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[Tianjiao Zeng](https://orcid.org/0000-0002-1286-0337), Lusi Chen, [Toru Yoshitomi](https://orcid.org/0000-0003-3847-1812), [Naoki Kawazoe](https://orcid.org/0000-0003-3916-0709), Yingnan Yang, [Guoping Chen](https://orcid.org/0000-0001-6753-3678)

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[Research Strategies and Methods of Hydrogels for Antitumor Drug Delivery](https://mdr.nims.go.jp/datasets/8be9b5d3-4d2a-4cae-8142-157c0bc96a63)

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Research Strategies and Methods of Hydrogels for Antitumor Drug DeliveryAcademic Editors: Ilaria Ottonelliand Ali NokhodchiReceived: 27 June 2025Revised: 25 July 2025Accepted: 1 August 2025Published: 4 August 2025Citation: Zeng, T.; Chen, L.;Yoshitomi, T.; Kawazoe, N.; Yang, Y.;Chen, G. Research Strategies andMethods of Hydrogels for AntitumorDrug Delivery. Biomedicines 2025, 13,1899. https://doi.org/10.3390/biomedicines13081899Copyright: © 2025 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license(https://creativecommons.org/licenses/by/4.0/).ReviewResearch Strategies and Methods of Hydrogels for AntitumorDrug DeliveryTianjiao Zeng 1 , Lusi Chen 1,2, Toru Yoshitomi 1 , Naoki Kawazoe 1 , Yingnan Yang 3 and Guoping Chen 1,2,*1 Research Center for Macromolecules and Biomaterials, National Institute for Materials Science,Namiki 1-1, Tsukuba 305-0044, Ibaraki, Japan; zeng.tianjiao@nims.go.jp (T.Z.); lschen.1214@gmail.com (L.C.);yoshitomi.toru@nims.go.jp (T.Y.); kawazoe.naoki@nims.go.jp (N.K.)2 Graduate School of Science and Technology, University of Tsukuba, Tsukuba 305-8577, Ibaraki, Japan3 Graduate School of Life and Environment Science, University of Tsukuba, 1-1-1 Tennodai,Tsukuba 305-8572, Ibaraki, Japan; yo.innan.fu@u.tsukuba.ac.jp* Correspondence: guoping.chen@nims.go.jp; Tel.: +81-029-860-4496AbstractTumor treatments have substantially advanced through various approaches, includingchemotherapy, radiotherapy, immunotherapy, and gene therapy. However, efficient treat-ment necessitates overcoming physiological barriers that impede the delivery of therapeuticagents to target sites. Drug delivery systems (DDSs) are a prominent research area, particu-larly in tumor therapy. This review provides a comprehensive overview of hydrogel-basedDDSs for tumor treatment, focusing on the strategies and designs of DDSs based on theunique pathophysiological characteristics of tumors. The design and preparation of hydro-gel systems for DDSs are summarized and highlighted. The challenges and opportunitiesfor translating hydrogel-based DDSs into clinical applications are discussed.Keywords: hydrogel; drug delivery system; antitumor therapy; combination therapy1. IntroductionDrug delivery systems (DDSs) have attracted considerable attention, particularly inthe context of antitumor treatments. A DDS is a formulation or device that enables theintroduction of therapeutic substances into the body and improves treatment efficiencyand safety by controlling the rate, timing, and location of drug release [1]. The sustaineddrug mechanism of DDSs is more conducive to eliminating tumor cells compared withthat using free drug administration [2–4]. Furthermore, during antitumor therapy, theDDS improves local drug accumulation, which enhances therapeutic efficacy and reducesoff-target toxicity to normal cells, thus alleviating safety issues [5,6].Currently, a variety of carriers are used in DDSs for tumor therapy, including lipo-somes, nanoparticles (NPs), and hydrogels [7–9]. Among them, liposomes have achievedclinical success and several formulations have already been approved for use. For example,Doxil was the first liposomal product approved by the FDA for ovarian tumor treatmentin 1995. More recently, Vyxeos is a new liposomal product that combines two currentlyused chemotherapies (daunorubicin and cytarabine) for leukemia treatment [10]. Othernanocarriers, such as dendrimers and inorganic NPs, have also been explored for DDSapplications [11,12]. Despite the differences in their mechanisms and applications, mostDDSs focus on three primary goals: achieving controlled drug release, targeting specificrelease sites, and maintaining the therapeutic potency of drugs. However, liposomes andNPs have limitations, including low drug entrapment efficiency, endosomal entrapment,Biomedicines 2025, 13, 1899 https://doi.org/10.3390/biomedicines13081899https://doi.org/10.3390/biomedicines13081899https://doi.org/10.3390/biomedicines13081899https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://www.mdpi.com/journal/biomedicineshttps://www.mdpi.comhttps://orcid.org/0000-0002-1286-0337https://orcid.org/0000-0003-3847-1812https://orcid.org/0000-0003-3916-0709https://orcid.org/0000-0001-6753-3678https://doi.org/10.3390/biomedicines13081899https://www.mdpi.com/article/10.3390/biomedicines13081899?type=check_update&version=2Biomedicines 2025, 13, 1899 2 of 33and rapid clearance by the mononuclear phagocyte system, which ultimately diminishestheir antitumor efficacy [13–16]. Therefore, alternative biomaterials capable of enablingcontrolled drug release are required to overcome these drawbacks.Hydrogels, a class of water-swollen polymer networks composed of macromoleculescrosslinked either physically or chemically, are promising candidates for DDSs. Hydrogelshave received substantial attention as drug delivery devices in medical applications, espe-cially in antitumor treatment, owing to their high water content, excellent biocompatibility,combinatorial optimization potential, and adaptable structure.For example, hydrophilic drugs, such as vitamin C (Vit C), often face challengesdue to rapid excretion, limiting their therapeutic effectiveness. To address this issue,Zhang et al. developed amphiphilic vitamin C self-assembled nanofiber hydrogels toachieve controlled release. By chemically modifying Vit C with a hydrophobic alkyl chainlinked via an ester bond that is enzymatically degradable in vivo, hydrogels are formedthrough hydrogen bonding and hydrophobic interactions and can be used as Vit C depots.Approximately 75% of Vit C was released over 10 days, demonstrating a controllable releaseprofile and improved antitumor effects [17]. In another example, alginate–pectin hydrogelswere prepared using ionic crosslinking for the oral delivery of resveratrol (RES) to theintestines. Alginate hydrogels remained stable in acidic environments but degraded underalkaline conditions, whereas pectin enhanced the mechanical strength of the hydrogels.This approach protects RES from stomach degradation, enables targeted delivery to theintestine, and improves therapeutic outcomes [18].Moreover, hydrogels combined with liposomes can markedly enhance drug deliveryefficiency. Li et al. developed a multilevel DDS platform in which drugs were encapsulatedin liposomes and subsequently coated with thiolate chitosan to form a liposomal hydrogel.Hydrophobic curcumin was loaded into liposomes to enhance its solubility, enablinglocalized drug delivery. This system demonstrated superior antitumor efficacy comparedwith that of free drugs or liposomal drugs alone, effectively inhibiting tumor recurrenceand promoting tissue repair [19]. Hydrogels have been extensively studied as drug deliverydevices for disease treatment.Despite extensive research on hydrogel-based DDSs, most existing reviews have fo-cused on their fundamental properties and general applications [20–22]. Few reviews havefocused on the strategies and methods for designing hydrogels specifically for antitumordrug delivery. Hence, this review summarizes the strategies and preparation methodsfor hydrogels used in antitumor therapy, with an emphasis on common approaches forevaluating hydrogel-based DDSs both in vitro and in vivo (Figure 1).Biomedicines 2025, 13, 1899 3 of 33Figure 1. Schematic illustration of various strategies used to prepare hydrogel-based drug deliverysystems (DDSs) for antitumor therapy. The illustration was created using BioRender, https://app.biorender.com/ (accessed on 27 June 2025).2. Design of Hydrogel-Based DDSsHydrogel materials are broadly categorized as naturally derived, semisynthetic, andsynthetic polymers [23]. Naturally derived polymers such as collagen, gelatin, and fib-rin exhibit advantageous properties, including biodegradability, low cytotoxicity, and aminimal immune response in vivo. Additionally, many naturally derived polymers facil-itate cellular adhesion, making them suitable for applications in tissue engineering andlocalized DDSs [24–26]. Commonly used synthetic polymers exhibit good mechanicalfeatures, plasticity, and variable properties for on-demand applications [27,28]. Followingthis development [29], polymers frequently used to prepare hydrogel-based DDSs aresummarized in Table 1.https://app.biorender.com/https://app.biorender.com/Biomedicines 2025, 13, 1899 4 of 33Table 1. Polymers and preparation methods for hydrogel-based DDSs after 2019.Polymers LoadingDrugs Preparing Method Degradation and Stability SterilizationMethodIn Vitro/In VivoAnalysisBiocompatibility ReferenceNaturallyderivedpolymersGelatin and alginate Doxorubicin(DOX)Chemicallycrosslinking by“Shift-Base”condensation reaction/No descriptionwhile Schiff base haspart ofantibacterial effectIn vitroGood