Engineered Hydrogels in Cancer Therapy and Diagnosis

Review

Engineered Hydrogels in Cancer Therapy and Diagnosis Mohammadmajid Sepantafar,1 Reihan Maheronnaghsh,2 Hossein Mohammadi,3 Fatemeh Radmanesh,4 Mohammad Mahdi Hasani-sadrabadi,5 Marzieh Ebrahimi,4 and Hossein Baharvand4,6,* Over the last decade, numerous investigations have attempted to clarify the intricacies of tumor development to propose effective approaches for cancer treatment. Thanks to the unique properties of hydrogels, researchers have made significant progress in tumor model reconstruction, tumor diagnosis, and associated therapies. Notably, hydrogel-based systems can be adjusted to respond to cancer-specific hallmarks and/or external stimuli. These wellknown drug reservoirs can be used as smart carriers for multiple cargos, including both naked and nanoparticle-encapsulated chemotherapeutics, genes, and radioisotopes. Recent works have attempted to specialize hydrogels for cancer research; we comprehensively review this topic for the first time, synthesizing past results and defining paths for future work.

The State of Hydrogels in Cancer Research Cancer is a worldwide health problem that reduces the quality of life and life expectancy. The two main characteristics of cancer development are abnormal cell proliferation and metastasis, which leads to cancer recurrence and is considered as the most important reason for mortality [1,2]. The dynamic cancer microenvironment causes changes in extracellular matrix (ECM) (see Glossary) properties over a long period (Box 1). However, exact cancer progression mechanisms and malignant cell responses to inhibitors are still not clear for physicians. In recent years, numerous hydrogel-based reconstructive tumor models have been introduced to be used for cancer investigations. The findings indicate that researchers are close to designing a comprehensive tumor model. Their common point is that these models mostly benefit from unique properties of the hydrogels to mimic the dynamic tumor ECM [3–5]. Furthermore, cancer drug delivery systems (DDSs) have extensively tended toward hydrogel-based approaches (Box 2). Hydrogel-based DDSs have been shown to improve chemotherapy results and gene therapy efficacy by increasing drug half-life, facilitating controlled and adjustable drug release, and subsequently decreasing nontargeted exposure. Hydrogels give researchers a wide range of possibilities to optimize cancer DDSs [6,7]. In addition, extensive research has been performed on tumor imaging, permanent tissue replacement, and temporary prosthesis – areas in which hydrogels play key roles [8,9]. Considerable efforts have been made to adjust the physical and chemical properties of hydrogels according to the requirements for cancer research. Nonetheless, there is a great need for a comprehensive focus on

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http://dx.doi.org/10.1016/j.tibtech.2017.06.015

Trends Cancer is a complicated disease that necessitates high-throughput research. Animal models are not adaptable for cancer progression in humans. However, hydrogel-based reconstructive models can properly simulate the tumor microenvironment for cancer research. Smart hydrogel-based carriers and drug reservoirs could overcome the weaknesses of conventional therapy methods and provide targeted, localized, and adjusted delivery systems for genes, drugs, and radioisotopes. Hydrogels provide correlative and/or complementary combined therapies, such as chemotherapy and hyperthermia, chemotherapy and radiotherapy, and chemotherapy and gene therapy. Tumor imaging via a long-term modified nanomagnet-loaded hydrogel has demonstrated a reliable biodegradable imaging platform for tumor imaging and anticancer drug screening.

1 Department of Cell Engineering, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran 2 Department of Genetics, Tehran Medical Sciences Branch, Islamic Azad University, Tehran, Iran 3 School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia, Engineering Campus, 14300 Nibong Tebal, Penang, Malaysia

Box 1. Alterations in the Cancer Microenvironment In brief, changes in the levels of structural components, hormones, and proteins, and subsequently temporal changes in stiffness and rigidity, stabilize and activate hypoxia-inducible factors. These in turn promote intratumoral oxygen deprivation (hypoxia) and regulate matrix metalloproteinase expression, which causes ECM degradation, and in turn changes the ECM architecture. These are just a few of the alterations that occur in cancer progression [10–13].

this matter, comparing the achievements, distinguishing the advantages and disadvantages, and elucidating what is still needed to brighten the way for the future. This review investigates various aspects of hydrogel-based systems for cancer therapy and diagnosis. First, we briefly explain the currently available cancer therapies, their insufficiency, and the need to improve them. Then, we outline a few key features of hydrogels. Finally, by taking into consideration the unique characteristics of hydrogels, we offer some suggestions that we believe could be beneficial. Next, we focus on the application of hydrogels to improve cancer DDSs, and we describe the vital role of hydrogels in tumor microenvironment (TME) reconstruction and the application of hydrogels as tissue substitutes. Finally, we explain how hydrogels enable current imaging approaches to diagnose solid tumors. We attempt to establish a strong conceptual structure that covers potential opportunities to design useful hydrogel-based platforms for future research.

4 Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran 5 Parker H. Petit Institute for Bioengineering and Bioscience, G.W. Woodruff School of Mechanical Engineering and School of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, GA, USA 6 Department of Developmental Biology, University of Science and Culture, Tehran, Iran

*Correspondence: [email protected] (H. Baharvand).

