery. Here, we report the applications of solid polymer-based ion-conductive porous microneedles (PMN) containing interconnected micropores for improving iontophoresis, which is a technique of enhancing transdermal molecular transport by a direct current through the skin. The PMN modified with a charged hydrogel brings three innovative advantages in iontophoresis at once: (1) lowering the transdermal resistance by low-invasive puncture of the highly resistive stratum corneum, (2) transporting of larger molecules through the interconnected micropores, and (3) generating electroosmotic flow (EOF). In particular, the PMN-generated EOF greatly enhances the transdermal molecular penetration or extraction, similarly to the flow induced by external pressure. The enhanced efficiencies of the EOF-assisted delivery of a model drug (dextran) and of the extraction of glucose are demonstrated using a pig skin sample. Furthermore, the powering of the PMN-based transdermal EOF system by a built-in enzymatic biobattery (fructose / O2 battery) is also demonstrated as a possible totally organic iontophoresis patch.

DNA-crosslinked alginate and layered microspheres to modulate the release of encapsulated FITC-dextran.

Alginate can be gently crosslinked by calcium into hydrogels and microspheres for the encapsulation and release of proteins and drugs. However, the release is often over short periods unless alginate is also covalently modified or crosslinked. This research aims to sustain the release of encapsulated model drug FITC-dextran by covalently crosslinking alginate with short oligomers DNA because evidence suggests that DNA may also interact with alginate to further increase effective crosslinking. Furthermore, modulating the release of drugs from alginate in response to specific proteins could tailor release profiles to improve patient treatment. This research develops a DNA-crosslinked alginate hydrogel and layered alginate microspheres to encapsulate and then sustain the release FITC-dextran (model drug). An aptamer sequence to hen egg-white lysozyme is included in one DNA strand to allow for the disruption of the crosslinks by interactions with human lysozyme. Alginate was covalently modified with complementary strands of DNA to crosslink the alginate into hydrogels, which had increased crosslinking density when re-swollen (in comparison to controls crosslinked with PEG) and could sustained the release of encapsulated FITC-dextran. When an aptamer sequence for hen lysozyme was included in the DNA crosslinks, the hydrogels decrosslinked when incubated in human lysozyme for 60 days. In addition, calcium alginate microspheres were coated with 3 alternating layers of poly-Lysine, DNA-crosslinked alginate, and poly-L-lysine. FITC-dextran loaded into the microspheres released in a sustained manner past 30 days (into PBS at 37 °C) and would likely continue to release for far longer had the studies continued. When incubated with 3 µM of human lysozyme, a burst release of FITC-dextran occurred from both the hydrogels and microspheres, with no changes in the controls. The increased release was in bursts followed by similar sustained release rates suggesting that the human lysozyme temporarily disrupted the DNA crosslinks which were then re-established or were influenced by interactions between DNA and alginate. Importantly, covalently bound complementary strands of DNA could crosslink the alginate and additional interactions appeared to further sustain the release of encapsulated therapeutics.

Weak Electric Current Treatment to Artificially Enhance Vascular Permeability in Embryonated Chicken Eggs.

Technologies that overcome the barrier presented by vascular endothelial cells are needed to facilitate targeted delivery of drugs into tissue parenchyma by intravenous administration. We previously reported that weak electric current treatment (ET: 0.3-0.5 mA/cm2) applied onto skin tissue in a transdermal drug delivery technique termed iontophoresis induces cleavage of intercellular junctions that results in permeation of macromolecules such as small interfering RNA and cytosine-phosphate-guanine (CpG) oligonucleotide through the intercellular space. Based on these findings, we hypothesized that application of ET to blood vessels could promote cleavage of intercellular junctions that artificially induces increase in vascular permeability to enhance extravasation of drugs from the vessels into target tissue parenchyma. Here we investigated the effect of ET (0.34 mA/cm2) on vascular permeability using embryonated chicken eggs, which have blood vessels in the chorioallantoic membrane (CAM), as an animal model. ET onto the CAM of the eggs significantly increased extravasation of intravenously injected calcein (M.W. 622.6), a low molecular weight compound model, and the macromolecule fluorescein isothiocyanate (FITC)-dextran (M.W. 10000). ET-mediated promotion of penetration of FITC-dextran through vascular endothelial cells was also observed in transwell permeability assay using monolayer of human umbilical vein endothelial cells without induction of obvious cellular damage. Confocal microscopy detected remarkable fluorescence derived from injected FITC-dextran in blood vessel walls. These results in embryonated chicken eggs suggest that ET onto blood vessels could artificially enhance vascular permeability to facilitate extravasation of macromolecules from blood vessels.

