POLYMER NANOMEDICINES

Abstract

Polymer nanomedicines (macromolecular therapeutics, polymer-drug conjugates, drug-free macromolecular therapeutics) are a group of biologically active compounds that are characterized by their large molecular weight. This review focuses on bioconjugates of water-soluble macromolecules with low molecular weight drugs and selected proteins. After analyzing the design principles, different structures of polymer carriers are discussed followed by the examination of the efficacy of the conjugates in animal models and challenges for their translation into the clinic. Two innovative directions in macromolecular therapeutics that depend on receptor crosslinking are highlighted: a) Combination chemotherapy of backbone degradable polymer-drug conjugates with immune checkpoint blockade by multivalent polymer peptide antagonists; and b) Drug-free macromolecular therapeutics, a new paradigm in drug delivery.

1. Introduction

There is no distinct classification of the term macromolecular therapeutics in the literature, but we shall focus on water-soluble polymer – drug conjugates and on macromolecular systems that hold therapeutic activity and do not contain low molecular weight drugs. Some reports in the literature use the term „nanoparticles“ for both vesicular carriers and water soluble macromolecular therapeutics. However, there is a clear distinction in size, shape, and flexibility of the two types of carriers.

The research on water-soluble macromolecules for drug delivery started more than 60 years ago. Jatzkewitz used a glycylglycine spacer to bind mescaline to poly(vinylpyrrolidone) (PVP) [1], Ushakov’s group conjugated several antibiotics to PVP and studied biological properties of the conjugates [2–4]. De Duve discovered the lysosomotropism of macromolecules [5] and Ringsdorf published a first comprehensive model of the potential of water-soluble polymers as drug carriers [6]. Maeda revealed the enhanced permeability and retention (EPR) effect – the enhanced deposition of macromolecules in solid tumors [7]. The early research of polymer-drug conjugates was reviewed in 1977 [8]. The interdisciplinary field developed dramatically due to the advantages of polymer-drug conjugates vs. free drugs [9]. The possibility to manipulate pharmacokinetics, biorecognition, distribution and subcellular location, as well as therapeutic efficacy was the driving force.

Numerous reviews on macromolecular therapeutics have been published [10–31]. This review focuses on the design and structure of macromolecular drug carriers, important research achievements that are prerequisites for future clinical translation and novel directions, including combination chemotherapy and immunotherapy and drug-free macromolecular therapeutics.

2. Design of macromolecular therapeutics

The major rationale for the use of water-soluble polymers as drug carriers is the potential to improve the efficacy of low molecular weight drugs. One of the main distinction of conjugates vs. free drugs is the different mechanism of cell entry [5,32]. Macromolecular therapeutics are composed from a suitable polymer carrier, spacer between the macromolecular carrier and drug, and optionally a targeting moiety [21]. Synthetic nondegradable macromolecules need to have the whole molecular weight below the renal threshold to ensure the elimination of the carrier by glomerular filtration [21,28]. Biodegradable carriers such as poly(amino acids) or polysaccharides need to preserve their degradability after covalently binding drugs. The structure modification (by drug attachment) impairs the formation of the enzyme-substrate complex resulting in decreased degradability [33,34]. When the major degradation mechanism is pure hydrolysis, the substitution of the degradable chain may not substantially impair degradability as in conjugates based on poly(β,L-malic acid) [35,36].

In addition to molecular weight, structure of the carrier, architecture, mechanism of drug release, and subcellular location impact the efficacy of polymer-drug conjugates.

2.1. Spacers

Spacers used in attachment of drugs to synthetic polymer carriers need to remain attached to the carrier in the blood stream but are cleaved off the carrier by enzymes in the lysosomal compartment [37–40]. Otherwise, the drug can be bound to the carrier via a hydrolytically cleavable bond to be released in late endosomes and lysosomes due to decreased pH in these subcellular compartments [41].

Self-immolative spacers are elongated spacers where the enzymatically sensitive bond is separated from the drug by a self-eliminating group. Rate controlling enzymatic cleavage is followed by fast 1,6-elimination as demonstrated in the design of cathepsin K sensitive spacers for the release of prostaglandin in osteoclasts [42]. Garrison and coworkers designed cathepsin S degradable spacers [43–45]. Santi and coworkers developed non-enzymatically cleavable linkers where the rate of release based on β-elimination reaction can be manipulated in a wide range [46,47]. An interesting design for self-immolative spacers is a combination of aromatic azobonds with a 1,6-elimination unit suitable for colon delivery [48]. Following azobond reduction in the colon the drug (9-aminocamptothecin) is released in addition to p-aminobenzylalcohol and CO₂ [49].

Notably, FDA approved recently ENHERTU® (trastuzumab deruxtecan) an antibody drug conjugate containing a tetrapeptide spacer GGFG, validating the concept of cathepsin B sensitive peptide spacers [50].

Spacer length is important for drug attachment. Proteases responsible for the cleavage of oligopeptide side chains terminated in drug have considerable large active sites which are capable of combining with a number of amino acid residues [51]. Generally, four amino acid residues toward the NH₂ end of the substrate (i.e., toward the polymer backbone) take part in the interactions that determine the formation of the enzyme-substrate complex and ultimately the drug release [37,38]. Another factor is the steric hindrance of the polymer chain on the formation of enzyme-substrate complex. An oligopeptide attached to the end of the polymer chains accommodates into the enzyme active site easier than the same sequence attached to the polymer as a side chain [52].

Spacer length can be important also in the attachment of targeting moieties. In a prostate cancer model, DUPA (3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid) attached to polymer carrier via a longer (dodecaethyelene glycol, 46 Å) spacer reaches easier the PSMA (prostate-specific membrane antigen) binding site via a ~ 20 Å funnel shaped tunnel than conjugates with a short spacer [53].

Coiled-coil forming peptides were used for stable non-covalent attachment of drugs [54,55] or recombinant scFv fragments [56,57] to polymeric carriers. A triheptad or tetraheptad peptide was attached to the polymeric carrier. The complementary peptide sequence is terminated in drug or scFv fragment. Decreased pH in the lysosomes causes the detachment of peptide and release of the modified drug.

2.2. Molecular weight of synthetic polymer carriers

Macromolecules accumulate into solid tumors by the EPR effect (see 3.1). The efficacy of the EPR process is molecular weight dependent – deposition of conjugates in solid tumor increases is directly proportional to molecular weight [58,59]. However, to ensure biocompatibility, the molecular weight of non-degradable synthetic macromolecules need to be below the renal threshold (about 50 kDa for random coil conformation of hydrophilic macromolecules). Consequently, first HPMA copolymer – doxorubicin (DOX) conjugates evaluated in clinical trials had a molecular weight of 28 kDa [60–62]. To keep polymer carriers biocompatible and long-circulating, degradable bonds can be inserted into the polymer backbone as described in 2.4.1.2.

2.3. Architecture

Molecular architecture (Figure 1) can exert an important effect on the biological properties of polymer-drug conjugates [25,63]. Several studies have described advanced methods for the synthesis of linear, star, graft, and branched copolymers [64,65] and evaluated the impact of architecture on the rate of drug release, IC50 doses, biodistribution and in vivo efficacy [66,67]. Star-like N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers can be synthesized by conjugating semitelechelic HPMA copolymer-drug conjugates to different generation (G2, G3, G4) PAMAM dendrimers [68].

Figure 1.

Different architectures of polymer conjugates. Adapted from ref. [25].

The conclusions of comparative studies are frequently difficult to generalize because the activity of conjugates depends on several factors, including molecular weight, architecture, hydrodynamic volume, type of bond used for drug attachment (enzymatically or hydrolytically cleavable), and rate of cellular uptake. Frequently, conjugates that were compared (e.g., linear vs. star) have different molecular weight as well as different size. For example, Nakamura et al. compared star-like PAMAM dendrimer-based HPMA copolymer-pirarubicin conjugate (400 kDa, 26 nm) with linear HPMA copolymer-pirarubicin conjugate (39 kDa, 8.2 nm; hydrazone bond used in both conjugates) [69]. The tumor growth inhibitory activity of the star-like conjugate was higher than that of the linear one in S-180 tumor-bearing mice. However, due to the higher molecular weight of star-like conjugate it is difficult to distinguish the impact of architecture from the molecular weight dependent accumulation in solid tumors [69]. Šírová et al. evaluated the efficacy of combination treatment of murine EL4 lymphoma using linear HPMA copolymer-doxorubicine (DOX) and -docetaxel (DTX) conjugates (28 kDa) as well as star DOX and DTX conjugates (250 kDa; all hydrolytically cleavable bonds). Whereas the combination of linear conjugates resulted in additive treatment effects, the star-like DTX conjugate decreased the treatment efficacy of the star DOX conjugate [70]. Sadekar et al. compared biodistribution of PAMAM dendrimers and linear HPMA copolymer (29 kDa) in tumor bearing mice using ¹²⁵I labeled macromolecules. Hydroxyl terminated generation 5, 6, and 7 dendrimers were taken up by the liver and kidney more than the HPMA copolymer that was mainly excreted into the urine [71].

Architecture impacts the cellular internalization – linear HPMA copolymer-meso-tetra(4-carboxyphenyl)porphyrin) (MTCP) conjugate possessed higher internalization rates as well as light induced cytotoxicity than MCTP attached to hyperbranched amine terminated PAMAM dendrimer [72].

Generally, one can assume if the drug is bound to the polymer carrier via a hydrolytically cleavable bond the rate of drug release (and corresponding activity) will not be substantially different when comparing linear and hyperbranched structures. However, for enzymatically cleavable spacers complex architectures may result in impairing the formation of the enzyme-substrate complex, and slower drug release with concomitant decrease in activity.

Structural factors have an important impact on the efficacy of polymer-drug conjugates. Different chemical structures of polymer backbones are discussed below.

2.4. Structure of polymer carrier

Different structures have been used as drug carriers. The factors important in the selection of a particular macromolecular structure are hydrophilicity (to keep the conjugate soluble after covalent attachment of hydrophobic drugs), biodegradability (to achieve long intravascular half-life) and biocompatibility (appropriate host response). The main polymer carriers are discussed in the following paragraphs.

