# Synthetic Polymers as Drug-Delivery Vehicles in Medicine

Authors: Eberhard W. Neuse
Journal: Metal-Based Drugs
 (2008)

---

## Abstract

Cancerous diseases present a formidable health problem worldwide. While the
chemotherapy of cancer, in conjunction with other treatment modalities, has reached a
significant level of maturity, efficacious use of such agents is still restricted by numerous
pharmacological deficiencies, such as poor water solubility, short serum circulation
lifetimes, and low bioavailability resulting from lack of affinity to cancer tissue and
inadequate mechanisms of cell entry. More critically still, most drugs suffer from toxic
side effects and a risk of drug resistance. The class of platinum anticancer drugs,
although outstandingly potent, is particularly notorious in that respect. Among the
countless methods developed in recent years in an effort to overcome these deficiencies,
the technology of polymer-drug conjugation stands out as a particularly advanced
treatment modality. The strategy involves the bioreversible binding, conjugating, of a
medicinal agent to a water-soluble macromolecular carrier. Following pharmacokinetic
pathways distinctly different from those of the common, nonpolymeric drugs, the
conjugate so obtained will act as a prodrug providing safe transport of the bioactive
agent to and into the affected, that is, cancerous cell for its ultimate cell-killing activity. The
present treatise will acquaint us with the pharmacological fundamentals of this drug
delivery approach, applied here specifically to the metalorganic platinum-type drug
systems and the organometallic ferrocene drug model. We will see just how this
technology leads to conjugates distinctly superior in antiproliferative activity to cisplatin,
a clinically used antitumor agent used here as a standard. Polymer-drug conjugation
involving metal-based and other medicinal agents has unquestionably matured to a
practical tool to the pharmaceutical scientist, and all indications point to an illustrious
career for this nascent drug delivery approach in the fight against cancer and other
human maladies.


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## Body

## 1. Introduction

The delivery of medicinal agents to the human body for healing purposes is probably as old as mankind. While
in the early days such agents, now generally known asdrugs, were simply delivered
from hand to mouth, perhaps also by rubbing into the skin, or placing on an open
wound, we are using more numerous and doubtlessly more complicated
modalities of delivery in modern times, simply prompted by the need to control and combat a
hugely increased variety of identified infirmities. A new stage of chemotherapeutic
development was attained, more than a quarter of a century ago, with the advent of
the polymer-drug conjugation paradigm, which teaches the use of polymeric, that
is, macromolecular compounds as pivotal helpers in drug delivery. Numerous delivery
techniques, taking advantage of the functional assistance provided by a macromolecular
partner, have since been successfully developed for various routes of
administration, not only the time-proven oral route (“just swallow it!”), but
more importantly the
parenteral routes involving intravenous, intraperitoneal, or intramuscular methods. It is
particularly these parenteral administration techniques that have led to the successful use of
partaking polymers. We will learn more about those developments further down the road
in this paper. Although metal-free drug systems will find due mentioning in this
treatise, emphasis will be on metal-containing compounds in accordance with this
journal’s mission. Before we delve into this core topic, we are prudently advised to
take a primer lesson on the pharmacokinetic pathways generally available to a drug—any drug—after its administration to the mammalian
organism, especially the human
body. Section 2 will guide us leisurely on this learning path.


## 2. What We Should Know about Drug  Administration

### 2.1. The Cancer Problem

Malignancies and
cardiac diseases together provide the main cause of death in the developed world. In
the United States, one in every three persons currently alive is estimated to contract
some form of cancer, and every fifth person will ultimately succumb to it. A
similar pattern of mortality obtains in other Western nations, and an even gloomier
prognosis is observed in many developing countries. In South Africa, for example, cancer is
the second-most common cause of death in the white, colored, and Asian population, and
the third-most common cause in the black population group. Despite major
variations among the different racial groups and incomplete registries especially in the
black group, statistical data leave no doubt that, on average, South African rates for
certain malignancies such as cervical, oesophageal, and skin cancers rank among the
highest worldwide. Cancers of the lung and prostate rate among the top six cancers with
white and nonwhite males, breast and cervical cancers are among the top six with
white and nonwhite females, and melanoma represents one of the top six cancers with
white females. Other types of cancer, including the various forms of leukaemia, although
of lower incidence, are nonetheless causative for serious concern. The problem will be
aggravated in the coming years as increasing urbanization is expected to create
environmental and dietary conditions conducive to further initiation and spread of
cancers; it will be further compounded by the finding that cancerous afflictions occur in
approximately one third of all male and female adult breadwinners in the age bracket of
15–54 years. A novel factor now coming into play is the rapid spread of AIDS;
immunodepressed patients will be at special risk to develop early cancerous lesions, and
virus-related cancers may well find particularly suitable targets among such patients.Chemotherapy
constitutes an important cancer treatment modality, alone or in conjunction with
other treatment regimens. However, despite much progress in drug research, results on
average have been highly discouraging. Let us pause for a moment and examine
this unsatisfactory state-of-affairs more closely.Presently used
anticancer drugs suffer from a multitude of severe pharmacological deficiencies, all of
which continue to contribute to the limitations of effectiveness generally experienced
in current clinical practice. Typical pharmacological shortcomings include the
following: (i)lack of cell specificity, with ensuing drug distribution
into both normal and
transformed cells, that is, in consequence, drug application will be
excessively wasteful; (ii) inadequate
water solubility, hampering swift, and efficacious drug distribution in the
body’s aqueous fluid system, with resulting enhanced exposure to macrophage activity;
(iii) decreased serum half-life as a consequence of catabolism, protein binding by the
reticuloendothelial system, or efficacious excretion mechanisms; (iv) monophasic salt-like
or charged structure, inhibiting membrane penetration and cell
entry through normal passive diffusion, and resulting from these deficiencies, only a
small fraction of the medicinal agent will successfully enter intracellular space
for interaction with nuclear DNA or proteinaceous constituents; (v) excessive systemic toxicity,
which grossly
diminishes therapeutic drug efficacy; and (vi) lack of long-term
efficaciousness because of inherent or induced drug resistance. Both (v)
and (vi) are clearly the
most aggravating factors added
to abovementioned deficiencies, they generally necessitate premature
discontinuation of drug-specific therapy, and thus, tend to reactivate
tumorous or metastatic growth. As an overall consequence of the cited drug
deficiencies, periods of regression tend to be limited, relapses occur frequently, and
complete cures of most types of cancer by chemotherapy alone are still the exception rather
than the rule.A rather
discomforting outlook indeed! Why then bring up this gloomy topic? Will you, the thoroughly
depressed reader, not be prompted to disembark speedily from the wrong ferry boat and
run for greener pastures elsewhere? The answer: worldwide efforts in biomedical
research are striving to overcome existing chemotherapeutic hurdles through the
expediency of developing efficacious cancer-fighting tools, and it is precisely this global
development drive in which macromolecular compounds are crucial participants
and find their true and foremost destiny. In Section2.2 we will explore the
background to this unique role played by polymers and their outstanding capabilities as
delivery vehicles in the chemotherapeutic treatment of cancerous diseases.


