Re-engineered imaging agent using biomimetic approaches

Abstract

Recent progress in biomedical technology, the clinical bioimaging, has a greater impact on the diagnosis, treatment, and prevention of disease, especially by early intervention and precise therapy. Varieties of organic and inorganic materials either in the form of small molecules or nano-sized materials have been engineered as a contrast agent (CA) to enhance image resolution among different tissues for the detection of abnormalities such as cancer and vascular occlusion. Among different innovative imaging agents, contrast agents coupled with biologically derived endogenous platform shows the promising application in the biomedical field, including drug delivery and bioimaging. Strategy using bio-components such as cells or products of cells as a delivery system predominantly reduces the toxic behavior of its cargo, as these systems reduce non-specific distribution by navigating its cargo toward the targeted location. The hypothesis is that depending on the original biological role of the naïve cell, the contrast agents carried by such a system can provide corresponding natural designated behavior. Therefore, by combining properties of conventional synthetic molecules and nanomaterials with endogenous cell body, new solutions in the field of bioimaging to overcome biological barriers have been offered as innovative bioengineering. In this review, we will discuss the engineering of cell and cell-derived-components as a delivery system for various contrast agents to achieve clinically relevant contrast for diagnosis and study underlining mechanism of disease progression.

1. Introduction

Better patient outcomes rely on how early and precisely the diagnosis and treatment of a disease can be. To date, medical imaging has become an essential tool for clinical practitioners to access disease pathology based on anatomical or functional evaluations. As the need of diagnostic imaging has grown significantly in treatment planning, a variety of imaging technologies such as positron emission tomography (PET), computed tomography (CT), optical imaging, magnetic resonance imaging (MRI), X-ray, and ultrasound are becoming routine procedures in the clinics.¹ Aligning with the development of imaging modalities, a wide range of small molecules, molecular complexes used as tracer or contrast agents are also maturing and diversifying over time. These contrast agents or imaging tracers are employed to assist the conventional imaging modality for a clearer visualization of abnormality allowing the diagnosis of previously undetectable pathologies. The advantages of using imaging probes are further pushed forth by incorporating into nanosized materials with the hope of continuing the improvement on the sensitivity, stability, and plasma residence times as compared to that of free small molecules. The benefits of using nanomaterial-based imaging probe have been well-documented in a wide range of imaging modalities.^2–7 For example, when thousands of iron oxide molecules clustered and rearranged to form iron oxide nanocrystals, it becomes strongly paramagnetic enabling it’s usage in MRI. Moreover, the confinement of this small molecule or particulate contrast agent in second stage nanoparticle (NP) system provides enhancement in contrast as well as increase in targeting specificity. For an instant, tiny iron oxide NPs when confined into sub 100 nm polymeric matrix enhance an order of magnitude of its magnetic relaxivity.⁸ Gao et al. encapsulated superparamagnetic iron oxide NPs into the polymeric micelles to enhance the imaging efficacy of tumor biomarker in vivo.⁹ Similarly, Gd-based contrast agent in mesoporous silica NPs is reported to be effective in enhancing contrast.¹⁰ In some other cases, the incorporation of gadolinium-based macromolecules into a liposomal system assist the brighter contrast compared to that of the small molecule counterpart.^11,12,10

Despite their potential for contrast enhancement, the application of nanomaterial-based contrast agent (NP-CA) is severely hampered due to the complexity of biological environment. One of the first obstacles that NP-CA encounters upon administration is opsonization and formation of protein corona. The interaction with these endogenous materials alters the designed properties NP-CAs preventing them from performing the desired task in physiologically relevant condition. For example, the binding of protein causing fluorescent quenching effect in optical imaging modality,^13,14 reduction in relaxivity of MRI contrast agent due to metal displacement competition with endogenous ions,^15,16 and accelerating the decay rate of nuclei in PET.¹⁷ Also, the recognition and oxidative destruction by the resident macrophages of the mononuclear phagocyte system becomes another major hurdle for the biological fate of NP-CA. The sequestration by mononuclear phagocyte system results in high accumulation of nanoparticles in organs, such as the spleen and the liver, preventing it to successfully navigate to the target site. In fact, despite extensive amount of research have been devoted to developing NP-CA, there were just a handful nanoparticulate contrast agents find their way to get into the FDA approval list. A clear indication can be seen by the discontinuation from the manufactures of FDA approved and commercially available iron oxide MRI contrast agents, such as Feridex® produced by Berlex-USA, Endorem™ produced by Guerbet-France, and Resovist® produced by Bayer Schering Pharma AG-Germany.⁶ The retardation in the clinical translation of NA-CA is a direct consequence of the nanoparticle’s inability to overcome many of above mention biological barriers.

As the field of nanotechnology mature overtime, we started to gain a better understanding about the process of bio-nano interfaces, motivating innovative features to be rationally introduced into the synthetic nanoparticulate platform. A broad spectrum of agents ranging from synthetic targeting moiety to biologically derived ligands (e.g., high-density lipoprotein, folate, glycan, etc.) has been taken into design consideration.^18,19 However, biological environment is highly intricate that individual type of ligand is not sufficient to provide multitasking effects. In other to achieve proper negotiation with biological barriers, a more inhomogeneous with multi-functionality is in need to be involved. As a basic unit of life, cell carries functionalities that developed throughout evolution, becomes a potential provider to carry (by itself) or enhance (by its extracted components) the functionality of synthetic nanomaterials.^19–24 Taking the design cues from cellular nature, integration of biologically derived components into synthetic nanoparticles has extended imaging agent’s usage beyond its ordinary feature. Particularly in bioimaging area, the combination of imaging contrast agent with bio-functionality of cells results in attractive imaging tools that enable unique effects for a broad range of biomedical applications.

In this review, a lesson learned from the endogenous cell is executed to design a biomaterial in which both cellular and synthetic components are collaborating to augment the systemic effect thereby representing one of the unique areas of bioengineering. While numbers of literature and reviews in drug delivery nanoparticles and membrane disguised delivery system can be seen, less attention has been given in the area of imaging contrast agents and strategies of inclusion of contrast agents in the system with the component of a cell or cell body. The review is divided into two main categories: cell based-engineering and cell-component-based engineering approach (Figure 1). In each section, we will highlight current engineering strategies, emphasizing the unique properties of each system, and summarize representative examples along with their principal advantages regarding molecular imaging and disease diagnosis. Finally, we will briefly discuss current challenges and potential risks in their clinical translational pathway.

Figure 1.

An overview of the cell or cell-based imaging system. A variety of imaging agents can be coupled with biologically derived endogenous platform such as cell and cell membrane to extended imaging agent’s usage beyond its ordinary feature. Depending on the original biological role of the naïve cell, the contrast agents carried by such a system can provide corresponding natural designated behavior.

2. Cell-based engineering approach

Cells is an actively living entity that continuously navigates to the targeted location in the body to perform its biological function. Depending on the original biological role of the naïve cell, the contrast agents carried by cells can provide corresponding natural designated behavior.

Human body consists trillion of cells which are highly specialized for their biological functions. Depending on the origin, type, and biological mission, characteristic of cells can be varied significantly which lead to the diversification of cell morphology, mode of action, and the way they encounter with exogeneous materials. Ultimately, the strategies that have been developed to engineer cell with contrast agent also varies along with the nature of cells and agents of interest. Some cells are inert to the substances in surrounding environment, while other cells take up substances instantly as a part of natural defense mechanism. When the strategy used to modify cell with contrast agent involved in active cellular uptake phenomenon, it is considered as phagocytosis based-engineering approach, which is widely used for monocyte, macrophage, stem cell, and cancer cells. On the other hand, when active uptake cannot be applied like in the case of red blood cell (RBC), non-phagocytosis engineering approach is implemented.

