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Poly(amidoamine)s: Past, Present, and Perspectives
Paolo Ferruti
1,2
1
Dipartimento di Chimica, via C. Golgi 19, 20133, Milano, Italy
2
Consorzio Nazionale Interuniversitario di Scienza e Tecnologia dei Materiali (INSTM), via G. Giusti 9, 50121, Firenze, Italy
Correspondence to: P. Ferruti (E-mail: paolo.ferruti@unimi.it)
Received 7 February 2013; accepted 8 February 2013; published online 15 March 2013
DOI: 10.1002/pola.26632
Poly(amidoamine)s (PAAs) are a family of synthetic polymers
obtained by stepwise polyaddition of prim-orsec-amines to
bisacrylamides. Nearly all conceivable bisacrylamides and
prim-orsec-amines can be employed as monomers endowing
PAAs of a structural versatility nearly unique among stepwise
polyaddition polymers. PAAs are degradable in aqueous
media, including physiological fluids. Many of them are
remarkably biocompatible notwithstanding their cationic char-
acter. PAAs are per se highly functional polymers and, in addi-
tion, can be further functionalized giving rise to an endless
variety of polymeric structures meeting the requisites for appli-
cations in such apparently disparate fields as inorganic water
pollutants scavengers, sensors, drug and protein intracellular
carriers, transfection promoters, peptidomimetic antiviral and
antimalarial agents. In this review, the unique chemistry of
PAAs is discussed and a vast library of PAA structures and
PAA applications from the beginning to the present days
reported. V
C2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A:
Polym. Chem. 2013,51, 2319–2353
KEYWORDS: biodegradable; biomimetic; poly(amidoamine)s;
step-growth polymerization; water-soluble polymers
INTRODUCTION The second half of the twentieth century has
witnessed the outstanding development of polymeric materials,
or “plastics,” in every aspect of life, from everyday housing com-
modities to biomedical devices. In the new century, one of the
most exciting frontiers of macromolecular science lies in the de-
velopment of purpose tailored, sophisticated functional poly-
mers, that is, polymers endowed with reactive chemical
functions whose type, number, and arrangement have been
planned to specific purposes. For instance, a large deal of poly-
mer therapeutics involve functional polymers, such as mem-
brane-active polymers with a potential as transfection
promoters, polymer drug carriers, polymers endowed with spe-
cific biological activities of their own, polymers able to promote
the lysosomal escape, and the intracellular trafficking of pro-
teins, polymeric hydrogels for cell culturing and tissue engineer-
ing, polymer nanoparticles, and their multiform uses. Besides
medicine, functional polymers find a number of technical appli-
cations, such as specific ion-exchange resins, noxious water pol-
lutants absorbers, active coatings of sensors for pollutants
detection, agents for surface modification, and many others.
The aim of this article is to provide an updated state-of-the-
art on chemistry and applications of a particular family of
synthetic functional polymers, poly(amidoamine)s (PAAs),
originally obtained by stepwise Michael-type polyaddition of
prim-monoamines [Scheme 1(a)] or sec-diamines [Scheme
1(b)] with bisacrylamides.
HISTORICAL BACKGROUND
The Michael-type addition of prim- and sec-amines to car-
bon–carbon double bonds activated by adjacent electron-
attracting groups is well known since the first half of the
twentieth century. The first intimation of a high polymer pre-
pared by polyaddition of sec-diamines with bisacrylamides is
in a patent application dating back to 1956, which appa-
rently was no further developed.
1
An extensive study on this
type of stepwise polyaddition was independently started in
the mid-sixties of the twentieth century at the Polytechnic
Institute of Milan and the first resultant polymers, called
PAAs, were first published in a series of papers on an Italian
Journal, together with structurally related polymers obtained
by substituting bisacrylic esters and divinylsulfone for bisa-
crylamides or phosphines and hydrazines for amines.
2–8
These early articles were soon collectively reviewed on a
British Journal.
9
Slightly afterwards, some PAAs were also in-
dependently reported elsewhere.
10
Subsequently, the chemis-
try of PAAs was reviewed at intervals.
11–13
Several new PAA-related polymers, such as for instance those
derived by stepwise polyaddition of aminated bis-thiols with
bisacrylamides,
14,15
bisacrylic esters,
16,17
divinylsulfone,
18
and bis-cyanovinyl compounds,
19
were later reported.
The self-polyaddition of 1-acrylamido-2-aminoethane hydro-
chloride gave rise in the early eighties of the nineteenth
V
C2013 Wiley Periodicals, Inc.
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century
20
to a highly successful family of hyperbranched, but
not crosslinked polymers also called poly(amidoamine)s but
initialed PAMAM, which were extensively studied and modi-
fied in view of a number of biotechnological applications,
reported in hundreds of articles and reviews.
21
PAMAMs
shouldnotbeconfusedwithPAAs,astheyconstituteadiffer-
ent polymer family, inasmuch as in their dendrimer-like back-
bone each amide group (a) is preceded and followed by an
amine (b) group and vice-versa, that is, with the sequence
…a…b…a…b…, whereas in the PAA chains the sequence of the
same groups is either …a…a…b…a…a…b…. or
…a…a…b…b…a…a…b…b. depending on whether prim-amines
or bis-sec-amines were involved in their preparation. Moreover,
the PAMAM multiple chains contain sec-andtert-amine groups
and the chain termini are prim-amine groups. On the opposite,
unless purposely functionalized or crosslinked, PAAs are linear,
single chain amine- (nearly always tert-amine-) polymers and
their chain termini are either vinyl- or sec-amine groups.
PAMAMs will not be included in this review.
SYNTHETIC FEATURES
The Michael polyaddition leading to PAAs is best performed
in aqueous media or, alternatively, alcohols. Ethylene glycol
is the best substitute for water as reaction solvent. Methanol,
ethanol, N-methyl-N,N-di-2-hydroxyethylamine and benzyl
alcohol can also be used to overcome solubility problems. A
source of protons is necessary to speed up the reaction and
obtain high molecular weight products in reasonable time,
whereas aprotic solvents are unsuitable as reaction media.
2,7
A comparative kinetic study on the polyaddition kinetics of
2-methylpiperazine and 2,5-dimethylpiperazine with 1,4-
bisacryloylpiperazine, chosen as model PAA monomers, was
performed in water, methanol, ethylene glycol, formamide,
and dimethylformamide.
22
In the protic solvents, the polyad-
dition involving 2-methylpiperazine proceeded through a
two-step mechanism, each step involving one of the two dif-
ferent sec-amine groups, whose reaction constants were sig-
nificantly different owing to the different steric hindrance by
the neighboring groups. Each step followed pseudo-second-
order kinetics. The kinetic constants included the catalytic
protic species. In dimethylformamide, the polyaddition pro-
ceeded through third-order kinetics. This accounted for the
autocatalytic activity of the amine groups. The apparent
kinetic constants in the protic solvents increased with the
increase of the autoprotolysis constant value and decreased
with the increase of the dipole moment.
Recently, it was reported that salts of earth alkali metals
exert a catalytic activity on the Michael reaction of prim-
amines to bis-acrylamides, whereas the salts of transition
metals are apparently inactive. In particular, the addition of
CaCl
2
to the reaction mixtures led to a significant increase in
the reaction rate and the PAAs obtained in its presence were
identical to those prepared by the conventional method.
Doubtless, this technique may represent an attractive
improvement in the preparation of PAAs from poorly reac-
tive amines.
23
Both prim-monoamines and sec-diamines lead to linear poly-
mers. prim-Diamines usually act as tetrafunctional monomers
and give crosslinked insoluble resins. However, by employing
low reactant concentrations, low initial temperatures and
excess bis-amines, soluble PAAs carrying sec- instead of tert-
Paolo Ferruti took his degrees at the University of Pavia and then was summoned by Giu-
lio Natta at the Polytechnic of Milan. In 1968, he worked with Melvin Calvin in Berkeley at
the Lawrence Radiation Laboratory of the University of California. In 1976, he became Full
Professor at the University of Naples and then commuted to Bologna, Brescia and finally
Milan. He authored more than 400 papers and 50 patents. Functional polymers for techni-
cal and biotechnological applications are his chief interest. His main scientific achievement
has been the discovery of poly(amidoamine)s.
SCHEME 1 Traditional synthesis of linear PAAs.
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amine groups in the main chain were obtained in some
instances. These PAAs allowed drug attachment and were
studied as soluble drug carriers.
24
PAAs are not only intrinsically functional polymers, but are
also amenable to further functionalization. A number of
chemical functions, such as hydroxy-, tert-amine-, allyl-, am-
ide-, and ether groups if present in the monomers do not
interfere in the polymerization process and remain as side
substituents in the resultant PAAs. Chemical groups capable
of reacting with activated double bonds, such as SH, NH
2
,
NHR, and PH
2
, cannot be directly introduced in PAAs as side
substituents, but can with special precautions. Additional
prim-orsec-amine groups, for instance, must be protected
first by groups cleaved by acids, but stable under basic con-
ditions. When the protonation constants of a prim-diamine
are widely different, monoprotonation may suffice without
any further protection. For instance, monoprotonated 1,2-
bis-aminoethane yielded soluble PAAs carrying prim-amine
groups as side substituents.
25
For the same reason, by react-
ing partially protonated poly-(L)-lysine with a large excess
N,N-dimethylacrylamide in aqueous media, the reaction
stopped when the unprotonated amine groups, and only
these, were saturated.
26
Accordingly, linear PAAs carrying
guanidine groups as side substituents are currently prepared
from 4-aminobutylguanidine in water at pH 9.
27
It was immediately realized that nearly all conceivable bisa-
crylamides, sec-diamines and, contrary to a first statement,
1
also prim-amines were eligible as monomers. Most bisacryla-
mides, monoamines, and bisamines employed so far in PAA
synthesis are reported in Tables 1–3, respectively. For the
sake of simplicity, in Tables 2 and 3 “monoamines” and
“bisamines” are defined as the amines that contribute to
chain building with one or two nitrogen atoms, respectively,
not considering the actual number of nitrogen atoms present
in their molecule.
Random or quasi-random copolymeric PAAs are obtained in
most cases starting from mixtures of monomers with no
further precautions. However, when a limited number of
poorly reactive amines need to be introduced in a PAA
chain, performing the reaction in a single pot, but in two
steps, is advisable. The less reactive amine should be
treated first with a large excess bisacrylamide, thus forcing
it to react completely. The polymerizing system, containing
vinyl-terminated trimers or short oligomers, is then added
with the more reactive amine in the amount needed to stoi-
chiometrically balance the reactant functions and the poly-
addition is let to proceed normally.
28
This technique
ensures a fair distribution of the less reactive amine moi-
eties in the final product by minimizing the risk of confin-
ing them at the chain ends of the lowest molecular weight
fractions.
From the above considerations it is clearly apparent that
PAAs, as a class, are endowed with versatility nearly unique
among stepwise polymers. Since, in addition, they are
assembled in a modular fashion both as homo- and copoly-
mers, they are particularly amenable to structure-tailoring
for specific purposes. There is little doubt that the already
large number of PAAs described in the literature still repre-
sents only a minor part of their synthetic potential.
The structures of most soluble PAAs described so far in the
literature, apart from two special cathegories (see below),
TABLE 1 Bisacrylamide Monomers
X-1 X-9
X-2 X-10
X-3 X-11
X-4 X-12
X-5 X-13
X-6 X-14
X-7 X-15
X-8 X-16
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TABLE 2 Monoamine Moieties
Y-1 Y-21
Y-2 Y-22
Y-3 Y-23
Y-4 Y-24
Y-5 Y-25
Y-6 Y-26
Y-7 Y-27
Y-8 Y-28
Y-9 Y-29
Y-10 Y-30
Y-11 Y-31
Y-12 Y-32
Y-13 Y-33
Y-14 Y-34
Y-15 Y-35
Y-16 Y-36
Y-17 Y-37
Y-18 Y-38
Y-19 Y-39
Y-20 Y-40
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are reported in Tables 4 and 5. In particular, the PAAs listed
in Table 4 contain monoamine-deriving units, defined as earlier,
either alone or as copolymers with diamines, whereas the PAAs
listed in Table 5 contain only diamine-deriving units.
PAAs characterized, respectively, by acidic functions impart-
ing them amphoteric properties or by SH or SAS groups, the
latter both as side substituents or inserted in the polymer
chain, will be treated separately.
Amphoteric PAAs
Amphoteric PAAs are PAAs carrying acid functions as side
substituents, usually carboxyl groups, but in a few instances
also sulfonic
9,44
or phosphonic groups.
85
Aminoacids or car-
boxylated bis-acrylamides can be used as monomers. In the
presence of acid groups, the polyaddition require triggering
by a stoichiometric amount of base. After that, the polyaddi-
tion proceeds normally with carboxylated bisacrylamides
and b-aminoacids or their higher analogs, such as b-alanine,
c-aminobutyl acid, and so forth. Glycine and peptides in
which the first aminoacid residue is glycyl react slower. Nat-
ural a-aminoacids other than glycine react very sluggishly, of-
ten requiring months at room temperature to reach
reasonably high molecular weight polymers.
Amphoteric PAAs present a unique interest as bioactive poly-
mers. By tuning the number and nature of the ionizable
groups, amphoteric PAAs with isoelectric points ranging
from 3 to 10 can be obtained.
44
The prevailingly basic ones
are often considerably more biocompatible than purely cati-
onic PAAs of similar net average positive charge at physio-
logical pH,
44
but maintain most polycation properties. For
instance, they form interpolyelectrolyte complexes with poly-
anions such as heparin and DNA, may exert membrane activ-
ity, and may act as transfection promoters (see later).
Amphoteric PAAs that are prevailingly anionic at pH 7.4 do
not exert membrane activity. However, in solution their net
average charge is a function of pH. By a proper choice of the
starting monomers the acid and basic strength of the amine
and the carboxyl groups can be tuned in such a way that the
resultant PAAs shift from a prevailingly anionic to a prevail-
ingly cationic state for relatively modest pH changes, as for
instance when they are internalized in cells and pass from
the extracellular fluids, where the pH is 7.4, into cellular com-
partments where the pH is 5.5 or whereabouts. As a rule, this
charge reversal renders these PAAs membrane active.
53
Most amphoteric PAAs described so far are reported in Table 6.
Thio- and Dithio-Functionalized PAAs
Considerable attention has been recently focused on poly-
mers carrying thiol groups. They are especially studied for
their mucoadhesive properties
99
and find, inter alia, applica-
tions in nanomedicine.
100
Polymers bearing dithio groups
are strictly related to the thiol-bearing ones since oxidation–
reduction processes can easily transform these groups into
each other. Thiol-functionalized PAAs were obtained by
employing N-mono-protected cystamine as co-monomer and
then reductively cleaving the SAS groups of the resultant
polymers
101
[Scheme 2(a)]. Alternatively, they were obtained
by reduction of the crosslinked PAAs obtained by substitut-
ing plain cystamine for mono-protected cystamine [Scheme
2(a)]. The thiol groups could be transformed into activated
TABLE 2 (Continued).
Y-41 Y-47
Y-42 Y-48
Y-43 Y-49
Y-44 Y-50
Y-45 Y-51
Y-46 Y-52
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dithio-derivatives by reaction with 2,20-dithiodipyridine.
Interestingly, the same activated dithio-derivatives were
straightforwardly prepared in the presence of triethylamine
by dithio–dithio exchange reaction of the crosslinked PAAs
obtained from cystamine with excess 2,20-dithiodipyridine
[Scheme 2(b)]. These activated dithio derivatives easily
underwent coupling reactions with added thiols, for instance
thiocholesterol, obtaining amphiphilic polymers that in aque-
ous media spontaneously gave nano-aggregates [Scheme
2(c)]. The aggregation property is shared by other PAAs con-
taining hydrophilic and hydrophobic moieties,
68
but in the
above derivatives the hydrophobic moieties are linked to the
PAA chain by SAS reductively cleavable bonds. Therefore,
the aggregates are liable to collapse in reducing
TABLE 3 Diamine Moieties
Z-1 Z-12
Z-2 Z-13
Z-3 Z-14
Z-4 Z-15
Z-5 Z-16
Z-6 Z-17
Z-7 Z-18
Z-8 Z-19
Z-9 Z-20
Z-10 Z-21
Z-11 Z-22
Z-23 Z-29
Z-24 Z-30
Z-25 Z-31
Z-26 Z-32
Z-27 Z-33
Z-28
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TABLE 4 Monoamine-Deriving PAAs
No. X Y/Z References
1 X-1 Y-1 29
2 X-1 Y-8 20, 31
3 X-1 Y-18 32
5 X-1 Y-21 33
6 X-1 Y-13 (10–0%) 1Y-49 (90–70%) 34
7 X-5 Y-1 4, 7, 9, 35
8 X-6 Y-1 4, 7, 9, 35
9 X-11 Y-1 9, 36–38
10 X-11 Y-1 9, 10, 35, 36, 39–41
11 X-11 Y-2 4, 7, 9
12 X-11 Y-3 4, 7, 9
13 X-11 Y-4 4, 7, 9
14 X-11 Y-5 4, 7, 9, 42
15 X-11 Y-7 35, 43
16 X-11 Y-9 9
17 X-11 Y-14 25, 44
18 X-11 Y-18 35, 37, 43, 45–48
19 X-11 Y-19 45, 49
20 X-11 Y-20 45, 49
21 X-11 Y-22 43
22 X-11 Y-23 43
23 X-11 Y-24 44
24 X-11 Y-26 43
25 X-11 Y-45 50
26 X-11 Y-46 51
27 X-11 Y-5 (10–50%) 1Y-10 (90–50%) 52
28 X-11 Y-5 (10–30%) 1Y-18 (70–90%) 52
29 X-11 Y-6 (10–30%) 1Y-10 (90–70%) 52
30 X-11 Y-9 (10–50%) 1Y-10 (90–50%) 52
31 X-11 Y-9 (30%) 1Y-18 (70%) 52
32 X-11 Y-13 (5%) 1Z-18 (47.5%) 1Z-15 (47.5%) 53
33 X-11 Y-14 (5%) 1Z-18 (47.5%) 1Z-15 (47.5%) 25
34 X-11 Y-44 (50%) 1Z-18 (50%) 54
35 X-1 Y-14 55, 56
36 X-1 Y-15 55, 56
37 X-1 Y-16 55, 56
38 X-7 Y-14 57
39 X-7 Y-15 57
40 X-7 Y-16 57
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environments, as for instance after internalization in cells.
PAA conjugates of SH-bearing peptides, such as glutathione,
were also prepared by the same technique [Scheme
2(d)].
102,103
Polymers bearing SAS bonds in the polymer chain are selec-
tively degraded after cell internalization. When orally admin-
istered, the same polymers are stable in the first sections of
the gastrointestinal tract, but degrade in the colon. PAAs are
particularly amenable to this structural modification, since
SAS containing amines and bisacrylamides are either com-
mercially available or easily synthesized, and if used as co-
monomers straightforwardly lead to SAS linkages in the
polymer chain. This concept was first put forward in a
pioneering paper reporting on the use of N,N0-bisacryloyl-
cystamine or N,N0-bisacryloylcystine as amidic monomers
for the specific purpose of obtaining bioreducible PAAs
(Scheme 3).
104
This pioneering study was followed by an extensive series of
articles on different SAS bearing PAAs based on bis-acryloyl-
cystamine, proposed as nucleic acid carriers, and transfection
promoters as well as, in some cases, as protein car-
riers.
