Anticancer nanoparticulate polymer‐drug conjugate

Abstract We review recent progress in polymer‐drug conjugate for cancer nanomedicine. Polymer‐drug conjugates, including the nanoparticle prepared from these conjugates, are designed to release drug in tumor tissues or cells in order to improve drugs’ therapeutic efficacy. We summarize general design principles for the polymer‐drug conjugate, including the synthetic strategies, the design of the chemical linkers between the drug and polymer in the conjugate, and the in vivo drug delivery barriers for polymer‐drug conjugates. Several new strategies, such as the synthesis of polymer‐drug conjugates and supramolecular‐drug conjugates, the use of stimulus‐responsive delivery, and triggering the change of the nanoparticle physiochemical properties to over delivery barriers, are also highlighted.


| Stimuli-sensitive PDC
Although it is suggested that the EPR effect exist in human tumors, 46,47 it is still questionable whether the EPR effect is sufficient to significantly improve the survival of cancer patients by nanomedicine. 48,49 Several delivery barriers limit the transport of NPs deep into tumors 4,50 ; (see Section 2.4) recent advances in biology show that abnormal tumor microenvironments help tumor progress and resist the

| Polymer
Polymers that have functional groups for the incorporation and release of drugs in PDCs must be well characterized ( Figure 1a). All in vivo metabolic products of PDCs should be nontoxic and nonantigenic.

| Block copolymer's composition
The relative ratio of the hydrophobic to hydrophilic block length profoundly affects the NP's morphology. [81][82][83] Typically the morphology of prepared amphiphilic block copolymer NP is spherical, particularly if the molecular weight of the hydrophilic block exceeds that of the hydrophobic block (so-called star micelles). However, if the copolymer is asymmetric in its relative block lengths (i.e., the hydrophobic block is considerably longer than the hydrophilic block) during the selfassembly process, varying morphologies can be obtained. 84,85 In addition, the copolymer's concentration in water-miscible solvent affects the final NPs' size. 86 The use of triblock polymers could improve the NP's stability. 54 Nevertheless, there lacks systemic studies on the block copolymer ratio or composition on the in vivo circulation and stability of the NPs' morphology, presumably due to the technical difficulty to monitor the sub-100 nm polymeric NPs in vivo. Recent in vivo pharmacokinetic studies using dual-radiolabeling of lipid and drug in liposomes could provide a valuable example for the study the biodistribution of copolymer-based PDCs in vivo. 87 Notably, recent studies have shown that the zwitterionic copolymers (i.e., polymers containing both cationic and anionic groups) are super-hydrophilic, which can prolong the circulation of NPs in the way similar to PEG. 88

| Synthetic strategy of PDC
There are three strategies for drug-polymer conjugates ( Figure 3). 89 The first is conjugating a drug to a pre-synthesized polymer, named as "conjugation to." The second is to conjugate a drug to a monomer prior to polymerization, namely "conjugation through." The last is the polymerization of drugs to prepare PDCs, where drugs are directly used in the polymerization as monomers or initiator. 90 The last two strategies have been recently developed to prepare PDCs, aiming to overcome the non-controlled drug conjugation problem in the "conjugation to" strategy (see Sections 3.1 and 3.2).

