Peanut Shaped Microrobots for Anticancer Therapy

Abstract

iron nanoparticles have been used in cancer research for their anticancer activity. The mechanism of action for this activity tends to be ferroptosis. Inspired by this we made hematite based peanut shaped microrobots and demonstrated their anticancer activity. We examined how peanut-shaped microrobots impacted HT1080 cancer cell lines, aiming to understand the mechanism behind cell death. Surprisingly, we discovered that unlike iron particles, these specific microrobots did not trigger ferroptosis. Additionally, when we tried to understand the mechanism behind the cell death by using ferroptosis inhibitors, they did not rescue the cells from death as expected. These results suggest that the cytotoxic effects of peanut-shaped microrobots on HT1080 cells may involve alternative pathways rather than ferroptosis.

I. INTRODUCTION

Extensive interest has been focused on employing microrobots in biomedical applications such as targeted drug delivery, microsurgery, or thrombolysis [1], [2]. Realizing these goals has the potential to revolutionize the healthcare industry. One specific use of microrobots for biomedical applications is for cancer therapy. Current conventional medical practices for cancer therapies rely on treatments such as radiation and chemotherapy which can create many adverse side effects. Therefore, targeting treatment to the relevant region while reducing the harmful dosage to the rest of the body would be a significant achievement. Microrobots are potentially well suited for this since, unlike passive drugs that cannot be easily targeted to a specific region, microrobots can be controlled, and therefore could be guided to a specific site for targeted treatment [1], [2], [3]. Indeed, proof of concept experiments have been performed in vitro that demonstrate the targeted delivery of drugs to kill cancer cells [4], [5], and in vivo showing the feasibility of maneuvering microrobots in the body [6], [7], [8]. However, attaching drugs to microrobots and releasing them at the site can be challenging since proper surface functionalities of the microrobot are needed and automatic or triggered release of drugs is generally difficult to achieve [9], [10], [11]. Therefore, other methods of triggering cancer cell death using microrobots is highly desired.

Aside from killing cancer cells via drug delivery, cell death can be triggered by other mechanisms, such as necrosis by mechanical or chemical stress [12], [13], ferroptosis by lipid peroxidation brought about by reactive-oxygen species (ROS) and iron [14], or apoptosis which is a programmed cell death that can be triggered by certain stresses, for example, the release of bioactive molecules, ROS, or other chemicals, as well as mechanical stress [15], [16].

Ferroptosis, characterized by a redox imbalance and elevated intracellular lipid peroxide levels, represents a distinctive form of regulated cell death dependent on iron. Key features include the inhibition of the lipid peroxide repair enzyme glutathione peroxidase 4, excess ferrous iron presence, and polyunsaturated fatty acid peroxidation. In cancer therapy research, iron-based nanoparticles (NPs) have emerged as promising agents for inducing ferroptosis. These nanoparticles release ferrous (Fe²⁺) or ferric (Fe³⁺) ions within acidic lysosomes, triggering ferroptotic cell death. Magnetic fields are often employed to target iron-based NP treatments to specific sites within the body. Additionally, studies have demonstrated the ability of magnetic fields to impede cancer cell proliferation, further highlighting the therapeutic potential of ferroptosis induction in cancer treatment [17], [18].

By exploiting the vulnerabilities of cancer cells, which often exhibit elevated iron levels and increased sensitivity to oxidative stress, ferroptosis induction represents a targeted strategy to selectively eliminate malignant cells while sparing normal tissues. The therapeutic potential of ferroptosis in cancer treatment has garnered considerable attention, with ongoing research focusing on elucidating its underlying mechanisms and exploring novel ferroptosis-inducing agents for clinical applications [19], [20], [21].

Large stresses that damage the cell can result in necrosis, which is cell death resulting from a compromise of cellular function or structural integrity. Microrobots can physically disrupt the cell membrane [22], [23], [24], cause hyperthermia [25], release toxic drugs [10], or produce ROS [26] that leads to oxidative stress and death of the cell.

In this work, hematite microrobots were used to induce cell death of Fibrosarcoma HT-1080 cancer cells on a 96 well plate. It was found that microrobots cultured with the cells result in a high degree of cell death compared to the control, therefore demonstrating the potential for these microrobots to be used in future cancer therapy treatments. Ferroptosis as the method of cell death was explored and ruled out, therefore future work is needed to elucidate the method of cell death and whether necrosis or apoptosis is occurring.

