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1.
Head Face Med ; 15(1): 27, 2019 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-31711509

RESUMO

BACKGROUND: Controlled release of proteins bound to conventional bone substitutes is still insufficient. Therefore, this study evaluates in-vitro release kinetics of the model protein FITC-BSA (fluorescein conjugated bovine serum albumine) from insoluble bovine collagenous bone matrices (ICBM) with different polymer coatings. Analyzes aim at comparing FITC-BSA release from uncoated versus coated ICBM over time to find bone substitute coatings with consistent release profiles. METHODS: Release kinetics of FITC-BSA from uncoated as well as coated ICBM with five different polymers (RESOMER R 203 H, RG 503 H, RG 504 H, RG 505, L 206 S) were measured over a period of 11 days (d). Measurements were conducted after 6 h (h), 12 h, 24 h, 3 d, 5 d, 7 d, 9 d and 11 d with six samples for each coated ICBM. Two groups were formed (1) with and (2) without medium change at times of measurement. For each group ANOVA with post-hoc Bonferroni testing was used. Scanning electron microscopy assessed morphologic differences between ICBM coating. RESULTS: In group 1 approx. 70% of FITC-BSA release from uncoated ICBM occurred after 6 h compared to approx. 50% in group 2. Only polymers with medium inherent viscosity, i.e. RESOMER RG 503 H, constantly showed significantly more FITC-BSA release throughout 11 d than uncoated ICBM (p = 0.007). The same was found for group 2 (p = 0.005). No significant differences between PLA and PLGA polymers were found. Scanning electron microscopy results indicate a weak adhesion of polymer coatings to ICBM explaining its rather weak retentive effect on overall FITC-BSA release. CONCLUSIONS: Medium molecular size polymers reduce the overall released FITC-BSA from ICBM over time. In clinical practice these polymers may prove ideal for bone substitute materials.


Assuntos
Substitutos Ósseos , Fluoresceína-5-Isotiocianato/análogos & derivados , Polímeros , Soroalbumina Bovina , Animais , Substitutos Ósseos/farmacocinética , Bovinos , Fluoresceína-5-Isotiocianato/farmacocinética , Cinética , Microscopia Eletrônica de Varredura , Soroalbumina Bovina/farmacocinética
2.
Invest Ophthalmol Vis Sci ; 60(10): 3547-3555, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31415078

RESUMO

Purpose: Current treatments for diabetic retinopathy (DR) have considerable limitations, underpinning the need for new therapeutic options. In this article, the ability of an engineered angiopoietin-1 variant (COMP-Ang1) to ameliorate the injurious effects of hyperglycemia on barrier integrity in a human retinal microvascular endothelial cell (HRMvEC) model is comprehensively investigated. Methods: Confluent HRMvECs were treated (0-72 hours) with d-glucose (5 or 30 mM) in the absence and presence of COMP-Ang1 (10-200 ng/mL). l-glucose (30 mM) was used as osmotic control. Posttreatment, intact cell monolayers were monitored for permeability to FITC-dextran 40 kDa. Cells were also harvested for analysis of interendothelial junction targets by RT-qPCR and Western blotting. The impact of receptor tyrosine kinase Tie2 gene silencing on COMP-Ang1 efficacy was also evaluated. Results: Treatment with 30 mM d-glucose (but not l-glucose) demonstrated a time-dependent elevation in the mean rate of FITC-dextran diffusion across intact HRMvEC monolayers, in parallel with significant reductions in mRNA/protein levels of occludin, claudin-5, ZO-1, and VE-Cadherin. These effects were all attenuated by COMP-Ang1 in a concentration-dependent fashion, with 200 ng/mL recovering barrier function by ∼88%, and recovering reduced interendothelial junction protein levels by more than 50%. Finally, Tie2 knockdown by small interfering RNA silencing blocked the ability of COMP-Ang1 to mitigate against hyperglycemia-induced permeabilization of HRMvECs and depletion of junctional expression levels. Conclusions: In summary, this article presents a reproducible in vitro cell study that quantifies the concentration-dependent efficacy of COMP-Ang1 to mitigate the injurious effects of hyperglycemic challenge on HRMvEC barrier properties via Tie2-mediated signaling.


