Recent Res. Devel. Polymer Science, 11(2012): 99 … PS B6.pdf · Polymer-metal complexes (PMC) for cancer therapy 101 of the conjugate to the normal sites of toxicity. Moreover,

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Review Article

Recent Res. Devel. Polymer Science, 11(2012): 99-128 ISBN: 978-81-7895-538-4

4. Polymer-metal complexes (PMC) for

cancer therapy

Andreia Valente1 and Philippe Zinck2 1Centro de Ciências Moleculares e Materiais, Departamento de Química e Bioquímica Faculdade de Ciências da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa

Portugal; 2Unity of Catalysis and Solid State Chemistry, UMR CNRS 8181, Polymerization Catalysis Group, Cité Scientifique, Bât C7, University of Lille 1, Science

and Technology, 59652 Villeneuve d’Ascq Cédex, France

1. Introduction

The finding of the EPR (“enhanced permeation and retention”) effect

100 years ago together with the discovery that macromolecules selectively

accumulate in the tumours relatively to healthy cells was a landmark in the

anticancer nanomedicine field. Indeed, these findings led to the development

of a new rationale to fight the cancer based on the EPR concept. This

phenomenon was first identified by Maeda et al. [1] and results in passive

accumulation of macromolecules and nanosized particles in solid tumour

tissues increasing the therapeutic index while decreasing side effects.

However, the Food and Drug Administration (FDA) has only

approximately one dozen of nano-therapeutics for cancer therapy approved

at the present [2-4]. Even if inorganic complexes offer more possibilities for

medicinal chemistry than organic drugs due to their vast coordination and

redox properties and ligand substitution rates, this area is much less explored

than organic polymeric drugs. Within this chapter we will essentially focus

Correspondence/Reprint request: Dr. Andreia Valente, Centro de Ciências Moleculares e Materiais

Departamento de Química e Bioquímica Faculdade de Ciências da Universidade de Lisboa, Campo Grande

1749-016 Lisboa, Portugal. E-mail: [email protected]

Andreia Valente & Philippe Zinck 100

in metallic polymer-drug conjugates bearing antitumor activity. There are

some PMCs in advanced phases of clinical trials, mainly, based on platinum

polymer conjugates [2-4]. In this frame, we will initially discuss the rationale

beyond the synthesis of the macromolecular drugs. We will describe the

different polymer synthesis and coordination to metal strategies as well as

the anticancer properties observed for these compounds.

1.1. Polymer-drug conjugate, definition

In polymer-drug conjugates (metallic and non-metallic) the drug is

covalently bound to a polymeric carrier, usually through a biodegradable

linker. This nanotechnology was proposed in the 1970s by Ringsdorf [5],

where he formulated the rationale for targetable polymeric drug delivery

(Figure 1) based on extensive analysis of earlier studies.

This knowhow was then developed pre-clinically in the 1980s [6,7] and

entered the clinical phase in the 1990s [2,8-11]. The main benefits of polymer-

drug conjugates compared to the parent free drug are: (a) passive tumour

targeting by the enhanced permeability and retention (EPR) effect; (b)

decreased toxicity; (c) capability of solubilisation in biological fluids of

insoluble low molecular weight compounds; (d) ability to overpass some

mechanisms of drug resistance; (e) ability to elicit immunostimulatory effects;

(f) stabilization and prolongation of the plasma half-life of the low molecular

weight drugs or proteins [12]. Nowadays, more than one dozen polymer-drug

conjugates have been clinically evaluated as anti-cancer therapeutics. The

reduced toxicity of the polymer-drug conjugate and the good anti-tumour

activities in resilient patients has clearly achieved a proof-of-concept status.

Most of these conjugates have used HPMA (N-(2-Hydroxypropyl)

methacrylamide) based polymers and copolymers as the water-soluble

polymer platform [2-4]. Drug conjugation to water-soluble polymers confines

cellular uptake to the endocytic pathway allowing tumour-specific targeting of

low molecular weight chemotherapeutic agents in addition to limiting the access

Cleavable linker

Drug

Solubilizing groupTargeting moiety

Figure 1. Concept of linear polymeric-drug conjugate systems with active targeting

functionality and solubilizing groups.

Polymer-metal complexes (PMC) for cancer therapy 101

of the conjugate to the normal sites of toxicity. Moreover, the use of a water-

soluble polymer also helps to solubilize hydrophobic drugs (e.g. doxorubicin

and paclitaxel), simplifying the formulation to administer intravenously.

The simplest polymeric drug delivery system is a complex formed between the drug and the polymer (Figure 2, linear polymeric-drug conjugate). The polymer must have suitable coordinating groups for the metal centre, for example, nitrogen or oxygen donors. The number of complexes that may be attached per polymer molecule depends on the number of coordinating groups present and the binding mode (denticity). The polymer-drug complexes should be water soluble and this can either be achieved using, for example, a diblock copolymer with a water soluble block such as poly(ethylene glycol) (PEG) or by lowering the loading of insoluble drugs to avoid precipitation and solubility issues. The drugs might also be linked to polymers through coordinating groups attached to the polymer via cleavable linking groups. This allows the release of the drug under specific conditions (for example, acidic pH in lysossomal/endossomal compartments). When the diblock copolymer comprises a hydrophilic and a hydrophobic chain, the polymer-drug conjugate can be self-assembled into micelles allowing high drug loading (Figure 2, Micelle). This multinuclearity can be also achieved using dendrimers, which are nanosized synthetic highly branched macromolecules (Figure 2, Dendrimers).

Hydrophilic block

Hydrophobic block

Drug

Self-assembled polymeric micelleDendrimer

Linear polymeric-drug conjugate

Polymer-drug conjugates

Figure 2. Polymer-drug conjugates covalently coordinated to each other.


The main variables in the design of polymer-drug conjugates are the

polymers molecular weight, coordinating groups number, the nature and size

of the solubilizing agents, and the drug loading onto the polymer. Ideally, a

polymeric system has low polydispersity, good biocompatibility and is made

via a reproducible synthetic procedure.

