Biomimetic Systems Involving Macrophages and Their Potential for Targeted Drug Delivery

1. Introduction

Due to the increased risk of infectious diseases, with the lack of ways to effectively treat oncology, the need to develop and improve treatment methods, in particular, drug therapy, increases. However, drug therapy of inflammatory diseases, including oncology and infectious diseases, has limitations, since drugs in current use often have significant drawbacks, such as toxicity to healthy tissues, immunoreactivity, short circulation time and low stability in the biological media. In this regard, systems of targeted drug delivery to the pathology area (site of inflammation, tumor) and/or to individual pathogens (viruses, bacteria, parasites, etc.) are of the greatest interest. Due to the selectivity of the drug in this case, it is possible to avoid its effect on healthy tissues and organs of the body and reduce the effective dose required for treatment.

Thanks to the use of nanoparticles, it became possible to partially overcome such problems as the low solubility of the drug in biological fluids, the low stability of biodegradable therapeutic agents and their toxic effect on biological systems [1,2,3]. However, despite the high ability of nanoparticles to cross many biological barriers and diffuse in intercellular and cellular media, the development of a targeted drug delivery method is still required. Recently, cell-mediated drug delivery using red blood cells, neutrophils, macrophages, stem cells and lymphocytes has attracted much attention, due to its multifunctionality and inherent stability in biological media.

During the course of diseases and inflammatory processes, a complex of immune reactions takes place in the body, among which infiltration and targeted transportation of immune system cells - leukocytes are primary. In particular, macrophages play an active role in the course of the immune response, the main function of which is the selective engulfment and utilization of pathogens and necrosis products of cells, as well as the activation of lymphocytes in the site of inflammation [4,5]. Since macrophages play an important role in a variety of pathological processes, many biomimetic approaches inspired by the biological functions of macrophages have recently appeared. Among them two distinct macrophage-mediated therapeutic strategies are developed: utilizing macrophage-like particles for targeted therapy, and utilizing therapeutic agents specifically targeting macrophages in vivo (Figure 1).

The first strategy is based on the targeting ability of macrophages, which is due to their functional proteins, namely Pattern-Recognition Receptors (PRRs), such as TLR4 and CD14, receptors which bind proinflammatory cytokines, such as CD120, CD126 and CD119, and receptors which bind factors of inflamed endothelium, such as CD44 and Mac-1. Applications of therapeutic agents, such as drugs, nucleic acids, proteins, encapsulated into macrophage-like particles or adhered to macrophages has proved to be a potent strategy to treat various diseases, especially cancer and infections, due to targeting ability, affinity to cytokines and biocompatibility of these biomimetic formulations inherited from macrophages. In this regard, biomimetically modified drug delivery systems using macrophages, macrophage membranes or macrophage-derived vesicles harness the long circulation time, abundant surface receptors, high biocompatibility, low immunogenicity and active targeting ability inherited from macrophages.

The potency of the second strategy is related to the close connection of macrophages with the development of pathological processes. The plasticity of macrophage is extraordinarily recruited, activated, and polarized under pathological conditions, playing crucial role in occurrence, development, and prognosis of various chronic diseases, such as atherosclerosis, and cancer. Moreover, some diseases, such as HIV infection, Tuberculosis, Leishmaniasis, granulomatosis, atypical pneumonia caused by Chlamydia pneumoniae etc are caused by microorganisms that use macrophages as safe reservoirs, which reduces exposure to chemotherapy and prevents immune detection. Therefore, macrophage has become an important therapeutic target. In this regard, immunomodulators, such as cytokines, siRNA, macrophage receptor agonists/antagonists and some other therapeutic agents have attracted attention. In addition, with the development of nanotechnologies, drug-loaded nanoparticles are presented to be potential macrophage-targeted therapeutic agents, especially when modified with ligands, such as mannose, galactose, glucans, hyaluronic acid and others, for enhanced macrophage uptake via recognition by macrophage receptors.

2. Macrophages as immune system cells

Macrophages are present in almost all tissues of the body [6]. The origin of macrophages in different tissues is not fully established [7,8,9], although it is generally believed that some macrophages originate during embryonic development, and other macrophages represent a mature form of monocytes. [10,11,12,13]. The lifetime of macrophages ranges from a few days to several years, and during this time they perform several different functions within the framework of innate and adaptive immunity [6,14].

The phagocytic function of macrophages consists in the absorption and utilization of pathogens and infected cells and can be carried out both without the participation of other cells of the immune system, and within the framework of the complement system; in both cases, macrophages act through specific receptors. Macrophages also play an important role in the participation and regulation of immune responses - macrophages induce inflammation by producing a variety of inflammatory mediators that activate cells of the immune system and involve them in the immune response.

Macrophages represent an important class of sensory cells capable of detecting pathogens and initiating an immune response through mediators. This is possible due to the presence of pattern recognition receptors (PRRs), which detect small molecules and/or regular pathogen-associated molecular patterns (PAMPs). These structures are usually mannose-rich oligosaccharides, peptidoglycan and lipopolysaccharides of the bacterial cell wall, as well as nucleic acids. Among PRRs membrane bound Toll-like receptors (TLRs) and Scavenger receptors (SRs) play significant role in phagocytosis and regulating immune responses.

2.1. Two macrophage phenotypes

Due to the variety of functions performed, two phenotypes are distinguished among macrophages: M1- and M2-macrophages [15,16]. It is known that macrophages are highly plastic cells and can change the phenotype depending on various environmental factors, such as cytokines, pathogens and stress factors [16,17,18,19].

M1 macrophages are the most characterized subpopulation and are known primarily for their phagocytic function [20,21]. These macrophages are activated during cell-mediated immune reactions through the action of stress factors [22,23] and cytokines, mainly IFN-γ [24], TNF-α [25], and the recognizing of PAMPs via TLRs [26,27]. Activation provokes the secretion of various cytokines (TNF-α, IFN-γ, IL-1, IL-6, IL-12, IL-23) [28,29,30] and enzymes (MMPS, MMP12, hyaluronidase, collagenase) [31,32,33,34] by macrophages, which leads to an amplification of the immune response and destruction of the extracellular matrix. The main functions of M1 macrophages are phagocytosis, utilization of the remnants of destroyed cells, regulation of the immune response, presentation of antigen and destruction of the extracellular matrix for tissue reorganization.

M2 macrophages are a subpopulation of alternatively activated macrophages [35], namely by IL-13 [36,37], IL-10 [38], TGF-β [39], glucocorticoids [40] and some other factors [41,42]. A distinctive feature of M2 macrophages is their homeostatic, regenerative and anti-inflammatory functions, which is due to their production of STAB-1 [43], IL-10 [44,45], fibronectin, collagen [46,47] and IL-1 receptor antagonists [48].

3. The use of macrophage-derived vesicles in therapy

Macrophages are differentiated cells of the immune system that are able to engulf microorganisms, particles and macromolecules. This property of macrophages attracted attention to them as potential carriers of various therapeutic agents that allow achieving such advantages as sustained drug release, targeting ability, prolonged half-life and circulation in the blood, high biocompatibility and low immunoreactivity [49] (Figure 2).

Interest in this area has increased with an improved understanding of the mechanisms of pathogen recognition by macrophages and their involvement in inflammatory processes due to the presence of PRRs receptors and cytokine receptors. In this regard, a large number of studies have been conducted aimed at developing biomimetic macrophage-mediated systems for selective drug delivery by means of macrophage-like nanoparticles obtained ex vivo.

3.1. Ex vivo preparation of macrophage-like carriers of therapeutic agents

In recent years, this concept has been developed in combination with advances in nanotechnology. Since macrophages are able to phagocytize nanoparticles (NPs), therapeutic NPs can be loaded into them ex vivo via simple incubation and then injected into an organism [50,51]. Moreover, in addition to incubation, special methods of introducing therapeutic agents into macrophages, such as hypotonic dialysis [52] and electroporation [53], have been presented. In order to preserve the biological functions of carrier macrophages, which are key to the benefits of a macrophage-mediated drug delivery system, methods of attaching therapeutic agents to the surface of the cell membrane are also used.