### 2.2. The Polymer-Drug Conjugation Concept

To begin with, using
text book knowledge, we will examine a typical medicinal agent’s pharmacokinetic fate
as it unfolds upon the drug’s administration by the common intravenous,
intramuscular, or oral techniques. For these, Figure1 depicts exemplifying curves,
given in terms of the agent’s serum concentration as a function of postadministration
time. In typical fashion, the intravenous route (curve A) delivers highest initial
concentrations, which decrease more or less asymptotically with time, ultimately to reach zero-concentration
level. Intramuscular administration (curve B) needs time for the
curve to ascend from zero to a maximum, after which it gradually descends to zero. The
oral route (curve C), while likewise providing a concentration maximum (generally
below that of curve B), requires time extension for that trend, if one considers the
long and obstacle-cluttered gastrointestinal pathway the drug has to follow before
entering the blood pool.Figure 1
Drug serum concentration versus time for three administration routes.At this point, we learn that for any administered drug system there are two critical serum concentration
limits, represented by the dashed lines in Figure2: a lower limit below which the drug
has ceased to produce any therapeutic effect, and an upper limit above which the drug
exerts various forms of toxicity in the organism. For a drug employed via intravenous
injection, which is the administration mode favored for polymer-drug
conjugates, curve A allows for drug application devoid of toxic side effects, yet
providing limited effectiveness time. In contrast, curve B, representing an increased initial
dosis, yields a considerably extended effectiveness period; this, however, does happen
at the cost of emerging toxicity. Hence, for a medicinal agent to remain
therapeutically efficacious without toxic effects, its serum concentration must stay between the
dotted lines over an optimally extended time span as schematically depicted by curve C.Figure 2
Drug serum concentration versus time for two different doses.An obvious approach toward achieving this goal of continuous, nontoxic bioactivity, although being tedious
and cumbersome, would involve repetitive drug administration within carefully identified
time intervals and with doses adjusted so as to maintain serum concentrations just
below toxic levels. Better still, one might design a drug delivery construct that would,
upon a single bolus administration, allow for a slow and gradual intraserum release of
the agent from some sort of depot system. An optimally efficacious
concentration trend for this model is schematically shown in Figure3.Figure 3
Drug serum
concentration versus time for slow-release modality.Needless to say, the pharmaceutical community, far from being asleep, has over the past two decades
focused research activities on a massive scale preferentially on this kind of drug delivery
system, generally with highly promising outcomes. Thus, microencapsulated
drugs in the form of microspheres, nanospheres, and liposomes have been developed
and successfully tested, and so have numerous implantable matrices capable of
liberating embedded drugs into the body’s fluid system and other body compartments
through bioleaching, bioerosion, or simply through matrix biodegradation. These
amply discussed drug delivery systems [1–3], all aiming at enhanced therapeutic
effectiveness of medicinal agents and by-and-large quite efficacious in their
own right, fall outside the definition of the narrow term of polymerdrug conjugation and,
hence, outside the scope of the present text.Deviating in itsmodus operandi from the aforementioned drug delivery constructs, the drug conjugation
modality embodies a symbiotic union between two equally contributing
partners: the polymer acts as a carrier and transport vehicle (it “knows” how to overcome
biological hurdles and deliver the drug load safely at the designated terminal), whereas
its “partner in healing”, the drug proper, enjoys the safe ride and exerts its bioactivity
in the target tissue. The notion of pairing up a bioactive agent with a transporter
vehicle, born in the early 1970s, was forcefully advanced by Ringsdorf [4], who defined and
refined this paradigm in palpable terms and set in motion broad-based
development activities in numerous laboratories, on the strength of which the notion has by now
matured to a highly sophisticated pharmaceutical tool. It utilizes the advantageous
pharmacokinetic behavior of polymeric compounds in the mammalian body, paired
with their enormous compositional versatility allowing for near-limitless structural
modifications of these large molecules. In practical terms, the technique comprises
the bioreversible binding (conjugation) of a bioactive agent, typically an antitumor
drug, to a water-soluble macromolecular carrier molecule designed and
synthesized in strict conformance with pharmaceutical guidelines. Specifically, the
carrier polymer will be composed of the following: (i) subunits containing some molecular entity permitting
facilitated cell entry; (ii) other subunits equipped with intra- or extrachain
water-solubilizing groups; (iii) still other units featuring a homing device capable of directing
the polymer-drug assembly, the conjugate, selectively to the target tissue; and (iv),
most importantly, units equipped with functional groups suitable for the critical conjugation
step involving bioreversible drug binding to the polymer. The conjugate represents
a prodrug, from which the active agent is hydrolytically or enzymatically
released into the predestined biological environment, which, for anticancer action,
are the cytoplasmic lysosomes and cell nuclei. A conjugate conforming in
structure to those specifications will demonstrate therapeutic superiority on account of the
following factors.(i)
Even if insoluble
in water, the medium providing the circulating blood pool and other fluids, the
intraveneously (IV) or intraperitoneally (IP) administered polymer-anchored drug, now perfectly
water-soluble, will be swiftly carried into central circulation or the intraperitoneum. This
will ensure efficient distribution of the bound drug in the fluid system, a requirement
most vital for drug transport within the body.(ii)
While in transit
in the central circulation system, the polymer-bound drug, notably if additionally modified
by inclusion of oligo(ethylene oxide) units, will experience temporary protection
from serum protein binding, enzymatic attack, and other scavenging processes.
As a result, renal clearance (i.e., excretion through the kidneys’ globular system) will
be minimized, while serum life-time and drug bioavailability will be extended as part of a
major change in biodistribution.(iii)
The dreaded
toxic side effects invariably caused by the common, nonpolymeric medicinal agents will
be minimized through carrier attachment, as now the “anchored” agent will be
constrained in body and organ distribution while in transit.(iv)
The connecting
groups incorporated into the polymer-drug linker for ultimate drug release can be
uniquely designed so as to remain intact while the conjugate is on its way to target in the
body’s ever so slightly basic (ph∼7.5) environment, yet
to undergo biofission once the
carrier-drug assembly has reached intracellular (specifically lysosomal) space.
Such biofission may be mediated by the lysosomal proteolytic enzymes, or else may
simply follow a hydrolytic pathway, taking advantage of the acidic (ph∼5) intralysosomal
environment. Reduced to monomeric size, the agent so liberated will now be
able to undergo extravasation through penetration of the lysosomal membrane (a
transport step not generally available to a macromolecular compound) and head
for its ultimate destination, which for most anticancer drugs is the cell’s nuclear DNA.(v)
The presence of a
“homing” (i.e., actively targeting) group will lead to enhanced drug affinity for the
transformed, that is,cancerous cell, thus facilitating guided drug transport. To
exemplify this process, we wish to take a look at a cancer-targeting system featuring
galactosamine as the homing device. This carbohydrate shows affinity for the
asialoglycoprotein receptor in hepatocytes and thus, if attached to a cytotoxic agent,
directs the latter efficiently to liver tissue, thereby providing cell-killing action against
hepatomas, a form of liver malignancies. Other successfully used homing groups are
represented by cationic moieties, tracking the conjugate preferentially to
neoplastic tissue as a consequence of electrostatic attraction to the negative surface
charge displayed by many cancer cells. Still other homing systems utilize the affinity
of monoclonal antibodies for specific cancer-associated antigens. This research area,
while still hampered by pharmacological problems of solid-state insolubility,
immunogenicity, and in-transit vulnerability to side reactions and degradation, has
proved eminently promising, and considerable progress has been achieved in recent
years.(vi)
The polymer-drug
conjugate will experience facilitated endocytotic cell entry, thus circumventing
potential problems caused by drug polarity or ionicity in the normal process of membrane
crossing by passive diffusion common to nonpolymeric solutes. The mechanism is
pinocytotic in nature and thus not subject to the limitations imposed by the reticuloendothelial
system on phagocytotically captured particulates. Ideally, adsorptive
pinocytosis is utilized by the conjugate for increased efficiency of translocation, and
cationic polymers are good examples of compounds so translocated. In cases where
excessive P-glycoprotein-mediated drug efflux from endocytic space and consequent
resistance problems have developed, the pinocytotic cell entry mechanism, through
replenishment of the intracellular drug pool, assumes unique importance as a means
of counteracting the dreaded phenomenon of drug resistance and restoring
chemotherapeutic drug activity.(vii)
Polymer conjugates,
in common with macromolecules in general, tend to accumulate in solid
tumors because of enhanced intratumoral vascular permeability, allowing for
substantial leakage of the polymeric molecules into the tumor tissue. Whereas, in normal
tissues, macromolecules in interstitial space are efficiently recovered and
eliminated by the lymphatic system, such lymphatic clearance is grossly retarded in
tumorous tissue and so represents a weighty factor contributing to the tumoritropic
characteristics of macromolecular compounds. Thisenhanced permeability and
retention (EPR)
effect associated with polymers provides passive targeting and renders
polymer-drug conjugate administration more efficacious while reducing systemic
toxicity in other organs.(viii)
Through the
expediency of introducing biofissionable segments into the backbone, a polymer
can be rendered biodegradable, thus effecting its gradual removal from the body in the
form of fragments qualifying for excretion through the body’s normal waste removal
systems. This process is crucial because high molecular-mass carriers are not
readily eliminated from the body and in the longer term may cause additional toxicity.A general flow chart
depicting the pharmacokinetic pathway of a carrier-anchored antineoplastic agent
constructed in conformance with the aforementioned specifications is schematically
reproduced in Scheme1. Most of the earlier papers in the field were concerned with
natural macromolecules as carriers, such as carbohydrates or proteinaceous
compounds; yet, as pointed out before, immunogenicity and backbone toxicity, premature
biodegradation (alternatively, strong resistance to biodegradability), poor stability in the
solid state, and other detrimental features are militating against major developments on
that front. More recently, in line with the pharmaceutical guidelines discussed
further above in this section, synthetic polymers have taken the lead in drug
conjugation studies for reasons of synthetic versatility and reproducibility, improved control of
physical and chemical behavior patterns, as well as minimization of systemic toxicity and
immunogenic properties. Parenthetically (no metal-based constituents being
involved here) we wish to introduce the reader to a related, rapidly growing branch of
drug conjugation science known as gene therapy. In this therapeutic modality, synthetic
polymers are used in place of viral vectors as vehicles for the in vivo transport of specific
nucleic acid agents for the therapeutic manipulation of pivotal genes involved in
malignancies and other diseases. Despite existing pharmacological shortcomings, gene
therapy is bound to take its well-deserved place in forth-coming therapeutic
technology developments [5, 6]. The immensely wide-ranging drug conjugation topic has been amply
surveyed over the years in the literature, and some of the most outstanding reviews
both of yesteryear [7, 8] and up-to-date [9–14], are recommended to the reader for in-depth studying.Scheme 1
Pharmacokineticin vivo pathway of polymer-drug conjugate.Having thoroughly
digested the fundamental principles of polymer-drug conjugation and the significant
pharmacological benefits to be derived from this mode of drug delivery, we are ready now for
a lecture on the practicalities. Out of a large number of variously structured carrier
types developed over the past decade, Section3 will introduce just three
types that have proved to be the predominant role players in the drug conjugation
domain, and their structural peculiarities will be highlighted as a helpful prerequisite
for the discussion of the drug anchoring strategies covered in Section 4.


## 2.1. The Cancer Problem

Malignancies and
cardiac diseases together provide the main cause of death in the developed world. In
the United States, one in every three persons currently alive is estimated to contract
some form of cancer, and every fifth person will ultimately succumb to it. A
similar pattern of mortality obtains in other Western nations, and an even gloomier
prognosis is observed in many developing countries. In South Africa, for example, cancer is
the second-most common cause of death in the white, colored, and Asian population, and
the third-most common cause in the black population group. Despite major
variations among the different racial groups and incomplete registries especially in the
black group, statistical data leave no doubt that, on average, South African rates for
certain malignancies such as cervical, oesophageal, and skin cancers rank among the
highest worldwide. Cancers of the lung and prostate rate among the top six cancers with
white and nonwhite males, breast and cervical cancers are among the top six with
white and nonwhite females, and melanoma represents one of the top six cancers with
white females. Other types of cancer, including the various forms of leukaemia, although
of lower incidence, are nonetheless causative for serious concern. The problem will be
aggravated in the coming years as increasing urbanization is expected to create
environmental and dietary conditions conducive to further initiation and spread of
cancers; it will be further compounded by the finding that cancerous afflictions occur in
approximately one third of all male and female adult breadwinners in the age bracket of
15–54 years. A novel factor now coming into play is the rapid spread of AIDS;
immunodepressed patients will be at special risk to develop early cancerous lesions, and
virus-related cancers may well find particularly suitable targets among such patients.Chemotherapy
constitutes an important cancer treatment modality, alone or in conjunction with
other treatment regimens. However, despite much progress in drug research, results on
average have been highly discouraging. Let us pause for a moment and examine
this unsatisfactory state-of-affairs more closely.Presently used
anticancer drugs suffer from a multitude of severe pharmacological deficiencies, all of
which continue to contribute to the limitations of effectiveness generally experienced
in current clinical practice. Typical pharmacological shortcomings include the
following: (i)lack of cell specificity, with ensuing drug distribution
into both normal and
transformed cells, that is, in consequence, drug application will be
excessively wasteful; (ii) inadequate
water solubility, hampering swift, and efficacious drug distribution in the
body’s aqueous fluid system, with resulting enhanced exposure to macrophage activity;
(iii) decreased serum half-life as a consequence of catabolism, protein binding by the
reticuloendothelial system, or efficacious excretion mechanisms; (iv) monophasic salt-like
or charged structure, inhibiting membrane penetration and cell
entry through normal passive diffusion, and resulting from these deficiencies, only a
small fraction of the medicinal agent will successfully enter intracellular space
for interaction with nuclear DNA or proteinaceous constituents; (v) excessive systemic toxicity,
which grossly
diminishes therapeutic drug efficacy; and (vi) lack of long-term
efficaciousness because of inherent or induced drug resistance. Both (v)
and (vi) are clearly the
most aggravating factors added
to abovementioned deficiencies, they generally necessitate premature
discontinuation of drug-specific therapy, and thus, tend to reactivate
tumorous or metastatic growth. As an overall consequence of the cited drug
deficiencies, periods of regression tend to be limited, relapses occur frequently, and
complete cures of most types of cancer by chemotherapy alone are still the exception rather
than the rule.A rather
discomforting outlook indeed! Why then bring up this gloomy topic? Will you, the thoroughly
depressed reader, not be prompted to disembark speedily from the wrong ferry boat and
run for greener pastures elsewhere? The answer: worldwide efforts in biomedical
research are striving to overcome existing chemotherapeutic hurdles through the
expediency of developing efficacious cancer-fighting tools, and it is precisely this global
development drive in which macromolecular compounds are crucial participants
and find their true and foremost destiny. In Section2.2 we will explore the
background to this unique role played by polymers and their outstanding capabilities as
delivery vehicles in the chemotherapeutic treatment of cancerous diseases.