1.2. The EPR effect

Most of the approaches to the development of macromolecular drugs are

based on the EPR effect, by which macromolecules selectively accumulate in

malignant tissues compared to healthy tissues by either passive or active

targeting, thus precluding the undesirable side effects generated by free drug.

Importantly, macromolecules internalize into cells by endocytosis [13]. This

process is recognized to overcome the multi-drug resistance phenomenon that

renders tumours refractory to chemotherapy, and confers poor prognosis for

patient survival. In addition, due to the defective vascular structure of tumour

blood vessels they are permeable to macromolecules. Because of the decreased

lymphatic drainage, the permeate macromolecules are not removed efficiently,

and are thus retained in the tumour, while most conventional low molecular

weight anticancer drugs traverse in and out of blood vessels freely. This

phenomenon, first identified by Maeda et al. [14,15], is called the “enhanced

permeation and retention (EPR) effect” which is schematically represented in

Figure 3 and can increase the drug concentration in tumour compared to that

of the blood as high as 10-100 times [16,17].

Polymer-drug conjugate

Blood vessel

Ineffective lymphatic vessel

Lymphatic vessel

Passive targeting by EPR effect

Tumour

Endothelial cell

Figure 3. Blood vessel showing the defective vascular structure of tumour blood

vessels and the decreased lymphatic drainage, which allows the polymer-drug

conjugate to enter the tumour cells by the “enhanced permeation and retention (EPR)

effect”.


2. Platinum-polymer conjugates

In order to improve stability and the release control of platinum

compounds, several formulations using polymers containing multicarboxylic

acids [18-20] and β-cyclodextrin [21] to form stable complexes, an

enzymatically degradable bond for the drug-linkage [22,23] or a pH sensitive

chelate [24] have been explored. Generally, the platinum-polymer conjugates

synthesis involves the coordination of the polymer chain to the platinum

moiety, rather than polymerization of monomers containing platinum

complexes.

2.1. Poly-N-(2-hydroxypropyl)methacrylamide (HMPA) based

conjugates

The most commonly water-soluble, biocompatible and nontoxic polymer

used for the formulation of platinum conjugates is a backbone consisting of

poly-N-(2-hydroxypropyl)methacrylamide, which, when coordinated to the

platinum centre, originates the AP5280 conjugate. The tetrapeptide [GPLG

(glycine-phenylalanine-leucine-glycine)] spacer with the COOH-terminal

glycine is bound to an aminomalonic acid chelating agent that binds the

bioactive platinum complex. Its molecular weight is approximately 25000

g/mol and the pharmaceutical product contains approximately 8.5% Pt by

weight (w/w) (Scheme 1) [18]. This polymer conjugate was developed

aiming a long-circulating time and to be a targeted-selective agent taking

advantage of the EPR effect. The goal was to synthesize an agent with low

systemic toxicity that could increase the delivery of a cytotoxic platinum

moiety to tumours relatively to the usual Pt-containing drugs. Release of the

cytotoxic moiety was expected to occur intratumorally through the action of

extracellular and lysosomal thiol-dependent proteinases known to be

elevated in human tumours [18]. Actually, the ability of N-(2-

hydroxypropyl)methacrylamide polymers to passively accumulate in high

concentrations to tumours is well documented for experimental tumour

models [22]. In accordance with these findings, AP5280 was able to deliver

11-fold more Pt to tumour DNA than the maximum tolerated dose of

carboplatin (Scheme 1), and exhibited a substantially better therapeutic

index than the small molecules Pt drugs [18].

The results already obtained from Phase I clinical trials (AP5280 was

tested preclinically in the B16 melanoma and Lewis lung murine tumour

models and the UMSCC10b squamous cell head and neck and 2008

carcinomas xenograft models) show that some of these goals were achieved:

i) high quantities of Pt could be administered with only modest systemic


AP5280 Carboplatin

AP5346 Oxaliplatin

Scheme 1. Chemical structure of carboplatin, oxaliplatin and the polymer-metal

conjugate AP5280 and partial chemical structure of AP5346 (random copolymer

conjugated with SACH-platinum, where q:r:s is approximately 36-47:2.3:1.

toxicity (indication that native AP5280 itself and any other forms of Pt

resulting from its degradation have very low cytotoxicity potency); ii)

prolonged half-life for total plasma Pt; and iii) renal clearance. However, it

remains to be determined whether AP5280 actually increases delivery of Pt

to tumour DNA in humans and has a superior therapeutic index relative to

low molecular weight Pt drugs in cancer patients [18]. Still, AP5280 seems

to induce nephrotoxicity and myelosuppression at high doses, besides

prompting changes in hepatic enzymes consistent with hepatotoxicity [18].

In the case of AP5346 (ProlindacTM

), the rationale behind its synthesis

was to bound 1,2-diaminocyclohexane (DACH)-platinum to a

macromolecular carrier polymer via a pH-sensitive chelate (Scheme 1). The

chosen water-soluble biocompatible polymer backbone was a 90:10 random

copolymer of HMPA and a methacrylamide monomer substituted with a

triglycine aminomalonate group providing the primary binding site for the

DACH-platinum moiety [24]. The conjugate contains approximately 10%


platinum by weight (w/w) and has a molecular weight of 25000 g/mol, being

large enough to benefit from EPR effect. Platinum release studies showed

that, while in neutral solutions the release is negligible, at low pH (like for

example the intracellular lysossomal compartment) the DACH-platinum

moiety release is increased [25,26]. The antitumor activity of AP5346 has

been evaluated in several syngeneic murine and human tumour xenograft

models, like B16F10 tumours, 16 melanoma, Lewis lung, 2008 ovarian and

HT29 colorectal, between others [27]. Results showed that AP5346 produced

greater tumour growth inhibition and prolonged growth delay compared to

equitoxic doses of oxaliplatin and/or carboplatin in many cases [25,28]

Pharmacokinetic studies revealed that a single dose of AP5346 delivered

16-time more platinum to the tumour and 14-times more platinum to tumour

DNA than an equitoxic dose of free oxaliplatin (Scheme 1) [29]. Once again,

for high doses impairment of renal function and myelosuppression could be

observed [28]. However, no adverse events different from those already

identified for platinum cytotoxic agents were described [26].