Assessment of Amphiphilic Poly-N-vinylpyrrolidone Nanoparticles' Biocompatibility with Endothelial Cells in Vitro and Delivery of an Anti-Inflammatory Drug.

Nanoparticles (NPs) produced from amphiphilic derivatives of poly-N-vinylpyrrolidone (Amph-PVP), composed of various molecular weight polymeric hydrophilic fragments linked into hydrophobic n-alkyl chains of varying lengths, were previously shown to exert excellent biocompatibility. Although routes of administration can be different, finally, most nanosystems enter the blood circulation or lymphatic vessels, and by this, they establish direct contact with endothelial cells. In this study, Amph-PVP NPs and fluorescently labeled Amph-PVP-based NPs, namely "PVP" NPs (Amph-PVP-NPs (6000 Da) unloaded) and "F"-NPs (Amph-PVP-NPs (6000 Da) loaded with fluorescent FITC), were synthesized to study Amph-PVP NPs interactions with HMEC-1 endothelial cells. PVP NPs were readily uptaken by HMEC-1 cells in a concentration-dependent manner, as demonstrated by immunofluorescence imaging. Upon uptake, the FITC dye was localized to the perinuclear region and cytoplasm of treated cells. The generation of lipopolysaccharide (LPS)-induced activated endothelium model revealed an increased uptake of PVPNPs, as shown by confocal microscopy. Both unloaded PVP NPs and F-NPs did not affect EC viability in the 0.01 to 0.066 mg/mL range. Furthermore, we focused on the potential immunological activation of HMEC-1 endothelial cells upon PVPNPs treatment by assessing the expression of their E-Selectin, ICAM-1, and VCAM-1 adhesion receptors. None of the adhesion molecules were affected by NP treatments of both activated by LPS and nonactivated HMEC-1 cells, at the utilized concentrations (p = NS). In this study, PVP (6000 Da) NPs were used to encapsulate indomethacin, a widely used anti-inflammatory drug. The synthesized drug carrier complex did not affect HMEC-1 cell growth and expression of E-selectin, ICAM-1, and VCAM-1 adhesion receptors. In summary, PVP-based NPs are safe for use on both basal and activated endothelium, which more accurately mimics pathological conditions. Amph-PVP NPs are a promising drug delivery system.

Correlations Between Skin Barrier Integrity and Delivery of Hydrophilic Molecules in the Presence of Penetration Enhancers.

PURPOSE: We investigated the potential correlations between skin barrier integrity and hydrophilic drugs distribution in skin in the presence of different types of penetration enhancers (PEs) and their combinations. METHODS: We measured skin conductivity to evaluate skin barrier integrity before and after the topical application of different chemical PEs, physical PE, peptide PE and their combinations in vitro. We also investigated their effect on the skin distribution profiles of two hydrophilic model drugs, Fluorescein sodium (376 Da) and Fluorescein isothiocyanate-dextrans 10 (10 KDa). RESULTS: The physical PE significantly increased the skin conductivity compared to all other PEs, while the peptide PE had no effect on it. The drug deposition in different skin layers was not only dependent on PE applied but also its own molecular weight. We further found two excellent correlations: one (R2 = 0.9388) between skin barrier integrity and total skin absorption of FNa and another one(R2 = 0.9212) between skin barrier integrity and the deposition of FNa in dermis and receptor in presence of chemical or physical PEs and their combinations. CONCLUSIONS: The total skin absorption or the deposition in dermis and receptor of small hydrophilic drug in the presence of chemical and physical PEs and their combinations show a good correlation with skin barrier integrity. However, such correlations hold true neither for large hydrophilic drug nor for peptide PE. All good relationships found in this work will allow screening suitable PEs or combinations by measuring the skin conductivity induced by corresponding PEs. Graphical Abstract The total skin absorption of small hydrophilic drug shows a good correlation with skin barrier integrity in the presence of chemical and physical penetration enhancers and their combinations. However, such a correlation hold true neither for large hydrophilic drug nor for peptide penetration enhancer.

Critical Role of TSLP Receptor on CD4 T Cells for Exacerbation of Skin Inflammation.

