cancers Review Stimuli-Responsive Hydrogels for Cancer Treatment: The Role of pH, Light, Ionic Strength and Magnetic Field Fernanda Andrade 1,2,3,† , Maria Mercé Roca-Melendres 1,† , Esteban F. Durán-Lara 4,5 , Diana Rafael 1,2, * and Simó Schwartz Jr. 1,2, * 1 Drug Delivery and Targeting Group, Molecular Biology and Biochemistry Research Centre for Nanomedicine (CIBBIM-Nanomedicine), Vall d’Hebron Institut de Recerca, Universitat Autònoma de Barcelona, 08035 Barcelona, Spain; fernanda.silva@vhir.org (F.A.); mroca.3@alumni.unav.es (M.M.R.-M.) 2 Networking Research Centre for Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN), Instituto de Salud Carlos III, 28029 Madrid, Spain 3 Department of Pharmacy & Pharmaceutical Technology, School of Pharmacy and Food Sciences, University of Barcelona, 08028 Barcelona, Spain 4 Bio and NanoMaterials Lab, Drug Delivery and Controlled Release, Universidad de Talca, Talca 3460000, Maule, Chile; eduran@utalca.cl 5 Departamento de Microbiología, Facultad de Ciencias de la Salud, Universidad de Talca, Talca 3460000, Maule, Chile * Correspondence: diana.fernandes_de_so@vhir.org (D.R.); simo.schwartz@vhir.org (S.S.J.) † These authors contribute equally to this work. Simple Summary: Cancer remains as the world second leading cause of death. The severe side effects associated to high doses of chemotherapy and the development of drug resistance are major   drawbacks for a successful cancer treatment. Therefore, new formulations that promote localized therapy at tumor sites are needed to improve the therapeutic outcomes and patient welfare. The use Citation: Andrade, F.; Roca-Melendres, of hydrogels is a very promising alternative, since they can be composed by smart materials able M.M.; Durán-Lara, E.F.; Rafael, D.; Schwartz, S., Jr. Stimuli-Responsive to respond to external factors, changing their properties accordingly and promoting a localized Hydrogels for Cancer Treatment: The drug delivery. As a result, a more specific, efficient, and less toxic local cancer treatment can be Role of pH, Light, Ionic Strength and accomplished. In this context, the most important characteristics of hydrogels recent studies regarding Magnetic Field. Cancers 2021, 13, the application of pH-, light-, ionic strength-, and magnetic-responsive hydrogels in cancer treatment 1164. https://doi.org/10.3390/ are here presented. cancers13051164 Abstract: Cancer remains as the second leading cause of death, worldwide. Despite the enormous Academic Editor: Jan Stenvang important advances observed in the last decades, advanced stages of the disease remain incurable. The severe side effects associated to systemic high doses of chemotherapy and the development Received: 15 January 2021 of drug resistance impairs a safe and efficiency anticancer therapy. Therefore, new formulations Accepted: 5 March 2021 are continuously under research and development to improve anticancer drugs therapeutic index Published: 9 March 2021 through localized delivery at tumor sites. Among a wide range of possibilities, hydrogels have recently gained special attention due to their potential to allow in situ sustained and controlled Publisher’s Note: MDPI stays neutral anticancer drug release. In particular, stimuli-responsive hydrogels which are able to change their with regard to jurisdictional claims in published maps and institutional affil- physical state from liquid to gel accordingly to external factors such as temperature, pH, light, iations. ionic strength, and magnetic field, among others. Some of these formulations presented promising results for the localized control and treatment of cancer. The present work aims to discuss the main properties and application of stimuli-responsive hydrogels in cancer treatment and summarize the most important advances observed in the last decades focusing on the use of pH-, light-, ionic strength- Copyright: © 2021 by the authors. , and magnetic-responsive hydrogels. Licensee MDPI, Basel, Switzerland. This article is an open access article Keywords: hydrogels; pH-responsive; photosensitive; ionic strength-responsive; magnetic-responsive; distributed under the terms and drug delivery; cancer treatment; local treatment conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Cancers 2021, 13, 1164. https://doi.org/10.3390/cancers13051164 https://www.mdpi.com/journal/cancers Cancers 2021, 13, 1164 2 of 17 1. Introduction In 2018 the World Health Organization (WHO) reported an estimated 9.6 million cancer deaths, being the second leading cause of death worldwide. Lung, prostate, and col- orectal are the most common types of cancer in men, while breast, colorectal and lung are the most common among women [1]. Unfortunately, despite all the advances achieved in the last decades in the pharmaceutical and biomedical fields, metastatic cancer is still considered an incurable disease. One of the major drawbacks regarding cancer treatment is related with the adverse effects associated with conventional chemotherapy [2–4]. Thus, sustained release local therapy emerged as promising alternative to reduce the systemic ex- posure and toxicity, thus increasing the therapeutic outcomes. In accordance to the National Cancer Institute definition, local therapy is conceived as a treatment directed to a specific organ or to a limited area of the body, embracing surgery, radiation therapy, cryotherapy, laser therapy, and topical therapy [5]. In recent years, a novel form of local cancer therapy using hydrogels have been proposed, owing to the favorable results achieved by these formulations in several other biomedical applications. The present review is focused on stimuli-responsive hydrogels (pH-, photo-, ionic strength-, and magnetic-sensitive) proposed in the last 10 years as controlled release formulations for cancer treatment. 2. Hydrogels for Biomedical Applications Different definitions for hydrogels have been suggested through the years. Often, they are described as a water-swollen and cross-linked polymeric network produced by the reaction/conjugation of one or more monomers. However, more recently hydrogels have been represented as three-dimensional networks which may absorb large amounts of water (from 10–20% up to thousands of times their dry weight) due to the presence of hydrophilic functional groups which fill the space among macromolecules and show high affinity for biological fluids [6]. The term hydrogel was firstly used by Wichterle and Lim in 1960 when they described a hydrogel based on poly-2-hydroxyethylmethacrylate (PHEMA), a synthetic biocompati- ble material useful for contact lens applications. In fact, in 1962 PHEMA lenses were firstly distributed in Western Europe, even though with limited success and acceptance. After a license buying agreement by the National Patent Development Corporation (NPDC) and a subsequent selling to Basuch and Lomb, that optimized the product, PHEMA lenses received Food and Drug Administration (FDA) approval [7]. Until today, hydrogels are routinely present in people using contact lenses, especially soft lenses based on silicon hydrogels. Moreover, hydrogels have been extensively used in biomedical applications regarding tissue engineering and wound healing [8,9]. Advanced dressings based on hydro- gels proved to be more effective in wound healing since they maintain a moist environment at the application site that avoid the spread of fluids to other healthy areas of the skin. Nowadays, many of them are commercially available, such as DermaFilm® , Kaltostat® , Condress® , and Sofargen® [10], being some of them filled with active ingredients such as iodine or zinc ions providing antimicrobial and cleansing properties, respectively [7]. Additionally, hydrogel-based formulations have been approved and used in the clinical practice for a variety of diseases. This includes Perseris® a risperidone hydrogel for acute and chronic schizophrenia, Sublocad® for buprenorphine induced analgesia, and Azasite® with lidocaine hydrochloric