Hydrogel-Based Cancer Drug Delivery Systems Delivering therapeutic agents to cancer tissues is a significantly challenging issue due to both the complex and dynamic TME and the instability and off-target effect of associated drugs (Box 3). Therefore, delivery systems need to be controlled and targeted [30,31]. Researchers have previously recommended incorporating genes and drugs in carriers to decrease unwanted toxicity, improve hydrophilicity, prolong duration of action, protect drug bioactivity, and control the rate and zone specificity of drug release. Interestingly, localized and targeted DDSs appear to be reliable solutions [6,32]. Although past research has reported the inefficiency of systemic pharmaceutical therapies and the need for an advanced DDS, the development of microsphere and nanoparticle-based systems as cancer treatments has progressed slowly. One of the most important challenges is the initial burst release of these systems, which frequently jeopardizes their efficiency. This initial burst release likely delays relapse rather than removing the tumor [33]. Another challenge is that although passive targeting of nanoparticulate delivery systems by the enhanced permeability and retention effect causes penetration and accumulation of nanoparticles into tumor tissues, it inefficiently increases in vivo drug accumulation at the tumor site. In addition, the enhanced permeability and retention effect strongly depends on the vascular permeability of the tumor. This effect is overestimated in clinical tumor therapy because of tumor heterogeneity and tumor tissue barriers, such as high cell density [34–36]. In addition, numerous limitations exist for clinical applications of some active targeting drugs because of their rapid elimination by the reticuloendothelial system and high interstitial fluid pressure in tumors [37]. Therefore, nanoformulations have limited capacity to locally affect cells for an extended time because they undergo rapid elimination from the desired sites due to their small size [38]. It is also possible that nanoparticles nonspecifically interact with normal cells. Therefore, the fraction of nanoparticles that penetrate the tumor cells may be much less than 0.7% of the applied nanoparticles [39]. Hydrogels are attractive for localized and targeted therapy in contrast to active and passive targeting due to their sustained or pulsed localized administration, regardless of the blood supply and microvasculature morphology of the tumor [7,27]. They have significantly improved

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RNAi delivery systems, resulting in better clinical outcomes [38,40]. Localized presentation of siRNA to nonmalignant tumors may result in a reduced functional dose and potential off-target effects. Furthermore, sustained release of siRNA may cause a long-term silencing effect [38]. A hydrogel-based system could extend the physical stability of chemotherapeutic agents without precipitation for some months [41]. Cancer cell vaccination is another hot topic, in which the excellent properties of hydrogels, particularly cryogels, have been recently demonstrated. For example, one study described a resilient vaccine platform with interconnected macroporous architecture to increase cellular infiltration. Interestingly, this system could easily be injected through a surgical needle [42]. Hydrogels are also capable of delivering two or more therapeutic agents concurrently. A single drug might not be able to efficiently kill all cancer cells due to the heterogenic nature of tumors and presence of malignant cells at diverse cell divisions or growth stages [43]. Concurrent delivery of multiple therapeutic agents with different molecular targets could be a promising strategy to overcome drug resistance and reduce the chance of tumor metastasis [44]. This approach could effectively improve treatment outcome and decrease adverse effects related to the administration of a highly toxic drug [43]. Recently, investigators observed a marked synergistic effect of lapatinib and paclitaxel after a peritumoral injection that resulted in a significantly steady accumulation of lapatinib in the tumors with an acceptable level of safety. They incorporated paclitaxel nanoparticles and lapatinib microparticles into a thermosensitive hydrogel to generate a localized codelivery system [45]. Combination therapy is specifically valuable for DNA or RNAi–chemotherapeutic drug combined delivery (Figure 1). RNAi–chemotherapeutic drug combinations can effectively overcome tumor angiogenesis and tumor resistance to chemotherapeutic agents by inhibiting multidrug resistance induced by efflux and non-efflux pumps [44]. In an interesting study, a poly (N-isopropylacrylamide-co-acrylamide) hydrogel with near-infrared absorbing silica–gold nanoshells successfully carried concurrent DOX and DNA [46]. In another study, protein kinase B (AKT)-targeted gene therapy along with conventional paclitaxel given as an injectable linoleic acid-coupled pluronic hydrogel showed possible synergistic anticancer effects by downregulation of AKT signaling and facilitation of apoptosis induction [30]. In addition, hydrogels can be effectively used to deliver radioisotope and chemotherapeutic agents for cancer treatment. For example, one group of researchers successfully codelivered a radioisotope with chemotherapeutic agents to cancer tissues using a single vehicle. They designed a macroscale thermosensitive injectable micellar hydrogel that could serve as a chemo-radioisotope drug reservoir. This hydrogel was internalized by tumor cells with rapid intracellular DOX release and immobilized radioisotopes at the internal irradiation hot focus. A peritumoral injection of this system significantly inhibited tumor growth with minimal adverse effects [47]. Therapeutic applications of combined drugs with synergistic effects require concurrent, separate encapsulation of multiple cargos in a single vehicle. One group employed a cooperative and incompatible assembly of PEGylated hydrocarbon nanoparticles with PEGylated fluorocarbon nanoparticles in an attempt to simultaneously, but separately, encapsulate waterinsoluble doxorubicin and paclitaxel into different compartments of the hydrogel [43]. Another proposed approach included nanoparticles to control diffusive release of cargo from the hydrogel and enhance transfer efficiency. Researchers introduced an implantable dextran dendrimer hydrogel carrier loaded with a gold nanoparticle base for combined delivery of miRNAs and cisplatin. Localized, selective, and sustained delivery of gene and drug in this delivery vehicle markedly shrank the primary tumor and suppressed metastasis in a mouse model of breast cancer [48].

