2.1. pH-Responsive Drug Delivery Systems
The variation of pH values in different regions of the human body can provide a suitable physiological stimulus for pH-responsive drug delivery in order to deliver the active substances in the area of interest [
82]. For example, the pH range of the stomach is between 1.5–3.5 pH, 5.5–6.8 pH of the intestine, 6.4–7 pH of the colon, and up to 7.4 pH of the blood [
83]. The pH of cancerous tissue exhibits a decreasing trend due to the Warburg effect, which explained that the hypoxic cells produce lactic acid due to glycolysis [
35]. Further, pH-responsive drug delivery systems have excellent advantages and have attracted much attention in the past decade because the pH values in tumors and inflammatory tissues are significantly lower than those in blood and normal tissues and could increase the therapeutic efficacy of administrated drugs [
33,
35,
84,
85,
86]. The main principle of pH-responsive drug delivery systems is to release the active substance when the pH trigger point is achieved, and the intracellular concentration of drugs is equal to the therapeutic dose needed [
34]. This release system time has the role of releasing the active substance preferentially, in order to eliminate the disadvantages of chemotherapy treatment.
One modality to obtain this type of release system is to introduce “ionizable” chemical groups, for example, amines, phosphoric acids, and carboxylic acids among others, with nanomaterials [
87]. These groups, with varying pKa values and chemical structures, have the ability to accept or donate protons and be subjected to pH-dependent alterations in the chemical or physical properties, such as solubility and swelling ratio, culminating in drug release [
34]. Various biomaterials have been reported in the obtaining of pH-responsive drug delivery systems, such as inorganic nanoparticles, core-shell nanoparticles, liposomes, hydrogels, and polymer micelles [
12,
88,
89,
90]. An example of some widely used materials for the production of pH-sensitive drug delivery systems are pH-sensitive polymers, which are polyelectrolytes with ionizable groups in their backbones, side groups, or end groups. When the pH of an aqueous solution changes, these pH-sensitive polymers are ionized, resulting in a change in their conformation. These “smart” polymers can either accept or donate H+ ions in response to the pH changes in the environment. The protonation or deprotonation of these ionizable groups can lead to changes in the structure of the polymer chain by electrostatic repulsion of the generated charges, which causes the transition of the chains from collapsed to an expanded state [
83]. For example, pH induces protonation/deprotonation in the -NH2 groups of chitosan, making it susceptible to use in obtaining pH-sensitive release systems [
91,
92]. At acidic pH (<6), primary amines are protonated and positively charged, making chitosan soluble in an aqueous solution [
93]. In addition to sensitivity to pH, chitosan is low toxicity, biocompatible, has antibacterial properties, mucoadhesive properties, and permeation-enhancing effects [
94,
95]. Due to these beneficial characteristics of chitosan, many researchers have used chitosan in various pH-sensitive release systems for liver cancer [
96,
97,
98,
99,
100]. Mi et al. [
96] reported pH-sensitive drug delivery for DOX based on Carboxymethyl-β-Cyclodextrin/Chitosan Nanoparticles. In vitro release showed higher cumulative release rates of DOX from HF-DOX-CD NPs at a low pH, than cumulative release at neutral pH or slightly basic pH due to the tendency of chitosan nanoparticles to swell at low pH, and also, due to the acidic pH, the protons might penetrate the interior and attack the inner secondary bonds of the nanoparticle. These two reasons lead to a release of DOX in an acidic medium. Zhan et al. [
79] developed pH-sensitive and self-healing properties based on 4armPEGDA and N-carboxyethyl chitosan for liver cancer. The degradation rate was observed to be higher in the case of exposure to an acidic environment indicating that hydrogels have pH-dependent degradation behavior. Cytotoxicity tests were performed on human hepatocytes (L02) and the therapeutic effect of DOX-loaded CEC/4armPEGDA hydrogels on HepG2 cells. The results showed no toxicity for human hepatocyte cells and even at a fairly small load of DOX the obtained system was able to kill tumor cells over time due to the continuous release of DOX in the hydrogel during this process. Qu et al. [
65] presented the obtaining of a pH-sensitive hydrogel that was able to release DOX into an acidic environment based on N-carboxyethyl chitosan (CEC) and dibenzaldehyde-terminated poly(ethylene glycol) (PEGDA) as a possible treatment for hepatocellular carcinoma. At an acidic pH, the amino groups of chitosan become protonated and positively charged, resulting in weaker -NH
2 and -CHO bonds, that also decompose and the release of the DOX takes place.
