AbstractPolyamidoamines (PAAs) are multifunctional polymers prepared from prim- or sec-amines by Michael-type polyaddition with bisacrylamides. The reaction is preferably carried out at room temperature in water and without added catalysts or organic solvents. The reaction is specific. The presence in the starting monomers of additional functions, leaving apart amine, thiol and phosphine groups, does not interfere with the polymerization process. Consequently, PAAs are a polymer class endowed with unusual structural versatility. Moreover, at pH > 7 they are hydrolytically degradable in aqueous media to harmless products mostly consisting of β-amino-propionic acids. Many PAAs are remarkably biocompatible notwith-standing their polycationic nature. These properties allow to prepare multifunctional polymeric structures suitable for many diversified applications in biotechnology, such as drug carriers, transfection promoters, antiviral and antimalarial agents, hydrogels scaffolds for tissue engineering. The objective of this paper is to fully report the PAA chemistry and the studied biotechnological applications of a vast PAA library.
The polymer family obtained by the Michael-type poly-addition of prim- or bis-sec-amines with bisacrylamides (Scheme 1) was first reported in 1967 and called “poly-amidoamines” [1]. This name was subsequently commonly abbreviated as PAAs.
Further work demonstrated that this type of polymeriza-tion is a general one and that, as a rule, bisacrylamides and prim- or bis-sec-amines give rise to linear PAAs, whereas multifunctional prim- or sec-amines give rise to crosslinked PAAs (Sect. 2.5). The publications on different PAAs and PAA analogues were collectively reviewed from time to time [2–5]. Later on, the synthesis of dendrimeric polymers containing amide and amine groups was reported [6, 7]. These polymers were named PAMAMs and subsequently extensively studied [8]. The classical synthesis of PAMAM dendrimers is mostly carried out by alternating two reac-tions: the Michael addition of the amine-terminated surface
with methyl acrylate, resulting in an ester-terminated outer layer, followed by coupling with ethylene diamine to achieve a new amine-terminated surface. After each step, extensive purification procedures is required. PAAs and PAMAMs have a substantial difference in the general structure of the repeat unit, since the sequence of amide and amine groups is, in the case of PAAs, -A-A-B-A-A-B- or -A-A-B-B-A-A-B-B-, depending on the nature (primary or bis-secondary) of the amine monomers, and in the case of PAMAMs -A-B-A-B-. In addition, PAAs and PAMAMs are differentiated by molecular architecture. PAMAMs have the globular shape typical of dendrimers with internal branching points whose number grows exponentially from generation to generation. They are also characterized by low polydispersity, precise structure and size control, and by a large number of surface sites relative to the total molecular volume.
PAAs are polydisperse step-wise polyaddition polymers whose polydispersity can be narrowed only by fractionation. The hallmark of PAAs is ease of synthesis, chemical versa-tility and the fact that polymerization reactions are normally carried out in water in one step, one pot processes. PAAs can be designed to have either linear or grafted or hyper-branched architectures. PAA prepolymers can give rise to block and graft copolymers as well as crosslinked hydrogels.
The innumerable chemical functions that can be inserted in the structure of PAAs allow them to assume important functions for biotechnological applications, such as cellular penetration or intrinsic bioactivity, for example as antima-larials, antivirals or antibacterial agents. This review is lim-ited to linear and crosslinked PAAs synthesized according to Scheme 1.
PAAs can easily be designed to be water-soluble and bio-compatible. Moreover, they are hydrolytically degraded in aqueous systems at a pH-dependent rate.
Besides biomedicine and biotechnology, PAAs have been studied for potential applications in different technical fields, including heavy metal ion adsorbing resins for water puri-fication [9–13] and as heterogeneous catalysts [14], high-performance nonlinear optical dyes [15, 16], coating for sensing applications [17–19] and flame retardant agents for cotton textiles [20, 21]. In this review, the potential of PAAs in biotechnology will be discussed.
PAAs are characterized by significant features making them suitable for countless biotechnological applications. Most remarkable is their structural versatility. Monomers bearing a variety of functional groups that do not interfere with the Michael reaction, such as hydroxy-, carboxy-, sul-phonic-, tert-amine- and allyl functions, can be employed in PAA synthesis. Another relevant feature of PAAs is that they are polyelectrolytes with tunable ionic species distri-butions. PAAs in which the only ionizable functions are the tert-amine groups placed in the polymer chain are fully cationic. The presence of carbonyl groups β to the amine groups lowers both basic strength and toxicity. Indeed, sev-eral amphoteric PAAs are almost biocompatible as dextran, in the absence of basic- or highly hydrophobic side substitu-ents. PAAs carrying different ionizable functions may show complex, pH dependent charge distributions. The possible combinations of chemical functions in PAA repeating units, coupled with their intrinsic polyelectolytic nature, can be planned to render them in some respects mimics of natural biomacromolecules such as peptides, sharing with them a number of chemical and physico-chemical properties.
A further relevant feature of PAAs is that they are nor-mally synthesized at room temperature in water through one-pot processes in the absence of added catalysts and organic
solvents. Thanks to these unique features, the PAA synthesis is “green”, safe and easily scalable.
The aim of this review is to illustrate the main biotech-nological applications of PAAs for which they were con-sidered worthy of attention. These include cell-penetrating polymers with potential as transfection promoters, polymer drug carriers, nanoparticles, nanovectors, intrinsically bioac-tive polymers, as for instance antiviral and antimethastatic polymers, promoters of intracellular trafficking of proteins and scaffolds for tissue engineering.
2 Chemistry of Polyamidoamines
2.1 Synthetic Features
The polymerization reaction is a typical two-component step-wise polyaddition between difunctional complementary functions, normally proceeding without side reactions, as demonstrated by the strict dependence of the polymerization degree on the monomer ratio (Fig. 1) [1].
The presence of chain end groups besides those expected from the monomer ratio, as well as of cyclic structure, was excluded by NMR analysis [22]. Proton transfer is the rate-determining step in the Michael addition reaction, which proceeds faster in protic solvents. Not surprisingly, the poly-addition reaction leading to PAAs is best performed, both in terms of attainable molecular weight and reaction rate, in water or, alternatively, alcohols whereas aprotic solvents are normally unsuitable [23]. The kinetics of the polymerization reaction of 2-methylpiperazine and 2,5-dimethylpiperazine with N,N′-bisacryloylpiperazine in water, methanol, ethyl-ene glycol, formamide and dimethylformamide [23] dem-onstrated that in the protic solvents, 2-methylpiperazine polyaddition proceeded through a two-step mechanism, each of them involving one of the two sec-amine groups. The reaction rate constants related to these steps significantly differed because of the different steric hindrance induced by the neighboring groups. Each step followed a pseudo-second-order reaction rate, whose rate constants included the concentration of the protic species acting as catalysts. In dimethylformamide, the reaction rate followed third-order
Scheme 1 Synthesis of linear PAAs
N R1N
O O
+
R2 R2
NR3
HHN R1
N
O O
R2 R2NR3
n
N R1N
O O
+R2 R2
H N R4N H
R3 R3
N R1N
O O
R2 R2nN R4
NR3 R3
∗∗
∗∗
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kinetics, due to the self-catalysis by the amine groups that represented the only source of mobile hydrogens.
Alkaline-earth metal ions, as for instance calcium, act in water as catalysts of the Michael reaction of amines with bisacrylamides [24].
The effect of temperature is to increase the reaction rate. However, its effect on the molecular weight is dual. In aprotic solvents, such as pyridine, it allows obtaining
relatively high polymerization degrees in reasonable time (Fig. 2a) [1, 2]. At room temperature, the polymerization proceeds lethargically (Fig. 2a). On the opposite, in water, at high temperatures after an initial fast increase, the molecular weight first levels off and then decreases (Fig. 2b). This was ascribed to the competition with the concurrent hydrolytic degradation, which at high temperatures eventually prevails [1, 2].
