Another therapeutic target of responsive nanomedicine is the TME hypoxia (Scheme 9). This is a direct consequence of heterogenic vasculature and fluctuating blood flow, and results in insufficient oxygen diffusion and perfusion within the tumor environment. The rapid proliferation rate of tumor and stromal cells creates excessive consumption of supplied oxygen, nutrients, and energy [155]. Imbalance in the diffusion mechanisms of oxygen supply is observed at depths after 70–150 μm from peripheral tumor blood vessels. This results in gas oxygen (gas-O₂) levels falling below 1–2% in hypoxic solid tumors. There are two types of hypoxia: the chronic, wherein oxygen’s concentration is characterized by a longitudinal gradient drop for a prolonged time period of several hours; and the acute, in which tumor ECs and stromal cells are attached to vasculature with deprived oxygen perfusion [156]. Extensive research has resulted in the understanding of hypoxia mechanisms and their effects on tumor biology by participating in the regulation of angiogenesis, metastasis, and multidrug resistance (Table 5) [157,158]. Hypoxia-regulated genes are expressed among various tumor types with high hypoxic gene expressions such as the squamous cell carcinoma (SCC) of the head and neck, lung, and cervix tumors; these have also been investigated [159].

3.4.1. GLUT Targeting Nanomedicines

A central factor in targeting TME hypoxia is effective exploitation of transcription factors related to various regulatory pathways of glycolysis, oxygen homeostasis, MDR, and resistance to apoptosis. Such factors within TME include the carbonic anhydrase IX (CA-IX), the glucose transporter-1 and 4 (GLUT-1, 4), and the hypoxia inducible factors (HIF) family (including HIF-1, HIF-2, HIF-3). These act as oxygen regulators to stabilize hypoxic conditions and promote tumor cell survival and angiogenesis through PDGF secretion [160]. Specifically, the blockage of GLUT-1 and GLUT-4 transporters with glucose-modified PLGA and chitosan nanoparticles was selected as an active targeting strategy to limit nutrient supply to tumor cells by Abolhasani et al. [161]. They found that this glucose-modified nanoparticle effectively inhibited GLUTs function and stimulated glucose deprivation and increased apoptotic enzyme expression. In a recent study by Sun et al. [162], the ATRP copolymerization of glucose-containing methacrylate (GluMA) and OEGMA was used for the conjugation of interferon-α (IFN-α) for effective GLUTs targeting. The study outlined the importance of optimizing glucose content. This is because excessive glucose concentration resulted in inhibition of the antitumor activity, while the optimal system showed enhanced tumor targeting and antitumor immunity; this was expressed by the secretion levels of TNF-α and IL-2 cytokines. In general, glycosylation of nanoparticles has been exploited for its increased GLUT-targeting ability, and it promotes metabolic changes and immune responses. This has been reviewed by Torres-Perez et al. [163]. Moreover, glycolysis is the main source of energy production in hypoxic and aerobic tumor sites (the Warburg effect). This demonstrates the importance of nanomedicines targeting the glycolytic mechanisms. In a recent paper by Geng et al. [164], the targeting of glycolytic enzymes and transporters, and combinational strategies based on nanomedicines were reviewed. Also, glucose metabolism in hypoxic tumor sites highly activates cancer stem cell (CSCs) phenotypes, and this promotes the activation of a stem-like polarization that is mainly regulated by the transcription factor HIF-1α and the Notch pathways [165]. Shibuya et al. [166] demonstrated the importance of modulating GLUT-1 transporter in regulating the stem-like phenotype in pancreatic, ovarian, and glioblastoma CSCs. By specifically inhibiting GLUT-1 with WZB117, the self-renewal and tumor-initiating activities of the CSCs were effectively suppressed in animal models upon implantation of CSCs.

Scheme 9. Tumor hypoxia represents a significant target for responsive nanomedicines. Important axes of nanomedicine research are GLUT targeting, multidrug resistance targeting, increase of chemo-sensitivity, M1/M2 macrophage polarization, increase of tumor oxygenation, and antioxidants. Biomaterials used for hypoxia targeting nanomedicines include polysaccharides, polymers, hybrid nanoparticles, metal oxides and hybrid metal–polymer nanoparticles, polymersomes, nanogels, and liposomes in combination with chemotherapeutic drug targeting (CDT), magnetic targeting, and PDT/PTT therapy. (Created with the assistance of BioRender.com https://app.biorender.com/user/signin?illustrationId=6156d45891063d00af8af51d (accessed on 1 September 2023 up to 31 November 2023), and Microsoft ppt).

