Perspectives of Methotrexate-Based Radioagents for Application in Nuclear Medicine

Methotrexate is a gold standard among disease modifying antirheumatic drugs and is also extensively used clinically in combination with oncological therapies. Thus, it is not surprising that nuclear medicine found an interest in methotrexate in the search for diagnostic and therapeutic solutions. Numerous folate-related radiopharmaceuticals have been proposed for nuclear medicine purposes; however, methotrexate radioagents represent only a minority. This imbalance results from the fact that methotrexate has significantly weaker affinity for folate receptors than folic acid. Nevertheless, radiolabeled methotrexate agents utilized as a tool for early detection and imaging of inflammation in rheumatoid arthritis patients gave promising results. Similarly, the use of multimodal MTX-release nanosystems may find potential applications in radiosynovectomy and theranostic approaches in folate receptor positive cancers.


INTRODUCTION
Folic acid (FA, vitamin B 9 , Figure 1) is a member of the folate family, known as a significant methyl-donor in the cellular formation of the essential amino acid methionine, and the syntheses of elements of nucleic acidspurines and thymidylates. However, even before the accurate determination of folates' regulation of cell growth, function, and proper division, folate deficiency has been studied in the context of leukemia remission in humans. 1 These beneficial effects have given the impulse to the search for compounds that are antagonists of FA action without causing FA deficiency. Indeed, the implementation of folate antagonists, known also as antifolates, provides similar and more selective effects on acute and subacute leukemia patients. 2,3 Over time, methotrexate (MTX, formerly amethopterin, Figure 1) has been distinguished in clinical oncology among other initial antifolates, primarily due to its acceptable toxicity profile. Currently, MTX is one of the most widely applied chemotherapeutics, used alone or in combination therapy for different types of cancer, mainly in acute lymphoblastic leukemia, but also trophoblastic neoplasms, breast, lung, head and neck cancers, osteosarcomas, cutaneous T-cell lymphomas, or non-Hodgkin's lymphomas. 4,5 Furthermore, it has found great application in autoimmune pathologies and inflammatory disorders such as psoriasis and rheumatoid arthritis (RA). 6,7 For most early and well diagnosed RA patients, MTX is the preferred initial disease-modifying antirheumatic drug.
MTX's complex mechanism of action is involved in antitumor and immunosuppressive activity of this drug. First, MTX shows a folate antagonism by blocking multiple enzymes on the folate metabolic pathway and thereby disturbing the metabolic balance of the cell. After crossing the cell membrane, MTX follows rapid intracellular bioconversion into polyglutamate derivatives (MTX-pGlu), via folylpolyglutamyl synthase (FPGS). 8 This bioactivation significantly enhances MTX pharmacological activity, 9,10 namely, extending the intracellular retention and expanding inhibitory activity. Free MTX inhibits noticeably only dihydrofolate reductase (DHFR), while polyglutamated MTX provides greater inhibition of that enzyme. DHFR catalyzes the reduction of dihydrofolate into tetrahydrofolatea significant proximal carbon atom donor in various transmethylation reactions, including synthesis of nucleosides. Furthermore, MTX-pGlu targets thymidylate synthase, amidophosphoribosyltransferase, and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase, 11−14 other key enzymes in de novo thymidylates and purines syntheses. Consequently, the cellular syntheses of DNA and RNA building blocks are highly inhibited, whereby the cell loses its proliferative ability and undergoes apoptosis. 15,16 This scenario applies primarily to highly proliferating cells that are most sensitive to the cytotoxic effect of MTX. 17,18 Furthermore, AICAR transformylase blockage results in accumulation of AICAR, the inhibitor of adenosine and adenosine monophosphate deaminases. 19,20 Therefore, intracellular elevation of AICAR promotes an adenosine increase followed by its extracellular efflux. 21 Adenosine is a local signal transmitter, acting by its specific receptors (adenosine receptors A 1 , A 2A , A 2B , A 3 ) present on the surface of the immune cell but also on the origin tissue. Activation of A 2A and A 3 receptors overexpressed on leucocytes and synoviocytes, specific macrophage-like and fibroblast-like cells in RA patients decreases production of inflammatory cytokines and downregulates the immune system. 22,23 Adenosine through A 2A also inhibits the action of activated neutrophils and macrophages including production of pro-inflammatory tumor necrosis factor and interleukins 24,25 as well as promoting leukocyte production of anti-inflammatory interleukins 4 and 10. 26 Thus, MTX indirectly induces an anti-inflammatory and immunosuppressive effect through adenosine-mediated action.