acid for the treatment of bacterial conjunctivitis [11]. Owing to the demonstrated success of hydrogels in a variety of biomedical applica- tions, it was speculated whether they could be used as drug delivery platforms for cancer treatment. Despite most of the research done in cancer treatment is dedicated to systemic and oral administration, local administration of drugs could be highly beneficial in cases of non-resectable or incomplete surgically removed tumors. In this context, the use of hydrogels, and especially stimuli-responsive hydrogels, has been proposed for in situ application at the tumor site/cavity, promoting a local sustained release of the drugs and a reduction of systemic exposure and off-target effects [12]. Moreover, administration of Cancers 2021, 13, 1164 3 of 17 depot systems reduces the needs for repeated anticancer therapy cycles and the associated drawbacks to the patients and the economic impact in the health care systems [11]. Stimuli-responsive hydrogels are promising smart materials able to change conforma- tion as a response to the surrounding environment variations like temperature, pH, light, ionic strength, and magnetic field [13]. This type of hydrogel has gained special importance due to the possibility to manipulate rheological behavior of the hydrogel according to the different tumor microenvironment conditions. In fact, cancer cells display several metabolic adaptations to ensure survival. Among them, glucose and nutrient uptake; production of lactic acid under aerobic conditions, and acclimatization to hypoxic and low-nutrient microenvironments. Moreover, an acidification of the extracellular milieu (low pH) and intracellular alkalization of the cytoplasm (high pH) occurs in cancer cells [14]. Thus, it is possible to take advantage of these characteristics to develop tumor targeted stimuli-responsive drug delivery systems. Over the years several hydrogel-based formulations have been designed for cancer diagnosis, prevention, and treatment, some of them enrolling clinical trials. Moreover, some have been granted with FDA and/or European Medicines Agency (EMA) approval, being translated into the clinical practice such as Lupron® depot or Eligard® , two poly lactic-co-glycolic acid (PLGA)-based hydrogels for in situ delivery of leuprolide acetate against advanced prostate cancer [11]. Another example is TraceIT® , a polyethylene glycol (PEG) hydrogel microparticles containing covalently bound iodine, approved as tissue marker. After being injected it allows exact tumor visualization using magnetic resonance imaging (MRI), computed tomography (CT) and ultrasounds, thus providing more accurate identification of cancer cells before surgical removal. Moreover, it is also used to define more accurate dose planning in radiation therapy, avoiding unnecessary radiation of cancer-free areas. The product remains dimensionally stable for 3 months being fully absorbed after 6 months [15]. The feasibility and efficacy of TraceIT® as organ spacer is under clinical evaluation in sparing vagina or prostate, in the treatment of rectal cancer patients, and also as a spacer between pancreas and duodenum in pancreatic cancer patients [16]. Another example is SpaceOAR® , an FDA approved PEG-based hydrogel organ spacer used to prevent rectum injury during radiation therapy sessions in prostate cancer patients [17]. 3. Classification of Hydrogels The majority of the developed hydrogels for biomedical applications are based on polymers with medium to high molecular weight; however, in recent years, low molec- ular weight self-assembling systems have been described and proposed. These systems are generally based on low molecular weight gelators, with especial focus on peptides that self-assemble into long, anisotropic structures, most commonly fibers. At a certain concentration, these fibers crosslink forming the gel network. Depending on the gelators properties they could be used to develop stimuli-responsive hydrogels [18,19]. Low molec- ular weight self-assembling peptide-based materials were the focus of a recent paper reviewing its properties and potential biomedical application [18]. In the present review we will focus on stimuli-responsive polymer-based hydrogels intended for cancer treat- ment. In Table 1 are presented some of examples of polymers used in the development of hydrogels. Hydrogels can be classified according to different parameters including: (i) composi- tion (homo or copolymeric), (ii) network size (macrogels, microgels, nanogels), (iii) elec- trical charge (non-ionic, cationic, anionic, amphoteric or zwitterionic), (iv) crosslinking (physical or chemical), among others [20]. Cancers 2021, 13, x 4 of 17 CancersCancers Cancers 2021, 13, 2021, 13, xx 13, 1164 2021, 444of of 17 17 4 of 17 Cancers2021, Cancers Cancers 2021,13, 2021, 13, 13, xxx 4 of17 of 4 of 17 17 Table 1. Chemical structure of some of the most commonly used polymers in hydrogels production. PEG—polyethylene Table 1. Table 1. Chemical Chemical structure structure ofof some some of of the the most most commonly commonly used used polymers polymers in in hydrogels hydrogels production. production. PEG—polyethylene PEG—polyethylene TableTable glycol, Table Table glycol, 1. 1. 1. Chemical Chemical Chemical PEGDA—poly structure 1. Chemical PEGDA—poly structure structure of some of of (ethylene some structure (ethylene some of the of of some glycol) of glycol) theof the most most most diacrylate, commonly the commonly diacrylate, most commonly PLGA—poly used commonly PLGA—poly used used polymers polymers polymers in hydrogels used polymers lactic-co-glycolic in in lactic-co-glycolichydrogels hydrogels acid, production. in hydrogels acid, production. production. PVA—poly(vinylPEG—polyethylene production. PVA—poly(vinyl PEG—polyethylene alcohol). The ox- PEG—polyethylene PEG—polyethylene alcohol). The ox- glycol, glycol, ygen (O) (O)PEGDA—poly PEGDA—poly atoms (ethylene (ethylene are highlighted highlighted glycol) glycol) in diacrylate, red anddiacrylate, and PLGA—poly PLGA—poly the nitrogen nitrogen lactic-co-glycolic lactic-co-glycolic (N)PLGA—poly atoms in blue. blue. acid, acid, PVA—poly(vinyl PVA—poly(vinyl alcohol). The alcohol).The ox- Theox- ox- The glycol, glycol, ygen PEGDA—poly glycol, PEGDA—poly PEGDA—poly atoms are (ethylene (ethylene glycol) (ethylene glycol) in red diacrylate, glycol) diacrylate, the PLGA—poly diacrylate, PLGA—poly (N) lactic-co-glycolic lactic-co-glycolic atoms in acid, PVA—poly(vinyl acid, PVA—poly(vinyl lactic-co-glycolic alcohol). acid, PVA—poly(vinyl alcohol). The ox- alcohol). ygen ygen (O) (O) atoms atoms are are highlighted highlighted in in red red and and the the nitrogen nitrogen (N) (N) atoms atoms in in blue. blue. ygen (O) ygen (O) atoms oxygen atoms are (O) highlighted atoms are in red red and are highlighted highlighted in and the in the nitrogen red nitrogen (N) atoms atoms and the nitrogen (N) inatoms (N)in blue. in blue. blue. Polymer Polymer Chemical Structure Chemical Structure Polymer Polymer Polymer Chemical Chemical Chemical Structure Structure Structure Polymer Polymer Chemical Chemical Structure Structure Acrylamide Acrylamide Acrylamide Acrylamide Acrylamide Polymer base Polymer base for for aa variety variety ofofAcrylamide derivatives including Acrylamide derivatives including polyacrylamide, polyacrylamide, Polymer Polymer Polymer base base base for for for a a a variety variety variety of of of derivatives derivatives derivatives including including including polyacrylamide, polyacrylamide, polyacrylamide, bisacrylamide, Polymer base forN-Isopropylacrylamide, a variety of derivatives Polymer base N-Isopropylacrylamide, bisacrylamide, N-N′-dimethylacrylamide including for a variety of derivatives including polyacrylamide, polyacrylamide, N-N′-dimethylacrylamide bisacrylamide, bisacrylamide, bisacrylamide, N-Isopropylacrylamide, N-Isopropylacrylamide, N-Isopropylacrylamide, N-N′-dimethylacrylamide 0 -dimethylacrylamide