Furthermore, many polymeric networks have been studied for showing pH-sensitive bioerodible properties such as Poly(Ortho Ester Amides) (POEAd) due to the hydrolysis of ortho esters at low pH [
61]. Yan et al. [
101] proposed a solution for the promotion of drug accumulation and efficient killing ability of tumoral cells via galactose-grafted on an ultra-pH-sensitive drug carrier (POEAd-g-LA-DOX micelles), which can respond to both intracellular and extracellular pH, to remain stable at pH 7, responding to extracellular tumor pH, conjugating receptors in the cell membrane of liver cancer through surface galactose-ligands of micelles, and being sensitive to intracellular tumor pH following further swelling for rapid drug release. The POEAd-g-LA copolymers were successfully obtained via facile polycondensation followed by the grafting of lactobionic acid.
Figure 2 shows the percentage of main chain hydrolysis over time, as well as size and accumulation release over time, at different pH values. In order to assess the influence of pH, Yan carried out an NMR analysis to follow the time-course of the hydrolysis of ortho esters in the main-chain for up to 72 h in deuterated water buffers (pH 7.4, 6.5, and 5.5). The hydrolysis of the ortho ester did not occur at neutral pH, while at pH 5.5 it was observed that the hydrolyzation of ortho esters was accelerated due to the acidic pH, which could lead to a size reduction and drug release. Furthermore, it was highlighted that the ultra-pH-sensitivity of ortho ester can influence the size, observing that at an acidic pH the size of the particles increases, swelling itself, which was probably due to the interaction between the hydrogen bonding interaction of the hydrophilic products with plenty of hydroxyl groups and amide, and gradually increasing hydration. Additionally, the rate of drug release was reported to be higher in the case of an acidic medium, which can thus be a benefit due to the acidic pH of the tumor tissue. The in vivo therapeutic efficacy of the reported formulations was tested in the mice bearing subcutaneous-inoculated H22 tumors, and it was observed that in relation to the mice treated with POEAD-g-LA20-DOX a decrease in the tumor size was reported, reducing the growth, and demonstrating an inhibitory effect on tumoral tissue.
Hyaluronic acid (HA) has been widely used in targeted drug delivery systems due to its good biocompatibility, good water solubility, high selectivity, and affinity to CD44 receptors found in different tumor cells [
101,
102]. In order to increase the drug/cargo accumulation specifically in CD44 over-expressing cancer cells, HA-attached pharmaceuticals and nanocarriers have been created. This is because HA has numerous functional groups available for chemical conjugation with anticancer medications or nanocarriers of drugs or genes [
103]. Further, it was reported that DOX could be covalently bounded on the backbone of HA through the hydrazone linkage [
62,
104]. Li et al. [
105] described that hydrazone bonds disintegrated in lysosomal pH and remained stable at neutral pH. Liao et al. [
62] reported a pH-responsive drug delivery of hyaluronic acid-hydrazone linkage-Doxorubicin (HA-hyd-DOX), which is illustrated in
Figure 3. Due to the amphiphilic structure between the hydrophilic glucopyranose ring and hydrophobic DOX segment, a series of HA-hyd-DOX NPs were generated by self-assembling in an aqueous solution. The results showed a burst release in the first 6 h in buffers at pH 5.0 due to a looser structure of particles with the cleavage of hydrazone bonds at a lower pH. The cell cytotoxicity was studied on HeLa and L929 cells for 48 h of incubation and a nontoxicity against HeLa or L929 cells was observed, suggesting that HA is nontoxic, having good biocompatibility. Additionally, an internalization was observed due to the receptor-mediated binding affinity of CD44 for HA with high specificity.