Pleiotropic action of MTX led to its wide application in clinical oncology and treatment of numerous inflammatory and autoimmune pathologies. In this review, we focus on the perspectives of MTX features for the needs of nuclear medicine. The overview of articles presented below concerns the application of MTX in the form of any radioactive agents to date. We will acquaint the reader with MTX's ability to act as a leading vector (in terms of diagnostic radiopharmaceuticals) or the ability to support the apoptotic effect of radiation (with regard to therapeutic radiopharmaceuticals). Both of these aspects are key issues in the design of new receptor radiopharmaceuticals for the ever more intensive development in nuclear medicine.

MECHANISMS OF INTRACELLULAR TRANSPORT
OF METHOTREXATE MTX is a small molecule that possesses two free carboxylic moieties from glutamate and two weak amino groups in the aromatic structure of 4-aminopteroic acid (Figure 1). It is slightly soluble in water, and if so, becomes strongly ionized and unable to penetrate biological membranes by itself. Thus, the distribution of MTX depends on bioavailability associated with an active transport system. First, MTX adsorption is clearly related to the route of drug administration, especially in the case of the oral form. The intestinal adsorption of the drug is mediated via proton-coupled folate transporters (PCFTs, more precisely, solute carrier family 46 member 1, SLC46A1), 27 specifically, symporters that exhibit high density in the upper gastrointestinal tract, where at a preferred low pH participate in specific uptake of both oxidized and reduced folates. 28 PCFTs also occur on many healthy tissues and cancer cells, which are barely active at physiological pH; however, during the internalization process they mediate active folate transport due to the acidic environment inside the endosome (Figure 2 and Figure 3). 27,28 Around neutral pH, cellular MTX transport is realized mainly by the reduced folate carrier 1 (RFC) or to some extent via endocytosis by the folate receptors (FRs) (Figure 2). RFC is a 12 transmembrane-domain exporter in the solute carrier transporter superfamily (solute carrier family 19 member 1, SLC19A1), ubiquitously expressed in human tissues and cancers. 29 It mediates the influx of 5-methyl-tetrahydrofolate  Expression of FRs may significantly increase in some cancer cell lines due to increased sensitivity to folates; thus, MTX endocytic transport also improves.

Molecular Pharmaceutics
pubs.acs.org/molecularpharmaceutics Review or the majority of DHFR antagonists in exchange for various organic anions. In comparison to PCFT, RFC demonstrates high specificity only for reduced folates (K m ≈ 1−10 μM for MTX), 30,31 whereas affinity for FA is 100-fold less (K m ≈ 100− 400 μM). Furthermore, the reduced folate carrier impairment is one of the most clinically important mechanisms responsible for cancer cell resistance to MTX therapy (Figure 3), 32 since antifolates are predominantly delivered intracellularly by RFC. However, MTX cellular uptake takes place also via FRs (formerly called the folate binding proteins), realized through receptor mediated endocytosis. This transport pathway is mostly dedicated to FA and oxidized folates (K d ≈ 0.01 nM for FA), while MTX and other DHFR inhibitors show above 3fold lower affinity to FRs (K d ≈ 10−100 nM for MTX). 31,33 Importantly, RFC and FRs operate independently and possess individual characteristics of action: different pH and temperature sensitivities, tissue expression, distinct chemical individuals causing transport blockade, and an inverse ratio of reduced/oxidized folates binding affinities. 34,35 Moreover, compared to FRs, RFC creates a stronger complex with MTX 36 and has a higher maximal velocity and Michaelis− Menten transport constant for MTX transport. 35 Despite these facts, the contribution of both types of transport may provide similar influx rates of MTX. This is possible only when FR density is significantly elevated (e.g., on the surfaces of cancer cells) and drug concentration in the blood is fairly low (e.g., radiopharmaceutical application).