biocompatibility (lessthan 10% decrease in cellviability of no-drug-loadinghydrogel)[30]Bisphosphonate-functionalizedhyaluronic acidDOX Chemical interaction ofbisphosphonate–zinc60% mass decrease indrug-loading hydrogel after316 h in medium/ In vitroand in vivoGood biocompatibility (lessthan 5% decrease in cellviability of no-drug-loadinghydrogel)[31]Alginate sodiumcarboxymethylcelluloseMethotrexate(MTX) andaspirin (AP)Physical crosslinking 42% mass decrease after8 days in PBS / In vitroGood biocompatibility (lessthan 5% decrease in cellviability of no-drug-loadinghydrogel)[32]Alginate andmagnetichydroxyapatite5-fluorouracil(5-FU) Physical crosslinkingIncreasing light andtemperature reduces thestorage stabilityFiltration In vitroGood biocompatibility (lessthan 15% decrease in cellviability of no-drug-loadinghydrogel)[33]SyntheticpolymersPoly (D, L-lactide-co-glycolide)-poly(ethylene-glycol)-poly (D, L-lactide-co-glycolide),beta-cyclodextrinDOX andcurcumin Physical crosslinking 13.3% mass decrease after24 h in PBS / In vitroand in vivoGood biocompatibility (lessthan 10% decrease in cellviability of no-drug-loadinghydrogel)[34]Methacrylicacid (MAA)Camptothecin(CPT)Chemicallycrosslinking throughdisulfide linkagebetween CPTand MAAStable in normalphysiological environment;quickly shrinks to a smallervolume in low pHenvironment of tumortissue or tumor cells/ In vitroand in vivoGood biocompatibility (lessthan 20% decrease in cellviability of no-drug-loadinghydrogel)[35]Polyethyleneglycol (PEG)DOX andcurcuminChemicallycrosslinking Instability / In vitroGood biocompatibility (lessthan 10% decrease in cellviability of no-drug-loadinghydrogel)[36]Biomedicines 2025, 13, 1899 5 of 33Table 1. Cont.Polymers LoadingDrugs Preparing Method Degradation and Stability SterilizationMethodIn Vitro/In VivoAnalysisBiocompatibility ReferenceSyntheticpolymersPolyacrylamide (PAM)and carbon nanotube DOXPhysicallycrosslinking byhydrogen bondStable in PBS after 80 h / In vitroGood biocompatibility (lessthan 20% decrease in cellviability of no-drug-loadinghydrogel)[37]Combinationand/ormodifiedpolymersChitosan, polypyrrole DOXChemicallycrosslinking by“Shift-Base”condensation reactionStable in PBS (both pH 7.4and 6.5) after 7 days / In vitroand in vivoGood biocompatibility (lessthan 3% decrease in cellviability of no-drug-loadinghydrogel)[38]PEG-modified bovineserum albuminPaclitaxel(PTX)Physical crosslinkingof PEG-BSA46% mass loss in PBS after50 days. Degraded completelywithin 200 days in vivo/ In vitroand in vivo No toxicity of hydrogel [39]Carboxymethylarabinoxylan 5-FU Physicallycrosslinking5 to 15% mass decrease after7 days in PBS, 37 ◦CThe nanocompositehydrogel hasantibacterial activityIn vitro Degradation leadsto cell death [40]Carboxymethylchitosan andpolyvinyl alcoholOxaliplatinChemicallycrosslinking byfree-radicalpolymerization/ / In vitroand in vivoGood biocompatibility byacute oral toxicity assay [41]Ultrasmall peptide DOX Dimerization of thePyKC peptideHigh stability after 18 daysin vivo / In vitroand in vivoGood biocompatibility (lessthan 5% decrease in cellviability of no-drug-loadinghydrogel)[42]Thiol-modifiedhyaluronic acid(HASH) and vinylsulfone-modifiedβ-cyclodextrinDOXChemicallycrosslinking of a “clickreaction” betweenthiol and vinylsulfone groupsKeep stable after 15 days inPBS while degraded after 5days in enzymaticconditions/ In vitro Good biocompatibility [43]N-isopropylacryl-amide (NIPAAm) andmaleic anhydride(MA) copolymer (poly(NIPAAm-co-MA))chitosanMTX andcurcumin Physical crosslinking Over 80% weight loss after28 days in PBS / In vitroGood biocompatibility (lessthan 10% decrease in cellviability of no-drug-loadinghydrogel)[44]Biomedicines 2025, 13, 1899 6 of 33Although simple polymer hydrogels have been successfully used in DDSs, hybridhydrogels composed of combinations of different polymers provide enhanced versatil-ity [45]. Regardless of the type of polymer used to prepare the hydrogels, environment-responsive functionalities and cargo-specific designs are the two key considerations thatdictate polymer selection and modification to achieve efficient and safe drug delivery inclinical applications.2.1. Stimulus-Responsive Drug Delivery HydrogelsThe tumor microenvironment (TME) significantly differs from normal tissues andis characterized by unique features, such as low pH, hypoxia, thermal sensitivity, andeven different stiffness or viscosity of the extracellular matrix (ECM) [46]. These distinctproperties of the TME not only influence the therapeutic response and clinical outcome [47]but also provide opportunities to design materials that specifically target the TME and phys-iological conditions, thereby improving safety and therapeutic efficacy [48]. Accordingly,stimuli-responsive materials have been extensively studied for DDSs.Using the acidic pH of the TME to develop pH-responsive hydrogels is one of the mostwidely studied strategies (Table 2). The extracellular pH in tumor sites typically rangesfrom 6.5 to 7.0, caused by the high metabolic rate of tumors [49], whereas the pH value ofnormal tissues maintains a pH of approximately 7.4 [50]. pH-sensitive hydrogels exploit thisdifference to enhance drug release, specifically at tumor sites, thereby improving localizeddrug concentration and minimizing systemic toxicity. For instance, Qu et al. developeda hydrogel for the delivery of doxorubicin (DOX), a commonly used first-line antitumoragent. The hydrogel was synthesized using N-carboxyethyl chitosan (CEC) via the Michaelreaction in an aqueous solution, combined with dibenzaldehyde-terminated poly (ethyleneglycol) (PEGDA). Under acidic conditions, the protonated and positively charged aminogroups of chitosan weaken the Schiff base bonds between -NH2 and -CHO, acceleratinghydrogel degradation and enabling faster drug release in an acidic environment than in aneutral environment [51]. Qing et al. designed a hydrogel using dopamine, carboxymethylcellulose (CMC), and hydroxyethyl cellulose to deliver the antibacterial drug, ciprofloxacin(CIP). In this system, the hydrolysis of the amide bond between CMC and CIP is theprimary mechanism controlling drug release and is highly sensitive to pH changes [52].Mukherjee et al. demonstrated that acidic environments not only increased the swellingratio of the hydrogel but also facilitated the breakage and degradation of ester bonds withinthe polymer matrix, thus enhancing drug release [53].The design of pH-responsive hydrogels typically aims to modulate the drug releaseprofile by accelerating hydrogel degradation or weakening the crosslinking bonds of thehydrogel under acidic conditions (Table 2). By targeting the mildly acidic pH of the TME,these hydrogels can effectively concentrate drugs at tumor sites and help reduce systemicside effects. However, the sensitivity of some hydrogels to pH changes is a critical chal-lenge. Some of the drug release curves exhibited significant differences with physiologicalconditions (pH around 7.4) only when the pH value decreased to 4.0, or even lower [52–55],which is much lower than the typical pH range of the TME (pH around 6.5 to 7.0). Suchlimitations may hinder their effectiveness, as the pH at most tumor sites may not be suf-ficiently low to induce drug release, potentially compromising therapeutic outcomes orcausing off-target effects.Biomedicines 2025, 13, 1899 7 of 33Table 2. pH-sensitive hydrogels for DDSs.LoadingDrugs Polymers DDS MechanismDrugReleasepHDegradation and Stability Sterilization MethodIn Vitro/In VivoAnalysisReferenceDOXNitrogen-doped carbonquantum dots andhydroxyapatiteDisintegration of the Schiffbase bond and dissolution ofhydroxyapatite inacidic conditions6.5–4.0 /The introduction of HA byeither chemical bonding orphysical incorporation canresult in hydrogels withenhanced antimicrobialactivityIn vitro [56]DOXN-carboxyethyl chitosan (CEC)and Di benzaldehyde-terminated poly (ethyleneglycol) (PEGDA)Disintegration of the Schiffbase bond 6.8–4.0Around 30% mass loss in PBS 7.4and 50% mass loss in PBS 5.5after 200 h/ In vitro [51]DOX Sericin, rice bran albumin, andgellan gumDegradation in the ester bondand increase in the swellingratio of the hydrogel5.0–4.0 / / In vitro [53]Prospidine Dextran phosphateDiffusion of the cytostaticbond and the destruction ofthe polymer4.0–1.2 100% mass loss after 30 days inPBS / In vitro andin vivo [54]DOX Glycol chitosan–Pluronic F127and α-CD Dissociation of the hydrogel 5.0 100% mass loss after 220 to 260 hin PBS / In vitro andin vivo [55]BortezomibA ‘ABA’ triblock copolymer ofphenylboronicacid-functionalizedpolycarbonate/poly(ethylene glycol)Dissociations of boronatedester bond 5.8At pH 7.4, the BTZ release fromthe micelle/hydrogel compositeremained low at 7%, an acidicenvironment, ∼85% of BTZ wasreleased over 9 days/ In vitro andin vivo [57]DOX Dextran-based nanogel Disintegration of the Schiffbase bond 5.0–2.0 / / In vitro [58]DOX Carboxymethyl chitosan Hydrolysis of ortho ester bond 6.5–5.0Around 30% mass loss after 300 hin PBS 7.4 and around 40% massloss after 300 h in PBS 5.0/ In vitro [59]Gemcitabine,paclitaxel(PTX)OE peptide (VKVKVOVK-VDPPT-KVEVKVKV-NH2)Disruption of the 3D networkof peptide from beta-sheet torandom coil5.8 / / In vitro andin vivo [60]Drug release pH: the pH condition used to measure the drug