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The second half of the twentieth century has witnessed the outstanding development of polymeric materials, or “plastics,” in every aspect of life, from everyday housing commodities to biomedical devices. In the new century, one of the most exciting frontiers of macromolecular science lies in the development of purpose tailored, sophisticated functional polymers, that is, polymers endowed with reactive chemical functions whose type, number, and arrangement have been planned to specific purposes. For instance, a large deal of polymer therapeutics involve functional polymers, such as membrane-active polymers with a potential as transfection promoters, polymer drug carriers, polymers endowed with specific biological activities of their own, polymers able to promote the lysosomal escape, and the intracellular trafficking of proteins, polymeric hydrogels for cell culturing and tissue engineering, polymer nanoparticles, and their multiform uses. Besides medicine, functional polymers find a number of technical applications, such as specific ion-exchange resins, noxious water pollutants absorbers, active coatings of sensors for pollutants detection, agents for surface modification, and many others.
The aim of this article is to provide an updated state-of-the-art on chemistry and applications of a particular family of synthetic functional polymers, poly(amidoamine)s (PAAs), originally obtained by stepwise Michael-type polyaddition of prim-monoamines [Scheme 1(a)] or sec-diamines [Scheme 1(b)] with bisacrylamides.
The Michael-type addition of prim- and sec-amines to carbon–carbon double bonds activated by adjacent electron-attracting groups is well known since the first half of the twentieth century. The first intimation of a high polymer prepared by polyaddition of sec-diamines with bisacrylamides is in a patent application dating back to 1956, which apparently was no further developed. An extensive study on this type of stepwise polyaddition was independently started in the mid-sixties of the twentieth century at the Polytechnic Institute of Milan and the first resultant polymers, called PAAs, were first published in a series of papers on an Italian Journal, together with structurally related polymers obtained by substituting bisacrylic esters and divinylsulfone for bisacrylamides or phosphines and hydrazines for amines.[2-8] These early articles were soon collectively reviewed on a British Journal. Slightly afterwards, some PAAs were also independently reported elsewhere. Subsequently, the chemistry of PAAs was reviewed at intervals.[11-13]
Several new PAA-related polymers, such as for instance those derived by stepwise polyaddition of aminated bis-thiols with bisacrylamides,[14, 15] bisacrylic esters,[16, 17] divinylsulfone, and bis-cyanovinyl compounds, were later reported.
The self-polyaddition of 1-acrylamido-2-aminoethane hydrochloride gave rise in the early eighties of the nineteenth century to a highly successful family of hyperbranched, but not crosslinked polymers also called poly(amidoamine)s but initialed PAMAM, which were extensively studied and modified in view of a number of biotechnological applications, reported in hundreds of articles and reviews. PAMAMs should not be confused with PAAs, as they constitute a different polymer family, inasmuch as in their dendrimer-like backbone each amide group (a) is preceded and followed by an amine (b) group and vice-versa, that is, with the sequence …a…b…a…b…, whereas in the PAA chains the sequence of the same groups is either …a…a…b…a…a…b…. or …a…a…b…b…a…a…b…b. depending on whether prim-amines or bis-sec-amines were involved in their preparation. Moreover, the PAMAM multiple chains contain sec- and tert-amine groups and the chain termini are prim-amine groups. On the opposite, unless purposely functionalized or crosslinked, PAAs are linear, single chain amine- (nearly always tert-amine-) polymers and their chain termini are either vinyl- or sec-amine groups. PAMAMs will not be included in this review.
The Michael polyaddition leading to PAAs is best performed in aqueous media or, alternatively, alcohols. Ethylene glycol is the best substitute for water as reaction solvent. Methanol, ethanol, N-methyl-N,N-di-2-hydroxyethylamine and benzyl alcohol can also be used to overcome solubility problems. A source of protons is necessary to speed up the reaction and obtain high molecular weight products in reasonable time, whereas aprotic solvents are unsuitable as reaction media.[2, 7] A comparative kinetic study on the polyaddition kinetics of 2-methylpiperazine and 2,5-dimethylpiperazine with 1,4-bisacryloylpiperazine, chosen as model PAA monomers, was performed in water, methanol, ethylene glycol, formamide, and dimethylformamide. In the protic solvents, the polyaddition involving 2-methylpiperazine proceeded through a two-step mechanism, each step involving one of the two different sec-amine groups, whose reaction constants were significantly different owing to the different steric hindrance by the neighboring groups. Each step followed pseudo-second-order kinetics. The kinetic constants included the catalytic protic species. In dimethylformamide, the polyaddition proceeded through third-order kinetics. This accounted for the autocatalytic activity of the amine groups. The apparent kinetic constants in the protic solvents increased with the increase of the autoprotolysis constant value and decreased with the increase of the dipole moment.
Recently, it was reported that salts of earth alkali metals exert a catalytic activity on the Michael reaction of prim-amines to bis-acrylamides, whereas the salts of transition metals are apparently inactive. In particular, the addition of CaCl2 to the reaction mixtures led to a significant increase in the reaction rate and the PAAs obtained in its presence were identical to those prepared by the conventional method. Doubtless, this technique may represent an attractive improvement in the preparation of PAAs from poorly reactive amines.
Both prim-monoamines and sec-diamines lead to linear polymers. prim-Diamines usually act as tetrafunctional monomers and give crosslinked insoluble resins. However, by employing low reactant concentrations, low initial temperatures and excess bis-amines, soluble PAAs carrying sec- instead of tert-amine groups in the main chain were obtained in some instances. These PAAs allowed drug attachment and were studied as soluble drug carriers.
PAAs are not only intrinsically functional polymers, but are also amenable to further functionalization. A number of chemical functions, such as hydroxy-, tert-amine-, allyl-, amide-, and ether groups if present in the monomers do not interfere in the polymerization process and remain as side substituents in the resultant PAAs. Chemical groups capable of reacting with activated double bonds, such as SH, NH2, NHR, and PH2, cannot be directly introduced in PAAs as side substituents, but can with special precautions. Additional prim- or sec-amine groups, for instance, must be protected first by groups cleaved by acids, but stable under basic conditions. When the protonation constants of a prim-diamine are widely different, monoprotonation may suffice without any further protection. For instance, monoprotonated 1,2-bis-aminoethane yielded soluble PAAs carrying prim-amine groups as side substituents. For the same reason, by reacting partially protonated poly-(l)-lysine with a large excess N,N-dimethylacrylamide in aqueous media, the reaction stopped when the unprotonated amine groups, and only these, were saturated. Accordingly, linear PAAs carrying guanidine groups as side substituents are currently prepared from 4-aminobutylguanidine in water at pH ≤ 9.
It was immediately realized that nearly all conceivable bisacrylamides, sec-diamines and, contrary to a first statement, also prim-amines were eligible as monomers. Most bisacrylamides, monoamines, and bisamines employed so far in PAA synthesis are reported in Tables 1-3, respectively. For the sake of simplicity, in Tables 2 and 3 “monoamines” and “bisamines” are defined as the amines that contribute to chain building with one or two nitrogen atoms, respectively, not considering the actual number of nitrogen atoms present in their molecule.
Table 1. Bisacrylamide Monomers
Table 2. Monoamine Moieties
Table 3. Diamine Moieties
Random or quasi-random copolymeric PAAs are obtained in most cases starting from mixtures of monomers with no further precautions. However, when a limited number of poorly reactive amines need to be introduced in a PAA chain, performing the reaction in a single pot, but in two steps, is advisable. The less reactive amine should be treated first with a large excess bisacrylamide, thus forcing it to react completely. The polymerizing system, containing vinyl-terminated trimers or short oligomers, is then added with the more reactive amine in the amount needed to stoichiometrically balance the reactant functions and the polyaddition is let to proceed normally. This technique ensures a fair distribution of the less reactive amine moieties in the final product by minimizing the risk of confining them at the chain ends of the lowest molecular weight fractions.
