The upregulation of glycolysis, a near-universal trait of primary and metastatic cancers indicated based on clinical tumor imaging can give rise to glucose consumption and the production of metabolite lactic acid, which in turn leads to microenvironmental acidosis. Significantly different pH values between plasma (pH = 7.4) and the tumor extracellular microenvironment (pH = 6.5–7.2) and between lysosomes (pH = 4.5–5.5) and endosomes (pH = 5.5–6.8) (Kuppusamy et al., 2002; Wojtkowiak et al., 2011) provide a good trigger for drug release. From this observation, a pH-responsive drug delivery system has been developed and deeply explored. The existing preparation strategies mainly include hydrophilic–hydrophobic conversion and the introduction of pH-sensitive bonds.

The tumor extracellular microenvironment is weakly acidic due to the combination of acidic byproducts produced via tumor metabolism and high glycolytic activity due to impaired acid clearance, where the pH-responsive groups are protonated to trigger hydrophilic–hydrophobic conversion, separating drug molecules from the carrier and releasing them in the tumor tissue, thereby improving the efficiency of nanoparticle delivery.

Yan et al. (Yan et al., 2017) reported a method to synthesize a hollow nanogel poly (l-glutamic acid)/chitosan (PLGA/CS) loaded with the water-soluble antitumor drug mitoxantrone using a nanotemplate method with good biocompatibility. As shown in Figure 2A, the author grafted PLGA onto silica nanoparticles and then used CS as a cross-linking agent to finally remove the template silica. As shown in Figure 2B, the nanogel has a particle size of 189.3 nm under neutral conditions, and the size of the nanogel is significantly reduced when the pH value is 5–9. As shown in Figure 2C, the use of the MTT assay to study the cytotoxicity of PLGA/CS nanogels and drug-loaded nanogels in vitro shows that the biocompatibility of nanogels is very good, and the MTX-loaded nanogels perform well on tumor cells compared with free MTX, which shows a significant inhibitory effect. As shown in Figure 2D, the endocytosis of nanogels was evaluated using a fluorescence microscope. Rhodamine B (RB) was used as a model dye. The fluorescence signal of cells incubated with RB-loaded nanogels was much stronger than that of RB-treated cells. This is because CS decorated on the surface of the nanogel can promote cell internalization. This will deliver higher concentrations of drugs into the cells (Wang et al., 2016). Therefore, this pH-sensitive nanogel synthesized by the nanotemplate method has great potential in smart drug delivery systems (Weigel and Yik, 2002).

Li et al. (Li et al., 2013) developed a polypeptide-based block ionomer complex composed of anionic methoxy( polyethylene glycol)-b-poly-l-glutamic acid and cationic doxorubicin hydrochloride for treating non–small cell lung cancer.

Compared with normal biological tissues, solid tumors can cause cell acidosis. Therefore, it is a current hotspot for introducing pH-sensitive bonds into nanogels.

Mackiewicz et al. (Mackiewicz et al., 2019) synthesized biocompatible degradable nanogels based on polyethylene glycol methyl methacrylate (OEGMA) and diethylene glycol methyl methacrylate cross-linked with redox-sensitive linker N,N-bis (methacryloyl) cystine to synthesize biocompatible and degradable nanogels. With the aid of the carboxyl group in N,N-bis (methacryloyl) cystine, the nanogels had become sensitive to pH, became more stable in the physiological environment, and binded 8% DOX loaded through electrostatic interaction. This degradable nanogel was non-toxic to cells; however, the drug-loaded nanogel has a killing effect on cancer cells, similar to free DOX.

Lu et al. (Lu et al., 2014) formed a pH-responsive peptide nanogel by the hydrazone self–cross-linking of poly (asparagine sultaine-co-aspartyl hydrazide) and poly (asparagine sultaine-co-2-oxyethyl aspartame) under mild conditions. Hydrazone has been extensively studied in biomedical research due to its preparation under mild conditions and hydrolysis under acidic conditions (Bae and Kataoka, 2009). This nanogel has excellent stability and anti-protein adsorption ability. The nanogel has been stable in the PBS solution for 3 weeks and in the protein solution for at least 12 h. When the pH drops to 4.0, the size of the PAsp nanogel will change rapidly. With 18% drug loading rate, doxorubicin (DOX) as a model drug has been loaded onto the nanogel, and the potential of the nanogel as a pH-sensitive drug delivery system was evaluated. When pH = 5.0, the nanogel released more than 70% of the drug in 12 h, and only about 25% of the drug was released at a pH of 7.4. In the in vitro cytotoxicity test, the nanogel showed good biocompatibility. Confocal microscopy results show that the loaded DOX can be successfully released and transported to the nucleus of the tumor cells. Therefore, this easily prepared nanogel is a promising, intelligent drug delivery system.