The most contemporary distinction of FRs is made between four isoforms: two glycosylphosphatidylinositol (GPI)-anchored receptor isoforms α and β, free soluble receptor isoform γ, 37 and newly discovered isoform δ (also known as JUMO or IZUMO1R), which is also the GPI-anchored receptor. 38,39 The noticeably up-regulation of folate receptor isoform α (FR-α) is observed in various epithelial cancers including breast and kidney carcinomas, 40,41 also brain cancer, 42 while significant FR-α overexpression is reported in lung and internal parts of female reproductive system carcinomas. 43−48 FRs-α physiologically occur to a small extent on the choroid plexus, fallopian tubes, colorectal and pulmonary epithelial cells, 43,44,48,49 and in particular on the renal proximal and distal tubules; 48,50 however, they are hardly accessible to folate conjugates circulating in the bloodstream, 51 and thus, folate imaging gives the only pronounced signal in the kidneys of healthy controls. 52,53 Second FR isoform β (FR-β) is expressed on surfaces of activated macrophages during chronic inflammatory diseases, 54 in spleen of patients with chronic and acute myelogenous leukemia, 55,56 and also on healthy placental tissue. 44 Both isoforms are beneficial in the clinical prognosis of cancer. FR-α is specified as reliable cancer marker, 49,57−59 that even may allow for the possibility of defining a tumor grade and subtype 55,57 or prognosis view for patient recovery. 41,58 Similarly, elevated expression of FR-β can be detected in the microenvironment of particular tumors due to a specific receptor presence on tumor-associated macrophages. 60 In addition, both isoforms show different binding affinities for some ligands that are folate analogues, 34,61 which may give an opportunity to design a selective tracer for a specific FR isoform in order to diagnose a particular type of cancer or inflammatory disease.
FR isoform γ (FR-γ) is a soluble protein related principally to hematopoietic and lymphoid systems. 37 Because of its inability to connect to GPI anchor, FR-γ is constantly secreted into the serum at the modest level; however, this secretion increases during dietary folate deficiency and especially in the case of leukemia. Therefore, the increase of FR-γ serum level predominantly indicates the presence of malignancies of bone marrow, spleen, or thymus. 37 FR isoform δ (FR-δ, FOLR4), commonly known as JUNO, originally has been reported on CD4+CD25+ regulatory Tcells responsible for ongoing tumor resistance in the murine immune system. 62 Moreover, mammalian JUNO serves as an egg cell plasma membrane receptor for IZUMO1 spermspecific protein. Creation of JUNO-IZUMO1 complexes results from an egg cell−sperm fusion essential for the fertilization process. 39 The foregoing ample description of MTX active transport shows the significance of folate action in mammalian physiology. The presence of so many pathways of FA derivative uptake cannot be accidental. Particular expression of presented receptors on healthy tissues and cancers or inflammation makes them attractive molecular targets. Simultaneously, this provides huge opportunities to search for new imaging and therapeutic solutions for laboratory diagnostics and nuclear medicine.
Molecular sciences provide numerous data on the correlation between FRs/RFC affinities or cellular influx capacities with structure of folates/MTX and theirs conjugates. 31,33,36,63−65 While MTX has a superior affinity toward ubiquitous RFC than FRs, MTX conjugates should provide a molecular recognition of FRs at the specific place of receptor overexpression. The choice of FRs as a molecular target in nuclear medicine has significant advantages such as great tumor/inflammation targeting or the possibility of endocytic delivery across the membrane of various types of conjugated molecules (proteins, immune agents, nucleotides, chromophores, radionuclides, nanostructures, liposomes, etc.).

RADIONUCLIDE-LABELED MTX AGENTS
The widespread use of MTX in oncology and the development of quantitative radionuclide tumor imaging have given rise to further research of radioconjugates based on FA and MTX analogues. Currently, numerous folate-based radiopharmaceuticals have been proposed for nuclear medicine purposes; 66,67 however, MTX radioagents represent only a minority in the field because MTX has more than three orders of magnitude weaker affinity toward folate receptors than FA. 31,33 The conversion of oxygen into nitrogen in the pteridine ring ( Figure 1) deprives MTX of a significant ability to form a hydrogen bond at the active center of folate receptors, which consequently decrease binding to FRs. 36 Despite the weakened affinity of MTX for FRs, there have been numerous studies describing multiple applications of radionuclide labeled MTX agents.
