A tunable microfluidic device toiInvestigate the influence of fluid-dynamics on polymer nanoprecipitation

Polymer drug-embedding nanocapsules are attracting increasing attention as effective tools for the targeted delivery of pharmaceutical molecules on specific biological tissues. Besides, it is well established that an effective selectivity of the delivery dictates that the size of the carrier particles be accurately controlled, thus maintaining the size dispersion of the particle population as low as possible. To this end, microfluidics-assisted precipitation provides a promising alternative to the traditional processes in that the structure of the flow - ultimately controlling the particle size distribution - can be reliably predicted from the solution of Navier-Stokes equations in the laminar regime. Notwithstanding the great potential provided by microfluidics techniques, much about the interaction between fluid-dynamics and polymer transport and precipitation is yet to be understood. In this work, we investigate polymer precipitation in a simple cross-junction inflow-outflow microchannel, which has proven a viable benchmark to gain insight into the physics of nanoprecipitation in that the particle size distribution is sensitively dependent on the flow operating conditions. Specifically, previous experimental work by some of these authors proved that average particle size can vary by an order of magnitude for operating conditions where the solvent flow rate varies by a factor of three, while keeping the non-solvent flow rate constant. The scope of this work is to show that such sensitive dependence on operating conditions finds direct correspondence in the kinematic structure of the flow, which undergoes abrupt qualitative changes in the same range of operating conditions, provided a fully three-dimensional solution of the incompressible Navier-Stokes equation (thus retaining the inertial term in momentum balance) is afforded

A modular microfluidic platform for the synthesis of biopolymeric nanoparticles entrapping organic actives

Microreactors have been shown to be a powerful tool for the production of nanoparticles (NPs); however, there is still a lack of understanding of the role that the microfluidic environment plays in directing the nanoprecipitation process and the synthetized nanoparticles size and morphology. We fabricated a novel capillary microfluidic device using a newly designed modular apparatus by assembling commercial stainless steel microcapillary tubes for HPLC through a cross junction. In this way, we realized a flow-focusing-based microdevice in which the dispersed organic phase is continuously focused by the continuous phase using a couple of syringe pumps. Our device is substantially distinct from others because of its modularity and flexibility, having the possibility to allocate, in its geometry, microchannels of different length, material, or internal diameter; moreover, it could be used to prepare NPs from different classes of pristine polymers independently from their nature (natural or synthetic ones), and no limitation can be envisaged due to the hydrophobic and hydrophilic character of the applied polymers. Using this capillary microfluidic flow-focusing device we fabricated monodisperse fluorescent-loaded nanoparticles from biodegradable polymers (i.e., poly-lactic-co-glycolic-acid and chitosan) with a one-step procedure. A flourescent probe (6 - coumarin) was incorporated within the biodegradable matrix of the particles. Various operating conditions, such as the polymer molecular weight and concentration, flow rate ratio, type of solvent phase, τmix, microreactor-focusing channel diameters and length, and temperature have been investigated. Their influences on the formation of NPs, have been correlated to the final particle size distribution, ranging from 20 to 300 nm, and morphological characteristics. NPs’ characterization was performed by Dynamic light scattering (DLS), size and Z-potential, and by means scanning electron microscopy (SEM), morphology. This technique allows the fast, low cost, easy, and automated synthesis of polymeric nanoparticles and it may become a useful approach in the progression from laboratory scale to pilot-line scale processes. The presence of the fluorescent probe enables to carry out preliminary studies of cellular uptake, transport and translocation of nanoparticles in cell cultures and in vivo systhems, in order to produce NPs containing organic actives for medical applications and ''plant medicine''. In the future, we will approach fluid dynamics and modeling studies to analyze the microfluidic behavior and mixing process within the capillary microchannels. These results may expand the current understanding of the nanoprecipitation mechanism of biopolymeric NPs and the role exerted by the microfluidic environment

Microfluidic-assisted nanoprecipitation of antiviral-loaded polymeric nanoparticles