Arroyo-Crespo et al. (Arroyo-Crespo et al., 2018) developed a family of biodegradable poly (l-glutamic acid) (PGA) conjugates based on pH response, to optimize anticancer effects. DOX-loaded conjugates prepared using different pH-sensitive linkers to connect DOX and amino glutamine could promote specific drug release from the main polymer chain in the tumor microenvironment (TME). In a preclinical metastatic triple-negative breast cancer (TNBC) mouse model, low DOX load and short linkers have the best effects on primary tumor growth, lung metastasis, and toxicologic properties. Juan et al. (Arroyo-Crespo et al., 2018) also determined the relevant molecular mechanisms through transcriptomics analysis, including differential immunomodulation between the conjugates and cell death pathways. Their efforts focus on the advantages of the targeted TME, the therapeutic value of polymer-based combined methods, and methods based on omics analysis that can enhance the anticancer performance of drug delivery materials.

Li et al. (Li Y. et al., 2018) proposed a new method to prepare pH-responsive nanogels by a simple one-step method. They used hydrophilic methoxy poly (ethylene glycol)-b-poly {N-[N-(2-aminoethyl)-2-aminoethyl]-l-glutamic acid} to construct a pH-responsive nanogel by a pH-sensitive benzimine bond. The acid-labile benzimine bond could be cleaved under weak acidic conditions (pH = 6.4) (Figure 3), and then the prepared nanogel could physically encapsulate the therapeutic molecules with high stability with the help of the mPEG shell, whereas, the core of the nanogel at physiological pH could minimize the non-specific absorption of its payloads and side effects. Once the drug-loaded nanogel system accumulated at the tumor site was exposed to the acidic conditions of the tumor, the core part could be quickly destroyed because of the rupture of the benzimine bond, resulting in enhanced drug accumulation at the tumor site. Compared with other pH-responsive nanogels, this nanogel has advantages in three aspects: 1) Preparation and drug loading of the nanogel can be completed in one step; 2) the polymer used is biodegradable and biocompatible, and the nanogel responds to subtle pH changes; and 3) a decrease in pH will trigger the release of the loaded drug and the dissociation of the nanogel, which makes it possible to remove the nanogel that has completed its mission.

Yan et al. (Yan et al., 2020) successfully synthesized a pH-responsive nanomaterial methoxy poly (ethylene glycol)-b-polyethyleneimine-poly (N_ε-Cbz-l-lysine) (mPEG-PEI-PBLL), further improving its controlled release ability and drug loading efficiency. Schiff’s base introduced into the polymer allowed the material to be responsive to changes in pH. mPEG-PEI-PBLL was degraded at pH = 5.0 and stabilized at pH = 7.4. mPEG-PEI-PBLL could self-assemble due to its amphiphilic property and effectively encapsulates the therapeutic agent by introducing PEI (hyperbranched copolymer). The drug loading index of the nanogel was about 25.8%. The PBLL block as a hydrophobic block can obtain a good degree of polymerization, and the polymer structure which was similar to the protein has very good biocompatibility Moreover, therapeutic agents such as DOX and DiD, a near-infrared dye, were effectively contained in the pH-responsive nanogels. In this study, the biological properties of mPEG-PEI-PBLL nanogels were also evaluated by investigating in vivo cell absorption, biocompatibility, and pharmacokinetics. Cell uptake studies indicated that the absorption rate of DOX which was wrapped in the nanogels was much slower than free DOX, subsequently reducing systemic toxicity in the body. The biodistribution of the drug shows that nanogels can reduce excessive damage to healthy cells and tissues. Moreover, in vivo treatment and pharmacokinetic studies indicated that the nanogels could accumulate at the tumor tissue to inhibit the growth of the tumor while improving the survival of mice. These findings reveal that the drug delivery system prolongs the circulation of the drug in the blood and allows the drug to effectively accumulate at the tumor tissue.

Therefore, the nanogels prepared by these two strategies have very broad application prospects.

GSH is an important chemical substance to determine the redox environment in the body. The concentration of GSH in normal tissue cells (2–10 mmol/L) is 100–1,000 times that of the extracellular concentration. Because tumor cells are often deprived of oxygen, the concentration of GSH in tumor cells is at least four times higher than that in normal tissues and cells. Therefore, the tumor cell microenvironment has strong reducibility (Meng et al., 2009; Park et al., 2010; Sun et al., 2013; Huo et al., 2014; Phillips and Gibson, 2014) To this end, numerous studies have been conducted.

