Detecting and Monitoring Hydrogels with Medical Imaging

Abstract

Hydrogels, water-swollen polymer networks, are being applied to numerous biomedical applications, such as drug delivery and tissue engineering, due to their potential tunable rheologic properties, injectability into tissues, and encapsulation and release of therapeutics. Despite their promise, it is challenging to assess their properties in vivo and crucial information such as hydrogel retention at the site of administration and in situ degradation kinetics are often lacking. To address this, technologies to evaluate and track hydrogels in vivo with various imaging techniques have been developed in recent years, including hydrogels functionalized with contrast generating material that can be imaged with methods such as X-ray computed tomography (CT), magnetic resonance imaging (MRI), optical imaging, and nuclear imaging systems. In this review, we will discuss emerging approaches to label hydrogels for imaging, review the advantages and limitations of these imaging techniques, and highlight examples where such techniques have been implemented in biomedical applications.

Graphical Abstract

1. INTRODUCTION

Hydrogels are solid materials mainly constituted of water, composed of a three-dimensional cross-linked polymer or colloidal network immersed in fluids, and they have found utility across many fields.^1,2 In the past decades, hydrogels have become increasingly important in diverse biomedical applications, such as drug delivery and tissue engineering, due to their unique characteristics, such as high water content, tunable properties, controllable degradation, and their potential responsiveness to extrinsic signals.^3–5 Hydrogels are fabricated by numerous polymerization and cross-linking strategies to form polymeric networks⁶ and are generally classified into two categories based on the nature of their cross-linking, namely, physically or chemically cross-linked hydrogels.^7,8 Of these, physically cross-linked hydrogels are assembled through noncovalent cross-linking (e.g., ionic interactions, hydrogen bonding),^6,9 whereas chemically cross-linked hydrogels involve covalent bonds within their cross-links.^10,11 The cross-links can be designed to be reversible so the hydrogels possess shear-thinning properties, which can be advantageous in biomedical applications.^12,13

Owing to these unique characteristics, hydrogels can be designed for minimally invasive delivery to a tissue site of interest, where accurate localization and monitoring of the hydrogel are of great importance.^3,14,15 Meanwhile, the in vivo degradation of hydrogels can play an important role in achieving desired therapeutic outcomes.¹⁶ Thus, the ability to image hydrogels is useful to confirm that the hydrogel has been successfully administered to the target site and in the correct quantity as well as to monitor the hydrogel erosion over time. In many instances, groups of animals are sacrificed either soon after injection or at staggered time points, and the residual hydrogel is removed for examination in order to monitor the state of implanted hydrogels in vivo.^17,18 Thus, serial postadministration imaging of hydrogels would yield improved information on hydrogel placement, degradation in vivo, or the release of therapeutics, due to the need for fewer animals and to allow comparisons across the same animal. To accomplish this, hydrogels can be rendered imageable with various medical imaging modalities by using intrinsically imageable polymers during hydrogel formation or by functionalizing hydrogels with contrast-generating materials through physical loading or chemical grafting.^1,15,19,20

To date, multiple imaging modalities and their combinations have been used for different clinical applications^21–30 as well as used for detecting and monitoring hydrogels in tissues, with each of them having their own advantages and limitations (Tables 1 and 2).^31–39 For example, CT is one of the most widely used clinical imaging modalities, where computerized X-ray imaging is used to produce three-dimensional, anatomic images with high spatial and temporal resolution.⁴⁰ To distinguish hydrogels from the surrounding soft tissues in CT, X-ray contrast agents are usually used. The currently approved contrast agents for X-ray imaging are iodinated small molecules or barium sulfate suspensions. Although both can be incorporated into the hydrogel to impart radiopacity, they can cause adverse events and have shortcomings in their specificity of detection.^41,42 With the same capability of rendering hydrogels radiopaque, nanoparticle-based contrast agents such as gold nanoparticles (AuNP) can produce more contrast than iodinated contrast agents, and their versatility also allows for a wider range of applications. While other X-ray based imaging modalities such as conventional radiography, spectral photon-counting computed tomography (SPCCT), mammography, and fluoroscopy would offer similar benefits to utilizing radiopaque hydrogels, there have been only a few studies reporting the use of these modalities for hydrogel imaging as compared to more commonly used CT.^43–45

Table 1.

Characteristics of Imaging Modalities Used for Hydrogel Imaging^a

imaging modality	advantages	limitations	source	contrast agents	common applications
CT	high spatial resolution anatomic imaging low cost	radiation poor soft tissue contrast common artifacts	X-rays	iodinated small molecules (A), barium sulfate suspension (A), gold nanoparticles	trauma, joints, cardiovascular diseases, gastrointestinal tract
MRI	high spatial resolution soft tissue contrast	high cost low sensitivity long scan times	magnetic fields, radiowaves	gadolinium chelates (A), iron oxide nanoparticles, exchangeable protons	brain and spinal cord, musculoskeletal imaging, prostate and breast cancer imaging
nuclear imaging	high sensitivity quantitative metabolic activity	high cost high radiation exposure	gamma-rays	radioisotopes (e.g., 18F, 124I) (A)	tumor imaging and cancer stage evaluation
optical imaging	high sensitivity multichannel imaging	complex relationship between signal and probe concentration poor tissue penetration	near-infrared (NIR) light	quantum dots, indocyanine green (A), methylene blue (A)	endoscopic and retinal imaging
photoacoustic	noninvasive high penetration depth	not available in the clinic limited field of view	nonionizing laser pulse/acoustic waves	quantum dots, gold nanoparticles, semiconducting polymers	hemodynamics monitoring
ultrasound	widely available low cost portability	common artifacts low resolution operator dependent	high-frequency acoustic waves	microbubbles (A)	fetus imaging, soft tissue imaging

^aNote that while the poor soft tissue contrast of CT is typically viewed as a disadvantage for imaging, in the context of hydrogel imaging it may be an advantage, since it allows the hydrogel to be clearly distinguished from background soft tissue. On the other hand, the excellent soft tissue contrast of MRI can render identification of the hydrogel challenging due to a confounding endogenous signal. Note: The contrast agents have been FDA-approved for clinical use are noted with (A).

Table 2.

Overview of Multimodal Imaging Techniques and Their Common Applications

imaging modalities	applications	advantages
PET/CT	tumor detection, cancer staging, and tumor response monitoring assessment of targeted therapies	clarification of equivocal CT findings improved spatial resolution and lesion characterization
SPECT/CT	musculoskeletal infection, trauma, and malignant disease imaging cardiac imaging	increased specificity through more precise localization and characterization
PET/MRI	tumor staging and presurgical planning for oncological conditions associated with soft tissues	reduced ionizing radiation provides simultaneous anatomic and molecular information
ultrasound/CT	soft tissue imaging breast cancer diagnosis	produces quantitative images of the acoustic properties of tissue

As another example of an imaging modality, MRI uses a large magnetic field and radio waves to perform anatomical imaging of soft tissues.⁴⁶ It is a noninvasive and nondestructive diagnostic tool with good potential for monitoring tissue implants.^47,48 Similar to CT, MRI uses contrast agents to enhance internal structures. Gadolinium chelates are the only type of MRI contrast agents both approved by the FDA and available on the market.^49,50 Superparamagnetic iron oxide nanoparticles have been FDA-approved as MRI contrast agents; however, all have been withdrawn from the market due to low sales.⁵¹ The one remaining iron oxide nanoparticle on the market, ferumoxytol, is approved for iron replacement therapy, so its applications as an MRI contrast agent are off-label uses. Hydrogels can either be functionalized with contrast agents such as these so that they can be distinguished from adjacent tissues with conventional MRI or certain types of hydrogel can be induced to produce contrast in the absence of exogenous contrast agents via techniques such as chemical exchange saturation transfer (CEST) MRI.^32,52,53

Fluorescence imaging relies on the absorption and scattering properties of light in tissue or biomaterial components.^54,55 Its high sensitivity, resolution, and high throughput render this technique popular for in vivo imaging.⁵⁶ However, its low tissue penetration (<1 cm) and limited quantitative accuracy limits its utility. Probes that absorb in the near-infrared (NIR) region(i.e., 650–900 nm) are often used for in vivo imaging as most tissues have relatively low absorbance in the NIR region.⁵⁷ Thus, noninvasive in vivo tracking of the distribution and degradation of hydrogels can be achieved by labeling the hydrogel with fluorescent probes such as fluorophores (indocyanine green and methylene blue are the only two NIR fluorophores approved by FDA), quantum dots, or upconversion nanoparticles.^58–62 In addition to these techniques, other imaging modalities such as nuclear imaging, ultrasound, and photoacoustic imaging can also be used to visualize the in vivo distribution and degradation of hydrogels, although these modalities remain relatively underexplored for these applications.^36,63,64

In the following sections, we first summarize the various strategies used in the cross-linking of hydrogels, which is important to inform hydrogel properties and subsequent imaging approaches. Next, the approaches that have been taken for the functionalization of hydrogels to render them imageable are reviewed. We then focus on recent developments in the imaging of hydrogels using modalities such as CT, MRI, and fluorescence imaging. For each imaging modality, the basic principles of the imaging method are first described, and then the biomedical applications using the technique are reviewed. Lastly, the potential and challenges of hydrogel imaging in clinical settings are addressed.