2.4.1. HPMA copolymers

2.4.1.1. First generation of HPMA copolymer carriers

Before using polyHPMA [73,74] as drug carrier, the biocompatibility of the homopolymer was established. Early studies revealed that polyHPMA (working name Duxon) was nontoxic, apyrogenic, did not exhibit any effect on LEP and HeLa cells cultures, and did not initiate formation of antibodies in mice [75,76]. ¹⁴C-Labeled polyHPMA (Mw=28,000) was eliminated from the organism of rabbits following intravenous administration [77]. PolyHPMA prolonged the survival of semiallogeneic skin grafts in mice and rats and did not inhibit the growth of lymphocytes in tissue culture [78,79]. Additionally, no effect of polyHPMA on the activation of porcine complement was observed [80]. Interestingly, a recent study on intracellular polymerization of HPMA in HeLa cells mentioned remarkable biocompatibility up to 250 mM of HPMA inside the cells [81].

Studies of HPMA copolymer-drug conjugates were reviewed numerous times [8–10,13,18,23,24,26,28,31]. Just briefly, the first generation of HPMA copolymer-drug conjugates with nondegradable backbone was active on numerous animal models of cancer [82]. The drugs were attached to the polymer backbone via lysosomally degradable oligopeptide spacers [37–39,42] or hydrolytically cleavable (e.g., hydrazone) bonds [41]. Several conjugates were evaluated in clinical trials [60–62,83–86]; the results clearly demonstrated a significant decrease of non-specific toxicity – the MTD of PK1 (HPMA copolymer-DOX conjugate) was 320 mg/m² (of DOX equivalent; MTD for free DOX is 60–80 mg/m²) and no congestive heart failure was observed despite doses up to 1680 mg/m² [60]. In phase II of PK1 (dose 280 mg/m²) the difference of PK1 efficacy [62] when compared to free DOX was smaller than in animal models [87–91]. Similar observations were made in other clinical trials [84–86]. See detailed analysis in ref. [10].

It was hypothesized that the reason for lower efficacy in humans was the short blood circulation time of the low molecular weight HPMA copolymer-drug conjugates. The intravascular half-life was sufficient in animal models where the tumor is up to 10% of body weight. In humans, a prolonged concentration gradient between vasculature and tumor is needed to ensure abundant extravasation of the conjugate into the tumor and efficacy [10,18,23]. This observation initiated the design of backbone degradable, 2^nd generation conjugates described in the next paragraph.

2.4.1.2. Backbone degradable, long-circulating HPMA copolymer carriers

To address the discrepancy between circulation time and biocompatibility of nondegradable carriers, new backbone degradable, long circulating HPMA copolymers were designed, synthesized, and evaluated (Figure 2). These carriers contain an enzymatically degradable sequence both in the main chain and in the spacer between polymer backbone and drug. They are synthesized by a combination of living radical polymerization and click reactions [92–94]. In particular, RAFT (reversible addition-fragmentation chain transfer) polymerization in the presence of a bifunctional chain transfer agent (peptide2CTA) is used to prepare a degradable HPMA diblock copolymer using a scalable, one step process [94]. Peptide2CTA (N^α,N^ε-bis(4-cyano-4-(phenylcarbonothioylthio)pentanoylglycylphenylalanylleucylglycyl)lysine) contains an oligopeptide (GFLGKGLFG) degradable sequence flanked by two dithiobenzoate groups. Monomer inserts at both positions with the same rate, producing a diblock copolymer with a very narrow molecular weight distribution in one scalable synthetic step. Further expansion of molecular weight can be achieved by click reactions, producing tetrablock and hexablock copolymers. This is a platform technology and numerous drugs can be attached to these carriers (Figure 2).

Figure 2.

Long-circulating, backbone degradable HPMA copolymer-drug conjugates. (A) Structure of non-degradable (1^st generation) and backbone degradable conjugates. (B) Synthesis of backbone degradable conjugates by RAFT copolymerization of HPMA and polymerizable derivative of drug containing degradable GFLG sequence (MA is methacryloyl). Bifunctional chain transfer agent (Peptide2CTA) is composed from a lysosomally degradable sequence GFLGKGLFG flanked by two dithiobenzoate groups. This permits the synthesis of degradable diblock copolymers with narrow polydispersity in one, scalable synthetic step. Post polymerization click reaction produces multiblock copolymers.

To optimize the molecular weight of the multiblock backbone degradable copolymers, diblock (Mw~100 kDa), tetrablock (Mw~200 kDa), and hexablock (Mw~300 kDa) HPMA copolymers containing gemcitabine (GEM) or paclitaxel (PTX) were used in the treatment of human ovarian carcinoma A2780 xenografts in mice [95]. Notably, degradable diblock conjugates 2P-GEM and 2P-PTX inhibited tumor growth more successfully than multiblock conjugates [95]. It is known that higher molecular weight enhances the accumulation of conjugates in solid tumors [59]. However, other factors, such as the conformation of the macromolecule [66,96,97], association of side-chains by hydrophobic interactions (formation of unimolecular micelles), or random association of flexible chains by “point-point” contacts [98–100], influence the rate of enzymatic drug release [37,38], penetration of the solid tumor [101] and, ultimately, the efficacy.

Similar approach based on RAFT polymerization and incorporation of enzymatically degradable sequences into the polymer backbone was used by Luo’s [102–104] and Garrison’s labs [43–45,105]. Pechar et al. used different chemistry, they prepared multiblock copolymers by interfacial polycondensation of telechelic PEG (2 kDa) and oligopeptide diamine from glutamic acid and lysine. The oligopeptide sequence contained GFLG-DOX side chains. The conjugate was cleavable by cathepsin B and possessed anticancer activity [106]. Additionally, multiblock HPMA copolymers were synthesized via polyaddition of homotelechelic polymer diazides with azo-compounds containing two alkyne groups [107]. Yang et al. synthesized an enzyme-sensitive, alkyne-functionalized, chain transfer agent (CTA-GFLG-alkyne) for RAFT polymerization and copolymerization of HPMA. Post-polymerization modification with 4,4’-azobis(azidopropyl 4-cyanopentanoate) resulted in the formation of heterotelechelic HPMA copolymers containing terminal alkyne and azide groups. Chain extension via click reaction resulted in high molecular weight multiblock copolymers [92].

2.4.1.3. Comparison of first generation (non-degradable) and second generation (backbone degradable) HPMA copolymer – drug conjugates

Numerous in vivo data proved the higher therapeutic efficacy of 2^nd generation conjugates when compared to 1^st generation conjugates. These results are a consequence of dramatically improved pharmacokinetics and tumor accumulation [10]. Comparison of epirubicin (EPI) conjugates [non-degradable ~40,000 Da P-EPI vs. degradable diblock ~100,000 Da 2P-EPI) on human ovarian carcinoma A2780 xenografts (5 mg/kg EPI equivalent doses administered i.v. on days 0, 4, and 8) revealed that complete tumor regression was observed for 100 days in all five evaluated mice treated with 2P-EPI, whereas four mice (out of five) treated with P-EPI started fast tumor regrowth from day 35 (Figure 3) [108]. Measured up to the 1^st generation conjugate (P-EPI, Mw~40kDa), 2P-EPI (Mw100 kDa) demonstrated outstandingly improved pharmacokinetics such as four-fold increase in terminal half-life (33.22±3.18 h for 2P-EPI vs 7.55±3.18 h for P-EPI) [108], which is primarily due to the increased molecular weight of the polymer carrier. Strikingly, complete tumor remission and long-term inhibition of tumorigenesis (100 days) were reached in the mice (n=5) treated with 2P-EPI (Figure 3). As a top product in our development pipeline, 2P-EPI (also called KT-1), was selected by Nanotechnology Characterization Laboratory (NCL) scientific review board, and has been evaluated with respect to physicochemical characterization, in vitro cytotoxicity, immunotoxicity, and cancer treatment efficacy [109]. 2P-EPI exhibited strongly superior antitumor activities over free EPI in various preclinical models including colon, lung, pancreatic, and breast cancers. Similar data were obtained with doxorubicin conjugates [110] and paclitaxel conjugates [111].

Figure 3.

Treatment of nude mice bearing A2780 human ovarian carcinoma xenogratfs with HPMA copolymer-epirubicin (EPI) conjugates. Comparison of 1^st generation conjugate (P-EPI) with backbone degradable, long circulating conjugate (2P-EPI); free EPI as control. The dose for each injection was 5 mg/kg EPI equivalent). (A) Dose schedule; Monitoring of tumor growth in conjugate treatment; Tumor volume after treatment with P-EPI and 2P-EPI at day 80 (saline and free EPI groups were terminated at day 20 due to large tumors). (B) Terminal half-life of EPI, P-EPI, and 2P-EPI. (C) Average body weights of mice in individual groups. Adapted from ref. [108].

Clinical treatment of cancer frequently uses combination of drugs that possess different mechanisms of action. One such combination is GEM + PTX. Based on a combination index study [112] synergistic doses were selected and mice bearing A2780 human ovarian carcinoma xenografts were treated with a combination of 2P-GEM and 2P-PTX. Combination of 1^st generation conjugates (P-GEM + P-PTX) and combination of free drugs (GEM + PTX) were used as controls. The data showed that 2^nd generation backbone degradable conjugates possessed prolonged blood circulation time, enhanced tumor accumulation, and improved anti-tumor efficacy as compared to 1^st generation low-Mw conjugates and free drugs. The new 2^nd generation conjugates were degradable in vivo and lacked noticeable systemic toxicity [113].

2.4.2. Poly(ethylene glycol)

Poly(ethylene glycol) (PEG) is the most widely used polymer in the clinics [30,114,115]. Its main application is the modification of proteins via one-point attachment using semitelechelic PEG derivates. The idea to modify proteins with PEG originated in the 1970s. Prof. Davies from Rutgers University has shown that such a modification results in prolonged blood circulation, no interactions with antibodies toward the native protein, better solubility, and resistance to proteolysis [116,117]. Numerous protein-PEG conjugates have been FDA approved [see Tables in refs. 30,114,115], starting with PEGylated adenosine deaminase (Adagen) [118] and PEGylated asparaginase (Oncaspar) [119]. Both conjugates contain multiple 5 kDa PEG molecules attached to ε-amino groups of lysines. Commercialized products include enzymes (uricase, phenylalanine ammonia lyase), interferons, granulocyte colony stimulating factor, factor VIII, and anti-tumor necrosis factor Fab’ fragment [30]. The binding chemistry improved dramatically, strategies to attach PEG specifically to cysteine, histidine, and tyrosine are available. Alternatively, engineered protein molecules (mutants) that incorporate a specific amino acid as an attachment point, for example cysteine in the Fab’ fragment of anti-TNF antibody. The molecular weight of PEG increased to 20 kDa (G-CSF conjugate), 30 kDa (epoetin conjugate) and branched 40 kDa (interferon α2a, anti-VEGF aptamer) [30,115].