Cross-linking is the main component to prepare reduction-responsive nanogels. These kinds of agents are active units that contain disulfide bonds that can be reduced and form a 3D cross-linked redox-responsive network through the disulfide bond to load therapeutic drugs. These nanogels can be reduced by the reducing environment, thereby releasing their payload followed by biodegradation. Many redox reactive crosslinkers used in the synthesis of nanogels contain cystine and cystamine as reductive reactive components, terminal acrylates are involved in free radical polymerization reactions, and PEG was used as the functional backbone of the nanogels (Ma et al., 2010; Yu et al., 2011; Dispinar et al., 2012; Zhang Q. et al., 2013). Disulfide bonds are the main reporting structural units and important structural unit of reduction-reactive materials, which have excellent activity. When GSH or dithiothreitol are present, disulfide bonds will be broken to provide biodegradability and rapid drug release. To this end, disulfide bonds have been used to develop many reduction-responsive nanogels (Wu et al., 2009; Abdullah Al et al., 2013). This section mainly focuses on disulfide-bonded cross-linked nanogels from the perspective of cross-linking agents.

The first strategy is to prepare a disulfide bond cross-linked polypeptide nanogel by the addition of a disulfide bond–containing cross-linking agent into the polypeptide.

Yao et al. (Yao et al., 2019) developed a polypeptide nanogel, poly (ethylene glycol)-block-poly (ϵ-propargyloxycarbonyl-l-lysine) (PEG₁₁₃-b-PPAL) coupled with reducible side chains to deliver antitumor drugs. PEG₁₁₃-b-PPAL was synthesized by ROP of alkyne-containing NCA. Then, they introduced the disulfide bond–containing side chain into the PEGylated polypeptide by the click reaction. The obtained polymer self-assembled to form a nanogel and exhibited a reduction reaction when treated with 10 mmol/L GSH (Figure 4A). Then, DOX was loaded onto the nanogel at a percentage of 6.73wt and a loading efficiency of 40.3%. The average diameters of the blank and polylysine-derived polymer nanogel loaded with DOX (LMs/DOX) were about 48.0 and 63.8 nm, respectively (Figure 4B). In vitro drug release shows that doxorubicin can be released much quickly in the presence of GSH. CLSM and flow cytometry analysis further proved the sensitive release behavior of LMs/DOX to GSH and its ability to enhance the intracellular delivery of DOX. MTT results showed negligible cytotoxicity of the polymer to normal cells L929 or cancer cells MCF-7 (Figure 4C). With the increase of DOX concentration, there was a better anti-proliferative activity in MCF-78 cells in LMs/DOX, LMs/DOX/GSH, and DOX HCl groups. When the concentration of DOX was lower than 2.5 mg/L, the cell viability of the nanogel group was lower than the free-drug group, and the cell viability of LMs/DOX with GSH was lower than LMs/DOX. This may be due to the fact that GSH pretreatment triggers faster release of DOX, resulting in a stronger inhibition of proliferation in MCF-7 cells (Figure 4D). These findings indicate that LMs/DOX have broad prospects in cancer treatment.

The second strategy is to use cystine with a disulfide bond to polymerize and to prepare cross-linked polypeptide nanogels.

Li et al. (Li S. et al., 2018) attempted to reinforce the inhibition of osteosarcoma and lung metastasis by drug-induced necrosis. As shown in Figure 5A, they used a sarcoma-targeting peptide-modified disulfide-bonded cross-linked polypeptide nanogels (STP-NG) to enhance the intracellular delivery of shikonin (SHK), thereby inhibiting the progression of osteosarcoma with minimal systemic toxicity. This study confirmed the ability of STP to target 143B cells. As shown in Figures 5B–D, STP could specifically bind to cell surface vimentin in osteosarcoma, but does not bind to cell surface vimentin–deficient hFOB1.19 cells and blood cells (Satelli et al., 2014). Due to this difference, an ideal efficacy and minimal toxicity can be obtained. In addition, due to the targeting effect of STP-NG/SHK, higher membrane binding force has been observed, thereby increasing the cellular uptake of NG/SHK that could selectively accumulate in 143B osteosarcoma in situ (Figure 5E). RIP1- and RIP3-dependent necrosis in vitro proved that the inhibitory effect of STP-NG/SHK on cell proliferation was enhanced (Figures 5F,G). This animal experiment showed excellent antitumor efficacy of STP-NG/SHK (Figures 6A–E), and metastatic osteosarcoma in the STP-NG/SHK group was mostly suppressed (Figures 6F,G). These data prove that STP-NG/SHK profoundly inhibits osteosarcoma metastasis to the lungs. This excellent anti-metastatic effect was caused by intracellular drug release and tumor targeting. STP-NG/SHK targeting cell surface vimentin was related to epithelial-to-mesenchymal transition, and it is critical for metastasis (Satelli and Li, 2011). As shown in Figure 6H, SHK mainly damages the myocardium, whereas the abnormalities in other organs were minimal. H and E staining showed that compared with free SHK, NG/SHK and STP-NG/SHK showed much lower myocardial damage. These results indicate that coating drugs with nanogels can reduce systemic toxicity and improve biological safety, and STP can further effectively enhance tumor-specific affinity. Therefore, the intelligent drug delivery system equipped with SHK has great significance for VIM-targeted malignant tumor chemotherapy.