Assuntos
Barreira Hematorretiniana/fisiologia , Células Endoteliais/efeitos dos fármacos , Hiperglicemia/prevenção & controle , Proteínas Recombinantes de Fusão/farmacologia , Vasos Retinianos/efeitos dos fármacos , Antígenos CD/genética , Western Blotting , Caderinas/genética , Permeabilidade Capilar/efeitos dos fármacos , Células Cultivadas , Claudina-5/genética , Dextranos/metabolismo , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Inativação Gênica/fisiologia , Glucose/farmacologia , Humanos , Hiperglicemia/metabolismo , Ocludina/genética , Fenótipo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor TIE-2/genética , Vasos Retinianos/metabolismo
3.
Int J Pharm ; 568: 118550, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31336152

RESUMO

Implants offer the opportunity to improve patient adherence and real-world outcomes. However, most polymers used today are hydrophobic and limit drug properties suitable for development. Thermoplastic poly(urethanes) (TPUs) form pores upon hydration and may facilitate the development of implants containing drugs exhibiting broadly different properties. We sought to investigate the effect of drug physicochemical properties on permeability through membranes of varying TPU mixture composition; leverage imaging to visualize microstructural changes to the membrane across the TPU mixture composition range; and quantitatively characterize the membrane microstructure using equivalent pore analysis. We observed a correlation between drug hydrophobicity and its permeability through hydrophobic-rich TPU membranes. Conversely, all compounds diffused through hydrophilic-rich TPU membranes at similar rates, regardless of drug properties. Imaging revealed significant microstructure differences between hydrophobic-rich and hydrophilic-rich TPU membranes, supporting hypotheses proposed in our previous study. The hydrated hydrophilic TPU membrane pore area was determined to be 0.583% and its equivalent pore radius was found to be 128 nm, suggesting that hydrophilic TPU membranes may be used to modify the release of small molecular weight drugs and macromolecules. These findings highlight the benefits of hydrophilic TPUs as rate-controlling membranes to modulate the release rate of drugs with varying physicochemical properties.


Assuntos
Membranas Artificiais , Poliuretanos/química , Dextranos/química , Difusão , Implantes de Medicamento , Emtricitabina/química , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Interações Hidrofóbicas e Hidrofílicas , Ibuprofeno/química , Metoprolol/química , Peso Molecular , Permeabilidade , Porosidade
4.
Nihon Yakurigaku Zasshi ; 153(6): 254-260, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31178529

RESUMO

Various physiological and pathological processes are accompanied with the local acidification of extracellular local pH. However, imaging tools to investigate the spatio-temporal dynamics as well as the functional significance of cell surface pH are limitedly available. We established a novel method of in vitro cell surface pH imaging by using a membrane-anchored pH probe, poly(ethylene glycol)-phospholipid conjugated with fluorescein isothiocyanate (FITC-PEG-lipid). PEG-lipid, amphiphilic synthetic polymer, is a biomaterial originally synthesized for cell-surface engineering for transplantation therapy. When added into the cell culture medium, FITC-PEG-lipid was spontaneously inserted into the plasma membrane via its phospholipid moiety. FITC-PEG-lipid was retained at the extracellular surface due to the hydrophobic PEG moiety. The ratiometric readout of FITC fluorescence was unique to the extracellular pH in the range of weakly alkaline and acidic pH (pH 5.0-7.5). The pH measurement with FITC-PEG-lipid was accurate enough to distinguish the difference of 0.1 pH unit for the external solutions at pH 5.9, 6.0 and 6.1, near the inflection point of fluorescence ratio. The response of FITC-PEG-lipid to the extracellular pH was reversible. Continuous alteration of extracellular pH was successfully visualized by time-lapse imaging analysis. Our study demonstrated that FITC-PEG-lipid is useful as a sensitive and reversible cell surface-anchored pH probe. The simple labeling procedure of FITC-PEG-lipid is advantageous especially when considering its application to high-throughput in vitro assay. Furthermore, PEG-lipid holds a great potential as the membrane anchor of various analytical probes to approach the juxtamembrane environments.


Assuntos
Membrana Celular/química , Concentração de Íons de Hidrogênio , Imagem Óptica , Fosfolipídeos , Polietilenoglicóis , Animais , Fluoresceína-5-Isotiocianato/análogos & derivados , Soroalbumina Bovina
5.
Eur J Pharm Sci ; 137: 104976, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31254642

RESUMO

This research aimed to manufacture and evaluate in vitro 3D printed microneedles for transdermal drug delivery. Firstly, microneedle arrays were fabricated using a polymer-based material. Subsequently, these arrays were tested for their mechanical strength applying axial load along their length, while prediction of the buckling load was performed using widely known arithmetic models. Additionally, the force required to pierce human skin was calculated in order to verify that microneedles insert human skin without buckling or fracturing. Finite Element Analysis (FEA) was used to simulate the insertion process and complement the experimental findings. Furthermore, permeation studies were carried out in order to compare diffusion of two model dyes with different molecular weight namely; FITC-Dextran (M.W.:4000 Da) and calcein (M.W.:622.54 Da) across full thickness human skin in vitro before and after skin treatment with microneedles. Finally, visualization studies enabled illustration of microneedle perforation sites. The results showed that the manufactured 3D printed microneedle arrays penetrate sufficiently human skin and can significantly enhance the transport of the dyes across human skin.