The improved chemistry resulted in more homogeneous conjugates [120]. However, the modification of proteins with multiple molecules of PEG will impact biorecognition and/or activity. Modification of enzymes with numerous PEG molecules did not have an impact on the efficacy of cleavage of low molecular weight substrates, but substantial decrease of cleavage efficiency of high molecular weight substrates occurred [121]. Modification of vesicles with PEG inhibits cellular uptake and endosomal release. To surmount this challenge targeting moieties and reversible (cleavable) attachment of PEG are being used [122,123].

PEG has also been studied as a carrier in PEG-drug conjugates as well as in micelle-forming block copolymers that incorporate drugs in the core. The latter will be evaluated in a special chapter in this volume. Several PEG-drug (camptothecin, docetaxel, irinotecan, paclitaxel, SN-38) conjugates reached phase I/phase II clinical trials [30,114]. Recently, a four-arm PEG-irinotecan conjugate (Onzeald) reached Phase III clinical trials to treat breast cancer. The drug is bound to the PEG via a slowly hydrolyzable ester bond. Improved pharmacokinetics was observed but longer survival following Onzeald administration was not reached [124]. Current research involves long-circulating PEG-SN-38 conjugates; their accumulation in experimental solid tumor was correlated with μPET/CT imaging of PEG labeled with zirconium-89 [125].

There are numerous concerns related to the biocompatibility of PEG [126]. The formation of IgM antibodies against PEG results in accelerated blood clearance [127] of PEGylated liposomes [128] or nanoparticles [129]. Following second injection, complement activation occurs as a result of IgM binding to the surface of PEGylated vesicles. Additionally, PEGylated proteins are not efficient in the treatment of patients that have anti-PEG antibodies in their blood. Treatment of a substantial fraction of patients with acute lymphoblastic leukemia (ALL) was not efficient due to rapid clearance of PEG-asparaginase [130]. Another concern is the formation of cytoplasmic vacuoles in cortical tubular epithelial cells [131] and the above the renal threshold hydrodynamic volume of PEGs used; the latter may impair elimination from the organism [132].

McSweeney et al. [133] have shown in experiments on mice that pre-administration of 40 kDa PEG can saturate circulating anti-PEG antibodies and restore the prolonged circulation of stealth liposomes.

Alternatives to PEG for protein or vesicle modification

Polymers with various compositions have been studied as PEG replacement [120], including polyoxazolines [134], poly(N-vinyl-2-pyrrolidone) (PVP) [129], poly(N-acryloylmorpholine) (PAcM) [129], poly(N,N-dimethylacrylamide) (PDMA) [129], and polyHPMA [135,136]. Repeated administration of nanoparticles coated with PVP did not result in the ABC phenomenon and antibodies against PVP were not found [129]. Notably, modification of vesicular carriers with PEG alternatives (as well as with PEG) increases the intravascular half-life of modified carriers as well as decreases the adsorption of blood plasma proteins [136–138]. Early results on nanosphere modification with semitelechelic polyHPMA [136] demonstrated molecular weight dependent increase of intravascular half-life in rats and decrease of protein adsorption (albumin, IgG, fibrinogen) on modified particles when compared to unmodified nanospheres [136]. Recent data on the interaction of HPMA copolymers with blood plasma proteins observed similar phenomena. Moreover, they contributed to the explanation of the mechanism of corona formation [137–139].

Szoka and coworkers evaluated the effect of backbone structure on the induction of accelerated blood clearance (ABC) of polymer modified liposomes [140]. PVP, PDMA, HPMA, and PAcM modified liposomes did not demonstrate ABC in rats, whereas PEG and poly(2-methyl-2-oxazoline) modified liposomes exhibited strong ABC effect (Figure 4). Apparently, a follow-up study on the potential immunogenicity of polyoxazolines is warranted.

Figure 4.

A) Elimination of a single dose of liposomes (100 nm) from blood of rats over 48 h (mean ± SEM, n = 3). B) Elimination of the second dose of liposomes from blood of rats administered one week after the first dose measured over 48 h after administration of the second dose (mean ± SEM, n = 3–4). PEG, poly(ethylene glycol); HPMA, poly[N-(2-hydroxypropyl)methacrylamide]; PMOX, poly(2-methyl-2-oxazoline); PVP, polyvinylpyrrolidone; PDMA, poly(N,N-dimethylacrylamide); PAcM, poly(N-acryloylmorpholine); molecular weight of polymers = 2.1 – 2.5 kDa. Reprinted from ref. [140].

Other macromolecular structures used in protein modification are zwitterionic peptides (alternating copolymers of E and K) [141] and polymers [142]. A novel approach, called “pepylation” is the modification of proteins with synthetic peptides [143].

Alternatively, proteins may be modified via multipoint attachment. For example, cobra venom acetylcholinesterase was modified with multifunctional HPMA copolymer. The copolymer-modified acetylcholinesterase demonstrated a substantially longer blood circulation time as well as higher stability to thermal inactivation when compared to the native enzyme [144].

Similarly, lateral attachment of multivalent HPMA copolymers to polyelectrolyte complexes of polycations (poly-L-lysine or polyethyleneimine) with DNA results in increased blood circulation time [145]. Seymour and coworkers demonstrated that multilateral modification of therapeutic viruses with HPMA copolymers provided protection against neutralization by antibodies and complement. In addition, the extended plasma circulation and the potential of targeting provide a real opportunity for intravenous delivery of gene and virotherapy agents to treat disseminated cancer [146]. Reversible multipoint coating was achieved with HPMA copolymers with thiazolidine-2-thione (TT) groups at side chain termini and attached to the polymer backbone via spacers containing hydrolytically cleavable hydrazone bonds [147].

2.4.3. Poly(malic acid)

Synthetic poly(β-malic acid) has been studied as a drug carrier in the 1990s [148]. In the last 15 years Ljubimova’s laboratory studies copolyesters derived from poly(β,L-malic acid) (PMLA) as a nanoconjugate platform in drug delivery [29,36]. PMLA is produced by biological fermentation from the slime of Physarium polycephalum. The unsubstituted PMLA backbone is degradable; it is metabolized into water and carbon dioxide. The complex conjugate used in numerous cancer models including glioma is called Polycefin. It is composed from the PMLA backbone, morpholino antisense oligonucleotides (AON; attached via disulfide bonds) targeting α4 and β1 chains of laminin-8 overexpressed in gliomas, monoclonal anti-transferrin antibody, and pH-sensitive membrane disrupting units [149,150]. The endosomolytic function of Polycefin has been optimized; both trilysine [151] and tritryptophan [152] units were evaluated. Obviously, a challenge for such multicomponent conjugates is the detailed analysis of each component. A quantitative analytical method composed of backbone cleavage, chromatography, and HPLC was developed [153].

The nanoconjugates were proven effective in breast cancer treatment. A conjugate using 12-mer peptide mimetic of trastuzumab was effective toward HER2-positive breast cancer model [154]. Another modification of the nanoconjugate composition was successful against triple negative breast cancer [155]. However, the main focus of the team is focused on brain tumors [36]. Attachment of Angiopep-2, a 19 amino acid peptide [156] recognized by the low-density lipoprotein receptor-related protein (LRP1) mediates the transcytosis of the conjugate across the blood brain barrier (BBB) due to high expression of LRP1 on capillary endothelial cells at the BBB surface [157]. Attachment of anti-CTLA-4 and anti-PD-1 checkpoint inhibitor antibodies to an angiopep-2 containing nanoconjugate was successful in the treatment of mice bearing intracranial GL261 glioblastoma. An increase of CD8+ T cells, NK cells and macrophages as well as a decrease of Tregs was observed in the brain tumor area. Survival of mice was significantly longer when compared to single antibody containing conjugates or free antibodies [158].

2.4.4. Poly(glutamic acid)

Synthetic poly(L-glutamic acid) (PG) was employed as a carrier of taxol (TXL) by Li et al. Excellent research and development efforts proved the efficacy of PG-PTX in numerous animal cancer models. PG-TXL possessed better antitumor activity than free paclitaxel, improved pharmacokinetics, and different mechanism of action. In addition to different pharmacokinetics and biodistribution, efficient uptake of PG-TLX by macrophages resulting in enhancement of necrotic tissue are important mechanistic phenomena associated only with macromolecular conjugate of PTX [159]. Cathepsin B was found to mediate selective proteolysis of the PG backbone and site-specific delivery and antitumor efficacy of PG-TLX [160,161]. Extensive preliminary data [reviewed in 159] provided a road map for clinical trials of PG-TLX (CT-2103, Xyotax^R). The clinical formulation of CT-2103 had a molecular weight 48,000 and contained 37% paclitaxel by weight. No Cremophor or alcohol were needed and the solution of CT2103 was infused within about 30 minutes. Based on successful Phase I and Phase II studies, more than 1700 patients with NSCLC took part in Phase III studies. The results revealed similar efficacy with controls, but CT-2103 lessened side effects and provided a more convenient administration [159,162]. Interestingly, evaluation of a subset of premenopausal women with normal estrogen levels indicated superior survival following administration of CT-2103. This may relate to estrogen role in pulmonary physiology and enhanced cathepsin B levels [163,164].

Further research on PG as carrier focused in the design of noninvasive imaging agents [165], phthalocyanine conjugates for photodynamic therapy [166], and PG-doxycycline conjugates as fibril disrupters for potential amyloidosis treatment [167]. Recently, star-shaped polyglutamates were designed as new carriers [168]. Star-shaped polyglutamate – curcuminoid conjugate was designed for the treatment of acute kidney injury [169].

2.4.5. Cyclodextrin-based polymers

Davis and coworkers designed linear polymers that contain cyclodextrins in their main chain [170]. For the delivery of nucleic acids, they synthesized positively charged polymers [171] for the delivery of anticancer drugs (camptothecin, CPT) they synthesized neutral copolymers of cyclodextrin and PEG [172] (Figure 5).

Figure 5.

Scheme of the synthesis of cyclodextrin-based polymer conjugate IT-101 (CRLX101). β-CD, beta-cyclodextrin; PEG-DISPA, poly(ethylene glycol) dipropanoic succinimide; CPT, camptothecin. Reprinted from ref. [170].

Following preclinical evaluation [173,174], the cyclodextrin polymer - CPT conjugate (first named IT-101, later CRLX101) was evaluated in several clinical trials [175, reviewed in 176]. The conjugate localized in human tumors and not in adjacent tissue following intravenous infusion [177]. Evaluation of immune response after IT-101 administration revealed decrease of adverse effects and modulation of immune responses resulting in enhanced drug efficacy [178]. Correlation of animal and human studies indicated that the behavior of CRLX101 in animals is translatable to humans [179].