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Glossary Active targeting: in this approach, various targeting ligands, such as peptides, antibodies, aptamers (oligonucleotide or peptide-based molecules that can bind to their target with high affinity and specificity), and other small molecules (e.g., folic acid) are conjugated to nanocarriers. These ligands bind to overexpressed receptors on the surface of their target cells, leading to enhanced intracellular uptake of nanocarriers by receptor-mediated endocytosis. Chemotherapy: cancer therapy using cytotoxic chemical substances. It has been applied to decrease the tumor burden, achieve prolonged disease control, and inhibit tumor recurrence. Coacervate: a spherical aggregate of colloidal viscous lipid molecules that are held together by hydrophobic forces. Cryogel: a gel matrix that is formed in frozen monomeric solutions and polymeric precursors. It possesses interconnected macropores that allow the unhindered mass transport of nanoparticles and microparticles. Doxorubicin: an anticancer chemotherapy drug that is administrated by injection into a vein. It interacts with DNA by intercalating between base pairs in the DNA helix, thereby preventing the process of DNA replication, and finally inhibiting protein synthesis. Drug delivery systems (DDSs): engineered pharmaceutical systems that are designed to transport required doses of therapeutic compounds to the targeted tissues in proper periods. Extracellular matrix (ECM): the collection of protein molecules that is secreted by cells to structurally and biochemically support the surrounding cells. Gene therapy: the insertion of nucleic acid constructs that play key roles in regulating gene expression inside the targeted cells to replace a mutated gene, silence a mutated gene, or directly kill malignant cells. Hyperthermia: the procedure of heating cancer tissue up to 40–45 C to increase tumor blood flow and improve therapeutic efficacy. Interstitial fluid: also called tissue fluid, the main component of the extracellular fluid, which is a waterlike solvent found in the tissue spaces

Poor oxygen and drug availability in inner cancer area leads to poor therapeutic outcomes. A promising approach to overcome this problem is hyperthermia, in which the tumor site is heated up to 41–45 C. Hyperthermia is usually employed in combination with chemotherapy and radiotherapy to improve cytotoxic effects by acidosis, energy deprivation, and increased tumor blood flow at the tumor site to enhance therapy efficacy. Although the results of hyperthermia are promising, damage to surrounding tissue is still challenging [49]. To make magnetic hyperthermia effective, it is necessary to elevate the concentrations of nanoparticles. These high concentrations are a major disadvantage that preclude its clinical use due to toxicity [50]. However, hydrogel nanocomposites that can be compressed into the tumor site provide local heating. This system can successfully overcome low chemical stability and inadequate biocompatibility of the naked metallic nanoparticle. The iron oxide content of the hydrogel and alternation in the applied magnetic field can control the system’s temperature. An advantage of this system is its potential for being a dual therapeutic system that carries chemotherapeutic agents [49]. Another group designed a thermally sensitive micelle composed of an elastin-like polypeptide (ELP) labeled with the radionuclide (radioisotope) 131I, which is formed and stabilized by two independent mechanisms. Initially, body heat triggers radioactive ELP micelles to rapidly develop a phase transition into an insoluble, viscous coacervate; then, elevated temperatures lead to cell death and destruction of blood microvasculature. These injectable hydrogels have been used to treat two aggressive tumor models. The ELP depots retained greater than 52% (in prostate tumor) and 70% (in pancreatic tumor) of their radioactivity over a period of 60 days with no appreciable radioactive accumulation in off-target tissues after 72 h [51]. In addition, metallic nanoparticles could be modified to more intelligently target the tumor cells [20]. Functionalizing hydrogels with tumor targeting ligands, and nanodevices that are sensitive to various tumor characteristics is another potentially smart platform designed for localized cancer treatment. For example, the folate receptor is a glycosylphosphatidylinositol receptor that upregulates in most cancer cell types compared with healthy cells. Folate-conjugated nanogels can target overexpressed receptors on the cancer cell surface to enhance intracellular uptake of nanocarriers by receptor-mediated endocytosis [52]. Another example is phosphoglycoprotein multidrug resistance protein 1 (MRP1), which is associated with resistance to a broad spectrum of anticancer drugs and belongs to the ATP-binding cassette of proteins. MRP1 is a cell surface energy-dependent efflux pump involved in the redox regulation of multidrug resistance by reducing the intracellular concentration of chemotherapeutic drugs. Recently, an anti-MRP1 system was introduced to overcome multidrug resistance. It employed dark-gold nanoparticles modified with nanobeacons as delivery vehicles nanodevices via an implantable hydrogel that was responsive to increased MRP1 expression levels. In this system, conformational reorganization of nanobeacons triggered the drug release [53]. The continuity or discontinuity of the delivery mechanism is another important question. Cancer cells have reportedly shown greater sensitivity to a short-term high dose of chemotherapeutic agents compared with sustained constant doses over extended periods. One study used an injectable alginate hydrogel that had the ability to self-heal damage induced by ultrasonic pulses to provide on-demand localized mitoxantrone delivery. This system could be externally controlled for dose and timing. Both in vitro and in vivo investigations indicated that a controllable short-term drug burst release might increase breast cancer cell mortality. According to the results of this study, daily drug release induced by external stimulation significantly decreased tumor growth compared with sustained drug release [27].

