Chemistry Africa

, Volume 2, Issue 2, pp 167–193 | Cite as

Polyamidoamines: Versatile Bioactive Polymers with Potential for Biotechnological Applications

  • Elisabetta RanucciEmail author
  • Amedea Manfredi
Invited Review


Polyamidoamines (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 notwithstanding 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.


Polyamidoamines Functional polymers Degradable polymers Bioactive polymers Biocompatible polymers 

1 Introduction

The polymer family obtained by the Michael-type polyaddition of prim- or bis-sec-amines with bisacrylamides (Scheme 1) was first reported in 1967 and called “polyamidoamines” [1]. This name was subsequently commonly abbreviated as PAAs.
Scheme 1

Synthesis of linear PAAs

Further work demonstrated that this type of polymerization 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, 3, 4, 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 reactions: 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 versatility 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 hyperbranched 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 antimalarials, antivirals or antibacterial agents. This review is limited to linear and crosslinked PAAs synthesized according to Scheme 1.

PAAs can easily be designed to be water-soluble and biocompatible. 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 purification [9, 10, 11, 12, 13] and as heterogeneous catalysts [14], high-performance nonlinear optical dyes [15, 16], coating for sensing applications [17, 18, 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-, sulphonic-, 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 distributions. 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, several amphoteric PAAs are almost biocompatible as dextran, in the absence of basic- or highly hydrophobic side substituents. 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 normally 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 biotechnological applications of PAAs for which they were considered worthy of attention. These include cell-penetrating polymers with potential as transfection promoters, polymer drug carriers, nanoparticles, nanovectors, intrinsically bioactive 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].
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

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 polyaddition 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, ethylene glycol, formamide and dimethylformamide [23] demonstrated 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 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].
Fig. 2

Trend of the intrinsic viscosity measured in chloroform at 30 °C of the N,N’-bisacryloylpiperazine/2-methylpiperazine mixture in pyridine and water at different polymerization temperatures

The Michael polyaddition is highly specific and allows obtaining, with the appropriate functional monomers, PAAs bearing many functional lateral substituents, including 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 ethylenediamine, yield soluble PAAs with prim-aminoethyl pendants [26] (Table 1).
Scheme 2

Synthesis of an AGMA1 copolymer bearing prim-amine pendants

Table 1

Structure of cationic PAAs

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. Due to steric hindrance, α-aminoacids other than glycine react slowly [30]. Amphoteric PAAs with different isoelectric points can be synthesized by adjusting the number and strength of the acid and basic groups in the starting monomer mixtures (Table 3) [27].
Table 2

Structure of amphoteric PAAs

Table 3

Acid-base properties of some amphoteric PAAs







< 2










< 1





< 1




Data from reference no. [27]

aAs in the original article

bIsoelectric point

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’-bisacryloylcystamine 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 promoters [35]. l-cystine [36] and N,N’-dimethylcystamine [37] were also used as difunctional diamine monomers, capitalizing 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].
Scheme 3

Synthesis of PAAs bearing disulfide groups in the main chain

PAAs bearing disulphide bonds in the main chain PAA are particularly interesting for their ability to give bioreducible 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 prepared in slightly alkaline media by dithio–dithio exchange reaction between crosslinked PAAs previously obtained using cystamine as crosslinking agent and excess 2,2′-dithiodipyridine (Scheme 4).
Scheme 4

Synthesis of PAAs bearing disulfide pendants

These products were amenable to coupling reactions with thiolated peptides, such as for instance glutathione [39], and thiocholesterol [40]. In the latter instance, amphiphilic polymers were obtained giving spontaneously in aqueous media nano-aggregates that collapsed in the presence of reducing agents (Fig. 3).
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

ISA23-cholesterol nanoparticles were succesfully used to formulate the lipophilic anticancer drug tamoxifen [41]. Electrospray allowed obtaining nanoparticles with homogeneous size distribution (Fig. 4) that could be loaded with 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 concentrations, blank nanoparticles were negligibly cytotoxic.
Fig. 4

SEM image of blank ISA23-cholesterol nanoparticles. Scale bar: 200 nm

Polymers bearing thiol groups are known to be mucoadhesive [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].
Scheme 5

Synthesis of ISA23-SH

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 \( \bar{X}_{n} \) of the resultant PAAs can be planned according to the well-know Carothers equation (Eq. 1) [45]
$$ \bar{X}_{n} = \frac{1 + r}{1 - r} $$
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-terminated PAA macromonomers, in turn obtained by treating amine-terminated PAAs with excess diisocyanate, onto commercial polyurethanes through allophanate group formation [48, 49, 50, 51, 52, 53, 54, 55, 56]. Tough coatings could be obtained by casting the reaction mixtures onto polyurethane, polyvinyl chloride and glass [56]. PUPA based materials proved able to adsorb significant heparin amounts, remarkably improving their hemocompatibility [56], thanks to the formation of polyelectrolyte complexes with the negatively charged heparin and the positively charged PAA segments [56]. Similarly, different amine-terminated PAA macromonomers were grafted onto polyetherurethaneamide 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 polymerization of 4-acryloylmorpholine in the presence of cysteamine acting a chain transfer agent [62]. Finally, the PAA/albumin graft copolymers were obtained from N,N’-bisacryloylpiperazine and piperazine under non-denaturing conditions [63].
Scheme 6

Synthesis of block- and graft-PAA copolymers

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-monoamines in the polymerizing mixture, while mantaining equimolarity between the complementary functions (Scheme 7) [2, 3, 4].
Scheme 7