release profile from the DDS. DDS, drug delivery system; DOX, doxorubicin.Biomedicines 2025, 13, 1899 8 of 33In contrast to pH-responsive hydrogels, thermosensitive hydrogels represent a class ofphysically stimuli-responsive hydrogels, designed with a particular focus on injectability.These hydrogels remain in a liquid state before injection and undergo gelation to the solidstate at physiological temperatures (37 ◦C) [61–64]. This property allows thermosensitivehydrogels to be directly injected into tumor sites, where they provide sustained drugrelease. The application of these hydrogels minimizes the toxicity caused by drug diffusionand, more importantly, avoids the extensive tissue damage caused by surgical implantation.Additionally, the swelling and collapsing processes of thermosensitive polymer networksnear their critical temperature have been used as a mechanism for drug delivery [65] dueto the fact that therapeutic agents are commonly encapsulated within hydrogels, whichresults in a drug release profile governed by constrained diffusion of the agents throughhydrogel networks [66].Poly (N-isopropylacrylamide) (PNIPAAm) is one of the most commonly used poly-mers for preparing thermosensitive hydrogels. The sol–gel transition is driven by reversiblechanges between the hydrated and dehydrated states of PNIPAAm as the temperaturecrosses its lower critical solution temperature (LCST), typically below 37 ◦C [67]. Liu et al.developed an alginate-g-poly (N-isopropylacrylamide) copolymer system by conjugatingPNIPAAm to alginate. This system remains dissolved in water or phosphate-bufferedsaline buffer at room temperature but self-assembles into a hydrogel when heated tobody temperature (37 ◦C). DOX loaded onto a hydrogel exhibited sustained drug releaseand achieved significant antitumor effects against multidrug-resistant AT3B-1 cells [61].Similarly, Luo et al. synthesized a PNIPAAm-coacrylic acid-g-F68 copolymer hydrogelfor triptolide delivery. This hydrogel maintained a liquid state at room temperature andshowed a marked increase in storage modulus when the temperature exceeded 35 ◦C. Theantitumor agent, triptolide, was encapsulated in nanomicelles at room temperature andreleased intratumorally after injection. In animal models, the hydrogel exhibited superiorantitumor efficacy and low toxicity compared with that of free drug administration [62].In addition to the sol–gel translation behavior of thermosensitive hydrogels, theswelling and shrinking properties of hydrogels at their LCST have been widely usedto control drug release [65,68,69]. For instance, PNIPAAm-based hydrogels typicallyexhibit LCST below 37 ◦C, collapsing and shrinking above the LCST while swelling andexpanding below the LCST. This reversible swelling and shrinking behavior can be usedin DDSs to control drug release profiles [70]. Lei et al. further advanced the design ofthermosensitive hydrogels by developing a near-infrared (NIR)-triggered hydrogel for on-demand drug delivery. This thermosensitive chitosan (chitosan-g-PNIPAAm) polymer wassynthesized by grafting PNIPAAm onto chitosan (chitosan-g-PNIPAAm) via free-radicalcopolymerization, followed by UV-induced crosslinking with methacryloyl groups. Theresulting composite hydrogels exhibited significant shrinkage at elevated temperatures(45 ◦C). By incorporating photothermal carbon, the hydrogel could trigger the shrinkage ofpolymer networks upon NIR irradiation and thus release the loaded drug [71]. Althoughthe composite hydrogels exhibited NIR-triggered behavior, the burst release profile ofDOX following each exposure to 45 ◦C may negatively impact treatment outcomes, andthe lack of detailed experimental data limits the evaluation of its clinical applicability. Acritical mechanism underlying drug release control via the LCST is that below the LCST,the swelling of the hydrogel increases the diffusion distance for the encapsulated drugs,thus slowing their release. Conversely, above the LCST, the hydrogel network collapses,reducing diffusion resistance and accelerating drug release [65,70]. Therefore, it is essentialto clarify the diffusion rate of drugs at various environmental temperatures because highenvironmental temperatures may accelerate drug release, potentially leading to misleadingBiomedicines 2025, 13, 1899 9 of 33conclusions regarding hydrogel performance. A detailed investigation of these factors iscrucial for optimizing LCST-based hydrogel systems for clinical use.2.2. Cargo-Based Drug Delivery HydrogelsThe properties of hydrogels can be designed for DDSs using different materials; how-ever, the encapsulation efficiency and release rates of drugs from hydrogels are influencedby multiple factors, even when using the same hydrogel matrix. Properties, such as hy-drophilicity or hydrophobicity [72], electrostatic interactions [73], and molecular weight [74]significantly affect the drug delivery efficiency and antitumor efficacy. Consequently, thedesign and preparation of hydrogel-based DDSs must consider the physicochemical prop-erties of the drugs and their specific delivery environment.Reportedly, over 40% of marketed drugs exhibit poor water solubility, and approx-imately 60% of the compounds emerging from pharmaceutical research laboratories areclassified as insoluble [75]. Among the challenges of hydrogel-based DDSs, the delivery ofhydrophobic drugs via high-water-content hydrogels remains one of the most significantobstacles. Poor drug encapsulation efficiency and a low release rate often result in reducedbioavailability and therapeutic outcomes [76,77].Paclitaxel (PTX), a widely used first-line antitumor agent with broad-spectrum an-titumor effects, addresses these challenges. PTX functions as an anti-microtubule agent,binding specifically to the β-subunit of tubulin to inhibit microtubule disassembly, therebyarresting mitosis and suppressing tumor proliferation. In contrast, DOX hydrochloride(DOX · HCl), a water-soluble anthracycline, can bond to nucleic acids and form complexeswith DNA by intercalating to base pairs to inhibit the function of topoisomerase II ac-tivity and then present antimitotic and cytotoxic effects. Owing to the hydrophilic andhydrophobic differences between these drugs, their release behaviors in the same DDS aremarkedly different. For example, in a study by Xu et al., the cumulative release of PTX froma hydrogel was only approximately 10% after 24 h, whereas DOX showed a much fasterrelease and resulted in higher cytotoxicity against tumors than PTX after 48 h of culture.Conversely, the inhibition of cell proliferation by PTX was notably weaker even after 72 h,likely because of its low drug release rate [76]. Similarly, docetaxel (DOC), another taxanederivative with potent antitumor effects, is commercially available only as an intravenousformulation owing to its poor water solubility [78]. To address these limitations, strategies,such as combining hydrogels with liposomes, NPs, or microspheres, are commonly usedas delivery tools for hydrophobic drugs. Furthermore, modifying the water solubility ofdrugs is commonly used (Table 3).Amphiphilic polymers are particularly advantageous for delivering hydrophobicdrugs. Their ability to self-assemble via hydrophobic interactions facilitates hydrogelformation and enhances drug encapsulation within the hydrogel. Rezazadeh et al. encapsu-lated PTX into mixed polymeric micelles composed of PF127 and tocopherol polyethyleneglycol 1000 succinate (TPGS), which were subsequently incorporated into a hyaluronicacid (HA)-based hydrogel for the co-delivery of PTX and DOX. TPGS, with its amphiphilicstructure, formed micelles in aqueous media and achieved a PTX entrapment efficiencyof approximately 51%. Although the initial PTX loading affected the release rate, morethan 50% of the drug was released from the hydrogel within 60 h, demonstrating efficientdrug loading and release capabilities [79]. Although the study did not directly evaluate theantitumor efficacy of the hydrogel, subsequent in vitro and in vivo studies of similar DDSsdemonstrated significant therapeutic effects [80].The host–guest effect of cyclodextrin (CD)–drug complexes has been extensivelyexplored as a strategy to improve the water solubility of hydrophobic drugs. CDs are cyclicoligosaccharides composed of D-glucopyranoside units linked via alpha-1,4 glycosidicBiomedicines 2025, 13, 1899 10 of 33bonds. The most commonly used CDs are alpha-CDs, beta-CDs, and gamma-CDs, whichconsist of six, seven, and eight glucose repeating units, respectively. The arrangement ofthese molecular structures forms a hydrophobic inner cavity and hydrophilic exterior [81],enabling CDs to encapsulate hydrophobic guest molecules within the cavity, therebyenhancing their physicochemical properties and aqueous solubility [82–84].Nieto et al. used a host–guest reaction to load PTX into beta-CDs to form PTX:beta-CD inclusion complexes, which were subsequently encapsulated within a gellangum hydrogel. The hydrogel was synthesized in different buffer systems (acetate andphosphate) and crosslinked with varying concentrations of L-cysteine using the crosslinkerEDC/NHS to provide redox-responsive properties to glutathione (GSH). In drug releasestudies, approximately 50% of PTX was released from the hydrogel within 50 h