Release Of Retinoic Acid Research Articles

Single metal-organic framework-embedded nanopit arrays: A new way to control neural stem cell differentiation.

Stable and continuous supply of essential biomolecules is critical to mimic in vivo microenvironments wherein spontaneous generation of various cell types occurs. Here, we report a new platform that enables highly efficient neuronal cell generation of neural stem cells using single metal-organic framework (MOF) nanoparticle–embedded nanopit arrays (SMENA). By optimizing the physical parameters of homogeneous periodic nanopatterns, each nanopit can confine single nMOFs (UiO-67) that are specifically designed for long-term storage and release of retinoic acid (RA). The SMENA platform successfully inhibited physical interaction with cells, which contributed to remarkable stability of the nMOF (RA⊂UiO-67) structure without inducing nanoparticle-mediated toxicity issues. Owing to the continuous and long-term supply of RA, the neural stem cells showed enhanced mRNA expressions of various neurogenesis-related activities. The developed SMENA platform can be applied to other stem cell sources and differentiation lineages and is therefore useful for various stem cell–based regenerative therapies.

Regulation of Myelination by Exosome Associated Retinoic Acid Release from NG2-Positive Cells.

In the CNS, oligodendrocytes are responsible for myelin formation and maintenance. Following spinal cord injury, oligodendrocyte loss and an inhibitory milieu compromise remyelination and recovery. Here, we explored the role of retinoic acid receptor-beta (RARβ) signaling in remyelination. Using a male Sprague Dawley rat model of PNS-CNS injury, we show that oral treatment with a novel drug like RARβ agonist, C286, induces neuronal expression of the proteoglycan decorin and promotes myelination and differentiation of oligodendrocyte precursor cells (NG2+ cells) in a decorin-mediated neuron–glia cross talk. Decorin promoted the activation of RARα in NG2+ cells by increasing the availability of the endogenous ligand RA. NG2+ cells synthesize RA, which is released in association with exosomes. We found that decorin prevents this secretion through regulation of the EGFR–calcium pathway. Using functional and pharmacological studies, we further show that RARα signaling is both required and sufficient for oligodendrocyte differentiation. These findings illustrate that RARβ and RARα are important regulators of oligodendrocyte differentiation, providing new targets for myelination.SIGNIFICANCE STATEMENT This study identifies novel therapeutic targets for remyelination after PNS-CNS injury. Pharmacological and knock-down experiments show that the retinoic acid (RA) signaling promotes differentiation of oligodendrocyte precursor cells (OPCs) and remyelination in a cross talk between neuronal RA receptor-beta (RARβ) and RARα in NG2+ cells. We show that stimulation of RARα is required for the differentiation of OPCs and we describe for the first time how oral treatment with a RARβ agonist (C286, currently being tested in a Phase 1 trial, ISRCTN12424734) leads to the endogenous synthesis of RA through retinaldehyde dehydrogenase 2 (Raldh2) in NG2 cells and controls exosome-associated-RA intracellular levels through a decorin–Ca2+ pathway. Although RARβ has been implicated in distinct aspects of CNS regeneration, this study identifies a novel function for both RARβ and RARα in remyelination.

Development of a novel pH-sensitive polymer matrix for drug encapsulation from maleated poly(vinyl alcohol) grafted with polyacrylamide

Hydrogels are widely used in medical, food and other applications due to their good water absorption without dissolution. The objective of this work was to synthesize a maleated poly(vinyl alcohol) (PVAM) grafted copolymer with anionic polyacrylamide (PAM) (PVAM-graft-PAM) using K2S2O8 as an initiator in an eco-friendly process. The chemical structure of the PVAM-graft-PAM was confirmed by FTIR. The influences of the PVAM/PAM ratio and the natural rubber-graft-cassava starch (NS) and PVAM/PAM ratio on the properties of the PVAM-graft-PAM were investigated. It was observed that the swelling ratio of the sample was enhanced with increasing PAM portions in the PVAM-graft-PAM. However, as the quantity of NS in the graft copolymer increased, the swelling ratio decreased. In addition, the PVAM-graft-PAM was sensitive to variation in pH due to the presence of carboxylic and amide groups on the grafting structure. After the preparation of the PVAM-graft-PAM, it was used in the controlled release of retinoic acid. After use, the resulting hydrogel degraded easily in natural soil.

