Divya Dheer

Julien Nicolas
email: julien.nicolas@u-psud.fr

Ravi Shankar
email: rshankar@iiim.ac.in

Cathepsin-Sensitive Nanoscale Drug Delivery Systems for Cancer Therapy and other Diseases

Keywords: Cathepsin-Sensitive Nanoscale Drug Delivery Systems for Cancer Therapy and other Diseases Cathepsins, enzymes, cancer, drug delivery, optical imaging

published or not. The documents may come

Introduction

Cathepsins are widely known proteolytic enzymes whose main function is to degrade proteins or peptides [START_REF] Klein | Proteolytic cleavage-mechanisms, function, and "Omic" approaches for a near-ubiquitous posttranslational modification[END_REF]. Nevertheless, this perception has changed over the past many years as they are being considered as important signaling molecules playing different crucial roles [START_REF] Turk | Targeting proteases: successes, failures and future prospects[END_REF][START_REF] Overall | In search of partners: linking extracellular proteases to substrates[END_REF]. There are dozens of Cathepsins which are classified according to their structure, catalytic mechanism and substrate. Based on the human genome draft sequence, the main Cathepsin categories are serine (Cathepsin A and G), aspartic (Cathepsin D and E) and lysosomal cysteine proteases (Cathepsin B,C,F,H,K,L1,L2/V,O,S,W,X/Z) [START_REF] Reiser | Specialized roles for cysteine cathepsins in health and disease[END_REF][START_REF] Turk | Cysteine cathepsins: From structure, function and regulation to new frontiers[END_REF]. They have multiple functions, as one finds digestive proteases (present in saliva, stomach and intestines) for food processing inside the gastrointestinal tract (GIT), lysosomal proteases for intracellular housekeeping or caspases for transduction of one-way signal in apoptosis [START_REF] Brömme | Role of Cysteine Cathepsins in Extracellular Proteolysis[END_REF][START_REF] Oikonomopoulou | Proteinases and their receptors in inflammatory arthritis: an overview[END_REF][START_REF] Cirman | Selective disruption of lysosomes in HeLa cells triggers apoptosis mediated by cleavage of Bid by multiple papain-like lysosomal cathepsins[END_REF]. Interestingly, lysosomal Cathepsins (i.e., intracellular enzymes) have been widely involved in drug targeting as they require a slightly acidic environment to exhibit optimal enzymatic activity [START_REF] Braulke | Sorting of lysosomal proteins[END_REF][START_REF] Michael | Lysosomal membrane proteins and their central role in physiology[END_REF][START_REF] Steenhuis | Proteolytic cleavage of the diseaserelated lysosomal membrane glycoprotein CLN7[END_REF]. Given the features of diseaseassociated proteolysis (i.e., cleavage of amide bond), different types of prodrugs, nanocarriers, biomaterials or probes, have been designed and synthesized to exert their activity in endosomal/lysosomal compartments [START_REF] Wei | Discovery of peptidomimetic antibody-drug conjugate linkers with enhanced protease specificity[END_REF][START_REF] Sun | Synthesis, self-assembly, and biomedical applications of antimicrobial peptide-polymer conjugates[END_REF][START_REF] Linke | Trafficking of lysosomal cathepsin B-green fluorescent protein to the surface of thyroid epithelial cells involves the endosomal/lysosomal compartment[END_REF]. For instance, Cathepsins can induce the release of active ingredients from nanocarriers, chemically or physically, leading to enhanced therapeutic activity or in situ imaging sensitivity [START_REF] Law | Proteolysis: A biological process adapted in drug delivery, therapy, and imaging[END_REF]. Kopecek, Duncan and others have shown the importance of protease-cleavable linkers, especially those sensitive to Cathepsin B, in polymerbased, nanoscale drug delivery constructs for enhancing the in vivo delivery of drugs to tumor tissues [START_REF] Kopeček | HPMA copolymer-anticancer drug conjugates: Design, activity, and mechanism of action[END_REF][START_REF] Ulbrich | Polymeric anticancer drugs with pH-controlled activation[END_REF][START_REF] Duncan | The dawning era of polymer therapeutics[END_REF].

Cysteine cathepsins and their substrate interaction have been well-identified on the basis of papain (Carica papaya) used as a model of lysosomal proteases, as first introduced by Schechter and Berger [START_REF] Schechter | On the size of the active site in proteases. I. Papain[END_REF]. In this model, the substrate residues (P) as well as the subsites (S) were given nomenclature based on their position bonded to the protease surface. Later, this model was revisited by Turk et al. [START_REF] Turk | Revised definition of substrate binding sites of papain-like cysteine proteases[END_REF]who showed that the subsites were positioned on the lefthand side (i.e., S2', S1 and S3) along with right-hand side of the active site (i.e., S1' and S2), and further composed of two L-domain loops consisting of Gln-19-Cys-25 as well as Arg-59-Tyr-67 residues and two R-domain loops consisting of Leu-134-His-159 as well as Asn-175-Ser-205 residues (Figure 1) [START_REF] Turk | Cysteine cathepsins: From structure, function and regulation to new frontiers[END_REF]. Among the different pathologies, Cathepsins have been largely employed as leverage to treat cancer from various Cathepsin-sensitive drug delivery systems because of its overexpression at the tumor sites. For instance, cysteine proteases have increased activity as well as aberrant localization within the tumour microenvironment, which contributes to cancer progression, proliferation and metastasis [START_REF] Olson | Cysteine cathepsin proteases: regulators of cancer progression and therapeutic response[END_REF]. Such findings led to the development of the glycylpenylalanyl-leucyl-glycine (GFLG) sequence that is hydrolyzed by Cathepsin B. In this area, poly(N-(2-hydroxypropyl)methacrylamide-doxorubicin (PHPMA-Dox, also called PK1) were the first clinically investigated conjugates for anticancer therapy that comprised Cathepsinsensitive degradable GFLG sequences [START_REF] Vasey | Phase I clinical and pharmacokinetic study of PK1 [N-(2-hydroxypropyl)methacrylamide copolymer doxorubicin]: First member of a new class of chemotherapeutic agents-drug-polymer conjugates. Cancer Research Campaign Phase I/II Committee[END_REF]. Since PK1, several PHPMA-drug conjugates have entered clinical trials [START_REF] Kopecek | HPMA copolymers: origins, early developments, present, and future[END_REF][START_REF] Julyan | Preliminary clinical study of the distribution of HPMA copolymers bearing doxorubicin and galactosamine[END_REF], which confirmed the great potential of these systems. Some of the structures of the different drug delivery conjugates are gathered in Figure 2 along with their cathepsin cleavable sites for better understanding [START_REF] Doronina | Novel peptide linkers for highly potent antibody-auristatin conjugate[END_REF][START_REF] Peterson | Cathepsin substrates as cleavable peptide linkers in bioconjugates, selected from a fluorescence quench combinatorial library[END_REF][START_REF] Ferber | Polymeric nanotheranostics for real-time non-invasive optical imaging of breast cancer progression and drug release[END_REF]. Whereas several reviews have already been published on targeted polymer-based drug delivery systems [START_REF] Nicolas | Design, functionalization strategies and biomedical applications of targeted biodegradable/biocompatible polymer-based nanocarriers for drug delivery[END_REF][START_REF] Larson | Polymeric conjugates for drug delivery[END_REF][START_REF] Ulbrich | Targeted drug delivery with polymers and magnetic nanoparticles:Covalent and noncovalent approaches, release control, and clinical studies[END_REF][START_REF] Wong | Mechanisms of drug release in nanotherapeutic delivery systems[END_REF], cysteine Cathepsins as imaging probes [START_REF] Verdoes | Improved quenched fluorescent probe for imaging of cysteine cathepsin activity[END_REF][START_REF] Wang | Lysosome-targeting fluorogenic probe for cathepsin B imaging in living cells[END_REF][START_REF] Bender | Design of a highly selective quenched activity-based probe and its application in dual color imaging studies of cathepsin S activity localization[END_REF][START_REF] Edem | Synthesis and evaluation of radioiodinated acyloxymethyl ketones as activity-based probes for cathepsin B[END_REF], ageing and neurodegeneration [START_REF] Stoka | Lysosomal cathepsins and their regulation in aging and neurodegeneration[END_REF], disease management [START_REF] Kramer | The future of cysteine cathepsins in disease management[END_REF] and other protease functions [START_REF] Deu | New approaches for dissecting protease functions to improve probe development and drug discovery[END_REF][START_REF] Bachovchin | The pharmacological landscape and therapeutic potential of serine hydrolases[END_REF], the dynamic involvement of Cathepsins in targeted drug delivery systems including their role in various diseased states and their clinical prospects have never been covered in a single Review Article.