underphysiological conditions. Furthermore, the presence of L-cysteine and GSH allowed foradjustable drug release rates, demonstrating promising control over drug delivery andsignificant antitumor efficacy in vitro [85]. In another study, Ma et al. developed a beta-CD/camptothecin inclusion complex that was incorporated into folic acid hydrogels. Thefolic acid was chemically modified with NaOH and then crosslinked with ZnCl2 using ametal-ion-induced gelation method. The prepared CPT-beta-CD complex was added to thefolic acid solution to form drug-loaded hydrogels [86]. This system leverages the selectiveaffinity of folic acid for tumor cells, enhancing the targeting and delivery efficiency of CPTwhile mitigating off-target effects.Notably, preclinical studies have demonstrated that CD–drug inclusion complexes of-ten exhibit superior therapeutic performance compared with that of free drugadministration [87–89]. However, most CD-based formulations are administered intra-venously rather than incorporated into hydrogels for local drug delivery. Intravenousadministration typically results in uncontrolled drug release, leading to systemic drugaccumulation in non-target tissues and organs, which increases the risk of toxicity [90,91].In contrast, combining CD–drug complexes with hydrogels provides an opportunity toleverage the sustained and localized release characteristics of hydrogels. This approach canenhance antitumor efficacy while minimizing systemic toxicity, particularly for hydropho-bic drugs that pose substantial formulation challenges.Furthermore, the application of CDs in local drug delivery commonly involves theformation of host–guest supramolecular hydrogels rather than the direct modification ofdrug properties. These supramolecular hydrogels have shown substantial potential fortumor therapy by providing tunable release kinetics, responsive behavior, and enhancedmechanical stability [92–94]. Combining drug complexes with hydrogel systems mayoptimize their therapeutic performance and clinical utility.Biomedicines 2025, 13, 1899 11 of 33Table 3. Hydrogels for hydrophobic drug delivery.HydrophobicDrugsMaterials to SynthesizeHydrogelsDrug-LoadingMechanismDrugEntrapmentEfficiencyDrug-LoadingEfficiency Degradation and Stability SterilizationMethodIn Vitro/In VivoAnalysisReferencesPTXMixed polymeric micellecomposed of PF127,tocopherol polyethyleneglycol 1000 succinate(TPGS), and hyaluronicacid (HA)Micelle formation byhydrophobicinteraction~51% ~12%Hydrogel maintained 75% of itsoriginal weight over 5 days at37 ◦C, low viscosity at 4 ◦Cconverted to a semisolid uponheating to 35 ◦C/[76] In vitro[77] In vitroand in vivo[79,80]PTX Gellan gum Formed CD: drugcomplex / / / Sterilized by UVradiation In vitro [85]PTX PEGylated star polymerand PNIPAAmFormed CD: drugcomplex ~90% ~3%Complex nanoparticlesexperienced fast sizebroadening underphysiological salt conditions(150 mM) within 30 min/ In vitro andin vivo [95]PTX Gellan gum modifiedby prednisoloneHydrophobicinteraction byprednisolone~40% / Good stability / In vitro [96]PTX IC1-R peptideBeta-folded structureformation byhydrophobicinteraction~98% /Parameters set to a frequency of0.1–100 rad/s, a sheer force of1%, and a test time of 15 min.Fixed frequency of 6.28 rad/s,0–5 min shear force set to 1%,and 5–7 min set to 100%. Theshear force was set to 1% at7–37 min, and the process wasrepeated at 37–70 minFiltration via0.22 µm filterIn vitro andin vivo [97]PTX,epirubicinHA and poly(e-caprolactone)-poly(ethylene glycol)-poly-(e-caprolactone)(PCL-PEG-PCL)Loading PTX toPCL-PEG-PCLnanoparticles~90% ~10%The HA-Gel embeddedsubcutaneously in micegradually degraded 3 dayspost-implantation, and theextent of degradation wassignificant on day 5. Almost noresidual hydrogel on day 9/ In vitro andin vivo [98]Biomedicines 2025, 13, 1899 12 of 33Table 3. Cont.HydrophobicDrugsMaterials to SynthesizeHydrogelsDrug-LoadingMechanismDrugEntrapmentEfficiencyDrug-LoadingEfficiency Degradation and Stability SterilizationMethodIn Vitro/In VivoAnalysisReferencesPTX PEG-PCL-PEG/DDP +MPEG-PCL/PTXLoading PTX bymonomethoxyPEG-PCL~99% ~4%When the temperatureexceeded 25 ◦C, the sampleschanged from a liquid to anelastic gel-like substance at thecrossover point of G and G; thecomposite’s viscosity was thestrongest at about 43 ◦CThe preparedPDMP hydrogelcomposite wassterilized usingCO-60 (20 Gy)before injectionIn vitro andin vivo [99]Curcumin Thiolated chitosan Encapsulated byliposomes ~88% ~4%[2] The deformation loss ratiowas 20.06% (CSSH Gel), 23.92%(100 µM), 20.95% (150 µM), and17.95 % (200 µM) afterfive cycles of compression [19].The best compressiveperformance is at 5%, thecompressive modulus of 5% is27.44 kPa, and the maximumcompressive strength is 32 kPa/ In vitro andin vivo [2,19]Curcumin Peptide (MAX8)Beta-hairpin structureformation byhydrophobicinteraction/ /Stable colloidal dispersionsystem and goodmechanical properties/ In vivo [100]CurcuminPolyethylene glycol (PEG)and polycaprolactone (PCL)polymer PCL-PEG-PCLHydrophobicinteraction byhydrophobic PCL47~74% 2~9% / / In vitro [101]Curcumin Alginate/chitosanhydrogel microparticlesLoading curcumin byhyaluronic acid/zein nanoparticles~70% /Cur/MPS in simulated gastricfluid (SGF) (pH 2) has noobvious change; NMPs candecompose rapidly incolonic fluid/ In vitro andin vivo [102]Camptothecin(CPT)Pluronic F127and alpha-CDsMicelle formationby hydrophobicinteraction/ / / Filtration via0.45 µm filterIn vitro andin vivo [92]Biomedicines 2025, 13, 1899 13 of 33Table 3. Cont.HydrophobicDrugsMaterials to SynthesizeHydrogelsDrug-LoadingMechanismDrugEntrapmentEfficiencyDrug-LoadingEfficiency Degradation and Stability SterilizationMethodIn Vitro/In VivoAnalysisReferencesCPT Folic acid and beta-CDs Formed CD:drug complex / /Good rheological mechanicalproperty and thermodynamicstability/ In vitro [86]Triptolide PNIPAm-g-pluronic F68Micelle formationby hydrophobicinteraction~84% ~5%0.45 mg/kg TPL-equivalentdose three times over 14 daysin 4T1 tumor-bearing miceFiltration via0.45 µm filterIn vitro andin vivo [62]DOX(DOX·HCldeprotonatedat pH 9.6)PEG-b-PCL and alpha-CDsMicelle formationby hydrophobicinteraction72~74% ~15% / / In vitro [103]DOCMixed polymeric micellecomposed of PF127, PL121,and hyaluronic acid (HA)Micelle formationby hydrophobicinteraction~99% ~2% / / In vivo [104]DOC, docetaxel; DOX, doxorubicin; PTX, paclitaxel.Biomedicines 2025, 13, 1899 14 of 33In addition to hydrophobic interactions and host–guest mechanisms, other advancedtools, such as NPs and liposomes, are commonly used in hydrogel-based DDSs [105,106].Compared with the individual use of these tools, integration with high-water-contenthydrogels for in situ delivery can provide dual benefits. The hydrogels enable localizedand controlled drug release and simultaneously serve as a postoperative filler, offeringadditional benefits, such as promoting tissue regeneration or reducing inflammation atsurgical sites.Compared with free drug administration, the application of hydrogels is expected tofulfill at least one of the following three primary functions: (1) mitigating burst release toextend the therapeutic window and improve treatment efficacy; (2) minimizing systemictoxicity by reducing the drug dosage or enhancing local drug accumulation, therebyimproving the survival curve; and (3) addressing drug incompatibility issues throughspatiotemporal control of drug release. The first two functions are easily understood;however, the third requires an understanding of the complexity of tumor therapy. It ishighly relevant to the common properties of tumors, including inherently heterogeneousand diverse genetic, epigenetic, and phenotypic variations that enable them to adapt anddevelop resistance to single-agent therapies. Moreover, not only can cancer cells developdrug resistance [107], but changes in the TMEs can contribute to single-drug resistance,thus leading to treatment failure (Figure 2) [108,109]. Therefore, combination therapy hasbeen widely adopted for the treatment of various tumors in clinical settings. Establishedclinical protocols, such as FOLFOX (folinic acid, fluorouracil, and oxaliplatin) for colorectalcancer (CRC) and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone)for non-Hodgkin lymphoma, exemplify the effectiveness of multi-agent regimens thatsignificantly improve patient outcomes [110,111].Figure 2. Extracellular matrix (ECM) stiffness affects chemoresistance of breast cancer cells todoxorubicin. Following encapsulation and culture in hydrogels, both cell viability and P-gp mRNAexpression were found to decrease in the soft hydrogel, whereas they increased in the hard hydrogel.Copyright: Guoping Chen. There is no copyright issue [108].Despite its advantages, combination therapy presents challenges, such as increasedcomplexity in pharmacokinetic and pharmacodynamic interactions, heightened risk ofoverlapping toxicities, and the need for precise dose optimization to prevent adverseeffects [112,113]. Therefore, the development of advanced DDSs, including hydrogel-based platforms, has become popular for ensuring the controlled and targeted release oftherapeutic agents in combination regimens.Biomedicines 2025, 13, 1899 15 of 332.3. Drug Delivery Hydrogels for Pathological Characteristics of TumorsThe distinct TME can