Moreover, the energy supply blockage promoted by specific starvation therapeutics is highly associated with hypoxia-targeting nanomedicines. This is caused by exploitation of biological metal ions (e.g., Ca²⁺, Zn²⁺, Cu²⁺) in tumor starvation mechanisms. Among them, Cu²⁺ and Zn²⁺ are the most prevalent components of enzymes, and they play a crucial role in energy metabolism, gene expression, and genomic stability. Yang et al. [167] studied the effect of copper-based ultra-small nanoparticles (Cu_2−xSe) modified with HIF-1α inhibitor and coated with tumor cell membrane. The nanoparticles were effectively transported through the blood–brain barrier upon focused ultrasound application, and accumulated at glioblastoma tumor sites where they exhibited significant antitumor activity by inhibiting HIF-1α expression and enhancing tumor sensitivity to disulfiram. In another study, Wu et al. [168] examined the effect of zinc imidazole metal-organic particles (ZIF-8) modified with hyaluronic acid for systemic glycolytic energy deprivation. The nanoparticles expressed CD44-targeting mechanism. This led to effective tumor accumulation and cellular endocytosis that promoted the disaggregation of zinc core by hyaluronidase. The latter further triggered a decrease of NAD⁺ and inactivation of GAPDH, and obtained strong glycolysis inhibition. Additionally, it suppressed GLUT1 regulation which promoted energy starvation in B16-F10 tumor-bearing C57BL/6 mice. The energy demand of tumor cells is critically supported by increased glucose production that leads to increased expression levels of GLUT1 and GLUT2, and abundant glycolytic enzymes. The phenotyping of tumor cells with elevated glucose expression levels represents a negative prognostic factor that can be exploited in diagnosis for the effective design of personalized nanomedicines. In this respect, glucose nanosensors have been investigated for application in diagnosis by Nascimento et al. [169]. They studied the effectiveness of nanopipette-based glucose sensors functionalized with glucose oxidase (GOx) to quantify single cell intracellular glucose levels.

3.4.2. Multidrug Resistance Targeting Therapeutics

The targeting of HIF transcription factors will not be broadly discussed here. This is because its role in hypoxia mechanisms and in responsive therapeutic nanomedicine has been extensively reviewed [170–172]. A comprehensive mini-review on engineered nanomedicines summarizing recent progress on HIF-1 targeting was presented by Zhang et al. [173]. HIF is highly associated with multidrug resistance in solid tumors. It develops as tumor cells develop a defense mechanism against drugs, and this results in drug efflux and decrease of intracellular drug concentration. MDR is an ultimately complex mechanism related to gene mutations, increased DNA repair ability, and epigenetic alterations involved in the regulation of gene expression such as silencing of tumor suppressor genes and overexpression of oncogenes. In MDR, drug efflux from tumor cells is mediated by efflux transmembrane pumps such as permeability-glycoprotein (P-gp) (also known as multidrug resistance protein 1 (MDR1)), and important transmembrane proteins that are ATP-dependent and regulated by HIF proteins [174]. Certain chemotherapeutic agents that act through mitochondrial ROS formation, e.g., cisplatin (CSP or DDP), can affect MDR either through inducing ROS-mediated cancer cell death by activating apoptotic signaling pathways or through promoting drug resistance and activation of HIF-1 cascade due to elevated ROS production. However, ROS-induced HIF-1 activation also leads to severe damage to the surrounding normal tissues. In a recent study, Zhang et al. [175] investigated the effect of cisplatin on inhibiting ROS induced HIF-1 activation and acquired resistance using chitosan-coated selenium/cisplatin nanoparticles. Chitosan provided long circulation and effective nanoparticles accumulation at the tumor site in cisplatin-resistant A549 tumor-bearing mice. At the tumor, cisplatin promoted HIF-1 expression in an ROS dependent function while the selenium antioxidant effect suppressed ROS formation and inhibited HIF-1 activation. The inhibitory effect of selenium nanoparticles was also confirmed by the downregulation of GCLM, P-gp, MDR2, and HIF-1α protein expression levels. The acquired drug resistance promoted by cisplatin application was examined by Zhang et al. [176] in organosilica-coated cisplatin nanoparticles that co-deliver the HIF-1 inhibitor acriflavine (ACF). The nanoparticles were efficiently accumulated at the tumor site, being susceptible to glutathione triggered biodegradation, and released ACF for inhibiting HIF-1 activity by preventing the formation of HIF-1α/β dimers via HIF-1α binding. The synergistic release of cisplatin resulted in a highly effective system suppressing tumor growth and metastasis. Reversing MDR was also studied by Li et al. [177] through the combined delivery of doxorubicin and the HIF inhibitor PX478 by silk fibroin nanoparticles. The nanoparticles were functionalized with folic acid (FA) for cancer cells targeting. The multi-responsive nanoparticles inhibited HIF gene expression by the synergistic action of FA receptor-mediated endocytosis and PX478 inhibition that effectively downregulated MDR1 expression levels, thus eliminating DOX efflux. Neuropilin-1 (NRP1) is another receptor involved in cellular processes related to MDR in tumor cells. The role of NRP-1 on the loss of therapeutic efficacy of lenvatinib was evaluated by Fernandez-Palanca et al. [178] in relation to hypoxia and modulation of HIF-1α. Mamnoon et al. [179] studied NRP-1 as a molecular target for the delivery of iRGD peptide and doxorubicin by hypoxia-responsive PLA-diazobenzene-PEG polymersomes. Evaluation in tumor-bearing animal models demonstrated the accumulation of the polymersomes at the tumor site and the antitumor effect of their drug cargo.