Thymic stromal lymphopoietin (TSLP) is a key cytokine that initiates and promotes allergic inflammation both in humans and mice. It is well known that TSLP is important in initial step of inflammation by stimulating dendritic cells to promote Th2 differentiation of naive T cells. However, TSLP is abundantly produced in the late phase of inflammation, as well; therefore, we focused on the function of TSLP in chronic Th2-type inflammation. By establishing a novel (to our knowledge) chronic allergic skin inflammation mouse model with repetitive challenges of hapten after sensitization, we demonstrated that CD4 T cell-specific deletion of TSLP receptor (TSLPR) resulted in near-complete ablation of ear swelling and infiltration of CD4 T cells and eosinophils, but after second challenge. Of note, TSLPR deletion on CD4 T cells did not affect acute inflammation. As expected, transfer of Ag-sensitized wild-type CD4T cells, but not of TSLPR-deficient CD4T cells, increased skin inflammation in the model upon challenge. Furthermore, production of IL-4 from TSLPR-deficient CD4T cells in inflamed ear lesions was markedly diminished, demonstrating that TSLP-dependent IL-4 production from CD4T cells was critical for the exacerbation of skin inflammation. Similar results were obtained in Th2-type allergic skin inflammation model using MC903. Collectively, these results indicate that TSLP acts directly on CD4 T cells to elicit pathogenesis of Th2 cells, thereby having a critical role in exacerbation of skin inflammation in the chronic phase.

Kidney targeted delivery of asiatic acid using a FITC labeled renal tubular-targeting peptide modified PLGA-PEG system.

Chronic kidney disease (CKD) is one of the leading public health problems worldwide and finally progresses to end-stage renal disease. The therapeutic options of CKD are very limited. Thus, development of drug delivery systems specific-targeting to kidney may offer more options. Here we developed an efficient kidney-targeted drug delivery system using a FITC labeled renal tubular-targeting peptide modified PLGA-PEG nanoparticles and investigated the intrarenal distribution and cell-type binding. We found that the modified nanoparticles with an approximate diameter of 200 nm exhibited the highest binding capacity with HK-2 cells and fluorescence and immunohistochemical analysis showed they mainly localized in renal proximal tubules by passing through the basolateral side. Furthermore, these kidney-specific nanoparticles could significantly enhance the therapeutic effects of asiatic acid, an insoluble triterpenoid compound as drug delivery carriers. In conclusion, these results suggest the potential of the peptide modified PLGA-PEG nanoparticles as kidneytargeted drug delivery system to proximal tubular cells in treatment of CKD.

Cationic Polylactic Acid-Based Nanoparticles Improve BSA-FITC Transport Across M Cells and Engulfment by Porcine Alveolar Macrophages.

This work described the development of a cationic polylactic acid (PLA)-based nanoparticles (NPs) as an antigen delivery system using dimethyldioctadecylammonium bromide (DDA) to facilitate the engulfment of BSA-FITC by porcine alveolar macrophages (3D4/2 cells) and heat-labile enterotoxin subunit B (LTB) to enhance the transport of BSA-FITC across M cells. The experimental design methodology was employed to study the influence of PLA, polyvinyl alcohol (PVA), DDA, and LTB on the physical properties of the PLA-based NPs. The size of selected cationic PLA NPs comprising 5% PLA, 5% PVA, and 0.6% DDA with or without LTB absorption was range from 367 to 390 nm with a polydispersity index of 0.26, a zeta potential of + 26.00 to + 30.55 mV, and entrapment efficiency of 41.43%. Electron micrographs revealed NPs with spherical shape. The release kinetic of BSA from the NPs followed the Korsmeyer-Peppas kinetics. The cationic PLA NPs with LTB surface absorption showed 3-fold increase in BSA-FITC transported across M cells compared with the NPs without LTB absorption. The uptake studies demonstrated 2-fold increase in BSA-FITC intensity in 3D4/2 cells with cationic NPs as compared with anionic NPs. Overall, the results suggested that LTB decreased the retention time of BSA-FITC loaded in the cationic PLA NPs within the M cells, thus promoting the transport of BSA-FITC across the M cells, and cationic NPs composed of DDA help facilitate the uptake of BSA-FITC in the 3D4/2 cells. Further studies in pigs with respiratory antigens will provide information on the efficacy of cationic PLA NPs as a nasal antigen carrier system.

Influence of molecular weight on transdermal delivery of model macromolecules using hydrogel-forming microneedles: potential to enhance the administration of novel low molecular weight biotherapeutics.

