ICG-loaded gold nano-bipyramids with NIR activatable dual PTT-PDT therapeutic potential in melanoma cells

Highlights

• The intrinsic PDT properties of uncoated AuBPs were studied using two NIR sources.

• By loading the ICG, the PTT and PDT performances of AuBPs were noticeably enhanced.

• The nanosystems’ dual PTT-PDT activity was validated in vitro in B16-F10 cells.

Abstract

A great amount of effort is directed towards the progress of cancer treatment approaches aspiring to develop non-invasive, targeted and highly efficient therapies. In this context, Photothermal (PTT) and Photodynamic (PDT) Therapies were proven as promising. This work aims to integrate the therapeutic activities of two near-infrared (NIR) photoactive biomaterials - gold nano-bipyramids (AuBPs) and Indocyanine Green (ICG) - into one single targeted hybrid nanosystem able to operate as dual PTT-PDT agent with higher efficiency compared with each one alone. Firstly, different aspect ratio’ AuBPs were systematically investigated in water solution for their intrinsic ability to efficiently generate toxic reactive oxygen species, namely oxygen singlet (¹O₂), under NIR laser irradiation, as this effect is less investigated in literature. Interestingly, the photodynamic activity of AuBPs measured by monitoring the photooxidation of 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA) – a well-known ¹O₂ sensor, is important, counting for 30 % decrease in ABDA optical absorbance for the most active AuBPs, well-correlating with the previously determined photothermal conversion efficiency. Furthermore, ICG was successfully grafted onto the Poly-lactic acid (PLA) coating of plasmonic nanoparticles and, consequently, the as-designed fully integrated hybrid nanosystem shows improved PTT-PDT performance in solution. Specifically, by triggering simultaneous PTT-PDT activities, the ¹O₂ amount is doubled, while the heating monitoring shows higher and faster increase in temperature compared to AuBPs alone. Finally, the efficiency of the combined PTT-PDT therapeutic activity was validated in vitro against B16-F10 cell line by covalent conjugation of the nanosystem with Folic Acid, which ensures the cellular recognition by overexpression of folate receptor.

Introduction

One of the main concerns of the biomedical research community remains the early diagnosis and treatment of cancer, which is one of the leading causes of human mortality worldwide. According to the World Health Organization in 2018, the incidence and death rates caused by all types of cancer are predicted to increase by 2040 with more than 60 % and 70 %, respectively. The International Agency for Research on Cancer reports that of a total of over 18 million new cases in 2018, roughly 1.6 % of the patients were diagnosed with skin melanoma and 21 % of them turned into fatalities [1]. An early detection ensures an overall 5-year survival rate of 95 % diminishing the probability of relapse by regional and/or distant metastasis, respectively [2]. To date, the most common therapeutic approaches include surgery, (bio)chemo-therapy, immunotherapy and, more recently, the photodynamic therapy was considered for clinical trials [3]. Nonetheless, these therapeutic strategies present some limitations related to their side effects leading to skin and gastrointestinal toxicity as well as reduced efficiency due to patient resistance. In this clinical context, new strategies with improved biochemical and therapeutic performances are considered an urgent need for the health care system.

At the time being, a lot of the scientific effort is put into the development of therapeutic applications based on (bio)materials with improved specificity, biocompatibility, targeting capabilities and localized distribution at the tumorous site, thus aiming to replace the current invasive cancer treatment strategies such as chemo-, radio- and immunotherapy [[4], [5], [6], [7]]. In this context, photosensitizers (PS) have gathered a lot of attention. Concretely, PS are able to absorb electromagnetic radiation in the visible and/or Near-Infrared (NIR) and transfer the energy to the local environment by non-radiative relaxation processes. Upon light exposure, the PS is located energetically on an excited singlet state, the return to the ground state can be realized based on two mechanisms: (i) non-radiative relaxation from a higher to the ground vibrational excited singlet state by heat generation followed by the transition to an excited triplet state where (ii) the occurrence of photochemical reactions via electron transfer are enabled [8]. These phenomena have led to the development of the Photothermal (PTT) and Photodynamic (PDT) therapies, which proved themselves to be minimally invasive, present fewer side effects and have improved selectivity [8,9]. Specifically, PTT relies on the light-to-heat conversion [10], while PDT occurs due to the electron transfer to the oxygen molecules in the environment, thus generating toxic reactive oxygen species (ROS), which oxidize the cancerous cells and induce the cellular death [11]. Conventional PTT and PDT are being employed using various PS molecules like porphyrins, chlorins, bacteriochlorins and phtalocyanines [8] as well as synthetic dyes, such as Methylene Blue (MB), Rhodamine B and cyanine dyes, are systemically used for ROS generation [12,13]. A symmetrical cyanine dye known to exhibit both PTT and PDT properties [14] is the Indocyanine Green (ICG) molecule, a Food and Drug Administration (FDA) approved drug [15] currently implemented in various imaging and therapeutic applications [16]. Despite its therapeutic activity, a lot of effort is put into overcoming two major limitations of its use, namely its low yield and high instability in aqueous solution [17], which make ICG hard to manipulate and graft. Considerable advances are being made in the synthetization of nanoparticles to bind or encapsulate the PS molecules in order to preserve their photodynamic activity. Yi et al. give an overview of the organic and inorganic nanocarrier systems designed based on polymers and phospholipids but also luminogens, which are efficiently used in PDT applications [18,19]. Several self-assemblies were proven to be efficient PTT and PDT nanodrugs based on biomolecules showing improved tumor ablation capabilities [[20], [21], [22], [23], [24]].