Gradient release of cardiac morphogens by photo-responsive polymer micelles for gradient-mediated variation of embryoid body differentiation.

Retinoic acid (RA) is a well-known morphogen in human development. However, how an RA gradient distribution influences cardiac development remains obscure due to the lack of appropriate experimental apparatus. To address this issue, a polymeric micelle system with covalently attached RA was engineered to deliver gradient quantities of RA upon photo-irradiation. A photo-degradable polymeric nanoparticle (NP) composed of an amphiphilic methoxy(polyethylene glycol)-b-poly(ε-caprolactone)-co-poly(azido-ε-caprolactone-g-ortho nitrobenzyl retinoic ester) copolymer was synthesized, and hanging RA was covalently attached through a photo-sensitive o-nitrobenzyl (ONB) linker. The ONB linker was efficiently cleaved when exposed to a light (365 nm)-gradient, and the consequent gradient release of RA from the micelles was demonstrated. The efficacy of the photo-gradient-mediated RA release was validated across different concentrations of polymer micelles over varied irradiation periods. It was confirmed that polymer micelles demonstrated minimal cytotoxicity when exposed to mouse embryoid bodies (EBs). Finally, when the photo-gradient release of polymer micelles was applied, GFP-cardiac troponin T reporter mouse EBs demonstrated a concurrent gradient-like pattern of cardiac differentiation, verifying the utility of our novel photo-gradient approach to study morphogen gradients not only for cardiac development but also for other potential biological microenvironments subject to morphogen presentation with highly defined spatial and temporal geometries.

Differentiation of neuronal stem cells into motor neurons using electrospun poly-l-lactic acid/gelatin scaffold

Neural stem cells (NSCs) provide promising therapeutic potential for cell replacement therapy in spinal cord injury (SCI). However, high increases of cell viability and poor control of cell differentiation remain major obstacles. In this study, we have developed a non-woven material made of co-electrospun fibers of poly l-lactic acid and gelatin with a degradation rate and mechanical properties similar to peripheral nerve tissue and investigated their effect on cell survival and differentiation into motor neuronal lineages through the controlled release of retinoic acid (RA) and purmorphamine. Engineered Neural Stem-Like Cells (NSLCs) seeded on these fibers, with and without the instructive cues, differentiated into β-III-tubulin, HB-9, Islet-1, and choactase-positive motor neurons by immunostaining, in response to the release of the biomolecules. In addition, the bioactive material not only enhanced the differentiation into motor neuronal lineages but also promoted neurite outgrowth. This study elucidated that a combination of electrospun fiber scaffolds, neural stem cells, and controlled delivery of instructive cues could lead to the development of a better strategy for peripheral nerve injury repair.

Controlled Assembly and Release of Retinoic Acid Based on the Layer-by-Layer Method

All-trans retinoic acid (RA) has been proved to play important roles in regulating cell growth in various types of cells. Yet most experiments were performed by adding RA in solution previously. In this Article, we focus on the incorporation of RA, as a negatively charged moiety, into layered polyelectrolyte films on surfaces by means of layer-by-layer (LbL) deposition, followed by adding of capping layers to regulate the release of RA from the films. The incorporated RA was designed to release over 5 days in buffer solution. The assembly and release of RA were verified by UV and QCM results. The controlled release of RA from multilayer films can serve as a model system to study the influence of small molecules on cell growth.

Nanofiber-mediated release of retinoic acid and brain-derived neurotrophic factor for enhanced neuronal differentiation of neural progenitor cells.

