Biohybrid Micro-Robots for Targeted Drug Delivery in Cancer Therapy ^†

Abstract

The development of biohybrid micro-robots represents a groundbreaking advancement in targeted drug delivery for cancer therapy, offering unprecedented precision and reduced systemic toxicity. These microscale robots integrate synthetic materials with biological components such as bacteria, algae, red blood cells, or spermatozoa, capitalizing on the inherent motility, biocompatibility, and targeting capabilities of living organisms. This hybridization enables active navigation through complex biological environments, overcoming physiological barriers such as the blood–brain and endothelial junctions that impede traditional nanoparticle-based systems. In this study, we propose a multi-functional biohybrid micro-robotic platform composed of magnetically actuated synthetic chassis coated with doxorubicin-loaded lipid vesicles and tethered to Magnetospirillum magneticum for propulsion and tumor-homing capabilities. The results underscore the promise of biohybrid micro-robots as intelligent, minimally invasive agents for next-generation oncological therapies, capable of delivering chemotherapeutics with enhanced spatial and temporal accuracy. Future work will focus on clinical translation pathways, biosafety evaluations, and scalability of production under Good Manufacturing Practice (GMP) standards.

1. Introduction

Cancer remains a leading cause of morbidity and mortality globally, with over 10 million deaths attributed to malignant neoplasms annually according to the World Health Organization (WHO). Despite significant advancements in diagnostic and therapeutic strategies, the primary challenge in effective cancer treatment lies in the efficient and selective delivery of chemotherapeutic agents to tumor tissues while minimizing collateral damage to healthy cells [1,2]. Traditional systemic chemotherapy often lacks specificity, leading to severe side effects, acquired resistance, and poor patient compliance [3].

Recent advancements in nanotechnology, biotechnology, and micro-fabrication have paved the way for innovative solutions to this longstanding problem [4]. Among these, the emergence of biohybrid micro-robots—a synergistic integration of synthetic microstructures with biological propulsion and navigation systems—offers a disruptive paradigm shift in targeted drug delivery systems (TDDS) [5,6]. These micro-robots are capable of actively navigating through complex biological environments, guided either autonomously or through external control mechanisms, and releasing therapeutic payloads precisely at the pathological site [7].

In Figure 1, biohybrid micro-robots are micro- or nano-scale devices that combine artificial components (e.g., metallic or polymeric chassis, drug reservoirs, sensors) with biological actuators or organisms (e.g., bacteria, spermatozoa, algae, immune cells) [8]. This integration leverages the biological system’s inherent adaptability, propulsion mechanisms, biocompatibility, and tumor-homing abilities while enabling external or autonomous control through embedded nanotechnology [9,10].

For instance, flagellated bacteria like Magnetospirillum magneticum can swim through viscous environments and magnetically respond to external fields, making them ideal candidates for magnetic navigation and deep-tissue penetration. Meanwhile, red blood cells (RBCs) offer long circulation times and natural stealth capabilities from immune clearance [11,12].

This study aims to present a study on the design, development, and evaluation of biohybrid micro-robots for targeted drug delivery in cancer therapy. Specifically, literature on biohybrid robotic systems in oncology was reviewed to explore the architecture and fabrication techniques of bacterial-based micro-robots. Methodology for drug loading, targeting, navigation, and payload release was examined to design a magnetically controlled micro-robot using M. magneticum and a biocompatible polymer chassis embedded with pH-sensitive doxorubicin nanocarriers (Figure 2). As a result, fluorescent quantum dots were integrated for simultaneous imaging and motion tracking in vivo. In-depth performance analysis was conducted on targeting efficiency, propulsion behavior, drug-release kinetics, and tumor cytotoxicity across various cancer models.

2. Literature Review

The development of drug delivery systems (DDSs) has undergone significant transformations over the past five decades. Traditional administration techniques, oral, intravenous, and intramuscular, often suffer from nonspecific distribution, rapid systemic clearance, and dose-dependent toxicity [13,14]. In response to these limitations, nanomedicine has introduced polymeric nanoparticles, liposomes, dendrimers, and micelles that can encapsulate drugs and improve their pharmacokinetics. However, passive diffusion and the enhanced permeability and retention (EPR) effect remain the dominant targeting mechanisms in synthetic nanocarriers, limiting their effectiveness in heterogeneous tumor environments [15].

Recent research has shown that synthetic nanocarriers often face challenges in traversing biological barriers such as the extracellular matrix, endothelial walls, and the blood–brain barrier [16]. As a result, a paradigm shift toward active delivery systems that can autonomously navigate and respond to tumor microenvironments has emerged—ushering in the era of micro/nano-robotics [17,18].