Wu et al. [
63] reported on the liver cancer-targeting mixed micelles based on hyaluronic acid–glycyrrhetinic acid conjugate and a hyaluronic acid-L-histidine conjugate S(HA–GA/HA–His) were prepared via ultrasonic dispersion, and the in vitro and in vivo investigation of antitumor effect of Doxorubicin (DOX)-loaded micelles (
Figure 4). It was related to the pH sensibility of DOX-loaded HA–GA/HA–His is micelles and the remarkable absorption of HepG2 cells. It was reported that the hydrophobic DOX molecules were efficiently encapsulated into the HA–GA/HA–His micelles in an aqueous solution because of the presence of a hydrophobic core in the micelles. It was shown that the obtained DOX-loaded micelles exhibited a sustained DOX release under the acid pH of hepatocellular carcinoma cells, due to protonation of His, resulting in the swelling of the core, followed by the DOX release.
Anirudhan et al. [
64] delivered DOX and cisplatin (CDDP) via pH-responsive drug delivery, also based on hyaluronic acid (HA) and chitosan (CS-NSA). The release was studied via immersion in two buffer media at pH 7.4 and 5.5 to mimic the intestinal fluid and tumor environment, at 37 °C, for 48 h. A better release of DOX was observed in comparison with cisplatin, for both pH levels. Further, a higher release was observed for both drugs at pH 5.5 (for CS-NSA/A-HA/DOX, the release was 89.0% at pH 5.5 and 42.0% at pH 7.4 and for CS-NSA/A-HA/CDDP, the release was 87.0% at pH 5.5 and 44.6% pH 7.4). Lei et al. [
106] developed a pH-responsive drug delivery based on HA showed a great release under the endosomal/lysosomal environment for DOX (56.5%) (
Figure 5).
Folic acid (FA) was reported as an effective targeting prodrug due to the selective binding of FA to the folate receptor (FR), which is overexpressed in cancer cells [
107]. Zeolitic imidazolate framework (ZIF) coordination bonds are sensitive to low pH and this could be used for the delivery of an active substance, such as DOX [
108]. Bi et al. [
66] reported the delivery of DOX for human hepatocellular carcinoma via folic acid-modified and zeolitic imidazolate framework (ZIF) nanoparticles. It highlighted the pH sensitivity of the obtained nanocarrier; thus, it was observed that drug release of the drug delivery system based on folic acid is increased by the acidic environment. Further, it was reported that the release mechanism of the DOX is influenced by the ZIF degradation in acid environments and the increased solubility of DOX at lower pH as a result of increased protonation of amino groups in DOX molecules. In addition to the previously listed advantages of pH-sensitive systems, the limitations of pH-responsive drug delivery systems are that the pH level is an endogenous stimulus and this makes it difficult to control, a narrow range of pH variation, and steady kinetics for the drug release [
39,
109,
110,
111]. These limitations can be overcome by the development of multi-responsive drug delivery systems, so that the release is not based only on a single stimulus, such as pH.
2.2. Temperature-Responsive Drug Delivery Systems
One of the most closely studied stimuli of controlled release systems is temperature. Temperature-sensitive polymers are used in order to obtain this responsive DDS, for different biomedical applications, such as temperature-sensitive gels, liposomes, micelles, colloidal particles, mRNA recovery, and gene delivery [
112,
113,
114]. These thermosensitive polymers are able to release the encapsulated active substance even at small temperature variations. Numerous thermosensitive compounds are used for obtaining thermoresponsive hydrogels in DDS applications, such as poly(N-isopro-pylacrylamide (PNIPAAm) derivatives, poly(ethylene oxide)-poly(propylene oxide) (PEO–PPO) pluronic copolymers), core–shell thermoresponsive NPs, polymeric nanotubes, polymeric micelles, layer-by-layer (LBL)-assembled nanocapsules, microbeads (MBs), and elastin-like polypeptides (ELPs) [
50,
115,
116]. However, thermo-responsive release systems can retain the load at systemic circulation temperatures of 37 °C, but release a load rapidly when the temperature exceeds 40 °C, due to the locally heated tumor. These DDS experience a reversible phase transition from a molecularly dissolved hydrated state in an aqueous solution (hydrophilic) to a dehydrated state (hydrophobic) as a reaction to the slight variation in temperature leading to an induced sharp globule-to-coil conversion that generates the release of encapsulated antitumoral agents from these polymeric nanocarriers [
117]. Depending on the critical solution temperature (CST), thermo-responsive polymers are classified into two categories: (i) polymers that have a low critical solution temperature (LCST), which means that these polymers are water-soluble and make homogenous systems below this temperature; and (ii) polymers that have an upper critical solution temperature (UCST), which means that these polymers are water-soluble and make homogenous systems above this temperature [
117].