In addition to methods of direct use of living cells, methods have been developed for encapsulating therapeutic agents in macrophage membranes or in vesicles derived from macrophages, since they can preserve the biological functions of macrophages necessary for effective drug delivery [54,55,56].

Figure 3. Two approaches to the use of macrophages in the development of delivery systems: (A) utilization of living cells (methods of incubation, hypotonic dialysis, electroporation and adhesion); (B) and utilization of macrophage-derived membrane structures (cell membranes and vesicles).

Figure 3. Two approaches to the use of macrophages in the development of delivery systems: (A) utilization of living cells (methods of incubation, hypotonic dialysis, electroporation and adhesion); (B) and utilization of macrophage-derived membrane structures (cell membranes and vesicles).

3.1.1. Sources of macrophages

As part of the use of macrophages in the design of drug delivery systems, researchers proceed either from primary macrophages directly isolated from the animal’s body, or from already cultured macrophages stored in appropriate banks. Macrophages isolated from the body are usually bone marrow macrophages [57], alveolar macrophages [58] or peritoneal macrophages [59]. Well-known cultured macrophage cell lines are murine cell lines RAW264.7 [60,61,62,63] and J774A.1 [64,65], and human monocytic cell line THP-1 [66,67].

In studies for the direct isolation of BMMs, BALB/c or C57BL/6 house mice are often used, from which bone marrow is isolated and dispersed in an environment containing factors that stimulate the proliferation of monocytes into macrophages (usually cytokines M-CSF, GM-CSF, CSF-1 or IL-3) [68]; this is followed by the isolation of macrophages and their incubation [51,69,70]. In the case of isolation of peritoneal macrophages, the serous contents of the peritoneum are collected [71,72].

3.1.2. Obtaining of macrophage-like carriers

In order to avoid the disadvantages of pure drugs, such as immunogenicity, non-selectivity, instability in biological media, low permeability in tissue, etc., an approach using carrier nanoparticles is often preferred. Thus, many studies in this area are devoted to drug delivery systems involving micelles [73], dendrimers [74] or microgels [75]. These particles are often modified by biomolecules, special ligands or synthetic polymers, for example PEG [76,77], to avoid the deactivation and degradation of drugs by mononuclear phagocyte system [3,78]. A different way to increase the therapeutic effectiveness of drugs is to use a cell-mediated delivery system, in particular with the involvement of macrophages.

Drugs can be loaded directly into living cells (encapsulation), or they can be bound to the outer surface of the cell membrane (adhesion). The methods currently used to obtain macrophages-like particles loaded with therapeutic agents are discussed below and summarized in Table 1.

3.1.2.1. Using of living cells

• Encapsulation of drugs in macrophages via incubation

The convenience of using macrophages in cell-mediated drug delivery systems is partly due to the fact that drugs, and, in particular, therapeutic nanoparticles, can penetrate macrophages through endocytosis mechanisms (phagocytosis, micropinocytosis, clathrin-mediated endocytosis, caveolar endocytosis) [90]. In this regard, the method of obtaining biomimetic therapeutic agents by incubating macrophages in the presence of drug particles became the first and widespread [51,91,92].

Due to the fact that the drug may have cytotoxicity, therapeutic agents are usually encapsulated in macrophages in the form of drug-loaded particles, which often enables preservation of biological functions of macrophages. For instance, Choi et al. [80] showed that doxorubicin-loaded liposomes enclosed into mouse peritoneal macrophages via simple incubation exhibited much less toxicity to macrophages than doxorubicin.

In addition to reducing cytotoxicity, loading the drug into special carriers enables to increase its stability and maintain therapeutic activity in the biological environment. In the assay [92], Batrakova et al. demonstrated that the macrophage-taken nanozime in the form of catalase enclosed in a block ionomer complex showed enzymatic activity after release, while pure catalase lost its activity after incubation with macrophages.

It is known that hydrophobic particles are better engulfed by macrophages [93]. However, modification of nanocarriers, which increases their hydrophilicity, makes it possible to improve the biocompatibility and stability of the therapeutic agent [94,95]. For example, Madsen et al. [96] utilized gold–silica nanoshells coated with PEG, since PEGylation prevented aggregation of particles and allowed efficient encapsulation into macrophages.

b.

Encapsulation of drugs in macrophages using hypotonic/resealing method

Osmotic shock of cells by hypotonic dialysis, using hypotonic buffers, induces cell swelling and the formation of pores, without causing apparent lysis. The presence of pores and the placing of cells in contact with an appropriate concentration of the substance allow the latter to be incorporated inside the cell by a passive mechanism. When normal conditions of osmotic pressure are reinstated later on, the pores close and the substance remains encapsulated inside the cell at a suitable proportion [97,98]. This method can be applied to create drug delivery systems with macrophages as drug carriers. For instance, the membrane-impermeable enzyme catalase was packaged into THP-1 cells using this method [52]; It is worth noting that to protect degradation by protease enzymes, encapsulation was carried out in the presence of protease inhibitors, thus enzymatic activity was preserved.

c.

Encapsulation of drugs in macrophage cell membranes using electroporation/resealing method

Electroporation is a technique enabling to increase the permeability of the cell membrane by applying an electrical field to cells; thus, using this method, drugs, chemicals and biomolecules can be loaded into cells [99], in particular, into macrophages. For example, in the assay [53] macrophages were electroporated, and doxorubicin diffused into the cells through the small pores; compared to passive loading, electroporation increased the loading yield of doxorubicin.

d.

Adhesion of therapeutic particles to the macrophage membrane (cellular backpacks)

Cellular backpacks, micron-scale patches of a few hundred nanometers in thickness, can be fabricated by layer-by-layer assembly and attached to the surface of cell-carriers [100]. Due to their shape, size and composition, cellular backpacks are not engulfed by macrophages, making them an attractive strategy that sequesters the cargo outside the cells to protect both the cell and the drug from degradation [83,101].

Such microscale structures are usually composed of 4-5 multilayer films, including payload region and cell attachment region [82,83]. For instance, in the research [82] catalase, a proposed therapeutic agent, was loaded into payload region of the backpack, which conjugated with macrophages via polyclonal antibodies inserted into cell attachment region. Most importantly, the attachment of cell backpacks to macrophages did not alter their major functions, including adherence capability, or cell activation.

3.1.2.2. Encapsulation of drugs in macrophage-derived membrane structures

• Encapsulation inside macrophage cellular membranes

The key properties of macrophages, due to which they can be effectively used in therapy as drug carriers, are mainly associated with proteins integrated into their cell membrane. For example, the membrane proteins CD14 and TLR4 bind lipopolysaccharides [102], and CD44 and Mac-1 bind inflamed endothelium expressing P-selectin and ICAM-1 [56]. Several membrane receptors bind proinflammatory cytokines: CD120a and CD120b bind tumor necrosis factor (TNF); CD126 and CD130, interleukin 6 (IL-6); and CD119, interferon-γ (IFN-γ) [54]. In this regard, the isolated cell membrane of macrophages, as well as macrophage-derived vesicles, can serve as the basis of macrophage-mediated drug delivery system, which enables high targeting ability and anti-inflammatory properties.

In order to remove cell contents extraction of macrophage membranes is usually carried out using a combination of hypo-osmotic swelling, mechanical destruction and several gradient centrifugation steps. In addition, to preserve the structure and activity of transmembrane proteins, protease inhibitors are often added to the medium with macrophages before extraction, and the process itself is carried out at a low temperature [54,84].

In the assay [54] it was shown that the membrane derivation process not only preserved the transmembrane PRRs (CD14 and TLR4) and receptors of cytokines (CD126, CD130, CD120a, CD120b and CD119), but also resulted in significant enrichment of these proteins, which enabled high therapeutic efficacy in vivo.

b.

Encapsulation inside macrophage-derived vesicles

In order to avoid the difficulties of isolating pure cell membranes of macrophages while preserving the structure and activity of transmembrane proteins, key to the biomimetic parameters of therapeutic particles, macrophage-derived vesicles can be used.

