Hybrid ultrasound-activated nanoparticles based on graphene quantum dots for cancer treatment

Abstract

Theranostic liposomes have recently found a broad range of applications in nanomedicine due to stability, the high solubility of biomacromolecules, bioavailability, efficacy, and low adverse effects. However, the limitations of liposomes concerning the short systemic circulation in the body, limited controllability of the release rate, and the inability of in vivo imaging remain challenging. Herein, the development of novel hybrid ultrasound-activated piezoelectric nanoparticles based on a hybrid liposome nanocarrier composed of poly(vinylidene fluoride‐trifluoroethylene), graphene quantum dots (GQDs), and Silibinin (a hydrophobic drug) is presented. The hybrid nanoparticles are an acoustically sensitive drug delivery platform that releases the biomacromolecules in a specific tissue area (through surface labeling with PD-1 antibody) in a non-invasive and controlled manner. We show that the developed hybrid nanoparticles (with an average outer diameter of ∼ 230 ± 20 nm) enable piezoelectric-stimulated drug delivery combined with simultaneous fluorescent imaging of cancer cells in vivo. Significant enhancement (>80 % up to 240 h) and tunable drug release from the nanocarrier through enhanced diffusion from the liposome membrane are demonstrated. Cytotoxicity assays using MCF-7, 4T1, and NIH3T3 cell lines exhibit no confrontational influence of nanoparticles on cell viability up to 125 µg/ml. The PD-1 antibody on the surface of the hybrid nanocarrier allows for selective delivery to breast cancer tumors and low biodistribution to other tissues. Our results affirm that the developed ultrasound-activated piezoelectric nanoparticles have great potential as multifunctional platforms with sustainable release profiles for the delivery of hydrophobic drugs to breast cancer, especially when the ability for adequate labeling and cell monitoring is valued.

Introduction

Over the past decade, one of the leading causes of global mortality with an estimation of > 10 million death has been a malignant tumor or malignant neoplasm known as cancer (Sung et al., 2021). One of the most challenging obstacles in cancer treatment is limiting the systemic toxicity effects of chemotherapeutic agents on the tumor environment, which contributes to the high mortality rates associated with cancer. Therefore, developing new approaches that selectively target cancer tissue seems essential (Hamblin, 2019, Mansoori et al., 2019). Nanomedicine based on customized nanoparticles have shown significant promise after several decades of scientific breakthroughs (Barjasteh et al., 2022).

Photodynamic therapy (PDT) is one of the most common methods in cancer treatment, combined with other forms of cancer therapy to improve treatment outcomes. In PDT, a chemical known as a photosensitizer (PS) kills cancer cells after being activated by light. The light may originate from a laser or any other source, such as light-emitting diodes (LEDs). This method boosts reactive oxygen species (ROS) levels, such as ¹O₂, within cells and their microenvironment (Agostinis et al., 2011). It is a non-invasive method and causes less destruction to surrounding tissues than other treatments. It also has fewer side effects due to its low toxicity. Studies have shown that ROS-responsive platforms, including PS, can destroy and kill cancer cells, usually with minimal damage to normal cells (Agostinis et al., 2011, Castano et al., 2006, 2004). Tetrapyrrole group (porphyrins, chlorins, bacteriochlorins, and phthalocyanine), synthetic dyes group (phenothiazinium, squaraine, and boron-dipyrromethene), and some natural molecular groups (such as hypericin, riboflavin, and curcumin) are examples of organic PS (Abrahamse and Hamblin, 2016). These systems not only enable us to treat cancer but also offer in vivo imaging simultaneously. The United States Food and Drug Administration has authorized some PS for pharmaceutical usage. However, these carriers have disadvantages, such as a lack of photostability, low solubility in water, and wavelength absorption range (O’Connor et al., 2009).