2.1. Non-phagocytic approach

Different methods have been proposed to engineer cell to appropriate delivery of imaging agent without using phagocytosis phenomenon. These methods can be classified into three main categories namely encapsulation, surface coupling, and fusion strategy (Figure 2). The ultimate goal of these methods is to push forth the performance of imaging agents while minimizing the cell modification to preserve the naïve plasma membrane integrity and functionality.

Figure 2.

Schematic represents three different strategies to modify cell with imaging contrast agents (red blood cell is used as a model cell).

2.1.1. Encapsulation strategy

Encapsulating agent of interest into cell inner compartment is one of the earliest technique that was developed to labelled RBC.²⁵ In order to achieve appropriate engineered RBC carriers, naïve RBCs have to undergo a sequential process of loading. RBCs are first collected from whole blood by centrifugation to remove the buffy coat and other blood components. Under hypotonic condition with the osmolarity ranging from 64 to 160 mOsm/L, RBCs are incubated with imaging agents. The controlled osmolarity condition permits the temporary opening of RBC’s membrane pores allowing an agent of interest to get in without altering the main features of natural cells. Once the encapsulation is completed, the cell membrane is resealed using successive isotonic treatment by changing the osmolarity to 280 mOsm/l to restore its natural membrane conformation. The obtained contrast agent encapsulated engineered RBC are washed extensively to remove any non-specifically absorbed agent on the surface (Figure 3A).

Figure 3.

RBC labeled with contrast agent using encapsulation approach. (A) Schematic illustrating the preparation process to load agent of interest using hypotonic treatment followed by resealing technique under isotonic condition. (B) Iron oxide nanoparticle loaded RBC (a) Ferucarbotran –a 50 nm iron oxide nanoparticle coated with carboxydextran, (b) TEM images showing the morphology of RBC before and after ferucarbotran loading, (c) ferucarbotran loaded RBC used for imaging of brain vasculature, (d) Anatomical features gallbladder can be detected upto 4 days post injection. (Adapted from ref. 26 and 27)

The technique has found its way to successfully load a variety of imaging agent into RBC ranging from small imaging molecule like Gadoteridol (or ProHance™) to nanoparticles such as iron oxide and gold nanoparticles.^26–29. In a pilot study conducted by Antonelli et al., some iron oxide nanoparticles with different sizes and coatings were investigated for their feasibility in human and murine RBC encapsulation.³⁰ By carefully controlling the hypotonic and resealing dialysis process, a percentage of RBC recovery can reach as high as 70%. The loading efficiency and T₂ contrast enhancement were varied significantly depending on the physiochemical properties of nanoparticles. About 19 types of SPIONs were investigated, among those coatings with dextran or carboxydextran showed higher compatibility with the RBC loading procedure with minimal cell disruption. The r₂ relaxivity of these formulations was also considerably higher in comparison to that of un-loaded one, presumably, due to the well dispersion of SPIONs intracellularly. The technique was further applied to load ferucarbotran (a hydrophilic colloidal suspension of superparamagnetic iron oxide nanoparticles coated with carboxydextran) into the RBC for the possible in vitro and in vivo characterization (Figure 3).^26,27 The ferucarbotran loaded RBC exhibit prolong circulation time with a half-life in bloodstream up to 4.5 days. Using the ferucarbotran loaded RBC in a mouse model, proposed SPIONs loaded RBC delineate features of brain vasculatures. The in vivo contrast enhancement of ferucarbotran loaded RBC can last up to 3 days post injection, whereas the free ferucarbotran reduce signal immediately after 20 minutes of administration (Figure 3). The long circulation time and contrast enhancement makes it stand out for possible application in early detection of ischemic stroke or monitor the cardiovascular medication over prolonged period .²⁷

In line with the development of nanoparticulate based contrast agent loaded RBC, the loading of a molecular contrast agent such as Gd-based contrast agent into RBC was also demonstrated. As early as 1998, Johnson et al. reported the encapsulation of Magnevist^® (gadolinium-diethylenetri amine pentaacetic acid, Gd-DTPA) into human and rat RBCs to create an MRI blood pool contrast agent using osmotic pulse sequence.³¹ During the procedure, a sudden osmotic condition was generated by the exchange of hyperosmolar dimethyl sulfoxide and Gd-DTPA solution. Under such condition, red cells were subsequently undergoing swelling and resealing process within 1s to formed Gd-DPTA loaded red cells. Even though the changing in red cells morphology happened instantly giving the advantages for conserving the RBC biological features in term of cell integrity, deformability, and stability; the loading efficiency of Gd-DTPA in RBC was found to be varied significantly from batch to batch with half of the red cells were lysed during the procedure. Also, the cell membrane damage during loading procedure could expose harmful antigens onto the cell surface causing the immunogenicity once used in vivo condition.³¹ The perfusion of the modified red cell is of great concern due to continuous “leakage” of potassium out of the red cells.³¹ Following the very first demonstration, Gd-loaded RBC concept was further expanded to widen its application into tumor vascular volumes assessment to gain quantitative information on tumor vascularization.²⁸ This approach could be utilized to link between the hallmark of tumor growth and the selection of personalized anti-tumor drugs that target tumor vasculature architecture. In this study, Ferrauto et al. utilized Gd-HPDO3A (Gadoteridol, marked as ProHance™) to encapsulate into murine RBC resulting in highly stable Gd-RBC while preserving the natural RBC’s deformability, fragility, and functionality with a high percentage of red cells recovery (>75%) (Figure 4A). The Gd-loaded RBC was intravenously injected into TS/A mammary breast cancer-bearing mice and vascularization degrees at two different tumor development stages were accessed by the ratiometric value of contrast enhancement at the tumor region. The vascular volume fraction obtained by signal enhancement method has shown to be independent of the concentration of the circulating Gd-RBC; yet providing distinctive insight into tumor vascularization in compare to that of extracellular fluid CAs such as small Gd-H PDO3A molecule or murine serum albumin binding Gd-based contrast agent (Figure 4B). With the advancement of confining into blood vessels and prolonging circulation time up to 5 days post injection, Gd-RBC could be used to monitor the vascularization of the tumor during its progression. As the size of tumor increase from 200 mm³ to 800 mm³, the vascular network also gets expanded from 12% to 45% measured by contrast enhancement using Gd-RBC (Figure 4C). This phenomenon is most likely due to the neoangiogenesis occur in the late stage of tumor development in tumor xenograft. Thus engineered Gd-RBC show great potential in monitoring the therapeutic response of clinically approved drug that targets tumor vasculature network.

Figure 4.

