Article pubs.acs.org/molecularpharmaceutics
Core−Shell Electrospun Fibers Encapsulating Chromophores or Luminescent Proteins for Microscopically Controlled Molecular Release Luigi Romano,†,‡ Andrea Camposeo,† Rita Manco,† Maria Moffa,*,† and Dario Pisignano*,†,‡ †
Istituto Nanoscienze-CNR, Euromediterranean Center for Nanomaterial Modelling and Technology (ECMT), via Arnesano, I-73100 Lecce, Italy ‡ Dipartimento di Matematica e Fisica “Ennio De Giorgi”, Università del Salento, via Arnesano, I-73100 Lecce, Italy S Supporting Information *
ABSTRACT: Core−shell fibers are emerging as interesting microstructures for the controlled release of drugs, proteins, and complex biological molecules, enabling the fine control of microreservoirs of encapsulated active agents, of the release kinetics, and of the localized delivery. Here we load luminescent molecules and enhanced green fluorescent proteins into the core of fibers realized by coaxial electrospinning. Photoluminescence spectroscopy evidences unaltered molecular emission following encapsulation and release. Moreover, the release kinetics is microscopically investigated by confocal analysis at individual-fiber scale, unveiling different characteristic time scales for diffusional translocation at the core and at the shell. These results are interpreted by a two stage desorption model for the coaxial microstructure, and they are relevant in the design and development of efficient fibrous systems for the delivery of functional biomolecules. KEYWORDS: fluorescent proteins, core−shell fibers, molecular delivery, coaxial electrospinning, controlled release
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INTRODUCTION Micro- and nanostructured materials are currently exploited in many technological applications due to their enhanced physicochemical properties compared to bulk systems,1,2 which are related to the small size, to the high surface-tovolume ratio and, especially for organic systems, to the unique assembly of macromolecules within tiny particles and fibers. Micro- and nanostructures with tailored structure and composition can perform multiple functions3 in the fields of sensors,4 optoelectronics,5 biotechnology,6 tissue engineering,7 and drug delivery.8 Indeed, nanoparticles, nanotubes, and nanofibers might improve the delivery of various compounds, allowing for concomitant releasing multiple molecules and for targeting specific cells, organs, or tissues, thus helping in avoiding systemic exposures of the organism to external molecular agents.9 For drug delivery applications, the release kinetics of bioactive molecules from micro- and nanomaterials has to be precisely known, preferably at single-nanoparticle scale, in order to control the administration of these compounds in the body. In general, a condition as close as possible to full delivery should be reached, and burst release at early times should be reduced. However, an initial release targeting specific sites with high concentrations, followed by a sustained release, could be advantageous in some applications, such as in postsurgery therapies based on antibiotics.10 Moreover, drugs and biomolecules must retain their functionality after release from © XXXX American Chemical Society
nanocarriers, and they have to be carefully tested since both material processing and delivery mechanisms can modify the molecular conformation of the involved compounds. In this framework, polymer fibers feature some interesting properties. They can be produced as nonwoven mats for straightforward in vivo implants,11 supporting a three-dimensional architecture, which resembles the complex extracellular matrix structure and potentially combines morphological cues directing cell behavior and capability of local release of growth factors, proteins, vitamins, and drugs.12 Nonwoven mats can be easily prepared by electrospinning, which allows continuous solid nanofibers to be realized by the electric field-assisted stretching of a polymer solution jet.13 This technology is highly versatile in terms of spun polymers, used solvents, and incorporated bioactive agents,14,15 which may include antibiotics,16,17 paracetamol,18 anticancer compounds,19 small interfering RNA,20 growth factors and genes,21 as well as viruses and bacteria.22,23 An effective approach to avoid issues related to material denaturation exploits core−shell fibers, whose internal region establishes a favorable microenvironment for encapsulated agents.24 These fibers can be produced by coaxial electrospinning25−27 using two concentrically aligned Received: July 16, 2015 Revised: January 9, 2016 Accepted: January 29, 2016
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DOI: 10.1021/acs.molpharmaceut.5b00560 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Article
Molecular Pharmaceutics needles and suitable solvents for the core solution embedding active compounds. Detrimental effects related to the presence of electric charges are minimized since these are mostly confined at the outer surface of spun jets.25 In this way, proteins,26,28,29 enzymes,30,31 liposomes,22 and bacteria32 have been successfully encapsulated. However, many properties of core−shell fibers still need to be clarified, especially concerning release mechanisms at individual f iber level. In fact, most of the available data on release kinetics are based on macroscopic spun mats, namely, on samples made of many (often, thousands of) fibers, where the resulting kinetics might be made very complex due to the convolution of many contributions. Instead, release properties at single fiber level are basically unexplored, due to the inherent difficulties in monitoring individual microstructures in a liquid environment over many hours or days. Here, we report on core−shell fiber carriers embedding rhodamine B isothiocyanate (RhB) chromophores or enhanced green fluorescent proteins (E-GFP), which allow the release kinetics from the core and from the shell to be unveiled in individual filaments by highly sensitive optical methods. This analysis evidences different diffusional characteristics with an initial fast release (with associated time scale of 102 minutes) mainly driven by molecules encapsulated in the shell, followed by a more sustained release (with associated time scale 103−104 minutes). These features are relevant for developing suitable in vitro models and for predicting in vivo kinetics33,34 and for designing next generation drug delivery systems35 based on core−shell fibers.
Figure 1. (a) Schematics of the coaxial electrospinning setup. Applied bias V = 10 kV. Flows QS = 4 mL/h, QC = 0.5 mL/h and QS = 1.5 mL/ h, QC = 0.5 mL/h for RhB-based and E-GFP based fibers, respectively. (b) Pendant solution drop at the coaxial spinneret. Scale bar: 5 mm. (c,d) Mats of core−shell fibers encapsulating RhB (c) and E-GFP (d), respectively. Scale bars: 1 cm. (e) SEM micrograph of the typical electrospun surface topology. Fibers obtained by PCL shell solutions with concentration, CS = 35%. Scale bar: 5 μm.
mL in water) and of a solution of E-GFP in PBS (20 μg/mL) was used for the core. The core/shell flow rate ratio was 1:3. Extensive experiments were performed to optimize the delivery of fluids into the jet (Figure 1b), by varying the applied voltage and the spinneret-to-collector distance in the ranges 10−20 kV and 15−20 cm, respectively. Samples used in the study were obtained by a positive voltage of 10 kV applied to the needle and with a spinneret-to-collector distance of 20 cm. Characterization of Core−Shell Fibers. The fiber morphology was characterized by scanning electron microscopy (SEM) using a Nova NanoSEM 450 System (FEI), operating at acceleration voltages 6−20 kV, and by atomic force microscopy (AFM), using a Multimode head equipped with a Nanoscope IIIa electronic controller (Veeco). The surface topography of individual fibers was imaged in tapping mode AFM, using phosphorus-doped Si cantilevers with resonance frequency 375 kHz and nominal tip radius
