2.2.1. Magnetic-Assisted Medical Applications

The size of nanomaterials is a critical factor for their behavior and distribution in the body. Small particles in the blood tend to aggregate or to be decorated with a protein corona and, because of their charge, create an electrical double layer. In this sense, the total size of particles characterized by the hydrodynamic diameter (dH) can be measured i.e., using dynamic light scattering (DLS) [138]. Both the hydrodynamic and physical size of MNPs determine their magnetic properties. In circulation, systemically injected MNPs circulate in the lumen of blood vessels, interacting with macrophages of the reticuloendothelial system (RES). Smaller USPIONs with dH > 10 nm were characterized by longer blood half-life than SPIONs dH > 50 nm [66,134]. Depending on the hydrodynamic size as well as the electrical charge and other properties, pharmacokinetics and biodistribution of MNPs are rendered [60,139]. MNPs with a dH below than 15 nm are filtered by the kidney; MNPs with a dH less than 100 nm accumulate in the liver in hepatocytes and Disse space; a dH of 100–150 nm leads to the primary accumulation in liver that is based on the uptake by Kupffer cells and these larger hydrodynamic particles can also be trapped by splenic macrophages.

Since the liver and spleen are primary targets for MPN accumulation, they can also be targeted with these particles. The hydrodynamic size of 10–50 nm seems optimal for longer circulation time, but it should be stressed that not only size matters, but properties of the surface such as zeta-potential and hydrophilic/hydrophobic properties are critical factors for biodistribution [60,134]. Non-specific biodistribution of MNPs was used for MRI enhancement of liver disease [139,140]. Ferrucci and Starkre

reported that 80% of intravenously injected non-specific SPIONs were internalized by Kupffer cells. Thereby, the MNPs create an MR-contrast that enables us to trace hepatic neoplasms [140].

We would like to highlight four non-exhaustive areas in which magnetic materials are important (Table 2). The first area includes the biggest success story for using magnetic fields for binding-mediated cell capturing. The chimeric antigen receptor (CAR) T cells have significantly advanced tumor therapies. This technology is based on the isolation of T cells from autologous donors and these cells are genetically engineered to target a specific antigen. The isolation of T cells is currently approved for treatment of B cell lymphoma, and it is expected that T cell engineering will enrich many other fields as well; clinical studies are ongoing in liver cancer. The group of Michael Sadelain pioneered T cell engineering and they have recently shown that macrophages play a key role in mediating the side effects of CAR T cell therapy [141]. Other groups also attempt to manipulate other immune cells, such as natural killer cells [142]. Microfluidics that facilitate binding-mediated cell capturing and release is a key technology for sorting cells in closed systems [143–145]. The microfluidic concept for manufacturing lab-on-a-chip devices use MNPs and these allow for a quick analysis and an automated composition of an individual treatment. Currently, the CAR T cell therapies are mainly focused on B cell malignancies, but other types of cancer are being explored in clinical trials.

Tissue engineering has been given further opportunities by magnetic fields, i.e., by facilitating the arrangement of different cellular layers [146–148]. Magnetic fields have been used to establish three-dimenstional cell culture arrays, to enable cell patterning for the evaluation of the effect of fibroblasts on their capability of infiltration [149–151]. Rieck et al. recently demonstrated that magnetic nanocarriers can be localized in specific areas of the body using magnetic fields. Their approach was done using complexes of lentivirus and MNPs in combination with magnetic fields. The group highlighted that using this method in the murine embryonic stem cell system offers the opportunity to site-specifically downregulate protein tyrosine phosphatase SHP2 by RNAi technology in selected areas with the pathogenic vessel formation [152]. Using this principle would allow us to selectively transmit a specific payload to immune cells based on phagocytic uptake to diseased sites in the liver. Muthana et al. demonstrated that even magnetic resonance imaging machines might be usable to control the spatial concentration of MNPs [153].

The second field of application that we would like to emphasize is mechanical cell control. Here, the aim is to get control over the mechanical properties of cells. In the near future, the technology may be used to measure the stiffness of biological molecules or cellular organelles in vivo. Magnetic tweezers are used as a simple tool to study mechanical forces of biological molecules and cells [154]. Low-frequency magnetic fields are applicable for directed cell destruction and induction of apoptosis [78,155,156]. The concept of mechanical cell control has already been used to function as a cellular remote control, to modulate the stem cells differentiation, or to modulate the behavior of single cells [157–159]. Magnetic micromanipulation based on the application of magnetic tweezers is a potent biophysical technique that is applicable for single-molecule unfolding, rheology measurements, and analyses of force-regulated processes in living cells [160,161].