Gadolinium chelates loaded RBC. (A) Chemical structure of Gd-HPDO3A, Madec^®, and Gd-HPDO3A loaded RBC and their corresponding MRI image of tumor vasculature (from top to bottom, respectively). (B) Enhanced voxels in tumor region upon the administration of contrast agents (left) and ratiometric values distribution obtained by normalizing tumor values to the from vessel voxels (right). (C) Vascular volume fraction at different tumor stages obtained upon the i.v. administration of Gd-RBCs (left) and its corresponding % during tumor development (right). (Adapted from ref. 28)

In addition to the application as a carrier for MRI based contrast agent, RBC was explored for the possibility to carry gold nanoparticles (AuNP) – an X-ray contrast enhancer.²⁹ Using typical pore opening technique under hypotonic treatment, 20 nm spherical AuNP with different surface-modifications were investigated for the possible mechanism of AuNP loading . The incorporation mechanism and loading efficiency of modified AuNP into RBC would changedepending on the chemistry of ligands on the surface of AuNPs. Particularly, ligands exhibiting negatively charged functional groups such as citric acid, thioglycolic acid, and 4-mercaptobenzoic acid shows negligible AuNp loading due to the charge repulsion caused by negatively charged RBC surface. However, by changing negatively charged ligand to positively charged like 6-thioguanine, the AuNP incorporation increase from 13% to 17%. The encapsulation efficiency was further pushed up when lignads become neutral with decreased in hydrophilicity. By monitoring the agglomeration stage and X-ray contrast enhancement of AuNP during RBC encapsulation process, three mains incorporation pathway was proposed. For 6-thioguanine capped AuNP, since X-ray contrast enhancement was observed while just a few intracellular AuNP was detected, the phenomenon suggested that 6-thioguanine capped AuNPs are predominantly hanging on the surface of RBC rather than getting into the cytosolic compartment. On the other hand, AuNPs were found to be reversibly transporting pathway through the channel into RBC when smaller with neutral hydrophilic ligands such as 2-mercaptoethanol were used to modified particle’s surface. When the ligand’s hydrophobicity increases, the mechanistic pathway was shifted to become an irreversible process using endocytosis as the primary mean of transportation across the cell membrane.

2.1.2. Surface coupling strategy

Surface coupling strategy to modify RBC is an emerging tool to attach agent of interest covalently on the surface of RBC. Even though this approach has been long used in the field of cell-based delivery system to tag small drug molecules onto the outer leaflet of the cell membrane, it was just lately introduced to engineer cell-based carrier for the contrast agent. In comparison to conventional encapsulation strategy, the surface modification technique offers advantages in many regards. The coupling of contrast agent onto cell can reduce harsh condition that cells have to undergo to facilitate loading of cargo into intracellular compartment.³² In addition, the attached amount and release of the agent after engineering into cell carrier can also be controlled by changing the feed amount and chemicals affinity of the linker.³²

The development of biointerface and biomaterials using cell surface coupling approach is catalyzed by biotin-avidin linkage (Figure 5A). Avidin-biotin complex is the strongest known non-covalent interaction between a protein and ligand with dissociation constant (K_d) of 10⁻¹⁴ to 10⁻¹⁵ M.^33–35 Biotin, also known as vitamin B7, is a small water-soluble molecule comprise of an ureido ring fused with a valeric acid substituted tetrahydrothiophene ring. The carboxylic acid in biotin makes it easy to conjugate to the N-terminus protein via various biocompatible chemical reactions. Among different biotin-binding proteins, avidin is one of the most commonly used owing to its ease of purification and economical source of production (egg white). Avidin is a highly glycosylated and water-soluble tetrameric protein that can interact with up to four biotin molecules yielding a stable bond over a wide range of pH and temperature.³⁶

Figure 5.

Cell surface modification strategy using the biotin-avidin bridge. (A) Structure of biotin and avidin protein. (B) Design and preparation of theranostic RBCs modified with IONPs. (C) Fluorescence imaging (Ce6) of mice bearing two subcutaneous 4T₁ tumors showing in vivo magnetic tumor targeting. (D) In vivo T₂ -weighted MR images of mice 12 h after i.v. injection of RBC-IONP-Ce6-PEG or IONP-Ce6-PEG and their corresponding MR signal intensities. (E) Tumor growth in different groups of mice after the various treatments. (Adapted from ref. 37)

The cell surface functionalization using biotin-avidin has found various applications in the delivery of imaging agent with combination therapy for intratumoral extra-vascularization to approach cancer cell. One such rational design was proposed by Wang et al., in which red blood cell based drug delivery system was equipped with chemo-, photodynamic and image-guided therapy that can respond to the external magnetic field (Figure 5B).³⁷ In this stimuli-responsive combination therapy design, RBCs were loaded with doxorubicin and cell surfaces were biotinylated using EDC/NHS coupling reaction. The engineered surface was further connected to photosensitizer modified iron oxide nanoparticles (IONPs) through an avidin protein. The obtained multifunctional RBCs provide a long circulating delivery system with an ability to diminish retention in RES organs and exert a synergistic effect of photodynamic and chemotherapy for tumor reduction (Figure 5C, D, and E). The study also highlights the opportunity to use the external magnetic field to drag IONPs decorated RBC selectively to the subcutaneous tumor in the mouse model. Hence, the accumulation of IONPs-RBC at tumor site was enhanced and localized under the external magnet, which further provides strong contrast enhancement in both optical and MR imaging modalities.

2.1.3. Fusion Strategy

The incorporation imaging agents into cell using encapsulation and attachment strategies could result in distress of cell membrane’s structural integrity, causing damaged cell. The damaged cell, hence, would trigger surface recognition and initiate the selective endocytosis of Kupffer macrophages. As an alternative, the approach involves in membrane fusion driven by molecules or liposomal structure have been exploited to achieve a practical and straightforward method for engineering surface-painted cell.

Membrane fusion driven by lipophilic molecules was proposed taking the inspiration from natural process of anchoring proteins to plasma membrane via palmitoyl, myristoyl, or glycosylphosphatidylinositol anchors.³⁸ During the process, the hydrophobic residue can interact with fatty acyl group of membrane phospholipid to submerge into hydrophobic core of lipid bilayer.³⁹ A variety of moieties ranging from gadolinium based contrast agent to fluorophores (DiI, FITC) can be conjugated to cell membrane permeable lipophilic molecules, hence giving cell imaging functionalities after insertion. The process of lipophilic anchoring RBC can be robustly accomplished within 15 to 30 minutes of incubation, facilitating the attachment of 8500 to 16000 target molecules onto RBC while keeping the long-circulating property of RBC up to 3 days in vivo condition. This lipophilic painting technique was further used to apply on studying the targeting and depletion of circulating leukocytes and cancer cells.⁴⁰ By partnering with membrane incorporated fluorescent dye (DiI or DiO), the targeting efficiency of antibody modified RBC toward leukocyte/cancer cell was revealed in vitro and in vivo. The technique further paves the way for future research in investigating cell-cell interaction using optical fluorescent imaging modality.

Recently, a new paradigm for functionalizing cell surface with imaging agents was established using artificial lipid bilayer nanovesicle as a vector. In general, the fusion process of lipid vesicle and cell membrane occurs naturally and plays a vital role in cell-cell communication via extracellular signaling.^41,42 Unlike natural fusion process that can be promoted by binding of extracellular vesicle’s proteins and cell surface receptors, the fusion between artificial nanovesicle such as liposome requires special design consideration. In order to initiate membrane fusion process, the designed liposome must (1) approach cell membrane in proximity following by (2) the destruction of the cellular lipid bilayer. Different strategies have been developed to assist such requirement by introducing divalent cations, polycations, positively charged amino acids, and membrane-disrupting peptides into the system.^43–45 Among these strategies, building a liposomal vector containing positively charged lipids as its building block has offered potential promises to re-engineer cell surface without changing the nature of cells. An example for turning naïve RBC into imageable RBC using fusogenic liposomes was first presented by incorporating gadolinium conjugated lipid (Gd-Lipid) into the liposomal vector (Figure 6A).⁴⁶ For the purpose, a liposomal nanoconstruct made up of L-α-phosphatidylcholine (Egg-PC), Gd-Lipid and cholesterol was used to fuse with RBC generating Gd labeled RBC (Gd-RBC). The fusion between Gd-liposome and RBC membrane was easily monitored using fluorescent resonance energy transfer and confocal imaging techniques. Gd-liposome fused RBC resulted in Gd-RBC containing tunable Gd content (Figure 6B). Being located on the outer layer of RBC surface, where glycosylated protein, carbohydrate, and other hydrophilic moieties are densely packed, macrochelated Gd³⁺ have excess interaction with water thereby enhance its relaxivity. As a result, Gd-RBCs exhibit a longitudinal relaxivity of 20 ± 2 mM⁻¹ s⁻¹ with an extended circulation time of 5 days post-injection (t_1/2=50h). When Gd-RBC was injected into tumor-bearing mice model, the abdominal aorta and tumor mass were glowed up under MRI (3T) with a minimal signal enhancement in kidney indicating the low accumulation of Gd-RBC in renal system (Figure 6C). Moreover, the contrast enhancement at tumor was clearly visible to 24h post-injection, which otherwise gets clear out rapidly as in the case of clinical Gd-based blood pool contrast agent.