influence drug delivery efficiency and is increasingly recognizedas a unique characteristic that can be used in the design of DDSs, such as hypoxia, acidity,ultrahigh GSH levels, and the overexpression of specific enzymes [114]. Another importantaspect is tumor heterogeneity, which also affects drug delivery and treatment efficiencyyet remains an unresolved challenge. Heterogeneity refers to the various genetic andnon-genetic phenotypes that emerge during tumorigenesis, progression, and treatment,as well as interpatient variability, even within the same tumor type [115,116]. Tumorvasculature is characterized by chaotic organization, high permeability, and substantialheterogeneity [117]. Additionally, cancer genotypes can vary across different microenviron-ments, possibly because of the different distributions of cytokines around tumor sites [118].Moreover, significant spatial discordance in programmed death ligand 1 (PD-L1) expres-sion has been observed between primary tumors and lymph node metastases in surgicallyresected gastroesophageal adenocarcinoma [119]. The heterogeneity of tumors not onlyreduces drug delivery efficiency by the disorganized vasculature [120] but also contributesto treatment failure through the emergence of drug-resistant cells and metastasis [121].It also results in conflicting therapeutic outcomes and the unpredictability of clinicalprognosis [119]. In single-agent DDSs, tumor heterogeneity leads to difficulties in precisetargeted therapy [122], thereby compromising the effectiveness of hydrogel-based DDSsand diminishing therapeutic returns. Therefore, the development of combination therapystrategies using hydrogels has become a prevailing direction in efforts to enhance treatmentefficacy [123–125].2.3.1. DDSs of Cytotoxic DrugsDOX is a model drug widely used in DDSs and acts via multiple mechanisms [126].Despite its effectiveness, the non-negligible cardiotoxicity of both dose- and schedule-dependent DOX significantly limits its clinical application to a substantial extent [127].Additionally, short-term treatment with DOX demonstrated a low antitumor effect [128];therefore, long-term treatment is recommended but is associated with cumulative toxic-ity, followed by reduced therapeutic utility and eventual exhaustion of antitumor effec-tiveness, as evidenced by tumor regrowth typically observed at approximately 20 dayspost-administration in most in vivo experiments [129–131]. Collectively, efforts to mitigatethe systemic toxicity of DOX, such as reduction or altered administration schedules, showpotential, but often compromise the required therapeutic efficacy. Conversely, high-doseadministration enhances antitumor outcomes but exacerbates toxicity [127,130,132]. Tobalance these competing factors, the combination of DOX with other antineoplastic agentshas emerged as a promising strategy, particularly for hydrogel-based DDSs (Table 4).Zhao et al. developed an injectable hydrogel system composed of glycol chitosan(GC) and amphiphilic hydrogels (OHC-PEO-PPO-PEO-CHO) to co-deliver DOX and PTX.The system showed a burst release of DOX and a sustained release of PTX owing todifferences in their water solubility. Although the combination of DOX and PTX did notyield significantly enhanced antitumor efficiency, which is likely due to the asynchronousrelease profiles of the two drugs, the toxicity profile was not exacerbated by the prolongedsurvival time in comparison with a single DDS [133]. In another study, PTX and DOXwere loaded with PECT and PEPF, respectively, and NPs prepared using nanoprecipitationtechnology were incorporated into hydrogels. A hydrogel containing drug-loaded NPswas then synthesized and achieved better antitumor efficacy and reduced systemic toxicitycompared with free drug administration [134].In addition to DOX, cisplatin is another important chemotherapeutic agent used totreat various malignancies, including sarcoma, carcinoma, and lymphoma. Cisplatin,Biomedicines 2025, 13, 1899 16 of 33or cis-diamminedichloroplatinum (II) (CDDP), is a platinum-based chemotherapeuticagent that exerts antitumor effects by forming DNA crosslinks, thereby disrupting DNAsynthesis and transcription to induce tumor cell apoptosis. Despite its antitumor effects, thewidespread uptake of CDDP by both normal and tumor cells via ubiquitously expressedtransporters results in significant side effects, thereby limiting its application [135]. Forinstance, the specific expression of organic cation transporter-2 (OCT2) in the kidneys mayexplain the nephrotoxicity of CDDP [136,137]. The high water solubility and rapid burstrelease of CDDP at the time of administration further contribute to its systemic toxicityand suboptimal antitumor efficacy. Hydrogel-based DDSs have been used to addressthese challenges by retarding the drug release rate and enabling the localized deliveryof drugs to tumor sites, thus improving therapeutic outcomes while avoiding off-targeteffects [138–140]. For example, a study by Li et al. demonstrated that incorporating CDDPinto thermosensitive hydrogels not only provided controlled and sustained release butalso enhanced tumor suppression while significantly reducing renal toxicity in animalmodels [141].As for the co-administration of CDDP and other therapeutic agents, Chen et al. devel-oped a co-delivery system using a biodegradable temperature-sensitive hydrogel (PDLLA-PEG-PDLLA, PLEL) for combination chemotherapy of gastric tumors. The thermosensitivehydrogel not only enhanced local combination therapy with 5-fluorouracil (5-FU) andCDDP but also demonstrated significantly better antitumor efficacy than that of the free ad-ministration of 5-FU and CDDP. Moreover, the hydrogel promoted the local accumulationof 5-FU and CDDP. Moreover, the hydrogel system promotes the localized accumulation ofdrugs, reduces systemic exposure, and extends the overall survival time of treated animalswith minimal tissue damage and reduced systemic toxicity [138]. Similarly, Shen et al.investigated a self-assembled poly (ethylene glycol) (PEG)/polyester copolymer hydrogelfor the co-delivery of CDDP and PTX to ovarian tumor models. This hydrogel-based DDSdemonstrated low systemic toxicity, as evidenced by minimal weight loss in the exper-imental animals. Although PTX showed a slower release rate than that of CDDP, bothdrugs maintained a steady release profile throughout the study, contributing to enhancedantitumor efficacy through sustained therapeutic concentrations at the tumor site [139].To further enhance antitumor efficiency, other chemotherapeutic agents with distinctmechanisms, such as topotecan, SN-38, and 5-FU, have also been explored in hydrogelcarrier systems [142–145]. These studies highlighted the multiple advantages of hydrogel-based DDSs for combination therapy. First, hydrogels mitigate burst release effects andreduce the systemic toxicity associated with free drug administration. By tailoring the hy-drophilic and hydrophobic properties of the hydrogel matrix, controlled and localized drugdelivery can be achieved, thereby improving the therapeutic index. Second, the differentialsolubility of the co-administered drugs can be leveraged to regulate their overall concentra-tions at the target site, ensuring sustained exposure and enhanced synergistic effects.Additionally, hydrogels enable the sequential or simultaneous delivery of multiple drugs,facilitating combination therapies that synergistically exploit distinct antitumor mechanisms.This approach is particularly effective for overcoming drug resistance because it disruptsadaptive cellular responses and maintains therapeutic pressure. Furthermore, hydrogels canbridge pharmacokinetic gaps in drug metabolism cycles by providing controlled release andpreventing therapeutic windows in which tumor cells can recover or proliferate.By co-delivering hydrophilic and hydrophobic drugs on a single platform, hydro-gel systems can maximize the efficacy of chemotherapeutic agents while minimizingoff-target effects. These systems exemplify a powerful strategy for achieving preciseand effective combination chemotherapy, paving the way for personalized and tolerablecancer treatments.Biomedicines 2025, 13, 1899 17 of 33Table 4. Combinations of different antineoplastic agents.Tumor Drugs Materials to SynthesizeHydrogels Effects ReferenceMelanoma DOX and PTXGlycol chitosanand benzaldehyde-terminated polymerHigh antitumor efficacyand safety [133]Colon carcinoma DOX and DOC Micelles by PL121, PF127,and HAHigh antitumor efficacyand safety [104]Bladder carcinoma DOX and CDDP PEG-b-PCLand alpha-CDsControllable drug releaseand injectable ability [103]Hepatoma DOX and Curcumin PCL-PEG-PCL High antitumor efficacyand safety [101]Lung tumor PTX and CDDP PEG-PCL-PEGand PMEG-PCLHigh antitumor efficacy,controllable drug release,and safety[99]Ovarian tumor PTX and CDDP Bi-mPEG-PLGA-Pt (IV) High antitumor efficacyand safety [139]Breast tumor PTX andEpirubicin (EPI) PCL-PEG-PCL and HAHigh antitumor efficacy,controllable drug release,and safety[98]Breast tumor PTX andGemcitabine (GEM) OE peptide hydrogel High antitumor efficacy [60]Breast tumor PTX and Honokiol PLGA-PEG-PLGA High antitumor efficacyand safety [146]Ovarian tumor PTX, Rapamaycin,LS301 PLGA-b-PEG-b-PLGA High antitumor efficacy [147]Colorectal