The chemical treatment called “chemotherapy of plants” consists in the in vivo administration of substances able to interfere with viral replication. The current availability of synthetic molecules with a high chemotherapeutic index, i.e. with a high ratio between the maximum concentration tolerated and the minimum effective, together with the possibility to further widen the therapeutic window by the use of appropriate nanocarriers, seems to open on the application level of a novel chemical approach to treat plant viral infections. Ribavirin, for example, is a synthetic water-soluble nucleoside that possesses broad spectrum activity against a variety of DNA and RNA plant viruses. As well known, water-soluble drugs are generally difficult to encapsulate in solid particles. Chemical modification of these drugs, such as esterification, may increase their encapsulation efficiency, but may also decrease bioactivity. In this work we have synthesized stable solid monodispersed PLGA (poly-D,L-lactic-co-glycolic acid) NPs with diameters ranging from 50 to 200 nm containing ribavirin by using a microfluidic reactor with a flow-focusing geometry. Previous work carried out in our lab showed an improvement of drug loading efficiency when using a microfluidic approach in comparison with traditional nanoprecipitation methods for nanoencapsulation. On this basis we optimized Ribavirin loading within PLGA NPs by investigating the influence of different operating conditions, such as polymer concentration, flow rate ratio, τmix, microreactor-focusing channel diameter and length, on nanoparticle size and morphology. NPs characterization was performed by Dynamic light scattering (DLS) measurements and by scanning electron microscopy (SEM). The synthesized NPs showed a significant drug loading efficiency

Uptake and internalization of nanoparticles in Vitis vinifera and phytopathogenic fungi (Botrytis cinerea and Aspergillus spp.)

In recent years, NPs have been applied in several fields of biomedicine. Only recently researchers have begun to explore the potential of nanocarriers in plant biology (1). In the near future, the development of NPs for plant research and agriculture will allow several new applications, includind treatments with pesticides and fertilizers. Poly(lactic-co-glycolic) acid-based nanoparticles (PLGA NPs) are currently considered among the most promising drug carriers (2). For the first time, in this work it has been evaluated the ability of cells and plants of Vitis vinifera to internalize, transport and accumulate PLGA NPs, with an without an outer shell of chitosan. To visualize the cellular uptake, we synthesized PLGA NPs tagged with the high fluorescent probe coumarin 6. The fluorescence-microscopy analysis has shown the ability of PLGA NPs to cross the cell wall and the membrane of in vitro V. vinifera cells. It has been observed a relatively weak and diffuse fluorescence in the cytoplasm and in the nucleus, while a very intense fluorescence signal in large spherical bodies (generally 1 or 2 per cell), whose nature at present remains unknown. Cell viability test has shown that PLGA NPs were not cytotoxic in grapevine cultured cells. Through the use of fluorescent probes and inhibitors of specific endocytic pathways, it has been demonstrated that the internalization involves both the clathrin-dependent and clathrin-independent pathways. TEM analysis on cultured cells showed that PLGA NPs with a diameter ≤ 50 nm were able to enter in grapevine cells, while the larger ones remained adherent to the cell wall. Furthermore, it was demonstrated that PLGA NPs can enter in leaf tissues of V. vinifera through the stomata openings and that they can be absorbed even by the root and transported to aerial organs via the xylem. The cellular uptake of PLGA NPs has been also studied in grapevine pathogenic fungi (Botrytis cinerea, Aspergillus carbonarius, Aspergillus niger), suggesting that PLGA NPs could be used as vectors to deliver antifungal compounds. In contrast to what observed for V. vinifera cells, the PLGA NPs coated with chitosan enter quickly into fungal cells as well as those without chitosan coat. These results suggest a possible role of PLGA NPs surface charge: NPs without chitosan coat, with negative surface charge, are able to enter both in plant and fungal cells; NPs with chitosan coat that made positive the surface charge, selectively enter into fungal cells; NPs with a thin chitosan coat, showing neutral charge, are internalized only in the in vitro grapevine cell suspensions. PLGA NPs could provide targeted delivery systems, using the developed electrostatic reactions between residues of the wall and the surface charge of NPs. These results suggest that PLGA NPs might play a crucial role in the future development of crop management techniques, offering the possibility to deliver chemicals to specific targets in a controlled manner (3). 1) Chronopoulou L., Cutonilli A., Cametti C., Dentini M., Palocci C. (2012). Colloid Surf B 97:117–123 2) De Oliveira J.L., Ramos Campos E. V., Bakshi M., Abhilash P. C., Fraceto L. F. (2014). Biotechnology advances; 32.8:1550-1561. 3) Ghormade V., Deshpande M.V., Paknikar K.M. (2011). Biotechnology Advances; 29(6):792-803

Bio-compatible and environmental-friendly nanoparticles as a possible strategy to deliver agrochemicals in planta and in phytopathogenic fungi

