Nanotechnology-Assisted Cell Tracking

Abstract

The usefulness of nanoparticles (NPs) in the diagnostic and/or therapeutic sector is derived from their aptitude for navigating intra- and extracellular barriers successfully and to be spatiotemporally targeted. In this context, the optimization of NP delivery platforms is technologically related to the exploitation of the mechanisms involved in the NP–cell interaction. This review provides a detailed overview of the available technologies focusing on cell–NP interaction/detection by describing their applications in the fields of cancer and regenerative medicine. Specifically, a literature survey has been performed to analyze the key nanocarrier-impacting elements, such as NP typology and functionalization, the ability to tune cell interaction mechanisms under in vitro and in vivo conditions by framing, and at the same time, the imaging devices supporting NP delivery assessment, and consideration of their specificity and sensitivity. Although the large amount of literature information on the designs and applications of cell membrane-coated NPs has reached the extent at which it could be considered a mature branch of nanomedicine ready to be translated to the clinic, the technology applied to the biomimetic functionalization strategy of the design of NPs for directing cell labelling and intracellular retention appears less advanced. These approaches, if properly scaled up, will present diverse biomedical applications and make a positive impact on human health.

1. Introduction

Cell tracking refers to the investigation of living cells over a period allowing the visual depiction, characterization, and quantification of biological processes at the cellular and subcellular levels within intact living organisms. A fundamental property of any real-world object is that it extends in both space and time. This is particularly true and applicable for living organisms in which the study of subjects in three dimensions and the possibility to track it over time (denoted as 3D or 4D) assumes great significance.

Research community interest has grown exponentially in the last decades concerning the possibility of tracking objects (Figure 1A) [1,2,3,4,5,6] to automatically follow hundreds to thousands of cells. Imaging has provided the elective approach to enable cell tracking and it assumes the labeling of the target cell with objects, otherwise defined as contrast agents, with the aim of achieving contrast between the cells of interest and the other cells of the organism. Interestingly, nanoparticle (NP) systems have received great attention as the contrast agents to be used for cell labeling and followed by means of imaging techniques. Although the first attempts to automate the tracking of cells using NPs date back at least 20 years, the development of more advanced tracking methods really took off in the past decade (Figure 1B).

Two main components have a big impact in defining the approaches for cell tracking: the NP systems and the imaging technologies adopted to assist in cell tracking. NP systems are characterized by particles or constituents with unique features making them attractive for tracking purposes: (1) a small size (range diameter between 1 and 100 nm) and (2) a surface to mass ratio, functional to adsorb and efficiently carry compounds including peptides, fluorescent probes, and drugs that can be used as tracers, therapeutic tools or cell uptake enhancers [7].

Due to their nanoscale dimensions, NPs can be easily transported across cell membranes and reach the crucial subcellular organelles [8]. Furthermore, a high surface area-over-volume ratio enhances their interaction with cellular components [9]. In addition, NPs offer attractive features since their structure and chemical properties can be modified to facilitate cellular incorporation and because they can carry a high payload of the relevant labels into cells, thus generating high contrast signals to be followed by imaging.

Aside from NPs, the selection of appropriate imaging modalities assumes a crucial role in tuning the effective cell tracking approaches. Many advances have been achieved by developing new optical imaging probes, which have been useful for setting up multimodal imaging systems, and by adopting sophisticated optics and optical systems able to overcome the resolution limitations imposed by the scattering and absorption properties of tissue [10].

Overall, NP systems and imaging techniques can be considered as two closely related components of any cell tracking approach, which, if well managed, have an important impact in the improvement of current imaging resolution and sensitivity. The cell-tracking imaging platform choice relies on the type of contrast agent used for cell labeling and vice versa. Currently, the most suitable platform categories for NP-based imaging are magnetic resonance imaging (MRI), X-ray (also known as X-ray computed tomography and abbreviated as CT), optical imaging (endoscopy and fluorescence near-infrared or bioluminescence-based imaging methodologies), ultrasound, radionuclide molecular imaging and photoacoustic imaging (PAI), a hybrid technology combining optical and ultrasound imaging [11].

Specifically, MRI and CT are the two most used and clinically approved techniques for in vivo cell tracking, whereas the other imaging approaches have been primarily investigated in the last decade in translational research protocols showing a great potential for their clinical application. Cell tracking applications can be found in different areas of biomedicine ranging from the imaging of tissue replacement cell therapy (regenerative medicine) to cancer diagnosis and treatment (cancer medicine).

In regenerative medicine, cell tracking is a key technology for the development and optimization of cell therapy for the replacement or renewal of damaged or diseased tissue using transplanted cells [12]. In the cancer context, it helps early-stage cancer diagnosis and therapeutic imaging of tumor foci by improving accuracy and sensitivity of the current clinical protocols [13].

Several NPs with unique magnetic and/or optical properties have been investigated preclinically for real-time, in vivo cell monitoring [11]. These NPs include inorganic (with metal, ceramic, or semiconductor elements), organic (with polymeric or lipidic elements) and hybrid/composite (with both inorganic and organic elements) NPs. Of note, since the current imaging technology of choice for clinical translation is MRI, NPs with magnetic properties have been also tested for clinical application. Consistently, 11 studies employing NPs with magnetic properties for intra-operative procedure monitoring and/or diagnosis purposes are currently in clinical trial phases (Supplementary Materials Table S1).

Different cell tracking approaches have been developed including the ability to track NPs enclosed in cell membranes, cell derivates (microvesicles) or actively and directly incorporated by the cells. The last, showing great potential for application in the field of cell tracking with imaging [14,15,16].

In the field of regenerative medicine, the tracking of stem cell transplantation procedures and regenerative performance through imaging have been investigated with success by employing direct cell labeling methods based on the introduction of the NPs working as a contrast agent into the cells to be transplanted prior to in vivo injection [17]. Stem cell tracking approaches enhancing the regenerative properties of stem cells to be transplanted have also been developed by exploiting the possibility of incorporating into these cells NPs bearing specific moieties with an immunomodulatory role [12] (Figure 2A).

Of note, this strategy emerged as also applicable in the field of cancer imaging, where the native attitude of stem cells to sense and home in on tumor foci as well as the ability of some immune cells (e.g., macrophages or T cells) to be recruited by tumor-derived inflammatory stimuli, were exploited by using these cells as vehicles for NPs and thus, as a tracer guiding the identification of cancerous districts [18,19,20,21]. Moreover, because of the availability of specific NP systems enclosing therapeutics within their structure, this approach was also found to be suitable for monitoring the therapeutic effects elicited by the NPs conveyed by the cargo cell or directly incorporated by the cancer cells to be targeted [22] (Figure 2B).

The application of cell tracking approaches has a big impact on the improvement of currently available diagnostic and therapeutic imaging protocols. In the context of regenerative medicine, NP-based cell tracking through imaging provides a safe way to monitor the viability, proliferation and differentiation of transplanted cells by enabling the guidance of cell replacement and testing the therapeutic success of the adopted transplantation procedure.

With respect to cancer medicine, NP-based cell tracking through imaging represents an invaluable tool to provide a rapid and sensitive detection of cancer foci, thus acquiring a relevant role in early tumor diagnosis [13,23], consequently yielding greater chances for cancer treatment, and extending life expectancy. Furthermore, it can be exploited to monitor the response to specific theranostics, allowing the assessment of disease state [24]. Interestingly, some recent evidence has shown a possible application in image-guided surgery, enabling the intra-operative detection of any residual tumor mass to be removed and/or therapeutically targeted [25,26,27]. Despite their great potential, there are currently some limitations regarding their application for in vivo cell tracking at the translational level yet to be overcome [28].