Figure 6.

RBC surface modification driven by fusogenic liposome. (A) Gadolinium lipid incorporation onto red blood cell membrane using liposome fusion technique and its corresponding fluorescent and confocal images. (B) The enhancement of r₁ relaxivity when Gd-lipid is inserted onto RBC surface and its corresponding phantom images in comparison to that of Gd-liposome. (C) T₁ weighted MRI images of Nu/Nu nude mice bearing orthotropic B16 melanoma tumors at pre-contrast and post-contrast showing Gd-RBC confining in circulatory system. (D) Strategic fusion mechanism showing gold nanoplating process. (E) Transmission electron microscopic images of RBC, AuNP, and Au-RBC and its stability various AuNPs input by measuring the released hemoglobin during nanoplating. (F) Retention of RBC surface protein in Au-RBC analyzed using gel electrophoresis. (G) CT images of Pure RBC, Au-RBC, and AuNPs embedded in agarose gel. (Adapted from ref. 46 and 47)

The versatility in liposome modification makes it stand out for diversifying RBC surface functionality when membrane fusion strategy is in used.⁴⁷ Besides introducing the contrast agent directly to the cell surface, the fusogenic liposome can also be utilized to incorporate nanoparticle-stabilized moieties, which would further be used as a substrate to plate contrast agent onto RBC surface (Figure 6D). The proof of concept was presented by fusing thiolated liposome with red blood cell resulting thiolated RBC.⁴⁷ Due to the strong interaction of thiol and AuNPs, thiolated RBC can stabilize AuNP on its surface producing an Au nanoplated RBC (Figure 6E). The double structural fusion approach provides higher RBC stability when incubated with AuNPs than that of nonspecific nanoparticle absorption strategy (Figure 6F). The Au nanoplated RBC showed a darker contrast enhancement with a lower attenuation coefficient than that of RBC (Figure 6G). Given a strong X-ray absorption, the Au-nanoplates RBC can be depicted as a bioengineering CT contrast agent that is detectable in the pool of blood for possible visualization of vascular abnormalities.

2.2. Phagocytic approach

Unlike non-phagocytic cell that needs specific strategies for cargo incorporation, phagocytic cells can actively uptake substances that are incubated in culture media or administrated in the body via phagocytosis (Figure 7). Compelling the advantages of this phenomenon, cells can be either labeled with imaging agents ex-vivo or in vivo.

Figure 7.

Phagocytic cell-based engineering approaches.

2.2.1. Ex vivo labeling

In ex vivo labeling strategy, cells are harvested, followed by co-incubation with imaging probes such as fluorophores, radiotracers, and paramagnetic nanoparticles (Figure 7). Once cells are labeled, they are re-injected into the animal and visualized by various imaging modalities such as CT, FLI, PET/SPECT, and MRI. For the purpose, both autologous and allogenic cells are in research , broadly defined as cell therapy achieved by engineering cell as a delivery vehicle. The significant advantage of direct labeling is a straightforward protocol with a particular population of targeted labeled cells. There are, however, many aspects of cell and imaging agents selections need to be carefully evaluated while labeling is executing. First, since the interface between imaging agent and cells plays a vital role in cellular interaction and uptake pathway, the contrast agent chemistry/physical properties and cell type directly influent cellular functionality and internalization efficiency.^48,49 Especially when the imaging tracers are nanoparticle-based contrast agents, criteria such as preservation of physiochemical properties upon internalization (size, shape, dispersity), and high payloads need to take into consideration to achieve an effective contrast agent labeled cell. Secondly, imaging agents must be highly biocompatible to ensure cell biological functions such as cell migration and adhesion, viability, and immunomodulation (in the case of immune cell).

An example emphasizing the important of size and surface coating is presented in a pilot study conducted by Chhour et al., where the small changes in nanoparticle characteristic can consequently affect cellular uptake, hence influence the imaging output (Figure 8). In these studies, monocyte, a large phagocytic leukocyte, was used to label with CT contrast agent ex vivo for imaging of atherosclerosis. Forty-four distinct AuNPs formulations with a diameter ranging from 15 to 150 nm blending with six different capping agents were examined to address the complex influence of AuNPs size and coating on monocyte uptake (Figure 8A).⁵⁰ The cellular uptake of AuNPs was found to be in a size-dependent manner, in which when the size of AuNP increase, cellular uptake also increase, while the coating in all these groups are of methoxy PEG. However, when these AuNPs are coated with carboxylated–PEG, monocyte uptake was significantly increase when the sizes of AuNP increase from 15 to 75 nm and then dropped down when the particle size reaches to 100 nm and 150 nm (Figure 8C). The nanoparticle was found to predominantly reside in the lysosomal compartment demonstrating that the NPs went through phagocytosis pathway to enter the cell (Figure 8B). Consequently, the CT attenuation enhancement (reductions in intensity of x-ray beam as it traverses matter either by absorption or deflection) of AuNP labeled monocytes showed a consistent pattern to cellular internalization efficiency (Figure 8D). Besides nanoparticle, physiochemical properties, in vitro parameters including cytotoxicity, cytokine production, and cell uptake with monocytes were also tested for a library of 15 nm AuNPs with five different ligand coatings (11-mercaptoundecanoic acid, 16-mecaptohexadecanoic acid, poly(ethyleneimine), 4-mercapto-1-butanol, and 1-mercaptoundecyl-tetra(ethylene gycol)).⁵¹ Even though all nanoparticle formulations exhibit a spherical core of 14.7 nm, the distinct chemical structure of surface ligands resulted in significant altered of hydrodynamic sizes and surface properties of the particle. The hydrodynamic size of nanoparticles proportionally increases as the ligand size increase, while the surface charge of AuNPs is directly reflected by the functional groups existing in capping agents. As such, the nanoparticle capped with ligands such as 11-mercaptoundecanoic acid, 16-mecaptohexadecanoic acid, and 4-mercapto-1-butanol exhibit negatively charge, whereas methoxy end group in 1-mercaptoundecyl-tetra(ethylene gycol) and amine end group in poly(ethyleneimine) provide neutral and positively charge to the nanoparticle, respectively. As a result, their cellular consequences were varied considerably. Among five different formulations tested in the study, AuNPs coated with 11-mercaptoundecanoic acid were chosen to labelled monocyte for in vivo cell recruitment tracking (Figure 8E). The effectiveness of CT on tracking AuNPs labelled monocyte migration to atherosclerotic lesion were studied on three different groups including atherosclerotic mice receiving gold labeled monocytes (AtT), atherosclerotic mice receiving non-labeled monocytes (AtN) and wild-type mice receiving gold labeled monocytes (WdT) (Figure 8F). The attenuation was seen to statistically significant during the experimental time (5 days) on the atherosclerotic mice receiving gold labeled monocytes group, whereas the increase for the wild-type and atherosclerotic mice receiving non-labeled monocytes groups were not significant. Overall, these studies point out important aspects for nanoparticle selection in cell labelling technique for possible monitoring AuNP labeled monocytes recruitment by CT imaging.