Reconstructing the Cancer Microenvironment for Basic Research In vivo investigations of tumorigenesis are problematic due to the limited numbers of available models and technical challenges related to the detection of metastasis. An animal model

and provides nutrients for the cells and removes their waste products. Micelle: the aggregation of molecules in a colloidal solution. Microfluidics: handling and controlling laminar fluids in networks of microchannels. Mitoxantrone: a drug used to treat some types of cancer and multiple sclerosis. It is an intercalating drug that disrupts the process of DNA synthesis and repair by inhibiting an enzyme called topoisomerase. Multidrug resistance: a phenomenon whereby cancer cells gain the capacity to develop crossresistance and survive a variety of structurally and functionally unrelated drugs. Organoid: a cell culture approach that employs different cell types to simulate heterogeneous microtissues and make more realistic spheroid models. Organ on a chip: a multichannel 3D microfluidic chip that can simplify and simulate an organ or tissue microenvironment to high-throughput screening. Paclitaxel: an anticancer chemotherapy drug that is administrated by injection into a vein. It is a tubulin-targeting drug that blocks the progression of mitosis process and cell division by its binding to beta-tubulin subunits of microtubules and stabilizing and preventing them from disassembly. Passive targeting: an enhanced permeability and retention effectmediated approach used as a target delivery system to transfer chemicals (i.e., drugs) to some pathological sites, such as tumor tissues and infarcted areas. Enhanced permeability and retention is a unique phenomenon of solid tumors by which various nanosized particles are transported, accumulated, and retained preferentially into tumor tissue due to the enhanced permeability of tumor vessels and reduced lymphatic draining. PEGylation: the technique of covalently bonding PEG to a given molecule to increase circulation time without affecting activity. Radiotherapy: applying ionizing radiation to affect genetic material to stop cells from growing and dividing in the exposed area. This approach can kill different tumor cells that are present in the exposed site. In addition, it can be used in combination with other cancer

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cannot entirely simulate human cancer due to the complexity of human cancer metastasis [58,59]. By contrast, numerous important in vivo tumor characteristics cannot be sufficiently recapitulated by 2D conventional culture models [9,60]. In 2D culture models, adherent cells form tight focal adhesions between the cells and plastic, in contrast to the poor adhesion that closely imitates cancer cell interactions [24]. Because this flat morphology affects cellular behaviors, it decreases the validity of 2D in vitro investigations. Here, adherent cells possess a sheetlike morphology on stiff and planar plastic surfaces, which influences their cell signaling, gene expression, and drug sensitivity [61]. Therefore, bioengineering of the TME is beneficial, and hydrogel materials play a role in this endeavor [4]. Previous studies have confirmed that cell behavior is more realistic in 3D cell cultures. In these systems, cancer cells undergo more realistic proliferation compared with 2D cultures. More importantly, the interactions between different cell types can be explored in 3D cultures [9,60]. Several 3D culture models have been designed to suitably imitate various aspects of human cancer progression [9] and drug screening [62]. Bioengineered 3D hydrogel models that study cell–cell and cell–drug interactions can provide a link between in vitro and in vivo systems [60,63]. They resemble the special characteristics of the TME well, including stiffness and ductility modulations, spatial heterogeneity, signaling responsive ability, programmed degradability, cancer-specific biomarker responsibility, and other properties; none of the other platforms reproduce all of these concurrently. These features make in vitro tumor remodeling realistic. The architecture, stiffness, and composition of the ECM could affect cell migration, invasion, and proliferation in human cancers as well as their sensitivity to anticancer drugs. Engineered models are powerful tools used to investigate the influence of cancer cell cocultures and ECM progressions. One example is a 3D matrix of reticulate hyaluronic acid (HA) hydrogel alone and with various coatings of collagen. Cancer cells that were noninvasive in the HA hydrogel became invasive in the simultaneous presence of collagen types I and II [64]. This finding is valuable for designing models that investigate tumor-associated chemotactic potentials. Another example is heparan sulfate proteoglycans (HSPGs), which are recognized as critical crossroads within the metastatic cascade. They interact with the structural protein of the ECM, act as intermediates in cell adhesion, and control factors related to growth and motility. Their function can change according to their location, such that sometimes they play roles as promoters and other times as inhibitors. The location of proteoglycans and signaling molecules that are bound to heparan sulfate determines the role of HSPG in the regulation of metastasis. Thus, research on the matrix to adjust the signaling factors would be valuable. In tumors, HSPGs bind to C-X-C motif chemokine 12 (CXCL12, also known as SDF1) and create a chemotactic gradient that directs cell migration. Taking this phenomenon into consideration, a patterning system was developed to spatially pattern cells within the ECM and investigate cancer cell migration. Matrigel has been shown to provide binding sites for CXCL12 factors, as well as significantly improve and direct cancer cell migration [65]. Recently, researchers reconstructed a tumor model that had controllable parameters such as the composition and density of ECM. Tumor invasion could be regulated through specific integrin binding sites that switched on mechanotransduction pathways. They identified different mechanisms of migration among cancer cell lines independent of ECM density or composition [66]. If the purpose is to deeply evaluate tumorogenesis in a specific tissue, reconstructing a comprehensive model specific to the native tissue is critical. For instance, the ECM of breast tissue is known to have a complex structure containing interwoven protein fibrils inside a network of glycosaminoglycan carbohydrate chains. The structural protein constituents, such