2.2. Poly(γ,L-glutamic acid)-citric acid (γ-PGA-CA) based

conjugates

A cisplatin-loaded nanoconjugate, poly(γ,L-glutamic acid)-citric acid-

cisplatin [γ-PGA-CA-CDDP] was synthesized from γ-PGA-CA and CDDP,

through the displacement of chlorine ions on CDDP by hydrogen of carboxyl

groups on γ-PGA-CA side-chains [30]. Though there may be many possible

structures of γ-PGA-CA-CDDP conjugate because of the different

conjugated manners of CDDP the average particle size is 107 ± 6.3 nm and

average molecular weight is 66000 g/mol (determined by light scattering,

DLS, and gel permeation chromatography, GPC, respectively). These

features might allow a good internalization by the EPR effect.

Nanoconjugate release studies revealed that the platinum is released in a

sustained manner in PBS (Phosphate Buffered Saline) at 37 ºC. MTT

(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in

human breast cancer cell line BcaP-37 and human liver cancer cell line Bel-

7402 showed that γ-PGA-CA-CDDP had about 2 times higher IC50 values

than the unconjugated CDDP at an equivalent dose, confirming that the

conjugation of CDDP to γ-PGA-CA polymer reduced its in vitro

cytotoxicity. However the in vivo studies showed that the maximum

tolerated dose (MTD) of γ-PGA-CA-CDDP was about 5 times higher than

CDDP. Using near-infrared fluorescence (NIRF) imaging system, it was

demonstrated that γ-PGA-CA-CDDP gradually accumulated at the tumour

and showed higher antitumor activity in H22-implanted mice as compared to


CDDP. The conjugate revealed to be more stable, less cytotoxic and to have

a prolonged in vivo retention time. This features could be explained by i) the

particle size of the conjugate ( 107 nm) which makes it less exposed to

filtration by nephrons, the ii) long time reaction with the DNA in the tumour

cells relatively to normal cells due to the EPR effect, and the iii) sustained

release of CDDP from the conjugate which made the use of CDDP adequate

with no saturation of chloride ions.

2.3. Micellar platinum systems

Block copolymers comprising both hydrophilic and hydrophobic blocks

can spontaneously assemble into polymeric micelles. The pharmacokinetic

parameters of polymeric micelles are controlled by the copolymer

architecture. In this frame, the length of the micelle, core-forming block, not

only determines the drug loading capacity of the micelle but also contributes

largely to the physicochemical properties of the micelles. Accordingly, a

variety of self-assembling block copolymers has been explored as micellar

drug carriers.

2.3.1. Poly(ethylene glycol)-block-poly(glutamic acid) (PEG-b-P(Glu))

based conjugates (NC-6004)

Several PEG-b-P(Glu) bearing different P(Glu) block lengths (degree of

polymerization DPn of 20, 40 and 70) were synthesized (Scheme 2)

according to the procedure used for poly(ethylene glycol)-block-

poly(aspartic acid) described in the next section, using β-benzyl L-glutamate

N-carboxy anhydride instead of β-benzyl L-aspartate N-carboxy anhydride

[31]. The in vitro and in vivo biological properties of DACHPt/m micelles

prepared with [PEG-b-P(Glu)] were studied with the aim of optimizing the

biological performance of these polymer-drug conjugates [31,32].

Biodistribution studies and antitumor activity experiments in mice bearing

the murine colon adenocarcinoma C-26 inoculated subcutaneously showed

20-fold greater accumulation of DACHPt/m at the tumour site than free

oxaliplatin. Concerning the length of the copolymer blocks several

conclusions could be drawn: i) the micelles prepared from PEG-b-P(Glu)

with a P(Glu) DPn of 20 exhibited the lower non-specific accumulation in

the liver and spleen to critically reduce non-specific accumulation, resulting

in higher specificity to solid tumours; ii) liver accumulation of CDDP-loaded

micelles was reduced for the micelle formulation from PEG-b-P(Glu) with

longer PEG segment (coverage of the nanoparticles with PEG palisades is

likely to be a crucial factor in the reduced liver accumulation); iii) micelles


Scheme 2. Synthesis of DACHPt-loaded micelle (DACHPt/m). Reproduced from

[31] under fair use.

prepared with PEG-b-P(Glu) 12-20 showed considerably reduced toxicity

and, in particular, allowed a dosage increase (the use of PEG-b-P(Glu) with

shorter P(Glu) segments may allow the formation of DACHPt/m with

effective surface coverage by PEG, leading to reduction of the liver

accumulation of the micelles) [31]; and iv) DACHPt/m prepared with PEG-

b-P(Glu) 12-40 or 12-20 presented a remarkable and statistically relevant

in vivo antitumor activity, whereas free oxaliplatin failed to supress tumour

growth. The improved performance of DACHPt/m in the tumour could be

due to:

a) High and preferential accumulation of DACHPt/m in the tumour due to

the prolonged circulation of micelles in the bloodstream (10 times higher

than free oxaliplatin) as well as the aforementioned EPR effect;

b) Avoidance of permanent drug inactivation by protein binding through

the complexation of the platinum to the carboxylic groups in the micelle

core - since discharge of DACHPt products from the micelle core occurs

only after cleavage of the polymer-metal complex by chloride ions, and

this release is enhanced at low pH. DACHPt/m probably set up

conditions that favour the formation of active complexes of oxaliplatin,

including the highly active [DACHPt(H2O)2]2+

, leading to an improved

efficacy of the drug. Moreover, selective intracellular release of

DACHPt complexes might occur after internalization of the micelles by

endocytosis in cancer cells. As a result, DACHPt complexes may avoid

extracellular inactivation and may readily induce intracellular damage

[31].


