Gold nanoparticles for the in situ polymerization of near-infrared responsive hydrogels based on fibrin

Abstract

Non-invasiveness and relative safety of photothermal therapy, which enables local hyperthermia of target tissues using a near infrared (NIR) laser, has attracted increasing interest. Due to their biocompatibility, amenability of synthesis and functionalization, gold nanoparticles have been investigated as therapeutic photothermal agents. In this work, hollow gold nanoparticles (HGNP) were coated with poly-l-lysine through the use of COOH-Poly(ethylene glycol)-SH as a covalent linker. The functionalized HGNP, which peak their surface plasmon resonance at 800 nm, can bind thrombin. Thrombin-conjugated HGNP conduct in situ fibrin polymerization, facilitating the process of generating photothermal matrices. Interestingly, the metallic core of thrombin-loaded HGNP fragmentates at physiological temperature. During polymerization process, matrices prepared with thrombin-loaded HGNP were loaded with genetically-modified stem cells that harbour a heat-activated and ligand-dependent gene switch for regulating transgene expression. NIR laser irradiation of resulting cell constructs in the presence of ligand successfully triggered transgene expression in vitro and in vivo.

Statement of significance

Current technological development allows synthesis of gold nanoparticles (GNP) in a wide range of shapes and sizes, consistently and at scale. GNP, stable and easily functionalized, show low cytotoxicity and high biocompatibility. Allied to that, GNP present optoelectronic properties that have been exploited in a range of biomedical applications. Following a layer-by-layer functionalization approach, we prepared hollow GNP coated with a positively charged copolymer that enabled thrombin conjugation. The resulting nanomaterial efficiently catalyzed the formation of fibrin hydrogels which convert energy of the near infrared (NIR) into heat. The resulting NIR-responsive hydrogels can function as scaffolding for cells capable of controlled gene expression triggered by optical hyperthermia, thus allowing the deployment of therapeutic gene products in desired spatiotemporal frameworks.

Introduction

Fibrin, a water insoluble, degradable naturally-occurring biopolymer generated from fibrinogen, is involved in the last step of the blood coagulation cascade as major component of the clot [1]. Fibrin clot formation is catalyzed by thrombin, a member of the serine protease family that removes fibrinopeptides A and B from fibrinogen to produce fibrin monomer. Noncovalent interactions of fibrin monomers result in intermediate polymers which aggregate to form the fibrin clot [2]. Fibrin plays an important and ubiquitous role during tissue repair serving as provisional extracellular matrix for infiltrating cells and acting as a reservoir for growth factors [3]. As biomaterial for tissue repair and regeneration, fibrin shows attractive features over synthetic polymers, often limited by biocompatibility concerns, their inability to support cell attachment, undesirable degradation rate and toxic degradation products, potential immune response and acute inflammation [4,5]. Key advantages of fibrin include ease of purification of fibrinogen from donors, possibility of recombinant production, high tunability of the matrix architecture and mechanical properties by controlling the conditions of fibrin gelation, in situ curability, biodegradability, and minimal inflammation or foreign body reaction. These features support the clinical interest of products based on fibrin to be used as sealants or bioadhesives in surgical procedures [6], [7], [8], [9], cellular scaffolds for reconstituting full thickness wounds [4,10] or carriers for the delivery of drugs, genes and growth factors [11]. As scaffold, fibrin has been employed for supporting pre-vascularized tissues in the treatment of ischemic strokes [12]. Thus, fibrin patches containing endothelial cells and smooth muscle cells derived from human embryonic stem cells were implanted in porcine infarcted hearts showing significant engraftment and functional improvement [13]. Also, composite materials based on fibrin and other biopolymers have improved myocardial function in several animal models [14,15]. Encouraging outcomes have been reported from a first clinical trial of transplantation of cardioprogenitor cells incorporated in fibrin patches in patients with severely impaired cardiac function [16,17]. Fibrin scaffolds have also been employed as three-dimensional matrices containing growth factors and embryonic stem cells prone to differentiate into neurons in animal models of spinal cord injury [18]. Tendon regeneration and bone-tendon junction repair have also been accelerated in animal models after the application of mesenchymal stem cells engrafted in fibrin matrices [19]. Accelerated healing of acute and chronic non-healing cutaneous wounds has been achieved in patients treated with fibrin-based sprays containing autologous mesenchymal stem cells [20]. Commercial available scaffolds based on fibrin hydrogels (i.e. Bioseed^Ⓡ-C, BioTissue Technologies or CHONDRON™, Sewon Cellontech) have been successfully used in combination with autologous cells in articular cartilage regeneration of knee [21,22].

