BB Release Research Articles

Exploring Brilliant Blue FCF Assembly with Beta Cyclodextrin, Characterizations and Applications in Biological Systems Using Physicochemical and Computational Methods

AbstractThis research delvedinto the BrilliantBlue FCF (BB) dye encapsulation within the nano hydrophobic cavity of β‐cyclodextrin (β‐CD) utilizing various spectroscopic and physicochemical techniques. Job's plot using UV‐visible and Fluorescence spectroscopy, surface tension, and conductance study confirmed 1 : 1 stoichiometry of theinclusion complex (BBIC).1H NMR and FT‐IR studies demonstrated the possible binding mode of BB into the β‐CD cavity, which wasin good agreement withthe DFT study. The degree of affinity of β‐CD for BB in the ground state and excited state were estimatedfromUV‐visible andFluorescence spectroscopy(binding constant=8113.9451 M−1and10199.563 M−1), andthe negative free binding energyshowedthe encapsulation process to bethermodynamically feasible. PXRD analysis and SEM image studies revealed the formation of the BBIC. After complexation with β‐CD, the photostability of BB was found to be enhanced. The interaction study of BB and BBIC with CT‐DNA revealed the sustained release of BB from the complex. Furthermore, neither BBICnor free BB exhibited a significant cytotoxic effect on the normal cell line HEK‐293. However, BBIC complex (IC50=6.15 μM) showed significant in vitrocytotoxic activity compared to pure BB (IC50=8.90 μM) towards cancer cell line A549.

MOFs-Based Nanoagents Enable Sequential Damage to Cancer-Associated Fibroblast and Tumor Cells for Phototriggered Tumor Microenvironment Regulation.

A composite nanoagent capable of phototriggered tumor microenvironment (TME) regulation is developed based on copper (II) metal-organic frameworks (MOFs) with encapsulation of blebbistatin (Bb) and surface modification of fibroblast activation protein-αtargeted peptide (Tp). Tp enables active targeting of the nanoagents to cancer-associated fibroblast (CAF) while near-infrared light triggers Cu2+ -to-Cu+ photoreduction in MOFs, which brings about the collapse of MOFs and the release of Bb and Cu+ . Bb mediates photogeneration of hydroxyl radicals (•OH) and therefore inhibits extracellular matrix production by inducing CAF apoptosis, which facilitates the penetration of nanoagent to deep tumor tissue. The dual-channel generation of •OH based on Bb and the Cu+ species, via distinct mechanisms, synergistically reinforces oxidative stress in TME capable of inducing immunogenic cell death, which activates the antitumor immune response and therefore reverses the immunosuppressive TME. The synergistic antitumor phototherapy efficacy of such a type of nanoagent based on the abovementioned TME remodeling is unequivocally verified in a cell-derived tumor xenograft model.

Monomeric α-synuclein induces blood–brain barrier dysfunction through activated brain pericytes releasing inflammatory mediators in vitro

Blood–brain barrier (BBB) disruption is often mediated by neuroinflammation, and occurs during various neurodegenerative diseases including Parkinson's disease (PD). PD is characterized by loss of dopaminergic neurons and aggregated α-synuclein protein in inclusions known as Lewy bodies. Misfolded α-synuclein has been implicated in neurodegeneration and neuroinflammation through activation of microglia and astrocytes. Pericytes are a key cellular regulator of the BBB, although it is not known if they participate in α-synuclein-associated PD pathology. Here, we investigated the impact of pericytes on BBB integrity in response to α-synuclein using rat brain endothelial cells (RBECs) co-cultured with rat brain pericytes (RBEC/pericyte co-culture). In RBEC/pericyte co-cultures, α-synuclein added to the abluminal chamber (where pericytes were grown) significantly increased RBEC permeability to sodium fluorescein. In contrast, it had no marked effect when added to the luminal chamber. In the absence of pericytes, both luminal and abluminal addition of α-synuclein failed to affect permeability of the RBEC monolayer. α-Synuclein did not self-assemble in culture media within 24 h, suggesting that monomeric α-synuclein can disrupt the BBB by interacting with pericytes. We found that in response to α-synuclein, pericytes, but not RBECs, released interleukin (IL)-1β, IL-6, monocyte chemotactic protein (MCP)-1, tumor necrosis factor (TNF)-α, and matrix metalloproteinase-9 (MMP-9). α-Synuclein did not affect platelet-derived growth factor (PDGF)-BB release from RBECs and PDGF receptor-β expression in pericytes. These results suggest that pericytes are more sensitive to monomeric α-synuclein than RBECs regarding release of various inflammatory cytokines/chemokines and MMP-9. Thus, monomeric α-synuclein-activated pericytes may contribute to BBB breakdown in patients with PD.