In conclusion, decreasing the length of the core-forming block of

DACHPt/m improved their tumour specificity and drastically diminished

their toxicity [31].

2.3.2. Poly(ethylene glycol)-block-poly(aspartic acid) copolymer (PEG-b-

P(Asp)) based conjugates

Poly(ethylene glycol)-block-poly(aspartic acid) is obtained from the

alkaline hydrolysis of poly(ethylene glycol)-block-poly(β-benzyl L-aspartate)

[33]. The latter block copolymer is formed by polymerization of β-benzyl

L-aspartate N-carboxy anhydride initiated by α-methyl ω-amino

poly(ethylene glycol) as represented in Scheme 3 (top).

CDDP-loaded block copolymer micelles can be formed through the

complexation between CDDP and PEG-b-P(Asp), in aqueous medium [34-

36] through the aspartic residues by ligand substitution reaction at platinum

atoms of CDDP (Scheme 3, bottom) [34]. At a molar ratio of CDDP and an

aspartic acid residue of 1:1, polymeric micelles are formed with an average

diameter of 16 nm by reaction in distilled water at 37 ºC for 48 h. The

polymeric micelle is easily purified by ultrafiltration and a micellar structure

of this fraction is stable in distilled water and NaCl solution at 37 ºC for 24 h.

Ligand exchange reactions by water molecules and Cl- ions were not

observed and studies revealed that bridge formation through Pt atoms

chelated by two carboxylates of different polymer chains contributed to

micelle formation. The polymeric micelle showed 1/8 to 1/5 cytotoxicity of

intact cisplatin against murine B16 melanoma cells during 24-72 h

incubation, suggesting release of platinum complexes from the micelle [34].

The CDDP-loaded micelles showed approximately sevenfold higher IC50

in Lewis lung carcinoma (LCC) cells, comparatively to free CDDP. Thus,

the micellization reduces the in vitro cytotoxicity activity of CDDP [35].

However, after preincubation in physiological saline, the CDDP-loaded

micelles nearly recovered the cytotoxic activity, indicating that Pt

compounds released from the micelles through the ligand substitution

reaction still preserve antitumor activity almost comparable with that of free

CDDP. Hence, CDDP-loaded micelles are expected to have a long

circulation in the bloodstream and eventually to accumulate in higher

concentrations in the solid tumour while maintaining the cytotoxic activity

of CDDP [35]. This was further confirmed by pharmacokinetic studies in

Lewis lung carcinoma-baring mice which revealed the tumour-specific

distribution of CDDP-loaded micelles while avoiding the accumulation and

cytotoxicity activity of CDDP in the kidney [35].


Scheme 3. Poly(ethylene glycol)-block-poly(aspartic acid) synthesis (top) and

introduction of cisplatin into PEG-b-P(Asp) block copolymer (bottom) [33,34].

2.3.3. Tri-block copolymers-based conjugates

The biodegradable amphiphilic tri-block copolymer MPEG-b-PCL-b-

PLL (monomethoxy poly(ethylene glycol)-block-polycaprolactone-block-

poly(N-hydroxy-succinimide), containing pendant amino groups was

coordinated to a Pt(IV) complex in order to form a cisplatin(II) prodrug,

MPEG-b-PCL-b-PLL/Pt(IV) [37]. The polymer synthesis is presented in Scheme 4. Briefly, monomethoxy poly(ethylene glycol) was used as initiator

for the Sn(Oct)2 catalyzed ring opening polymerization of ε-caprolactone.


Scheme 4. Biodegradable amphiphilic tri-block copolymer MPEG-b-PCL-b-PLL

synthesis, reproduced from [37] under fair use.

Upon activation of the hydroxyl end group of the resulting MPEG-block-

PCL with mesyl chloride and reaction with NaN3, terminal azido groups

were formed. The latter were reduced to amino groups by catalytic

hydrogenation over Pd(OH)2/C. The resulting amino end functionalized

MPEG-block-PCL was then used as initiator for the ring opening

polymerization of ω-carbobenzoxy L-lysine N-carboxy anyhydride. The

resulting triblock copolymer was finally deprotected using

CF3COOH/HBr/CH3COOH to yield MPEG-b-PCL-b-PLL.

After the Pt(IV) complex synthesis [38] (with a COOH-functionalized

axial ligand, Scheme 5), the amphiphilic block copolymer which synthesis

was described here above was covalently coupled [37]. The polymer-drug


Scheme 5. Pt(IV) complex used to the MPEG-b-PCL-b-PLL/Pt(IV) conjugate

synthesis.

conjugate was able to self-assemble into micelles with a mean diameter of

150-160 nm, a surface potential less than + 10 mV, a CM of ca. 0.01 g/L

(critical micelle concentration) and a platinum content over 10 wt%. As

observed in aforementioned cases, the drug release from the polymer-Pt(IV)

micelles follows an acid responsive and oxidation-reduction sensitive kinetics

(data from HPLC-ICP-MS analysis, HPLC-Inductively Coupled Plasma Mass

Spectrometry); these conditions can be fulfilled inside a cancerous cells. In

vitro MTT assays showed that the polymer-Pt(IV) micelles display higher

cytotoxicity against SKOV-3 ovarian carcinoma cells than both cisplatin and

polymer-Pt(IV) complex. This enhanced cytotoxicity was attributed to the

effective internalization of the micelles into the cells by endocytosis (cellular

uptake was observed by fluorescence imaging).

2.4. Platinum based dendrimers

The idea behind the synthesis of metallodendrimers is the concept of

multinuclearity, i.e., the cytotoxicity of a drug can be improved by the

increase of the metal centres in a molecule. In this frame, dendrimers are an

emerging field in metal-based drugs due to their multimeric scaffolds [39].