II. MOTIVATION

The investigation into the anticancer properties of microrobots is a burgeoning area of research with promising potential. Microrobots, owing to their unique capabilities such as precise targeting and controlled manipulation, have emerged as innovative tools in cancer therapy. These microrobots can be engineered to deliver therapeutic payloads directly to tumor sites, enabling localized treatment while minimizing systemic toxicity. Additionally, their ability to navigate through complex biological environments offers opportunities for enhanced drug delivery and therapeutic efficacy. By harnessing the strengths of microrobotic technology, researchers aim to develop novel strategies for combating cancer, including targeted drug delivery, localized therapy, and minimally invasive interventions. While still in the early stages of development, the anticancer effect of microrobots holds immense promise for revolutionizing cancer treatment paradigms in the future [3], [27], [28].

III. MATERIALS AND METHODS

A. Synthesis of Hematite Particles

Figure 1 illustrates the synthesis procedure of the peanut shaped microrobots. We followed a procedure that was described elsewhere [29], [30]. To initiate the synthesis, a glass bottle was filled with a 100 mL solution of Fe₃Cl₃ (at a concentration of 56% w/v). To this, 90 mL of 6M NaOH was added. Subsequently, 10 mL of 0.6 M Na₂SO₄ was introduced, and the mixture was vigorously agitated for approximately 10 minutes. Following this agitation period, the bottle was subjected to an oven environment at 100°C for a duration of 8 days, facilitating the discernible separation between solid particles and the liquid supernatant. The sample was then washed thrice via centrifugation, supernatant removal, and replacement with DI water. Characterization via SEM imaging (refer to Fig. 2) illustrates the resultant peanut-shape of the particles which have an average length of roughly 2 μm and a diameter measuring 0.5 μm. The peanut-shaped particles possess a magnetic moment along their short axis, as noted in previous reports [31], [32].

Fig. 1:

Fabrication of peanut shaped microrobot

Fig. 2:

(a) Schematic of the experiments showing the microrobots and the electromagnetic setup (b) A cartoon of the microrobots showing their magnetic moment which, in the absence of other torques of similar magnitude, aligns with an externally applied magnetic field. (c) SEM image of the peanut shaped microrobots.

B. Cell Culture

The Fibrosarcoma HT-1080 cell line was cultured in Dulbecco’s Modified Essential Medium/Nutrient Mixture F-12 (DMEM/F-12, Gibco, BenchStable, USA), supplemented with 10% fetal bovine serum (FBS, Sigma, USA) and 1% penicillin-streptomycin, and maintained under standard physiological conditions (37°C with 5% CO₂). Cultures were regularly passaged upon reaching approximately 90% confluency using TrypLE (12604-013, GIBCO, USA) and subcultured. Continuous exponential growth was observed until the eighth passage under these conditions.

C. Measurement of Cell Viability with the MTT Assay

To evaluate cell toxicity and inhibitor profiles, cells were seeded onto 96-well plates at a seeding density 2×10⁴ and 1×10⁵ cells and cells were incubated with different concentration (400μg/ml-0.62μg/ml) of peanut shape microrobots, and ferroptosis inhibitors. After a 24-hour treatment period, the culture medium was removed. Cell viability was assessed by adding complete growth medium supplemented with 1/10 volume of 5 mg/mL MTT (Sigma-Aldrich dissolved in PBS) to each well and incubating for 2 hours until formazan crystals developed. Following incubation, the medium was aspirated, and the formazan crystals were dissolved in DMSO with an additional 10-minute incubation at 37°C. Absorbance of the formazan solution was then measured at 560 nm using a microplate spectrophotometer.

D. Electromagnetic Setup

Rotating magnetic fields were used to move the microrobots by a rolling motion. The fields were created by electromagnetic coils along three axes, as described elsewhere [33]. The fields produced by the coils are of a sinusoidal nature and are controlled via a custom computer software that modulates the amount and direction of current that is sent to each coil. A joystick is used to communicate with the computer and set the rolling direction. The field strength is approximately 5 mT in the region of interest.

E. Statistical analyses

We employed Prism and Excel software for data management and analysis. Statistical analyses were conducted using T-tests and one-way ANOVA to assess significant differences among experimental conditions. For all assays P<0.05 was considered significant.