Feng et al. (Feng et al., 2021) synthesized three kinds of mPEG-P (LP-co-LC) nanogels with different l-cysteine scales, including mPEG-P (LP₁₀-co -LC₁₅) (NG_10-15), mPEG-P (LP₁₀-co-LC₁₀) (NG_10-10), and mPEG-P (LP₁₀-co-LC₅) (NG_10-5). They evaluated the pharmacokinetic properties, tissue distribution, and antitumor efficiency of these nanogels with similar surface charge, morphology, and reductive reaction characteristics. As the degree of polymerization of l-cystine in the nanogels increased from five to 15, the size of the nanogels also increased from 105 to 256 nm. Researchers found that NG_10-5 has the best stability in physiological environments. Compared with other two nanogels, NG_10-5 could migrate to the tumor faster and accumulate for a longer time after intravenous injection. These results indicate that the size of the nanoparticles was more influential than that of polymers. Compared with the other two nanogels, NG_10-5/DOX maintained a higher blood concentration and retained more drugs in the tumor. The improvement in the pharmacokinetics of NG_10-5/DOX may result from slower liver clearance and more accumulation in the tumor. In addition, among these three preparations, NG_10-5/DOX has optimal antitumor efficacy on 4T1 breast cancer. On this basis, Feng et al. (Feng et al., 2021) used mPEG-P (LP-co-LC) to co-encapsulate DOX and 1-methyl-DL-tryptophan (1 MT) referred as NG/(DOX + 1 MT) for 4T1 breast cancer treatment. By reducing the dose of DOX required to induce immunogenic cell death–inducing (ICD), the author achieved the goal of reducing the toxicity of mice and inducing the ICD of cancer cells at the same time. The drugs in NG/(DOX + 1 MT) were released in the tumor cells at the same time, and they have a synergistic antitumor effect. Nanogels exhibit excellent performance by downregulating the expression and activity of indoleamine 2,3-dioxygenase, limiting the recruitment of regulatory T cells and myeloid suppressor cells, and enhancing the antitumor activity of CD8⁺ T cells (Holmgaard et al., 2015; Peng et al., 2018; Huang et al., 2019).

Liu et al. (Liu et al., 2015) prepared a reduction-reactive nanogel methoxy poly (ethylene glycol)-poly (l-phenylalanine-co-l-cystine) (mPEG-P (LP-co-LC)) with a medium drug loading of 10.2 wt% and a diameter of less than 110 nm. DOX was used as a model antitumor drug loaded onto the nanogels to treat liver cancer in rodent models. The drug-loaded nanogels accumulated in the tumor tissues and released their payload rapidly due to the redox environment in tumor cells after the nanogels entered the cells. The toxicity of the loaded drug was mitigated because of stable encapsulation with the nanogels, and less drug leakage enhanced the retention and permeability effect. Compared with free drugs, the selective intratumoral release of DOX targeting intracellular reduction enhanced the antitumor efficacy of the drug-loaded nanogels.

He et al. (He et al., 2016) used mPEG-P (LP-co-LC) nanogels for RM-1 prostate cancer chemotherapy. DOX acted as a conventional chemotherapeutic drug to be embedded in the nanogels. Based on the drug-loaded nanogels labeled as NG/DOX, GSH induced cell swelling and promoted the release of DOX. Subsequently, NG/DOX presented with effective cell uptake and proliferative inhibition. In addition, NG/DOX showed enhanced antitumor efficacy and safety in the mouse model of RM-1 prostate cancer transplantation, indicating that it has a huge therapeutic potential in clinical practice.