This review aims to provide an overview of the nanotechnologies and methodologies eligible for cell tracking which have been investigated in the last decade, the window period identified as critical for the development of NP-based tracking strategies, with emphasis on (1) the specific NP characteristics responsible for effects on cell labeling, consequently defining the effectiveness of cell tracking, and (2) the approaches and mechanisms of NP incorporation into cells.

Consequently, the next paragraphs will focus on the following key elements: the types of NPs currently adopted in cell imaging (Section 2); the NP functionalization strategies most used to improve cell targeting, cell uptake, therapeutic tracking, and imaging performance (Section 3); the approaches suitable for in vitro, in vivo and ex vivo tracking of NP delivery (Section 4); the uptake mechanisms of the NPs (Section 5) and the cellular systems mostly described for NP loading (Section 6).

All the above key aspects have been inferred from research evidence collected with a systematic literature review performed in Web of Science (WoS) database during the last decade which focused on the investigation of NP-based cell tracking strategies.

2. NP-Based Cell Tracking Derived Bibliographic Database

Literature data published in peer-reviewed international papers indexed in the advanced search of WoS [v.5.35] “Core collection” archive (https://apps.webofknowledge.com/WOS_AdvancedSearch (accessed on 3 July 2021) in the last 10 years were considered.

Specifically, three lists were created by searching for the following key words and filtering for original research articles:

• Nanoparticles, cell tracking methods, fluorescent dyes;
• Nanoparticles, cellular uptake, cell labelling, staining;
• Tomography, magnetic resonance, nanoparticles, stem cell homing.

The words “AND” and “OR” were used as Boolean operators, “TS” as field tag.

The lists of papers originating from each key word search were then matched to create the final database of citations.

Overall, the adopted bibliographic approach allowed us to collect 151 original studies focusing on the investigation of strategies for direct cell labelling and tracking with NPs (Supplementary Materials Table S2). Ninety-eight of the total citations (64.5%) characterized the approaches to incorporate NPs into vehicle cells to be used to sense target cells or body districts after in vivo delivery into the body. In these studies, the aim was to find the most suitable way to improve uptake efficiency without affecting cell cargo wellness.

Fifty-four of the total citations (35.5%) investigated the effect of NP incorporation into cell models to be therapeutically targeted. In these studies, the aim was to find a protocol that could be effective in targeting desired cells, monitoring NP uptake and the cytotoxic effect of NP cell internalization (see Supplementary Materials Table S2).

Furthermore, based on the adopted cell models, the collected studies could find application in the field of cancer and tissue regeneration imaging. Specifically, 72 of the total citations were applied to cancer imaging, 57of the total citations to tissue regeneration imaging, whereas 23 of the total citations adopted models suitable in both fields (see Supplementary Materials Table S2).

3. NP Features and Cell Tracking Applications for Tissue Regeneration and Cancer Imaging

The literature survey allowed us to identify four distinctive classes of NPs as useful for developing suitable cell tracking platforms: inorganic, polymeric, hybrid and lipid based. The inorganic category resulted in the most represented NP typology in this survey, accounting for the 79.6% of the total collected citations, followed by polymeric (12.5%), hybrid (4.6%) and lipid based (3.3%), respectively (Figure 3). The prominent representation of the inorganic typology reflects the fact that these NPs are the ones best suited to cell tracking through the most characterized imaging platforms to date.

3.1. Inorganic

Inorganic NPs are characterized by a typical core/shell structure which can assume different shapes: spherical, rods, wires, tubes, pyramids, and stars. The core size of these nanoparticles is approximately 3–6 nm and a coated system (shell) increases its size to 20–150 nm. The core can contain metals, other chemical elements or fluorescent dyes encapsulated in silica. The core defines the magnetic, electronic, fluorescence and optical properties of the NP [28]. The shell is usually made of metals or organic polymers that (I) protect the core from chemical interactions with the external environment, (II) serves as a substrate for conjugation with biomolecules (antibodies, peptides or oligonucleotides) and (III) preserves NP stability avoiding aggregation [29,30]. Due to their magnetic, radioactive, X-ray absorption or plasmonic properties, inorganic NPs are used for diagnostics and imaging purposes and most of them display good stability [31] and biocompatibility [32].

Four main subcategories of inorganic NPs applicable for cell tracking were identified by this survey, based on the nature of the core element:

(I)

Metal-based NPs, accounting for 31.5% of the collected inorganic NPs, include silica, manganese, gold, silver, lanthanide, molybdenum, ruthenium, rubidium, gadolinium, and zinc elements.

(II)

Metal oxide-based NPs, representing 64.5% of the collected inorganic NPs, enclose iron oxide, superparamagnetic iron oxide (SPIO), ultrasmall superparamagnetic iron oxide (USPIO), titanium oxide and cobalt iron oxide elements.

(III)

Metal sulfide or phosphide-based NPs, accounting for 2.4% of the collected inorganic NPs, comprise quantum dots.

(IV)

Mineral-based NPs, accounting for 2.4% of the collected inorganic NPs, take in hydroxyapatite and selenium elements.

3.2. Polymeric

Polymeric NPs are particles within the size range from 1 to 1000 nm and are constituted by a polymeric matrix core which can be loaded with bioactive molecules. They are categorized into two forms, spheres (the bioactive molecule is dispersed within a polymer matrix) and capsules (the bioactive compound is placed in the core of the particle covered by a layer of polymer). Polysaccharides and proteins are the commonly used materials for the fabrication of polymeric nanoparticles. The polymer provides biocompatibility and protection to the active molecules to the delivery site [33].

The most used polymers for the constitution of polymeric NPs collected by this survey were poly(ethylene glycol) (PEG; 33% of the collected polymeric NP), poly(lactide-co-glycolide) (PLGA; 24%) and polystyrene (PS; 13%). Although to a lesser extent, other polymers were also identified: poly(epsilon-caprolactone) (PCL), poly(lactide) (PLA), poly(2-methacryloyloxy) ethyl phosphorylcholine (PMPC), poly(glycerol-co-sebacate) (PGS), poly(diphenylamine)-poly(ethylene oxide) (PDPA-PEO) and poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV). Of note, these polymers have been used alone or in combination to design polymeric NPs for cell tracking.

3.3. Hybrid

Hybrid NPs are constructed from at least two different NPs, to overcome the limits of single-component nanoparticles, to improve properties, to achieve new properties not possible for single nanoparticles, and/or to achieve multiple functionalities for single nanoparticles. Size range can be variable depending on the NP typologies used for their fabrication [34].

3.4. Lipid-Based

Lipid-based NPs, defined as colloidal carriers for bioactive molecules, are assembled to constitute different structures: liposomes with size < 200 nm, solid lipid NP (SLN, solid lipids), and nanostructured lipid carriers (NLC, a combination of liquid and solid lipids) with sizes ranging from 10–1000 nm [35,36,37,38].

Liposomes are spherical vesicles with an aqueous core and bilayer lipid membrane. They have the capacity to encapsulate diverse bioactive compounds, which can be included into the aqueous core or at the bilayer interface [39].