From the above considerations it is clearly apparent that PAAs, as a class, are endowed with versatility nearly unique among stepwise polymers. Since, in addition, they are assembled in a modular fashion both as homo- and copolymers, they are particularly amenable to structure-tailoring for specific purposes. There is little doubt that the already large number of PAAs described in the literature still represents only a minor part of their synthetic potential.
The structures of most soluble PAAs described so far in the literature, apart from two special cathegories (see below), are reported in Tables 4 and 5. In particular, the PAAs listed in Table 4 contain monoamine-deriving units, defined as earlier, either alone or as copolymers with diamines, whereas the PAAs listed in Table 5 contain only diamine-deriving units.
PAAs characterized, respectively, by acidic functions imparting them amphoteric properties or by SH or SS groups, the latter both as side substituents or inserted in the polymer chain, will be treated separately.
Amphoteric PAAs are PAAs carrying acid functions as side substituents, usually carboxyl groups, but in a few instances also sulfonic[9, 44] or phosphonic groups. Aminoacids or carboxylated bis-acrylamides can be used as monomers. In the presence of acid groups, the polyaddition require triggering by a stoichiometric amount of base. After that, the polyaddition proceeds normally with carboxylated bisacrylamides and β-aminoacids or their higher analogs, such as β-alanine, γ-aminobutyl acid, and so forth. Glycine and peptides in which the first aminoacid residue is glycyl react slower. Natural α-aminoacids other than glycine react very sluggishly, often requiring months at room temperature to reach reasonably high molecular weight polymers.
Amphoteric PAAs present a unique interest as bioactive polymers. By tuning the number and nature of the ionizable groups, amphoteric PAAs with isoelectric points ranging from 3 to 10 can be obtained. The prevailingly basic ones are often considerably more biocompatible than purely cationic PAAs of similar net average positive charge at physiological pH, but maintain most polycation properties. For instance, they form interpolyelectrolyte complexes with polyanions such as heparin and DNA, may exert membrane activity, and may act as transfection promoters (see later).
Amphoteric PAAs that are prevailingly anionic at pH 7.4 do not exert membrane activity. However, in solution their net average charge is a function of pH. By a proper choice of the starting monomers the acid and basic strength of the amine and the carboxyl groups can be tuned in such a way that the resultant PAAs shift from a prevailingly anionic to a prevailingly cationic state for relatively modest pH changes, as for instance when they are internalized in cells and pass from the extracellular fluids, where the pH is 7.4, into cellular compartments where the pH is 5.5 or whereabouts. As a rule, this charge reversal renders these PAAs membrane active.
Most amphoteric PAAs described so far are reported in Table 6.
Considerable attention has been recently focused on polymers carrying thiol groups. They are especially studied for their mucoadhesive properties and find, inter alia, applications in nanomedicine. Polymers bearing dithio groups are strictly related to the thiol-bearing ones since oxidation–reduction processes can easily transform these groups into each other. Thiol-functionalized PAAs were obtained by employing N-mono-protected cystamine as co-monomer and then reductively cleaving the SS groups of the resultant polymers [Scheme 2(a)]. Alternatively, they were obtained by reduction of the crosslinked PAAs obtained by substituting plain cystamine for mono-protected cystamine [Scheme 2(a)]. The thiol groups could be transformed into activated dithio-derivatives by reaction with 2,2′-dithiodipyridine. Interestingly, the same activated dithio-derivatives were straightforwardly prepared in the presence of triethylamine by dithio–dithio exchange reaction of the crosslinked PAAs obtained from cystamine with excess 2,2′-dithiodipyridine [Scheme 2(b)]. These activated dithio derivatives easily underwent coupling reactions with added thiols, for instance thiocholesterol, obtaining amphiphilic polymers that in aqueous media spontaneously gave nano-aggregates [Scheme 2(c)]. The aggregation property is shared by other PAAs containing hydrophilic and hydrophobic moieties, but in the above derivatives the hydrophobic moieties are linked to the PAA chain by SS reductively cleavable bonds. Therefore, the aggregates are liable to collapse in reducing environments, as for instance after internalization in cells. PAA conjugates of SH-bearing peptides, such as glutathione, were also prepared by the same technique [Scheme 2(d)].[102, 103]
Polymers bearing SS bonds in the polymer chain are selectively degraded after cell internalization. When orally administered, the same polymers are stable in the first sections of the gastrointestinal tract, but degrade in the colon. PAAs are particularly amenable to this structural modification, since SS containing amines and bisacrylamides are either commercially available or easily synthesized, and if used as co-monomers straightforwardly lead to SS linkages in the polymer chain. This concept was first put forward in a pioneering paper reporting on the use of N,N′-bisacryloylcystamine or N,N′-bisacryloylcystine as amidic monomers for the specific purpose of obtaining bioreducible PAAs (Scheme 3).
This pioneering study was followed by an extensive series of articles on different SS bearing PAAs based on bis-acryloylcystamine, proposed as nucleic acid carriers, and transfection promoters as well as, in some cases, as protein carriers.[66, 105, 106] Alternatively, soluble SS PAAs were obtained by employing l-cystine as diamine comonomer, taking advantage of the different reactivity of the amine hydrogens of substituted α-aminoacids (Scheme 4).
Shortly later, N,N′-dimethylcystamine was also used for the same purpose. More recently, PAAs containing both SS and acetal acid-labile groups in the main chain were also reported.
Most thio-and dithio-functionalized PAAs described so far in the literature are reported in Table 7.
Since PAAs are prepared by stepwise polyaddition of amines with bisacrylamides (Scheme 1), driving the polymerization of stoichiometrically unbalanced monomer mixtures to completion leads to PAAs whose polymerization degree can be calculated from to the following well-know equation
where r is the ratio of the number of defect functions to the number of excess functions, but with qualifications. A purposely planned NMR study performed on the bisacryloylpiperazine/2-methylpiperazine PAA (Table 5, No. 34) as model showed that the eq (1) was strictly obeyed only with excess amine, whereas with excess bis-acrylamide the products invariably had molecular weight higher than expected, as further confirmed by unpublished results obtained with several other PAAs. An older viscometric study had pointed to the same conclusion. A possible explanation lies in the occurrence, during polymerization, of some hydrolytic cleavage of the terminal acrylamide groups. This would partly substitute amine groups for acrylamide groups, thus adjusting to some extent the stoichiometric balance, as under the basic polymerization conditions and at room temperature the double bond of the resultant acrylic acid is practically unreactive toward amines and does not act as chain-terminator.
It is apparent that stoichiometrically unbalanced monomer mixtures lead to PAAs doubly terminated with the excess function. Both vinyl- and sec-amine end capped PAAs (V-PAA and sec-A-PAA) have been prepared. In turn, V-PAAs can be converted into prim-amine end-capped PAAs (prim-A-PAA) by treating sec-A-PAAs with a large excess ammonia. All these PAAs can be regarded as macromonomers and were employed for preparing hybrid block- and graft copolymers with polymeric structures other than PAAs.
The first example of soluble and moldable PAA block copolymer involved the radical polymerization of styrene in the presence of a V-PAA (Scheme 5).