Assuntos
Corantes/administração & dosagem , Dextranos/administração & dosagem , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceínas/administração & dosagem , Agulhas , Pele/metabolismo , Administração Cutânea , Feminino , Análise de Elementos Finitos , Fluoresceína-5-Isotiocianato/administração & dosagem , Humanos , Microinjeções , Pessoa de Meia-Idade , Impressão Tridimensional , Absorção Cutânea , Tecnologia Farmacêutica
6.
Int J Pharm ; 567: 118458, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31247277

RESUMO

Successful treatment of age-related macular diseases requires an effective controlled drug release system with less invasive route of administration in the eye to reduce the burden of frequent intravitreal injections for patients. In this study, we developed an episcleral implantable device for sustained release of ranibizumab, and evaluated its efficacy on suppression of laser-induced choroidal neovascularization (CNV) in rats. We tested both biodegradable and non-biodegradable sheet-type devices consisting of crosslinked gelatin/chitosan (Gel/CS) and photopolymerized poly(ethyleneglycol) dimethacrylate that incorporated collagen microparticles (PEGDM/COL). In vitro release studies of FITC-labeled albumin showed a constant release from PEGDM/COL sheets compared to Gel/CS sheets. The Gel/CS sheets gradually biodegraded in the sclera during the 24-week implantation; however, the PEGDM/COL sheets did not degrade. FITC-albumin was detected in the retina during 18 weeks implantation in the PEGDM/COL sheet-treated group, and was detected in the Gel/CS sheet-treated group during 6 weeks implantation. CNV was suppressed 18 weeks after application of ranibizumab-loaded PEGDM/COL sheets compared to a placebo PEGDM/COL sheet-treated group, and to the intravitreal ranibizumab-injected group. In conclusion, the PEGDM/COL sheet device suppressed CNV via a transscleral administration route for 18 weeks, indicating that prolonged sustained ranibizumab release could reduce the burden of repeated intravitreal injections.


Assuntos
Inibidores da Angiogênese/administração & dosagem , Neovascularização de Coroide/tratamento farmacológico , Implantes de Medicamento/administração & dosagem , Ranibizumab/administração & dosagem , Inibidores da Angiogênese/química , Animais , Quitosana/administração & dosagem , Quitosana/química , Colágeno/administração & dosagem , Colágeno/química , Implantes de Medicamento/química , Liberação Controlada de Fármacos , Olho/efeitos dos fármacos , Olho/metabolismo , Olho/patologia , Fluoresceína-5-Isotiocianato/administração & dosagem , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Gelatina/administração & dosagem , Gelatina/química , Lasers , Masculino , Metacrilatos/administração & dosagem , Metacrilatos/química , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Ranibizumab/química , Ratos Sprague-Dawley , Albumina Sérica/administração & dosagem , Albumina Sérica/química
7.
Methods Mol Biol ; 1965: 261-279, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31069681

RESUMO

Histiotrophic nutrition is a process whereby the rodent visceral yolk sac (VYS) internalizes exogenous macromolecules, degrades them, and sends the degradation products to the embryo. Quantification and visualization of histiotrophic nutrition can be accomplished using fluorescent tracer molecules such as fluorescein isothiocyanate-conjugated albumin (FITC-albumin). The methods are simple and can provide complimentary functional and structural information in studies of the effects of embryotoxicants on visceral yolk sac function.


Assuntos
Embrião de Mamíferos/citologia , Fluoresceína-5-Isotiocianato/análogos & derivados , Corantes Fluorescentes/metabolismo , Albumina Sérica/metabolismo , Saco Vitelino/metabolismo , Animais , Técnicas de Cultura Embrionária , Embrião de Mamíferos/metabolismo , Endocitose , Fluoresceína-5-Isotiocianato/metabolismo , Microscopia de Fluorescência , Proteólise , Ratos
8.
Carbohydr Polym ; 217: 160-167, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31079673

RESUMO

Polysaccharides can be modified by reactive functional groups to enable chemical crosslinking. We studied how different methods of crosslinking methacrylate-functionalized chitosan affected the network structures and various properties relevant for utilization of the chemically crosslinked hydrogels in biomedical applications, including tissue engineering and delivery of therapeutic agents. Four chitosan hydrogels were made by either the free radical polymerization with varying initiation kinetics and an addition of chain transfer agents or the based-catalyzed Michael-type addition reaction. Four chitosan hydrogels having identical polymer fractions at equilibrium swelling exhibited marked differences in shear moduli, dextran diffusion rate, and especially enzymatic degradation behaviors. Hydrogels made by the free radical polymerization with no chain transfer agent were highly resistant to complete degradation by enzyme for an extended period. We inferred that such resistance originated from chain bundles characterized by densely branched networks of chitosan chains, which was determined by small-angle X-ray scattering analysis.