3.4.3. Increasing Chemo-Sensitivity

On the basis of chemotherapy and radiotherapy resistance, the researched solutions focused on sensitizers and in enhancing TME oxygenation. The sensitizers are, in general, chemical compounds (originally nitro-aromatic ring compounds) that act by elevating the tumor cellular sensitivity to ionizing radiation, and produce free radicals and promote DNA damage [180]. A great drawback of the application of chemical sensitizers was the dose-dependent toxicity and adverse effects related to ROS and RNS expression levels. However, more effective sensitizers with low toxicity were designed by exploiting small molecules (O₂, NO), macromolecules (proteins, peptides, miRNA, siRNA), and nanomaterials (noble metals, ferrite, heavy metals) that act without distressing ROS expression levels [181]. In tumors, TRPA-1 is a channel plasma membrane protein that promotes extracellular Ca²⁺ influx and represents the most abundant redox sensor upregulated in metastatic cells of solid tumors such as human oral squamous cell carcinoma, colorectal cancer adenocarcinoma, and glioblastoma multiform. The TRPA-1 was evaluated as a ROS sensor, since its upregulation-mediated enhanced ROS-induced Ca²⁺ influx as a result of oxidative stress. This promoted anti-apoptotic signaling pathways [182]. Wang et al. [183] studied TRPA-1 inhibition in order to increase tumor chemosensitivity in tumor-bearing mice. They used hyaluronic acid nanogels functionalized with DSPE-PEG nano-micelles conjugated with a tumor homing penetrating tLyP-1 peptide for co-delivering doxorubicin and TRPA-1 inhibitor (AP-18). The combined effects of HA and penetrating peptide resulted in enhanced intratumoral and intracellular localization becasue the HA nanogels disassembled in irregular fragments releasing peptide conjugated nano-micelles upon hyaluronidase effect in TME. The TRPA-1 inhibitor enhanced tumors sensitivity to DOX by suppressing Ca²⁺ uptake and AKT phosphorylation, and promoting regulation of EMT-related proteins.

A redox enzyme that is highly associated with tumor hypoxia is nitroreductase (NTR). This catalyzes the reduction of nitro compounds in the mitochondria by using NADPH. Jang et al. [184] examined the effect of NTR in self-assembled nanoparticles of glycol chitosan loaded with doxorubicin, functionalized with folic acid andmodified with 4-nitrobenzyl chloroformate (4NC). The nanoparticles effectively accumulated at the tumor site due to the combined effects of CS and FA targeting, and disassembled due to the reduction of the chitosan bonded 4NC under hypoxic conditions by the NTR/NADPH cascade. This efficiently promoted DOX antitumor activity. Another study exploiting the hypoxia-mediated bond reduction was by Luo et al. [185]. They examined self-assembling thioether linked dihydroartemicin (DHA) prodrug nanoparticles loaded with chlorin e6 and further encapsulated in core-shell amphiphilic carboxymethyl chitosan polymer particles, grafted with maleimide and 2-nitroimidazole (NI) groups. The nanoparticles showed significantly long circulation time and accumulation ability through the combined chitosan and EPR effect. Once PDT was applied, oxygen consumption was increased and the NI groups were reduced. This destabilized the structure of the chitosan particles, and further promoted drug release at the tumor site. The synergistic effect resulted in suppression of HIF-1α and VEGF expression levels, inhibition of tumor metastasis, and an improved therapeutic effect in LLC tumor-bearing rats and mice.