With a view to improve the current monoclonal antibody-based therapies dominating the pharmaceutical market, low molecular weight (MW) protein-based macromolecules, such as recombinant antibody fragments, typically within the range of 10-70 kDa, have been developed. Previously, our group successfully delivered Avastin®, a monoclonal antibody (mAb) across the skin using hydrogel-forming microneedles (MN). However, it is thought that this delivery system can be further enhanced using novel, lower MW biomolecules. To address this perception, in the current study, FITC-dextran of different MWs (10, 70 and 150 kDa) was used to model the transdermal delivery of low MW biotherapeutics and mAbs with MWs of approximately 150 kDa. Conversely, fluorescein sodium was the compound selected to model hydrophilic, low MW drugs. As expected, fluorescein sodium produced the greatest cumulative permeation (637.4 ± 42.69 µg). The amounts of FITC-dextran 10 kDa and 150 kDa which permeated across neonatal porcine skin in vitro were 462.17 ± 65.85 µg and 213.54 ± 15.19 µg after 24 h, respectively. The results collated here suggest that the delivery of emerging novel biotherapeutics, via'super swelling' hydrogel-forming MNs, have the potential to result in greater permeation across human skin, compared to the delivery of mAbs delivered via the same route.

Efficacy of SPM-NONOate following intrapulmonary delivery in promoting absorptions of poorly absorbed macromolecules in rats and the underling mechanism.

This research aims to investigate the potential of N-[4-[1-(3-Aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl]-1,3-propanediamine (SPM-NONOate) for promoting the absorption of poorly absorbed macromolecules delivered by intrapulmonary route. Influence of SPM-NONOate on the drug absorption was characterized by using a series of fluorescein isothiocyanate-labeled dextrans (FDs) as affordable models of hydrophilic macromolecules with established tools for quantitative analysis. SPM-NONOate increased concentration-dependently within 1-10 mM the pulmonary absorptions of FDs in rats. Moreover, this promoting effect varied with the molecular weight of FDs, and the largest absorption enhancement effect was obtained for FD70. SPM-NONOate also showed promising enhancement potential on the absorption of some therapeutic peptides, where obvious hypoglycemic and hypocalcemic effects were observed after intrapulmonary delivery of insulin and calcitionin, respectively, with SPM-NONOate to rats. The safety of SPM-NONOate was confirmed based on measurement of some biological markers in bronchoalveolar lavage fluid (BALF) of rats. Additionally, mechanism underling the absorption enhancement action of SPM-NONOate was explored by combinatorial administration of FD4 and SPM-NONOate with various scavengers and generator to rat lungs. Results indicated that NO released from SPM-NONOate induced the enhancement in the drug absorption, and peroxynitrate, a NO metabolite, possibly participated in the absorption enhancing action of SPM-NONOate.

FDM-printed pH-responsive capsules for the oral delivery of a model macromolecular dye.

To this day, the oral delivery of biomacromolecules remains a major developmentally-oriented challenge. A combinatorial approach was followed at this study, to formulate an efficient carrier for the in vitro delivery of a model macromolecule, fluorescein isothiocyanate-dextran 4 kDa (FD4). The model macromolecule was formulated in a self-assembling peptide hydrogel (ac-(RADA)4-CONH2), prior to deposition in a hydroxypropyl methylcellulose-phthalate (HPMCP)-based 3D-printed capsule. Loading of FD4 was investigated for potential alterations on the structural (AFM) and gelling properties of the peptide carrier. Thermal analysis and morphological properties of the 3D-printed capsules were assessed by TGA, DSC and microscopy studies. For the peptide hydrogel, similar release profiles of FD4 were recorded in simulated gastric fluid pH 1.2 and phosphate buffer saline pH 7.4, indicating the need for a structural barrier, to protect the peptide carrier from the acidic environment of the stomach. The pH responsive character of the HPMCP-based capsule was evidenced in the release profiles of FD4 in a sequence of release media, i.e. simulated gastric fluid pH 1.2, simulated intestinal fluid pH 6.8 and phosphate buffer saline pH 7.4. The results supported the combinatorial formulation approach as a promising system for the efficient oral delivery of biomacromolecules.

A strategy for iron oxide nanoparticles to adhere to the neuronal membrane in the substantia nigra of mice.