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treatments. Some drugs, called radiosensitizers, make the cells more sensitive to radiation. Radiotherapy may be applied either externally or internally, for both curative and palliative treatments. Reconstructive tumor model: engineered 3D tumor models to recapitulate the native cancer microenvironment to study cancer progression and drug screening. Reticuloendothelial system: a part of the immune system. It consists of phagocytic cells, including primary monocytes and macrophages, distributed in different tissues, such as the liver, spleen, lymph nodes, lungs, and central nervous system. It removes damaged and senescent erythrocytes and leukocytes by phagocytosis and digestion. It also plays an important role in defense against different foreign microorganisms. Spheroid: a structure that employs individual cells to model a microtissue. Cells are coated on plastic dishes without being allowed to form 2D adhesion foci. Consequently, cells grow away from the surface and form a spherical balllike cell mass.

as laminin, fibronectin, and collagens, resist tensile forces, whereas the carbohydrates (primarily consisting of hyaluronan chains, which chelate water) resist compressive forces. One recently engineered hydrogel platform included both the carbohydrate components (hyaluronan) and protein (collagen, laminins, and fibronectin) of human breast tissue. The ECM-based platform has two times as much elasticity than collagen gels alone, probably because the additional ECM components partially disrupt the rigidity of the collagen network, thereby resulting in a more elastic hydrogel that more closely approximates the elasticity of breast tissue in vivo [12]. Although mechanics alone do not control malignant cell migration, a combination of integrated cell adhesion to hydrogels and manipulated mechanics could have an effect [67]. Research has shown that HA-based hydrogel particles may provide a mechanically and biologically relevant

Box 2. Hydrogels: Valuable Biomaterials The introduction of hydrogels in 1954 [14] has led to the growth of hydrogel-based technologies in numerous fields. Hydrogels are 3D networks composed of hydrophilic polymers that absorb large amounts of water. They remain insoluble in aqueous solutions because of their chemical or physical crosslinking polymer chains [15–17]. There are various gelation mechanisms for hydrogels, which enable researchers to flexibly design their experiments (for a review, see [18]). Currently, hydrogels are one of the most appealing polymeric systems for biomaterial development because they are responsive to stimuli and possess adjustable properties. They have a reasonable similarity to soft tissues due to their high water content. Therefore, they can potentially be a suitable substitute for the ECM. Hydrogels have been used as 3D reconstructive models to investigate cellular responses to changes in the tumor microenvironment. The application of such artificial ECMs, particularly with well-defined physicochemical properties, has enabled researchers to gain insight into the crosstalk between cells, microenvironmental components, and drug influences (Figure I) [3,5]. Hydrogels are also outstanding candidates to encapsulate biomacromolecules that control the therapeutic agent release rate by making systematic changes in their physical and chemical structure. Under certain conditions, surface or bulk erosion of hydrogels can control the rate of drug release [18,19]. A hydrogel-based DDS can improve the penetration of therapeutic agents into tumor tissues by both localized and targeted deliveries. This system can also overcome the low permeability of hydrophobic therapeutic agents [6,20]. Anticancer drugs can be encapsulated in hydrogel matrices to overcome delivery complications, inhibit their denaturation, and increase their half-life and effectiveness at the tumor site (Figure II) [7,21]. There are two classes of hydrogels: synthetic and natural. Naturally based hydrogels can play valuable biological roles to retain cell viability and, in turn, enhance specific cellular functions. Disadvantages of naturally based hydrogels include batch-to-batch variations and uncontrolled biodegradation, which lead to difficulties in controlling their mechanical properties [18]. By contrast, synthetic polymers usually have well-defined structures [18,19]. They are highly reproducible, which allows for incorporation of tailored functions [22], biological molecules [10], or inorganic nanometer-scale objects [23]. Although synthetic polymers do not provide extracellular cues and ligands like the native ECM [24], protease cleavage and integrin cell-binding sites can be chemically incorporated to overcome this drawback [25]. In addition, the reversible behavior of hydrogels is a key factor. Hydrogels can be sensitive to environmental stimuli such as pH, drugs, antigens, peptides, proteins, and enzymes along with external stimuli such as temperature, light, and electric, magnetic and electromagnetic fields. The system can be adjusted in such a way that a hydrogel maintains its expected behavior when facing some of the aforementioned stimuli to intelligently manage a therapeutic procedure [18,26,27]. For example, in thermosensitive cases, aqueous polymeric solutions are liquid at room temperature and form a firm pliable hydrogel after injection [28]. Similarly, tumor-associated enzymes are hallmarks that can be exploited as triggers to control the release of encapsulated chemotherapeutic drugs [29]. Tumor-sensitive systems that respond to invasion, angiogenesis, and recurrence can be used to activate and/or release therapeutic agents. Matrix metalloproteinases (MMPs) are proteolytic enzymes that are expressed and activated during advanced cancer stages. Therefore, MMP-sensitive delivery systems can be regarded as potential platforms to achieve activated and/or controlled release of drugs for cancer treatment (Figure III) [10].