## 2.2. The Polymer-Drug Conjugation Concept

To begin with, using
text book knowledge, we will examine a typical medicinal agent’s pharmacokinetic fate
as it unfolds upon the drug’s administration by the common intravenous,
intramuscular, or oral techniques. For these, Figure1 depicts exemplifying curves,
given in terms of the agent’s serum concentration as a function of postadministration
time. In typical fashion, the intravenous route (curve A) delivers highest initial
concentrations, which decrease more or less asymptotically with time, ultimately to reach zero-concentration
level. Intramuscular administration (curve B) needs time for the
curve to ascend from zero to a maximum, after which it gradually descends to zero. The
oral route (curve C), while likewise providing a concentration maximum (generally
below that of curve B), requires time extension for that trend, if one considers the
long and obstacle-cluttered gastrointestinal pathway the drug has to follow before
entering the blood pool.Figure 1
Drug serum concentration versus time for three administration routes.At this point, we learn that for any administered drug system there are two critical serum concentration
limits, represented by the dashed lines in Figure2: a lower limit below which the drug
has ceased to produce any therapeutic effect, and an upper limit above which the drug
exerts various forms of toxicity in the organism. For a drug employed via intravenous
injection, which is the administration mode favored for polymer-drug
conjugates, curve A allows for drug application devoid of toxic side effects, yet
providing limited effectiveness time. In contrast, curve B, representing an increased initial
dosis, yields a considerably extended effectiveness period; this, however, does happen
at the cost of emerging toxicity. Hence, for a medicinal agent to remain
therapeutically efficacious without toxic effects, its serum concentration must stay between the
dotted lines over an optimally extended time span as schematically depicted by curve C.Figure 2
Drug serum concentration versus time for two different doses.An obvious approach toward achieving this goal of continuous, nontoxic bioactivity, although being tedious
and cumbersome, would involve repetitive drug administration within carefully identified
time intervals and with doses adjusted so as to maintain serum concentrations just
below toxic levels. Better still, one might design a drug delivery construct that would,
upon a single bolus administration, allow for a slow and gradual intraserum release of
the agent from some sort of depot system. An optimally efficacious
concentration trend for this model is schematically shown in Figure3.Figure 3
Drug serum
concentration versus time for slow-release modality.Needless to say, the pharmaceutical community, far from being asleep, has over the past two decades
focused research activities on a massive scale preferentially on this kind of drug delivery
system, generally with highly promising outcomes. Thus, microencapsulated
drugs in the form of microspheres, nanospheres, and liposomes have been developed
and successfully tested, and so have numerous implantable matrices capable of
liberating embedded drugs into the body’s fluid system and other body compartments
through bioleaching, bioerosion, or simply through matrix biodegradation. These
amply discussed drug delivery systems [1–3], all aiming at enhanced therapeutic
effectiveness of medicinal agents and by-and-large quite efficacious in their
own right, fall outside the definition of the narrow term of polymerdrug conjugation and,
hence, outside the scope of the present text.Deviating in itsmodus operandi from the aforementioned drug delivery constructs, the drug conjugation
modality embodies a symbiotic union between two equally contributing
partners: the polymer acts as a carrier and transport vehicle (it “knows” how to overcome
biological hurdles and deliver the drug load safely at the designated terminal), whereas
its “partner in healing”, the drug proper, enjoys the safe ride and exerts its bioactivity
in the target tissue. The notion of pairing up a bioactive agent with a transporter
vehicle, born in the early 1970s, was forcefully advanced by Ringsdorf [4], who defined and
refined this paradigm in palpable terms and set in motion broad-based
development activities in numerous laboratories, on the strength of which the notion has by now
matured to a highly sophisticated pharmaceutical tool. It utilizes the advantageous
pharmacokinetic behavior of polymeric compounds in the mammalian body, paired
with their enormous compositional versatility allowing for near-limitless structural
modifications of these large molecules. In practical terms, the technique comprises
the bioreversible binding (conjugation) of a bioactive agent, typically an antitumor
drug, to a water-soluble macromolecular carrier molecule designed and
synthesized in strict conformance with pharmaceutical guidelines. Specifically, the
carrier polymer will be composed of the following: (i) subunits containing some molecular entity permitting
facilitated cell entry; (ii) other subunits equipped with intra- or extrachain
water-solubilizing groups; (iii) still other units featuring a homing device capable of directing
the polymer-drug assembly, the conjugate, selectively to the target tissue; and (iv),
most importantly, units equipped with functional groups suitable for the critical conjugation
step involving bioreversible drug binding to the polymer. The conjugate represents
a prodrug, from which the active agent is hydrolytically or enzymatically
released into the predestined biological environment, which, for anticancer action,
are the cytoplasmic lysosomes and cell nuclei. A conjugate conforming in
structure to those specifications will demonstrate therapeutic superiority on account of the
following factors.(i)
Even if insoluble
in water, the medium providing the circulating blood pool and other fluids, the
intraveneously (IV) or intraperitoneally (IP) administered polymer-anchored drug, now perfectly
water-soluble, will be swiftly carried into central circulation or the intraperitoneum. This
will ensure efficient distribution of the bound drug in the fluid system, a requirement
most vital for drug transport within the body.(ii)
While in transit
in the central circulation system, the polymer-bound drug, notably if additionally modified
by inclusion of oligo(ethylene oxide) units, will experience temporary protection
from serum protein binding, enzymatic attack, and other scavenging processes.
As a result, renal clearance (i.e., excretion through the kidneys’ globular system) will
be minimized, while serum life-time and drug bioavailability will be extended as part of a
major change in biodistribution.(iii)
The dreaded
toxic side effects invariably caused by the common, nonpolymeric medicinal agents will
be minimized through carrier attachment, as now the “anchored” agent will be
constrained in body and organ distribution while in transit.(iv)
The connecting
groups incorporated into the polymer-drug linker for ultimate drug release can be
uniquely designed so as to remain intact while the conjugate is on its way to target in the
body’s ever so slightly basic (ph∼7.5) environment, yet
to undergo biofission once the
carrier-drug assembly has reached intracellular (specifically lysosomal) space.
Such biofission may be mediated by the lysosomal proteolytic enzymes, or else may
simply follow a hydrolytic pathway, taking advantage of the acidic (ph∼5) intralysosomal
environment. Reduced to monomeric size, the agent so liberated will now be
able to undergo extravasation through penetration of the lysosomal membrane (a
transport step not generally available to a macromolecular compound) and head
for its ultimate destination, which for most anticancer drugs is the cell’s nuclear DNA.(v)
The presence of a
“homing” (i.e., actively targeting) group will lead to enhanced drug affinity for the
transformed, that is,cancerous cell, thus facilitating guided drug transport. To
exemplify this process, we wish to take a look at a cancer-targeting system featuring
galactosamine as the homing device. This carbohydrate shows affinity for the
asialoglycoprotein receptor in hepatocytes and thus, if attached to a cytotoxic agent,
directs the latter efficiently to liver tissue, thereby providing cell-killing action against
hepatomas, a form of liver malignancies. Other successfully used homing groups are
represented by cationic moieties, tracking the conjugate preferentially to
neoplastic tissue as a consequence of electrostatic attraction to the negative surface
charge displayed by many cancer cells. Still other homing systems utilize the affinity
of monoclonal antibodies for specific cancer-associated antigens. This research area,
while still hampered by pharmacological problems of solid-state insolubility,
immunogenicity, and in-transit vulnerability to side reactions and degradation, has
proved eminently promising, and considerable progress has been achieved in recent
years.(vi)
The polymer-drug
conjugate will experience facilitated endocytotic cell entry, thus circumventing
potential problems caused by drug polarity or ionicity in the normal process of membrane
crossing by passive diffusion common to nonpolymeric solutes. The mechanism is
pinocytotic in nature and thus not subject to the limitations imposed by the reticuloendothelial
system on phagocytotically captured particulates. Ideally, adsorptive
pinocytosis is utilized by the conjugate for increased efficiency of translocation, and
cationic polymers are good examples of compounds so translocated. In cases where
excessive P-glycoprotein-mediated drug efflux from endocytic space and consequent
resistance problems have developed, the pinocytotic cell entry mechanism, through
replenishment of the intracellular drug pool, assumes unique importance as a means
of counteracting the dreaded phenomenon of drug resistance and restoring
chemotherapeutic drug activity.(vii)
Polymer conjugates,
in common with macromolecules in general, tend to accumulate in solid
tumors because of enhanced intratumoral vascular permeability, allowing for
substantial leakage of the polymeric molecules into the tumor tissue. Whereas, in normal
tissues, macromolecules in interstitial space are efficiently recovered and
eliminated by the lymphatic system, such lymphatic clearance is grossly retarded in
tumorous tissue and so represents a weighty factor contributing to the tumoritropic
characteristics of macromolecular compounds. Thisenhanced permeability and
retention (EPR)
effect associated with polymers provides passive targeting and renders
polymer-drug conjugate administration more efficacious while reducing systemic
toxicity in other organs.(viii)
Through the
expediency of introducing biofissionable segments into the backbone, a polymer
can be rendered biodegradable, thus effecting its gradual removal from the body in the
form of fragments qualifying for excretion through the body’s normal waste removal
systems. This process is crucial because high molecular-mass carriers are not
readily eliminated from the body and in the longer term may cause additional toxicity.A general flow chart
depicting the pharmacokinetic pathway of a carrier-anchored antineoplastic agent
constructed in conformance with the aforementioned specifications is schematically
reproduced in Scheme1. Most of the earlier papers in the field were concerned with
natural macromolecules as carriers, such as carbohydrates or proteinaceous
compounds; yet, as pointed out before, immunogenicity and backbone toxicity, premature
biodegradation (alternatively, strong resistance to biodegradability), poor stability in the
solid state, and other detrimental features are militating against major developments on
that front. More recently, in line with the pharmaceutical guidelines discussed
further above in this section, synthetic polymers have taken the lead in drug
conjugation studies for reasons of synthetic versatility and reproducibility, improved control of
physical and chemical behavior patterns, as well as minimization of systemic toxicity and
immunogenic properties. Parenthetically (no metal-based constituents being
involved here) we wish to introduce the reader to a related, rapidly growing branch of
drug conjugation science known as gene therapy. In this therapeutic modality, synthetic
polymers are used in place of viral vectors as vehicles for the in vivo transport of specific
nucleic acid agents for the therapeutic manipulation of pivotal genes involved in
malignancies and other diseases. Despite existing pharmacological shortcomings, gene
therapy is bound to take its well-deserved place in forth-coming therapeutic
technology developments [5, 6]. The immensely wide-ranging drug conjugation topic has been amply
surveyed over the years in the literature, and some of the most outstanding reviews
both of yesteryear [7, 8] and up-to-date [9–14], are recommended to the reader for in-depth studying.Scheme 1
Pharmacokineticin vivo pathway of polymer-drug conjugate.Having thoroughly
digested the fundamental principles of polymer-drug conjugation and the significant
pharmacological benefits to be derived from this mode of drug delivery, we are ready now for
a lecture on the practicalities. Out of a large number of variously structured carrier
types developed over the past decade, Section3 will introduce just three
types that have proved to be the predominant role players in the drug conjugation
domain, and their structural peculiarities will be highlighted as a helpful prerequisite
for the discussion of the drug anchoring strategies covered in Section 4.


## 3. The Leading Carrier Systems

The design and construction of a drug carrier represents a pivotal task in polymer-drug conjugation as both
the anchoring chemistry and the therapeutic activity of the derived conjugate depend
critically on the carrier’s solubility behavior and resulting “staying power” in solution
upon drug conjugation. Steric accessibility and reactivity of the anchoring sites
additionally contribute to the carrier’s functional efficacy. A nontoxic chain construction
amenable to slow biodegradation in the spent state (i.e., upon drug liberation) is a
further requirement for optimal efficaciousness. Lastly, high main-chain flexibility depending
on freely rotating single bonds in the backbone and the absence of aromatic or
heteroaromatic, sterically blocking units, will be required as this will increase the entropy
of solution, thus reinforcing the aforementioned staying power of the assembly in
solution. In the following text we will find out if, and to what extent, these prerequisites
are met in the structural compositions of the three selected carrier types: (1) theα,β-DL-polyaspartamides,
(2) the poly(amidoamines), and (3) the 2-hydroxypropyl-methacrylate
polymers (HPMA). In addition, polyamides obtained by polycondensation
processes will cursorily be discussed because of their sporadic use as carriers.


### 3.1.α,β-DL-Polyaspartamides

This carrier type has
moved into the limelight of polymer research some three decades ago [15–17]. It is readily
accessible synthetically by high-temperature condensation polymerization of
DL-aspartic acid in orthophosphoric acid medium, leading to a polysuccinimide
intermediate (see Scheme 2).Scheme 2
Synthesis
of polysuccinimide from aspartic acid.In subsequent
polymer-homologous reactions conveniently performed in a dipolar aprotic solvent such
as N,N-dimethylformamide, the intermediary polysuccinimide is treated sequentially
with two amine nucleophiles (NH2–R1, NH2–R2;
occasionally a third amine, NH2–R3, may be employed in
this operation), whereby imide ring opening gives rise to the
generation of the ultimate polyaspartamides [18, 19].
These linear polyamides are composed of
randomly distributed aspartamide repeat units bearing N-attached substituents (R1, R2, etc.) as introduced
by the amine nucleophiles chosen. Scheme 3 depicts this sequence of ring opening steps.Scheme 3
Aminolytic ring opening in polysuccinimide.It should be noted
that here and in subsequent polyaspartamide representations, only theα-peptidic forms are
shown for convenience, although the isomeric β-forms, with two carbon atoms
(rather than one only) separating –CO and NH–, are also present.Let us now redraw the
structural arrangement in Scheme3 by substituting a solubilizing entity S for NH–R1, a homing device H
for NH–R2, and a drug-binding functional group F for NH–R3 (see Figure 4).Figure 4
Copolyaspartamide equipped with solubilizing, target-directing, and drug-binding moieties.It should be evident
from the detailed discussion in the foregoing of structure-function relationships that
polyaspartamides complying with this schematic representation will be able to provide an
excellent “workhorse” service in drug conjugation studies. Specifically, the
following hold. (i) The vital prerequisite of water solubility will be afforded
by the numerous freely rotating
aliphatic main-chain bonds and, in particular, by the extrachain S units chosen so as to
containtert-amine, hydroxyl, or, perhaps, oligo(ethylene oxide) groups [20]. These
functionalities will be prone to aquation, that is,hydrogen bond
formation with the aqueous
solvent, and/or protonation with generation of cationic sites. (ii) Steric accessibility of the
anchoring site F is optimized by insertion of a suitably long (say, 8–10 atomic
constituents) aliphatic spacer link between F and backbone, thus reducing any steric hindrance
potentially provided by the main chain proper. (iii) The chain construction has been
rendered nontoxic through use of aspartic acid as the principal molecular
backbone source. (iv) The chain is biodegradable hydrolytically at the CO–NH links for
catabolic fragment elimination through the globular system of the kidneys upon drug
release, a vital detoxification process in light of the fact that, as mentioned before,
large nondegradable macromolecules, such as vinyl-type polymers and other carbochain
macromolecules, strongly resist excretion and tend to induce toxicity. Such
backbone degradation must be slow and controlled, however, so as to ensure stability
during serum exposure, yet fragmentation in the endosomal compartment. (See
also remarks in Section 2.2). In the polyaspartamide structure, these conditions are
ideally met: in the serum environment (pH∼7.5) only minimal hydrolytic CO–NH bond
fission is observed, whereas in the endosomal space (pH∼5) such fragmentation
proceeds as expected, albeit slowly. Superimposed on hydrolytic bond fission is the
process of enzymatic bond braking, effective both at main-chain NHCO sites and, to a
variable extent, also at drug-binding links. Whereas natural polypeptides are
prone to rapid degradation (unzipping) as a consequence of α-peptidase activity in
the serum, such unzipping is grossly impeded by the presence of D-configurated CH
groups and β-peptide units in the chain that are inert to
α-peptidase attack [21]. Section 4 will show how proper use can be made of these structural features.