Assuntos
Quitosana/química , Hidrogéis/química , Quitosana/síntese química , Reagentes para Ligações Cruzadas/síntese química , Reagentes para Ligações Cruzadas/química , Dextranos/química , Difusão , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Humanos , Hidrogéis/síntese química , Hidrólise , Metacrilatos/síntese química , Metacrilatos/química , Muramidase/química , Polimerização
9.
Int J Pharm ; 564: 48-58, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-30999045

RESUMO

Fractional CO2 laser treatment has been used in some clinical trials to promote topical drug delivery. Currently, there is no standard for laser settings to achieve a feasible therapy. The cutaneous recovery following laser treatment and its influence on drug absorption have not been well explored. This study evaluated the kinetics of laser-treated skin-barrier restoration and drug permeation in nude mice. The skin recovery and observation of the process were characterized by transdermal water loss (TEWL), erythema measurement, gross appearance, optical microscopy, and scanning electron microscopy (SEM). The skin absorption of a lipophilic small permeant (tretinoin), a hydrophilic small permeant (acyclovir), and a large molecule (fluorescein isothiocyanate dextran 4 kDa, FD4) was examined in vitro using Franz cell. TEWL suggested that the laser-treated skin restored its barrier function at 16 h after irradiation. The fractional laser produced microchannels of about 150 µm in diameter and 25 µm in depth that were surrounded with thermal coagulation. The bright-field imaging indicated that the micropores were progressively closed during the recovery period but had not completely closed even after a 16-h recovery. The laser treatment led to a rapid tretinoin penetration across the skin immediately after irradiation, with a 5-fold enhancement compared to intact skin. This enhancement was gradually reduced following the increase of recovery time. Conversely, the acyclovir and FD4 permeation peaked at 1-2 h post-irradiation. The FD4 flux was even elevated as the recovery time increased. The reasons for this could have been the subsequent inflammation after laser exposure and the deficient tight junction (TJ) barrier. The confocal imaging demonstrated the perpendicular diffusion of rhodamine B and FD4 through microchannels immediately after laser exposure. The lateral diffusion from the microchannels was observed at 2 h post-irradiation. Our results revealed a time-dependent recovery of skin permeation. The time frame for applying the drugs after laser irradiation was dependent upon the permeants and their various physicochemical properties.


Assuntos
Sistemas de Liberação de Medicamentos , Lasers , Absorção Cutânea/efeitos da radiação , Aciclovir/administração & dosagem , Aciclovir/farmacocinética , Administração Cutânea , Animais , Dextranos/administração & dosagem , Dextranos/farmacocinética , Feminino , Fluoresceína-5-Isotiocianato/administração & dosagem , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/farmacocinética , Camundongos Nus , Rodaminas/administração & dosagem , Rodaminas/farmacocinética , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/efeitos da radiação , Pele/ultraestrutura , Absorção Cutânea/efeitos dos fármacos , Proteínas de Junções Íntimas/metabolismo , Tretinoína/administração & dosagem , Tretinoína/farmacocinética
10.
J Colloid Interface Sci ; 548: 151-159, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991181

RESUMO

Light-responsive nanocarriers are applicable as non-invasive, highly tunable and precisely controlled drug delivery systems. Here, we report a new nanocarrier system, achieved by doping D1, a type of green light-responsive donor acceptor Stenhouse adduct (DASA), into a lipid-based lyotropic liquid crystalline system. Time-resolved small angle X-ray scattering was used to confirm that the matrix underwent a rapid and fully reversible phase transition from lamellar to inverse cubic phase upon irradiation with green light (532 nm), reverting back on removal of light. Fluorescein isothiocyanate-dextran (FD4) was used as a model hydrophilic cargo. The release of cargo upon varying irradiation parameters was investigated in vitro which showed that irradiation can trigger a burst release of FD4 upon phase transition. This additive shows promise for the development of new visible light-activated, "on demand" drug delivery systems.