Poly(N-isopropyl acrylamide) (PNIPAA)-based materials show thermo–thermo responsiveness behavior which could be very useful in the development of different temperature-sensitive release systems [
118]. Cheng et al. [
67] developed a PEGylated star-shaped polymer based on the conjugation of the β-CD core with thermosensitive poly(N-isopropyl acrylamide) (PNIPAAm) and biocompatible poly(oligo(ethylene glycol) acrylate) (POEGA) arms in order to obtain temperature-sensitive drug delivery systems for liver cancer. The DOX was used as a model chemotherapeutic in order to study its in vitro cellular uptake. It was shown that only DOX and β-CD-g-(PNIPAAm-b-POEGA)x/DOX at 25 °C have a slow cellular uptake, but when the tempera ture was increased to 37 °C, a faster and higher cellular uptake was reported. Interestingly, at a temperature above LCST, PNIPAAm becomes hydrophobic, having a tendency to aggregate, forming the core of nanoparticles, meanwhile, the POEGA chains help maintain the integrity of the formed nanoparticles. In addition, it was reported at 80% in the first 6 h and 90% release after 24 h of DOX at 37 °C condition; meanwhile, at the 25 °C condition, a slow release of DOX was observed, where after 24 h, the release decreased to 53.9%. In
Figure 6, the decrease in the cell viability is reported in the case of β-CD-g-(PNIPAAm-b-POEGA)x/DOX in comparison with neat DOX. The test was performed on HepG2 and H460 cancer cells. The same results were shown in the case of hydrophobic paclitaxel (PTX), which is a hydrophobic anticancer drug. Kunene et al. [
68] reported pH and temperature-responsive based on magnetic graphene nanosheets (MGNSs), functionalized by poly(N-isopropylacrylamide) (PNIPAM) and polyethylenemine (PEI) nanogel for DOX delivery for liver cancer. The cell viability was reported at above 90% against HEK293 cells and HepG2 cancerous cells. The DOX release was reported higher at pH 5.4 than at pH 7.4 and above, the LCST was rapid, due to the swelling and de-swelling of the PNIPAM/ PEI nanogel at variations of the medium’s temperature.
Furthermore, other studies were reported in order to release DOX for liver cancer through temperature-responsive drug delivery. Mdlovu et al. [
69] described a dual-responsive drug delivery system (pH- and thermo-responsive drug delivery system) based on magnetic iron oxide (MIO) nanoparticles functionalized with Pluronic F127 (PF127) and branched polyethylenimine (bPEI) and loaded with DOX. The DOX releases were dependent on temperature and pH as the highest release rate (54.8%) was in acidic conditions (pH 5.4) and when the temperature was increased from 37 °C to 42 °C an increased rate release (51%) was reported due to the LCST of the PF127 polymer which is 42 °C [
119]. Furthermore, Mdlovu et al. [
120] described pH- and thermo-sensitive Doxorubicin-conjugated magnetic SBA-15 mesoporous for hepatocellular carcinoma with a release rate of 70% in acidic conditions and 69% at 42 °C (pH = 7.4), which in comparison with previous work, showed an increase in the release values of DOX. Sebeke et al. [
70] reported thermo-responsive drug delivery based on phosphatidylglycerol (DPPG2) via magnetic resonance-guided high-intensity focused ultrasound (MR-HIFU)-mediated hyperthermia. A rapid release was reported at 42 °C due to the melting temperature of DPPG2-TSL. Classical methods of chemotherapy agents are hemofiltration or plasma filtration [
121,
122]. Removal of ~30% of the administered dose was reported and a reduction in the toxicity of the remaining drug in the organism.