SLNs are generally spherical in shape and consist of a solid lipid core stabilized by a surfactant. This construct can be used to deliver both hydrophilic and hydrophobic bioactive molecules [40].

Mixing solid lipids with small amounts of liquid lipids allows structural rearrangements of the matrix-generating NLC formulations to be produced, improving their properties in this respect, while maintaining the original benefits of SLNs. Of note, recently, cell-derived membrane lipidic vesicles with a small size range (40–100 nm) have been considered as alternative naturally derived lipid NPs versus the synthetic lipidic formulation [16].

With respect to the application of the different NP typologies in the field of cancer imaging or tissue regeneration imaging, the literature survey revealed that polymeric and lipid-based NPs are the most represented in the studies, pointing to the development of cancer imaging and therapeutic protocols; the hybrid category was applied for tissue regeneration imaging whereas use of the inorganic category was well documented for setting up both cancer and regenerative imaging protocols (see Supplementary Materials Table S1). Importantly, some of the collected studies, which will be further discussed below, moved a step forward in the characterization of the best NP platform for cell tracking by providing the adoption of in vitro and/or in vivo imaging procedures to be applicable in cancer or regenerative imaging.

3.5. NP-Based Cell Tracking for Imaging of Tissue Regeneration

In the context of tissue replacement cell-therapy imaging, protocols assuring the long-term monitoring of transplanted cells without affecting cell viability and/or properly modulating their state of differentiation depending on the tissue/organ to be targeted, have yet to be identified. Indeed, at present, clinical cell-tracking trials have only provided information on immediate cell delivery and short-term cell retention [41]. Overall, many key issues concerning labeling solutions able to augment the possibility of tracking cells enclosing NPs, avoiding label dilution during cell proliferation and improving the uptake and retention of NPs, as well as preserving the proper functions of the transplanted cell and their correct homing to the target site, remain to be addressed [42].

3.6. Labeling Solution Improving Cell Imaging

Over the last decade, direct cell labeling with inorganic NPs engineered with multiple tracers has received increased attention for improving the tracking and imaging performance of tissue and/or organ regeneration [39]. Indeed, the use of a combination of two or more chemical elements to be traced by means of different imaging techniques, known as the multi-labeling approach, represents a way to overcome the imaging detection limits of a specific technique. As some examples, inorganic gold NPs with red-emitting firefly luciferase (RfLuc)-based bioluminescence (BL) tags [43] and inorganic NPs with gold and gadolinium elements [44] have been developed for the noninvasive labeling and tracking of human mesenchymal stem cells (hMSCs) in a mouse model of pulmonary disease. Specifically, multimodal imaging with CT, MRI and BL was successfully adopted to characterize the in vivo migration, homing, functions, and survival of transplanted hMSCs, giving further insight for the treatment of pulmonary fibrosis.

Inorganic iron oxide NPs modified with melanin and Fe ions were successfully used for dual modal imaging through MRI and photoacoustic (PA) methods of engrafted bone marrow-derived mesenchymal stem cells in mouse models [45]. Multilayered inorganic NPs constituted by a combination of mesoporous silica, iron oxide and gold elements were successfully used to multilabel umbilical cord mesenchymal stem cells in ischemic mouse brain models [46]. Moreover, inorganic iron oxide labeled with a fluorophore have been used to trace in utero hematopoietic cell transplantation in a canine animal model, offering a potential approach for early intervention for the treatment of diseases before birth [47].

Interestingly, Geburek and colleagues experimented on an additional way for multilabeling by using adipose-derived mesenchymal stem cells traduced with a reporter gene for GFP and co-labelled with iron particles for tracking after the intralesional treatment of artificial equine tendon lesions [48]. A similar approach was used to efficiently track the homing of magnetically labeled adipose-derived stem cells [49] and human umbilical cord-derived mesenchymal stem cells [50] expressing GFP into mouse carotid artery and cutaneous injury sites, respectively. In contrast, Wu and colleagues suggested combining the reporter gene directly with the NPs. In this case, the adoption of inorganic iron oxide NPs complexed with polymers acting as a cargo for the luciferase system and or a fluorescence protein as reporter genes was shown to be effective for an MRI-visible gene delivery agent which could effectively label MSCs, providing the basis for bimodal bioluminescence and MRI tracking of transplantation to solve acute liver injury [51].

Hybrid NPs have also been adopted for generating a platform for multimodal imaging. Fluorinated polymeric NPs combined with fluorine as the MRI contrast agent and functionalized with a fluorophore (N-fluoresceinyl) maleimide) as optical contrast moiety were used for tracking MCS in vivo [52].

Advances in multimodal labeling and tracking of cell transplantations have also been made by functionalization of polymeric NPs. Zhang et al. identified how the fluorescent imaging of human mesenchymal stem cells that have endocytosed fluorescent polymeric NPs can be directly and clearly captured in the one-photon and two-photon modes, offering the possibility of the direct monitoring of stem cells with high resolution and quantifying NP uptake, encouraging future quantitative clinical assessment in imaging-guided cell therapies [53]. Similarly, the exploitation of the one-photon and two-photon imaging properties of polymeric NPs loaded with red phosphorescence dye of bis(2-methyldibenzo[f,h]quinoxaline) (acetylacetonate) iridium(III) (Ir(MDQ)2acac) provided a reliable approach for the efficient and sensitive tracking and monitoring of transplanted neural stem cells (NSCs) [54]. Multimodal tracking was also achieved by functionalizing NPs with fluorine, which served as contrast agent for MRI and as an effective platform to properly direct the transplanted MSC fate in a non-obese diabetic/severe combined immunodeficient mouse model [52], as well as for the tracking of immune and stem cells to be adopted for cellular therapies [55].

3.7. Labeling Solution Improving Cell Uptake and Homing

The labeling solutions aimed at improving cell uptake and homing to the desired site of the body have been investigated by functionalizing NPs with different biomaterials. Accordingly, the literature survey identified inorganic iron oxide NPs functionalized with several moieties including poly-lysine, dextran, zinc, protamine sulfate, heparin or poly-(l-lactide) as good platforms for direct label, home and track MSCs in preclinical models of wound healing [56], traumatic brain injury [57,58,59,60,61], after renal transplantation in mice [62], hepatic cirrhosis [63] and photothrombotic cerebral infarction [64] with MRI. Poly (dopamine)-coated iron oxide NPs were also used for noninvasive labeling, tracking, and targeted delivery of adipose tissue-derived stem cells in mouse models of liver diseases [65].

Efficient strategies for the traceable homing of therapeutic NPs were developed by using hybrid NPs composed of glucocorticoid betamethasone phosphate (BMP) and the fluorescent dye DY-647 (BMP-IOH-NPs). Uptake of these NPs into macrophages was proved to efficiently drive treatment of inflammation with simultaneous in vivo monitoring of NP delivery [66].

3.8. Labeling Solutions for Long-Term Imaging and Modulation of Stem Cell Differentiation State

Protocols assuring long-term monitoring and targeted modulation of transplanted stem cell differentiation have been investigated by functionalizing NPs with moieties such as bioactive factors and/or nucleic acids able to manage cell fate upon their in situ release. Consistently, the literature survey identifies studies in which inorganic mesoporous silica NPs functionalized with dexamethasone [67] or a specific miRNA (miRNA-26a-5p) [68] (p. 2) resulted in effective tracking of bone marrow stem-cell homing and enhancing osteogenic differentiation in preclinical mouse models. Furthermore, polymeric NPs bearing the stromal factor-1α (SDF-1α) as the bioactive molecule known to regulate MSC homing and localization and the antimir138, with a key role in the promotion of osteogenic differentiation proved to be efficient for driving cranial bone regeneration by means of MSCs in preclinical mouse models [69].

