In recent years, robotic research groups worldwide have actively participated in the development of microrobots for efficient drug delivery in anticancer therapy applications. To enhance drug delivery, externally controlled microrobots have been developed. Artificial microrobots have been most commonly developed using various fabrication techniques. Especially with the fast development of 3D/4D printing technologies, these microrobots are easily fabricated with different sizes, shapes, and materials in a short amount of time and are capable of being mass produced ^{[1][2][3][4][5][6]}[1,2,3,4,5,6]. However, these microrobots lack the intrinsic sensing ability needed to target and, more importantly, penetrate tumors since their actuations entirely rely on external sources, limiting the therapeutic efficacy and potential clinical applications. To address this limitation, biohybrid microrobots have recently been developed by biohybridizing microorganisms or biological cells with synthetic materials. Various techniques of fabricating biohybrid microrobots have been introduced in the literature ^[7][8][9][7,8,9]. Flagellum bacteria have been extensively used in the design and fabrication of these robots, as they can be bioconjugated by researchers with micro-to-nano-scale structures that can carry therapeutic agents ^{[10][11][12][13][14][15][16][17][18][19]}[10,11,12,13,14,15,16,17,18,19]. These bacteria possess high levels of motility and some exhibit tumor-targeting abilities [14]. However, the use of bacteria is associated with several limitations, such as toxicity, ineffective bioconjugation, and small actuation forces ^[8][9][8,9]. Alternatively, macrophages can be used in macrophage-based microrobots to deliver the drugs to tumors ^[10][11][12][10,11,12]. Drugs or drug-loaded nanoparticles can easily be functionalized with macrophages through internalization via phagocytosis or surface conjugation with the cells [13]. Other important advantages of using macrophages for drug delivery to tumors include a reduced immune response as they are recognized as immune cells, tumor-homing ability due to their migration and chemotaxis properties [20], and their ability to traverse blood barriers, infiltrate tumors, and become tumor-associated macrophages, accounting for up to 80% of the tumor mass [14].

2. Preparation of Macrophage-Based Microrobots

2.1. Macrophage Cells

Macrophages belong to a group of mononuclear phagocytic cells of the innate immune system that plays an important role as the first line of defense against foreign objects, harmful pathogens, and tumorous cells ^[21][22][23][21,22,23]. Due to their characteristics, these cells can be utilized to create macrophage-based microrobots. To make such microrobots, commercial monocytes (precursors of macrophages, which become macrophages after migrating from capillaries to tumors) and macrophage-like commercial cell lines are commonly used to construct macrophage-based microrobots. These cells include the mouse cell lines RAW 264.7 ^{[24][25][26][27][28]}[24,25,26,27,28] and J774A.1 ^[29][30][29,30], rat alveolar macrophages [31], and human original cells such as THP-1 ^[32][33][32,33]. However, to enhance the biocompatibility of these microrobots for in vivo use, primary macrophages are obtained both from animals, such as peritoneal [34], spleen-derived [29], and bone marrow cell-derived macrophages ^{[35][36][37][38]}[35,36,37,38], and humans, such as human macrophages [39].

2.2. Preparation of the Microrobots

There are two main methods that are widely used to prepare microrobots. In the first method, which is used in a majority of related studies, the payloads are “eaten” or engulfed by macrophages when co-incubated in culture media for a certain period of time due to the strong phagocytosis ability of the macrophages [40]. In the second method, the payloads are chemically bound to the surface of the macrophage [41]. Mitragotri and colleagues introduced the attachment of disk-shaped backpacks (BPs) with a diameter of 7 μm and a thickness of 500 nm. They showed that the BPs were not phagocytosed by monocytes and were strongly attached to the cell surfaces due to their particular shape and flexibility. As a result, the BP-laden monocytes could hitchhike to the inflamed skin or lung in vivo [42]. In a recent study, Yang et al. prepared biotin-modified liposomes loaded with Dox and attached them to the surfaces of RAW 264.7 macrophages, which were modified with streptavidin-conjugated (polyethylene glycol) PEGylated lipids, via high-affinity biotin-streptavidin (MA-Lip). They showed that the MA-Lip infiltrated deeper, enhanced Dox accumulation, and increased the antitumor immune response [43]. Each method has its own advantages and disadvantages. While the first one provides a simple means for preparing a microrobot, the premature released drugs from the payloads may affect the functions of the macrophages upon their engulfment. In the second one, the preparation is more complicated because of the surface modification of the macrophages and the functionalization of the payloads. However, since no therapeutics are internalized into the macrophages, the cells will have a higher chance of survival when reaching the targeted sites.

2.3. Payloads

Several materials have been adopted as payloads for macrophage-based microrobots to induce therapeutic effects. Depending on the specific applications or experimental settings, macrophages can engulf and transport multiple types of payloads.