The so-called “smart” hydrogels, i.e. those which have the ability to respond to environmental changes in temperature, pH, light, magnetic and electric fields, ionic strength, mechanical load, enzymatic activity, antigen concentration, etc., are among the leading candidates to provide controlled delivery of therapeutic agents in regenerative medicine applications [23]. Photothermal heating induced by NIR irradiation has shown great potential as an external stimulus to regulate the activity of “smart” hydrogels. For example, NIR has been used to trigger on-demand cell release upon irradiation of composite hydrogels based in graphene oxide and poly(N-isopropylacrylamide) or to trigger sol-gel transitions in gold nanorod-filled polypeptide hydrogels to modulate drug release [24,25]. Recently, we developed a technological approach which employs “smart” hydrogels based on fibrin to define spatiotemporal patterns of transgene expression in response to incident NIR light [26]. Briefly, during fibrin polymerization catalyzed by soluble thrombin, we included HGNP tailored to show a surface plasmon resonance absorption in the NIR region. These plasmonic nanoparticles (NP) act as phototransducers that convert incident NIR light into heat, increasing the temperature of the surrounding region. The distance over which thermal diffusion and temperature rise occur depend on the exposure time and the thermal diffusion time, defined as the ratio of the thermal conductivity of the irradiated material to the product of the heat capacity and density [27]. Therefore, thermal diffusion distance from the nanoparticulated absorber will depend on the laser operation mode, continuous or pulsed, having longer heat diffusion distances when using continuous lasers. In the NIR region, light scattering prevails over absorption, and light penetrates underneath the subcutaneous tissue at depths that depend on the nature of the irradiated tissue [28]. Thus, NIR light can be used as a non-invasive trigger for activating internally implanted absorbers and generate local hyperthermia. We used these fibrin-based plasmonic hydrogels as scaffolds for genetically modified cells that harbour a heat-activated and ligand-dependent gene circuit that regulates the expression of transgenic growth factors. This regulatory circuit is based on the promoter of the highly heat-inducible HSPA7 gene [29], which drives the expression of a ligand-dependent chimeric transactivator that activates a promoter to which the transgene of interest is linked. Irradiation of photothermal scaffolds with NIR laser can increase the local temperature to trigger, in the presence of ligand, the gene circuit. After implanting NIR-responsive scaffolds populated by stem cells which harbour the gene circuit, transgenic expression of growth factors such as VEGF can be remotely patterned in the host using NIR light [26]. In the present work, we used a covalent coupling to coat HGNP with poly-L-lysine (PLL) through the use of COOH-poly(ethylene glycol)-SH as a linker (COOH-PEG-SH). The thiol group of the heterobifunctional PEG strongly binds to the gold surface while its carboxyl group binds to the amino groups in PLL via amide bond. These functionalized HGNP can efficiently load thrombin, enabling them to polymerize fibrin in situ by mixing the resulting NP and fibrinogen, and therefore simplifying the process of generating plasmonic fibrin matrices. The structural, mechanical and photothermal properties of these matrices were compared to those reported in our previous study [26], which were prepared using three independent components (fibrinogen, HGNP and soluble thrombin) and were used as control in the present study. Interestingly, we found that the metallic core of thrombin-loaded HGNP fragmentates in aqueous medium. This finding represents an exciting advantage for the clinical use of gold nanostructures, whose non-biodegradability has raised concerns regarding long-term toxicity [30], [31], [32].