Other studies have noticed a strong influence of intrinsic physicochemical properties of some types of NPs in directing stem cell fate by modulating the differentiation and proliferation processes. Accordingly, inorganic gold NPs modified with 11-mercaptoundecanoic acid as well as inorganic upconverting NPs have shown the ability to modulate lineage differentiation of rat bone marrow MSCs [70,71].

3.9. NP-Based Cell Tracking for Imaging of Cancer

A NP-based cell tracking system represents a critical element, which if well managed, might allow the improvement of the resolution and sensitivity of current imaging techniques applied for targeted cancer diagnosis and/or therapy. Currently, the most widely used diagnostic imaging tools for the clinical detection of cancer, such as X-ray, magnetic resonance imaging (MRI), computed tomography (CT), endoscopy, and ultrasound, can only detect cancer when there is a visible change to the tissue. By that time, thousands of cancer cells may have proliferated and even metastasized. In addition, current imaging methods cannot distinguish benign lesions from malignant lesions [72]. Furthermore, cytology and histopathology examinations are usually adopted to characterize malignant cells and/or tissues primarily identified through imaging techniques such as CT or MRI and cannot be effectively and independently applied to detect cancer at an early stage [73].

The exploitation of the different physicochemical properties of NPs and the possibility of functionalizing NPs with a plethora of bioactive molecules have been demonstrated to be crucial in innovating the procedures for cancer imaging and to allow targeted therapeutic NP uptake.

3.10. Labeling Strategies to Improve Targeted Cell Uptake and Tracking

Improved targeted cancer cell tracking has been achieved by functionalizing several types of NPs with moieties which enhance tracking resolution.

In this context, inorganic silica NPs conjugated with innovative fluorescent nanomaterials bearing excellent photostability and with specific miRNAs and/or active molecules with a high affinity for cancer cells were successfully adopted to trace cancer foci. In detail, the synthesis of fluorescent dye-doped silica NPs carrying miRNA-21 or PEG peptide were demonstrated to be effective in NP delivery and imaging in breast [74] and liver [75] cancer cells. miRNA21 was found to be one of the most represented in breast cancer tumors [76], whereas PEG has been found to effectively recognize tumor-marker carcinoembryonic antigens (CEAs) [77]. Furthermore, inorganic iron oxide NPs carrying therapeutic siRNAs were efficiently used for tracking siRNA-based gene therapy targeting the BCL2 and BIRC5 of oral cancer and glioblastoma cells [78].

Another class of NPs, polymeric NPs conjugated with liposoluble fluorescent probes (fluorescein, coumarin 6 or DiR probes) as contrast agents and with oleanolic acid [79] or emodin and heparin sodium [80] as targeting moieties, were also used to track liver cancer cells to be therapeutically targeted. The multifunctionalization of polymeric NPs with coumarin 6 as labeling moieties and with either a therapeutic moiety and a peptide (peptide-22) with a special affinity for the low-density lipoprotein receptor (LDLR) was also successfully used to facilitate drug penetration of the blood–brain barrier and to improve the visualization of targeted cellular uptake, avoiding a non-selective brain drug accumulation in a preclinical mouse model of glioblastoma [81].

Furthermore, the tracking of polymeric NPs labeled with a near-infrared dye (NIR) and conjugated with either a chemotherapeutic drug paclitaxel (PTX) and transferrin as the targeting moieties allowed the biodistribution of tumor cells incorporating NPs and the antitumor effect to be studied in a preclinical mouse model of cancer overexpression in the transferrin receptor [82]. Conjugation of polymeric NPs with NIR fluorophore dye has also been proved efficient in mediating the long-term tracking of cancer cells in vivo [83].

Strategies for cancer imaging have also been developed by adopting other types of NPs. Hybrid NPs constituted by glyceryl monooleate-coated magnetic nanoparticles (MNPs) combined with glyceryl monooleate were found to act as a better labeling and efficient tracking agent without affecting the inherent properties of the MSCs to be used for tumor homing in preclinical rat models [84].

Lipid-based micelle NPs carrying a fluorescent-tagged antibody targeting the breast cancer cell antigen HER2 showed highly efficient internalization into the target cell, good tracking performance and a cytotoxic effect [85].

3.11. Labeling Strategies to Improve Imaging Sensitivity

Methods to augment the possibility of localizing the homing of stem cells or other cell types bearing NPs to tumors are being investigated with the goal of enhancing tracking and treatment efficacy.

As some examples, cationic magneto-liposomes were used to magnetically label human blood outgrowth endothelial cells (BOECs) and follow their homing to map cancer foci by magnetic resonance imaging (MRI) [86]. Importantly, these cells have shown extensive promise as gene-delivery vehicles for cancer cell treatment.

Furthermore, the two- and three-photon luminescence capabilities and strong optical absorption of an innovative class of gold NP structures, known as gold nanocages, enabled the quantitative tracking of labeled hMSCs using two imaging procedures: two-photon microscopy and photoacoustic microscopy. Importantly, this bi-modal tracking enabled the locating of cells that had migrated to glioblastoma regions formed by subcutaneous injection in nude mice in vivo [87].

In addition, luciferase-expressing human adipocyte-derived stem cells (ADSCs) were also used as a vehicle for Indium-111 radiolabeled inorganic iron oxide NPs to produce cells with tri-modal imaging capabilities. The ADSCs’ homing and fate was monitored by using mouse models of breast cancer with bioluminescence imaging (BLI) as a measure of cell viability, magnetic resonance imaging (MRI) for cell localization and single photon emission computer tomography (SPECT) for cell quantification [88].

Dual-modal imaging has been investigated for improving the sensitivity of detection and accurate evaluation of benign and malignant lymph nodes by adopting hybrid NPs comprising superparamagnetic polymeric micelles conjugated with a fluorescent dye. These NPs showed efficient targeting and uptake by lymph node macrophages [89].

4. NP Functionalization Strategies for Cell Targeting, Cell Uptake, Therapeutic and Imaging Tracking

4.1. Key Factors for an Efficient and Targeted NP Uptake into Cells

Accurate cell tracking occurs if the NPs are correctly taken up by the cell to be targeted. The targetability and efficiency of the uptake are governed by the interactions between the NP surface groups and the plasma membrane antigens and/or receptors, which in turn depend on the density of the ligands and the antigens/receptors present on an NP and a cell, respectively [90]. NP functionalization with specific bioactive molecules (hereinafter referred as moieties) have been investigated to that purpose and those collected with the bibliographic survey were summarized in Table 1. Specifically, these moieties can elicit a targeted NP uptake by capturing specific cell biomarkers such as antigens/receptors (moieties with active action) or by enhancing NP permeation and retention (moieties with passive action) (Table 1).

4.2. Targeting and Uptake Moieties

Antibodies [91,92,93,94] with high specificity and affinity towards cancer cell surface molecules have been identified as active targeting and uptake moieties. Likewise, folic acid and riboflavin vitamins [95,96,97,98,99,100] have represented an attractive strategy for efficient NP uptake by cancer cells to be targeted, due to both overexpression of the folate and riboflavin receptors on the cancer cells and the rapid internalization of the receptor by receptor-mediated endocytosis [101,102].