The earliest reports refer to tritiated MTX derivative 3′,5′,7- 68 which has been applied in MTX uptake studies of murine leukemia L210 cell line 69−71 and in vivo on rodents. 30,72 Subsequently, the natural evolution of this research has been in pharmacokinetic and metabolic studies of MTX in small mammalians, rhesus monkeys, 73,74 and humans. 75−77 In the meantime, the first [ 3 H]-MTX radioimmunochemical methods for the determination of MTX in biological matrix were developed. 78,79 In a short period of time, practical proposition of radioimmunoassay using MTX labeled with gamma emitters (selenium-75 and iodine-125) was reported for more convenient clinical monitoring of patients  82 Generally, in most MTX cellular transport studies, tritiated derivative [ 3 H]-MTX is the preferred reference compound. This was observed in affinity studies for FR-α, RFC, and PCFT of newly designed antifolate drugs, 28,31,83,84 in PCFT-mediated transport research on mesothelioma cell lines, 85 and FR-βmediated transport investigation on synovial macrophages from RA patients. 54 Similarly, [ 3 H]-MTX was applied to monitor in vitro/in vivo enzymatic polyglutamylation rate and thereby the ability to inhibit the thymidylate synthase in murine leukemia cells, 86 or DHFR inhibition in human nonsmall-cell lung carcinoma. 87 Additionally, [ 3 H]-MTX uptake studies were performed on MTX-resistant osteogenic sarcoma cell line variants. 88 In parallel to cellular transport research, new methods for desirable MTX distribution have been investigated. For this purpose, a number of various types of liposomes (lipophilic forms of the delivery drug system) containing entrapped [ 3 H]-MTX have been developed and tested due to their ability to modify tissue distribution or metabolic rate of MTX. 89−93 Similarly, conjugates of [ 3 H]-MTX with functionalized polypeptides (hydrophilic forms of drug carriers) were examined specifically for their analogical aspects. 94,95 For instance, simple modification of the primary drug, namely, [ 3 H]-MTX conjugation with individual peptide phenylalanine (via α-carboxyl group of glutamine moiety in MTX structure), produced a less toxic prodrug, capable of revealing its intrinsic cytotoxicity using tumor targeted monoclonal antibody conjugated with specific hydrolyzing enzyme. 96 Additionally, a significantly larger hydrophilic drug carrier in the form of antitransferrin-receptor monoclonal antibody (anti-TFR-mAb) and [ 3 H]-MTX conjugate has been proposed to enable migration of MTX across the blood−brain barrier (BBB). 97 Another antibody-based carrier, [ 3 H]-MTX conjugated with mAb against mouse mammary tumor antigen, demonstrated its ability toward selective tumor delivery of MTX. 98 It is also worth noting the concept of conjugating MTX with modified serum albumin for more intended drug delivering. [ 125 I]-Iodinated conjugate of MTX with maleylated-bovine serum albumin (BSA) was capable of increasing deposit of the radioagent in macrophages, which resulted in a superior antileishmanial effect compared to free MTX. 99 103 Authors showed that these MTX-HSA conjugates aimed at both FR and albumin receptors provided the ability to induce a significant antitumor effect, even in originally MTX-resistant tumors.
Reports on simple and ingenious possibilities of MTX targeting (to a specific target or selected systemic compartment) provided an insight into new possibilities in MTX therapy and the impetus for further development of MTXbased radiopharmaceuticals.
Recently, two MTX derivatives with two glutamine or lysine molecules attached to the amino groups at positions 2 and 4 of the pteridine ring have been presented. 104 116 both obtained in direct labeling, showed negligible human breast tumor targetability (tumor-to-blood ratios below 1) throughout in vivo biodistribution. Bombesin analogue and MTX conjugate complexed with 99m Tc 117 was found to demonstrate high affinity and internalization grade toward FRs (FR-positive KB cell line K d = 3.95 ± 0.80 nM; internalization at 31.0 ± 2.10%) or bombesin receptors expressed on tumor cell lines. However, further biodistribution on the xenograft murine models with the same cell lines revealed little tumor uptake of peptide-MTX radioconjugate and proved its inadequacy for intended imaging use.
