Smart Stimuli-Responsive and Mitochondria Targeting Delivery in Cancer Therapy

Introduction

In addition to cancer prevention and early detection, an effective cancer treatment strategy is undoubtedly very important.^1,2 In order to achieve maximum efficacy and minimal side effects, it seems to be necessary to precisely control drug location and maintenance at the destined tumor cells.^3,4 Mitochondria (Mc) is linked to many hallmarks of cancer cells and its dysfunction may induce tumor cell death, so Mc has been considered as a pharmacological target over the last decades.⁵ Scientists have developed various smart stimuli-responsive (St) and Mc-targeting drug delivery systems (DDSs) (St-Mc-DDSs) (Figure 1). These systems have better efficacy and higher safety compared to Mc-DDSs or Sm-DDSs.^5–10

Figure 1 Schematic diagram of the therapeutic mechanism of St-Mc-DDSs. Notes: (A) Endogenous and exogenous St types of St-Mc-DDSs. (B) Smart St-Mc-DDSs are achieved by combining St- and Mc-targeting strategies. (C) Five St-Mc-DDS types classified according to the spatial location relationships of different types of St- or Mc-DDSs. (D) Two types of Mc-targeting modes according to the structure.Abbreviation: Mc, mitochondria; Mc-DDSs, mitochondria-targeting drug delivery systems; St, stimuli-responsive; St-DDSs, stimuli-responsive drug delivery systems; St-Mc-DDSs, smart stimuli-responsive and mitochondria-targeting drug delivery systems.

St-Mc-DDSs have the added advantages of St-DDS and Mc-DDS (Table 1), including (1) hierarchical targetability. St-Mc-DDSs deliver drugs into tumor cells and cellular Mc organelles successively. (2) Controlled drug release. St-DDSs only release drugs at the target tumor tissue under the stimuli trigger but does not release or leak drugs at nontarget tissue sites. After entering the cells, St-Mc-DDSs further accurately deliver drugs to the target organelle Mc under the guidance of the Mc-targeting group. (3) This system can achieve maximum therapeutic efficacy and produce minimal adverse effects. Drug molecules accumulate as much as possible at the precise target site organelle to exert efficacy while spending as little time as possible in normal cells and other organelles to reduce nonspecific toxicity. St-Mc-DDSs avoid the respective design based-deficiencies of the single St-DDSs (such as low drug concentration at the Mc) or Mc-DDS (such as high drug leakage at nontarget tissue/cell).

Table 1 A Comparison of the Characteristics Among St-Mc-DDS, St-DDS and Mc-DDS

The Mc presenting in most eukaryotic cells has been well recognized as an organelle target.^11,12 The Mc is an organelle with two-layered membrane. It is approximately 0.5 to 1.0 µm in diameter and of variable length. In addition to energizing cells, Mc also play a crucial role in cell differentiation, signaling and apoptosis.^5,13 Mc dysfunction induces various hallmarks of cancer cells, such as limitless proliferation, damaged apoptosis, reduced autophagy and increased anabolism.^14,15 However, Mc-targeted therapy is a tedious task.

Before the drug molecule finally reaches the Mc, it needs not to be eliminated from blood circulation, selectively accumulate in the target tissue, pass through the cellular membrane barrier and escape from lysosomal endocytosis.⁵ The St-DDS seems to be able to increase the stability of the nanocarriers and reduce nontarget drug release in systemic circulation. The responsiveness of St-DDS to endogenous (En) stimuli (also called biostimulators, include factors such as pH, redox potentials, enzymes and glucose)^7,16,17 and exogenous (Ex) stimuli (also called externally applied triggers, include factors such as temperature, light and ultrasound) shows potential for a variety of biomedical applications^8,18. The St-DDS senses the stimuli in the surrounding environment, analyzes the stimulus and responds accordingly.⁹ In reaction to the differences between tissue/cell microenvironments or blood systems of tumor and normality.

St-DDSs change the physicochemical characteristics (hydrophilicity, diameter or charge) accordingly to acquire greater in-depth tumorpenetration, increased cell ingestion, controlled drug delivery, and useful endosomal flee.^7,9 Cellular Mc-specific DDSs may be achieved or strengthened by anchoring cell-specific homing ligands (which bind to overexpressed antigens on tumor cell outside) along with Mc-selective groups on its surface.^19,20 The receptors currently utilized for active cancer cell targeted induction contain epidermal growth factor receptor (EGFR),²¹ folate^22,23 and transferrin receptors.^24,25 For further endocytosis, a series of cell penetrating peptides (CPPs), such as cationic trans-activating transcriptional activator (TAT),²⁶ polyarginine,²⁷ amphipathic MPG (unabbreviated notation),²⁸ Pep-1,²⁹ Pep-7,³⁰ and hydrophobic C105Y,³¹ have been developed to modify drug molecules or nanocarriers. After entering the organelle-rich cytoplasm, bioactive drug molecules need to be delivered directly to their Mc action site to acquire high therapeutic efficacy and low off-target result.^10,32,33 The Mc-targeted groups that are able to carry drugs include micromolecule ligands,³⁴ hydrophobic cations,^35,36 Mc protein import machinery,^37,38 Mc-penetrating peptides^10,39 and Mc inner/outer membrane-targeting molecules.^40,41 Through the above analyses, St-Mc-DDS deliver the medicines as much as possible to targeted organelle/cell/tissues and acquire perfect treatment effect and medication safety.

Obviously, there are many ways to combine St- and Mc- DDSs, and we have given a whole list of the documented pathways to achieve smart St-Mc-DDSs by combining St- and Mc-targeting strategies (Figure 2). Here, we systematically collected and analyzed the design theories, implementation method and action mechanisms of St-Mc-DDSs in cancer therapy. We present their smart features and physicochemical and pharmacokinetic properties (Tables 2 and 3), which are closely related to their antitumor efficacy and safety (Tables 4 and 5). Compared to conventional DDSs, St-Mc-DDSs exhibit improved curative efficacy and safety. We propose the challenges and future perspectives of St-Mc-DDSs. This is the first review of the combined application of stimuli-responsive St- and Mc-targeted DDSs. This review will help to evaluate and choose the appropriate smart DDS to cure cancer.

Table 2 Smart Features of St-Mc-DDS

Table 3 Physicochemical and Release Properties of St-Mc-DDS

Table 4 In vitro Antitumor Efficacy and Mechanisms of St-Mc-DDS

Table 5 In vivo Antitumor Efficacy and Safety Evaluation of St-Mc-DDS

Figure 2 A list of documented pathways to achieve smart St-Mc-DDS by combining St- with Mc-targeting methods.