Aptamers, consisting of small molecule DNA or RNA fragments that fold into well-defined 3D structures, have also been found to recognize specific receptors on the cell surface and mediate NP internalization [43,74,81,85,103,104,105,106,107]. The same is true for carbohydrates such as dextran and carbodextran [47,48,57,64,108,109,110,111,112,113,114,115,116], chitosan [69,117], glucose [118,119], beta cyclodextrin [120], and the glycoprotein transferrin [82] that have been mostly used for NP coating as a strategy to avoid immune response and, furthermore, to introduce active-targeting capabilities as well as increase cellular uptake [121].

Passive targeting and NP uptake have been efficiently achieved by polymers [56,60] and heparin [55,62] which, based on their biocompatibility, allow for enhanced NP permeation and retention.

Other types of moieties with passive action were quaternary ammonium cations [87], the polypeptide polylysine [49,58,63,64,122,123,124,125,126], and amino acids such as histidine [127,128], which, thanks to their positive charge, stabilize NPs and mediate the electrostatic interaction with the cell membrane, improving the endocytosis mechanisms. Similarly, some derivatives of vitamins, as an example TPGS compound [80], have been found to improve NP uptake by increase membrane permeability [129].

Table 1. NP surface targeting and uptake moieties.

Targeting and Uptake Moieties	Active or Passive Action	References
Antibodies, peptides	Active	[91,92,93,94]
Aminoacids	Passive	[127,128]
Aptamers	Active	[43,74,81,85,103,104,105,106,107]
Carbohydrates and glycoproteins (chitosan, beta cyclodextrin, dextran, transferrin, glucose)	Active	[47,48,57,64,69,82,108,109,110,111,112,113,114,115,116,117,118,119,120,121]
Vitamins (folate, riboflavin)	Active	[95,96,97,98,99,100]
Polymers (poly-L-lactide; hyaluronic acid, cholanic acid)	Passive	[56,60]
Polypeptide (Polylysine)	Passive	[49,58,63,64,122,123,124,125,126]
Heparin	Passive	[55,62]
D-α tocopheryl polyethylene glycol 1000 succinate (TPGS)	Passive	[80]
Quaternary ammonium cations	Passive	[87]

Table 1. NP surface targeting and uptake moieties.

Targeting and Uptake Moieties	Active or Passive Action	References
Antibodies, peptides	Active	[91,92,93,94]
Aminoacids	Passive	[127,128]
Aptamers	Active	[43,74,81,85,103,104,105,106,107]
Carbohydrates and glycoproteins (chitosan, beta cyclodextrin, dextran, transferrin, glucose)	Active	[47,48,57,64,69,82,108,109,110,111,112,113,114,115,116,117,118,119,120,121]
Vitamins (folate, riboflavin)	Active	[95,96,97,98,99,100]
Polymers (poly-L-lactide; hyaluronic acid, cholanic acid)	Passive	[56,60]
Polypeptide (Polylysine)	Passive	[49,58,63,64,122,123,124,125,126]
Heparin	Passive	[55,62]
D-α tocopheryl polyethylene glycol 1000 succinate (TPGS)	Passive	[80]
Quaternary ammonium cations	Passive	[87]

4.3. Key Factors for an Efficient Imaging of NPs in Cells

Identification of tissue alterations or lesion characterization as well as the diagnosis and treatment of disease through NP-based imaging techniques requires functionalization with contrast agents to be followed with imaging devices. Different molecules with optical properties such as fluorophore and bioluminescent dyes, isotopes or chemical elements with high molecular weight have been investigated as contrast agents. It is noteworthy that a considerable part of the reviewed studies suggested a combinatorial usage of contrast agents as an effective strategy for multimodal in vitro and/or in vivo tracking, as it could allow the limitations found with the use of a single-tracking approach to be overcome [130]. For this purpose, several protocols for the surface modification of inorganic NPs with specific elements have been developed. Because of this, the chemical element in the core of the NP represents one contrast moiety, and the molecule used for surface functionalization with an additional label. another. As an example, bi-modal tracking with MRI and PA has been performed by adopting NPs with two different metallic elements, such as gold and iron oxide [45,46]. A combination of fluorophores and metallic gold and iron oxide contrast agents was successfully used for optical and MRI tracking [47,48,49,50,87,88]. A review of the surface modification moieties for in vitro and/or in vivo tracking of the NPs identified by our bibliographic survey is included in

Table 2. Moieties for labeling. NPs can be functionalized with labeling moieties allowing both in vitro and in vivo NP tracking delivery.

Moieties for Labeling	In Vitro	In Vivo
Fluorophores (fluorescein, rhodamine, squaraine, acridine, DY674, cianine, AxHD dye, SR-FLIVO dye, IR780, selenium, NIR dyes, becon probe, RuBpy, IRGD, I-BODIPY, Fluo4, PKH26)	[43,52,53,54,66,74,75,83,95,98,105,107,115,120,123,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176]	[43,52,54,61,66,83,95,96,105,115,123,157,158,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179]
Radionuclides (Indium-111; Gallium-68; Lutetium-177)	-	[55,88,180,181,182,183,184,185,186]

.

4.4. Key Factors for Tracking Therapeutic NPs through Imaging

Advances in the field of NP-based cell tracking have led to extraordinary progress, with several strategies being deployed to also track therapy delivery through imaging. Several therapeutic NPs that are capable of both self-reporting disease and/or tissue damage and delivering therapy have been developed to date and play a central role for the implementation of currently available diagnostic and therapeutic protocols [24]. As an example, therapy followed by imaging might be useful to test reactions in order to treat and identify patients in which therapy has an effect with the goal of providing personalized therapy for individual patients. Functionalization allowing the use of these NPs as therapeutic agents, referred to as therapeutic moieties consist of molecules managing the proliferative and inflammation status of the target cell.

Therapeutic moieties identified by the present literature survey include miRNA [68,69], siRNA [78,187], genes [164] and drugs or compounds with key roles in the modulation of cell proliferation and differentiation [66,67,79,80,104,146,166,188] (Table 3).

Some examples of the adoption of these moieties are listed as follows. The miRNA AntimiR-138 was shown to significantly promote the expression of osteogenesis-related genes and its conjugation to the NPs used to label the MSCs was found to efficiently enhance the osteogenic differentiation of transplanted MSCs and direct cranial bone regeneration in preclinical mouse models [69]. Similarly, Hosseinpour and colleagues adopted NPs functionalized with the miRNA -26a-5p to label MSCs to be transplanted for tissue regeneration purposes [68].

siRNAs targeting genes whose expression is enhanced in tumors, such as marker B-cell lymphoma-2 (BCL2) and baculoviral IAP repeat-containing 5 (BIRC5) as well as genes with regulatory roles in sustaining cancer cell proliferation and migration, were used for NP functionalization and were found to selectively blockade the cancer cell proliferation of oral [115] and glioblastoma [78] cancer cell models.

NP functionalization with a model gene, consisting of a plasmid containing the green fluorescent protein, enabled gene transfer to the hMSCs showing the potential use of NPs for gene therapy delivery [121].