N-N′-dimethylacrylamide N-N′-dimethylacrylamide bisacrylamide, N-Isopropylacrylamide, N-N bisacrylamide, N-Isopropylacrylamide, N-N′-dimethylacrylamide Methacrylate Methacrylate Methacrylate Methacrylate Methacrylate Polymer base Polymer base for base for a variety of derivatives derivatives including Methacrylate for aa variety variety of Methacrylate including poly-2- poly-2- Polymer Polymer Polymer base base for for a a variety variety of of of derivatives derivatives derivatives including including including poly-2- poly-2- poly-2- hydroxyethylmethacrylate, Polymer base for Polymer hydroxyethylmethacrylate, basepoly(N,N′-dimethyl a for variety of derivatives a variety aminoethyl of derivatives poly(N,N′-dimethyl including methacrylate), poly-2- including aminoethyl methacrylate), tert- tert- hydroxyethylmethacrylate, hydroxyethylmethacrylate, hydroxyethylmethacrylate, poly(N,N′-dimethyl poly(N,N′-dimethyl poly(N,N′-dimethyl poly-2-hydroxyethylmethacrylate, poly(N,N aminoethyl 0 aminoethyl aminoethyl -dimethyl methacrylate), methacrylate), methacrylate), aminoethyl tert- tert- tert- butyl methacrylate, hydroxyethylmethacrylate, butylmethacrylate), methacrylate, n-butyl methacrylate, poly(N,N′-dimethyl n-butylmethacrylate, methacrylate, n-butyl acrylate, aminoethyl n-butyl and methyl methacrylate), acrylate, and and methyl tert- butyl butyl butyl methacrylate, methacrylate, methacrylate, n-butyl tert-butyl n-butyl n-butyl methacrylate, methacrylate, methacrylate, n-butyl n-butyl n-butyl n-butyl acrylate, methacrylate, acrylate, acrylate, and and methyl n-butyl methyl methyl methacrylate n-butyl acrylate, and methyl butyl methacrylate, n-butyl methacrylate, methacrylate methacrylate acrylate, and methyl methacrylate methacrylate methacrylate methacrylate PLGA PLGA PLGA PLGA PLGA PLGA (x == number (x number of of units units of of lactic lactic acid acid and and PLGA y == number y number of of units units ofof glycolic glycolic acid) acid) (x = (x = number number of of units units of of lactic lactic acid acid and and yy = number ofofunits of of glycolic acid) (x = number (x == number (x number ofof units of units of units of lactic of lactic acid lactic acid and acid and and yyy ==== number number number of number of units of units units of units glycolic of glycolic of acid) glycolic acid) glycolic acid) acid) PEG PEG PEG PEG PEG PEG (n (n (n= = number = of numberof number units PEG ofunits units of ethylene of of ethyleneglycol) ethylene glycol) glycol) (n (n (n === number number number of of of units units units of of of ethylene ethylene ethylene glycol) glycol) glycol) (n = number of units of ethylene glycol) PEGDA PEGDA PEGDA PEGDA PEGDA PEGDA (n (n = = numberof number of units units PEGDA of of ethylene ethyleneglycol) glycol) (n == number (n number of of units units of of ethylene ethylene glycol) glycol) (n (n = = number number of of units units of of ethylene ethylene glycol) glycol) (n = number of units of ethylene glycol) Chitosan Chitosan Chitosan Chitosan Chitosan Chitosan (n == number (n number of (n = of units of number units ofunits of β-(1→4)-linked Chitosan of β-(1 β-(1→4)-linked D-glucosamine →4)-linked andand D-glucosamine D-glucosamine and N-acetyl-D- N-acetyl-D- (n (n ==== number (n number number of number of of units of units units of units of β-(1→4)-linked of β-(1→4)-linked of β-(1→4)-linked β-(1→4)-linked D-glucosamine and D-glucosamine and D-glucosamine N-acetyl-D- and N-acetyl-D- and N-acetyl-D- N-acetyl-D- (n glucosamine) N-acetyl-D-glucosamine) glucosamine)D-glucosamine glucosamine) glucosamine) glucosamine) glucosamine) Alginate Alginate Alginate Alginate Alginate Alginate (x = number (x == number number of units of units units of (1,4)-linked of (1,4)-linked (1,4)-linked β-D-mannuronate and Alginate β-D-mannuronate and y y == number number of of units units (x (x (x = = (x = number number of number of of of units units of units of of of (1,4)-linked (1,4)-linked β-D-mannuronate (1,4)-linked β-D-mannuronateand β-D-mannuronate β-D-mannuronate andyy and and y y == == number number number of number of of of units units units (x = number of units of (1,4)-linkedof α-L-guluronate) of units α-L-guluronate) of β-D-mannuronate α-L-guluronate) and y = number of units of of α-L-guluronate) α-L-guluronate) of α-L-guluronate) α-L-guluronate) of PVA PVA PVA PVA PVA PVA (n == number (n number of of units units PVA of vinyl of vinyl alcohol) alcohol) (n (n= =number numberof (n === number (n ofunits number of number of units of of units of units of units of vinyl ofvinyl alcohol) vinylalcohol) vinyl alcohol) alcohol) (n vinyl alcohol) Poloxamer Poloxamer Poloxamer Poloxamer Poloxamer Poloxamer (x (x = = number number of of units units of of ethylene ethylene glycol and y Poloxamer glycol and yand = number number of units units of of propylene (x (x (x (x === number number number = number of of of units units units of of of of units of ethylene ethylene ethylene ethylene glycol glycol glycol glycol and and and y yy ==== number number number of y = number of of of units units of of units units of of of propylene propylene propylene propylene (x = number of units of ethylene glycol) glycol propyleneand y = number of units of propylene glycol) glycol) glycol) glycol) glycol) glycol) covalent bonds and the hydrogels present phase-reversibility. The main physical interac- tions between polymer chains described in physically crosslinked hydrogels are based on hydrogen bonding, ionic/electrostatic interactions, self-assembly of amphiphilic polymers by hydrophobic interactions, crystallization, protein interactions, and metal coordination Cancers 2021, 13, 1164 [21]. For example, polyacrylic acid and polymethacrylic acid forms complexes with 5PEG of 17 through a hydrogen bonding. They are only formed when exists a protonation of carbox- ylic acid groups, which would result in the formation of a pH-sensitive hydrogel [22]. Another example Regarding theistype alginate, a naturalhydrogels of crosslinking, polymer canthatbejellifies prepared through ionic interactions via physical or chemical with divalent cations such as calcium (Ca 2+) or magnesium (Mg2+) [21]. On the other hand, crosslinking (Figure 1). When a physical crosslinking is used, there is no formation of amphiphilic covalent bonds polymers and thesuch as poloxamers hydrogels suffer thermally The present phase-reversibility. induced main phase transition physical interac- forming hydrogels due to hydrophobic interactions above lower tions between polymer chains described in physically crosslinked hydrogels are based critical solution temper- on ature (LCST) hydrogen [23]. Polyvinyl bonding, alcohol (PVA)-based ionic/electrostatic interactions,hydrogels self-assemblyare an ofexample of crystalliza- amphiphilic polymers tion induced physical by hydrophobic crosslinking interactions, as polymerprotein crystallization, crystallization and gelation interactions, and metaloccurs when coordina- the solution repeatedly undergoes freeze-thawing cycles [24]. tion [21]. For example, polyacrylic acid and polymethacrylic acid forms complexes with PEGThe biggest through drawbackbonding. a hydrogen of physical crosslinking They are only hydrogels formed when relies on their exists possible lack a protonation of or reduced stability under physiological conditions. Therefore, chemical carboxylic acid groups, which would result in the formation of a pH-sensitive hydrogel [22]. crosslinking is being Another preferred examplefor is in vivo applications alginate, [22]. that jellifies through ionic interactions with a natural polymer Chemically divalent cations crosslinked such as calcium hydrogels (Ca2+ )are more stable (Mg or magnesium under2+ ) physiological conditions [21]. On the other hand, and exhibit excellent amphiphilic polymersmechanical properties such as poloxamers owing suffer to the covalent thermally induced bonds in-between phase transition the form- polymer chains. Additionally, their degradation behavior can be ing