Furthermore, for positron emission tomography (PET) imaging few 18 F radiotracers based on MTX have been developed. These include MTX derivatives conjugated with selected prosthetic groups already labeled with 18 118 In further biodistribution studies on xenograft mice MTX radiotracers exhibited tumor uptake comparable with concentrations of the tracer in blood and sensitive to FA blocking, however, significantly worse uptake than that observed in the case of folate analogues. The third MTX-radioligand, [ 18 F]FDG-oxime-carbohydrazide-MTX, proved to be the most promising. 119 It had not only higher binding affinity than previous MTX-tracers (FR-positive KB cell line K d = 4.74 ± 0.71 nM), but a similar in vivo imaging study demonstrated a noticeable tumor uptake at the same level as in other organs such as spleen, lungs, or intestines. At the same time, comparable [ 18 F]FDG-oxime-carbohydrazidefolate demonstrated superior binding affinity (FR-positive KB cell line K d = 1.83 ± 0.15 nM) than [ 18 F]FDG-oximecarbohydrazide-MTX as well as significant tumor uptake, 3fold higher than in the case of MTX radioagent. These reports clearly indicated the superiority of folate-based radiotracers in the biodistribution on FR-positive tumor bearing rodents against MTX-based radioligands.
[ 99m Tc]Tc-MTX complex was also investigated as a diagnostic agent for RA and compared to [ 99m Tc]Tc-MDP (methylene diphosphonate) complex. MDP complexed with 99m Tc is one of the most commonly applied diphosphonatebased radiopharmaceutical for skeletal scintigraphy in patients with bone metastases. 120,121 For decades, it has been considered as the standard single-photon emission computed tomography (SPECT) imaging agent with superior sensitivity compared to conventional radiography, as well as due to limited availability of PET facilities in numerous nuclear medicine departments. [ 99m Tc]Tc-MDP skeletal uptake mechanism is based on the bone surface chemisorption associated with the local blood flow and metabolic activity of the osteoblasts, factors easily altered by the tumor presence. Clinical experiments conducted on 5 patients with RA and 19 healthy volunteers compared [ 99m Tc]Tc-MTX and [ 99m Tc]Tc-MDP complexes in RA lesions imaging. 122 In reported results, the authors present scintigraphy evaluation of both radioagents only for two patients with regard to whole volunteer group. Thus, the obtained outcome for particular subjects indicated that [ 99m Tc]Tc-MTX demonstrated effective detection of early and small articular rheumatoid nodules, while [ 99m Tc]Tc-MDP exhibited noticeable differences in uptakes of healthy and severe inflammation altered joints only.
Another study investigated the ability of [ 99m Tc]Tc-MTX complex to detect severe (chemically induced) inflammations. 123 An ex vivo study performed on a small group of three mice with experimentally induced inflammation showed very high uptake of radiocomplex in kidneys and significant accumulation in bones. In parallel, [ 99m Tc]Tc-MTX binding to hydroxyapatite at the level of about 45% after 10 min of incubation was determined. Furthermore, dynamic imaging of turpentine-irritated mouse pointed to a high instant uptake of [ 99m Tc]Tc-MTX in the induced-inflammatory tissue and unfortunately also in liver. Simultaneously, control mice imaging showed first of all high bladder and kidney uptake, but also highlighted radiocomplex uptake in scull and in hindlimb joints. Despite authors demonstrating elevated uptake of [ 99m Tc]Tc-MTX in the target inflamed tissue, MTX radiocomplex had limited usability of chemically induced inflammation imaging due to low selectivity. Nevertheless, at the same time, this very study provided firm evidence of [ 99m Tc]Tc-MTX affinity to the hydroxyapatite present in skeletal tissue.