66,105,106
Alternatively, soluble SAS PAAs were obtained
by employing L-cystine as diamine comonomer, taking
advantage of the different reactivity of the amine hydrogens
of substituted a-aminoacids
107
(Scheme 4).
Shortly later, N,N0-dimethylcystamine was also used for the
same purpose.
60
More recently, PAAs containing both SAS
and acetal acid-labile groups in the main chain were also
reported.
63
Most thio-and dithio-functionalized PAAs described so far in
the literature are reported in Table 7.
PAA-Based Block and Graft Copolymers
Since PAAs are prepared by stepwise polyaddition of amines
with bisacrylamides (Scheme 1), driving the polymerization
of stoichiometrically unbalanced monomer mixtures to com-
pletion leads to PAAs whose polymerization degree
Xncan
be calculated from to the following well-know equation
118
Xn511r
12r(1)
where ris the ratio of the number of defect functions to the
number of excess functions, but with qualifications. A pur-
posely planned NMR study performed on the bisacryloylpi-
perazine/2-methylpiperazine PAA (Table 5, No. 34) as model
showed that the eq 1 was strictly obeyed only with excess
amine, whereas with excess bis-acrylamide the products
invariably had molecular weight higher than expected,
77
as
further confirmed by unpublished results obtained with sev-
eral other PAAs. An older viscometric study had pointed to
the same conclusion.
2
A possible explanation lies in the
occurrence, during polymerization, of some hydrolytic cleav-
age of the terminal acrylamide groups. This would partly
substitute amine groups for acrylamide groups, thus
TABLE 5 Diamine Deriving PAAs
No. X Z References
1 X-1 Z-1 24
2 X-1 Z-2 58
3 X-1 Z-3 12, 29, 59
4 X-1 Z-8 12, 60
5 X-1 Z-9 24
6 X-1 Z-10 24
7 X-1 Z-11 24
8 X-1 Z-15 61, 62
9 X-1 Z-16 63
10 X-1 Z-17 1, 30, 31, 61, 64
11 X-1 Z-18 59, 61, 65
12 X-1 Z-21 66, 67
13 X-1 Z-29 24
14 X-2 Z-17 10
15 X-3 Z-3 29
16 X-4 Z-17 3, 7, 9
17 X-6 Z-3 3, 7, 9
18 X-6 Z-17 3, 7, 9
19 X-6 Z-18 3, 7
20 X-7 Z-2 60
21 X-8 Z-2 60
22 X-10 Z-3 68
23 X-10 Z-20 68
24 X-11 Z-2 60
25 X-11 Z-3 3, 7, 9, 35–41, 44–47, 69, 70
26 X-11 Z-5 3, 7, 9
27 X-11 Z-6 35, 37, 41, 45, 47, 70, 71
28 X-11 Z-7 41, 45, 47, 70–72
29 X-11 Z-13 36, 39–41, 73
30 X-11 Z-14 32, 35, 37, 72
31 X-11 Z-15 61, 74–76
32 X-11 Z-17 2, 7, 9, 38–40, 61, 65, 74
33 X-11 Z-18 2, 7, 9, 22, 44, 61, 65, 77
34 X-11 Z-19 22
35 X-11 Z-21 66
36 X-11 Z-23 37
37 X-11 Z-30 78
38 X-11 Z-15 (50%) 1
Z-18 (50%)
53, 79–84
39 X-12 Z-17 9
40 X-12 Z-18 9
41 X-13 Z-2 60
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TABLE 6 Amphoteric PAAs
No. X Y/Z References
1 X-1 Y-18 (50%)1Y-33 (50%) 61
2 X-11 Y-29 9, 72, 86
3 X-11 Y-30 9, 35, 44, 72, 86
4 X-11 Y-31 9
5 X-11 Y-32 9, 72
6 X-11 Y-33 72
7 X-11 Y-34 9, 44
8 X-11 Y-35 9
9 X-11 Y-36 9
10 X-11 Y-37 9
11 X-11 Y-38 9
12 X-11 Y-39 85
13 X-11 Y-40 87
14 X-11 Y-41 87
15 X-11 Z-25 44, 85
16 X-11 Z-26 9
17 X-11 Z-27 9
18 X-11 Y-14 (7.5%) 1Y-39 (92.5%) 85
19 X-11 Y-13 (7.5%) 1Y-39 (92.5%) 85
20 X-11 Y-13 (7.5%) 1Z-25 (92.5%) 85
21 X-11 Y-18 (50%)1Y-29 (50%) 48
22 X-11 Y-18 (50%)1Y-33 (50%) 61
23 X-11 Y-18 (50%)1Z-27 (50%) 48
24 X-11 Y-29 (50%) 1Z-17 (50%) 52
25 X-11 Y-39 (95%) 1Y-44 (5%) 85
26 X-14 Y-1 29
27 X-14 Y-14 25, 44, 85
28 X-14 Y-24 27, 44, 88–92
29 X-14 Y-30 44
30 X-14 Y-34 44
31 X-14 Y-39 85
32 X-14 Z-3 29, 44, 59, 93
33 X-14 Z-4 93
34 X-14 Z-6 93
35 X-14 Z-8 93
36 X-14 Z-15 61
37 X-14 Z-17 74
38 X-14 Z-18 44, 53, 59, 61, 65, 79–84, 90, 91, 93–96
39 X-14 Z-25 44, 85
40 X-14 Y-13 (7.5%) 1Y-39 (92.5%) 85
41 X-14 Y-13 (7.5%) 1Z-25 (92.5%) 85
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adjusting to some extent the stoichiometric balance, as under
the basic polymerization conditions and at room tempera-
ture the double bond of the resultant acrylic acid is practi-
cally unreactive toward amines and does not act as chain-
terminator.
It is apparent that stoichiometrically unbalanced monomer
mixtures lead to PAAs doubly terminated with the excess
function. Both vinyl- and sec-amine end capped PAAs (V-PAA
and sec-A-PAA) have been prepared. In turn, V-PAAs can be
converted into prim-amine end-capped PAAs (prim-A-PAA)
by treating sec-A-PAAs with a large excess ammonia. All
these PAAs can be regarded as macromonomers and were
employed for preparing hybrid block- and graft copolymers
with polymeric structures other than PAAs.
The first example of soluble and moldable PAA block copoly-
mer involved the radical polymerization of styrene in the
presence of a V-PAA (Scheme 5).
The addition of chain-transfer agents and the use of fairly
high molecular weight samples of V-PAA prevented crosslink-
ing.
119–121
Most subsequent preparations on PAA block- and
graft-copolymers employed prim-A-PAAs and sec-A-PAAs.
Graft copolymers were prepared by condensation with chlor-
osulfonated polyethylene
122–125
and with ethylene/vinylalco-
hol/vinylacetate terpolymer, the latter after activation of the
hydroxyl groups with N,N0-carbonyldiimidazole.
126
Graft
copolymers were also obtained by reacting prim-A-PAA with
poly(urethaneamide)s containing fumaric- or maleic acid
moieties, that is, activated double bonds in their main
chain.
127
All the above grafting reactions lead to soluble
polymers only by employing a large excess A-PAA to mini-
mize the formation of intermolecular bridges. This recurrent
problem was only recently overcome by synthesizing hetero-
difunctional PAA dimers and polymers (see later).
Mixed poly(urethane-PAA) networks, named PUPA, were
more extensively studied.
128–136
These materials were pre-
pared by first treating A-PAA with excess diisocyanate in
chloroform solution and subsequently coupling the resultant
isocyanate-terminated PAA with a commercial polyurethane
via the CONH groups of the latter (Scheme 6).
Tough films were cast from the reaction mixture, which
could also be used for coating purposes. In another process,
adding A-PAAs to the monomer mixtures leading to polyur-
ethanes gave linear polyurethane-PAA block copolymers.
Both prim-and sec-A-PAAs were used, but the former gave
better results.
Block and graft copolymers of PAAs containing segments of
different nature were also straightforwardly obtained by
copolymerization techniques with amine-terminated oligom-
ers, such as a-bis(sec-amino)polyoxyethylenes [Scheme
7(a)],
78
a-methyl-x-amino-polyoxyethylenes [Scheme 7(b)],
50
or x-amine-terminated poly(4-acryloylmorpholine) [Scheme
7(c)],
51
in turn obtained by chain transfer technique by radi-
cal polymerization of 4-acryloylmorpholine in the presence
of cysteamine).
137
Surface-Grafting PAAs Onto Inorganic and Organic
Materials
PAAs have been surface-grafted on many organic and inor-
ganic commercial materials, such as poly(ethyleneterephta-
late),
138
polyurethanes,
139
plasticized poly(vinylchloride),
140
glass,
141
and silica.
142–145
All PAA surface-grafting processes
involved reactive groups already present on the substrate or
purposely introduced, capable of giving coupling reactions
with amine or acrylamide groups. For instance, glass and
silica were grafted after a previous treatment with 3-amino-
propyltriethoxy silane. PAAs can be grafted onto surface-ami-
nated materials by two different processes. The first one
consists of carrying out the polyaddition of amines with bisa-
crylamides in the presence of the aminated material in heter-
ogeneous phase [Scheme 8(a)]. The reaction involves the
surface amine groups and a part of the resultant PAA
remains covalently grafted, the ungrafted part being easily
washed away. In the second method, an acrylamido-termi-
nated PAA prepolymer is prepared, purified from the low
TABLE 6. (Continued).
No. X Y/Z References
42 X-14 Y-14 (5%) 1Z-18 (95%) 25
43 X-14 Y-14 (7.5%) 1Z-25 (92.5%) 85
44 X-14 Y-18 (50%)1Y-33 (50%) 61
45 X-14 Y-44 (7.5%) 1Z-25 (92.5%) 85
46 X-14 Y-24 (20–50%) 1Z-18 (50–80%) 27
47 X-14 Y-27 (10 or 40%) 1Z-18 (90 or 60%) 96, 97
48 X-14 Y-42 (10%)1Z-18 (90%) 243
49 X-14 Y-43 (10%)1Z-18 (90%) 243
50 X-14 Y-39 (95%) 1Y-44 (5%) 85
51 X-14 Y-44 (10–20%) 1Z-18 (10–20%) 54, 98
52 X-14 Y-28 (5%) 1Z-18 (95%) 28
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molecular weight fractions and then reacted with the ami-
nated material [Scheme 8(b)].
These two methods proved not equivalent. For instance, by
grafting a Cu
21
-complexing PAA (Table 5, No. 26) on finely
subdivided aminated silica, the resultant PAA-silica conju-
gates contained 20% and 11% PAA on a wt/wt basis,
respectively. Up to this point, the first method seemed more
effective, but subsequent investigation revealed that the
Cu
21
-complexing capacity of the resultant conjugate was
remarkably lower than expected. By contrast, the PAA-silica
conjugate prepared by the second method displayed the
expected Cu
21
complexing capacity, that is, one Cu
21
ion
per repeating unit. Possibly, with the first method the
lower-molecular-weight PAA species, being at all times
the prevailing ones by numbers and, therefore, possessing
the larger share of acrylamide end groups, had the higher
probability of undergoing grafting reaction. Once immobi-
lized, they might not exhibit the same chemical behavior as
long-chain PAAs. With the second procedure, only long
chain PAAs were present and, once grafted, behaved like
their high molecular weight soluble conterparts. It is worth
mentioning that soluble
146
and crosslinked PAA-grafted al-
bumin samples were also prepared by the same
methods.
147,148
Crosslinked PAAs
Crosslinked PAAs can be prepared by partially substituting
multifunctional amines for sec-bisamines or prim-
SCHEME 2 Syntheses of PAAs bearing SH and SAS groups as pendants: (a) cysteamine deriving PAAs; (b) PAAs with activated
side SAS-groups; (c) self-assembling bioreducible amphiphilic PAA-cholesterol conjugates; (d) bioreducible PAA-glutathione
conjugates.
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monoamines employed in the preparation of linear PAAs
11–13
(Scheme 9).
In the case of amphoteric PAAs carrying carboxyl groups in
the acrylamide moieties, as for instance (Table 6, No. 28)
this method has the serious drawback that the acid–base
properties of the resultant hydrogels may be grossly altered
with respect of its linear counterpart, since the amine/car-
boxyl ratio diminishes by increasing the amount of multi-
functional amine in the monomer mixture.
PAA resins obtained from moltifunctional amines are usually
very highly swellable in aqueous media unless for extreme
crosslinking degrees, giving hydrogels with poor mechanical
properties. However, exceptions exist. PAAs endowed of
structure-forming properties may give hydrogels presenting
good mechanical properties even in the swollen state.
149
An
alternative crosslinking method consists of triggering the
radical polymerization of V-PAAs by UV irradiation, water-
soluble diazo compunds, or redox systems. These methods
lead to hydrogels whose crosslinking degree depends on the
Xnof the starting oligomer (Scheme 10).
150
These hydrogels
maintain intact the acid–base properties of their linear coun-
tarparts, are moderately swellable in aqueous media and, as
a rule, exhibit better mechanical properties in the swollen
state than those derived from multifunctinal amines. The
presence in their network of hydrophobic polyvinyl chains
connecting the PAA chains probably explains this.
Resins coupling long PAA chains with high crosslinking
degree can be obtained by partly substituting allylamine for
the same quantity (on a molar basis) of amine monomers in
the starting PAA. Mixed networks were also obtained by
copolymerizing V-PAAs with traditional vinyl monomers such
as N-vinylpyrrolidinone.
151
Hetero-Diterminated PAA Dimers and Polymers
It is apparent that in order to obtain high molecular weight
PAAs, the functions involved, that is, activated double bonds
and amine hydrogens, must be stoichiometrically balanced. A
perfectly balanced mixture contains three types of macromo-
lecules, a—
.
—a, b—
.
—b and a—
.
—b in 1:1:2 ratio. Unbal-
anced mixtures contain the same molecular species, albeit in
different ratios, until the minority function is completely con-
sumed. Only at this point the product will be entirely consti-
tuted of molecules doubly terminated with the excess
function. There is no way, by the traditional method, to
straightforwardly obtain PAAs with controlled hetero-difunc-
tional chain terminals, that is, PAAs solely containing mole-
cules of “a—
.
—b” type. This precluded or rendered it
difficult to PAAs the access to the remarkable number of bio-
technological applications, as for instance liposome prepara-
tion, drug conjugation, and protein modification, which have
been so far nearly uniquely mastered by hetero-difunctional
poly(ethyleneglycol)s (PEGs) that, however, are far to be
endowed with the functional versatility of PAAs. Recently, a
SCHEME 4 Synthesis of soluble PAAs bearing SAS linkages in
the main chain using L-cystine as diamine comonomer.
SCHEME 3 Syntheses of PAAs bearing SAS linkages in the
main chain and their thiol-exchange mediated degradation.
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simple and straightforward preparation method of PAAs with
hetero-difunctional chain ends and of several previously
hardly obtainable PAA derivatives of biotechnological inter-
est, such as for instance PAAs of controlled molecular weight
and narrow polydispersity mono-functionalized at one end
with an acrylamide group, PAAs with star-like molecular
architecture, graft-PAA-protein conjugates, “tadpole-like” PAA
conjugates with hydrophobic moieties able to self-assemble
into nanoparticles in aqueous media, has been reported.
152
The key step was to design suitable building blocks consist-
ing of hetero-difunctional dimers (HDDs), that is the mono-
addition products of sec-diamines and bisacrylamides of the
“a—a—b—b” type, obtained as hydrochlorides or trifluoroa-
cetates. In this form, they could be indefinitely kept dormant
at 0–5C in the dry state, whereas at room temperature and
in aqueous media at pH >7.5 they polymerized according to
Scheme 11.
The synthetic scope of HDDs is manifold. They can be poly-
merized to high molecular weight PAAs without bothering
with stoichiometric balance. PAAs of controlled average mo-
lecular weight and mono-functionalized with an acrylamide-
or a sec-amine group at one end can be prepared by adding,
respectively, a controlled amount of monofunctional acryla-
mides or sec-amines, whereas the addition of multifunctional
acrylamides or amines will lead to star-like PAAs [Scheme
12(a)]. Block PAA-PAA or PAA-PEG copolymers with con-
trolled structure will be easily obtained. “Velvety-like” graft-
ing of PAA chains to properly functionalized surfaces can be
achieved. PAA chains of controlled average length can be
grafted to proteins with no risk of undesirable side reactions
such as protein–protein coupling or crosslinking [Scheme
12(b)]. “Tadpole-like” PAA conjugates carrying hydrophobic
moieties forming in aqueous media liposomes or nanopar-
ticles can be prepared as functional drug carriers.
152
Sequence-Defined PAAs by Polycondensation
of Diacids with Polyamines
Linear polymers obtained by polycondensation of dicarbox-
ylic acids or their activated derivatives with polyamines fol-
lowing synthetic procedures not consuming all the amine
groups of the latter, such as those obtained by condensation
of succinic or adipic acids with pentaethylenehexamine,
other short ethyleneimine oligomers or peptides, might be
defined as “inverted” PAAs. It should be observed, in fact,
that compared with traditional PAAs the relative positions
along the chain of the amide and amine nitrogens are
inverted. Whereas in traditional PAAs a hypothetical ob-
server leaving a chain amine group and proceeding along the
chain would find the amidic CO, in these polycondensation
polymers he would find the amidic NH. These polymers
TABLE 7 Thiol and Dithio-Containing PAAs
No X Y/Z References
1 X-1 Z-32 60
2 X-7 Z-32 60
3 X-8 Z-32 60
4 X-11 Z-32 60
5 X-11 Z-33 44, 107
6 X-13 Z-32 60
7 X-14 Z-33 44, 107
8 X-14 Y-47 (10%) 1
Z-18 (90%)
101
9 X-14 Y-48 (10–30%) 1
Z-18 (70–90%)
102, 103
10 X-15 Y-11 105, 108–113
11 X-15 Y-12 108, 113
12 X-15 Y-14 105, 108
13 X-15 Y-15 106, 108
14 X-15 Y-16 108, 114
15 X-15 Y-17 108
16 X-15 Y-19 108
17 X-15 Y-20 115
18 X-15 Y-25 108, 112
19 X-15 Y-45 110
20 X-15 Y-50 108
21 X-15 Y-51 108
22 X-15 Y-52 108
23 X-15 Z-1 105, 116
24 X-15 Z-2 60
25 X-15 Z-8 60
26 X-15 Z-10 105, 117
27 X-15 Z-12 117
28 X-15 Z-16 63
29 X-15 Z-18 104
30 X-15 Z-21 66, 108
31 X-15 Z-28 117
32 X-15 Z-18 104
SCHEME 5 Synthesis of PAA-polystyrene block copolymers.
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deserve attention because they are amenable to sequential
solid-phase supported synthesis hardly suitable for tradi-
tional PAAs. The latter, in fact, rely on the Michael addition
that typically benefits of a protic micro-environment and is
highly biased by steric hindrance, making it difficult in a
solid-phase supported synthesis to bring each step to com-
pletion. By contrast, the condensation polymers from dicar-
boxylic acids and polyamines can employ resins and
automatic equipment commonly marketed to this purpose
for peptide synthesis. Side functional short chains can be
also introduced in definite positions. Linear oligomers were
also obtained by polymerization of ethyl acrylate and N-
methyl-1,3-diaminopropane, catalyzed by the Candida antarc-
tica lipase. The enzyme catalyzes both the formation of the
amide groups by reaction of the ester function with amines
and the Michael addition involving the activated vinyl
groups.