Assuntos
Portadores de Fármacos/química , Corantes Fluorescentes/química , Cristais Líquidos/química , Nanopartículas/química , Dextranos/química , Liberação Controlada de Fármacos , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Interações Hidrofóbicas e Hidrofílicas , Isomerismo , Ácidos Láuricos/química , Luz , Lipídeos/química , Imagem Óptica/métodos , Tamanho da Partícula , Transição de Fase , Processos Fotoquímicos , Propriedades de Superfície
11.
Lab Chip ; 19(10): 1747-1754, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-30964485

RESUMO

The successful intracellular delivery of exogenous macromolecules is crucial for a variety of applications ranging from basic biology to the clinic. However, traditional intracellular delivery methods such as those relying on viral/non-viral nanocarriers or physical membrane disruptions suffer from low throughput, toxicity, and inconsistent delivery performance and are time-consuming and/or labor-intensive. In this study, we developed a single-step hydrodynamic cell deformation-induced intracellular delivery platform named "hydroporator" without the aid of vectors or a complicated/costly external apparatus. By utilizing only fluid inertia, the platform focuses, guides, and stretches cells robustly without clogging. This rapid hydrodynamic cell deformation leads to both convective and diffusive delivery of external (macro)molecules into the cell through transient plasma membrane discontinuities. Using this hydroporation approach, highly efficient (∼90%), high-throughput (>1 600 000 cells per min), and rapid delivery (∼1 min) of different (macro)molecules into a wide range of cell types was achieved while maintaining high cell viability. Taking advantage of the ability of this platform to rapidly deliver large molecules, we also systematically investigated the temporal biostability of vanilla DNA origami nanostructures in living cells for the first time. Experiments using two DNA origami (tube- and donut-shaped) nanostructures revealed that these nanostructures can maintain their structural integrity in living cells for approximately 1 h after delivery, providing new opportunities for the rapid characterization of intracellular DNA biostability.


Assuntos
Membrana Celular/química , DNA/administração & dosagem , DNA/química , Sistemas de Liberação de Medicamentos/instrumentação , Sistemas de Liberação de Medicamentos/métodos , Hidrodinâmica , Nanoestruturas/administração & dosagem , Dextranos/química , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Humanos , Células K562 , Tamanho da Partícula , Propriedades de Superfície
12.
Eur J Pharm Biopharm ; 139: 232-239, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30954658

RESUMO

Fast in situ forming, chemically crosslinked hydrogels were prepared by the amidation reaction between N-succinimidyl ester end groups of multi-armed poly(ethylene glycol) (PEG) and amino surface groups of poly(amido amine) (PAMAM) dendrimer generation 2.0. To control the properties of the PEG/PAMAM hydrogels, PEGs were used with different arm numbers (4 or 8) as well as different linkers (amide or ester) between the PEG arms and their terminal N-succinimidyl ester groups. Oscillatory rheology measurements showed that the hydrogels form within seconds after mixing the PEG and PAMAM precursor solutions. The storage moduli increased with crosslink density and reached values up to 2.3 kPa for hydrogels based on 4-armed PEG. Gravimetrical degradation experiments demonstrated that hydrogels with ester linkages between PEG and PAMAM degrade within 2 days, whereas amide-linked hydrogels were stable for several months. The release of two different model drugs (fluorescein isothiocyanate-dextran with molecular weights of 4·103 and 2·106 g/mol, FITC-DEX4K and FITC-DEX2000K, respectively) from amide-linked hydrogels was characterized by an initial burst followed by diffusion-controlled release, of which the rate depended on the size of the drug. In contrast, the release of FITC-DEX2000K from ester-containing hydrogels was governed mainly by degradation of the hydrogels and could be modulated via the ratio between ester and amide linkages. In vitro cytotoxicity experiments indicated that the PEG/PAMAM hydrogels are non-toxic to mouse fibroblasts. These in situ forming PEG/PAMAM hydrogels can be tuned with a broad range of mechanical, degradation and release properties and therefore hold promise as a platform for the delivery of therapeutic agents.


Assuntos
Dendrímeros/química , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Hidrogéis/química , Polietilenoglicóis/química , Animais , Linhagem Celular , Dendrímeros/toxicidade , Dextranos/administração & dosagem , Dextranos/farmacocinética , Portadores de Fármacos/toxicidade , Composição de Medicamentos/métodos , Estabilidade de Medicamentos , Fibroblastos , Fluoresceína-5-Isotiocianato/administração & dosagem , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/farmacocinética , Hidrogéis/toxicidade , Camundongos , Polietilenoglicóis/toxicidade , Reologia , Fatores de Tempo , Testes de Toxicidade
13.
Int J Pharm ; 562: 218-227, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30902707