The treatment of an injured central nervous system using stem-cell-based regenerative medicine still faces considerable hurdles that need to be overcome. Chief among which is the lack of efficient strategies to generate functional neurons from stem cells. The sustained delivery of biochemical cues and synergistic topographical signaling from electrospun nanofibrous scaffolds may be a potential strategy to enhance neuronal differentiation of stem cells for therapeutic purposes. In this study, retinoic acid (RA) and brain-derived neurotrophic factor (BDNF) were encapsulated into a copolymer of ε-caprolactone and ethyl ethylene phosphate to form a multifunctional, electrospun nanofibrous scaffold. Sustained release of RA and BDNF was achieved for at least 7 and 14 days, respectively. Despite lower cumulative release of drugs as compared to bolus delivery to plain nanofibers (at least 2× and 50× lower for RA and BDNF, respectively), nanofiber-mediated delivery of RA and/or BDNF resulted in similar capacity for neuronal differentiation of mouse neural progenitor cells (NPCs). In addition, nanofiber topography significantly increased neuronal differentiation (with BDNF, 47.4% Map2(+) cells on 2D vs. 53.4 to 56.5% on nanofibers, p < 0.05) and reduced glial cell differentiation. BDNF was a more potent inducer of neuronal differentiation than RA. RA supplementation alone resulted in minimal effect on NPC differentiation, and dual supplementation of RA and BDNF did not further enhance the neuronal differentiation of NPCs. Collectively, the results suggest that synergistic effects of nanofiber topography and sustained delivery of RA and/or BDNF may contribute towards the design of a multifunctional artificial stem cell niche for NPC neuronal differentiation.

Microsphere size effects on embryoid body incorporation and embryonic stem cell differentiation

Differentiation of pluripotent embryonic stem cells (ESCs) in vitro via multicellular spheroids called embryoid bodies (EBs) is commonly performed to model aspects of early mammalian development and initiate differentiation of cells for regenerative medicine technologies. However, the three-dimensional nature of EBs poses unique challenges for directed ESC differentiation, including limited diffusion into EBs of morphogenic molecules capable of specifying cell fate. Degradable polymer microspheres incorporated within EBs can present morphogenic molecules to ESCs in a spatiotemporally controlled manner to more efficiently direct differentiation. In this study, the effect of microsphere size on incorporation into EBs and ESC differentiation in response to microsphere- mediated morphogen delivery were assessed. PLGA microspheres with mean diameters of 1, 3, or 11 microm were fabricated and mixed with ESCs during EB formation. Smaller microspheres were incorporated more efficiently throughout EBs than larger microspheres, and regardless of size, retained for at least 10 days of differentiation. Retinoic acid release from incorporated microspheres induced EB cavitation in a size-dependent manner, with smaller microspheres triggering accelerated and more complete cavitation than larger particles. These results demonstrate that engineering the size of microsphere delivery vehicles incorporated within stem cell environments can be used to modulate the course of differentiation.

Poly(lactic- co-glycolic acid) electrospun fibrous meshes for the controlled release of retinoic acid

Poly(lactic- co-glycolic acid) (PLGA) meshes loaded with retinoic acid (RA) were prepared by applying the electrospinning technique. The purpose of the present work was to combine the biological effects of RA and the advantages of electrospun meshes to enhancing the mass transfer features of controlled release systems and cell interaction with polymeric scaffolds. The processing conditions for the fabrication of three-dimensional meshes were optimized by studying their influence on mesh morphology. Tensile testing showed that RA loading influenced the meshes’ mechanical properties by increasing their strength and rigidity. Moreover, the drug release and degradation profiles of the electrospun systems were compared to analogous RA-loaded PLGA films prepared by solvent casting. The results of this study highlight that the electrospun meshes preserved their fibrous structure after 4 months under in vitro physiological conditions and showed a sustained controlled release of the loaded agent in comparison to that observed for cast films. The bioactivity of the loaded RA was investigated on murine preosteoblasts cells by evaluating its influence on cell proliferation and morphology.

All-trans retinoic acid release from surfactant-free nanoparticles of poly(DL-lactide-co-glycolide)

In this study, we prepared all-trans retinoic acid (ATRA)-encapsulated, surfactant-free, PLGA nanoparticles. The nanoparticles were formed by nanoprecipitation process, after which the solvent was removed by solvent evaporation or dialysis method. When a nanoparticle was prepared by the nanoprecipitation - solvent evaporation method, the nanoparticles were bigger than the nanoparticles of the nanoprecipitation - dialysis method, despite the higher although loading efficiency. Nanoparticles from the nanoprecipitation - dialysis method were smaller than 200 nm in diameter, while the loading efficiency was not significantly changed. Especially, nanoparticles prepared from DMAc, 1,4-dioxane, and DMF had a diameter of less than 100 nm. In the transmission electron microscopy (TEM) observations, all of the nanoparticles showed spherical shapes. The loading efficiency of ATRA was higher than 90 % (w/w) at all formulations with exception of THF. The drug content was increased with increasing drugfeeding amount while the loading efficiency was decreased. In the drug release study, an initial burst was observed for 2∼6 days according to the variations of the formulation, after which the drug was continuously released over one month. Nanoparticles from the nanoprecipitation - dialysis method showed faster drug release than those from the nanoprecipitation — solvent evaporation method. The decreased drug release kinetics was observed at lower drug contents. In the tumor cell cytotoxicity test, ATRA-encapsulated, surfactant-free, PLGA nanoparticles exhibited similar cytotoxicity with that of ATRA itself.