Recent studies have indicated that carbon nanostructures (CNs) are superior to PS concerning water dispersion and photostability(Han et al., 2019, Nie et al., 2020). Mainly, graphene quantum dots (GQDs) are an auspicious material for PDT and imaging applications due to their photoluminescence (PL) properties (Chen et al., 2020, Fan et al., 2019, Ge et al., 2014, Kuo et al., 2020, Mangalath et al., 2021, Mushtaq et al., 2022, Santos et al., 2021, Tabish et al., 2018, Chong et al., 2016, Ge et al., 2014, Jovanović et al., 2015, Kuo et al., 2017, Markovic et al., 2012, Ristic et al., 2014, Thakur et al., 2017, Zhang et al., 2018, Zhou et al., 2017). Ge et al. have demonstrated the potential of GQDs to generate singlet oxygen under blue, green, or even wide-spectrum light irradiation (such as xenon lamp spectrum). They have shown that GQDs can act as an efficient PS (through high singlet oxygen generation) and imaging agent for cancer cell monitoring (Ge et al., 2014). Despite these advantages, the biocompatibility of GQDs in clinical trials has been questioned (Hore et al., 2022, Maity et al., 2017, Sangam et al., 2022, Sapkota et al., 2017). As a result, numerous strategies, including surface modification of GQD with biocompatible polymers, have been used to reduce toxicity. For instance, Chandra et al.(Chandra et al., 2014) have shown that the agent’s toxicity could be mitigated by polyethylene glycol (PEG). Although PEG modification reduces cytotoxicity and ROS generation, intracellular penetration becomes more effective. In another study by Awad et al. (Awad et al., 2021), liposomes were employed to reduce the toxicity of GQDs. They have prepared liposome-encapsulated GQDs and triggered their release using ultrasound for delivering fluorescent probes to cancer cells. Their results have determined that GQDs can be delivered effectively to tumors with minimal toxicity by using low-frequency ultrasound (LFUS).

Combination therapy is a cornerstone of cancer treatment because it can overcome the challenges of tumor heterogeneity and drug resistance of cancer cells (Fan et al., 2017, Shrestha et al., 2019). Combining chemotherapy and PDT is an effective strategy that have recently attracted interest in clinical trials (Chen et al., 2019). For successful treatment, drug carriers should not be eliminated by the reticuloendothelial system (RES) to attain a high therapeutic index at the tumor (Abdel-kader, 2016, DeVita and Chu, 2008, Zimmermann et al., 2007). As a result, multifunctional drug delivery systems based on nanotechnology have emerged. To this end, liposomes have attracted much attention due to biocompatibility, portability of hydrophilic and hydrophobic drugs, good dispersion, ease of preparation, selectivity, physicochemical properties, and superior cellular uptake efficiency (An et al., 2017, Yoon et al., 2018, Yoon et al., 2017, Zhang et al., 2019, Zhao et al., 2018). The encapsulated drug must be released from liposomes before it can be absorbed by the surrounding cells in the tumor environment. Therefore, a rapid release is essential for fast-growing tumors (Drummond et al., 1999). Therefore, stimuli-responsive liposomes have been considered to improve therapeutic efficacy by increasing drug availability in the tumor. It is also possible to modify liposomes or individual constituents to have different surface structures for specific purposes, for example, targeting cells of tissues or imparting a “smart” character (Kim et al., 2019, Kou et al., 2020). Stimuli-responsive liposomes are very attractive for drug delivery because the macromolecules are released on demand when external or internal stimuli are presented (Lin et al., 2019). Several attempts over the last decade have been made to optimize local therapeutic delivery by stimulating drug release from liposomes. The hypothesis is that an environmental stimulus (such as enzymatic, hyperthermia, and pH) rearranges the structure of the liposome membranes and releases the encapsulated drug. Among different external techniques, ultrasound waves offer specific advantages for targeted drug delivery regardless of the depth of tissue (e.g., liver, brain, pancreas, and breast) (Ahmadi et al., 2020, Delaney et al., 2022, Lin et al., 2020, Walsh et al., 2021). Their membranes restrict the penetration of drugs into cells. Intensifying drug delivery within cells is challenging. However, employing ultrasonic waves is a promising strategy to overcome this problem. Ultrasonic waves penetrate soft tissues and induce stable cavitation, leading to increased cell membrane permeability. Although the thermal and chemical effects induced by ultrasonic waves are equally valuable for enhancing the permeability of the cell membrane, sonoporation is the primary mechanism by which drug permeability is enhanced in cancer cells (Awad et al., 2021, Sundaram et al., 2003). Liposomes, like cell membranes, are similarly susceptible to sonoporation. Consequently, drug release is controlled temporally and spatially (AlSawaftah et al., 2021, Awad et al., 2021, Awad et al., 2019, Ben Daya et al., 2021, Elamir et al., 2021, Salkho et al., 2018). Combining stimuli types, such as HIFU (high-intensity frequency ultrasound), radiofrequency, or microwave, with thermosensitive liposomes (TSL) in local hyperthermia of cancerous tumors increases the membrane permeability and thus the drug release kinetics. Drug release from TSL usually occurs at T_c and above (40–45 °C). However, one drawback of thermosensitive liposomes is that the entrapped therapeutics can be prematurely released into the blood circulation before reaching the cancer cells. In order to overcome this limitation, a non-thermal ultrasound strategy has been proposed to improve the efficiency of drug delivery to cancerous tumors. For this purpose, liposomes must be sensitized to ultrasonic waves by piezoelectric materials (Cotero et al., 2022, Lin et al., 2014, Lin et al., 2019, Marino et al., 2018, Rwei et al., 2017, Shalaby et al., 2020, Suzuki et al., 2010, Yin et al., 2013, Zhang et al., 2020, Cotero et al., 2022).