RESUMO

PURPOSE: To develop a three-dimensional visualization method for evaluating the distribution of pulmonary drug delivery systems and compare four tissue-clearing techniques (ClearT2, CUBIC, ScaleS, and SeeDB2) using intrapulmonary liposomes as drug carriers. METHODS: Rhodamine B-labeled liposomes were administered intrapulmonarily to mice using a MicroSprayer, and then fluorescent-labeled tomato lectin was administered intravenously to visualize the general lung structure. Tissue-clearing treatment of the mouse lungs was performed using the standard protocols of the ClearT2, CUBIC, ScaleS, and SeeDB2 techniques. Lung clearing was clarified using laser-scanning confocal microscopy, and three-dimensional images were reconstructed. RESULTS: Fluorescent-labeled tomato lectin was preserved using ClearT2 and SeeDB2 but not using CUBIC and ScaleS. In addition, the liposomes were stable in ClearT2 reagent, but they were mostly degraded in other reagents by surface-active agents. ClearT2 treatment enabled the three-dimensional visualization of intrapulmonary rhodamine B-labeled liposomes at the alveolar scale. CONCLUSIONS: These results suggest that the ClearT2 tissue-clearing technique was appropriate for the three-dimensional visualization of intrapulmonary liposomes at the alveolar scale. This study provides important information for selecting and optimizing suitable optical tissue-clearing techniques in lungs for evaluating the distribution of pulmonary drug delivery systems.


Assuntos
Imagem Tridimensional/métodos , Lipossomos/administração & dosagem , Alvéolos Pulmonares/metabolismo , Animais , Dextranos/administração & dosagem , Fluoresceína-5-Isotiocianato/administração & dosagem , Fluoresceína-5-Isotiocianato/análogos & derivados , Corantes Fluorescentes/administração & dosagem , Masculino , Camundongos Endogâmicos ICR , Lectinas de Plantas/administração & dosagem , Rodaminas/administração & dosagem , Distribuição Tecidual , Xantenos/administração & dosagem
14.
Bull Exp Biol Med ; 166(3): 369-372, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30627900

RESUMO

Natural response to hypoxia critically depends on rapid stabilization of hypoxia-inducible factor (HIF). Under normoxic conditions, HIF-prolyl hydroxylases mark α-subunits of HIF for degradation, while hypoxia results in stabilization of HIF-α. Oxyquinoline derivatives suppress activity of HIF-prolyl hydroxylases leading to HIF activation in the cell. Here we show that 24-h incubation of BeWo b30 choriocarcinoma cells (a model of trophoblast in the placental barrier) with oxyquinoline derivative leads to a decrease in transepithelial electrical resistance (TEER) of the cell monolayer, while the permeability of the monolayer for FITC-dextran (70 kDa) remains unchanged. These findings suggest that the overall barrier function is preserved, while the structure of intercellular tight junctions can undergo minor changes. Using Affymetrix Human Transcriptome Array 2.0, we showed that the treatment with oxyquinoline derivative was followed by a decrease in the expression of claudins 6 and 7 (CLDN6, CLDN7), occludin (OCLN), contact adhesion molecule 3 (JAM3), and angiomotinlike protein 1 (AMOTL1).


Assuntos
Claudinas/genética , Ocludina/genética , Oxiquinolina/farmacologia , Junções Íntimas/efeitos dos fármacos , Trofoblastos/efeitos dos fármacos , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Hipóxia Celular/genética , Linhagem Celular Tumoral , Claudinas/metabolismo , Dextranos/metabolismo , Impedância Elétrica , Feminino , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Ocludina/metabolismo , Permeabilidade/efeitos dos fármacos , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Gravidez , Junções Íntimas/genética , Junções Íntimas/metabolismo , Trofoblastos/metabolismo , Trofoblastos/patologia
15.
Colloids Surf B Biointerfaces ; 175: 713-720, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30612047

RESUMO

Mechanical properties of nanoparticles are an important characteristic for drug delivery and therefore, they have gained interest in pharmaceutical research during the last years. Among others, cellular uptake, blood circulation time and accumulation in organs are influenced by the elastic modulus of nanoparticles. Thus, by varying the stiffness of nanoparticles a more specific drug targeting might be achieved. Gelatin nanoparticles (GNPs) show advantageous characteristics in respect to encapsulation and delivery of hydrophilic drugs such as antibodies or other biologicals. Furthermore, the GNPs as hydrogel-nanoparticles offer adjustable elastic behavior. In this study, a method for GNP sample preparation and the determination of the mechanical properties by nanoindentation experiments using atomic force microscopy (AFM) was developed. The obtained force-distance curves were evaluated and fitted with the Hertzian model in order to calculate the Young's modulus. GNPs were crosslinked with glutaraldehyde (GTA) for different incubation times to investigate a possible modification of the Young's modulus. In addition, this study addresses the influence of storage on the mechanical characteristics of GNPs. The results provide first insights about the elastic properties of GNPs and their development over time. In the tested range of crosslinking times no notable differences in the mechanical properties occurred. In turn, the influence of the storage on the mechanical particle properties was observed: particle stiffness raised over time. Furthermore, it could be observed that the cellular uptake in a model cell line (A549) was increased for harder particles.


