Full length articleLipogels responsive to near-infrared light for the triggered release of therapeutic agents
Graphical abstract
Introduction
Hydrogels and other polymer-based carriers have emerged as promising systems to provide reservoirs for pharmaceuticals acting in a variety of anatomical regions. Highly biodegradable and biocompatible fibrin gels, obtained by thrombin-catalyzed polymerization of fibrinogen, have been used since last century with remarkable success as post-surgery sealants and stand out for their potential as controlled delivery systems of various pharmaceutical agents [1]. After addition of thrombin to a fibrinogen solution, fibrin networks can be prepared in the form of macroscopic gels, films, threads, microbeads or nanoparticles. The final properties of the gels, including porosity and degradability, can be tailored by the process and formulation parameters [2]. The release of drugs from fibrin gels occurs via diffusion once the pores in the network are bigger than the hydrodynamic size of the entrapped therapeutics [3]. Moreover it has been shown that the release can be modulated by entrapment of protease inhibitors in the protein matrix that slow down the in vivo degradation, by crosslinking with bifunctional reagents that enhance the retention of the therapeutic agents in the protein network or by covalent linking of the payloads to the matrix [4], [5], [6]. Entrapment of drug-loaded nanoparticles, e.g. liposomes, can remarkably lower the release rates of water-soluble therapeutics that readily diffuse out from fibrin gels [7], [8].
Liposomes are sphere-shaped, unilamellar or multilamellar vesicles typically made from phospholipids. Hydrophobic drugs can be solubilized in the lipid bilayer while more hydrophilic drugs can be loaded in the aqueous core of liposomes. Pharmaceutical agents retained by liposomes become bioavailable only after being released. Most attempts to use liposomes as drug delivery vehicles are based on considering liposomes as entities that circulate in the blood, being taken up by certain cells or tissues to slowly release their therapeutic cargo as they degrade [9], [10], [11]. Optimization of the release rate of entrapped drug is essential to control local drug concentration and increase the therapeutic outcome of liposomes [12], [13]. Several drug delivery systems aiming sustained release of therapeutic peptides, proteins or drugs from liposomes have been proposed, including their encapsulation in fibrin networks [7], [8], [10], [11]. Also, triggering modalities for site-specific release of therapeutics have been developed to improve the therapeutic efficacy of liposomal formulations. These strategies include local triggers which are specific to target sites (e.g., pH and enzymes) and remote triggers (e.g., temperature, ultrasound, magnetic field, radiofrequency and light) through the use of specific lipid compositions and coatings [14], [15], [16], [17]. Among the latter approaches, heat-based delivery appears to be the most promising to date [18]. TSL are special formulations capable of releasing encapsulated cargos in response to an increase of temperature, whilst preventing premature leakage at physiological temperature [19]. Incorporation of lysolipids, phospholipids with one hydrocarbon chain, in the formulation of TSL can lower the phase transition temperature of the lipid bilayer to the mild hyperthermia range [20], [21], [22]. Upon heating, lysolipids create and stabilize water-filled pores along liquid-solid boundaries formed in the lipid bilayer of liposomes, enhancing its permeability [18]. An example of LTSL is ThermoDox®, a heat-activated liposomal formulation of doxorubicin that upon intravenous administration releases the entrapped drug at temperatures above 40 °C. This formulation, the first and only thermosensitive liposome formulation to reach clinical development to date [23], is currently being evaluated in combination with radiofrequency thermal ablation for the treatment of hepatocellular carcinoma in the OPTIMA trial [24], a clinical phase III trial based on the promising findings obtained from post-hoc analysis of the HEAT trial [25]. A major drawback of systemically administered liposomes, as Thermodox®, is their poor and inhomogeneous distribution at distances away from the tumor vasculature which can compromise clinical outcomes. Furthermore, radiofrequency energy is difficult to focus on a discrete target site. These limitations may be circumvented by technologies that allow direct disposal of thermosensitive liposomes in the tumor region and that employ focusable sources of heat to trigger drug release.
In the present work, we exploit the ability of fibrin hydrogels to incorporate in their structure inorganic photoabsorbers such as hollow gold nanoparticles (HGNP) [26] or copper sulfide nanoparticles (CuSNP) [27] during gelation [28]. Both NPs exhibit strong light absorption at near-infrared (NIR) wavelengths (650–1050 nm) and can convert light energy into thermal energy through localized surface plasmon resonance, and in the case of CuSNP, also through the excitation of direct (band-to-band) or indirect electronic transitions due to their semiconductor character [29]. We hypothesized that incorporating NIR nanotransducers (NIR-NT) in fibrin matrices that include DOX-loaded LTSL (LTSL-DOX), referred to as “lipogels” [30], [31], may result in an advanced drug delivery platform exogenously regulated by NIR electromagnetic energy, which has its maximum depth of penetration in biological tissue (Fig. 1).
Section snippets
Chemical reagents
The phospholipids 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-2000 (DSPE-PEG2000) were provided by Lipoid (Ludwigshafen, Germany). Monostearoylphosphatidylcholine (MSPC) was purchased from Avanti Polar Inc (Alabaster, AL, USA). Other chemical reagents were purchased from Sigma Aldrich unless otherwise specified.
DOX-loaded liposomes
LTSL or cholesterol-containing LTSL (LTSL-Ch) were
Characterization of LTSL-DOX
Fig. 2A shows cryo-TEM images of LTSL-DOX prepared by the lipid film hydration and extrusion method and then loaded with the drug by a transmembrane proton gradient technique. As previously described for DOX-loaded liposomes using citrate-gradient [37], accumulation of the drug into the acidic interior of the liposomes loaded with 240 mM ammonium sulfate buffer (pH 5.5) led to the generation of linear, circular and curved bundles of DOX fibers that change liposomal shape, resulting in
Discussion
Methods for delivering therapeutic agents at a controlled rate and in a targeted manner have been pursued vigorously in the last decade. In this regard, much research has been focused on the development of stimuli-responsive hydrogels. Such systems consist of three-dimensional networks of crosslinked hydrophilic polymers of which the physical properties, e.g. a dramatic volume transition, are able to change abruptly in response to environmental stimuli such as pH, chemicals, electric or
Conclusions
The in situ polymerization of fibrinogen allows the simultaneous encapsulation of different LTSL formulations and NIR-NT within fibrin networks. The resulting lipogels efficiently transform NIR energy into heat. The rise of local temperature above the transition temperature of the entrapped liposomes promotes DOX release from the lipogel regions concentric to the irradiation site. This effect leads to the increase of drug levels in the media conditioned by NIR-responsive composites. DOX release
Disclosure
The authors declare no competing financial interest.
Acknowledgements
The authors thank to D. Morales (Confocal Microscopy Laboratory, Universidad Autónoma de Madrid, Spain (UAM)) as well as F. Urbano and C. Aguado (Transmission Electron Microscopy Laboratory, UAM) for excellent technical support. This work was supported by grant PI15/01118 from the Instituto de Salud Carlos III-Fondos FEDER, Ministry of Economy and Competitiveness, Spain (MINECO); grant SAF2013-50364-EXP from MINECO; grant S2013/MIT-2862 from Comunidad Autónoma de Madrid, Spain (CAM) and grant
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These authors contributed equally.