Synthesis of crosslinked PAAs from multifunctional amines

However, soluble PAA drug carriers bearing sec- in place of tert-amine groups in the macromolecular backbone 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 fragile 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 polymerization 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 copolymerizing 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 normally tougher than hydrogels obtained from multifunctinal amines. It should be observed that, regardless of the preparation method, PAA hydrogels were toughened by different methods, either using inorganic fillers [69] or biodegradable electrospun non-woven mats (Sect. 4.5) [70].
Scheme 8

Synthesis of crosslinked PAAs by radically polymerizing vinyl-end-capped PAA oligomers

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).
Scheme 9

Hydrolytic degradation of PAAs

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 chromatography (Fig. 5) and/or by meauring the intrinsic viscosity. The degradation rate depends to some extent on the structure of the bisacrylamide moiety. PAAs deriving by the polyaddition of 2-methylpiperazine with different bisacrylamides exhibited degradation rates in the order N,N′-bisacryloylpiperazine > methylenebisacrylamide > 2,2-bisacrylamidoacetic acid based PAAs [71]. Also crosslinked PAA are subject to hydrolytic degradation. Hydrogels completely eroded within weeks in Dulbecco medium at pH 7.4 and 37 °C [72]. When tested, PAAs proved insensitive to lysosomal enzyme degradation at pH 5.5 [73].
Fig. 5

Hydrolytic degradation of ARGO7 at pH 7.4 monitored by size exclusion chromatrography

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 properties, 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 several 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)
$$ pH = pK - \beta log\frac{1 - \alpha }{\alpha } $$
where K is the weak acid dissociation constant; a is the dissociation 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 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 distinct 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 polyelectrolyte since interactions between neighbouring units are still present (Fig. 6):
Fig. 6

Structure of poly(1,4-piperazinediyl-1-oxo-trimethylene)

However, amphoteric PAAs exhibited a typical polyelectrolyte 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).
Fig. 7

Speciation diagrams of AGMA1

2.8 Chiral PAAs

It is generally recognized that chirality governs biomaterial-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. 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 scattering analysis showed that all PAACs structured in tiny nanoparticles (about 1–2 nm) that turned to be stable at different ionic strength for prolonged time periods. Molecular dynamics indicated that all chiral PAACs considered assumed compact but rapidly pH-responsive folded structures stabilized by intramolecular interactions. These structures were characterized by transoid arrangements of the main chain reminiscent 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.
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)

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, 84, 85] PAAs has been extensively investigated. The hallmark of PAA is their reduced cytotoxicity despite being cationic. Synthetic polycations, including poly-l-lysine (PLL) and polyethyleneimine (PEI), 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 cytotoxic 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 concentration (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 conventional polycations has been ascribed to their less pronounced cationic character, that is, to the lower positive charge density 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 polymer toxicity was significantly lowered (Table 4) [89].
Table 4

IC50 values for modified PLL


IC50 (µg mL−1)


IC50 (µg mL−1)






> 500



> 500


aCell line

bPLL modified as in ref. no. [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-diethylethylenediamine, and from α- and β-aminoacids were two orders of magnitude less cytotoxic than conventional polycations [27, 30, 32] confirms this statement (Table 5).
Table 5

Cytotoxicity of some amphoteric PAAs


IC50 (mg mL−1 ± SD)



> 5



> 5



> 5



> 5



> 5



2.5 ± 0.31



0.23 ± 0.06



> 5



0.05 ± 0.01



0.01 ± 0.01


The amphoteric AGMA1 (Table 2) differs from most other PAAs because, despite having isoelectric point > 10 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 tissues (enhanced permeability and retention (EPR) effect [91].

3.2 PAA-anticancer drug conjugates

ISA1-mitomycin (MMC) (Table 1 for ISA1 structure) conjugates [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 bearing 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 complexes 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), transition 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 Plasmodium yoelii (Fig. 10). In addition, AGMA1 was intrinsically 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 intraperitoneally 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.
Fig. 10

In vitro confocal fluorescence micrograph showing the internalization of ISA23 in Plasmodium yoelii: RBC plasma membrane (red); Plasmodium nuclei stained with DAPI (blue) was used to indicate parasitized RBCs; localized fluorescein isothiocyanate (FITC)-labeled ISA23 (green). Reproduced with permission from [151]

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 potential as NMR imaging contrast agents [99].
Fig. 11

Structure of the ISA23-TEMPO copolymer

An ISA23 derivative bearing 10% thiol-functionalized units, ISA23SH10%, was obtained using 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 solution. The rhenium complexes were soluble at pH 7.4 and proved highly stable even in the presence of excess cysteamine. 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 phenanthroline moieties.
Fig. 12

Structure of PhenISA rhenium complexes

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 diffused 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 hydrodynamic 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 segregated 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 + fragments with 4-(butyl-4-amino)-1,10-phenanthroline). The PhenIsa-iridium complex proved an efficient photoluminescent 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 copolymer bearing 17% repeat units modified by inserting catechol moieties (Fig. 13) [102]. The size of the nanocomposites, 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 ISA23 stealthiness. SPION@ISA23-ND was easily internalized in HeLa cells. 1H-NMR relaxivity measurements showed relaxivity values superior to that of NMR commercial contrast agents in fields relevant for magnetic resonance imaging.
Fig. 13

TEM image of SPION@ISA23-ND nanoparticles

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, plasmid 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, absorbing protons within the endosome, where pH is 5.5, swelling and causing membrane rupture, has since long been under debate [104]. It was postulated that passing from the extracellular fluid to the endosomal intracellular compartments, 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 provided 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 endosomal 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 mediated the pSV β-galactosidase transfection of HepG2 cells [107]. The PAA deriving from the polyaddition of methylenebisacrylamide with dymethylethylenediamine and its PEG copolymers proved able to efficiently deliver DNA delivery systems [109, 110, 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 cytotoxic [112, 113, 114].