Figure I. Hydrogel-Mediated Delivery. The therapeutic influences of drugs can be screened excellently on hydrogel-based tumor reconstructive models in vitro.

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Figure II. Simulated Cancer Environment As an In Vitro Evaluation of Anticancer Therapeutics. Gene and/or drug administration to the (A) 3D and (B) 2D cell cultures.

Figure III. Smart Activation and Release of Chemotherapy Drug Molecules by Soluble Proteases. (A) Tumor. (B) Injection of a drug-loaded hydrogel into the peripheral of the tumor. (C) Drug binding with matrix metalloproteinase (MMP)-sensitive peptides. (D) Loosening of drug-binding peptides in the presence of MMP followed by drug release. (E) MMP expression by cancerous tissue, influence on drug-loaded MMP-sensitive peptides, drug release, drug penetration, and influence on tumor tissue.

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Box 3. Conventional Pharmaceutical Therapies against Cancer Each cancer pharmaceutical therapy approach has its pros and cons. Chemotherapeutic drugs are often highly toxic and poorly specific. Because these agents cannot distinguish between normal and malignant cells, a small fraction of the chemotherapeutic drug affects the tumor, whereas the remainder targets healthy cells, resulting in adverse effects [32,54]. Further, due to the short half-life of these medications, patients frequently need repetitive injections, which may intensify physical and mental inconvenience. In addition, poor water solubility reduces therapeutic effects and limits efficacy of a number of chemotherapeutic drugs [55]. Overall, about 70% of cancer patients do not respond to chemotherapeutic drugs, and the response to some of the prescriptive drugs remains less than 15% due to resistance mechanisms. Another strategy to inhibit tumor growth and invasion is to deliver nucleic acid constructs – DNA, siRNA, short hairpin RNA, miRNA, and antisense oligonucleotides – that modulate gene expression [6,7,56]. miRNAs are potentially important targets for cancer treatment because of their widespread expression and ability to regulate gene expressions and signaling pathways. Replacement of tumor-suppressive miRNAs and inhibition of overexpressed oncogenes are two different routes through which the miRNA system may be controlled [57]. However, challenges exist for the in vivo delivery of siRNA/miRNA because of their biological instability, short-term gene silencing effects, stimulation of the host immune system, and off-target effects [30,31]. Some researchers recommend incorporating genes and drugs in carriers to decrease unwanted toxicity, improve hydrophilicity, prolong duration time, protect drug bioactivity, and control the rate and zone specificity of drug release. Interestingly, localized and targeted DDSs appear to be reliable solutions [6,32].

microenvironment that is similar to the tumor tissue environment [68]. Investigating the independent roles of mechanical characteristics of the microenvironment in the regulation of cancer invasion is important. One way to address this matter is to adjust a Matrigel scaffold’s rigidity by applying a crosslinked polyethylene glycol (PEG) network. Increasing the microenvironmental rigidity restricted epithelial growth; however, the dissemination remained unchanged [13]. Another group embedded a growing spheroid in microbeads to evaluate mechanical stress on cancer progression. The results indicated that the inhomogeneous mechanical properties of confining tissue induce morphological changes in tumor growth and cell migration. More compressive stress is associated with more apoptotic cells, whereas less compressive stress is associated with a higher rate of cell proliferation [69]. Similarly, the role of specific biochemical factors in cancer regulation is still an important question for which researchers are trying to find an answer. Thanks to collagen protein, mouse

Figure 1. RNAi–Chemotherapeutic Drug Combinations. The combination of RNAi with drugs may exhibit a synergistic effect against tumors.

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epithelial cell spheroids were produced four decades ago [70]. Although the results produced valuable insights, the biology of mouse mammary tissue is significantly different from that of human mammary tissue [12,71]. To address this issue, researchers have developed 3D cultures supporting organoid growth from human cell lines. However, it has been proven that the growth of tissues from primary human mammary cells is challenging due to limited ductal growth of cells seeded into collagen and Matrigel matrices. Thus, a more elastic structure like ECM-derived hydrogels is needed [12]. The multicellular tumor spheroid model is recognized as the gold standard for tumor modeling because of its simplicity. Hydrogels can facilitate spheroid formation and allow tumor cells to establish tight cell–cell connections [72]. Nonetheless, spheroid models are unable to replicate some key features of the native TME itself due to the lack of surrounding ECM components and heterogeneity in shape, size, and aggregate forming tendencies. To this end, a PEG–fibrinogen microsphere tumor model has been developed with a higher degree of size and shape homogeneity to encapsulate and maintain a range of different tumor cells (metastatic and non-metastatic) for long-term culture [5]. Another research group established a microwell array method, based on photo-reactive hydrogels, to precisely investigate the effects of distinct compositions and stiffness on breast tumor formation. They successfully formed size-controllable tumor spheroids and examined tissue-specific tumor progression of mammary organoids in their tunable system [3]. Such high-throughput platforms make the simulation of organspecific diverse TMEs possible. Like spheroid models, organoid models have their own limitations. Most organoid models are only a partial representation of tissue, so it is frequently difficult to handle cell types, organization, and cell–cell/cell–matrix interactions inside the systems [73]. Nevertheless, despite these setbacks, the organoid culture approach is still valid, even when compared with precise engineering strategies such as organs on chips. Organoid culture systems could provide more tissue mass and facilitate experiments that usually require a large number of samples. They can generate heterogeneous and highly complex tissues with macroscale architecture by a simple method, while organ-on-chip methods intensively depend on high-level technology [74]. Despite many useful applications of organoid models, their applications in recapitulating various interactions between multiple tissues in the human body are limited. Hydrogels can be applied to develop 3D organization and further enhance the utility of organoid models in research and therapies. For example, incorporating organoids in hydrogels enable them to be printed in 3D tissuelike masses to resemble native tissues [75].