153
Several studies on this subject have been pub-
lished in the last years providing evidence of the potential of
this novel synthetic tool,
154–160
and no doubt others will
appear in the near future.
PHYSICO-CHEMICAL PROPERTIES
Molecular Weight
Most PAAs mentioned in this review have
Mnand
Mwin
the range 5,000–30,000 and 10,000–50,000, respectively.
The polydispersity index of unfractionated samples was
approximately 2, with the exception of some PAAs obtained
from HDDs, where it was remarkably lower and in some
instances approached monodispersity,
152
and the “inverted”
PAAs prepared by solid-state supported synthesis. Obtaining
high molecular weight PAAs by the traditional procedure is
mainly a matter of solvent, monomer concentration, mono-
mer steric hindrance, reaction temperature, and patience.
As regards the reaction solvent, this topic has been already
discussed in the “Synthetic features” paragraph. As regards
concentration and steric hindrance, for the former the higher
the better and for the latter, at least as the reaction rate is
concerned, the lower the better. This is not surprising, as the
Michael addition is an equilibrium reaction mostly affected
by these parameters. As regards the reaction temperature,
its influence is double-edged. By increasing the temperature,
the polymerization rate increases as expected, but the
SCHEME 6 Synthesis of poly(urethane-PAA) copolymeric networks (PUPA).
SCHEME 7 Synthesis of PAA block and graft copolymers: (a)
linear PAA-PEG copolymers; (b) graft PAA-PEG copolymers; (c)
graft PAA-poly(4-acryloylmorpholine) (PAcM) copolymers.
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molecular weight tends to level off at a progressively lower
limiting value.
9
This effect is minimal at room temperature,
that is, 18–25C. Therefore, patience is the most important
requisite when dealing with sterically hindered monomers.
At room temperature, the polymerization may go on even for
months, but finally reasonably high molecular weight prod-
ucts can be obtained in nearly all conceivable cases. The use
of CaCl
2
as catalyst, recently proposed,
23
may help speeding
up these lethargic reactions.
Solution Properties
All PAAs synthesized so far are soluble or swellable in water.
As a rule, amphoteric PAAs dissolve only in water, but most
non-amphoteric ones are soluble also in chloroform, lower
alcohols, dimethylformamide, dimethylsulfoxide, and other
polar solvents.
The intrinsic viscosities of PAAs in organic solvents or aque-
ous media usually range from 0.15 to 1 dL/g. As indicated
by their viscometric values, PAAs usually exhibit larger
hydrodynamic volumes in solution than most polyvinyl poly-
mers of similar molecular mass. For instance, the results of a
study performed on two typical PAAs (Table 5, No. 33 and
Table 6, No. 38, that is, ISA23), are reported here. The Mark–
Houwink–Sakurada (MHS) constants for samples of the for-
mer PAA of
Mn<10,000 were k51.76 310
25
dL/g and
SCHEME 8 PAA grafting onto silica: (a) polyaddition of amines with bisacrylamides in the presence of the aminated silica; (b)
reaction of an acrylamido-terminated PAA prepolymer with aminated silica.
SCHEME 9 Synthesis of crosslinked PAAs by employing multi-
functional amines as crosslinkers.
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a51.14.
77
The amphoteric ISA23 was studied more detail-
edly within a wide range of molecular weights. The initial
slope (a) of its MHS plot for Mlower than 20,000 was very
high, that is >1. The plot exhibited a significant downward
curvature at higher molecular masses. For a molar mass of
60,000 g/mol the ISA23 MHS constants were: k59.525 3
10
25
dL/g; a50.846.
93
This PAA, and probably other am-
photeric PAAs, reversibly aggregates in solution, whereas
non-amphoteric PAAs of similar structure do not.
59
ISA23
bears positive and negative charges in relatively distant loca-
tions along the polymer chain and electrostatic interactions
are doubtless responsible for its tendency to aggregate.
Crystallinity
Owing to their regular structure, many PAAs are partially
crystalline in the solid state. Quite a few of them crystallize
spontaneously on isolation, but in some instances, especially
for PAAs deriving from primary amines, crystallization must
be induced by solvent treatment.
9
When cyclic structures
without side substituents are present, such as in the bisacry-
lopylpiperazine/piperazine PAA (Table 5, No. 32) the poly-
mer may crystallize even from water during polymerization.
The melting point of this PAA is 270C (with decomposition),
the highest so far determined for these polymers.
9,149
It is
worth mentioning that the same PAA when crosslinked by
the multifunctional amine method (see above) and for low to
moderate crosslink density usually establishes crystalline
domains, probably involving the linear chain segments in
between the crosslink points. These domains are stable in
aqueous systems and act as reinforcing fillers greatly
enhancing the mechanical strength of the resultant hydro-
gels. Paradoxically, but not entirely unexpectedly, the modu-
lus of these hydrogels up to a certain degree decreases by
increasing the crosslink density, probably due to an increas-
ing difficulty in establishing ordered domains.
149
Thermal and Shelf-Stability
The thermal stability of PAAs is as expected for polymeric b-
dialkylaminoethyl-acrylamides, considering that the non-poly-
meric ones are known to undergo b-elimination on heating.
Performing thermogravimetric analyses of some linear PAAs,
for instance, the weight loss started at about 200Cunder
vacuum and at slightly higher temperatures under nitrogen.
9
The shelf-stability of PAAs is usually good, provided some pre-
cautions are taken. Most PAAs absorb moisture in open air
both as free bases and as salts. PAA free bases must be thor-
oughly dried and protected from moisture and oxygen to
avoid discoloration, whereas PAA salts are indefinitely stable
in the dry state and only need to be protected from moisture.
Degradation of PAAs in Aqueous Media
In principle, all PAAs contain cleavable bonds in the main
chain. However, as stated above, in concentrated solution at
pH 8–10 and at 18–25C most PAAs are sufficiently stable
for weeks and even months to be kept polymerizing and
SCHEME 10 Synthesis of crosslinked PAAs by radical polymer-
ization of vinyl terminated oligomers.
SCHEME 11 Synthesis of hetero-difunctional bisacrylamide/
amine dimers.
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therefore increasing their molecular weight. Several of them,
however, were specifically tested for degradability in very
dilute solution at pH 7.4 and 37C by viscometric and chro-
matographic methods. In many cases the degradation rate
markedly depends on the structure of the bisacrylamide moi-
ety, as it was found, for instance, in a study performed on 2-
methylpiperazine deriving PAAs, the degradation rates were
in the order: (Table 5, No. 33) >(Table 5, No. 11) >ISA23
(Table 6, No. 38) (Fig. 1).
This trend was confirmed by further studies on other PAAs
in which the same amide monomers had been combined
with different amine monomers.
61,65
Broadly speaking,
increasing temperature and pH speeded up the PAA degrada-
tion in aqueous media. By contrast, isolated lysosomal
enzymes at pH 5.5, where tested, proved ineffective.
74
Acid–Base Properties of PAAs
All PAAs are polyelectrolytes, since the prim-or sec-amine
groups involved in polyaddition reactions leading to PAAs
give rise to tert-amine groups in the polymer chain and retain
their basic character. The polyelectrolytic character can be fur-
ther enhanced by introducing ionizable side substituents.
Many relevant properties of PAAs, including toxicity and abil-
ity to interact with components of the biological environ-
ments, such as nucleic acids, proteins, and living cells, are
strongly dependent on their acid–base properties, hence on
their ionization state in different biological districts. There-
fore, this subject was the object of several investigations.
Normally, the acid–base dissociation equilibria of polyelectro-
lytes are best interpreted by the generalized Henderson–Has-
selbach (eq 2)
pH 5pK2blog 12aðÞ
a(2)
where Kis the weak acid dissociation constant being pH-
determining in the buffer titration zone considered; ais the
dissociation degree of the considered acidic function, and b
is the Katchalsky and Spitnik parameter
161
accounting for
possible interactions between ionizable groups being adja-
cent either structurally or on account of conformational
effects (random-coil structure). To describe this behavior, the
concept of “apparent” constant (for polyelectrolytes) as op-
posite to “real” constant (for low molecular weight electro-
lytes) is often adopted.
Unlike traditional polyelectrolytes, PAAs usually behave simi-
larly to low molecular weight amines as regards protonation
as well as heavy metal ion complex formation (see
later),
32,35–41,45,69,71,73,162,163
in that the ionizable groups of
each repeating unit exhibit “real” or quasi “real” protonation
constants, as if each unit was an isolated molecule. This
equals to say that for PAAs the Katchalsky and Spitnik pa-
rameter bis 1 or whereabouts. It is noteworthy that the
number of the protonation constants of PAAs is always equal
to the number of the ionizable groups per unit and apart
from entropic effects their values are similar to those found
for the non-macromolecular models prepared by Michael
addition of 4-acryloylmorpholine with the same amines used
in the preparation of the corresponding PAAs. A linear rela-
tionship was found for both PAAs and their models between
the protonation enthalpies and the net charge of the nitrogen
atom to be protonated. This is a further indication that no
significant interactions exist between different repeating
units. This behavior is probably due to the relatively long
SCHEME 12 Star-like PAAs (a) and protein-PAA conjugates (b) from HDDs.
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distance between the ionizable groups belonging to different
units, combined with the extended conformation of PAAs in
solution and the high charge-sheltering efficiency of the two
amide groups interposed. In fact, a single amide group
bound to a piperazine ring is not sufficient to minimize
interactions between neighboring units, as in the case of
poly(1,4-piperazinediyl-1-oxo-trimethylene) (Fig. 2), which
was found to exhibit a typical polyelectrolyte behavior.
164
Amphoteric PAAs of traditional chain structure, but deriving
from aminoacids and therefore carrying a carboxyl group
attached to the amine moiety behave anomalously in the
PAA domain, but normally in the polyelectrolyte domain,
since the carboxyl group exhibits a typical polyelectrolyte
behavior.
72
This tendency, however, is minimal for ampho-
teric PAAs in which the carboxyl groups is attached to the
bisacrylamide moiety, in particular ISA23 (Table 6, No. 38).
44
Viscometric titrations in 0.1 M NaCl
41,71
proved that the con-
formational freedom is reduced on protonation. For PAA No.
25 of Table 5, for instance, the first protonation led to the
formation of a strong hydrogen bond between “onium” ions
and carbonyl groups belonging to the same monomeric unit.
When the first protonation of all the monomeric units was
complete, the above effect strongly reduced the conforma-
tional freedom of the whole polymer, which tended to
assume a rigid structure. This was clearly shown by a pro-
nounced jump of its reduced viscosity at a50.5 (a5degree
of protonation). The second protonation led to an electro-
static repulsion between the positively charged onium ions
belonging to the same unit, and this effect further compelled
the polymer to adopt a more rigid structure. Consequently, a
second jump was observed at a51 (Fig. 3).
The reduction of conformational freedom became less pro-
nounced by lengthening the aliphatic chain separating the
amine groups of the repeating units. For example, PAAs Nos.
27 and 28 of Table 5 showed the same jumps progressively
reduced. In contrast, their isomers (Table 4, Nos. 18–20),
equally carrying two tert-amine groups per unit, but only one
of which inserted in the polymer chain, did not show any vis-
cosity jump either after the first or after the second protona-
tion step. This was explained by the fact that the single
“onium” ion per unit present in the polymer chain of the lat-
ter PAAs can interact indifferently with two neighboring car-
bonyl groups, thus enjoying a greater conformational freedom.
Consequently, viscometric titrations did not show any jump
throughout the whole titration curve.
41,71
Recently, a library of acid–base properties of PAAs, many of
which carrying multiple ionizable groups and never previ-
ously considered in this respect, has been published.
44
The
pKdeterminations were performed following three distinct
approaches,
165,166
which gave congruent results. All previous
statements about the “real” or “quasi real” protonation con-
stants of PAAs were confirmed. In all cases, the bparameter
was found ranging from 1 to 1.1, only exceptionally rising to
1.2–1.3.
For many PAAs, the protonation constants as well as the av-
erage distribution of the charged species, hence the net av-
eragechargeasafunctionofpH,weredetermined.As
typical examples, the speciation diagrams for two typical
amphoteric PAAs (Table 6, Nos. 28 and 38) are reported in
Figure 4.
Metal Complexes of Linear PAAs
In early studies, many PAAs proved to form coordination
complexes with some heavy metal ions, namely Cu
21
,Ni
21
,
Co
21
. As with protonation, “real” stability constants could be
determined for the complexes.
29,46,47,49,86,167
It is noteworthy
that metal complexation, similarly to protonation, led to stiff-
ening of the PAA conformation in solution.
41
In the absence
of additional side amine or carboxyl groups, only PAAs carry-
ing at least two amine nitrogens per unit that neither
belonged to a cyclic structure nor were separated by more
than three carbon atoms showed complexing ability. The
electronic and electron paramagnetic resonance (EPR) spec-
tra of both polymeric and non-polymeric complexes,
FIGURE 1 Degradation behavior of 2-methylpiperazine deriving
PAAs. (a) Monitoring of the reduced viscosity, g
sp
/C, of PAAs
No. 33, Table 5 (䊉); No. 11, Table 5 (䊊); and No. 38, Table 6
(‡) in 0.2 M phosphate buffer pH 7.5 at 37C. (b) Variation of
SEC tracing of PAA No. 11, Table 5 with time under the same
degradation conditions as above. Eluent 5TRIS buffer pH 8.2
with 0.2 M NACl. Elution flow rate 51 mL/min.
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obtained from PAA models, were similar and consistent with
an octahedral tetragonally distorted structure, in agreement
with the substantial independence of the polymer repeating
units in complex formation.
Technical Applications of PAAs
PAAs as Inorganic Pollutants Sorbing Materials for Water
Purification
It was quite early ascertained that crosslinked PAAs
70,75,76
and PAA-grafted materials, for example silica,
141–144
retained
the ion-complexing ability of their linear counterparts. This
concept was more recently resumed by studying crosslinked
amphoteric PAAs as specific heavy metal ion absorbers for
water purification from inorganic pollutants. An amphoteric
PAA with inter-segmented structure obtained by synthesizing
ISA23 in the presence of a second pre-synthesized PAA car-
rying primary amino groups as side substituents (Table 6,
No. 27) that acted as a macromolecular crosslinking agent
was studied as a Co
21
,Ni
21
, and Cu
21
-sorbing material.
168
This resin exhibited a remarkable sorption capacity and
sorption rate for the three ions considered, which were in
situ monitored by cyclic voltammetry. The metal-ion uptake
was very fast and quantitative. Dilute acids quantitatively
eluted the absorbed metal ions, thus allowing metal recover-
ing and resin regeneration. In a subsequent study, two novel
crosslinked PAAs named LYMA and LMT85 (Fig. 5), contain-
ing amine and carboxyl groups, were reported as highly
effective heavy metal ions absorbers.
169
In particular, LYMA contained one carboxyl and two amine
groups and was a mimic of L-lysine, whereas LMT85 con-
tained two amine and five carboxyl groups and was a mimic
of EDTA. The heavy metal ion set adopted as benchmark was
Cu
21
,Cd
21
,Pb
21
,Zn
21
,Ni
21
, and Co
21
. LYMA proved selec-
tive for Cu
21
and Ni
21
, the other ions tested being negligibly
absorbed, whereas LMT85 proved capable of rapidly and
quantitatively absorbing all the ions tested either singly or in
mixed solution (Fig. 6).
In a subsequent investigation, both resins proved capable of
absorbing Mn
21
as well.
170
As observed for other PAA resins,
the absorption process was fast and reversible and the res-
ins were easily regenerated by acidification. Moreover, the
metal ion absorption imparted intense coloring to the resins,
a feature possibly exploitable for analytical purposes (Fig. 7).
Crosslinked PAAs as Matrices for High-Performance
Nonlinear Optical Dyes
Highly crosslinked PAAs obtained from reaction systems con-
sisting of stoicheiometrically balanced mixtures of bisacryla-
mides and multifunctional amines with minor amounts of
difunctional amines are very hard and rigid materials with
limited swelling in aqueous media. They have found applica-
tion as superior matrices for high-performance nonlinear op-
tical (NLO) heterocycle-based cationic dyes. In particular,
prim-amino-alkyl substituted dyes were prepared and added
as co-monomers to reaction systems leading to highly cross-
linked PAA, thus obtaining PAA networks with NLO moieties
covalently attached as side substituents. The very mild prep-
aration conditions combined with the wide-ranging tuneabil-
ity of optical and mechanical properties made these matrices
a valuable alternative to conventional high-T
g
thermoplastic
polymers.
171–173
Moreover, it was found that a stable align-
ment of the second order covalently attached NLO-phores
could be obtained by a three steps procedure (swelling–
poling–de-swelling) performing the last two steps under
poling conditions.
174
This procedure appears widely applica-
ble for producing composite polymeric materials with a
rather stable second harmonic generation. It is of particular
interest for NLO-phores sensitive to temperature, since both
synthesis and poling were best performed at room
temperature.
Sensing Applications of PAAs
Owing to their hydrophilicity and multifunctionality, PAAs
were also considered for sensing applications. Early papers
reported on the use of amorphous, rubber-like PAAs (Table
4, Nos. 14, 15, 18, 21, 22, and 24) as coatings of quartz
microbalance in gravimetric sensors for the detection of CO
2
and SO
2
in gaseous mixtures. The rubber-like properties,
being obviously related to structural flexibility, were apt to
facilitate the diffusion of analyte molecules through the poly-
mer layer.
42,43
A subsequent article reported on the develop-
ment of vapor detectors formed from composites of
conductive carbon black and PAAs. The new materials were
tailored to produce increased sensitivity toward specific
classes of analyte vapors.
175
Subsequent articles reported on
the use of PAAs as the active components of a flow type
quartz crystal microbalance chemical sensor suitable for
determining heavy metal ions in aqueous solution, hence
having potential for environmental monitoring. This sensor
FIGURE 3 Trend of the reduced viscosity, g
sp
/C, of PAAs No.
25 (䊉), No. 27 (䊊), and No. 28 (䉫) of Table 5 as a function of
the protonation degree a. Experimental conditions: 0.17 g/dL in
0.1 M NaCl aqueous solution, 25C.
FIGURE 2 Structure of poly(1,4-piperazinediyl-1-oxo-trimethylene).
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was based upon surface chelation of the metal ions at PAA-
modified gold electrodes on 9 MHz AT-cut quartz resonators,
functioning as a quartz crystal microbalance.
176,177
BIOMEDICAL AND BIOTECHNOLOGICAL APPLICATIONS
OF PAAS
The biotechnologial applications of PAAs explored so far can
be divided in two main categories: those employing soluble
PAAs and those regarding PAA hydrogels or PAA-based solid
materials in which the PAA portion is the active component.
Bioactive Soluble PAAs
PAAs with Antimetastatic Activity
As early as 1973 it was found that some copolymeric PAAs
(Table 4, No. 29 and Table 6, No. 24) were active as antime-
tastatic agents after intravenous administration, reducing
both the number and the average size of metastases in
mice.
52
PAAs No. 29 of Table 4 contained variable amounts
of long chain aliphatic side substituents. They were purely
basic, amphiphilic, and significantly toxic, nevertheless could
be administered to mice at a dose up to 20 mg/kg and
showed activity in reducing the number and average weight
of Lewis lung (but not Sarcoma 180) tumor metastases. By
contrast, the PAA No. 24 of Table 6 was amphoteric and
non-toxic. It could be administered at a dose of 200 mg/kg
and proved to reduce the number and average weight of
both Sarcoma 180 and Lewis lung tumor metastases. How-
ever, none of the above PAAs was active against the primary
tumor. Whereas the hydrophobically substituted PAAs car-
ried long aliphatic chains and their activity could be ascribed
to cell membrane interactions, by analogy with non-ionic
detergents of the Triton series previously found to be
equally active,
178
the amphoteric PAA lacked hydrophobic
moieties, suggesting a different mode of action. Regrettably,
this point was no further investigated.