Assuntos
Portadores de Fármacos/química , Endocitose/fisiologia , Gelatina/química , Hidrogéis/química , Nanopartículas/química , Células A549 , Reagentes para Ligações Cruzadas/química , Dextranos/química , Composição de Medicamentos/métodos , Módulo de Elasticidade , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Glutaral/química , Dureza , Humanos , Microscopia de Força Atômica , Nanopartículas/ultraestrutura , Imagem Óptica
16.
J Neurol Sci ; 396: 247-253, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30529802

RESUMO

Glioblastoma (GBM) is a typical malignant tumor, and there are no effective drugs capable of improving patient survival. Docosahexaenoic acid (DHA), a nutrient essential to animal health and neurodevelopment, exerts an anticancer effect in several types of cancer. However, the function of DHA in GBM is still unclear. Here, we showed that DHA could repress the migration and invasion of GBM U251 cells and promote their apoptosis in a dose- and time-dependent manner, indicating that DHA has an anticancer effect on GBM cells. Whole-transcriptome analysis indicated that DHA treatment mainly regulates the genes associated with receptor binding, oxidoreductase activity, organic acid transmembrane transporter activity, and carboxylic acid transmembrane transporter activity. Long non-coding RNAs (LncRNAs) involved in the regulation network of DHA were also identified, and their targets were assigned to the Gene Ontology (GO) categories. In silico analysis was conducted to predict the pathways related to the differentially expressed genes by DHA treatment. Our findings suggest that DHA acts as an antitumor agent in GBM, which may provide a suitable means of improving the efficacy of GBM treatment in the future.


Assuntos
Antineoplásicos/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Sequenciamento Completo do Exoma/métodos , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Biologia Computacional , Relação Dose-Resposta a Droga , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Glioblastoma/patologia , Humanos , Invasividade Neoplásica , RNA Mensageiro/metabolismo , Fatores de Tempo , Cicatrização/efeitos dos fármacos
17.
Nanotechnology ; 30(14): 145601, 2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30524021

RESUMO

The development of the tumor-targeting ability of nanocarriers is of paramount importance for gene delivery into tumor lesions as well as to avoid biotoxicity. Here we report the synthesis of the polyethyleneimine-fluorescein isothiocyanate-folic acid (PEI-FITC-FA) polymer, which could condense the tumor suppressor pp53 to form nanocomplexes. These targeted nanocomplexes exhibited favorable physical properties including a small size of <100 nm, exploiting the enhanced permeability and retention effect and tumor-targeting ability by binding to the overexpressed FA receptors on tumor cell surfaces. In addition, once the nanocomplexes are accumulating in the tumor tissue, the target functional ligand, FA, can selectively recognize the over-expressed FA receptor and subsequently remain on the tumor cell surface, which can significantly promote the tumor cell uptake because of the time- and concentration-dependent internalization caused by the enhanced interaction between nanocomplex and tumor cell. Our results indicated that PEI-FITC-FA/pp53 nanocomplexes could be efficiently delivered into tumor cells, and subsequently induce tumor cell apoptosis. Thus, the targeted cationic polymer PEI-FITC-FA could be used as an advanced nanocarrier for gene delivery.


Assuntos
Receptores de Folato com Âncoras de GPI/metabolismo , Ácido Fólico/metabolismo , Nanoconjugados/química , Neoplasias/metabolismo , Proteína Supressora de Tumor p53/genética , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/química , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Ácido Fólico/química , Terapia Genética , Vetores Genéticos/farmacologia , Células HeLa , Células Hep G2 , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Tamanho da Partícula , Plasmídeos/genética , Plasmídeos/metabolismo , Polietilenoimina/química , Proteína Supressora de Tumor p53/metabolismo
18.
Mol Pharm ; 16(1): 60-70, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30422668