AGMA1 was easily internalized in HT-29 cells, condensed DNA in spherical, positively charged nanoparticles and protected it from enzymatic degradation [25, 115]. Whereas FITC-labeled AGMA1 localized in the perinuclear region, its DNA/AGMA1 polyplexes showed extensive intranuclear localization. DNA/AGMA1 polyplexes intravenously administered to mice exhibited negligible toxicity and promoted gene expression in liver, but not in other organs (Fig. 14).
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)

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 introducing guanidine pendants either by homopolymerizing 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].
Scheme 10

Synthesis of hyperbranched bioreducible PAAs

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 internalization pathways investigated [123].

Hyperbranched bioreducible PAAs were obtained from 1-(2-aminoethyl)piperazine and N,N’-cystaminebisacrylamide (Scheme 10b) [124, 125, 126, 127].

Hyperbranched PAAs modified with folate units exhibited 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 environmental 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].

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 different 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 characterized by DNA condensation capacity, cellular uptake, strong nuclear localization ability, high transfection efficiency 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 remarkably improved the killing efficacy of BODIPY upon irradiation 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 concentrations of BODIPY of 1.0 and 0.1 μM in the case of Escherichia 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 carcinoma implanted intramuscularly and the formation of lymphnodal metastases from Erlich carinanoma implanted intratibially was investigated [134]. In particular, the polyaddition 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 surfactant-like PAAs, though fairly toxic, could be administered to mice up to 20 mg kg−1 and reduced the number and average weight of Lewis lung tumor metastases. Meanwhile, the amphoteric and non-toxic polyaddition product of N,N’-bisacryloylpiperazine with 1:1 piperazine/glycine 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 metastases (Fig. 15b). The activity of the hydrophobically substituted PAAs was ascribed to their cell membrane interaction ability, [135], whereas the mode of action of the amphoteric PAA is still open to question.
Fig. 15

Structure of amphiphilic PAAs with antimethastatic activity

4.3 PAAs with Antiviral Activity

An ISA23 copolymer carrying 10% β-cyclodextrin pendants, ISA23-CD, was obtained by replacing in the ISA23 synthetic 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 initially postulated that the cationic nature of AGMA1 was responsible for its antiviral activity; the inefficacy as infection 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 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 effective 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 infection 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 inactive towards Lactobacillus spp.

Immature dendritic cells and macrophages express the C-type lectin DC-SIGN that acts as receptor of the mannose 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 inhibitory 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.
Fig. 16

Structure of the ISA23 (a) and AGMA1 (b) copolymers bearing mannosyl-triazolyl pendants

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).
Fig. 17

HIV, HPV-16 and HSV-2 infection inhibitory activity. Data represent the percent infection following polymer treatment in comparison 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

4.4 PAAs as Cell Adhesion Promoters

PAAs bearing bisphosphonate pendants obtained by the polyaddition of N,N’-bisacryloylpiperazine with pamidronate or neridronate (Fig. 18) proved endowed with potential as osteoblast proliferation promoters [144].
Fig. 18

Structure of PAAs bearing bisphosphonate pendants

More recently, AGMA1 surface-adsorbed on cell culturing 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 morphologically 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 neuron spontaneous activity on AGMA1 comparable to that of neurons grown on poly-l-lysine coated medium, with miniature excitatory and inhibitory electric signals, indicating that neurons cultured on AGMA1 are characterized by spontaneous activity.
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 postsynaptic current (mEPSCs) and miniature inhibitory postsynaptic potential (mIPSCs) recorded from 16 div hippocampal neurons plated on AGMA1 and PLL thin coating

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 aqueous 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].

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).
Fig. 20

Tubular PAA hydrogels

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, 148, 149].

Amphoteric ISA23-based hydrogels were obtained from 2,2-bisacrylamidoacetic acid, 2-methylpiperazine and primary bis-amines as crosslinking agents [72]. Alkyl pendants 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 prepared. 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 cytotoxicity. 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 differently 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 synthesized 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 solubilized 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 layers 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 surface, ISA23 hydrogels showed a much lower cell adhesion (Fig. 21).
Fig. 21

Immunofluorescence analysis of cytoskeleton and focal contacts (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

Soluble AGMA1 adsorbed on glass and polystyrene plates acted as growth promoter of Schwann and Dorsal Root Ganglion neurons proving in this application competitive with poly-l-lysine, with the additional advantage of lack of toxicity [150]. This result represented the starting 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 regeneration 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 evidence of in vivo degradability of crosslinked AGMA1 [67].

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 adequate 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 different 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 scaffold for the growth of mouse calvaria-derived pre-osteoblastic 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.
Fig. 22

Confocal laser scanning micrographs of MC3T3-E1 cells cultured on AGMA1-MMT hydrogels. Scale bar: 30 μm

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 was to favor chemical grafting by the AGMA1 oligomers. The resultant mixture was cured by UV-inititiated radical polymerization of the PAA terminals affording macroscopically homogeneous and tough PLLA-AGMA1 composite hydrogels (Fig. 23) that absorbed large amounts of water and, when swollen in water, were translucent, soft, and pliable, yet as strong as the parent PLLA mat. The fact that the hydrogel was covalently bound to the embedded nanofibers imparted superior toughness. PLLA-AGMA1 composite hydrogels proved capable of maintaining short-term undifferentiated cultures of human pluripotent stem cells in feeder-free conditions.
Fig. 23

SEM micrograph of an AGMA1-PLL composite hydrogel

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 popular polycations such as polyl l-lysine and poly-l-ornithine, provided there are no strongly basic or long-chain hydrophobic substituents. Amphoteric, but at pH 7.4 prevailingly cationic PAAs, are in many cases nearly deprived of cytotoxicity. However, many of these are are able to interact with polyanions producing polyplexes and may be used as transfection 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 unprotonated. Therefore, at acidic pH, PAAs proved stable over long periods of time. Crosslinked PAAs are more hydrolytically 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 challenges 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 building blocks in PAA synthesis, biomimetic chiral polymers may be designed with selective interactions with biomolecules 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 considerations, it is reasonable to conclude that the potential of PAAs for biotechnological applications, and probably not just biotechnology, is still far from being fully explored.