Role of Myoendothelial Gap Junctions in the Regulation of Human Coronary Artery Smooth Muscle Cell Differentiation by Laminar Shear Stress

Background/Aims: Smooth muscle cells may dedifferentiate into the synthetic phenotype and promote atherosclerosis. Here, we explored the role of myoendothelial gap junctions in phenotypic switching of human coronary artery smooth muscle cells (HCASMCs) co-cultured with human coronary artery endothelial cells (HCAECs) exposed to shear stress. Methods: HCASMCs and HCAECs were seeded on opposite sides of Transwell inserts, and HCAECs were exposed to laminar shear stress of 12 dyn/cm² or 5 dyn/cm². The myoendothelial gap junctions were evaluated by using a multi-photon microscope. Results: In co-culture with HCAECs, HCASMCs exhibited a contractile phenotype, and maintained the expression of differentiation markers MHC and H1-calponin. HCASMCs and HCAECs formed functional intercellular junctions, as evidenced by colocalization of connexin(Cx)40 and Cx43 on cellular projections inside the Transwell membrane and biocytin transfer from HCAECs to HCASMCs. Cx40 siRNA and 18-α-GA attenuated protein expression of MHC and H1-calponin in HCASMCs. Shear stress of 5 dyn/cm² increased Cx43 and decreased Cx40 expression in HCAECs, and partly inhibited biocytin transfer from HCAECs to HCASMCs, which could be completely blocked by Cx43 siRNA or restored by Cx40 DNA transfected into HCAECs. The exposure of HCAECs to shear stress of 5 dyn/cm² promoted HCASMC phenotypic switching, manifested by morphological changes, decrease in MHC and H1-calponin expression, and increase in platelet-derived growth factor (PDGF)-BB release, which was partly rescued by Cx43 siRNA or Cx40 DNA or PDGF receptor signaling inhibitor. Conclusions: The exposure of HCAECs to shear stress of 5 dyn/cm² caused the dysfunction of Cx40/Cx43 heterotypic myoendothelial gap junctions, which may be replaced by homotypic Cx43/Cx43 channels, and induced HCASMC transition to the synthetic phenotype associated with the activation of PDGF receptor signaling, which may contribute to shear stress-associated arteriosclerosis.

Disruption of the alternative pathway convertase occurs at the staphylococcal surface via the acquisition of factor H by Staphylococcus aureus

Staphylococcus aureus is a significant human pathogen that causes skin-structure, invasive, and hospital-associated infections worldwide. The complement system is vital to innate defense against many bacterial infections. As shown with other pathogens, mechanisms for circumventing complement attack may include recruitment of the complement regulatory protein factor H (fH). In the present study, we show that S. aureus binds fH in a dose-dependent and time-dependent manner. Interestingly, this interaction does not require complement activation nor C3-fragment presence and occurs efficiently in the absence of other serum components suggesting a mechanism other than bridging between intermediary molecules. However, fH binding is greater when incubated with normal human serum compared to heat-inactivated serum, which suggests that complement activation may enhance fH binding. S. aureus-bound fH was found to inhibit the alternative pathway through disruption of the alternative pathway C3 convertase as shown by an increase in Bb release and a decrease in total C3-fragment deposition. Furthermore, S. aureus-bound fH retains cofactor activity for factor-I mediated cleavage of C3b. These studies show that the acquisition of fH to the S. aureus surface inhibits complement-mediated opsonization via disruption of the alternative pathway convertase; thus, we report an immune-evasion mechanism not previously described for S. aureus.