peritonealcarcinomatosis CDDP and 5-FURing-openingcopolymerization tosynthesize ε-CL and PEGand chitosanHigh antitumor efficacyand safety [145]Gastric tumor CDDP and 5-FU PDLLA-PEG-PDLLAHigh antitumor efficacy,safety, and high efficacy toinhibit tumor recurrence[138]Pancreatic tumor CDDP and GEM PDLLA-PEG-PDLLA High antitumor efficacy [148]Colorectal tumor Oxaliplatin andHesperetin Cationic Okra gum High cytotoxicity [149]Brain tumor Carmustine andCurcumin PCL-PEG High efficacy to inhibittumor recurrence [150]DOC, docetaxel; DOX, doxorubicin; HA, hyaluronic acid; PTX, paclitaxel.2.3.2. DDSs of Anti-Angiogenesis DrugsGenerally, tumor growth is characterized by uncontrolled proliferation, which leads toa high metabolic demand, rapid nutrient consumption, and increased production of wasteproducts. Unlike blood cancers and other nonsolid tumors, the growth of solid tumorsgenerally requires vascularized connective tissue stroma to sustain expansion beyondtheir minimum size [151]. It is widely accepted that the vasculature in tumors is chaoticwith high permeability and heterogeneity compared with that in normal tissues [152,153].As tumors rely on angiogenesis for their continued growth and metastasis, angiogenesistherapy has been recognized as a critical target in tumor treatment, promoting the devel-Biomedicines 2025, 13, 1899 18 of 33opment of numerous anti-angiogenic agents, such as bevacizumab, cediranib, axitinib,and regorafenib [154,155]. Other anti-angiogenic agents, such as inhibitors of tyrosinekinase receptors, including sunitinib, sorafenib, and pazopanib, have found broad clinicalapplications [156]. Anti-angiogenic strategies transiently normalize tumor vasculatureand enhance the delivery and efficacy of chemotherapeutic agents [157,158]. Furthermore,targeting tumor angiogenesis facilitates metronomic chemotherapy, a regimen of chronic,low-dose drug administration aimed at preventing angiogenesis and inducing sustainedantitumor effects [159]. According to these theories, the combination of anti-angiogenesisand chemotherapy has become a widely adopted therapeutic model [160].To address the toxicity and limited efficiency of anti-angiogenesis therapies, hydrogelshave been explored for localized drug delivery and the controlled release of the drugs.Li et al. used a classic injectable thermosensitive PLGA-PEG-PLGA hydrogel to co-deliverthe vascular disruptive agents, combretastatin A-4 (CA4) disodium phosphate (CA-4DP)and epirubicin. Combination therapy not only exhibited superior antitumor effects com-pared with that of single-drug administration but also prolonged the survival of treatedanimals [161].CA4P is a water-soluble prodrug of CA4, which demonstrates robust antitumor effi-cacy, particularly in combination therapies for platinum-resistant ovarian tumors. This is amechanism that induces vascular congestion and reduces tumor blood flow by changing theendothelial cell morphology to achieve treatment outcomes [162]. However, the vasculareffects of CA4 can negatively affect the efficacy of concurrently administered drugs, whichnecessitates high requirements for controlling the sequence and timing of drug release [163].Wei et al. addressed this challenge by designing a polypeptide-based hydrogel capable ofsequentially releasing CA4 and DOX. By utilizing the distinct hydrophobic properties ofthese drugs, their hydrogel facilitated the faster release of DOX and the slower release ofCA4, minimizing potential negative interactions and optimizing therapeutic efficacy [63].Conversely, Wang et al. reported a different release profile using an injectable PLGA-PEG-PLGA triblock polymer hydrogel. They found that CA4P, which is more hydrophilic thanDOX, was released at a faster rate. Despite this reversed release sequence, combination ther-apy yielded superior antitumor effects compared with single-drug treatments, highlightingthe versatility and potential of hydrogel-based co-delivery systems [164].Regardless of the agent that should be released first, it is important to avoid druginteractions with anti-angiogenic and antineoplastic agents. Owing to the different mecha-nisms of anti-angiogenic and antineoplastic agents, the design and synthesis of hydrogelsmust be considered. In addition to enhancing the antitumor effect, avoiding high toxicity,preventing negative drug interactions, and the time-dependent induction of drug resistanceby anti-angiogenic therapies [165,166] increase requirements for hydrogel-based DDSs. Thepreparation of smart hydrogels for the combination of antineoplastic and anti-angiogenicagents is urgently required.2.3.3. DDSs of Immune Checkpoint InhibitorsCancer treatment includes various strategies beyond conventional chemotherapy, suchas surgery, radiotherapy, immunotherapy, and gene therapy. Combination therapy lever-ages the complementary mechanisms of different approaches to enhance efficacy, mitigateresistance, and improve overall outcomes. In the case of advanced non-small-cell lungcancer, the combination of immune checkpoint inhibitors with chemotherapy in a first-linetreatment prolongs the survival of patients in phase III trials [167]. Hydrogel-based DDSscan facilitate the combination of cytotoxic drugs for cancer treatment and other treatmentapproaches by providing controllable and localized release of different therapeutic agents.As an example of chemotherapy combined with immunotherapy, Chao et al. developed anBiomedicines 2025, 13, 1899 19 of 33alginate-based hydrogel to deliver the chemotherapeutic agent oxaliplatin (OXA) and im-mune adjuvant imiquimod (R837) as a “cocktail” of chemoimmunotherapeutic composites.The composite was administered intratumorally to form a hydrogel for localized treatment.In addition, an immune checkpoint blockade antibody (anti-PD-1) was included in theinjection solution to enhance local immune modulation. The combination of OXA, R837,and anti-PD-1 within the hydrogel matrix showed superior antitumor efficiency comparedwith that of the free administration of these agents [168]. In another study, a nanogel wasdesigned for combined chemoimmunotherapy by crosslinking carboxymethyl chitosan-derived polymetformin loaded with DOX. This system efficiently inhibited tumor growth,as polymetformin reshaped the TME and DOX killed the tumor cells more effectively [169].Owing to the complexity of immune responses, designing hydrogels for combiningchemotherapy and immunotherapy often requires meticulous consideration of both drugrelease profiles and treatment sequences. Animal studies typically involve multifacetedvalidation to assess the additive or synergistic effects of therapeutic strategies. Sun et al.used immunogenic cell death (ICD) to develop a hydrogel capable of administering im-mune adjuvants to tumors in a controlled manner during each cycle of chemotherapy andradiotherapy. The hydrogel is designed to release immunotherapeutic factors in responseto low doses of oxaliplatin and X-rays. This approach results in a marked synergistic effectbetween chemotherapy and immunotherapy. By avoiding the simultaneous administrationof chemotherapy and immunotherapy, this design effectively reduces systemic toxicity.More importantly, because ICD is induced by the release of chemotherapeutic agentsor radiation therapy, it leads to the generation of a robust immune response, which hasdemonstrated a potent therapeutic effect in animal models. Hydrogel-based DDSs havebeen extensively reported as a means of minimizing the adverse effects associated withsystemic immunotherapy. These systems facilitate the controlled release of therapeuticagents, thereby improving both safety and efficacy [170].From a fundamental perspective, one of the most valuable attributes of hydrogel-basedDDSs in combination therapies is their ability to control drug release based on the distinctsolubility profiles of the incorporated drugs. Engineering hydrogels with appropriateand tunable affinities for specific drugs may be critical to achieve optimal DDS perfor-mance. Other types of hydrogels, such as hydrophobic hydrogels [171], tough adhesivehydrogels [172], and specialized formulations, are also under investigation. Owing totheir high elasticity, water content, plasticity, and tunable gelation properties, hydrogelsrepresent an indispensable DDS platform with immense potential for research and clinicalapplications. Furthermore, hydrogels can be used to carry NPs and serve as fillers fortreatments. Magnetic NPs exhibit stronger thermal effects in hydrogels than conventionalscaffold materials (Figure 3) [173], making hydrogels not only effective DDSs but also po-tentially transformative as post-surgical fillers with enhanced therapeutic outcomes. Thismultifunctionality broadens the scope of hydrogel applications in oncology, highlightingits versatility as both a drug carrier and therapeutic material.Biomedicines 2025, 13, 1899 20 of 33 Figure 3. Anticancer experimental scheme of free Fe3O4 NPs (A), agarose/Fe3O4 hydrogels (B–D),and gelatin/Fe3O4 porous scaffolds (E–G). Three modes (sitting, Transwell, and adhesion modes)were used to simulate the cells near or far away from or directly adhered to the matrices. Copyright:Guoping Chen. There is no copyright issue [173].3. Methods for In Vitro and In Vivo Evaluations3.1. Method for In