Transporter-to-Trap Conversion: a Disulfide Bond Formation in Cellular Retinoic Acid Binding Protein I Mutant Triggered by Retinoic Acid Binding Irreversibly Locks the Ligand Inside the Protein

Transport proteins must bind their ligands reversibly to enable release at the point of delivery, while irreversible binding is usually associated with the extreme cases of ligand sequestration. Protein conformational dynamics is an important modulator of binding kinetics, as increased flexibility in the regions adjacent to the binding site may facilitate both association and dissociation processes. Ligand entry to, and exit from, the internal binding site of the cellular retinoic acid binding protein I (CRABP I) occurs via a flexible portal region, which functions as a dynamic aperture. We designed and expressed a CRABP I mutant (A35C/T57C), in which a small-scale conformational switch caused by the ligand binding event triggers formation of a disulfide bond in the portal region, thereby arresting structural fluctuations and effectively locking the ligand inside the binding cavity. At the same time, no formation of the disulfide bond is observed in the apo form of the mutant, and most characteristics of the mutant, including protein stability, are very similar to those of the wild-type protein in the absence of retinoic acid. The mutation does not alter the kinetics of retinoic acid binding to the protein, although the disulfide formation makes the binding effectively irreversible, as suggested by the absence of retinoic acid transfer from the holo form of the mutant to lipid vesicles in the absence of a reducing agent. Taken together, these data suggest that the disulfide bond formation in the portal region arrests large-scale structural fluctuations, which are required for retinoic acid release from the protein. The unique properties of the CRABP I mutant described in this work can be used to inspire and guide a design of nanodevices for multiple tasks ranging from sequestering small-molecule toxins in both tissue and circulation to nutrient deprivation of pathogens.

Drug–polymer interaction in the all-trans retinoic acid release from chitosan microparticles

Abstract Chitosan microparticles were prepared with the purpose of incorporating all-trans retinoic acid (ATRA). Morphology, drug content, release behavior and the interaction between chitosan and ATRA were investigated. Chitosan microparticles presented irregular and rough surface and drug content of 47±3%. The results of DSC and IR spectroscopy demonstrated interaction between drug and polymer resulting from retinoate or retinoamide formation. The drug release study showed that approximately 90% of drug was not released from microparticles until the end of experiment (48 h). That release behavior was probably due to the strong drug–polymer interaction and the more compact network of microparticles formed.

Polyethylenimine Complexes with Retinoic Acid: Structure, Release Profiles, and Nanoparticles

In this paper we discuss the preparation of complexes formed by polyethylenimine (PEI) and retinoic acid. The molecular weights of the PEIs were 600, 2000, 25 000, and 750 000 g/mol. All complexes form smectic A-like structures whose orders decrease with increasing molecular weight. The complexes in the bulk material obey Porod's law. Macroscopically oriented multilayer films of the complexes were prepared in a single-step procedure by spin coating and were characterized by X-ray reflectivity. The release of retinoic acid from the films was investigated by FTIR and surface tension measurements. It was found that the release from the complexes with the high molecular weight PEIs is faster than that from the complexes with low molecular weight. Steric stabilized nanoparticles of the complexes were prepared with sizes ranging from 170 to 580 nm by using Poloxamer 188 as the dispersing agent in aqueous media. Their characterization was carried out using dynamic light scattering and atomic force microscopy. It...

In vitro retinoic acid release and skin permeation from different liposome formulations

The effect of including charged phospholipids (both negative and positive) and/or cholesterol in liposomal formulations on retinoic acid (RA) in vitro release and permeation through human stratum corneum and epidermis was studied. No significant difference in RA release was observed comparing the different liposomal formulations tested. On the contrary, positively charged liposomes provided significantly higher RA skin permeation compared to negatively charged vesicles which, in turn, showed RA permeation through the skin similar to that obtained from neutral liposomes. Furthermore, the inclusion of cholesterol in charged liposomes did not significantly affect RA skin permeation.