The Michael polyaddition is highly specific and allows obtaining, with the appropriate functional monomers, PAAs bearing many functional lateral substituents, includ-ing hydroxy, tert-amine, allyl, amide, carboxyl, and ether groups. Monomers bearing extra functional groups capable of interfering with the Michael reaction, such as thiols, prim- and sec-amines and phosphines, cannot be directly used in PAA synthesis unless protected with cleavable groups, such as for instance the tert-butyloxycarbonyl group (Scheme 2) [25]. Monoprotonated prim-diamines with largely different basicity constants, as for instance monoprotonated ethylen-ediamine, yield soluble PAAs with prim-aminoethyl pen-dants [26] (Table 1).
2.2 Amphoteric PAAs
PAAs carrying acidic functions as side substituents (Table 2), as for instance carboxyl-, phosphonic- [29] and sulphonic groups [27] are amphoteric. Amphoteric PAAs are easily obtained by employing amino acids or carboxylated bis-acrylamides as monomers, provided the carboxyl group is neutralized with the addition of a base, either sodium-, potassium- or lithium hydroxide, as well as triethylamine.
Fig. 1 Dependence of the intrinsic viscosity, measured in chloroform at 30 °C, of the polymerization product of N,N’-bisacryloylpiperazine and 2-methylpiperazine on the monomer molar ratio
Fig. 2 Trend of the intrinsic viscosity measured in chloroform at 30 °C of the N,N’-bisacryloylpiperazine/2-methylpiperazine mixture in pyri-dine and water at different polymerization temperatures
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Due to steric hindrance, α-aminoacids other than glycine react slowly [30]. Amphoteric PAAs with different isoelec-tric points can be synthesized by adjusting the number and strength of the acid and basic groups in the starting mono-mer mixtures (Table 3) [27].
The biological activity of polyelectrolytes is related to their pH-dependent ionic species distribution (Sect. 2.7). Amphoteric PAAs with, on average, excess positive charge per repeat units at pH 7.4 are normally considerably less cytotoxic than cationic PAAs of similar charge [27]. In addition, they maintain many relevant properties typical of polycations, such as the ability to form interpolyelectrolyte complexes with negatively charged biomacromolecules, including heparin and DNA, to exert membrane activity and to act as transfection promoters (Sect. 3.5). Amphoteric PAAs that are prevailingly negatively charged at pH 7.4 are not normally membrane active but, once cell-internalized, become so after reaching acidic subcellular compartments with pH below their isoelectric point, such as endosomes (pH 5.6–6) and lysosomes (pH 5–5.5) [28].
2.3 PAAs Bearing Disulfide Functions
Polymers bearing disulfide groups either in the main chain or as side substituents are endowed with a wide potential for biological applications, because are amenable to reductive cleavage in biological environments and can participate in thiol-disulphide exchange reactions [33]. For instance, after oral administration these polymers pass unaltered through the upper gastrointestinal tract, but their disulphide groups are reductively cleaved in the colon. If the disulfide group is located in the main chain, the polymers degrade. If they are in the side substituents, they release on cleavage any attached moieties.
The reaction of 2-methylpiperazine with N,N’-bisacry-loylcystamine or N,N’-bisacryloylcystine (Scheme 3a), in turn obtained from acryloyl chloride and the corresponding amines [34], gave PAAs bearing in the backbone disulfide groups that were reductively cleaved by 2-mercaptoethanol. N,N’-bisacryloylcystamine-based PAAs were proposed as carriers of nucleic acid, of protein and as transfection pro-moters [35]. l-cystine [36] and N,N’-dimethylcystamine [37] were also used as difunctional diamine monomers, capitaliz-ing on the different reactivity of the N–H hydrogens on NH2 functions (Scheme 3b). PAAs with both disulfide and acetal acid-labile groups in the backbone were described [38].
PAAs bearing disulphide bonds in the main chain PAA are particularly interesting for their ability to give bioreduc-ible polyplexes with DNA and siRNA. These polyplexes are stable in extracellular fluids, with glutathione concentrations 0.001–0.01 mM but sensitive to reductive degradation in the intracellular environment, with glutathione concentrations 1–10 mM (Sect. 3.5).
PAA bearing lateral activated dithio-derivatives were pre-pared in slightly alkaline media by dithio–dithio exchange reaction between crosslinked PAAs previously obtained using cystamine as crosslinking agent and excess 2,2′-dithi-odipyridine (Scheme 4).
These products were amenable to coupling reactions with thiolated peptides, such as for instance glutathione [39], and thiocholesterol [40]. In the latter instance, amphiphilic poly-mers were obtained giving spontaneously in aqueous media nano-aggregates that collapsed in the presence of reducing agents (Fig. 3).
ISA23-cholesterol nanoparticles were succesfully used to formulate the lipophilic anticancer drug tamoxifen [41]. Electrospray allowed obtaining nanoparticles with homo-geneous size distribution (Fig. 4) that could be loaded with
Scheme 2 Synthesis of an AGMA1 copolymer bearing prim-amine pendants
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Table 1 Structure of cationic PAAs
Code a) Structure of the repeat unit Reference
PAA1
[17]
PAA2
[17]
PAA3
[17]
PAA4
[17]
PAA5 [17]
BP-MP [27]
BP-DM [27]
BP-AG [27]
BP-ED [27]
ISA1 [28]
a) As in the original article
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Table 2 Structure of amphoteric PAAs
Codea) Structure of the repeat unit Reference
M-GLY
[20]
AGMA1
[25]
BAC-TA
[27]
BAC-AL
[27]
BAC-EDD
[27]
ARGO7
[30]
ISA23
[28]
M-L-Ala
[31]
M-L-Val [31]
M-L-Leu [31]
DMEDA-BAC
[32]
DEEDA-BAC [32]
DMEPDA-BAC
[32]
DMEXA-BAC
[32]
a) As in the original article.
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40% drug. Tamoxifen-loaded nanoparticles were easily internalized in African green monkey kidney Vero cells and showed higher cytotoxicity than tamoxifen against MCF-7 human breast adenocarcinoma cells. At the same concentra-tions, blank nanoparticles were negligibly cytotoxic.
Polymers bearing thiol groups are known to be mucoad-hesive [42] and find applications in nanomedicine [43]. An ISA23 (Table 2) copolymers bearing thiol pendants, ISA23-SH, was synthesized from N-mono-protected cystamine, subsequently cleaving the disulfide groups introduced as polymer pendants (Scheme 5) [44].
2.4 PAA Block and Graft Copolymers
Polyaddition reactions carried out with stoichiometrically unbalanced amine- or bisacrylamide mixtures lead PAAs
terminated at both chain ends by the excess function. If the reaction is carried out to completion, the number-average polymerization degree X̄n of the resultant PAAs can be planned according to the well-know Carothers equation (Eq. 1) [45]
where r is the ratio of the number of defect functions to the number of excess functions. Both amine- and acrylamide-terminated PAA oligomers are macromonomers that can be used as building blocks in the synthesis of block- and graft PAA copolymers and, limited to the bisacrylamide-terminated PAAs, with vinyl polymers obtained by radical polymerization, for instance polystyrene [46].
Linear polyurethane-polyamidoamine (PUPA) block copolymers were obtained [47]. Films cast from these copolymers contained a relatively small PAA content (6 and 15 wt%) nevertheless proved able to complex up to 1.6 pg cm−2 heparin. PUPA samples with a crosslinked architecture were prepared by grafting isocyanate-termi-nated PAA macromonomers, in turn obtained by treating amine-terminated PAAs with excess diisocyanate, onto commercial polyurethanes through allophanate group formation [48–56]. Tough coatings could be obtained by casting the reaction mixtures onto polyurethane, polyvi-nyl chloride and glass [56]. PUPA based materials proved able to adsorb significant heparin amounts, remarkably
(1)X̄n =1 + r
1 − r
Table 3 Acid-base properties of some amphoteric PAAs
Data from reference no. [27]a As in the original articleb Isoelectric point
Scheme 3 Synthesis of PAAs bearing disulfide groups in the main chain
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improving their hemocompatibility [56], thanks to the formation of polyelectrolyte complexes with the nega-tively charged heparin and the positively charged PAA
segments [56]. Similarly, different amine-terminated PAA macromonomers were grafted onto polyetherurethaneam-ide surfaces functionalized with fumaric or maleic acid through the Michael type addition [57].