3.4.4. Targeting M1/M2 Macrophage Polarization

In TME, hypoxia is responsible for inducing the reprogramming of pro-inflammatory M1 macrophages to take a M2 anti-inflammatory phenotype; this promotes tumor aggressiveness and MDR. M1 macrophages play critical roles in innate host immunity and tumor cell death through the production of ROS/RNS and pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α. M2 macrophages polarization induced by Th2 cytokines such as IL-4, IL-10, and IL-13 play a critical immunosuppressive role in immune responses, under the activation of immune complexes (IC) and TLR ligands, producing anti-inflammatory cytokines such as IL-10, IL-13, and TGF-β to promote tumor development. By altering macrophage responses, hypoxia downregulates the expression of antigens and the release of pro-angiogenetic factors. This exerts immunosuppressive effects that promote cell proliferation. While the M1/M2 macrophage states have been identified, the regulation of macrophage phenotypes remains a complex mechanism. The production of distinct functional phenotypes in macrophages (polarization), as a consequence of hypoxia, is essentially regulated by HIF-1α, which plays a key role in the expression of several genes [186]. Among them, the PTGS gene encoding cyclooxygenase-2 (COX-2) is of major importance, since it is related to increased accumulation of pro-inflammatory signals, and represents an indicator associating inflammation with cancer [187]. COX-2 is secreted by CAFs, M2 macrophages and TME cells inducing cancer stem-like cells activity, MDR, and metastasis. COX-2 induced HIF-1α activity is responsible for biosynthesis of prostanoids, like prostaglandin E2 (PGE2); this is overexpressed in a variety of cancers including breast cancer, osteosarcoma, and colorectal cancer. The HIF-1α/COX2 signaling axis is highly related to resistance, invasion, and metastasis [188]. Karpisheh et al. [189] studied the effect of HIF-1α silencing siRNA and PGE2 receptor antagonist (EP4-antagonist) as a combinational therapy with hyperthermia in tumor-bearing BALB/c nude mice. For this purpose, superparamagnetic iron oxide nanoparticles (SPIONs) functionalized with hyaluronic acid and trimethyl chitosan were used for the delivery of siRNA and the EP4 antagonist. The synergistic effect of hyperthermia, CS and HA resulted in increased SPIONs biodistribution at the tumor site, and effectively delivered the siRNA. The combined effect of HIF-1α siRNA and the EP4 antagonist resulted in (i) suppression of proliferation and migration of tumor cells by decreasing the expression levels of ki-67 gene, anti-apoptotic protein Bcl-2, and MMP-2/-9; and (ii) inhibition of VEGF, FGF, and TGF-β. This promoted suppression of angiogenesis and inhibition of tumor growth.

3.4.5. Combinational Targeting for Increasing Tumor Oxygenation

In order to increase tumor oxygenation, various strategies have been investigated. These include: (i) modulation of tumor blood flow with compounds, such as noradrenaline, benzyl nicotinate, nicotinamide, and pentoxifylline; (ii) high oxygen breathing through hyperbaric chambers, and carbogen breathing; (iii) targeting of tumor vasculature with anti-angiogenetic therapies and vascular disruptive agents [190]. Hyperbaric oxygen (HBO) treatment was used to suppress hypoxia by effectively elevating the oxygen concentration in plasma and, subsequently, enhancing oxygen delivery in tumor tissues independently of hemoglobin. This resulted in reduced tumor growth in breast cancer; however, cervical and bladder cancers appear to be insensitive to HBO [191]. In a recent study by Wang et al. [192], HBO was applied in combination with Abraxane and gemcitabine (GEM) to trigger antitumor activity against murine PDAC tumors, expressing inhibitory activity over ECM by decreasing fibril deposition of collagen I and fibronectin. As CAFs are the main cellular components of ECM, HBO significantly inhibited their activity by suppressing tumor hypoxia that, in combination with abraxane and GEM, resulted in inhibition of CSCs. This is evidenced by the decreased expression levels of CD133 and Sox2 CSCs-related biomarkers, and this further promoted the antitumor activity of the drugs in primary and in metastatic PANC02 tumors.