hydrogels due to hydrophobic interactions above lower critical solution temperature tunable, even though they (LCST) are[23]. generally irreversible. Polyvinyl A potential disadvantage alcohol (PVA)-based hydrogels arecompared an example to of physically cross- crystallization linked induced hydrogels physicaliscrosslinking the frequently as required polymer use of organic solvents crystallization and catalysts and gelation occurs that when often the raise biocompatibility solution and environmental repeatedly undergoes freeze-thawingconcerns [21]. cycles [24]. Figure 1. Schematic representation of hydrogels formation via chemical and physical crosslinking. RAFT—reversible addition-fragmentation chain transfer polymerization, FRP—free radical poly- Figure 1. Schematic merization. representation O—oxygen, of hydrogels N—Nitrogen, formation C—Carbon, via chemical H—Hydrogen. and physical Created crosslinking. with BioRender.com RAFT—reversible addition-fragmentation (accessed on 15 January 2021). chain transfer polymerization, FRP—free radical The biggest drawback of physical crosslinking hydrogels relies on their possible lack or reduced stability under physiological conditions. Therefore, chemical crosslinking is being preferred for in vivo applications [22]. Chemically crosslinked hydrogels are more stable under physiological conditions and exhibit excellent mechanical properties owing to the covalent bonds in-between the polymer chains. Additionally, their degradation behavior can be tunable, even though they are generally irreversible. A potential disadvantage compared to physically crosslinked hydrogels is the frequently required use of organic solvents and catalysts that often raise biocompatibility and environmental concerns [21]. Covalent bonds in chemically crosslinked hydrogels are generally promoted by click chemistry including Schiff base formation, Michael addition or Diels–Alder reaction, pho- topolymerization, free radical polymerization (FRP), reversible addition-fragmentation chain transfer (RAFT) polymerization or enzyme catalyzed reactions [21]. Cancers 2021, 13, 1164 6 of 17 Due to the presence of certain chemical groups such as NH2 -, COOH-, OH- in the skeleton of hydrophilic polymers, the amine-carboxylic acid, isocyanate-OH/NH2 reaction or Schiff base formation are commonly used to create a covalent linkage between polymers to form the hydrogel network. Schiff base is commonly used for hydrogels formation and generally obtained via the nucleophilic attack of amines on the electrophilic carbon atoms of aldehydes or ketones. For example, glycol chitosan have been crosslinked with PEG or poly(N-isopropylacrylamide)-co-poly(acrylic acid) forming promising hydrogels for anticancer drug delivery [21]. In photopolymerization crosslinking, acrylate derivatives (e.g., poly (ethylene glycol) diacrylate (PEGDA) and glycidylmethacrylate (GMA)) are generally used as pre-polymers due to the presence of unsaturated groups. These groups react with photo-initiators (UV or visible light) like Irgacure 2959, lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) or Eosin-Y, among others. In the presence of a light source, photo-initiators are cleaved by photons forming free radical molecules that react with the vinyl bonds in the pre-polymer promoting the crosslink between the polymer chains [25]. Enzyme catalyzed crosslinking can be used, for example, to create of PEG- based hydrogels using a transglutaminase, which catalyzes the reaction between the γ-carboxamide group of the PEG-functionalized with glutaminyl groups and the ε-amine group of poly(lysine-co-phenylalanine) resulting in the formation of an amide bond [26]. To avoid the irreversibility while maintaining the higher mechanical strength of chemically crosslinked hydrogels, dynamic covalent chemistry is being applied to generate hydrogels via both physical and chemical crosslinking [21]. For example, boronate esters prepared from boronic acids and 1,2- or 1,3-diols in aqueous solutions results in reversible covalent bonds. Importantly, the strength and reversibility of the bond largely depends on the pH of the solution and the pKa of the boronic acid used as the boronate ester formation is favored at pH values above the pKa of the boronic acid. By changing the boronic acid derivative and the 1,2- or 1,3-diols used, is possible to produce reversible pH-sensitive hydrogels with tunable mechanical properties [21]. 4. Stimuli-Responsive Hydrogels Some hydrogels can be sensitive to environmental stimulus such as temperature, pH, light, ionic force or pressure, suffering sol-to-gel transitions that can allow controlled gelation and drug release at specific sites. On the other hand, non-sensitive hydrogels swell due to water absorption without responding to environmental changes. Thermo- responsive hydrogels are the most commonly used being the main subject of a bibliographic revision performed by our group (under publication). Here we report some of the pH-, light-, ionic strength-, magnetic-sensitive, as well as examples of dual responsive hydrogels developed in recent years for cancer treatment. 4.1. pH-Sensitive Hydrogels Cancer cells are characterized by the acidification of the extracellular milieu and increased alkalization of the cytoplasm. Some studies suggest that alkaline intracellular pH increases glycolysis, promotes cellular adaptation to hypoxia and maximizes cancer cell proliferation [14]. Taking advantage of the pH alteration at the tumor environment, new pH-sensitive hydrogels have been developed in order to release the cytotoxic drug exclusively in the cancer cells area, reducing the occurrence of adverse side-effects in healthy tissues. To create pH-sensitive hydrogels, ionizable groups including amines, carboxylic acids, imines, etc., can be introduced in their structure. These groups, depending on the pKa values and the environmental pH, present the capability to donate or accept protons and undergo alterations of their physical and chemical properties [27,28]. Cationic hydrogels swell at low pH (pH < pKa) due to protonation of amino/imine groups and consequent repulsion of the positively charged moieties on the polymer chains. On the contrary, an- ionic hydrogels swell at higher pH (pH > pKa) due to ionization of the acidic groups and chain repulsion (Figure 2) [29]. pH-sensitive conformational changes of polymer can occur values and the environmental pH, present the capability to donate or accept protons and undergo alterations of their physical and chemical properties [27,28]. Cationic hydrogels swell at low pH (pH < pKa) due to protonation of amino/imine groups and consequent repulsion of the positively charged moieties on the polymer chains. On the contrary, ani- onic hydrogels swell at higher pH (pH > pKa) due to ionization of the acidic groups and Cancers 2021, 13, 1164 7 of 17 chain repulsion (Figure 2) [29]. pH-sensitive conformational changes of polymer can occur via: (a) destabilization (via collapse or swelling), (b) dissociation, and (c) alteration of the drug/vehicle partition coefficient. Conformational changes or solubility alterations will affect via: the release of encapsulated (a) destabilization drugs. (via collapse The kinetics or swelling), of release could (b) dissociation, be alteration and (c) controlledofbythe manipulating drug/vehicle thepartition degree and ratio ofConformational coefficient. the conformational changes changes [27,28]. For or solubility example, alterations will cationic affect hydrogels the releaserelease the drug when of encapsulated swell drugs. Theatkinetics low pHof(Figure release2A), while could anionic hy-by be controlled drogels release the manipulating thedrug at higher degree pH of and ratio values (Figure 2B). Forchanges the conformational this reason, anionic [27,28]. For hydro- example, gels are preferred cationic hydrogelsforrelease controlled intracellular the drug drug when swell delivery at low in tumor pH (Figure 2A),cells, whilewhile