Antiviral Activity of AGMA1
The already mentioned PAA named AGMA1 (Table 6, No. 28)
proved very active both in vitro and in vivo against Herpes
Simplex Viruses Types 1 and 2 (HSV-1 and HSV-2), closely
related pathogens of the Herpesviridae family of DNA
viruses, without eliciting adverse side effects.
88
Both HSV-1
and HSV-2 are capable of infecting mucosal sites causing
lesions on the lips, eyes or genitalia, encephalitis, and others.
AGMA1 is water-soluble and amphoteric, but prevailingly
cationic at pH 7.4. Its repeating unit is reminiscent of the
RGD peptide sequence (Fig. 8).
The IC
50
of AGMA1 was found >5 mg/mL and its intrave-
nous maximum tolerated dose (MTD) in mice was >0.5 g/
kg. It was deprived of hemolytic activity and easily entered
cells localizing in the perinuclear region.
88,89
It proved active
not only against HSV-1 and HSV-2, but also against Papilloma
virus (HPV-16), Cytomegalovirus, and Murid Herpesvirus 68
(MHV-68). The inhibitory effect of AGMA1 was exerted at
low polymer concentration. For instance, EC
50
values of 0.74
mg/mL and 1.14 mg/mL, respectively, were found for HSV-1
for HSV-2 (Fig. 9).
AGMA1 acted with a different mechanism than conventional
antiviral drugs as Acyclovir, in that it was not virucidal, that
is, it did not kill viruses, but blocked the transmission of the
infection from cell to cell. Its antiviral activity was not
directly related to its prevailingly cationic charge, as other
cationic PAAs, for example ISA1 (Table 5, No. 38), under the
same conditions proved inactive. AGMA1 did not affect the
growth of Lactobacillus spp. responsible for maintaining the
correct pH in the vaginal fluids of healthy animals and its ac-
tivity was not pH-dependent within the physiological range.
AGMA1 also inhibited HSV-1 and HSV-2 infection in vitro on
cultivated human epithelial tissue (EpiVaginal
TM
) without
any detectable inflammatory effect (Fig. 10).
In Figure 10, the gray bars refer to control infected with
HSV-2 1000 pfu. The barely detectable black bars refer to
epivaginal tissue infected in the same way and treated with
100 mg/mL AGMA1 solution. At this dose no cytokine release
(indicating inflammatory reactions) was detected after 24 h.
The same inhibitory effect and lack of inflammatory activity
FIGURE 4 Speciation diagrams calculated using experimentally determined pK
a
values of two typical amphoteric PAAs, that is,
No. 28 (left panel) and No. 38 (right panel) of Table 6. L,L
11
,L
12
,L
21
represent the differently charged repeating units. The
extracellular pH value (pH 7.4) is evidenced by the vertical black bar. The isoelectric point is the pH at the crosslink of the L
11
/L
21
curves. Figure adapted with permission (reference 44).
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was subsequently confirmed in vivo after topical administra-
tion to female mice.
88
Antimalarial Activity of PAAs
Studies in progress have demostrated that AGMA1 and
ISA23 tested on Plasmodium falciparum 3D7 are endowed
with antimalarial activity per se.
90,91
Significant results are
reported in Table 8.
It is apparent that for both polymers the higher molecular
weight fractions were significantly more active than their
lower molecular weight analogs. Moreover, the antimalarial
activity of AGMA1 was always remarkably superior to that of
ISA23.
PAAs as Promoters of Cell Adhesion on Substrates
for Cell Culturing
In spite of their toxicity, cationic polyaminoacids such as
poly-L-ornitine, poly-L-lysine, and poly-D-lysine are widely
used since many years as enhancers of cell adhesion, prolif-
eration, and differentiation on solid substrates for cell cultur-
ing. AGMA1 was found to be equi-active in this respect to
poly-L-lysine, but with a vastly inferior toxicity
179
and is
presently sold for this application under the trade mark of
Cell GRIP
V
R
.
Soluble PAAs as Osteoblast Proliferation Promoters
PAAs synthesized by polyaddition of pamidronate or neridro-
nate with bisacryloylpiperazine (Table 6, Nos. 13 and 14)
were recently proposed as osteoblast proliferation
promoters.
87
Soluble PAAs as Carriers for Bioactive Substances
Soluble PAAs have been studied as polymer-drug conjugates
(particularly as anticancer-drug conjugates) and, more exten-
sively, as endosomolytic vectors for intracellular delivery of
nucleic acids and toxins. The early studies on this subject
FIGURE 6 Absorption experiment with LMT85 resin in multiple
heavy metal ion solution: in situ square wave voltammetry
(SWV) monitoring of metal absorption from a 0.000025 M solu-
tion at pH 6.8 (phosphate buffer) in 10:1 resin repeating unit/
metal ratio. Current reduction indicates ion absorption. Figure
adapted with permission (reference 169).
FIGURE 5 Structure of LYMA and LMT85 resins.
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have been reviewed elsewhere.
180
They were soon followed
by more extensive investigations reported in the following
paragraph.
Biocompatibility and Biodistribution Studies of Soluble
PAAs
Many polycations other than PAAs explored as drug and oli-
gonucleotide delivery systems such as poly-L-lysine, polye-
thyleneimine, and unmodified PAMAM,
181–183
are generally
toxic to cells in culture. For example, poly-L-lysine displays
IC
50
values in the range 1–60 mg/mL depending on the cell
type and incubation time
184
and poly-L-lysine, polyethylenei-
mine and unmodified PAMAMs showed significant hemolytic
activity which is molecular weight- (generation-) depend-
ant.
182,183
In early studies, PAAs Nos. 31 and 32 of Table 5
were found consistently less cytotoxic than poly-L-lysine.
74
Many amphoteric PAAs carrying a carboxyl group per unit
proved even less toxic. For instance, PAAs Nos. 32, 33, and
38 of Table 6 were >100 times less cytotoxic than the tradi-
tional amine polymers used as a reference.
44,93
Equally, PAAs
deriving from a- and b-aminoacids were in general highly
cytobiocompatible.
44
All these PAAs carried excess negative
charges at pH 7.4.
12,44
This charge effect was thought to
explain their lack of toxicity, supported by the observation
that the amphoteric, but at pH 7.4 prevailingly cationic PAAs
(Table 6, Nos. 34 and 35) were significantly more cytotoxic
than their prevailingly anionic lower homologs. Up to this
point, basicity, hence the net cationic charge at pH 7.4,
seemed to be the main factor affecting PAA toxicity. Accord-
ingly, the reduced toxicity of most purely cationic PAAs com-
pared with poly-L-lysine (PLL) and polyethyleneimine (PEI)
could be explained by their generally lower basicity. This
general conclusion was further confirmed by the fact that
the Michael addition of N,N-dimethylacrylamide to the side
amino groups of poly L-lysine reduced in the mean time both
its basicity and its toxicity.
26
Later studies, however, demon-
strated that, whereas in most instances this assumption held
true, exceptions existed. In AGMA1, the presence of side gua-
nidine groups in addition to the chain tert-amine groups
(Fig. 8) imparted significant basicity not accompanied by
cell-mediated toxicity. AGMA1, notwithstanding its isoelectric
FIGURE 9 In vitro inhibition of HSV-1 (a) and HSV-2 (b) infec-
tion by AGMA1.
FIGURE 8 Structure of AGMA1 repeating unit and RGD motif.
FIGURE 7 Coloring of LYMA and LMT85 resins upon heavy
metal ion absorption (403magnification). LMT85 exhibits a
wider range of efficiently absorbed metal ions. Figure adapted
with permission (reference 169).
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point >10 and its 0.55 average positive charges per unit at
pH 7.4, proved highly biocompatible (IC50 5 mg/mL on
several cell strains) and deprived of significant hemolytic ac-
tivity in the pH range 4–7.4.
27
This contrasted with the
behavior of ISA23 that had been previously found non-hemo-
lytic at pH 7.4, where it was prevailingly anionic, but mem-
brane active below its isoelectric point, that is 5.5.
Interestingly, AGMA1-ISA23 copolymers showed a small, but
noticeable hemolytic activity that increased by decreasing
the pH of the medium and increasing the ISA23 propor-
tion.
27
As reported below, the introduction of AGMA1 units
in ISA23 hydrogels imparted them an otherwise lacking abil-
ity of inducing cell adhesion and proliferation. This was
attributed to the structural similarity of the AGMA1 repeat-
ing units to the RGD peptide, probably imparting a similar
aptitude to interact with cell membranes. It might be postu-
lated that AGMA1, owing to its peculiar structure, exerted a
stabilizing action on cell membranes overshadowing the
membranolytic effect of the excess positive charges. In
AGMA1-ISA23 copolymers, the stabilizing effect was partially
superseded by the known hemolytic activity at low pH val-
ues of the ISA23 portion.
To gather information on PAA biodistribution, the analogs of
two typical PAAs, namely ISA1 and ISA23 (Table 5, No. 38
and Table 6, No. 38), were synthesized to contain approxi-
mately 1 mol % 2-p-hydroxyphenylethylamine-deriving units
amenable to iodination. These polymers were named ISA4
and ISA22, respectively.
53
After intravenous injection to rats
125
I-labelled ISA 4 was rapidly taken up by the liver (>80%
recovered dose at 1 h) whereas
125
I-labelled ISA22 was not
(liver uptake <10% recovered dose at 5 h). The so-called
“stealth like” properties of ISA22, probably due to its zwit-
terionic nature with prevailingly negative charge at pH 7.4,
provided opportunity for tissue targeting either by the incor-
poration of ligands or, as regards tumors, by passive means
such as the enhanced permeability and retention (EPR)
effect.
185–189
In fact, biodistribution studies in mice bearing
subcutaneous B16F10 melanoma showed that
125
I-labelled
ISA22 was still accumulating in tumor tissue after 5 h (2.5%
dose/g).
PAA and PAA Anticancer Drug Conjugates
PAA-anticancer drug conjugates have been prepared and
tested. In early studies, PAAs were developed as water solu-
ble carriers for known anticancer agents including mitomy-
cin C (MMC)
190
and platinates.
54
Two PAA-MMC adducts
were synthesized from hydroxylated PAAs such as ISA1
using carbonyldiimidazole as coupling agent. After in vitro
studies, preliminary in vivo experiments were carried out on
TABLE 8 Plasmodium Growth Inhibition Using Different Molecular Weight Fractions of AGMA1 and ISA23
Sample
a
Concentration (mg/mL) % Inhibition
b
Standard Deviation
AGMA1 10–30K 1.00 65.4 8.74
0.50 54.8 9.87
0.25 25.2 9.99
0.12 2.20 3.91
AGMA1 50–100K 1.00 81.5 11.96
0.50 73.2 9.90
0.25 50.3 9.61
0.12 9.30 5.47
ISA23 10–30K 2.00 17.5 3.40
1.00 12.5 1.80
0.50 5.40 1.10
0.25 1.80 0.60
ISA23 50–100K 2.00 34.8 5.90
1.00 16.3 2.30
0.50 8.50 1.90
0.25 2.50 2.10
a
Figures refer to molecular weight values.
b
Percentage parasitemia reduction with respect to controls.
FIGURE 10 AGMA1 inhibition of HSV-2 infection in human epi-
vaginal tissue.
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mice bearing L1210 tumor cells. The mice were treated with
free MMC or PAA-MMC conjugates by a single i.p. dose the
day after tumor inoculation. The PAA-MMC conjugates
proved equi-active compared to MMC given intraperitoneally
and a long-term survivor was observed in each group
treated with conjugate. PAA-MMC conjugates proved less
toxic than free MMC when administered at a MMC-equivalent
dose of 5 mg/kg.
190
PAA-platinates were also prepared by
reaction of ISA23 and two PAAs containing pendant b-cyclo-
dextrin moieties with cisplatin.
54
The conjugates contained
8–70 wt % platinum and released low molecular weight
platinum species in vitro at pH 5.5 and pH 7.4 (0–20%/72
h). The PAA-platinates were generally less toxic than cispla-
tin toward lung tumor cell lines, but in vivo proved equi-
active compared to cisplatin in an i.p. L1210 leukemia
model.
54
The antitumor activity of ISA23-platinates was simi-
lar to that reported for other platinum conjugates of similar
molecular weight.
191–193
In a subsequent investigation,
79
PAA conjugates containing
the membrane disrupting peptide melittin were prepared
and tested. It was hypothesized that PAA conjugation would
reduce the hemolytic activity of melittin at pH 7.4, but upon
delivery to tumors by the EPR effect, the polymer would
uncoil in an acidic endosomal environment exposing melittin
and allowing it to interact with membranes. The melittin
content of the conjugates was 6–19% (wt/wt). They were
obtained using melittin as a comonomer in the preparation
of ISA1 and ISA23.
Under the conditions adopted, the terminal amine group of
melittin, being a weaker base than its side amine groups
deriving from lysine, was the only un-ionized, hence the only
amenable to the addition reaction. Although the ISA1 conju-
gate improved gelonin delivery and showed pH-dependent
hemolytic activity at a polymer concentration of 0.05 mg/
mL, it also displayed high hemolytic activity at pH 7.4. By
contrast, the ISA23 conjugate did not deliver gelonin. How-
ever, this conjugate lacked hemolytic activity at pH 7.4 while
retaining the melittin cytotoxicity and could have potential
as a novel polymer anticancer agent.
More recently,
80
ISA1 and ISA23 with amine pendant groups
(Table 4, No. 33 and Table 6, No. 42) were prepared. Dansyl
cadaverine and doxorubicin were bound to the former and
doxorubicin to the latter via an acid-labile cis-aconityl spacer.
Release of dansyl cadaverine and doxorubicin at physiologi-
cal and acidic pH varied from 0 to 35% over 48 h and was
pH dependent. Whereas the ISA1 conjugate apparently did
not present significant promise as anticancer agent, the
ISA23 conjugate proved to release biologically active doxoru-
bicin in vitro and might be suitable for further development.
PAA-Antiviral Drug Conjugates
An ISA23 sample carrying b-cyclodextrin pendants, obtained
by copolymerization with 6-deoxy-6-amino-b-cyclodextrin
was employed as carrier for the antiviral drug Acyclovir. Up
to 11% wt/wt of Acyclovir was solubilized. In cell cultures,
the Acyclovir b-cyclodextrin–PAA complex exhibited a signifi-
cantly higher antiviral activity than the free drug against
Herpes simplex virus Type I.
98
PAAs—Imaging Probes Conjugates
The ease of PAA functionalization allowed envisaging their
use as carriers of probes for imaging applications. The pre-
ferred PAA was ISA23, owing to its biocompatibility and
stealth-like properties leading to preferential tumor localiza-
tion by the EPR effect.
53
The introduction of paramagnetic N-oxyl groups in PAAs is
easy, since 4-amino-2,2,6,6-tetramethyl-piperidine N-oxyl (4-
amino-TEMPO, Table 2, Y-27) behaves as co-monomer in PAA
polymerization mixtures leading straightforwardly to
TEMPO-labeled products. Two PAA-TEMPO conjugates based
on ISA23, ISA23-TEMPO1 and ISA23-TEMPO2 (Table 6, No.
47) with 10 and 40% TEMPO-carrying units per polymer
chain, respectively, were prepared. Their relaxivity values
were, respectively, 0.4 and 1.8 mM
21
s
21
. These values indi-
cated that PAA-TEMPO adducts have a definite potential as
NMR imaging contrast agents. This was confirmed by prelim-
inary magnetic resonance imaging (MRI) determinations.
97
In order to provide a suitable carrier for radioactive tracers,
an ISA23 sample with 10% thiol-functionalized units (Table
7, No. 8) was synthesized by adding mono-N-boc-cystamine
as co-monomer during polymerization and then reductively
cleaving the SAS bond in the resultant polymer.
101
This thio-
mer gave stable rhenium complexes and was reasonably con-
sidered worth of attention as carrier for radioactive rhenium
and technetium. In particular, two rhenium complexes con-
taining 0.5 and 0.8 equiv of rhenium per thiol groups,
respectively, were obtained by reacting the cysteamine-func-
tionalized ISA23 with [Re(CO)
3
(H
2
O)
3
](CF
3
SO
3
) in aqueous
solution at pH 5.5. The chelation occurred through the S and
N atoms present in the PAA carrier and the rhenium content
was governed by the stoichiometric ratio between rhenium
and thiol groups. Both the cysteamine-functionalized ISA23
and its rhenium complexes were soluble in water under
physiological conditions. The complexes proved highly stable
in solution even in the presence of excess cysteamine. Nei-
ther cysteamine-functionalized ISA23 nor its rhenium com-
plexes showed hemolytic activity up to a concentration of 5
mg/mL. No cytotoxic effects were observed on Hela cell after
48 h at a concentration of 100 ng/mL. In vivo tests showed
that cysteamine-functionalized ISA23 was highly biocompati-
ble. Moreover, the rhenium complexes did not elicit detecta-
ble toxic effects on mice after intravenous injection in doses
up to 20 mg/kg.
More recently, in order to obtain strongly luminescent mac-
romolecular probes, a new ISA23 copolymer with 6% phe-
nanthroline-containing repeating units (Table 6, No. 52), was
obtained by copolymerization with 4-(40-aminobutyl)21,10-
phenanthroline (Table 2, No. 28).
28
The copolymer showed
excellent solubility in water. The phenanthroline pendants
stably coordinated either Re(CO)
3
1
or Ru(phen)
221
frag-
ments, affording luminescent complexes emitting from
3MLCT excited states with k
em
5608, 571, and 614 nm and
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U
em
50.7%, 4.8%, and 4.1%, respectively, in aerated water
solution. The complexes were stable under physiological con-
ditions even in the presence of excess competing cysteine.
Both complexes were non-toxic in the concentration range
0.5–50 lM (calculated on the metal-containing unit) toward
HEK-293 cells. Moreover, preliminary studies showed that
the ruthenium complex entered HEK-293 cells by endocyto-
sis and then homogeneously diffused within the cytoplasm
across the vesicle membranes, thus confirming the results of
previous studies on ISA23 as an endosomolytic carrier.
53
PAAs as Non-Viral Vectors for Intracytoplasmic Delivery
Background and Basic Investigations
Considerable attention in the last decades has been and is
being focused on the exploitation of the unique combination
of properties of PAAs for designing membrane active poly-
mers able to promote intracytoplasmic delivery of high mo-
lecular weight therapeutics. Polymers widely explored as
anticancer conjugates such as hydroxypropylmethacrylamide
(HPMA) copolymers and PEG are not eligible for these appli-
cations. Viral systems, whereas mediating high transfection
efficiencies in vitro, have proven unstable and at times cata-
strophically immunogenic.
194,195
Many non-viral delivery sys-
tems such as cationic lipids and polymers are relatively toxic
and, moreover, rapidly localize to lung or liver after intrave-
nous administration thus making it difficult to target other
tissues. It has been suggested that polymeric branched trans-
fection agents with normal polyelectrolyte behavior, such as
polyethyleneimine, act as transfection promoters according
to the so-called “proton sponge” hypothesis, that is, by
absorbing protons within the endosome, where the pH is 5.5
or whereabouts, they swell and cause membrane rupture.