Dendrimers are nanosized synthetic highly branched macromolecules which

arise from a central core in a fan out or in a concentric, layered and well-

defined architecture. Dendrimers are also characterized by having narrow

molecular weight distributions and being easily functionalizable.

Metallodendrimers contain metals, which ancilliary ligands are coordinated

to the periphery of the dendrimer, interspersed throughout the dendritic

framework or encapsulated within the dendrimer (Figure 4). Through

synthesis, the size, molecular weight or the introduction of other molecules

of interest can also be controlled or tuned at nanometer scale.


(b) (c)(a)

Figure 4. Schematic representation of metallodendrimers containing metals at (a) the

periphery, (b) interspersed throughout the framework or (c) encapsulated within the

framework; = metal centre.

In accordance with other polymer-drug conjugate therapeutics, the area

of metallodendrimers is mostly dedicated to platinum with some reports in

initial study phases using cooper [40], palladium [41], gold [42], tungsten

[43] and ruthenium (the latter will be discussed in section 3.1.1).

In order to amplify the effect of cisplatin on DNA through binding of the

metal to the guanine bases, researchers aimed to increase the number of

interstrand crosslinks between the drug and DNA, i.e., increasing the number

of DNA-binding moieties. In this frame the trinuclear complex [µ-trans-

Pt(NH3)2{trans-PtCl(NH3)2{NH2(CH2)6-NH2}}2][NO3]4 (BBR3464, Scheme

6) [44], seemed adequate. Unfortunately, the good results obtained

overcoming resistance to cisplatin were not enough to complete phase II of

clinical trials.

The nontoxic anionic polyamidoamine (PAMAM) dendrimer with a

sodium carboxylate surface of generation 3.5 was coordinated to cisplatin

through the dendrimer carboxylate groups affording one of the first

dendrimer-platinum conjugates with a Pt loading of almost 25 wt.-% [45].

Selective accumulation of this macromolecular drug in the solid tumour was

observed and was explained on the basis of the EPR effect.

The first tetranuclear platinum-functionalized metallodendrimer DAB(PA-tPt-Cl)4 was based on the first-generation PPI dendrimer [1,4-diaminobutanepoy(propyleneimine)] (Scheme 6) [46].

Similarly to BB3464,

DAB(PA-tPt-Cl)4 was able to bind to four molecules of the model nucleobase GMP (guanine-5’-minophosphate) at the N7 position and showed moderate cytotoxicity against L1210/0 (IC50 = 12.4 µM) and L1210/2 (IC50 = 9.3 µM) mouse leukemia cell lines and seven human tumour cell lines (IC50 = 9 µM). The fairly low cytotoxicity was attributed to the high charge and branching of the metallodendrimer, which could hinder the passive diffusion of the molecule across the hydrophobic cell membrane and into the cell. However these results seem somehow contradictory since the trinuclear complex BBR3464 (with a charge of +4) showed a more efficient uptake in L1210 cells.


BBR3464

DAB(PA-tPt-Cl)4

(m-4F-PtDMSO)4DAB(PA)4 Scheme 6. Structures of BBR3464, DAB(PA-tPt-Cl)4 and (m-4F-PtDMSO)4

DAB(PA)4

The tetranuclear metallodendrimer (m-4F-PtDMSO)4DAB(PA)4 based

on the first-generation PPI dendritic scaffold, functionalized with {1,2-bis(4-

fluorophenyl)ethylenediamine}platinum(II) moieties in the periphery

(Scheme 6) showed an increased selectivity for breast tumours [47]. The

compound presents a cytotoxicity of IC50 = 5 µM in MCF7 human breast

cancer cell line, comparable to cisplatin (IC50 = 2 µM). Cellular uptake and


DNA-binding experiments showed that the metallodendrimer has a 20-fold

higher cellular uptake and 700-fold higher DNA binding than cisplatin.

Likewise, a recent report describes the peripheral functionality of platinum

(II) moieties to a series of polyamidoamine denditric scaffolds [48].

2.5. Platinum (IV)-coordinate polymers

Platinum (IV)-coordinate polymers were synthesized by condensation

polymerization using diaminedichlorodihydroxyplatinum (DHP) or its

dicarboxyl derivative diaminedichlorodisuccinatoplatinum (DSP) as

comonomers (Scheme 7) [49]. These conjugates were synthesized with high

platinum contents (27.7 % for P(DSP-EDA) and 29.6 % for P(DSP-PA)).

The Pt(IV) polymers were found to be very stable in water and thus should

be stable during transport in the bloodstream. Pt(IV) dicarboxylates are

intracellularly reduced to Pt(II) and lose their two carboxylate ligands.

Therefore, the authors proposed that the Pt(IV) conjugates might be

degraded by similar intracellular reduction and released to Pt(II). The

reduction potentials of DSP and the three platinum(IV)-coordinated

polymers at two different pH values (pH 6.0 and pH 7.4) were thus

determined by cyclic voltammetry studies. The reduction of DSP was not

sensitive to the acid environment. However, the reduction potentials of

P(DSP-EDA) were -0.43 V at pH 6.0 and -0.62 at pH 7.4 and those of

P(DSP-PA) were even lower, -0.31 V at pH 6.0 and -0.58 V at pH 7.4. These

data suggest that Pt(IV) centre in the polymers was much easier to be

reduced to Pt(II) than DSP, particularly at acidic pH, for instance the pH at

Scheme 7. Synthesis of Pt(IV)-conjugated polymers [49].


the endosomes/lysosomes. Thus, these polymers could be explored as

intracellular reduction-responsive backbone-type polymer conjugates that

could be degraded and release Pt(II). The cellular uptake and intracellular

localization of P(DSC-EDA) in SKOV3 ovarian cancer cells was observed

using a confocal laser scanning microscopy (CLSM). The conjugate was

found to be mainly localized in the lysosomes. MTT studies in ovarian and

breast cancer cell lines (MDAMB468, SKOV3 and MCF7) demonstrate that

P(DSP-EDA) and P(DSP-PA) exhibited significantly higher cytotoxicity

than platinum (IV)-coordinated DSP monomer (Table 1). However, caution

must be taken when comparing these data due to the multinuclearity of these

Pt(IV) coordinate polymers. In vivo, the conjugate showed a longer blood

circulation time, better tumour accumulation and faster blood clearance than

DSP monomer.