### 3.2. Polyamidoamines

Pioneered in Ferruti’s laboratory [22, 23], these macromolecules have proved as versatile as the
polyaspartamides, allowing for the construction of a multitude of unique carrier compositions. In the
simplest mode of synthesis, a monomer equipped with two activated vinyl
groups as in a bis(acryloylamido) compound exemplified by methylenebisacrylamide
or bisacryloylpiperazine, is allowed to undergo aqueous-phase Michael addition
polymerization with a comonomer that either contains two secondary amino groups for
single substitution on each N (see Scheme 4(a)) or else features a primary amine (see Scheme
4(b)), the latter now being doubly N-substituted in the process. The reactions are
preferably conducted in aqueous solution and give rise to polymer structures comprising
both amide and amine functionalities. In the author’s laboratory these studies were
extended to include comonomers featuring two primary amino groups that are
allowed to enter polymerization by single-step Michael addition at each –NH2 under modified,
carefully controlled conditions. This will produce polyamidoamines possessing secondary
amino groups in the main chain (see Scheme 4(c)), which, in turn, are
destined for drug binding [24]. With R2 standing for an
ethylene residue, the
resulting polymer possesses an intrachain –NH–(CH2)2–NH segment important for cis-N,N
chelation of platinum, as will be shown in Section 4.Scheme 4
Different modes of Michael addition polymerization of a bisacrylamide.A special case of sequence (b) in Scheme4, in which a mono-N-protected diamine takes the place of H2N–F3, leads to a polymer
possessing the mono-N-protected moiety as a side group. This
strategy thus compels primary diamine monomers to be incorporated through
just one amine functionality instead of two as in (c). Subsequent N-deprotection then
provides a polymer featuring a free –NH2 side-chain terminal directly available
for drug anchoring [25]. Scheme 5 depicts this reaction course (n = 3–21).Scheme 5
Michael addition polymerization involving mono-N-protected diamine.It is obvious from these described synthetic strategies that the resultant polyamidoamines in
all cases are linear and follow the established rules of construction, in so far as one may
vary the intrachain-type segments as well as—more usefully—the extrachain-type side
groups, in an effort to provide the vital functions of solubility, target homing, and drug
conjugation. In Section4 we will learn more about the versatile use of the amidoamine
polymers.


### 3.3. N-(2-Hydroxypropyl)Methacrylamides (HPMA)

The HPMA polymers have followed a glorious development path since early beginnings in 1970s [26]. On the strength of
preceding methacrylate polymer research in numerous
laboratories, this polymer class, developed almost singularly in the collaborating
laboratories of Kopeček (synthesis) and Duncan (biomedical evaluation), has by now most
successfully entered the forum of global research in polymer therapeutics [27–29].The synthetic approach, illustrated in Scheme6, involves the free-radical copolymerization of
two or more methacrylamide monomers bearing substituents S for provision of water
solubility (almost always a 2-hydroxypropyl moiety), other substituents, F, for
drug anchoring reactions, and still other functionalities, H, as required for specific
purposes serving facilitated cell entry, homing to the cancerous target tissue, or in
vivo detection and tracing.Scheme 6
Synthesis of methacrylamide polymers.The polymers are thus
characterized by a carbochain-type backbone composed of substituted
1,2-propylene repeat units, and these are randomly distributed along the main chain. In
contrast to the polyaspartamide situation, all basic functionalities are generally preintroduced
into the monomers and are automatically polymer-incorporated as the free-radical
propagation proceeds. In order to facilitate the drug conjugation step, the
respective precursor monomers are methacrylamides substituted with a
spacer-separatedpara-nitrophenolate group, the latter acting as a
convenient leaving group in the
ultimate addition-elimination step leading to the introduction of amine-functionalized
drug species with loss of para-nitrophenol. A nitrophenolate-terminated repeat unit and its
reaction product, the drug-conjugate unit, are shown in Scheme 7.Scheme 7
Drug conjugation with HPMA copolymer.Selected HPMA conjugates derived from the here-described carriers will be dealt with in Section4.


### 3.4. Polyamides Prepared by Polycondensation Reactions

Although of less importance, polycondensation products resulting from interfacial or high-temperature
condensation reactions should be included here because monomer variation results in
a variety of carrier structures featuring introduction of –NH− groups useful for drug
conjugation. Typically, exemplified in Scheme8, a diacid chloride, such as succinyl chloride,
is allowed to react interfacially with a diamine like 1,2-bis(3-aminopropylamino)ethane
or the solubilizing commercial Jeff ED-600 (an oligo(ethylene oxide) terminated at
both ends with a primary amino group) [30]. High-temperature solution
polymerization in polyphosphoric acid provides analogous polyamides, if free diacids are employed
in place of the acid chlorides [31].
The –NH–(CH2)2–NH– segments in these
structures have conveniently been introduced for platinum chelation as pointed out
before.Scheme 8
Synthesis of polyamides by interfacial polymerization.


## 3.1.α,β-DL-Polyaspartamides

This carrier type has
moved into the limelight of polymer research some three decades ago [15–17]. It is readily
accessible synthetically by high-temperature condensation polymerization of
DL-aspartic acid in orthophosphoric acid medium, leading to a polysuccinimide
intermediate (see Scheme 2).Scheme 2
Synthesis
of polysuccinimide from aspartic acid.In subsequent
polymer-homologous reactions conveniently performed in a dipolar aprotic solvent such
as N,N-dimethylformamide, the intermediary polysuccinimide is treated sequentially
with two amine nucleophiles (NH2–R1, NH2–R2;
occasionally a third amine, NH2–R3, may be employed in
this operation), whereby imide ring opening gives rise to the
generation of the ultimate polyaspartamides [18, 19].
These linear polyamides are composed of
randomly distributed aspartamide repeat units bearing N-attached substituents (R1, R2, etc.) as introduced
by the amine nucleophiles chosen. Scheme 3 depicts this sequence of ring opening steps.Scheme 3
Aminolytic ring opening in polysuccinimide.It should be noted
that here and in subsequent polyaspartamide representations, only theα-peptidic forms are
shown for convenience, although the isomeric β-forms, with two carbon atoms
(rather than one only) separating –CO and NH–, are also present.Let us now redraw the
structural arrangement in Scheme3 by substituting a solubilizing entity S for NH–R1, a homing device H
for NH–R2, and a drug-binding functional group F for NH–R3 (see Figure 4).Figure 4
Copolyaspartamide equipped with solubilizing, target-directing, and drug-binding moieties.It should be evident
from the detailed discussion in the foregoing of structure-function relationships that
polyaspartamides complying with this schematic representation will be able to provide an
excellent “workhorse” service in drug conjugation studies. Specifically, the
following hold. (i) The vital prerequisite of water solubility will be afforded
by the numerous freely rotating
aliphatic main-chain bonds and, in particular, by the extrachain S units chosen so as to
containtert-amine, hydroxyl, or, perhaps, oligo(ethylene oxide) groups [20]. These
functionalities will be prone to aquation, that is,hydrogen bond
formation with the aqueous
solvent, and/or protonation with generation of cationic sites. (ii) Steric accessibility of the
anchoring site F is optimized by insertion of a suitably long (say, 8–10 atomic
constituents) aliphatic spacer link between F and backbone, thus reducing any steric hindrance
potentially provided by the main chain proper. (iii) The chain construction has been
rendered nontoxic through use of aspartic acid as the principal molecular
backbone source. (iv) The chain is biodegradable hydrolytically at the CO–NH links for
catabolic fragment elimination through the globular system of the kidneys upon drug
release, a vital detoxification process in light of the fact that, as mentioned before,
large nondegradable macromolecules, such as vinyl-type polymers and other carbochain
macromolecules, strongly resist excretion and tend to induce toxicity. Such
backbone degradation must be slow and controlled, however, so as to ensure stability
during serum exposure, yet fragmentation in the endosomal compartment. (See
also remarks in Section 2.2). In the polyaspartamide structure, these conditions are
ideally met: in the serum environment (pH∼7.5) only minimal hydrolytic CO–NH bond
fission is observed, whereas in the endosomal space (pH∼5) such fragmentation
proceeds as expected, albeit slowly. Superimposed on hydrolytic bond fission is the
process of enzymatic bond braking, effective both at main-chain NHCO sites and, to a
variable extent, also at drug-binding links. Whereas natural polypeptides are
prone to rapid degradation (unzipping) as a consequence of α-peptidase activity in
the serum, such unzipping is grossly impeded by the presence of D-configurated CH
groups and β-peptide units in the chain that are inert to
α-peptidase attack [21]. Section 4 will show how proper use can be made of these structural features.


## 3.2. Polyamidoamines

Pioneered in Ferruti’s laboratory [22, 23], these macromolecules have proved as versatile as the
polyaspartamides, allowing for the construction of a multitude of unique carrier compositions. In the
simplest mode of synthesis, a monomer equipped with two activated vinyl
groups as in a bis(acryloylamido) compound exemplified by methylenebisacrylamide
or bisacryloylpiperazine, is allowed to undergo aqueous-phase Michael addition
polymerization with a comonomer that either contains two secondary amino groups for
single substitution on each N (see Scheme 4(a)) or else features a primary amine (see Scheme
4(b)), the latter now being doubly N-substituted in the process. The reactions are
preferably conducted in aqueous solution and give rise to polymer structures comprising
both amide and amine functionalities. In the author’s laboratory these studies were
extended to include comonomers featuring two primary amino groups that are
allowed to enter polymerization by single-step Michael addition at each –NH2 under modified,
carefully controlled conditions. This will produce polyamidoamines possessing secondary
amino groups in the main chain (see Scheme 4(c)), which, in turn, are
destined for drug binding [24]. With R2 standing for an
ethylene residue, the
resulting polymer possesses an intrachain –NH–(CH2)2–NH segment important for cis-N,N
chelation of platinum, as will be shown in Section 4.Scheme 4
Different modes of Michael addition polymerization of a bisacrylamide.A special case of sequence (b) in Scheme4, in which a mono-N-protected diamine takes the place of H2N–F3, leads to a polymer
possessing the mono-N-protected moiety as a side group. This
strategy thus compels primary diamine monomers to be incorporated through
just one amine functionality instead of two as in (c). Subsequent N-deprotection then
provides a polymer featuring a free –NH2 side-chain terminal directly available
for drug anchoring [25]. Scheme 5 depicts this reaction course (n = 3–21).Scheme 5
Michael addition polymerization involving mono-N-protected diamine.It is obvious from these described synthetic strategies that the resultant polyamidoamines in
all cases are linear and follow the established rules of construction, in so far as one may
vary the intrachain-type segments as well as—more usefully—the extrachain-type side
groups, in an effort to provide the vital functions of solubility, target homing, and drug
conjugation. In Section4 we will learn more about the versatile use of the amidoamine
polymers.


## 3.3. N-(2-Hydroxypropyl)Methacrylamides (HPMA)

The HPMA polymers have followed a glorious development path since early beginnings in 1970s [26]. On the strength of
preceding methacrylate polymer research in numerous
laboratories, this polymer class, developed almost singularly in the collaborating
laboratories of Kopeček (synthesis) and Duncan (biomedical evaluation), has by now most
successfully entered the forum of global research in polymer therapeutics [27–29].The synthetic approach, illustrated in Scheme6, involves the free-radical copolymerization of
two or more methacrylamide monomers bearing substituents S for provision of water
solubility (almost always a 2-hydroxypropyl moiety), other substituents, F, for
drug anchoring reactions, and still other functionalities, H, as required for specific
purposes serving facilitated cell entry, homing to the cancerous target tissue, or in
vivo detection and tracing.Scheme 6
Synthesis of methacrylamide polymers.The polymers are thus
characterized by a carbochain-type backbone composed of substituted
1,2-propylene repeat units, and these are randomly distributed along the main chain. In
contrast to the polyaspartamide situation, all basic functionalities are generally preintroduced
into the monomers and are automatically polymer-incorporated as the free-radical
propagation proceeds. In order to facilitate the drug conjugation step, the
respective precursor monomers are methacrylamides substituted with a
spacer-separatedpara-nitrophenolate group, the latter acting as a
convenient leaving group in the
ultimate addition-elimination step leading to the introduction of amine-functionalized
drug species with loss of para-nitrophenol. A nitrophenolate-terminated repeat unit and its
reaction product, the drug-conjugate unit, are shown in Scheme 7.Scheme 7
Drug conjugation with HPMA copolymer.Selected HPMA conjugates derived from the here-described carriers will be dealt with in Section4.