Mc Targeting Characteristics of St-Mc-DDSs

St-Mc-DDSs usually have 2 types of Mc-targeting modes according to the component structure: an Mc group or an Mc molecule (lipophilic cation, peptide or aptamer), and a material (Figure 1D).

Mc Group and Molecule-Based St-Mc-DDSs

Lipophilic Cation-Based St-Mc-DDSs

Mitochondriotropics are low-molecular weight compounds with high intrinsic affinity towards Mc. Mitochondriotropic molecules often have delocalized positive charge and enough hydrophilicity. Once entering inside mammalian cells, mitochondriotropics accumulate either at or inside Mc without requiring the assistance of any Mc-targeted delivery system.⁸² Owing to the high Mc targeting of mitochondriotropics, they are widely used as Mc targeting groups to facilitate drug delivery.^19,20,42,54 Hydrophobic cations such as triphenylphosphonium (TPP),⁴² (4-carboxybutyl) triphenylphosphonium bromide (CTPP),²⁰ IR780⁵⁴ and 9-O-octadecyl¹⁹ have been used as Mc targeting groups of St-Mc-DDS to deliver a variety of small molecule drugs. TPP consists three phenyl groups. It has positive charge and sufficient lipophilicity that facilitates transportation across the Mc membrane.^83,84 TPP and CTPP moieties are usually conjugated with small molecules^20,42 or decorated on the surface of nanoparticles.^43,44 Owing to their sufficient lipophilicity and delocalized positive charge, TPP-carrying molecules can get easily across lipophilic Mc membrane.⁸⁵ The merits of TPP-molecules over other hydrophilic cation-molecules include its good biological stability, amphiphilic properties, ease of modification, inertia toward cellular materials, and deficiency in ultraviolet (UV)-visible light and fluorescence absorption. TPP-ubiquinone is safe for human, thus shows good clinical potential and the translational significance of TPP.³⁴ IR-780 is a lipophilic heptamethine cyanine dye. It is automatically ingested by tumor cells through organic-anion polypeptide transporters, and accumulated in Mc sites due to its hydrophobic cation.⁸⁶ IR-780 is typically used in nanocarriers for photothermography, photodynamic and photothermal therapies (PDT and PTT).^54,56,86

In addition to the above lipophilic cations, rhodamine 123,^87,88 flupirtine,⁸⁹ MKT-077⁹⁰ and anthracyclines^91,92 have demonstrated specific affinity to Mc in different Mc-DDSs, but they have not been used in St-Mc-DDSs.

Mc Targeting Peptide-Based St-Mc-DDS

Two Mc-targeting peptides, ie, KLA peptide (_D[KLAKLAK]₂)⁴⁷ and MQ peptide (MLFNLRILLNNAAFRNGHNFMVRNFRCGQPLQ),³⁷ have been used in St-Mc-DDSs for subcellular-targeted delivery of chemical drugs and DNA, respectively. KLA is an Mc-penetrating peptide (MPP) that can be decorated on the surface of liposomes. Positively charged KLA targets Mc, and its lysine unit facilitates cellular uptake since lysine interacts with the cellular membrane via a hydrogen bond and electrostatic force to accelerate internalization.⁴⁷ In addition, KLA further disrupts the Mc membrane and initiates apoptotic cell death.⁹³ MPPs have been confirmed to delocalize lipophilic cations to deliver bioactive compounds to the Mc.^94–97 MPPs usually have positively charged (lysine, arginine) and lipophilic (isoleucine, phenylalanine, tyrosine) amino acids.^98,99 For example, the aromatic cation-containing Szeto-Schiller (SS) peptide¹⁰⁰ was mainly designed to deliver tyrosine or dimethyl tyrosine as an antioxidant motif to the Mc. The SS peptide is taken up by the cell and inner Mc membrane by an energy-independent mechanism.⁵ In order to meet different investigative requirements, MPPs are designed to have different functions and structures (eg, different amino acid sequences, hydrophobicities and peptide charges). For example, incorporating the D-isomer of arginine into MPPs protects the peptides from enzymatic degradation,^34,101 integrating the targeting motif for cell ingestion and the proapoptotic motif into MPPs for enhanced anticancer effects.^10,102,103

The MQ peptide sequence is an Mc-targeting sequence (MTS, also called an Mc signal peptide).³⁷ MTSs usually consist of 20~30 amino acids, which reach the Mc through the use of a translocase from the outer and inner membrane complexes by mimicking the cellular mechanism of Mc protein delivery.^34,104,105 That is, MTSs create chimeric proteins and are taken up via the Mc protein import machinery.⁵ The multifunctional peptide/DNA complex facilitates DNA targeting to the Mc.³⁷ In a peptide chain having lysine-histidine fragment, cation contributes to cross cellular membrane, lysine condenses DNA for cellular ingestion, and histidine helps DNA to reach cytosol by endosomal lysis through proton sponge effect. Once this type of peptide is attached to a Mc-specific peptide sequence, it will effectively facilitates the targeting of DNA to the Mc.

However, the deep mechanism of Mc-targeting characteristics of peptides is unclear. In contrast to hydrophobic cations, peptides are degradable and cause slight cytotoxicity after Mc deposition. Peptides have large molecular weights, so they might induce undesired immune responses; peptide degradation might cause Mc-targeting failure;¹⁰⁶ some peptides are difficult to synthesize or to be conjugated with targeting materials; and the characteristics and abilities of some targeting materials conjugated with Mc-targeting peptides might be affected.¹⁰⁷

Mc Aptamer-Based St-Mc-DDSs

The Mc cytochrome (Cyt) C aptamer^67,71 is employed to make St-Mc-DDSs efficiently accumulate in the Mc of cancer cells. The Cyt C aptamer is a short single-stranded oligonucleotide sequence that specifically recognizes CytC^108,109 which is normally located in the inner Mc membrane and plays a key role in ATP synthesis.^67,71 Cyt C is secreted into the cytosol and induces apoptosis through a Mc pathway.^59,110,111 Cyt C aptamers have been attached on the outside of smart mesoporous silica-encapsulated gold nanorods⁶⁷ and bacterial magnetic nanoparticles⁷¹ to allow them to efficiently accumulate in the Mc of cancer cells. Mc-targeting aptamers are a prospective strategy for St-Mc-DDSs. Compared to peptides, aptamers are much easier to synthesize while harder to be biodegraded and denatured by further modification. Aptamer has large molecule, so its conjugation efficiency with targeting material is low and targeting efficiency is probably influenced. Aptamer is so expensive that the clinical application of aptamer-based St-Mc-DDSs is severely hindered.¹⁰⁷