2. OVERVIEW OF HYDROGEL FUNDAMENTALS

Hydrogels can be classified based on the polymer source (natural, synthetic), polymer composition (homopolymer, copolymer, interpenetrating, nanocomposite), polymer configuration (amorphous, semicrystalline, crystalline), network degradability (biodegradable, nondegradable), and type of cross-linking.⁴ These variables give rise to hydrogels with a wide range of biochemical and biophysical properties that can be tailored for specific applications.

Natural polymers such as polysaccharides (e.g., hyaluronic acid (HA), chitosan, heparin) and proteins (e.g., gelatin) are derived from natural sources and often inherently incorporate important functional features, such as being biocompatible, biodegradable, and presenting critical biological cues. These natural polymers can be modified with various functional groups for cross-linking into networks for hydrogel formation. Synthetic polymers such as polypeptides, polyesters, polyanhydrides, and polyphosphazenes can be designed with specific features in mind, such as tunable degradation rates, mechanical properties, and microstructures.⁶⁵ Furthermore, they are designed to incorporate functional groups during synthesis to allow for cross-linking. Nondegradable synthetic hydrogels have been prepared from the copolymerization of various vinylated monomers or macromers such as 2-hydroxyethyl methacrylate (HEMA), acrylamide (AAm), acrylic acid (AAc), N-isopropylacrylamide (NIPAm), and polyethylene glycol (PEG).⁶⁶ For example, the end hydroxyl groups of PEG can be modified with functional groups (e.g., thiols, acrylates), allowing many different cross-linking methods to form hydrogels.⁶⁷ Similarly, poly(vinyl alcohol) (PVA) is another common synthetic polymer with pendant hydroxyl groups that can be modified for cross-linking with different chemical methods⁶⁸ as well as with freeze/thaw cycles to induce physical cross-linking.⁶⁹

Chemically cross-linked networks traditionally have permanent cross-linking points or junctions, while physical networks have reversible junctions that are formed through polymer chain entanglements or physical interactions (e.g., ionic interactions, metal coordination, hydrogen bonds). Recently, dynamic covalent networks have also emerged as a means to chemically cross-link hydrogels, whereby bond cleavage can occur under mechanical shear and reform to exhibit self-healing properties. In this section, we review the common methods of physical and chemical cross-linking to form imageable hydrogels.

2.1. Physical Cross-Linking.

Physical associations that are used to form hydrogels from polymer chains include ionic interactions,⁷⁰ metal–ligand coordination,⁷¹ hydrogen bonding,⁷² hydrophobicity,⁷³ and supramolecular host–guest⁷⁴ interactions. The self-assembly of physically cross-linked hydrogels enables rapid hydrogel formation while avoiding the use of any external cross-linking agents. These physical associations are reversible and contribute to the shear-thinning and self-healing properties of many hydrogels, which allows injectability and processing using techniques such as 3D printing. These features are reviewed extensively elsewhere.⁷⁵

One of the simplest approaches to hydrogel formation is the ionic cross-linking of charged biopolymers with multivalent ions. For example, alginate is a naturally occurring, anionic, and hydrophilic polymer that cross-links in the presence of cations, most typically Ca²⁺, and is commonly used for tissue engineering and drug delivery applications. Recently, radionuclides such as In³⁺ and Zr⁴⁺ were used to cross-link alginate, providing a novel and simple hydrogel radiolabeling approach.⁶³ Hydrogen bonding, represented as an attractive interaction between hydrogen atoms and electronegative atoms such as nitrogen, oxygen, and fluorine, has also been used in hydrogel formation. For example, many binding motifs such as ureiodopyrimidone (UPy),⁷⁶ benzene-1,3,5 tricarboxamide,⁷⁷ and catechols⁷⁸ have been used to functionalize polymers for the formation of hydrogels through hydrogen bonding. Recently, PEG end-functionalized with UPy moieties as well as DOTA-Gd(III) were used to form fibrous hydrogels based on pH that could be visualized via MRI after injection into the myocardium.⁴⁸

Metal–ligand coordination complexes offer near-covalent stabilities with pH-tunable kinetics and have been implemented in hydrogel cross-linking. The inherent reversibility of metal–ligand coordination renders the resulting hydrogels as shear-thinning and self-healing. While catechol–Fe^3+79,80 and histidine–Ni²⁺⁸¹ are some common metal–ligand complexes used previously, many other complexations exist and offer a unique opportunity to form radiopaque hydrogels utilizing the coordination between heavy metals and polymers.⁷¹ Nanocomposite hydrogels have also been formed where polymer chains are physically cross-linked to assemble with nanoparticles or nanostructures.⁸² Various nanoparticles such as carbon nanotubes, silicates, and metal/metal oxide nanoparticles can be combined with polymers to obtain nanocomposite hydrogels.⁸³ In addition to physical incorporation of nanoparticles into hydrogel systems,⁸⁴ recent studies have highlighted the use of nanoparticles as cross-linkers.⁸⁵ Along with metal coordination, nanocomposites offer significant potential in the formation of radiopaque hydrogels, particularly as nanoparticles can be used for both cross-linking and radiopacity.

Hydrophobic associations play a critical role in the formation of hydrogels via physical associations and usually involve incorporation of hydrophobic domains into polymers for self-assembly. Peptide amphiphiles and amphiphilic block copolymers rely largely on these hydrophobic associations and are reviewed elsewhere.^86–88

2.2. Chemical Cross-Linking.

Chemical cross-linking relies on covalent bonding between polymer chains. When compared to physical interactions, chemical cross-linking generally increases hydrogel stability and mechanical properties and allows enhanced control of variables such as gelation time and degradation properties. There are numerous types of chemical reactions that are employed to form hydrogels, including radical polymerizations, thiol–ene cross-linking, Michael addition reactions, and enzymatic cross-linking. Each approach uses specific reactive groups and has their own advantages in the formation of biomedical hydrogels.

Radical chain polymerizations involve formation of a radical through an initiator and initiation source (e.g., light, temperature, redox reaction), which then reacts with functional groups on molecules to form polymers. Acrylates and methacrylates are the most common reactive groups used in radical polymerizations,⁸⁹ and a wide variety of initiators have been used, such as photoinitiators and the ammonium persulfate (APS)/tetramethylethylenediamine (TEMED) system as a common nonlight-activated initiator/accelerator system. Many synthetic (polyanhydrides, PEG, polypropylene fumarates, poly(α-hydroxy esters), PVA) and natural (HA, dextran, chitosan, chondroitin sulfate) polymers have been functionalized with reactive groups to render them crosslinkable into hydrogels using radical polymerizations and have been explored for a variety of biomedical applications.⁹⁰ While this method allows rapid and robust hydrogel formation, the polydispersity in kinetic chains and cross-linking density results in heterogeneous networks.⁹¹

Light-activated free-radical cross-linking has gained particular interest in biomedical applications due to its temporal and spatial control of reactions and potential for in situ gelation.⁹² Photo-cross-linking can be carried out at physiological temperatures and pH, enabling their use in minimally invasive surgical procedures.⁹³ Photo-cross-linking of biomedical hydrogels is achieved via a photoinitiator and irradiation at adsorption wavelengths to generate radicals.⁹² These radicals attack carbon–carbon double bonds on precursor macromolecules, forming covalent bonds that cross-link into a network rapidly upon light exposure. A variety of photoinitiators are commercially available and continue to be developed.⁹⁴

Thiol–ene photo-cross-linking, a reaction between a thiol and an alkene (e.g., norbornene) in the presence of light and a photoinitiator, represents a different mechanism involving step-growth of a network.⁹⁵ Combining the advantages of both radical polymerization and bio-orthogonal click reactions, thiol–ene photo-cross-linking has enhanced control of network homogeneity. Furthermore, unlike other photo-cross-linking techniques, thiol–ene reactions are not inhibited by the presence of oxygen. As an example, the thiol-norbornene reaction has been used extensively to form,⁹⁶ photopattern,⁹⁷ and further functionalize⁹⁸ hydrogels with peptides, proteins, and small molecules.