Batrakova et al. [55] attained efficient enzyme incorporation into macrophage-derived vesicles and properties of obtained nanocarriers, including targeting ability, indicated that key proteins of macrophage cell membrane were preserved in the vesicles.

Pang et al. [56] developed this approach by using cytochalasin B to stimulate macrophages to produce many microvesicles for nanoparticle cloaking. Analysis proved that the key membrane proteins, such as those involved in self-tolerance (CD45 and CD14) and in adhesion to the inflamed endothelium (CD44, CD18, and Mac-1) were maintained in the obtained microvesicles.

3.2. Macrophage- derived membranes (or particles) as anti-inflammatory agents

Inflammation is a complex, local and general protective and adaptive process that occurs in response to pathology or the presence of a pathogen in the body [103]. Monocytes and tissue macrophages circulating in the blood play a major role in the occurrence of inflammation and its course [104]. Important inflammatory mediators are pro-inflammatory cytokines [105], most of which are secreted by M1 macrophages at the site of inflammation and lead to an amplification of the immune response [106].

Transmembrane proteins, in particular endotoxin and cytokine receptors, can be preserved in macrophage-like particles, which means they can be used as therapeutic agents that reduce inflammatory processes by binding inflammatory mediators and endotoxins [54].

Thus, a group of researchers [54] developed a therapeutic detoxification strategy to treat sepsis via biomimetic macrophage-mediated system; for this purpose, macrophage-derived membranes were used as a coating for polymeric nanoparticles. It is reported that the designed macrophage-like particles showed perfect absorption of LPS and cytokines both in vitro and in vivo due to the preservation of key macrophage transmembrane proteins (PRRs and cytokine-receptors) after membrane isolation. The assay indicated that macrophage-like nanoparticles represent a promising biomimetic detoxification strategy aimed at relieving inflammation by neutralizing endotoxins and LPS.

Using macrophage derived membranes Tan et al. [107] demonstrated that they can be effectively used to design therapeutic agents reducing the level of pro-inflammatory cytokines at the site of inflammation. Obtained nanoparticles inherited the membrane antigenic profile from macrophages and disguised as a mini macrophage to absorb multiple pro-inflammatory substances competitively. It was shown that these macrophage-like particles can effectively suppress cytokine-induced activation of macrophages and neutrophils, acting as decoy for cytokines and other inflammatory mediators, thus offering a promising strategy to alleviate inflammatory processes and prevent cytokine storm.

In addition to anti-inflammatory properties of macrophage-like particles caused by transmembrane proteins inherited from macrophages, these nanoparticles can be employed as the carriers of drugs. For instance, in the assay [108], macrophage-mediated system was successfully used to deliver a model drug (atorvastatin) to atherosclerotic lesion area in mice: biomimetic approach allowed drug loaded nanoparticles coated with macrophage membranes to evade MPS and endowed them with targeting ability. Due to the fact that cooperative binding of cytokines and releasing of atorvastatin by macrophage-like nanoparticles decreased atherosclerotic lesion area this technique was proposed to be a promising way to treat various inflammatory diseases.

3.3. Macrophage- derived membranes (or particles) particles as anti-tumor agents

At present, anti-tumor drugs often show low efficiency, mainly because of their lack of tumor targeting and their high toxicity to healthy tissues. Since macrophages, as immune antigen-presenting cells, have a long blood half-life and can specifically bind to tumor tissue (Figure 4), applying macrophages in drug delivery can lead to a substantial drug accumulation in tumors (Table 2).

• Therapeutic effect obtained from macrophages

In accordance with their functions, the cell membrane of M1 macrophages contains pro-inflammatory cytokines, which lead to the suppression of tumor growth. Thus, after coating polymeric nanoparticles with macrophage derived membranes, enriched with TNFα, Bhattacharyya and Ghosh [109] showed that the fabricated nanoassemblies triggered apoptosis in cancer cells after treatment in vitro. Therefore, a method of cancer treatment using macrophage-like nanoparticles of the core-shell type with a membrane shell isolated from macrophages and containing inflammatory mediators with antiproliferative activity was proposed.

b.

Therapeutic effect due to drug loaded nanoparticles inside macrophages

In addition to the inflammatory properties of macrophage-like particles and their protein profile, which determines their anti-cancer activity, they can also serve as carriers of therapeutic agents such as drugs, genes, nanoparticles etc. Many studies are devoted to anti-tumor properties of ex vivo obtained macrophages or their membrane structures loaded with a therapeutic agent in the form of drug molecules or nanoparticles. Fu et al. [63] reported that therapeutically significant amount of doxorubicin (DOX) could be loaded into macrophages without evident cytotoxicity. The DOX loaded macrophages exhibited tumor-tropic capacity towards 4T1 cancer cells and showed anti-cancer efficacy via tumor suppression, life-span prolongation and metastasis inhibition.

Loading of drug molecules into macrophages, however, may have a limitation in the form of cytotoxicity. In this regard, the use of nanoparticles is more effective, due to which it is possible to achieve low cytotoxicity for healthy cells, high stability of the therapeutic agent in biological media and controlled release of the drug. Tao et al. [110] encapsulated polymer nanoparticles loaded with PTX in macrophages and used them in the treatment of glioma. It was shown that nano-PTX-loaded macrophages had a stronger anti-cancer effect on U87-tumor-cells than naked nano-PTX. It is also worth noting that the use of nanoformulations made it possible to reduce the cytotoxicity of PTX for healthy cells and preserve the biological functions of macrophages, thanks to which it is possible to achieve the penetrating ability of macrophage-like carriers of antitumor drugs. Thus, the ability of DOX-PLGA nanoparticles encapsulated in macrophages to cross the blood-brain-barrier and accumulate in glioma region was demonstrated in vivo [117].

It is known that M1 macrophages can selectively accumulate in hypoxic areas of tumors, which are known for their key role in tumor development and resistance to chemotherapy. In the assay [111], Mitragotri et al. demonstrated the tropism of PLGA nanoparticles containing tirapazamine (TPZ) and internalized in macrophages (MAC-TPZ) towards hypoxic regions of 4T1 tumors; eventually it resulted in a 3.7-fold greater reduction in tumor weight compared to TPZ alone.

c.

Therapeutic effect due to surface engineering of macrophages

Therapeutic agents encapsulated in macrophages can be exposed to phagosomes, which causes restrictions on the utilization of macrophages as drug carriers; drug can affect biological functions of macrophages, and its isolation in phagosomes can lead to its degradation and a reduced drug release rate. For this reason, phagocytosis-resistant drug loaded backpacks that are able to attach to the outer membrane of macrophages have been developed [83,118,119,120]. There are known methods of modifying the cell membrane of macrophages with other particles, for example, quantum dots and dendrimers [121]. Sugimoto et al. [113] showed that the surface modification of macrophages with nucleic acid aptamers improved the capture of T-cell acute lymphoblastic leukemia cells and enhanced their anticancer immune response.

d.

Therapeutic effect due to bioengineered species

In addition to the use of nanoparticles, bioengineered organisms capable of having an anti-cancer effect can also be loaded into macrophages. For instance, in the assay [79] researchers showed that macrophage mediated tumor-targeted delivery of modified bacteria VNP20009 substantially suppressed melanoma in mice. Muthana et al. [114] utilized macrophages as carriers of oncolytic adenovirus, which effectively accumulated in hypoxic tumor areas, inhibited tumor growth and reduced pulmonary metastases of prostate cancer in mice.

e.

Photothermal therapy

Macrophage-like particles can also be used in photothermal cancer therapy, in which a nanomaterial with high photothermal conversion efficiency is injected into the body. Such nanomaterials, when used by themselves, often accumulate in healthy tissues and organs, which is why they can cause long-term harmful effects [122]. Encapsulating such materials into macrophage membranes can solve these this problem and lead to drug delivery systems with good photothermal conversion ability, biocompatibility, ability to escape immune responses, and ability to target tumors. In many studies, this has been proven by the example of Au nanorods (AuNRs) encapsulated in macrophages [123]. Moreover, photothermal therapy can be carried out in conjunction with other types of anti-cancer therapy; for instance, it is reported that the joint encapsulation of DOX loaded into temperature-sensitive liposomes and AuNRs into macrophages results in synergetic chemo−photothermal therapy enabling to target and kill tumor cells in vivo [61].