The addition of chain-transfer agents and the use of fairly high molecular weight samples of V-PAA prevented crosslinking.[119-121] Most subsequent preparations on PAA block- and graft-copolymers employed prim-A-PAAs and sec-A-PAAs. Graft copolymers were prepared by condensation with chlorosulfonated polyethylene[122-125] and with ethylene/vinylalcohol/vinylacetate terpolymer, the latter after activation of the hydroxyl groups with N,N′-carbonyldiimidazole. Graft copolymers were also obtained by reacting prim-A-PAA with poly(urethaneamide)s containing fumaric- or maleic acid moieties, that is, activated double bonds in their main chain. All the above grafting reactions lead to soluble polymers only by employing a large excess A-PAA to minimize the formation of intermolecular bridges. This recurrent problem was only recently overcome by synthesizing hetero-difunctional PAA dimers and polymers (see later).
Mixed poly(urethane-PAA) networks, named PUPA, were more extensively studied.[128-136] These materials were prepared by first treating A-PAA with excess diisocyanate in chloroform solution and subsequently coupling the resultant isocyanate-terminated PAA with a commercial polyurethane via the CONH groups of the latter (Scheme 6).
Tough films were cast from the reaction mixture, which could also be used for coating purposes. In another process, adding A-PAAs to the monomer mixtures leading to polyurethanes gave linear polyurethane-PAA block copolymers. Both prim-and sec-A-PAAs were used, but the former gave better results.
Block and graft copolymers of PAAs containing segments of different nature were also straightforwardly obtained by copolymerization techniques with amine-terminated oligomers, such as α-bis(sec-amino)polyoxyethylenes [Scheme 7(a)], α-methyl-ω-amino-polyoxyethylenes [Scheme 7(b)], or ω-amine-terminated poly(4-acryloylmorpholine) [Scheme 7(c)], in turn obtained by chain transfer technique by radical polymerization of 4-acryloylmorpholine in the presence of cysteamine).
Surface-Grafting PAAs Onto Inorganic and Organic Materials
PAAs have been surface-grafted on many organic and inorganic commercial materials, such as poly(ethyleneterephtalate), polyurethanes, plasticized poly(vinylchloride), glass, and silica.[142-145] All PAA surface-grafting processes involved reactive groups already present on the substrate or purposely introduced, capable of giving coupling reactions with amine or acrylamide groups. For instance, glass and silica were grafted after a previous treatment with 3-aminopropyltriethoxy silane. PAAs can be grafted onto surface-aminated materials by two different processes. The first one consists of carrying out the polyaddition of amines with bisacrylamides in the presence of the aminated material in heterogeneous phase [Scheme 8(a)]. The reaction involves the surface amine groups and a part of the resultant PAA remains covalently grafted, the ungrafted part being easily washed away. In the second method, an acrylamido-terminated PAA prepolymer is prepared, purified from the low molecular weight fractions and then reacted with the aminated material [Scheme 8(b)].
These two methods proved not equivalent. For instance, by grafting a Cu2+-complexing PAA (Table 5, No. 26) on finely subdivided aminated silica, the resultant PAA-silica conjugates contained 20% and 11% PAA on a wt/wt basis, respectively. Up to this point, the first method seemed more effective, but subsequent investigation revealed that the Cu2+-complexing capacity of the resultant conjugate was remarkably lower than expected. By contrast, the PAA-silica conjugate prepared by the second method displayed the expected Cu2+ complexing capacity, that is, one Cu2+ ion per repeating unit. Possibly, with the first method the lower-molecular-weight PAA species, being at all times the prevailing ones by numbers and, therefore, possessing the larger share of acrylamide end groups, had the higher probability of undergoing grafting reaction. Once immobilized, they might not exhibit the same chemical behavior as long-chain PAAs. With the second procedure, only long chain PAAs were present and, once grafted, behaved like their high molecular weight soluble conterparts. It is worth mentioning that soluble and crosslinked PAA-grafted albumin samples were also prepared by the same methods.[147, 148]
Crosslinked PAAs can be prepared by partially substituting multifunctional amines for sec-bisamines or prim-monoamines employed in the preparation of linear PAAs[11-13] (Scheme 9).
In the case of amphoteric PAAs carrying carboxyl groups in the acrylamide moieties, as for instance (Table 6, No. 28) this method has the serious drawback that the acid–base properties of the resultant hydrogels may be grossly altered with respect of its linear counterpart, since the amine/carboxyl ratio diminishes by increasing the amount of multifunctional amine in the monomer mixture.
PAA resins obtained from moltifunctional amines are usually very highly swellable in aqueous media unless for extreme crosslinking degrees, giving hydrogels with poor mechanical properties. However, exceptions exist. PAAs endowed of structure-forming properties may give hydrogels presenting good mechanical properties even in the swollen state. An alternative crosslinking method consists of triggering the radical polymerization of V-PAAs by UV irradiation, water-soluble diazo compunds, or redox systems. These methods lead to hydrogels whose crosslinking degree depends on the of the starting oligomer (Scheme 10). These hydrogels maintain intact the acid–base properties of their linear countarparts, are moderately swellable in aqueous media and, as a rule, exhibit better mechanical properties in the swollen state than those derived from multifunctinal amines. The presence in their network of hydrophobic polyvinyl chains connecting the PAA chains probably explains this.
Resins coupling long PAA chains with high crosslinking degree can be obtained by partly substituting allylamine for the same quantity (on a molar basis) of amine monomers in the starting PAA. Mixed networks were also obtained by copolymerizing V-PAAs with traditional vinyl monomers such as N-vinylpyrrolidinone.
Hetero-Diterminated PAA Dimers and Polymers
It is apparent that in order to obtain high molecular weight PAAs, the functions involved, that is, activated double bonds and amine hydrogens, must be stoichiometrically balanced. A perfectly balanced mixture contains three types of macromolecules, a—.—a, b—.—b and a—.—b in 1:1:2 ratio. Unbalanced mixtures contain the same molecular species, albeit in different ratios, until the minority function is completely consumed. Only at this point the product will be entirely constituted of molecules doubly terminated with the excess function. There is no way, by the traditional method, to straightforwardly obtain PAAs with controlled hetero-difunctional chain terminals, that is, PAAs solely containing molecules of “a—.—b” type. This precluded or rendered it difficult to PAAs the access to the remarkable number of biotechnological applications, as for instance liposome preparation, drug conjugation, and protein modification, which have been so far nearly uniquely mastered by hetero-difunctional poly(ethyleneglycol)s (PEGs) that, however, are far to be endowed with the functional versatility of PAAs. Recently, a simple and straightforward preparation method of PAAs with hetero-difunctional chain ends and of several previously hardly obtainable PAA derivatives of biotechnological interest, such as for instance PAAs of controlled molecular weight and narrow polydispersity mono-functionalized at one end with an acrylamide group, PAAs with star-like molecular architecture, graft-PAA-protein conjugates, “tadpole-like” PAA conjugates with hydrophobic moieties able to self-assemble into nanoparticles in aqueous media, has been reported.
The key step was to design suitable building blocks consisting of hetero-difunctional dimers (HDDs), that is the mono-addition products of sec-diamines and bisacrylamides of the “a—a—b—b” type, obtained as hydrochlorides or trifluoroacetates. In this form, they could be indefinitely kept dormant at 0–5°C in the dry state, whereas at room temperature and in aqueous media at pH > 7.5 they polymerized according to Scheme 11.