196
Unlike many other polyamines (e.g., polyethyleneimine and
poly L-lysine), protonation and de-protonation of the repeat-
ing units along the PAA backbone are independent events.
PAAs bearing two amine nitrogens in the repeating unit
show two distinct pK
a
values and undergo sharp conforma-
tional changes at the corresponding pH.
71
Therefore, by
properly selecting the starting monomers it is possible to tai-
lor PAAs that passing from the extracellular fluid to the
endosomal intracellular compartments change conformation
and unravel their latent endosomolytic properties, thus ena-
bling the endosomal escape toward the cytosol of a high mo-
lecular weight therapeutic payload such as a protein or a
nucleic acid that would be otherwise destroyed by the endo-
somal enzymes. While the polycation-mediated membrane
damage is known since a long time,
197–200
the peculiarity of
most PAAs is precisely the sharp increase of their protona-
tion degree and the consequent conformational change in
the pH interval 7.4–5.5.
The “bioresponsiveness” of PAAs was first demonstrated by
synthesizing a hydroxylated PAA (Table 5, No. 8) modified by
covalently attaching the membrane lytic non-ionic detergent
Triton X-100 as side substituent
62
conjugate. Subsequent
experiments performed with amphoteric PAAs not carrying
pendant detergent moieties, namely Nos. 32–35 and 38 of Ta-
ble 6, demonstrated that by lowering pH below 7.4 they could
become inherently membrane active.
53,93
The ability of PAAs to
mediate DNA or toxins delivery was studied.
81,94
At 10:1 poly-
mer excess, ISA1 and ISA23 formed with DNA toroid shaped
interpolyelectrolyte complexes of diameter 80–150 nm in di-
ameter) which were visible using transition electron micros-
copy (TEM).
94
The complexes displayed retarded
electrophoretic mobility and also the ability to protect DNA
from DNase II degradation. In transfection experiments, the
PAAs demonstrated the ability to mediate pSV b-galactosidase
transfection of HepG2 cells. After these breakthrough experi-
ments, the use of both amphoteric and purely cationic PAAs as
transfection promoters was extensively investigated and will
be reported in a separate paragraph.
The ability of some PAAs to promote the endosomal escape
and intracellular trafficking of proteins was investigated
using as models two non-permeant ribosome-inactivating
toxins, namely ricin A chain and gelonin. Ricin is a highly cy-
totoxic protein in the native dimeric form, consisting of an
A-chain (RTA) and a B-chain (RTB) linked by a disulfide
bridge.
201
The RTB binds to cell membranes promoting cyto-
solic entry of the RTA moiety which acts by cleavage of the
N-glycosidic bond of adenosine
4324
nucleoside leading to in-
hibition of protein synthesis.
202
Gelonin has similar activity
to RTA, but lacks the equivalent of RTB and therefore is non-
toxic to intact cells.
203
When PAAs were incubated with
B16F10 melanoma cells in vitro in combination with RTA or
gelonin (at non-toxic concentrations of toxin) it was found
that tyramine-modified ISA1 could restore toxin cytotoxic-
ity,
94
whereas the amphoteric, but prevailingly anionic tyra-
mine-modified ISA23 did not. The ability of ISA1 to promote
intracellular delivery of non-permeant toxins was confirmed
by comparing in this respect ISA1 with its random and block
copolymers with ISA23. It was found that only ISA1 and the
block copolymer ISA23:ISA1 having a 2:1 molar ratio were
able to promote intracellular delivery.
82
These findings were
further confirmed by studying the covalent conjugates of the
same PAAs with the membrane disrupting peptide melittin.
79
In a parallel study, the ability of ISA23 to establish interac-
tion with model membrane vesicles was investigated using
EPR in conjunction with SANS.
95
Besides pH, also the type of
counterion determined the gyration radius of ISA23,
204
as
well some important biological properties, such as toxicity
and hemolytic properties of both ISA1 and ISA23.
83
For EPR,
16-DSE was dissolved in the vesicle membrane to measure
its dynamics and polarity, whereas a spin-labeled ISA23 (Ta-
ble 6, No. 47) analogue was used to give a measure of the
polymer flexibility. No interaction was found adding ISA23 to
the external vesicle surface.
96
This observation conflicts with
the reported ability of ISA23 to lyse the membrane of red
blood cells (RBC) at pH 5, but is in agreement with previ-
ous studies showing no effect on membrane permeability
when this PAA was added to an incubation medium contain-
ing isolated lysosomal vesicles, whereas destabilized the
lysosomal membrane if internalized into the lysosomal com-
partment.
205
All the above studies point to the conclusion
that linear PAAs can be designed to exhibit minimal non-spe-
cific toxicity, display pH-dependent membrane lysis, and
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deliver genes and toxins in vitro. A related study was aimed
at measuring PAA cellular uptake using ISA1-Oregon Green
conjugate (ISA1-OG) [and as a reference ISA23-Oregon Green
conjugate (ISA23-OG)] in B16F10 cells in vitro and, by sub-
cellular fractionation, quantitate intracellular trafficking of
125
I-labelled ISA1-tyrosine in liver cells after intravenous
administration to rats.
84
ISA1-OG displayed 60-fold greater
B16F10 cell uptake than ISA23-OG. Passage of ISA1 along
the liver cell endocytic pathway caused a transient decrease
in vesicle buoyant density. Increasing ISA1 dose from 10
mg/kg to 100 mg/kg increased both radioactivity and N-ace-
tylglucosamine levels in the cytosolic fraction (5- to 10-fold)
at 1 h. Moreover, internalized ISA1 provoked N-acetylglucos-
amine release from an isolated vesicular fraction in a dose-
dependent manner. These results provide direct evidence, for
the first time, of PAA permeabilization of endocytic vesicular
membranes in vivo, and they have important implications for
potential efficacy/toxicity of such polymeric vectors. It is
worth mentioning that one of the important observations in
this study is that PAA endosomolytic activity is probably due
to physical PAA–membrane interaction, rather than to the
proton sponge effect as hypothesized for PEI and other tradi-
tional polycations.
206
Most of these latest studies have been
recently reviewed and discussed elsewhere.
207
PAAs as DNA Carriers and Transfection Promoters
Triggered by the basic investigations reported in the previ-
ous paragraph, several papers on the use of PAAs other than
ISA1 and ISA23 as DNA condensing agents and transfection
promoters have been published in recent years. For the sake
of clarity, these papers will be grouped in two categories
dealing, respectively, with PAAs or PAA-PEG copolymers
whose polymer chain contains only carbon, oxygen, and
nitrogen (“traditional” PAAs) and PAAs with reducible SAS
bonds in the polymer chain.
Traditional PAAs
PAA No. 3 of Table 5 and its PEG copolymers have first been
the object of independent studies as DNA delivery systems
with remarkable success.
208–210
Later on, an extensive study
was performed on AGMA1. AGMA1 easily entered HT-29
cells, gave stable complexes with DNA and showed good
transfection efficiency suggesting the ability to transport in
the cytoplasm a DNA payload. AGMA1 probably established
non-disruptive membrane interactions allowing membrane
crossing of AGMA1 together with its payload without exert-
ing membranolytic activity. Further investigation on three
AGMA1 samples, AGMA5, AGMA10, and AGMA20 of
Mn
5100, 10,100, and 20,500, respectively, as DNA non-viral car-
riers were performed. All samples condensed DNA in spheri-
cal, positively charged nanoparticles and protected it against
enzymatic degradation. AGMA10 and AGMA20 polyplexes
had average diameters lower than 100 nm. AGMA5 poly-
plexes were larger. All polyplexes showed negligible cytotox-
icity and were internalized in cells. AGMA10 and AGMA20
effectively promoted transfection, whereas AGMA5 was inef-
fective, suggesting a MW dependence of the transfection effi-
ciency. Fluorescein isothiocyanate (FITC)-labeled AGMA10
was also prepared and its intracellular trafficking, as well as
that of its DNA polyplex, studied. FITC-AGMA10 concentrated
in the perinuclear region, but did not enter the nucleus,
whereas DNA/FITC-AGMA10 polyplex largely localized inside
the nucleus. DNA/AGMA10 polyplex intravenously adminis-
tered to mice promoted gene expression in liver, but not in
other organs and proved exempt from detectable toxic side
effects.
89,92
The transfection and intracellular trafficking of three PAAs
with pendant primary amine groups obtained from mono-
protected diamines followed by deprotection have been
described (Table 4, Nos. 35–40).
55–57
These PAAs exhibited
good DNA-binding capacities and even higher transfection
efficiencies than commercial PEI of molecular weight 25,000,
being in the meantime less cytotoxic. Their favorable per-
formance was attributed to efficient cell uptake and intracel-
lular trafficking. In a different study, branched PAAs were
developed by polyaddition of 1-(2-aminoethyl)piperazine
with N,N0-methylenebisacrylamide and in a water/N,N-dime-
thylformamide mixture. By increasing the branching degree,
the polymers became more compact and their DNA conden-
sation ability increased, whereas their cytotoxicity decreased.
Correspondingly, their efficiency as transfection promoters
improved by more than three orders of magnitude.
211
A differently conceived PAA-based carrier consisted of PAA-
grafted carbon multiwalled nanotubes.
67
The PAA chains
were attached to chemically oxidized nanotubes by amide
bonds. The adducts, besides giving stable suspensions,
showed lower cytotoxicity and comparable or even higher
transfection efficiency than both PEI of molecular weight
25,000 and the parent PAA.
“Inverted” PAAs obtained by sequential polycondensation of
diacids with polyamines are also considered as DNA carriers,
possibly susceptible of considerable development.
156
PAAs with SAS Bonds in the Main Chain (SASAPAAs)
Reduction-sensitive biodegradable polymers and conjugates
soon appeared highly promising functional biomaterials with
enormous potential in formulating sophisticated drug and
gene delivery systems.
108
In fact, the SAS bond is reductively
cleaved after cell internalization or, if administered orally, af-
ter reaching the lower portions of the gastrointestinal tract.
As regards PAAs, this technique, besides obviously facilitating
the selective intracellular release of a drug, protein or plasmid
payload, greatly increased biocompatibility even in the pres-
ence of structural features apt to enhance transport efficiency,
but in the meantime normally imparting toxicity, such as
hydrophobic side substituents and high density of positive
charges. In particular, the effects of variation in charge density
and hydrophobicity on the gene delivery properties of
SASAPAAs with aminobutyl side chains were investigated by
varying the degree of acetylation and benzoylation of the side
amine groups. Hydrophobic benzoyl groups imparted higher
transfection efficiencies.
115
In a parallel study, a SASAPAA
was modified by introducing long-chain alkyl groups, thus
apparently enhancing its ability to condense DNA into
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compact nanoparticles with positive surface charges.
212
Bio-
reducible copolymeric PAAs with tunable charge densities
were prepared by polyaddition of N,N0-bisacryloylcystamine
with variable ratios of 4-amino-1-butanol and ethylenedia-
mine or triethylenetetramine and optimized as silencing RNA
(siRNA) vectors. It was found that whereas 20–30% ethylene-
diamine or triethylenetetramine units was needed to encapsu-
late siRNA into small and stable polyplexes, higher
proportions of these units did not significantly improve their
performance in this respect, but resulted in higher cytotoxic-
ity and hemolytic activity, probably owing to the increased
cationic charge.
105
siRNA transfecting properties were also
revealed by bioreducible PAAs with x-aminohexyl pend-
ants.
114
Interestingly, bioreducible PAAs with polyamine moi-
eties inserted in the main chain proved to condense DNA
forming nanoparticles and efficiently in vitro transfect the
murine capillary endothelial cells forming the blood brain
barrier.
117
Recently, the gene delivery properties of new bioreducible
branched PAAs were investigated in comparison with their
linear analogs. The branched PAAs were prepared from N,N0-
bisacryloylpiperazine and cystamine or ethylenediamine
using unbalanced monomer mixtures to avoid crosslinking.
Their linear counterparts were obtained by substituting N,N0-
dimethylcystamine or N,N0-dimethylethylenediamine for cyst-
amine and ethylenediamine, respectively. All PAAs were ter-
minated with 4-aminobutanol or 2-aminoethanol. All PAAs
formed polyplexes with plasmid DNA with sizes around 200
nm and positive zeta potentials. Remarkably, little to negligi-
ble cytotoxicity was observed in all cases. Branched N,N0-
bisacryloylcystamine-based PAAs showed higher gene
expression in DNA transfection tests with COS-7 cells than
their linear analogues and up to two times higher than linear
PEI used as reference polymer. Moreover, the transfection
efficiencies of branched PAAs were generally enhanced by
the presence of serum.
213,214
An original modification of disulfide-containing PAAs was
recently described. It consisted of introducing phenylboronic
acid moieties either by grafting 4-carboxyphenylboronic acid
on a PAA sample with aminobutyl side chains, or incorporat-
ing 2-aminomethylphenylboronic acid units in the PAA chain
by copolymerization. The ratio of phenylboronic-substituted
units versus residual aminobutyl units was 30:70 for both
PAAs. Compared with non-boronate benzoylated PAA samples
(see above), both polymers were approximately as effective as
gene delivery vectors, but more highly cytotoxic, possibly due
to increased membrane disruptive interactions.
106
PEG-ylated bioreducible PAAs prepared by polyaddition of
N,N0-bisacryloyl cystamine with a mixture of 4-amino-1-buta-
nol and mono-tert-butoxycarbonyl-diamino PEG were eval-
uated as gene delivery vectors in comparison with their de-
protected analogs carrying x-prim-amine groups. These PAA-
PEG copolymers proved to condense DNA into nanoscaled
polyplexes (<250 nm) with a stability in buffer suspension
significantly higher than their non-PEG-ylated counterparts.
The PEG-ylated polyplexes, however, remarkably biocompati-
ble were less effective as transfection promoters, possibly
because the PEG substituent biased the endosomal escape of
the polyplexes.
215
The cellular uptake and intracellular traf-
ficking of bioreducible PAA–gene complexes in cells of the
retinal pigment epithelium, considered good targets for ocu-
lar gene therapy, was studied in a few recent papers. The
polyplexes exhibited a cationic surface, attached to cell sur-
face proteoglycans of the cells, and were subsequently inter-
nalized via a phagocytosis-like mechanism.
109,216,217
The introduction of disulfide linkages for achieving con-
trolled degradation has been employed also for “inverted”
PAA-PEG block copolymers. In particular, a single disulfide
moiety was incorporated between the two blocks. This tech-
nique was used to establish a two-phase process for releas-
ing active substances inside cells and tested by analyzing the
complexation behavior of the system with plasmid DNA,
before and after reductive degradation of the block copoly-
mer.
155
SASAPAAs have also been used for localized gene
delivery. Multilayered films composed of bioreducible cati-
onic micelles, obtained from amphiphilic disulfide-containing
PAAs and DNA, were prepared and employed as transfection
promoters. Films with 10 bilayers prepared from PAAs with
28% of alkyl side chains showed the fastest release of DNA
in the presence of 2.5 mM glutathione and the highest trans-
fection efficiency toward 293T cells cultured on the film
surface.
218
SASAPAAs obtained from N,N0-cystamine bisacry-
lamide, 4-amino-1-butanol, and ethylene diamine are promis-
ing carriers also as delivery systems of siRNA. In particular,
the effects of the percentages of butanolic side chains and
the density of basic sites in the main chain on siRNA com-
plexation, cellular uptake, gene silencing, and toxicity were
investigated. More than 80% knockdown efficiency, com-
bined with low cytotoxicity, was found for polyplexes formed
with polymers containing 25% or 50% ethylenediamine
(EDA).
219
It is finally worth mentioning that PAA-PEG nanoparticulated
constructs crosslinked with SAS bonds, to which DNA end-
capped with SH group had been grafted, were also employed
for a purpose widely different from transfection, that is, as
environmentally safe and extremely sensitive tracers for
detecting sources of water pollution.
220
Comments on PAAs as DNA Carriers and Transfection
Promoters
By considering the remarkable wealth of positive results by
many authors on polyplex forming and transfection promot-
ing ability of PAAs, it can be concluded that these are virtu-
ally general properties of this class of polymers, probably
related to the fundamentals of their structure. Going deeper
into details, favorable elements are, in the order, the basicity,
the presence of some proportions of hydrophobic substitu-
ents, and of other structural elements enhancing cell mem-
brane interaction and cell internalization followed by
endosomal escape, probably including branched molecular
architectures and presence of guanidine side substituents
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owing to the well-known chaotropic properties of this
group.
221,222
The presence of SAS linkages in the polymer
chain with the consequent selective degradation inside cells
is a structural feature reducing cytotoxicity to acceptable lev-
els even in the presence of other features acting in the oppo-
site sense, such as the first two listed above as favorable to
transfection efficiency. However, its positive effect goes far-
ther, in that intracellular degradation triggers the release of
the DNA loading in the right place and at the right moment.
Protein Delivery
Linear SASAPAAs were also studied as carriers for intracel-
lular protein delivery. To this purpose, a SASAPAA bearing
citraconic acid side moieties was prepared. This polymer
gave nanosized complexes with proteins of opposite charge
by electrostatic interaction. After cell internalization, the
release of the protein payload was triggered by a dual mech-
anism, the reduction of the disulfide bond, and the inversion
of the protein–polymer interaction at the endosomal pH, due
to the charge-reversal of the citraconic side group.
223
A se-
ries of functionalized PAAs were synthesized, two of which
contained disulfide bonds in the main chain. They self-
assembled into cationic, and essentially non-toxic nanocom-
plexes with oppositely charged proteins, as for instance b-ga-
lactosidase. The results indicated that these PAAs were
highly potent and non-toxic intracellular protein carriers.
111
Bioreducible SASAPAAs containing multiple disulfide link-
ages in the polymer backbone were used to form nanocom-
plexes by self-assembly with human insulin, used as a
negatively charged model protein at neutral pH. They were
analyzed upon adsorption on model membranes.
110
Imidaz-
ole pendants were also introduced in cationic SASAPAAs for
the same purpose, by employing histamine as comono-
mer.
112,113
Moreover, two PAAs, one of which containing SAS
bonds in the main chain, both capable of forming self-
assembled cationic nanocomplexes with oppositely charged
proteins such as albumin, were recently prepared and the
uptake of the resultant albumin-PAA nanoparticles by
human-derived intestinal mucus secreting cells was studied.
Both types of nanoparticles acted as intracellular protein
transporters, but the SASAPAA based nanoparticles were
more effective owing to their mucoadhesive properties.
224
The delivery of the hypoxia-inducible vascular endothelial
growth factor (RTP-VEGF) plasmid assisted by a soluble SAS
PAA obtained by polyaddition of excess 1,2-diaminoethane
with bisacryloylcystamine was also studied.
116
Comments on PAAs as Protein Carriers
The results of the above papers on PAAs as protein carriers
are in agreement with the previously mentioned results of
basic research in the same field
82–84,95,96,204–207
and, not
unexpectedly, point to a similar conclusion. By a proper
design of the acid/base properties and the hydrophobic/
hydrophilic balance, there is little doubt that a number of
PAAs may display intracellular protein transport ability. As
in the case of transfection, the presence of SS bonds in the
main chain is advisable or even determinant, and for the
same reasons outlined there.