3.4. Macrophage-derived particles for the treatment of infectious diseases

Macrophages mediate a wide range of infectious diseases. They play a key role in protecting the body against many pathogens, including viruses, bacteria and parasites. Due to the functional activity of transmembrane proteins of macrophages, macrophage-like particles can serve as targeting carriers of antimicrobial agents.

• Treatment of viral infections

In the already mentioned study [107], in addition to alleviating inflammation by absorbing inflammatory mediators, polymer nanoparticles wrapped in a macrophage membrane reduced virus replication, thereby increasing the survival rate among mice infected with the SARS-CoV-2 model. It was shown that the membrane shell of the particles contained the receptor ACE II which is essential for SARS-CoV-2 targeting.

Due to the ability of macrophages to pass through the blood-brain barrier, macrophage-derived particles loaded with antiviral drugs can be used for antiviral therapy of neurological complications of AIDS. For instance, macrophages loaded with indinavir were used to treat mice with HIV-1 encephalitis (HIV), and the study revealed a steady accumulation of the drug and a decrease in HIV-1 replication in HIVE brain regions [124].

b.

Wound healing and treatment of bacterial infections

Macrophage membranes, which contain TLRs, can be used for developing targeted drug delivery systems to bacteria. In this regard, before wrapping therapeutic agents in the membrane, macrophages are often pre-exposed to pathogen in order to enrich the membrane with the necessary proteins.

Using hydrogels or particles surrounded by a macrophage membrane, it is possible to increase the effectiveness of healing of bacteria-infected wounds with photothermal therapy. Liu et al. [125] developed AuNRs-containing hydrogel coated with bacteria-pretreated macrophage membrane. The resultant hydrogels could recognize specifically the source bacteria, and destroy 98% bacteria in vitro under NIR irradiation. Moreover, the hydrogels implanted on the dorsal area of rats could facilitate the infected wound healing and avoid secondary damage during peeling. Similarly, Zhang et al. [126] showed that applying a pretreated macrophage membrane to the surface of a gold–silver nanoparticles increased targeting ability and prolonged blood circulation time. The developed membrane-coated nanoparticles also were proposed as potential drug delivery vehicles, so that photothermal therapy could be applied synergistically with drug therapy.

Due to their affinity to inflammatory mediators and ability to bear drugs macrophage-like particles have high therapeutic potential in the treatment of inflammatory diseases, such as atherosclerosis and sepsis. For instance, to simultaneously realize the pathogen elimination and inflammation resolution in the region infected with pathogen in periodontitis, Xu et al. [127] utilized simvastatin-loaded nanoparticles coated with pathogen-pretreated macrophage membranes, which simultaneously diminished the atheromatous plaque formation in atherosclerosis and rejuvenated the alveolar bone loss in periodontitis. In the study on the treatment of sepsis [128], macrophage membrane coated antimicrobial peptide nanoparticles effectively delivered the drug and retained at the site of infection, and eventually reduced the level of inflammatory factors and ultimately gave infected mice a significant survival advantage.

4. Macrophage-mediated therapy via macrophage targeting

4.1. Design of therapeutic agents targeting macrophages

Macrophages mediate the pathological processes of inflammatory diseases, including oncology and infectious diseases. Since macrophages are closely related to tumor development, as well as due to the existence of pathogens acting through macrophages, studies aimed at the design of drug-loaded micro- and nanoparticles targeting macrophages are of great interest.

Therapeutic agents in the form of drug-loaded particles can be delivered to particular organs or cells based on their physicochemical properties, such as size, shape, charge and solubility (passive targeting); or can be delivered to macrophages via specific targeting ligands (active targeting).

4.1.1. Passive macrophage-targeting therapeutic agents

Passive delivery is based on the pharmacokinetics of NPs, enhanced permeability and retention (EPR) effect, and immune responses of the targeted tissue, leading to the accumulation of NPs. The efficiency of capture of particles by macrophages as part of passive delivery strategy is affected by their following parameters: size, shape, surface charge and hydrophilicity.

• Size

The size of the particles can affect the efficiency of their capture by macrophages, although the cellular uptake depends on environmental conditions. For instance, the uptake of non-modified liposomes by rat alveolar macrophages in-vitro increased with an increase in particle size over the range of 100–1000 nm, and became constant at over 1000 nm, while the uptake of non-modified liposomes by alveolar macrophages after pulmonary administration to rats in-vivo increased with an increase in particle size in the range 100–2000 nm due to the increasing extent of opsonization by lung surfactant proteins [129]. In addition, it is reported that the size of the particles affects the phagocytic capacity, endocytosis speed and endocytosis mechanism of the cell [130,131].

It is also known that the bio-distribution in RES organs is affected by the particle size. For example, large NPs (up to 500 nm) mainly accumulate in the liver and lungs; medium NPs (10–300 nm) accumulate predominantly in the liver and spleen; and small NPs (1–20 nm) are usually degraded by macrophages in the kidneys [132,133]. In addition, epithelial destruction and vascular leakage occur in areas of inflammation and in solid tumors [103,134]. Therefore, NPs with proper size can preferentially extravasate from the blood into the interstitial spaces and accumulate in inflammation sites or tumor tissues via EPR effect [135,136].

b.

Shape

Particle shape has significant impact on macrophage cellular uptake and can be exploited for controlling the efficacy of drug delivery to macrophages. Smith et al. [137] proved that particle shape independently influences binding and internalization by macrophages. Interestingly, they found that the attachment of particles to macrophages could be ranked in the following order: prolate ellipsoids>oblate ellipsoids>spheres, but internalization of particles followed a different rank: oblate ellipsoids>>spheres>prolate ellipsoids. The effect of the particle shape can be explained by the fact that endocytosis is an actin-dependent process, and therefore the internalization of particles with a larger aspect ratio requires more energy to perform the cytoskeleton remodeling [133,137,138].

c.

Surface charge and hydrophilicity

Surface charge is another factor that influences macrophage uptake and many assays both in vitro and in vivo indicate that charged particles are more likely to be taken up by macrophages than neutral particles. Despite the fact that the absorption of positively charged particles by cells is usually easier as a result of electrostatic interactions [139], it has been shown that the same increase in cellular uptake by macrophages can be achieved with an increase in both the negative and positive charge of particles [139,140,141]. However, the role of charge on macrophage uptake is still controversial with contradictory observations in the literature [142].

Hydrophilicity is another parameter that strongly affects the capture of particles by macrophages in vivo. Hydrophilicity, as well as surface charge, can impact the adsorption of opsonin, thus influencing the uptake of NPs by macrophages. Increased hydrophilicity results in a lower degree of protein adsorption and reduced uptake by macrophages [143,144,145]. It is often used to hide NPs from MPS via covering them with PEG [146].

4.1.2. Active macrophage-targeting therapeutic agents

Active targeting can significantly enhance the selectivity of macrophage-mediated therapy due to specific interactions between the therapeutic agent and the cell. This approach involves the direct use of agonists or antagonists of macrophage receptors, or modification of the surface of NPs with ligands or antigen in order to establish selective interaction with macrophage receptors. Many studies demonstrate the advantages and therapeutic potential of active targeting of macrophages in the treatment of oncological and infectious diseases. In this regard, promising results can be attained by using therapeutic agents specifically delivering drugs to macrophages (Table 3).