In addition to Cyt C aptamers, other Mc-targeted aptamers have been applied. An aptamer containing folding guanine-rich RNA/DNA was designed to modify nanocomplexes for Mc imaging and decrease the Mc membrane potential.¹¹² A dual-ligand liposomal system was decorated with a Mc RNA aptamer (RNase P) that enhanced cellular uptake and achieved Mc targeting.¹¹³ The short RNA aptamer Mitomer 2 showed good binding affinity to the Mc and resistance to degradation by nucleases.¹¹⁴

Mc Materials-Based St-Mc-DDSs

Mc materials such as colloidal dequalinium vesicles³⁶ and carbon nanomaterials^{64,68,70,115,116} have been used in St-Mc-DDSs. Amphiphilic dicationic dequalinium may self-assemble to form aggregates DQAsomes.⁵ DQAsomes display a positive surface charge in aqueous environments and accumulate in the Mc in response to the electrochemical gradient across the Mc membrane system.³⁶ DQAsomes serve as a mitochondriotropic carriers to deliver hydrophobic drugs and genes to the Mc.^117,118

Carbon-based nanomaterials (such as graphene, carbon dots and carbon nanotubes) have been emerging as new biomaterials to design and fabricate St-Mc-DDSs due to their high tunability, good biocompatibility and other unique physicochemical characteristics.^{64,68,70,116,119,120} They selectively target the Mc based on their polarization and positive charge. In graphene oxide (NGO)-based St-Mc-DDSs, graphene serves not only as a carrier material for Mc targeting but also as a phototherapy agent.⁶⁴ The hypericin-functionalized NGO nanoparticles enhance Mc-targeting and the synergistic anticancer effects of phototherapy and chemotherapy.¹¹⁵ A novel type of fluorescent carbon dot achieved Mc imaging and Mc-targeted PDT without further modification by other mitochondriotropic ligands (such as TPP).¹¹⁶ A dual Mc-targeting moiety (TPP and carbon dot)-modified biocompatible platform (magnetic mesoporous silica nanovesicles) achieved long-term imaging and magnetic field-enhanced cellular uptake.⁷⁰ Mc-targeting single-walled carbon nanotubes (SWCNs) are used for cancer photothermal therapy.⁶⁸

Stimuli-Responsive Characteristics of St-Mc-DDSs

Endogenous Stimuli-Responsive St-Mc-DDSs

En stimuli-responsive St and Mc-targeted platforms (En/St-Mc-DDS) have gradually been used to enhance cancer treatment efficacy. En stimuli in the cancerous microenvironment provide signals for anticancer DDSs to accumulate in tumor tissues/cells and release drugs in an on-demand manner.¹¹⁹ The physiological signals facilitating tumor targeting include an acidic media,¹²¹ overexpressed enzymes (such as matrix metalloproteinase, cathepsin, phospholipase, and oxidoreductase)¹²² or membrane-protein makers,¹²³ ATP,¹²⁴ intracellular glutathione (GSH)¹²⁵ or hypoxic features.¹²⁶ According to the En/St type,^127,128 En/St-Mc-DDSs are mainly classified into three types (pH-, redox- and enzyme-responsive St-Mc-DDSs). Their recent advances are described as follows. To date, there have been no reports of En (other biomolecules outside of our list) stimuli-responsive St-Mc-DDSs.

pH-Responsive St-Mc-DDSs

Compared to normal tissues, tumors have higher metabolism and most of them have lower pH values of extracellular and intracellular fluids (6.5~7.0 vs ~7.4; 5.0~6.0 vs ~7.2).¹²⁹ The organelles of tumor cells such as Mc (~8.0),^43,130,131 lysosomes (4.5~5.0), endosomes (5.5~6.0) and cytosol (7.4)¹³² have different pH values. Therefore, Mc-targeted nanocarriers with a particular responsiveness to pH can intently release drugs at tumor locations, simultaneously preventing the unwanted release in normal tissue. Weakly acidic/basic compounds are suitable constituents for the preparation of pH-responsive Mc-targeted platforms. For example, compounds containing -COOH, -NH₂ or -SO₃H groups may alter from their neutral to ionized forms,¹³³ and further induces dramatic alteration in the interaction or affinity between the drug molecule and drug vesicle. The pH gradients are stimuli that release drug from pH-sensitive Mc-targeted systems.^36,42–49

Two easy measures have been employed to design pH-responsive St-Mc-DDSs.¹³⁴ The first is based on acid-cleavable linkers such as a Schiff-base, hydrazone, acetal/ketal, amide or cis-aconityl. The second is based on the degradation of the polymer and destabilization of the nanocarrier in a pH-sensitive manner. pH-sensitive St-Mc-DDSs usually have the following functional features: they expose the carrier core or overturn the positive charge in the tumor extracellular environment to promote carrier uptake and they degrade the carrier inside the cells to achieve rapid drug release or proton sponge action to promote endosome escape.

The polyethylene glycol (PEG)-Schiff base-cholesterol derivate was synthesized and attached to the liposomes (PSLP).⁴² Schiff base bond is hydrolyzed in acidic media, and the PEG shell is removed from the liposomes. The remaining lipophilic PSLP is exposed and easily internalized by tumor cells. Dioleoyl phosphoethanolamine (DOPE) is a constituent of lamellar PSLP that exists in a hexagonal phase at physiological pH but is deformed in acidic medium. After internalization, DOPE merges with the lysosomal membrane and releases the drugs into the cytosol. Then, the drug accumulates in Mc through the guidance of TPP. In addition to the combination of stimuli-triggered St- and Mc-targeted strategies, other active targeted strategies (such as Eph receptor A10 (EphA 10)-mediated cellular endocytosis) and passive targeted strategies (such as the enhanced permeability and retention effect, EPR) have been applied simultaneously to achieve better St-Mc delivery.⁴²

A pH-responsive St-Mc nanohybrid comprised a N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer shell and a positively charged nanovesicle core was fabricated via electrostatic interactions.⁴⁵ Under mildly acidic environment of the tumor, the first-stage pH-responsiveness took place when the hydrolysis of the amide bonds in the HPMA copolymers occurred and the charge of the copolymer changed from negative to positive, which was beneficial for cellular ingestion. The second-stage occurred in endosomes/lysosomes due to the proton sponge effect, which facilitated Mc location.