Table 1. IC50 values (μM) found for DSP, P(DSP-EDA) and P(DSP-PA) in human

MCF7, MDAMB468 breast and SKOV3 ovarian cancer cells. The conjugates were

incubated with cells for 72 h and post-treated for another 24 h [49].

MCF7 MDAMB468 SKOV3

DSP 319.6 46.9 351.4

P(DSP-EDA) 156.2 19.9 104.9

P(DSP-PA) 198.9 16.7 47.5

3. Ruthenium-polymer conjugates

Ruthenium complexes are nowadays established alternatives to Pt-based

complexes in cancer therapy showing different mechanisms of action and

spectrum of activity, and possessing potential to overcome platinum-

resistance, as well as lower toxicity [50-55]. To date there are not yet

commercially available ruthenium drugs, however, there are two important

examples which are now in Phase II clinical trials, namely KP1019

([HInd][trans-RuIII

Cl4(Ind)2], Ind = indazole) [56] and NAMI-A

([HIm][trans-RuIII

Cl4(DMSO)Im], Im = imidazole) [57].

Ruthenium complexes present a high potential in the medicinal field,

since they present several properties that make them attractive within this

area:


i) Multiple oxidation states (II, III and IV) accessible under physiological

conditions;

ii) Favourable ligand-exchange kinetics with low toxicity;

iii) Antitumour activity both in vivo and in vitro studies, as well as

antimetastatic and intrinsic angiostatic activity;

iv) Multiple cytotoxic routes involving the competing processes of

extracellular protein binding (active transport), and/or cellular uptake

(passive diffusion).

3.1. Multinuclear approaches

3.1.1. Ruthenium based dendrimers

Recently, a series of first- and second-generation monodentate (N-donor)

ruthenium(II)-arene (arene = p-cymene or hexamethylbenzene)

metallodendrimers based on poly(propyleneimine) dendritic scaffolds was

published in order to exploit the EPR effect [58]. The dinuclear arene

ruthenium complexes [(arene)RuCl2]2 reacts with the dendritic scaffolds by

stirring at room temperature in dichloromethane to yield the neutral

tetranuclear and octanuclear ruthenium metallodendrimers (Scheme 8). The

cytotoxicity of the metallodendrimers was evaluated against A2780 human

ovarian cancer cells after an incubation period of 72 h; the complexes showed

moderate anti-proliferative activity (between 20-40 µM per

metallodendrimer) relative to cisplatin (1.5 µM) for the same cell line. Since

there are studies showing that cationic dendrimers are able to interact with

the cell surface, enter the cytoplasm and reach the nucleus over a short

period of time [59], a second series of metallodendrimers, containing

tetranuclear and octanuclear chelating neutral (N,O) and cationic (N,N) first-

and second-generation ruthenium(II) arene (arene = p-cymene or

hexamethylbenzene) metallodendrimers based on poly(propyleneimine)

dendritic scaffolds, were synthesized from dinuclear arene ruthenium precursors by reactions with salicylaldimine and iminopyridyl dendritic

ligands (Scheme 9) [60]. The antiproliferative activity of the chelating

ruthenium-arene dendrimers was also evaluated against the A2780 (cisplatin

sensitive) and A2780cisR (cisplatin resistant) human ovarian cancer cell

lines. All complexes showed moderate anticancer activity, the octanuclear

complexes being more cytotoxic than the tetranuclear ones suggesting a

correlation between the size of the metallodendrimer and the cytotoxicity.

DNA-binding experiments by gel electrophoresis with pBR322 plasmid

DNA showed that the second-generation N,N-ruthenium-hexamethylbenzene

metallodendrimer interacted more efficiently with DNA than cisplatin.


R = p-cymene or hexamethylbenzene

Scheme 8. Tetra- and octanuclear arene ruthenium dendritic systems [58].

3.1.2. Ruthenium based metalla-cage conjugates

In the search of new anticancer compounds, two generations of

lipophilic pyrenyl functionalized poly(benzylether) dendrimers have been

encapsulated into the arene-ruthenium metalla-cage, [Ru6(p-cymene)6(tpt)2

(donq)3]6+

, [P1 1]6+

(tpt = 2,4,6-tri-(pyridine-4-yl)-1,3,5-triazine; donq = 5,8-

dioxydo-1,4-naphthoquinonato) [61]. The host-guest systems [Pn 1]6+

were


R = p-cymene or hexamethylbenzene

n = 4 or 8 Scheme 9. Tetra- and octanuclear chelating neutral (N,O) and cationic (N,N)

ruthenium(II) metallodendrimers [60].

prepared using a two-step strategy. Firstly, the dinuclear complex [Ru2(p-

cymene)2(donq)Cl2] was reacted with AgCF3SO3 affording the dinuclear

intermediate. Then, the tpt and the guest molecule (Pn) were added to obtain

the corresponding inclusion compounds. The resulting hexacationic host-guest

systems were obtained in good yield (80 %) as triflate salts [Pn 1][CF3SO3]6

(Scheme 10). Both the metalla-cage [1]6+

and the [P1 1]6+

host-guest system

are water-soluble and exhibit a similar cytotoxicity in the A2780 cell line. The

results did not clarify if the guest is released or not after cellular

internalization of the host-guest systems. “Per metal” [1]6+

and [P1 1]6+

are

less cytotoxic than cisplatin.

[Pn 1]6+

Scheme 10. Ruthenium metalla-prism [Pn 1]6+ [61].