Cathepsin-sensitive drug delivery systems 2.1 Anticancer drug delivery systems

In the past few decades, anticancer drug delivery has attracted extensive interest from both academia and industry. A considerable effort is being spent on the design of nanoscale systems having suitable properties for drug delivery purposes such as stealthiness, non-immunogenicity, biocompatibility as well as biodegradability. The fate of stealth nanoscale systems is governed, at least in part, by the enhanced permeability and retention (EPR) effect (also called passive targeting). It allows for their preferential accumulation at the tumor site because of leaky vasculatures and lack of lymphatic drainage [START_REF] Rosenblum | Progress and challenges towards targeted delivery of cancer therapeutics[END_REF][START_REF] Shi | Cancer nanomedicine: Progress, challenges and opportunities[END_REF]. Interestingly, a variety of different Cathepsins have been reported to be overexpressed in many types of cancers; mostly found in cancer cells but also in cancer-associated leukocytes, fibroblasts, osteoclasts, myoepithelial cells as well as endothelial cells [START_REF] Kallunki | Cancer-associated lysosomal changes: friends or foes?[END_REF]. The list of cancer overexpressing Cathepsins is given below (Table 1). Hence, the intimate relationships between Cathepsins and cancer stimulated the conception of (macro)molecules sensitive to the presence of Cathepsins for enhanced therapeutic effect.

Cathepsin D Lysosomes

Breast, colorectal, ovarian [START_REF] Johnson | Noncaspase proteases in apoptosis[END_REF][START_REF] Dian | Expression of cathepsin-D in primary breast cancer and corresponding local recurrence or metastasis: An immunohistochemical study[END_REF][START_REF] Mehrotra | Expression and localization of cathepsins B, D and G in cancer stem cells in liver metastasis from colon adenocarcinoma[END_REF][START_REF] Pranjol | Cathepsin D nonproteolytically induces proliferation and migration in human omental microvascular endothelial cells via activation of the ERK1/2 and PI3K/AKT pathways[END_REF][START_REF] Lösch | Cathepsin D in ovarian cancer: Prognostic value and correlation with p53 expression and microvessel density[END_REF][START_REF] Pranjol | Tumour secreted factors cathepsins D and L induce pro-angiogenic changes in human omental microvascular endothelial cells (HOMECs) in ovarian cancer metastasis[END_REF][START_REF] Glondu | Downregulation of cathepsin-D expression by antisense gene transfer inhibits tumor growth and experimental lung metastasis of human breast cancer cells[END_REF] In the following, we have covered Cathepsin-sensitive drug delivery systems for anticancer therapy, by distinguishing five different types of systems: (i) polymeric; (ii) inorganic; (iii) dendritic/comb-like; (iv) lipidic and (v) protein-based/peptidic.

Polymeric systems

Different types of polymeric systems have been utilized to develop drug-polymer conjugates for anticancer drug delivery [START_REF] Nicolas | Design, functionalization strategies and biomedical applications of targeted biodegradable/biocompatible polymer-based nanocarriers for drug delivery[END_REF][START_REF] Hans | Biodegradable nanoparticles for drug delivery and targeting[END_REF][START_REF] Dheer | Polysaccharides based nanomaterials for targeted anti-cancer drug delivery[END_REF][START_REF] Panyam | Biodegradable nanoparticles for drug and gene delivery to cells and tissue[END_REF][START_REF] Brannon-Peppas | Recent advances on the use of biodegradable microparticles and nanoparticles in controlled drug delivery[END_REF][START_REF] Cabral | Supramolecular nanodevices: From design validation to theranostic nanomedicine[END_REF][START_REF] Couvreur | Nanotechnology: Intelligent design to treat complex disease[END_REF][START_REF] Sukhorukov | Nanoengineered polymer capsules: Tools for detection, controlled delivery, and site-specific manipulation[END_REF][START_REF] Miyata | Rational design of smart supramolecular assemblies for gene delivery: Chemical challenges in the creation of artificial viruses[END_REF]. Given Cathepsin B is a lysosomal cysteine protease overexpressed in the microenvironment of advanced tumors [START_REF] Loser | Cysteine cathepsins: their role in tumor progression and recent trends in the development of imaging probes[END_REF], this feature has been widely exploited in cancer therapy using polymer-based drug delivery systems bearing the Cathepsin Bsensitive GFLG sequence [START_REF] Yang | Backbone degradable N-(2-Hydroxypropyl)methacrylamide copolymer conjugates with gemcitabine and paclitaxel: impact of molecular weight on activity toward human ovarian carcinoma xenografts[END_REF]. This area was pioneered by Kopeck who developed PHPMAbased drug conjugates containing GFLG sequences on the polymer backbone as well as on the side-chains,giving enhanced therapeutic efficacy while still maintaining their biocompatibility.

This system was further extended to a two-drug combination approach using gemcitabine (Gem, unstable in vivo) and paclitaxel (Ptx, poorly water soluble) linked to either diblock, tetrablock or hexablock PHPMA copolymers obtained by a combining RAFT polymerization and "click" chemistry (Figure 3). The diblock copolymer (M n ~100 kDa) was found to be the most efficient one in vivo on A2780 human ovarian carcinoma xenografts in nude mice. It indeed showed a more pronounced synergistic antitumor effect compared to other structures, thus overcoming the limitations of the free drug. The strongest synergistic interactions in acute myeloid leukemia (AML) was also observed as assessed in HL-60 human AML cells when cytarabine and GDC-0980 were linked to similar GFLG-bearing PHPMA copolymers, conversely to daunorubicin or JS-K [START_REF] Rui | N-(2-Hydroxypropyl)methacrylamide copolymer-drug conjugates for combination chemotherapy of acute myeloid leukemia[END_REF]. Similarly, another study reported on the combination of GDC-0980 (P13K/mTOR inhibitor) and docetaxel against prostate cancer and showed promising results (Figure 4) [START_REF] Zhou | Combination therapy of prostate cancer with HPMA copolymer conjugates containing PI3K/mTOR inhibitor and docetaxel[END_REF]. Several other combinations directed against cancer have also been explored from PHPMA copolymer bearing GFLG sequences [START_REF] Duangjai | Combination cytotoxicity of backbone degradable HPMA copolymer gemcitabine and platinum conjugates toward human ovarian carcinoma cells[END_REF][START_REF] Zhang | Sequential combination therapy of ovarian cancer with degradable N-(2-Hydroxypropyl)methacrylamide copolymer paclitaxel and gemcitabine conjugates[END_REF][START_REF] Peng | Synthesis and activity of tumor-homing peptide iRGD and histone deacetylase inhibitor valproic acid conjugate[END_REF][START_REF] Zhou | HPMA copolymer-based combination therapy toxic to both prostate cancer stem/progenitor cells and differentiated cells induces durable antitumor effects[END_REF][START_REF] Larson | Biodegradable multiblock poly(N-2-hydroxypropyl)methacrylamide gemcitabine and paclitaxel conjugates for ovarian cancer cell combination treatment[END_REF].

Figure4. GFLG-containing PHPMA prodrugs for combination therapy (GDC-0980 and docetaxel) for prostate cancer exhibiting effective anti-Cancer Stem Cell (CSC) effect and in vitro increased anti-bulk tumor effect. Adapted with permission from Ref. [START_REF] Zhou | Combination therapy of prostate cancer with HPMA copolymer conjugates containing PI3K/mTOR inhibitor and docetaxel[END_REF].

In a more mechanistic study, two PHPMA-based multiblock S-CMP (small copolymer block size) and L-CMP (long copolymer block size) have been synthesized [START_REF] Fan | Investigation into the Biological Impact of Block Size on Cathepsin S-Degradable HPMA Copolymers[END_REF]. Both the copolymer blocks and the peptide linkers were tagged with 125 I and 177 Lu, respectively (Figure 5). S-CMP showed increased cleavage rates by Cathepsin S compared to L-CMP resulting from the lower steric hindrance as assessed by in vitro studies. The cleavage and clearance of the different blocks were both greater inside the tumor and the liver, as observed from radioisotopic ratios.

Figure5. Chemical structure of PHPMA-based, dual-labeled small copolymer block size (S-CMP) and long copolymer block size (L-CMP) showing cleavage sites for CathepsinS [START_REF] Fan | Investigation into the Biological Impact of Block Size on Cathepsin S-Degradable HPMA Copolymers[END_REF].

Dox has been conjugated to different polymeric architectures via Cathepsin-sensitive linkers. For instance, Dox was linked to an octa-guanidine-based peptide sequence (Phe-Lys) via 4aminobenzyloxy carbonyl (PABC) as a self-immolative linker, resulting in a G8-PP1-FK-PABC-Doxprodrug. It was able to be cleaved by lysosomal Cathepsin B and inducing selective toxicity against HeLa cells without affecting healthy cells [START_REF] Nair | Novel lysosome targeted molecular transporter built on a guanidinium-poly-(propylene imine) hybrid dendron for efficient delivery of doxorubicin into cancer cells[END_REF].On the contrary, small-molecule (MW<500 g.mol -1 ) self-assemblies have also been utilized to develop a generic cross-linked micellar drug delivery system based on gemcitabine (Gem) prodrugs (Figure 6a). This system proved to be advantageous as compared to well-known polymeric micellar systems in terms of composition, colloidal stability, drug payload (~58 wt.%), biosafety, as well as ease of synthesis, functionalization and in vitro/in vivo anticancer activity [START_REF] Kazunori | Block copolymer micelles as vehicles for drug delivery[END_REF][START_REF] Matsumura | Poly (amino acid) micelle nanocarriers in preclinical and clinical studies[END_REF][START_REF] Tong | Smart chemistry in polymeric nanomedicine[END_REF]. Infact, nearly 60% of the drug was released from the micelles by Cathepsin B in phosphate buffer saline (PBS) at pH 5.5 for 240 h conversely to <7% without Cathepsin B because of the amide bond in between the drug and the promoiety (Figure 6b) [START_REF] Liao | Cross-linked small-molecule micellebased drug delivery system: Concept, synthesis, and biological evaluation[END_REF].