RESUMO

Drugs and proteins with poor intestinal permeability have a limited oral bioavailability. To remediate this problem, a receptor-mediated endocytosis and transcytosis approach was explored. Indeed, the nontoxic ß subunit of cholera toxin (CTB) can cross the intestinal barrier by binding to receptor GM1. In this study, we explored the use of GM1-binding peptides and CTB as potential covalent carriers of poorly permeable molecules. GM1-binding peptides (G23, P3) and CTB were conjugated to poorly permeable fluorescent probes such as fluorescein isothiocyanate (FITC) and albumin-FITC using triethylene glycol spacers and click chemistry. The affinity of the peptide conjugates with receptor GM1 was confirmed by isothermal titration calorimetry or microscale thermophoresis, and the results suggested the involvement of nonspecific interactions. Conjugating the model drugs to G23 and P3 improved the internalization into Caco-2 and T84 cells, although the process was not dependent on the amount of GM1 receptor. However, conjugation of bovine serum albumin FITC to CTB increased the internalization in the same cells in a GM1-dependent pathway. Peptide conjugates demonstrated a limited permeability through a Caco-2 monolayer, whereas G23 and CTB conjugates slightly enhanced permeability through a T84 cell monolayer compared to model drugs alone. Since CTB can improve the permeability of large macromolecules such as albumin, it is an interesting carrier for the improvement of oral bioavailability of various other macromolecules such as heparins, proteins, and siRNAs.


Assuntos
Toxina da Cólera/metabolismo , Mucosa Intestinal/metabolismo , Peptídeos/metabolismo , Animais , Células CACO-2 , Linhagem Celular Tumoral , Citometria de Fluxo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Humanos , Ligação Proteica , Soroalbumina Bovina/química
19.
Pharm Dev Technol ; 24(5): 575-583, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30457420

RESUMO

The aim of this study was to investigate intravitreal injection of silk fibroin nanoparticles (SFNs) encapsulating bio-macromolecules, achieving enhanced drug bioavailability, and extended retention in retina. SFNs were prepared with regenerated silk fibroin using desolvation method with fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) as bio-macromolecular model drug encapsulated. In vitro physicochemical properties and in vitro drug release of FITC-BSA loaded SFNs (FITC-BSA-SFNs) were evaluated. Cytotoxicity, cellular uptake, and retention of FITC-BSA-SFNs were determined in human retinal pigment epithelial cell line (ARPE-19). In addition, in vivo distribution and safety of intravitreally administered FITC-BSA-SFNs were investigated in New Zealand white rabbits. The particle size of FITC-BSA-SFNs was 179.1 ± 3.7 nm with polydispersity index of 0.102 ± 0.033 and the zeta potential was greater than -25 mV. FITC-BSA-SFNs exhibited excellent biocompatibility with no cytotoxicity observed within 24 and 48 h in AREP-19 cells. Compared to FITC-BSA solution, FITC-BSA-SFNs showed enhanced cellular uptake and prolonged retention. Furthermore, FITC-BSA-SFNs achieved accumulated distribution and extended retention in retina in vivo following intravitreal injection compared to a single administration of free drug solution. Therefore, this bio-macromolecule delivery platform based on SFNs could have great potential in the treatment of posterior segment disorders.


Assuntos
Portadores de Fármacos/química , Fibroínas/química , Fluoresceína-5-Isotiocianato/análogos & derivados , Nanopartículas/química , Retina/metabolismo , Soroalbumina Bovina/administração & dosagem , Soroalbumina Bovina/farmacocinética , Animais , Bovinos , Linhagem Celular , Liberação Controlada de Fármacos , Feminino , Fluoresceína-5-Isotiocianato/administração & dosagem , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/farmacocinética , Humanos , Injeções Intravítreas , Coelhos , Retina/citologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Soroalbumina Bovina/química
20.
Int J Pharm ; 554: 420-428, 2019 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-29933061

RESUMO

UV and NIR-responsive monoolein cubic phase was prepared by including poly(ethyleneimine) (PEI)/cinnamic acid (CA) conjugate and gold nanoparticle (GNP) within its structure. UV irradiation elevated significantly the release % of Auramine O loaded in cubic phase containing PEI/CA, possibly because of the trans-to-cis isomerization of CA. NIR irradiation also increased significantly the release % of FITC-dextran loaded in cubic phase containing PEI/CA. The release % of the dye loaded in cubic phase containing PEI/CA and GNP was elevated more markedly by NIR irradiation, possibly due to the phase transition of cubic phase and the disassembling of PEI/CA assembly.


Assuntos
Cinamatos/química , Glicerídeos/química , Nanopartículas Metálicas , Polietilenoimina/química , Benzofenoneídio/administração & dosagem , Benzofenoneídio/química , Dextranos/administração & dosagem , Dextranos/química , Liberação Controlada de Fármacos , Fluoresceína-5-Isotiocianato/administração & dosagem , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/administração & dosagem , Ouro , Raios Infravermelhos , Raios Ultravioleta
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