Effective attachment of magnetic nanoparticles to neuronal membranes has far-reaching significance in activating ion channels and treating neurodegenerative diseases. Superparamagnetic iron oxide nanoparticles (SPIONs) synthesized by the polyol pyrolysis method have the advantages of rich surface functional groups, excellent magnetic properties, controllable particle size and water dispersibility. We propose that perfusion of biotin into the targeted brain area should be initially performed because it tends to be adsorbed by cell membranes, followed by injection of streptavidin (SA)-modified SPIONs into the same area of the brain. By means of the strong binding force between SA and biotin, the SPIONs may subsequently adhere to the cell surfaces in the brain area. In this work, fluorescein isothiocyanate-streptavidin (FITC-SA) was modified on the surface of polyethylene imine (PEI)-SPIONs by the EDC-NHS method and stereotaxically injected into the biotin-supplemented substantia nigra of mice. The combination of fluorescence detection with transmission electron microscopy (TEM) confirmed that FITC-SA/PEI-SPIONs adhered to neuronal membranes in the substantia nigra of mice 24 h after injection. The results show that our strategy can promote the attachment of SPIONs to neuronal membranes.

Shear-Induced Encapsulation into Red Blood Cells: A New Microfluidic Approach to Drug Delivery.

Encapsulating molecules into red blood cells (RBCs) is a challenging topic for drug delivery in clinical practice, allowing to prolong the residence time in the body and to avoid toxic residuals. Fluidic shear stress is able to temporary open the membrane pores of RBCs, thus allowing for the diffusion of a drug in solution with the cells. In this paper, both a computational and an experimental approach were used to investigate the mechanism of shear-induced encapsulation in a microchannel. By means of a computational fluid dynamic model of a cell suspension, it was possible to calculate an encapsulation index that accounts for the effective shear acting on the cells, their distribution in the cross section of the microchannel and their velocity. The computational model was then validated with micro-PIV measurements on a RBCs suspension. Finally, experimental tests with a microfluidic channel showed that, by choosing the proper concentration and fluid flow rate, it is possible to successfully encapsulate a test molecule (FITC-Dextran, 40 kDa) into human RBCs. Cytofluorimetric analysis and confocal microscopy were used to assess the RBCs physiological shape preservation and confirm the presence of fluorescent molecules inside the cells.

The All but Forgotten Mascagni-Sappey Pathway: Learning from Immediate Lymphatic Reconstruction.

BACKGROUND: Upper extremity lymphedema occurs in 25 to 40% of patients after axillary lymph node dissection (ALND). Immediate lymphatic reconstruction (ILR) or the lymphatic micro- surgical preventative healing approach has demonstrated a significant decrease in postoperative rates of lymphedema (LE) from 4 to 12%. Our objective was to map the Mascagni -Sappey pathway, the lateral upper arm draining lymphatics, in patients undergoing ILR to better characterize the drainage pattern of this lymphosome to the axilla. METHODS: A retrospective review of our institutional lymphatic database was conducted and consecutive breast cancer patients undergoing ILR were identified from November 2017 through June 2018. Patient demographics, clinical characteristics, and intraoperative records were retrieved and analyzed. RESULTS: Twenty-nine consecutive breast cancer patients who underwent ILR after ALND were identified. Patients had a mean age of 54.6years and body mass index (BMI) of 26.6 kg/m2. Fluorescein isothiocyanate (FITC) was injected at the medial upper arm and isosulfan blue was injected at the cephalic vein, or lateral upper arm, prior to ALND. After ALND, an average 2.5 divided lymphatics were identified, and a mean 1.2 lymphatics were bypassed. In all patients, divided FITC lymphatics were identified. However, in only three patients (10%), divided blue lymphatics were identified after ALND. CONCLUSION: In this study, variable drainage of the lateral upper arm to the axillary bed was noted. This study is the first to provide a description of intraoperative findings, demonstrating variable drainage patterns of upper extremity lymphatics to the axilla. Moreover, we noted that the lateral- and medial-upper arm lymphosomes have mutually exclusive pathways draining to the axilla. Further study of lymphatic anatomy variability may elucidate the pathophysiology of lymphedema development and influence approaches to immediate lymphatic reconstruction.

One injection for one-week controlled release: In vitro and in vivo assessment of ultrasound-triggered drug release from injectable thermoresponsive biocompatible hydrogels.

Episodic release of bioactive compounds is often necessary for appropriate biological effects under specific physiological conditions. Here, we aimed to develop an injectable, biocompatible, and thermosensitive hydrogel system for ultrasound (US)-triggered drug release. An mPEG-PLGA-BOX block copolymer hydrogel was synthesized. The viscosity of 15 wt% hydrogel is 0.03 Pa*s at 25 °C (liquid form) and 34.37 Pa*s at 37 °C (gel form). Baseline and US-responsive in vitro release profile of a small molecule (doxorubicin) and that of a large molecule (FITC-dextran), from the hydrogel, was tested. A constant baseline release was observed in vitro for 7 d. When triggered by US (1 MHz, continuous, 0.4 W/cm2), the release rate increased by approximately 70 times. Without US, the release rate returned to baseline. Baseline and US-responsive in vivo release profile of doxorubicin was tested by subcutaneous injection in the back of mice and rats. Following injection into the subcutaneous layer, in vivo results also suggested that the hydrogels remained in situ and provided a steady release for at least 7 d; in the presence of the US-trigger, in vivo release from the hydrogel increased by approximately 10 times. Therefore, the mPEG-PLGA-BOX block copolymer hydrogel may serve as an injectable, biocompatible, and thermosensitive hydrogel system that is applicable for US-triggered drug release.