2.3.1. Gold-Based Nanoparticles

Gold nanoparticles are biocompatible and have a good ability to absorb near-infrared (NIR) light. In a pioneering work, Choi et al. used Au nanoshells phagocytosed by monocyte-derived macrophages to penetrate intratumorally into tumor spheroids and induce cell death in both the macrophages and tumor cells through photoinduction [39]. In addition, gold nanorods (AuNRs) have been widely used as payloads for macrophage-based microrobots due to their longitudinal surface plasmon resonance peak in the NIR window, allowing them to efficiently convert NIR light energy into heat [44]. Li et al. utilized RAW 264.7 macrophages that engulfed small AuNRs with a size of 7 nm, which showed high cell viability after engulfment. The AuNR-laden macrophages were found to enhance tumor coverage and improve phototherapy in vivo [45]. In another study, An et al. prepared macrophages loaded with AuNRs of different surface charges (cationic, neutral, anionic), showing promising photoacoustic imaging of tumor hypoxia and enhanced in vivo photothermal therapy of the tumor [24].

2.3.2. Liposomes

Liposomes are vesicles consisting of lipid bilayers. They are biocompatible and biodegradable materials that can be loaded with both hydrophobic and hydrophilic therapeutics. By adjusting the lipid composition, liposomes can be designed to release therapeutics in a controlled manner. Based on these merits, liposomes are widely adopted as payloads for macrophage-based microrobots since they protect the macrophages from cell death and premature release of the payloads before reaching the targeted sites. Choi et al. prepared 150 nm liposomes loaded into peritoneal macrophages and observed high in vivo migration and positive therapeutic effects in an A549 tumor-bearing mouse after administering five doses [34]. Fujita and colleagues incorporated magnetic lipoplexes (SPION-incorporated cationic liposome/pDNA complexes) into RAW 264.7 macrophages. They showed that cytokine release was similar in engineered and pristine macrophages, but the production of nitric oxide was significantly enhanced in the engineered cells. In addition, under a magnetic field, the engineered cells exhibited strong attachment to a Caco-2 cell layer and the colon of mice, suggesting improved colonic delivery and potential therapy for colonic inflammation [46].

2.3.3. Magnetic Nanoparticles (MNPs)

MNPs have been widely used in biomedical applications such as biomedical imaging agents due to their excellent biocompatibility and magnetic properties [47]. In addition, they have been used to enhance the functionality of the systems carrying them, specifically through their controllability via a magnetic field [27]. Recently, Li et al. modified MNPs by incorporating them with bioengineered bacterial outer membranes, generating biogenic macrophage-based microrobots (MΦ-OMV robots). These robots were able to be manipulated in vitro in a confined space, and in vivo in a mouse tumor model [48]. Researchers used poly-(vinyl alcohol)-coated (PVA-coated) MNPs encapsulated in paclitaxel liposomes and engulfed by J774A.1 macrophages, enabling dual controllability of the macrophages through an external magnetic field and chemotaxis [49]. In addition, MNPs show responsiveness to near-infrared (NIR) light from a laser [50]. Therefore, they can be used as therapeutic agents that convert light energy into heat when irradiated using an NIR laser ^{[51][52][53][54]}[51,52,53,54].

2.3.4. Polymeric Nanoparticles

Biocompatible polymeric nanoparticles offer many advantages for drug delivery, such as nontoxicity and a prolonged controlled release of the encapsulated drugs. These properties make polymeric nanoparticles ideal candidates for encapsulation into macrophages since they can prolong the lifespan of macrophages due to their slow drug release rates. Therefore, many researchers utilize these nanoparticles as payloads for macrophage-based microrobots ^{[32][35][55][56][57]}[32,35,55,56,57]. Xie et al. used biodegradable photoluminescent poly-(lactic acid) decorated with muramyl tripeptide, loaded with a drug (PLX4032), and engulfed the nanoparticle complex to macrophages. The engineered macrophages could carry the drug to the cancer cells via cell–cell binding. The authors proved that the system effectively killed the cancer cells [32]. Researchers' group created a macrophage-based microrobot by utilizing the phagocytosis of poly-lactic-co-glycolic acid loaded with MNPs and docetaxel anticancer drugs that allowed for hybrid control of the microrobot and the ability to destroy cancer cells using the drug released from the microrobot [55]. Shi and coworkers internalized hyaluronic acid nanogels, prepared using a double-emulsion method, encapsulated with DOX and polypyrrole into RAW 264.7 macrophages to create macrophage-based microrobots (MAs-NGs). As a result, upon treatment with the MAs-NGs followed by laser irradiation, a subcutaneous cancer model was significantly inhibited [56].

2.3.5. Free Drugs

The engulfment of free drugs into macrophages has also been studied in the literature. Fu et al. engineered RAW 264.7 macrophages with a high concentration of Dox solution (400 μg/mL, 1.8 mL for 1 million cells). As a result, a therapeutically meaningful amount (100 μg) of Dox was encapsulated in one million macrophages. In addition, the Dox-modified macrophages displayed good tumor-homing abilities and promising metastasis inhibition [58]. Guo et al. loaded Dox directly into M1 macrophages (M1–Dox) stimulated from RAW 264.7 cells. The authors reported that the engineered macrophages significantly enhanced tumor-homing ability by upregulating CCR2 and CCR4 compared to un-engineered cells. In addition, M1–Dox prevented tumor invasion induced via Dox. Moreover, compared with a commercial liposomal product (Lipo–Dox), M1–Dox showed a superior penetration and deeper accumulation within disseminated neoplastic lesions, leading to a critical reduction in metastatic tumors and an increase in the survival rate [28].