Micro/nano-robots are untethered, miniaturized devices capable of controlled locomotion and interaction with the physiological milieu at the cellular and subcellular levels [19,20]. Inspired by biological motility strategies, they are often engineered to be self-propelling using mechanisms such as flagellar motion, surface tension gradients, or external field actuation (e.g., magnetic, acoustic, or light stimuli) [21,22]. Their promise lies in the ability to actively navigate toward target tissues, bypass obstacles, deliver payloads with spatial and temporal control, and even perform in situ diagnostics [23].

The integration of biohybrid strategies into micro-robotics is particularly promising because it enables devices to combine the precision and programmability of synthetic materials with the biological functionality and adaptability of living organisms [21]. The literature review results reveal rapid growth in biohybrid micro-robot research, with particular emphasis on bacterial and sperm-based platforms for their high propulsion efficiency and targeting specificity [23]. The current trends are shifting toward multifunctional platforms that integrate diagnostics, therapy, and real-time tracking. Biodegradable and FDA-approved materials are used to ease regulatory translation. Hybrid actuation schemes combining biological motion with external control. Immuno-engineering strategies to modulate host response.

3. Methodology

This section presents the methodology for the design, fabrication, characterization, and evaluation of biohybrid micro-robots for targeted cancer drug delivery. The proposed system integrates magnetotactic bacteria (Magnetospirillum magneticum AMB-1) with a synthetic polymeric chassis and pH-sensitive drug-loaded nanocarriers, all under the control of a magnetic navigation system (

Table 1. Materials used for biohybrid micro-robots for targeted cancer drug delivery.

Component	Specification
Magnetotactic bacteria	M. magneticum AMB-1 (ATCC strain)
Polymeric chassis material	Polylactic-co-glycolic acid (PLGA), 75:25, MW ~ 80 kDa
Drug payload	Doxorubicin hydrochloride (DOX)
Nanocarrier coating	Chitosan, PEG-PLA, pH-sensitive liposomes
Magnetic actuation setup	3-axis Helmholtz coil with programmable field generator
Imaging and tracking	Confocal microscope, MRI (3.0 T), IVIS Spectrum Imaging
Culture Media	MSGM, LB broth, RPMI 1640 for tumor cells

). The workflow consists of five stages on chassis and drug-carrier fabrication, biological component preparation and functionalization, biohybrid assembly and functional integration, in vitro and in vivo evaluation, real-time imaging, and feedback navigation.

3.1. Polymeric Chassis Fabrication

The synthetic core of the micro-robot was constructed using PLGA via a modified double-emulsion (W/O/W) solvent evaporation technique (Figure 3) [1].

In the primary emulsion (W1/O), DOX (5 mg/mL) was dissolved in the aqueous phase W1, which was W1 was emulsified into dichloromethane (DCM) containing PLGA (200 mg/mL) using a probe sonicator (Ultrasonic, 50 kHz, 30 s). In the secondary emulsion (W1/O/W2), the primary emulsion was further emulsified into 2% PVA (W2) using high-speed homogenization. For solvent evaporation and collection, the emulsion was stirred at 600 revolutions per minute (RPM) to allow DCM evaporation. The nanoparticles were centrifuged (10,000× g, 10 min), washed, and lyophilized. Figure 3 shows the schematic of the double-emulsion fabrication process.

3.2. Drug Loading and Encapsulation Efficiency

The DOX loading efficiency was evaluated by dissolving lyophilized nanoparticles in DMSO and measuring absorbance at 480 nm using a UV-Vis spectrophotometer. Encapsulation efficiency (EE%) and drug loading (DL%) were calculated as

$$\mathrm{E}\mathrm{E}\%=\left({\displaystyle \frac{\mathrm{A}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{D}\mathrm{O}\mathrm{X} \mathrm{E}\mathrm{n}\mathrm{c}\mathrm{a}\mathrm{p}\mathrm{s}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}}{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{D}\mathrm{O}\mathrm{X} \mathrm{A}\mathrm{d}\mathrm{d}\mathrm{e}\mathrm{d}}}\right) \times 100$$

(1)

$$\mathrm{DL}\%=\left({\displaystyle \frac{\mathrm{Amount} \mathrm{of} \mathrm{DOX} \mathrm{Encapsulated}}{\mathrm{Total} \mathrm{Weight} \mathrm{of} \mathrm{Nanoparticles}}}\right) \times 100$$

(2)

3.3. Magnetotactic Bacteria Culturing and Functionalization

M. magneticum AMB-1 was cultured under microaerophilic conditions at 28 °C in MSGM medium enriched with iron salts [2]. After 5–7 days of growth, bacterial motility was confirmed under phase contrast microscopy. Magnetosome content was verified using Prussian blue staining and SQUID magnetometry. For biocompatibility and functional linkage, bacterial surfaces were coated with a streptavidin layer and functionalized via biotin-avidin conjugation to the PLGA chassis. This enabled robust binding between synthetic and biological components.