Release characteristics of brilliant blue from calcium-alginate beads coated with quaternized chitosan

Calcium-alginate beads coated with quaternized chitosan were prepared in a neutral environment, and morphologies were observed by SEM. Optimum conditions for the encapsulation and retention of a model drug (brilliant blue, BB) in acid were obtained from studies of preparation conditions, including alginate and quaternized chitosan concentration, calcium chloride (CaCl2) concentration in the gelling medium and by comparing one-step and two-step preparation methods. Results showed that very high BB encapsulation efficiency (99%, w/w) and low leakage in acid (8%, w/w) was achieved from dry beads when 2.0% (w/v) alginate was dropped into 1.0% (w/v) CaCl2 containing 0.3% (w/v) quaternized chitosan by a one-step method. The release of BB in 0.9% (w/v) NaCl was modulated by coating calcium-alginate with different weight average molecule weight (Mw) and degree of substitution (DS) of quaternized chitosan. A decreased of Mw accelerated the release of BB and a high DS value significantly decreased the release in 0.9% (w/v) NaCl.

Synthesis and application of nanoparticles by a high gravity method

Synthesis and application of nanoparticles by a high gravity method

Fabrication of porous hollow silica nanoparticles and their applications in drug release control

Preparation and characterization of porous hollow silica nanoparticles (PHSN) for controlled release applications were investigated. Through orthogonally designed experiments, the optimal synthesis conditions for the preparation of PHSN were obtained and the produced PHSN were characterized by BET, SEM, TEM and IR. Scanning and transmission electron microscopy images revealed their hollow shell–core structure and also demonstrated that the size and shape of PHSN are determined by the templating CaCO 3 nanoparticles. The produced PHSN were applied as a carrier to study the controlled release behaviors of Brilliant Blue F (BB), which was used as a model drug. Being loaded into the inner core and on the surfaces of the nanoparticles, BB was released slowly into a bulk solution for about 1140 min as compared to only 10 min for the normal SiO 2 nanoparticles, thus exhibited a typical sustained release pattern without any burst effect. In addition, higher BET of the carriers, lower pH value and lower temperature prolonged BB release from PHSN, while stirring speed showed little influence on the release behavior. It showed that PHSN have a promising future in controlled drug delivery applications.

Preparation of novel double liposomes using the glass-filter method

The glass-filter method, a newly developed preparative method for liposomes, was applied for preparation of novel double liposomes. Double liposomes were prepared by filtering a suspension of liposomes prepared using a G4 filter (pore size: 10–16 μm) into a G3 filter (pore size: 40–100 μm) coated with a similar lipid layer. The morphological structure of the double liposomes was confirmed using scanning electron microscopy by the freeze-fracture method to be multivesicular vesicles consisting of small liposomes enveloped in larger liposomes. The diameter of liposomes prepared using the G4 filter was 0.8–2 μm and that of liposomes prepared using the G3 filter or double liposomes was 5–10 μm. These results suggested that the particle size of liposomes is dependent on the pore size of the glass-filter. The encapsulation efficiencies of double liposomes for brilliant blue FCF (BB) and erythrosine (ER) were higher than those of liposomes prepared by the standard Bangham method. Double liposomes showed suppressed release of BB or ER compared with normal liposomes. In particular, no release of BB was observed from the double liposomes prepared with stearylamine. These findings implied that the outer lipid layer protects the inner liposomes. The glass-filter method is the only method that we can get the double liposomes in a short period, and double liposomes prepared by this novel method had adequate size and good stability in vitro.

The release behavior of brilliant blue from calcium–alginate gel beads coated by chitosan: the preparation method effect

The aim of this study is to reveal how the release behavior of a model drug (brilliant blue, BB) from chitosan coating calcium–alginate gel beads (CCAGB) was influenced by the preparation methods. The CCAGB were prepared by dropping alginate solution into CaCl 2/chitosan solution (method 1(a)), or into chitosan solution then gelled by CaCl 2 (method 1(b)), or into CaCl 2 solution then coated by chitosan (method 2). Scanning electron microscopy was used for morphology observation, and elemental analysis was applied to determine the chitosan content bound on calcium–alginate gel beads (CAGB). Compared to CAGB, the dried CCAGB had poorer shape and rougher surface morphology especially in methods 1(a) and (b); moreover, CCAGB was found to be more instable in 0.9% NaCl and serious burst of beads occurred when high concentration of alginate (3.0 and 5.0% w/v) was used. The influence on BB release from the beads by chitosan coating was not only related to the chitosan density on bead surface, but also preparation method and other factors. Under un-dried bead state in method 1(a), the increase of chitosan content prolonged BB release in 0.9% (w/v) NaCl; while in method 2, the increase of chitosan concentration over 0.1% (w/v) (3.0% (w/v) alginate concentration was used) resulted in more serious burst of beads and hence facilitated BB release. Furthermore, in both methods 1(a) and 2, the increase of alginate from 1.5 to 3.0 or 5.0% (w/v) usually resulted in the significant burst of beads and accelerated BB release when 0.3 or 0.5% (w/v) chitosan was used for coating. Drying process greatly influenced BB release profile due to the destroying of alginate–chitosan film. The acceleration of BB release from CCAGB by drying process was more significant in the case of method 1 than of method 2.