Vitro Antitumor Effect EvaluationThe evaluation of antitumor efficiency is critical for determining the applicability ofdrug-loaded hydrogels. Currently, four primary methods are commonly used to assessthe cytotoxicity of drug-loaded hydrogels (Figure 4). The most widely used approach,reported in over 70% of the studies, involves treating cells with a medium containinghydrogel. Approximately 20% of the studies used a method in which cell suspensions weredeposited directly into the hydrogel for co-culture. Among the 106 studies analyzed, sixused hydrogel extracts for treatment and seven studies used Transwell systems to analyzecytotoxicity. Each of these methods have distinct advantages and limitations.Adding hydrogels or their extracts directly to the culture medium is convenient, whichis not different from methods that add hydrogel extracts with drugs into the culture mediumand allow the simultaneous evaluation of hydrogel biocompatibility and cytotoxicity.However, these tests are typically conducted over a short period (48–72 h), which isconsiderably shorter than the extended duration of drug release observed in practicalapplications. Therefore, these methods may not accurately represent the actual antitumorefficacy of hydrogels. The method involving the deposition of cell suspensions on/intothe hydrogels provides a closer approximation of the real-world applications of drug-loaded hydrogels. However, this approach inherently reflects the behavior of cells in directcontact with the hydrogel without considering the effects of remote DDSs. Moreover, drugdiffusion from the hydrogel can significantly affect antitumor efficiency, necessitating theoptimization of culture conditions, such as prolonging the incubation period, to bettersimulate in vivo drug release dynamics.The Transwell assay focuses on assessing the cytotoxicity of drugs released fromhydrogels. Although this provides valuable insights into drug delivery and release efficacy,it lacks the ability to directly evaluate hydrogel biocompatibility. The primary purpose ofBiomedicines 2025, 13, 1899 21 of 33using a hydrogel-based DDS is to enhance the safety of drug administration and achievelocalized drug concentrations; a comprehensive evaluation strategy is essential. It wouldbe more reasonable to test the toxicity of the hydrogels and the optimal concentration ofantitumor drugs.Figure 4. Summary of the methods used to analyze antitumor effects at the cellular level.3.2. Method for In Vivo Antitumor Effect EvaluationThe results of animal experiments for DDSs are more generalizable for clinical usethan those of in vitro experiments. Although hydrogel-based DDSs have been extensivelystudied, some studies are still in the preclinical stage [174]. To reveal the real effects ofhydrogel-based DDSs, in vivo experiments are indispensable before preclinical analysis.Numerous studies have reported the antitumor effect of hydrogels in vivo, with somefocusing on their efficacy in animal models of recurrence. Unlike free drug administration,the dosage regimen of hydrogels typically involves intratumoral, peritumoral, or subcuta-neous injections. These methods require tailoring of the rheological properties of hydrogelsto meet the requirements of injectability and sol–gel transition under in vivo conditions(Figure 5).Figure 5. Summary of the methods used for animal experiments.Biomedicines 2025, 13, 1899 22 of 33Peritumoral or intratumoral injections maximize local drug concentrations and anti-tumor efficacy, minimize drug loss through the circulatory system, and reduce accumu-lation in non-target organs, thereby lowering systemic toxicity compared to intravenousinjections [134,144,175,176].In contrast, in situ administration is more frequently used in brain tumor research [177–179].For brain tumor treatments, the blood–brain barrier presents a considerable challenge,reducing effective drug concentrations at the target site and potentially causing off-targeteffects and systemic toxicity [180,181]. Compared with NP-based DDSs, in situ injectionis more important for analyzing the function and availability of hydrogel-based DDSs inbrain tumors, and subcutaneous tumor models cannot replicate the challenges posed bythe unique environment of the brain.Moreover, for specific therapies, such as immunotherapy, hydrogel carriers havedemonstrated higher efficacy than that of free drug administration [182]. This may be at-tributed to the rapid clearance or degradation of immune-related agents, such as antibodies,during free administration, which reduces their therapeutic activity [183]. The applicationof hydrogels mitigates this issue by protecting these agents and enabling their sustainedrelease, thereby enhancing their efficacy.In addition to a single method of administration, in most studies, researchers haveused more than one experimental method to evaluate the treatment potential of hydrogel-based DDSs. Jin et al. investigated the tumor ablation effects of injectable peptide hydrogelsco-delivering DOX and Melttin. Their study compared intratumoral, peritumoral, and insitu injection methods and demonstrated significant antitumor effects across all approachesusing hydrogel-based DDSs. In a footpad model used to study lymphatic metastasis, thein situ injection of hydrogels also achieved effective tumor suppression [184]. For thesame hydrogel material, intratumoral and peritumoral injections showed similar results,likely due to the close proximity of the injection sites to the tumor [104]. In various studies,intratumoral or peritumoral administration consistently exhibited higher antitumor efficacythan that of free drug administration via intravenous injection [134,175,185]. Even whentumor growth inhibition was comparable, localized administration methods resulted inlower systemic toxicity, as reflected by the reduced body weight loss in treated animals [176].These findings underscore the significant advantages of hydrogel-based DDSs in improvingtherapeutic outcomes while minimizing adverse effects.Despite the gradual comprehensive analysis of hydrogel-based DDSs, challengesremain in translating these systems into practical applications. One issue is the discrepancybetween the administration methods used in the experiments and those applicable topreclinical models. For disease treatment, intravenous, intramuscular, and subcutaneousinjections remain the most common routes, whereas intratumoral injection is primarily usedin antitumor immunotherapy at the preclinical stage [186–188]. Hydrogel injection wouldinvolve more problems than single immune agent injection. Furthermore, the feasibility ofinjection imposes additional constraints on the selection of hydrogel materials.Apart from administration approaches, the variability in animal experiments in var-ious studies poses substantial challenges. Differences in tumor volume at the start ofexperiments and research-dependent injection sites for peritumoral administration makeit difficult to precisely evaluate treatment efficiency. Therefore, it is necessary to developstandardized and reliable animal models and experimental protocols to facilitate accuratecomparison and reproducibility in hydrogel-based DDS research.Finally, the lack of attention to sterilization methods in many studies is another is-sue. Only a few studies provided detailed descriptions of the sterilization process [189].Although some hydrogels incorporate antibacterial drugs [190] or are composed of inher-ently antibacterial materials [191], most studies have not described sterilization methods,Biomedicines 2025, 13, 1899 23 of 33particularly for long-term animal experiments. Although Schiff base interactions have an-tibacterial effects [192], programmed sterilization remains essential for both in vivo studiesand further applications.4. Challenges and Developments of Hydrogel-Based DDSs4.1. Preclinical Evaluation and Clinical TrialsNo matter how innovative or imaginative a concept may be, it ultimately needs to betranslated into practical applications. Only when favorable therapeutic efficacy is demon-strated can a hydrogel-based system proceed to clinical trials and eventual implementation.Currently, the major limitations hindering the clinical translation of hydrogel-based DDSsinclude suboptimal therapeutic efficiency, safety concerns, and insufficient objective pre-clinical evaluations.For hydrogel-based DDSs, the most critical factor is drug delivery efficiency, whichdirectly affects treatment effectiveness. Macroscopically, this efficiency is influenced by twokey factors: the design of the hydrogel and the biological characteristics of the tumor. Interms of the hydrogel design, the mesh size of the hydrogel polymers, interactions betweendrugs and hydrogels, and drug concentration significantly affect the overall DDS perfor-mance. These factors have been extensively discussed in previous reviews [66,193,194].However, pathophysiological characteristics of tumors also play a crucial role in drugdelivery efficiency and cannot be ignored. The development of DDSs that adapt to theTME, along with combination therapy-based delivery strategies, is a major research topicin tumor treatment research. Specifically, with the application of novel materials and thedevelopment of innovative antitumor agents, the