2.4.6. Poly(2-oxazoline)s and poly(2-oxazine)s

Monomers 2-oxazoline and 2-oxazine are cyclic iminoethers. They can be polymerized by living cationic ring opening polymerization (CROP) initiated by strong alkylating agents, such as alkyl halides [180]. However, the synthesis of uniform high-molecular weight polymers is troubled by chain transfer. Recently, polymerization conditions were established that minimize side reactions and produce 50 kDa polymers with polydispersity 1.05 [180]. The use of polyoxazolines and polyoxazines as drug carriers has been reviewed [181]. Early work focused on protein-polyoxazoline conjugates [182], recent studies concentrated on conjugates with low molecular weight drugs, doxorubicin [183] and rotigotine [184]. The latter conjugate, poly(2-ethyloxazoline) with rotigotine bound via ester bonds, was evaluated in Phase I clinical trials as a potential therapeutic to treat Parkinson disease. Methods to control the kinetics of drug release from polyoxazoline conjugates were published [185,186]; this should encourage more studies on polyoxazoline-drug conjugates.

2.4.7. Other polymer structures

Numerous water-soluble polymer structures that have been used as carriers of drugs, including poly(vinyl pyrrolidone) [1–4,187], polysaccharides [188–191], polypeptoids [192,193], and polyacetals [194]. Fleximer is a biodegradable polyacetal macromolecule (1-hydroxymethylethylene hydroxymethyl formal); its conjugate with camptothecin is being evaluated in clinical trials [195]. Mersana Therapeutics also developed a polymer (Fleximer)-based antibody-drug conjugate platform including XMT-1536, antibody-Fleximer-auristatin F-hydroxypropylamide conjugate targeting the sodium-dependent phosphate transport protein NaPi2b expressed on NSCLC adenocarcinoma and ovarian cancer [196].

3. Examples of efficacy of polymer-drug conjugates in animal models

Polymer-drug conjugates possess abundant advantages when compared to low molecular weight drugs [9]. The major gains from attaching drugs to polymer carriers are the different mechanism of cellular uptake and subcellular fate [21,33,197–201], changed pharmacokinetics [113,174], accumulation in solid tumors [59,113], lessened non-specific toxicity of the conjugated drugs [60], surmounting multidrug resistance [91], modulation of cellular signaling and apoptotic pathways [202,203], different stress responses [204], multivalency of targeted conjugates [150,205,206], induction of immunogenic cell death and immunostimulating properties [14], enhanced efficacy in the treatment of cancer and other diseases [110,113,167,173,207–210], and prevention of metastasis [211,212].

3.1. Enhanced permeability and retention (EPR) effect

Enhanced permeability and retention (EPR) effect was proposed by Maeda and coworkers to explain enhanced accumulation of macromolecules in solid tumors [7]. The phenomenon was attributed to fenestrations in tumor vasculature and impairment of lymphatic drainage, but numerous other factors contribute [213]. Indeed, the EPR effect was detected in plentiful animal models and became a leading scientific hypothesis behind the design of various nanomedicines. However, clinical trials revealed a complex situation and a large interpatient heterogeneity [214–216]. The main problem in the translation of animal data to the clinics was the belief that EPR is a generally occurring phenomenon. In contrast to the development of antibodies or other targeted therapies, where patients have been selected based on the expression of the target antigen on cancer cells, no patient selection based on tumor susceptibility to EPR has been made in clinical trials with nanomedicines [60,62,217,218].

The incongruity between animal and human data on the treatment of solid tumors led to suggestions that the EPR effect does not work in humans [219,220]. Apparently, this conclusion is premature. Future research needs to take into account the fact that tumors are highly variable with large interpatient, intertumor and intratumor variability [218]. Inspiring approaches how to improve delivery of nanomedicines to solid tumors were suggested. Jain and coworkers have shown that small size nanomedicines (~12 nm) can take advantage of the normalization of tumor blood vessels [221]. Such size is typical for water-soluble macromolecular therapeutics, in contrast to the majority of nanoparticles. A recent review summarized factors that have the potential to improve patient outcomes, namely anti-angiogenic therapy, administration of tumor necrosis factor-alpha (TNF-α), angiotensin II receptor blockers, and ionizing radiation [222]. Additionally, NO, CO, and hyperthermia augment EPR effect [223–227].

Indeed, basic research and clinical data provide hope for the future of macromolecular therapeutics [228]. Nearly 20 years ago, Harrington et al. have shown the successful accumulation of PEGylated liposomes in patients with progressed tumors. Simultaneously, a wide interpatient heterogeneity was observed [214]. CRLX101, a cyclodextrin-containing polymer conjugate of camptothecin (CPT), self-assembled into nanoparticles of about 30 nm was administered to nine cancer patients with gastric, gastroesophageal, or esophageal cancer. CPT was detected in the tumor of five patients 24 – 48 h post administration. Evidence of biological activity was detected as well as of intact deposition of CRLX101 in the tumor tissue [176]. Two Merrimack clinical trials provided evidence and quantitative data on the accumulation of nanomedicines in human metastatic tumors using positron emission tomography. The ferumoxytol iron nanoparticles uptake as detected by MRI was correlated with the tumor response to liposomal irinotecan [215,216].

These [214–216] and other reports [218,229–231] clearly indicate that it is of highest significance for the future success of nanomedicines to develop diagnostic tests to select patients that will benefit from the treatment as it is done routinely for molecularly targeted therapies. Identification of predictive “biomarkers” is crucial for the development of this treatment modality.

Clinical imaging including magnetic resonance imaging, positron emission tomography, single photon emission computerized tomography is an important tool that can accelerate the translation of nanomedicines into the clinic [229,232]. In particular, it can provide techniques for the selection of patients whose tumors will be EPR responsive. Generally, there are two approaches to the combination of therapeutic effects and imaging. One is to combine the nanomedicine with the imaging moiety in one conjugate [233]. This is valuable for basic research, but may not be favored for clinical use. The other approach is to develop diagnostic nanoconjugates [43,125,215,216,231,234,235] that will be used for the selection of patients, followed by treatment of selected patients.

Additional important topics to study are tumor-stromal interactions [236–238], intratumoral pressure [239], and tumor penetration [101,240,241].

3.2. Targeting

There are several targeting terms used in the literature: passive targeting, (active) targeting, negative targeting, and subcellular targeting. Passive targeting is being used for the passive accumulation of macromolecular therapeutics in solid tumors. It is discussed in 3.1. Negative targeting is sometimes used to illustrate the fact that polymer-bound drugs possess a different body distribution than free drug and might avoid the major organ where free drug exerts toxic effects. For example, polymer-bound doxorubicin avoids heart tissue [242] the main toxic site for free doxorubicin [243].

Active targeting of polymer-drug conjugates can be achieved by attachment of cell-specific ligands, such as antibodies [88,244], antibody fragments [57,245–247], affibodies [205], saccharides [248–251], lectins [252], peptides [253–255], and aptamers [256] resulting in increased biorecognition and cellular uptake [257–261]. Combinatorial approaches, such as phage display [262] and synthetic peptide (one-bead-one-compound) libraries [263] are being used for identification of peptides complementary to a receptor, systematic evolution of ligands by exponential enrichment (SELEX) technique is being used for selection of aptamers [264]. An ideal target is expressed predominantly on the target cells and minimally in other locations. Upon binding of the targeting moiety to the relevant receptor the complex can internalize followed by subcellular trafficking via the endosomal/lysosomal route; it can be recycled recovering the surface receptor and releasing the ligand, or it can stay for an extended time at the surface. The internalization and subcellular trafficking processes can be manipulated by receptor crosslinking [265]. Multivalent ligands not only possess higher binding avidity [266], but they initiate internalization mediated by receptor crosslinking [267]. Very slowly internalizing CD20 and HER2 receptors will internalize by crosslinking-mediated endocytosis [268,269]. Crosslinking of PD-L-1 receptors on breast cancer cells by multivalent PD-L1 antagonist modified the subcellular fate of the PD-L1/antagonist complex; instead of recycling it back to the surface it biases the PD-L1 into the lysosomal route [270]. The design of macromolecular therapeutics for a particular receptor must match the receptor property [257].

Antibodies have been frequently used as targeting moieties. A widely studied field are antibody drug conjugates that reached clinics [271]. Recent designs incorporate polymers in the design of ADCs. To avoid using very toxic drugs (calicheamycins, auristatin monomethyl ester, IC50 <1 nM) that could do damage in off-site locations, semitelechelic polymer-drug conjugates are attached to antibodies. This permits to increase the drug to antibody ratio and employ less potent drugs, such as epirubicin [272,273].

Subcellular targeting relates to subcellular manipulation of drug fate. It is known that subcellular location impacts the efficacy of drugs [274]. Smart designs of conjugates permit endosomal escape if degradation in lysosomes needs to be avoided [275,276], basic studies on the passive [201] and chaperoned [277] transport through the nucleopore complex help the design of conjugates for nuclear targeting [278,279].

Mitochondrial targeting can be mediated by utilizing the negative mitochondrial potential and employing positively charged triphenylphosphonium groups as targeting moieties [201,280]. Triphenylphosphonium containing HPMA copolymer-bound photosensitizer, mesochlorin e₆ (Mce₆), enhanced cytotoxicity toward human ovarian SKOV3 cells as compared to non-targeted HPMA copolymer-Mce₆ conjugates [280].

Nuclear targeting can be mediated by steroid hormone receptors. For example, when glucocorticoid receptor (GR) binds to its steroid ligand, it dimerizes and transfers into the nucleus. Rebuffat et al. used this approach to enhance the steroid-mediated transport of transfected DNA into the nucleus of GR positive cells [281]. This approach can be also used for the delivery of photosensitizer Mce₆ into the nucleus. To this end, photosensitizer Mce₆ was modified with cortisol (attachment moiety for GR) to produce Mce₆-cortisol. The latter was attached to the HPMA copolymer via a lysosomally degradable GFLG spacer. The HPMA copolymer - Mce₆-cortisol conjugate was incubated with 1471.1 cells transfected with GR labeled with green fluorescent protein, GFP. Following internalization of the HPMA copolymer conjugate by endocytosis, Mce₆-cortisol was enzymatically released by cathepsin B inside the lysosomes, translocated to the cytoplasm, and bound to GR (mediated by cortisol); this initiated GR dimerization and nuclear transport of the modified photosensitizer [282].

