Polyamidoamines: Versatile Bioactive Polymers with Potential for Biotechnological Applications
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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.
KeywordsPolyamidoamines Functional polymers Degradable polymers Bioactive polymers Biocompatible polymers
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 . 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 , 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 presence of chain end groups besides those expected from the monomer ratio, as well as of cyclic structure, was excluded by NMR analysis . 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 . 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  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 .
Structure of cationic PAAs
2.2 Amphoteric PAAs
Structure of amphoteric PAAs
Acid-base properties of some amphoteric PAAs
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 . 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) .
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 . 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.
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).
2.4 PAA Block and Graft Copolymers
Linear polyurethane-polyamidoamine (PUPA) block copolymers were obtained . 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 . PUPA based materials proved able to adsorb significant heparin amounts, remarkably improving their hemocompatibility , thanks to the formation of polyelectrolyte complexes with the negatively charged heparin and the positively charged PAA segments . Similarly, different amine-terminated PAA macromonomers were grafted onto polyetherurethaneamide surfaces functionalized with fumaric or maleic acid through the Michael type addition .
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).
2.6 Hydrolytic Degradation of PAAs
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].
2.8 Chiral PAAs
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 . Their cytotoxicity has been ascribed to the destabilization of the cell membrane followed by disruption of the mitochondrial functions that causes apoptosis .
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 . 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 . 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.
IC50 values for modified PLL
IC50 (µg mL−1)
IC50 (µg mL−1)
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 body distribution of ISA1 and -ISA23 containing 1% on a molar basis units deriving from radioactive 125I-labelled tyrosine was studied in rats . 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 . 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 .
3.2 PAA-anticancer drug conjugates
ISA1-mitomycin (MMC) (Table 1 for ISA1 structure) conjugates  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 . PAA conjugates with pamidronate and platinum complexes showed in vitro anticancer activity combined with inferior general cytotoxicity compared to the free drug .
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 . 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 .
3.3 PAAs as Nanocarriers of Antimalarial Drugs
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 . 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
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) . 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.
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 .
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 , 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.
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 . 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 . 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  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 . 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 . This research provided direct evidence that ISA1 permeabilizes the vesicular membranes of endosomes by moderate physical interaction without inducing the proton sponge effect.
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 . 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].
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 .
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 .
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 .
Interestingly, PAA-PEG nanosized constructs crosslinked with disulfide functions, grafted with thiol-end-capped DNA, were also employed as tracers of environmental organic pollutants .
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 .
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 .
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 . 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
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 .
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 . 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 . Accordingly, AGMA1 did not kill the virus, but blocked the infection transmission from cell to cell .
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 . Moreover, AGMA1 did not influence the vaginal pH, since proved inactive towards Lactobacillus spp.
4.4 PAAs as Cell Adhesion Promoters
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 .
4.6 PAA Hydrogels and as Scaffolds for Tissue Engineering
Their comprehensive structural characterization can be performed by high-resolution magic angle spinning (HRMAS) NMR spectroscopy . 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 . 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 . 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 . 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.
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 . 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 .
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 . 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.
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.Danusso F, Ferruti P, Ferroni G (1967) Chimica e Industria 49:271–278Google Scholar
- 6.Tomalia DA, Dewald JR (The Dow Chemical Corporation) U.S. Patent 4 507 466, 1983Google Scholar
- 9.Barbucci R, Casolaro M, Beni MC, Ferruti P, Pesavento M, Soldi T, Riolo C (1981) JCS Dalton 2559–2564Google Scholar
- 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
- 35.Jeong JH, Kim TI, Bae JW, Park KD (2016) In: Torchilin VP (ed) Smart pharmaceutical nanocarriers. Imperial College Press, LondonGoogle Scholar
- 41.Cavalli R, Bisazza A, Bussano A, Trotta M, Civra A, Lembo D, Ranucci E, Ferruti P (2011) J. Drug Delivery 587604:9Google Scholar
- 45.Flory PJ (1953) Principles of polymer chemistry. Cornell University Press, LondonGoogle Scholar
- 46.Ferruti P, Arnoldi D, Marchisio MA, Martuscelli E, Palma M, Riva F, Provenzale L (1977) J Polym Sci 15:2151–2162Google Scholar
- 53.Barbucci R, Tempesti F, Benvenuti M, Magnani A, Albanese A (1992) Adv Biomat Sci 10:217–228Google Scholar
- 68.Marchisio MA, Ferruti P, Longo T, Danusso F, (Zambon S.p.A.) US Patent 3865723, October 18 1973Google Scholar
- 77.Martell AE, Motekaitis RJ (1992) Determination and use of stability constants, 2nd edn. Wiley-VCH, New YorkGoogle Scholar
- 78.De Levie R (1999) Aqueous acid-base equilibria and titrations. Oxford University Press, New YorkGoogle Scholar
- 91.Matsumura Y, Maeda H (1986) Cancer Res 46:6387–6392Google Scholar
- 92.Schacht E, Ferruti P, Duncan R Chem. Abstr. 1995, 595, 248301a, WO 9505,200Google Scholar
- 114.Min L, Liu BC, Yanan X, Jie H, Liming Z, Huang S, Li Q, Zhijun Z (2001) Bioconj Chem 22:2237–2243Google Scholar
- 122.Sun Y, Liu H, Xing H, Lang L, Cheng L, Yang T, Yang L, Ding P Polymer International (2018) https://doi.org/10.1002/pi.5728
- 150.Ranucci E, Ferruti P, Lenardi C, Matteoli M (Neurozone s.r.l.) WO2010099962, September 10 2010Google Scholar