Block- and graft-PAA copolymers were also obtained by copolymerization with amine-terminated oligomers, such as α,ω-bis(sec-amino)polyoxyethylenes (Scheme 6a) [58, 59], α-methyl-ω-amino-polyoxyethylenes (Scheme 6b) [60], or ω-amine-terminated poly-4-acryloylmorpholine (Scheme 6c) [61], in turn prepared by radical polymeriza-tion of 4-acryloylmorpholine in the presence of cysteamine acting a chain transfer agent [62]. Finally, the PAA/albumin
Scheme 4 Synthesis of PAAs bearing disulfide pendants
NH
NH
O O
NHNHH2N S
SNH2+ +
NH
NH
OO
NmN N N
HNH
OO
n
SS
HN
HN
O O
N NNHN
HN
O O∗∗
∗∗
nm
N S S N
O OH
O OH O OH
O OH O OH
NN N
SS
N
n m
∗ ∗
OO
NH
NH
OHOOO
NH
NH
OHO
Fig. 3 Reductive cleavage of PAA-cholesterol particles revealed by size decrease, with increasing concentration of reduced glutathione: 0.30, 0.41, 0.46, and 0.50% (w/v) at pH 7.4 and 30 °C. Measurements were taken after 30 min incubation
Fig. 4 SEM image of blank ISA23-cholesterol nanoparticles. Scale bar: 200 nm
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graft copolymers were obtained from N,N’-bisacryloylpiper-azine and piperazine under non-denaturing conditions [63].
2.5 Crosslinked PAAs
Crosslinked PAAs, unless for too high crosslinking degrees, give in water soft highly swollen hydrogels succesfully tested as substrates for cell culturing and tissue engineering (Sect. 4.5).
Crosslinked PAAs were obtained by partially substituting multifunctional amines for sec-bisamines or prim-monoam-ines in the polymerizing mixture, while mantaining equi-molarity between the complementary functions (Scheme 7) [2–4].
However, soluble PAA drug carriers bearing sec- in place of tert-amine groups in the macromolecular back-bone could be synthesized from multifunctional amines in dilute solutions and excess bis-amines in the cold [64]. The sec-amines introduced in the main backbone allowed complexation with heavy metal ions of therapeutic interest [65]. Crosslinked PAAs synthesized using multifunctional amines as crosslinking agensts are normally soft and frag-ile hydrogels, although in some instances PAAs endowed with structure-forming properties gave sufficiently tough swollen hydrogels when crosslinked by this procedure [66]. Crosslinked PAAs were also obtained by radical polymeri-zation of vinyl-end-capped PAA oligomers, through either UV irradiation, or using thermal- or redox initiators [67].
The crosslinking degree of products mostly depended on the molecular weight of the oligomeric precursor (Scheme 8). Mixed crosslinked networks were obtained by copolymeriz-ing vinyl-end-capped PAA oligomers with vinyl monomers, as for instance N-vinylpyrrolidone [68]. Hydrogels obtained by this procedure retain the acid–base properties of their linear precursors, moderately swell in water and are nor-mally tougher than hydrogels obtained from multifunctinal amines. It should be observed that, regardless of the prepa-ration method, PAA hydrogels were toughened by different methods, either using inorganic fillers [69] or biodegradable electrospun non-woven mats (Sect. 4.5) [70].
2.6 Hydrolytic Degradation of PAAs
PAAs degrade hydrolytically at a pH-, concentration- and temperature-dependent rate. The PAA amide groups appear more prone to hydrolytic degradation than normal amides, due to the presence of tert-amines β to the amide functions. The ultimate degradation products are small molecules with γ-aminopropionic structure (Scheme 9).
The hydrolytic degradation of PAAs was studied by monitoring the molecular weight decrease in dilute aqueos solutions at pH 7.4 and 37 °C by size exclusion chroma-tography (Fig. 5) and/or by meauring the intrinsic vis-cosity. The degradation rate depends to some extent on the structure of the bisacrylamide moiety. PAAs deriv-ing by the polyaddition of 2-methylpiperazine with
water, LiOH
CaCl2 cat. 10 mol%
NH
NH
O O+
NHN
HN
OO
H2NS
SNH2
+
SS
.2 HCl
HN
HN
OO
HOSH
O OH
OHO OHO
excess
NH
HN
HN
HN
OO
∗
OHO
N N NN NHN
HN
OO
SS
∗
OHO
NHN
HN
OO
HN
HN
OOOHO OHO
HN
HN
OO
∗
OHO
N N NN NHN
HN
OO∗
OHO
NHN
HN
OO
SH
HN
HN
OOOHO OHO
HN
HN
OO
∗
OHO
N N NN NHN
HN
OO
SH
∗
OHO
Scheme 5 Synthesis of ISA23-SH
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Scheme 6 Synthesis of block- and graft-PAA copolymers
(a)
(b)
(c)
Scheme 7 Synthesis of crosslinked PAAs from multi-functional amines
NR1
N
O O
R2 R2
N
R5
*N
R1N
O O
R2 R2
* NR3
N
R4 R4
NR1
N
O O
R2 R2
HN
R3N
H
R4 R4+ H2N
R5NH2+
NR1
N
O O
R2 R2
N*
NR1 N
O O
R2 R2
* NR3
N
R4 R4
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different bisacrylamides exhibited degradation rates in the order N,N′-bisacryloylpiperazine > methylenebisacryla-mide > 2,2-bisacrylamidoacetic acid based PAAs [71]. Also crosslinked PAA are subject to hydrolytic degrada-tion. Hydrogels completely eroded within weeks in Dul-becco medium at pH 7.4 and 37 °C [72]. When tested, PAAs proved insensitive to lysosomal enzyme degradation at pH 5.5 [73].
2.7 PAAs as Polyelectrolytes
PAAs are polyelectrolytes, thanks to the ionizable tert-amine groups in the main chain, and occasionally other ionizable functions as side substituents. The PAA solution proper-ties, their ability to interact with biomolecules, biological structures and cells and their cytotoxicity depend on their ionization state. Understanding the polyelectrolyte behavior of PAAs is therefore of paramount relevance to interpret PAA solution behavior. The protonation constants of sev-eral PAAs have been obtained by several techniques, such as carbon nuclear magnetic resonance (13CNMR), solution calorimetry and potentiometry [27, 74, 75].
The generalized Henderson–Hasselbalch equation have been used to describe the acid–base dissociation equilibria of polyelectrolytes (Eq. 2)
where K is the weak acid dissociation constant; a is the dis-sociation degree of the weak acid and β is the Katchalsky and Sputnik parameter [76] that takes into account possible ionic interactions between neighboring groups. The effective protonation constants of polyelectrolytes normally depend on the protonation degree of the macromolecule and only apparent constants are determined. However, in the amine
(2)pH = pK − �log1 − �
�
group protonation of many cationic PAAs, β approaches 1 and “real” basicity constants were determined [27, 75]. The pKas of a library of PAAs were assessed [27] following dis-tinct approaches [77, 78]. The tert-amine group present in PAA polymer chain backbone, β to one or two amide groups, had pKa values in the 7.25–8.25 range [27]. The pKa values of the second tert-amine group on PAAs deriving from sec-diamines are usually in the range 3.25–7.5. The β parameters associated to both pKas normally ranged from 1 to 1.1, only exceptionally rising to 1.2–1.3. The peculiar behavior of PAAs as polyelectrolytes was ascribed to a combination of factors, namely the distance between tert-amines placed in adjacent repeat units and the charge-sheltering effect of the two amide groups interposed between them. This hypothesis is in line with the observation that poly(1,4-piperazinediyl-1-oxo-trimethylene), [79] where a single amide group is linked to a single piperazine ring, is a typical polyelectro-lyte since interactions between neighbouring units are still present (Fig. 6):
However, amphoteric PAAs exhibited a typical polyelec-trolyte when their carboxyl groups were located far from the chain backbone [31, 80]. The speciation diagrams of many PAAs have been determined (see for instance Fig. 7).