Compliance with Ethical Standards

Conflict of Interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.


  1. 1.
    Danusso F, Ferruti P, Ferroni G (1967) Chimica e Industria 49:271–278Google Scholar
  2. 2.
    Danusso F, Ferruti P (1970) Polymer 11:88–113Google Scholar
  3. 3.
    Ferruti P, Marchisio MA, Barbucci R (1985) Polymer 26:1336–1348Google Scholar
  4. 4.
    Ferruti P, Marchisio MA, Duncan R (2002) Macromol Rapid Commun 23:332–355Google Scholar
  5. 5.
    Ferruti P (2013) J Polym Sci A 1(51):2319–2353Google Scholar
  6. 6.
    Tomalia DA, Dewald JR (The Dow Chemical Corporation) U.S. Patent 4 507 466, 1983Google Scholar
  7. 7.
    Tomalia DA, Baker H, Dewald J, Hall M, Kallos G, Martin S, Roeck J, Ryder J, Smith P (1985) Polym J 17:117–132Google Scholar
  8. 8.
    Tomalia DA (2012) Nanomedicine 7:953–956Google Scholar
  9. 9.
    Barbucci R, Casolaro M, Beni MC, Ferruti P, Pesavento M, Soldi T, Riolo C (1981) JCS Dalton 2559–2564Google Scholar
  10. 10.
    Ferruti P, Bertoglio Riolo C, Soldi T, Pesavento M, Barbucci R, Beni MC, Casolaro M (1982) J Appl Polym Sci 27:2239–2248Google Scholar
  11. 11.
    Ferruti P, Ranucci E, Bianchi S, Falciola L, Mussini PR, Rossi M (2006) J Polym Sci, Part A: Polym Chem 44:2316–2327Google Scholar
  12. 12.
    Ferruti P, Ranucci E, Manfredi A, Mauro N, Ferrari E, Bruni R, Colombo F, Mussini PR, Rossi M (2012) J Polym Sci, Part A: Polym Chem 50:5000–5010Google Scholar
  13. 13.
    Manfredi A, Ranucci E, Morandi S, Mussini PR, Ferruti P (2013) J Polym Sci, Part A: Polym Chem 51:769–773Google Scholar
  14. 14.
    Ferruti P, Ranucci E, Tempesti E, Giuffrè L, Arlati P, Airoldi G (1990) J Appl Polym Sci 41:1923–1927Google Scholar
  15. 15.
    Abbotto A, Beverina L, Chirico G, Facchetti A, Ferruti P, Gilberti M, Pagani GA (2003) Macromol Rapid Commun 24:397–402Google Scholar
  16. 16.
    Proutiere S, Ferruti P, Ugo R, Abbotto A, Bozio R, Cozzuol M, Dragonetti C, Emilitri E, Locatelli D, Marinotto D, Pagani G, Pedron D, Roberto D (2008) Mater Sci Eng, B 147:293–297Google Scholar
  17. 17.
    Ranucci E, Putelli L, Ferruti P, Ferrari V, Marioli D, Taroni A (1995) Mikrochim Acta 120:257–270Google Scholar
  18. 18.
    Ranucci E, Ferruti P, Ferrari V, Marioli D, Taroni A (1996) Polym Advan Technol 7:529–535Google Scholar
  19. 19.
    Sartore L, Penco M, Della Sciucca S, Borsarini G, Ferrari V (2005) Sensors Actuat B Chem 111:160–165Google Scholar
  20. 20.
    Manfredi A, Carosio F, Ferruti P, Ranucci E, Alongi J (2018) Polym Degrad Stabil 151:52–64Google Scholar
  21. 21.
    Manfredi A, Carosio F, Ferruti P, Ranucci E, Alongi J (2018) Polym Degrad Stabil 156:1–13Google Scholar
  22. 22.
    Bignotti F, Sozzani P, Ranucci E, Ferruti P (1994) Macromolecules 27:7171–7178Google Scholar
  23. 23.
    Manfredi A, Ranucci E, Suardi M, Ferruti P (2007) J Bioact Compat Pol 22:219–231Google Scholar
  24. 24.
    Zintchenko A, van der Aa LJ, Engbersen JFJ (2011) Macromol Rapid Commun 32:321–325Google Scholar
  25. 25.
    Ferruti P, Franchini J, Bencini M, Ranucci E, Zara GP, Serpe L, Primo L, Cavalli R (2007) Biomacromol 8:1498–1504Google Scholar
  26. 26.
    Malgesini B, Verpilio I, Duncan R, Ferruti P (2003) Macromol Biosci 3:59–66Google Scholar
  27. 27.
    Ranucci E, Ferruti P, Lattanzio E, Manfredi A, Rossi M, Mussini PR, Chiellini F, Bartoli C (2009) J Polym Sci, Part A: Polym Chem 47:6977–6991Google Scholar
  28. 28.
    Richardson S, Ferruti P, Duncan R (1999) J Drug Targeting 6:391–404Google Scholar
  29. 29.
    Baldi G, Bonacchi D, Innocenti F, Lorenzi G, Bitossi M, Ferruti P, Ranucci E, Ricci A, Comes Franchini M (Colorobbia Italia S.p.A.) PCT Int Appl WO 2008074804 A2 20080626, June 27 2008Google Scholar
  30. 30.
    Ferruti P, Mauro N, Falciola L, Pifferi V, Bartoli C, Gazzarri M, Chiellini F, Ranucci E (2014) Macromol Biosci 14:390–400Google Scholar
  31. 31.