Development and Validation of Method for Determination ofIn VitroRelease of Retinoic Acid from Creams

AbstractThe objective of this study was to develop and validate an in vitro test method that can be used as a tool for accessing batch-to-batch uniformity of finished topical products. The studies were performed by utilizing topical creams containing the 13-cis isomer of retinoic acid. Various physicochemical factors which may affect drug release from topical cream formulations were evaluated including drug concentration, internal phase droplet size distribution, viscosity and the composition of the emulsion internal and external phases. Utilizing a modified Franz diffusion cell with a cellulose membrane, the in vitro drug release profile from various cream formulations was establishedThe effect of varying the concentrations of various key ingredients by 30% does affect the viscosities and release rates compared to a standard formulation. However, there is no correlation between the viscosities and the release rates. No significant differences in pH and droplet size distribution were observed in these cre...

A quantitative analysis of the effect of all- trans-retinoic acid on the pattern of chick wing development

Small, positively charged beads that slowly release known amounts of all- trans-retinoic acid have been implanted below the apical ectodermal ridge at the anterior margin (opposite somite 16) of wing buds of 3 1 2 - day-old chick embryos. The continuous release of retinoic acid is shown to create an anteroposterior concentration gradient of retinoic acid in the limb field that is stable with time, despite the fact that this compound is metabolized by the limb tissue. With beads that release increasing amounts of retinoic acid, the normal 234 digit pattern is progressively altered to a 2234, to a 32234, and then to a 432234 pattern. The tissue concentrations of all- trans-retinoic acid required to change the digit pattern in this way range between 1 and 25 n M. When the same amounts of retinoic acid are released from posteriorly implanted beads (placed below the apical ectodermal ridge opposite somite border 19 20 or somite 20), the normal digit pattern is unaffected. Implantations of beads that release all- trans-retinoic acid are thus identical in their effect to grafts of cells from the limb polarizing region, which cause similar dose-dependent changes in the digit pattern when grafted to the anterior margin of the bud (but not when grafted opposite somites 19 or 20). Because of the low concentrations of retinoic acid required for its biological effect, the graded response observed, and the fact that a concentration gradient is established across the limb field, all- trans-retinoic acid closely mimics the putative morphogen that has been postulated to be emitted by polarizing region cells during normal development.

Microcontrolled release of biologically active compounds in chick embryos: Beads of 200-μm diameter for the local release of retinoids

A method of controlled release that allows the continuous local application of retinoids (vitamin A derivatives) in living tissues has been developed. Several biocompatible 200-μm-diameter polymeric beads have been tested as possible carriers. Each type of bead was loaded by soaking in an isotopically labeled retinoid solution, washed, and then transferred into tissue culture medium for quantitative release measurements. Positively-charged ion-exchange resins of the Dowex 1 type were found to be the most suitable for the controlled release of retinoic acid, a negatively charged compound. For the controlled release of uncharged retinoids such as retinyl acetate, uncharged acrylic ester polymer beads are preferred; these beads can also be used to release the negatively charged compounds retinoic acid and prostaglandin E 1. In all cases, a prolonged release is obtained that persists for more than a day. During this interval, the release is diffusion-controlled, and the total amount of compound released is directly proportional to the amount of the compound that the bead is exposed to during the initial loading step. High-performance liquid chromatography has been used to analyze the nature of the released retinoid. When the positively charged beads are loaded with all- trans-retinoic acid, there is a time-dependent decrease in the proportion of the all- trans isomer released which is due to an increased release of two cis isomers. This isomerization reaction occurs at a considerably slower rate when the uncharged beads are used as carriers. To mimic the conditions under which the local release of retinoic acid causes striking pattern duplications in developing chick wings, beads loaded with isotopically labeled retinoids were manually implanted into a slit cut into wing buds of stage-20 chick embryos. The release rate obtained was comparable to that found in vitro, and a time-dependent accumulation of the released radioactive compound was measured that was confined to the tissue near the site of implantation. All of the beads tested were readily accommodated by the tissue and could be easily removed at any time to terminate the treatment. It is believed that the controlled release of chemicals from such tiny biocompatible implants has a wide potential range of applications in biology.