In a recent review by Wu et al. [65], nanoparticle-based systems targeting TME were presented. These include hypoxia-responsive nanomedicines that effectively target hypoxic TME to promote tumor oxygenation. Tumor oxygenation in order to inhibit hypoxia was examined by Song et al. [193], with the application of manganese dioxide nanoparticles (MnO₂) coated with hyaluronic acid and modified with mannan-PE ligands in combination with priming of pro-inflammatory M1 macrophages phenotype. The nanoparticles caused elevation of tumor oxygenation. This is shown by the suppression of HIF-1α and VEGF expression levels, and expressed immune-toxicological effects in reprogramming the antitumor M1 phenotype. Furthermore, the nanoparticles had synergistic effect upon administration with doxorubicin in tumor-bearing mice; in which they inhibited tumor growth and cell proliferation. Importantly, the MnO₂ nanoparticles express a redox-active catalytic behavior toward hydrogen peroxide (H₂O₂) to produce oxygen and regulate acidic pH. This is because their Mn²⁺ decomposing products are excellent T1-shortening MRI agents; this is described by Lin et al. [194] in human serum albumin MnO₂ nanoparticles conjugated with Ce6 photosensitizer. The evaluation of the nanoparticles in tumor-bearing mice showed increased tumor-targeting ability and accumulation efficacy, with elevated oxygenation levels. Additionally, treatment with PDT-enhanced cellular apoptosis, resulted in a large tumor necrosis area. Another effective system promoting oxygenation was studied by Jiang et al. [195], who used MnO₂ albumin nanoparticles delivering indocyanine green (ICG), a hydrophilic anion drug, for effective PDT of hypoxic tumors. The nanoparticles were evaluated in both CT26 and B16F10 tumor-bearing mice and showed enhanced tumor accumulation efficacy and successful responsive release of ICG in the presence of hydrogen peroxide. The combined effect with MnO₂ and PDT resulted in enhanced oxygen production and attenuation of hypoxic TME, while elevated distribution of CD3⁺ and CD8⁺ T cells was promoted at tumor sites. In advanced hepatocellular carcinoma (HCC) the anti-angiogenetic agent sorafenib is a first-line treatment. However, the hypoxic TME of advanced HCC regulated anti-apoptotic signaling pathways and promoted immunosuppressive reprogramming, resulting in MDR against sorafenib. Ren et al. [196] studied the synergistic effect of tumor oxygenation and PDT by a strategic hypoxia relieving nanodrug (SHRN). SHRN was based on pegylated liposomes encapsulating MnO₂-BSA nanoparticles, the oxygen consumption inhibitor atovaquone (ATO), and the photosensitizer hypericin (HY). In this system, oxygen production was effectively increased by MnO₂ nanoparticles and the synergistic action of ATO with mitochondrial complex III resulted in the blockage of aerobic respiratory chain and the suppression of oxygen consumption. By suppressing hypoxia in TME of tumor-bearing mice, PDT application essentially promoted HY action to react with oxygen and promote increased antitumor effect. The antitumor activity of PDT originated from the elevated generation of ROS as an outcome of electron transfer mechanisms of the photosensitizer and the molecular oxygen intracellularly. A review of the mechanisms associated with PDT efficacy and the limitations induced by hypoxia has been authored by Zhou et al. [197]. In another study, Chang et al. [198] investigated the effect of lipid-PLGA particles co-delivering MnO₂ nanoparticles and sorafenib as an effective approach for HCC. The nanoparticles promoted oxygen production in orthotopic HCC tumor mice xenografts, and this resulted in suppression of hypoxia and of MDR to sorafenib. As a result, inhibition of tumor cells proliferation and suppression of angiogenesis and metastasis were observed.