The synthetic scope of HDDs is manifold. They can be polymerized to high molecular weight PAAs without bothering with stoichiometric balance. PAAs of controlled average molecular weight and mono-functionalized with an acrylamide- or a sec-amine group at one end can be prepared by adding, respectively, a controlled amount of monofunctional acrylamides or sec-amines, whereas the addition of multifunctional acrylamides or amines will lead to star-like PAAs [Scheme 12(a)]. Block PAA-PAA or PAA-PEG copolymers with controlled structure will be easily obtained. “Velvety-like” grafting of PAA chains to properly functionalized surfaces can be achieved. PAA chains of controlled average length can be grafted to proteins with no risk of undesirable side reactions such as protein–protein coupling or crosslinking [Scheme 12(b)]. “Tadpole-like” PAA conjugates carrying hydrophobic moieties forming in aqueous media liposomes or nanoparticles can be prepared as functional drug carriers.
Sequence-Defined PAAs by Polycondensation of Diacids with Polyamines
Linear polymers obtained by polycondensation of dicarboxylic acids or their activated derivatives with polyamines following synthetic procedures not consuming all the amine groups of the latter, such as those obtained by condensation of succinic or adipic acids with pentaethylenehexamine, other short ethyleneimine oligomers or peptides, might be defined as “inverted” PAAs. It should be observed, in fact, that compared with traditional PAAs the relative positions along the chain of the amide and amine nitrogens are inverted. Whereas in traditional PAAs a hypothetical observer leaving a chain amine group and proceeding along the chain would find the amidic CO, in these polycondensation polymers he would find the amidic NH. These polymers deserve attention because they are amenable to sequential solid-phase supported synthesis hardly suitable for traditional PAAs. The latter, in fact, rely on the Michael addition that typically benefits of a protic micro-environment and is highly biased by steric hindrance, making it difficult in a solid-phase supported synthesis to bring each step to completion. By contrast, the condensation polymers from dicarboxylic acids and polyamines can employ resins and automatic equipment commonly marketed to this purpose for peptide synthesis. Side functional short chains can be also introduced in definite positions. Linear oligomers were also obtained by polymerization of ethyl acrylate and N-methyl-1,3-diaminopropane, catalyzed by the Candida antarctica lipase. The enzyme catalyzes both the formation of the amide groups by reaction of the ester function with amines and the Michael addition involving the activated vinyl groups. Several studies on this subject have been published in the last years providing evidence of the potential of this novel synthetic tool,[154-160] and no doubt others will appear in the near future.
Most PAAs mentioned in this review have and in the range 5,000–30,000 and 10,000–50,000, respectively. The polydispersity index of unfractionated samples was approximately 2, with the exception of some PAAs obtained from HDDs, where it was remarkably lower and in some instances approached monodispersity, and the “inverted” PAAs prepared by solid-state supported synthesis. Obtaining high molecular weight PAAs by the traditional procedure is mainly a matter of solvent, monomer concentration, monomer steric hindrance, reaction temperature, and patience. As regards the reaction solvent, this topic has been already discussed in the “Synthetic features” paragraph. As regards concentration and steric hindrance, for the former the higher the better and for the latter, at least as the reaction rate is concerned, the lower the better. This is not surprising, as the Michael addition is an equilibrium reaction mostly affected by these parameters. As regards the reaction temperature, its influence is double-edged. By increasing the temperature, the polymerization rate increases as expected, but the molecular weight tends to level off at a progressively lower limiting value. This effect is minimal at room temperature, that is, 18–25°C. Therefore, patience is the most important requisite when dealing with sterically hindered monomers. At room temperature, the polymerization may go on even for months, but finally reasonably high molecular weight products can be obtained in nearly all conceivable cases. The use of CaCl2 as catalyst, recently proposed, may help speeding up these lethargic reactions.
All PAAs synthesized so far are soluble or swellable in water. As a rule, amphoteric PAAs dissolve only in water, but most non-amphoteric ones are soluble also in chloroform, lower alcohols, dimethylformamide, dimethylsulfoxide, and other polar solvents.
The intrinsic viscosities of PAAs in organic solvents or aqueous media usually range from 0.15 to 1 dL/g. As indicated by their viscometric values, PAAs usually exhibit larger hydrodynamic volumes in solution than most polyvinyl polymers of similar molecular mass. For instance, the results of a study performed on two typical PAAs (Table 5, No. 33 and Table 6, No. 38, that is, ISA23), are reported here. The Mark–Houwink–Sakurada (MHS) constants for samples of the former PAA of < 10,000 were k = 1.76 × 10−5 dL/g and a = 1.14. The amphoteric ISA23 was studied more detailedly within a wide range of molecular weights. The initial slope (a) of its MHS plot for M lower than 20,000 was very high, that is > 1. The plot exhibited a significant downward curvature at higher molecular masses. For a molar mass of 60,000 g/mol the ISA23 MHS constants were: k = 9.525 × 10−5 dL/g; a = 0.846. This PAA, and probably other amphoteric PAAs, reversibly aggregates in solution, whereas non-amphoteric PAAs of similar structure do not. ISA23 bears positive and negative charges in relatively distant locations along the polymer chain and electrostatic interactions are doubtless responsible for its tendency to aggregate.
Owing to their regular structure, many PAAs are partially crystalline in the solid state. Quite a few of them crystallize spontaneously on isolation, but in some instances, especially for PAAs deriving from primary amines, crystallization must be induced by solvent treatment. When cyclic structures without side substituents are present, such as in the bisacrylopylpiperazine/piperazine PAA (Table 5, No. 32) the polymer may crystallize even from water during polymerization. The melting point of this PAA is 270°C (with decomposition), the highest so far determined for these polymers.[9, 149] It is worth mentioning that the same PAA when crosslinked by the multifunctional amine method (see above) and for low to moderate crosslink density usually establishes crystalline domains, probably involving the linear chain segments in between the crosslink points. These domains are stable in aqueous systems and act as reinforcing fillers greatly enhancing the mechanical strength of the resultant hydrogels. Paradoxically, but not entirely unexpectedly, the modulus of these hydrogels up to a certain degree decreases by increasing the crosslink density, probably due to an increasing difficulty in establishing ordered domains.
Thermal and Shelf-Stability
The thermal stability of PAAs is as expected for polymeric β-dialkylaminoethyl-acrylamides, considering that the non-polymeric ones are known to undergo β-elimination on heating. Performing thermogravimetric analyses of some linear PAAs, for instance, the weight loss started at about 200°C under vacuum and at slightly higher temperatures under nitrogen. The shelf-stability of PAAs is usually good, provided some precautions are taken. Most PAAs absorb moisture in open air both as free bases and as salts. PAA free bases must be thoroughly dried and protected from moisture and oxygen to avoid discoloration, whereas PAA salts are indefinitely stable in the dry state and only need to be protected from moisture.
Degradation of PAAs in Aqueous Media
In principle, all PAAs contain cleavable bonds in the main chain. However, as stated above, in concentrated solution at pH 8–10 and at 18–25°C most PAAs are sufficiently stable for weeks and even months to be kept polymerizing and therefore increasing their molecular weight. Several of them, however, were specifically tested for degradability in very dilute solution at pH 7.4 and 37°C by viscometric and chromatographic methods. In many cases the degradation rate markedly depends on the structure of the bisacrylamide moiety, as it was found, for instance, in a study performed on 2-methylpiperazine deriving PAAs, the degradation rates were in the order: (Table 5, No. 33) > (Table 5, No. 11) > ISA23 (Table 6, No. 38) (Fig. 1).