2.3. Redox-Responsive Drug Delivery Systems
At this moment, redox-responsive drug delivery systems have been intensely studied, to improve the controlled release of the antitumoral agents, via targeting the tumoral tissue through redox-response in the presence of glutathione (GSH) [
71]. The cancerous tissue presents particular abnormal cellular environments, such as the presence of enzymes and reducing environments [
123]. The main principle of redox-responsive drug delivery systems is employing the distinct differences in redox potentials between tumors and normal tissues. The reducing environment of cancerous tissue is based on the reduction and oxidation state of NADPH/NADP
+ and glutathione (GSH, GSH/GSSG) [
123,
124]. The most popular redox couple is GSH/glutathione disulfide (GSSG) [
125]. The GSH is a tripeptide of glutamate, cysteine, and glycine found at an increased concentration in ovarian, breast, lung cancer, head and neck cancer, and in lower concentration in brain and liver tumors compared to healthy tissue [
126]. In addition, it was reported that the GSH plays an important role in cell differentiation, proliferation, and apoptosis, and imbalanced values may indicate cancer presence [
127]. The GSH levels can be influenced by oxidative stress, and can act as a biomarker, to indicate the severity of cancer. The highest concentration of GSH values for healthy tissues is in the liver and hepatic GSH plays an important role in interorgan GSH homeostasis by being the main source of plasma GSH. Liver disease can decrease the concentration of GSH values due to multiple factors, such as reduction during oxidative stress, increased utilization and export, and decreased synthesis [
128].
The main advantages of redox-responsive delivery systems are the stability in contact with the normal tissue, which can decrease the systematic toxicity and side effects, the prompt response to high values of GSH concentration in tumoral cells to release the therapeutic agents, and the release of the therapeutic agent in the cytoplasm, which improves the therapeutic effect [
123]. The main categories of redox-responsive drug delivery systems are disulfide bonds (which are able to break down via reducing glutathione to sulfhydryl group and break the drug delivery system and facilitate the therapeutic agent release) and diselenide bonds (the Se–Se bond and C–Se bond are with lower bond energy than that of the S–S bonds) [
123,
129,
130]. Luo et al. [
131] reported the pH and redox-responsive drug delivery for DOX via deprotonation/protonation under acidic pH and cleavage of disulfide bonds (
Figure 7).
These linkages express great stability in the oxidative extracellular medium, the therapeutic agent is released in the increased reductive intracellular compartments by thiol–sulfide exchange reactions [
72]. Chen et al. [
71] developed a new antibody-targeted and redox-responsive drug delivery system by binding the anti-carbonic anhydrase IX antibody (A-CAIX Ab) on the surface of mesoporous silica nanoparticles (MSNs) via disulfide linkages used as the vehicle to load the chemotherapy drug, Doxorubicin (DOX). In
Figure 8, it is shown how the MSNs are used as a vehicle to load the DOX and CAIX grafted on MSNs by disulfide bonds. The in vivo tests performed on mice showed a reduction in tumor weight after only 11 days as well as MSNs loaded with DOX and DOX@MSNs-CAIX.
Wang et al. [
132] developed a redox-responsive liposome, capable of probably leading both cancer stem cells and bulk cancer via the incorporation of Salinomycin (Sal), which is a hydrophobic drug, into the lipid layers of the obtained liposomes, and Doxorubicin (DOX), a hydrophilic drug, encapsulated into the aqueous cavity of the liposomes. It was shown that GSH potentially affects the disulfide bonds of obtained liposome lipid layers destabilizing the liposomal nanostructure and the presence of GSH may influence the fast release of DOX and Sel, leading to the synergistic inhibition of tumor growth and reduction in cancer stem cells’ stemness. Mezghani et al. [
72] described that the introduction of disulfide linkages acts as a burst release constituent and the hydrophobic groups of the glycyrrhetic acid were covalently linked to the hydrophilic backbone of hyaluronic acid over amide bond development, with the mixture of an intermediate disulfide bond as a major component. The resulting swelled nanoparticles from the reduction in the disulfide bonds lead to hydrophobicity modification of the core of nanoparticles conducting to the production of aggregates, which can lead to the deconstruction of the nanoparticle in the deeply reductive environment. In addition, it was observed that the size of the nanoparticles remains unchanged in the absence of GSH, which indicates the stability of the obtained nanoparticles in the non-reductive media. Furthermore, the performed in vivo test showed that the obtained DDS accumulated in hepatic tissue, approving their targeting abilities.