integration of bioactive compounds withhydrogel polymers offers expanded possibilities for tumor therapy.Hydrogels exhibit excellent biocompatibility, supporting their broad clinical potential.However, the introduction of chemical crosslinking agents and the degradation of hydrogelpolymers may inadvertently trigger undesirable biological responses, leading to safetyconcerns regarding their use [195,196]. For example, the incorporation of crosslinkers, suchas glutaraldehyde, alters macrophage polarization during the foreign body response. Thisresponse is typically characterized by ECM deposition around the implanted biomaterial,which can impair device function and induce chronic inflammation [196,197]. Moreover,the degradation products of certain synthetic hydrogels, such as polyesters, poly (glycolicacid), and poly (L-lactic acid), may generate low-molecular-weight byproducts with cyto-toxic or immunogenic properties [198,199]. These byproducts can induce oxidative stress,promote pro-inflammatory cytokine release, or interfere with local tissue homeostasis, rais-ing concerns regarding long-term biocompatibility. However, most degradation analysesare conducted over short durations and the characterization of the degradation products isoften lacking (Tables 1–3). Therefore, understanding and controlling the immunotoxicol-ogy profiles of both crosslinking agents and degradation products is essential for the safetranslation of hydrogel-based DDSs into clinical use.Despite these uncertainties, progress in clinical trials continues to demonstrate thestrong potential of hydrogel-based DDSs for clinical applications. Currently, a hydrogeldeveloped for the effective release of fibroblast growth factor-binding protein 1 (FGFBP1)inhibitors to treat pancreatic adenocarcinoma is in progress at CNR Nanotec Lecce [200].Additionally, a thermosensitive hydrogel for 5-FU delivery for CRC treatment is in therecruitment phase [201]. The FDA has approved the chemotherapy gel, Jelmyto, for thetreatment of low-grade upper tract urothelial cancer. Jelmyto is a thermally responsivehydrogel used to deliver the cytotoxic chemotherapeutic agent mitomycin [202]. Anotherpromising candidate, UGN-102, a reverse thermal hydrogel with a mechanism similar tothat of Jelmyto, has been developed for the primary nonsurgical treatment of low-grade,Biomedicines 2025, 13, 1899 24 of 33intermediate-risk, non-muscle-invasive bladder cancer. It has shown a high completeresponse rate in Phase III trials and may serve as a potential alternative to surgery [203].Notably, both Jelmyto and UGN-102 offer minimally invasive approaches tochemotherapy [203]. Other non-drug delivery methods using hydrogels, such as the rectalhydrogel spacer system SpaceOAR [204], achieve a therapeutic effect by injection intothe site of action. In line with the clinical requirements, most in vivo experiments havefocused on the development of injectable hydrogels (Figure 5). However, their degra-dation behavior and immunogenicity have attracted less attention. More importantly,infection has also been reported as one of the complications associated with hydrogel injec-tion, whereas sterilization methods are rarely described in hydrogel preparation protocols(Tables 1–3). In summary, although several hydrogel-based DDS candidates have enteredclinical trials [205,206], insufficient preclinical data hinders the clinical translation of manypromising systems. Completing in vivo studies with robust and sufficient data, particu-larly regarding the degradation safety and preparation methods, would be substantiallybeneficial for accelerating the entry of hydrogel-based DDSs into clinical use.4.2. Future Directions and ConclusionsIn this review, we summarized the strategies and methods commonly used to preparehydrogel-based DDSs for antitumor therapies. Unlike traditional administration, hydrogel-based DDSs not only avoid blast drug release but are also applicable for combinationtherapy design. Hydrogel-based DDSs represent a promising frontier in antitumor therapyowing to their unique properties, such as high water content, biocompatibility, tunablephysical and chemical characteristics, and the ability to encapsulate a variety of therapeuticagents. These features make hydrogels well-suited for addressing key challenges in cancertreatment, including enhancing local drug concentrations, reducing systemic toxicity, andenabling controlled drug release. Despite significant progress, several avenues remainunexplored, providing opportunities for future research.One critical area of advancement is the integration of smart and responsive hydrogeltechnologies. Hydrogels that respond to external stimuli, such as temperature, pH, andmagnetic or electric fields, are increasingly being investigated for their potential to achievespatiotemporal drug release. Future studies should focus on developing multi-responsivehydrogels capable of adapting to the dynamic TME, thereby improving the precision ofdrug delivery and therapeutic outcomes.Another promising direction is the application of hydrogels based on the clinicaldemand for tumor treatment, such as the combination delivery of different antitumor drugs,the combination delivery of antitumor and anti-angiogenic agents, and the combination ofchemotherapy and other therapeutics. These hybrid systems can leverage the advantagesof each component to improve the targeting specificity and achieve multilevel drug releaseprofiles. Additionally, because of their good biocompatibility and designable mechanicalproperties using different polymers, hydrogel systems can further broaden their applicationas postoperative fillers for tissue regeneration.Despite rapid developments, several challenges need to be addressed for the transla-tion of hydrogel-based DDSs from the laboratory to clinical settings. Biodegradability andbiocompatibility must be meticulously evaluated to ensure that the hydrogel degradationproducts are nontoxic and do not elicit adverse immune responses. Furthermore, the scala-bility and reproducibility of hydrogel synthesis must be optimized for mass productionunder good manufacturing practice standards.The versatility and adaptability of hydrogels make them well-positioned to play apivotal role in personalized medicine. As research continues to reveal the complexities oftumor biology, hydrogel-based systems are expected to evolve into more sophisticated andBiomedicines 2025, 13, 1899 25 of 33effective tools for cancer therapy, with promising improvements and reduced treatmentburdens for patients worldwide.Author Contributions: Conceptualization, G.C. and N.K.; methodology, G.C., N.K., T.Y., Y.Y. and T.Z.;software, T.Z. and N.K.; validation, G.C., N.K., T.Z. and L.C.; formal analysis, T.Z. and L.C.; investi-gation, T.Z. and L.C.; resources, G.C., N.K. and T.Y.; data curation, T.Z. and L.C.; writing—originaldraft preparation, T.Z. and G.C.; writing—review and editing, T.Z., L.C., T.Y., N.K., Y.Y. and G.C.;visualization, T.Z. and L.C.; supervision, G.C.; project administration, G.C. and N.K.; All authorshave read and agreed to the published version of the manuscript.Funding: This research was funded by JSPS KAKENHI Grant Number 19H04475, and 24K03289.Conflicts of Interest: The authors declare no conflict of interest.References1. 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MDPI and/or the editor(s) disclaim responsibility for any injury topeople or property resulting from any ideas, methods, instructions or products referred to in the content.https://doi.org/10.1093/annonc/mdx683https://doi.org/10.1016/S0939-6411(03)00095-Xhttps://doi.org/10.1016/j.actbio.2015.03.024https://www.ncbi.nlm.nih.gov/pubmed/25818947https://doi.org/10.1021/acsami.6b03041https://www.ncbi.nlm.nih.gov/pubmed/27530316https://doi.org/10.1016/j.carbpol.2019.115207https://doi.org/10.1016/j.ijbiomac.2025.140010https://www.ncbi.nlm.nih.gov/pubmed/39828168https://doi.org/10.3390/genes1030413https://doi.org/10.3390/jcs9060305https://doi.org/10.1021/acsbiomaterials.1c01304https://doi.org/10.1002/adhm.201801451https://doi.org/10.1002/jab.770050208https://doi.org/10.1081/ESE-100103475https://www.ncbi.nlm.nih.gov/pubmed/11413830https://www.clinicaltrials.gov/study/NCT06616688?term=inhibit&viewType=Table&checkSpell=&rank=7https://www.clinicaltrials.gov/study/NCT06616688?term=inhibit&viewType=Table&checkSpell=&rank=7https://clinicaltrials.gov/study/NCT06385418https://clinicaltrials.gov/study/NCT06385418https://doi.org/10.1080/20415990.2025.2480535https://doi.org/10.1097/JU.0000000000004296https://www.ncbi.nlm.nih.gov/pubmed/39446087https://doi.org/10.1259/bjr.20220947https://clinicaltrials.gov/study/NCT04688931https://clinicaltrials.gov/study/NCT04062721 Introduction  Design of Hydrogel-Based DDSs  Stimulus-Responsive Drug Delivery Hydrogels  Cargo-Based Drug Delivery Hydrogels  Drug Delivery Hydrogels for Pathological Characteristics of Tumors  DDSs of Cytotoxic Drugs  DDSs of Anti-Angiogenesis Drugs  DDSs of Immune Checkpoint Inhibitors  Methods for In Vitro and In Vivo Evaluations  Method for In Vitro Antitumor Effect Evaluation  Method for In Vivo Antitumor Effect Evaluation  Challenges and Developments of Hydrogel-Based DDSs  Preclinical Evaluation and Clinical Trials  Future Directions and Conclusions  References