In the study by Guo et al. (Guo et al., 2017), poly (l-lysine)-poly (l-phenylalanine-co-l-cystine), delivered 10-hydroxycamptothecin (HCPT) into BC cells in situ. The drug-loaded nanogel was labeled as NG/HCPT. NG/HCPT increases retention time and improves tissue permeability, thereby promoting cancer cell uptake and intracellular activation of HCPT. Positively charged poly (l-lysine) allows the nanogel to bind to the negatively charged bladder mucosa. Thereby, NG/HCPT was given a mucosal adhesion. In addition, the amphiphilic nanogel with l-lysine residues will allow HCPT to enter cells similarly to amphiphilic cell–penetrating peptides (CPPs) (Shin et al., 2014). Cystine offers an opportunity that the disulfide bonds in the NG/HCPT core can be selectively degraded by intracellular GSH, which further accelerates the release of HCPT, thereby enhancing cell apoptosis (Wang J. et al., 2014; Lin TY. et al., 2016). This feature can reduce the dosage and avoid the potential risk of drug resistance due to high-dose administration. Based on this, Guo et al. (Guo et al., 2020) prepared a positively charged disulfide bond cross-linked nanogel, oligoarginine-PEG-P (LP-co-LC) (R9-PEG-P (LP-co-LC)), which further increases the residence time and enhanced the penetration ability of chemotherapeutics into the bladder wall. CPPs were less cytotoxic and can penetrate through the cell membrane to transport the “cargo” (Douat et al., 2015; Meloni et al., 2015). The combination of CPPs and antitumor drugs is becoming an attractive opinion to enhance the treatment effect (Shi and Qi, 2017). Arginine-rich CPPs have been confirmed with a high-efficient cellular uptake, especially the oligoarginine which contains nine arginine residues (Li et al., 2015; Zhang N. et al., 2016). Therefore, R9 upregulates the mucosal adhesion of the HCPT-loaded R9-PEG-P (LP-co-LC) (R9 NG/HCPT) through an electrostatic interaction with the negatively charged bladder, further promoting the permeability of the drug-loaded nanogel in the bladder wall. In addition, as a CPP, R9 effectively penetrates the cell membrane and transports the drugs. The disulfide bond gives the nanogel the function of selectively delivering HCPT in the cell through the reducing microenvironment. R9 NG/HCPT exhibits excellent cytotoxicity to human BC 5637 cells in vitro and significantly enhances the antitumor activity in mouse and rat orthotopic BC models in vivo.

Chen et al. (Chen et al., 2017b) synthesized morpholine and phenylboronic acid dual-modified polypeptide nanogels (PMNG) to conform receptor-mediated targeting and microenvironment-mediated targeting into a drug delivery system. Phenylboronic acid was used for selective receptor-mediated targeting in the highly metastatic cells, overexpressing sialyl on the membrane (Matsumoto et al., 2010; Sanjoh et al., 2014). Charge-transformable morpholine was neutral at physiological pH, and it has a positive charge in tumor tissues, which can be used to enhance cellular internalization in the tumor microenvironment (Zhang Y. et al., 2013; Guo et al., 2015). The nanogels were cross-linked by the core of disulfide bonds, which can be disassembled by intracellular GSH to selectively release DOX. PMNG has an excellent ability to target primary and metastatic B16F10 tumors, overexpressing sialyl in vitro and in vivo. In addition, PMNG/DOX has shown great advantages in inhibiting the growth of primary and metastatic tumors.

Xing et al. (Xing et al., 2012) prepared reduction-sensitive polymer nanocarriers with near-infrared fluorescent probes. They first synthesized a disulfide bond cross-linked polypeptide nanogel (NIRF nanogels) with near-infrared fluorescence properties and then encapsulated DOX in the core of the nanogels to prepare a near-infrared fluorescent drug carrier (NIRF prodrug). In vitro drug release studies of NIRF prodrugs have shown that there was an accelerated release in the environment where exists 10 mmol/L GSH. Studies on cell uptake of NIRF nanogels and NIRF prodrugs have indicated that they can enter cells through cellular endocytosis. NIRF labeling can directly describe the drug release from the NIRF nanogels using imaging methods, and subsequently the released drug molecules migrate to the nuclei, while the NIRF nanogel remains in the cytoplasm. The in vivo distribution of NIRF nanogels and NIRF prodrugs on tumor-bearing mice indicates that they all accumulate at the tumor site 24 h after injection through enhanced retention effect and permeability. This accumulation may be attributed to the highly permeable vascular structure of the tumor, which leads to the accumulation of nanomaterials (Ichikawa et al., 2004). The NIRF prodrugs that were prepared in these studies have antitumor potential.

The third strategy is to oxidize polypeptides containing sulfhydryl groups to prepare disulfide bond cross-linked polypeptide nanogels.