This trend was confirmed by further studies on other PAAs in which the same amide monomers had been combined with different amine monomers.[61, 65] Broadly speaking, increasing temperature and pH speeded up the PAA degradation in aqueous media. By contrast, isolated lysosomal enzymes at pH 5.5, where tested, proved ineffective.
Acid–Base Properties of PAAs
All PAAs are polyelectrolytes, since the prim-or sec-amine groups involved in polyaddition reactions leading to PAAs give rise to tert-amine groups in the polymer chain and retain their basic character. The polyelectrolytic character can be further enhanced by introducing ionizable side substituents. Many relevant properties of PAAs, including toxicity and ability to interact with components of the biological environments, such as nucleic acids, proteins, and living cells, are strongly dependent on their acid–base properties, hence on their ionization state in different biological districts. Therefore, this subject was the object of several investigations.
Normally, the acid–base dissociation equilibria of polyelectrolytes are best interpreted by the generalized Henderson–Hasselbach (eq 2)
where K is the weak acid dissociation constant being pH-determining in the buffer titration zone considered; α is the dissociation degree of the considered acidic function, and β is the Katchalsky and Spitnik parameter accounting for possible interactions between ionizable groups being adjacent either structurally or on account of conformational effects (random-coil structure). To describe this behavior, the concept of “apparent” constant (for polyelectrolytes) as opposite to “real” constant (for low molecular weight electrolytes) is often adopted.
Unlike traditional polyelectrolytes, PAAs usually behave similarly to low molecular weight amines as regards protonation as well as heavy metal ion complex formation (see later),[32, 35-41, 45, 69, 71, 73, 162, 163] in that the ionizable groups of each repeating unit exhibit “real” or quasi “real” protonation constants, as if each unit was an isolated molecule. This equals to say that for PAAs the Katchalsky and Spitnik parameter β is 1 or whereabouts. It is noteworthy that the number of the protonation constants of PAAs is always equal to the number of the ionizable groups per unit and apart from entropic effects their values are similar to those found for the non-macromolecular models prepared by Michael addition of 4-acryloylmorpholine with the same amines used in the preparation of the corresponding PAAs. A linear relationship was found for both PAAs and their models between the protonation enthalpies and the net charge of the nitrogen atom to be protonated. This is a further indication that no significant interactions exist between different repeating units. This behavior is probably due to the relatively long distance between the ionizable groups belonging to different units, combined with the extended conformation of PAAs in solution and the high charge-sheltering efficiency of the two amide groups interposed. In fact, a single amide group bound to a piperazine ring is not sufficient to minimize interactions between neighboring units, as in the case of poly(1,4-piperazinediyl-1-oxo-trimethylene) (Fig. 2), which was found to exhibit a typical polyelectrolyte behavior.
Amphoteric PAAs of traditional chain structure, but deriving from aminoacids and therefore carrying a carboxyl group attached to the amine moiety behave anomalously in the PAA domain, but normally in the polyelectrolyte domain, since the carboxyl group exhibits a typical polyelectrolyte behavior. This tendency, however, is minimal for amphoteric PAAs in which the carboxyl groups is attached to the bisacrylamide moiety, in particular ISA23 (Table 6, No. 38).
Viscometric titrations in 0.1 M NaCl[41, 71] proved that the conformational freedom is reduced on protonation. For PAA No. 25 of Table 5, for instance, the first protonation led to the formation of a strong hydrogen bond between “onium” ions and carbonyl groups belonging to the same monomeric unit. When the first protonation of all the monomeric units was complete, the above effect strongly reduced the conformational freedom of the whole polymer, which tended to assume a rigid structure. This was clearly shown by a pronounced jump of its reduced viscosity at α = 0.5 (α = degree of protonation). The second protonation led to an electrostatic repulsion between the positively charged onium ions belonging to the same unit, and this effect further compelled the polymer to adopt a more rigid structure. Consequently, a second jump was observed at α = 1 (Fig. 3).
The reduction of conformational freedom became less pronounced by lengthening the aliphatic chain separating the amine groups of the repeating units. For example, PAAs Nos. 27 and 28 of Table 5 showed the same jumps progressively reduced. In contrast, their isomers (Table 4, Nos. 18–20), equally carrying two tert-amine groups per unit, but only one of which inserted in the polymer chain, did not show any viscosity jump either after the first or after the second protonation step. This was explained by the fact that the single “onium” ion per unit present in the polymer chain of the latter PAAs can interact indifferently with two neighboring carbonyl groups, thus enjoying a greater conformational freedom. Consequently, viscometric titrations did not show any jump throughout the whole titration curve.[41, 71]
Recently, a library of acid–base properties of PAAs, many of which carrying multiple ionizable groups and never previously considered in this respect, has been published. The pK determinations were performed following three distinct approaches,[165, 166] which gave congruent results. All previous statements about the “real” or “quasi real” protonation constants of PAAs were confirmed. In all cases, the β parameter was found ranging from 1 to 1.1, only exceptionally rising to 1.2–1.3.
For many PAAs, the protonation constants as well as the average distribution of the charged species, hence the net average charge as a function of pH, were determined. As typical examples, the speciation diagrams for two typical amphoteric PAAs (Table 6, Nos. 28 and 38) are reported in Figure 4.
Metal Complexes of Linear PAAs
In early studies, many PAAs proved to form coordination complexes with some heavy metal ions, namely Cu2+, Ni2+, Co2+. As with protonation, “real” stability constants could be determined for the complexes.[29, 46, 47, 49, 86, 167] It is noteworthy that metal complexation, similarly to protonation, led to stiffening of the PAA conformation in solution. In the absence of additional side amine or carboxyl groups, only PAAs carrying at least two amine nitrogens per unit that neither belonged to a cyclic structure nor were separated by more than three carbon atoms showed complexing ability. The electronic and electron paramagnetic resonance (EPR) spectra of both polymeric and non-polymeric complexes, obtained from PAA models, were similar and consistent with an octahedral tetragonally distorted structure, in agreement with the substantial independence of the polymer repeating units in complex formation.
Technical Applications of PAAs
PAAs as Inorganic Pollutants Sorbing Materials for Water Purification
It was quite early ascertained that crosslinked PAAs[70, 75, 76] and PAA-grafted materials, for example silica,[141-144] retained the ion-complexing ability of their linear counterparts. This concept was more recently resumed by studying crosslinked amphoteric PAAs as specific heavy metal ion absorbers for water purification from inorganic pollutants. An amphoteric PAA with inter-segmented structure obtained by synthesizing ISA23 in the presence of a second pre-synthesized PAA carrying primary amino groups as side substituents (Table 6, No. 27) that acted as a macromolecular crosslinking agent was studied as a Co2+, Ni2+, and Cu2+-sorbing material. This resin exhibited a remarkable sorption capacity and sorption rate for the three ions considered, which were in situ monitored by cyclic voltammetry. The metal-ion uptake was very fast and quantitative. Dilute acids quantitatively eluted the absorbed metal ions, thus allowing metal recovering and resin regeneration. In a subsequent study, two novel crosslinked PAAs named LYMA and LMT85 (Fig. 5), containing amine and carboxyl groups, were reported as highly effective heavy metal ions absorbers.
In particular, LYMA contained one carboxyl and two amine groups and was a mimic of l-lysine, whereas LMT85 contained two amine and five carboxyl groups and was a mimic of EDTA. The heavy metal ion set adopted as benchmark was Cu2+, Cd2+, Pb2+, Zn2+, Ni2+, and Co2+. LYMA proved selective for Cu2+ and Ni2+, the other ions tested being negligibly absorbed, whereas LMT85 proved capable of rapidly and quantitatively absorbing all the ions tested either singly or in mixed solution (Fig. 6).