The third type of application is drug delivery. MNPs are a powerful tool for therapy to target killing of injured or infected cells, which may be achieved by, for example, native toxicity of the MNPs, thermomagnetic effect in magnetic hyperthermia, or targeted drug delivery and release [135,162,163]. In particular, the field of drug delivery can potentially be highly enriched by magnetism-based applications. In addition to the above-mentioned application of MRI scanners to spatially concentrate magnetic particles, magnetic fluids, meaning magnetic particles in a dispersion, might be controlled magnetically. The release of drugs, for example, mediated by magnetism-enhanced thermosensitive polymers, might also be enriched by magnetic actions, for example, to trigger the release of different drugs from a single carrier from porous materials, remote-controlled drug release using azo-functionalized iron oxide nanoparticles, or in order to trigger heat and drug release from magnetically controlled nanocarriers [133,164,165]. In addition to particulate or crystal-based systems, surface immobilized iron might be controlled using noninvasive magnetic stimulation [166–169].

The fourth area in which magnetism has been used intensively is applications based on imaging. For instance, MRI-based imaging of liver fibrosis can be enabled by visualizing fibrosis based on non-invasive analysis of collagen or elastin. Fibrosis might even be visualized by labeling HSC, i.e., by monitoring a specific surface marker such as the folate-receptor, that has already been used for this purpose. Folate receptor-targeted particles have also led to improved specificity of tissue binding [66,124,170]. In order to enable an early therapy of fibrosis, it is critical that it is also diagnosed at early stages. However, there is currently no established early stage marker. In the future, improved early recognition techniques such as circulating collagen fragments, i.e., the N-terminal propeptide of collagen III (Pro-C3) will very likely enable to perform "liquid biopsies" for early detection of fibrosis. Screening of Pro-C3 in plasma of patients has already been done in hepatitis C patients and has been demonstrated to be able to predict fibrosis progression [171]. Other biomarkers might be circulating micro-RNA, and other types of RNA, such as long non-coding RNA (LncRNA), epigenetic analyses, and microbiome studies. Usually, biomarkers work best if they are combined to calculate a specific score such as the NAFLD fibrosis score, the fibrosis 4 (FIB-4) index, or the aspartate aminotransferase to platelet ratio index (APRI) score.

Non-invasive imaging techniques have high potential to confirm the findings from biomarker screening. Consequently, there is a high unmet need for the development of improved techniques for non-invasive diagnosis of liver fibrosis [116]. In ref. [116], the molecular MRI was used for distinguishing different fibrosis stages in a CCI4-based rat model. Enhanced accuracy of detection was achieved by using a targeted USPIO-based contrast agent for MRI. The MRI contrast depends on the time of relaxation of protons that alters in the presence of a magnetic field generated by MNPs and it depends on the degree of interaction of the MNPs with protons. Furthermore, direct imaging of the distribution of MNPs is possible by magnetic particle imaging (MPI) introduced in 2005 and based on measurements of the nonlinear magnetic signal of MNPs [172,173]. Potentially, MPI can enable an improved spatial and temporal resolution than the resolution of the MRI and because of the native biodistribution of iron oxide-based MNPs, the use of this technique for liver seems promising. Improved detection methodologies may also further improve the usage of magnetic particles as biosensors [174]. Imaging probes such as those monitoring Elastin will very likely also improve imaging of liver fibrosis at later stages of the disease, as Elastin appears in late-stage fibrosis [175] (Table 2).

The tripeptide arginine-glycine-aspartic acid (RGD) binds to integrin αvβ3 expressed on HSCs [124]. Conjugation with RGD significantly improved targeting of administrated USPIONs [124]. This allowed authors of ref. [124] to differentiate various liver fibrosis stages with MRI. The relaxivity of developed nanoparticles was higher than, for example, the earlier reported collagen-specific contrast agent based on Gd3<sup>+</sup>. Further improvement was done by multimodal imaging with nanohybrids [123]. In this study, it was suggested that the conjugation of USPIONs-SiO2 with indocyanine green (ICG) dye may further improve near-infrared fluorescence imaging and RGD for targeting. This combination of imaging modalities enables it to perform multimodal imaging (NIR and MRI). To establish theranostic platforms, an additional drug should be attached [176,177].