Chemotherapy drugs such as doxorubicin and methotrexate as well as molecules with the ability to modulate cell proliferation and differentiation events such as dexamethasone, betamethasone, caspase inhibitors, ceramide, oleanolic acid, emodin, heparin, aspirin, curcumin and sulforaphane were also adopted as therapeutic moieties for NP functionalization and found to be effective in (1) blocking cancer cell proliferation in lymph node [183] glioma [188], ovarian [146], hepatocellular [189], liver [79,80], cervical [95], pancreatic ductal [190] tumor models; (2) exerting anti-inflammatory actions [66]; (3) directing stem cell differentiation [62]; and (4) targeting apoptotic events of cerebral ischemia [166].

Importantly, in all the mentioned studies the functionalization of NPs with tracking moieties also working as contrast agents enabled a solid strategy for monitoring the effect of NP-cell based therapy to be set up.

Table 3. NP surface moieties with therapeutic effects.

Therapeutic Moieties	References
miRNA	[68,69]
siRNA	[78,187]
Drugs (doxorubicin, dexamethasone, betamethasone, methotrexate, caspase inhibitors, ceramide, oleanoic acid, emodin, heparin, aspirin, curcumin and sulforaphane)	[66,67,79,95,103,132,147,167,189]
Gene	[164]

Table 3. NP surface moieties with therapeutic effects.

Therapeutic Moieties	References
miRNA	[68,69]
siRNA	[78,187]
Drugs (doxorubicin, dexamethasone, betamethasone, methotrexate, caspase inhibitors, ceramide, oleanoic acid, emodin, heparin, aspirin, curcumin and sulforaphane)	[66,67,79,95,103,132,147,167,189]
Gene	[164]

The surface modifications for NP delivery, diagnostic imaging and tracking of therapeutic effects are summarized in Figure 4.

5. Approaches and Devices for In Vitro, In Vivo and Ex Vivo Tracking of NP Delivery and for Assessing NP Stability

5.1. In Vitro and In Vivo Tracking of NP Delivery

Based on the present literature review, fluorophore labeling moieties have been found to be suitable for both in vitro and in vivo tracking. Conversely, labeling with radionuclides was only applied for in vivo tracking.

In detail, optical imaging instruments such as confocal scanning, fluorescence microscopy and flow cytometry have been adopted for in vitro fluorophore tracking whereas the fluorescence imaging devices such as the NIR fluorescence IVIS Spectrum system, fluorescence molecular tomography imaging and single- and two-photon fluorescence imaging have been applied for in vivo fluorescence tracking. Conversely, radionuclide-labeled NP monitoring has been achieved in vivo by means of nuclear molecular imaging tools such as single photon emission computed tomography (SPECT) and positron emission tomography (PET).

The multimodality of imaging has also been investigated by incorporating labeled NPs with cells bearing ectopic expression of the luciferase gene [43,115,170] and provided a non-invasive and more effective imaging technology to track transplanted cells [43,170] and stem cell homing [115].

The multilabeling system represents an advantageous approach as it provides different molecules which can act as contrast agents to be distinctively used for in vitro and in vivo NP delivery characterization. Importantly, multilabeling possibilities rely on the definition of the NP material and type. Indeed, differently from polymeric and lipid-based NPs, the tracking of inorganic NPs also takes advantage of the intrinsic attitude of the constituent core element to work as a contrast agent. In that case, both the metallic core and the surface labeling moiety enable in vitro and in vivo tracking of NP delivery.

In vitro tracking of the core element can be achieved by means of transmission electron microscopy (TEM) and scanning electron microscopy (SEM), whereas NP surface-labeling moieties such as fluorophores and cell-labeling moieties such as luciferin can be followed in vitro by using confocal laser microscope and luminometer, respectively (Figure 5A and Supplementary Materials Table S3). On the other hand, in vivo tracking of the core element can be accomplished with MRI and tomography imaging such as computed tomography (CT), microcomputed tomography (µCT), photoacoustic tomography (PA) and X-ray fluorescence tomography (XFT) imaging.

It is noteworthy that, according to NP classification, our literature survey identified the MRI (52%), followed by CT (25%), optical (19%) and radionuclide (4%) imaging as the most used in vivo imaging devices (Figure 5B and Supplementary Materials Table S4).

Two different aspects bind the choice of the imaging technique: (I) the tissue to be targeted and (II) the sensitivity of the technique.

MRI represents the imaging method with the best soft tissue contrast, providing multiplanar capability without the use of ionizing radiation. It has emerged as the preferred modality for evaluating soft tissue masses and vascular structures, and it has been applied for checking soft tumors and inflammatory foci [191]. Iron oxide and gadolinium have been described as the most used contrast agents for MRI. While an MRI takes excellent pictures of soft tissue and blood vessels, a CT scan shows hard tissues much better, so it is often used to image bone structures [192]. Gold elements represent the elective contrast agent of CT. Many studies have shown that due to their strong X-ray absorption coefficient, good colloidal stability and biocompatibility, sustained contrast, shape and size controllability, surface modifiability, and few negative impacts on the marked cells, gold nanomaterials are ideal CT nano tracers for tracking [44]. As examples, with the use of this strategy, it was possible to demonstrate non-invasive cell tracking towards tumor sites [118], understand the fate of transplanted mesenchymal stem cells in certain diseases such as pulmonary fibrosis [43], and allow non-invasive and long-term CT monitoring of mesenchymal stem cells in tissue repair [123].

In contrast to MRI and CT, the two most used and clinically approved techniques for in vivo tracking, optical imaging is well suited for non-clinical use. However, it retains the potential for clinical application as it could exploit an enormous range of contrast agents such as molecules with fluorescent, near infrared or bioluminescent properties, that provide information about the structure and function of tissues ranging from single cells to entire organisms. An additional benefit of optical imaging that is often underexploited is its ability to acquire data at high speeds; a feature that enables it to not only observe static distributions of contrast, but to probe and characterize dynamic events related to physiology, disease progression and acute interventions in real time [193].

A few studies applied nuclear molecular imaging for NP tracking in vivo. Although this technique provides highly sensitive and quantitative imaging and shows good tissue penetration depth, it is limited by its low spatial resolution [194]. This was reflected in the small number of research articles collected by our bibliographic approach.

Apart from these limitations, nuclear molecular imaging holds great potential for preclinical research and clinical applications as it might be a valuable tool granting long-term monitoring of cells, as well as the observation of cell clearance from various organs and homing at the tumor site.

NPs can be labeled with several radionuclides and have been proposed as innovative tools for imaging with single-photon emission computerized tomography (SPECT) and positron emission tomography (PET) and enhanced radioisotope therapy for several cancer types with limited toxicity [195].

Radionuclide-labeled NPs mostly characterized for diagnostic imaging and/or therapy purposes include technetium-99m (99mTc) [196], fluorine-18 (18F) [197], gallium-68 (68Ga) [198], indium-111 (111In) [199], and lutetium-177 (177Lu) [200].

The imaging and therapeutic applications of an isotope depend on its particle emission. Gamma (γ)-ray emitters such as 99mTC, 111In and positron emitters such as 18F, 68Ga are commonly used for diagnostic purposes using SPECT and PET imaging, respectively, and β- emitters such as 177Lu are mostly, but not exclusively, used for radionuclide therapy.