3.3. Brain delivery

The majority of polymer-drug conjugates is designed for intravenous delivery. Brain delivery has an additional hurdle to overcome, i.e., the nanomedicine needs to cross the blood brain barrier (BBB) [283]. To enhance the transport across the BBB either physical methods, such as osmotic pressure [284] and focused ultrasound [285] are being used. Alternatively, the BBB can be circumvented by nasal administration [286].

Recent designs use biorecognition at the BBB for transport into the brain [29]. Transferrin receptor antibody has been used for many years to mediate transport of therapeutics into the brain [287,288]. An advanced design focuses on peptides that bind to receptors and transcytose bound cargo into the brain. LDLR (low-density lipoprotein receptor)-related protein (LRP)-1, a member of LDLR family binds Kunitz domain-related peptides and initiates transcytosis. Demeule et al. [156] designed 96 Kunitz domain-related peptides, evaluated their potential for transport through BBB and angiopep-2 (AP-2; TFFYGGSRGKRNNFKTEEY) was found to be the best. AP-2 was reported to highly improve therapeutic efficiency in brain targeting studies [289,290]. Recently, the Ljubimova group has used polymer-attached trastuzumab-mimetic anti-HER2 peptide to successfully target and treat HER2+ breast cancer and metastases in the brain, as well demonstrated AP-2-polymer privileged ability for enhancing BBB penetration [157]. AP-2 is also useful for brain tumor targeting as LRP-1 is overexpressed in human glioma cells [291] and in the treatment of brain metastases of breast cancer [292]. AP-2 successfully transcytosed across the BBB conjugates based on poly(β,L-malic acid) [151] and HPMA copolymers [293]. Interestingly, a recent Phase II clinical study of ANG1005, a conjugate of AP-2 with 3 molecules of PTX, demonstrated that all patients, particularly patients with leptomeningeal carcinomatosis, showed symptomatic improvements and prolonged overall survival compared to controls [294].

3.4. Multidrug resistance

Nanomedicines can surmount the efflux pump-type of multidrug resistance [295]. They are internalized in the cells by endocytosis [198] in contrast to diffusion of free low molecular weight drugs. Internalization in membrane limited organelles avoids interaction with P-glycoprotein (Pgp), an ATP-dependent efflux pump that constricts the transport of drugs into the cytoplasm [296]. Polymer-drug conjugates reach the lysosomal compartment located in the perinuclear region of the cell. Following enzymatically mediated release from the carrier they diffuse to the cytoplasm out of reach of the efflux pump. In vitro and in vivo comparison of DOX and HPMA copolymer-DOX conjugate in sensitive A2780 and DOX-resistant A2780/AD human ovarian carcinoma cells/xenografts validated the hypothesis. In contrast to free DOX the IC50 dose [297] and tumor treatment efficacy [91] of the polymer conjugate was very similar for both cell types. Additionally, chronic exposure of A2780 cells to HPMA copolymer-DOX conjugate did not induce resistance, whereas chronic exposure to DOX resulted in a new phenotype expressing the MDR1 gene that encodes Pgp [298]. Similarly, polymer bound mesochlorin e₆ (Mce₆) also did not induce multidrug resistance following chronic exposure to human ovarian carcinoma cells [299].

Another approach is the design of a HPMA copolymer that contains both DOX and Pgp inhibitor R121 bound via hydrolytically cleavable hydrazone bonds. The conjugate was efficient in the treatment of resistant P388/MDR and CT26 tumors [300]. Alternatively, anti-Pgp antibody targeted polymer bound photosensitizer Mce₆ provided an efficient conjugate. An important design factor was the activity of polymer-bound Mce₆ to produce singlet oxygen before internalization [244].

3.5. Combination therapy with polymer-drug conjugates

Treatment of cancer in the clinics is frequently performed with combination of drugs. Combinations of polymer-bound drugs have been evaluated for several decades [301]. The factors that were studied include combination of two polymer conjugates vs. polymer conjugate that contains both drugs covalently attached; sequence of administration (which drug first, or simultaneous administration), and dosing in synergistic concentrations. Another approach is the combination of polymer-drug conjugates (chemotherapy) with physical stimuli – focused ultrasound, photothermal, and photodynamic therapy (activation by light).

Satchi-Fainaro’s group found that treating murine model of mammary adenocarcinoma with a PGA conjugate containing two drugs, PTX and DOX (PGA-PTX-DOX), was more efficient than a mixture of two separate conjugates containing one drug each [302,303]. Obviously, the evaluation of a combination of two separate conjugates is easier since changing of dose ratio is easy; for conjugates containing two drugs a change in concentration ratio requests a new synthesis. One can assume that if the two drugs have similar hydrophobicity, then the biodistribution of respective conjugates will be similar and two conjugates may be advantageous. However, for drugs with different properties one conjugate should be evaluated.

Numerous combinations of polymer-drug conjugates were evaluated with enhanced efficacy of the combination when compared to the monotreatment [70,113,304]. An interesting combination treatment is the combination of two conjugates that target different cell subpopulations, cancer stem cells and differentiated cells. The eradication of stem cells would prevent their self-renewal, cancer recurrence, and metastasis. In a prostate cancer model, the stem cells could be downregulated by blocking the hedgehog pathway; the differentiated cells are sensitive to the action of clinically used docetaxel. Indeed, the combination of two HPMA copolymer conjugates, one containing cyclopamine, a hedgehog pathway inhibitor, the second containing docetaxel (DTX) were active in vitro [305] and demonstrated long-term tumor growth inhibition in nude mice bearing PC-3 prostate cancer xenografts. [306]. Similarly, a combination of a DTX conjugate with a conjugate containing GDC-0980, a dual PI3K/mTOR inhibitor, was effective in the PC-3 prostate cancer model [307].

Combination treatments that involve physical stimuli are efficient in cancer treatment

In combination chemotherapy with photodynamic therapy (PDT), polymer-bound chemodrugs are combined with polymer-bound photosensitizers (PS)s. The latter are not active in dark, but when irradiated with a characteristic wavelength the PS gets into its first excited singlet state. Intersystem crossing may convert the singlet state PS into its triplet state. Exchange of energy with ground state triplet state oxygen produces singlet oxygen (¹O₂) a reactive species indeed. The ¹O₂ has a short half-life in aqueous environment, it interacts with biological molecules close to the location where it originated. Photosensitizers are active both when covalently bound to the polymer carrier or when released inside the cell. However, the quantum yield of singlet oxygen formation is substantially higher when released from the carrier [301]. Consequently, enzymatically degradable spacers are preferred for PS attachment and PS that are activated with near infrared wavelengths are more efficient due to deeper penetration of light into the tissue. Combination of HPMA copolymer-DOX and HPMA copolymer-Mce₆ (mesochlorin e₆) conjugates was more active in the treatment of Neuro 2A neuroblastoma in A/J mice [301], treatment of human ovarian OVCAR-3 xenografts in female athymic mice than each of the individual therapies [308]; OV-TL-16 antibody targeted HPMA copolymer-DOX and - Mce₆ conjugates were more active in the OVCAR-3 model than non-targeted conjugates [309].

Larson et al. used moderate (43 °C, 30 min) gold nanorod induced tumor hyperthermia to increase the delivery and efficacy of heat shock protein (HSP) targeted (mediated by peptide WDLAWMFRLPVG) HPMA copolymer-DTX and -aminohexylgeldanamycin conjugates in the treatment of DU145 prostate cancer in vitro and in DU145 tumor bearing mice. Hyperthermia induced the expression of cell surface HSP glucose regulated protein 78 kDa (GRP78) and synergistically enhanced the efficacy of the polymer-drug conjugates [310]. Using the same DU145 prostate cancer model in nu/nu mice the hyperthermia was induced by high intensity focused ultrasound. Enhancement of efficacy of HPMA copolymer-DTX was observed [311].

3.6. Potential immunogenicity/immunostimulation of polymer carriers

When evaluating polymer-drug conjugates both the cytostatic and immunomodulating effects have to be evaluated [24]. Due to the complex structure of these nanomedicines, test on the immunogenicity of the carrier, immunogenicity of the oligopeptide sequence used for drug attachment or self-assembly, the possibility that the drug attached to the side-chain termini may act as hapten need to be performed. Also, potential immunosuppression by depleting immune cells needs to be evaluated. When homopolymers are used for protein modification the situation is simpler – the immunogenicity of the polymer is the main factor. Evaluation of potential immunotoxicity of nanomedicines is an important part of research [312].

Detailed studies were performed for PEG [124–129] and HPMA copolymers [14,313–318]. Whereas administration of PEG results in the production of IgM antibodies, HPMA homopolymer is not recognized as an antigen and does not act as an immunogen. The attachment of oligopeptide spacers induces mild immunogenicity, but several orders of magnitude lower than bovine gamma-globulin [318]. Doxorubicin bound to GFLG spacers did not act as a hapten [318]. Interestingly, HPMA copolymer-DOX conjugate induces immunogenic cell death [24].

Notably, immunostimulatory properties of HPMA copolymer-drug conjugates were observed. Mice previously cured with HPMA copolymer-DOX conjugates survived cancer re-challenge without treatment [14]. Thus, these conjugates produce a double attack on cancer cells – cytotoxicity and immunostimulation. Importantly, clinical data revealed that similarly to experiments on animals both cytotoxic and immunostimulating role of HPMA copolymer-DOX-IgG conjugates was observed [313–315]. Similarly, camptothecin conjugated to cyclodextrin-based polymer (IT-101) modulated the immune response to enhance drug efficacy [178].

HPMA copolymer-peptide conjugates described in Section 5 contain peptide grafts 38 amino acid residues long. Detailed studies in vitro and in vivo using both D- and L- peptides have shown no immune response to free peptides and their copolymer conjugates in vitro. In vivo (in BALB/c mice) minor production of antibodies was detected and no cellular response observed when administered with complete Freund’s adjuvant [319].

3.7. Clinical experience

Polymer-drug conjugates and similar nanomedicines (frequently named nanoparticles) possess numerous advantages over free drugs [9,10,320,321]. However, the clinical potential of conjugates with low molecular weight drugs has not been fully exploited [10,217,322]. Yet, data from activity on patients [163,164,313,314,323,324] and the development of long-circulating backbone degradable HPMA copolymer-drug conjugates [108,109,113,270] bode well for the future. As discussed in 3.1 an important part of speeding up the translation is the development of biomarkers that will permit patient stratification before clinical trials.