Figure 8.

Gold nanoparticle labeled monocyte using ex-vivo labeling approach for CT application. (A) Schematic illustrates monocyte being labeled with AuNPs with different surface coating and sizes. (B) TEM images showing the internalization of AuNPs. (C) Influence of surface coating on cellular uptake. (D) CT contrast enhancement of AuNPs (75 and 150 nm) coated with PCOOH (left) and theirs corresponding attenuation value (right). (E) Evaluation of in-vitro biocompatibility (left) and immunomodulation response (right) of AuNPs with monocytes. (F) CT application of AuNPs labeled monocyte, (a) in vitro attenuation of monocyte labeled with different ligands capped AuNPs, (b) CT images showing the accumulation of AuNP labeled monocyte after 5 days post-injection, (c) The significant increment of attenuation at atherosclerotic lesion observed in atherosclerosis mice injected with AuNPs labeled monocytes. (Adapted from ref. 50 and 51)

In addition to particle size, shape, and surface coating, the effect of cell culture media such as the formation of protein corona on the nanoparticle surface also play a crucial role in cell uptake; hence, directly affect cell labelling efficiency. One such example is presented in a study conducted by Nejadnik et al, in which the formation of protein corona around ferumoxytol nanoparticles (30 nm iron oxide nanoparticle coated with carboxymethyldextran) was found to facilitate the uptake of human mesenchymal stem cells.⁵² Three different type of protein corona covered nanoparticle was formed in media containing human serum, fetal bovine serum, cGMP compliant proteins (StemPro®). After the protein corona formation, the surface zeta potentials of corona covered NPs were significantly shifted from negative (−37.03±0.59 mV for bare nanoparticles) to neutral value (−9 to −5 mV), while their hydrodynamic size was larger ranging from 13 to 35 nm in compared to the bare nanoparticles (16.53±0.94 nm). The coronal layer surrounding ferumoxytol nanoparticles mainly consist of small proteins with a molecular weight of less than 30 kDa, in which 10–20% of corona is made up of apolipoproteins. As a result, the cellular uptake of these protein corona covered nanoparticle also varied significantly leading to the different in shortening T₂ relaxation time. Among them, ferumoxytol nanoparticles incubated in human serum show the most efficient stem cell internalization and T₂ contrast enhancement, which further enabling the delineation of labelled stem cell after implantation into pig knee joints.

2.2.2. In vivo labelling

In contrast to ex vivo labeling, in vivo cell labeling occurs after systemic injection of imaging agents. The specific interaction between imaging agents and cells in situ is facilitated by two factors, the phagocytosis nature of phagocytic cell (including circulating blood monocytes and tissue macrophages, which are frequently found at inflammatory sites) to eliminate foreign materials (in this case imaging probes) or the active binding of imaging agents to biomarkers present in the disease-associated cells. When the imaging agents undergo phagocytosis for in vivo cell labeling, the process is considered as passive targeting that frequently applied for nanosized contrast agents. This passive labeling strategy relies on the in vivo opsonization of imaging agents with CD3b, antibodies and other proteins to trigger phagocytosis. Under this approach, representative examples include the use of perfluorocarbon, USPIOs, ¹⁸F-polyglucose for labeling macrophage-associated with cardiovascular diseases (atherosclerotic plague, myocarditis, myocardial infarction, and aneurysm), inflammatory and ischemia, and tumor microenvironments to allow them glowed up under ¹⁹F-MRI, proton MRI, PET/MRI imaging modalities.^53–56 From another standpoint, when the imaging agent is tagged with a targeting moiety, the labeling of imaging agents become active toward one type of phagocytic cell in vivo. The involvement of specific biomarkers present on cell-associated disease makes this approach more specific in compare to that of passive in vivo cell labeling. A wide range of biomarkers presents on disease-associated cells have been demonstrated for the feasibility for directing the homing of the imaging probe such as P-selectin (CD62P) and VCAM-1 on endothelial cell, glucose and class A macrophage scavenger receptors on macrophage.⁵⁷

The strategy to track in vivo plague macrophage can be perform using macrophage-targeted molecule linked to imaging agents. In this context, mannose and glucose are the most frequently used to target plague macrophage owing to its ability to be taken up by macrophages through glucose transporters.^58–60 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG), a PET imaging tracer, has long been standing out as a gold standard of clinical imaging modality to identify the atherosclerotic plaques inflammatory state. By coupling with CT, PET of [¹⁸F]FDG can be used to score the severity of vulnerable in patient.⁶¹ On the other hand, mannose, a C-2 epimer of glucose, is another potential candidate to access high-risk plague due to the ability of targeting upregulated mannose receptor on macrophage subsets associated with high-risk plaques.⁶² The capability to monitor those macrophages subset by targeting mannose receptors can be performed in a wide range of imaging modalities by changing the imaging agent labeling strategies. In the case of molecular imaging agent, mannose can be modified directly with radioactive isotope ¹⁸F in the form of ¹⁸F-labeled mannose (2-deoxy-2-[¹⁸F] fluoro-ᴅ-mannose) for the application in PET. The specificity of ¹⁸F-labeled mannose toward mannose receptor bearing macrophages subset was investigated by titrating the binding of an antibody specific for the mannose receptor (CD206) in the competition with ¹⁸F-labeled mannose, in which the presence of ¹⁸F-labeled mannose reduced the antibody binding significantly . The result further implies the specific binding of ¹⁸F-labeled mannose to the macrophage’s mannose receptor presented in high-risk atherosclerotic plaques.⁶² In vivo administration of [¹⁸F] FDG and [¹⁸F]FDM into atherosclerotic rabbit revealed the similar manner of macrophage uptake at the atherosclerotic lesion. The ability of targeting mannose receptor in macrophage was further put forth by using mannose as a targeting moiety constructed in macromolecules, nanobodies, and nanoparticles labelled with ¹¹¹In, ^99mTc, and Cy5.5 to track them under SPECT/CT, PET, and intravascular optical imaging modalities, respectively.^63–65

In line with active targeting strategy, macrophage also can be labeled by nanoparticles owing to its ability in taking up particles as a part of phagocytes job in the body. The main challenge of this strategy is to enhance target-to-background ratios in the vascular system, especially in the case of atherosclerotic, since the long-circulating nanoparticle tends to increase background noise. To address the limitation, in a study conducted by E. J. Keliher et al., a polyglucose nanoparticle was designed to have a size below the renal excretion threshold for a rapid renal excretion and with a high affinity for macrophages residing in cardiovascular organs.⁵⁵ The polyglucose used in this study is a class of lysine-crosslinked low molecular weight carboxymethyl polyglucose polymers, each containing 22 glucose units and labeled with ¹⁸F to create a PET imaging agent (termed Macroflor). After intravenous injection, Macroflor rapidly accumulated in kidneys and bladder with a blood half-life of 21.7 min as it was intentionally designed.⁵⁵ While the blood circulation half-life of Macroflor is very short, nanoparticle was rapidly up-taken by cardiac macrophages. As a consequent, in an atherosclerotic mouse model, Macroflor enrichment was observed in the aortic root and arch which are affected by inflammatory atherosclerosis. The in vivo Macroflor uptake profile measured by flow cytometry when the cells were retrieved from the aortic tree of ApoE^−/− mouse shows significant accumulation in macrophages over other leukocytes such as lymphocytes and neutrophils. Similar to the atherosclerotic lesion, Macroflor was also found to be accumulated in ischaemic myocardium macrophages.⁵⁵

3. Cell component-based engineering approach

The biomimetic surface modification of the synthetic nanoparticle using mammalian cell-ghosts has shown the installment of the biological complexity of the original cells. The biomimicry strategy has pushed the boundary to reveal unexplored properties of imaging agent thereby open-up new opportunities in medical imaging. The advantages of incorporating biological materials with synthetic materials include biocompatibility, resistivity, cellular interaction, enhanced circulation half-life, and cellular retention.