A series of large pyrenyl-containing dendrimers of different generations

have been also encapsulated into the hydrophobic cavity of a hexanuclear

arene-ruthenium cage complex [Ru6(p-cymene)6(tpt)2(donq)3]6+

and [Ru6

(p-cymene)6(tpt)2(dotq)3]6+

(dotq = 6,11-dioxydo-5,12-naphtacenedionato)

[62,63]. The IC50 values in A2780 and A2780cisR human ovarian cancer cell

lines revealed that all the different generations were cytotoxic against these

cancer cell lines. The toxicity seemed to be inversely related with the size of

the dendrimer in the majority of cases, i.e., the smaller the dendrimer, the

lower the cytotoxicity.

3.1.3. Ruthenium(II)-coordinate polymers

In a preliminary study, RAPTA-C, [RuCl2(p-cymene)(PTA)] bearing the

water soluble 1,3,5-triaza-7-phosphaadamantane (PTA) ligand was attached

to the polymer moieties to create water-soluble macromolecular drugs [64].

Poly(2-chloroethyl methacrylate) (PCEMA) and Poly(2-chloroethyl

methacrylate-co-N-(2-hydroxypropyl) methacrylamide) (P(HPMA-CEMA))

were synthesized by RAFT (Reversible Addition-Fragmentation chain

Transfer) polymerization using cumyldithiobenzoate as RAFT agent

(Scheme 11). The chloride end-group was converted to an iodide end-group

Scheme 11. Synthesis of macromolecular ruthenium complex copolymer-RAPTA-C

in DMSO-d6 [64].


by reaction with NaI, yielding PIEMA and P(HMAP-co-IEMA) that were

recovered by dialysis. Two pathways were employed to conjugate RAPTA-

C to the PIEMA, either by synthesis of the complex and subsequent

conjugation to the polymer, or by attachment of PTA to the polymer and

subsequent complexation to give the polymer RAPTA-C macromolecule.

However, the high temperature needed in the direct reaction of RAPTA-C

with the polymer led to the loss of the p-cymene ligand, while in the two

step reaction the amount of iodide reacted was only 50%. Yet, this way was

clearly identified as the preferred pathway. The two-step reaction with PTA

and RuCl2(p-cymene) dimer led to a water-soluble polymer, P(HPMA172-

IEMA44-(RAPTA-C-EMA)44) (Scheme 11), with an intense orange colour,

indicative of the pendant RAPTA-C complex (the macromolecular

ruthenium complexes were synthesized in DMSO-d6 and purified by

dialysis). The cytotoxicity profile for the copolymer-RAPTA-C product was

measured on the ovarian cancer cell line OVCAR-3 and compared with the

profile of the lone RAPTA-C drug. The results revealed high IC50 values and

further work is needed to enhance toxicity.

3.2. Mononuclear approaches

Most of the problems encountered within the synthesis of drug-carrier

conjugates are related to the requirement of a chemical bond between the

drug and the polymer or the encapsulation of drugs into drug carriers. Since

these synthesis procedures are generally multi-step and complex, sometimes

poorly reproducible, and with loss of drug activity in some cases, only some

drug-carrier conjugates have been clinically used, despite the promise of

enhancing in vivo drug efficacy. It is thus of prime importance to develop

new and simpler strategies to conjugate drugs to the carriers without using

tedious processes [65].

Taking into account previous good results obtained with RuIICp (Cp =

cyclopentadienyl) low molecular weight drugs [66-71], new polymer-conjugates having this organometallic moiety started to be studied. In fact, cytotoxic studies against human colon adenocarcinoma (LoVo and HT29), pancreatic (Mia PaCa), promyelocytic leukemia (HL-60), breast (MDAMB231 and MCF7), prostate (PC3) and ovarian (A2780 and A2780cisR) cancer cell lines, revealed IC50 values in the nano- and micromolar range for this family of Ru

IICp drugs [66-71]. Apoptosis was

found to be the dominant process, similar to that of cisplatin, in all the studied cases. In addition, the good stability in aqueous solutions verified for the [CpRu(P)(bpy)]

+ family (P = phosphane coligand, bpy = bipyridine)

stimulated the use of this organometallic core to search for new polymer-


metal complexes as potential anticancer agents. Thus, the unprecedented synthesis of RuPMC and its preliminary in vitro results have been recently published [72]. [CpRu(P)(bpyPLA)]

+ (Cp = cyclopentadienyl, P =

triphenylphosphane and bpyPLA = 2,2’-bipyridine-4,4’-D-glucose end-capped polylactide) has been synthesized in good yield by halide abstraction from [Ru(η

5-C5H5)(PPh3)2Cl] with silver triflate, refluxing for 3 h in

dichloromethane in the presence of the bpyPLA macroligand (Scheme 12). The bpyPLA was obtained in good yield (74 %) by the synthesis between 2,2'-bipyridine-4,4'-dicarbonyl dichloride and D-glucose functionalized PLA, which synthesis was adapted from [73]. Initially, the three secondary hydroxyl groups of D-glucose were selectively protected. The resulting monosaccharide yielding a primary hydroxyl group was used as initiator for the organocatalyzed ring-opening polymerization of lactide, leading to D-glucose end-capped PLA. The hydroxyl group located at the other chain end of the polymer was then involved in a coupling reaction with a bypiridine dithionyl chloride derivative leading to the targeted macroligand. The molecular weight of the polymer can be easily tuned via controlled polymerization processes, such as ring-opening polymerization, by playing on the monomer /initiator ratio. An acidic pH dependent hydrolysis of the polylactide was advanced by UV-Vis. studies at pH 7.4 and pH 5 (in 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer). Such behaviour might be important considering drug delivery since i) the measured pH of most solid tumours range from pH 5.7 to pH 7.2 while in normal blood it remains well-buffered and constant at pH 7.4 [74]; ii) this feature of the polymer degradation discards the need for a biodegradable linker and provides the opportunity for site specific drug delivery, mainly within endosomal/lysosomal compartments [75-77].

Scheme 12. D-Glucose end-capped polylactide ruthenium-cyclopentadienyl (RuPMC)

synthesis [72].