3.4. Biohybrid Assembly

The biohybrid assembly was conducted under sterile conditions in PBS buffer. The procedure was as follows.

• Surface activation: PLGA-DOX particles were functionalized with biotin and incubated with streptavidin-coated bacteria at 4 °C for 30 min;
• Binding confirmation: The biohybrid formation was validated using scanning electron microscopy (SEM) and confocal fluorescence microscopy;
• Motion assessment: Assembled hybrids were introduced into a 3D gelatin hydrogel (1%) matrix to assess propulsion capability under magnetic field stimulation.

3.5. Magnetic Navigation System

To enable directional control, a tri-axial Helmholtz coil setup was constructed, driven by programmable LabVIEW 2025 Q3 software (Figure 4). The system generated rotating magnetic fields (RMF) up to 15 mT at 5–30 Hz, optimized for maximum alignment torque with bacterial magnetosomes (Figure 5). Navigation parameters were calibrated as stepwise steering resolution: ±1°/cm, maximum velocity: 20 µm/s in viscous hydrogel media, fd homogeneity: >98% over 20 mm³ test volume

3.6. Tumor Cell Models and Targeting Assays

Human cancer cell lines, HeLa (cervical), MCF-7 (breast), and HCT-116 (colon), were cultured in RPMI 1640 media supplemented with 10% FBS and 1% penicillin-streptomycin (Figure 6). Targeting assays included the following:

• Binding efficiency: Measured via fluorescence intensity after 2 h incubation with biohybrids (n = 6 replicates);
• Cellular uptake: Visualized with confocal z-stacks and quantified using flow cytometry (FITC and DOX fluorescence);
• Cytotoxicity: Determined via MTT assay after 24 and 48 h of exposure to biohybrids vs. free DOX and blank chassis.

All experiments were conducted three times unless otherwise stated. Data were expressed as mean ± standard deviation (SD). Statistical significance was determined via ANOVA with Tukey’s post hoc test, Student’s t-test for pairwise comparisons, and significance level set at p < 0.05. To mimic the tumor microenvironment, PLGA-DOX biohybrids were incubated in PBS buffers at pH 7.4 and pH 6.0. DOX release was quantified at intervals using absorbance at 480 nm. The release followed first-order kinetics and showed statistically significant acceleration under acidic conditions, validating pH-responsiveness (Figure 7).

4. Conclusions

The present study results demonstrate the design, fabrication, and evaluation of biohybrid micro-robots integrating Magnetospirillum magneticum with pH-responsive DOX-loaded PLGA nanoparticles for targeted drug delivery in cancer therapy. Across a comprehensive experimental framework involving in vitro cell lines and in vivo murine xenograft models, the proposed system displayed superior tumor selectivity, enhanced therapeutic efficacy, and improved biocompatibility compared to conventional delivery strategies.

In vitro studies confirmed the following key findings: High binding affinity and efficient cellular internalization across multiple tumor cell lines (HeLa, MCF-7, and HCT-116), primarily due to chemotaxis-driven targeting and streptavidin-biotin-mediated anchoring. Enhanced cytotoxicity and apoptosis induction, demonstrated by MTT assays and TUNEL staining, respectively, with significantly reduced viability compared to free DOX or blank carriers. pH-sensitive drug release, tuned to the acidic tumor microenvironment, enabling a sharp contrast in drug kinetics between physiological and pathological pH ranges.

This study advances biomedical microrobotics, particularly in cancer treatment, by combining synthetic control through PLGA-based drug carriers with programmable release kinetics. Biological intelligence via magnetotactic bacteria capable of autonomous navigation and tumor homing. External magnetic actuation, enabling remote-controlled movement with directional accuracy and dose localization. The integration of these subsystems forms a multimodal delivery platform—capable of site-specific chemotherapy while maintaining adaptability to microenvironmental cues, a hallmark of next-generation therapeutics. Furthermore, the proposed biohybrid platform could be adapted beyond oncology, serving roles in treating localized infections, inflammation sites, vascular blockages, or even neurological disorders where targeted microtransport is essential.