Figure6. Chemical structure of the gemcitabine prodrug (a) and in vitro drug release profile from the gemcitabine prodrug micellar system (b). Adapted with permission from Ref. [START_REF] Liao | Cross-linked small-molecule micellebased drug delivery system: Concept, synthesis, and biological evaluation[END_REF].

Another report focused on the construction of PEGylated, enzyme-sensitive,macrocyclic pillar [START_REF] Turk | Cysteine cathepsins: From structure, function and regulation to new frontiers[END_REF]arene amphiphiles which self-assembled in water into micelles with high Dox loading capacity [START_REF] Gao | Enzyme-responsive pillar[5]arene-based polymer-substituted amphiphiles: Synthesis, self-assembly in water, and application in controlled drug release[END_REF]. The micelles had enzyme-cleavable amide bonds that were cleaved by Lasparaginase (L-ASP) used here as a mimic of intracellular Cathepsin B because it can catalyze the hydrolysis of asparagine to aspartic acid (Figure 7). The Dox-loaded micelles led to significant cytotoxicity on MCF-7 and multidrug-resistant MCF-7/ADR cells, comparatively to drug-free micelles. Folic acid (FA) surface-functionalized, biodegradable poly(ethylene oxide)-b-poly( L -glutamic acid) (FA-PEG-b-PLG) block copolymer vesicles loaded with cisplatin were also reported [START_REF] Shirbin | Cisplatininduced formation of biocompatible and biodegradable polypeptide-based vesicles for targeted anticancer drug delivery[END_REF].

The drug was released intracellularly from the rigid block due to overexpressed Cathepsin B which cleaved the nanostructure because of the increased activity of this proteolytic enzyme in metabolizing PLG acid residues. The enzyme was also responsible for the higher activity in metabolizing polyglutamate (PGA) residues. The nanovesicles exhibited surface-positioned FA moieties for active targeting via selective cell binding and led to enhanced cytotoxicity towards HeLa cells.

PGA was also used as a polymer scaffold to link both Ptxand an integrin-targeted ligand

(E-[c(RGDfK) 2 ]) on the side chains, to give PGA-Ptx-E-[c(RGDfK) 2 ]
). The resulting conjugate gave significant enhancement in anticancer activity compared to free Ptx [START_REF] Eldar-Boock | Integrinassisted drug delivery of nano-scaled polymer therapeutics bearing paclitaxel[END_REF]. As assessed by the in vitro drug release profile, Ptx was released in the presence of Cathepsin B but PGA-Ptx-E-[c(RGDfK) 2 ] was found to be stable in plasma. Interestingly, incorporation of a targeting ligand towards integrin expressing cells led to anti-angiogenic mechanism to overcome multi-drug resistance.

Another targeted drug delivery system was reported and consisted in a heterobifunctional oligomeric PEG chains embedding octreotide as a ligand for the targeting of somatostatin receptors and either an anticancer drug (Dox) tethered via a dipeptidic substrate for Cathepsin B, or a fluorescent dye [START_REF] Tian | Dual-targeting delivery system for selective cancer cell death and imaging[END_REF]. This oligomeric prodrug system was suitable for tumor cell imaging expressing both Cathepsin B and somatostatin receptors and led to selective cytotoxicity towards cancer cells.

Inorganic systems

Inorganic materials (e.g., silica, gold, iron oxide, quantum dots, etc.)is also an attractive family of materials that have been extensively investigated for anticancer drug delivery [START_REF] Setyawati | Mesoporous silica nanoparticles as an antitumoralangiogenesis strategy[END_REF][START_REF] Mout | Surface functionalization of nanoparticles for nanomedicine[END_REF][START_REF] Laurent | Magnetic iron oxide nanoparticles: Synthesis, stabilization, vectorization, physicochemical characterizations, and biological applications[END_REF][START_REF] Lee | Designed synthesis of uniformly sized iron oxide nanoparticles for efficient magnetic resonance imaging contrast agents[END_REF][START_REF] Reddy | Magnetic nanoparticles: Design and characterization, toxicity and biocompatibility, pharmaceutical and biomedical applications[END_REF][START_REF] Vivero-Escoto | Silica-based nanoprobes for biomedical imaging and theranostic applications[END_REF][START_REF] Dreaden | The golden age: gold nanoparticles for biomedicine[END_REF]. In this area, a Cathepsin B-induced tumor targeted drug delivery system loaded with Dox was developed by immobilizing cleavable rotaxanes onto mesoporous silica nanoparticles (MSNs) [START_REF] Cheng | Enzyme-induced and tumor-targeted drug delivery system based on multifunctional mesoporous silica nanoparticles[END_REF]. Nano-constructs comprising a rotaxane moiety and a GFLG sequence linked to the RGDS peptide were used as Cathepsin B-cleavable stoppers for the cyclodextrin valves by means of "click" chemistry (Figure 8). Thanks to the targeting ligand displayed at its surface, such system demonstrated efficient receptor-mediated tumor cell uptake and selective enzymatic digestion of GFLG peptide. MSNs were also coated with Cathepsin B-sensitive peptide sequences (alkynyl-GIVRAKEAEGIVRAK-OH) through triazole rings and led to efficient Dox release (Figure 9).

The study also proved that this peptide sequence was selectively cleaved by Cathepsin B as assessed by in vitro experiments [START_REF] Cristina | Cathepsin-B induced controlled release from peptide-capped mesoporous silica nanoparticles[END_REF]. This peptide sequence was also anchored onto silica supports to develop nanoparticles with prevented release the loaded [Ru(bipy) 3 ] 2+ dye unless specific proteases are present [START_REF] Carmen | Enzyme-mediated controlled release systems by anchoring peptide sequences on mesoporous silica supports[END_REF]. In another study, an enzyme-cleavable peptide precursor conjugated to Dox was further linked onto the surface of silica-coated magnetic nanoparticles by using "click" chemistry (Figure 10) [START_REF] Yang | Enzymeresponsive multifunctional magnetic nanoparticles for tumor intracellular drug delivery and imaging[END_REF].

The nanocarriers exhibited efficient Dox release and selective intracellular Dox delivery into tumors with high Cathepsin B expression together with imaging of cancer cells.

Figure10. Synthesis of Dox-peptide-coated, magnetic silica nanoparticles cleaved by Cathepsin B for Dox release inside cancer cells. Adapted with permission from Ref. [START_REF] Yang | Enzymeresponsive multifunctional magnetic nanoparticles for tumor intracellular drug delivery and imaging[END_REF].

A dual enzymatic responsive nanoconstruct for pancreatic cancer therapy was engineered and relied on surface functionalization of CdSe/ZnS quantum dots (QDs) by an amphiphilic PEG-GGPLGVRGK-NH 2 polymer sensitive to matrix metalloproteinases (MMP-9), and by Gem via a GELG Cathepsin B substrate sequence [START_REF] Han | Dual enzymatic reaction-assisted gemcitabine delivery systems for programmed pancreatic cancer therapy[END_REF]. Some of the PEG chains were also functionalized by cyclo-RGD as a tumor-homing ligand. The nanocarrier exhibited long circulating features and increased drug accumulation at tumor sites, resulting in successful delivery of Gem in BxPD-3 cells because of their inherently elevated concentrations of Cathepsin B (Figure 11).

Figure11. Synthesis of Gem-loaded, decorated QDs and their dual enzymatic behavior. Adapted with permission from Ref. [START_REF] Han | Dual enzymatic reaction-assisted gemcitabine delivery systems for programmed pancreatic cancer therapy[END_REF].