Wang et al. (Wang et al., 2012) prepared poly (ethylene glycol)-b-poly (l-cysteine)-b-poly (l-phenylalanine) (PEG-PCys-PPhe) by ROP. As shown in Figure 7A, self-assembled into a micelle with a core shell structure in an aqueous solution, the shell has self-crosslinked by the oxidation of the thiol group, and finally a disulfide-bonded cross-linked nanogel with reduction responsiveness was obtained. Glue, even in harsh environments can maintain the stability of the nanogels. As shown in Figure 7B, the hydrodynamic radius measured by DLS was about 150 nm, whereas the average diameter measured by TEM was about 50 nm. As shown in Figure 7C, in vitro drug release studies have shown that nanogels can reduce drug loss and accelerate drug release due to high GSH levels in the cell. As shown in Figure 7D, the cell uptake experiment results showed that the nanogels can be successfully internalized into HeLa cells because all cells exhibited green fluorescence.

In summary, because the disulfide bond contained in cystine can be quickly broken under a high GSH environment, thereby releasing a large amount of drug, this method can greatly improve the therapeutic efficiency of the drug.

ROS play an important role in cell signal transduction. They are involved in various signal paths, such as mediating inflammation, cell growth and differentiation, and regulating enzyme activity. Reactive oxygen species mainly exist in hydrogen peroxide, superoxide, and hydroxyl radicals (Lee et al., 2013) and are highly reactive. It is worth noting that normal cells have a stable redox environment and have a series of systems to remove and balance ROS levels. An increase in the ROS level may destroy intracellular homeostasis and cause oxidative damage to lipase and DNA, thereby leading to a series of diseases. Cancer cells with abnormal regulation of redox homeostasis and stress adaptability are more susceptible to oxidative stress induced by ROS generators, which is a significant basis for ROS therapy to work. ROS-responsive drugs can kill cancer cells selectively and reduce the damage to normal cells. At present, ROS-responsive nanodrugs, microspheres, and polymers have been well-studied. However, there are only a few reports on ROS-responsive nanogels (Liu JN. et al., 2017; Bawa and Oh, 2017; Dharmaraja, 2017).

Deepagan et al. (Deepagan et al., 2016) developed in situ diselenide–cross-linked nanogels (DCMs) that, as anticancer drug carriers, can respond to abnormal ROS levels in tumor areas. The DCMs were derived spontaneously from selenol-loading triblock copolymers that were composed of polyethylene glycol and polypeptide derivatives. As shown in Figure 8A, DOX was effectively encapsulated in the hydrophobic core during the formation of nanogels. The DCMs maintained their stability in blood. However, when DCMs enter into ROS-rich tumor tissues, the hydrophobic diselenide bond of the DCMs was broken into hydrophilic selenic acid derivatives, which triggers the drug release from DOX-encapsulating DCMs (DOX-DCMs). As shown in Figure 8B, both DCMs and non-crosslinked micelles (NCMs) were spherical with the feature of a single peak size distribution. The average diameter of NCM and DCMs was about 65.48 and 85.10 nm, respectively. Least absorption of these nanoparticles by the reticuloendothelial system was observed (Blanco et al., 2015). The DCMs have great structural stability, even in the presence of destabilizing agents, and the DCMs maintained structural stability for at least 6 days under physiologic conditions. The diselenide cross-linking of DOX-DCMs, as a diffusion barrier, can effectively control the release of DOX. However, more DOX was released from DOX-DCMs within 3 days in the presence of H₂O₂ (100 μM). Moreover, enhanced drug release inhibited the efflux of p-glycoprotein (Wang et al., 2011). As shown in Figure 8C, compared with other treatments, DOX-DCM treatment greatly reduced tumor volume. As shown in Figure 8D, the hematoxylin–eosin staining images confirmed that DOX-DCM treatment resulted in the presence of a larger amount of dead cells in tumors, but the damage to the main organs can be ignored.

The enzyme is a kind of biological-related stimulant that can be overexpressed at the edge of tumor invasion and can also be used as a trigger to control the release of the drug. Currently, the main targets are matrix metalloproteinase and cathepsin B.