3.1. Cell-membrane engineering strategy

In cell membrane coating technology the lipid bilayer and proteins of cell membrane were translocated onto a synthetic nanoparticle surface to create a core-shell nanoconstruct.^{20,22,24,66,67} At early day, red blood cell membrane coated PLGA was investigated for various applications from detoxification to cancer therapy.^24,66–68 Since then, the cell membrane coating technology has expanded into different research area, transforming ordinary materials into functional materials by introducing multifaceted functionalities to conquer complex biological system. These applications cover a broad spectrum of nanotechnology ranging from drug delivery,²⁰ vaccination,⁶⁹ immunotherapy,^70,71 to micromotor⁷² and now starting to expand toward bioimaging area.^73,74

The process of coating cell membranes onto synthetic materials can be simply described by 3 steps process (1) cell membrane extraction, (2) fabrication of nanoparticle core, and (3) coating of cell membrane onto synthetic core. The imaging agents can be introduced as a part of cell membrane or core materials. The translocation process of cell membrane onto synthetic core is facilitated by extrusion, sonication, or microfluidic electroporation (Figure 9).^73,75,76 Under the mechanical stress condition, both membrane and particle core were suggested to undergo instability transition process to promote the re-arrangement of two entities into a single core-shell structure leading to the formation of stable nanoconstruct.^21,77 The membrane coating process has shown to favor the negatively charged core over neutral and positively charge counterpart.⁷⁷ The preservation of right-side-out membrane conformation after translocation onto synthetic core attributes to the conservation of biological function of proteins domains located on the outer leaflet of cell membrane, thereby acquiring biomimetic action.

Figure 9.

Incorporation of imaging agent into cell membrane vesicle using extrusion (A), sonication (B), and electroporation (C) techniques. (Adapted from ref. 73, 75, 76)

3.1.1. Cell membrane coated metallic nanoparticulate imaging agents

Exciting application of cell membrane camouflaged contrast agent was depicted with variety of cell types and core materials. Solid nanoparticulate contrast agents can be directly utilized as core materials in the biomimetic core-shell structure. Examples include the use of red blood cells, platelet, stem cell, and cancer cell membrane coating over iron oxide, gold, and upconversion nanoparticles.

Among different metallic nanoparticulate contrast agent, iron oxide nanoparticle (IONP) appears as an attractive core material to build up biomimetic nanoparticulate contrast agent due to its ease of synthesis with controllable size and tunable magnetic properties. Recent researches have also demonstrated that when IONPs are clustered, the clustered IONPs exhibits significant enhance in T₂ magnetic relaxivity and photothermal property when exposed to near infrared laser.^8,78,79 These potentials can be further improved as clustered IONPs are enveloped in cell membrane vesicles. Taking the advantages of IONPs clustering phenomenon under ultrasonic condition, Pei-Ying et al co-sonicated IONPs with cell membrane derived from mesenchymal stem cell (MSC) to obtain stem cell membrane-camouflaged IONP cluster for possible MRI and photothermal therapy application (Figure 9B).⁸⁰ Owing the presence of vascular cell adhesion molecule and ICAM-1, the cell membrane derived from MSC can interact with endothelial cells at inflammatory and cancer site allowing the designed nanoconstruct to acquire tumor targeting capacity.^81–83

Beyond standard biomimetic nanoparticle fabrication strategies, more sophisticated technique with high-throughput outcome was also developed to reduce the number of cells being used while maintaining the high coating efficiency over solid IONP core. For example, Rao et al. engineered a microfluidic device coupled with electroporation to facilitate the translocation of cell membrane onto the surface of iron oxide nanoparticle (Figure 9C).⁷⁵ Under such electroporation condition, multiple temporary transient pores are opened on the cell membrane vesicles allowing the iron oxide nanoparticle to go through. With the optimized pulse voltage, duration, and flow velocity, the obtained RBC coated iron oxide nanoparticle revealed a core-shell structure as normally observed in biomimetic nanosystem with highly stable colloidal stability. As a result, the RBC coated IONP display photothermal and magnetic properties from IONP while capable of evading immune system due to the presence of RBC characteristics on the surface.

In addition to iron oxide nanoparticle, upconversion nanoprobe made up of lanthanide‐doped nanocrystals with hexagonal shape can also be modified with cell membrane to solve the existing problems occur during bio-nano interaction. Upconversion nanoprobe is a class of nanoparticle capable of converting low energy photon into higher energy.^84,85 Owing to its emission in in-fared region, narrow emission band, higher photostability, low autofluorescence background, and wide Stroke shift, upconversion nanoparticle has been proven its great potential and can be pictured as the next generation imaging probe for optical imaging.⁸⁶ Similar to many other synthetic materials, the upconversion nanoparticle also faces to biological barrier such as lack on in vivo stability and targeting capability. In order to overcome such limitations, Rao et al. uztilized extrusion technique to enclose upconversion nanoprobes with in cancer cell and red blood cell membranes to improve tumor targeting and prolong circulation time for specific tumor imaging, respectively (Figure 9A).^76,87 Upon coating with cell membrane, a typical core-shell structure was observed under TEM with the shell thickness of 6–10 nm. The cell membrane coating has minimal influence on the fluorescence emission peaks at 543 and 655 nm in compare to un-coated nanoparticles. Exerting the biomimicry properties on the surface and exceptional fluorescent emitting core, the cell membrane coated upconversion nanoprobe provide significant signal at tumor site up to 24hr when coated with cancer cell and 48 hr in the case of RBC coating.

3.1.2. Cell membrane incorporated molecular based imaging agents

Compared to nanoparticle-based contrast agent, the size of molecular imaging probe is much smaller with diverse polarity allowing them to locate in different compartment of biomimetic nanoconstruct. Two different strategies of imaging agent incorporation, encapsulating in the synthetic core and infusing on the cell membrane layer, were accessed.

The loading of hydrophobic imaging probe can be achieved by mixing contrast agent with polymer in polar organic phase, followed by the nanoprecipitation. During nanoprecipitation, the nucleation of polymer trapping indocyanine green (ICG) molecules in the polymeric matrix. Once the imaging agent labeled cores are formed, the subsequent extrusion or sonication process will allow the coating of cell membrane materials to take place. In the first example of this incorporation strategy, Chen et. al., fabricated a cancer cell membrane coated ICG labelled nanoparticles that can acquire both imaging and photothermal therapy at tumor site.⁸⁸ Taking the advantages of intercellular homologous binding capability of cancer cells, cancer cell membrane coated ICG-loaded nanoparticles could recognize and home back to the same lineage. The dye used in the study acquires two important functions (1) emitting fluorescent signal at targeted site and (2) enabling the photothermal therapy by absorbing light at 800 nm and convert it into heat. Owing to the naked-eye visibility and cancer homing property, the use of ICG dye in biomimetic nanoconstruct would be advantages in the practice of image-guided surgery. In another example conducted by Li et al., a highly NIR absorbing semiconducting polymer, poly(cyclopentadithiophene-alt-benzothiadiazole), was co-nanoprecipitated with PEGylated triblock copolymer, to generate a polymeric core that can subsequently undergo extrusion with cell membrane derived from activated fibroblast.⁸⁹ Due to the presence of semiconducting π-conjugated system, the semiconducting polymer turn the designed biomimetic nanoconstruct into an optically active material, permitting the use in fluorescence and photoacoustic imaging modalities.