2.8 Chiral PAAs
It is generally recognized that chirality governs biomate-rial-cell interaction and chiral polymers may have chirality-induced selectivity towards organic structures [81]. The polyaddition of N,N′-methylenebisacrylamide with amino acids bearing hydrophilic and hydrophobic residues, namely D,L-, D-, and l-arginine (D,L-, D-, L-ARGO7, Table 2) [30, 31]; l-alanine (M-L-Ala), l-valine (M-L-Val) and l-Leucine (M-L-Leu) [31] gave chiral polyamidoamino acids (PAACs) characterized by circular dichroism spectra of similar shape.
N R1 N
O O
R2 R2
NR3
N R1 N
O O
+
R2 R2
NR3
HHN R1 N
O O
R2 R2
NR3
n N R1 N
O O
R2 R2
N R1 N
O O
R2 R2
NR3
N R1 N
O O
R2 R2
NR3
n NR1 N
O O
R2 R2
N R1N
O O
R2 R2
NR3
N R1N
O O
R2 R2
NR3
nN
R1N
O O
R2 R2
radical initiation
∗
∗
∗
∗
Scheme 8 Synthesis of crosslinked PAAs by radically polymerizing vinyl-end-capped PAA oligomers
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The molar ellipticities of these chiral polymers promptly shifted along the wavelength axis and changed intensity with pH but showed little dependence on temperature, ionic strength and denaturating agents. All spectral changes were completely reversible (Fig. 8), suggesting the establishment of dynamic dissimmetric conformations. Dynamic light scat-tering analysis showed that all PAACs structured in tiny nan-oparticles (about 1–2 nm) that turned to be stable at different ionic strength for prolonged time periods. Molecular dynam-ics indicated that all chiral PAACs considered assumed com-pact but rapidly pH-responsive folded structures stabilized by intramolecular interactions. These structures were charac-terized by transoid arrangements of the main chain reminis-cent of the protein hairpin motif (Fig. 9). Morphologies and dipole moments significantly and reversibly changed with
pH. All structural features were correlated to the ionization state of macromolecules.
3 PAAs as Nanocarriers of Bioactive Compounds
3.1 Biocompatibility and Biodistribution Studies of PAAs
The cytotoxicity of linear [25, 27, 28, 30, 32, 44, 73, 82] and crosslinked [69, 70, 72, 83–85] PAAs has been exten-sively investigated. The hallmark of PAA is their reduced cytotoxicity despite being cationic. Synthetic polycations, including poly-l-lysine (PLL) and polyethyleneimine (PEI),
Scheme 9 Hydrolytic degradation of PAAs
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are generally cytotoxic with dose- and molecular weight dependence [86]. Their cytotoxicity has been ascribed to the destabilization of the cell membrane followed by disruption of the mitochondrial functions that causes apoptosis [87].
Purely cationic PAAs, as the reaction products of N,N’-bisacryloylpiperazine with piperazine and N,N′-dihydroxyethylamine, respectively, proved much less cyto-toxic than poly-l-lysine towards human hepatocellular carcinoma (HepG2) and human lymphoblastoid leukaemic (CCRF) cells [73]. PAA growth inhibition never fell below 50% up to 2 mg mL−1 concentration, wherease that of poly-l-lysine were 50 and 10 µg mL−1, respectively. The PAA deriving from the polyaddition of methylenebisacrylamide with N,N-dimethylethylenediamine underwent LDH assay to assess the integrity of the plasma membrane following cell treatment [88]. The LDH release in response to the above cited PAA was negligible, at least up to the concentration of 1 mg mL−1, whereas PEI had half maximal effective concen-tration (EC50), i.e., the concentration that caused 50% LDH release, as low as 8.7 μg mL−1.
The scarce cytotoxicity of PAAs compared to conven-tional polycations has been ascribed to their less pronounced cationic character, that is, to the lower positive charge den-sity per repeating unit. This hypothesis was corroborated by the observation that when the lateral NH2 groups of poly-l-lysine were modified by Michael addition with N,N-dimethylacrylamide, reducing the basic strength, the poly-mer toxicity was significantly lowered (Table 4) [89].
Amphoteric PAAs carrying a carboxyl group per unit proved the least toxic [27, 32]. The fact that PAAs deriving from 2,2-bisacrylamidoacetic acid and 2-methylpiperazine, N,N-dimethylethylenediamine and N,N-diethylethylenedi-amine, and from α- and β-aminoacids were two orders of magnitude less cytotoxic than conventional polycations [27, 30, 32] confirms this statement (Table 5).
The amphoteric AGMA1 (Table 2) differs from most other PAAs because, despite having isoelectric point > 10
Fig. 5 Hydrolytic degradation of ARGO7 at pH 7.4 monitored by size exclusion chromatrography
Fig. 6 Structure of poly(1,4-piperazinediyl-1-oxo-trimeth-ylene)
N N
On
∗∗
Fig. 7 Speciation diagrams of AGMA1
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and, at pH 7.4, + 0.55 average positive charge per unit, proved little cytotoxic (IC50 ≥ 5 mg mL−1 on different cell strains; maximum tolerated dose 50%, MTD50, in mice > 0.5 g kg−1 upon intravenous administration) and negligibly hemolytic from pH 4 to 7.4 [82, 90].
The biocompatibility of AGMA1 hydrogels and of their degradation products was demonstrated by in vitro [83] and in vivo tests [67].
The body distribution of ISA1 and -ISA23 containing 1% on a molar basis units deriving from radioactive 125I-labelled tyrosine was studied in rats [28]. The results indicated that whereas ISA21 rapidly left blood and accumulated in liver,
ISA23 showed longer circulation in the blood and was cleared through kidneys up to 60% in 5 h. The same polymer, unlabeled, completely degraded at pH 7.4 and 37 °C to small molecules [71]. These results demonstrated the “stealth” properties of the long circulating ISA23, in turn ascribed to its polyanionic nature at pH 7.4 (isoelectric potential 5.5), which allowed passive targeting to the fenestrated tumor tis-sues (enhanced permeability and retention (EPR) effect [91].
Fig. 8 Reversibility with respect to a pH change of L-ARGO7 CD spectra
Fig. 9 Solvent accessible surface area in water of M-L-Ala decamer. C atoms (dark grey); H atoms (light gray); N atoms (blue); O atoms (red)
ISA1-mitomycin (MMC) (Table 1 for ISA1 structure) con-jugates [92] were found less toxic than free MMC when adminstered by intraperitoneal route at a MMC-equivalent dose of 5 mg kg−1. Their anticancer activity was comparable to that MMC and induced long-term survival of mice bear-ing L1210 tumour cells.
Cisplatin complexes containing 8 to 70 wt% platinum were obtained from ISA23, ISA2350-CD50 (an ISA23 copolymer containing 50% amino-β-cyclodextrin units) and a PAA derived from N,N’-bisacryloylpiperazine and a 1:1 amino-β-cyclodextrin/2-methylpiperazine mixture. All these PAA-platinates were less cytotoxic in vitro against lung tumor cells than cisplatin and, in addition, ISA23/Pt and ISA2350-CD50/Pt showed in vivo the same activity as cisplatin against an intraperitoneal L1210 leukaemia model [93]. PAA conjugates with pamidronate and platinum com-plexes showed in vitro anticancer activity combined with inferior general cytotoxicity compared to the free drug [94].
ISA1- and ISA23-doxorubicin conjugates bearing an acid-labile cis-aconityl spacer containing 28–35 mg mg−1 drug showed the ability to release biologically active Dox in the endosomal compartment of murine melanoma B16F10 cells [95]. Mixed bioredicible disulphide-containing PAA/PEG/PCL gels degraded releasing 4-fluoroacil in acidic, basic, enzymatic, and slightly reducing environments. The same mixed hydrogels gave a limited extent of drug release at pH 7.4 [96].