| Drug release and the conjugation linker
An ideal PDC for cancer treatment should be able to release the drug in tumor tissues or cells, but not to the normal tissues or cells. Two types of linker can be positioned between drug and polymer: cleavable linker and non-cleavable linker. Non-cleavable linkers, such as thioether linkers, have been seen in the antibody-drug conjugates (ADCs). 91 The release of the drug from these ADCs requires complete hydrolysis of the polypeptide backbone of the antibody in cell lysosomes. 92 One example is T-DM1 (Kadcyla TM ), an ADC to treat metastatic breast cancer, which has the thioether linker, and exhibited better antitumor efficacy than the same ADC but with disulfide linker. 93 However, the use of non-cleavable linker of degrading the delivery platform after cell uptake might not be feasible in more complex NP systems.
Enormous synthetic efforts have been devoted to design stimulussensitive cleavable linkers to trigger drug release ( Figure 4). During the NP's extravasation, local tumor microenvironmental factors, such as pH (6.7-7.0), 94 redox state (hypoxic tumor microenvironment 95 and elevated reactive oxygen species generated by tumor cells 96 ) and specific molecules overexpressed in tumor (e.g., matrix metalloproteinases 97 can be utilized to disrupt PDCs' structures to release loaded drugs, or induce NPs size or morphology change for enhanced penetration (see Section 3.5). Besides the endogenous stimuli, the external stimuli-such as magnetic field, temperature, light, and ultrasound-can be applied in a spatiotemporal manner to control drug release. [98][99][100][101][102] More importantly, cleavable linkers have to result in direct release of the drug from the remaining linker fragment upon the cleavage, that is, no prodrug released. For systemically delivered PDCs, these linkers should be stable in circulation to avoid the side effects from the free drug and/or the decreased drug accumulation in tumors. We mainly discuss each type of stimulus-sensitive linker, focusing on the general chemistry, in vivo stability and some preclinical successful examples.

| Redox sensitive linker
The difference in redox potential between normal and tumor tissues, and between the intracellular and extracellular environment, can be exploited for triggered drug delivery. 115 In the nanomedicine field, it is generally believed that the concentration of glutathione, a reducing tripeptide with thiol group, in cancer cells is 100-to 1,000-fold higher than in the blood, and in a tumor mass the glutathione concentration is also markedly (100fold) higher than the extracellular level of glutathione in normal tissue. 116 However, studies showed that in mice model a total fourfold higher level of glutathione in tumor tissues compared with normal tissues, and there exists significant heterogeneity of redox status in the tumor tissue. 117 In human cancer patients, glutathione levels tend to be elevated in breast, ovarian, head and neck, and lung cancers compared with disease-free peritumoral or healthy tissue; conversely, brain and liver tumors patients exhibit lower tissue level of glutathione in tumor compared with that in healthy tissue. 118 In addition, two studies concluded that glutathione levels did not differ between parenchymal tissue sampled from healthy patients and uninvolved parenchymal tissue from lungs with tumors. 119,120 Therefore, right preclinical models and tumor types have to be rationally chosen when applying redox-sensitive PDCs.
The reducing materials in vivo could facilitate the cleavage of redox-sensitive bonds such as disulfide bond and diselenide bond. 121 For example Kopeček  1 day in ADCs, respectively, which is much shorter than the parent antibody moieties. 108 The in vivo circulation stability PDCs containing redox-sensitive linker should be evaluated carefully in future.

| Enzyme sensitive linker
The increased expression of certain local enzymes in cancer, such as MMP, not only can be regarded as a biomarker for disease diagnosis and prognosis, but also represents a means for enzyme-triggered drug release in tumor. [123][124][125][126] Early studies on detailed degradation studies of oligopeptide sequences attached to polyHPMA-based PDCs identified the short peptide GFLG, specific for cathepsin B. 127 The poly-HPMA PDCs with such linker have shown efficacy in various preclinical efficacy study and have entered the clinical trials. 128,129 Another widely used short peptide linker is citrulline-valine, which can be cleaved by specific lysosomal proteases but impart greater stability in plasma. Of note Brentuximab vedotin (Adcetris TM approved for the use in Hodgkin lymphoma) is an ADC containing such dipeptide linker to facilitate release of the drug, monomethylauristatin E. 130 Other short peptides linkers include PVGLIG (cleaved by MMP-2/MMP-9), 131 and SSKYQL (cleaved by prostate-specific antigen). 132 One can envision that the presence of certain enzymes as biomarkers potentially could be utilized to design a PDC for personalized medicine, once the concentration of enzymes at the tumor site should also be sufficient for the disruption of the PDC.