The major clinical use of polymer conjugates are the PEG-modified proteins. Numerous FDA approved conjugates [30,114,115] and the intensive research on PEG alternatives [129,140] demonstrate the success of this field. Generally, nanomedicines are progressing to clinics with an acceptable speed. Recent reviews [30,320,321,325,326] and opinions [327–329] summarize the numerous clinical trials and FDA approved nanomedicines. Very recent successes include Onpattro®, Givlaari®, and Onivide®.

4. New directions in macromolecular therapeutics

In spite of several negative reports on the future of nanomedicine [219,220,330], we and others [327–329,331–333] strongly believe in the bright future of macromolecular therapeutics. Obviously, as any new science field that is well funded, nanomedicine experienced a period of inflated expectations. Using Gartner “hype cycle” terminology [334] nanomedicine is beyond the period of disillusionment and basic research by devoted scientists brought it back to a plateau of excellent productivity. This is one important aspect of future developments. Excellent progress will be made by scientists devoted to targeted basic research without the disturbing positive or negative hype.

Another important approach to macromolecular therapeutics will be the design of new paradigms based on the knowledge of mechanisms of physiological processes. The design of new paradigms is an extremely important part of scientific progress. However, it is important to avoid packaging old concepts in more complex approaches/structures [335] and elude superficiality [336]. Examples of two new designs, both based on employing receptor crosslinking, are presented below: a) improvement in combination chemotherapy and immunotherapy by designing a new multivalent anti PD-L1 antagonist; and b) design of drug-free macromolecular therapeutics.

4.1. Combination chemotherapy and immunotherapy with macromolecular therapeutics

Many chemotherapeutic drugs have significant immunosuppressive side effects, either directly, by inhibiting or killing effector cells, or indirectly, by provoking allergy or immune paralysis [337]. However, over the past decades, considerable evidence demonstrated cytotoxic drugs have also favorable function in the activation of the immune system [338]. There are two ways to elicit the immune system: some therapeutics can stimulate specific cellular responses that render tumor-cell death immunogenic, resulting in enrichment of T-cell infiltration in the tumor, whereas other drugs may have inhibition effects on regulatory T cell activity and foster an immunosuppressive tumor microenvironment, therefore improve immunotherapy response rate [339,340].

Immune checkpoint blockade (ICB) is a formidable strategy for cancer treatment. Immune checkpoints are regulators of immune activation. Tumor cells often overexpress immune checkpoint proteins to escape immunosurveillance and avoid attack by the immune system. T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) promote immune evasion and result in cancer immunoresistance [341,342]. The blockade of immune checkpoints, e.g., blockade of the interaction of programmed cell death ligand 1 (PD-L1 on cancer cells) with PD-1 on T cells using anti-PD-1 or anti-PD-L1 antibodies is a successful cancer treating strategy [343]. In addition to antibodies, small molecules, peptides, and macrocycles are efficient checkpoint inhibitors [344].

A myriad of factors contributes to the low response rate of immune checkpoint blockade (ICB). For example, tumor-infiltration of T lymphocytes is associated with prognostic implication. There is increasing evidence that patients with low effector T cell recruitment in tumor microenvironment fail to such immunotherapies. Therefore, to exploit ICB for cancer treatment, it is important to expand tumor-infiltrating lymphocytes (TIL). One of such strategies is immunogenic cell death induced by specific chemotherapy drugs.

Combining chemotherapies with immunotherapies creates the possibility of synergistic and/or additive effects and therefore, offers great promise in modulating tumor microenvironment and treating cancers successfully. Comparing with free drugs, polymer-drug conjugates have advantages in ‘priming’ tumors attributed to their long-circulation in blood stream and favorable accumulation in tumors. However, the combined approach is effective in inflamed “hot tumors” (lung, melanoma, liver, bladder, head and neck cancers); the treatment outcome is considerably poorer in non-inflamed “cold tumors” (estrogen receptor positive breast and prostate cancers) [345]. The distinction between hot and cold tumors relates to the amount of T cells inside tumor and expression of PD-1 and PD-L1. Fortunately, some anticancer drugs and polymer-drug conjugates can initiate immunogenic cell death (ICD) and convert cold tumors into warm or hot ones [270,338,346,347].

4.1.1. Immunogenic cell death by polymer-drug conjugates

Cytotoxic drugs not only exhibit chemotherapy effects on tumor cells, but they also affect the immune system to contribute to tumor regression. For example, anthracyclines such as doxorubicin and epirubicin induce tumor-specific immune response [338,339] by depleting regulatory T cells (Treg)s, stimulating dendritic cells (DC), and generating tumor immunogenic cell death (ICD). Cells which undergo ICD are characterized by pre-apoptotic translocation of calreticulin to cell surface and post-apoptotic release large amounts of ATP and high-mobility group box 1 (HMGB1) protein into the extracellular space [348,349]. The HMGB1 released from immunogenically dying tumor cells binds to several receptors such as Toll-like receptors 2 and 4, which are expressed on antigen-presenting cells, and functions as a “danger” signal to promote activation of DCs and boost antigen-specific T cell responses. Consequently, ICD can be utilized not only for killing cancer cells but also for “heating-up” the antitumor immunity, priming the tumor for consecutive immune checkpoint inhibition therapy.

Polymer-drug conjugates demonstrated their efficiency in inducing ICD. HPMA copolymer-doxorubicin conjugates demonstrated immunostimulating potential. In EL-4 lymphoma cancer model mice survived re-transplantation with cancer cells without further treatment [14,350]. Backbone degradable HPMA copolymer-epirubicin conjugate (2P-EPI; KT-1) triggered an increase of calreticulin expression and release of HMGB1 in 4T1 cells in vitro. In vivo in BALB/c mice bearing 4T1 tumors, 2P-EPI produced higher enhancement of calreticulin expression and HMGB1 release than free EPI (Figure 6). Importantly, 2P-EPI attracted considerably more CD8+ cells into the tumor than free EPI (Figure 6F). Both free EPI and 2P-EPI enriched PD-L1 expression on tumor cells (Figure 6G). The efficacy of 2P-EPI is supported by CD*+ T cells. Coadministration of anti-CD8+ antibodies considerably weakened 2P-EPI mediated tumor regression (compare Figures 6D and 6E) [270].

Figure 6.

Immunogenic cell death of 4T1 murine breast cancer cells/ BALB/c mice bearing 4T1 tumors treated with backbone degradable, long circulating HPMA copolymer-epirubicin conjugate (2P-EPI). (A) Conversion of cold tumor into hot tumors as detected by calreticulin expression (B), high mobility group box 1 protein (HMGB1) release (C) and T-lymphocyte infiltration. (D) Tumor growth curves following treatment with saline, EPI, or 2P-EPI (KT-1); (E) Treatment with saline, or 2P-EPI (KT-1) plus anti-CD8 antibody (to disable CD8 positive T cells). (F) Tumor recruitment of CD8+ cells; (G) PD-L1 expression on tumor cells after chemotherapy with EPI or 2P-EPI (KT-1). Adapted from ref. [270].

Moon’s lab has shown ICD and robust T cell response in CT26 and MC38 colon carcinoma cells exposed to DOX containing high density lipoprotein-mimicking nanodiscs [351]. Priming tumors by ICD for subsequent checkpoint inhibition therapy seems to be effective when using nanomedicines of various structures and combinations, such as nab-paclitaxel [352], mitoxantrone combined with multilamellar lipid-hyaluronic acid adjuvant nanodepots [353], and Doxil [354].

4.1.2. Immune checkpoint blockade by multivalent polymer peptide antagonists (MPPA)

Combination of ICD inducing chemotherapy with immune checkpoint blockade using anti-PD-1 or anti-PD-L1 antibodies has shown marked clinical success in melanoma, prostate, kidney, and lung cancer. However, the response rate in breast cancer is low due to the immunologically “cold” characteristics including low amount of cytotoxic T lymphocytes inside the cancer tissue. Recent studies demonstrated active redistribution of PD-L1 back to the cell surface following application of anti-PD-L1 antibodies, suggesting traditional mAb-based approaches produce inefficient blockades of PD-L1 and lead to adaptive therapy resistance [355,356].

To generate a more effective PD-L1 elimination strategy the subcellular trafficking of PD-L1 needs to be changed from recycling to the lysosomal route. Wang et al. have shown that HIP1R (Huntingtin interacting protein 1 related protein) targets PD-L1 to lysosomal degradation (through a lysosomal targeting signal) and alters T cell mediated cytotoxicity [357]. Another approach to decrease the PD-L1 surface expression is to inhibit palmitoylation of the intracellular domain of PD-L1 by palmitoyltransferase ZDHHC3. Palmitoylation of proteins acts as a mechanism to regulate protein localization and function. Inhibition of PD-L1 palmitoylation decreased the PD-L1 expression on tumor cells [358]. Additionally, CMTM6 depletion prevents PD-L1 recycling and decreases PD-L1 surface expression [359].

We proposed a new design of a PD-1/PD-L1 inhibition based on multivalency and receptor crosslinking - multivalent polymer-peptide antagonist (MPPA). It is well known that receptor crosslinking manipulates subcellular trafficking of receptor-bound ligands to the endosomal/lysosomal route [265]. Peptides have become interesting alternatives to block PD-1/PD-L1 interactions [360]. Chang et al. developed a peptide-based antagonist to the PD-1/PD-L1 pathway using phage display. This antagonist (PPA-1, sequence NYSKPTDRQYHF) has been shown not only to possess a high binding affinity to PD-L1 in vitro, but also to effectively disrupt the PD-1/PD-L1 interaction both in vitro and in tumor-bearing mice [361]. MPPA is a linear HPMA copolymer containing multiple PPA-1 grafts. Such a construct will hyper-crosslink the cell surface PD-L1 receptors and efficiently traffic the complex to the lysosome for degradation by lysosomal enzymes. This results in decreased PD-L1 expression at cell surface and enhanced antitumor activity [270].