Poly(lactic-co-glycolic) acid (PLGA)-based NPs are currently considered among the most promising drug carriers, nevertheless their use in plants has never been investigated. Our study shows the ability of PLGA NPs to cross the plant cell wall and membrane both of in vitro Vitis vinifera cells and some grapevine-pathogenic fungi. Viability tests demonstrated that PLGA NPs were not cytotoxic for V. vinifera-cultured cells. By means of fluorescence microscopy and TEM analysis, we established that PLGA NPs ≤ 50 nm can enter in grapevine cells, while bigger ones remained attached to the cell wall. Fluorescence analysis suggested that PLGA NPs penetrate through endocytic vesicles but endocytosis in plants has been poorly studied. To date, the research has been mainly focused on clathrin-mediated endocytosis and only a limited amount of evidence on clathrin-independent pathways is available, partly because there are no specific markers for non-clathrin vesicles. Through the combined use of TEM and confocal analysis, fluorescent PLGA NPs loaded with coumarin-6, probe FM4-64 specific for clathrin vesicles, and clathrin endocytosis inhibitors, we demonstrated the involvement of clathrin-indipendent endocytosis in the internalization of PLGA NPs. Consequently, PLGA NPs are accumulated in the cytoplasm and not directed to the vacuole in which should be digested. Moreover, experiments on protoplasts highlighted the different selectivity of the cell wall and membrane on nanoparticle size. Moreover, we performed experiments in planta. PLGA NPs can enter into leaf tissues through stomata, in the roots through root hairs and transported to the shoot through vascular tissues. Uptake of PLGA NPs by Aspergillus spp and Botrytis cinerea cells has been also observed. The obtained results suggest that PLGA NPs could represent a novel strategy to deliver gradually agrochemicals in the prevention of damages caused by pathogenic fungi in agriculture

Microfluidic synthesis of methyl jasmonate-loaded PLGA nanocarriers as a new strategy to improve natural defenses in Vitis vinifera

The objective of the present work was to synthesize biopolymeric nanoparticles (NPs) entrapping the resistance-inductor methyl jasmonate (MeJA) to be employed as a novel and alternative strategy in integrated pest management. NPs were prepared by using a continuous fow microfuidic reactor that allows to precisely control some features that are crucial for applications such as size, polydispersion, morphology and reproducibility. Poly(lactic-co-glycolic acid) (PLGA), a biopolymer largely studied for its use in biological applications, was chosen for the production of NPs entrapping MeJA, a biotic endogenous elicitor able to trigger plant’s defense responses. The efect of diferent fuid-dynamic conditions, PLGA molecular weight and concentration on NP properties (dimensions, polydispersion, morphology, stability) was evaluated. DLS and SEM were employed to characterize the obtained NPs. MeJA-loaded PLGA NPs ranging from 40 to 70nm were administered to Vitis vinifera cell cultures, in order to evaluate the biological response in terms of stilbene biosynthesis. HPLC investigations showed a faster response when the elicitor was administered by PLGA NPs in comparison with free MeJA. This result demonstrates that the encapsulation in PLGA NPs signifcantly promotes MeJA cell uptake and the activation of MeJA-induced responses

Endocytic pathways involved in PLGA nanoparticle uptake by grapevine cells and role of cell wall and membrane in size selection

In the last years, many studies on absorption and cell uptake of nanoparticles by plants have been conducted, but the understanding of the internalization mechanisms is still largely unknown. In this study, polydispersed and monodispersed poly(lactic-co-glycolic) acid nanoparticles (PLGA NPs) were synthesized, and a strategy combining the use of transmission electron microscopy (TEM), confocal analysis, fluorescently labeled PLGA NPs, a probe for endocytic vesicles (FM4-64), and endocytosis inhibitors (i.e., wortmannin, ikarugamycin, and salicylic acid) was employed to shed light on PLGA NP cell uptake in grapevine cultured cells and to assess the role of the cell wall and plasma membrane in size selection of PLGA NPs. The ability of PLGA NPs to cross the cell wall and membrane was confirmed by TEM and fluorescence microscopy. A strong adhesion of PLGA NPs to the outer side of the cell wall was observed, presumably due to electrostatic interactions. Confocal microscopy and treatment with endocytosis inhibitors suggested the involvement of both clathrin-dependent and clathrin-independent endocytosis in cell uptake of PLGA NPs and the latter appeared to be the main internalization pathway. Experiments on grapevine protoplasts revealed that the cell wall plays a more prominent role than the plasma membrane in size selection of PLGA NPs. While the cell wall prevents the uptake of PLGA NPs with diameters over 50 nm, the plasma membrane can be crossed by PLGA NPs with a diameter of 500–600 nm