Additionally, Yang et al. [
25] reported the obtaining of a redox-responsive drug delivery system based on a hyaluronic acid (HA)-grafted polymer loaded with DOX. The HA is conjugated with folic acid (FA) through a reduction-sensitive disulfide linkage in order to design an amphiphilic polymer (HA-ss-FA). Further, cystamine (CYS) was used as a cross-linking agent to link HA and FA. The DOX release was studied in a phosphate buffer solution (PBS), at physiological pH and temperature, in the presence of GSH, in order to simulate the environment of the tumor cells and it was observed that the presence of GSH influenced a faster release of DOX because of the disruption of the disulfide bond in the HA-ss-FA molecules in the reductive environment. Pandey et al. [
133] presented a dual-stimuli-responsive drug-delivery system based on micelles for cancer therapy using Doxorubicin. The micelles are represented by an amphiphilic biocompatible miktoarm star copolymer comprising two hydrophobic poly(ε-caprolactone) (PCL) blocks, a short poly(propargyl glycine) middle block, and a hydrophilic glycopolypeptide (GP) block containing galactose units for targeting liver cancer cells and were tested for responsiveness via two stimuli, such as redox and enzymes. It was observed that with a higher concentration of GSH, the release of DOX was increased, and in the case of the absence or lower concentration of GSH in the media, the release was radically decreased probably due to the presence of a few lightly cross-linked micelles. Additionally, the TEM images showed that after 24 h, the obtained micelles showed aggregation, and after 48 h the micelles disappeared in the presence of GSH treatment showing that the micellar assembly is only rattled by GSH-mediated cleavage of disulfide bonds. A multifunctional dual-responsive drug delivery system for targeting tumor therapy based on hollow mesoporous nanosilica loaded with Doxorubicin was proposed by Huang et al. [
73]. Cytochrome C (CytC) was used as a sealing agent for mesoporous nanosilica and also as a mediator of apoptosis by recruiting and activating caspase once it is released from the cell mitochondria to the cytoplasm via conjugation with Apoptotic protease activating factor-1 (Apaf-1) in the presence of Deoxyadenosine triphosphate (dATP) which is a nucleotide used for DNA synthesis as a substrate in DNA polymerase [
134,
135]. In addition, the usage of lactobionic acid (LA) was utilized as a targeting agent, especially for HepG2 cells due to the particular ligand binding to the asialoglycoprotein receptor (ASGP-R) of HepG2 cells. The purpose of this dual-responsive drug delivery was to create a special delivery of DOX in the presence of glutathione (GSH) and acidic pH in the tumor microenvironment. The drug-release results showed that an increased release was observed in the case of an acidic environment and in the presence of a higher concentration of GSH via the simultaneous breakage of disulfide bonds and disassociation of boronated ester bonds of the system, which can lead to cell apoptosis and tumor growth inhibition [
73]. Saedi et al. [
136] described a dual-sensitive drug delivery (redox and pH-sensitive) based on a folate-modified star-like amphiphilic copolymer based on castor oil for DOX. The drug release was studied in PBS (pH = 7.4, 2 μM GSH) and ABS (pH = 5.5), with or without 10 mM GSH, and the results showed that at pH 7.4 the release of DOX was slow and inefficient, but at pH 7.4 and 2 μM GSH, the release was approximately 20% and by increasing the concentration of GSH (10 mM GSH), the release was increased up to 39%. The acidic condition also favors the release, such as when at pH 5.5 and 10 mM GSH, the biggest release of DOX was observed. Yan et al. [
137] described polyethyleneimine (disulfide cross-linked PEI, PSP)/tetrahedral DNA (TDNs)/Doxorubicin (DOX) nanocomplexes (NCs)-based redox-responsive drug delivery system.
Figure 9 details the gradually disassembled drug delivery system through the breakage of the disulfide when it interacts with the intracellular high concentration of GSH at the tumor site. Further, the disassembled DDS penetrated the tumoral tissue, improving the therapeutic efficacy. An increase in the release was observed in the presence of an acidic environment and GSH (50%) due to cleavage of the disulfide linkages in the high concentration of GSH.