4.1.3. Efficacy of combination of 2P-EPI (KT-1) with MPPA

As an example of the efficacy of combination chemotherapy and immunotherapy the treatment of triple negative 4T1 breast cancer with 2P-EPI followed by MPPA is presented (Figure 7). The 4T1 syngeneic cancer model shares genomic features of basal-like breast cancer which is a non-immunogenic tumor. The treatment strategy involves (a) immunogenic chemotherapy with long-circulating backbone degradable epirubicin conjugate KT-1, and (b) PD-L1 degradation immunotherapy with multivalent HPMA polymer-peptide antagonist to PD-L1 (MPPA). First, KT-1 immunologically “heated up” 4T1 tumor mediated by ICD. In the second step, multivalent MPPA was administered. This approach resulted in 10/10 complete tumor regressions (Figure 7F). For comparison, treatment with 2P-EPI (KT-1) and anti-PD-L1 antibodies produced 8/10 complete tumor regressions (Figure 7C) and treatment with 2P-EPI (KT-1) and peptide PPA 2/10 complete tumor regressions (Figure 7F). Treatment with only immunotherapy, PPA or MPPA was not effective (Figure 7F). The excellent efficacy of the combination with MPPA is a consequence of different subcellular fate of PD-L1 following receptor crosslinking (Figure 7D). The decreased expression of PD-L1 after treatment with MPPA (Figure 7E) validates the hypothesis on the impact of receptor crosslinking on the efficacy of immunotherapy.

Figure 7.

Combined chemotherapy and immunotherapy; design of multivalent polymer peptide PD-L1 antagonist (MPPA). (A) Scheme of PD-1/PD-L1 immune checkpoint blockade (ICB). (B) Treatment of BALB/c mice bearing 4T1 tumor with combination chemotherapy (EPI or 2P-EPI (KT-1)) and immunotherapy (anti-PD-L1 antibodies): schedule of dosing and tumor growth curves. (C) Individual tumor growth curves following administration of KT-1 and anti-PD-L1 antibodies; 8/10 complete tumor regressions. (D) MPPA changes the subcellular fate of PD-L1. Following binding of anti-PD-L1 antibodies the PD-L1 receptor recycles back to the surface. In contrast, binding of multivalent MPPA crosslinks the PD-L1 receptors and biases its subcellular fate into lysosomes, where they degrade. (E) Recovery of surface PD-L1 after treatments with anti-PD-L1 antibodies, PPA, or MPPA. (F) Individual tumor volumes for combination treatment of 4T1 BALB/c mice tumors with saline, PPA peptide, multivalent MPPA, 2P-EPI (KT-1) followed by PPA, and 2P-EPI (KT-1) followed by MPPA. Combination of 2P-EPI (KT-1) with MPPA produced 10/10 complete tumor regressions. Adapted from ref. [270].

4.2. Drug-free macromolecular therapeutics (DFMT)

4.2.1. First generation of DFMT

The concept of DMFT is a new paradigm in macromolecular therapeutics – treating diseases by biorecognition at cell surface and crosslinking of receptors without the need for low molecular weight drug [362,363]. Its concept emerged from the study of self-assembly of hybrid macromolecules composed of HPMA backbone and peptide grafts. Two pentaheptad peptides (CCE and CCK) with differing charge were bound to HPMA copolymer, respectively. Separately, the conformation of CCE and CCK is random coils, but their equimolar mixture forms antiparallel coiled-coil heterodimers and could serve as physical crosslinkers. Indeed, HPMA graft copolymers, P-(CCE)_x and P-(CCK)_y, self-assemble into hybrid hydrogels with a high degree of biorecognition [364].

Efforts to use the pair of peptides in biological processes logically focused on crosslinking of receptors – crosslinking of hydrogels is based on similar physicochemical phenomena. The CD20 receptor is one of the most reliable cell surface markers of B lymphocytes. It has a low rate of internalization; when crosslinked it relocates to lipid rafts, resulting in calcium influx, mitochondrial depolarization, and caspase 3 activation [365–367]. The new DMFT system is composed of two nanoconjugates: a) bispecific engager: anti-CD20 Fab’ antibody fragment attached to CCE (Fab’-CCE); and b) crosslinking (effector) component: HPMA copolymer containing multiple grafts of the complementary peptide, CCK (P-(CCK)_x). Fab’ was chosen over Ab to avoid crosslinking of CD20 bound Abs by immunocompetent cells.

The DFMT system translates the biomaterial biorecognition principles to nanomedicine [368]. Its three main features are: a) absence of a low molecular weight drug; b) pretargeting – the two-step treatment permits to manipulate the pharmacokinetics by separating the targeting modality from the effector modality; and c) multivalency – the second nanoconjugate is multivalent; its efficiency is valency dependent (Figure 8). The term DFMT is based on experimental observations that both parts of the system, the bispecific engager and the crosslinking effector do not initiate apoptosis when used individually. Only when both nanoconjugates colocalize at cell surface, resulting in receptor crosslinking, apoptosis is initiated.

Figure 8.

Design of drug-free macromolecular therapeutics (DFMT). Exposing B cells to a conjugate of one motif with a CD20 targeting ligand (Fab’-motif1) decorates the cells with this motif. Further exposure of decorated cells to a macromolecule (synthetic polymer or human serum albumin) grafted with multiple copies of the complementary motif [P-(motif2)_x or HSA-(motif2)_x] results in receptor crosslinking and apoptosis induction [362,363].

The design is active both in vitro [368] and in vivo on disseminated Raji B cell lymphoma in CB17 SCID mice [369]. The structure of the crosslinking effector nanoconjugate was modified; HPMA copolymer was replaced by human serum albumin, HSA-(CCK)_x and high apoptotic levels were obtained [370].

Advanced design employed complementary 25 base pair morpholino oligonucleotides instead of peptides, resulting in Fab’-MORF1 and P-(MORF2)_x (or HSA-(CCK)_x) nanoconjugates. Detailed studies of apoptotic mechanism in Raji B cells revealed that, following biorecognition of two nanoconjugates at cell surface, two pathways are involved in apoptosis initiation: a) CD20 crosslinking→calcium influx→mitochondrial depolarization; b) Bcl-2 inhibition→mitochondrial depolarization→cytochrome c release→caspase 3 activation [371]. These data are supported by visualization of biorecognition at cell surface by super resolution imaging [372,373]. The morpholino based system was very active with both effectors, based on HPMA copolymer [267,371] and on HSA [268]. DMFT overpowers rituximab resistance by amplifying surface CD20 crosslinking through enhancing surface CD20 expression following pretreatment with gemcitabine; by enhancing surface CD20 accessibility due to absence of Fc fragment; and by multivalency of P-(MORF2)_x) [374].

Efficacy of DMFT in cells isolated from patients with various B cell malignancies

Apoptosis induction by DFMT in cells from 44 patients with different B-cell malignancies, chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL), marginal zone lymphoma (MZL), follicular lymphoma (FL), mantle cell lymphoma (MCL), and Burkitt’s lymphoma (BL) was investigated. DFMT induced apoptosis in 65.9% of patient samples [375]. High-risk mutations such as 13q14, 17p13 and 11q22 deletions, which are considered poor prognostic factors in CLL, did not hamper the therapeutic efficacy of DFMT treatment [375]. On the other hand, in patient samples with poor response to DFMT treatment, low CD20 expression level was observed. Pre-treatment with gemcitabine enhanced surface CD20 expression and restored the cell responsiveness to DFMT [374]. DFMT effectively increased apoptosis of tumor cells from patients with a variety of B cell malignancies, irrespective of genomic aberrations. The results, in agreement with our early data [267,376], suggest that DFMT is a capable modality for the treatment of various B cell malignancies.

4.2.2. Second generation of DFMT

Rituximab (RTX) and other anti-CD20 antibodies (ofatumumab and obinutuzumab (OBN)) dramatically improved treatment of Non-Hodgkin lymphoma (NHL) and CLL. On their own or in combination with chemotherapy (e.g. R-CHOP, a combination with cyclophosphamide, doxorubicin, vincristine, and prednisone) they produced better clinical outcomes [366,377]. However, frequent relapses and poor patient outcomes demonstrate the need for improved treatment strategies. To this end, the second generation of DFMT for the treatment of B-cell malignancies was designed.

Type I (RTX) and Type II (OBN) anti CD20 antibodies have distinctive patterns of binding to the CD20 receptor. RTX binds between CD20 tetramers resulting in accumulation in lipid rafts, calcium influx and caspase activation [377]. OBN binds within one tetramer with the conformation consistent with homotypic adhesion regions, steering to actin cytoskeleton remodeling and lysosome disruption [378,379]. The second generation DFMT enhances the activity of Type II OBN by triggering the apoptosis activation pathways of both types of antibodies. The system consists of two nanoconjugates: a) bispecific engager, OBN-MORF1 (OBN conjugated to one morpholino oligonucleotide MORF1); and b) a crosslinking (effector) component HSA-(MORF2)_X (human serum albumin grafted with multiple copies of complementary MORF2). Modification of OBN with one MORF1 does not affect the binding of OBN-MORF1 to CD20 and following binding Type II effects occur. Further exposure to multivalent HSA-(MORF2)_X results in receptor crosslinking and clustering the OBN-MORF1-CD20 complexes into lipid rafts and Type I effects occur (Figure 9). This new approach, called “clustered OBN (cOBN)” combines effects of both antibody types resulting in very high apoptotic readings.

Figure 9.

Mechanism of apoptosis induction of second-generation drug-free macromolecular therapeutics (DFMT) (clustered obinutuzumab, cOBN): combination of Type I and II effects into one system). (A+B) Mechanism of action of cOBN. Following binding of OBN-MORF1 to CD20 Type II effects occur. Further exposure of cells with multivalent HSA-(MORF2)_x results in crosslinking of CD20 receptors, clustering the OBN-MORF1-CD20 complexes into lipid rafts and onset of Type I effects. (C) Caspase activity after Raji cell treatment with RTX, cRTX (clustered rituximab), OBN, or cOBN. *P < 0.05, n.s, not significant, by students t-test. (D) Comparison of whole antibodies (RTX and OBN) and their Fab’ fragments after multivalent binding of HSA-(MORF2)₁₀ to induce apoptosis. (E) Reactive oxygen species (ROS) generation after Raji cells were treated with RTX, cRTX, OBN, or cOBN. (G) Treatment of NRG mice bearing disseminated Raji B-cell lymphoma by clustered obinutuzumab (OBN-MORF1 followed by HSA-(MORF2)₁₀). Treatment schedule (dosing: OBN (0.5 nmol), or cOBN (OBN-MORF1, 0.5 nmol→HSA-(MORF2)₁₀ and 1.5 nmol MORF2, 5 h later); Paralysis-free survival; Residual Raji cells (human CD10+CD19+) in bone marrow (BM) following exposure to saline, OBN, or cOBN. BM cells isolated from native NRG mouse served as the negative control, and Raji cells served as the positive control. *P<0.05, **P<0.01, by students t-test. Adapted from ref. [380].

The design is scientifically novel and has great translational potential. Also, it provides a new paradigm for the design of macromolecular therapeutics applicable to other diseases beyond lymphomas [380].