Thermo-responsive release from interpenetrating porous silica–poly( N-isopropylacrylamide) hybrid gels

Novel thermo-responsive inorganic–organic hybrid gels were prepared by hybridizing porous silica and poly( N-isopropylacrylamide) gels (PNIPAAm gel). The internal pores of the silica were filled with PNIPAAm gel to give a thermo-responsive drug reservoir. Brilliant blue FCF (BB) was also added to the hybrid gels for release. The BB release rate was faster above the lower critical solution temperature (LCST) of the PNIPAAm gel than below the LCST. When the temperature changed across the LCST, reversible and thermo-responsive release behavior was observed. The transition of the release behavior upon changing the temperature was similar to the behavior of the PNIPAAm gel itself. The BB release rate can be controlled simply by changing the amount of PNIPAAm gel loaded into the silica.

Nicotine reorganizes cytoskeleton of vascular endothelial cell through platelet-derived growth factor BB.

Cigarette smoking has been directly linked to atherosclerosis formation and vascular graft failures but the role of nicotine in these processes is not yet completely understood. We investigated the release of platelet-derived growth factor BB (PDGF BB) by the bovine aortic endothelial cell (EC) after nicotine administration at concentrations similar to those found in plasma of active and passive smokers and the role of PDGF BB, autocrinally released, in EC cytoskeletal modification. EC were stimulated in a serum-free medium for 72 h with (-)-nicotine (from 6 x 10(-4) to 6 x 10(-8) M). The release of PDGF BB was assessed by inhibition antibody-binding assay and confirmed by Western blotting. Mitogenic activity of nicotine on EC was also determined. The EC cytoskeleton was studied with specific antibodies anti-alpha-actin fibers and anti-vimentin and the modification induced by PDGF BB was assessed by blocking PDGF BB activity with specific antibodies. The greatest PDGF BB release was noted at a (-)-nicotine concentration of 6 x 10(-6) M (P < 0.001). The addition of antibody anti-PDGF BB to EC exposed to (-)-nicotine decreased tritiated thymidine uptake by 20% (P < 0.001). EC exposed to (-)-nicotine concentrations of 6 x 10(-6) and 6 x 10(-8) M had a significant alteration in the expression of alpha-actin fibers and vimentin as compared with control. Administration of the antibody anti-PDGF BB in the culture medium reversed cytoskeletal alteration. Nicotine enhanced the release of PDGF BB by EC which in turn caused an alteration in cytoskeletal organization.

Growth Factor Expression of Human Arterial Smooth Muscle Cells and Endothelial Cells in a Transfilter Coculture System

By releasing growth factors, vascular cells can modulate proliferation and migration of neighboring cells in the arterial wall. Previous histological studies in transfilter cocultures, a culture model aimed to simulate vessel wall architecture, indicated that human arterial endothelial cells (haEC) can influence human arterial smooth muscle cell (haSMC) growth significantly. The aim of this study was to investigate the expression and secretion of various growth factors in order to better define the functional interactions between haEC and haSMC. Protein levels of platelet-derived-growth factor-AB (PDGF-AB), transforming-growth factor-β1 (TGF-β1), and tumor-necrosis factor-α(TNF-α) in mono- and cocultures were determined by ELISA 6, 12, 24, 48, 72 h after serum reduction. Highest PDGF-AB levels were found in monocultures with proliferative haEC, showing a peak after 24 h. In cocultures of haEC and haSMC, PDGF-AB levels were significantly lower. In contrast, neither proliferative, nor confluent haSMC released PDGF-AB significantly. Highest TGF-β1 concentrations were detected in cocultures, followed by monocultures of haSMC and monocultures of haEC. In all cultures, TGF-β1 levels increased in parallel with cultivation time and cell numbers, showing a maximum after 72 h. TNF-αcould not be detected in any culture. Northern blots demonstrated a strong expression of PDGF-B chain-mRNA in haEC, but not in haSMC. PDGF-A chain and TGF-β1-mRNA were expressed by haSMC and haEC. Addition of PDGF-AB to haSMC resulted in a potent growth stimulation, whereas TGF-β1 and TNF-αexerted only moderate, divergent effects on haSMC. Histological observations in transfilter cocultures demonstrated that proliferative haEC induce the formation of fibromuscular plaques. These results suggest that proliferative haEC act as potent growth stimulators for haSMC, predominantly by PDGF-AB or -BB release.