On the other hand, some natural plant compounds have shown anticancer properties, which have substantially impacted cancer treatment (Tiwari and Mishra, 2015). For instance, Silibinin, a natural flavonoid, can cause apoptosis when it targets cellular signaling pathways, including mitochondrial apoptosis, ROS/receptor-mediated apoptosis, growth and transcription factors, angiogenesis, and metastasis. Thus, the drug presents a tremendous therapeutic potential for selective and efficient cancer treatment (Li et al., 2010, Singh et al., 2005). Previous studies have shown that Silibinin affects the production of ROS in MCF-7 breast cancer cells (Jiang et al., 2015, Wang et al., 2010a, Wang et al., 2010b, Zheng et al., 2017).

In this work, we have hypothesized that the hybridization of GQDs and Silibinin with an electroactive biopolymer addresses the cytotoxicity issues while enabling us with an ultrasound-stimulated drug delivery system. This study presents a novel nanocarrier for combination therapy based on dual stimuli-responsive liposomes enabling it to be activated by light and ultrasonic waves. The novelty of this work relies on the design of a hybrid ultrasound-activated piezoelectric nanoparticles enabling triggered and tunable release rate of natural-derived therapeutics. The nanoparticles have been designed based on piezoelectric macromolecules (P(VDF-TrFE)) and graphene quantum dots (GQDs). The concept is schematically shown in Fig. 1. The combination of P(VDF-TrFE) nanoparticles and CQDs offers the capacity of dual-sensitivity (light and ultrasound) for better release rate control. The piezoelectric organic nanoparticles are conjugated with Silibinin, a well-known antioxidant material and antitumor agent (Binienda et al., 2020, Deep and Agarwal, 2010, Dheeraj et al., 2018, García-Maceira and Mateo, 2009). The hybrid nanoparticles, including Silibinin@P(VDF-TrFE) and GQD@P(VDF-TrFE), are encapsulated in liposomes by dehydration of the dry lipid film. We have hypothesized that the hybrid nanocarrier damages the membrane of cancer cells by generating ROS, and silibinin disrupts the function of the cell nucleus. The application of the dual-stimuli responsive nanocarrier with a high drug loading capacity and sustained release ability is demonetarized. The biocompatibility and biodistribution of the developed materials in the body are presented by in vitro and in vivo assays. The stimuli-responsive character of the hybrid nanocarrier and its tunability pave the way for new tactics to develop novel drugs based on piezoelectric nanostructures.