Dendrimeric/comb-like systems

Dendrimers, which are perfectly monodisperse and highly branched 3D macromolecules, have been the topic of great attention especially as drug carriers [START_REF] Kesharwani | Recent advances in dendrimer-based nanovectors for tumortargeted drug and gene delivery[END_REF][START_REF] Medina | Dendrimers as carriers for delivery of chemotherapeutic agents[END_REF][START_REF] Tekade | Dendrimers in oncology: An expanding horizon[END_REF]. For example, peptide dendrimers surface-functionalized by methoxy polyethylene glycol (mPEG) and Dox through the GFLG sequence have been designed [START_REF] Zhang | Dendrimer-doxorubicin conjugate as enzyme-sensitive and polymeric nanoscale drug delivery vehicle for ovarian cancer therapy[END_REF]. The resulting enzyme-responsive dendrimer-GFLG-Dox nanocarrier gave greater accumulation and retention in ovarian tumor cells(SKOV-3), leading to improved anticancer effect and no obvious systemic toxicity. Similarly, mPEG-PAMAM dendrimers of different chain lengths for the formation of Dox-loaded magnetite nanoparticles have also been reported [START_REF] Li | Amphiphilic peptide dendritic copolymer-doxorubicin nanoscale conjugate self-assembled to enzyme-responsive anti-cancer agent[END_REF]. In this system, Cathepsin B was used to selectively degrade the dendritic shell to trigger sustained Dox release near the tumor cells. The concept of enzymatic breakdown of the nanocarrier may represent a new approach for controlled drug delivery systems. Also, Cathepsin B-responsive and amphiphilic PEGylated dendritic polymerdrug conjugates (PEGylated dendron-GFLG-Dox) were obtained by "click" chemistry and led to enhanced antitumor efficacy (Figure 12). Another study reported on the combination of undecapeptide KKLFKKILKKL-NH 2 with the GFLG sequence for the delivery of chlorambucil (CLB) [START_REF] Soler | Enzyme-triggered delivery of chlorambucil from conjugates based on the cellpenetrating peptide BP16[END_REF]. The free drug was inactive (IC 50 = 73.7 to >100 μM) conversely to its prodrug (IC 50 = 3.6 -16.2 μM) on various cancer cell lines including MCF-7, PC-3, CAPAN-1, 1BR3G and SKMEL-28. CLB-Gly-OH was indeed released when Cathepsin B was present as evidenced by Cathepsin B enzymatic assays. Also, these studies supported the fact that CLB would be released in the lysosomal compartment. A comparative study was reported between dendrimers based on mPEG conjugated to Dox via a Cathepsin B-cleavable Gly-Phe-Leu-Gly sequence and GFLG-free dendrimers [START_REF] Lee | Enzymeresponsive doxorubicin release from dendrimer nanoparticles for anticancer drug delivery[END_REF]. The GFLG sequence-bearing nanoconstructs were formulated into nanoparticles exhibiting Cathepsin Bsensitive drug delivery properties. The enhanced anticancer activity compared to that of free Dox was validated in vivo in a CT26 tumor xenograft mouse model.

Asorbitol scaffold functionalized by octa-guanidine moieties and conjugated to Dox via a GLPG sequence, another peptidic substrate of Cathepsin B, was produced (Figure 13) [START_REF] Maniganda | A lysosome-targeted drug delivery system based on sorbitol backbone towards efficient cancer therapy[END_REF].

This conjugate was efficiently taken up by the cells via electrostatic interaction between guanidine moieties and negatively-charged phospholipids/sulphates exposed at the surface of the

Lipidic systems

A great amount of work is also currently been carried out to design lipid-based drug delivery systems either as drug-loaded lipidic nanocarriers or lipidic prodrug nanocarriers [START_REF] Kalepu | Oral lipid-based drug delivery systems -An overview[END_REF][START_REF] Mura | Lipid prodrug nanocarriers in cancer therapy[END_REF].

However, examples of Cathepsin-sensitive lipidic drug delivery systems are rather scarce. For instance, when a lipidated Cathepsin B inhibitor (NS-629) was anchored into a liposome bilayer (Figure 14),its selective targeting and internalization into tumors and stromal cells was shown ex vivo and in vivo, confirming that using Cathepsin B as an efficient leverage for cancer diagnosis and treatment [START_REF] Mikhaylov | Selective targeting of tumor and stromal cells by a nanocarrier system displaying lipidated cathepsin B inhibitor[END_REF]. Combination therapy, that relies on the simultaneous administration of at least two different drugs, is increasingly used to treat various diseases, including cancer [START_REF] Al-Lazikani | Combinatorial drug therapy for cancer in the post-genomic era[END_REF][START_REF] Hu | Nanoparticle-based combination therapy toward overcoming drug resistance in cancer[END_REF]. Combination therapy from cathepsin-sensitive lipidic systems was illustrated by the conception of methotrexate-methoxypoly(ethylene glycol)-1,2-distearoyl-snglycero-3-phosphoethanolamine (Mtx-MePEG-DSPE) prodrug micelles loaded with mitomycin C-soybean phosphatidylcholine (SPC-MMC) prodrugs [START_REF] Li | Orthogonally functionalized nanoscale micelles for active targeted codelivery of methotrexate and mitomycin C with synergistic anticancer effect[END_REF]. This micellar system exhibited synergistic anticancer activity in presence of Cathepsin B because of the amide linker in between the polymer and the drugs, as opposed to the action of individual drugs.

Protein-based/peptidic systems

Drug delivery systems based on proteins or peptides represent an appealing class of materials especially because of their biocompatibility [START_REF] Sood | Peroral route: An opportunity for protein and peptide drug delivery[END_REF][START_REF] Pan | Protein-based multifunctional nanocarriers for imaging, photothermal therapy, and anticancer drug delivery[END_REF][START_REF] Habibi | Self-assembled peptide-based nanostructures: Smart nanomaterials toward targeted drug delivery[END_REF][START_REF] Spicer | Peptide and protein nanoparticle conjugates: versatile platforms for biomedical applications[END_REF][START_REF] Law | Peptide-based biomaterials for protease-enhanced drug delivery[END_REF]. For instance, proteinticles, which are proteins that can self-assemble inside cells into nanoscale particles, can be employed in many different biomedical applications owing to their enhanced biocompatibility, conversely to synthetic nanomaterials [START_REF] Ahn | Engineered protein nanoparticles for in vivo tumor detection[END_REF]. Conferring cathepsin-sensitivity to such systems have also been reported, especially for small interfering RNA (siRNA) delivery where it showed great potential against various cancers. For instance, proteinticles based on human ferritin were genetically engineered to display at their surface different functional peptides in a simultaneous manner, such as cationic peptides for self-assembling siRNA, cancer cell-targeting or cell penetrating peptide [START_REF] Jung | Engineered proteinticles for targeted delivery of siRNA to cancer cells[END_REF]. They led to enhanced siRNA capture, cancer cell targeting together with enhanced penetration into the cytoplasm of tumor cells. They were eventually cleaved by Cathepsin B for intracellular release of siRNA inside tumor cells, leading to efficient gene silencing. One of the greatest advantages of proteinticles is that such functional peptides of different nature can be evenly placed on their surface, depending on the tumor cell type through a simple genetic modification, thus making it a very versatile system for targeted siRNA delivery. Another study revealed the development of a polyglutamate amine (APA) nanocarriers containing miRNA and siRNA polyplexes which showed great accumulation into pancreatic tumor cells [START_REF] Gibori | Amphiphilic nanocarrier-induced modulation of PLK1 and miR-34a leads to improved therapeutic response in pancreatic cancer[END_REF]. It was also shown that the release of miRNA occurred from APA-containing polyplex in the presence of Cathepsin B.

Given the poor water-solubility of many anticancer drugs, a considerable amount of research has been done to improve their hydrophilicity by conjugation to hydrophilic moieties via Cathepsinsensitive linkers. For instance, Ptx has been conjugated to a highly water-soluble nucleolin aptamer (NucA) for the targeting of ovarian cancer with reduced off-site toxicity [START_REF] Li | A water-soluble nucleolin aptamer-paclitaxel conjugate for tumor-specific targeting in ovarian cancer[END_REF]. The resulting bioconjugate proved to be biologically stable as assessed by fluorescence resonance energy transfer (FRET) (Figure 15) and also inactive in the blood circulation. NucA was conjugated to the hydroxyl group at position 2' of the drug via a dipeptide bond sensitive to Cathepsin B, which then got cleaved once inside the cells by Cathepsin B, thus triggering the anticancer mechanism. The GFLG sequence was also embedded into a star-shaped peptidic prodrug structures that can be cleaved by Cathepsin B. This feature has been used to develop drug delivery vehicles for 2methoxyestradiol (2ME) which is a natural metabolite of estradiol with antiproliferative and antiangiogenic activities (Figure 16) [START_REF] Shankar | Cathepsin B degradable star-shaped peptidic macromolecules for delivery of 2-methoxyestradiol[END_REF].

Figure16. Representation of the degradation of star-shaped peptidic prodrug structures that can be cleaved by Cathepsin B.Adapted with permission from Ref. [START_REF] Shankar | Cathepsin B degradable star-shaped peptidic macromolecules for delivery of 2-methoxyestradiol[END_REF].

In the context of combination therapy, a dual-functionalized linker bearing Dox and Ptx, and comprising a maleimide moiety for its subsequent coupling to albumin through its cysteine-34 position, was designed [START_REF] Ajaj | Development of protein-binding bifunctional linkers for a new generation of dual-acting prodrugs[END_REF]. Each drug was linked by a self-immolative para-aminobenzyloxy carbonyl linker and a cleavable dipeptide (Phe-Lys) sensitive to Cathepsin B, leading to drug release at the tumor site (Figure 17).A similar approach combining a polymer prodrug and a polymer-enzyme bioconjugate was used to selectively and rapidly deliver a cytotoxic drug to the target site [START_REF] Satchi-Fainaro | PDEPT: polymerdirected enzyme prodrug therapy. 2. HPMA copolymer-β-lactamase and HPMA copolymer-C-Dox as a model combination[END_REF].

Figure17. Structure of Cathepsin B-sensitive, dual-functionalized linker bearing Dox and Ptx, and comprising a maleimide moiety for its coupling to albumin. Adapted with permission from Ref. [START_REF] Ajaj | Development of protein-binding bifunctional linkers for a new generation of dual-acting prodrugs[END_REF].