## 3.4. Polyamides Prepared by Polycondensation Reactions

Although of less importance, polycondensation products resulting from interfacial or high-temperature
condensation reactions should be included here because monomer variation results in
a variety of carrier structures featuring introduction of –NH− groups useful for drug
conjugation. Typically, exemplified in Scheme8, a diacid chloride, such as succinyl chloride,
is allowed to react interfacially with a diamine like 1,2-bis(3-aminopropylamino)ethane
or the solubilizing commercial Jeff ED-600 (an oligo(ethylene oxide) terminated at
both ends with a primary amino group) [30]. High-temperature solution
polymerization in polyphosphoric acid provides analogous polyamides, if free diacids are employed
in place of the acid chlorides [31].
The –NH–(CH2)2–NH– segments in these
structures have conveniently been introduced for platinum chelation as pointed out
before.Scheme 8
Synthesis of polyamides by interfacial polymerization.


## 4. Selected Carrier-Bound Drug Models

Having acquired by now the requisite conceptual tools, we are duly prepared for the ultimate topic: the
strategies required to put the carrier-drug assembly together along biomedically
prescribed routes. As we delineate the scope of this effort in the forth-coming
discussions, we are guided by the requirement to confine the treatment to metal-based polymer
systems and, there again, to those systems with a medicinal, specifically
therapeutic application potential. Among the considerable number of metal-containing macromolecules that
have attracted the bioscientists’ interest, we will uniquely focus our
attention to the two most actively researched classes: the metal-coordination polymer
class belonging to the platinum drug family, and the organometallic class of
ferrocene-type iron-containing conjugates. Both classes, as we will see, turn out to be
just the right types of “guinea pig” as we set out to explore the broad field of medicinal
agents conjugated to macromolecular adjuvants. In addition (you guessed it!), it so
happens that it is the development of these two classes to which the author’s group has
been particularly devoted over the past four decades. Other metals, such as titanium or
ruthenium, continue to play a role in the field marginal enough as yet to permit us to ignore
them within the scope of this text.


### 4.1. Polymer-Platinum Conjugates

As metal-based
medicinal agents found their way into the oncologist’s arsenal of antitumor drugs,
several decades ago, platinum containing compounds immediately moved to the forefront
with the advent of the prototype,cisplatin (cis-diamminedichloroplatinum(II)), and its
second-generation cousins, carboplatin and oxaliplatin (see
Figure 5).Leading platinum drugs in clinical use.
(a)(b)(c)Their general
mechanism of action involves the formation of aquated species upon parenteral
administration and subsequent intra- and interstrand crosslinking with intracellular DNA,
leading to irreversible lesions in the double-helix and ultimate cell death. The compounds
are highly potent against numerous malignanciesper se and in combination with
other antitumor drugs. Set against these highly acclaimed performance criteria,
the platinum compounds, just like the metal-free drugs, suffer from severe
pharmacological shortcomings, curtailing clinical effectiveness. Thus, residence times in
central circulation are generally short, and this is paired with rapid excretion through the
urinary tracts and consequent unpredictable variation of drug concentration. Drug
action is nonselective, normal and transformed cells being equally affected; this leads
to significant, dose-limiting acute and chronic toxicities, and in consequence, therapy
discontinuation will frequently be required long before regression-free cure
rates are attained. Lastly, and most importantly, various forms of drug resistance tend
to build up in the cancerous tissue, which, again, requires treatment
termination. In view of these serious deficiencies, active research worldwide has been
focused on structural modifications designed to enhance the overall performance
profile [32–35]. The polymer-drug conjugation modality is playing an outstanding part in
these research endeavors, and, as we will see in the following section, encouraging
progress has been made until now. Before going into details, let us remember that the
present treatise, far from presenting a comprehensive review, is intended, first and
foremost, to provide a didactic lecture on the topic; hence, it will cover only a small
number of exemplifying conjugate types selected to mirror general trends in this
rapidly expanding field of pharmaceutics. For the student wishing to “dig deeper”, Carraher’s
excellent review chapter with original literature quotes is strongly recommended [36].


#### 4.1.1. Metal Coordination Involving Carrier-Bound Nitrogen Donor Ligands

By strict definition
of the polymer-drug conjugation concept, the presynthesis of a suitable carrier
polymer of biomedically “approved” structure is followed in another reaction step by the
bioreversible attachment of the drug. Before we study the exemplifying
macromolecules compliant with this definition of drug conjugation, it is only fair to Charles
Carraher, the pioneering scientist, that we are acquainted first with a separate synthetic
approach deviating from that routine, yet significant in its own right because it is one of
the earliest major assaults on the problem of providing biomedically important metal
compounds in polymeric form. This approach, developed in meticulous detail in Carraher’s
laboratory, embodies the aqueous-phase polycondensation of the tetrachloroplatinate(II)
dianion with diamines, giving polymers with the metal coordinated directly
into the main chain incis-diamine fashion, as shown in Scheme 9. The link R in this
process stands for a variety of aliphatic, aromatic, and heteroaromatic units, or simply for
a direct bond, and molecular masses attain the 106 Dalton  level. Biological test data,
notably for antiviral and antiproliferative performance against a number of cancer
cells, at that time demonstrated a “leap forward” in platinum-drug cancer therapy.Scheme 9
Polycondensation of diamines with the tetrachloroplatinate dianion.This brings us to the main stream of drug conjugate development: the polymerhomologous drug anchoring to
presynthesized carrier polymers. In this principal mode, polymer attachment of
the square-planar structural element ofcisplatin can be achieved via several
reaction paths, in the design of which care must be taken to include suitable
cleavage sites in the assembly for in vivo liberation of the bioactive agent from the
carrier vehicle. In principle, two approaches of metal-binding are available. In the
first, the metal center is bound via amine ligands attached to the carrier. In the
second mode, the metal is polymer-bound via the leaving group ligands. In the first case,
the result will be a structure comprising a Pt center tightly bound to the carrier, and release
of the active complex will require cleavage of a suitable anchoring segment in the side
chain. This is a time-dependent process, and the conjugates so constructed should
prove most efficacious in long-term administration. In the second case, the active
complex is released from the conjugate through (generally hydrolytic) cleavage of the bonds
connecting the metal with the leaving groups, resulting in aquation. Release
rates will be higher in this model, which should, hence, find optimal utilization in
short-term administration, whenever a boost is required for rapid effects. Let us look at the
first case: conjugation via carrier-bound amine ligands (see Scheme 10).Scheme 10
Platination of amine-functionalized carriers.In the simplest fashion (type A, with R = CH2CH2−), the >PtCℓ2 moiety is incorporated into a carrier
structure containing ethylenediamine as a main-chain segment. Such carriers may be
prepared by Michael addition polymerization with selected tetramine comonomers, and
Scheme 11 provides an illustrating example. A related metal bonding pattern
exists in certain polyethyleneimines prepared by Carraher’s group. In conjugates of these
structures the cis-diamine ligand system is strain-free and thus smoothly generated.Scheme 11
Platinum
atom coordinated by intrachain ethylenediamine segment.Platination of
carriers of type B (see Scheme10) leads to conjugates in which the entire cisplatin skeleton is located
outside the main chain. With R standing for a direct bond or, less favorably,
no more than a single atomic constituent, metal coordination through chelation with both
amino groups would appear to be sterically feasible. Exemplifying product structures
have indeed been obtained in Carraher’s laboratory [36] by platination of a
vinylamine/vinylsulfonate copolymer and of the natural (and hence,
biodegradable) polymer chitosan. A
special case of type B has been reported by Howell and Walles [37]. These authors
inter alia presynthesized a cisplatin analog with both NH3 ligands replaced by the two
NH2 groups in 1,2-diamino-4-hydroxybenzene. This reactant was then allowed to bind to
poly(vinylpyrrolidone) by molecular complexation, that is, in a form permitting its
release from the carrier by simple dissociation [37].Appreciably more
research has been performed with conjugates falling into the pattern of types C and D (see
Scheme10). In the author’s laboratory, polyaspartamides prepared per Scheme 3 (yet
including only two R-substituted units) were converted to platinated species by treatment
with tetrachloroplatinate dianion in aqueous phase. The two platination runs in
Scheme 12 exemplify this reaction type. Whereas the upper reaction leads to a conjugate
with cis-N,N′-coordinated platinum [38, 39], the lower one gives
rise to the formation of a structure wherein the metal is polymer-bound through a single N donor ligand [40].
In both cases, the first depicted, potentially cationic repeat unit provides both solubilizing and
homing functions. Cell culture tests showed both conjugate types to be equally
antitumor-active, and while with the upper conjugate ranked first in a series of cytotoxicity
tests [41], the lower one ranked first in an in vivo toxicological study, demonstrating
some 100-fold lower toxicity than determined for the cisplatin standard [42].Scheme 12
Platinum conjugation through carrier-attached amine donors.Similar platination patterns were reported from Ruth Duncan’s prolific laboratory (reviewed [36]), with HPMA polymers
used as the carriers. Broad-based biological investigations
provided excellent results with respect to release rates, toxicological behavior, and
cell-killing properties both in vitro and in vivo.Somewhat related to type D is a conjugate synthesized by Gianasi et al. [43] and further investigated by
Howell [44, 45], in which only one nitrogen donor ligand, in cooperation with an oxygen donor,
partakes in platinum coordination (See Figure 6(a)). The compound, an HPMA derivative, is
noteworthy as it features and N,N,N′,O-coordination square around the Pt atom. The
compound underwent in vitro and Phase I clinical tests with highly promising results,
low toxicities being paired with excellent antiproliferative activities [44, 45].Platinum
conjugation through repeat units featuring carrier-attached N- and O-donors.
(a)(b)A differently
structured polymer type featuring N,O-coordination of the metal has just been reported by
Bariyanga [46]. Representative
compounds contain the Fc−CH=N(R)−Pt(Cl2)−O− segment (Fc = ferrocenyl) connected to a poly(ethylene oxide) terminal (see
Figure 6(b)). These polymers are thus characterized by the presence of both a
ferrocene and a platinum complex. While no biomedical test data are available, it
would be interesting to find out in future studies whether such compounds display
cooperative cytotoxicity between the two drug species.


#### 4.1.2. Metal Coordination through Carrier-Bound Oxygen Donor Ligands

In the polymer
described in this section the Pt complex is conjugated through two oxygen donor ligands
provided by the carrier. Contrasting with those covered in the preceding section,
these conjugates follow a release mechanism involving hydrolytic Pt bond cleavage,
thus requiring no special biofissionable links in the carrier for drug liberation. In
structures of this kind, the anchoring mechanism must be designed with a view to counteracting
the inherently limited stability of the O−Pt bond. The preferred approach to this goal
involves the construction of 5–7-membered chelate rings connecting the metal
atom with the carrier-attached donor ligands. Scheme13 offers acceptable choices,
for which the literature provides exemplifying experiments (reviewed by Carraher [36]); whereas types A
and D exemplify carrier binding through directly
backbone-attached hydroxylato- or carboxylato-type oxygen donors, the types B, C, and E represent
structures with spacers interposed between carrier backbone and oxygen donor.
Further work will determine the extent to which biological performance will be
affected by the coordinating atoms’ special remoteness from the main chain.Scheme 13
Platinum chelation patterns.Figure7 provides an example each of type A- and type D-derived conjugates without
further comments on the synthetic methodology [47].Examples of O,O-coordinated platinum in close main-chain proximity.
(a)(b)Considering now the B- and C-type platination reactions of Scheme13, the literature provides numerous
reaction sequences originating from uniquely functionalized carriers. Scheme 14
represents a prototype reaction starting out from polysuccinimide [47]. In all these
exemplifying cases, the platination agent has been trans-1,2-diaminocyclohexanediaquaplatinum(II)
dinitrate (DACH-Pt), a reactant preferred by us and others over
the tetrachloroplatinate dianion on grounds of favorable biological behavior, notably the
lack of crossresistance with cisplatin.Scheme 14
Synthesis
of conjugate featuring extrachain-type 1,2-carboxylatohydroxylato coordination of
platinum.We will now focus our attention to the bonding pattern of type E (see Scheme13) which underlies the largest
body of recent carrier-Pt conjugation research. Thus, Ohya et al. [48] prepared a
water-soluble poly(ethylene oxide) carrying a methoxy terminal and, at the other end, a
propylmalonate moiety suitable for platination with hydrolyzed cisplatin. The release behavior
was examined and the cytoxicity against lymphocytic leukemia cells was determined.
Good activity, although slightly below that of cisplatin, was observed. A study by the
afore-mentioned group of Duncan et al. produced numerous dicarboxylatoplatinum
compounds, among others, HPMA-derived conjugates possessing
carrier-attached dicarboxylato ligands [43]. Upon treatment with hydrated cisplatin, these converted to
highly bioactive cis-dicarboxylato-chelated platinum polymers as
exemplified in Scheme 15 for the affected repeat unit.Scheme 15
HPMA-derived dicarboxylato-coordinated platinum.A recent patent, outstanding among the many studies of polymeric phosphazene drugs [49], describes
polyphosphazene derivatives of dicarboxylato-coordinated platinum with activity inter
alia against the gastric tumor YCC-3 [50]. A simplified repeat unit structure is
reproduced in Figure 8.Figure 8
Simplified
structure of polyphosphazene repeat unit bearing sidechain-coordinated platinum.Numerous conjugates
of type E have in recent years been synthesized in the author’s laboratory, of which
only a single example is reproduced in Scheme16. The carrier educt in this
reaction is a polyamidoamine equipped with a solubilizing
N,Ndimethylaminopropyl side chain and an
aspartic acid-derived dicarboxylate, and platination once more
involves treatment with DACH-Pt [47, 51]. This polymer and related structures
have shown outstanding antiproliferative performance, on the strength of which, a
major development program has been initiated.Scheme 16
Polyamidoamine-derived dicarboxylato-coordinated platinum.This section would be
incomplete without a brief treatment of the comprehensive research efforts by
the group of Bilha Schechter and collaborators. These workers at the Weizmann
Institute of Science have been involved for some decades now in studies aiming at the
provision of platinum-containing polymers antibody-modified for immunotargeting to
human tumors. Representative compounds are a polyglutamic acid providing chelating
capacity of type E (see Scheme13) and some carboxymethyl derivatives of
dextran with similar, albeit less structurally defined metal coordination patterns. Excellent
biomedical test results on a broad front attest to the soundness of the development
program in Schechter’s highly productive laboratory. For in-depth reviews of the
Weizmann group’s activities, see Siegmann-Louda and Carraher [36], and Chen et al. [52].