In addition to MnO₂, iron oxide nanoparticles were also studied as Fenton catalysts triggering reduction of hydrogen peroxide to oxygen by ferrous ions. He et al. [199], described the in vitro and in vivo effectiveness of solid lipid calcium dioxide (CaO₂) nanocarriers (SLNs) for co-delivery of doxorubicin and iron-oleate. At the tumor site, the SLNs dissociated by lipase overexpressed in cancer cells, enabling the release of iron-oleate and CaO₂ particles. These, in response to acidic cellular environment, released DOX and produced hydrogen peroxide molecules. The Fe³⁺ ferrous ions released from iron-oleate reacted with H₂O₂ molecules to produce oxygen and the Fe²⁺ ions created hydroxyl radicals for antitumor chemodynamic therapy (CDT). The latter can induce oxidative damage to tumor tissues through Fenton or Fenton-like reactions of metal catalysts. In another interesting study by Ou et al. [200], the antitumor effect of CDT based on Fenton reaction was exploited. Βlack phosphorus nanosheets (BPNS) functionalized with active photosynthetic Chlorophyceae (Chl) cells and Fe³⁺ ions were synthesized. In this smart system, Chl cells exploited their inherent photosynthetic ability to produce O₂ and metabolites that, in combination with the BPNS, improved ¹O₂ and O₂ generation. The presence of Fe³⁺ ions resulted in the simultaneous consumption of glutathione and created hydroxyl radicals (·OH) through the reaction with hydrogen peroxide. The ultimate goal was the synergistic PDT/CDT and immune response, since Chl cells stimulate the proliferation and maturation of dendritic cells. Recently, Dong et al. [201] prepared Zn/Cu responsive nanoparticles with improved blood circulation time and increased tumor accumulation in animal models through the EPR effect. The pH-responsiveness of Cu/Zn nanoparticles originated from the ZnO particles which, at acidic pH, dissolve to Zn²⁺ ions. Enhanced CDT efficacy was observed at the acidic tumor pH, since Cu/Zn nanoparticles dissolved to Cu²⁺ ions that were further reduced by glutathione to Cu⁺. This effectively generates hydroxyl radical from hydrogen peroxide through a Fenton-like reaction. Moreover, CaO₂ nanoparticles coated with a pH-sensitive methacrylate based copolymer were studied by Sheng et al. [202] to enable tumor oxygen generation and improved PDT therapy in MIA PaCa-2 tumor-bearing mice. The combination of CDT with immunotherapy was examined by Zhang et al. [203]. They applied liposome nanoparticles carrying copper-oleate and the HIF-1 inhibitor acriflavine (ACF), for combined antitumor immune responses. The liposomes dissociated in the acidic hypoxic TME and the released copper ions catalytically reduced hydrogen peroxide to highly active hydroxyl radicals. The activity of copper ions was supported by ACF that effectively inhibited the HIF-1/glutathione pathway. This suppressed the expression of programmed death ligand-1 (PD-L1), reduced the extracellular expression levels of lactate and adenosine, and promoted immunogenic cell death (ICD).

3.4.6. Synergistic Targeting with Antioxidants

The synergistic effect of hypoxia-targeting nanomedicines in combination with chemotherapeutic agents and antioxidants has been extensively investigated, due to their effects in reoxygenation and ROS expression levels [204–206]. The overproduction of ROS can promote oxidative stress (OS) and induce oxidative damage of biomolecules as DNA, lipids and proteins. This eventually promotes the death of normal cells while in tumor tissues, due to increased energy demand and metabolic modifications, the demand on ROS production is excessively increased. The reduction in antioxidant level and/or the disruption of redox equilibrium within TME can further promote tumor cell growth and progression. Among various antioxidants studied in tumor therapeutics, polyphenols are associated with cancer cell apoptosis, inhibition of proliferation, and downregulation of COX-2 and tumor gene expression. Vitamins and minerals elicit their antioxidant action by maintaining DNA methylation inhibiting cancer cell proliferation and progression [207]. Vitamins such as Vitamin C (ascorbic acid, AA) are capable of scavenging and neutralizing tumor-generated ROS, providing normalization of OS within local tumor sites. This interaction promotes the production of dehydroascorbic acid (DHAA) that is related with GLUT-1,3, and 4 transporters for effective and rapid cell influx. Intracellularly, DHAA is converted into AA depleting glutathione and ATP enzymes. This affects the expression levels of hypoxia signaling regulation factors [208]. However, the therapeutic exploitation of AA is limited by the ultra-high doses that are required for efficacy and by its chemical instability [209]. Thus, palmitoyl ascorbate (PA; an acylated derivative of AA) also features antitumor activities, and has been incorporated in ROS-scavenging nanomedicines. Sawant et al. [208] investigated the effect of palmitoyl ascorbate PEGylated liposomes (PEG-PAL) in vitro and in vivo in BALB/c mice bearing 4T1 tumors. PEG-PAL liposomes exhibited enhanced effectiveness in suppressing tumor growth compared to free ascorbic acid. The mechanism of action of PEG-PAL was similar to that of ascorbic acid, since liposomes acted as ROS scavengers inhibiting extracellular ROS. In another study, Yang et al. [209] examined the combinational delivery of PA and doxorubicin by liposomes for efficient synergistic effect in suppressing tumor growth. This is because PA has a similar mechanism of action with DOX associated with reduced cardiotoxicity without delaying DOX activity. The evaluation in BALB/c mice and SD rats showed the synergistic antitumor effect of the PA-DOX liposomes, which caused an increased expression of tumor apoptotic cells and a suppression of Ki-67 and CD31 protein expression levels.