| Light sensitive linker
There has recently been growing interest in light-responsive NPs for triggered drug delivery. The use of an optical stimulus is appealing because it could provide a greater selectivity in terms of control over the moment and the location of drug release, and potentially transfer photonic energy to heat, acoustic wave, or induce reactive species such as singlet oxygen in photodynamic therapy. 133 In terms of light-  Figure 4). 134 However, many photocaging groups required the irradiation by UV light or short wavelength visible light, which restricted primarily to superficial lesions unless fiberoptics or near-infrared (NIR) light can be used. Of note, NIR light, with wavelengths in the range of about 700-1000 nm, is more suitable for biomedical applications than UV or visible light; the irradiation is less detrimental to healthy cells, and the absorption and scattering by water and biological substances are reduced, which results in a greater tissue penetration depth (on the order of millimeters to centimeters). [135][136][137] One way to use NIR light is to use two-photon excitation for many UV-light absorbing photosensitive linkers. 138 (Figure 4). 142 151 In addition, the NP should not bind with proteins in blood that could lead to aggregation, or be uptaken by the macrophages in the mononuclear phagocytic system, all of which can lower the dose of NPs reaching tumors. 152 Coating of NPs with PEG that mimics a cell's glococalyx, 153-155 known as "PEGylation," can suppress protein absorption to NPs and delay the rate of NP uptake and clearance, greatly prolonging circulation time. 156 The NP's circulation half-life is impacted by the extent of PEGylation on NPs surface, 157,158 and may be reduced upon repetitive administration, which has been reviewed elsewhere. 159 163 and NP's cell uptake. 164 Other strategies of improving NP's tumor penetration include coinjecting drugs to reduce tumor's extracellular matrix density, 165,166 and conjugating tumor-homing or tumor penetration ligands. 167,168

| Tumor cell uptake
After reaching the tumor cells, NPs may need to cross the barrier of the cell membrane to deliver the loaded drugs into specific organelles to achieve efficacy. The surface modification of NPs with cell targeting ligands, 169 cell penetration peptides, 170 or lysosome-destabilizing agents 171 can greatly enhance intracellular uptake. Generally cancer cells may contain certain receptors or targets, such as transferrin, EGFR/HER-2, PSMA, VCAM, that can mediate the corresponding enhanced cellular uptake of targeted NPs. 172 Of note, the use of targeting ligands can enhance NPs' cellular uptake but not necessarily increase the tumor accumulation of NPs when compared with EPR-mediated accumulation. [173][174][175] Conversely, the introduction of targeting ligands onto NPs not only requests synthetic efforts but also sometimes compromises the prolonged circulation of PEGylated NPs. 176,177 The surface density of targeting ligands should be closely monitored to provide a desirable targeting effect without reducing NP's circulation or tumor penetration capability.
For NPs without targeting ligands, intracellular NPs are found mainly within endosomes or lysosomes. These organelles have acidic pH, and contain proteases for degradation. The rate of uptake and intracellular localization of NPs have been studied by many research groups. [178][179][180] Currently it is difficult to draw general conclusions about optimal physicochemical properties of NPs for rapid cellular uptake, since the rate and mechanism of uptake are cell-type dependent and could vary between NPs with different size, charge, and other surface properties. However, some reports show that NPs of 20-50 nm are taken up more rapidly than smaller or larger NPs. 178,181 For hydrophilic polymer based PDCs, it is found that some PDCs, such as polypeptides 182 or dextran, 183 cannot be naturally degraded into small fragments that can cross the lysosomal membrane; the accumulated polymers in the lysosome increase the osmotic pressure and adversely affect the biocompatibility. 184 Another study shows that most polyHPMA-based PDCs quickly and evenly diffuse throughout the cytoplasm and remain excluded from membrane-bound organelles; only strongly cationic HPMA copolymers can bound to microtubules; the nuclear entry kinetics were affected by the ratio of the HPMA to comonomer compositions. 185

| N E W ST R AT E G I E S I N P DC
The purpose of this section is to highlight some novel ways in which chemistry and nanotechnology are being applied to tackle challenges in PDC development.

| "Conjugation through" PDC
The "conjugation through" method in PDCs requires monomer-drug conjugates not interfere the polymerization. 89 The "conjugation through" method could address the drawbacks in the "conjugation to" strategy, such as inconsistent and uncontrolled site conjugation along the polymer backbone. 89 The drug loading can be controlled by adjusting the feed ratio of monomer-drug conjugates; and the drug release can be controlled by the judicious selection of the linker between the drug and the monomer, which could be stimulus-responsive ( Figure   3b). 186 Such method thus allows for even higher drug-loadings than the "conjugation to" approach, by avoiding steric hindrance and accessibility limitation during the conjugation.
A few of monomer-drug conjugates have been synthesized to prepare PDCs and corresponding NPs. Ring-opening metathesis polymerization (ROMP) is often unitized in the "conjugation through" strategy.