The cytotoxicity of RuPMC was assayed in human MCF7 and MDAMDB231 breast and A2780 ovarian adenocarcinoma and was found to be active against all tested cancer cell lines and is 7-fold more cytotoxic than cisplatin for the MCF7 breast cancer cell line. Although the direct comparison of the IC50 values between RuPMC and its low molecular weight parent drug [Ru

II(

5-C5H5)(bipy)(PPh3)]

+ (TM34, Scheme 13) reveals a slight decrease

on the cytotoxicity of RuPMC (3.9 vs. 0.29 μM), it might be expected that the prolonged plasma half-life of the RuPMC can considerably improve the chemotherapeutic efficacy, and thus, decrease the necessary dose for treatment, allowing a positive final outcome, as it has been described for many platinum-related compounds (see platinum section). The cellular distribution of RuPMC was studied with the MCF7 breast cancer cells by ICP-MS. The total ruthenium uptake by cells was mostly localized in the nucleus (50.5 %) and in the membrane (39.8 %). Only 2.9 % was found in the cytosol and 6.7 % in the cytoskeletal. These results can forecast RuPMC potential targets, such as DNA. It also indicates that RuPMC should have quite a different mechanism of action from TM34 since this compound is mainly found in the membrane (ca. 80 %) [71]. This new RuPMC seems a viable candidate for the intended drug-delivery application, however further studies are needed to prove its higher in vivo accumulation in cancer cells.

TM34

Scheme 13. Structure of the low molecular weight TM34 [68].

4. Conclusion

Cancer therapy must be seen as a multidisciplinary challenge demanding

close collaboration among clinicians, biological, chemical, biochemical and

material scientists. In this frame, Polymer-Metal Complexes can potentially

be seen as efficient drug-delivery systems in order to shorten the gap between

drug discovery and drug delivery. Despite all the good advances in the area of

polymer-metallic drug conjugates there is still the need to develop high-


molecular-weight biodegradable polymeric carriers that can better exploit

EPR-mediated tumour targeting. There is a serious need to move away from

heterogeneous and random-coiled polymeric carriers towards better defined

polymer structures. In this frame, controlled/living polymerizations, such as

controlled radical polymerization (e.g. ATRP, RAFT) and ring-opening

polymerization have been used providing stimuli-responsive polymers with

narrow molecular weight distributions. Polymers that start to degrade under

acidic conditions are being increasingly explored since the drug release can be

triggered by the slightly acidic tumour environment or after the internalization

by cells resulting in the accumulation of the polymer in the acidic endosomes

and lysosomes. In this frame, PEG-polyacetals that show pH-dependent

degradation and cyclodextrins that are degraded by amylase might be two

practical options for such carriers.

Another approach has been the introduction of multinuclearity trying a

synergic effect between the EPR affect and an increased cytotoxicity.

Dendrimers and dendronized polymers combine the features of

monodisperse nanoscale geometry with high end-group density at their

surface, and appear as attractive options for coordination of anticancer drugs.

Also, other supramolecular assemblies (non-polymeric) like arene-ruthenium

nano-prismatic cages [78] and ruthenium-based nanoparticles [79] showed

cytotoxicity against several cancer cell lines and can be considered

promising alternatives in chemotherapy. In the first case, the IC50 values for

the SK-hep-1 (liver cancer), HeLa (ovary cancer), HCT15 (colon cancer),

A549 (lung cancer) and MDAMDB231 (breast cancer) were sometimes

lower than cisplatin [78], while the nanoparticles showed moderate activity

towards A2780 ovarian cancer cell line and the cisplatin resistant variant

A2780cisR [79]. In the frame of photodynamic therapy Ru(II) porphyrins

conjugates showed cytotoxic activity in human cervical carcinoma (HeLa),

in MDAMB231 human breast cancer cells and Me300 melanoma cells,

constituting also an encouraging alternative [80].

As explained above, some of the problems encountered in the

development of new covalently bound polymer-metal conjugates lies on the

multi-step, complicated and with poor reproducibility synthesis, which cause

many times an inevitable loss of drug activity. In this frame it is urgent to

develop a simple strategy for coordinating drugs to the carriers. This was

overcome using a one-step coordination strategy in ruthenium

cyclopentadienyl derivatives, where the cytotoxicity of the final polymer-

ruthenium conjugate was maintained [72].

As it would be expected, the literature concerning polymer-metal

complexes for drug delivery applications is mainly dedicated to platinum

drugs. Between them, the polymer–metal complex of oxaliplatin has been


approved for treatment of malignant tumours including colorectal cancer in

2003 [81]. Nevertheless, ruthenium-conjugates seem a promising alternative

though many studies must be done. Almost all the cytotoxic studies were

performed only over one cancer cell line (namely human ovarian A2780)

and there is still the need to present in vivo studies in order to have a proof of

concept, i.e., if these new polymeric drugs are indeed better than their low

molecular weight parenteral drugs by the so-called EPR effect.

Finally, it is hoped that the use of receptor-targeting ligands will lead to

improved tumour targeting through the EPR effect achieving polymer-drug

conjugates acting as the well-aimed arrows of William Tell: the basic

“polymeric structures” being the “well-aimed arrows” which accurate shots

will be guaranteed by specific compounds on their arrowheads (targeting

vectors) that will be recognized by the cancer cell (Figure 5). As the well-

aimed arrow of William Tell thrashing the apple, these accurate “metallic-

arrows” tailored with targeting vectors will efficiently shot to the specific

site: THE CANCER CELL.

“Metallic moiety”

Polymeric Chain

Targeting Biomolecule

Figure 5. “Metallic-arrows” tailored with targeting vectors which will efficiently

shot to the cancer cells.

5. Acknowledgments

Professor M. Helena Garcia and Dr. Fernanda Marques are gratefully

acknowledged for careful reading. Andreia Valente thanks the Portuguese

Foundation for Science and Technology for her postdoctoral scholarship

(SFRH/BPD/80459/2011).


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