In order to further visualize some selected applications of magnetic fields, we have illustrated these with a sketch. In particular, cell sorting technology which is currently enabling breakthroughs in cancer therapy is largely based on magnetic forces to sort cells, i.e., the magnetic-assisted cell sorting technology (MACS). These technologies are based on magnetic fields and enable sorting and manipulate cells in closed systems. Mechanical cell control is still in its infancy, but may significantly enrich single cell analysis methods. The field of drug delivery may assumingly expect high potential for breakthroughs due to the potential localized targeting of tissues or cells in vivo, to reduce side effects on non-target cells with enhanced specificity. Imaging applications are already profiting from steadily improved probes, improved methods for signal detection, and better biological targets. In fibrosis imaging, non-invasive assessment of Collagen and Elastin might in future enable significantly improved and more specific assessment of liver fibrosis (Figure 2).


#### **Table 2.** Selected areas of applications for magnetic nanoparticles.

<sup>1</sup> MNP: Magnetic nanoparticle(s).

**Figure 2.** Applications of magnetic nanoparticles in medicine and biotechnology. We would like to highlight four main fields of application for magnetic materials and have chosen some representative schemes for each field of application.

2.2.2. Selective Targeting of Hepatic Stellate Cells—Mission Impossible?

Cell type-specific treatment of disease was first proposed by Paul Ehrlich, who suggested the development of magic bullets which aim at the pathogens only [178]. Ehrlich assumed that people would in the near future be able to specifically target cells or tissues to cure disease with unmet specificity. However, such directed therapies for cancer have been challenged in the past with unexpected problems. One example of a company focusing on cell targeting is BIND therapeutics which went bankrupt in 2016 [179]. Through re-investments, its drug, encapsulated Docetaxel targeting the prostate-specific membrane antigen (PSA), has entered clinical testing again and is currently evaluated in a phase 2 trials in patients with metastatic castration-resistant prostate cancer [180]. It can be assumed that targeting liver fibrosis might face similar difficulties like cancer. However, without any doubt, HSCs as the key cells for fibrosis, represent an ideal target in this regard since they are the main source of activated myofibroblasts and portal fibroblasts that control the fibrogenic process [181].

However, reaching HSCs with drugs is intrinsically difficult since they are located in the perisinusoidal space between hepatocytes and sinusoidal endothelial cells. Under normal physiological conditions, one major function of qHSCs is a depository for vitamin A [182]. In response to liver damage, inflammatory mediators promote HSC activation and their subsequent differentiation into myofibroblasts [183]. Activated HSCs (aHSC) are the main source of collagen in the liver and can abundantly secrete ECM proteins, tissue inhibitors of metalloproteinases and matrix metalloproteinases (MMPs), which cause remodeling of the liver architecture [184]. It is important to note that HSCs are responsible for 80% of the total fibrillar collagen I in the fibrous liver [185]. Thus, inactivation or modulation of HSC is a one of the key aspects in the development of innovative fibrosis therapy.

HSC and MFB express/upregulate diverse specific receptors, such as the mannose-6-phosphate/ insulin-like growth factor II receptor (M6P/IGFII) [186,187], PPAR [188], integrins [189], platelet-derived growth factor receptor (PDGFR) [181], or a peptide receptor of the relaxin 1 family (RXFP1) [182]. Various receptors of HSC have been engaged with specific drugs directed to these. The mannose 6-phosphate/Insulin-like growth factor receptor (M6P/IGFR) is among the most popular targeting structures for HSC. The strategy of eliminating the HSC was investigated in a study 2006 by Greupink et al., in which a nanocarrier consisting of human serum albumin (HSA) was used as a carrier, and functionalized with mannose-6-phosphate (M6P) for this purpose. In order to eliminate these cells, the authors employed doxorubicin, which is used in cancer therapy to kill tumor cells [190]. However, side effects were noted from using the M6P-directed carriers, and in search for a better target, researchers have turned towards PDGFβR since it was also observed that it is specifically upregulated in liver fibrosis [191]. It is known that interferon γ (IFNγ) has exhibited side effects when administered systemically. The research group of Klaas Poelstra therefore created a fusion protein composed of IFNγ and PDGFβR bicyclic peptide. These fusion proteins were demonstrated to inhibit liver fibrogenesis in vivo, based on a significant increase in pSTAT1α activation, compared to the single unit of the fusion protein [192]. A drawback of these fusion proteins is their limited bioavailability, and therefore, a strategy to deposit them in the body is desirable. Van Dijk et al. thus developed biodegradable polymeric microspheres for the sustained release of such protein-based drugs [193].