Receptor targeting	Carrier formulation	Ligand modification/coating	Cargo	Purpose	Result	Ref.
Mannose receptor	Liposomes	Mannose	DNA	Stimulation of immune response	Mannosylated cationic liposomes exhibited improved DNA delivery	[147]
	Polymeric micelles		siRNA	TAMs repolarization	Modified micelles could selectively deliver efficacious amounts of functional siRNA into TAMs	[148]
	Liposomes		64Cu	PET imaging of TAMs	High selective accumulation of the liposomes in TAMs was observed	[149]
	Selenium NPs		Isoniazid	Treatment of tuberculosis	The NPs preferentially entered macrophages and accumulated in lysosomes releasing Isoniazid	[150]
Galactose receptor	Dextran NPs	Galactose	CpG, anti-IL-10 and anti-IL-10 receptor oligonucleotides	TAMs repolarization	NPs accumulated in the tumor and was taken up predominantly by TAMs	[151]
	Chitosan-cysteine NPs		siRNA	Treatment of ulcerative colitis	Galactose modification significantly facilitated the uptake by macrophages and targeting ability of the NPs	[152]
	Poly(lactic-co-glycolic acid) NPs		Dexamethasone	Developing of the strategy to catch macrophages during intestinal inflammation	NPs were effectively captured by macrophages	[153]
Dectin-1	Polymer-lipid hybrid NPs	Yeast cell wall microparticles, containing β-1,3-D-glucan	Cabazitaxel	Developing of oral targeted drug delivery	The microparticles were rapidly and efficiently taken up by macrophages	[154]
	Mesoporous silica NPs		Doxorubicin	Developing of anti-tumor therapy	Drug delivery to macrophages was enhanced compared to uncoated silica NPs	[155]
Fc receptor	Alginate NPs	Tuftsin	DNA	Developing of anti-inflammatory agents	Tuftsin-modified NPs were rapidly internalized in murine macrophages	[156]
Folate receptor-β (FRβ)	-	Anti-mouse FRβ monoclonal antibody	Pseudomonas exotoxin A	TAMs depletion	Direct eliminating of TAMs was attained	[157]
	Poly(amidoamine) dendrimers	Folic acid	Methotrexate	Alleviating of the inflammatory disease of arthritis	High degree of specific binding and internalization of the dendrimers into macrophages was observed	[158]
	Human serum albumin nanocapsules		-	Evaluating targeting ability of folic acid-modified agents	The internalization of nanocapsules was enhanced via FR specificity	[159]
CD44	Hyaluronic acid-tocopherol succinate micelles	Hyaluronic acid	Rifampicin	Developing of tuberculosis treatment	Micelles exhibited a significant phygocytosis and CD44-dependent uptake in comparison to free drug	[160]
	Liposomes		Prednisolone	Developing of rheumatoid arthritis therapy	Enhanced cellular uptake, mainly mediated by caveolae- and clathrin-dependent endocytosis, was acheived	[161]
	Poly(lactic-co-glycolic acid) NPs		Curcumin	Alleviating of ulcerative colitis	Enhanced drug delivery to intestinal macrophages and selective accumulation in inflamed colitis tissue with minimal accumulation in healthy colon tissue was observed	[162]
Siglec-1	Liposomes	Sialic acid	Epirubicin	Tumor therapy	The tumor targeting efficiency and the accumulation of epirubicin in monocytes was improved	[163]
			Zoledronic acid	TAMs depleting and repolarization	Good targeting ability was observed	[164]

Below are main approaches to active macrophage targeting based on the interaction of therapeutic agents with different macrophage receptors (Figure 5).

• Toll-like receptors targeting

Toll-like receptors (TLRs) are well-defined pattern recognition receptors responsible for pathogen recognition and induction of innate immune responses via signaling pathways. TLRs can detect various endogenous damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) [165]. The activation of TLRs initiate a variety of downstream signaling cascades and signaling pathways, leading to production of inflammatory cytokines or type I IFNs [166]. The activation of TLR signaling is also crucial to the induction of antigen-specific adaptive immune responses by activating the adaptive immune cells for the clearance of invading pathogens [165].

Among identified in humans functional TLRs, some are localized on the cell surface (TLR1, TLR2, TLR4, and TLR5) and others in intracellular compartments (TLR3, TLR7, TLR8, and TLR9) [165]. Cell surface TLRs mainly detect membrane components of the pathogens such as proteins, lipoproteins, lipids and lipopolysaccharides (LPS), while intracellular TLRs mainly recognize nucleic acids derived from pathogens or self-nucleic acids in a pathological condition [165].

Since TLRs are involved in the production of pro-inflammatory mediators and activation of immune responses, TLRs present an attractive target for more precise manipulation of the function of macrophages [167]. Recent studies demonstrate that TLRs –pathways play an important role in polarizing macrophages. Therefore, TLRs can serve as a target for modeling macrophage phenotype, for example, as part of tumor treatment via TAMs reprogramming [168]. In addition, TLR ligands have found application in the context of infectious diseases, inflammatory and autoimmune diseases [167].

b.

Scavenger receptors targeting

Scavenger receptors (SRs) are a diverse superfamily of cell surface receptors. They are expressed by myeloid cells (macrophages and dendritic cells) and certain endothelial cells. Being a subclass of the membrane-bound pattern recognition receptors (PRRs), they play an important role in cellular uptake and clearance of endogenous host molecules and apoptotic cells, and exogenous components marked with pathogen-associated molecular patterns (PAMPs) [169]. Removal is often carried out by simple endocytosis but might entail more complex processes, such as micropinocytosis or phagocytosis, which both require elaborate signal transduction [169,170].

Due to the fact that SRs are involved in phagocytosis and in endocytosis, these receptors are potential intermediaries in macrophage-targeting therapy, which can facilitate the selective delivery of therapeutic agents into macrophages. C-type lectin receptors (CLRs), which recognize conserved carbohydrate structures, attract a lot of attention [171]. In this regard, many assays are devoted to developing drug nanocarriers targeting mannose receptor (also known as CD206) [172,173]. Thus, mannosylated therapeutic agents are of great interest and many researchers show that high targeting ability can be achieved via modification of drug nanocarriers with mannose [174,175,176]. Other pathogen-associated components which are used for SRs-mediated targeted delivery are galactose [177,178,179], dextran and its derivatives [180,181,182]. Selective delivery can also be achieved by encapsulating therapeutic agents in glucan particles derived from yeast cell wall, which can be recognized by CLR Dectin-1 [154,155]. In addition, bio-nanocapsules (BNCs) derived from pathogens, such as virus envelope particles [183] or bacteria-like particles [184,185], can be directly used as macrophage-targeted drug carriers.

c.

Fc-receptors targeting

Cellular receptors for the different immunoglobulin isotypes (IgA, IgE, IgM, and IgG), so-called Fc-receptors (FcRs), are involved in regulating and executing antibody-mediated responses [186]. FcRs are widely expressed on cells of the immune system, including macrophages [186]. These receptors recognize antibodies that are attached to infected cells or invading pathogens and stimulate phagocytosis and endocytosis [187].

Since FcR activation stimulates phagocytosis and endocytosis, FcR-mediated drug delivery strategies targeting macrophages have been developed. In this regard, tuftsin - a tetrapeptide formed by enzymatic cleavage of the Fc portion of the immunoglobulin (IgG) molecule, has gained a lot of attention due to its ability to activate FcR [156,188,189,190]. For instance, Jain et al. [156] developed tuftsin-modified NPs and noted much higher cellular uptake by macrophages in vitro than non-modified or scrambled peptide-modified NPs. In addition, tuftsin derivative, tuftsin tetramer, can dramatically enhance uptake into macrophages [191].

d.

Other receptors targeting

In addition to the above-mentioned important receptors expressed by macrophages, the folate receptors [192] and CD44 [193] are considered to be potential mediators in macrophage-targeting therapy strategies.

Folate receptors are expressed on the surface of activated macrophages, known to be upregulated in the macrophages in rheumatoid arthritis and pulmonary fibrosis [194]. High macrophage-targeting ability can be achieved by modification of therapeutic agents with folic acid. Thus, folate-conjugated particles, such as dendrimers [158], chitosan NPs [195], liposomes [196] and human serum albumin NPs [159] exhibited enhanced macrophage uptake when compared with non-folated particles.