    Manfredi A, Mauro N, Terenzi A, Alongi J, Lazzari F, Ganazzoli F, Raffaini G, Ranucci E, Ferruti P (2017) ACS Macro Lett 6:987–991Google Scholar
  32. 32.
    Ferruti P, Manzoni S, Richardson SCW, Duncan R, Pattrick NG, Mendichi R, Casolaro M (2000) Macromolecules 33:7793–7800Google Scholar
  33. 33.
    Gyarmati B, Némethy Á, Szilágyi A (2013) Eur Polym J 49:1268–1286Google Scholar
  34. 34.
    Emilitri E, Ranucci E, Ferruti P (2005) J Polym Sci, Part A: Polym Chem 43:1404–1416Google Scholar
  35. 35.
    Jeong JH, Kim TI, Bae JW, Park KD (2016) In: Torchilin VP (ed) Smart pharmaceutical nanocarriers. Imperial College Press, LondonGoogle Scholar
  36. 36.
    Emilitri E, Ferruti P, Annunziata R, Ranucci E, Rossi M, Falciola L, Mussini P, Chiellini F, Bartoli C (2007) Macromolecules 40:4785–4793Google Scholar
  37. 37.
    Piest M, Lin C, Mateos-Timoneda MA, Lok MC, Hennink WE, Engbersen JFJ (2008) J Control Release 130:38–45Google Scholar
  38. 38.
    Yu Z, Yan J, You Y (2011) J Control Release 152:e179–e181Google Scholar
  39. 39.
    Ranucci E, Suardi MA, Annunziata R, Ferruti P, Chiellini F, Bartoli C (2008) Biomacromol 9:2693–2704Google Scholar
  40. 40.
    Ranucci E, Ferruti P, Manfredi A, Suardi MA (2007) Macromol Rapid Commun 28:1243–1250Google Scholar
  41. 41.
    Cavalli R, Bisazza A, Bussano A, Trotta M, Civra A, Lembo D, Ranucci E, Ferruti P (2011) J. Drug Delivery 587604:9Google Scholar
  42. 42.
    Bernkop-Schnürch A (2005) Adv Drug Deliv Rev 57:1569–1582Google Scholar
  43. 43.
    Albrecht K, Bernkop-Schnürch A (2007) Nanomed 2:41–50Google Scholar
  44. 44.
    Donghi D, Maggioni D, D’Alfonso G, Amigoni A, Ranucci E, Ferruti P, Manfredi A, Fenili F, Bisazza A, Cavalli R (2009) Biomacromol 10:3273–3282Google Scholar
  45. 45.
    Flory PJ (1953) Principles of polymer chemistry. Cornell University Press, LondonGoogle Scholar
  46. 46.
    Ferruti P, Arnoldi D, Marchisio MA, Martuscelli E, Palma M, Riva F, Provenzale L (1977) J Polym Sci 15:2151–2162Google Scholar
  47. 47.
    Tanzi MC, Levi M (1989) J Biomed Mat Res 23:863–881Google Scholar
  48. 48.
    Barbucci R, Benvenuti M, Dal Maso G, Ferruti P, Tempesti F, Lemm WG (1987) Biomaterials 8:306–307Google Scholar
  49. 49.
    Barbucci R, Magnani A (1989) Biomaterials 10:429–432Google Scholar
  50. 50.
    Barbucci R, Casolaro M, Magnani A, Roncolini C (1991) Polymer 32:897–903Google Scholar
  51. 51.
    Barbucci R, Albanese A, Magnani A, Tempesti F (1991) J Biomed Mater Res 25:1259–1274Google Scholar
  52. 52.
    Barbucci R, Magnani A, Albanese A, Tempesti F (1991) Int J Artificial Organs 14:499–507Google Scholar
  53. 53.
    Barbucci R, Tempesti F, Benvenuti M, Magnani A, Albanese A (1992) Adv Biomat Sci 10:217–228Google Scholar
  54. 54.
    Barbucci R, Magnani A (1994) Biomaterials 15:955–962Google Scholar
  55. 55.
    Cimmino S, Martuscelli E, Silvestre C, Barbucci R, Magnani A, Tempesti F (1993) J Appl Polym Sci 47:631–643Google Scholar
  56. 56.
    Albanese A, Barbucci R, Belleville J, Bowry S, Eloy R, Lemke HD, Sabatini L (1994) Biomaterials 15:129–136Google Scholar
  57. 57.
    Tanzi MC, Barzaghi B, Anouchinsky R, Bilenkis S, Penhasi A, Cohn D (1992) Biomaterials 13:425–431Google Scholar
  58. 58.
    Ranucci E, Ferruti P (1999) Synth Commun 20:2951–2957Google Scholar
  59. 59.
    Ranucci E, Ferruti P (1991) Macromolecules 24:3747–3752Google Scholar
  60. 60.
    Vansteenkiste S, Schacht E, Ranucci E, Ferruti P (1992) Makromol Chem 193:937–943Google Scholar
  61. 61.
    Ushakova V, Panarin E, Ranucci E, Bignotti F, Ferruti P (1995) Macromol Chem Phys 196:2927–2939Google Scholar
  62. 62.
    Ranucci E, Spagnoli G, Sartore L, Ferruti P, Caliceti P, Schiavon O, Francesco MV (1994) Macromol Chem Phys 195:3469–3479Google Scholar
  63. 63.
    Ranucci E, Bignotti F, Paderno PL, Paolo Ferruti P (1995) Polymer 36:2989–2994Google Scholar
  64. 64.