Another pH-responsive St-Mc-DDS was prepared to induce cellular apoptosis.⁴⁷ This liposome contains a hybrid lipid by synthesizing Mc peptide KLA with dimethylmaleic anhydride via amide bond and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). This liposome exhibited positive charge at pH ~6.8 of extracellular media to facilitate its entrance into cancerous cells. After that, KLA purposely delivered cargo to Mc.

An alkaline pH-responsive St-Mc-DDS was constructed.⁴³ Lipophilic CTPP conjugated with glucolipid-like conjugates formed micelles in aqueous solution and encapsulated celastrol in the hydrophobic core. These micelles selectively responded to the Mc alkaline pH (pH 8.0), while reduced drug leakage occurred in the cytoplasm (pH 7.4) and lysosomes (pH 5.0). The acidity/basicity of the loaded drug was relevant to the drug release rate and solubility in different environments.

Redox-Responsive St-Mc-DDSs

The redox/oxidation states between the intracellular and extracellular matrices of tumors are very different. For example, the GSH level in the cytosol (2–10 mM) of tumor cells is ~1000 times greater than that in the extracellular matrix (2–20 μM) or >4 times greater than that in the normal cells, which renders the tumor intracellular redox potential.¹³⁵ Redox-responsive functional bonds or groups⁸ such as disulfide, phenylboronic acid and ester bonds, and singlet oxygen-responsive imidazoles have been used to design redox-responsive St-Mc-DDSs.

Disulfide bonds are incorporated into either the crosslinker or polymer structure of DDSs. Disulfide bonds are converted to thiol groups by various intracellular stimuli, such as the reducing agent GSH.⁸ A Sm-Mc-DDS for overcoming multidrug resistance (MDR) had a dendrimer core co-decorated with paclitaxel (PTX) via a disulfide bond and TPP via an amido bond, and a shell covered with PEG layer via a MMP2-sensitive peptide.⁷⁸ Once the core detached from the PEG layer, it targeted the Mc via TPP guidance, and PTX was rapidly released through a reductive reaction. Another Sm-Mc-DDS was TPP-oligomeric hyaluronic acid-S-S-curcumin-loaded micelles.⁵⁰

Phenylboronic acid and ester bonds are sensitive to H₂O₂. Arylboronic esters and thioketal linkers are oxidized by H₂O₂ at 50 μM¹³⁶ and 100 μM,¹³⁷ respectively. The lipophilic neutral ferrocene/lipophobic cation ferrocenium redox pair was utilized for the design of cancer-specific, Mc-targeting moieties to trigger reactive oxygen species (ROS)-mediated drug release.¹³⁸

A polymeric micelle with an imidazole group is singlet oxygen-responsive and able to deliver pyropheophorbide A (PPA)-TPP (a photosensitizer).⁷⁶ The imidazole moiety was oxidized to hydrophilic urea upon triggering with light. The amphiphilicity of micelles changed, followed by rapid photosensitizer release and Mc inhibition via TPP. The PDT efficacy was then enhanced.

Generally, these redox-responsive St-Mc-DDSs disassemblied and released drug in response to ROS, through lipophobic-lipophilic transition or cleavage of ROS-responsive linkers. The high sensitivity and specificity have been confirmed by the above St-Mc-DDSs. However, there are still some challenges to be addressed: the degraded linkers should be histobiocompatible, nontoxic and non-immunogenic. Otherwise, they may lead to unwanted side effects and a varied redox state inside tumor cells, which is associated with phenylboronic acid and ester bonds. Due to the ROS dynamic and heterogeneity of tumor cells, it is difficult to control redox balance and understand related molecular mechanism.¹³⁹

Enzyme-Responsive St-Mc-DDSs

Enzymes such as esterases, hyaluronidases (HAases) and alkaline phosphatases (APases) are concentrated inside the cellular cytoplasm or lysosomes or overexpressed in the extracellular environment of tumor sites.¹⁴⁰ Catalytic reactions refer to the cleavage/formation of chemical bonds or the oxidation/reduction of substrates. Enzymatic activation¹⁴¹ using different enzymes as stimuli has been applied to design enzyme-responsive St-Mc-DDSs.

A new stable and monodispersing nonisocyanate polyurethane nanocapsule (NIPU) are developed.⁵² Their shells had a polyurethane-based¹⁴² polymeric backbone with embedded ester linkages in response to esterases. Their core loaded the hydrophilic drug doxorubicin (DOX) during the polymer synthesis and NIPU preparation process. NIPU was further post-grafted with phosphonium ions to achieve Mc-targeted release of the drug. Song et al¹⁹ first designed a Mc-targeted nanodrug (a positively charged 9-O-octadecyl substituted berberine derivative, BD) that was dually modified with DSPE-PEG2000 to increase the stability and the negatively charged hyaluronic acid to achieve tumor targeting and lysosomal escape through recognition by HAase in tumor tissue and lysosomes. TPP was attached to phosphorylated tetrapeptide enantiomers to obtain oligomers that would self-assemble to form nanosized assemblies in response to APase enzyme-catalyzed reaction.⁵³ These assemblies further caused Mc dysfunction and killed cancer cells while minimizing acquired drug resistance.

In summary, enzymes as stimuli of St-Mc-DDSs have intrinsic merits: as endogenous components, they have inherent biocompatibility and biosafety; they have extraordinary selectivity for substrates and high catalytic efficiency; and the same enzyme family, such as matrix metalloproteinases, in tumor cells usually have similar active pockets that may lead to similar substrate preferences.¹⁴³

Exogenous Stimuli-Responsive St-Mc-DDSs

Ex stimuli such as magnetism and light have been employed to control drug release within a Mc-DDS (Ex/Sm-Mc-DDS). Compared to En stimuli, Ex stimuli seem to have been more successful in overcoming the individual variability of controlled drug release.^139,144 We will discuss the rationales and applications of light and magnetic responsive Ex/St-Mc-DDS. To date, the combination of other Ex stimuli (temperature, ultrasound, electric pulse, etc.) and Mc-DDS has not been reported.

Light-Responsive St-Mc-DDS

UV (200~400 nm) light only has weak penetrating power, and it is harmful to normal cells and tissues. In contrast, NIR light (650~900 nm) has better tissue penetration and an improved safety profile.¹⁴⁵ Therefore, NIR-responsive DDSs with good spatiotemporal control are potential nanocarriers for practical therapy. Based on the NIR-responsive mechanism, there are three types of light-responsive St-Mc-DDS: NIR light-activated PDT-, PTT- and PDT/PTT-based St-Mc-DDSs.