CD44 is a receptor for hyaluronic acid-mediated motility (RHAMM). Nanoparticles modified with hyaluronic acid (HA) can be recognized by CD44 and be taken by macrophages [193]. In recent assays such modification of drug-loaded nanoparticles, such as micelles [160], liposomes [161] and polymeric NPs [162], enabled efficient cellular uptake by macrophages.

e.

Tumor-associated macrophage targeting

Due to their significant role in tumor development and progression, tumor-associated macrophages (TAMs) are considered to be a therapeutic target. In this regard many strategies are proposed, including blocking CCL2/CCR2 axis [197] and CD47-SIRPα pathway [198].

CCR2 is predominantly expressed by monocytes/macrophages with strong proinflammatory functions. CCR2 is a CC chemokine receptor for monocyte chemoattractant protein-1 (CCL2) which is involved in macrophage recruitment. In this regard, in order to inhibit TAMs recruitment, CCR2 antagonists, such as RS 504393 [199], BMS CCR2 22 [200] and CCX872 [201], can be used. For instance, CCX872 has exhibited good inhibition of macrophage recruitment due to its high affinity to CCR2 [201].

Signal regulatory protein α (SIRPα) is a regulatory membrane glycoprotein from SIRP family expressed mainly by myeloid cells (macrophages, monocytes, granulocytes, and myeloid dendritic cells) [202]. SIRPα acts as inhibitory receptor and interacts with a broadly expressed transmembrane protein CD47 also called the “don’t eat me” signal, which inhibits phagocytosis [203]. Therefore, SIRPα inhibitors, such as CD47 analogues [204] or anti-SIRPα antibodies [205,206], are proposed as therapeutic agents that promote phagocytosis of tumor cells by macrophages. For example, the monoclonal antibody KWAR23, which binds human SIRPα with high affinity and disrupts its binding to CD47, has been shown to be a promising candidate in therapy, though in combination with tumor-opsonizing monoclonal antibodies [206].

In addition, TAM-targeted delivery of therapeutic agents is another promising strategy. In this regard, Siglec, cell surface receptors that bind sialic acid (SA), are a potential target. Among the Siglec family receptors, the SA adhesion protein Siglec-1 is one of the most abundant superficial receptors of TAMs and can mediate endocytosis after binding to SA. Recently, modification of drug-loaded liposomes [163,164] with sialic acid has enabled high targeting ability of drug nanocarriers. For instance, Deng et al. [207] demonstrated that the cellular uptake of liposomes modified with sialic acid-cholesterol conjugate was increased compared with other formulations.

4.2. Macrophage-targeting in anti-inflammation therapy

Since macrophages play indispensable role in initiating and developing of inflammatory processes, they are a potential target in the anti-inflammatory therapy. Important factors which promote macrophage recruitment into the site of inflammation are cell adhesion molecules, such as ICAM-1 and VCAM-1, inhibiting of which can suppress the inflammation. For instance, Sager et al. [208] showed that silencing endothelial cell adhesion molecules using siRNA reduced monocyte recruitment into atherosclerotic lesions.

Anti-inflammatory cytokines, such as IL-10, and anti-inflammatory drugs can also be applied to suppress inflammation. Thus, IL-10 delivered by polymeric nanocarriers was bioactive and reduced the production of pro-inflammatory cytokine IL-1β in the atherosclerotic lesion and led to significant regression in the plaque size [209]. Local inflammation treatment based on mannose-modified nanoparticles loaded with anti-inflammatory diclofenac was successfully applied to wound healing [210]; drug-loaded macrophage-targeted nanoparticles showed an enhanced anti-inflammatory effect in the wound healing compared to free drug-coated suture.

Overactivation of TLRs may lead to the production of high levels of IFN and other cytokines, leading to chronic inflammation [211]. Moreover, the chronic activation of TLRs exerted by DAMPs may stimulate T- and B-cells responses and, together with the release of cytokines, contributes to the development of autoimmunity [211]. Therefore, TLR antagonists, such as TLR2 antagonists AT1-AT8 [212], have been proposed as agents to attenuate inflammation.

4.3. Macrophage-targeting in anti-tumor therapy

In the case of oncological diseases, due to the influence of macrophages on tumor development, macrophages are the preferred target for various therapeutic agents. On the one hand, M1 macrophages inhibit tumor growth and metastasis, on the other hand, TAMs (M2 macrophages) provoke tumor growth and angiogenesis. Therefore, a promising strategy is to increase the ratio of M1 macrophages/TAMs at the tumor site, which can be achieved by inhibiting macrophage recruitment, direct depletion of TAMs, blocking “don’t eat me” signals, reprogramming TAMs.

• Inhibition of macrophage recruitment

Biomolecules recruiting monocytes, such as VEGF, CSF-1, CCL2 and CCL5, are involved in macrophage recruitment to the tumor area and, as a result, in increasing the number of TAMs. Inhibitors of these chemoattractants and their receptors can suppress macrophage recruitment and monocyte proliferation in TAMs, thereby reducing tumor growth and dissemination [213].

Approach to blocking CCL2/CCR2 axis through inhibition of the mRNA translation is shown in the research [214]. Wang et al. used cationic nanoparticles for targeted delivery of CCR2siRNA. Due to the charge modification, the particles were effectively engulfed by monocytes, due to which an efficient inhibition of macrophage recruitment and TAM infiltration was achieved.

b.

Targeting Anti-Phagocytic Checkpoints

Being important immune cells, macrophages are able to engulf tumor cells and present tumor-specific antigens to induce adaptive immunity. However, tumor cells can evade phagocytosis by macrophages due to the high expression of ”don’t eat me” signals [215].

Among ”don’t eat me” signals, CD47 is the most studied antiphagocytic signal, and it is known that it prevents phagocytosis through interaction with the SIRPα integrated into the macrophage membrane [216]. Blocking the CD47-SIRPα pathway via anti-CD47 therapy or anti-SIRPα therapy is a way to restore the antitumor activity of TAMs [217].

Chao et al. [218] demonstrated that a blocking monoclonal antibody against CD47 enabled phagocytosis of acute lymphoblastic leukemia (ALL) cells by macrophages in vitro and inhibited tumor engraftment in vivo. Moreover, anti-CD47 antibody eliminated ALL in the peripheral blood, bone marrow, spleen, and liver of mice engrafted with primary human ALL. It has been shown that in addition to antibodies, SIRPα analogues can be used to neutralize CD47. For instance, Koh et al. [219] utilized exosomes containing SIRPα variants, which induced significantly enhanced tumor phagocytosis and primed cells for an effective anti-tumor T cell response. Encouraging results can be achieved with the CD47 inhibitor, magrolimab, the ongoing phase 2 trial of which evaluates its tolerability, safety and effectiveness in the treatment of myeloma, especially in combination with other anti-cancer therapies [220].

CD47 is expressed in all types of cells, while SIRPα is only expressed on the surface of myeloid cells (macrophages, monocytes, granulocytes, and myeloid dendritic cells). Therefore, in some cases anti-SIRPα therapy is preferable [221]. In this regard, monoclonal antibodies which bind SIRPα with high affinity can be used. For instance, Ring et al. [206] showed that the anti-SIRPα antibody KWAR23 in combination with tumor-opsonizing monoclonal antibodies greatly augmented myeloid cell-dependent killing of human tumor-derived cell lines in vitro and in vivo.

c.

TAMs depletion

TAMs depletion is another approach to macrophage-targeted therapy that can help reduce angiogenesis, reactivate immune surveillance, and ultimately suppress tumor growth. For this purpose, various anti-cancer drugs or colony stimulating factor inhibitors, such as CSF-1, can be used.

Diphtheria toxin treatment during tumor initiation or in established tumors depletion of TAMs prevented pancreatic cancer initiation [222]; in the case of pre-established tumors, TAMs depletion inhibited tumor growth and, in some cases, induced tumor regression.