    Caldwell G, Neuse E, Stephanou A (1993) J Appl Polym Sci 50:393–401Google Scholar
  65. 65.
    Caldwell G, Neuse EW, van Rensburg CEJ (1997) J Inorg Organomet P 7:217–231Google Scholar
  66. 66.
    Mauro N, Manfredi A, Ranucci E, Procacci P, Laus M, Antonioli D, Mantovani C, Magnaghi V, Ferruti P (2012) Macromol Biosci 13:332–347Google Scholar
  67. 67.
    Magnaghi V, Conte V, Procacci P, Pivato G, Cortese P, Cavalli R, Pajardi G, Ranucci E, Fenili F, Manfredi A, Ferruti P (2011) J Biomed Mater Res, Part A 98A:19–31Google Scholar
  68. 68.
    Marchisio MA, Ferruti P, Longo T, Danusso F, (Zambon S.p.A.) US Patent 3865723, October 18 1973Google Scholar
  69. 69.
    Mauro N, Chiellini F, Bartoli C, Gazzarri M, Laus M, Antonioli D, Griffiths P, Manfredi A, Ranucci E, Ferruti P (2017) J Tissue Eng Regen Med 11:2164–2175Google Scholar
  70. 70.
    Gualandi C, Bloise N, Mauro N, Ferruti P, Manfredi A, Sampaolesi M, Liguori A, Laurita R, Gherardi M, Colombo V, Visai L, Focarete ML, Ranucci E (2016) Macromol Biosci 16:1533–1544Google Scholar
  71. 71.
    Ferruti P, Ranucci E, Sartore L, Bignotti F, Marchisio MA, Bianciardi P, Veronese FM (1994) Biomaterials 15:1235–1241Google Scholar
  72. 72.
    Ferruti P, Bianchi S, Ranucci E, Chiellini F, Caruso V (2005) Macromol Biosci 5:613–622Google Scholar
  73. 73.
    Ranucci E, Spagnoli G, Ferruti P, Sgouras D, Duncan R (1991) J Biomat Sci Polym Ed 2:303–315Google Scholar
  74. 74.
    Barbucci R, Ferruti P, Micheloni M, Delfini M, Segre AL, Conti F (1980) Polymer 21:81–85Google Scholar
  75. 75.
    Barbucci R, Casolaro M, Ferruti P, Barone V, Lelj F, Oliva L (1981) Macromolecules 14:1203–1209Google Scholar
  76. 76.
    Katchalsky A, Spitnik P (1947) J Polym Sci 2:432–446Google Scholar
  77. 77.
    Martell AE, Motekaitis RJ (1992) Determination and use of stability constants, 2nd edn. Wiley-VCH, New YorkGoogle Scholar
  78. 78.
    De Levie R (1999) Aqueous acid-base equilibria and titrations. Oxford University Press, New YorkGoogle Scholar
  79. 79.
    Barbucci R, Casolaro M, Ferruti P, Tanzi MC, Grassi L (1984) MC, Barozzi C. Makromol Chem 185:1525–1535Google Scholar
  80. 80.
    Barbucci R, Casolaro M, Nocentini M, Corezzi S, Ferruti P, Barone V (1986) Macromolecules 19:37–42Google Scholar
  81. 81.
    Wang X, Gan H, Sun T, Su B, Fuchs H, Vestweber D, Butz S (2010) Soft Matter 6:3851–3855Google Scholar
  82. 82.
    Tonna N, Bianco F, Matteoli M, Cagnoli C, Antonucci F, Manfredi A, Mauro N, Ranucci E, Ferruti P (2014) Sci Technol Adv Mater 15:045007Google Scholar
  83. 83.
    Ferruti P, Bianchi S, Ranucci E, Chiellini F, Piras AM (2005) Biomacromol 6:2229–2235Google Scholar
  84. 84.
    Jacchetti E, Emilitri E, Rodighiero S, Indrieri M, Gianfelice AC, Ranucci Podestà A, Ferruti P (2008) J Nanobiotech 6:14. Google Scholar
  85. 85.
    Dos Reis G, Fenili F, Gianfelice A, Bongiorno G, Marchesi D, Scopelliti PE, Borgonovo A, Podestà A, Indrieri M, Ranucci E, Ferruti P, Lenardi C, Milani P (2010) Macromol Biosci 10:842–852Google Scholar
  86. 86.
    Monnery BD, Wright M, Cavill R, Hoogenboom R, Shaunak S, Steinke JH, Thanou M (2017) Int J Pharm 521:249–258Google Scholar
  87. 87.
    Hunter AC, Moghimi SM (2010) Biochim Biophys Acta 1797:1203–1209Google Scholar
  88. 88.
    Almulathanon AAY, Ranucci E, Ferruti P, Garnett MC, Bosquillon C (2018) Pharm Res 35:86Google Scholar
  89. 89.
    Ferruti P, Knobloch S, Ranucci E, Duncan R, Gianasi E (1998) Macromol Chem Phys 199:2565–2575Google Scholar
  90. 90.
    Franchini J, Ranucci E, Ferruti P, Rossi M, Cavalli R (2006) Biomacromol 7:1215–1222Google Scholar
  91. 91.
    Matsumura Y, Maeda H (1986) Cancer Res 46:6387–6392Google Scholar
  92. 92.
    Schacht E, Ferruti P, Duncan R Chem. Abstr. 1995, 595, 248301a, WO 9505,200Google Scholar
  93. 93.
    Ferruti P, Ranucci E, Trotta F, Gianasi E, Evagorou EG, Wasil M, Wilson G, Duncan R (1999) Macromol Chem Phys 200:1644–1654Google Scholar