Michael addition reactions involve the nucleophilic addition of thiol- or amine-bearing molecules to an α-β-unsaturated carbonyl compound (e.g., acrylates, methacrylates, vinyl sulfones).⁹⁹ Compared to chain growth polymerizations, step growth reactions allow more homogeneous network structures and offer a simple strategy to incorporate functional peptides into hydrogels.⁹¹ Michael addition reactions between thiols and acrylates/vinyl sulfones have been extensively used for the in situ formation of hydrogels due to the reaction’s mild conditions, tunability, and high chemical yield. Since Elbert et al.’s early study on protein delivery,¹⁰⁰ many PEG-, HA-, and dextran-based hydrogels have been formed via Michael addition reactions for drug delivery applications.^101–104 Recently, divinyl sulfone-,⁵³ carbazate and aldehyde-,¹⁰⁵ and thiol- and diacrylate-⁵² modifications have been utilized to form imageable hydrogels via this reaction.

Enzymatic cross-linking has emerged as an approach to form in situ hydrogels due to mild reaction conditions.¹⁰⁶ Transglutaminases,¹⁰⁷ peroxidases,¹⁰⁸ and tyrosinases¹⁰⁹ have been reported for cross-linking hydrogels. Horseradish peroxidase (HRP) has been particularly attractive for hydrogel formation, whereby cross-linking is induced by the addition of hydrogen peroxide to solutions of tyramine-modified monomers after which phenolate radicals isomerize and dimerize to form C–C bonded dityramine adducts. Tyramine-modified gelatin has been recently functionalized with AuNP and cross-linked with HRP.¹¹⁰

Dynamic covalent bonds are able to be formed, broken, and reformed, either autonomously or under stimuli. These dynamic properties are particularly attractive as hydrogels are being developed for minimally invasive clinical applications. Compared to physical associations, dynamic covalent reactions have slower kinetics of bond cleavage and formation, giving rise to more stable materials while still allowing injectability.¹¹¹ Schiff base reactions including imine derivatives (between amine and aldehyde groups) as well as hydrazone bonds (between aldehyde and hydrazide groups) have been used extensively for tissue engineering and drug delivery applications.¹¹² Oxime bonds, formed through the condensation of hydroxylamine with a ketone or aldehyde, exhibit improved hydrolytic stability over hydrazones and imines.^113,114 Disulfides, formed by the reaction of two thiol groups, require the presence of an oxidation agent to form and can be broken down or reform via physiologically relevant reduction/oxidation reactions.^115,116

3. FUNCTIONALIZED IMAGEABLE HYDROGELS

Several approaches have been developed to render hydrogels detectable by various imaging techniques without affecting their rheological properties and biocompatibility, while limiting their synthetic complexity. These include hydrogels that are intrinsically imageable or where the hydrogel is rendered imageable via the encapsulation of contrast agents via dynamic interactions (e.g., ionic interactions, H-bonding) or by the formation of covalent or coordination bonds with contrast agents (Figure 1). In this section, we review the methods of functionalizing imageable hydrogels with a particular emphasis on methods used to incorporate imaging features for biomedical applications, and the formulations used for such purposes are summarized in Table 3.

Figure 1.

Schematic of various strategies to image hydrogels. This figure was created with BioRender.com.

Table 3.

Representative Hydrogel Formulations and Modifications for Imaging^a

imaging modality	imaging functionalization	polymer	cross-linking method	refs
Intrinsically Imageable Hydrogels
CEST MRI	N/A	alginate/liposomes	ionic cross-linking (Ca2+)	Han et al. (2020)117
CEST MRI	N/A	HA-SH/gelatin-SH	Michael addition (PEG-diacrylate)	Zhu et al. (2019)52
CEST MRI	N/A	peptide conjugate (glutamic acid + phenylalanine)	amphiphilic peptide self-assembly	Lock et al. (2017)118
CEST MRI	N/A	HA	Michael addition (divinyl sulfone)	Shazeeb et al. (2018)53
CEST MRI	N/A	hydroxyethyl methacrylate-modified HA (HeMA-HA)	radical polymerization	Dorsey et al. (2015)119
fluorescence	N/A	polyacrylamide	radical polymerization	Xu et al. (2019)120
fluorescence	N/A	multiarm PEG acrylates	Michael addition	Tsou et al. (2018)121
fluorescence	N/A	bovine serum albumin (BSA)/ human serum albumin (HAS)	enzymatic (glutaraldehyde)	Ma et al. (2016)122
Physical Loading of Contrast Agents
CT	gold nanoparticles	gelatin-tyramine	enzymatic	Lee et al. (2018)110
CT	gold nanoparticles	PNAGA-PAAm copolymer N-acryloyl glycinamide	radical polymerization	Wu et al. (2018)123
CT	gold nanoparticles	alginate	ionic cross-linking	Keshavarz et al. (2018)124
CT	gold nanoparticles	gelatin methacrylamide (GelMA)	radical polymerization	Celikkin et al. (2019)125
CT	iopidamol and tantalum oxide particles	gelatin-tetrazine/gelatin-norbornene	dynamic covalent (Diels–Alder)	Hong et al. (2016)31
CT	platinum nanoparticles	alginate	ionic cross-linking	Wang et al. (2016)126
CT	barium sulfate suspensions	polyacrylonitrile (PAN)	radical polymerization hydrogen bonding	Zhang et al. (2018)127
fluoroscopy	iodinated contrast agents	chitosan	ionic cross-linking	Coutu et al. (2012)128
MRI	M-ferrite nanoparticles	AMPEG550	hydrophobic association	Kim et al. (2012)129
MRI	DTPA-Gd (III)	chitosan-DTPA	Schiff base (PEG-dialdehyde)	Liu et al. (2016)32
MRI	DOTA-Gd(III)	polyethylene glycol (PEG)	hydrogen bonding	Bakker et al. (2018)48
MRI	ferumoxytol	chitosan	hydrogen bonding	Chen et al. (2020)130
MRI	iron oxide nanoparticles	dehydrodipeptides	peptide self-assembly	Carvalho et al. (2019)131
fluorescence	carbon nanodots	N-methacryloyl chitosan	radical polymerization	Wang et al. (2017)132
fluorescence	indocyanine green (ICG)	RADA16-I peptide	peptide self-assembly	Jin et al. (2017)133
fluorescence	CdTe and CdSe quantum dots	Fmoc-diphenylalanine	peptide self-assembly	Kim et al. (2010)134
fluorescence	NaYF4:Yb,Er@PAA upconversion nanoparticles	GelMA	radical polymerization	Dong et al. (2017)61
fluorescence	NaLuF4:Er3+, Yb3+ @graphene oxide (NGO) nanoparticles	gelatin	hydrogen bonding	Li et al. (2019)135
fluorescence	NaLuF4:Er3+, Yb3+ @graphene oxide (NGO) nanoparticles	silk fibrin	peptide self-assembly	He et al. (2018)136
photoacoustic	ferrous/ferricyanide ions	dibenzaldehyde-terminated telechelic PEG (DF-PEG)/chitosan	dynamic covalent (Schiff base)	Cheng et al. (2017)137
ultrasound	SonoVue microbubbles	silk fibrin	peptide self-assembly	Leng et al. (2015)34
fluorescence/photoacoustic	Ag2S quantum dots	polypeptide PC10	peptide self-assembly	Jin et al. (2019)36
MRI/fluorescence/ultrasound	Zn ferrite magnetic nanoparticles (MNPs) and ICG	PEG diacrylate (PEG-DA)	radical polymerization	Wu et al. (2017)138
Chemical Grafting of Contrast Agents
CT	2,3,5-triiodobenzaldehyde	polyvinyl alcohol (PVA)	radical polymerization	Ashrafi et al. (2017)15
MRI	F19	HA-carbazate	Michael addition	Yang et al. (2014)105
MRI	gadolinium complex	HA-aldehydeHA-hydrazide	dynamic covalent (Schiff base)	Bermejo-Velasco et al. (2018)139
MRI	GdDOTA nanoparticles	chitosan/HA	ionic cross-linking	Courant et al. (2012)140
MRI	iron oxide nanoparticles	cellulose nanocrystal/silk fibroin	peptide self-assembly	Chen et al. (2018)16
fluorescence	near IR-gelatin	HA-tyramine gelatin	Enzymatic (tyraminadase)	Park et al. (2019)33
fluorescence	bodipy-aldehyde	chitosan	dynamic covalent (Schiff base)	Belali et al. (2017)62
fluorescence	glucose-responsive fluorescent monomer	PEG-bonded polyacrylamide (PAM)	radical polymerization	Heo et al. (2011)141
fluorescence	protoporphyrin	PCL-PEG-PPOR-PEG-PCL	hydrophobic associations	Dong et al. (2016)142
SPECT/CT	Ba2+In3+Zr4+	alginate	ionic cross-linking	Patrick et al. (2020)63
SPECT/CT	DOTA-In3+	amphipathic hexapeptide H-FEFQFKNH2	amphipathic peptide (β sheet)	Oyen et al. (2017)143
SPECT/CT	99mTc	carboxymethyl-cellulose	hydrogen bonding	Lauren et al. (2014)144
SPECT/CT	indium-111 (111In)	polyisocyanopeptide (PIC)	peptide self-assembly	Op’t Veld et al. (2019)145
scintigraphy	131-I	chitosan	ionic cross-linking (TPP)	Kim et al. (2014)35
fluorescence/MRI/ultrasound	rhodamine B and CoFe2O4 nanoparticles	PLGA-PEG-PLGA	amphiphilic block copolymers	Chen et al. (2020)146

^aN/A: not available.