Alternatively, the imaging agents can also be infused into the shells compartment of the nanoconstruct. Using cell membrane-resorbing method, Wei et al. incorporate gadolinium-based contrast agent onto the biomimetic nanoconstruct. Using Gd-DTPA-bis(stearylamide) (Gd-DTPA-BSA), a gadolinium based contrast agent structure that exhibit both hydrophobic and hydrophilic regions, the imaging probe can align with platelet membrane protein and phospholipid to generate pre-labelled cell membrane prior to co-sonication with preformed PLGA core.⁹⁰ The Gd-labelled platelet nanoparticles was used to detect atherosclerotic plague aortic arch area after 1 h of injection. The ability to provide a distinctive positive contrast was attributed to the biological function of platelet in atherogenesis development including immune cell recruitment and de-stabilize sub-endothelial matrix.

3.1.3. Cell membrane based multi-modal imaging agents

Combining multimodal imaging probes in a single setting offers opportunity to achieve precision diagnosis by exploiting the strength of each imaging modality while overcoming their respective limitations. The combination of multiple imaging probes in a single biomimetic nanoplatform can be achieved by the rational arrangement of two or more molecular and nanoparticulate contrast agents. A common combination can be found in dual/hybrid imaging system, such as MRI/PET, PET/CT, MRI/NIR, and SPECT/CT, that can reveal both anatomical and functional features of diagnostic subject.⁹¹

Aligning with this approach, a NIR dye (DiR) together with gadolinium lipid conjugate (Gd-Lipid) were used to incorporate into a Natural Killer cell biomimetic system (Figure 10). With reactive functionality such as carboxylic acid, gadolinium macromolecules such as Gd-DOTA can be conjugated with hydrophilic head of phospholipid, yielding a more gadolinium lipid conjugate (Gd-Lipid) in compare to the linear DTPA-Gd system.^11,46,73 Owing to the lipophilic character, both DiR dye and gadolinium lipid conjugate spontaneously become a building block of particle’s shell via hydrophilic and hydrophobic assembly when Natural Killer cell (NK-92) membrane, Gd-lipid and PLGA nanoparticle were co-extruded.⁷³. Using this technique, the MRI relaxivity is tunable by varying the amount of Gd-lipid in the system, exhibiting r₁ ranging from 2.1 ± 0.17 to 5.3 ± 0.5 mm⁻¹ s⁻¹ under 14.1 T. The hybrid nanoplatform acquire the properties of NK-92 cell to drive imaging agents toward tumor site, thereby improving the diagnostic efficiency in targeted cancer fluorescent and MRI bioimaging.

Figure 10.

Multimodal biomimetic imaging nanoconstruct made up of natural killer cell membrane and polymeric PLGA core capable equipped with gadolinium lipid conjugate and NIR dye that can acquire MRI and Fluorescent bioimaging. (Adapted from ref. 73)

Similarly, an effort has been made to engineer a cancer cell membrane coated IONPs and Chlorin e6 (Ce6) dye for possible application in MR/NIR fluorescence dual modal imaging of tumor.⁹² In the study, an electrostatic interaction was explored as an alternative way for coating cell membrane over nanoparticle. To achieve a dual imaging system, IONPs was embedded into styrene and acrylic acid block co-polymer to create a IONPs nanocluster core for MRI T₂ contrast enhancement (r₂=163.67 mM^-1.s⁻¹). The surface of IONPs nanocluster was modified with polyethyleneimine (PEI) providing a positively charge on the nanoparticle core. This positively charge layer serve as an intermediate substrate for the absorption of Ce6 and cancer cell membrane via electrostatic interaction. Taking the advantages of IONPs confinement effect, the ability to emit NIR fluorescence of Ce6, and tumor homing capacity of cancer cell membrane, the engineered cell membrane coated dual imaging nanosystem exhibited better tumor targeting effect with superior dark contrast enhancement and specific fluorescent detection at tumor site.

3.2. Exosome derived engineering strategy

Exosomes are cell-derived nanovesicles that play an important role in cell-cell communication. Secreted by parent cell and equipped with the original biological identity and information, exosomes can overcome the biological barriers, travel in the body fluid until they get to target cell to deliver the payloads via membrane fusing mechanism. With this natural transporting property, exosomes can be seen as a “mailman” of biological system that can be used as a drug, gene, and imaging agent carrier.

In other to push forth the potential of exosome in those applications, the knowledge regarding biological function of circulating exosomes, such as which cells take them up in vivo, what is their biodistribution, and how they contribute to the biological microenvironment modulation, is required. The ability to track exosomes in vivo using different imaging modalities offer the opportunity to fulfill the current knowledge gap and can be further expand the knowledge to diagnostic and therapeutic applications.⁹³ further the purpose, the exosome can be labelled with imaging probe by engineering indirectly via donor cell or directly using exogenous manipulation techniques.

The formation of exosome takes place in endosome, a membrane bound compartment generated during endocytosis. During early stage of endosome formation, the endosomal membrane can undergo inward budding to produce multiple intraluminal vesicles . As a result, endosomes turn into multivesicular bodies and fuse with the cell membrane to release internal intraluminal vesicles called as exosomes. Considering the relation between plasma membrane internalization and exosome formation mechanism, an approach of using endocytosis as a possible pathway to label exosome before isolation has been implemented. Under pre-exosome isolation labelling approach, a wide range of contrast agent based nanosized materials have been investigated as their main cellular uptake pathway is endocytosis.^94–96 Ultra-small superparamagnetic iron oxide nanoparticles (USPIONs) are advantageous to use under this approach. Exosome labeled with USPIONs can be simply achieved by co-incubating of USPION with adipose stem cell (ASC).⁹⁴ After internalization, the USPIONs cluster in exosome vesicle while undergoing secretion process. Under in vivo condition, the MRI detection limit of USPION labeled exosome can be achieved with 0.032 µg Fe. Furthermore, the ability of precisely tracking exosome both in vitro and in vivo can be significantly improved by adding other imaging modality onto USPIONs. For example, the inclusion of rhodamine B dye on USPIONs offer the opportunity to incorporate fluorescent imaging modality along with MRI into exosome to explore intracellular fate of exosome using super-resolution microscopy, flow cytometry or immunostaining.⁹⁵ Although pre-exosome isolation labelling approach is simple and does not interfere exosome morphology and physiological characteristics, just a small number of cargo loaded-MVBs can escape from lysosome degradation pathway to fuse to plasma membrane and release exosome. As a result, predominant uptake materials get digested in the lysosomal compartment leading to a modest exosome labelling yield.