cationic anionic hydro- gels release hydrogels are the drug atfor preferred higher pH values delivery (Figure 2B). For at the extracellular this reason, matrix in tumor anionic tissues hydrogels [29,30]. are preferred for controlled intracellular drug delivery in tumor cells, while cationic hydrogels are preferred for delivery at the extracellular matrix in tumor tissues [29,30]. Figure 2. Schematic representation of pH-sensitive hydrogels swelling. Swelling of the hydrogel occurs Figure via ionization 2. Schematic of the pendant representation groups with of pH-sensitive consequent hydrogels increase swelling. hydrophilic Swelling nature of the of the hydrogel polymer chains and electrostatic chain repulsion. ECM—extracellular matrix, H + —Hydrogen ions, occurs via ionization of the pendant groups with consequent increase hydrophilic nature of the OH− —Hydroxide polymer chains and electrostatic chain ions. The red repulsion. circles ECM—extracellular represent the drugs loaded matrix, H+—Hydrogen in the hydrogels. ions, Created with OHBioRender.com −—Hydroxide ions. The red circles represent the drugs loaded in the hydrogels. Created with (accessed on 15 January 2021). BioRender.com (accessed on 15 January 2021). pH-sensitive hydrogels can be also produced by formation of polyelectrolyte com- plexes between hydrogels pH-sensitive can alginate, anionic (e.g., be also produced dextran) byandformation of polyelectrolyte cationic (e.g., com- chitosan) polymers, plexes thusbetween avoidinganionic the use(e.g., alginate, and of chemical dextran) and cationic possibly (e.g., chitosan) toxic crosslinkers polymers, [29,31,32]. The thus use of avoiding the use acid-labile of chemical chemical bonds and that possibly are stabletoxic crosslinkers [29,31,32]. at physiological (neutral) pH Theandusedegrade of acid-or hydrolyze labile chemicalatbonds low pH values that (e.g.,attumor are stable microenvironment) physiological (neutral) pHisand alsodegrade a goodor strategy hydro- to produce lyze at low this pH “smart” hydrogels, values (e.g., especially in the fieldisof tumor microenvironment) cancer also a goodtreatment strategy[27]. to produce One ofhydrogels, this “smart” the most used polymers especially in thetofield produce pH-responsive of cancer treatment hydrogels [27]. is chitosan. It is a natural, non-toxic, biodegradable, and biocompatible polymer One of the most used polymers to produce pH-responsive hydrogels is chitosan.obtained from chitin. It is However,non-toxic, a natural, the limited solubility in water biodegradable, and and other organic biocompatible solventsobtained polymer is its weakness. To over- from chitin. come this, different derivatives of chitosan presenting a good solubility in physiological conditions such as N-trimethyl chitosan, N-carboxymethyl chitosan, or N-carboxyethyl chitosan (CEC) have been developed [29,33]. For example, amine groups from CEC and benzaldehyde groups from PEGDA create a dynamic covalent Schiff-base linkage forming a doxorubicin-loaded hydrogel with self-healing performance [33]. Moreover, the hydro- gel presented adequate physical, rheological, and biological properties for the targeted delivery of doxorubicin in hepatic cancer. Of note, since the pKa value of D-glucosamine residue of chitosan is about 6.2~7.0, the hydrogel promoted drug release at low pH val- ues (92% of doxorubicin was released at pH 5.5 over 42% at pH 7.4 after 7 days), due to the decomposition of the Schiff-base (14% mass loss at pH 7.4 and 40% at pH 5.5) and Cancers 2021, 13, 1164 8 of 17 swelling/increase in pore size. More importantly, at low doses, the hydrogel loaded with doxorubicin significantly inhibited the proliferation of HepG2 cells when compared to the free drug [33]. A similar behavior was obtained with a CS/PVA hydrogel loaded with 5-fluoracil. The hydrogel retained the drug at pH 7.4, thus preventing possible drug release to normal cells and the consequent side effects, promoting a continuous and controllable drug release at pH 5 [34]. Many other natural polymers (e.g., alginate, cellulose, guar gum, carrageenan, dextran, xanthan, among others) and synthetic polymers (e.g., polyamines like poly(acrylamide), poly(N,N0 -dimethyl aminoethyl methacrylate) or poly(ethylene imine), acrylic derivatives like poly(acrylic acid), sulfonic acid derivatives like 2-acrylamido-2-methylpropylsulfonic acid, sulfonamides like sulfamethazine, pyridine derivatives like poly(2-vinyl pyridine), imidazole derivatives like poly(N-vinyl imidazole), PEG, PVP, PLA, among others) have been proposed for production of pH-sensitive hydrogels [29,35]. Beyond injectable hydrogels, pH-responsive hydrogels are also an interesting tool for oral delivery of anticancer drugs as the gastrointestinal tract is characterized by variations in pH. Therefore, it is possible to tune formulations to promote the release of drugs in affected regions of the digestive tract [35]. As example, different pH-responsive nanogels based on poly(methacrylic acid-g-polyethylene glycol-co-hydrophobic monomer) were developed and assessed as platforms for intestinal delivery of doxorubicin for colorectal cancer treatment [36]. The different hydrophobic monomers used to adjust pH-responsiveness were tert-butyl methacrylate (tBMA), n-butyl methacrylate (nBMA), n-butyl acrylate (nBA), and methyl methacrylate (MMA), while polyethylene glycol was added to provide better integrity of the hydrogel and confer P-gp efflux inhibition. Among the formulations the one composed by poly(methacrylic acid-g-polyethylene glycol-co-methyl methacrylate) presented the best performance, namely, doxorubicin loading and release at simulated intestinal pH, and transport across intestinal epithelia (Caco-2 cell monolayers) [36]. pH-sensitive hydrogels can be also applied for gastrointestinal protection and reg- ulate acute radiation syndrome, a consequence of tumor radiation therapy. A hydrogel constituted by polycaprolactone grafted with poly(methacrylic acid-co-ethyl acrylate) (PCL-g-MAC) was proposed for intestinal delivery of amifostine, and FDA approved radioprotective agent [37]. MAC is an FDA approved excipient for enteric coating due to its pH-responsiveness, while PCL was used to afford hydrophobicity to the system, improving hydrogel stability and performance. The hydrogel protected amifostine from gastric degra- dation (pH 1.2 release of 36.5% for 5.5 h), releasing the drug only at pH 7.4 (70.7% release at 0.6 h), as the pKa of the carboxyl groups is around 4.5, the hydrogel tends to dissociate and swell for pH > 4.5. Moreover, in vivo studies suggested that the formulation promotes pro- tection and enhances survival upon γ-radiation by inhibiting hematopoietic cell apoptosis and accelerating cell proliferation, and also by reducing weight loss [37]. Some examples of pH-sensitive hydrogels developed in the last decade proposed as drug delivery systems for cancer treatment are summarized in Table 2. 4.2. Photosensitive Hydrogels Photosensitive hydrogels suffer a chemical and/or physical alteration as a conse- quence to light exposure (UV, visible or near infrared (NIR)) (Figure 3). An incident light triggers modifications such as free-radical polymerization reaction, chemical link- age cleavage or isomerization, volume changes via swelling or shrinkage, etc. [51,52]. By application of light at the desired site of action is possible to promote a spatial and temporal controlled drug release avoiding systemic exposure. There are mainly three strategies used in the development of light-responsive hydrogels as drug delivery sys- tems: (i) photochemical cleavage of or crosslinking points (Figure 3A) polymer backbones (Figure 3B), (ii) photo-induced isomerization of specific motifs that alter gel properties like the crosslinking density, charging state, or hydrophilicity (Figure 3C), and (iii) incorpo- ration of photothermal agents that generate heat through irradiation and induce phase transition of thermo-responsive hydrogels (Figure 3D) [53]. Photoisomerization usually Cancers 2021, 13, 1164 9 of 17 originates reversible hydrogels, while photocleavage is usually irreversible. Ruthenium, coumarin nitrophenyl, or o-nitrobenzyl derivatives, are examples of photocleavable com- pounds used in the development of hydrogels, while azobenzene and spiropyran promote isomerization under light exposure [51,54]. On the other hand, photo-thermal agents include porphyrins, cyanines, gold, silver, oxide, and carbon nanoparticles [51]. Regarding photothermal induced hydrogels, NIR-responsive hydrogels are of great interest due to their deep tissue penetration and harmlessness; however, overheating and consequent tissue damage is one of the main concerns that must be properly addressed regarding its biomedical application [51]. Table 2. Examples of pH-sensitive hydrogels for cancer therapy. n.a.