Thermo-responsive behavior of a methacryloyl-DL-alanine methyl ester polymer gel prepared by radiation-induced polymerization

Loosely cross-linked poly(methacryloyl- DL-alanine methyl ester, MA- DL-AlaOme) gels, which were prepared by radiation-induced polymerization, exhibited a reversible low-temperature swelling and high-temperature deswelling when cycled in water at different temperatures at 24-h intervals, in the range of 0 and 40°C. The thermo-response strongly depended upon irradiation condition. Increasing the irradiation dose resulted in a formation of lower molecular weight polymer owing to the scission of polymer chain, in which it gave a high swelling ability at low temperature, in contrast to a sluggish shrinkage at high temperature. On the other hand, no irradiation temperature affects the thermo-response of the gel. An important characteristic of the MA- DL-AlaOMe polymer gel is the formation of the membrane barrier at the surface with the rise in temperature, but it disappears in reswelling, to be termed “surface-controlling on-off switch system”. Brilliant blue FCF (BB) as a model compound was corporated into the gel to evaluate the capability as a thermo-responsive carrier for application in drug delivery systems, and it was found that the reversibly surface-controlling on-off switch function responsible to temperature changes plays an important role in a pulsatile release of BB of BB from the gel in vivo.

Influence of erosion of calcium-induced alginate gel matrix on the release of Brilliant Blue

The relationship between the release of a dye (Brilliant Blue; BB) incorporated in calcium-induced alginate gels and the erosion of the gel itself was investigated. The release rate of BB from gel formed from low-molecular-weight alginate was faster than that from high-molecular-weight alginate, and the erosion of the gel was also faster in the former case. As the alginate concentration in the gel was increased, the release of both BB and alginate from the gel was controlled. No remarkable differences in the release of BB were found among two kinds of gel formed from alginates with different monomeric composition. Furthermore, some gels formed from high-molecular-weight alginate also decayed in aqueous media containing sufficient levels of univalent cations. It was confirmed that the erosion of gels accelerates the rate of BB release from them.

The C3/C5 convertase of the alternative pathway of complement: Stabilization and restriction of control by lanthanide ions

The alternative pathway C3 convertase (C3b,Bb) is a Mg-dependent, labile enzyme with a t/2 of 3 min at 37°C and of 14 min at 24°C (at half physiological ionic strength). To stabilize the enzyme. metal ions of the lanthanide series were tested. Formation and decay of the enzyme as well as binding of radiolabeled Factor B, Factor H or properdin to C3b were measured using C3b-bearing sheep erythrocytes (EC3b). Binding of Factor B to EC3b in presence of 40 μ M gadolinium (Gd) was two to three times greater than in presence of 1 m M Mg. Binding of Factor H and of properdin to EC3b was partially inhibited by Gd. Although it enhanced Factor B uptake by EC3b, Gd was unable to substitute for Mg in enzyme formation by Factor D and completely inhibited (at 10 μ M) Mg-dependent enzyme activation. However, the preformed enzyme was not inhibited by Gd. Instead, exposure of EC3b,Bb to 40–100 μ M Gd increased the t/2 at 37°C from 3 min to 12–28 min, and at 24°C from 14 min to 32min. The slow decay of the enzyme correlated with slow release of Bb. Similar enzyme stabilization was observed using terbium, ytterbium, dysprosium and lanthanum. The Gd stabilized enzyme was also less susceptible to control by Factor H and properdin than the unstabilized enzyme. Furthermore, Gd protected surface bound-C3b from being cleaved by Factor I. The Gd effects were instantaneously reversed upon addition of 10 m M EDTA. Thus, Gd is able to stabilize preformed C3b,Bb and to render the enzyme refractory to control by Factors H and I.