Pep42, which is a cyclic 13-mer oligopeptide, specifically binds to glucose-regulated protein 78

and translocates into the lysosomal compartment [START_REF] Kim | Targeting heat shock proteins on cancer cells: Selection, characterization, and cell-penetrating properties of a peptidic GRP78 ligand[END_REF][START_REF] Liu | Mechanistic studies of a peptidic GRP78 ligand for cancer cell-specific drug delivery[END_REF]. In this context, Pep42 was advantageously used to efficiently deliver Ptx and Dox into cancer cells for enhanced cytotoxicity [START_REF] Yoneda | A cell-penetrating peptidic GRP78 ligand for tumor cell-specific prodrug therapy[END_REF]. More specifically, Pep42-prodrug bioconjugates containing a Cathepsin Bsensitive linker were synthesized and facilitated the uptake of both cytotoxic agents for their delivery into cancer cells.

Nanoconstructs with methotrexate (Mtx) linked to a tuftsin-like peptide carrier via a GFLG spacer and several copies of a chemotactic targeting agent were designed [START_REF] Bai | Design, synthesis, and in vitro activity of novel drug delivery systems containing tuftsin derivatives and methotrexate[END_REF]. These conjugates led to greater cytotoxic effect than free Mtx and represented potential candidates for the specific targeting of cancer cells. Similarly, Dox-based dipeptide conjugates were designed and tethered to monoclonal antibodies (mAbs) recognizing tumor associated antigens on renal cell carcinoma and anaplastic large cell lymphoma [START_REF] Jeffrey | Dipeptide-based highly potent doxorubicin antibody conjugates[END_REF]. The dipeptides were substrates for Cathepsin B and got cleaved with comparable kinetics. Importantly, both prodrugs were 70-fold more potent than free Dox.

In another study, cytotoxic drug-carrying filamentous bacteriophages were chemically modified to tunedifferent key parameters (e.g., pharmacokinetics, biodistribution, immunogenicity)and compared to bare phages [START_REF] Bar | Killing cancer cells by targeted drug-carrying phage nanomedicines[END_REF]. Anti-ErbB2 and anti-ERGR antibodies were used as targeting entities, whereas Dox was tethered to phages through an amide linkage and also to genetically-engineered Cathepsin-B (Figure 18). In vitro studies explained the good penetration into tumors cells by their needle-like structure. This conjugate can be seen as a novel drug-delivery platform which might solve many issues related to the hydrophobicity of drugs at the target specific sites.

Figure18. Genetically engineered Cathepsin B-modulated bacteriophage conjugated to Dox. Adapted with permission from Ref. [START_REF] Bar | Killing cancer cells by targeted drug-carrying phage nanomedicines[END_REF].

Bone-targeting drug delivery systems

The most common skeleton disorders are arthritis, osteoporosis, osteomyelitis, osteosarcoma as well as metastatic bone cancer [START_REF] Kramer | The future of cysteine cathepsins in disease management[END_REF][START_REF] Rotman | Drug delivery systems functionalized with bone mineral seeking agents for bone targeted therapeutics[END_REF]. Bone metastasis is one of the most devastating stages of cancer [START_REF] Vinay | Potential of targeted drug delivery system for the treatment of bone metastasis[END_REF]. In addition, there are several limitations associated with the systemic administration of drugs for bone treatment and bone-related diseases such as poor drug uptake at the target site, potential systemic toxicity as well as suboptimal efficacy [START_REF] Rotman | Drug delivery systems functionalized with bone mineral seeking agents for bone targeted therapeutics[END_REF]. Interestingly, there are examples in the literature describing Cathepsin-sensitive polymer conjugates for bone targeting purposes [START_REF] Xie | Design, synthesis, and pharmacokinetics of a bone-targeting dual-action prodrug for the treatment of osteoporosis[END_REF][START_REF] Wang | Synthesis and evaluation of water-soluble polymeric bone-targeted drug delivery systems[END_REF][START_REF] Wang | Bone-targeting macromolecular therapeutics[END_REF][START_REF] Low | Targeting polymer therapeutics to bone[END_REF]. Therefore, drug delivery systems targeted towards bones can be adapted to bone diseases where the drug can be selectively delivered with minimal side effects [START_REF] Newman | Local and targeted drug delivery for bone regeneration[END_REF].

In a similar fashion to what has been reported for anticancer therapy, HPMA was conjugated to prostaglandin E 1 (PGE 1 ) via a spacer sensitive to Cathepsin K, which is an enzyme overexpressed in osteoclasts [START_REF] Pan | Water-soluble HPMA copolymer-prostaglandin E1 conjugates containing a cathepsin K sensitive spacer[END_REF]. The Cathepsin K-sensitive spacer comprised Gly-Gly-Pro-Nle as the tetrapeptide sequence and a self-eliminating 4-aminobenzyl alcoholmoiety.

Copolymerization of the resulting PGE 1 -containing HPMA macromonomer with HPMA yielded the desired PHPMA-PGE 1 conjugates,that released unmodified PGE 1 after incubation with Cathepsin K. PHPMA was also post-functionalized by a D-aspartic acid octapeptide targeting ligand. Therefore, this new drug delivery system might be a solution to treat osteoporosis and other bone-related pathologies.

Targeting inflammatory joints in rheumatoid arthritis (RA) was achieved by AWO54, a new prodrug that binds to endogenous albumin and was composed of Mtx, a spacer based on lysine and an enzyme-sensitive peptide linker linked to a maleimide moiety for further linkage to albumin [START_REF] Fiehn | Targeted drug delivery by in vivo coupling to endogenous albumin: An albumin-binding prodrug of methotrexate (MTX) is better than MTX in the treatment of murine collagen-induced arthritis[END_REF]. The prodrug was cleaved by two enzymes, Cathepsin B and plasmin, that exist in high concentrations in synovial effusion under RA condition, thus releasing Mtx lysine derivatives. The in situ coupling of endogenous albumin, AWO54 was found to be better in terms of dosage and efficacy than administration of the parent drug for treating collagen-induced arthritis.

Immune cell-targeting drug delivery systems

Lysosomal peptidases are part of innate and adaptive immune responses [START_REF] Perišić Nanut | Cysteine cathepsins as regulators of the cytotoxicity of NK and T Cells[END_REF][START_REF] Gupta | Cysteine cathepsin S as an immunomodulatory target: Present and future trends[END_REF][START_REF] Zavasnik-Bergant | Cysteine cathepsins in the immune response[END_REF]. Hence, modulation of such responses with Cathepsin-sensitive prodrugs can further enhance the immunological action and regulate cytotoxicity issues related to NK and T cells. For instance, influence of superparamagnetic iron oxide nanoparticles (SPIONs) from both a physiological and immunological point of view was investigated on cell function and their interaction with oxysterol laden cells [START_REF] Laskar | Degradation of superparamagnetic iron oxide nanoparticle-induced ferritin by lysosomal cathepsins and related immune response[END_REF]. Iron-loaded nanoparticles upregulated Cathepsin, membranous ferroportin (cellular efflux channel for iron) and ferritin degradation, which further altered cellular immune functions, resulting in secretion of pro-(TNF-α) and anti-inflammatory (IL-10) cytokines and ferritin. Importantly, this study highlighted a specific relationship between SPION metabolism and atheroma cellfunction that might conduct to innovative approaches to treat atherosclerotic plaques.

Immunoconjugates were also prepared from cytotoxic agents using a valine-alanine-paminobenzyl-amine linker which was well-adapted for the bioconjugation to monoclonal antibody and further specific cleavage by proteases [START_REF] Burke | Novel immunoconjugates comprised of streptonigrin and 17-amino-geldanamycin attached via a dipeptide-p-aminobenzyl-amine linker system[END_REF]. The linker efficiently released aminogeldanamycin and streptonigrin upon protease-mediated hydrolysis, emphasizing the activity and specificity of the conjugates in vitro and in vivo. In another study, different immunoconjugates comprising lysosomally cleavable peptides (i.e., Phe-Lys and Val-Cit), were synthesized [START_REF] Dubowchik | Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: Model studies of enzymatic drug release and antigen-specific in vitro anticancer activity[END_REF].

The monoclonal antibody BR96 that is known to bind to Lewis y -related tumor-associated antigen expressed at the surface of cancer cells was linked to Dox via a p-aminobenzyloxycarbonyl (PABC) spacer. Interestingly, the conjugates bearing the Phe-Lys sequence exhibited a 30-fold greater drug release kinetics in the presence of Cathepsin B than its counterpart with the Val-Cit linker.

Cathepsins as probes for imaging and theranostic

Different types of enzymes (e.g., caspases, secretases, furinases, phosphatases, etc.) have been exploited for cancer diagnosis. Furthermore, imaging probes utilizing these proteases have rapidly evolved [START_REF] Leriche | Fluorescent analogue of batimastat enables imaging of α-secretase in living cells[END_REF][START_REF] Darragh | Tumor detection by imaging proteolytic activity[END_REF][START_REF] Edgington | Noninvasive optical imaging of apoptosis by caspase-targeted activity-based probes[END_REF][START_REF] Gade | Imaging of alkaline phosphatase activity in bone tissue[END_REF]. It has been shown that the monitoring of protease activity was closely related to cancer progression especially in case of Cathepsin B [START_REF] Yhee | Optical imaging of cancer-related proteases using near-infrared fluorescence matrix metalloproteinase-sensitive and cathepsin B-sensitive probes[END_REF]. Among the numerous studies on proteases for such a purpose, hollow mesoporous silica nanoparticles loaded with Dox and conferred with a dual-enzyme sensitivity were conceived for the in situ imaging of Cathepsin B and the release of Dox mediated by proteases (Figure 19) Adapted with permission from Ref. [START_REF] Zheng | Peptide-mediated core/satellite/shell multifunctional nanovehicles for precise imaging of cathepsin B activity and dual-enzyme controlled drug release[END_REF].