—not applicable; β-CD—β-cyclodextrin; 5-FU—5- fluorouracil; DOX—doxorubicin; FACS—fluorescence-activated cell sorting; PTX—paclitaxel. Date Drug In vitro model In vivo model Hydrogel formulation Highlights References Presence of β-CD increase drug loading. Swelling and drug release is low at pH 1.2 HT-29 cells β-CD-graft-gelatin and high at pH 7.4, being 2014 5-FU (colorectal n.a. crosslinked with [38] appropriate for drug release adenocarcinoma) oxidized dextran at colon. Hydrogels were biocompatible and increase the efficacy of 5-FU. The release mechanism followed non-Fickian MC3T3E1 diffusion. FACS analysis (osteoblast) and Alginate-conjugated 2019 Bortezomib n.a. revealed cell apoptosis [39] NIH-3T3 polydopamine defined by loss of cell (fibroblast) cells viability for colon cancer cells. At low pH, the swelling of hydrogels is 4.6–12.3 times lower than at pH 7.4. HeLa (cervical Susceptible to degradation by adenocarcinoma) the simulated physiological Zajdel hepatoma 2019 Prospidin and HeP-2 (human Dextran phosphate (DP) conditions. The amount of [40] Mongrel white rats hepatocellular liver drug release is dependent on carcinoma) cells the pH of outer media and decreases with the growth of phosphoric group content in DP hydrogels N-N0 -dimethylacrylamide monomers polymerized in Drug release is always higher presence of methacrylic acid in the presence of a magnetic 2015 5-FU n.a. n.a. [41] or 2-aminoethyl methacrylate field and generally increases hydrochloride containing with its intensity. ferro-nanoparticles Exhibited in vitro HepG2 cells pH-dependent gel (human N-carboxyethyl degradation and doxorubicin 2017 DOX Sprague-Dawley rat [33] hepatocellular chitosan + PEGDA release. No hydrogel liver carcinoma) diffusion after subcutaneous injection. In vitro controlled release of PTX at pH 5.5 for 6 days. In vivo hydrogel retention at HepG2 (human H22 subcutaneous the tumor site. Increased hepatocellular liver 2019 PTX xenograft BALB/c Self-assembling octapeptides antitumor efficacy compared [42] carcinoma), H22 mice to free PTX (reduced tumor (murine hepatoma) weight and volume), and reduced toxicity (low weight loss). pH-induced separation of the nucleic acid duplex units MDA-MB-231 cells DNA hairpin conjugated 2017 DOX n.a. causing DOX release at pH [43] (breast cancer) with polyacrylamide 5.0. Increase DOX cellular uptake and efficacy. Cancers 2021, 13, 1164 10 of 17 Table 2. Cont. Date Drug In vitro model In vivo model Hydrogel formulation Highlights References 87% DOX release over 60 h at HCT116 cells Chitosan-grafted- pH 5.5 over 52% at pH 7.4. 2019 DOX (colorectal Kunming Mice dihydrocaffeic In vitro enhancement of DOX [44] adenocarcinoma) acid/oxidized pullulan therapeutic efficacy. In vivo adhesion in the injection site. Improved release at pH 7.4 MDA-MB-231 and Zein, acrylic acid, N, over pH 1.2. Improved 2019 5-FU and Rutin MCF-7 cells n.a. N-methylene bisacrylamide, in vitro pharmacological [45] (breast cancer) and ammonium persulphate activity by apoptosis induction by oxidative stress. Ehrlich Cholesterol and span 60 carcinoma cell Controlled release of drug Triaryl-(Z)- niosomes in chitosan and 2020 n.a. subcutaneous and improved [46] olefin glyceryl xenograft Swiss tumor regression monooleate-based hydrogels albino mice Improved DOX cellular HeLa cells (cervical Zein nanoparticle 2020 DOX n.a. internalization [47] adenocarcinoma) crosslinked pectin and cytotoxicity A549 (lung cancer) Improved release at pH < 6.8. and HepG2 (human Long-chain hexadecyl amine Improved PTX 2020 PTX n.a. [48] hepatocellular liver modified nanocellulose internalization by cells and carcinoma) cells therapeutic efficacy. Degradation occurs at pH 5.6 HepG2 cells which contributes for HepG2 xenograft (human 4armPEG-benzaldehyde and controlled DOX release. 2020 DOX BALB/c nude [49] hepatocellular N-carboxyethyl chitosan In vivo biocompatibility, mouse model liver carcinoma) degradation over 5 days, and improved tumor inhibition Improved drug release at pH Carboxyethyl modified HeLa cells (cervical < 6.8. Biocompatibility and 2019 DOX n.a. chitosan and aldehyde [50] adenocarcinoma) biodegradability. In vitro Cancers 2021, 13, x modified hyaluronic acid 11 of 17 pharmacological efficacy Figure 3. Schematic Figure 3. Schematic representation representation of hydrogel. of photosensitive photosensitive hydrogel. After exposure to light, theAfter formu- exposure to light, the formula- lation suffers a chemical and/or physical alteration including (A,B) photoclevage, (C) photoiso- tion suffers meration, and (D)a photo-thermal chemical and/ortransition.physical In the case ofalteration including photo-thermal (A,B) transition, the photoclevage, increase in (C) photoisomera- the temperature tion, and (D) promoted by the activation photo-thermal of photo-thermal transition. agents In the could case oflead to a sol-to-gel or a transition, the increase in the photo-thermal gel-to-sol transition, accordingly the nature of the thermo-responsive polymer. The red circles temperature represent the drugs promoted loaded in theby the activation hydrogels. Created withofBioRender.com photo-thermal agents (accessed could lead to a sol-to-gel or a gel-to- on 15 January 2021). sol transition, accordingly the nature of the thermo-responsive polymer. The red circles represent the drugs 4.3. Ionicloaded Strength in andthe hydrogels. Created Magnetic-Responsive Hydrogelswith BioRender.com (accessed on 15 January 2021). Besides temperature, pH and light, which are the most commonly used stimuli for As anofexample development of photo-thermal sensitive hydrogels, hydrogels, there are other parameters such asblack phosphorus ionic strength or nanosheets incorpo- magnetic field that can also modulate the physical and chemical attributes of stimuli-re- rated into an sponsive hydrogels. agarose hydrogel loaded with doxorubicin demonstrated to be a promising platform for cancer Magnetic-sensitive treatment hydrogels [55].composed are generally NIR irradiation promote heat production by phos- by hydrogels incorporated with iron oxide nanoparticles possessing paramagnetic properties (Figure 4). These vi- brate under exposure to a magnetic field and can dramatically increase local temperature promoting a therapeutic efficacy by thermal-ablation mechanisms. Moreover, these sys- tems are generally associated with thermo-sensitive hydrogels in which temperature in- crease triggers drug release, thus promoting a synergic efficacy of thermal and chemo- therapeutic cytotoxicity. As NIR, this strategy enjoys of spatial and temporal activity with Cancers 2021, 13, 1164 11 of 17 phorus nanosheets and gel-to-sol transition of the agarose hydrogel by hydrolysis that triggers temporal and spatial doxorubicin release. The hydrogel showed to be biocompati- ble in vitro in MDA-MB-231 breast cancer cells, A549 lung carcinoma cells, HeLa cervical cancer cells, and B16 melanoma cells. In vivo experiments demonstrated that this system is capable of accurately controlling the release of drugs and eradicate the tumor in contrast to animals treated with free dox or the non-irradiated