Wei et al⁶⁴ used NGO as a smart carrier, which was dually modified with a monoclonal antibody (mAb) for αvβ3-positive tumor cell location and a PPA-PEG conjugate for phototoxicity in the organic environment of the Mc. The nanodrug PPA-NGO-mAb significantly enhanced Mc-mediated PDT apoptosis by yielding ROS such as singlet oxygen and free radicals. Hou et al⁶⁵ designed titanium dioxide (TiO₂, shell)-decorated upconversion nanosystems.⁵⁷ These core/shell nanocomposites (inorganic crystalline nanoparticles, 1–100 nm) transformed NIR light to UV emission, which triggered the cytotoxicity of TiO₂. Therefore, under NIR irradiation, these nanocomposites served as an effective photosensitizer and generated intracellular ROS in the Mc to kill tumor cells.

PTT usually uses a photothermal conversion agent to convert NIR light into thermal energy for hyperthermia in the tumor region. Chen et al coencapsulated Mc-targeting gold nanostars (AuNSs) and DOX in a hyaluronic acid protective shell to fabricate a St-Mc-DDS, which was ingested into cancer cells upon recognition by CD44 receptor. DOX was then released for chemotherapy. The AuNSs codecorated with the cationic peptide R8 and proapoptotic peptide TPP-KLA acted as a Mc-targeting nanoheater for NIR-triggered PTT.⁵⁸ Kong et al¹⁴⁶ designed a microhybrid with two-photon absorption characteristics through coordination interactions between silver and a fluorescent cyano-carboxylic acid derivative. The decreased quantum fluorescence and improved two-photon absorption caused by the surface plasmon resonance effect led to good photothermal output in Mc of HeLa cancer cells when radiation at 780 nm.

Zhang et al⁵⁴ designed a nanosystem by integrating IR780 into perfluorooctyl bromide (PFOB)-based nanoliposomes for synergistic PDT/PTT under NIR irradiation at 808 nm. Mc-targeting IR780 is easily to be encapsulated into nanoliposomes due to its hydrophobicity. IR780 had PTT/PDT effects, and the PDT effect was enhanced by the oxygen carried by PFOB. This Mc-targeting nanoliposome was better than the one consisting of indocyanine green (ICG) and PFOB.¹⁴⁷ The latter had PTT/PDT effects but did not have a Mc-targeting effect. Luo et al¹⁴⁸ synthesized a Mc-targeted NIR photosensitizer for jointly PTT/PDT by modifying heptamethine cyanine dye with different side-chain N-alkyl.

In summary, in order to conquer resistance to chemotherapy, PTT and PDT are often applied jointly in a NIR-responsive St-Mc-DDS to achieve synergistic antitumor effects. However, the biocompatibility and biodegradability of the photosensitizer (especially inorganic nanoparticles) used in such delivery systems must be considered for clinical implications.¹⁴⁹ In addition, the light-responsive St-Mc-DDS is only suitable for the treatment of superficial tumors such as skin surface cancer and breast cancer due to limited light penetration.

Magnetic Field-Responsive St-Mc-DDSs

The application of magnetic materials along with external magnetic fields was first introduced to medicine by Freeman et al¹⁵⁰ in 1960. Magnetic stimuli-triggered St-DDSs features advantages over chemotherapy:¹⁵¹ they are a noninvasive approach to control drug release;¹⁵² they scarcely have any physical interaction with the body and are effective over a distance as long as a few centimeters; the nanocarriers can overcome blood flow resistance and arrive at the tumor region under the influence of a magnetic field spatially focused at desired sites; and upon removal of the external magnetic field, there is no residual magnetism or drug effects. Magnetic field-responsive St-Mc-DDSs usually use superparamagnetic iron oxide nanoparticles (SPIONs) 10~20 nm in size. Surface functionalization of SPIONs may overcome their drawbacks, such as a short blood circulation time due to aggregation and oxidization.

Kim et al⁶⁹ modified SPIONs with PK11195 (C₂₁H₂₁ClN₂O) and chitosan-graft-PEI to fabricate a Mc-targeting gene carrier, which effectively condensed and protected DNA. Under an exterior magnetic field, the transfection productivity of this gene carrier was comparable to PEI 25 K. PK11195 facilitated the accumulation in the Mc and activated apoptosis. This magnetofection of the magnetic-responsive St-Mc-DDS led to an enhanced therapeutic effect on tumor cells. Zhang et al⁷⁰ constructed a Mc-targeting nanoplatform of iron oxide silica nanovesicles decorated with TPP. The cellular uptake efficiency of the nanoplatform was enhanced upon application of a magnetic field of 0.30 T. SPIONs increased temperature under magnism to kill tumor cells by hyperthermia.¹⁵³

Multi-Responsive St-Mc-DDSs

Compared to single stimuli, multiple stimuli, such as the combination of Ex and En stimuli, may have synergistic effects. Wang et al⁷³ combined magnetic stimulus and NIR light irradiation with redox responsiveness into a multistage targeted nanocarrier to enhance the efficacy of cancer therapy. The core-shell-S-S-shell nanocarrier was composed of an Fe₃O₄ core, an inward polydopamine (PDA, a photosensitizer) shell connected to TPP, and an outward methoxy PEG (mPEG) shell linked to PDA via a disulfide bond. The magnetic Fe₃O₄ core increased nanocarrier location in tumor. Once entering the tumor cells, the outer mPEG shell is detached by redox reaction to disclose TPP for Mc targeting. Upon NIR light irradiation, PDA generated a photothermal effect, and the loaded DOX rapidly entered the Mc, resulting in cell apoptosis. Chen et al⁵⁸ combined NIR light stimuli and enzyme responsiveness to deliver dual peptide (Mc-targeting)-decorated AuNSs and DOX to cure tumors. Zhang et al⁷⁶ combined NIR light stimuli and redox responsiveness to coprepare a Mc-targeting polymeric micelle.