In a subsequent investigation, both resins proved capable of absorbing Mn2+ as well. As observed for other PAA resins, the absorption process was fast and reversible and the resins were easily regenerated by acidification. Moreover, the metal ion absorption imparted intense coloring to the resins, a feature possibly exploitable for analytical purposes (Fig. 7).
Crosslinked PAAs as Matrices for High-Performance Nonlinear Optical Dyes
Highly crosslinked PAAs obtained from reaction systems consisting of stoicheiometrically balanced mixtures of bisacrylamides and multifunctional amines with minor amounts of difunctional amines are very hard and rigid materials with limited swelling in aqueous media. They have found application as superior matrices for high-performance nonlinear optical (NLO) heterocycle-based cationic dyes. In particular, prim-amino-alkyl substituted dyes were prepared and added as co-monomers to reaction systems leading to highly crosslinked PAA, thus obtaining PAA networks with NLO moieties covalently attached as side substituents. The very mild preparation conditions combined with the wide-ranging tuneability of optical and mechanical properties made these matrices a valuable alternative to conventional high-Tg thermoplastic polymers.[171-173] Moreover, it was found that a stable alignment of the second order covalently attached NLO-phores could be obtained by a three steps procedure (swelling–poling–de-swelling) performing the last two steps under poling conditions. This procedure appears widely applicable for producing composite polymeric materials with a rather stable second harmonic generation. It is of particular interest for NLO-phores sensitive to temperature, since both synthesis and poling were best performed at room temperature.
Sensing Applications of PAAs
Owing to their hydrophilicity and multifunctionality, PAAs were also considered for sensing applications. Early papers reported on the use of amorphous, rubber-like PAAs (Table 4, Nos. 14, 15, 18, 21, 22, and 24) as coatings of quartz microbalance in gravimetric sensors for the detection of CO2 and SO2 in gaseous mixtures. The rubber-like properties, being obviously related to structural flexibility, were apt to facilitate the diffusion of analyte molecules through the polymer layer.[42, 43] A subsequent article reported on the development of vapor detectors formed from composites of conductive carbon black and PAAs. The new materials were tailored to produce increased sensitivity toward specific classes of analyte vapors. Subsequent articles reported on the use of PAAs as the active components of a flow type quartz crystal microbalance chemical sensor suitable for determining heavy metal ions in aqueous solution, hence having potential for environmental monitoring. This sensor was based upon surface chelation of the metal ions at PAA-modified gold electrodes on 9 MHz AT-cut quartz resonators, functioning as a quartz crystal microbalance.[176, 177]
BIOMEDICAL AND BIOTECHNOLOGICAL APPLICATIONS OF PAAs
The biotechnologial applications of PAAs explored so far can be divided in two main categories: those employing soluble PAAs and those regarding PAA hydrogels or PAA-based solid materials in which the PAA portion is the active component.
Bioactive Soluble PAAs
PAAs with Antimetastatic Activity
As early as 1973 it was found that some copolymeric PAAs (Table 4, No. 29 and Table 6, No. 24) were active as antimetastatic agents after intravenous administration, reducing both the number and the average size of metastases in mice. PAAs No. 29 of Table 4 contained variable amounts of long chain aliphatic side substituents. They were purely basic, amphiphilic, and significantly toxic, nevertheless could be administered to mice at a dose up to 20 mg/kg and showed activity in reducing the number and average weight of Lewis lung (but not Sarcoma 180) tumor metastases. By contrast, the PAA No. 24 of Table 6 was amphoteric and non-toxic. It could be administered at a dose of 200 mg/kg and proved to reduce the number and average weight of both Sarcoma 180 and Lewis lung tumor metastases. However, none of the above PAAs was active against the primary tumor. Whereas the hydrophobically substituted PAAs carried long aliphatic chains and their activity could be ascribed to cell membrane interactions, by analogy with non-ionic detergents of the Triton series previously found to be equally active, the amphoteric PAA lacked hydrophobic moieties, suggesting a different mode of action. Regrettably, this point was no further investigated.
Antiviral Activity of AGMA1
The already mentioned PAA named AGMA1 (Table 6, No. 28) proved very active both in vitro and in vivo against Herpes Simplex Viruses Types 1 and 2 (HSV-1 and HSV-2), closely related pathogens of the Herpesviridae family of DNA viruses, without eliciting adverse side effects. Both HSV-1 and HSV-2 are capable of infecting mucosal sites causing lesions on the lips, eyes or genitalia, encephalitis, and others. AGMA1 is water-soluble and amphoteric, but prevailingly cationic at pH 7.4. Its repeating unit is reminiscent of the RGD peptide sequence (Fig. 8).
The IC50 of AGMA1 was found >5 mg/mL and its intravenous maximum tolerated dose (MTD) in mice was >0.5 g/kg. It was deprived of hemolytic activity and easily entered cells localizing in the perinuclear region.[88, 89] It proved active not only against HSV-1 and HSV-2, but also against Papilloma virus (HPV-16), Cytomegalovirus, and Murid Herpesvirus 68 (MHV-68). The inhibitory effect of AGMA1 was exerted at low polymer concentration. For instance, EC50 values of 0.74 µg/mL and 1.14 µg/mL, respectively, were found for HSV-1 for HSV-2 (Fig. 9).
AGMA1 acted with a different mechanism than conventional antiviral drugs as Acyclovir, in that it was not virucidal, that is, it did not kill viruses, but blocked the transmission of the infection from cell to cell. Its antiviral activity was not directly related to its prevailingly cationic charge, as other cationic PAAs, for example ISA1 (Table 5, No. 38), under the same conditions proved inactive. AGMA1 did not affect the growth of Lactobacillus spp. responsible for maintaining the correct pH in the vaginal fluids of healthy animals and its activity was not pH-dependent within the physiological range. AGMA1 also inhibited HSV-1 and HSV-2 infection in vitro on cultivated human epithelial tissue (EpiVaginal™) without any detectable inflammatory effect (Fig. 10).
In Figure 10, the gray bars refer to control infected with HSV-2 1000 pfu. The barely detectable black bars refer to epivaginal tissue infected in the same way and treated with 100 µg/mL AGMA1 solution. At this dose no cytokine release (indicating inflammatory reactions) was detected after 24 h. The same inhibitory effect and lack of inflammatory activity was subsequently confirmed in vivo after topical administration to female mice.
Antimalarial Activity of PAAs
Studies in progress have demostrated that AGMA1 and ISA23 tested on Plasmodium falciparum 3D7 are endowed with antimalarial activity per se.[90, 91] Significant results are reported in Table 8.
Table 8. Plasmodium Growth Inhibition Using Different Molecular Weight Fractions of AGMA1 and ISA23
Percentage parasitemia reduction with respect to controls.
It is apparent that for both polymers the higher molecular weight fractions were significantly more active than their lower molecular weight analogs. Moreover, the antimalarial activity of AGMA1 was always remarkably superior to that of ISA23.
PAAs as Promoters of Cell Adhesion on Substrates for Cell Culturing
In spite of their toxicity, cationic polyaminoacids such as poly-l-ornitine, poly-l-lysine, and poly-d-lysine are widely used since many years as enhancers of cell adhesion, proliferation, and differentiation on solid substrates for cell culturing. AGMA1 was found to be equi-active in this respect to poly-l-lysine, but with a vastly inferior toxicity and is presently sold for this application under the trade mark of Cell GRIP®.