IV. RESULTS AND DISCUSSION

A. Peanut shaped microrobots and their motion

The presence of hematite gives the microrobots a magnetic moment (as shown in Fig. 2(b)) which, in the absence of other torques of similar magnitude, aligns with an externally applied magnetic field. Therefore, a rotating magnetic field can induce a rolling motion (Fig. 2(a)). Fig. 2(c) shows an SEM image of the peanut shaped microrobots demonstrating their size and shape. There have been various studies demonstrating the motion and microrobotic behavior of these particles [32], [34]. Fig. 3 shows the motion of the 2 μm microrobots in an external magnetic field and the supporting video (2x playback) shows the peanut shaped microrobots being driven to a HT-1080 cell.

Fig. 3:

Series of images showing a peanut shaped microrobot being moved to a cancer cell by magnetic rolling at a frequency of 30 Hz. The microrobot is circled as a visual aid.

B. Cytocompatibility of microrobots

Magnetic microrobots have garnered sustained interest in biomedicine due to their capacity to maneuver within biological samples without causing harm to cells or tissues [1], [35]. Previous experiments have shown that these peanut shaped particles are cytocompatible with cells especially NIH/3T3 cells [36]. The NIH/3T3 is a fibroblast cell line that is isolated from a mouse NIH/Swiss embryo. This cell line (ATCC^® CRL-1658) is highly sensitive to sarcoma virus focus formation and leukemia virus propagation. Thus, it serves as an effective control for demonstrating the anticancer effect of these peanut shaped microrobots since we use a fibrosarcoma cell line (HT-1080) for the experiments.

C. Anticancer effect of the peanut microrobots

HT1080 (ATCC^® CCL-121^™) represents a human fibrosarcoma cell line that originated from a 35-year-old Caucasian male donor. These cells exhibit adherent growth patterns and display an epithelial morphology. Additionally, HT1080 cells have been observed to efficiently form primary tumors and distant metastases upon implantation into immunocompromised mice. Their robust characteristics render HT1080 cells suitable for a wide range of applications, encompassing both in vitro and in vivo experimentation [37], [38]. Fig. 4 shows the anticancer activity of the peanut shaped microrobots in HT-1080 cells using MTT assay. Fig. 4(a) is the control with only HT-1080 cells (seeding density = 2 × 10⁴). Fig. 4(b) shows the cancer cells in contact with the 2 μm peanut shaped microrobots (50 μg/ml) for 24 hours. As seen from the figure 67% of the HT-1080 cells die after co-culturing with the microrobots showing the anticancer activity of the robots.

Fig. 4:

(a) Control showing only HT-1080 cells (b) Anticancer activity of 2 μm peanut shaped microrobots showing the decrease in cell viability using MTT assay.

D. Effect of concentration on cell death

Next, we study the effect of the concentration of the microrobots on HT-1080 viability using the MTT assay. The HT1080 cell line was seeded in 96-well plates (1×10⁵ cells/well). In the test groups, microrobot concentrations of 400 μg/ml–12.5 μg/ml dosage significantly showed reduced cell viability (p < 0.05) compared to the control group (0 μg/ml). In contrast, there were no differences between microrobot concentrations of 10 μg/ml – 0.6 μg/ml doses and control group (Fig. 5(a)). We repeated the experiments and again in the test groups, 100 μg/ml–12.5 μg/ml microrobot concentration significantly showed reduced cell viability (p < 0.05) compared to the control group (0 μg/ml). 6.2 μg/ml was weakly significant (Fig. 5(b))

Fig. 5:

Microrobot concentration effect on HT-1080 cell (1×10⁵ cells) viability. The two panels show two MTT assays showing the decrease in cell viability corresponding to different microrobot concentrations.

As seen from these experiments’ higher concentration of microrobots is needed for effective cell death. Below 12.5 μg/ml there is no significant effect on cell viability, after 200 μg/ml there is only a small decrease in cell viability with increasing microrobot concentrations.

E. Mechanism studies

HT-1080 serves as a commonly employed cellular model in ferroptosis investigations due to its notable susceptibility to compounds known to trigger ferroptosis, such as Erastin and RSL3 [39]. RSL-3 (RAS-selective lethal) is a ferroptosis inducing agent and promotes ferroptosis induced cell death in cancer cells [40].