The combination of different En stimuli (such as redox or pH and enzyme stimuli) may result in the sequential release of drugs or polymers at precise times.¹⁵⁴ Zhou et al⁵¹ constructed lipid polymer nanocarriers containing PTX, which is composed of an amphipathic copolymer containing TPP, poly (D,L-lactide-co-glycolide) (PLGA) and an amphipathic copolymer having redox-responsive property. The hydrophobic drug core (CCM) was decorated with hydrophilic shell (OHA and TPP) via a disulfide bond. The micelles were ingested by cancerous cells through CD44 recognition, entered the Mc and released CCM due to disulfide bond cleavage in response to high levels of GSH. The long-acting PEGylated nanocarriers accumulated in the tumor. PEG4000 detached via redox-triggered activation after uptake by cancer cells. The nanocarriers recovered to carry positive charge, and then enhanced anticancer efficacy was achieved through precise localization at the Mc.

Relationship Characteristics of St-Mc-DDSs

According to the spatial location relationships of different types of St- or Mc-DDSs, St-Mc-DDS are mainly classified as one of 5 types (Figure 1C): St material loaded with a Mc group or molecule-drug St-Mc-DDS; St material-Mc group or molecule loaded with drug St-Mc-DDS; St material-Mc material loaded with drug St-Mc-DDS; Mc group or molecule-drug St-Mc-DDS; or normal material-loaded with St-Mc-DDS.

St Material Loaded with Mc Group or Molecule-Drug

The drug molecules conjugated with Mc-targeting groups or molecules are further encapsulated in St materials to form different types (such as pH-, redox-, enzyme- or light-responsive) of St-Mc-DDSs. The Mc group or molecule-drug will be released in response to the tumor microenvironment or local Ex stimulus, which overcomes the nonspecific drug uptake by normal cells to a certain extent, thereby reducing toxicity. However, the linkage of the Mc group or molecule to the drug molecule may have a negative effect on anticancer efficacy. Therefore, an anticancer efficacy comparison between the Mc group or molecule-drug and free drug group is necessary and valuable.

Zhang et al⁴² synthesized a docetaxel-TPP (a Mc-targeting molecule) conjugate, and incorporated it into liposomes composed of PEG-Schiff base-cholesterol (a pH-sensitive St material) and DSPE. PEG-Schiff base-cholesterol was hydrolyzed in pH ~6.0 (tumor microenvironment) to get rid of PEG shell, and DSPE merged with the tumor lysosomal membrane (pH ~5.0), resulting in fast drug release into the cytoplasm and accumulation into the Mc under the guidance of TPP. Xing et al⁴⁶ developed amphiphilic quercetin-TPP conjugates into a self-assembled nanoparticles composed of phenylboronic acid-PEG (a pH-sensitive St material) via boric acid ester bonds for tumor therapy. Zhang et al⁷⁶ encapsulated PPA (a photosensitizer)-TPP (a Mc-targeting molecule) into imidazole (a redox-responsive St material)-bearing polymeric micelles. Song et al¹⁹ constructed a nanodrug self-assembled from a 9-O-octadecyl (a Mc-targeting group)-substituted berberine derivative, further modified with DSPE-PEG2000 to increase stability and coated the nanodrug with HA (an enzyme HAase-sensitive St material) to achieve tumor targeting. Chen et al coencapsulated DOX and TPP-KLA (a Mc-targeting molecule and peptide)-decorated AuNSs (a light-responsive Sm material) into a HA (an enzyme HAase-sensitive St material) shell.⁵⁸

St Material-Mc Group or Molecules Loaded with Drug

The Mc targeting group or molecule is directly bonded to a St material to form a composite material that will further encapsulate drug molecules to form different types (such as pH-, redox- or enzyme-responsive) of St-Mc-DDSs. Tan et al⁴³ conjugated a lipophilic cation CTPP (a Mc-targeting molecule) with CSOSA (an alkaline pH-sensitive St material) to produce a St-Mc-material (CTPP-CSOSA) that formed micelles that simultaneously encapsulated celastrol into the hydrophobic core. Jiang et al⁴⁷ conjugated the Mc peptide KLA with DSPE and DMA (a pH-responsive material) to yield a DSPE-KLA-DMA lipid to prepare liposomes containing PTX. Zhou et al⁵¹ mixed C18-PEG2000-TPP (a Mc group) and DLPE-SS-mPEG4000 (a reductive responsive polymer) with PLGA to prepare nanoparticles loaded with PTX. Pramanik et al⁵² selected a TPP-modified nonisocyanate polyurethane (an esterase-responsive polymer) to construct biodegradable nanocapsules containing DOX.

As far as St-Mc-DDSs with St material-Mc group or molecules loaded with drugs are concerned, the antitumor effects of the drug can retain intact since it has an unmodified structure. However, Mc groups or molecules are usually positively charged, easily absorbed by non-specific proteins and rapidly eliminated by the reticuloendothelial system in circulation.^47,51,57 Therefore, it is necessary to cover a Mc material with a negatively charged St material. In addition, when reaching the tumor site, the St material should be able to expose the Mc groups or molecules in response to stimuli, which can promote the electrostatic interaction between the nanoparticles and cancer cell membrane for cell internalization.

St Material-Mc Material Loaded with Drug

St material-Mc material can be a compound material composed of St- and Mc-material⁴⁵ or a material that has both smart properties and Mc-targeting abilities.^115,119

Shi et al³⁶ incorporated the HER-2 peptide-PEG2000-Schiff base-cholesterol (HPSC) derivative (a pH-responsive material) on the surface of DQAsomes (dicationic dequalinium vesicle, a Mc material) containing DOX to treat drug-resistant breast cancer. Li et al⁴⁵ encapsulated docetaxel in a positively charged mesoporous silica nanoparticle core (MSN, a Mc material) and then wrapped it with a pH-responsive HPMA copolymer shell to cover the positive charge of the mesoporous silica MSN. When St-Mc-DDS is prepared using a compound of St material-Mc material,³⁶ its structural characteristics and in vivo processes are similar to those of St-Mc-DDSs containing a drug loaded in St material-Mc group or molecule.^47,51 St material-Mc material St-Mc-DDSs usually respond to stimulation of the tumor microenvironment or local Ex stimuli and induce physical property changes, such as particle size and charge. Then, the St-Mc-DDS arrives at the Mc under the guidance of the Mc material.