hydrogel. Agarose is approved by FDA and black phosphorus degrades into nontoxic phosphate and phosphonate that could be easily excreted through urine, which favors the clinical translation of the system [55]. Sodium selenite (Se)-directed crosslinked hydrogels based on hyaluronic acid-dopamine, loaded with indocyanine green (ICG) as photo-thermal agent, were developed for local therapy of breast cancer [56]. After NIR irradiation, ICG promote an increase in the tem- perature superior to 7 ◦ C, responsible for both in vitro and in vivo photo-thermal activity against MDA-MB-231 breast cancer cells and in tumor-bearing BALB/c nude mice models, respectively. Additionally, the system enjoyed from the combinational effect of ICG and pro-oxidant effect of Se, resulting in a significant tumor inhibition without significant toxic effects [56]. The use of photosensitizer compounds incorporated into hydrogels for anticancer photodynamic therapy is also being proposed. For example, phthalocyanine zinc (ZnPc) was used both as photo-initiator and photosensitizer in the formulation of a hybrid hydro- gel containing PEGDA, PEG 400 and phosphotungstic acid [57]. When irradiated with NIR laser light, ZnPC become excited forming a singlet oxygen (1 O2 ), that promotes a strong decrease on HeLa cells. Moreover, the hydrogel presents a good biocompatibility and offers the possibility to be loaded with anticancer drugs for a combined and synergic ther- apy [57]. More recently, similar results have been obtained with a PEGDA-based hydrogel containing methylene blue sensitized mesoporous titanium (IV) oxide (TiO2 ) nanocrystals prepared in situ via NIR photopolymerization [58]. TiO2 was used as photo-initiator and photosensitizer while methylene blue works as photosensitive additive to improve the effects of TiO2 [58]. Combinatory effects of photo-thermal activity and photodynamic therapy has also been investigated. For example, a biocompatible agarose-based hydrogel that incorpo- rates sodium humate (SH) as photothermal agent, chlorin e6 (Ce6) as photosensitizer, and manganese oxide (MnO2 ) for the catalytic decomposition of H2 O2 and modulation of hypoxia at the tumor microenvironment, associated to treatment resistance [59]. Under NIR irradiation, SH promote an increase in the temperature that besides the photothermal ac- tivity, promote degradation of the agarose gel forming H2 O2 . This will be decomposed into O2 which will convert into 1 O2 by the released Ce6. In vivo studies in 4T1 BALB/c mice bearing demonstrated a strong anticancer activity of the system with a significant tumor regression (TGI of 93.8%), without causing toxicity to the animals. Accordingly, this hydrogel should be considered as a promising platform for tumor microenvironment modulation and cancer therapy [59]. 4.3. Ionic Strength and Magnetic-Responsive Hydrogels Besides temperature, pH and light, which are the most commonly used stimuli for development of sensitive hydrogels, there are other parameters such as ionic strength or magnetic field that can also modulate the physical and chemical attributes of stimuli- responsive hydrogels. Magnetic-sensitive hydrogels are generally composed by hydrogels incorporated with iron oxide nanoparticles possessing paramagnetic properties (Figure 4). These vibrate under exposure to a magnetic field and can dramatically increase local temperature pro- moting a therapeutic efficacy by thermal-ablation mechanisms. Moreover, these systems are generally associated with thermo-sensitive hydrogels in which temperature increase triggers drug release, thus promoting a synergic efficacy of thermal and chemotherapeu- tic cytotoxicity. As NIR, this strategy enjoys of spatial and temporal activity with low invasiveness and deep tissue penetration [35]. lized by a PEGylated phospholipid encapsulated with paclitaxel and doxorubicin [61]. The rheological properties of the hydrogel allow to easily inject the hydrogel at the surgi- cal site after tumoral resection, which facilitates local delivery of its dual cargo due to magnetocaloric gel-to-sol transition under application of a magnetic field. In vivo experi- ments in 4T1-tumor bearing Balb/c mice demonstrated an improvement in the survival Cancers 2021, 13, 1164 12 of 17 rate and reduction in tumor recurrence of animals treated with the proposed hydrogel [61]. Figure 4. Schematic representation Figure 4. of magnetic-responsive Schematic representation hydrogels. They of magnetic-responsive incorporate hydrogels. nanoparticles, They incorporate which under a nanoparti- magnetic fieldcles, which vibrate dueunder a magnetic to their field vibrate paramagnetic due toAtheir properties. paramagnetic temperature properties. increase A temperature is induced triggering drug release. The red circlesincrease representis induced the drugstriggering loaded indrug release. The the hydrogels. red circles Created withrepresent the drugs BioRender.com loadedon (accessed in15 theJanuary hy- 2021). drogels. Created with BioRender.com (accessed on 15 January 2021). As an example, ferromagnetic vortex-domain iron oxide nanorings (FVIOs) were in- Ionic strength-sensitive hydrogels refer tohydrogel corporated into chitosan-PEG-based hydrogels that with loaded suffer conformational doxorubicin. Both in vitro and in vivo studies changes in response demonstrated to cations such as K+a, Na synergic +, and therapeutic Ca2+ (Figureefficacy 5). Theyof are chemotherapeutic generally mecha- produced withnism of doxorubicin ionizable and thermal and zwitterionic activity polymers suchof FVIOs upondeacetylated as alginate, application of a magnetic field. gellan After tumor gum, carboxymethyl surgical dextran, removal acid), poly(acrylic and formulation poly(itaconicadministration was possible acid), sulfobetaine and car-to observe al- most complete inhibition of tumor growth 21 days post-surgery, boxybetaine derivatives, polypeptides, among others [11,62]. Up to now, few ionic showing the efficacy of the system in strength-responsive reducingas hydrogels tumor recurrence platforms [60]. Further, for cancer treatment another example have been is a magnetic-sensitive proposed. As hydrogel an example, poly composed (L-glutamic by PEGylated Fe3 O4hydrogels acid-co-L-lysine)-based nanoparticles andwith loaded doxorubicinstabilized by α-cyclodextrin have shown a anPEGylated phospholipid ionic strength sensitivity encapsulated by an increasewith paclitaxel in the swelling andratiodoxorubicin of the hydro- [61]. The rhe- gel proportional to the increase of the ionic strength of the surrounding medium [63]. At surgical site ological properties of the hydrogel allow to easily inject the hydrogel at the after tumoral high ionic strength, Cl− andresection, Na+ shieldwhich facilitates the NH local 3+ and the COOdelivery of its dual − polypeptidic cargo groups due to magne- pre- tocaloric gel-to-sol transition under application of a magnetic field. venting electrostatic interactions and promoting hydrogel swelling. The presented formu- In vivo experiments in 4T1-tumor bearing Balb/c mice demonstrated lation also presented pH and enzymatic sensitiveness [63]. an improvement in the survival rate and reduction in tumor recurrence of animals treated with the proposed hydrogel [61]. Ionic strength-sensitive hydrogels refer to hydrogels that suffer conformational changes in response to cations such as K+ , Na+ , and Ca2+ (Figure 5). They are generally pro- duced with ionizable and zwitterionic polymers such as alginate, deacetylated gellan gum, carboxymethyl dextran, poly(acrylic acid), poly(itaconic acid), sulfobetaine and carboxybetaine derivatives, polypeptides, among others [11,62]. Up to now, few ionic strength-responsive hydrogels as platforms for cancer treatment have been proposed. As an example, poly (L-glutamic acid-co-L-lysine)-based hydrogels loaded with doxoru- bicin have shown an ionic strength sensitivity by an increase in the swelling ratio of the hydrogel proportional to the increase of the ionic strength of the surrounding medium [63]. At high ionic strength, Cl− and Na+ shield the NH3+ and the COO− polypeptidic groups preventing electrostatic interactions and promoting hydrogel swelling. The presented formulation also presented pH and enzymatic sensitiveness [63]. 