An appealing strategy that has proved beneficial for cell imaging with radionucleotides was its adoption in combination with other imaging tools such as those with magnetic and/or X-ray properties to combine the advantages of different imaging modalities and minimize the limitations [201]. Of note, multimodality imaging systems such as SPECT/MRI, PET/MRI, SPECT/CT, and PET/CT are currently under investigation. As some examples, the conjugation of 99mTc with silica [202], iron oxide [203] or gold [204] NPs was exploited for SPECT/MRI or SPECT/CT imaging of tumors in preclinical mouse models. Likewise, the conjugation of 18F with iron oxide NP was found effective for dual imaging with PET/MRI of stem cell biodistribution [205] as well as for monitoring anti-angiogenic therapeutic effects in breast cancer xenograft mouse models [206] and diagnosis of rectal cancer in clinics [207]. Importantly, since PET/MRI systems have become operational worldwide, several imaging protocols involving the use of 18F for whole body scanning in oncology are currently under standardization for patient management in clinics [208].

Radionuclide imaging for tumor diagnosis was also performed by adopting NPs conjugated with radiopharmaceutical formulations of the (γ)-ray emitter 111In (such as indium-111-oxine) allowing the monitoring of migration and homing of intravenously given stem cell-bearing NPs to the tumor location [55,88,180].

Advantageous strategies for simultaneous diagnostics and therapy are also being characterized. In this context, a promising strategy is the pairing of radionuclides of different elements [195]. As an example, 68Ga for PET imaging and 177Lu as β-emitter for a therapy couple have been investigated as a radiopharmaceutical tool to face prostate [181] colon [182], lymph node [183,184,185] and glioblastoma [186] cancers.

Overall, based on these premises, it would be helpful to find a compromise between invasiveness and diagnostic resolution to improve actual NP tracking methods.

5.2. Ex Vivo NP Tracking of NP Delivery

Specific ex vivo protocols have been developed to verify and quantify NP uptake on tissue and cell specimens prior to being applied for in vivo protocols. Several histochemical dyes can be adopted for that purpose (

Table 4. Dyes used for ex vivo quantification of NP uptake. Dyes can react with elements characterizing the NPs or can be applied for NP coating.

Dye	Type	References
Histochemical staining	Prussian blue	[48,49,51,57,58,59,64,65,88,109,114,116,117,124,127,138,154,155,160,177,187,189,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224]
	Oil Red O	[58,225]
	Alizarin red	[225]
Dyes for NP coating	Propidium iodide (PI)	[118]
	Nile red	[89]

). Inorganic NP uptake has been reported to be obtained by using Prussian blue or alizarin red dyes as their ability to stain ferric ions and minerals, respectively. Conversely, Oil Red O dye, with its affinity to lipids, was successfully applied for staining lipidic NPs.

Moreover, ex vivo checking, and quantification of cell uptake was performed by coating NPs with PI or Nile red dye. As example, NP coating with PI was successfully adopted to detect cells incorporating gold NPs through flow cytometry [118], whereas Nile red labeling of hybrid iron oxide/polymeric NPs was used to check in vitro uptake into macrophages [89].

5.3. NP Stability Assessment

Another key element influencing the performance of any NP-based cell tracking strategy is the stability of the NPs. Any NP formulation to be applied for tracking purposes needs to preserve a particular nanostructure property including aggregation, composition, shape, size, and surface chemistry.

Different environmental stresses such as extended storage, pH and mineral composition, thermal processing, freeze–thaw cycling, dehydration, mechanical stress and light exposure have been described as responsible for influencing NP stability [29].

The ability to measure the stability of the NP formulation prior to utilization for tracking purposes thus represents a key step to be pursued. Methods that provide quantitative metrics for measuring and modeling nanoparticle stability in terms of core composition, shape, size, and surface chemistry are distinguished by their physical- and chemical- [226,227] based approaches and are summarized in

Table 5. Key parameters defining NP stability and strategies to determine stability preservation.

NP Stability	Definition	Approaches Used for Characterization of NP Stability
		Physical	Chemical
Aggregation	Preservation of NPs upon collisions	Dynamic light scattering	Single particle inductively coupled plasma mass spectrometry UV–visible spectroscopy
Core Composition	Unchanged chemistry of the core during the use	X-ray diffraction	Single particle inductively coupled plasma mass spectrometry UV–visible spectroscopy Surface-enhanced Raman scattering X-ray photoelectron spectroscopy Energy dispersive X-ray
Shape	Preservation of NP architecture during the use	Transmission electron microscopy Scanning electron microscopy X-ray diffraction Atomic force microscopy	Single particle inductively coupled plasma-mass spectrometry UV–visible spectroscopy
Size	Preservation of NP dimension during use or storage	Dynamic light scattering Scanning electron microscopy Transmission electron microscopy Small-angle X-ray scattering Atomic force microscopy	Single particle inductively coupled plasma-mass spectrometry UV–visible spectroscopy
Surface chemistry	Preservation of the native surface functionality	Low energy ion scattering X-ray photoelectron spectroscopy	Single particle inductively coupled plasma-mass spectrometry UV–visible spectroscopy Surface-enhanced Raman scattering X-ray photoelectron spectroscopy Energy dispersive X-ray

.

6. NP Uptake Strategies and Mechanisms

The effects of the physicochemical properties of NPs, such as size, shape, charge and surface chemistry, are crucial for determining the cellular uptake mechanism and the NP function exerted upon the cells [228]. Importantly, knowledge of the underlying mechanisms involved in cellular NP incorporation is found to be relevant for the fate of the NPs and their cytotoxicity [90], as well as for safe and efficient therapeutic applications [229]. Our bibliographic survey identified the endocytosis mechanism as the most representative for the internalization of NPs (97%). Endocytosis can be accomplished through two main mechanisms: phagocytosis and pinocytosis. Phagocytosis is the preferred uptake for larger nanoparticles (≥100 nm), while pinocytosis dominates the uptake of smaller nanoparticles (≤100 nm) [230,231]. Of note, pinocytosis can be subdivided into clathrin-mediated and caveolae-dependent endocytosis, to date, considered as some of the most important mechanisms for NP uptake [229,230]. However, since the variation in endocytic mechanisms depends on cell and NP types, it is still challenging to generalize the current findings [229].

Another key aspect to be considered when testing cell uptake efficiency is the NP incubation time, which is influenced by the type and density of the cultured cells [232] and the shape, size and concentration of the NPs [233,234]. Our literature survey identified 24 h as the most validated incubation time for different types of cell target. However, it has been also proved that the addition of some compounds to NPs might shortening the time required for cell uptake: consistently, Mishra and colleagues showed that the use of protamine sulfate for inorganic iron oxide NP coating reduced the incubation time to 6 h in MSCs [59].

Cell density also influences NP uptake; the most suitable cell density reported in the reviewed studies refers to 10⁴ cells/cm² with high variability defined by the type of cells adopted for the uptake assay. However, several uptake protocols are currently under investigation to increase the quantity of the recovered cells with the aim of having a bulk cell preparation to be used for in vivo NP delivery and tracking. Even in this case, tracking sensitivity reflects the resolution of the imaging technique applied; thus, it was not possible to identify a reference interval for cell number to be injected in in vivo mouse models. Generally, based on the reviewed studies, in vitro protocols pointed to the recovery of at least totally 10⁶ cells. Likewise, NP concentration parameters were highly fluctuating and were strictly correlated to the NP architecture and size.