Carbon nanomaterials can be used not only as Mc-targeted nanocarriers but also as St photosensitizers to induce phototherapy.¹¹⁹ Han et al¹¹⁵ constructed a hypericin-functionalized NGO to deliver DOX, which had enhanced Mc targeting and synergistic anticancer effects. St-Mc-DDSs prepared using a single material having both Sm properties and a Mc targeting ability usually first accumulates in the Mc, and then energy conversion occurs under Ex stimulation.^115,119

Mc Group or Molecule-St Material-Drug

When the Mc group or molecule, St material and drug are connected together, they form a St-Mc-DDS with a 100% encapsulation rate. Wang et al⁵⁰ proposed TPP-OHA-S-S-CCM micelles (an Mc molecule-enzyme/redox-sensitive multifunctional micelle) to exert anticancer efficacy. Li et al⁴⁴ presented TPP-fluorogen-hydrazone bond-PEG micelles (an Mc molecule-pH sensitive micelle), and fluorogen underwent aggregation-induced emission (AIE). The PEG moiety increased blood circulation stability. Guan et al⁵⁷ decorated AIE copolymers (PAIE, a photosensitizer able to be photoactivated upon 980 nm laser irradiation to yield ROS) with TPP to form PAIE-TPP, which was further conjugated with mPEG-CHO (a pH-responsive material) via a benzoic imine bond. Wang et al⁷³ prepared TPP-PDA (a photothermal agent)-S-S (redox sensitive)-mPEG nanoparticles using a similar strategy.

St-Mc-DDSs formed by directly connecting a drug with the Mc group or molecule-St material may reduce the problem of drug leakage during circulation. These compounds can not only change the physical properties in response to stimulation of the tumor microenvironment but also deliver therapeutic agents to produce PDT^44,57 and PTT⁷³ effects under light irradiation.

Normal Material Loaded with St-Mc-Drug

(St-Mc) drugs mainly refer to small molecule drugs with Mc targeting ability and light conversion performance. They can be encapsulated inside common materials to form St-Mc-DDSs. IR-780 is a St-Mc drug that can selectively accumulate in the Mc and is commonly used in photothermography, PTT, and PDT.^54,56,86 Zhang et al⁵⁴ encapsulated IR780 into nanoliposomes based on perfluorooctyl bromide to form an artificial nanoRBC. These liposomes delivered oxygen to the tumor to alleviate tumor hypoxia and enhanced Mc-targeted phototherapy and multiple imaging guidance/monitoring. The iridium (Ir) complex is another St-Mc drug that targets the Mc and can achieve photoredox reaction in tumor.^55,155–157 Huang et al¹⁵⁷ prepared an Ir photocatalyst. This complex localizes in the Mc and depletes NADH, unbalances intracellular redox and causes immunogenic cellular death upon light irradiation. In another work, Ir and Fe₃O₄ cooperated to form photothermogenic nanozyme Ir@Fe₃O₄ NPs, which could increase the local temperature of the tumor, thereby catalyzing H₂O₂ to generate OH.⁵⁵ Similarly, PEGylated SWCNTs were used for Mc-targeted PTD.⁶⁸

Compared with the above four types of St-Mc-DDSs, St-Mc-DDSs with St-Mc drug loading in common materials has a simpler design and preparation requirements for dosage forms. They can even be administered directly.¹⁵⁷ In general, it is necessary to prepare St-Mc-DDSs with suitable structural properties based on the properties of the given drug and material.

Clinical Research Progress

PDT is considered a prospect effective therapy without obvious side effects. Photofrin^® (a hematoporphyrin-derived photosensitizer) is a powder injection to cure various cancers such as colorectal carcinoma,¹⁵⁸ esophageal cancer,¹⁵⁹ and malignant cutaneous neoplasms.¹⁶⁰ The application of micelle nanotechnology further enhances Photofrin^® delivery and efficacy at the cellular level.¹⁶¹ Visudyne^® (a liposomal photosensitizer containing a second-generation photosensitizer derived from porphyrin) is clinically used to treat subfoveal choroidal neovascularization^162,163 and has been found to be preclinically effective for cancer treatment.¹⁶⁴ Ameluz^® (containing 10% of the photosensitizer 5-aminolevulinic acid) is a nonsterile white-to-yellow nanoemulsion-based gel for topical use to clinically detect and treat bladder cancer^165,166 or cure melanoma skin cancer.¹⁶⁷ In addition to light-responsive St-DDSs, other stimuli-responsive St-DDSs are developed in different clinical phases. They are thermoresponsive liposomes ThermoDox to cure breast tumor (phase II) and liver cancer (phase III); the enzyme-responsive polymeric nanoparticles Opaxio to cure ovarian carcinoma; and the magnetic field-responsive iron oxide NanoTherm for the treatment of glioblastoma. One significant characteristic of these promising St-DDSs is their simple formulation, which favors their preclinical to clinical transformation.^168,169

Some synthesized Mc-targeted compounds, such as MitoQ and SkQ1 (also called Visomitin), have already entered the clinical trial stage. Both are lipophilic antioxidants with TPP groups.¹⁷⁰ MitoQ is used to treat aging,¹⁷¹ pulmonary hypertension, etc.,¹⁷² while SkQ1 is used to treat conjunctivitis¹⁷³ in completed clinical trials. MitoQ is currently undergoing other clinical trials for pulmonary hypertension, etc. Unfortunately, all antioxidant tests are not for antitumor therapy. However, it is worth trying to perform the clinical transformation of other Mc-targeted compounds with TPP groups and their DDSs.³⁴ Some novel compounds with potent anticancer activity have been identified to use the Mc as a target and act on its metabolism in recent years. Venetoclax represents a first-in-class selective and effective Bcl-2 inhibitor.¹⁷⁴ It was approved by the United States FDA in 2016 to treat relapsed-refractory chronic lymphocytic leukemia. Its clinical dosage form is ordinary tablets. Ganetespib is an injectable small molecule drug. It has a favorable safety profile and promising early results by inhibiting heat shock protein 90. It has been investigated in multiple clinical trials of various tumors, such as metastatic pancreatic cancer (phase II),¹⁷⁵ relapsed-refractory small cell lung cancer (phase Ib/II),¹⁷⁶ and advanced carcinomas and sarcomas (phase I).¹⁷⁷

To date, there are still no clinical antitumor investigations of St-Mc-DDSs or Mc-DDSs. In addition, there are only a few St-DDSs in the clinic or the clinical trial stage. Most of these compounds are still at the basic research or preclinical stages. The possible reasons are listed as follows.