4.4. Dual-Responsive Hydrogels Dual-responsive hydrogels, namely thermo- and pH sensitive hydrogels have at- tracted high attention in the biomedical field due to their wide range of possible applica- tions and functionalities. In the case of cancer, it is of great interest a system able to be easily injected that suffer a sol-to-gel transition in situ in response to body temperature and promote drug release at specific tumoral pH. For instance, a dual thermo- and pH-sensitive injectable hydrogel based on chitosan with poly(N-isopropylacrylamide-co-itaconic acid) was developed for delivery of doxorubicin for breast cancer treatment [64]. Due to the protonation of the amino groups of chitosan at acidic pH, doxorubicin was released more Cancers 2021, 13, 1164 13 of 17 rapidly at the pH 5.5 than at pH 7.4. The in vitro cytotoxicity studies demonstrated cyto- compatibility of the aforementioned hydrogels and the enhancement of the doxorubicin activity against MCF-7 breast cancer cells [64]. Another formulation with similar proper- ties was proposed based on a nano-hydrogel of lysine-modified poly(vinylcaprolactam) loading doxorubicin [65]. Lysine was conjugated with poly(vinylcaprolactam) via re- versible addition-fragmentation chain transfer (RAFT) polymerization while doxorubicin was conjugated through Schiff-base reaction, responsible for the pH sensitiveness property. The maximum release of doxorubicin for 72 h was observed at the simulated tumor microen- vironment, namely 40 ◦ C and pH of 5 [65]. A crosslinker-free thermo- and pH-responsive hydrogel based on poly(ethylene glycol) methyl ether methacrylate and acrylic acid was proposed for oral delivery of 5-fluoracil in colorectal cancer treatment [66]. The swelling behavior of the proposed hydrogels shown to be highly dependent on the pH and temper- ature, with higher swelling at low temperatures, thus retaining the drug at 37 ◦ C, being the release profile mainly dependent on the pH. Contrary to what happens at pH 1.2 and 4, the hydrogel swells due to the electrostatic repulsion between the ionized carboxyl groups at pH > 6 and release the drug (<20% at pH 1.2 and ~50% at pH 7.4 for 37 ◦ C). This avoids drug release at the gastric environment and promotes drug availability in the intestinal Cancers 2021, 13, x lumen. Moreover, the developed hydrogel shown to be biocompatible and promote 13 of 17 a controlled release of 5-fluouracil presenting pharmacological activity against HepG2 tumor cells [66]. Figure 5. Schematic representation of ionic strength responsive hydrogels. Swelling of the hydrogel Figure 5. Schematic representation of ionic strength responsive hydrogels. Swelling of the+ hydro- occurs when the ionic strength of surrounding medium increases. O—Oxygen, Na —Sodium. gel occurs when the ionic strength of surrounding medium increases. O—Oxygen, Na+—Sodium. The The redredcircles circlesrepresent representthe thedrugs drugsloaded loaded in in the the hydrogels. hydrogels. Created Created with withBioRender.com BioRender.com(accessed (ac- on 15 January 2021). cessed on 15 January 2021). More recently,Hydrogels 4.4. Dual-Responsive it was proposed a system for co-delivery of granzyme B (GrB) and docetaxel (DOC) loaded in pH-sensitive mini micelles incorporated in a thermo-sensitive Dual-responsive hydrogel hydrogels, [67]. Micelles namely by were composed thermo- and pH sensitive poly (γ-glutamic hydrogels have(PGA- acid)-poly(L-histidine) at- tracted high attention in the biomedical field due to their wide range of PLH) and the hydrogel composed by poly (ethylene glycol)-poly(γ-ethyl-L-glutamate) possible applica- tions and copolymer diblock functionalities. In the case ofAfter (mPEG-b-PELG). cancer, it is from release of great interest aby the hydrogel system able to proteinase be degra- easily injected dation, micellesthatshown suffertoa be sol-to-gel transitionininthe able to penetrate situ in response tumor to body and release temperature the cargo at pH 5.5. and Moreover, it was demonstrated that both drugs were able to maintain activity and promote drug release at specific tumoral pH. For instance, a dual thermo- afterpH- lyso- sensitive injectable somal escape and hydrogel promote abased on chitosan synergic antitumorwith poly(N-isopropylacrylamide-co-ita- efficacy as improve the tumor inhibition conic acid)inwas capacity B16developed tumor-bearingfor delivery of doxorubicin female C57BL/6 mice modelfor breast [67]. cancer treatment [64]. Due to the protonation of the amino groups of chitosan at acidic pH, doxorubicin was released more rapidly at the pH 5.5 than at pH 7.4. The in vitro cytotoxicity studies demon- strated cytocompatibility of the aforementioned hydrogels and the enhancement of the doxorubicin activity against MCF-7 breast cancer cells [64]. Another formulation with sim- ilar properties was proposed based on a nano-hydrogel of lysine-modified poly(vi- nylcaprolactam) loading doxorubicin [65]. Lysine was conjugated with poly(vinylcapro- Cancers 2021, 13, 1164 14 of 17 Even most of the developed dual-responsive hydrogels are pH and thermo-sensitive, other types of dual responsiveness have been proposed. As an example, a novel formulation of a photo and thermo-responsive multicompartment hydrogel for cancer treatment [68]. The formulation is based on the triblock copolymer poly(N-isopropylacrylamide)-b-poly(4- acryloylmorpholine)-b-poly(2-((((2-nitrobenzyl)oxy)carbonyl)amino)ethyl methacrylate) (PNBOC-b-PNAM-b-PNIPAM), which forms a PNBOC-cored micelles and consequently hydrogels after being exposed to high concentrations and temperatures. Because of both hydrophilic and hydrophobic domains, it was loaded with gemcitabine and doxorubicin. After administration, the hydrogel suffers a sol-to-gel transition retaining both drugs at the administration site. A significant drug release is obtained when UV irradiation is applied at the desired site of action due to sol-to-gel transition as a consequence of crosslinking of micellar cores and hydrophobic-to-hydrophilic transition [68]. 5. Conclusions The design of new strategies to obtain new and effective local cancer treatments, to improve the therapeutic outcome and to reduce the systemic toxicity associated with the conventional drugs, are of utmost importance. Hydrogels bring the possibility to create an in situ sustained and controlled delivery system of anticancer drugs at the tumoral site. In particular, stimuli-responsive hydrogels open a wide range of new options to specifically target the tumor site. Drug release at tumor site can be modulated by taking advantage of the specific physiologic characteristics in the tumor microenvironment, namely the differential extracellular and intracellular pH values. Moreover, the application of light, magnetic field, or even alterations in the ionic strength, can regulate the rate and location of drug release. Despite not being widely explored, these are promising approaches to the future of local cancer therapy, especially in the case of non-surgically resectable tumors. It is expected that in a near future new smart materials and new formulations designed and validated with this propose will hopefully enroll in clinical trials and reach the clinical practice. Author Contributions: F.A. writing—review and editing, writing—original draft preparation; M.M.R.-M., writing—review and editing, writing—original draft preparation; E.F.D.-L., writing—original draft preparation; D.R., conceptualization, writing—review and editing, writing—original draft prepara- tion; S.S.J., writing—original draft preparation. All authors have read and agreed to the published version of the manuscript. 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