Drug-carrying nanoparticles modified for targeted delivery to macrophages can also be effectively used to deplete TAMs. Zhou et al. [207] utilized sialic acid–cholesterol conjugate modified liposomes loaded with epirubicin (EPI-SAL) and showed that EPI-SAL achieved enhanced accumulation of the drug into TAMs; the antitumor studies indicated that EPI-SAL provided strong antitumor activity via modulating the tumor microenvironment with the depletion of TAMs. Another anti-cancer drug, doxorubicin, was loaded into liposomes modified with PEG-D-mannose and PEG-L-fucose conjugates as macrophage receptor ligands [223]; the dual-ligand modified PEGylated liposomes achieved an increased distribution of DOX in tumor tissues and the superior tumor inhibitory rate via modulation of the tumor microenvironment with the exhaustion of TAMs was shown.

d.

Reprogramming of TAMs

Under the influence of various factors, TAMs can switch their phenotype between tumoricidal M1- and protumorigenic M2 macrophages, which inspires the design of therapeutic agents targeting this macrophage plasticity. Thus, one of the promising immunotherapeutic strategies for cancer therapy is repolarization (reprogramming) of TAMs towards anti-tumor M1 phenotype [224]. Drugs, cytokines, immunoagonists, CpG oligonucleatids, siRNA and ROS/O2 generating nanoparticles can be used to reprogram TAMs.

In many studies, liposomes are used to encapsulate drug and its targeted delivery to macrophages. Sousa et al. [225] showed that the effect of liposome-encapsulated zoledronate on macrophages cultured in a conditioned environment of breast cancer cells increased the content of markers of the M1 phenotype of macrophages (iNOS and TNF-α). Later, Tan et al. [164] designed liposomes modified with sialic acid (SL) and loaded zoledronic acid (ZA) into them; thanks to the modification, these drug nanocarriers (ZA-SL) could effectively deliver ZA to TAMs. In vivo experiments showed that ZA-SL cancer treatment increased the M1/M2 ratio, which was partly caused by phenotypic remodeling of M2-like TAMs.

Polymer-based nanoparticles can also be used as nanocarriers of drugs targeted at macrophages for TAMs repolarization. Wang et al. [226] developed poly β-amino ester-based NPs which could adopt by systemic administration and release IL-12 in the tumor microenvironment, subsequently re-educating TAMs. The nanocarriers loaded with IL-12 exhibited enhanced tumor accumulation, extended the circulation half-life and therapeutic efficacy of encapsulated IL-12 compared to free IL-12. Cheng et al. [227] proposed a multifunctional macrophage targeting system to deliver CpG oligodeoxynucleotides to macrophages; they utilized mannosylated carboxymethyl chitosan/protamine sulfate/CaCO3/CpG nanoparticles, which were efficiently taken by macrophages and exerted a polarizing effect on them increasing the production of proinflammatory cytokines including IL-12, IL-6, and TNFα.

Downregulation of CSF-1R is known to reprogram the immunosuppressive M2 macrophages to the immunostimulatory phenotype, M1 macrophages. Sialic acid-targeted cyclodextrin-based nanoparticles were developed to deliver CSF-1R siRNA to TAMs [228]; in in vitro experiments the nanoparticles achieved cell specific delivery to TAMs, eventually polarized M2-like TAMs to M1 phenotype, which enhanced the level of apoptosis in the prostate cancer cells.

Since reactive oxygen species (ROS) are important modulators of macrophage activation and polarization towards tumoricidal M1 phenotype, ROS-generating NPs can be used as therapeutic agents modulating tumor microenvironment. Nascimento et al. [229], using breast cancer models in vitro, found that polyaniline-coated maghemite (Pani/γ-Fe2O3) nanoparticles could be easily taken by M2-like macrophages and could re-educate them towards a pro-inflammatory profile via ROS generating. Immunotherapy can be enhanced by using tumor-derived antigenic microparticles loaded with nano-Fe3O4 and CpG-containing liposomes, which can convert TAMs to M1 macrophages and induce abundant infiltration of cytotoxic T lymphocytes at the tumor site in vivo [230].

Additionally, due to TAM recruitment driven by hypoxia and accumulation of TAMs in hypoxic regions of solid tumors, oxygen-generating NPs can regulate TAM repolarization by reducing hypoxia. Thus, Youn et al. [231] developed mannose-decorated/macrophage membrane–coated upconverting nanoparticles which contained particles generating ROS and oxygen under light irradiation.

4.4. Macrophage-targeting in the therapy of infectious diseases

Macrophages, as crucial components of immune system, can engulf and digest microbes. However, some pathogenic microorganisms have the ability to survive the digestion and utilize macrophages as reservoirs for safe haven avoiding the action of other cells of the host immune system [232,233,234]. These microorganisms can circumvent the effectiveness of antibiotics by surviving inside host macrophages. Since, therapeutics in current use have varying ability to enter macrophages, there is an interest in using special drug delivery systems via drug-loaded nanoparticles to treat intracellular infections. The targeted delivery of drugs to macrophages is considered below on the example of HIV, tuberculosis and leishmaniasis.

• Viral infectious diseases

Human immunodeficiency virus (HIV) is a Lentivirus that leads to acquired immunodeficiency syndrome (AIDS), an immunocompromised condition that increases susceptibility to macrophage resident diseases. Since The major cellular HIV reservoirs are macrophages and CD4+ T cells with macrophages being responsible for carrying and spreading the virus, the development of methods for direct delivery of anti-HIV drugs to macrophages is of great interest [235,236].

Recently, drug-loaded nanoparticles, such as liposomes, polymer NPs and dendrimers, have been considered as potential therapeutic agents for treating HIV. In order to achieve high targeting ability, Jain et al. [237] conjugated efavirenz-loaded dendrimers with tuftsin; the obtained therapeutic agents not only exhibited excellent cellular uptake but also possessed relatively low cytotoxicity with simultaneous high antiviral activity. Similarly, Jain et al. [238] developed stavudine-loaded mannosylated liposomes, which also exhibited high targeting ability and increased biocompatibility in comparison with pure drug. The study of the kinetics of release, the effectiveness of loading antiviral drugs in polymeric nanoparticles and their targeting ability revealed their potential as anti-HIV drug carriers [239,240,241]. Thus, Krishnan et al. [242] , using chitosan carriers loaded with saquinavir, demonstrated a drug encapsulation efficiency of 75% and cell targeting efficiency greater than 92%; as compared to the soluble drug control, the saquinavir-loaded chitosan carriers caused superior control of the viral proliferation.

b.

Tuberculosis

Tuberculosis (TB) is a lung infection caused by Mycobacterium tuberculosis (Mtb). Mtb primarily infects host macrophages, developing special survival and reproduction strategies in these highly specialized cells [243]. Most of the known anti-tubercular agents are less effective in vivo due to the low macrophage permeability and rapid degradation of these drugs [244]. The use of antibiotic-loaded, macrophage-targeted nanoparticles can enable a prolonged and systemic dose of antibiotics [244].

Many assays are devoted to the development of nanoparticles for active targeted delivery, for example, by modification with mannose [172,173]. For instance, Huang et al. [174] designed mannose-modified solid lipid nanoparticles (SLNs) containing pH-sensitive prodrug of isoniazid (INH) for the treatment of latent tuberculosis infection. In vitro assay showed a fourfold increase of intracellular antibiotic efficacy and enhanced macrophage uptake compared to pure drug solution; for the in vivo antibiotic efficacy test, the SLNs group displayed an 83% decrease of the colony-forming unit while the free INH group only showed a 60% decrease. Later, in order to design macrophage-targeted delivery in TB, Ambrus et al. [245] developed nanomediated isoniazid-loaded dry powder for inhalation, based on mannosylated chitosan and hyaluronic acid hybrid nanoparticles, which were found to be a promising vehicle for targeting TB-infected macrophages. Pi et al. [150] first reported the bactericidal effects of selenium nanoparticles and introduced a novel nanomaterial-assisted anti-TB strategy manipulating Isoniazid-incorporated mannosylated selenium (Ison@Man-Se) NPs for synergistic drug killing and phagolysosomal destructions of Mtb. They found that Ison@Man-Se NPs preferentially entered macrophages and accumulated in lysosomes releasing Isoniazid. Furthermore, Ison@Man-Se/Man-Se NPs promoted the fusion of Mtb into lysosomes for synergistic lysosomal and Isoniazid destructions of Mtb.

c.