### 4.2. Polymer-Ferrocene Conjugates

Contrasting with the platinum drugs, which have firmly established their prominent position as antitumor
agents in clinical use, research in the realm of ferrocene-based chemotherapy is still
at an experimental stage. However, biomedical test data, preliminary as they
are, have shown overwhelming promise, stimulating comprehensive investigations
currently in progress worldwide. The core organoiron compound, di-(η5-cyclopentadienyl)iron(II),
has been under scientific and technical scrutiny for more than half a
century, and numerous topical surveys, including fundamental physical and chemical
applications, have appeared in the literature (surveyed in two books [53, 54]). The fascinating
ferrocene chemistry, pivotally involving the compound’s redox equilibrium
with its one-electron oxidation product, the ferricenium cation, has prompted wide-ranging
investigations of this electrochemical ferrocene/ferricenium couple in the
biological environment, with ultimate ramification into the topic of
carrier-bound ferrocene conjugates.The bulk of contributions to these research developments has materialized in the author’s laboratory,
as attested by more than 50 pertinent papers published by our group until now. The
reader is referred to some reviews [54–57] covering this prominent development trend.Let us look now in detail at the large family of polymer-bound ferrocenes designed for biomedical
applications. As we embark on this theme, we realize that, in the “old days” of ferrocene polymer
development spanning the 1950–1970 decades, macromolecular ferrocenes and other
metallocenes had been synthesized in huge numbers (reviewed in the book [54] and three book
chapters [58–60]), yet most of those
were hydrophobic and lacked
hydrosolubility and, hence, were not considered in later work. An entirely new concept had to be
developed for the purpose of rendering any metallocene polymers qualified for
biological examinations. That concept, as we learned in Section 2, originated a decade
later. It embodies the technique of conjugation so successfully utilized in the
design of polymeric platinum and other drugs, and in the present section it will be seen to be
instrumental in the synthesis of carrier-bound ferrocene compounds.Earlier exploratory studies [61] demonstrated the feasibility of carrier binding and provided a range of variously
structured conjugates meeting the crucial requirement of water solubility.
Ferrocenylamines, initial candidate agents for ferrocenylation because of their strongly
nucleophilic functionality, were soon omitted on electrochemical grounds. Ferrocenyl-carboxylic
acids, on the other hand, notably 4-ferrocenylbutanoic acid, emerged in those
earlier investigations as suitably functioning compounds providing electrochemically
stable ferricenium counterparts [62–64]. The butanoic acid derivative soon became the
ferrocenylation agent of choice in all subsequent conjugation developments in
preference over other chemically feasible coupling mechanisms. Again, the
polyaspartamides introduced in Section 3.1 turned out to be the most versatile and functional
carriers; equipped with NH2-terminated side chains, they soon became, and still
are, the “workhorse” carrier polymers in our laboratory, conveniently undergoing coupling
reactions in aprotic medium with the ferrocenyl acid via carboxyl group activation, or
directly with the aid of strong coupling agents, such as HBTU (O-[1H-benzotriazol-1-yl]-1,
1,3,3-tetramethyluronium hexafluorophosphate). Scheme 18 shows a typical
reaction step of this type, singled out here on account of the conjugate’s particularly
rewarding cytotoxic behavior; in vitro screens against the HeLa human cervix
epithelial cancer revealed a cell-killing activity (on metal-to-metal equivalent basis)
twice that of the cisplatin standard, and a fourfold superiority over cisplatin against Colo
320 DM, a notoriously drug-resistant human colon cancer line [65]. Interestingly,
similar conjugates possessing nonbasic hydroxyalkyl side groups in place of the strongly
basic tert-amine-terminated solubilizing and homing moiety of the drawn structure gave
rather poor results, reflecting the lowered affinity to the cancerous cell (see Section
2.2) because of lacking basicity.Among the great variety of other carrier types, we cite the primary amine-functionalized amidoamine polymer
synthesized by Michael polyaddition (see Section3.2); readily ferrocenylated with
HBTU mediation [66], it converts to a conjugate featuring a long (8 atomic constituents)
spacer connecting the metallocene to the main chain as depicted in Scheme 19.On the other hand, a related amidoamine-type carrier characterized by an additional intrachain −NH−NH− group yet lacking the
NH2-spacer side chain converts to a conjugate by direct N-acylation of the main chain [67] (see Scheme 20). Subjected to the
same cell culture tests as cited above, this ferrocene-loaded polymer, again, performed distinctly better
than the cisplatin standard.Lastly, we mention an anchoring step involving O-acylation. To this end, various hydroxyalkyl-substituted
polyaspartamides were treated in aprotic medium with ferrocenylbutanoic
acid in the presence ofN,N′-dicyclohexylcarbodiimide
coupling agent and 4-(dimethylamino)pyridine (DMAP) catalyst [68]. Scheme 21 shows a representative run,
with x/y varying from about 3 to 10 as a function of molar reactant ratios.A conjugate of this
type, withx/y = 2.6, subjected to an in vivo toxicity test, gave a maximum tolerated
dose of 0.43 mmol Fe/kg, more than 20-times higher than determined for the cisplatin standard [69].
The polymer is thus some 20-fold less toxic than the standard,
and comparable toxicity behavior is also displayed by selected amide-linked analogs.
These findings, in conjunction with cell-killing properties, augur exceptionally well
for the class of ferrocene conjugates as cancer-chemotherapeutic agents, and their
future looks highly promising indeed.


## 4.1. Polymer-Platinum Conjugates

As metal-based
medicinal agents found their way into the oncologist’s arsenal of antitumor drugs,
several decades ago, platinum containing compounds immediately moved to the forefront
with the advent of the prototype,cisplatin (cis-diamminedichloroplatinum(II)), and its
second-generation cousins, carboplatin and oxaliplatin (see
Figure 5).Leading platinum drugs in clinical use.
(a)(b)(c)Their general
mechanism of action involves the formation of aquated species upon parenteral
administration and subsequent intra- and interstrand crosslinking with intracellular DNA,
leading to irreversible lesions in the double-helix and ultimate cell death. The compounds
are highly potent against numerous malignanciesper se and in combination with
other antitumor drugs. Set against these highly acclaimed performance criteria,
the platinum compounds, just like the metal-free drugs, suffer from severe
pharmacological shortcomings, curtailing clinical effectiveness. Thus, residence times in
central circulation are generally short, and this is paired with rapid excretion through the
urinary tracts and consequent unpredictable variation of drug concentration. Drug
action is nonselective, normal and transformed cells being equally affected; this leads
to significant, dose-limiting acute and chronic toxicities, and in consequence, therapy
discontinuation will frequently be required long before regression-free cure
rates are attained. Lastly, and most importantly, various forms of drug resistance tend
to build up in the cancerous tissue, which, again, requires treatment
termination. In view of these serious deficiencies, active research worldwide has been
focused on structural modifications designed to enhance the overall performance
profile [32–35]. The polymer-drug conjugation modality is playing an outstanding part in
these research endeavors, and, as we will see in the following section, encouraging
progress has been made until now. Before going into details, let us remember that the
present treatise, far from presenting a comprehensive review, is intended, first and
foremost, to provide a didactic lecture on the topic; hence, it will cover only a small
number of exemplifying conjugate types selected to mirror general trends in this
rapidly expanding field of pharmaceutics. For the student wishing to “dig deeper”, Carraher’s
excellent review chapter with original literature quotes is strongly recommended [36].


### 4.1.1. Metal Coordination Involving Carrier-Bound Nitrogen Donor Ligands

By strict definition
of the polymer-drug conjugation concept, the presynthesis of a suitable carrier
polymer of biomedically “approved” structure is followed in another reaction step by the
bioreversible attachment of the drug. Before we study the exemplifying
macromolecules compliant with this definition of drug conjugation, it is only fair to Charles
Carraher, the pioneering scientist, that we are acquainted first with a separate synthetic
approach deviating from that routine, yet significant in its own right because it is one of
the earliest major assaults on the problem of providing biomedically important metal
compounds in polymeric form. This approach, developed in meticulous detail in Carraher’s
laboratory, embodies the aqueous-phase polycondensation of the tetrachloroplatinate(II)
dianion with diamines, giving polymers with the metal coordinated directly
into the main chain incis-diamine fashion, as shown in Scheme 9. The link R in this
process stands for a variety of aliphatic, aromatic, and heteroaromatic units, or simply for
a direct bond, and molecular masses attain the 106 Dalton  level. Biological test data,
notably for antiviral and antiproliferative performance against a number of cancer
cells, at that time demonstrated a “leap forward” in platinum-drug cancer therapy.Scheme 9
Polycondensation of diamines with the tetrachloroplatinate dianion.This brings us to the main stream of drug conjugate development: the polymerhomologous drug anchoring to
presynthesized carrier polymers. In this principal mode, polymer attachment of
the square-planar structural element ofcisplatin can be achieved via several
reaction paths, in the design of which care must be taken to include suitable
cleavage sites in the assembly for in vivo liberation of the bioactive agent from the
carrier vehicle. In principle, two approaches of metal-binding are available. In the
first, the metal center is bound via amine ligands attached to the carrier. In the
second mode, the metal is polymer-bound via the leaving group ligands. In the first case,
the result will be a structure comprising a Pt center tightly bound to the carrier, and release
of the active complex will require cleavage of a suitable anchoring segment in the side
chain. This is a time-dependent process, and the conjugates so constructed should
prove most efficacious in long-term administration. In the second case, the active
complex is released from the conjugate through (generally hydrolytic) cleavage of the bonds
connecting the metal with the leaving groups, resulting in aquation. Release
rates will be higher in this model, which should, hence, find optimal utilization in
short-term administration, whenever a boost is required for rapid effects. Let us look at the
first case: conjugation via carrier-bound amine ligands (see Scheme 10).Scheme 10
Platination of amine-functionalized carriers.In the simplest fashion (type A, with R = CH2CH2−), the >PtCℓ2 moiety is incorporated into a carrier
structure containing ethylenediamine as a main-chain segment. Such carriers may be
prepared by Michael addition polymerization with selected tetramine comonomers, and
Scheme 11 provides an illustrating example. A related metal bonding pattern
exists in certain polyethyleneimines prepared by Carraher’s group. In conjugates of these
structures the cis-diamine ligand system is strain-free and thus smoothly generated.Scheme 11
Platinum
atom coordinated by intrachain ethylenediamine segment.Platination of
carriers of type B (see Scheme10) leads to conjugates in which the entire cisplatin skeleton is located
outside the main chain. With R standing for a direct bond or, less favorably,
no more than a single atomic constituent, metal coordination through chelation with both
amino groups would appear to be sterically feasible. Exemplifying product structures
have indeed been obtained in Carraher’s laboratory [36] by platination of a
vinylamine/vinylsulfonate copolymer and of the natural (and hence,
biodegradable) polymer chitosan. A
special case of type B has been reported by Howell and Walles [37]. These authors
inter alia presynthesized a cisplatin analog with both NH3 ligands replaced by the two
NH2 groups in 1,2-diamino-4-hydroxybenzene. This reactant was then allowed to bind to
poly(vinylpyrrolidone) by molecular complexation, that is, in a form permitting its
release from the carrier by simple dissociation [37].Appreciably more
research has been performed with conjugates falling into the pattern of types C and D (see
Scheme10). In the author’s laboratory, polyaspartamides prepared per Scheme 3 (yet
including only two R-substituted units) were converted to platinated species by treatment
with tetrachloroplatinate dianion in aqueous phase. The two platination runs in
Scheme 12 exemplify this reaction type. Whereas the upper reaction leads to a conjugate
with cis-N,N′-coordinated platinum [38, 39], the lower one gives
rise to the formation of a structure wherein the metal is polymer-bound through a single N donor ligand [40].
In both cases, the first depicted, potentially cationic repeat unit provides both solubilizing and
homing functions. Cell culture tests showed both conjugate types to be equally
antitumor-active, and while with the upper conjugate ranked first in a series of cytotoxicity
tests [41], the lower one ranked first in an in vivo toxicological study, demonstrating
some 100-fold lower toxicity than determined for the cisplatin standard [42].Scheme 12
Platinum conjugation through carrier-attached amine donors.Similar platination patterns were reported from Ruth Duncan’s prolific laboratory (reviewed [36]), with HPMA polymers
used as the carriers. Broad-based biological investigations
provided excellent results with respect to release rates, toxicological behavior, and
cell-killing properties both in vitro and in vivo.Somewhat related to type D is a conjugate synthesized by Gianasi et al. [43] and further investigated by
Howell [44, 45], in which only one nitrogen donor ligand, in cooperation with an oxygen donor,
partakes in platinum coordination (See Figure 6(a)). The compound, an HPMA derivative, is
noteworthy as it features and N,N,N′,O-coordination square around the Pt atom. The
compound underwent in vitro and Phase I clinical tests with highly promising results,
low toxicities being paired with excellent antiproliferative activities [44, 45].Platinum
conjugation through repeat units featuring carrier-attached N- and O-donors.
(a)(b)A differently
structured polymer type featuring N,O-coordination of the metal has just been reported by
Bariyanga [46]. Representative
compounds contain the Fc−CH=N(R)−Pt(Cl2)−O− segment (Fc = ferrocenyl) connected to a poly(ethylene oxide) terminal (see
Figure 6(b)). These polymers are thus characterized by the presence of both a
ferrocene and a platinum complex. While no biomedical test data are available, it
would be interesting to find out in future studies whether such compounds display
cooperative cytotoxicity between the two drug species.