3.1. Intrinsically Imageable Hydrogels.

Hydrogels can display intrinsic contrast in medical imaging without the need for orthogonal functionalization with a probe, such as semiconducting organic polymers that are fluorescent,¹⁴⁷ carbonyl-containing hydrogels that universally present autofluorescence,¹²⁰ and polyaromatic networks that display aggregation-induced emission in optical imaging.¹⁴⁸ Although the monitoring of label-free hydrogels requires sophisticated design to access polymers or small molecules that display high fluorescence quantum yield or near-infrared (NIR) optical properties and to understand complex relationships between changes in signal and hydrogel quantity, this approach holds significant benefits since it does not typically require modification of the hydrogel formulation for imaging. As an example, biocompatible polyester oligomers can be obtained by enzymatic catalyzed transesterification and can be further cross-linked with polytopic polyethyleneglycol acrylates to afford hydrogels displaying intrinsic fluorescent contrast and eventually degrade via ester bond hydrolysis.¹²¹

On the other hand, hydrogels bearing exchangeable protons (e.g., protons involved in a covalent bond with a heteroatom such as hydroxy-, amino-, or amido- functions) can be visualized by chemical exchange saturation transfer (CEST), which is an approach in MRI that requires exchangeable protons that can be saturated and transferred for indirect detection through changes in the water signal.^52,117 For example, pemetrexed (Pem), can be functionalized with a rationally designed peptide and enable its self-assembly by π–π stacking interaction into a filamentous hydrogel that display intrinsic contrast in CEST-MRI.¹¹⁸

3.2. Physical Loading of Contrast Agents.

Contrast agents that are physically loaded into hydrogels are typically retained via hydrogen bonding, coordination bond, ionic, hydrophobic, or other noncovalent interactions, or simply physically restrained in covalently cross-linked hydrogels.^{129,145,149,150} The procedure of physical loading of contrast agents in the hydrogel is either via incubation with preformed hydrogels by passive diffusion through the network or entrapment in the hydrogel during formation. Such a strategy allows a wide range of chemical entities to be loaded into the hydrogel, leveraging the extensive developments in the field of contrast agents with a multitude of probes with excellent properties in terms of strong contrast production, high stability, and chemical diversity. The simplicity of this approach also allows excellent contrast properties for medical imaging without the need for new chemical compositions. Polymers with chelating ability, such as polytopic alginate, can be cross-linked with several cationic metals, e.g., ¹¹¹In and ⁸⁹Zr, that are active in single photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging, affording hydrogels that can be tracked in vivo.⁶³ This approach has been used to monitor oral drug delivery to the stomach in the form of metal cross-linked alginate based oral formulations and as cross-linked hydrogel suspensions for nasal administration of small molecules to facilitate their delivery to the brain. Additionally, some nanoparticles such as gold nanoparticles (AuNP) can be assembled via supramolecular interactions to form a hydrogel with potential for biomedical applications.¹⁵¹ This approach is especially useful as they are well established contrast agents in CT, photoacoustics, and other imaging modalities.^56,152–154 Aggregation is possible for nanoparticle-based contrast agents such as AuNP, which might lead to unwanted retention in tissues and toxicity.¹⁵⁵ To prevent aggregation, capping agents such as thiol-terminated polyethylene glycol (PEG) is commonly used to functionalize the surface of AuNP in order to improve their in vivo stability and to avoid uptake by the reticular endothelial system (RES).¹⁵⁶ Besides, other factors such as hydrogel pH and nanoparticle concentration can also contribute to the possibility of aggregation, and these factors should be taken into consideration when formulating contrast agent loaded hydrogels. Additionally, upconverting nanoparticles (UCNP) and fluorescent dyes (e.g., indocyanine green (ICG) and methylene blue (MB)) are of particular interest in optical imaging. For instance, a chitosan-HA hydrogel cross-linked with β-glycerophosphate and genipin and loaded with LiYF₄:Yb³⁺/Tm³⁺ UCNP can be monitored by optical imaging using NIR excitation.¹⁵⁷ A nanofiber hydrogel formed by self-assembly of the melittin peptide could be loaded by physical mixing with ICG and be detected by both photoacoustic imaging and NIR fluorescence.¹³³ Additionally, m-ferrite nanoparticles, gadolinium chelates, and iron oxide nanoparticles have been physically loaded in various hydrogel formulations and be detected by MRI.^{20,32,158,159}

One drawback of long-term in vivo monitoring of the morphology and degradation of hydrogels that are physically loaded with contrast agents is that the passive diffusion of the agents from the hydrogel may occur. This can result in gradients of the contrast agents from the surface to the center of the hydrogel, leading to possible underestimation of hydrogel volumes. However, multiple reports have addressed this by investigating the contrast agents’ elution from hydrogels in in vitro or ex vivo models and have developed mathematical models to predict this phenomenon in living systems or designed hydrogels to limit the passive diffusion of the contrast agent.^132,148,160 Furthermore, physical loading can allow the same hydrogel to be imaged with different medical imaging modalities, simply by using multiple contrast agents that leverage different imaging modalities. This can be of particular interest when switching from use in superficial to deeply located tissues due to the varying depth penetration of imaging techniques and has been used with a range of contrast agents, including small probes active in fluorescence^112,161 or aggregation induced emission,¹⁴⁸ metal complexes monitored by PET/CT¹⁶² or MRI,²⁰ and nanosized contrast agents including metal nanoparticles,^{16,36,157,163} proteins,¹⁶⁴ or inorganic assemblies.¹³²

3.3. Chemical Grafting of Contrast Agents.

Another strategy for hydrogel visualization in vivo is the chemical grafting of contrast agents to the hydrogel via a covalent bond.^{33,142,160,165–168} The chemical grafting of contrast agents has potential for monitoring both the hydrogel degradation and elimination route, which is valuable for biodegradable hydrogels. One of the factors that is critically important for monitoring hydrogels in vivo is the longevity of the contrast generation from the hydrogels. Unlike loading contrast agents through physical loading, the formation of a high-energy bond (e.g., a covalent bond) renders the leaching of the contrast agent from the hydrogel unlikely, thereby increasing the accuracy of quantifying remaining hydrogel. Furthermore, functionalization with a contrast agent by stimuli-responsive bonds, or so-called dynamic covalent bonds, enables the monitoring of stimuli-triggered degradation or payload delivery.

For instance, mPEG-PLA can be functionalized with 2,3,5-triiodobenzoic acid (TIB) by an ester bond enabling the formation of a thermogellable hydrogel that displayed strong radiopacity in CT.¹⁶⁹ The formation and biodegradation of hydrogel obtained by thermogellation of micelles composed of amphiphilic poly(ε-caprolactone-co-1,4,8-trioxa[4.6]spiro-9-undecanone)-b-poly(ethylene glycol)-b-poly(ε-caprolactone-co-1,4,8-trioxa[4.6]spiro-9-undecanone) (PECT) can be imaged with fluorescence resonance energy transfer (FRET) in real-time.¹⁷⁰

However, fabricating contrast agent functionalized hydrogels through covalent bonds involves sophisticated chemical reactions and purification processes, as opposed to the straightforward approach of physical loading of contrast agents. By covalently introducing contrast agents into the hydrogels, the chemical structure is modified, thereby influencing the properties of hydrogels.¹⁷ For example, covalent linkage of iodine moieties to thermogelling hydrogels to allow tracking with X-ray based imaging could reduce their gelation threshold to below body temperature or create a sol–gel transition that results in phase separation in living systems.^169,171 Thus, efforts have been made to avoid significant effects on their physical gelation properties by finding a specific grafting site on polymers or maintaining a low chemical modification proportion.¹⁷

Overall, we herein described three approaches to access hydrogels that can be visualized and monitored in vivo, including the rational design of intrinsically imageable hydrogels, physical loading of contrast agents by passive diffusion or entrapment in the hydrogel during the gelation process, or functionalization of the hydrogel with contrast agents by covalent bonds. These three strategies each offer different advantages, such as circumventing the need for labeling in the case of intrinsically imageable hydrogels, the ability to load a wide array of payloads in physical loading, or better correlation of signal with residual hydrogel in chemical grafting. Yet, they each suffer from limitations, for example, there is a relatively small number of intrinsically imageable hydrogels, while physical loading decouples signal from hydrogel erosion and chemical grafting has effects on mechanical properties. Hence, the different strategies offer complementary benefits and limitations that must be considered when selecting the best strategy for an individual application.