It was also recently discovered that indocyanine green (ICG)-containing PGA nanoparticles can be digested byCathepsin B and induce a sentization of the endo-lysosomal membrane mediated by the NIR properties of the released ICG (Figure 20) [START_REF] Sam | Cathepsin B-degradable, NIR-responsive nanoparticulate platform for target-specific cancer therapy[END_REF]. The system was combined with a ribosome-inactivating protein (saporin) which showed synergistic cytotoxicity because of the photo-induced release of saporin from endosomes or lysosomes. An enzymatically dependent FRET process was also used to monitor the payload release from PHPMA prodrug nanocarriers [START_REF] Zhang | FRET imaging of enzyme-responsive HPMA copolymer conjugate[END_REF]. PHPMA was functionalized with donor Cy5 and acceptor Cy7, thus inducing FRET. However, since only Cy7 was linked to the polymer via the GFLG sequence, presence of Cathepsin B was accurately measured because of the change in the FRET signal during the Cathepsin B-mediated Cy7 release (Figure 21). The in vitro results showed that the high level of expression of Cathepsin B in cancer cells induced effective release of the dye while in vivo observations resulted in a faster release in the ovarian tumor as compared to normal tissues. The specific presence of cysteine Cathepsins has also been exploited to perform radiolabeled drug delivery for nanoscale conjugates with the aim of inducing enhanced diagnostic and radiotherapeutic efficacy. For instance, PHPMA was radiolabeled with Lutetium-117 ( 117 Lu) via a peptide sequence made of two consecutive metabolically active linkers (MALs) sensitive to Cathepsin B and S, that are overexpressed in the liver and the spleen [START_REF] Ogbomo | 177 Lu-labeled HPMA copolymers utilizing cathepsin B and S cleavable linkers: Synthesis, characterization and preliminary in vivo investigation in a pancreatic cancer model[END_REF]. The MALs were shown to be metabolized by enzymes into single metabolites. The 117 Lu-peptide-PHPMA conjugate showed a substantial retention decrease in the long run in the liver and the spleen, compared to non-cleavable counterparts on human pancreatic adenocarcinoma xenograft mouse model. In another study, the Garrison's group developed the synthesis of cathepsin S-susceptible 177 Lu-labeled or FRET-capable multiblock PHPMA copolymers, which resulted into fast in vitro cleavage of both copolymers. Quicker clearance and lower non-target retention without reducing tumor targeting was also shown on pancreatic ductal adenocarcinoma mouse model [START_REF] Fan | Cathepsin Scleavable, multi-block HPMA copolymers for improved SPECT/CT imaging of pancreatic cancer[END_REF]. This study therefore took benefit of the presence of Cathepsin S in MPS tissues to lower non-target accumulation.

A targeted, theranostic prodrug relying on Cathepsin-B-sensitive Gem release and activation of a caspase-3 specific probe was designed (Figure 23) [START_REF] Han | A cascade enzymatic reaction activatable gemcitabine prodrug with an AIE-based intracellular light-up apoptotic probe for in situ self-therapeutic monitoring[END_REF]. The targeting relied on the RGD peptide for accumulation into pancreatic cancer cells with overexpressed α v β 3 integrin.

The GFLG peptide was then hydrolyzed by Cathepsin B leading to Gem release as well as the apoptotic probe. This system showed promising properties as a platform for both pancreatic cancer cell targeting and real-time, non-invasive imaging.

Figure23. Schematic structure of consecutive enzymatic reaction using a gemcitabine-based prodrug along with apoptotic probe for the killing and monitoring of pancreatic cancer cells. Adapted with permission from Ref. [START_REF] Han | A cascade enzymatic reaction activatable gemcitabine prodrug with an AIE-based intracellular light-up apoptotic probe for in situ self-therapeutic monitoring[END_REF].

In tumor imaging, many proteases can be used for the activation of fluorescent probes including near-infrared emitting dyes. Therefore, in vivo molecular profiling of protease activity can be performed with such probes in endoscopy or tomographic optical imaging [START_REF] Shah | Molecular imaging of gene therapy for cancer[END_REF]. For instance, it has been reported the design of quenched activity-based probe (qABP) mediated by Cathepsin S [START_REF] Verdoes | A nonpeptidic cathepsin S activity-based probe for noninvasive optical imaging of tumor-associated macrophages[END_REF]. It showed high tumor-specific fluorescence in a syngeneic breast cancer model. Other activity-based probes targeting Cathepsin X have been designed [START_REF] Paulick | Development of activity-based probes for cathepsin X[END_REF]. Cathepsin X is involved in a many different biological mechanisms, such as aging, cancer, neurodegenerative disorders, inflammation, etc. [START_REF] Kos | The role of cathepsin X in cell signaling[END_REF][START_REF] Jevnikar | The role of cathepsin X in the migration and invasiveness of T lymphocytes[END_REF][START_REF] Fonović | Identification and characterization of the novel reversible and selective cathepsin X inhibitors[END_REF]. These probes were successfully used for the selective labeling and imaging of Cathepsin X in vitro and in vivo, thus making them a valuable tool for examining protease activity and functions.

Malarial parasites are known to generate significant concentrations of mobile ferrous iron [START_REF] Li | Proteases as regulators of pathogenesis: examples from the Apicomplexa[END_REF].

In this context, parasite-specific, Fe II -sensitive delivery of a potent dipeptidyl aminopeptidase inhibitor through Cathepsin C was demonstrated by using activity-based probes [START_REF] Deu | Ferrous iron-dependent drug delivery enables controlled and selective release of therapeutic agents in vivo[END_REF].

Production of Fe II was triggered in the presence of 1,2,4-trioxolone moiety leading to instant drug release prior to the fragmentation of the aforesaid moiety. Further in vivo evaluation was performed using Plasmodium berghei model of murine malaria which showed selective drug targeting in parasitic infections.

Cathepsin D-conjugated peptides were self-assembled into nanoparticles with the help of gelatin to bypass early nonspecific dissolution as well as off-target Dox release and is useful for optical imaging in animal models [START_REF] Kulsharova | In vitro and in vivo imaging of peptide-encapsulated polymer nanoparticles for cancer biomarker activated drug delivery[END_REF]. Cathepsin D is an enzyme for breast cancer cell secretion, which got triggered by degrading the nanoparticles coated with peptide strands through hydrolytic cleavage, thus releasing Dox. The nanoparticles were evaluated under ultrasound imaging both in vitro and in vivo, and were found to be localized in the bladder and the tumors of mice as a result of the fluorescent profile of Dox. Synthesis of Cathepsin Bsensitive, near-infrared fluorescent probe was also carried out (Figure 24) [START_REF] Bao | DCPO based nanoparticles as a near-infrared fluorescent probe for Cathepsin B[END_REF]. The probe was found to be water-soluble but still self-assembled into nanoparticles having potential for tumor- Figure24. Chemical structure of NIR fluorescent probe sensitive to CathepsinB [START_REF] Bao | DCPO based nanoparticles as a near-infrared fluorescent probe for Cathepsin B[END_REF].

Similarly, a Cathepsin B-sensitive nanoparticulate probe comprising a Cathepsin B substrate peptidic probe linked to chitosan nanoparticles was reported [START_REF] Ryu | Cathepsin B-sensitive nanoprobe for in vivo tumor diagnosis[END_REF]. According to the study, this [START_REF] Poreba | Selective imaging of cathepsin L in breast cancer by fluorescent activity-based probes[END_REF]. This approach enabled to differentiate Cathepsin L activity from that of other Cathepsins such as Cathepsin B.

Miscellaneous

As shown in the previous sections, Cathepsins have been exploited in targeted drug delivery systems and imaging. However, Cathepsin inhibitors can also be exploited in regard to their role in numerous diseased conditions, mainly cardiovascular diseases such as myocardial infarction, atherosclerosis, cardiac hypertrophy, cardiomyopathy and hypertension based on animal models [START_REF] Liu | Cysteine protease cathepsins in cardiovascular disease: from basic research to clinical trials[END_REF]. Cathepsin inhibitors are also used against immune responses, osteoporosis, arthritis, inflammation and neurodegenrative disorders [START_REF] Stoka | Lysosomal cathepsins and their regulation in aging and neurodegeneration[END_REF][START_REF] Bird | Endolysosomal proteases and their inhibitors in immunity[END_REF][START_REF] Abdel-Magid | Inhibition of cathepsin K: A novel and promising treatment for osteoporosis[END_REF][START_REF] Falcão | Dipeptidyl vinyl sulfone as a novel chemical tool to inhibit HMGB1/NLRP3-Inflammasome and Inflamma-miRs in Aβ-Mediated microglial inflammation[END_REF]. A selection of representative examples is discussed below.

