Highlights
- •GemIPs deliver the chemotherapeutic gemcitabine to continously hold local concentration profiles within a therapeutically relevant range.
- •GemIP treatment of brain tumors grown on an avian host model using GemIPs induces apoptotic events and cell cycle arrest.
- •GemIP treatment interferes with tumor growth and vascularization and outperforms conventional metronomic therapies.
- •Iontronics enable on-site and on-demand administration of so far unconsidered chemotherapeutics for brain tumors.
Abstract
Local and long-lasting administration of potent chemotherapeutics is a promising therapeutic intervention to increase the efficiency of chemotherapy of hard-to-treat tumors such as the most lethal brain tumors, glioblastomas (GBM). However, despite high toxicity for GBM cells, potent chemotherapeutics such as gemcitabine (Gem) cannot be widely implemented as they do not efficiently cross the blood brain barrier (BBB). As an alternative method for continuous administration of Gem, we here operate freestanding iontronic pumps – “GemIPs” – equipped with a custom-synthesized ion exchange membrane (IEM) to treat a GBM tumor in an avian embryonic in vivo system. We compare GemIP treatment effects with a topical metronomic treatment and observe that a remarkable growth inhibition was only achieved with steady dosing via GemIPs. Daily topical drug administration (at the maximum dosage that was not lethal for the embryonic host organism) did not decrease tumor sizes, while both treatment regimes caused S-phase cell cycle arrest and apoptosis. We hypothesize that the pharmacodynamic effects generate different intratumoral drug concentration profiles for each technique, which causes this difference in outcome. We created a digital model of the experiment, which proposes a fast decay in the local drug concentration for the topical daily treatment, but a long-lasting high local concentration of Gem close to the tumor area with GemIPs. Continuous chemotherapy with iontronic devices opens new possibilities in cancer treatment: the long-lasting and highly local dosing of clinically available, potent chemotherapeutics to greatly enhance treatment efficiency without systemic side-effects.
Significance statement
Iontronic pumps (GemIPs) provide continuous and localized administration of the chemotherapeutic gemcitabine (Gem) for treating glioblastoma in vivo. By generating high and constant drug concentrations near the vascularized growing tumor, GemIPs offer an efficient and less harmful alternative to systemic administration. Continuous GemIP dosing resulted in remarkable growth inhibition, superior to daily topical Gem application at higher doses. Our digital modelling shows the advantages of iontronic chemotherapy in overcoming limitations of burst release and transient concentration profiles, and providing precise control over dosing profiles and local distribution. This technology holds promise for future implants, could revolutionize treatment strategies, and offers a new platform for studying the influence of timing and dosing dependencies of already-established drugs in the fight against hard-to-treat tumors.
1. Introduction
1.1. Local chemotherapy approaches for GBM treatment
Chemotherapy relying on cytotoxins and targeted drugs is a powerful tool to intervene in tumor progression and plays a vital role in clinical tumor management [1,2]. Efficiency of a chemotherapeutic regime strongly depends on the choice of the therapeutic, as well as the timing and dosing profiles of the medication [[3], [4], [5]]. Local therapeutic regimes offer the possibility to administer high concentrations of selected drugs at controlled times, thus increasing their efficiency while simultaneously lowering systemic burden and associated side effects. Hence, radically new local therapeutic regimes, especially for hard-to-treat tumors such as pancreatic adenocarcinoma, soft tissue sarcomas, and GBM, have the potential to substantially improve therapeutic outcomes. New drug delivery strategies including drug-loaded scaffolds and externally controllable drug delivery devices have been developed in order to exploit these benefits [[6], [7], [8]]. In the case of GBM, a local drug release system could be beneficial because of the high local recurrence rate of GBM. Tumors recur within an area of 3 cm around the resection margins in almost every case [[9], [10], [11]], which causes an overall survival prognosis of only 15 months [12,13]. In addition, the BBB hinders many potent chemotherapeutics from reaching the cancerous tissue, limiting chemotherapy almost exclusively to the BBB-passing drug temozolomide (TMZ). Unfortunately, however, approx. 50% of GBM patients develop drug resistance to TMZ [14]. These facts suggest a local cancer therapy with alternative chemotherapeutics as a favorable method to intervene with this rapidly growing type of tumor [8]. Several local treatment technologies have been developed and are partially used in the clinical setting, including convection-enhanced delivery (CED) of targeted cytotoxins [[15], [16], [17]], laser interstitial thermal therapy, tumor treating fields (TTFs) [18], immunotherapy [19], opening the BBB with electroporation [20,21] or high-intensity focused ultrasound [22,23], as well as using drug-loaded scaffolds such as Gliadel or Cerebraca wafers [24,25].
Among local drug delivery methods, CED has become one of the most prominent methods to administer otherwise BBB-restricted therapies via injections to the target tissue in the brain. This offers the advantage of accumulating high local concentrations of the administered therapeutics and therefore increased treatment efficiency, and at the same time, reduced systemic toxicity. In parallel, iontophoretic tools have been shown to be able to generate high drug concentrations in the tumor and induce anti-tumor effects, while maintaining low systemic concentrations [26,27]. Currently, iontophoretic tools for chemotherapy are applied predominantly for transdermal drug administration and intravesical delivery. Besides the clinical success of these local drug delivery methods, both CED and iontophoretic systems leave much room for improvement for brain tumor treatment. In the case of iontophoresis, the technology relies on electric potential gradients to generate a combination of electrophoretic and electroosmotic drug delivery, and thus a mix of diffusional drug delivery and volume infusion. To reach effective delivery, devices are usually operated in the milliampere range [27], which could potentially lead to unwanted muscle contractions and paresthesia when applied in the brain [28]. For safe operation, the generated pressure gradients from the volume infusion must be small and carefully monitored. This is an issue that is shared with the CED technique. CED can also, due to high flow rates and huge infusate volumes, cause complications due to backflow along the cannulas [[29], [30], [31]] (i.e., fluid flowing “back up” along the outside of the cannula rather than into the intended target tissue). Also, CED requires complex time- and personnel-consuming clinical protocols and is limited to the duration of the neurosurgery itself, which promoted CED to become predominantly a tool for clinical studies rather than an administration method for long-lasting GBM chemotherapy.