It is worth highlighting skeletal imaging performed on rats and rabbits using 99m Tc radiocomplex of MTX-bisphosphonate conjugate. 124 Although the precise structure of the complex was unknown in study, radioconjugate exhibited very high binding to bones and joints, most likely through a bi-

Molecular Pharmaceutics
pubs.acs.org/molecularpharmaceutics Review sphosphonate fragment. The authors of the study indicated promising prognostic utility of researched radioconjugate, but also the potential application of a similar complex containing therapeutic radionuclide 186 Re for the treatment of osteosarcoma or bone metastases. Above reports 111−119 provided limited uptake of MTX radioagents by FR-positive cancer cell lines especially in comparison to folate-based radioagents. However, results of in vivo or ex vivo biodistribution showed detectable MTX radioagents uptake at tumor sites, but also substantial uptake in spleen. On the other hand, reports on inflammatory lesions imaging 122,123 indicated that [ 99m Tc]Tc-MTX complex enabled clear detection of articular rheumatoid nodules or chemically induced inflammation. Interestingly, the favorable quality of these visualizations obtained using MTX radioagents may be due to pro-inflammatory M1 macrophages that highly express not only FR-β but also RFC on their surfaces. 125 These macrophages were recognized as granulocyte macrophage colony-stimulating factor-driven macrophages and demonstrated substantial contribution in tumor microenvironment and in close proximity inflamed tissue. 126 Moreover, it was determined that these pro-inflammatory M1 macrophages showed a specific response for methotrexate action. 127 More recently, to improve the outcome of the wellestablished therapeutic strategies, novel nanomaterials that combine immune agent MTX and radionuclides (for diagnosis or therapy) were developed. For targeted treatment of rheumatoid-affected joints, radiolabeled biodegradable nanoparticles entrapped with MTX were proposed. 128 These nanoparticles made of polylactic-co-glycolic acid (PLGA) polymer have the ability to release encapsulated drug in a controlled manner. Additionally, they possess hyaluronic acid (HA) molecules conjugated on their surface, which specifically guides molecules to the inflamed synovial tissue cells, and the macrocyclic chelator DOTA chelating cation of β − emitting radionuclide 177 Lu, which is favorable for mild irritation of macrophages in inflamed synovial tissue. In vitro assay on murine macrophages cell line was proven to display controlled and specific cytotoxicity of this multifunctional nanopolymeric system that was superior than MTX alone or PLGA polymer without HA, MTX, or [ 177 Lu]Lu-DOTA addition. Nevertheless, further in vivo examinations must be performed before these nanoparticles will find clinical application in radiosynovectomy of RA patients.
Another multimodal system based on multiwalled carbon nanotubes (MWCNTs) was developed for theranostic application in FR positive cancers. 129 Carbon nanotubes were surface-functionalized with Alexa-fluor-488/647 fluorochrome, diagnostic radionuclide 99m Tc, tumor-targeting FA, and MTX as anticancer agents. These nanotubes had the ability to release MTX under the lysosomal acidic conditions inside the cell, enabling MTX to exert its therapeutic action. Moreover, it was shown that these carbon nanotubes had superior binding affinity to FR positive human lung or breast cancer cell lines than MTX. Similarly, FR positive xenograft mice biodistribution studies showed that the microparticles demonstrated significant tumor uptake (however, also with very high uptake in liver, spleen and lungs) and tumor volume inhibition greater than free MTX. Proposed dual-imaging MWCNT system confirmed designed targeting and therapeutic functions; however, this still needs to improve the ability of tumor-specific delivery.
Presented reports on nanomaterial multimodal systems 128,129 seem very perspective and are designed rationally with properly established functions of the nanosystems. The selected biovectors (hyaluronic acid and folic acid) turned out to be appropriate and crucial for research outcomes. Moreover, a simultaneous application of immune agent and radiation (in reference 129 γ-emitting 99m Tc may be replaced by some β − emitter, as 186 Re) produced a synergistic effect in antiinflammatory action against cancer or/and immune cells. 130 In another study, an efficient in vitro delivery of the radiolabeled MTX conjugate to the targeted erythroid leukemia cells line expressing the transferrin receptor (TFR) was performed using a two-stage targeting procedure. 131 First, anti-TFR monoclonal antibody conjugated with recombinant DHFR enzyme (anti-TFR-mAb-rDHFR) was pretargeted to the tumor cells, followed by [ 111 In]In-DTPA-hydrazide-MTX We were surprised when we started our own research on [ 99m Tc]Tc-MTX radiocomplex and found great difficulty in the radiosyntheses of [ 99m Tc]Tc-MTX according to reports provided in this review (paper under preparation), which gives us food for thought related to the credibility of the presented above research.
On the other hand, the utilization of MTX in therapeutic radiopharmaceuticals is very limited to the component of multifunctional nanoparticles. Radiolabeled with 177 Lu, biodegradable PGLA nanoparticles entrapped with MTX are an elaborate concept for radiosynovectomy, 128 where decreased inflammation of the synovial tissue is induced synergistically by a disease-modifying antirheumatic drug and soft beta radionuclide guided by hyaluronic acid. Despite the promising results, the presented research is only the first step of the preclinical evaluation and requires further in vivo trials.