Integrins have also been explored as potential targeting moieties in this regard. Integrins fulfil important roles by connecting the intracellular cytoskeleton of cells to the ECM. Many integrins were reported to activate the transforming growth factor β (TGF-β), which is another critical factor in fibrogenesis [194]. However, direct inhibition of TGF-β is critical since it is involved in many anti-inflammatory processes. Thus, integrin targeting is in fact a type of indirect targeting of TGF-β. This was pioneered by Henderson et al. in 2013, showing that the pharmacological inhibition of αv integrins by a small molecule (CWHM-12) attenuated both liver and lung fibrosis [195]. Bansal and colleagues, in 2017, further explored the role of integrins in liver fibrosis. They observed that integrin α 11 (IαV) is critically involved in the regulation of the myofibroblast phenotype and that is colocalizes with α-smooth muscle actin-positive myofibroblasts. They further assumed that IαV apparently is involved in fibrogenic signaling and might act downstream of the hedgehog signaling pathway [196]. However, a drawback of integrin targeting is that integrins are important for many vital functions in the body. A scheme for several different ligands to target HSC receptors has been generated (Figure 3).

The hormone relaxin represents a potential anti-fibrotic ligand which binds to its receptor on HSC [197]. One of the important effects of relaxin is the widespread remodeling of the extracellular matrix, which includes altered secretion and degradation of its components [188]. The role of relaxin as an anti-fibrotic agent has been demonstrated in experiments on genetically engineered mice that are deficient in the relaxin gene. These mice spontaneously developed age-related fibrosis of the lungs, heart, skin, and kidneys [198]. It turned out that relaxin also affects the processes of fibrogenesis in the liver. Treatment with relaxin in rats caused acute changes in the microcirculation of the liver [198], and morphological changes were found in non-parenchymal sinusoidal cells [199]. The effects of relaxin were mediated by activation of the receptor for the peptide 1 family of relaxins (RXFP1), which is expressed predominantly in HSC in the liver [197]. Finally, using an in vivo model of experimental liver fibrosis, it was shown that relaxin prevented the content of liver collagen and was effective in treating established liver fibrosis [200]. Thus, relaxin has established itself as an active therapeutic agent in the treatment of liver fibrosis. However, free relaxin has a very short half-life due to its small

size (6 kDa) and rapid renal clearance [201]. Frequent administration of relaxin in chronic fibrosis increases systemic vasodilation, which adversely affects the general condition of the body [199].

When studying the effects of relaxin on fibrosis, it was shown that relaxin conjugated nanoparticles significantly inhibited differentiation, TGF-β-induced migration, and contractile ability of human HSC in contrast to the control, where free relaxin was used; in addition, they drastically reduced collagen gel contraction with maximum inhibitory effects [176].

The evolving and expanding field of RNA technologies has a huge potential to bring up novel drugs. Recently, the first siRNA-based drug, Patisiran, was approved [202]. In the area of liver fibrosis research, Bangen and colleagues demonstrated that inhibiting the cell cycle protein cyclin E1 during liver fibrosis progression in CCl4-induced fibrosis significantly reduced disease severity [203]. RNA interference may also make use of nucleic acids to manipulate micro-RNA, which is known to also regulate the inflammatory processes in liver fibrosis [204]. Local release of therapeutic RNA might further improve efficiency of RNA interference technology.

**Figure 3.** Ligand-based targeting of hepatic stellate cells. Hepatic stellate cells can be targeted based on their expression of receptors on their surface, in the cytoplasm, or inside the cell nucleus. The endocytic route allows the transport of HSC-directed sncRNA, small molecules, or liposomal carriers.

In conclusion, the path towards an efficient treatment of fibrosis remains a rocky road equipped with many pitfalls. However, the high number of drugs evaluated for novel therapies is encouraging. Targeted drugs that might be further improved by magnetism may represent anti-fibrotic drugs of the upcoming generations.

**Author Contributions:** Writing—original draft preparation, K.L., A.O. and M.B.; writing—review and editing, M.B., V.R., R.W.

**Funding:** Research in the group of M.B. was funded by the German Research Foundation (DFG), BA 6226/2-1 and the Wilhelm Sander Foundation (2018.129.1). R.W. is supported by the German Research Foundation (SFB/TRR57, projects P13 and Q3) and the Interdisciplinary Centre for Clinical Research (IZKF) within the Faculty of Medicine at the RWTH Aachen University (Project O3-1). K.L., A.O. and V.R., are supported by the Ministry of Education and Science of the Russian Federation in the framework of government assignment 3.4168.2017/4.6 and by the support of the 5 top 100 Russian Academic Excellence Project at the Immanuel Kant Baltic Federal University.

**Conflicts of Interest:** The authors declare no conflict of interest.