In this work we use a high precision and electronically controlled drug delivery technology that does not generate increased pressure, nor require high currents: organic electronic iontronic pumps. These “iontronic” pumps are devices that can be used for the controllable electromigration of charged molecules through an IEM [32]. We recently used a free-standing iontronic pump to deliver the chemotherapeutic Gem in vitro [33]. Gem is a potent chemotherapeutic and standard of care for different solid tumors, however, for GBM unused so far because of its BBB impermeability. It has been shown by us and others, that Gem itself however strongly affects proliferating tumor cells, while leaving neural tissue unaffected at therapeutic concentrations [[33], [34], [35]]. In the present work, we use a GemIP device with an anionically-functionalized hyperbranched polyglycerol (A-HPG) IEM [36]. Unlike our previous studies, which relied on stochastically organized linear polyelectrolytes to for the IEM, the hyperbranched structure of the A-HPG membrane allows for rational designes and optimization for high ionic conductivity. Its high stability is achieved by carefully balancing fixed charged groups in hydrophilic regions and stabilizing hydrophobic regions. First, we test GemIP performance in situ, followed by a characterization in an avian embryonic in vivo model. This model, also known as the chick chorioallantoic membrane (CAM) model, is a well-established model to study tumor proliferation, migration, and invasion in vivo [37,38]. The CAM model provides a preclinical miniature oncological model capable of evaluating tumorigenesis after therapeutic intervention, representing an intermediate model to bridge developmental hurdles between in vitro cell culture settings and rodent models. Engrafted CAM tumors are equipped with a rich vascularization and assemble into a three-dimensional tumor morphology, which provides an excellent model environment to observe tumor growth and pharmacodynamics – and to test bioelectronic drug delivery systems [[38], [39], [40]].
We hypothesized that a continuous dosing of Gem via an iontronic pump (GemIP) would interfere with GBM tumor growth significantly and causes relevant anti-tumor effects. We operated the GemIPs in close proximity to CAM-grown GBM tumors and compared the treatment performance with topical metronomic administration of Gem. In addition to experimental readouts evaluating tumor biology and drug accumulation, we use numerical models to simulate the drug concentration profiles for both transient and continuous treatment methods in a virtual CAM environment. Combining the in situ and in vivo experiments with the in silico models intertwines GemIP stimulation profiles with treatment performance.
2. Materials and methods
2.1. Synthesis of A-HPG and fabrication of GemIPs
All the commercial chemicals were used as received without any further purification. Bruno Bock Chemische Fabrik GmbH & Co. KG donated the chemical Thiocure 333 (ethoxylated trimethylolpropane tris(3-mercaptopropionate)). The A-HPG polyelectrolyte membrane material was synthesized as previously described by Abrahamsson et al. [36] HPG starting material (Mw: 10000–12,000 Da, ĐM: 1.0–2.4, DB: 50–60%) functionalized with sodium 1-butanesulfonate (80%) as fixed anionic charge groups and allyl groups (20%) for UV cross-linking capability [41]. Devices were fabricated as previously described [42]. Briefly, glass and polyimide-coated glass capillaries (25 & 50 μm inner diameter) were filled with an IEM solution consisting of A-HPG (200 mg), Thiocure 333 (83 mg), 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methylpropan-1-one (2 mg) in methanol (347 μL) and water (250 μL). The glass capillaries filled with IEM solution were photo-exposed at 254 nm for 1 h, while polyimide-coated counter-parts were photo-exposed at 365 nm for 24–67 h. The capillaries were then cut into individual sections of 15 mm in length, embedded in a heat shrink tube and stored in 10 mM KCl solution. As electrodes, Ag/AgCl wires were used, the reference electrode was operated in a pipette tip filled with 4 w% Agar in 3 M KCl (agar bridge). Devices were operated via an in-house developed Arduino-based voltmeter.
2.2. GemIP operation and determination of fixed charges and delivery rates
As described previously by Waldherr et al. [33], a solution of 100 mM Gem(aq) at pH 4 was placed in the GemIP source reservoir and devices were operated at 25–100 nA (depending on device geometry, limited to 5 V) using custom Arduino-based devices. The channel loading was performed at 50 nA, indicated by increasing voltage over time, which reached a steady state value at which the channel was considered fully loaded with Gem, and devices were deemed ready for further experiments. Delivery rates were determined by operating GemIPs in a target volume of deionized water, PBS or 120 mM NaCl(aq). After operation, Gem concentration was measured via absorption at 267 nm with a NanoDrop (Thermo Fisher Scientific, USA). The pH of the target solutions was measured under stirring using an HI 221 pH/ORP meter (Hanna Instruments, Germany) connected to an XS Sensor Micro S7 pH electrode (XS Instruments, Italy)….