3.3 PAAs as Nanocarriers of Antimalarial Drugs
AGMA1, ISA23 and ISA1 were tested as nanocarriers for the selective delivery of the antimalarial drugs primaquine and chloroquine to parasitized red blood cells (pRBCs) [97]. Fluorescence assisted cell-sorting data (CFM), transi-tion electron microscopy (TEM) and confocal microscopy
(CFM) analyses indicated that both AGMA1 and ISA23 preferentially bind to and internalize into pRBCs with respect to RBCs, in both Plasmodium falciparum and Plas-modium yoelii (Fig. 10). In addition, AGMA1 was intrin-sically active as antimalarial, with IC50 = 13.7 μM against Plasmodium falciparum. Furthermore, AGMA1 and ISA23 encapsulated primaquine and chloroquine with a maximum payload of 29.4 and 15.1 wt%, respectively, for primaquine and 14.2 and 32.9 wt% for chloroquine. Both intraperito-neally administered chloroquine-AGMA1 and -ISA23-formulations cured Plasmodium yoelii–infected mice at a chloroquine dose equal to half a dose at which the animals treated with the free chloroquine died.
Several PAA/chloroquine formulations obtained with ISA23, ISA1, AGMA and ARGO7 cured Plasmodium yoelii-infected mice, improved the activity of the free drug and induced in the animals immunity against malaria [98]. PAA adhesiveness to Plasmodium falciparum proteins was ascribed as responsible for the preferential binding of PAAs to Plasmodium-infected erythrocytes with respect to non-infected red blood cells. Fluorescein isothiocyanate (FITC)-labeled PAAs were fed to females of the malaria mosquito vectors, producing persistent fluorescence in the midgut and in other insect’s tissues.
3.4 PAAs as nanocarriers of imaging probes
PAAs bearing paramagnetic N-Oxyl pendants were obtained from 4-amino-2,2,6,6-tetramethyl-piperidine-N-oxyl (4-amino-TEMPO). ISA23-TEMPO (Fig. 11) conjugates with 10 and 40% TEMPO-carrying units, respectively, exhibited relaxivities of 0.4 and 1.8 mM−1 s−1, respectively. Preliminary magnetic resonance imaging (MRI) studies demonstrated that PAA-TEMPO conjugate warranted poten-tial as NMR imaging contrast agents [99].
An ISA23 derivative bearing 10% thiol-func-tionalized units, ISA23SH10%, was obtained using
Fig. 10 In vitro confocal fluorescence micrograph showing the inter-nalization of ISA23 in Plasmodium yoelii: RBC plasma membrane (red); Plasmodium nuclei stained with DAPI (blue) was used to indi-
cate parasitized RBCs; localized fluorescein isothiocyanate (FITC)-labeled ISA23 (green). Reproduced with permission from [151]
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mono-N-boc-cystamine as comonomer and then reducing the disulfide functions in the copolymer (Scheme 5) [44]. Rhenium complexes with 0.5 and 0.8 rhenium equivalent/SH unit were obtained from the reaction of ISA23SH10% with [Re(CO)3(H2O)3](CF3SO3) in pH 5.5 aqueous solu-tion. The rhenium complexes were soluble at pH 7.4 and proved highly stable even in the presence of excess cysteam-ine. Neither ISA23SH10% nor its rhenium complexes were cytotoxic against Hela cells over 48 h at 100 ng mL−1. No hemolytic activity was observed up to 5 mg mL−1. Both ISA23SH10% and its rhenium complexes induced negligible toxic effects on mice after intravenous injection in doses up to 20 mg kg−1. ISA23SH10% warrants potential as carrier of radioactive rhenium and technetium.
An ISA23 derivative bearing 6% phenanthroline-functionalized units, (PhenISA, Fig. 12), was prepared by introducing 4-(4′-aminobutyl)-1,10-phenanthroline as co-monomer in the polymerizing mixture [100]. PhenISA showed excellent solubility in water and both Re(CO)3
+ or Ru(phen)2
2+ were stably bound by coordination to phenan-throline moieties.
The resultant complexes were luminescent with emission in water λem = 608, 571 and 614 nm and Φem = 0.7, 4.8 and 4.1%, respectively. Excess cystein, a potential competing agent, had no effect on the complexes. The complexes lacked toxicity up to 50 μM, with respect to the metal-containing unit, against HEK-293 cell lines. The ruthenium complexes were endocytically internalized by HEK-293 cells and dif-fused within the cytoplasm across the vesicle membranes, as previously observed with ISA23 [28].
A PhenISA iridium complex, synthesized by binding bis(cyclometalated)Ir(2-phenylpyridyl)2
+ fragments to the copolymer bearing 6% on a molar basis phenanthroline pendants [101], gave in water nanoparticles with hydrody-namic diameter 30 nm. It was speculated that, due to the amphiphilic nature these nanoparticles had a core–shell architecture, with the lipophilic metal centers being segre-gated in the core. NMR data supported this hypothesis. This provided an explanation for the greater photoluminescence quantum yield exhibited by the PhenISA-iridium complex compared to the low molecular weight model prepared by reacting bis(cyclometalated)Ir(2-phenylpyridyl)2
+ frag-ments with 4-(butyl-4-amino)-1,10-phenanthroline). The
PhenIsa-iridium complex proved an efficient photolumines-cent cell staining endowed with two-photon excitation (TPE) imaging ability that localized in the perinuclear region of HeLa cells. Photodynamic therapy analyses showed that both the PhenIsa-iridium complex and its low molecular weight model induced cell apoptosis upon exposure to Xe lamp irradiation, but the polymer complex was less cytotoxic in the absence of irradiation.
Superparamagnetic iron oxide nanoparticles (SPION) were stabilized and decorated using an ISA23 copoly-mer bearing 17% repeat units modified by inserting cat-echol moieties (Fig. 13) [102]. The size of the nanocom-posites, SPION@ISA23-ND, as determined by TEM was 21.1 ± 2.9 nm and the hydrodynamic size by DLS was 100 ± 28 nm. After lyophilization, nanoparticles easily re-dispersed in water reverting to their pristine dimensions. SPION@ISA23-ND poorly interacted with bovine serum albumin, suggesting that nanoparticles could retain the
NH
NH
N
O OHO O
N∗ NH
NH
O O
N∗
OHO
O
mn
N
Fig. 11 Structure of the ISA23-TEMPO copolymer
Fig. 12 Structure of PhenISA rhenium complexes
Fig. 13 TEM image of SPION@ISA23-ND nanoparticles
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ISA23 stealthiness. SPION@ISA23-ND was easily inter-nalized in HeLa cells. 1H-NMR relaxivity measurements showed relaxivity values superior to that of NMR commer-cial contrast agents in fields relevant for magnetic resonance imaging.
3.5 PAAs as Non‑Viral Vectors for Intracytoplasmic Delivery
PAAs are membrane active polymers able to promote the cell delivery of proteins and of genetic material, that is, plas-mid DNA or siRNA constructs.
The membrane damage caused by polycations with high charge density has been recognized for a long time [103]. In this regard, the “proton sponge” hypothesis, that is, absorb-ing protons within the endosome, where pH is 5.5, swell-ing and causing membrane rupture, has since long been under debate [104]. It was postulated that passing from the extracellular fluid to the endosomal intracellular compart-ments, that is, passing from pH 7.4 to 5.5, PAAs undergo pH-induced conformational changes [75] that activate latent endosomolytic properties favoring the endosomal escape into the cytosol of sensitive drugs that would be otherwise digested by the endosomal enzymes.
The first hint of the pH-dependent responsiveness of PAAs was provided by a PAA conjugate with the membrane lytic non-ionic detergent Triton X-100 [105]. Subsequently, it was demonstrated that the amphoteric ISA23 became membrane active at pH < 7.4 [28, 32].
The cellular uptake of PAAs was investigated using the ISA1-Oregon Green conjugate in B16F10 cells in vitro and the intracellular trafficking of 125I-labelled ISA1-tyrosine in rats’ liver cells assessed in vivo [106]. This research pro-vided direct evidence that ISA1 permeabilizes the vesicular membranes of endosomes by moderate physical interaction without inducing the proton sponge effect.