### 4.1.2. Metal Coordination through Carrier-Bound Oxygen Donor Ligands

In the polymer
described in this section the Pt complex is conjugated through two oxygen donor ligands
provided by the carrier. Contrasting with those covered in the preceding section,
these conjugates follow a release mechanism involving hydrolytic Pt bond cleavage,
thus requiring no special biofissionable links in the carrier for drug liberation. In
structures of this kind, the anchoring mechanism must be designed with a view to counteracting
the inherently limited stability of the O−Pt bond. The preferred approach to this goal
involves the construction of 5–7-membered chelate rings connecting the metal
atom with the carrier-attached donor ligands. Scheme13 offers acceptable choices,
for which the literature provides exemplifying experiments (reviewed by Carraher [36]); whereas types A
and D exemplify carrier binding through directly
backbone-attached hydroxylato- or carboxylato-type oxygen donors, the types B, C, and E represent
structures with spacers interposed between carrier backbone and oxygen donor.
Further work will determine the extent to which biological performance will be
affected by the coordinating atoms’ special remoteness from the main chain.Scheme 13
Platinum chelation patterns.Figure7 provides an example each of type A- and type D-derived conjugates without
further comments on the synthetic methodology [47].Examples of O,O-coordinated platinum in close main-chain proximity.
(a)(b)Considering now the B- and C-type platination reactions of Scheme13, the literature provides numerous
reaction sequences originating from uniquely functionalized carriers. Scheme 14
represents a prototype reaction starting out from polysuccinimide [47]. In all these
exemplifying cases, the platination agent has been trans-1,2-diaminocyclohexanediaquaplatinum(II)
dinitrate (DACH-Pt), a reactant preferred by us and others over
the tetrachloroplatinate dianion on grounds of favorable biological behavior, notably the
lack of crossresistance with cisplatin.Scheme 14
Synthesis
of conjugate featuring extrachain-type 1,2-carboxylatohydroxylato coordination of
platinum.We will now focus our attention to the bonding pattern of type E (see Scheme13) which underlies the largest
body of recent carrier-Pt conjugation research. Thus, Ohya et al. [48] prepared a
water-soluble poly(ethylene oxide) carrying a methoxy terminal and, at the other end, a
propylmalonate moiety suitable for platination with hydrolyzed cisplatin. The release behavior
was examined and the cytoxicity against lymphocytic leukemia cells was determined.
Good activity, although slightly below that of cisplatin, was observed. A study by the
afore-mentioned group of Duncan et al. produced numerous dicarboxylatoplatinum
compounds, among others, HPMA-derived conjugates possessing
carrier-attached dicarboxylato ligands [43]. Upon treatment with hydrated cisplatin, these converted to
highly bioactive cis-dicarboxylato-chelated platinum polymers as
exemplified in Scheme 15 for the affected repeat unit.Scheme 15
HPMA-derived dicarboxylato-coordinated platinum.A recent patent, outstanding among the many studies of polymeric phosphazene drugs [49], describes
polyphosphazene derivatives of dicarboxylato-coordinated platinum with activity inter
alia against the gastric tumor YCC-3 [50]. A simplified repeat unit structure is
reproduced in Figure 8.Figure 8
Simplified
structure of polyphosphazene repeat unit bearing sidechain-coordinated platinum.Numerous conjugates
of type E have in recent years been synthesized in the author’s laboratory, of which
only a single example is reproduced in Scheme16. The carrier educt in this
reaction is a polyamidoamine equipped with a solubilizing
N,Ndimethylaminopropyl side chain and an
aspartic acid-derived dicarboxylate, and platination once more
involves treatment with DACH-Pt [47, 51]. This polymer and related structures
have shown outstanding antiproliferative performance, on the strength of which, a
major development program has been initiated.Scheme 16
Polyamidoamine-derived dicarboxylato-coordinated platinum.This section would be
incomplete without a brief treatment of the comprehensive research efforts by
the group of Bilha Schechter and collaborators. These workers at the Weizmann
Institute of Science have been involved for some decades now in studies aiming at the
provision of platinum-containing polymers antibody-modified for immunotargeting to
human tumors. Representative compounds are a polyglutamic acid providing chelating
capacity of type E (see Scheme13) and some carboxymethyl derivatives of
dextran with similar, albeit less structurally defined metal coordination patterns. Excellent
biomedical test results on a broad front attest to the soundness of the development
program in Schechter’s highly productive laboratory. For in-depth reviews of the
Weizmann group’s activities, see Siegmann-Louda and Carraher [36], and Chen et al. [52].


## 4.1.1. Metal Coordination Involving Carrier-Bound Nitrogen Donor Ligands

By strict definition
of the polymer-drug conjugation concept, the presynthesis of a suitable carrier
polymer of biomedically “approved” structure is followed in another reaction step by the
bioreversible attachment of the drug. Before we study the exemplifying
macromolecules compliant with this definition of drug conjugation, it is only fair to Charles
Carraher, the pioneering scientist, that we are acquainted first with a separate synthetic
approach deviating from that routine, yet significant in its own right because it is one of
the earliest major assaults on the problem of providing biomedically important metal
compounds in polymeric form. This approach, developed in meticulous detail in Carraher’s
laboratory, embodies the aqueous-phase polycondensation of the tetrachloroplatinate(II)
dianion with diamines, giving polymers with the metal coordinated directly
into the main chain incis-diamine fashion, as shown in Scheme 9. The link R in this
process stands for a variety of aliphatic, aromatic, and heteroaromatic units, or simply for
a direct bond, and molecular masses attain the 106 Dalton  level. Biological test data,
notably for antiviral and antiproliferative performance against a number of cancer
cells, at that time demonstrated a “leap forward” in platinum-drug cancer therapy.Scheme 9
Polycondensation of diamines with the tetrachloroplatinate dianion.This brings us to the main stream of drug conjugate development: the polymerhomologous drug anchoring to
presynthesized carrier polymers. In this principal mode, polymer attachment of
the square-planar structural element ofcisplatin can be achieved via several
reaction paths, in the design of which care must be taken to include suitable
cleavage sites in the assembly for in vivo liberation of the bioactive agent from the
carrier vehicle. In principle, two approaches of metal-binding are available. In the
first, the metal center is bound via amine ligands attached to the carrier. In the
second mode, the metal is polymer-bound via the leaving group ligands. In the first case,
the result will be a structure comprising a Pt center tightly bound to the carrier, and release
of the active complex will require cleavage of a suitable anchoring segment in the side
chain. This is a time-dependent process, and the conjugates so constructed should
prove most efficacious in long-term administration. In the second case, the active
complex is released from the conjugate through (generally hydrolytic) cleavage of the bonds
connecting the metal with the leaving groups, resulting in aquation. Release
rates will be higher in this model, which should, hence, find optimal utilization in
short-term administration, whenever a boost is required for rapid effects. Let us look at the
first case: conjugation via carrier-bound amine ligands (see Scheme 10).Scheme 10
Platination of amine-functionalized carriers.In the simplest fashion (type A, with R = CH2CH2−), the >PtCℓ2 moiety is incorporated into a carrier
structure containing ethylenediamine as a main-chain segment. Such carriers may be
prepared by Michael addition polymerization with selected tetramine comonomers, and
Scheme 11 provides an illustrating example. A related metal bonding pattern
exists in certain polyethyleneimines prepared by Carraher’s group. In conjugates of these
structures the cis-diamine ligand system is strain-free and thus smoothly generated.Scheme 11
Platinum
atom coordinated by intrachain ethylenediamine segment.Platination of
carriers of type B (see Scheme10) leads to conjugates in which the entire cisplatin skeleton is located
outside the main chain. With R standing for a direct bond or, less favorably,
no more than a single atomic constituent, metal coordination through chelation with both
amino groups would appear to be sterically feasible. Exemplifying product structures
have indeed been obtained in Carraher’s laboratory [36] by platination of a
vinylamine/vinylsulfonate copolymer and of the natural (and hence,
biodegradable) polymer chitosan. A
special case of type B has been reported by Howell and Walles [37]. These authors
inter alia presynthesized a cisplatin analog with both NH3 ligands replaced by the two
NH2 groups in 1,2-diamino-4-hydroxybenzene. This reactant was then allowed to bind to
poly(vinylpyrrolidone) by molecular complexation, that is, in a form permitting its
release from the carrier by simple dissociation [37].Appreciably more
research has been performed with conjugates falling into the pattern of types C and D (see
Scheme10). In the author’s laboratory, polyaspartamides prepared per Scheme 3 (yet
including only two R-substituted units) were converted to platinated species by treatment
with tetrachloroplatinate dianion in aqueous phase. The two platination runs in
Scheme 12 exemplify this reaction type. Whereas the upper reaction leads to a conjugate
with cis-N,N′-coordinated platinum [38, 39], the lower one gives
rise to the formation of a structure wherein the metal is polymer-bound through a single N donor ligand [40].
In both cases, the first depicted, potentially cationic repeat unit provides both solubilizing and
homing functions. Cell culture tests showed both conjugate types to be equally
antitumor-active, and while with the upper conjugate ranked first in a series of cytotoxicity
tests [41], the lower one ranked first in an in vivo toxicological study, demonstrating
some 100-fold lower toxicity than determined for the cisplatin standard [42].Scheme 12
Platinum conjugation through carrier-attached amine donors.Similar platination patterns were reported from Ruth Duncan’s prolific laboratory (reviewed [36]), with HPMA polymers
used as the carriers. Broad-based biological investigations
provided excellent results with respect to release rates, toxicological behavior, and
cell-killing properties both in vitro and in vivo.Somewhat related to type D is a conjugate synthesized by Gianasi et al. [43] and further investigated by
Howell [44, 45], in which only one nitrogen donor ligand, in cooperation with an oxygen donor,
partakes in platinum coordination (See Figure 6(a)). The compound, an HPMA derivative, is
noteworthy as it features and N,N,N′,O-coordination square around the Pt atom. The
compound underwent in vitro and Phase I clinical tests with highly promising results,
low toxicities being paired with excellent antiproliferative activities [44, 45].Platinum
conjugation through repeat units featuring carrier-attached N- and O-donors.
(a)(b)A differently
structured polymer type featuring N,O-coordination of the metal has just been reported by
Bariyanga [46]. Representative
compounds contain the Fc−CH=N(R)−Pt(Cl2)−O− segment (Fc = ferrocenyl) connected to a poly(ethylene oxide) terminal (see
Figure 6(b)). These polymers are thus characterized by the presence of both a
ferrocene and a platinum complex. While no biomedical test data are available, it
would be interesting to find out in future studies whether such compounds display
cooperative cytotoxicity between the two drug species.