  94. 94.
    Ndamase AS, Aderibigbe BA, Sadiku ER, Labuschagne P, Lemmer Y, Ray SS, Nwamadi M (2018) J Drug Deliv Sci Tec 43:267–273Google Scholar
  95. 95.
    Lavignac N, Nicholls JL, Ferruti P, Duncan R (2009) Macromol Biosci 9:480–487Google Scholar
  96. 96.
    Nutan B, Chandel AKS, Bhalani DV, Jewrajka SK (2017) Polymer 111:265–274Google Scholar
  97. 97.
    Urbán P, Valle-Delgado JJ, Mauro N, Marques J, Manfredi A, Rottmann M, Ranucci E, Ferruti P, Fernàndez-Busquets X (2014) J Contr Release 177:84–95Google Scholar
  98. 98.
    Martí Coma-Cros E, Biosca A, Marques J, Carol L, Urbán P, Berenguer D, Riera MC, Delves M, Sinden RE, Valle-Delgado JJ, Spanos L, Siden-Kiamos I, Pérez P, Paaijmans K, Rottmann M, Manfredi A, Ferruti P, Ranucci E, Fernàndez-Busquets X (2018) Polymers 10:225Google Scholar
  99. 99.
    Gussoni M, Greco F, Ferruti P, Ranucci E, Ponti A, Zetta L (2008) New J Chem 32:323–332Google Scholar
  100. 100.
    Maggioni D, Fenili F, D’Alfonso L, Donghi D, Panigati M, Zanoni I, Marzi R, Manfredi A, Ferruti P, D’Alfonso G, Ranucci E (2012) Inorg Chem 51:12776–12788Google Scholar
  101. 101.
    Maggioni D, Galli M, D’Alfonso L, Inverso D, Dozzi MV, Sironi L, Iannacone M, Collini M, Ferruti P, Ranucci E, D’Alfonso G (2015) Inorg Chem 54:544–553Google Scholar
  102. 102.
    Galli M, Rossotti B, Arosio P, Ferretti AM, Panigati M, Ranucci E, Ferruti P, Salvati A, Maggioni D (2019) Colloid Surface B 174:260–269Google Scholar
  103. 103.
    Nevo A, De Vries A, Katchalsky A (1955) Biochim Biophys Acta 17:536–547Google Scholar
  104. 104.
    Benjaminsen RV, Mattebjerg MA, Henriksen JR, Moghimi SM, Andresen TL (2013) Mol Ther 21:149–157Google Scholar
  105. 105.
    Duncan R, Ferruti P, Sgouras D, Tuboku-Metzger A, Ranucci E, Bignotti F (1994) J Drug Targeting 2:341–347Google Scholar
  106. 106.
    Richardson S, Pattrick NG, Lavignac N, Ferruti P, Duncan R (2010) J Control Release 142:78–88Google Scholar
  107. 107.
    Pattrick NG, Richardson SCW, Casolaro M, Ferruti P, Duncan R (2001) J Control Release 77:225–232Google Scholar
  108. 108.
    Lavignac N, Lazenby M, Franchini J, Ferruti P, Duncan R (2005) Int J Pharm 300:102–112Google Scholar
  109. 109.
    Hill IRC, Garnett MC, Bignotti F, Davis SS (2001) Anal Biochem 291:62–68Google Scholar
  110. 110.
    Rackstraw BJ, Stolnik S, Davis SS, Bignotti F, Garnett MC (2002) Biochim Biophys Acta 1576:269–286Google Scholar
  111. 111.
    Parkhouse SM, Garnett MC, Chan WC (2008) Bioorgan Med Chem 16:6641–6650Google Scholar
  112. 112.
    Peng L, Liu M, Xue Y-N, Huang S-W, Zhuo R-X (2009) Biomaterials 30:5825–5833Google Scholar
  113. 113.
    Liu M, Chen J, Cheng Y-P, Xue Y-N, Zhuo R-X, Huang S-W (2010) Macromol Biosci 10:384–392Google Scholar
  114. 114.
    Min L, Liu BC, Yanan X, Jie H, Liming Z, Huang S, Li Q, Zhijun Z (2001) Bioconj Chem 22:2237–2243Google Scholar
  115. 115.
    Cavalli R, Bisazza A, Sessa R, Primo L, Fenili F, Manfredi A, Ranucci E, Ferruti P (2010) Biomacromolecules 11:2667–2674Google Scholar
  116. 116.
    Cavalli R, Primo L, Sessa R, Chiaverina G, di Blasio L, Alongi J, Manfredi A, Ranucci E, Ferruti P (2017) J Drug Targeting 25:891–898Google Scholar
  117. 117.
    Coué G, Engbersen JFJ (2010) J Control Release 148:e9–e10Google Scholar
  118. 118.
    Sun M, Wang K, Oupický D (2018) Adv Healthcare Mater 7:1701070Google Scholar
  119. 119.
    Elzes MR, Akeroyd N, Engbersen JFJ, Paulusse JMJ (2016) J Contr Release 244:357–365Google Scholar
  120. 120.
    Yang Z, Sun Y, Xian L, Xun Z, Yu J, Yang T, Zhao X, Cai C, Wang D, Ding P (2019) J Cell Biochem 119:1767–1779Google Scholar
  121. 121.
    Won Y-W, Ankoné M, Engbersen JFJ, Feijen J, Kim SW (2016) Macromol Biosci 16:619–626Google Scholar
  122. 122.
    Sun Y, Liu H, Xing H, Lang L, Cheng L, Yang T, Yang L, Ding P Polymer International (2018)