ISA1 and ISA1-ISA23 copolymers promoted the endo-somal escape and intracellular trafficking of different model toxins, namely inactivated ricin, gelonin [107] and melittin [108].
3.6 PAAs as DNA/siRNA Transfection Promoters
ISA1 and ISA23 formed toroidal polyplexes with DNA 80 -150 nm in size at a 10:1 polymer/DNA ratio and medi-ated the pSV β-galactosidase transfection of HepG2 cells [107]. The PAA deriving from the polyaddition of methylen-ebisacrylamide with dymethylethylenediamine and its PEG copolymers proved able to efficiently deliver DNA delivery systems [109–111].
Cationic PAAs with NH2 pendants were more efficient than PEI with molecular weight 25000 as DNA-complex
forming and transfecting agent, being meanwhile less cyto-toxic [112–114].
AGMA1 was easily internalized in HT-29 cells, con-densed DNA in spherical, positively charged nanoparticles and protected it from enzymatic degradation [25, 115]. Whereas FITC-labeled AGMA1 localized in the perinu-clear region, its DNA/AGMA1 polyplexes showed extensive intranuclear localization. DNA/AGMA1 polyplexes intra-venously administered to mice exhibited negligible toxic-ity and promoted gene expression in liver, but not in other organs (Fig. 14).
AGMA1 forms stable polyplexes of size depending on its molecular weight. In particular, AGMA1 with Mn 7800 formed with siRNA small nanoparticles of size ≤ 50 nm, whereas the size of the nanoparticles obtained from AGMA1 with Mn 3700 was about 100 nm. Mn 7800 AGMA1/siRNA polyplexes induced Akt1 gene silencing in HeLa and PC3 cells. The transfection efficiency was comparable with that of commercial siRNA transfection promoters, as for instance JetPEIVR and OligofectamineVR [116].
Linear bioreducible PAAs bearing disulfide linkages in the main chain backbone formed nanocomplexes by self-assembly with DNA, siRNA and proteins and were studied as carriers for their intracellular delivery [117, 118]. Linear bioreducibles PAA with different steric hindrance, hence with diversified chemical stability, near the disulfide were synthesized (Scheme 10a) [119]. Bioreducible PAAs with increased cell penetration ability were obtained by intro-ducing guanidine pendants either by homopolymerizing
Fig. 14 Cartoon representing the intracellular trafficking and nuclear localization of FITC-labeled AGMA1 and FITC-labeled DNA/AGMA1 polyplex according to ref. no. [115]. a Nucleus staining by DAPI marker (blue). b Visualization of labeled AGMA1 and DNA/AGMA1 (green)
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N,N’-cystaminebisacrylamide with 4-aminebutylguanidine [120] or by copolymerizing N,N’-cystaminebisacrylamide with 4-aminebutanol/4-aminebutylguanidine [121] or hystidine/4-aminebutylguanidine mixtures [122].
Bioreducible PAAs with guanidine groups in the main chain were also recently obtained by polymerizing N,N’-cystaminebisacrylamide with guanidine hydrochloride and chlorhexidine and their intracellular distribution and inter-nalization pathways investigated [123].
Hyperbranched bioreducible PAAs were obtained from 1-(2-aminoethyl)piperazine and N,N’-cystaminebisacryla-mide (Scheme 10b) [124–127].
Hyperbranched PAAs modified with folate units exhib-ited lower cytotoxicity, higher hemocompatibility and gene
delivery efficiency than PEI in the presence of serum, led to decrease in MMP-9 protein expression and apoptosis of MCF-7 cells [125].
Interestingly, PAA-PEG nanosized constructs crosslinked with disulfide functions, grafted with thiol-end-capped DNA, were also employed as tracers of envi-ronmental organic pollutants [128].
Shell-core nanoparticles with a gold nanosized core and a multilayer shell made of stratifications of PEI and a reducible PAA obtained from allowed co-delivery of DNA and siRNA. This nanovector led to exogenous DNA expression and siRNA-mediated knockdown with efficacy higher than of Lipofectamine® 2000 [129].
Scheme 10 Synthesis of hyperbranched bioreducible PAAs
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Fluorinated bioreducible PAA-PEG copolymers exhibited superior serum stability of polyplexes and enabled efficient siRNA delivery [130, 131].
A series of amphoteric bioreducible copolymers were obtained from N,N’-cystaminebisacrylamide and differ-ent agmatine/γ-aminobutyric acid molar ratios to find the optimum balance between the active positively charged guanidinium groups of agmatine and the negatively charged carboxyl groups of γ-aminobutyric acid used to decrease cytotoxicity. The best copolymer composition corresponded to 80:20 agmatine/γ-aminobutyric acid units and was char-acterized by DNA condensation capacity, cellular uptake, strong nuclear localization ability, high transfection effi-ciency and low cytotoxicity [132].
4 Bioactive PAAs
4.1 PAAs with Antibacterial Activity
Borondipyrromethenes (BODIPY) photosensitizers proved effective antibacterials in photodynamic therapy against both Gram-positive and Gram-negative bacteria. The cationic PAAs BP-DM and BP-AG (Table 1), and the amphoteric AGMA1 were studied for their adjuvant effect on BODIPY [133]. BP-DM and AGMA1 exhibited limited toxicity against the Gram-negative bacterium Escherichia coli, but this effect was negligible at concentrations < 5 μg mL−1. At nontoxic concentrations (1 or 10 μg mL−1) all PAAs remark-ably improved the killing efficacy of BODIPY upon irra-diation with a green LED device (480–580 nm with λmax 525 nm) up to an energy rate of 16.6 J cm−2. A 6–7 log unit decrease in bacteria survival was observed with concentra-tions of BODIPY of 1.0 and 0.1 μM in the case of Escheri-chia coli and Staphylococcus aureus, respectively.
4.2 PAAs with Antimethastatic Activity
The effectiveness of copolymeric amphiphilic PAAs with different charge distributions against the dissemination of cancer cells from Sarcoma 180 implanted intracerebrally, against the formation of lung metastases from Lewis car-cinoma implanted intramuscularly and the formation of lymphnodal metastases from Erlich carinanoma implanted intratibially was investigated [134]. In particular, the pol-yaddition products of N,N’-bisacryloylpiperazine with mixtures of 1-dodecaneamine and N-hydroxyethylamine at different ratios were cationic and contained variable amounts of hydrophobic side chains (Fig. 15a). These sur-factant-like PAAs, though fairly toxic, could be adminis-tered to mice up to 20 mg kg−1 and reduced the number and average weight of Lewis lung tumor metastases. Mean-while, the amphoteric and non-toxic polyaddition product
of N,N’-bisacryloylpiperazine with 1:1 piperazine/gly-cine mixture, which could be administered up to a dose of 200 mg kg−1, proved able to reduce the number and average weight of both Sarcoma 180 and Lewis lung tumor metas-tases (Fig. 15b). The activity of the hydrophobically substi-tuted PAAs was ascribed to their cell membrane interaction ability, [135], whereas the mode of action of the amphoteric PAA is still open to question.
4.3 PAAs with Antiviral Activity
An ISA23 copolymer carrying 10% β-cyclodextrin pendants, ISA23-CD, was obtained by replacing in the ISA23 syn-thetic recipe 10% on a molar basis 2-methylpiperazine with 6-deoxy-6-amino-β-cyclodextrin. ISA23-CD loaded 11% w/w of Acyclovir creating stable solutions in water. The Acyclovir ISA23-CD inclusion complex showed superior anti-Herpes simplex virus type I (HSV-1) than the free drug [136].