## 4.1.2. Metal Coordination through Carrier-Bound Oxygen Donor Ligands

In the polymer
described in this section the Pt complex is conjugated through two oxygen donor ligands
provided by the carrier. Contrasting with those covered in the preceding section,
these conjugates follow a release mechanism involving hydrolytic Pt bond cleavage,
thus requiring no special biofissionable links in the carrier for drug liberation. In
structures of this kind, the anchoring mechanism must be designed with a view to counteracting
the inherently limited stability of the O−Pt bond. The preferred approach to this goal
involves the construction of 5–7-membered chelate rings connecting the metal
atom with the carrier-attached donor ligands. Scheme13 offers acceptable choices,
for which the literature provides exemplifying experiments (reviewed by Carraher [36]); whereas types A
and D exemplify carrier binding through directly
backbone-attached hydroxylato- or carboxylato-type oxygen donors, the types B, C, and E represent
structures with spacers interposed between carrier backbone and oxygen donor.
Further work will determine the extent to which biological performance will be
affected by the coordinating atoms’ special remoteness from the main chain.Scheme 13
Platinum chelation patterns.Figure7 provides an example each of type A- and type D-derived conjugates without
further comments on the synthetic methodology [47].Examples of O,O-coordinated platinum in close main-chain proximity.
(a)(b)Considering now the B- and C-type platination reactions of Scheme13, the literature provides numerous
reaction sequences originating from uniquely functionalized carriers. Scheme 14
represents a prototype reaction starting out from polysuccinimide [47]. In all these
exemplifying cases, the platination agent has been trans-1,2-diaminocyclohexanediaquaplatinum(II)
dinitrate (DACH-Pt), a reactant preferred by us and others over
the tetrachloroplatinate dianion on grounds of favorable biological behavior, notably the
lack of crossresistance with cisplatin.Scheme 14
Synthesis
of conjugate featuring extrachain-type 1,2-carboxylatohydroxylato coordination of
platinum.We will now focus our attention to the bonding pattern of type E (see Scheme13) which underlies the largest
body of recent carrier-Pt conjugation research. Thus, Ohya et al. [48] prepared a
water-soluble poly(ethylene oxide) carrying a methoxy terminal and, at the other end, a
propylmalonate moiety suitable for platination with hydrolyzed cisplatin. The release behavior
was examined and the cytoxicity against lymphocytic leukemia cells was determined.
Good activity, although slightly below that of cisplatin, was observed. A study by the
afore-mentioned group of Duncan et al. produced numerous dicarboxylatoplatinum
compounds, among others, HPMA-derived conjugates possessing
carrier-attached dicarboxylato ligands [43]. Upon treatment with hydrated cisplatin, these converted to
highly bioactive cis-dicarboxylato-chelated platinum polymers as
exemplified in Scheme 15 for the affected repeat unit.Scheme 15
HPMA-derived dicarboxylato-coordinated platinum.A recent patent, outstanding among the many studies of polymeric phosphazene drugs [49], describes
polyphosphazene derivatives of dicarboxylato-coordinated platinum with activity inter
alia against the gastric tumor YCC-3 [50]. A simplified repeat unit structure is
reproduced in Figure 8.Figure 8
Simplified
structure of polyphosphazene repeat unit bearing sidechain-coordinated platinum.Numerous conjugates
of type E have in recent years been synthesized in the author’s laboratory, of which
only a single example is reproduced in Scheme16. The carrier educt in this
reaction is a polyamidoamine equipped with a solubilizing
N,Ndimethylaminopropyl side chain and an
aspartic acid-derived dicarboxylate, and platination once more
involves treatment with DACH-Pt [47, 51]. This polymer and related structures
have shown outstanding antiproliferative performance, on the strength of which, a
major development program has been initiated.Scheme 16
Polyamidoamine-derived dicarboxylato-coordinated platinum.This section would be
incomplete without a brief treatment of the comprehensive research efforts by
the group of Bilha Schechter and collaborators. These workers at the Weizmann
Institute of Science have been involved for some decades now in studies aiming at the
provision of platinum-containing polymers antibody-modified for immunotargeting to
human tumors. Representative compounds are a polyglutamic acid providing chelating
capacity of type E (see Scheme13) and some carboxymethyl derivatives of
dextran with similar, albeit less structurally defined metal coordination patterns. Excellent
biomedical test results on a broad front attest to the soundness of the development
program in Schechter’s highly productive laboratory. For in-depth reviews of the
Weizmann group’s activities, see Siegmann-Louda and Carraher [36], and Chen et al. [52].


## 4.2. Polymer-Ferrocene Conjugates

Contrasting with the platinum drugs, which have firmly established their prominent position as antitumor
agents in clinical use, research in the realm of ferrocene-based chemotherapy is still
at an experimental stage. However, biomedical test data, preliminary as they
are, have shown overwhelming promise, stimulating comprehensive investigations
currently in progress worldwide. The core organoiron compound, di-(η5-cyclopentadienyl)iron(II),
has been under scientific and technical scrutiny for more than half a
century, and numerous topical surveys, including fundamental physical and chemical
applications, have appeared in the literature (surveyed in two books [53, 54]). The fascinating
ferrocene chemistry, pivotally involving the compound’s redox equilibrium
with its one-electron oxidation product, the ferricenium cation, has prompted wide-ranging
investigations of this electrochemical ferrocene/ferricenium couple in the
biological environment, with ultimate ramification into the topic of
carrier-bound ferrocene conjugates.The bulk of contributions to these research developments has materialized in the author’s laboratory,
as attested by more than 50 pertinent papers published by our group until now. The
reader is referred to some reviews [54–57] covering this prominent development trend.Let us look now in detail at the large family of polymer-bound ferrocenes designed for biomedical
applications. As we embark on this theme, we realize that, in the “old days” of ferrocene polymer
development spanning the 1950–1970 decades, macromolecular ferrocenes and other
metallocenes had been synthesized in huge numbers (reviewed in the book [54] and three book
chapters [58–60]), yet most of those
were hydrophobic and lacked
hydrosolubility and, hence, were not considered in later work. An entirely new concept had to be
developed for the purpose of rendering any metallocene polymers qualified for
biological examinations. That concept, as we learned in Section 2, originated a decade
later. It embodies the technique of conjugation so successfully utilized in the
design of polymeric platinum and other drugs, and in the present section it will be seen to be
instrumental in the synthesis of carrier-bound ferrocene compounds.Earlier exploratory studies [61] demonstrated the feasibility of carrier binding and provided a range of variously
structured conjugates meeting the crucial requirement of water solubility.
Ferrocenylamines, initial candidate agents for ferrocenylation because of their strongly
nucleophilic functionality, were soon omitted on electrochemical grounds. Ferrocenyl-carboxylic
acids, on the other hand, notably 4-ferrocenylbutanoic acid, emerged in those
earlier investigations as suitably functioning compounds providing electrochemically
stable ferricenium counterparts [62–64]. The butanoic acid derivative soon became the
ferrocenylation agent of choice in all subsequent conjugation developments in
preference over other chemically feasible coupling mechanisms. Again, the
polyaspartamides introduced in Section 3.1 turned out to be the most versatile and functional
carriers; equipped with NH2-terminated side chains, they soon became, and still
are, the “workhorse” carrier polymers in our laboratory, conveniently undergoing coupling
reactions in aprotic medium with the ferrocenyl acid via carboxyl group activation, or
directly with the aid of strong coupling agents, such as HBTU (O-[1H-benzotriazol-1-yl]-1,
1,3,3-tetramethyluronium hexafluorophosphate). Scheme 18 shows a typical
reaction step of this type, singled out here on account of the conjugate’s particularly
rewarding cytotoxic behavior; in vitro screens against the HeLa human cervix
epithelial cancer revealed a cell-killing activity (on metal-to-metal equivalent basis)
twice that of the cisplatin standard, and a fourfold superiority over cisplatin against Colo
320 DM, a notoriously drug-resistant human colon cancer line [65]. Interestingly,
similar conjugates possessing nonbasic hydroxyalkyl side groups in place of the strongly
basic tert-amine-terminated solubilizing and homing moiety of the drawn structure gave
rather poor results, reflecting the lowered affinity to the cancerous cell (see Section
2.2) because of lacking basicity.Among the great variety of other carrier types, we cite the primary amine-functionalized amidoamine polymer
synthesized by Michael polyaddition (see Section3.2); readily ferrocenylated with
HBTU mediation [66], it converts to a conjugate featuring a long (8 atomic constituents)
spacer connecting the metallocene to the main chain as depicted in Scheme 19.On the other hand, a related amidoamine-type carrier characterized by an additional intrachain −NH−NH− group yet lacking the
NH2-spacer side chain converts to a conjugate by direct N-acylation of the main chain [67] (see Scheme 20). Subjected to the
same cell culture tests as cited above, this ferrocene-loaded polymer, again, performed distinctly better
than the cisplatin standard.Lastly, we mention an anchoring step involving O-acylation. To this end, various hydroxyalkyl-substituted
polyaspartamides were treated in aprotic medium with ferrocenylbutanoic
acid in the presence ofN,N′-dicyclohexylcarbodiimide
coupling agent and 4-(dimethylamino)pyridine (DMAP) catalyst [68]. Scheme 21 shows a representative run,
with x/y varying from about 3 to 10 as a function of molar reactant ratios.A conjugate of this
type, withx/y = 2.6, subjected to an in vivo toxicity test, gave a maximum tolerated
dose of 0.43 mmol Fe/kg, more than 20-times higher than determined for the cisplatin standard [69].
The polymer is thus some 20-fold less toxic than the standard,
and comparable toxicity behavior is also displayed by selected amide-linked analogs.
These findings, in conjunction with cell-killing properties, augur exceptionally well
for the class of ferrocene conjugates as cancer-chemotherapeutic agents, and their
future looks highly promising indeed.


## 5. Summary and Conclusions

Mankind, along its arduous development path from early hominids viaHomo erectus to the present “status”
of Homo sapiens, has never been spared the experience of bodily injuries and the
suffering from debilitating or even lethal diseases. Yet the human spirit has at all times
searched for therapeutic weapons to overcome, or at least deter, those inimical adversaries.
Even at the sophisticated level of human enterprise attained by now, we have come to
accept the limited efficacy of those therapeutic weapons against many serious
infirmities and the pressing need for continued forceful research activities to eliminate existing
threats to our well-being. Cancerous diseases stand out amid those threats as
particularly dreaded adversaries, and on these we have focused our attention in the
present discourse.We have acquainted
ourselves with the cancer problemper se, with the current chemotherapeutic treatment
limitations, and, getting into the core topic, with the promising and highly
challenging present research endeavors in the field of polymer-drug conjugation. In doing
so, we have followed the development of one of the leading polymer-based chemotherapeutic
tools in the fight against cancer, with emphasis on platinum- and
iron-based drug systems. We have found out that the expediency of using goal-designed,
water-soluble, and biologically acceptable macromolecules as temporary transport
vehicles for powerful, yet potentially toxic, and resistance-promoting medicinal agents has,
beyond early doubts and objections, matured to a practicable technology. It is to
be hoped that the facts and arguments here-presented will encourage many students of the
biochemical and pharmaceutical sciences to participate in the
global efforts to promote and refine the drug conjugation modality for the future benefit of
the victims of neoplastic and other diseases Scheme 17.Scheme 17
The ferrocene/ferricenium redox couple.Scheme 18
Polyaspartamide-derived ferrocene conjugate.Scheme 19
Poly(amidoamine)-derived ferrocene conjugate.Scheme 20
Ferrocenylation of amidoamine polymer featuring intrachain-type −NH− group.Scheme 21
Ferrocenylation of polyaspartamide featuring extrachain-type hydroxyl groups; R = aliphatic segment.



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