Alternative strategies identified with the presented literature review include the adoption of electroporation and transfection accounting, respectively, for about 2% and 1% of the total citations. Electroporation was reported to be useful in assisting the passage of NPs larger than 100 nm through the cell membrane [44] or to enhance the quantity of incorporated NPs [235].

Furthermore, the adoption of transfectant agents such as commercially available lipofectamine formulations has been proved to increase uptake efficiency. Accordingly, Wan et al. designed a new approach involving the surface modification of gold NPs with layers of silica and Transfectin 3000 (TS) to reduce cytotoxicity and improve the incorporation in bone marrow-derived stem cells (BMSCs) [236]. Uptake strategies identified in the literature survey are summarized in Figure 6.

7. Cell Types Suitable for Safe NP Delivery

As previously mentioned, improved NP delivery can be achieved by using cells as the vehicle for NPs. Based on our literature survey, different types of cells have been investigated in the last decade as potential carriers for an efficient NP release.

Stem cells, with their intrinsic ability to home in on cancer or injury sites, represent the most suitable cell type source allowing NP delivery, with a 60% of the reviewed studies investigating their potential for biomedical application for cancer and regenerative medicine purposes. Conversely, a smaller percentage of studies (31%), focusing on the development of clinical protocols for precision cancer medicine, adopted cancer cells as the target of NP delivery. Only a few original research articles, pointing to the elucidation of inflammatory homing mechanisms, used other cell sources such as macrophages (7%) or T cells (2%) (Figure 7).

7.1. Stem Cells

Stem cell homing mechanisms are currently being investigated to provide further insights for their potential use as vehicles for contrast agents to be detected with currently available imaging techniques to improve the diagnosis and therapy of many diseases [237].

Different subtypes of stem cells were identified by reviewing our bibliographic dataset. In detail, mesenchymal stem cells (MSCs) accounted for 80% of the total citations using stem cells, 6% involved the use of adipose tissue-derived stem cells (ADSCs), and 14% instead involved the use of other different subtypes such as blastema cells [160], neuronal stem cells (NSC) [54], bone marrow dendritic cells (BMDCs) [55,219] and mouse embryonic stem cells (mESCs) [170].

Furthermore, stem cells derived from different species have been identified. Of note, many studies involved MSCs of human origin (84%), followed by those derived from rat (14%) and monkey (2%) species.

7.2. Cancer Cells

The most used cancer cell model identified with the present literature review is the HeLa cell line, with 44% of the total citations exploiting cancer cells for NP delivery purposes. HeLa was the first human cell line established in culture and has since become the most widely used human cell line in cancer research.

The choice of the cancer cell model relies on the type of cancer to be targeted. Thirteen percent of the studies focused their attention on the investigation of nanotechnology supporting hepatocellular carcinoma resolution by means of hepatocarcinoma-derived cell lines such as hepatoma cell line Hepa1–6 [91], HepG2 cells and HCa-F [80,100,136] and hepatocarcinoma cell line SMMC-7721 [111].

Cancer cell lines derived from glioma or glioblastoma, such as human GBM cell lines U-87 and U251 [78,180], U118 glioma [238] and F98 rat glioma [239] cells were represented by 13% of the total citations describing the application of cancer cell lines.

Similarly, 10% of studies aimed to identify therapeutic approaches against breast cancer with the MDA-MB-231 human breast cancer cell line [179,215,221] and breast cancer 4T1 cell line [162].

Ten percent of studies have investigated efficient protocols to cure lung cancer by means of lung cancer cell models such as A529, A549, NCI H441 and A2780 [134,141,152,169].

To a lesser extent, studies have been carried out with cell lines derived from pancreatic cancer such as Panc-1 and MIA Paca-2 cells [190] and human pancreatic cancer cell line BxPc-3 [94] and also derived from gastric cancer such as human gastric cancer cells (MGC80-3) [132,138], both of these cell types account for 5% of the total citations concerning the use of cancer cell line models for NP delivery.

7.3. Macrophages

Macrophage-bearing NPs have been adopted mostly for tracking. Macrophages were used to evaluate uptake of nanoparticles using imaging techniques [147,240] to investigate the metabolic conversion and elimination of nanoparticles [218] and since these macrophages are able to migrate to sites of inflammation [112], they have been labeled with NPs and their distribution assessed by clinically relevant imaging techniques [89,109,116,222]. Although little is known about NP delivery efficiency of macrophages, this cell source might represent a promising tool for inflammation-targeting purposes in the fields of both regenerative medicine and cancer diseases [241].

7.4. T Cells

T cells have been shown to kill malignant cells in vitro and in vivo, therefore, several studies have labeled these cells with gold NPs or SPIONs to follow their fate on CT or MRI. For example, T cells were transduced to express a melanoma-specific T cell, and then computed tomography analysis was performed, once labeled with gold NPs. CT efficiently detected the accumulation of these transduced and labeled T cells at the tumor site, leading to tumor regression [118]. Another study preferred to label T cells with ultrasmall SPIOs to follow their fate through MRI [242]. Overall, although T cells encapsulating NPs have been poorly characterized to date, their use in nanomedicine deserves attention for improving the efficacy of T-cell based immunotherapy [172].

8. Conclusions and Outlook for Effective NP-Based Cell Tracking

Issues determining the effectiveness of NP-assisted cell tracking such as NP design, material properties and functionalization with specific targeting, labeling (tracking) and/or uptake-enhancing moieties have been identified and almost exhaustively characterized during the last decade. Nevertheless, an in-depth analysis of the features relating to NPs and the interaction dynamics with the target body area, which in turn is influenced by the cell to be targeted, the NP loading time and concentration, still needs to be achieved.

The nexus of NPs with the biology is currently an area of intense study in the field of NP-based cell tracking as the success of NP incorporation into the cell and consequently the success of direct cell labeling in terms of non-invasive long-term targeted monitoring through imaging depends on the type of biological response triggered by NP uptake.

The investigation of mechanisms determining the efficacy of the active incorporation of NPs by the cell holds a prominent role in NP-assisted cell tracking as it keeps the potential clinical traceability for a targeted, sensitive, and long monitoring of cells with the current clinically approved imaging devices improving the performance of the currently used iodinated, barium or gadolinium-based contrast agents [14].

Much effort has been made to better characterize the biological cues of NP–cell dialogue with the aim of improving uptake and retention mechanisms to be exploited for direct cell tracking; despite its simple and straightforward nature, this approach has several drawbacks which deserve further investigation to improve their effective clinical use.

Specifically, the biggest limitation of any direct labeling method is the failure in the assessment of cell viability and division damping retention and the right delivery of NPs. Firstly, if the cells die, the NPs may be retained at the target site, being taken up by macrophages consequently leading to erroneous detection of cells. The NPs may also dissociate from the cells through efflux and exocytosis, leading to false quantification and monitoring of free labels instead of the cells of interest. Secondly, cell division dilutes the number of NPs, affecting the sensitivity of the detection of daughter cells and limiting the long-term tracking of labeled cells [243]. Overall, these aspects can be managed by deepening the characterization of cell-fate mechanisms and how they are modulated by NP incorporation.

Considering these premises, the definition of the impact of NP physicochemical properties on cell internalization mechanisms, together with the characterization of biological cell response might pave the way for effective NP-delivery solutions for cell tracking, finally providing future perspectives for a bench to bedside translation (Figure 8).