AGMA1 exhibited intrinsic antiviral activity both in vitro and in vivo. Its inhibitory efficiency towards HSV viruses was considerable, with EC50 values 0.74 and 1.14 μg mL−1 for HSV-1 and HSV-2, respectively, with no obnoxious side effects. Besides HSV-1 and HSV-2, AGMA1 inhibited a series of viruses, namely human papillomavirus-16 (HPV-16), cytomegalovirus (CMV), syncytial virus (RSV) and Murid Herpesvirus 68 (MHV-68), that recognize heparan sulfate proteoglycans (HSPGs) as cell receptors [137]. In particular, as regards HSV and HPV, virus entry is ascribed to the electrostatic interaction between the cationic HSV envelope and HPV capsid and the anionic sulfate/carboxyl groups of cellular HSPGs [138, 139]. Therefore, it was ini-tially postulated that the cationic nature of AGMA1 was responsible for its antiviral activity; the inefficacy as infec-tion inhibitor of the amphoteric, prevailingly anionic ISA23 seemed to confirm this hypothesis. However, under the same conditions, the polycationic ISA1, having at pH 7.4 the same
Fig. 15 Structure of amphiphilic PAAs with antimethastatic activity
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net average charge as AGMA1 per repeat unit, was inactive. This suggested that specific structural features of AGMA1, such as the presence of guanidine residues, favored its effec-tive binding to HSPGs. To confirm this, it was demonstrated that AGMA1 interacts with either immobilized heparin or cellular heparan sulfates, in the latter case preventing HPV attachment to the cell surface [140]. Accordingly, AGMA1 did not kill the virus, but blocked the infection transmission from cell to cell [141].
The AGMA1 activity against HSV-1 and HSV-2 infec-tion was also ascertained on reconstructed human epithelia, namely ectocervico-vaginal tissue (EpiVaginal™), without eliciting inflammation. The same inhibitory effect and lack of inflammatory activity was subsequently confirmed in vivo after topical administration to female mice [141]. Moreover, AGMA1 did not influence the vaginal pH, since proved inac-tive towards Lactobacillus spp.
Immature dendritic cells and macrophages express the C-type lectin DC-SIGN that acts as receptor of the man-nose clusters on the human immunodeficiency virus HIV-1 gp120 envelope glycoprotein. By this way, they mediate HIV infection. It was speculated that the presence, in AGMA1, of mannosylated units would impart anti-HIV activity while preserving its HPV-16 and HSV-2 infection inhibi-tory activity [142]. The rationale was that mannosylated glycodendrimers had proven able to block DC-SIGN thus acting as HIV entry inhibitors [143], AGMA1 and ISA23 conjugates bearing different amounts of mannosyl-triazolyl pendants (Fig. 16), were therefore prepared by reaction of differently propargyl-substituted AGMA1 and ISA23 with 2-(azidoethyl)-α-D-mannopyranoside.
Both mannosylated PAAs inhibited HIV infection with a mannosyl-dose dependence. Moreover, mannosylated AGMA1 retained the anti-HPV-16 and HSV-2 activity of AGMA1, thus proving to be a broad-spectrum, dual action mode virus inhibitor (Fig. 17).
4.4 PAAs as Cell Adhesion Promoters
PAAs bearing bisphosphonate pendants obtained by the pol-yaddition of N,N’-bisacryloylpiperazine with pamidronate or neridronate (Fig. 18) proved endowed with potential as osteoblast proliferation promoters [144].
More recently, AGMA1 surface-adsorbed on cell cultur-ing coverslips promoted the growth of primary brain cells, including microglia, hippocampal neurons and astrocytes [82]. Mixed cultures of primary astrocytes and neuronal cells grown on coverslips coated with AGMA1 and poly-l-lysine, employed as reference substrate, were morphologi-cally undistinguishable, in that neurons differentiated axon and dendrites and established functional synaptic contacts. Also immunocytochemical staining revealed no difference between AGMA1 and poly-l-lysine (Fig. 19). Furthermore, electrophysiological experiments allowed recording neu-ron spontaneous activity on AGMA1 comparable to that of neurons grown on poly-l-lysine coated medium, with miniature excitatory and inhibitory electric signals, indicat-ing that neurons cultured on AGMA1 are characterized by spontaneous activity.
4.5 Supramolecular PAA Micelles
Spherical supramolecular polymeric micelles with a core–shell structure were obtained by mixing hyperbranched PAA-dextran (HPA-Dex) conjugate with the hydrophilic (−)-epigallocatechin-3-gallate (EGCG) antioxidant in aque-ous solution. HPA-Dex was in turn obtained via host–guest interaction of hyperbranched PAA containing β-cyclodextrin and adamantyl-modified dextran. The stability of EGCG-HPAM-Dex was ascribed to a combination of interactions, namely electrostatic and hydrophobic interactions and hydrogen bonding. The release behavior of the EGCG-HPAM-Dex micelles was studied [145].
(a) (b)
Fig. 16 Structure of the ISA23 (a) and AGMA1 (b) copolymers bearing mannosyl-triazolyl pendants
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4.6 PAA Hydrogels and as Scaffolds for Tissue Engineering
Crosslinked PAAs are hydrogels that normally show crosslinking degree-dependent water absorption, from 100 to 1000% of their own dry weight. They are easily molded in different shapes by injection molding (Fig. 20).
Their comprehensive structural characterization can be performed by high-resolution magic angle spinning (HRMAS) NMR spectroscopy [146]. Advanced NMR techniques allowed elucidating their interaction with water molecules both in the absence and presence of inorganic ions [147–149].
Amphoteric ISA23-based hydrogels were obtained from 2,2-bisacrylamidoacetic acid, 2-methylpiperazine and pri-mary bis-amines as crosslinking agents [72]. Alkyl pendants
Fig. 17 HIV, HPV-16 and HSV-2 infection inhibitory activity. Data represent the percent infection following polymer treatment in com-parison with the untreated control. For Man-AGMA and Man-ISA samples, concentrations refer to the mannosylated units. In case of
plain ISA23 and AGMA1, the same w/v concentrations of Man-ISA7 and Man-AGMA6.5 were used, respectively. Values represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001
OO
N N Nn
∗
(CH2)
H2O3P PO3H2OH
n
∗
n = 2 oligoPAMIDRONATE
n = 5 oligoNERIDRONATE
Fig. 18 Structure of PAAs bearing bisphosphonate pendants
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of different length, i.e. dimethyl, butyl and octyl residues, were introduced by copolymerizing 2,2-bisacrylamidoacetic acid and 2-methylpiperazine with N,N-dimethyl-, N-butyl- and N-octylacrylamide, respectively. These monofunctional acrylamides allowed to introduce alkyl grafts on the PAA hydrogel. Hybrid PAA/albumin hydrogels were also pre-pared. All the amphoteric PAA hydrogels considered were cytobiocompatible. In addition, they completely eroded within two weeks in Dulbecco medium at pH 7.4 and 37 °C, whereas hybrid PAA/albumin hydrogels did not erode within an 8 month period. All samples exhibited negligible cyto-toxicity. None of tested hydrogels proved cell-adhesive as demonstrated in proliferation tests with fibroblast cell lines.
Electron beam microlithography was applied to the surface of a non-adhesive ISA23 based hydrogel placed in dry form inside a scanning electron microscopy
vacuum chamber [85]. The aim was designing patterns for the ordered growth of PC12 neural cells. Following e-beam exposure, the hydrogel surface morphology was substantially modified, as demonstrated by the differ-ently localized swollen zones highlighted by atomic force microscopic. Labelled proteins, namely epidermal growth factor (FITC, green), bovine serum albumin (Alexa) and fibronectin (Alexa) selectively adhered on the e-beam modified areas, whose chemical composition turned to be modified with respect to the remaining portion of the hydrogel surface, due to the de-carboxylation of the 2,2-bisacrylamidoacetic acid moieties of the hydrogel. No cell growth outside the patterns was observed.
AGMA1-based hydrogels were prepared from 2,2-bisacrylamidoacetic acid and 4-aminobutylguanidine crosslinked by adding multifunctional prim-amines, for instance either α,ω-bisaminododecane or a purposely syn-thesized PAA containing NH2 pendants [83]. Cytotoxicity and proliferation tests carried out on BALB/3T3 Clone A31 mouse embryo fibroblasts cell lines demonstrated that both hydrogels were adhesive to cell membranes and non-cytotoxic. Both hydrogels eroded and eventually solu-bilized in aqueous media; their dissolution times at pH 7 and 37 °C in Dulbecco medium were approximately 10 and 40 days, respectively. The degradation products exhibited negligible cytotoxicity.