| Polymerizable drug
The use of drug as the monomer could significantly increase the drug loading. However, not many drug molecules fit for such strategy.
Often, drug molecules contain two functional groups that allow for pol- Similar 10-hydroxycamptothecin-loaded polycarbonate caged with redox disulfide linker was also reported. 196

| Photosensitizer conjugate
Photodynamic therapy is a photochemistry-based approach for treating tumors or other diseases such as macular degeneration. It involves the administration of nontoxic dyes known as photosensitizers systemically or topically, followed by illumination of the lesion with visible or NIR light, 197 and then photosensitizers generate cytotoxic oxygen species (either singlet oxygen or oxygen radicals). 198 Most photosensitizers bind to normal cells as well as to cancer cells, leading to unwanted offtarget activation from environmental (ambient) light. [199][200][201][202] The conjugation of photosensitizer to polymeric delivery vehicles is designed to improve photosensitizer's performance by increasing specificity and/or uptake in tumors, or decreasing phototoxicity to normal tissue. 203,204 Early photosensitizer-drug conjugates include polyHPMA, PEG and antibody conjugates. [205][206][207][208] Factors such as the charge and hydrodynamic size of the conjugates affect the cellular uptake rate and tumor accumulation of hydrophilic polymer-photosensitizer conjugates. 209,210 In many cases the covalent linkage between photosensitizer and polymer significantly reduced the quantum yield. 211 The enzymaticcleavable linker, or environmental-sensitive linker, was introduced to enhance both the selectivity of photosensitizer and the quantum yield; The use of lipid-photosensitizer conjugates to formulate lightsensitive liposomes that combine photothermal therapy with chemotherapy has recently garnered interest. Photothermal therapy may potentially improve the chemotherapy efficacy of polymeric NPs containing drugs. 215 For example, nanoliposomes composed of lipid conjugates of pyropheophorbide (a chlorin analogue) can efficiently absorb and transfer light energy into heat for photothermal therapy, as well as release the loaded drugs inside liposome. 216 Of note, the use of another porphyrin-lipid conjugates could also induce the transient increased permeability of the nanoliposome upon NIR light triggering; its mechanism remains unknown but not due to the photothermal effect. 217 accumulate in the locally heated tumor region, which was confirmed by intravital fluorescence microscopy. 226 The use of acid-or redoxsensitive linkers in ELP-drug conjugates allowed for the intratumoral drug release. 227,228  An alternative delivery approach was proposed to use relatively larger NPs with initial size (still sub-100 nm NPs), but once docking at tumor sites, NPs were switchable to small particles to facilitate tumor penetration. 233 The stimuli-responsive NPs that are able to shrink their sizes by responding to enzymes or light exhibited the enhanced tumor penetration of NPs and improved efficacy. 234,235 Recently, a new pH sensitive NP was prepared by poly(caprolactone)-co-poly(amidoamine)