Interestingly, peptide-based pseudosubstrates for Cathepsin G and elastase were developed. These substrates can decrease the activated interleukin-36 (IL-36) family cytokines especially in case of inflammatory diseases(e.g., psoriasis, arthritis)because such cytokines are proteolytically processed in the presence of Cathepsin G and other proteases [START_REF] Sullivan | Suppressing IL-36-driven inflammation using peptide pseudosubstrates for neutrophil proteases[END_REF]. In another study, it was proven that amodiaquine, an antimalarial drug, inhibited host Cathepsin B to protect host cells against infection with multiple toxins or viruses [START_REF] Zilbermintz | Identification of agents effective against multiple toxins and viruses by host-oriented cell targeting[END_REF]. Cathepsin K has also been involved in diabetes-associated cardiac abnormalities. Wild-type as well as Cathepsin K knockout mice-induced diabetes exhibited severe cardiac dysfunctions in the form of dampened calcium handling intracellularly, cardiac morphology alterations and also increase in cardiomycyte apoptosis [START_REF] Guo | Cathepsin K knockout protects against cardiac dysfunction in diabetic mice[END_REF][START_REF] Yang | Deficiency and inhibition of cathepsin K reduce body weight gain and increase glucose metabolism in mice[END_REF]. Hence, Cathepsin K may be a suitable target in the aforementioned conditions. One study also investigated cysteine Cathepsin inhibitors such as GB111-NH 2 (that blocks the activity of Cathepsin B, L and S) as trigger in macrophage cell death especially in case of tumor-associated macrophages (TAMs) [START_REF] Salpeter | A novel cysteine cathepsin inhibitor yields macrophage cell death and mammary tumor regression[END_REF].

Clinical data on cathepsins

As seen throughout this review article, PHPMA has been extensively used for the design of Cathepsin-sensitive nanocarriers. This is explained by the favorable properties of PHPMA regarding biomedical applications. PHPMA is indeed hydrophilic, non-immunogenic, chemically inert, non-toxic (even at the dose of 30 g/kg rat), biocompatible and exhibits relatively long circulation time which is dependent on its molar mass [START_REF] Vasey | Phase I clinical and pharmacokinetic study of PK1 [N-(2-Hydroxypropyl)methacrylamide copolymer doxorubicin]: First member of a new class of chemotherapeutic agents-drug-polymer conjugates[END_REF]. Among the different conjugates based on PHPMA that have been synthesized and evaluated so far, some of them entered clinical trials (Table 2). In particular,PK1 (Prague-Keele-1) has shown very promising results in oncology [START_REF] Lammers | Drug targeting to tumors: Principles, pitfalls and (pre-) clinical progress[END_REF] and reached phase II trials in 2002 but clinical studies for both PK1 and PK2

were discontinued in 2008 because of lack of efficacy [START_REF] Yang | The light at the end of the tunnel-second generation HPMA conjugates for cancer treatment[END_REF]. Other polymeric systems also entered clinical trials (Table 2). For instance, XYOTAX (based on polyglutamate) has shown encouraging results in phase III trials in women with non-small-cell lung cancer [START_REF] Li | Complete regression of well-established tumors using a novel water-soluble poly(L-glutamic acid)-paclitaxel conjugate[END_REF][START_REF] Sabbatini | Phase II study of CT-2103 in patients with recurrent epithelial ovarian, fallopian tube, or primary peritoneal carcinoma[END_REF][START_REF] Nemunaitis | Phase I study of CT-2103, a polymer-conjugated paclitaxel, and carboplatin in patients with advanced solid tumors[END_REF][START_REF] Galic | Paclitaxel poliglumex for ovarian cancer[END_REF][START_REF] Langer | Phase III trial comparing paclitaxel poliglumex (CT-2103, PPX) in combination with carboplatin versus standard paclitaxel and carboplatin in the treatment of PS 2 patients with chemotherapy-naive advanced non-small cell lung cancer[END_REF][START_REF] Bonomi | Paclitaxel poliglumex (PPX, CT-2103): macromolecular medicine for advanced non-small-cell lung cancer[END_REF][START_REF] Singer | Paclitaxel poliglumex (XYOTAX; CT-2103): an intracellularly targeted taxane[END_REF].

Among the different PHPMA clinical candidates, PNU166945 is based on Ptx for the treatment of advanced breast cancer and PNU166148 is based on camptothecin for the treatment of metastatic solid tumors but both were stopped in Phase I trials because of severe neurotoxicity and lack of anticancer action, respectively [START_REF] Meerum Terwogt | Phase I clinical and pharmacokinetic study of PNU166945, a novel water-soluble polymer-conjugated prodrug of paclitaxel[END_REF][START_REF] Schoemaker | A phase I and pharmacokinetic study of MAG-CPT, a water-soluble polymer conjugate of camptothecin[END_REF].Also, AP5280 prodrug has been introduced

for PHPMA-carboplatinate and entered clinical phase II trial whereas PHPMA-oxiplatinate (ProLindac, also named AP5346) was in clinical phase II trial against ovarian cancer [START_REF] Gianasi | HPMA copolymers platinates containing dicarboxylato ligands. Preparation, characterisation and in vitro and in vivo evaluation[END_REF][START_REF] Rademaker-Lakhai | A Phase I and pharmacological study of the platinum polymer AP5280 given as an intravenous infusion once every 3 weeks in patients with solid tumors[END_REF][START_REF] Campone | Phase I and pharmacokinetic trial of AP5346, a DACH-platinumpolymer conjugate, administered weekly for three out of every 4 weeks to advanced solid tumor patients[END_REF].

PHPMA polymer conjugated to Dox using GFLG linker has also been attempted with proteinbound Ptx (abraxane), carbohydrate residues such as galactosamine, lactose and also amino acids like phenylalaninelysine (Phe-Lys), and are currently in preclinical studies [START_REF] Shiah | Biodistribution of free and N-(2hydroxypropyl)methacrylamide copolymer-bound mesochlorin e6 and adriamycin in nude mice bearing human ovarian carcinoma OVCAR-3 xenografts[END_REF][START_REF] Shiah | Biodistribution and antitumour efficacy of long-circulating N-(2-hydroxypropyl)methacrylamide copolymerdoxorubicin conjugates in nude mice[END_REF][START_REF] Kunath | HPMA copolymer-anticancer drug-OV-TL16 antibody conjugates. 3. The effect of free and polymer-bound adriamycin on the expression of some genes in the OVCAR-3 human ovarian carcinoma cell line[END_REF][START_REF] David | Design of a multivalent galactoside ligand for selective targeting of HPMA copolymer-doxorubicin conjugates to human colon cancer cells[END_REF].On the contrary, high molar mass PHPMA was investigated to enhance the anticancer efficacy which also reached preclinical settings [START_REF] Etrych | New HPMA copolymers containing doxorubicin bound via pH-sensitive linkage: synthesis and preliminary in vitro and in vivo biological properties[END_REF]. A second series of blockbuster polymers that entered clinical trials are PEG-based conjugates, namely, EZ-246 conjugated to camptothecin but its phase II trials has been stopped due to lack of efficacy. However, NK911 in the form of PEGylated micelles with aspartic acid and Dox had showed promising efficacy against various solid malignancies likely thanks to the EPR effect and is currently in phase II [START_REF] Posey | Phase 1 study of weekly polyethylene glycol-camptothecin in patients with advanced solid tumors and lymphomas[END_REF][START_REF] Rowinsky | A phase I and pharmacokinetic study of pegylated camptothecin as a 1-hour infusion every 3 weeks in patients with advanced solid malignancies[END_REF][START_REF] Matsumura | Phase I clinical trial and pharmacokinetic evaluation of NK911, a micelle-encapsulated doxorubicin[END_REF][START_REF] Nakanishi | Development of the polymer micelle carrier system for doxorubicin[END_REF].

Carboxymethyldextrans another synthetic polymer forming prodrugs with exatecan and camptothecin [START_REF] Li | Polymer-drug conjugates: recent development in clinical oncology[END_REF], and which entered Phase I clinical trials, showed prominent results especially against colon cancer [START_REF] Soepenberg | Phase I and pharmacokinetic study of DE-310 in patients with advanced solid tumors[END_REF][START_REF] Veltkamp | Clinical and pharmacologic study of the novel prodrug delimotecan (MEN 4901/T-0128) in patients with solid tumors[END_REF]. Polyglutamate has been conjugated using Ptx by Cell Therapeutics Inc. company and is currently in Phase II trials [START_REF] Li | Complete regression of well-established tumors using a novel water-soluble poly(L-glutamic acid)-paclitaxel conjugate[END_REF][START_REF] Sabbatini | Phase II study of CT-2103 in patients with recurrent epithelial ovarian, fallopian tube, or primary peritoneal carcinoma[END_REF]. Other Cathepsinsensitive drug delivery systems are under preclinical investigations, including polymeric dendritic systems containing Dox (KTB Tumorforschungs GmbH company) [START_REF] Calderón | Development of enzymatically cleavable prodrugs derived from dendritic polyglycerol[END_REF][START_REF] Calderón | Functional dendritic polymer architectures as stimuli-responsive nanocarriers[END_REF].

