Polylactic acid-based patterned matrixes for site-specific delivery of neuropeptides on-demand: functional NGF effects on human neuronal cells

The patterned microchamber arrays based on biocompatible polymers are a versatile cargo delivery system for drug storage and site- and time specific drug release on demand. However, functional evidence of their action on nerve cells, in particular their potential for enabling patterned neuronal morphogenesis, remains unclear. Recently, we have established that the polylactic acid (PLA)-based microchamber arrays are biocompatible with human cells of neuronal phenotype and provide safe loading for hydrophilic substances of low molecular weight, with successive site-specific cargo release on-demand to trigger local cell responses. Here, we load the nerve growth factor (NGF) inside microchambers and grow N2A cells on the surface of patterned microchamber arrays. We find that the neurite outgrowth in local N2A cells can be preferentially directed towards opened microchambers (site-specific NGF release). These results suggest the PLA-microchambers can be an efficient drug delivery system for the site- and time-specific delivery of neuropeptides, potentially suitable for guidance of human nerve cells

Molecular nature of breakdown of the folic acid under hydrothermal treatment: a combined experimental and DFT study

Using a combination of experimental Raman, FTIR, UV–VIS absorption and emission data, together with the corresponding DFT calculations we propose the mechanism of modification of the folic acid specifically under the hydrothermal treatment at 200 °C. We established that folic acid breaks down into fragments while the pteridine moiety remains intact likely evolving into 6-formylpterin with the latter responsible for the increase in fluorescence emission at 450 nm. The results suggest that hydrothermal approach can be used for production of other purpose-engineered fluorophores

Polymer microchamber arrays for geometry-controlled drug release: a functional study in human cells of neuronal phenotype

Polyelectrolyte multilayer (PEM) microchambers can provide a versatile cargo delivery system enabling rapid, site-specific drug release on demand. However, experimental evidence for their potential benefits in live human cells is scarce. Equally, practical applications often require substance delivery that is geometrically constrained and highly localized. Here, we establish human-cell biocompatibility and on-demand cargo release properties of the PEM or polylactic acid (PLA)-based microchamber arrays fabricated on a patterned film base. We grow human N2A cells (a neuroblastoma cell line widely used for studies of neurotoxicity) on the surface of the patterned microchamber arrays loaded with either a fluorescent indicator or the ubiquitous excitatory neurotransmitter glutamate. The differentiating human N2A cells show no detrimental effects on viability when growing on either PEM@PLA or PLA-based arrays for up to ten days in vitro. Firstly, we use two-photon (2P) excitation with femtosecond laser pulses to open individual microchambers in a controlled way while monitoring release and diffusion of the fluorescent cargo (rhodamine or FITC fluorescent dye). Secondly, we document the increases in intracellular Ca2+ in local N2A cells in response to the laser-triggered glutamate release from individual microchambers. The functional cell response is site-specific and reproducible on demand and could be replicated by applying glutamate to the cells using a pressurised micropipette. Time-resolved fluorescence imaging confirms the physiological range of the glutamate-evoked intracellular Ca2+ dynamics in the differentiating N2A cells. Our data indicate that the nano-engineering design of the fabricated PEM or PLA-based patterned microchamber arrays could provide a biologically safe and efficient tool for targeted, geometrically constrained drug delivery

Biodegradable Microcapsules Loaded with Nerve Growth Factor Enable Neurite Guidance and Synapse Formation

Neurological disorders and traumas often involve loss of specific neuronal connections, which would require intervention with high spatial precision. We have previously demonstrated the biocompatibility and therapeutic potential of the layer-by-layer (LbL)-fabricated microcapsules aimed at the localized delivery of specific channel blockers to peripheral nerves. Here, we explore the potential of LbL-microcapsules to enable site-specific, directional action of neurotrophins to stimulate neuronal morphogenesis and synaptic circuit formation. We find that nanoengineered biodegradable microcapsules loaded with nerve growth factor (NGF) can guide the morphological development of hippocampal neurons in vitro. The presence of NGF-loaded microcapsules or their clusters increases the neurite outgrowth rate while boosting neurite branching. Microcapsule clusters appear to guide the trajectory of developing individual axons leading to the formation of functional synapses. Our observations highlight the potential of NGF-loaded, biodegradable LbL-microcapsules to help guide axonal development and possibly circuit regeneration in neuropathology

High-fluorescent product of folic acid photodegradation: Optical properties and cell effect

Folic acid (FA) is a natural body component essential for normal cell growth and division. Uptake of FA into cells is mediated by the folate receptors with particularly large amount of folate receptors found on the surface of cancer cells. This feature traditionally is used for selective drug delivery systems targeting in cancer cells or visualization of the border between healthy and tumor tissue based on FA-modified fluorescent labels. However, preparation of FA-conjugates can be an elaborate procedure, while FA itself shows low photostability under illumination. Here we use photodegradation of FA colloid to produce high fluorescent product and compare its effects on the cells grows and division with the ones of FA. It was found UV-irradiation of FA colloid for 60 min allows obtaining water soluble fluorophore with quantum yield 30 ± 2 %. We also show that the resulting fluorescent product retained FA biological activity in relation to cell growth and division, comparable to native FA

Laser-triggered drug release from polymeric 3-D micro-structured films via optical fibers.

Photosensitive polymeric three-dimensional microstructured film (PTMF) is a new type of patterned polymeric films functionalized with an array of sealed hollow 3D containers. The microstructured system with enclosed chemicals provides a tool for the even distribution of biologically active substances on a given surface that can be deposited on medical implants or used as a cells substrate. In this work, we proposed a way for photothermally activating and releasing encapsulated substances at picogram amounts from the PTMF surface in different environments using laser radiation delivered with a multimode optical fiber. The photosensitive PTMFs were prepared by the layer-by-layer (LbL) assembly from alternatively charged polyelectrolytes followed by covering with a layer of hydrophobic polylactic acid (PLA) and a layer of gold nanoparticles (AuNPs). Moreover, the typical photothermal cargo release amounts were determined on the surface of the PTMF for a range of laser powers delivered to films placed in the air, deionized (DI) water, and 1% agarose gel. The agarose gel was used as a soft tissue model for developing a technique for the laser activation of PTMFs deep in tissues using optical waveguides. The number of PTMF chambers activated by a near-infrared (NIR) laser beam was evaluated as the function of optical parameters

In Vitro and in Vivo Visualization and Trapping of Fluorescent Magnetic Microcapsules in a Bloodstream

Remote navigation and targeted delivery of biologically active compounds is one of the current challenges in the development of drug delivery systems. Modern methods of micro- and nanofabrication give us new opportunities to produce particles and capsules bearing cargo to deploy and possess magnetic properties to be externally navigated. In this work we explore multilayer composite magnetic microcapsules as targeted delivery systems in vitro and in vivo studies under natural conditions of living organism. Herein, we demonstrate magnetic addressing of fluorescent composite microcapsules with embedded magnetite nanoparticles in blood flow environment. First, the visualization and capture of the capsules at the defined blood flow by the magnetic field are shown in vitro in an artificial glass capillary employing a wide-field fluorescence microscope. Afterward, the capsules are visualized and successfully trapped in vivo into externally exposed rat mesentery microvessels. Histological analysis shows that capsules infiltrate small mesenteric vessels whereas large vessels preserve the blood microcirculation. The effect of the magnetic field on capsule preferential localization in bifurcation areas of vasculature, including capsule retention at the site once external magnet is switched off is discussed. The research outcome demonstrates that microcapsules can be effectively addressed in a blood flow, which makes them a promising delivery system with remote navigation by the magnetic field