4. APPLICATIONS OF HYDROGEL IMAGING

There are numerous modalities used for biomedical imaging applications, but only a subset of them are widely used for hydrogel imaging. Considering several parameters, such as penetration depth, image resolution, source of contrast, and the goal of the imaging, the most relevant imaging modalities are largely CT, MRI, fluorescence imaging, and several other less commonly used modalities, such as nuclear imaging, photoacoustic imaging, and ultrasound.¹⁷² These imaging modalities, with the exception of photoacoustics and fluorescence imaging, are extensively utilized as diagnostic tools in clinical settings, and each of them has their own advantages and limitations. For example, CT is much faster and considerably lower cost than MRI, and it allows for accurate detection of calcified structures.^173,174 On the other hand, MRI does not use ionizing radiation and has a much greater range of available soft tissue contrast and higher anatomical resolution.¹⁷⁵ In this section, the principles of several imaging technologies and their hydrogel imaging applications will be reviewed and discussed.

4.1. X-ray Imaging.

4.1.1. X-ray Imaging Principles.

X-ray imaging was the first medical imaging modality, being invented in 1895. Despite its age, it is still by far the most widely used imaging technique and sees ubiquitous clinical use throughout the world. Moreover, it is an accessible, economical, and a widely used research tool, of particular value in imaging bones and joints, which are two of the organs for which there is the greatest interest in using hydrogels for tissue regeneration. Such advantages grant radiopaque hydrogels good potential for future clinical translation, and X-ray imaging of hydrogels is of considerable interest. X-rays are a form of electromagnetic radiation that is typically produced by two mechanisms, namely, Bremsstrahlung radiation and characteristic radiation.^176,177 There are several imaging modalities that use X-rays, including planar X-ray imaging that provides 2-dimensional images of objects such as fractured bones or the gastrointestinal tract;¹⁷⁸ CT which uses rotating X-ray sources to generate 3-dimensional images of anatomy;¹⁷⁹ fluoroscopy that uses mobile X-ray sources to obtain real-time moving images of anatomy;^180,181 SPCCT for material-specific multi-contrast imaging;^182,183 and mammography for breast imaging.¹⁸⁴

Despite the fact that X-rays are used differently in these modalities, the principles of X-ray contrast generation are similar for each. An X-ray beam’s intensity is reduced as it traverses matter, as a result of tissue absorption or scattering of X-rays.¹⁸⁵ However, most soft tissues are difficult to distinguish on the basis of the contrast produced by the tissues themselves, since they are all composed of similar, weakly attenuating elements.^179,186 Meanwhile, hydrogels, with water as their main component and polymers that are usually made up of elements close in atomic number to organic materials, tend to produce contrast that is indistinguishable with soft tissues.¹⁸⁷ As a result, X-ray contrast agents typically need to be embedded in hydrogels in order to differentiate hydrogels from adjacent tissues.

X-ray contrast agents are typically biocompatible, water-soluble, and highly stable in aqueous media, and such properties are beneficial to make them compatible with hydrogels.^154,188 A wide array of X-ray contrast agents have been utilized in X-ray hydrogel imaging, namely, AuNP,^{110,123–125} tantalum oxide microparticles,³¹ platinum nanoparticles (PtNP),¹²⁶ barium sulfate suspensions,¹²⁷ and iodine molecules.^128,189,190 Among these, barium sulfate suspensions and iodinated small molecules are the only contrast agents that are currently FDA-approved, although the other agents are either heavily investigated preclinically or in clinical trials.^41,191 For a hydrogel labeled with one of the FDA-approved agents, an easier translation of the approach to the clinic could be achieved. However, iodinated small molecules may rapidly leave the hydrogel, thus providing only a relatively short period of contrast during which the hydrogel can be monitored. On the other hand, non-FDA approved contrast generating materials such as AuNP will typically remain entrapped in the hydrogel longer, thereby allowing the hydrogel to be monitored for a long duration in preclinical settings. Barium sulfate suspensions, while FDA-approved, are designated for oral use and if administered via injection or surgically may never be cleared from the body, which would be a concern for clinical use.

4.1.2. Radiopaque Markers.

The encapsulation of these contrast agents in hydrogels is critical to the enhancement of hydrogel radiopacification, and numerous studies have demonstrated the use of such radiopaque hydrogels in different biomedical applications. For instance, radiopaque hydrogels have been used as radiopaque markers, known as fiducials, to aid with targeting of local therapy and radiographic localization of tumors and normal tissues.¹⁹² Indeed, a PEG hydrogel containing covalently bound iodine has been approved by the Food and Drug Administration (FDA) for this purpose.¹⁹³ This radiopaque PEG hydrogel was designed to identify the lumpectomy cavity during oncoplastic breast-conserving surgery with a CT scan, thus improving radiotherapy target definition in the lumpectomy cavity.¹⁹⁴ Similarly, it has been repurposed to visualize targets via CT scanning during brachytherapy in gynecologic malignacies¹⁹³ and during minimally invasive thoracic surgery in thoracic malignancies.¹⁹²

4.1.3. Wound Healing.

Alternatively, radiopaque hydrogels can play important roles in wound healing, for instance, to prevent postoperative adhesions (painful internal scarring). Lei et al. developed a thermoreversible injectable PET/polyester hydrogel with X-ray opacity. The system was fabricated based on mixing monomethoxyl poly(ethylene glocol)-poly(d,l-lactic acid-co-glycolic acid) (mPEG-PLGA) deblock copolymer with is 2,3,5-TIB end-capped derivative. The performance on the prevention of postoperative adhesions was evaluated using a rat model with cecum and abdominal defects. The animals underwent sequential CT scans during the week after receiving the treatment of the radiopaque thermoreversible hydrogels, and the hydrogels can be clearly observed in the abdomen and distinguished from the surrounding soft tissues from sectional views of the scans and reconstructed 3-D models at various time points (Figure 2A,B).¹⁷¹

Figure 2.

(A) Transverse CT images of a rat during the week after receiving radiopaque hydrogel treatments. (B) 3D reconstructions of CT images of a rat after treatment with the radiopaque hydrogel at the indicated time points. This figure is reproduced with permission from ref 171. Copyright 2017 Elsevier.

4.1.4. Tissue Engineering.

In addition to wound healing applications, tissue engineering is another field that can benefit from radiopaque hydrogels, whereby effective imaging methods can be used to evaluate new tissue formation and the fate of scaffolds. Hydrogels such as gelatin methacrylate are widely used for tissue regeneration owing to their ability to induce the formation of extracellular matrix. AuNP embedded gelatin hydrogels showed evident osteogenic features and were used to evaluate bone formation in bone regeneration.¹²⁵ More recently, a radiopaque alginate hydrogel formed by substituting calcium ions with barium ions for cross-linking was fabricated, which provided information on the rate of implant degradation and thus showed great potential as tissue engineering constructs.¹⁹⁵

In the case of hydrogel imaging, the low sensitivity of X-ray imaging methods does not usually present a problem, since it is straightforward to load hydrogels with sufficient contrast generating material to render them easily visible compared to soft tissues. In the case of highly attenuating tissues such as bone, new multienergy based X-ray imaging methods offer the ability to distinguish hydrogels that contain payloads such as AuNP via “k-edge imaging”. Indeed, in the context of hydrogel imaging, aspects of X-ray imaging that are sometimes viewed as a weakness, such as the lack of soft tissue contrast, can be a strength, since there should be no uncertainty as to the signal arising from the hydrogel. In comparison, for MRI, which provides a lot of soft tissue contrast, hydrogel administration can lead to scarring, an influx of inflammatory cells, and tissue movement, all of which can lead to contrast that may confound efforts to monitor the hydrogel. An additional strength of X-ray imaging is the linear correlation between signal and concentration, which is not the case for other methods such as fluorescence or MRI.

Another benefit for X-ray imaging is that FDA-approved iodinated contrast agents often have chemical groups that can be used for covalent attachment to hydrogels. Therefore, hydrogels can either be physically loaded or chemically grafted with these agents. Overall, X-ray imaging is an ascendant technique to evaluate the performance and degradation of tissue implants or to assess drug delivery.