Structural versatility has been already reported as a general
property of PAAs. Two specific elements, however, deserve to
be underlined here. First of all, as already mentioned, PAAs can
be assembled in a modular fashion by simply employing the
right amines and bisacrylamides, either singly or in combina-
tion with other monomers of the same category. Secondly,
nearly all PAAs carry in the polymer chain a tert-amine group,
only rarely a sec-amine group, flanked by one or two carbonyl
group in b-position. The pK
a
values of these groups are without
exceptions in the 7.25–8.25 (frequently in the 7.4–7.8) range.
44
PAAs deriving from sec-diamines carry a second amine group
per unit whose pK
a
is usually in the range 3.25–7.5. Therefore,
the omnipresent tert-amine group in the repeating unit or, in
the case of diamine-deriving units, at least one of them at the
pH of extracellular fluids (7.5) is in proximity of the mid-flex
point, hence the maximum slope of its titration curve. Remind-
ing that PAAs behave as regards ionization as if each repeating
unit were an isolated molecule, most PAAs change abruptly,
albeit predictably, their positive charge density passing from
the body fluids to intracellular endosomal compartments (pH
5.5). This brings about conformational changes and allows
displaying their latent endosomolytic activity.
BIOTECHNOLOGICAL APPLICATIONS OF CROSSLINKED PAAS
AND PAA BLOCK AND GRAFT COPOLYMERS
Heparin Absorbing Resins
Heparin is a natural mucopolysaccharide containing car-
boxyl- and sulfonic groups commonly used in clinics as anti-
coagulant agent. It is a polyanion with a high density of
negative charges. In some cases, the anticoagulant activity of
heparin must be inhibited when no longer needed. Tradition-
ally, this is achieved by administering salmin, a polymer per-
taining to a family of highly basic natural polypeptides
indicated with the generic term of protamine. Several PAAs
proved capable of neutralizing the anticoagulant activity of
heparin in solution much as salmin does (but with vastly in-
ferior toxicity) undoubtedly through the formation of PAA–
heparin polyelectrolyte complexes.
48
Not unexpectedly, cross-
linked PAAs display the same heparin-complexing ability as
their linear counterparts, but, being insoluble, act as heparin
removing resins from aqueous media, including biological
fluids such as plasma or blood.
151,225–229
Since heparin is
commonly administered to hemodialyzed patients to mini-
mize blood coagulation and this can result in morbidity in
patients at risk of bleeding, heparin-absorbing PAA resins
have been proposed to achieve regional deheparinization in
hemodialysis and extracorporeal circuits in general. These
resins displayed remarkable heparin-absorbing capacity,
even when incubated with very dilute solutions of heparin.
One of the best-performing resins was a hybrid PAA-N-vinyl-
pyrrolidinone copolymer prepared by applying the above
reported V-PAA radical polymerization method in the pres-
ence of N-vinylpyrrolidone. These resins absorbed heparin to
an extent ranging from 30 to 100 wt % (calculated on dry
resin) according to their PAA content and apparently did not
adversely affect any normal blood parameter. In particular,
they were not hemolytic and did not alter recalcification
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time, prothrombin time, partial thromboplastin time, and
fibrinogen content. The retentive power of a typical resin at
various pH values after heparin loading was investigated by
eluting with M/15 phosphate buffers at pH 4.8, 7.4, and 8.5.
No heparin was released.
229
The resin was then eluted with
carbonate buffers. All heparin was desorbed between pH
10.8 and 11.4 and the remarkably sharp elution peak was
centered at pH 11.0.
Heparinizable Materials
Non-physiological materials usually induce thrombus forma-
tion when placed in contact with blood. The development of
permanently non-thrombogenic materials for cardivascular
prosteses was a long-coveted, but never completely achieved
target in the second half of the twentieth century. One of the
earliest and most promising approaches was to ionically
adsorb heparin on polymeric materials, a process hindering
thrombus formation as long as heparin was present. The
PAA property of giving stable complexes with heparin both
in solution and in the hydrogel state, coupled with the pres-
ence in them of reactive end-functions amenable to coupling
reactions, was soon exploited in this direction. Two ways
were explored, the surface-grafting of PAAs on commercial
polymers and the preparation of PAA block—an graft copoly-
mers with medical grade commodity plastics. The prepara-
tion of these modified materials has been reported above in
the “PAA-based block and graft copolymers” and “Surface-
grafting of PAAs onto organic and inorganic materials”
sections.
The previously mentioned polyurethane-PAA block copoly-
mers nicknamed PUPA were extensively studied. PUPA sam-
ples containing 5–30 wt % PAA showed approximately the
same mechanical properties and hydrolytic stability of parent
polyurethane,
230
even if their tensile strength was somewhat
lower and the elongation at break higher. PUPA samples
were capable of adsorbing, and probably to some extent also
absorbing relatively large amounts of heparin (0.002–0.7
mg/cm
2
) and stably retained it.
128–136
No heparin was stably
retained by the corresponding native polyurethane. PUPA
samples proved an excellent coating for poly(vinylchloride),
virgin polyurethanes, and other materials.
138
Segmented pol-
yurethanes containing quaternary ammonium groups behave
in a similar way to PAA-polyurethane block and graft copoly-
mers,
231
but unlike the latter were liable to display hemo-
lytic properties.
The biocompatibility of all the PAA-modified materials was
evaluated.
134,136
Heparinization significantly improved their
blood compatibility, especially as regards clotting formation.
They showed a definite potential for the fabrication of
thromboresistant medical devices addressed to short- and
medium-term applications involving contact with blood.
Blood biocompatible PAA-polyamide block-copolymer were
also synthesized by reacting amine end-capped polyamides
with N,N-methylenebisacrylamide in m-cresol solution. The
block copolymers absorbed heparin. After heparinization,
they showed significant improvement in blood compatibil-
ity.
58
PAA-polymethylmethacrylate block copolymers were
also prepared and studied for the same purpose.
30
After hep-
arinization, the resultant materials showed acquired non-
thrombogenic properties. PAAs could not be used directly in
the making of blood-contacting materials due to their poor
mechanical strength. Characterization studies indicated that
the PAAs have been suitably incorporated into the MMA ma-
trix. The relative hydrophilic nature of the synthesized
copolymers was established from the measurement of water
contact angle. Two mixed PAA-PVP hydrogels were also
obtained from piperazine, cyclohexylamine, and N,N0-methyl-
enebisacrylamide by copolymerizing N-vinylpyrrolidone with
the terminal double bonds. After heparinization, these mate-
rials exhibited non-thrombogenic and non-hemolytic proper-
ties.
31
The maximum achievable duration of the
thromboresistance in vivo of all the above reported heparin-
izable materials was apparently never determined, but could
be hardly expected to be indefinite. However, the discovery
of permanently thromboresistant synthetic materials does
not seem anymore a priority biomedical target, possibly
owing to the improved methods of controlling blood coagula-
tion presently available coupled by the widespread use of
fast eliminable low molecular weight heparin, or simply
because of the diffuse feeling that reaching it is virtually
impossible.
Release of Bioactive Substances from Tailored
PAA-Based Hydrogels
Horseradish peroxidase (HRP)-mediated crosslinking of PAA
copolymers was recently applied in the preparation of in situ
forming degradable hydrogels. In particular, PAA copolymers
containing different amounts of tyramine residues were syn-
thesized. The gelation time varied with the amount of tyra-
mine residue, the HRP, and H
2
O
2
concentration. These
hydrogels completely degraded under physiological condi-
tions within ten weeks. They proved suitable for the sus-
tained release of model low molecular weight substances
and proteins.
34
A novel application of PAA-based copolymers as injectable
pH- and temperature-sensitive hydrogels has been proposed.
A series of poly(amidoamine)-poly(ethyleneglycol)-poly(ami-
doamine) (PAA-PEG-PAA) triblock copolymers were designed
and prepared to examine the factors affecting their sol–gel
transition behavior. The PAA-PEG-PAA copolymers under-
went sol–gel transitions in solution (10–15 wt %) in
response to changes in both the pH and temperature. Adjust-
ing the molecular weights of the PEG and PAA blocks and
changing the PAA-PEG-PAA concentration allowed to control
the sol–gel phase transition behavior. These copolymers
demonstrated stronger bioadhesive properties than chitosan
and poly(acrylic acid) in an aqueous solution. The in vitro
release of flubiprofen, as a model drug, from these hydrogels
was tested and found to be controllable.
232,233
Recently, a new pH sensitive, biocompatible, and biodegrad-
able polymer hydrogel obtained from the combination of
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Shellac (a natural polymer secreted by lac insect) and PAA
by a photopolymerization process has been reported. This
new material has been proposed for the controlled release of
colon specific therapeutic agents.
64
PAA Hydrogels and as Scaffolds for Cell Culturing and
Tissue Engineering Applications
As stated above, PAAs can be easily obtained in crosslinked
form either directly, by introducing multifunctional monomers
in the polymerizing system, or in two steps consisting in pre-
paring first a,x-divinyl-terminated PAA oligomers with unbal-
anced monomer mixtures, and then triggering a vinyl
polymerization by radical initiators. All crosslinked PAAs in
aqueous media absorb high amounts of water, ranging from
100 to 1000% of their own dry weight, or even more, depend-
ing from the crosslinking degree and the crosslinking method
adopted, and give rise to hydrogels. A comprehensive struc-
tural characterization of crosslinked insoluble PAA networks
can be performed by high-resolution magic angle spinning
(HRMAS) NMR spectroscopy.
234
The interaction of water with
PAA hydrogels in the absence and presence of inorganic ions
was carefully studied by NMR techniques.
235–237
The potential of crosslinked PAAs as scaffolds for cell cultur-
ing and tissue regeneration started only recently. In a first
paper, amphoteric PAA hydrogels were obtained from 2,2-
bisacrylamidoacetic acid, 2-methylpiperazine, and primary
bis-amines as crosslinking agents. In some instances, mono-
acrylamides as modifiers were added.
238
These hydrogels
were essentially crosslinked versions of ISA23. Hybrid PAA/
albumin hydrogels were also prepared. Cytotoxicity tests
demonstrated that all the amphoteric PAA hydrogels consid-
ered were cytobiocompatible both as free bases and salts.
Pure PAA hydrogels completely dissolved within two weeks
in Dulbecco medium at pH 7.4 and 37C, but hybrid PAA/al-
bumin hydrogels did not dissolve within eight months, sug-
gesting a way to tune the degradation time in vitro. The
degradation products of all samples turned to be completely
non-cytotoxic. Shortly after, two new hydrogels (PAA-AG1
and PAA-AG2) were prepared by polyaddition of 2,2-bisacry-
lamidoacetic acid with 4-aminobutylguanidine (agmatine)
239
employing two different crosslinkers, namely 1,10-decanedi-
amine for PAA-AG1 and PAA-NH2, that is a PAA containing
pendant NH
2
groups,
25
for PAA-AG2. These hydrogels were
differently crosslinked versions of AGMA1 (Table 6, No. 28).
Both PAA-AG1 and PAA-AG2 proved non-cytotoxic and adhe-
sive to cell membranes. Compared with PAA-AG1, PAA-AG2
exhibited improved mechanical strength. The dissolution
times of PAA-AG1 and PAA-AG2 under the conditions
reported for ISA23 hydrogels were approximately 10 and 40
days, respectively. As in the previous case, the degradation
products were completely non-cytotoxic. These data were
later substantially confirmed.
240
In a parallel study,
241
nanometric hydrogel layers based on
ISA23 and AGMA1 crosslinked with 1,2-diaminoethane sup-
ported on transparent substrates for cell culture were pre-
pared by in situ polymerization carried out on glass
substrates purposely modified with c-aminopropyltriethoxy-
silane, followed by swelling in water, which invariably led to
spontaneous delamination of the external bulk of the hydro-
gel. AGMA1 hydrogel layers exhibited a level of cell adhesion
toward epithelial cells (MDCK) comparable to that of com-
mercial plastic substrates. On the contrary, ISA23-hydrogels
showed a vastly inferior cell adhesion, thus demonstrating
that epithelial cell adhesion was probably due to the side
guanidine groups of the former hydrogel coupled with its
prevailingly cationic character at pH 7.4. Slightly later, it was
found that the treatment of plastic or glass wells for cell cul-
ture with a solution of linear AGMA1 rendered them good
substrates for neuronal cell culturing, promoting Schwann
and Dorsal Root Ganglion neurons cell adhesion and/or pro-
liferation to the same extent as poly-L-lysine, but with a
vastly inferior toxicity.
179
These results provided the ration-
ale for proposing tubular scaffolds made of AGMA1 hydro-
gels as guides for peripheral nerve regeneration in vivo. The
first reported AGMA1 hydrogels prepared with polyamines
as crosslinking agents were mechanically too weak to be
inserted in animals, but hydrogels prepared by radical poly-
merization of vinyl-a,x-terminated AGMA1 oligomers showed
improved mechanical properties and proved amenable to be
glued to living tissues by commercial fibrin glue. Rats with
severed sciatic nerve were implanted with AGMA1 tubes of
1.2 mm internal lumen. The animals were analyzed at 30,
90, and 180 days post-surgery. The tubing made nerve
regeneration remarkably easier. Good surgical outcomes
were achieved with no inflammation or neuroma signs.
Moreover, nerve regeneration was morphologically sound
and the quality of functional recovery satisfactory. At the end
of the experiment (90 days) the PAA tubing had faded away.
The weak point of the AGMA1 hydrogels, however prepared,
consisted of mechanical properties that, though barely suffi-
cient to perform animal experiments, were still unfit to be
proposed for human use. A strategy for obtaining PAA hydro-
gels combining mechanical strength with ability to promote
neuronal, in particular Schwann and DRG cell adhesion and
proliferation, was to capitalize on structure-forming proper-
ties exhibited by some linear PAAs, which could be expected
to be shared by their crosslinked analogs.
149
In particular,
the PAA No. 32 of Table 5 was highly crystalline and during
polymerization in water showed a strong tendency to precip-
itate by crystallization. Indeed, moderately crosslinked PAA
samples maintained to a considerable extent the same
structure-forming properties in aqueous media of their
linear counterpart. They formed hydrogels that, notwith-
standing their high swelling degree, contained crystalline
domains acting as effective reinforcing agents. Consequently,
their mechanical properties were remarkably superior to
those of all other PAA hydrogels described so far. Interest-
ingly, this property vanished by increasing the crosslinking
degree, probably because the length of the linear segments
connecting the crosslink points became too short to allow
extensive crystalline structuring in a tight network. In vitro
experiments on the structured hydrogels showed that
Schwann and Dorsal Root Ganglion neurons adhered and
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grew satisfactorily on them, warranting potential for in vivo
applications.
A parallel study dealt with the direct micro fabrication of to-
pographical clues for the guided growth of neural PC12 cells
along patterns sculptured by E-beam microlithography on
the prevailingly anionic, non-adhesive ISA23 hydrogels de-
posited on a solid surface. The cells grew only along the
sculptured patterns and a neural network of single cells con-
nected by neuritis extending along microchannels was
obtained.
242
The reason why the cells grew only along the
channels neglecting the rest of the surface was not fully
explained, but it might be supposed that the electron beam
induced de-carboxylation of the 1,1-bisacrylamidoacetic acid
moieties of the hydrogel, locally reversing from anionic to
cationic its prevailing charge.
CONCLUSIONS
PAAs are a polymer family endowed with a rarely equalled
combination of relevant properties making them eligible to a
variety of applications mainly, but not exclusively, in the bio-
medical field. The fundamentals of their structure are tert-
amine groups and amide groups regularly arranged along
the the polymer chain, of which they constitute integral
parts. The carbonyl groups, in b-position to the amine
groups, significantly lower both the basicity and the toxicity
of PAAs (indeed, some PAAs are nearly as biocompatible as
dextran), provided strongly basic- or long-chain hydrophobic
side substituents are absent. Notwithstandingly, most PAAs
are able to interact with polyanions yielding polyplexes. In
dilute aqueous solution, at pH 7.5 and T30C, PAAs de-
grade also in the absence of specific enzymes, whereas cross-
linked PAAs under the same conditions are apparently much
more stable even if ultimately dissolve both in vitro and,
more rapidly, in vivo. Their degradation rate is largely tune-
able according to needs. Both linear and crosslinked PAAs
are remarkably more stable in slightly acidic media.
In addition to the above general properties, PAAs are
endowed with a structural versatility enabling to finely tune
their acid–base properties, to introduce additional functions
for the sake of specific properties, to prepare PAA block
copolymers with other PAAs or with most other polymer
families, to surface-graft PAA chains on a number of organic
or inorganic materials. Last but not least, the PAA prepara-
tion is usually simple and environmentally friendly.
PAAs have been and are being successfully considered for a
number of disparate applications, such as selective absorbers
of inorganic water pollutants, as active componens of sen-
sors, as matrices for immobilizing NLO probes, as hydrogel
scaffolds for cell culturing and peripheral nerve regeneration,
as selective heparin absorbers, as polymer-drug conjugates,
as antiviral and antimalarial agents, as protein intracellular
carriers, as nucleic acids complexing agents and transfection
promoters. Possibly, from a practical standpoint, PAAs have
somewhat resented of having been developed mostly in the
Academy without having been sponsored ab initio by a
strong research-oriented Company. Nevertheless, it may be
expected that continuing the present trend their impact will
steadily increase in the next years.
REFERENCES AND NOTES
1Hulse, G. E. (Hercules Powder Co.). U.S. Patent 2,277,913, Au-
gust 21, 1956.
2F. Danusso, P. Ferruti, G. Ferroni, Chim. Ind. (Milano) 1967,
49, 271–278.
3F. Danusso, P. Ferruti, G. Ferroni, Chim. Ind. (Milano) 1967,
49, 453–457.
4F. Danusso, P. Ferruti, G. Ferroni, Chim. Ind. (Milano) 1967,
49, 587–590.
5F. Danusso, P. Ferruti, G. Ferroni, Chim. Ind. (Milano) 1967,
49, 826–830.
6P. Ferruti, R. Alimardanov, Chim. Ind. (Milano) 1967,49, 831–
834.
7F. Danusso, P. Ferruti, Chim. Ind. (Milano),1968,50, 71–80.
8P. Ferruti, Z. Brzozowski, Chim. Ind. (Milano) 1968,50, 441–
445.
9F. Danusso, P. Ferruti, Polymer 1970,11, 88–113.
10 D. L. Murfin, K. Hayashi, L. E. Miller, J. Polym. Sci. Part A-1:
Polym. Chem. 1970,8, 1967–1980.
11 P. Ferruti, M. A. Marchisio, Barbucci, R. Polymer 1985,26,
1336–1348.
12 P. Ferruti, In Polymeric Materials Encyclopedia; J. C. Sala-
mone, Ed.; CRC Press Inc.: Boca Raton, Florida, 1996; Vol. 2,
pp. 3334–3359.
13 P. Ferruti, M. A. Marchisio, Duncan, R. Macromol. Rapid
Commun. 2002,23, 332–355.
14 M. Casolaro, E. Ranucci, F. Bignotti, P. Ferruti, Makromol.
Chem. 1993,194, 3329–3339.
15 P. Ferruti, E. Ranucci, L. Depero, Polym. Commun. 1989,30,
157–160.
16 E. Ranucci, P. Ferruti, Polymer 1991,32, 2876–2879.
17 P. Ferruti, E. Ranucci, J. Polym. Sci. Part C: Polym. Lett. Ed.
1988,26, 357–360.
18 P. Ferruti, E. Ranucci, L. Depero, Makromol. Chem. Rapid
Commun. 1988,9, 807–811.