Novel Liposome Aggregate Platform (LAP) system for sustained retention of drugs in the posterior ocular segment following intravitreal injection.

A novel Liposome Aggregate Platform (LAP) system for prolonged retention of drugs in the posterior eye segment after intravitreal injection (IVT) was developed and evaluated. Calcein, FITC-dextran-4000 (FD4) and Flurbiprofen (FLB), were encapsulated in negatively charged liposomes, and mixed with protamine to produce the LAP. The lipid/protamine ratio was fixed, in order to have a convenient aggregation rate permitting IVT injection and also a sustained release of liposome-entrapped molecules (in vitro) from LAP. In vitro release studies confirmed the potential of LAP system consisted of HPC/DPPG/Chol liposomes and protamine (at 1:1 w/w to lipid), to delay calcein, FD4 and FLB release, compared to free liposomes. In vivo studies demonstrated increased vitreous retention of liposomes and LAP for all molecules, compared to the corresponding solutions; however the retention of FD4 is similar for non-aggregated liposomes and LAP, and calcein retention is only slightly increased by LAP compared to liposomes. The later result may be connected with the visible ocular inflammation caused by both dyes; interestingly inflammation was moderately reduced when dyes were entrapped in liposomes and even more when in LAP. No visible inflammation was demonstrated for FLB, and the LAP system significantly increased the retention of FLB in the ocular tissues (compared to non-aggregated liposomes). Taking into account the capability of the novel LAP system to decrease inflammatory reactions towards calcein and FD4, and prolong the retention of FLB in ocular tissues, it is concluded that such systems, after further optimization, may be considered as promising effective and safe approaches for treatment of posterior segment ocular pathologies.

Establishment of a multilayered 3D cellular model of the retinal-blood barrier.

Retinal disorders are leading causes of blindness. Still, treatment strategies are limited and the challenging anatomical barriers of the eye limit the evaluation and development of new therapeutics. Among these layers of barriers is the blood-retinal barrier, which separates the retina from the choroid by the Bruch's membrane. This work aimed to establish a 3D cellular model that recapitulates barrier properties of the BRB and diffusion through the vitreous, the main barriers encountered upon intravitreal injection. Several parameters were evaluated namely co-culture time of ARPE-19 and HUVECs and different biomaterial compositions of hydrogels to better mimic the human vitreous. The developed vitreous mimic has viscoelastic properties similar to human vitreous. Co-culture of human retinal and endothelial cells showed increased transepithelial resistance with longer co-culture times concomitant with reduced permeability to FITC-dextran 40 kDa. The proposed models lay the foundation of a platform for faster assessment of a large number of samples and without the use of animals.

In vitro testing of flash-frozen sublingual membranes for storage and reproducible permeability studies of macromolecular drugs from solution or nanofiber mats.

Sublingual drug delivery allows systemic delivery of drug without difficulties connected with the gastrointestinal pathway. We developed a new simple protocol for easy-to-use processing and storage of porcine sublingual mucosal membrane for in vitro studies using "flash freezing" in liquid nitrogen. All the dextrans used as mucosal membrane integrity and permeability markers permeated only slowly through sublingual mucosa illustrating usability both the "fresh" and "flash frozen" sublingual membranes whereas conventional cold storage "frozen" membranes have shown significantly higher permeabilities for macromolecules due to the sustained damage. The permeability values were too low to expect dextrans to be potential carriers at this context. To test albumin as a drug carrier we compared FITC-albumin permeation from solutions vs. nanofiber mats donors. To increase the amounts and prolong the transport, we manufactured nanofiber mats loaded with fluorescently marked albumin using well-scalable electrospinning technology. Nanofiber mats have allowed albumin passage through the sublingual membrane in similar amounts as from the pure artificial saliva solution. Since salivary washout strictly limits the duration of liquid dosages, nanofiber mats may thus permit prolonged sublingual administration.