Nanometric ISA23- and AGMA1-based hydrogel lay-ers were surface-grafted on cell culturing glass coverslips, previously treated with γ-aminopropyltriethoxysilane, by incubating with polymerizing mixtures leading to crosslinked ISA23 and AGMA1 [84]. By this way, the amine group introduced on the glass surface participated in the polymerization reaction covalently attaching the PAA chains on the glass. Swelling in water induced the spontaneous detachment of the hydrogel bulk and left on the glass surface a residual thin layer of hydrogel. Whereas AGMA1 hydrogel layers promoted cell adhesion towards Madin-Darby Canine Kidney (MDCK) epithelial cells comparable to that of petri dish plate plastic sur-face, ISA23 hydrogels showed a much lower cell adhesion (Fig. 21).
Soluble AGMA1 adsorbed on glass and polystyrene plates acted as growth promoter of Schwann and Dorsal Root Ganglion neurons proving in this application com-petitive with poly-l-lysine, with the additional advantage of lack of toxicity [150]. This result represented the start-ing point for the synthesis of swellable crosslinked AGMA1 hydrogels in tubular form as bioresorbable guides for the in vivo regeneration of sciatic nerve in rats. Nerve regenera-tion was complete within 90 days leaving no residues and without eliciting any detectable local inflammation. It may be observed that the same in vivo experiment provided evi-dence of in vivo degradability of crosslinked AGMA1 [67].
Fig. 19 a Quantitative analysis of the immunofluorescence staining of 16 div primary hippocampal neurons plated on AGMA1 and PLL with synaptic markers SV2, Bassoon (Bsn) and PSD-95: average size of SV2, Bassoon and PSD-95 puncta; percentage of area of PSD-95 puncta colocalized with SV2 and Bassoon vs total area of PSD-95 puncta. b Representative traces of miniature excitatory postsynap-tic current (mEPSCs) and miniature inhibitory postsynaptic poten-tial (mIPSCs) recorded from 16 div hippocampal neurons plated on AGMA1 and PLL thin coating
Fig. 20 Tubular PAA hydrogels
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Despite their excellent functional properties, AGMA1 hydrogels were still unsuitable for being further developed in view of human use because of their poor mechanical properties. Different strategies were therefore adopted for designing tough PAA hydrogels capable to combine ade-quate response to mechanical stimuli with cell adhesiveness and the ability to promote tissue growth.
The PAA deriving from N,N’-bisacryloylpiperazine and piperazine was highly crystalline and, when moderately crosslinked, maintained the structure-forming ability of its linear counterpart, notwithstanding its high swelling degree. Consequently, this PAA hydrogel was the toughest among those so far described [66]. At high crosslinking degrees, probably due to the short length of the linear segments between adjacent crosslink points, the toughening effect vanished. In vitro experiments showed that these structured hydrogels induced growth of Schwann cells and Dorsal Root Ganglion neurons.
Tough PAA hydrogels were prepared by reinforcing with nanosized inorganic fillers, namely montmorillonite (MMT) [69]. AGMA1/MMT nanocomposite hydrogels with differ-ent crosslink density had shear storage modulus, G′, when fully swollen in water, up to 200 kPa, i.e. 20 times higher than the virgin hydrogels and at least of the same order of other hydrogel-based composites proposed for orthopaedic applications. AGMA1/MMT hydrogels proved a good scaf-fold for the growth of mouse calvaria-derived pre-osteoblas-tic MC3T3-E1 cells and induced differentiation towards the osteoblastic phenotype (Fig. 22). AGMA1–MMT hydrogels degraded in water at pH 7.4 with no evidence of cytotoxicity.
Stitchable PAA hydrogels reinforced with electrospun poly-l-lactic acid (PLLA) nanofibrous mats were prepared [70]. PLLA mats were first surface functionalized with amine groups by nitrogen plasma and then embedded into aqueous solutions of oligomeric acrylamide-end capped AGMA1. The role of the amine functions on PLLA surface
Fig. 21 Immunofluorescence analysis of cytoskeleton and focal con-tacts (arrows) of MDCK cells cultured on PAA hydrogels and TCPS. Actin stress fibers (left panel), vinculin stains (middle panel) and
their merge (right panel) of MDCK cells 2 days after seeding. Nuclei are labeled with DAPI (blue). Scale bar: 10 μm
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was to favor chemical grafting by the AGMA1 oligomers. The resultant mixture was cured by UV-inititiated radical polymerization of the PAA terminals affording macroscopi-cally homogeneous and tough PLLA-AGMA1 composite hydrogels (Fig. 23) that absorbed large amounts of water and, when swollen in water, were translucent, soft, and pli-able, yet as strong as the parent PLLA mat. The fact that the hydrogel was covalently bound to the embedded nanofib-ers imparted superior toughness. PLLA-AGMA1 compos-ite hydrogels proved capable of maintaining short-term undifferentiated cultures of human pluripotent stem cells in feeder-free conditions.
5 Conclusions and Perspectives
PAAs are a a family of polymers characterized by tert-amine and amide groups placed in regular sequence along the macromolecular chain. They are endowed with a rarely matched combination of properties making them eligible to
a variety of applications, mainly, but not exclusively, in the biomedical field. The carbonyl groups β to the amine groups significantly reduce their basic strength and cationic charge density. As a result, PAAs are less cytotoxic than more popu-lar polycations such as polyl l-lysine and poly-l-ornithine, provided there are no strongly basic or long-chain hydro-phobic substituents. Amphoteric, but at pH 7.4 prevailingly cationic PAAs, are in many cases nearly deprived of cyto-toxicity. However, many of these are are able to interact with polyanions producing polyplexes and may be used as trans-fection promoters. In dilute aqueous solution, at pH ≥ 7.5 and temperature equal or higher than 30 °C, PAAs degrade also in the absence of specific enzymes. This was ascribed to the presence of tert-amine groups β to the amide groups favoring hydrolytic cleavage of the amide group. Obviously, the tert-amine groups could exert this activity only if unpro-tonated. Therefore, at acidic pH, PAAs proved stable over long periods of time. Crosslinked PAAs are more hydro-lytically stable than linear PAAs, nevertheless they proved completely erodible in vivo.
The wealth of results reported in this review lets envisage that the virtually unlimited structural versatility of PAAs allows the planning of polymers capable to match specific needs. Based on the most recent results, different future directions of PAA research can be foreseen and new chal-lenges may be met. PAA deserve to be further studied to stabilize and decorate metals or metal oxides to obtain smart nanovectors able to respond to different external stimuli (pH, redox potential, temperature variations). Moreover, they can act as macromolecular ligands of luminescent heavy metal ions producing quantum yields superior to those of non-macromolecular ligands. Countless combinations of PAA structures, heavy metal ions, ancillar non-macromolecular ligands are potentially available for designing efficient in vitro and in vivo imaging probes that may capitalize on the biocompatiblity and cell penetration ability of PAAs. In tissue engineering, there is a need for the design of scaffolds combining the good functional properties already shown by PAA hydrogels with tougness and tunable degradation. Since amino acids and peptides can be easily used as build-ing blocks in PAA synthesis, biomimetic chiral polymers may be designed with selective interactions with biomol-ecules and intracellular localization. General topics in PAA technology, as the in vivo fate and the long-term toxicity of PAAs, if any, as well as their effects on the environment, specifically in terms of degradation/biodegradation in soil and in aquifers, deserve deeper insights. A completely new field of investigation would be highly hydrophobic PAAs as potential technical materials or as surface conditioners of conventional materials. Based on all previous considera-tions, it is reasonable to conclude that the potential of PAAs for biotechnological applications, and probably not just bio-technology, is still far from being fully explored.
Fig. 22 Confocal laser scanning micrographs of MC3T3-E1 cells cul-tured on AGMA1-MMT hydrogels. Scale bar: 30 μm
Fig. 23 SEM micrograph of an AGMA1-PLL composite hydrogel
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Compliance with Ethical Standards
Conflict of Interest On behalf of all authors, the corresponding author states that there is no conflict of interest.
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