Different from indirect labelling approach, direct exosome labelling technique can be executed after exosome isolated from cell culture media. The incorporation of imaging probe into exosome is mediated by covalent bonding, physical insertion, electroporation, or exosome membrane mediated-active transportation via specific protein receptors.⁹⁷ Electroporation is a traditional way to transfer cargos across lipid bilayer by creating temporary pores under electric field. In exosome imaging labeling application, the opened membrane pores create temporary entrance to facilitate the translocation of cargo such as 5 nm iron oxide nanoparticle into intravesical compartment.^98,99 In order to avoid the aggregation of exosome during electroporation process, trehalose is introduced into exosome suspension as a biocompatible disaccharide membrane protectant.⁹⁹ The resulted SPIONs labelled exosome derived from B16-F10 melanoma cell line showed accumulation within lymph nodes of C57BL/6 mouse model and can be imaged using standard MRI approaches.⁹⁸ Similarly, a mild technique such as sonication was also introduced to enforce the insertion or loading of imaging agents.¹⁰⁰ Sonication, with the involvement of sound energy vibration, decreases the rigidity of exosomal membranes and facilitate the incorporation hydrophobic cargo into lipid bilayers.^100,101 Abello et. al. adapted sonication technique to label exosome with (Gd³⁺)-chelated DSPE-DOTA (Gd-Lipid) and DiR dye for dual fluorescent and MRI modalities tracking of exosome derived from human umbilical cord mesenchymal stromal cells (HUC-MSC) in vivo (Figure 11).¹⁰⁰ The lipid insertion mediated by sonication allows the hydrophobic tail of Gd-Lipid and DiR dye to anchor into exosomal membrane while the positively charge of the hydrophilic head ensure the intactness of imaging probe on exosome while traveling in the bloodstream via electrostatic interaction. The labeled HUC-MSC-exosomes exhibited tumor targeting properties in an in vivo osteosarcoma mouse model. Similar approach was also proposed by Rayamajhi et. al. where Gd-conjugated liposomal system was incorporated into macrophage cell-derived extracellular vesicles framework forming a gadolinium infused hybrid extracellular vesicles (Gd-HEVs).^102,103 Thanks to the presence of immune cell-derived EV proteins, the Gd-HEVs not only able to disguise themselves as a biological entity to improve tumor homing capability, but also assist in Gd contrast enhancement by reducing global tumbling motion of Gd ions. As such, the approach helps to increase conventional r1 relaxivity of Magnevist from 3.98 mM⁻¹.s⁻¹ to 9.86 mM⁻¹.s⁻¹ at an equivalent Gd concentration, when Gd conjugated into Gd-HEVs construct.¹⁰² These outstanding Gd-HEVs tumor targeting and contrast enhancement properties allow the platform to be used at much lower Gd concentration while safely achieving clinically relevant MRI contrast effect.

Figure 11.

Post-isolation labelling of exosome using sonication technique to incorporate gadolinium based MRI contrast agent and DiR fluorescent probe for in vivo exosome tumor homing tracking. (Adapted from ref. 100)

Aside from physical manipulation, chemistry technique can also be adapted to covalently attach imaging probe on the surface of exosomes. Click chemistry between azide and alkyne using copper catalyst and coupling reaction using EDC/NHS activating reagents are often employed for exosome surface modification.^104–106 These aqueous based chemistry reactions were implemented to attach Azide-fluor 545 fluorescent probe on the outer membrane of exosome in a study conducted by Smyth et al.¹⁰⁷ The surface of exosome was first functionalized with terminal alkyne group via an amide bond between carboxylic acid of 4-pentynoic acid and amine end terminus of protein. Subsequently, azide-fluor 545 was introduced to the modified exosome in the presence of copper to facilitate azide alkyne cycloaddition. The resulted exosome exhibited approximately 1.5 alkyne modifications for every 150 kDa of exosomal protein while maintaining original size and function.¹⁰⁷ The chemistry approach for surface modification of exosome can be further expanded to utilize other of functional groups available on exosome membrane that are readily react to each other under mild aqueous condition such as sulfo-maleimide, amine-anhydride.^108–110 In addition, technique such as “bio-orthogonal” chemistry in which functional group can be introduced via metabolic pathway can also be implemented to diversify chemical reactive moieties on exosome membrane.^104,111

Generating from the double-inward budding event of parent cell, exosome shares functionality that is similar to the outer leaflet of parent cell membrane, these include membrane transport proteins. These membrane transport proteins can be used to mediate the active transportation of imaging agents into exosome endocytosis pathway. Under this approach, Betzer et. al. employed glucose-coated gold nanoparticle to label purified exosomes derived from mesenchymal stem cell and visualized by CT using ischemic mouse model.¹¹² The glucose-coated gold nanoparticle was actively transported into exosome by glucose transporter GLUT-1 via an energy dependent pathway. The removal of protein receptors, use of GLUT-1 inhibitor or introduce excessive free ligand show inhibition on exosome uptake phenomenon. Using intranasal administration, a superior brain accumulation especially at stroke area, of glucose-coated gold nanoparticle labeled exosomes was observed.

4. Opportunities, challenges, and future outlook

The cell-based delivery system has been long used for the delivery of various bioactive molecules for disease management. While a significant amount of work has been done to cell-based drug delivery, researchers have recently started to realize its potential in the diagnosis when coupled with synthetic contrast agents. Research toward this direction, in the perspective of molecular imaging and disease diagnosis, is moving at a considerable rate. Results obtained from cellular and synthetic harness shows several advantages over their non-cellular synthetic counterparts. One of the major advantage is communication with endogenous cells and their ability to “talk” like cell. Such system rather than attempt to stealth themselves from the immune system, directly interacts with immune cells in order to convince the body that they belong to a common biological lineage. Due to the unique biologically derived surface properties, diseased sites are recognized and prevent rapid elimination and nonspecific distribution of the active agent to a wide range of tissues and organs. A precision in the system is due to its biomimetic properties that would accelerate early disease detection, monitoring treatment outcome through real-time imaging observations, finding answer key questions about disease pathologies, and would facilitate our understanding of the mechanisms underlying cell and body biology.As seeing is believing, these valuable information will not only help us to improve current disease management strategy but also shed the light on current black-box in biological processes.

While many other systems are under investigation, opportunities always come with challenges for design consideration to practical application. Especially, when a biologically derived material is used, the clinical translational pathway become more complicated with strict requirement on material selection, biological influence, technical imaging acquisition protocol, and manufacturing process. First of all, to improve the imaging outcome, the selected contrast agents must fulfill minimum clinical requirement such as preservation of (1) physicochemical properties, (2) sensitivity, and (3) resolution pre and post cell labeling. The imaging agents should be non-toxic and must not interfere with cell biology. Meanwhile, the cell or cell component engineering strategies should enhance or at least not alter the performance of imaging agents. Therefore, through in vitro evaluation and mapping of safety profile is an essential component prior to its implementation in animal models. For example, while taking account of the small molecular probe, the relationship between chemical structure and cell biology is a major concern and is a great challenge to avoid non-specific interaction and conversion due to the presence of biologically active components (enzyme, reductive and oxidative agents). Similarly, the properties of the nanoparticulate system such as surface coating, size, shape, decomposition upon cell interaction, and protein corona formation in vitro and in vivo have to be studied carefully.

Next, as the transporter and biological function provider, cell type selection will directly impact the outcome of imaging. The appropriate cell should be chosen for specific application depending on the need for biological information. Also, upon interaction with imaging agent and after undergoing modification process, cell profiles may be altered at the molecular level; thereby, impacting overall diagnostic outcomes. Therefore, the fundamental studies focusing on the impact of cell type, cargos, and modification methods would contribute significantly to extend our existing knowledge in the field. Most importantly, for the successful translation of these approaches to clinically relevant tools, cell-based imaging agents need to answer big question existing in the field regarding its potential immunogenicity.

Finally, broad vision contributing to the process of transforming the cell-based imaging agent from “bench to bedside” should take into account at the beginning stage of development, these includes feasible manufacture processes, large-scale synthesis, and fulfillment the needs of clinician. Unlike molecular and nanoparticulate imaging agents, cell-based imaging agents cannot easily be seen as an “off-the-shelf” product but rather to be considered as personalized medicine. With the recent advent in artificial intelligence for image analysis and the acceptance of cell-based immunotherapy for cancer treatment,^113–116 the field of cell-based imaging agents would have great promises in disease diagnosis and offer opportunities to understand the mechanism of disease progression.