19 F. Fenili, C. Rigamonti, A. Bossi, P. Ferruti, A. Manfredi, S.
Maiorana, C. Baldoli, S. Cauteruccio, E. Licandro, E. Ranucci, J.
Polym. Sci. Part A: Polym. Chem. 2010,48, 4704–4710.
20 D. A. Tomalia, J. R. Dewald (The Dow Chemical Corpora-
tion). U.S. Patent 45,074,667, January, 1983.
21 D. A. Tomalia, Nanomedicine 2012,7, 953–956.
22 A. Manfredi, E. Ranucci, M. Suardi, P. Ferruti, J. Bioact.
Compat. Polym. 2007,22, 219–231.
23 A. Zintchenko, L. J. van der Aa, J. F. Engbersen, J. Macro-
mol. Rapid Commun. 2011,32, 321–325.
24 G. Caldwell, E. Neuse, A. Stephanou, J. Appl. Polym. Sci.
1993,50, 393–401.
25 B. Malgesini, I. Verpilio, R. Duncan, P. Ferruti, Macromol.
Biosci. 2003,3, 59–66.
26 P. Ferruti, S. Knobloch, E. Ranucci, R. Duncan, E. Gianasi,
Macromol. Chem. Phys. 1998,199, 2565–2575.
27 J. Franchini, E. Ranucci, P. Ferruti, M. Rossi, R. Cavalli, Bio-
macromolecules 2006,7, 1215–1222.
JOURNAL OF
POLYMER SCIENCE WWW.POLYMERCHEMISTRY.ORG HIGHLIGHT
WWW.MATERIALSVIEWS.COM JOURNAL OF POLYMER SCIENCE, PART A: POLYMER CHEMISTRY 2013,51, 2319–2353 2349
28 D. Maggioni, F. Fenili, L. D’Alfonso, D. Donghi, M. Panigati,
I. Zanoni, R. Marzi, A. Manfredi, P. Ferruti, G. D’Alfonso, E.
Ranucci, Inorg. Chem. 2012,51, 12776–12788.
29 M. Casolaro, F. Bignotti, L. Sartore, M. Penco, Polymer 2001
42, 903–912.
30 R. K. Dey, A. R. Ray, J. Appl. Polym. Sci. 2003,90, 4068–
4074.
31 R. K. Dey, A. R. Ray, Biomaterials 2003,24, 2985–2993.
32 R. Barbucci, M. Casolaro, M. C. Beni, P. Ferruti, M. Pesa-
vento, T. Soldi, C. Riolo, J. Chem. Soc. Dalton Trans. 1981,
2559–2564.
33 P. Y. Vuillaume, M. Brunelle, M.-R. Van Calsteren, S. Lau-
rent-Lewandowski, A. Begin, R. Lewandowski, B. G. Talbot, Y.
El Azhary, Biomacromolecules 2005,6, 1769–1781.
34 Y. Y. Sun, Z. Deng, Y. Tian, C. Lin, J. Appl. Polym. Sci. 2013,
127, 40–48.
35 P. Ferruti, R. Barbucci, Adv. Polym. Sci. 1984,58, 55–92.
36 R. Barbucci, P. Ferruti, C. Improta, M. Delfini, A. L. Segre, F.
Conti, Polymer 1978,19, 1329–1334.
37 R. Barbucci, P. Ferruti, Polymer 1979,20, 1061–1062.
38 R. Barbucci, P. Ferruti, M. Micheloni, M. Delfini, A. L. Segre,
F. Conti, Polymer 1980,21, 81–85.
39 R. Barbucci, V. Barone, P. Ferruti, Atti Accad. Naz Lincei
1978,64, 481–484.
40 R. Barbucci, V. Barone, P. Ferruti, M. Delfini, J. Chem. Soc.
Dalton Trans. 1980,2, 253–256.
41 R. Barbucci, M. Casolaro, N. Danzo, M. C. Beni, V. Barone,
P. Ferruti, Gazz. Chim. Ital. 1982,112, 105–113.
42 E. Ranucci, L. Putelli, P. Ferruti, V. Ferrari, D. Marioli, A.Tar-
oni, Mikrochim. Acta 1995,120, 257–70.
43 E. Ranucci, P. Ferruti, V. Ferrari, D. Marioli, A. Taroni,
Polym. Adv. Technol. 1996,7, 529–535.
44 E. Ranucci, P. Ferruti, E. Lattanzio, A. Manfredi, M. Rossi, P.
R. Mussini, F. Chiellini, C. Bartoli, J. Polym. Sci. Part A: Polym.
Chem. 2009,47, 6977–6991.
45 R. Barbucci, M. Casolaro, P. Ferruti, V. Barone, Polymer
1982,23, 148–151.
46 P. Ferruti, L. Oliva, R. Barbucci, M. C. Tanzi, Inorg. Chim.
Acta 1980,41, 25–29.
47 R. Barbucci, M. Casolaro, V. Barone, P. Ferruti, M. Tramon-
tini, Macromolecules 1983,16, 1159–1164.
48 M. A. Marchisio, C. Sbertoli, G. Farina, P. Ferruti, Eur. J
Pharm. 1970,12, 236–242.
49 R. Barbucci, V. Barone, L. Oliva, P. Ferruti, T. Soldi, M. Pesa-
vento, C. Bertoglio Riolo, In Polymeric Amines and Ammonium
Salts; E. J. Goethals, Ed.; Pergamon Press: Oxford and New
York, 1980; p 263.
50 S. Vansteenkiste, E. Schacht, E. Ranucci, P. Ferruti, Makro-
mol. Chem. 1992,193, 937–943.
51 V. Ushakova, E. Panarin, E. Ranucci, F. Bignotti, P. Ferruti,
Macromol. Chem. Phys. 1995,196, 2927–2939.
52 P. Ferruti, F. Danusso, G. Franchi, N. Polentarutti, S. Garat-
tini, J. Med. Chem. 1973,16, 496–499.
53 S. Richardson, P. Ferruti, R. Duncan, J. Drug Target. 1999,6,
391–404.
54 P. Ferruti, E. Ranucci, F. Trotta, E. Gianasi, E. G. Evagorou,
M. Wasil, G. Wilson, R. Duncan, Macromol. Chem. Phys. 1999,
200, 1644–1654.
55 L. Peng, M. Liu, Y.-N. Xue, S.-W. Huang, R.-X. Zhuo, Bioma-
terials 2009,30, 5825–5833.
56 M. Liu, J. Chen, Y.-P. Cheng, Y.-N. Xue,; R.-X. Zhuo, S.-W.
Huang, Macromol. Biosci. 2010,10, 384–392.
57 W.-M. Liu, M. Liu, Y.-N. Xue, N. Peng, X.-M. Xia, R.-X. Zhuo,
S.-W. Huang, J. Biomed. Mater. Res. A 2012,100, 872–881.
58 J. P. Singhal, A. R. Ray, Biomaterials 2002,23, 1139–1145.
59 R. Mendichi, P. Ferruti, B. Malgesini, Biomed. Chromatogr.
2005,19, 196–201.
60 M. Piest, C. Lin, M. A. Mateos-Timoneda, C. M. C. Lok, W. E.
Hennink, J. F. J. Engbersen, J. Control. Release 2008,130, 38–45.
61 P. Ferruti, E. Ranucci, F. Bignotti, L. Sartore, P. Bianciardi,
M. A. Marchisio, J. Biomat. Sci. Polym. Ed. 1994,6, 833–844.
62 R. Duncan, P. Ferruti, D. Sgouras, A. Tuboku-Metzger, E.
Ranucci, F. Bignotti, J. Drug Target. 1994,2, 341–347.
63 Z. Yu, J. Yan, Y. You, J. Control. Release 2011,152, e179–
e181.
64 R. K. Dey, G. S. Tiwary, T. Patnaik, U. Jha, J. Appl. Polym.
Sci. 2012,125, 2626–2635.
65 P. Ferruti, E. Ranucci, L. Sartore, F. Bignotti, M. A. Marchi-
sio, P. Bianciardi, F. M. Veronese, Biomaterials 1994,15, 1235–
1241.
66 C. Lin, Z. Zhong, M. C. Lok, X. Jiang, W. E. Hennink, J. Fei-
jen, J. F. J. Engbersen, J. Control. Release 2006,116, 130–137.
67 R. B. Wang, L. Z. Zhou, Y. F. Zhou, G. L. Li, X. Y. Zhu, H. C.
Gu, X. L. Jiang, H. Q. Li, J. L. Wu, L. He, X. Q. Guo, B. S. Zhu,
D. Y. Yan, Biomacromolecules 2010,11, 489–495.
68 D. Wang, Y. Liu, C.-Y. Hong, C.-Y. Pan, Polymer 2006,47,
3799–3806.
69 R. Barbucci, P. Ferruti, C. Improta, M. La Torraca, L. Oliva,
M. C. Tanzi, Polymer 1979,20, 1298–1300.
70 P. Ferruti, C. Bertoglio Riolo, T. Soldi, M. Pesavento, J.
Appl. Polym. Sci. 1982,27, 2239–2248.
71 R. Barbucci, M. Casolaro, P. Ferruti, V. Barone, F. Leli, L.
Oliva, Macromolecules 1981,14, 1203–1209.
72 R. Barbucci, M. Casolaro, M. Nocentini, S. Corezzi, P. Ferruti,
V. Barone, Macromolecules 1986,19, 37–42.
73 R. Barbucci, V. Barone, P. Ferruti, L. Oliva, J. Polym. Sci.
Polym. Symp. 1981,69, 49–66.
74 E. Ranucci, G. Spagnoli, P. Ferruti, D. Sgouras, R. Duncan,
J. Biomater. Sci. Polym. Ed.,1991,2, 303–315.
75 M. Pesavento, T. Soldi, P. Ferruti, R. Barbucci, M. Benvenuti,
J. Appl. Polym. Sci. 1983,28, 3361–3368.
76 R. Barbucci, M. Benvenuti, M. Pesavento, P. Ferruti, Polym.
Commun. 1983,24, 26–29.
77 F. Bignotti, P. Sozzani, E. Ranucci, P. Ferruti, Macromole-
cules 1994,27, 7171–7178.
78 E. Ranucci, P. Ferruti, Macromolecules 1991,24, 3747–3752.
79 N. Lavignac, M. Lazenby, J. Franchini, P. Ferruti, R. Duncan,
Int. J. Pharm. 2005,300, 102–112.
80 N. Lavignac, J. L. Nicholls, P. Ferruti, R. Duncan, Macromol.
Biosci. 2009,9, 480–487.
81 S. C. W. Richardson, N. G. Pattrick, Y. K. S. Man, P. Ferruti,
R. Duncan, Biomacromolecules 2001,2, 1023–1028.
82 N. Lavignac, M. Lazenby, P. Foka, I. Verpilio, B. Malgesini, P.
Ferruti, R. Duncan, Macromol. Biosci. 2004,4, 922–929.
83 K.-W. Wai, B. Malgesini, I. Verpilio, P. Ferruti, P. Griffiths, P.
Alison, A. C. Hann, R. Duncan, Biomacromolecules 2004,5,
1102–1109.
84 S. C. W. Richardson, N. G. Pattrick, N. Lavignac, P. Ferruti,
R. Duncan, J. Control. Release 2010,142, 78–88.
HIGHLIGHT WWW.POLYMERCHEMISTRY.ORG
JOURNAL OF
POLYMER SCIENCE
2350 JOURNAL OF POLYMER SCIENCE, PART A: POLYMER CHEMISTRY 2013,51, 2319–2353
85 G. Baldi, D. Bonacchi, F. Innocenti, G. Lorenzi, M. Bitossi, P.
Ferruti, E. Ranucci, A. Ricci, M. Comes Franchini (Colorobbia
Italia S. p. A.). PCT Int. Appl., WO 2008074804 A2 20080626,
June 27, 2008.
86 R. Barbucci, M. J. M. Campbell, M. Casolaro, M. Nocentini,
G. Reginato, P. Ferruti, J. Chem. Soc. Dalton Trans. 1986,
2325–2330.
87 M. Casolaro, I. Casolaro, A. Spreafico, C. Capperucci, B. Fre-
diani, R. Marcolongo, N. Margiotta, R. Ostuni, R. Mendichi, F.
Samperi, T. Ishii, Y. Ito, Biomacromolecules 2006,7, 3417–
3427.
88 P. Ferruti, R. Cavalli, E. Ranucci, D. Lembo, (CISI, Manfredi
A.). PCT Int. Appl. WO2011145056 A120111124, November 24,
2011.
89 R. Cavalli, A. Bisazza, R. Sessa, P. Luca, F. Fenili, A. Man-
fredi, E. Elisabetta Ranucci, P. Ferruti, Biomacromolecules
2010,11, 2667–2674.
90 E. Ranucci, P. Ferruti, F. Fenili, A. Manfredi, N. Mauro, X.
Fernandez-Busquets, P. Urban, (Universita’ di Milano, Fundacio
Privada Institut de Bioenginyeria De Catalunya, Centre De
Recerca En Salut Internacional De Barcelona), EP12192633.1,
November 14, 2012.
91 X. Fernandez-Busquets, P. Urban, J. J. Valle-Delgado, E.
Moles, J. Marques, P. Marin-Garcia, M. B. Jimenez-Diaz, C.
Diez, E. Ranucci, A. Diez, A. Puyet, I. Angulo-Barturen, J. M.
Bautista, J. Estelrich, FEBS J. 2012,279 (SI Suppl. 1), 329–329.
92 P. Ferruti, J. Franchini, M. Bencini, E. Ranucci, G. P. Zara, L.
Serpe, L. Primo, R. Cavalli, Biomacromolecules 2007,8, 1498–
1504.
93 P. Ferruti, S. Manzoni, S. C. W. Richardson, R. Duncan, N.
G. Pattrick, R. Mendichi, M. Casolaro, Macromolecules 2000,
33, 7793–7800.
94 N. G. Pattrick, S. C. W. Richardson, M. Casolaro, P. Ferruti,
R. Duncan, J. Control. Release 2001,77, 225–232.
95 P. C. Griffiths, A. Paul, Z. Khayat, K. W. Wai, S. M. King, I.
Grillo, R. Schweins, P. Ferruti, J. Franchini, R. Duncan, Bioma-
cromolecules 2004,5, 1422–1427.
96 P. C. Griffiths, N. Renuka, E. Carter, P. Dodds, D. M. Murphy,
Z. Zeena Khayat, E. Lattanzio, P. Ferruti, R. K. Heenan, S. M.
King, R. Duncan, Macromol. Biosci. 2010,10, 963–973.
97 M. Gussoni, F. Greco, P. Ferruti, E. Ranucci, A. Ponti, L.
Zetta, New J. Chem. 2008,32, 323–332.
98 M.Bencini,E.Ranucci,P.Ferruti,F.Trotta,M.Donalisio,M.Cor-
naglia,D.Lembo,R.Cavalli,J. Control. Release 2008,126, 17–25.
99 A. Bernkop-Schn€
urch, Adv. Drug Deliv. Rev. 2004,57, 1569–1582.
100 K. Albrecht, A. Bernkop-Schn€
urch, Nanomedicine 2007,2, 41–50.
101 D. Donghi, D. Maggioni, G. D’Alfonso, A. Amigoni, E.
Ranucci, F. Ferruti, A. Manfredi, F. Fenili, A. Bisazza, R. Cavalli,
Biomacromolecules 2009,10, 3273–3282.
102 E. Ranucci, P. Ferruti, A. Manfredi, M. A. Suardi, Macromol.
Rapid Commun. 2007,28, 1243–1250.
103 E. Ranucci, M. A. Suardi, R. Annunziata, P. Ferruti, F. Chiel-
lini, C. Bartoli, Biomacromolecules 2008,9, 2693–2704.
104 E. Emilitri, E. Ranucci, P. Ferruti, J. Polym. Sci. Part A:
Polym. Chem. 2005,43, 1404–1416.
105 L. J. van der Aa, P. Vader, G. Storm, R. M. Schiffelers, J. F.
J. Engbersen, J. Control. Release 2011,150, 177–186.
106 M. Piest, J. F. J. Engbersen, J. Control. Release 2011,155,
331–340.
107 E. Emilitri, P. Ferruti, R. Annunziata, E. Ranucci, M. Rossi,
L. Falciola, P. Mussini, F. Chiellini, C. Bartoli, Macromolecules
2007,40, 4785–4793.
108 F. H. Meng, W. E. Hennink, Z. Zhong, Biomaterials 2009,
30, 2180–2198.
109 D. Vercauteren, M. Piest, M. Al Soraj, A. T. Jones, J. F. J.
Engbersen; S. C. De Smedt, K. Braeckmans, J. Control. Release
2010,148, e99–e100.
110 R. Frost, G. Cou
e, J. F. J. Engbersen, M. Zaech, B. Kesimo,
S. Svedhem, J. Colloid Interface Sci. 2011,362, 575–583.
111 G. Cou
e, J. F. J. Engbersen, J. Control. Release 2011,152,
90–98.
112 G. Cou
e, J. Feijen, J. F. J. Engbersen, J. Control. Release
2008,132, e2–e3.
113 C. Lin, Z. Y. Zhong, M. C. Lok, H. K. de Wolf, W. E. Hen-
nink, J. Feijen, J. F. J. Engbersen, J. Control. Release 2008,
132, e8–e10.
114 S. W. Kim, M. Ou, D. A. Bull, S. W. Kim, Macromol. Biosci.
2010,10, 898–905.
115 M. Piest, J. F. J. Engbersen, J. Control. Release 2010,148,
83–90.
116 L. V. Christensen, C.-W. Chang, J. W. Yockman, R. Con-
ners, H. Jackson, Z. Zhong, J. Feijen, D. A. Bull, S. W. Kim, J.
Control. Release 2007,118, 254–261.
117 H. Zhang, S. V. Vinogradov, J. Control. Release 2010,143,
359–366.
118 P. J. Flory, Principles of Polymer Chemistry; Cornell Uni-
versity Press: London, 1953.
119 P. Ferruti, L. Provenzale, Transplant. Proc. 1976,1, 103–105.
120 E. Martuscelli, M. Palma, F. Riva, P. Ferruti, L. Provenzale,
Chim. Ind. (Milan) 1976,58, 542–545.
121 P. Ferruti, D. Arnoldi, M. A. Marchisio, E. Martuscelli, M.
Palma, F. Riva, L. Provenzale, J. Polym. Sci. Part A: Polym.
Chem. 1977,15, 2151–2162.
122 P. Ferruti, E. Martuscelli, L. Nicolais, M. Palma, F. Riva,
Polymer 1977,18, 387–390.
123 E. Martuscelli, L. Nicolais, F. Riva, P. Ferruti, L. Provenzale,
Polymer 1978,19, 1329–1334.
124 M. C. Tanzi, G. Tieghi, P. Botto, C. Barozzi, P. Cardillo, Bio-
materials 1984,5, 357–361.
125 M. C. Tanzi, C. Barozzi, T. Tieghi, R. Ferrara, Biomaterials
1985,6, 273–276.
126 R. Barbucci, M. Benvenuti, A. Magnani, F. Tempesti, Mak-
romol. Chem. 1992,193, 2979–2988.
127 M. C. Tanzi, B. Barzaghi, R. Anouchinsky, S. Bilenkis, A.
Penhasi, D. Cohn, Biomaterials 1992,13, 425–431.
128 R. Barbucci, M. Benvenuti, G. Dal Maso, P. Ferruti, F. Tem-
pesti, W. G. Lemm, Biomaterials 1987,8, 306–307.