Conclusion

As shown in this Review Article, Cathepsins could be very efficient tools, leverages or even actuators for the design of advanced drug delivery systems. It has been shown that these systems were sensitive to the presence of a broad spectrum of different Cathepsins, leading to enhanced therapeutic benefit and imaging capabilities. Among the numerous Cathepsin-sensitive conjugates reported so far, some of them have shown promising results and even reached advanced clinical trials. However, a great deal of work remains especially regarding the lack of site specificity and the still limited understanding of the biological roles of some proteases.

These limitations must be resolved to develop optimized conjugates and offer more prominent clinical candidates.

Also, it has been demonstrated that cysteine Cathepsin proteases can act as regulators for cancer progression as well as therapeutic response [START_REF] López-Otín | Emerging roles of proteases in tumour suppression[END_REF]. It means that they can either promote tumor growth or suppress tumor depending upon the environment. However, more clinical investigations must be performed to have a complete and accurate picture of the situation in vivo.

Conflict of interest

The authors confirm that there are no known conflicts of interest associated with the content of this article.

Figure 1 .

 1 Figure 1.Schematic representation of the revised papain model showing different subsites based on substrate-mimicking inhibitors bonded to the active-site cleft. Reproduced with permission from Ref. [5].

Figure 2 .

 2 Figure 2. Structure of different cathepsin-sensitive drug-linker bioconjugates along with their indicated cleavable sites.

Figure 3 .

 3 Figure 3. Illustration of GFLG-containing PHPMA prodrugs. Adapted with permission from ref. [87].

Figure 7 .

 7 Figure 7. Structure of Dox-loaded, PEGylated, enzyme-sensitive, macrocyclic pillar[5]arene amphiphiles and their self-assembly into micelles. Adapted with permission from Ref. [101].

Figure 8 .

 8 Figure 8. (A)Mesoporous silica nanoparticle (MSN) functionalization. (B) Cathepsin-sensitive Dox-loaded MSNs. (C) Cell integrin receptor-mediated targeting by RGDS (Arg-Gly-Asp-Ser). (D) Endocytosis. (E) Drug release mediated by Cathepsin B. (F) Tumor cell death. Adapted with permission from Ref. [112].

  Figure 9. Synthesis of gatekeeper-supported functionalization with 3-(azidopropyl)triethoxysilane capped with peptide sequence and further delivery of Doxby action of Cathepsin B. Adapted with permission from Ref. [113].

Figure 12 .

 12 Figure 12. Synthesis of amphiphilic PEGylated dendron-GFLG-Dox conjugate followed by its self-assembly into NPs. Adapted with permission from Ref. [121].

  cells. Dox was then released into lysosomes via selective cleavage by Cathepsin B. Enhanced cytotoxicity compared to that of free Dox was obtained on HeLa cells that are known to express Cathepsin B.

Figure13.

  Figure13. Schematic representation and proposed action mechanism of a sorbitol scaffold functionalized by octa-guanidine moieties and conjugated to Dox. Adapted with permission from Ref.[START_REF] Maniganda | A lysosome-targeted drug delivery system based on sorbitol backbone towards efficient cancer therapy[END_REF].

Figure14.

  Figure14. Schematic representation showing conjugation of the lipidated Cathepsin B inhibitor (NS-629) at the surface of a liposome to target extracellular (EC) CathepsinB. Adapted with permission from Ref. [127].

Figure 15 .

 15 Figure 15. Schematic illustration of the in vivo tracking of the degraded Cathepsin B-labile dipeptide bond linker exploiting FRET with fluorescein amidate (FAM) and dual-labeled rhodamine B (Rh) NucA-Ptx bioconjugate. Adapted with permission from Ref. [139].

  [START_REF] Zheng | Peptide-mediated core/satellite/shell multifunctional nanovehicles for precise imaging of cathepsin B activity and dual-enzyme controlled drug release[END_REF]. The peptide-based satellite/shell structures secured Dox inside the nanoparticles thus acting as three-dimensional gatekeepers and Dox release subsequently occurred upon incubation with Cathepsin B.

Figure 19 .

 19 Figure 19. Illustration of Dox-loaded, hollow mesoporous silica nanoparticles for in situ imaging of Cathepsin B and protease-mediated Dox release. (a) Nanoparticle synthesis. (b) Nanoparticle

Figure 20 .

 20 Figure 20. (a) Sentization of endo-lysosomal membrane in the presence of dye released by enzymatic digestion of the nanoparticles. (b) Endo-lysosomal disruption by NIR laser leading to saporinrelease. Adapted with permission from Ref. [170].

Figure 21 .

 21 Figure 21. Structure of dual-functionalized PHPMA nanocarriers with Cy5 and Cy7 dyes for further Cathepsin B-mediated release of Cy7. Adapted with permission from Ref. [171].

Figure22.

  Figure22. Schematic structure of PHPMA functionalized by Cy5 and EPI for further FRETmonitoring of the EPI release mediated by Cathepsin B. Adapted with permission from Ref. [172].

  targeted imaging. A fluorescent molecule, DCPO (dicyanomethylene-4H-pyran), was released by Cathepsin B, leading to in vitro imaging Cathepsins of various tumor cells during incubation with different cell lines.

  probe was successfully delivered into tumor cells after nanoparticle accumulation and exhibited fluorescent signals inside the cytosol in presence of Cathepsin B. It thus showed increased potential for the optical detection of biological activities especially related to tumor growth or metastasis (Figure 25).

Figure25.

  Figure25. Cathepsin B-sensitive nanoparticulate probes and tumor diagnosisin vivo. Adapted with permission from Ref.[START_REF] Ryu | Cathepsin B-sensitive nanoprobe for in vivo tumor diagnosis[END_REF].

Table 1 .

 1 List of Cathepsin Overexpressing Cancer Types.

	Family	Cathepsin	Location	Tumour site	Reference
		General	Intracellular, lysosomes	Most	[42-44]
		Cathepsin K	Extracellular	Breast, bone	[45-49]
				Breast, cervix,
	Cysteine Proteases	Cathepsin B	Extracellular and pericellular conditions pathological under	colon, colorectal, gastric, head and pancreatic, ovarian, melanoma, neck, liver, lung,	[50-61]
				prostate, thyroid
		Cathepsin L		Breast, colorectal [62-65]
	Aspartic		Endosomal	Cervical, gastric,
	Proteases	Cathepsin E	structures, ER,	lung, pancreas	[61, 66-70]
			Golgi bodies	adenocarcinomas

Table 2 .

 2 List of Cathepsin B-Cleavable Prodrugs Evaluated inClinical Trials.

						Clinical
	Entry	Name	Composition	Spacer	Linker	Trial	Company	Refs
						status
							Pfizer
	1	PK1; FCE28068	PHPMA copolymer-Doxorubicine	Gly-Phe-Leu-Gly	Amide	Phase II (discontinu ed)	Inc., Cancer Campaign Research	[196, 225]
							, UK
							Pfizer
	2	PK2; FCE28069	PHPMA-Dox-Galatosamine	Gly-Phe-Leu-Gly	Amide	Phase I/II(disconti nued)	Inc., Cancer Campaign Research	[226, 227]
							, UK
	3	PNU166945/H PMA-Ptx	PHPMA copolymer-Paclitaxel	Gly-Phe-Leu-Gly	Ester	Phase ed) I(discontinu	Pharmacia	[206]
	4	PNU166148/H PMA-CPT CPT/MAG-	PHPMA Camptothecin copolymer-	Glycine residue/ spacer exanoyl Glycylaminoh	Ester	Phase ed) I(discontinu	Pharmacia	[207]
	5	CT-2103/PGA-Ptx-XYOTAXTM/ OPAXIO®	Polyglutamate-Paclitaxel	L-glutamic acid	Ester	Phase II/III	Cell Therapeut ics Inc.	[199-205]
	6	CT-2106/PGA-CPT	Polyglutamate-Camptothecin	L-glutamic acid	Ester	Phase II discontinue d	Cell Therapeut ics Inc.	[228-230]
	7	AP5280	PHPMA copolymer-Carboplatinate	Gly-Phe-Leu-Gly	Aminoma lonate	Phase I	Access Inc. Pharmace uticals	[208, 209]
	8	AP5346/ ProlindacTM	PHPMA copolymer-DACH Oxiplatinate	GGG-carboxylate-Pt coordination	Aminoma lonate	Phase II	Access Inc. Pharmace uticals	[210]
	9	EZ-246/PEG-CPT/Pegamote can/ProthecanT M	PEG-Camptothecin	Glycine	Ester	Phase II discontinue d	Enzon Pharmace Inc. uticals,	[216, 217]
	10	NK911	PEG-aspartic acid-Doxorubicinmi celle	Aspartic acid	Amide	Phase II	National Cancer Institute Japan	[97, 218, 219]
	11	P-Dox	PHPMA copolymer-	Gly-Phe-Leu-Gly	Amide	Preclinical	-	[211, 212]

Acknowledgement

D.D. thank to Indo French Centre for Promotion of Advanced Research (IFCPAR/CEFIPRA) for Raman Charpak Fellowship 2017 and CSIR-New Delhi, India for her research fellowship. This research work was financially supported by DST-EMR/2016/002304 and DST-EE2/2016/000102 and IIIM communication no. IIIM/2244/2018. CNRS and Université Paris-Sud are also acknowledged for financial support.