4.2. MRI Imaging.

4.2.1. MRI Imaging Principles.

MRI imaging makes use of a strong magnetic field (e.g., 0.2–7 T) in which hydrogen nuclei in water molecules are forced to align. These protons are excited by application of radiofrequency (RF) pulses and return to equilibrium, referred to as relaxation, once the RF signal ceases. The RF signal emitted from the nuclei as they relax is recorded to reconstruct images. Pulse sequences can be developed to focus on longitudinal relaxation(i.e., T₁ relaxation) or transversal relaxation (i.e., T₂ relaxation), which results in T₁-weighted or T₂-weighted images, respectively.^175,196 Unlike CT where tissue contrast mainly depends on electron density, contrast in MRI is a complex function of proton density, T₁ relaxation, T₂ relaxation, and local chemical environment.¹⁹⁷ The majority of MRI contrast agents are either paramagnetic (i.e., gadolinium ion complexes and manganese chelates), which are used for T₁ weighted imaging or superparamagnetic (i.e., iron oxide nanoparticles).¹⁹⁸ On the other hand, CEST imaging, a relatively new MRI contrast approach, enables certain endogenous compounds containing protons exchangeable with surrounding water molecules to be directly detected.^199,200

To render MRI-visible hydrogels, FDA-approved gadolinium chelates can be physically loaded into hydrogels. While there have been reports of covalent grafting of gadolinium chelates, this has to be done with chemically modified gadolinium chelates.¹³⁹ This chemical modification leads to two drawbacks. First, the chelate is no longer an FDA-approved entity, limiting translation. Second, gadolinium chelates are structures that are very carefully engineered to tightly bind gadolinium and prevent the release of this toxic ion. Modification of the structure can compromise the stability of the chelate. Moreover, given the concerns over gadolinium safety, such as nephrogenic systematic fibrosis and gadolinium brain retention, the approval of using a hydrogel in patients that is effectively a long-residing depot of gadolinium will face significant hurdles. Alternatives for MR imaging of hydrogels include labeling with iron oxide nanoparticles and CEST imaging. There are FDA-approved iron oxide nanoparticles, which is a benefit for clinical translation, but only ferumoxytol is still currently on the market. Additionally, there has only been one report to date of labeling hydrogels with this agent which points to challenges in incorporating ferumoxytol into hydrogels.¹³⁰ An additional challenge for labeling hydrogels with iron oxides is that they cause signal loss in MR images. However, there can be many factors that result in signal loss in MRI, such as gas or hemorrhages, therefore a site of contrast cannot be definitively ascribed to the presence of hydrogel. CEST is attractive as a label-free approach for hydrogel imaging, although it is limited to hydrogels that have appropriate exchangeable protons and the contrast yielded by CEST is typically quite modest. Moreover, for all types of approaches to imaging hydrogels with MRI, contrast is not directly correlated to concentration, which is a limitation for quantification and monitoring degradation. Nevertheless, MRI can be used to form images of exceptional quality and the ability to use natural or various FDA-approved materials for CEST MRI is appealing.

4.2.2. Conventional MRI.

One of the earliest efforts to visualize hydrogels using MRI with gadolinium contrast agents was reported by Courant et al.¹⁴⁰ Gadolinium complexes were encapsulated in chitosan and HA-based hydrophilic polymer matrix and exhibited contrast generation in T₁- and T₂-weighted images. Since then, monitoring hydrogel degradation using MRI has been extensively studied. Gadolinium complexes were modified with dithiopyridyl groups and used to label an HA derivative for longitudinal tracking in vivo.¹³⁹ In situ forming hydrogels are also of great interest in tissue regeneration as “MRI reporter gels”, which serve as smart and responsive polymer implants to deliver drug in vivo and are evaluated by MRI over time. A pH sensitive, injectable, and self-healing chitosan-based hydrogel functionalized with gadolinium chelate was able to self-heal within a pH range based on Schiff-base linkages (Figure 3 A). In vivo, the hydrogels were injected into rats subcutaneously and could be detected on T₁-weighted MRI upon injection and at 35 days after injection with declining intensity (Figure 3B,C).³² Similarly, a supramolecular in situ forming ureidopyrimidinone (UPy)-based hydrogel was functionalized with a gadolinium-DOTA complex for monitoring treatments postmyocardial infarction, where sequential measurements of specimens to determine structure, location, and degradation could be also achieved.⁴⁸ Alternatively, iron oxide nanoparticles have been investigated for visualizing hydrogels in tissue engineering and drug delivery as well. For example, ultrasmall superparamagnetic iron oxide nanoparticles (SPIONs) were labeled on cellulose nanocrystal/silk fibroin-blended hydrogel system to monitor hydrogel degradation during cartilage regeneration.¹⁶ The same SPIONs were incorporated into the dehydrodipeptide-based hydrogels. Upon magnetic excitation, the SPIONs were able to generate a significant amount of heat achieving magnetic hyperthermia, which can be used as a remote trigger for drug releases from hydrogels.¹³¹

Figure 3.

(A) Illustration of the formation of the self-healing hydrogel. (B) MRI image of rat after subcutaneous injection of hydrogels. (C) Transverse cross sections of pseudocolored MR images of rat after subcutaneous injection of hydrogels at different time-points. This figure is reproduced with permission from ref 32. Copyright 2013 Royal Society of Chemistry.

4.2.3. CEST MRI.

On the other hand, as mentioned previously, hydrogels can be characterized in vivo using CEST MRI if they have exchangeable protons. CEST imaging has the unique benefit of allowing the evaluation of hydrogels without the need of metal-based contrast agents, which can potentially lead to side effects such as nephrogenic systemic fibrosis.²⁰¹ Fortuitously, there are polymers that are CEST active and also FDA-approved. For example, HA and gelatin-based hydrogels are readily detectable with CEST MRI as they are rich in exchangeable protons, and they are of particular interest due to this reason. Dorsey et al. reported injectable HA hydrogels and illustrated the CEST signal tuning by manipulating hydrogel properties (i.e., macromer concentration) (Figure 4A).¹¹⁹ Subsequently, Shazeeb et al. assessed the degradation profiles and residence time of chemically cross-linked HA hydrogels with CEST MRI in vivo. The CEST signal showed a gradual decrease with time in response to the degradation of the hydrogels (Figure 4B,C), and a loss in CEST contrast indicated degradation, as supported by histology performed at the end of study.⁵³

Figure 4.

(A) Principles of CEST for hydrogel imaging. Hydrogels can be differentiated based on their dominant exchangeable proton groups. (B) CEST maps and corresponding T₂-weighted MRI images from a dithiobis(ethylamine) (DTEA)-cross-linked HA hydrogel over time. (C) Quantification of CEST signal from the corresponding images over different time courses. Panel A is reproduced with permission from ref 119. Copyright 2015 American Chemical Society. Panels B and C are reproduced with permission from ref 53. Copyright 2018 Elsevier.

Furthermore, taking advantage of the presence of an exchangeable aromatic amine proton with a chemical shift of 5.2 ppm in the backbone of pemetrexed (Pem, an FDA-approved chemotherapeutic that is highly hydrophobic) and loading this drug through π–π stacking interactions yielded a filamentous hydrogel that has intrinsic contrast in CEST-MRI (Figure 5A–D).¹¹⁸ This approach enabled a high drug content of 42% for glioma therapy and was used to visualize the injected hydrogel and monitor its degradation. The gradient-driven release of the drug to the surrounding tissues was visible up to 4 days postinjection and could be quantified by CEST-MRI.

Figure 5.

(A) Structure of the pemetrexed-peptide conjugate, which (B) serves as a CEST probe with a chemical shift of 5.2 ppm and (C) self-assembles into a filamentous hydrogel as the concentration increases. (D) Monitoring the diffusion of the hydrogel by MRI (top) and CEST at 5.2 ppm (bottom) before and 2 h or 5 h postinjection in brain tumor. This figure is reproduced with permission from ref 118. Copyright 2017 American Chemical Society.

Thus far, several other studies have taken advantage of CEST MRI for label-free imaging of hydrogels.^118,202,203 This method is particularly appealing as it does not perturb the intrinsic MR properties of native tissues and provides a practical approach to visualize polymer-mediated drug delivery without the use of imaging probes. Overall, MRI has been demonstrated to allow in vivo hydrogel assessment in several settings, and there is the option to use either exogenous contrast agents or intrinsic imageable materials (in certain cases). However, the wide use of MRI for hydrogel imaging is limited by its high cost, long scan times, and need for technical expertise compared to other methods such as X-ray and CT.