Soluble PAAs as Osteoblast Proliferation Promoters
PAAs synthesized by polyaddition of pamidronate or neridronate with bisacryloylpiperazine (Table 6, Nos. 13 and 14) were recently proposed as osteoblast proliferation promoters.
Soluble PAAs as Carriers for Bioactive Substances
Soluble PAAs have been studied as polymer-drug conjugates (particularly as anticancer-drug conjugates) and, more extensively, as endosomolytic vectors for intracellular delivery of nucleic acids and toxins. The early studies on this subject have been reviewed elsewhere. They were soon followed by more extensive investigations reported in the following paragraph.
Biocompatibility and Biodistribution Studies of Soluble PAAs
Many polycations other than PAAs explored as drug and oligonucleotide delivery systems such as poly-l-lysine, polyethyleneimine, and unmodified PAMAM,[181-183] are generally toxic to cells in culture. For example, poly-l-lysine displays IC50 values in the range 1–60 µg/mL depending on the cell type and incubation time and poly-l-lysine, polyethyleneimine and unmodified PAMAMs showed significant hemolytic activity which is molecular weight- (generation-) dependant.[182, 183] In early studies, PAAs Nos. 31 and 32 of Table 5 were found consistently less cytotoxic than poly-l-lysine. Many amphoteric PAAs carrying a carboxyl group per unit proved even less toxic. For instance, PAAs Nos. 32, 33, and 38 of Table 6 were >100 times less cytotoxic than the traditional amine polymers used as a reference.[44, 93] Equally, PAAs deriving from α- and β-aminoacids were in general highly cytobiocompatible. All these PAAs carried excess negative charges at pH 7.4.[12, 44] This charge effect was thought to explain their lack of toxicity, supported by the observation that the amphoteric, but at pH 7.4 prevailingly cationic PAAs (Table 6, Nos. 34 and 35) were significantly more cytotoxic than their prevailingly anionic lower homologs. Up to this point, basicity, hence the net cationic charge at pH 7.4, seemed to be the main factor affecting PAA toxicity. Accordingly, the reduced toxicity of most purely cationic PAAs compared with poly-L-lysine (PLL) and polyethyleneimine (PEI) could be explained by their generally lower basicity. This general conclusion was further confirmed by the fact that the Michael addition of N,N-dimethylacrylamide to the side amino groups of poly l-lysine reduced in the mean time both its basicity and its toxicity. Later studies, however, demonstrated that, whereas in most instances this assumption held true, exceptions existed. In AGMA1, the presence of side guanidine groups in addition to the chain tert-amine groups (Fig. 8) imparted significant basicity not accompanied by cell-mediated toxicity. AGMA1, notwithstanding its isoelectric point >10 and its 0.55 average positive charges per unit at pH 7.4, proved highly biocompatible (IC50 ≥5 mg/mL on several cell strains) and deprived of significant hemolytic activity in the pH range 4–7.4. This contrasted with the behavior of ISA23 that had been previously found non-hemolytic at pH 7.4, where it was prevailingly anionic, but membrane active below its isoelectric point, that is ∼5.5. Interestingly, AGMA1-ISA23 copolymers showed a small, but noticeable hemolytic activity that increased by decreasing the pH of the medium and increasing the ISA23 proportion. As reported below, the introduction of AGMA1 units in ISA23 hydrogels imparted them an otherwise lacking ability of inducing cell adhesion and proliferation. This was attributed to the structural similarity of the AGMA1 repeating units to the RGD peptide, probably imparting a similar aptitude to interact with cell membranes. It might be postulated that AGMA1, owing to its peculiar structure, exerted a stabilizing action on cell membranes overshadowing the membranolytic effect of the excess positive charges. In AGMA1-ISA23 copolymers, the stabilizing effect was partially superseded by the known hemolytic activity at low pH values of the ISA23 portion.
To gather information on PAA biodistribution, the analogs of two typical PAAs, namely ISA1 and ISA23 (Table 5, No. 38 and Table 6, No. 38), were synthesized to contain approximately 1 mol % 2-p-hydroxyphenylethylamine-deriving units amenable to iodination. These polymers were named ISA4 and ISA22, respectively. After intravenous injection to rats 125I-labelled ISA 4 was rapidly taken up by the liver (>80% recovered dose at 1 h) whereas 125I-labelled ISA22 was not (liver uptake <10% recovered dose at 5 h). The so-called “stealth like” properties of ISA22, probably due to its zwitterionic nature with prevailingly negative charge at pH 7.4, provided opportunity for tissue targeting either by the incorporation of ligands or, as regards tumors, by passive means such as the enhanced permeability and retention (EPR) effect.[185-189] In fact, biodistribution studies in mice bearing subcutaneous B16F10 melanoma showed that 125I-labelled ISA22 was still accumulating in tumor tissue after 5 h (2.5% dose/g).
PAA and PAA Anticancer Drug Conjugates
PAA-anticancer drug conjugates have been prepared and tested. In early studies, PAAs were developed as water soluble carriers for known anticancer agents including mitomycin C (MMC) and platinates. Two PAA-MMC adducts were synthesized from hydroxylated PAAs such as ISA1 using carbonyldiimidazole as coupling agent. After in vitro studies, preliminary in vivo experiments were carried out on mice bearing L1210 tumor cells. The mice were treated with free MMC or PAA-MMC conjugates by a single i.p. dose the day after tumor inoculation. The PAA-MMC conjugates proved equi-active compared to MMC given intraperitoneally and a long-term survivor was observed in each group treated with conjugate. PAA-MMC conjugates proved less toxic than free MMC when administered at a MMC-equivalent dose of 5 mg/kg. PAA-platinates were also prepared by reaction of ISA23 and two PAAs containing pendant β-cyclodextrin moieties with cisplatin. The conjugates contained 8–70 wt % platinum and released low molecular weight platinum species in vitro at pH 5.5 and pH 7.4 (0–20%/72 h). The PAA-platinates were generally less toxic than cisplatin toward lung tumor cell lines, but in vivo proved equi-active compared to cisplatin in an i.p. L1210 leukemia model. The antitumor activity of ISA23-platinates was similar to that reported for other platinum conjugates of similar molecular weight.[191-193]
In a subsequent investigation, PAA conjugates containing the membrane disrupting peptide melittin were prepared and tested. It was hypothesized that PAA conjugation would reduce the hemolytic activity of melittin at pH 7.4, but upon delivery to tumors by the EPR effect, the polymer would uncoil in an acidic endosomal environment exposing melittin and allowing it to interact with membranes. The melittin content of the conjugates was 6–19% (wt/wt). They were obtained using melittin as a comonomer in the preparation of ISA1 and ISA23.
Under the conditions adopted, the terminal amine group of melittin, being a weaker base than its side amine groups deriving from lysine, was the only un-ionized, hence the only amenable to the addition reaction. Although the ISA1 conjugate improved gelonin delivery and showed pH-dependent hemolytic activity at a polymer concentration of 0.05 mg/mL, it also displayed high hemolytic activity at pH 7.4. By contrast, the ISA23 conjugate did not deliver gelonin. However, this conjugate lacked hemolytic activity at pH 7.4 while retaining the melittin cytotoxicity and could have potential as a novel polymer anticancer agent.