In vivo activity of cOBN was tested on NOD/SCID-Rag1^null γ^null (NRG) mice bearing systemically disseminated Raji B-cell lymphoma. Vehicle (saline) treated mice became hind limb paralyzed at 19 days (median survival). OBN significantly extended the median survival to 57 days. Significantly, cOBN further prolonged the mice survival with the median survival of 83 days, and half of the mice were still paralysis-free after 100 days (Figure 9) without loss of body weight [380].

4.2.3. Other potential targets for DFMT

An excellent example of suitable targets for DMFT are death receptors 4 and 5 (DR4, DR5). The clinical inefficiency of therapeutic anti-DR5 antibodies stimulated the design of multivalent receptor crosslinking agonists [381–386]. Such multivalent nanoconjugates induce apoptosis without the need for FcγR-based crosslinking in contrast to antibodies that require crosslinking for exhibition of activity. Successful examples of multivalent conjugates are: Crosslinking of DR5 by drug-free immunoliposomes decorated by multiple copies of anti-DR5 Ab (TRA-8) or Fab’ antibody fragments triggers the employment of the death-inducing signaling complex and initiates apoptosis [381]. Huet et al. prepared multivalent nanobody DR agonists. Increasing the valency of domains to tetramer and pentamer resulted in enhanced potency for cell killing. Multivalent DR5 nanobodies were efficient in the treatment of patient derived pancreatic and colon cancer models [382]. A nanoconjugate composed of six single chain TRAIL-receptor-binding domains (scTRAIL-RBD) is a potent inducer of apoptosis in vitro and exhibited dose dependent anticancer activity in mice bearing Colo205 xenografts [385]. The augmentation of biorecognition of multivalent conjugates is strong. Trimeric and hexameric forms of DR5-binding peptide attached to adamantane-based dendrons augmented affinity about 1500 × and 20,000 ×, respectively when contrasted to the monomer. Only multivalent constructs induced apoptosis in DR5 expressing cells [386].

DMFT has a potential with additional receptors that are slowly internalizing, such as CD45 [387], prostate stem cell antigen [388], carcinoembryonic antigen [389], CD79b [390], and CD22 [391]. DMFT is also suitable for the treatment of diseases where CD20 crosslinking and B cell depletion therapy is suitable, such as rheumatoid arthritis [392,393], systemic lupus erythematosus [394], multiple sclerosis [395] and organ transplantation [396].

DMFT based on murine anti-CD20 Fab’-MORF1 and P-(MORF2)_X was effective in the treatment of collagen-induced rheumatoid arthritis in a mouse model [392].

4.2.4. E-selectin targeted drug-free macromolecular conjugates

Macromolecular therapeutics have a great potential to treat endothelial disorders in cancer and other diseases [397]. Efficient E-selectin binding peptides were identified and established their ability to manipulate biological processes in need of E-selectin interactions [398,399]. Notably, employing multivalent peptide – E-selectin interactions, the concept of drug-free therapeutics was applied also for prevention of metastasis and healing of microvascular and vascular inflammation.

David and coworkers used HPMA copolymer grafted with multiple copies of E-selectin binding peptide (ESBP; CDITWDQLWDLMK-CO-NH₂) to demonstrate that this conjugate hindered metastasis development in vivo by interrupting the interaction between cancer and endothelial cells [400]. In two independent experiments it was demonstrated that the drug-free conjugate was as efficient as the ESBP containing conjugate with drug also attached. P-ESBP (P is the HPMA copolymer backbone) was as efficient as the conjugate containing dexamethasone (P-ESBP-DEX) in the treatment of microvascular and vascular inflammation and in the inhibition of development and progression of atherosclerosis in atherosclerotic ApoE^(−/−) mice [401]. Similarly, P-(A5G27)_x an HPMA copolymer grafted with peptide A5G27 (RLVSYNGIIFFLK) binding to CD44v3 and CD44v6, inhibited cancer cell migration [211] and was as proficient in tumor growth inhibition as the P-(A5G27)_x-paclitaxel conjugate [402].

5. Beyond cancer

Polymer-drug conjugates are suitable for the treatment of non-cancerous diseases, such as musculoskeletal and infectious diseases [403]. Examples of such nanomedicines follow:

5.1. Bone delivery

Nanomedicines are an important part of bone therapeutics development. Bone and bone diseases can be targeted by nanomedicines containing acidic oligopeptides, bisphosphonates, tetracycline, and chelating compounds [403–405]. The location of binding to bone depends in the crystallinity of hydroxyapatite. D-aspartic acid octapeptide (D-Asp₈) would recognize resorption sites in skeletal tissues, whereas alendronate would direct bound cargo to both bone resorption and formation sites. This selectivity relates to the strength of the binding force between targeting moiety and bone as determined by atomic force microscopy [406].

HPMA copolymer – prostaglandin E₁ (PGE₁) conjugates targeted by D-Asp₈ were designed for the treatment of osteoporosis. The PGE₁ was bound to the carrier via a self-immolating spacer containing a cathepsin K sensitive Gly-Gly-Pro-Nle oligopeptide [42]. When the conjugate attaches to bone, the anabolic agent PGE₁ will be released at sites of higher osteoclast activity by cathepsin K mediated cleavage. EP receptors on bone cell surfaces will be activated by released PGE1 resulting in bone formation [407]. Pharmacokinetic and biodistribution studies as well as evaluation of bone formation in ovariectomized rats demonstrated efficacy of the PGE1 conjugates [208,408]. Interestingly, second generation, long-circulating, backbone degradable HPMA copolymer carriers of PGE1 were more effective in promoting bone growth in ovariectomized rats than first generation nondegradable conjugates [409].

Alendronate-targeted HPMA copolymer-TNP470 antiangiogenic conjugates [410,411] as well as alendronate-HPMA copolymer-paclitaxel conjugates [412] were successful in the treatment of animal models of bone cancer [410,411]. Similarly, a poly(oligoethylene glycol)methacrylate – combretastatin A-4 - alendronate conjugate exhibited bone recognition and antiangiogenic activity against HUVEC and U2-OS cells [413].

5.2. Inflammation

Water soluble polymer-drug conjugates are effective in the treatment of persistent and chronic inflammation, such as rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, and others [414]. Macromolecular therapeutics favorably build up in inflammatory tissues [403] especially in arthritis [415–417]. This phenomenon was named “ELVIS” (Extravasation through Leaky Vasculature and the subsequent Inflammatory cell-mediated Sequestration) suggesting the potential of polymer-drug conjugates in the treatment of inflammatory diseases [416,418]. Different biologically active molecules were attached to polymeric carriers to treat rheumatoid arthritis [419] including cathepsin K inhibitors [420], dexamethasone [421], and Janus kinase inhibitor [422].

A comparative study evaluated four DEX containing nanomedicines: liposome, core-crosslinked micelle, slow (drug) releasing HPMA copolymer-DEX conjugate, and fast-releasing HPMA copolymer-DEX conjugate. Following a single i.v. injection, nanomedicines with slower drug release kinetics (micelle and slow releasing HPMA copolymer-DEX conjugate) maintained longer duration of therapeutic activity in a rat model of adjuvant-induced arthritis than those with fast DEX release [423].

HPMA copolymer – tofacinitib conjugate [424] and HPMA copolymer – DEX (P-DEX) conjugate [425] were efficient in ameliorating dextran sulfate sodium induced ulcerative colitis. P-DEX also demonstrated more effective anti-inflammatory effects in the treatment of murine lupus nephritis in mice when compared to free DEX. P-DEX eliminated albuminuria, reduced macrophage recruitment to the kidney and extended the life span of mice [426]. The relationship between structure of P-DEX (molecular weight, DEX content) on one hand and pharmacokinetics and biodistribution on the other hand in mice in an aseptic implant loosening mouse model was recently evaluated [427].

Dextran [428] and PEG conjugates [429,430] were also effective in the treatment of inflammatory diseases on animal models.

6. Conclusions and future prospects

A young drug delivery scientist might be disappointed when reading several negative articles on the status and future prospects of nanomedicines [219,330]. To get a balanced non-biased picture we recommend to read opinions that represent the majority [228,327–329,331–333]. The facts are well described in recent reviews that summarize the status of the nanomedicine field and show successful current translation efforts, an area of frequent discussions [30,320,321,326]. Obviously, the drug delivery field, as any research area that is well funded, attracts “me-too” scientists. However, the majority of research in the area is based on serious efforts to move the science and translation of the research forward. As shown in several reports [328,329,331] not only results of basic research but also clinical translation has numerous success stories to present. Universities are not structured to translate research data into the clinics. This is the task of the industry. Enormous activities of start-up companies keep the pipeline strong. However, large pharma should not wait for the results of phase II clinical trials before getting involved. There should be a concerted effort to help start-up companies to overcome the “valley-of-the-death”. It would be naïve to anticipate that big pharma would do this voluntary. May be tax credit for such support could initiate the process.

The field of polymer-drug conjugates developed enormously and principles of design are matched with the knowledge of biological and immunological effects and delivery barriers these conjugates are exposed in vivo. Based on the results of early clinical trials second generation backbone degradable polymeric carriers were designed. A plentiful of polymer carriers with different structures are available for the creation of new nanomedicines. The general knowledge of the biological attributes of cancer, including heterogeneity of cancer cells, tumor microenvironment, and metastasis has significantly advanced [11,101,236–241]. In addition to systematic research in improving new conjugates and modifying the tumor and its microenvironment, imaging agents are being developed to select patients for clinical evaluation that will be amenable to the treatment with particular macromolecular therapeutics [229,231,232].

In addition, new paradigms in macromolecular therapeutics are emerging. They are an important part of the development and will open new horizons. Two new designs, based on receptor crosslinking, are discussed in this review. The first is the design of a multivalent polymer peptide antagonist (MPPA) to PD-L1. MPPA changes the subcellular fate of PD-L1. Instead of recycling it biases the PD-L1 to the endosomal/lysosomal route for targeted degradation in the lysosomes. The second example are drug-free macromolecular therapeutics (DFMT), a paradigm where apoptosis is initiated by receptor crosslinking without the need for a low molecular weight drug. This concept is already expanding by the design of E-selecting binding polymer-peptide conjugates active in prevention of metastasis and treatment of vascular inflammation, and by constructing the second generation of DFMT, a single platform that triggers two mechanisms of cell death.

We are confident that many new, more efficient macromolecular therapeutics will appear in the literature as a result of systematic basic research and new innovative designs of the future.