We evaluated the compatibility of the microrobots with HT1080 cells and potential effect of the ferroptosis inhibitors in alleviating cell death by subjecting them to cytotoxicity tests. We used two inhibitors that inhibit ferroptosis activity Ferrostatin-1 and Liproxtanin-1. After a 24-hour incubation period with the microrobots (0.75μg/ml-50 μg/ml) in presence and absence of inhibitors - Ferrostatin-1 (5μM) and Liproxstatin-1 (3μM)) cell viability was assessed using an MTT assay, accompanied by the capture of brightfield images to examine cellular morphology and growth patterns. Our findings indicated that the cells maintained their normal morphology and growth (Fig. 6). This suggested that the presence of ferroptosis inhibitors did not make a difference compared to the absence of inhibitors thus showing that ferroptosis was not the mechanism of cell death. In contrast, we performed cell viability assays with RSL-3, which is a ferroptosis inducer. When the cells were incubated for 24 hours with RSL-3 (0.078μg/ml-2.5 μg/ml), significant cytotoxicity was seen even at low concentrations as expected (Fig. 7). The addition of Ferrostatin-1 in Fig. 7 leads to an increase in cell viability since Ferrostatin-1 is a ferroptosis inhibitor.

Fig. 6:

Ferroptosis inhibitors effect on HT1080 cells. HT1080 cell viability (2×10⁴ cells) after a 24-hour incubation period with the microrobots (0.75μg/ml-50 μg/ml) in present and absent of inhibitors - Ferrostatin-1 (5 μM) and Liproxstatin −1 (3 μM). Fer = Ferrostatin, Lip = Liproxstatin. Data represent the mean ± sd. The experiment was replicated at least three times. Statistical analysis was performed using an unpaired two-tailed Student t-test.

Fig. 7:

Concentration dependent effect of RSL-3 on the viability of HT-1080 cells. The addition of Ferrostatin-1 shows an increase in cell viability since Ferrostatin-1 is a ferroptosis inhibitor and inhibits RSL-3 activity. Fer = Ferrostatin. Data represent the mean ± sd. The experiment was replicated at least three times. Statistical analysis was performed using an unpaired two-tailed Student t-test.

The results in Figure 5 show that peanut shaped micro-robots induce cell death in a dose-dependent manner. However, the application of Ferrostatin-1 (Fer-1) and Liproxstatin −1 (Lip-1), potent antioxidants known for neutralizing free radicals, did not prevent cell death. These antioxidants are specific for countering cell damage caused by ferroptosis, prompting their use with the micro-robots to determine if ferroptosis was the cause of cell death. The protective mechanism of these antioxidants is related to their radical trapping activity, they both can directly scavenge lipid peroxyl radicals and Lip-1 can also modulate enzymes in lipid metabolism and antioxidant pathways. A possible explanation for the lack of ferroptosis induction by the peanut-shaped hematite micro-robots could be their size.

For ferroptosis to occur, a smaller particle size is necessary to release iron, which, in the presence of H₂O₂, can catalyze lipid peroxidation in the membrane, creating pores that cause cellular contents to leak out. The large size and water insolubility of the microrobots hinder cellular membrane uptake, unlike iron oxide nanoparticles, which cells more readily assimilate. Hematite micro-robots might still cause oxidative stress, but this needs more investigation. It seems more likely that the hematite is causing physical stress on the cells, leading to membrane disruption or possibly triggering apoptosis. We also recognize other inhibitors, like Z-VAD for apoptosis, Nec-1 for necroptosis, and Baf-1 for autophagy, that block different cell death pathways. We plan to assess these inhibitors in future experiments to elucidate the pathways involved in micro-robot induced cell death. Through a combination of experimental techniques such as transcriptomics, proteomics, and functional assays, we can unravel the specific cellular processes triggered by microrobots and identify potential alternative mechanisms of cell death.

V. CONCLUSION

In this study, we observed the cytotoxic effects of peanut-shaped microrobots. We demonstrated the design, synthesis, and actuation of 2 μm peanut shaped microrobots in the presence of fibrosarcoma cells (HT-1080). We examined how peanut-shaped microrobots and RSL3 impacted HT1080 cell lines to understand the mechanism of cell death. Unexpectedly, we found that unlike iron particles, these specific microrobots did not initiate ferroptosis [20], [41]. Moreover, attempts to decipher the mechanism of cell death using ferroptosis inhibitors failed to rescue the cells from death. Further investigation is warranted to explore alternative mechanisms underlying the observed cytotoxic effects.