Temporal Substitutes and Tissue Replacements In some cancer types, not only does the possibility of recurrence exist following removal of the tumor tissue, but also the potential for deformity can affect patient’s quality of life. For example, mastectomy in breast cancer patients produces undeniable effects on patients’ social lives. The use of currently available prostheses is arguably unsafe due to health risks to patients. Both prosthesis and injectable tissue replacements are used in mammoplasty. One of the most popular nonresorbable soft-tissue fillers is polyacrylamide, which has been widely used for breast augmentation. However, these fillers can pose challenges due to confusion between radiologic images of injected polyacrylamide and infected galactoceles [76,77]. Although clinical trial data indicate that naturally based HA poses a low risk for allergic reaction, the interaction between HA and the CD44 cell receptor improves anchorage-independent proliferation, migration, invasion, and metastasis of the tumor cell. Nonetheless, it is unclear whether HA injected into the breast tissue is a trigger for breast cancer or if it affects cancer progression [78]. At first glance, it seems that the presently used material might not be ideal to replace the

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tissue removed in mastectomy, though thorough studies have not been performed so far in this regard. Apart from permanent substitution, temporal substitution may play an important role in cancer therapy. Temporal hydrogel spacing for radiotherapy could be beneficial. It has been reported that rectal spacers during prostate radiotherapy might be tolerable with significantly lower adverse effects. Hydrogels could help to reduce rectal toxicity during radiotherapy (Figure 2) [79,80].

Cancer Imaging Imaging studies could be performed in conjunction with conventional cancer treatments such as surgery, chemotherapy, and radiotherapy. Tumor imaging can be a valuable early stage cancer diagnostic tool. An injectable magnetic resonance imaging (MRI)-monitored long-term therapeutic hydrogel (MLTH) has been used in brain tumors. This thermosensitive magnetic poly(organophosphazene) hydrogel could potentially be a biodegradable imaging platform for anticancer drugs [8], which would be easy to administer in contrast with implantable Gliadel Wafers that require surgery. In this therapeutic method, a drug–nanomagnetic complex could be encapsulated within the hydrogel that surrounds the tumor. Conventional MRI cannot detect drug diffusion within a tissue; these MRIs provide information about the tissue itself, while MLTH can show the release and diffusion of nanomagnets into tissues, which provides essential data that can estimate the amount of drug that enters the tumor tissue. Importantly, this method can determine drug efficiency, which varies from patient to patient. Drugs may inefficiently diffuse into tissues, or they may diffuse into the tissue with an ineffective method of action. However, for effective treatment, it is necessary to distinguish between these deficiencies. MLTH enables physicians to investigate the amount of drug diffused and its efficiency. This technique can promote personalized therapy by allowing changes in dosage or drug when needed. MLTH has shown in vivo specificity to CD44-expressing stem cells, similar to cancer cells [81]. Interestingly, magnetic nanoparticles can be loaded with multiple drugs to target and inhibit tumor growth [82]. Another approach involves employing dye-conjugated microspheres or micelles [28]. A similar method is real-time labeling of a resected tumor during a surgical procedure with hydrogel nanoparticles. In this method, a nanoparticle-based visible contrast agent provides an in vivo

Figure 2. Hydrogel As a Rectal Spacer. A perirectal hydrogel injection can be used to reduce the effects of prostate radiotherapy.

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Table 1. Brief Summary of the Hydrogels Most Applicable to Cancer Research

*

Hydrogel

Advantages and disadvantages

Refs

PEG

PEG is a water-soluble and biocompatible polymer and is the most common nonionic drug cargo for cancer DDSs. Also, it is the most applicable unit for polypeptide modification.

[43]

N-isopropylacrylamide (NIPAAM)

Poly(NIPAAM) is a reversible thermally responsive biopolymer. It has a lower critical solution temperature (LCST)* of 32 C, which is near the physiologically relevant body temperature. Furthermore, by polymerizing with other hydrophilic biopolymers, its LCST can be raised above 40 C.

[18,28,46]

Poly(lactic-coglycolic acid) (PLGA)

PLGA is a well-known DDS platform for cancer therapy due to the biocompatibility and biodegradability. Nonetheless, PLGA-based nanotherapeutics display inert surfaces, low plasma stability, premature drug release, and poor tumor accumulation and retention. Finally, inefficient tumor cell uptake reduces their efficiency.

[88]

Polyacrylamide (PA)

PA hydrogel is a stable, nontoxic highly hydrophilic material and thought to be nonimmunogenic, viscous elastic, with biocompatible properties. It is a jellylike colorless substance, which includes 2.5% cross-linked polyacrylamide and 97.5% water. It is a wellknown material for breast augmentation.