1. Insufficient in vitro pharmaceutical data. The formulation consists of some excipients or critical materials that are not pharmaceutical grade and not approved for pharmaceutical application, and their components, purity, quality, function and toxicity are unclear. Most preparation processes are too sophisticated for industrial production scale-up. The drug payload and encapsulation efficiency of most St-Mc-DDSs have not been determined, and some may not be high. Formulation stability has not been considered and investigated.
2. Insufficient in vitro release, in vivo pharmacokinetics and biodistribution data. Some in vitro release in suitable release media should be supplemented, and the media type should be chosen based on administrative pathways and designed release microenvironment. There is no in vivo pharmacokinetic behavior research or analysis of the bioavailability or pharmacokinetic parameters, such as the area under the plasma drug concentration versus time curve, peak time, clearance, etc. The relationship between the in vivo pharmacokinetics and in vitro release should be investigated. The biodistribution in different normal tissues and tumors, tissue fluids, cells, and organelles, particularly the Mc, should be clarified.
3. Insufficient pharmacodynamic data. Most studies are limited to one or two cell lines and/or several animal experiments and are still in the proof-of-principle stage. The effects of stimuli and Mc targeting on intracellular trafficking, action pathways, and clinical efficacy require further research. When targeting the Mc alone does not achieve the desired therapeutic effect, designing nanocarriers that target multiple organelles at the same time may be an alternative strategy since in-depth studies on lysosomes, the endoplasmic reticulum and other organelles have shown that these organelles are closely related to apoptosis or autophagy, which can affect tumorigenesis.
4. Insufficient safety data. No preliminary safety evaluation of the Sm-Mc-DDS including toxicity, immunogenicity and side effects in animals has been reported. Additionally, the positive stimuli to rats or mice may not be suitable or safe for patients.
5. The mutual interference or promotion of St stimuli and Mc-targeting in one DDS requires more experimental data. Theoretically, each Sm type may combine with each Mc targeting type or subtype, but in fact, there have been no reports of some En (other biomolecules outside our list) or Ex (temperature, ultrasound, electric pulse, etc.) stimuli-responsive St-Mc-DDSs. The chosen criterion is not clear.
6. Compared to single stimulus-response St-Mc-DDSs, multiple stimuli-response St-Mc-DDSs are more difficult to achieve transformation. The latter is still in its infancy, their design principles are more complex than the former, and their fabrication and assembly processes are more difficult. Sometimes, failure to respond to one stimulus may lead to ineffectiveness of the whole system. Therefore, the sequence and degree of response to each stimulus should be further assessed.

Summary and Outlook

Mc dysfunction plays a vital role in programmed cell death, such as apoptosis and necrosis. It is reasonable to choose the Mc as a novel target for an antitumor strategy. However, most drugs or nanocarriers on the market do not have Mc targeting functions, and they need to overcome many obstacles before reaching the tumor tissue, cells and Mc due to the complexity of the tumor tissue environment. The concept of Mc targeting, while seemingly simple in theory, has multiple subtly different practical approaches. Lipophilic cation such as TPP and IR-780 have good safety and clinical potential. IR-780 are also used for PDT/PTT therapy. Mc-targeting peptides such as KLA and MQ peptides, have positively charged and lipophilic amino acids. Mc-targeting aptamers are too expensive to make clinical transformation difficult. Peptides and aptamers may be unstable and cause unwanted side-effects due to large molecular weights. Mc materials form vesicles to deliver drug. NGO nanoparticles may achieve PDT/PTT effects. There are differences on the future status of various Mc-targeted approaches to cancer therapy. In this context, structurally modified and programmed micro/nanoparticles, which can be programmed using computation techniques to obtain En (pH, redox and enzyme) or Ex (light and magnetism) stimuli responsive to increase their accumulation in the Mc. Multifunctional nanocarriers with single/multiple stimuli-responsiveness and Mc targeting properties may better protect and deliver drugs, reduce normal tissue accumulation and enhance therapeutic effects. Stimuli-responsive system may be an effective way to improve Mc targeting delivery. An important biological hypothesis is that the tissue microenvironment can trigger a desirable event to a large extent from stimuli-responsive behavior. However, until now, no evidence supports this. The responsiveness should be considered as an important contributor to therapeutic efficacy, and an urgent necessity to assess in depth in vivo responsiveness that is intimately relevant for the functionality. Some strategies had been proposed to amplify the responsiveness and improve the functionality.^178–180

Undoubtedly, enormous efforts have been exerted in the design and basic research of St-Mc-DDS for cancer therapy, and the preliminary results are encouraging. However, the clinical transformation of St-Mc-DDSs is comparatively motionless. In order to address the challenges in clinical transformation, there is a great need to consider and perform the following measures in the future, which include but are not limited to choosing the drug using the Mc as a main target; choosing approved pharmaceutical excipients to formulate the St-Mc-DDS; choosing suitable stimuli according to the tumor site (superficial or deep tumor); optimizing the preparation method and choosing a method that is as simple as possible; and performing the necessary experiments (such as stability, pharmacokinetics and safety tests) needed for clinical trial application. Recently, advances in machine learning and artificial intelligence immensely decode and empower the cell-nanomaterial interaction modelling, which give modern to nanomedicine to predict the targeting and efficacy of payload to intracellular compartment^181–183 using in-silico methods.⁶¹ This potentially decipher the quantitative nanostructure activity-relationship (Nano-QSAR) and promote the understanding of bio-physicochemical identity at the nano-bio interface. In this context, structurally modified and programmed micro/nanoparticles, which can be programmed using computation techniques to stimuli responsive and increase their accumulation in the mitochondria. The predictability of targeting and effectiveness, coupled with the clarity of the mechanism of action, may accelerate the clinical transformation of nanomedicine. We should follow innovative advantages and conduct prospective research. In addition, considering the fact that more than 10% of all cancer drugs in use today are nanodrug and irony is less 10% of clinical oncologist know this, future concept must evolve around the why there is a need for education and training in nanomedicine for future doctors? Emphasis must be their incorporation into the general medical curriculum the key concept in nanomedicine.

Role of EPR in cancer barrier is somewhat oversell considering less than 1% nanomedicine formulations follow the trend designed by using EPR. In the last decades, it has been increasingly recognized that there is large inter- and intra-individual heterogeneity in EPR-mediated tumor targeting, explaining the heterogeneous outcomes of clinical trials in which nanomedicine formulations have been evaluated. To address this heterogeneity, as in other areas of oncology drug development, we have to move away from a one-size-fits-all tumor targeting approach, towards methods that can be employed to individualize and improve nanomedicine treatments.

Overall, St-Mc-DDSs may be innovative and sensitive precision medicines. They provide great potential for enhanced cancer treatment. This new strategy is expected to be applied in clinical practice as soon as possible and to open up new ideas for precision medicine soon.