Kim et al. (Kim et al., 2013) synthesized partially hydrophobically modified polypeptide poly (ethylene glycol)-b-poly (l-glutamic acid) (PEG-b-PPGA) with l-phenylalanine methyl ester moieties and then used it for the synthesis of the template of nanogels. Then, Ca²⁺ was added to make it condense (Figure 9A). The resultant nanogels exhibited features that were similar to those of the hydrogels due to the protonation of the carboxyl group and the pH-dependent helical performance of the PPGA segment. Then, they loaded DOX onto the nanogel at high drug capacity. Under strong destabilization conditions (urea), nanogels maintained their firm structure, but they can be destroyed rapidly by enzymatic degradation. As shown in Figure 9B, the DOX-loaded nanogel can regulate the release of drugs and control the of the nanogel by lysosomal capture. DOX-loaded cross-linked nanogels show lower cytotoxic activity than free DOX. The reduction of cytotoxicity was consistent with the degradation of the nanogels. In addition, as shown in Figure 9C, biodegradable PEG nanogels provided sufficient DOX concentration to inhibit tumor growth in xenograft mouse models of ovarian cancer. Due to the retention effect and enhanced permeability, nanogel particles accumulated in solid tumors. The increase in the circulation time of nanogels (Oberoi et al., 2012) also increases the contact between the tumor and drug. Nanogels have stronger antitumor activity than free adriamycin. In addition, as shown in Figure 9D, there was no significant change between the body weight of the control and treatment groups, suggesting that all treatments were well tolerated.

Guo et al. (Guo et al., 2018) successfully synthesized a tri-block copolymer mPEG-Peptide-PCL, a kind of novel enzyme-responsive nanoparticle. The copolymer can respond to matrix metalloproteinase which is active and overexpressed in cancer, and, therefore, it can be used to guide targeted therapy. In this copolymer, the role of PCL was to load drugs. The peptide was used to target the tumor. The peptide GPLGIAGQ was designed to be degraded by matrix metalloproteinase-2 (Kratz et al., 2001). Cell-penetrating peptide R9 could promote the cellular uptake of nanoparticles (Wang HX. et al., 2014). PEGylation can improve the stability of the nanogel and extend its circulation time in vivo. In vitro, these scholars also evaluated the cumulative release rate of curcumin, as a model drug, under different pH conditions. In addition, they also assessed the cytotoxicity and cellular uptake in L929 and NSCLC A549 cells. In vivo, Guo et al. (Guo et al., 2018) characterized the selective targeting and biodistribution of mPEG-peptide-PCL using an in vivo imaging system because of the strong fluorescence effect of curcumin.

In summary, the enzyme-responsive nanogel is released in response to biological enzymes, which has very good biological targeting and safety properties.

Nanogels can also be designed to release drugs under exogenous stimulation (especially temperature) (Gao and Dong, 2017). The molecular structure of temperature-responsive nanogels usually contains functional groups, such as amides, ether bonds, and hydroxyl groups. Nanogels can respond to changes in external temperature through rapid volume changes, and a discontinuous volume phase transition occurs at a specific temperature, leading to the controlled release of drugs from these nanogels (Lin J.-Y. et al., 2016). This temperature is called lower critical solution temperature (LCST). When the temperature is below the LCST, nanogels are in a swollen state. When the temperature is above the LCST, the nanogels rapidly lose water and shrunk, thereby exhibiting a response to temperature (Kujawa and Winnik, 2001).

Nguyen et al. (Nguyen et al., 2018) synthesized a thermosensitive copolymer heparin-Pluronic F127 to co-encapsulate cisplatin and curcumins to form a dual-drug delivery system. The interaction between hydrated cisplatin and curcumins and the properties of amphiphilic heparin-Pluronic F127 greatly improved not only the working efficacy of the controlled release system but also the drug loading efficiency. The anti-proliferative activity determined by in vitro and xenograft tumor tests revealed that in-depth studies were needed to investigate the uses of this delivery system against various cancers and drug-resistant cancers because of the synergistic activity of bioactive phytochemical and anticancer drugs in nanocarriers.

To deliver fluorescein isothiocyanate–conjugated bovine serum albumin, Ko et al.(Ko et al., 2015) designed temperature-sensitive tri-block nanogels using ionic complexes of hyaluronic acid (HA) and poly (ethylene glycol)-poly (l-lysine)-poly (l-alanine) which are called PEG-PK-PA. The PA block formed a hydrophobic nanocore. The positively charged PK block was compounded with the negatively charged HA block. PEG formed the outer shell of nanogels and prevented random aggregation of nanogels during the formation of ionic complexes. As shown in Figures 10A–C, nanogels have unique reversible temperature sensitivity. Therefore, nanogels shrink when heated. The internalization efficiency of nanogels could be controlled by adjusting the Zeta potential and size of nanogels and by greatly reducing the cytotoxicity of positively changed nanogels through the formation of ionic complexes with negatively charged HA. As shown in Figure 10D, the internalization of the model drug increased greatly if the ζ⁺ and ζ⁰ systems were used, and it was very low if the ζ⁻ system was used. Moreover, the use of the ζ⁰ system led to the most excellent internalization efficacy. As shown in Figure 10E, the MTT assay showed that the ζ⁰ and ζ⁻ systems have a significant improvement in cell compatibility compared with the ζ⁺ system. This occurred because the ionic interaction between the positively charged ζ⁺ system and the negatively charged cell membrane damages the cell membrane and induces cytotoxicity (Hunter, 2006; Kurosaki et al., 2009). Therefore, the negatively charged HA shield was combined with the positively charged PEG-PK-PA nanogel to reduce cytotoxicity. Filipin, rottlerin, and chlorpromazine are specific inhibitors of caveolae-mediated endocytosis, micropinocytosis, and clathrin-mediated endocytosis, respectively (Xiang et al., 2012; Saha et al., 2013). As shown in Figure 10F, flow cytometry results have confirmed that fluorescence was greatly reduced by the chlorpromazine-treated ζ⁰ system. This suggests that clathrin-mediated endocytosis led to the internalization of the ζ⁰ system.