Protozoan infectious diseases

Leishmaniasis is a wide array of clinical manifestations caused by Leishmania, a parasitic protozoan [246]. The intracellular localization of Leishmania inside the phagolysosome of host macrophages limits chemotherapy treatment. In addition, the use of antileishmanial drugs is often compromised because of their toxicity and limited bioavailability [247]. Macrophage targeted therapeutic agents can solve these problems [247].

Compounds of pentavalent antimony, such as sodium stibogluconate (SSG), used in the treatment of leishmaniasis have high toxicity; encapsulation of the drug in nanocarriers can help to overcome this disadvantage. For instance, Khan et al. [248] developed nano-deformable liposomes (NDLs) for the dermal delivery of SSG against cutaneous leishmaniasis; compared with pure drug solution NDLs displayed an increase in the selectivity index, a decrease in the cytotoxicity and a higher anti-leishmanial activity due to effective healing of the lesion and successful reduction of the parasitic load in vivo. Recently, targeting nanoparticles loaded with other antileishmanial drugs, such as amphotericin B (AmB) and paromomycin (PM) have also been utilized for treatment of leishmaniasis. Heli et al. [249] investigated the effect of ligand modification of PM-loaded chitosan NPs on their anti-leishmanial activity and found the mannosylated formulation to be a suitable targeted drug delivery system for uptake into Leishmania-infected macrophages without any cytotoxic activity. Similarly, Das et al. [250] prepared a mannose containing composite hydrogel loaded with AmB and showed it to be a suitable candidate for the treatment of Leishmaniasis due to its injectability, biodegradability, non-cytotoxicity and efficient drug delivery properties.

4.5. Potency of macrophage targeting via CD206 receptor and future perspectives

The use of mannosylated targeted drug delivery systems using the idea of biomimetics for oligosaccharidic patterns of microorganisms to target macrophage mannose receptors has been studied in detail by our scientific group in a series of papers [172,251,252,253,254,255] (Figure 6), where we aimed at creation of targeted drug delivery system for the treatment of both infectious and oncological diseases. We propose the macrophage CD206 receptor as a target (Figure 6 shows a macrophage with its receptors recognizing mannosylated polymers) [172,253,255,256,257,258,260,261,262,263], because it provides high selectivity of drug delivery and does not require the use of immune-active compounds (interleukins, proteins, microRNAs, etc.). Systematic studies of CD206 ligands conducted by our group allowed us to develop a series of specific molecular container of different molecular architecture carrying an oligomannoside ligand of complex structure with optimal affinity to the mannose receptors of macrophages. We modeled the interaction of CD206 with more than a hundred relevant carbohydrate structures [251], about two dozen of them were studied experimentally. As a result, optimized polymeric ligands has been developed based on polyethylenimine, mannan, chitosan, grafted with cyclodextrins (Figure 6 – center) provided the effect of accumulation of a therapeutic “cargo” in macrophages, which significantly increased organ bioavailability (and accumulation of drugs in the lungs) and the permeability of bacterial cells to drugs has been developed.

Additionally, a significant increase in efficiency can be achieved by using adjuvants which enhance the effect of the antibiotic by inhibiting efflux and increase the permeability of the bacterial membrane [255,256,261]. Currently, the direction of biocompatible medicine is actively developing, in other words, the use of safe natural extracts and essential oils, which have a number of remarkable biological effects, including analgesic, antibacterial, anti-inflammatory, antitumor, antioxidant and regenerating properties. As adjuvants, in our scientific group, compounds of the terpenoids, flavonoids and allylbenzenes series (Figure 6 - in the center from the bottom), series are extensively studied, which show synergism with the main drug—antibiotics fluoroquinolones, rifampicin, etc. For the combination of fluoroquinolone–terpenoid, we observed a 2–3-fold increase in the effectiveness of the antibiotic (a 2–3-fold decrease in MIC) [255,256,260]. Recently we showed that adjuvants (allylmethoxybenzenes, terpenoids, flavonoids) have an enhancing effect on antibacterial drugs, including LF and MF [172,253,255,256,260,261].

However, the binding constants of the antibiotics and adjuvants with the developed molecular containers—polymer ligands—are not high enough (about 10⁴ M), which will cause the dissociation of the complexes upon intravenous administration and will not provide for a prolonged action of the antibiotic. So, we also created a moxifloxacin (fluoroquinolone) prodrug—a covalent conjugates of the antibiotic with mannosylated polymers (dendrimers) enhanced by a terpenoid adjuvant (limonene) with the function of prolonging drug action. Due to application of such “intelligent” prodrug system the selectivity has been achieved: in microbiological experiments an increased antibacterial effect on E. coli and B. subtilis cells is observed, while the effect on “good” Lactobacillus cells is reduced. We have developed pH, thermo- and stimulus-sensitive micelles [256,257,258], which are smart molecular containers that release drugs in a slightly acidic environment and in the presence of glutathione, corresponding to the microenvironment of tumors or in an inflammatory focus potentially applicable for antibacterial and anticancer drug delivery to macrophages. The pharmaceutical compositions developed [251-260] could significantly increase the effectiveness of therapy for a number of infectious and other diseases, reduce the dosage of antibiotics, shorten the duration of treatment, and reduce the risk of developing resistance.

The interaction of macrophages with ligands and the study of macrophage-like particles is classically studied by several methods, including flow cytometry, fluorescence microscopy, immunological methods. We have developed techniques that allow us to study macrophages from a different point of view. In particular, we demonstrated the phenomenon of efflux in bacteria and eukaryotes and were able to inhibit it, which made it possible to increase the effectiveness of antibiotics and anti-cancer drugs (Figure 6 - top left). The authors’ works present classical and original techniques (Table 4) of spectral approaches (FTIR, UV, NMR, fluorescence spectroscopy), computer modeling (molecular dynamics and neural network analysis), microscopy (atomic force, confocal scanning), biological and pharmacokinetic experiments to study both the fundamental aspects of biomimetics for pathogenic patterns recognized by macrophages, and practical applications to improve existing treatment regimens for macrophage-associated diseases. FTIR spectroscopy was used for high-throughput screening of lectin-ligand interactions using concanavalin A as a model mannose receptor to optimize the components and molecular architecture of the delivery system [172,251,253,254,259,260]. FTIR spectroscopy potentially allows you to monitor the individual status of therapy: whether drug compositions interact with bacteria, macrophages, tumor cells or they are indifferent. Using FTIR spectroscopy, we demonstrated the selectivity of chitosan-based micellar systems, observed the effect on A549 cells and, conversely, the protection of normal HEK293 cells [256,257], and a similar effect was observed for bacterial E. coli cells vs Lactobacilli. The technique allows testing drug delivery systems: if there is no effect on cells, then there are no changes in the spectra, and vice versa. The use of UV spectroscopy, as well as fluorescence quenching, makes it possible to determine the parameters of ligand-receptor complexation at concentrations of reagents 1-2 orders of magnitude lower than those described in the literature, including by isothermal calorimetry, with the possibility of analyzing a wide range of ligands and reducing the complexity of the process. Using fluorescent methods, we studied the interaction of ligands with cells, adsorption and permeability over time, and the effect of efflux inhibitors on drug permeability and retention. With the help of flow cytometry, the existence of fluorescent nanoparticles with the drug (not debris) was proved, and adsorption on E. coli cells was studied, and quantified living cells by DAPI staining.

Thus, targeting macrophages using the biomimetics of pathogen patterns is a very effective strategy for creating therapeutic systems for a range of diseases. In addition, using macrophage-derived particles, it is possible to selectively target therapeutic cargo to tumor cells, which makes it possible to bypass biological barriers, “switch” the tumor microenvironment (hot/cold) and regulate the status of inflammation. In other words, targeting macrophages and using macrophage-derived membranes as drug carriers has huge prospects for creating a golden bullet for the treatment of infectious, oncological, neurodegenerative, and autoimmune diseases.