| Surface charge
Positively charged NPs often have short circulation half-life compared with PEGylated or anionic NPs. [236][237][238][239] However cationic NPs may penetrate tumors deeper than neutral or anionic NPs due to the attractive electrostatic forces between cationic NPs and anionic endothelial glycocalyx. 240 Positively charged NPs also generally have better cellular uptake than negatively ones. 241,242 A pH-sensitive PDC-based NP was designed to achieve multi-stage charge changing to improve delivery efficiency: NP's surface charge maintained slightly anionic at pH 7.4; in tumor tissues with pH $ 6.8, the pH-sensitive cis-aconityl group was cleaved from the surface and expose the positive amine groups, which It is known that amphiphlic or lipid-like molecules could potentially self-assemble into supramolecular nanostructures. 247 Taking advantages of the self-assembly properties of these small molecules, an amphiphilic prodrug conjugate was synthesized by conjugating two hydrophobic Cpt molecules to a short oligo(ethylene glycol) as the hydrophilic segment via a biodegradable b-thioester bond. Such amphiphilic prodrug conjugates have high drug loading and form stable 100 nm nanoliposome (Figure 8a). 248 Similar approach was applied to synthesize amphiphilic PEG-block-dendritic polylysine-CPT conjugate that could assemble to nanorod. 249 Another reported strategy is to con-jugate hydrophobic squalene to hydrophilic drugs or prodrugs to construct NPs. 250 Doxo, gemcitabine and other drugs were "squalenoylated" and formulated into $100-150 nm sized NPs ( Figure   8b). 251 An extreme strategy is recently reported to synthesize an amphiphilic drug-drug conjugate by directly connecting the hydrophilic anticancer drug irinotecan to the hydrophobic anticancer drug chlorambucil via a hydrolyzable ester linker, which can be assembled to NPs with $80 nm size (Figure 8c). 252 Similar conjugate was synthesized between the hydrophilic drug floxuridine with the hydrophobic drug bendamustine. 253 Besides amphiphilic molecules or lipids, another interesting molecule to prepare prodrug conjugates is the small peptide. It is known that small peptides can assemble into filamentous supramolecular structures. 254 Such peptide-drugs conjugates have been intensively studied to formulate hydrogel system, and are reviewed elsewhere. 250,[255][256][257][258] A recent study demonstrate that a rationally designed peptide-Cpt conjugate be formulated to nanostructures for drug delivery. A b-sheet-forming peptide sequence derived from the tau protein was conjugated to Cpt via a redox-sensitive disulfylbutyrate linker, and the resulting nanostructures could vary from long filaments to short filaments and then to nanotubes with high drug loadings (Figure 8d). [259][260][261] Studies also showed the choice of the degradable linker between the peptide and Cpt affect the nanostructure. The carbonate linker is more preferred than ester since it minimizes the potential aggregation in cell culture, which could compromise Cpt's potency. 262

| O U T L O O K
The routine clinical use of PEGylated proteins since 1990s and the recent large investments in ADCs overshadow the development of PDCs. Although so many interesting designs and impressive data presented in this review, there seems a long and arduous journey to bring more PDCs or NPs into clinical practice. 129,263 Several recent publications try to provide their solutions for the whole nanomedicine field. [264][265][266] Progress in the field will depend on a fundamental understanding of chemistry, materials science, biology, and clinical practice to allow rational design of optimized NPs of PDCs, tools for delivering them and measure outcomes. In terms of chemistry design, one has to bear in mind the clinical application and whole-organism pharmacokinetics. One example is the in vivo studies in ADCs revealed an in vitro stable linker may have unexpected instability in vivo and cause reduced efficacy. 267,268 Many of the first generation PDCs were developed before the concept of nanomedicine and the study of the relationship between NPs' sizes and their in vivo circulation and intratumoral accumulation; thus such PDCs have moderate molecular weight and small particle sizes (Table 1), which may result in some of the failure in clinical trials. In addition, there lack standard or optimized preclinical or clinical protocols to evaluate PDCs' stability, tumor penetration, metabolism, and toxicity. 269 The development of labeling/imaging technique and nano-device system may help monitor the in vivo use of PDCs. Of all note that many data obtained in animal models cannot be FIG URE 8 Four representative supramolecular drug conjugates that can assemble into nanoparticle for drug delivery easily translated into humans; in the frequent-used subcutaneous tumor xenografts the access of the blood to the tumor interstitium is greater than that in solid tumors in patients. 270,271 Furthermore, the advances in cancer biology can change the landscape of the field rapidly, as seen in the recent promising therapeutics in cancer immunology. 272,273 Last but not least, nontrivial optimization and engineering has a bearing on the eventual translation of NPs from preclinical experimental models to daily clinical practice. 274 The PDC and NP preparation should not require complex multistep processes; the scalability of NPs should not represent a problem in industry; the NP product should be sufficiently stable under storage and easy to use in clinics, that is, no complex administration protocols or regimens. 172,275 All of these prudent considerations will ensure that the field of PDC-based NPs reaches its full potential for clinical impact in cancer therapy.

CONFLICT OF INTERESTS
The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.