[76,77]

HA

HA has strong binding affinity to overexpressed tumor cell-surface markers, so it is widely used for target cancer DDS. It is not immunogenic and has excellent biodegradability and biocompatibility. The injection of HA is a common noninvasive breast enlargement method. Increased HA levels upregulate hyaluronan synthesis, which affects tumor cell behaviors.

[78]

Collagen

Collagen is a well-known biodegradable material for 3D cell cultures, spheroid formation, DDS, and so on. It is the major structural protein of connective tissues and possesses good mechanical properties. However, its biological performance is limited compared with that observed for ECM-derived hydrogels. Its gelation mechanism is temperature sensitive.

[12,18]

Alginate

Alginate is derived from brown seaweed and is known as a low-cost material. It is a nontoxic, biocompatible, biodegradable, and weakly immunogenic material that has obtained Food and Drug Administration approval for broad biomedical applications. It is a commonly investigated delivery vehicle and shows poor interaction with proteins.

[18,28,89]

Chitosan

Chitosan is a biocompatible, biodegradable, and weakly immunogenic material that is derived from naturally available chitin and is widely employed for ECM substitution and DDS application. A dose-dependent anticancer activity is reported for chitosan.

[18,89]

ECM-derived hydrogel

The ECM is a complex mixture of structural and functional proteins. ECM-derived hydrogels possess excellent biological performance. They are nontoxic, biocompatible, and biodegradable materials. However, they suffer from batch-to-batch variation. They can effectively recapitulate the tumor microenvironment. Their mechanical performance can be modified through co-polymerization with other biopolymers.

[12,18]

LCST is the temperature at which hydrophobic–hydrophilic transition behavior occurs [18].

colored tumor tissue that can be visualized without any additional equipment. Polyacrylamide can covalently link to Coomassie blue molecules and assist with cancer diagnosis [83]. Apart from real-time tumor imaging, some immediate and informative analyses can be provided by microfluidic technology, which opens a new window in diagnostic research. Hydrogels are interesting materials in lab-on-a-chip-based research due to their unique chemical and mechanical properties [84]. The use of hydrogel-based protein microchips in clinical experiments is a cost-effective approach to monitor several tumor markers [60]. A primary advantage of the microchip-based assay is its capability to simultaneously analyze numerous samples for different antigens. The development of gel microchips allows for the detection of tumor markers [60,85]. In this system, the influence of drugs, genes, and nanoparticles can also be evaluated. Recently, a research group demonstrated the effects of photothermal therapy using a gold nanorod on human glioblastoma cells to migrate through hydrogel cross-shaped microchannels [86]. This finding successfully showed the risk of cancer cell migration due to photothermal therapy.

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Outstanding Questions How could designing a hydrogel that effectively mimics tumor microenvironments assist researchers to find comprehensive tumor models and compare tumor research outcomes universally? Does the combination of a hydrogel and nanoparticle lead to a flawless cancer drug delivery system? What would be the state of tumor imaging in clinics using hydrogels? Can we design fillers and prostheses that not only restore apparent beauty but also inhibit tumor recurrence?

Figure 3. The Most Prevalent Applications of Hydrogels in Cancer Research. Hydrogels are frequently used in cancer research for cancer drug delivery systems (DDSs), tumor reconstruction, cancer diagnosis, and tissue replacement. MMP, matrix metalloproteinase.

Concluding Remarks and Future Perspectives It is anticipated that continued adaptation and improvement of materials’ knowledge in the context of cancer biology understanding could facilitate cancer treatment. A number of clinical and experimental studies have demonstrated the potential use of hydrogels in cancer research. Nonetheless, there are some needs still unsatisfied (see Outstanding Questions). The unique features of hydrogels highlight them as broadly applicable biomaterials (Figure 3 and Table 1) [18]. Previous studies have demonstrated the feasibility of the use of hydrogel vehicles as smart and/or local delivery of multiple cargos [24,47,87]. Hydrogels also have the capability to carry nanoparticles that potentially could play a role as on/off molecular nanoswitches triggered by tumor hallmarks [53]. Hydrogels are the best suited materials to reconstruct the TME. They enable researchers to evaluate ECM changes through cancer progression, clarify how tumor cells correlate with normal cells, and elucidate information about hidden aspects of cancer cell behavior and drug uptake [63]. Further, the applications of hydrogels in cancer research are not restricted to DDSs and simulating the ECM. Promising results indicate tremendous capacity for cancer imaging, which could provide a broad perspective for the next treatment steps [8]. It seems that in situ implantation of biodegradable hydrogels as temporary fillers within abdominal viscera and at the periphery of major organs could potentially decrease the adverse effects of radiotherapy [80]. In this regard, it would be beneficial to develop a special class of biodegradable energy-absorbing hydrogels. By considering the superior properties of hydrogels, one promising approach is developing fillers and prostheses that not only can restore apparent beauty after a mastectomy but also can prevent tumor recurrence and induce normal tissue regeneration, which will provide physicians with game-changing resources against cancer. Disclaimer Statement The authors declare that they have no conflicts of interest.

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Acknowledgments This review study was funded by grants provided from the Royan Institute, the Iran National Science Foundation and Iran Science Elites Federation. The authors would like to express their appreciation to Mr Kazemi-Ashtiani for his critical comments.

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