129 R. Barbucci, A. Magnani, Biomaterials 1989,10, 429–432.
130 R. Barbucci, M. Casolaro, A. Magnani, C. Roncolini, Poly-
mer 1991,32, 897–903.
131 R. Barbucci, A. Albanese, A. Magnani, F. Tempesti,
J. Biomed. Mater. Res. 1991,25, 1259–1274.
132 R. Barbucci, A. Magnani, A. Albanese, F. Tempesti, Int. J.
Artif. Organs 1991,14, 499–507.
133 R. Barbucci, F. Tempesti, M. Benvenuti, A. Magnani, A.
Albanese, Adv. Biomater. Sci. 1992,10, 217–228.
134 R. Barbucci, A. Magnani, Biomaterials 1994,15, 955–962.
135 S. Cimmino, E. Martuscelli, C. Silvestre, R. Barbucci, A.
Magnani, F. Tempesti, J. Appl. Polym. Sci. 1993,47, 631–
643.
136 A. Albanese, R. Barbucci, J. Belleville, S. Bowry, R. Eloy, H.
D. Lemke, L. Sabatini, Biomaterials 1994,15, 129–136.
JOURNAL OF
POLYMER SCIENCE WWW.POLYMERCHEMISTRY.ORG HIGHLIGHT
WWW.MATERIALSVIEWS.COM JOURNAL OF POLYMER SCIENCE, PART A: POLYMER CHEMISTRY 2013,51, 2319–2353 2351
137 E. Ranucci, G. Spagnoli, L. Sartore, P. Ferruti, P. Caliceti, O.
Schiavon, F. M. Veronese, Macromol. Chem. Phys. 1994,195,
3469–3479.
138 R. Barbucci, M. Benvenuti, P. Ferruti, G. Casini, F. Tem-
pesti, Biomaterials 1985,6, 102–104.
139 R. Barbucci, G. Casini, P. Ferruti, F. Tempesti, Polymer
1985,26, 1349–1351.
140 P. Ferruti, R. Barbucci, N. Danzo, A. Torrisi, O. Puglisi, S.
Pignataro, P. Spartano, Biomaterials 1982,3, 33–37.
141 P. Ferruti, I. Domini, R. Barbucci, M. C. Beni, E. Dispensa,
S. S. Sancasciani, M. A. Marchisio, M. C. Tanzi, Biomaterials
1983,4, 217–221.
142 E. Ranucci, L. Sartore, E. Tempesti, P. Ferruti, Mater. Eng.
1993,4, 37–52.
143 L. Sartore, M. Penco, F. Bigotti, C. Pedrotti, S. D’Antone,
Macromol. Symp. 2004,218, 221–229.
144 L. Sartore, M. Penco, S. Della Sciucca, A. D’Amore; S.
D’Antone, Macromol. Symp. 2007,247, 162–171.
145 L. Sartore, M. Penco, F. Bignotti, I. Peroni, M. H. Gil, M. A.
Ramos, A. D’Amore, J. Appl. Polym. Sci. 2002,85, 1287–1296.
146 E. Ranucci, F. Bignotti, P. L. Paderno, P. Ferruti Polymer
1995,36, 2989–2994.
147 W. Lin, M. C. Garnett, M. C. Davies, P. Ferruti, S. S. Davis,
L. Illum, Biomaterials 1997,18, 559–565.
148 W. Lin, M. C. Garnett, S. S. Davis, E. Schacht, P. Ferruti, L.
Illum, J. Control. Release 2001,71, 117–126.
149 N. Mauro, A. Manfredi, E. Ranucci, P. Procacci, M. Laus, D.
Antonioli, C. Mantovani, V. Magnaghi, P. Ferruti, Macromol.
Biosci. 2012, DOI: 10.1002/mabi.201200354.
150 V. Magnaghi, V. Conte, P. Procacci, G. Pivato, P. Cortese,
R. Cavalli, G. Pajardi, E. Ranucci, F. Fenili, A. Manfredi, P. Fer-
ruti, J. Biomed. Mater. Res. Part A 2011,98, 19–31.
151 M. A. Marchisio, P. Ferruti, T. Longo, F. Danusso, (Zambon
S.p.A.) U.S. Patent 3,865,723, Oct 18, 1973.
152 P. Ferruti, N. Mauro, A. Manfredi, E. Ranucci, J. Polym. Sci.
Part A: Polym. Chem. 2012,50, 4947–4957.
153 L. N. Monsalve, M. Kaniz Fatema; H. Nonami, R. Erra-Bal-
sells, A. Baldessari, Polymer 2010,51, 2998–3005.
154 L. Hartmann, E. Krause, M. Antonietti, H. G. B€
orner, Bioma-
cromolecules 2006,7, 1239–1244.
155 L. Hartmann, S. Haefele, R. Peschka-Suess, M. Antonietti,
H. G. Boerner, Macromolecules 2007,40, 7771–7776.
156 L. Hartmann, S. Haefele, R. Peschka-Suess, M. Antonietti,
H. G. Boerner, Chem.—Eur. J. 2008,14, 2025–2033.
157 L. E. Prevette, M. L. Lynch, T. M. Reineke, Biomacromole-
cules 2010,11, 326–332.
158 D. Schaffert, N. Badgujar, E. Wagner, Org. Lett. 2011,13,
1586–1589.
159 L. Hartmann, Macromol. Chem. Phys. 2011,212, 8–13.
160 F. Wojcik, S. Mosca, L. Hartmann J. Org. Chem. 2012,77,
4226–4234.
161 A. Katchalsky, P. Spitnik, J. Polym. Sci. 1947,2, 432–446.
162 P. Ferruti, In Polymeric Amines and Ammonium Salts; E. J.
Goethals, Ed.; Pergamon Press: Oxford and New York, 1980;p
305.
163 P. Ferruti, N. Danzo, L. Oliva, R. Barbucci, V. Barone, J.
Chem. Soc. Dalton Trans 1981,1, 539–542.
164 R. Barbucci, M. Casolaro, P. Ferruti, L. Tanzi, M. C. Grassi,
C. Barozzi, Makromol. Chem. 1984,185, 1525–1535.
165 A. E. Martell, R. J. Motekaitis, Determination and Use of
Stability Constants, 2nd ed.; Wiley-VCH: New York, 1992.
166 R. De Levie, Aqueous Acid–Base Equilibria and Titrations;
Oxford University Press: New York, 1999.
167 R. Barbucci, M. Casolaro, S. Corezzi, M. Nocentini, P. Fer-
ruti, Polymer,1985,26, 1353–1358.
168 P. Ferruti, E. Ranucci, S. Bianchi, L. Falciola, P. R. Mussini,
M. Rossi, M. Rossi, J. Polym. Sci. Part A: Polym. Chem. 2006,
44, 2316–2327.
169 P. Ferruti, E. Ranucci, A. Manfredi, N. Mauro, E. Ferrari, R.
Bruni, F. Colombo, P. R. Mussini, M. Rossi, J. Polym. Sci. Part
A: Polym. Chem. 2012,50, 5000–5010.
170 A. Manfredi, E. Ranucci, S. Morandi, P. R. Mussini, P. Fer-
ruti, J. Polym. Sci. Part A: Polym. Chem. 2013,51, 769–773.
171 A. Abbotto, L. Beverina, G. Chirico, A. Facchetti, P. Ferruti,
G. A. Pagani, Synth. Met. 2003,139,629–632.
172 A. Abbotto, L. Beverina, G. Chirico, A. Facchetti, P. Ferruti,
M. Gilberti, G. A. Pagani, Macromol. Rapid Commun. 2003,24,
397–402.
173 A. Abbotto, L. Beverina, A. Facchetti, P. Ferruti, M. Gilberti,
G. A. Pagani, Polym. Prepr. (Am. Chem. Soc., Div. Polym.
Chem.) 2003,44, 1006–1007.
174 S. Proutiere, P. Ferruti, R. Ugo, A. Abbotto, R. Bozio, M.
Cozzuol, C. Dragonetti, E. Emilitri, D. Locatelli, D. Marinotto, G.
Pagani, D. Pedron, D. Roberto, Mater. Sci. Eng. B 2008,147,
293–297.
175 L. Sartore, M. Penco, S. Della Sciucca, G. Borsarini, V. Fer-
rari, Sens. Actuat. B Chem. 2005,111, 160–165.
176 L. Sartore, M. Barbaglio, L. Borgese, E. Bontempi, Sens.
Actuat. B Chem. 2011,155, 538–544.
177 L. Sartore, M. Barbaglio, M. Penco, P. Bergese, E. Bon-
tempi, P. Colombi, L. E. Depero, J. Nanosci. Nanotechnol.
2009,9, 1164–1168.
178 G. Franchi, L. Morasca, I. Reyers, S. Garattini, Eur. J. Can-
cer 1971,7, 533–544.
179 E. Ranucci, P. Ferruti, C. Lenardi, M. Matteoli, (Neurozone
s.r.l.). WO2010099962, Sept. 10, 2010.
180 P. Ferruti, S. Richardson, R. Duncan, In Targeting of Drugs:
Stealth Therapeutic Systems; G. Gregoriadis, B. McCormack,
Eds.; Plenum Press: New York,1998; pp 207–224.
181 C. F. Chu, S. B. Howell, J. Pharmacol. Exp. Ther. 1981,219,
389–393.
182 G. Citro, C. Szczylik, C. Ginobbi, P. Zupi, B. Calabretta, Br.
J. Cancer 1994,69, 463–467.
183 N. Malik, R. Wiwattanapatapee, R. Klopsch, K. Lorenz, H.
Frey, J. W. Weener, E. W. Meijer, W. Paulus, R. Duncan, J. Con-
trol. Release 2000,65, 133–148.
184 D. Sgouras, R. Duncan, J. Mater. Sci. Mater. Med. 1990,1,
61–68.
185 H. Maeda, In Polymeric Site Specific Pharmacotherapy; A.
J. Domb, Ed.; Wiley: New York, 1994.
186 H. Maeda, M. Ueda, T. Morinaga, T. Matsumotog, J. Med.
Chem. 1985,28, 455–461.
187 Y. Matsumura, H. Maeda, Cancer Res. 1986,46, 6387–6392.
188 L. W. Seymour, K. Ulbrich, P. S. Styger, M. Brereton, V.
Subr, J. Strohalm, R. Duncan, Br. J. Cancer 1994,70, 636–641.
189 R. Duncan, Pharm. Sci. Technol. Today 1999,2, 441–449.
190 E. H. Schacht, P. Ferruti, R. Duncan (European Community,
Luxembourg). WO 9,505,200, February 23, 1995.
191 E. Gianasi, M. Wasil, E. G. Evagorou, A. Keddle, G. Wilson,
R. Duncan, Eur. J. Cancer 1999,35, 994–1002.
192 Y. Ohya, T. Masunaga, T. Baba, T. Ouchi, J. Macromol. Sci.
A Pure Appl. Chem. 1996,33, 1005–1016.
HIGHLIGHT WWW.POLYMERCHEMISTRY.ORG
JOURNAL OF
POLYMER SCIENCE
2352 JOURNAL OF POLYMER SCIENCE, PART A: POLYMER CHEMISTRY 2013,51, 2319–2353
193 Y. S. Sohn, H. Baek, Y. H. Cho, Y. Lee, O. Jung, C. O. Lee,
Y. S. Kim, Int. J. Pharm. 1997,153, 79–91.
194 R. Heilbronn, S. Weger, Handb. Exp. Pharmacol. 2010,197,
143–170.
195 D. C. Gorecki, Emerg. Drugs 1999,4, 247–261.
196 O. Boussif, F. Lezoualch, M. A. Zanta, M. D. Mergny, D.
Scherman, B. Demeneix, J.-P. Behr, Proc. Natl. Acad. Sci. USA
1995,92, 7297–7301.
197 A. Nevo, A. De Vries, A. Katchalsky, Biochim. Biophys.
Acta 1955,17, 536–547.
198 A. Katchalsky, D. Dannon, A. Nevo, A. De Vries, Biochim.
Biophys. Acta 1959,33, 120–138.
199 J. Suh, H. Paik, B. K. Hwang, Bioorg. Chem. 1994,22, 318–
327.
200 W. Hartmann, H. J. Galla, Biochim. Biophys. Acta 1978,
509, 474–490.
201 Y. Endo, K. Mitsui, M. Motizuki, K. Tsurugi, J. Biol. Chem.
1987,262, 5908–5912.
202 M. Lord, L. Roberts, J. Robertus, FASEB J. 1994,8, 201–208.
203 F. Stirpe, S. Olsnes, A. Pihl, J. Biol. Chem. 1980,14, 6947–
6953.
204 P. C. Griffiths, Z. Khayat, S. Tse, R. K. Heenan, S. M. King,
R. Duncan, Biomacromolecules 2007,8, 1004–1012.
205 R. Duncan, P. Ferruti, N. Pattrick, Proc. Int. Symp. Control.
Release Bioact. Mater. 2001,28, 864–865.
206 P. Kinam, J. Control. Release 2010,142, 1–1. “COVER
STORY”.
207 M. W. Pettit, P. Griffiths, P. Ferruti, S. C. W. Richardson,
Ther. Deliv. 2011,2, 907–917.
208 I. R. C. Hill, M. C. Garnett, F. Bignotti, S. S. Davis, Anal.
Biochem.,2001,291, 62–68.
209 B. J. Rackstraw, S. Stolnik, S. S. Davis, F. Bignotti, M. C.
Garnett, Biochim. Biophys. Acta,2002,1576, 269–286.
210 S. M. Parkhouse, M. C. Garnett, W. C. Chan, Bioorgan.
Med. Chem.,2008,16, 6641–6650.
211 L. Min, B. C. Liu, X. Yanan, H. Jie, Z. Liming, S. Huang, Q.
Li, Z. Zhijun, Bioconjug. Chem. 2011,22, 2237–2243.
212 Y.-N. Xu, L. Liu, X. Ji, S.-W. Huang, R.-X. Zhuo, J. Control.
Release 2011,152, e177–e179.
213 F. Martello, J. F. J. Engbersen, P. Ferruti, J. Control.
Release 2010,132, e10–e12.
214 F. Martello, J. F. J. Engbersen, P. Ferruti, J. Control.
Release 2012,164, 372–379.
215 C. Lin, J. F. J. Engbersen, Mater. Sci. Eng. C 2011,31,
1330–1337.
216 J. F. J. Engbersen, M. Piest (Universiteit Twente, Nether-
lands). PCT Int. Appl WO 2011159161, Dec. 22, 2011.
217 D. Vercauteren, M. Piest, L. J. van der Aa, M. Al Soraj, A.
T. Jones, J. F. J. Engbersen, S. C. De Smedt, K. Braeckmans,
Biomaterials 2011,32, 3072–3084.
218 N. Peng, Y.-N. Xue, X.-M. Xia, S.-W. Huang, R.-X. Zhuo, J.
Control. Release 2011,152 (Suppl. 1), e166–e167.
219 P. Vader, L. J. van der Aa, J. F. J. Engbersen, G. Storm, R.
M. Schiffelers, Pharmaceut. Res.,2011,28, 1013–1022.
220 M. C. Garnett, P. Ferruti, E. Ranucci, M. Suardi, M. Heyde,
R. Sleat, Biochem. Soc. Trans,2009,37, 713–716.
221 C. N. Pace, D. V. Laurents, J. A. Thomson, Biochemistry
1990,29, 2564–2572.
222 F. A. Marston, D. L. Hartley, Method. Enzymol. 1990,182,
264–276.
223 G. Cou
e, J. F. J. Engbersen, J. Control. Release 2010,148,
e9–e10.
224 S. Cohen, G. Cou
e, D. Beno, R. Korenstein, J. F. J. Eng-
bersen, Biomaterials 2012,33, 614–623.
225 M. A. Marchisio, T. Longo, P. Ferruti, Experientia 1973,29,
93–95.
226 M. A. Marchisio, P. Ferruti, T. Longo, Eur. Surg. Res. 1972,
4, 312–313.
227 M. A. Marchisio, P. Ferruti, S. Bertoli, G. Barbiano di Bel-
giojoso, C. M. Samour, K. D. Wolter, Prog. Biomed. Eng. 1988,
5, 111–120.
228 M. A. Marchisio, P. Ferruti, S. Bertoli, G. Barbiano di Bel-
giojoso, C. M. Samour, K. D. Wolter, In Polymers in Medicine
II; C. Migliaresi, E. Chiellini, Eds.; Elsevier Science Publishers
B.V.: Amsterdam, 1988; pp 111–118.
229 P. Ferruti, G. Casini, F. Tempesti, R. Barbucci, R. Mastacchi,
M. Sarret, Biomaterials 1984,5, 234–236.
230 R. Barbucci, M. Benvenuti, G. Dal Maso, P. Ferruti, M.
Nocentini, R. Russo, F. Tempesti, R. Duncan, J. F. Bridges, L. A.
McCormick, In Polymers in Medicine; C. Migliaresi, E. Chiellini,
Eds.; Elsevier Science Publishers: Amsterdam, 1988; pp. 3–18.
231 W. Marconi, A. Martinelli, A. Piozzi, D. Zane, Biomaterials
1992,13, 432–438.
232 M. K. Nguyen, D. S. Lee, Macromol. Res. 2010,18, 284–
288.
233 M. K. Nguyen, D. K. Park, D. S. Lee, Biomacromolecules
2009,10, 728–731.
234 R. Annunziata, J. Franchini, E. Ranucci, P. Ferruti, Magn.
Reson. Chem. 2007,45, 51–58.
235 L. Calucci, C. Forte; E. Ranucci, Biomacromolecules,2007,
8, 2936–2942.
236 L. Calucci, C. Forte; E. Ranucci, J. Chem. Phys. 2008,129,
064511. http://dx.doi.org/10.1063/1.2968606.
237 L. Calucci, C. Forte; E. Ranucci, Langmuir 2009,25, 2449–
2455.
238 P. Ferruti, S. Bianchi, E. Ranucci; F. Chiellini, V. Caruso,
Macromol. Biosci. 2005,5, 613–622.
239 P. Ferruti, S. Bianchi, Ranucci E.; Chiellini F.; Piras, A. M.
Biomacromolecules 2005,6, 2229–2235.
240 E. Emilitri, F. Guizzardi, C. Lenardi, M. Suardi, E. Ranucci,
P. Ferruti, Macromol. Symp. 2008,266, 41–47.
241 E. Jacchetti, E. Emilitri, S. Rodighiero, M. Indrieri, A. Gian-
felice, C. Lenardi, A. Podest
a, E. Ranucci, P. Ferruti, P. Milani,
J. Nanobiotechnol. 2008,6, 14. doi:10.1186/1477-3155-6-14.
242 G. Dos Reis,; F. Fenili, A. Gianfelice, G. Bongiorno, D. Mar-
chesi, P. E. Scopelliti, A. Borgonovo, A. Podest
a, M. Indrieri, E.
Ranucci, P. Ferruti, C. Lenardi, P. Milani, Macromol. Biosci.
2010,10, 842–852.
243 M. Casali, S. Riva, P. Ferruti, J. Bioact. Compat. Polym.
2001,16, 479–491.
JOURNAL OF
POLYMER SCIENCE WWW.POLYMERCHEMISTRY.ORG HIGHLIGHT
WWW.MATERIALSVIEWS.COM JOURNAL OF POLYMER SCIENCE, PART A: POLYMER CHEMISTRY 2013,51, 2319–2353 2353