Kim et al. (Kim et al., 2018) designed a biodegradable and thermoresponsive linear-dendritic nanogel containing poly (l-lysine), poly (l-lactic acid), and poly (N- isopropylacrylamide). The LCST of the nanogel was very low when it is in water at temperatures of 30–37°C. When the concentration is between 0.05 and 1 mg/ml (Kim et al., 2006), the integration of the poly (l-lactic acid) component provides the necessary hydrophobicity, and the drug reservoir was loaded with lipophilic reagents through hydrophobic–hydrophobic interactions. The nanogel has good biocompatibility. The nanogel itself and its degradative products were non-cytotoxic to neuron-like PC12 cells for at least 1 month. The nerve growth factors were added to the nanoparticles via aqueous phase mediation, which were slowly released from the nanogel for about 12 and 33 days at 25 and 37°C, respectively. The released nerve growth factor exhibits biological activity by promoting the neurite growth of PC12 cells. This study reveals a new concept that using biodegradable and thermoresponsive nanogels for the treatment of neurologic diseases through thermal targeting and sustained release of nerve growth factors and other protein drugs.

In summary, the temperature-sensitive nanogel is stimulated by external factors to achieve the purpose of the release and has better controllability.

Light has become a stimulus that has attracted a lot of attention, is particularly suitable for the design of advanced biomedical platforms, improves the spatiotemporal control of the behavior of biomaterials, and dynamically adjusts their performance (Rapp and DeForest, 2020). In addition, the light-responsive platform also provides numerous opportunities for sequential degradation of implanted devices or remotely controlled drug delivery in a safe and non-invasive manner (Li et al., 2019).

Inspired by the design of a “Trojan horse”, Chen et al. (Chen et al., 2019) developed a photocontrollable nanogel, which is called SiPING, through simple self-assembly of three functional materials. The SiPING was composed of three functional components, including 1) long-circulating biocompatible polymer poly (ethylene glycol)-b-poly (l-glutamic acid), which prevents the nanogels from being detected by drug efflux transporters (DETs) on the membrane to enhance the accumulation and circulation of the nanogels; 2) photodegradable indocyanine green, which enables the photoactivatable disassembly for photo-controllable drug release; and 3) green fluorescent organosilica nanodot, which can be used as the bridge connecting the other two components and bioimaging probes. The traceable “SiPING” plays the role of a “Trojan horse” and has a very broad application prospect as a multifunctional nanoplatform. As shown in Figure 11, due to the existence of DET, the plasma membrane acts as the gate of the cell, making it difficult for DOX to cross.

By hiding inside the nanogel with a suitable surface coating to avoid the recognition of DETs, lots of DOX molecules were transported into multidrug-resistant cells via endocytosis pathways. This allows DOX to form a high local concentration near the nucleus to prepare for further transport to the nucleus. Under the condition of photo-triggered degradation of the nanogels, a large amount of “auxiliaries” were released from the nanogels, allowing the drug to quickly occupy the nucleus under the condition of DETs and were still on present on the nuclear membrane; thus, multidrug resistance can be effectively circumvented. Therefore, the multidrug-resistant cells could be killed by DOX-loaded nanogels in two steps: 1) DOX-loaded nanogels’ behavior like a “Trojan horse” and escape from DETs on the plasma membrane, effectively transporting DOX into the cytoplasm and preventing drug efflux 2) Irradiation with near-infrared light leads to the decomposition of the nanogel, releasing a large number of DOX that can escape from DETs on the plasma membrane, thereby exerting its nuclear effect in multidrug-resistant cells.

There are few studies on light-responsive nanogels. Generally, photo responsiveness and thermal responsiveness are used in combination to achieve better results.