  123. 123.
    Zhang J, Wang C, Lu M, Xing H, Yang T, Cai C, Zhao X, Wei M, Yu J, Ding P (2018) Asian J Pharm Sci 13:360–372Google Scholar
  124. 124.
    Wan L, You Y, Zou Y, Oupicky D, Mao G (2009) J Phys Chem B 113:13735–13741Google Scholar
  125. 125.
    Li M, Zhou X, Zeng X, Wang C, Xu J, Ma D, Xue W (2016) J Mater Chem B 4:547–556Google Scholar
  126. 126.
    Tang Q, Ma X, Zhang Y, Cai X, Xue W, Ma D (2018) Acta Biomater 69:277–289Google Scholar
  127. 127.
    Chen J, Wu C, Oupicky D (2009) Biomacromol 10:2921–2927Google Scholar
  128. 128.
    Garnett MC, Ferruti P, Ranucci E, Suardi M, Heyde M, Sleat R (2009) Biochem Soc Trans 37:713–716Google Scholar
  129. 129.
    Bishop CJ, Tzeng SY, Green JJ (2015) Acta Biomater 11:393–403Google Scholar
  130. 130.
    Chen G, Wang KK, Wang YX, Wu PK, Sun MJ, Oupicky D (2018) Adv Healthcare Mater 7:1700978Google Scholar
  131. 131.
    Xing H, Lu M, Yang T, Liu H, Sun Y, Zhao X, Xu H, Yang L, Ding P (2019) Acta Biomaterialia. Google Scholar
  132. 132.
    Sun Y, Liu H, Yang T, Lang L, Cheng L, Xing H, Yang L, Ding P (2019) Colloid Surface B 175:10–17Google Scholar
  133. 133.
    Caruso E, Ferrara S, Ferruti P, Manfredi A, Ranucci E, Orlandi VT (2018) Lasers Med Sci 33:1401–1407Google Scholar
  134. 134.
    Ferruti P, Danusso F, Franchi G, Polentarutti N, Garattini S (1973) J Med Chem 16:496–499Google Scholar
  135. 135.
    Franchi G, Morasca L, Reyers I, Garattini S (1971) Eur J Cancer 7:533–544Google Scholar
  136. 136.
    Bencini M, Ranucci E, Ferruti P, Trotta F, Donalisio M, Cornaglia M, Lembo D, Cavalli R (2008) J Control Release 126:17–25Google Scholar
  137. 137.
    Donalisio D, Ranucci E, Cagno V, Civra A, Manfredi A, Cavalli R, Ferruti P, Lembo D (2014) Antimicrob Agents Chemother 58:6315e6319Google Scholar
  138. 138.
    Shukla D, Spear PG (2001) J Clin Investig 108:503e510Google Scholar
  139. 139.
    Bousarghin L, Touze A, Combita-Rojas L, Coursaget P (2003) J Gen Virol 84:157–164Google Scholar
  140. 140.
    Cagno V, Donalisio M, Bugatti A, Civra A, Cavalli R, Ranucci E, Ferruti P, Rusnati M, Lembo D (2015) Antimicrob Agents Chemother 59:5250e5259Google Scholar
  141. 141.
    Donalisio M, Quaranta P, Chiuppesi F, Pistello M, Cagno V, Cavalli R, Volante M, Bugatti A, Rusnati M, Ranucci E, Ferruti P, Lembo D (2016) Biomaterials 85:40–53Google Scholar
  142. 142.
    Mauro N, Ferruti P, Ranucci E, Manfredi A, Berzi A, Clerici M, Cagno V, Lembo D, Palmioli A, Sattin S (2016) Sci. Rep. 6:33393. Google Scholar
  143. 143.
    Sánchez-Navarro M, Rojo J (2010) Drug News Perspect 23:557–572Google Scholar
  144. 144.
    Casolaro M, Casolaro I, Spreafico A, Capperucci C, Frediani B, Marcolongo R, Margiotta N, Ostuni R, Mendichi R, Samperi F, Ishii T, Ito Y (2006) Biomacromol 7:3417–3427Google Scholar
  145. 145.
    Hu B, Pei F, Sun X, Liang Y, He Z, Zhang L (2018) New J Chem 42:19600–19607Google Scholar
  146. 146.
    Annunziata R, Franchini J, Ranucci E, Ferruti P (2007) Magn Reson Chem 45:51–58Google Scholar
  147. 147.
    Calucci L, Forte C, Ranucci E (2007) Biomacromol 8:2936–2942Google Scholar
  148. 148.
    Calucci L, Forte C, Ranucci E (2008) J Chem Phys 129:064511Google Scholar
  149. 149.
    Calucci L, Forte C, Ranucci E (2009) Langmuir 25:2449–2455Google Scholar
  150. 150.
    Ranucci E, Ferruti P, Lenardi C, Matteoli M (Neurozone s.r.l.) WO2010099962, September 10 2010Google Scholar
  151. 151.
    Urbán P, Valle-Delgado JJ, Mauro N, Marques J, Manfredi A, Rottmann M, Ranucci E, Ferruti P, Fernàndez-Busquets X (2014) Use of poly(amidoamine) drug conjugates for the delivery of antimalarials to Plasmodium. J Control Release 177:84–95Google Scholar

Copyright information

© The Tunisian Chemical Society and Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Dipartimento di ChimicaMilanItaly

Personalised recommendations