2.4. Enzyme-Responsive Drug Delivery System
In recent years, many intelligent systems have been studied for the release of Doxorubicin based on enzyme responsiveness [
138,
139,
140,
141]. Enzymatic-responsive drug delivery systems represent a very promising category, due to the fact that changes in the expression can be found in tumor cells of specific enzymes, such as proteases, phosphatases, and glycosidases, which can be very easily targeted by enzyme-mediated drugs’ release [
130]. The enzyme-responsive drug delivery systems have ester bonds in their composition or the peptide structure that can be degraded by various enzymes specific to inflammation of the tumor location [
142,
143]. The main properties of enzyme-responsive drug delivery systems are biorecognition, selectivity, and catalytic efficacy [
142]. Generally, enzymatic-responsive drug delivery systems are obtained from peptide hydrogels, polymers and polymer conjugates, and polymeric nanoparticles, but also from mesoporous silica nanoparticles, metal nanoparticles, and semiconducting nanoparticles [
144,
145,
146]. Enzyme-responsive polymers are used to incorporate the therapeutic agent, and in the presence of the enzyme found in the body, to release the therapeutic agent in a targeted way [
147]. The most widely used enzymes for drug delivery systems are the hydrolases, including proteases, lipases, and glycosidases, due to the simple design requiring the attachment of bioactive moieties to the carrier through enzyme cleavable unit [
146]. Proteases are enzymes that break down the peptides at the level of amino acids, being involved in many physiological processes such as tissue remodeling, wound healing, and tumor invasion [
148]. An overexpression of proteases has been associated with cancer, so proteases can be used to allow for the selective activation of smart drug delivery platforms [
149]. Yildiz et al. [
74] reported core–shell nanoparticles based on amphiphilic copolymers poly(lactic-co-glycolic acid)-b-poly-l-lysine and poly(lactic acid)-b-poly(ethylene glycol) for Doxorubicin-loaded protease-activated drug delivery systems. The cytocompatibility was evaluated on MDA-MB-231 breast cancer cells and a significantly reduced cell viability was observed at drug concentrations of 0.10 µM.
Lipase, such as phospholipase, are enzymes that hydrolyze fats. However, in this case, it has also been observed that it can play the role of a pathological indicator for various conditions, such as many kinds of cancers and other conditions such as thrombosis, congestive heart failure, inflammation, neurodegeneration, and infectious pathogens [
148].
Another enzyme intensively studied in the enzyme-responsive drug delivery field is azoreductase, which is an enzyme produced by micro-organism species generally present in the colon [
148].
Another studied enzyme is azoreductase, which is a reductase enzyme extensively studied in the case of liver cancer, so that Medina et al. [
75] reported the development of enzyme-activated nanoconjugates for the treatment of liver cancer through the release of DOX by using L1-L4 azo-linkers to conjugate a generation of 5 of poly(amidoamine) dendrimer and designed to be able to bind to hepatic azoreductase enzymes. The obtained enzymatic-responsive system was tested on Hep G2 and Hep 3B cells. The results showed a non-toxicity for cardiomyocytes, comparing with the silenced toxicity after the classical administration of DOX at the same concentration and are readily cleavable by intracellular azoreductase enzymes proven to be effective in killing liver cancer cells, having IC
50 value similar to free DOX. Sun et al. [
150] reported an NTR-responsive 4-nitrobenzyl group, hydrophobic AIE, tetraphenyl ethylene (TPE), and polyethylene glycol hydrophilic moieties for DOX drug delivery trough nitro reductase (NTR)–catalyzed. It was reported that 4 HeLa and HEK 293T cells were used in order to evaluate the cytotoxicity. The TNP-based drug delivery system showed that the DOX release was possible in the presence of NADH due to high sensitivity and selectivity to NTR, leading to the breakdown of the micelles and DOX release.
The main disadvantage of enzyme-responsive drug delivery systems is the release of the therapeutic agent before reaching the target area due to possible exposure to an enzyme trigger, or a closely related enzyme could release the load prematurely. This limitation can be overcome by obtaining a release system sensitive to dual stimuli, with a component sensitive to the pH variation, which favors a much more targeted release [
151]. Gao et al. [
152] reported a dual-responsive drug delivery based on hydrophobic-modified sodium alginate. In vitro cellular uptake and cytotoxicity were tested on HepG2 or Hela cells, and the results showed had good growth inhibition effects on HepG2 cells or Hela cells and also a slow release effect was shown. In
Figure 10, the schematic synthesis and stimuli-responsive release is shown. This dual-stimulus drug delivery system is capable of releasing DOX in a much more targeted manner, due to lysosomal enzyme which is present in all mammalian cells.