Plasmonic nanoparticles, due to their unique optical properties, especially, the Localized Surface Plasmon Resonance (LSPR), represent a class of nano-systems worth exploiting. The LSPR is highly dependent on the composition, size and shape of the nanostructures, therefore, the high variety of designs and dimensions leads to tunable and specific optical features, which, subsequently, allow their efficient implementation in biomedical applications from biosensing and diagnostics [17] to therapy [25]. For instance, metallic nanostructures were demonstrated to play an important role in innovative cancer treatment strategies [26,27]. Taking advantage of their highly reactive surface, efficient therapeutic agents were developed showing multi-modal activation: (i) internal stimulation by factors like pH, temperature [28] and (ii) external triggers [29]. Concretely, plasmonic nanostructures were successfully employed as nanocarriers for localized drug delivery by simultaneously triggering the drug release using pH-and thermal responsive molecules as well as the intrinsic capabilities of the nanoparticles to dissipate heat upon external irradiation [26,27]. Furthermore, different surface modifications ensure not only improved biocompatibility and specificity by cellular recognition functionalization, but also encourage the integration of multiple functionalities onto the same platform leading to theragnostic and synergistic plasmon-assisted PTT and PDT therapeutic nanoplatforms as reports are found in the specialty literature using various geometries like nanospheres [28,29], nanoclusters [30], nanorods [[31], [32], [33]], nanostars [34,35], combined protein-nanoclusters [[36], [37], [38]], nanoshells [39] and nanorings [40].

Nonetheless, nanostructures with absorption bands located in the Near Infra-Red (NIR) region are of interest for two main reasons: (i) in the NIR the tissues are optically transparent ensuring a higher penetration depth and (ii) the nanoparticles present better absorption/scattering features. Gold bipyramidal-shaped nanoparticles (AuBPs) exhibit attractive optical properties by allowing the fine tuning of their LSPR along the electromagnetic spectrum as well as presenting intrinsic non-radiative properties, thus conveying their suitability for therapeutic applications.

Previously, we have demonstrated the intrinsic property of the AuBPs to efficiently convert light-to-heat using two excitation laser lines in NIR window thus determining high photothermal conversion efficiencies spanning from 40 to 97 % with regard to the excitation wavelength employed [41]. The high conversion rates are registered for nanoparticles with LSPR close to the resonance condition: AuBPs with LSPR at 802 nm for the 785 nm laser line and AuBPs 812 nm for the 808 nm source, both exceeding the biologically relevant temperature (37 °C) with 9 and 15 °C, respectively, proving their capability to further induce intrinsic hyperthermia.

Few studies address the possibility of using these sharp elongated nanostructures in PDT applications. By the functionalization with silica layers to ensure MB grafting [42] or directly with the sulfonated alphthalocyanine (AIPcS) PS [43,44], AuBPs were proven to be efficient nanoplatforms for PDT. To our knowledge, currently, there is only one study approaching the use of AuBPs as PDT agents in the absence of a PS molecule, which demonstrates the generation of oxygen singlet (¹O₂) by polyelectrolyte-coated AuBPs at low power laser densities and the influence of their absorption characteristics [45]. Nonetheless, the intrinsic photodynamic contribution of the AuBPs themselves, without coating, was not addressed as well as the nanoparticles’ dual PTT-PDT activity.

In this work, we aim to design a single AuBPs-based hybrid nanosystem able to integrate the dual PTT-PDT in order to develop an efficient innovative targeted plasmon-assisted therapeutic strategy. In this context, different aspect ratio’ AuBPs were tested in the presence of the 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA) molecule - a well-known oxygen singlet (¹O₂) sensor, in order to evaluate their intrinsic photodynamic activity with regard to two NIR laser lines, 785 and 808 nm, respectively. The ability to generate ¹O₂ was evidenced by the monitorization of the photooxidation reaction of ABDA – spectroscopic measurements indicate a 30 % decrease in ABDA optical response for the most efficient AuBPs, being in good agreement with the photothermal conversion efficiency determinations. Sequent, to ensure the biocompatibility of the nanosystem, the AuBPs were coated with Poly-lactic acid (PLA), which also provide the hydrophobic matrix for the Indocyanine Green (ICG) molecule grafting. The combined effect of the nanostructures’ and ICG– herein used as enhancer of the AuBPs intrinsic photothermal and photodynamic properties, is demonstrated by the doubling of the ¹O₂ amount as well as by the additional 2 °C increase in temperature. Finally, after the conjugation of the as-designed hybrid nanoplatform with folic acid as cellular recognition of the folate receptor overexpressed by the B16-F10 melanoma cell line, the efficiency of the combined PTT-PDT therapeutic activity was successfully validated in vitro.