Table 5. Nanomedicines based on biomaterials targeting tumor hypoxia.

Tumor and stromal cells use aerobic glycolysis for their amplified energy supply requirements, as a direct consequence of hypoxia and defective vasculature. Aerobic glycolysis is an oxygen-independent process known as the Warburg effect. However, even in normally oxygenated tumor regions, the main energy supplier remains aerobic glycolysis in about 80% of solid tumors [210]. In aerobic glycolysis, glucose constitutes the main macronutrient of tumor cells for their biosynthetic requirements and follows the lactate metabolic pathway, through GLUT transporters producing amplified levels of lactic acid (lactate). The main transcriptional factors of glycolytic activity regulating lactate production are the HIF-1α and c-Myc regulatory genes [211] that promote the overexpression of varied glycolytic enzymes such as lactate dehydrogenase A (LDHA), and monocarboxylate transporters (MCTs) such as MCT1 and MCT4 [212]. Mainly, the upregulation of LDHA gene favors the activity of LDH-5 and inhibits the activity of LDH-1, and promotes the conversion of pyruvate to lactate. Through this metabolic pathway, elevated amounts of lactate, protons (H⁺), and carbon dioxide (CO₂) are secreted into TME. This leads to acidosis [213]. Acidosis regulates the metabolism of innate and adaptive immune cells by: (i) hindering the function of CD8⁺ T, natural killer (NK), natural killer T (NKT) and dendritic (DC) cells; (ii) supporting regulation of FOXP3⁺ T cells (Treg); and (iii) promoting M2 activated macrophage polarization. Overall, the acidic TME is an immunosuppressive incubator of pro-oncogenic and tumorigenic factors, and has been extensively studied for targeted nanomedicine applications [214–216]. The glycolytic metabolic pathway and acidic pH gradient are key participating factors in MDR due to their activation of enzymes and proteins responsible for resistance, efflux of drugs through P-gp, and stimulation of migration [217,218].

In tumors, a unique pH-gradient effect is established, with extracellular pH levels (pH_e) being more acidic (6.4–7.0) and intracellular pH (pH_i) being more alkaline (7.25–7.50) (Scheme 10). Distinct pH variations exist in tumor cell organelles, and they can be divided into acidic, such as nucleosomes and lysosomes with a pH of 5.5 and 5.0, respectively; or alkaline, such as mitochondria and cytoplasm, which have a corresponding pH of 8.0 and 7.2, respectively. The pH gradient is associated with the expression of membrane transporters such as MCT1, MCT4, carbonic anhydrases, and sodium-bicarbonate co-transporter (NBC). These participate in the translocation of lactic acid, CO_2, and its bicarbonate ion byproducts. Other mechanisms influencing TME acidity are the efflux of endosomes acidic cargo and the release of the acidic intracellular comportments of necrotic cells. In stimuli responsive nanomedicine (Table 6), pH sensitivity has been highly exploited and reviewed [219–223]. Apart from drug delivery systems, tumor acidosis was targeted by pH-regulating molecular systems at various stages of clinical trials (this is described by Corbet et al.) [224], and by TME sensitive platforms for combined endogenous stimuli responsive effects (as reviewed by Wang et al. [225]).