4.3. Fluorescence Imaging.

4.3.1. Fluorescence Imaging Principles.

Fluorescence imaging is a type of noninvasive imaging technique that can be used to study a wide variety of molecular entities in both living cells and ex vivo tissue samples, via fluorescent probes. The use of NIR wavelengths (i.e., 650–900 nm) has advantages over visible-range light, including relatively deep photon penetration into tissues, less tissue autofluorescence, and higher optical contrast when exogenous NIR fluorophores are introduced.²⁰⁴ Coupled with advances in detectors and dye technologies, it offers exceptional visual monitoring of life processes with relatively minimal perturbation to biological samples.^56,205,206 To date, clinical applications employing NIR fluorescence are being explored and range from assessing blood flow and detecting sentinel lymph nodes to visualizing tumor lesions, with the help of nonspecific or specific fluorescent agents.²⁰⁷ Fluorescent materials such as quantum dots, small molecule fluorophores, intrinsically fluorescent polymers or proteins that emit light upon excitation can be used in fluorescence imaging.^208,209 Fluorescent hydrogels differ from conventional hydrogels in their light emitting properties but retain their extended polymeric networks. Hydrogels can have fluorescent properties due to the polymer of choice or via physically loaded fluorescent materials.²¹⁰ The drawbacks of fluorescence imaging include the low penetration depth, complex relationships between signal and concentration, and absence of FDA-approved fluorescence imaging systems.

4.3.2. Drug Delivery.

As an example of hydrogels with intrinsic fluorescent contrast, biocompatible polyester oligomers obtained by enzymatic catalyzed transesterification were cross-linked with PEG acrylates due to the intrinsic autofluorescence of sericin polypeptide, with high quantum yields ranging from 16.42% to 36.41%.¹²¹ The hydrogel degraded via ester bond hydrolysis, which could be monitored noninvasively after subcutaneous injection in nude mice via fluorescence imaging under 488 nm excitation and 520 nm emission wavelength. Additionally, this intrinsically imageable hydrogel successfully enabled the delivery of various payloads, highlighting its potential for theranostic and drug delivery applications. As another example in drug delivery, a nanofiber hydrogel formed by the self-assembly of the melittin peptide was loaded with 1% w/w of indocyanine green (ICG), which is an optically active small molecule, without affecting its rheological properties (Figure 6A,B). This hydrogel was detected in vivo by photoacoustic imaging and NIR fluorescence (Figure 6C) and displayed strong tumor growth inhibition activity against glioblastoma by photothermal therapy.¹³³ This strategy has found a broad application in the field of drug delivery, enabling both the coloading of two or more contrast agents and drugs and monitoring the in vivo drug release by the decrease in contrast in the hydrogel over time.^{36,146,211,212}

Figure 6.

(A) Photograph before (left) and after (right) gelation of the ICG loaded melittin-based hydrogel and (B) its ICG release profile. (C) Visualization by photoacoustic imaging of the ICG loaded melittin-based hydrogel (red) and solution of free ICG (green) after intratumoral injection. (D) NIR fluorescence monitoring of the biodistribution of the hydrogel (top) compared to free ICG solution (bottom) at various time points postinjection (5 min, 3 h, 24 h). This figure is reproduced with permission from ref 133. Copyright 2017 American Chemical Society.

4.3.3. Implant Monitoring.

Similar to other imaging modalities as mentioned earlier, fluorescent hydrogels have been explored for monitoring implants and other theranostic applications. Indeed, hydrogels conjugated with upconversion nanoparticles (UCNPs) have been reported for long-term in vivo tracking of the distribution and degradation of hydrogels.⁶¹ Besides their fluorescent properties, UCNPs can be used for photodynamic therapy (PDT) and photothermal therapy (PTT) for cancer, as they can activate surrounding photosensitizer molecules to generate reactive oxygen species and heat to kill tumor cells.²¹³ The combination of UNCPs and hydrogels can therefore not only act as tumor imaging probes but also serve as therapeutic agents. For instance, doxorubicin-loaded gelatin hydrogels containing UCNPs were used for antitumor chemophotothermal therapy and upconversion fluorescence imaging.¹³⁵ Similarly, an injectable silk fibroin nanofiber hydrogel hybrid system was developed for tumor upconversion luminescence imaging and photothermal therapy.¹³⁶ Apart from UCNPs, fluorophores can be used to give hydrogels fluorescent properties as well. Park et al. engineered fluorescent HA and gelatin-based hydrogels by conjugating an 800 nm indocyanine NIR fluorophore ZW800-3a through its carboxylic functional group to the amine groups in gelatin (Figure 7A).³³ They were able to simultaneously monitor scaffold degradation and brain tissue regeneration by imaging the hydrogel using the 800 nm channel and observing brain tissue ingrowth with the 700 nm channel, by use of a 700 nm active brain-specific contrast agent (Figure 7B). Furthermore, others have shown the incorporation of different fluorescent probes in hydrogels for similar purposes in hydrogel tracking, drug delivery, and fluorescence-guided surgery.^122,132,142

Figure 7.

(A) Schematic of the strategy of dual-channel fluorescence imaging for the in vivo assessment of brain tissue ingrowth and hydrogel scaffold degradation. (B) Fluorescent hydrogel and brain tissue targeted contrast agent Ox1 were administered to the animal. Dual-channel imaging was performed 1-h postinjection. Brain tissue ingrowth (red) and hydrogel (green) degradation can be observed in the merged image. This figure is reproduced with permission from ref 33. Copyright 2019 Ivyspring International Publisher.

4.3.4. Biosensors.

Hydrogels themselves also have drawn attention as biosensors as they can respond to external stimuli. For instance, when interacting with target analytes, they can undergo physical changes, which can be monitored by observing the hydrogel deformation.²¹⁴ However, directly observing such changes in vivo is challenging. To this end, fluorescent hydrogels find advantages as biosensing platforms by producing changes in fluorescence signal generated by chemical reactions.²¹⁵ Kim et al. presented a self-assembled, photoluminescent peptide hydrogel consisting of N-fluorenyl-methoxycarbonyl diphenylalanine (Fmoc-FF) for detection of analytes, such as glucose and phenolic compounds, via coencapsulation of enzymes as bioreceptors and quantum dots as fluorescent reporters.¹³⁴ Similarly, glucose-responsive PEG-bonded polyacrylamide (PAM) fluorescent hydrogel fibers were used for long-term in vivo glucose monitoring, and they could continuously respond to blood glucose concentration changes for up to 140 days.¹⁴¹ Selective and rapid fluorescence sensing of iron ions with detection limit of as little as 0.228 ppm could be achieved with a chitosan-based fluorescent hydrogel, which is valuable since iron ions are one of the most important and abundant metal ions in the human body. Additionally, fluorescent hydrogels have also been used to detect microRNA,²¹⁶ bacteria,²¹⁷ proteins,²¹⁸ and heavy metal ions.²¹⁹

4.4. Other Imaging Modalities.

In addition to the imaging modalities mentioned above, there are instances of hydrogels imaged via modalities such as nuclear imaging, photoacoustic imaging, and ultrasound, although this is less common. Nuclear imaging is a specialized form of radiological imaging which uses radioactive materials (i.e., radiotracers) to examine the body. Unlike X-ray or CT imaging that use ionizing radiation from an external source, nuclear imaging relies on ionizing radiation sources inside the body, which are then detected by a gamma camera.²²⁰ By radiolabeling hydrogels or incorporating radiotracers in hydrogels, several biomedical applications can be achieved.¹⁴³ Kim et al. developed a radiolabeled chitosan-based vascular endothelial growth factor (VEGF) delivery system for acute myocardial infarction.³⁵ Stuckey et al. incorporated the radio-metal indium-111 into the alginate hydrogels, enabling in vivo imaging of hydrogel delivery and retention.⁶³ In addition, aiming at the important roles hydrogel dressings play in wound care, Op’t Veld et al. used polyisocyanopeptide (PIC) thermosensitive hydrogel labeled with indium-111 to facilitate and monitor wound healing using SPECT/CT imaging. It was discovered that the hydrogel can stay localized at the site of application (i.e., the wounds and surrounding skin) for at least 7 days and generate a consistent signal (Figure 8).¹⁴⁵

Figure 8.

Left panel (L): overview SPECT/CT scan of mice. SPECT signal from ¹¹¹In in the kidney and bladder is indicated with arrows. Middle panel (M): the different treatment conditions indicated by the dashed circles in the SPECT/CT scans to the right. Right panel (R): representative SPECT/CT images for mice under different treatment conditions. Green arrows indicate that the activities of hydrogels leaked away from the wound area but stayed close to the site of application. This figure is reproduced with permission from ref 145. Copyright 2013 Royal Society of Chemistry.

4.4.1. Nuclear Imaging.

Nuclear imaging of hydrogels provides advantages of high sensitivity and specificity. However, as mentioned above, hydrogel applications often do not need such high sensitivity since they are concentrated depots of material. Moreover, a radiolabeled hydrogel represents significant logistical challenges related to safety, both from the perspective of material preparation and animal handling over a protracted period. In addition, while nuclear imaging is widely used in the clinic, this is mostly with relatively short-lived isotopes. The long-lived isotopes needed for longitudinal hydrogel monitoring, would present a risk to the health of a patient due to lengthy, continuous radiation exposure. These factors may explain the relative unpopularity of nuclear methods for hydrogel imaging. However, for applications where sensitivity is needed or imaging at only short postadministration is desired, nuclear imaging may be an appealing option.

4.4.2. Ultrasound.

Ultrasound is a diagnostic medical imaging method that uses high-frequency sound waves to produce dynamic images of tissues and blood flow inside the body.²²¹ Ultrasound is used for many applications and is well-known for its use in viewing the fetus during pregnancy. Such an application is attractive as it does not require the use of radiation like CT.²²² Ultrasound also has advantages of low cost, good soft tissue contrast, and portability but has drawbacks of requiring sophisticated training for both operation and image interpretation and providing only localized images, as opposed to the whole body imaging of CT or nuclear methods. The emergence of contrast-enhanced ultrasound (CEUS) has broadened its use, especially in cardiac and abdominal imaging as well as evaluation in tissues, tumors, and implants, when combined with ultrasound contrast agents (i.e., microbubbles).^223–225 Imaging of hydrogels can therefore be achieved for different theragnostic purposes. For example, Leng et al. used CEUS to characterize the biodegradation and neovascularization of silk protein hydrogel implants in rats. Both ultrasound and CEUS imaging revealed the change of shape and size of hydrogels over time (Figure 9).³⁴ In another application, alginate-based hydrogels displayed the ability to self-heal damage triggered by applying ultrasound pulses to disrupt ionically cross-linked hydrogels, enabling on-demand delivery of mitoxantrone for breast cancer therapy. Hydrogels were implanted near breast tumors and showed effective inhibition of tumor growth in vivo.²²⁶ Similar ultrasound-mediated self-healing hydrogels were reported elsewhere as well.⁶⁴

Figure 9.

(A) 2D greyscale ultrasound images of rat thigh with silk hydrogel implants. The echogenicity increased over time (a–f). (B) CEUS imaging of the hydrogel implants at different time points (a–e). More microbubbles infused into the gel matrix over time, indicating the progression of neovascularization. Red arrows indicate the outline of hydrogel implants. This figure is reproduced with permission from ref 34. Copyright 2015 John Wiley and Sons.

4.4.3. Photoacoustic Imaging.

Photoacoustic imaging, or optoacoustic imaging is a hybrid imaging technique based on the photoacoustic effect, where pulsed laser light is applied to the subject, which results in conversion of absorbed optical energy into acoustic energy. With typical optical imaging, the light diffusion limits the spatial resolution in deep-tissue imaging. However, since acoustic waves scatter much less than optical waves in tissue, photoacoustic imaging can generate high-resolution images in both the optically ballistic and diffusive regimen and provide greater tissue differentiation.^227,228 Although this imaging technique has scant availability in clinical practice, it shows great potential in preclinical research. Loading hydrogels with photoacoustically active materials allows their detection with photoacoustic imaging. For example, Cheng et al. reported a pH-responsive chitosan hydrogel encapsulating Prussian blue for photoacoustic imaging-guided photothermal therapy of tumors.¹³⁷ The precursors to Prussian blue, namely, ferrous and ferricyanide ions were released in the acidic tumor environment from the hydrogel, allowing the in situ formation of Prussian blue in the tumor area, which serves as a photoacoustic contrast agent and a photothermal ablation agent at the same time.

4.4.4. Multimodality Imaging.

Lastly, hydrogel monitoring can be achieved by multimodality imaging, which can help to overcome the limitations of separate techniques as each imaging modality has its own unique strength and intrinsic limitations. For example, a multifunctional hydrogel based on an engineered polypeptide and loaded with Ag₂S quantum dots and paclitaxel was developed for fluorescence/photoacoustic imaging.³⁶ Alternatively, a thermosensitive magnetic nanoemulsion hydrogel was used for MRI and fluorescence imaging guided thermoablative cancer therapy, while the injection was monitored by ultrasound.¹³⁸ In addition, the degradation and biomaterial–tissue-interaction of gelatin hydrogels were investigated in vivo with MRI, optical imaging, and PET.²²⁹ Multimodality imaging expands the scope of information that can be collected regarding hydrogel localization, retention, degradation, and activity. For example, CT and MRI offers superior structural imaging, whereas other imaging modalities such as PET and optical imaging could provide information at the molecular level, so that when they are used together, more detailed anatomical and biological information can be obtained.^230,231 One of the most commonly used combinations are PET/CT scan and SPECT/CT scan, which not only shows anatomic details but also images biochemical or physiological phenomena.^{63,143,144,232} Besides, optical imaging usually has poor tissue penetration, but this problem can be mitigated when combined with MRI, CT, or PET imaging, which do not have a tissue penetration limit.²³³ Overall, ideally the goal of any multimodal imaging is to provide the localization, extent, and metabolic activity of target tissue, and such a goal applies to hydrogel imaging as well.

5. SUMMARY AND FUTURE OUTLOOK

In the current review, we have highlighted various imaging techniques that can assess and monitor the performance of hydrogels after implantation or injection. Hydrogels can be rendered imageable by using intrinsically imageable polymers, physical loading of contrast agents, or functionalizing with contrast agents by covalent or coordination bonds. Using noninvasive imaging modalities to image hydrogels has offered opportunities to allow for direct access to information about their status in vivo and enabled study of the release of therapeutics or encapsulated cell migrations. At present, the mainstream techniques used for imaging hydrogels include CT, MRI, fluorescence imaging, nuclear imaging, ultrasound, or the combination of two or more modalities, and their applications can range from monitoring drug delivery, tissue regeneration, and imaging guided surgery to cancer therapy. Unlike traditional post-mortem assessment of implanted hydrogels with histological methods, in vivo imaging allows for longitudinal study of hydrogels without unnecessary animal sacrifices at different points in time, and, in the meantime, offer opportunities for combined imaging and therapy, namely, theranostics. Combining imaging diagnostic and therapeutic capabilities into a single platform can be beneficial to develop more individualized and specific therapies.

Currently, the majority of imaged hydrogels are coloaded with therapeutic agents and contrast generating materials. The resulting hydrogel systems that are capable of diagnosis, drug delivery and monitoring of therapeutic response have been previously well investigated. However, this has the disadvantage that the imaging agent is a proxy for the therapeutic and matching properties between the two is challenging. In comparison, there have so far been few hydrogel systems that are developed where the contrast generating material and the therapeutic are the same entity. Many contrast generating materials, especially nanoparticle-based agents, have inherent therapeutic properties, such as radiosensitization or photothermal heating. Moreover, nanoparticles can be readily converted to be theranostic agents by attaching therapeutic moieties to them, due to their tunable surface chemistry. For instance, therapeutics of various forms such as small molecules, proteins, can be conveniently tethered into nanoparticle-based contrast agents.²³⁴ The formation of all-in-one contrast agent-hydrogel theranostic platforms will likely be a greater focus of investigation in the coming years.

Taking the above concept one step further, if the intention is to image the hydrogel, in an ideal world, the imaging would provide information on the hydrogel structure and the therapeutic entity. This might point to developing hydrogels where the substance that both generates contrast and provides therapeutic effects is also part of the hydrogel structure itself. For example, a gold nanoparticle could provide both contrast and therapy and be engineered into the polymer backbone or be the cross-linker. Such a hydrogel has rarely been reported and would require quite specific chemistry for the theranostic agent; however, it would enable precise information to be gathered in it in vivo.

The fact that imageable hydrogels are usually heavily dependent on contrast agents can be a limitation, as only a relatively small subset of contrast agents are clinically approved and available for use for each imaging modality. For instance, only iodinated small molecules and barium sulfate suspensions are approved and available for CT and gadolinium-chelated contrast agents for MRI. Therefore, ideally using clinically approved contrast agents in hydrogel formulation can bolster the clinical translation of these hydrogels. Similar to contrast agents, there is a limit number of clinically approved hydrogel products for each biomedical application. Currently there are over 30 injectable hydrogel-based products that have been approved by the FDA, and most of them are used in intradermal injections.²³⁵ Thus, the most ideal imageable hydrogel formulation would consist of FDA-approved contrast agents and hydrogels, so that they have a higher chance to be approved and translated into clinics. Nevertheless, carefully designed systems composed either entirely or in part of unapproved materials may also progress toward clinical translation.

Overall, detecting and monitoring hydrogels in vivo using imaging techniques provides important information on implantation functional status and is of relevance to numerous clinical applications. We expect to see this field grow and become more routine in the coming years to allow systematic investigations of hydrogels in vivo as well as in patients, which will promote future clinical translation of these therapeutic systems.