More recently, ISA1 and ISA23 with amine pendant groups (Table 4, No. 33 and Table 6, No. 42) were prepared. Dansyl cadaverine and doxorubicin were bound to the former and doxorubicin to the latter via an acid-labile cis-aconityl spacer. Release of dansyl cadaverine and doxorubicin at physiological and acidic pH varied from 0 to 35% over 48 h and was pH dependent. Whereas the ISA1 conjugate apparently did not present significant promise as anticancer agent, the ISA23 conjugate proved to release biologically active doxorubicin in vitro and might be suitable for further development.
PAA-Antiviral Drug Conjugates
An ISA23 sample carrying β-cyclodextrin pendants, obtained by copolymerization with 6-deoxy-6-amino-β-cyclodextrin was employed as carrier for the antiviral drug Acyclovir. Up to 11% wt/wt of Acyclovir was solubilized. In cell cultures, the Acyclovir β-cyclodextrin–PAA complex exhibited a significantly higher antiviral activity than the free drug against Herpes simplex virus Type I.
PAAs—Imaging Probes Conjugates
The ease of PAA functionalization allowed envisaging their use as carriers of probes for imaging applications. The preferred PAA was ISA23, owing to its biocompatibility and stealth-like properties leading to preferential tumor localization by the EPR effect.
The introduction of paramagnetic N-oxyl groups in PAAs is easy, since 4-amino-2,2,6,6-tetramethyl-piperidine N-oxyl (4-amino-TEMPO, Table 2, Y-27) behaves as co-monomer in PAA polymerization mixtures leading straightforwardly to TEMPO-labeled products. Two PAA-TEMPO conjugates based on ISA23, ISA23-TEMPO1 and ISA23-TEMPO2 (Table 6, No. 47) with 10 and 40% TEMPO-carrying units per polymer chain, respectively, were prepared. Their relaxivity values were, respectively, 0.4 and 1.8 mM−1 s−1. These values indicated that PAA-TEMPO adducts have a definite potential as NMR imaging contrast agents. This was confirmed by preliminary magnetic resonance imaging (MRI) determinations.
In order to provide a suitable carrier for radioactive tracers, an ISA23 sample with 10% thiol-functionalized units (Table 7, No. 8) was synthesized by adding mono-N-boc-cystamine as co-monomer during polymerization and then reductively cleaving the SS bond in the resultant polymer. This thiomer gave stable rhenium complexes and was reasonably considered worth of attention as carrier for radioactive rhenium and technetium. In particular, two rhenium complexes containing 0.5 and 0.8 equiv of rhenium per thiol groups, respectively, were obtained by reacting the cysteamine-functionalized ISA23 with [Re(CO)3(H2O)3](CF3SO3) in aqueous solution at pH 5.5. The chelation occurred through the S and N atoms present in the PAA carrier and the rhenium content was governed by the stoichiometric ratio between rhenium and thiol groups. Both the cysteamine-functionalized ISA23 and its rhenium complexes were soluble in water under physiological conditions. The complexes proved highly stable in solution even in the presence of excess cysteamine. Neither cysteamine-functionalized ISA23 nor its rhenium complexes showed hemolytic activity up to a concentration of 5 mg/mL. No cytotoxic effects were observed on Hela cell after 48 h at a concentration of 100 ng/mL. In vivo tests showed that cysteamine-functionalized ISA23 was highly biocompatible. Moreover, the rhenium complexes did not elicit detectable toxic effects on mice after intravenous injection in doses up to 20 mg/kg.
More recently, in order to obtain strongly luminescent macromolecular probes, a new ISA23 copolymer with 6% phenanthroline-containing repeating units (Table 6, No. 52), was obtained by copolymerization with 4-(4′-aminobutyl)−1,10-phenanthroline (Table 2, No. 28). The copolymer showed excellent solubility in water. The phenanthroline pendants stably coordinated either Re(CO)3+ or Ru(phen)22+ fragments, affording luminescent complexes emitting from 3MLCT excited states with λem = 608, 571, and 614 nm and Φem = 0.7%, 4.8%, and 4.1%, respectively, in aerated water solution. The complexes were stable under physiological conditions even in the presence of excess competing cysteine. Both complexes were non-toxic in the concentration range 0.5–50 μM (calculated on the metal-containing unit) toward HEK-293 cells. Moreover, preliminary studies showed that the ruthenium complex entered HEK-293 cells by endocytosis and then homogeneously diffused within the cytoplasm across the vesicle membranes, thus confirming the results of previous studies on ISA23 as an endosomolytic carrier.
PAAs as Non-Viral Vectors for Intracytoplasmic Delivery
Background and Basic Investigations
Considerable attention in the last decades has been and is being focused on the exploitation of the unique combination of properties of PAAs for designing membrane active polymers able to promote intracytoplasmic delivery of high molecular weight therapeutics. Polymers widely explored as anticancer conjugates such as hydroxypropylmethacrylamide (HPMA) copolymers and PEG are not eligible for these applications. Viral systems, whereas mediating high transfection efficiencies in vitro, have proven unstable and at times catastrophically immunogenic.[194, 195] Many non-viral delivery systems such as cationic lipids and polymers are relatively toxic and, moreover, rapidly localize to lung or liver after intravenous administration thus making it difficult to target other tissues. It has been suggested that polymeric branched transfection agents with normal polyelectrolyte behavior, such as polyethyleneimine, act as transfection promoters according to the so-called “proton sponge” hypothesis, that is, by absorbing protons within the endosome, where the pH is 5.5 or whereabouts, they swell and cause membrane rupture. Unlike many other polyamines (e.g., polyethyleneimine and poly l-lysine), protonation and de-protonation of the repeating units along the PAA backbone are independent events. PAAs bearing two amine nitrogens in the repeating unit show two distinct pKa values and undergo sharp conformational changes at the corresponding pH. Therefore, by properly selecting the starting monomers it is possible to tailor PAAs that passing from the extracellular fluid to the endosomal intracellular compartments change conformation and unravel their latent endosomolytic properties, thus enabling the endosomal escape toward the cytosol of a high molecular weight therapeutic payload such as a protein or a nucleic acid that would be otherwise destroyed by the endosomal enzymes. While the polycation-mediated membrane damage is known since a long time,[197-200] the peculiarity of most PAAs is precisely the sharp increase of their protonation degree and the consequent conformational change in the pH interval 7.4–5.5.
The “bioresponsiveness” of PAAs was first demonstrated by synthesizing a hydroxylated PAA (Table 5, No. 8) modified by covalently attaching the membrane lytic non-ionic detergent Triton X-100 as side substituent conjugate. Subsequent experiments performed with amphoteric PAAs not carrying pendant detergent moieties, namely Nos. 32–35 and 38 of Table 6, demonstrated that by lowering pH below 7.4 they could become inherently membrane active.[53, 93] The ability of PAAs to mediate DNA or toxins delivery was studied.[81, 94] At 10:1 polymer excess, ISA1 and ISA23 formed with DNA toroid shaped interpolyelectrolyte complexes of diameter 80–150 nm in diameter) which were visible using transition electron microscopy (TEM). The complexes displayed retarded electrophoretic mobility and also the ability to protect DNA from DNase II degradation. In transfection experiments, the PAAs demonstrated the ability to mediate pSV β-galactosidase transfection of HepG2 cells. After these breakthrough experiments, the use of both amphoteric and purely cationic PAAs as transfection promoters was extensively investigated and will be reported in a separate paragraph.
The ability of some PAAs to promote the endosomal escape and intracellular trafficking of proteins was investigated using as models two non-permeant ribosome-inactivating toxins, namely ricin A chain and gelonin. Ricin is a highly cytotoxic protein in the native dimeric form, consisting of an A-chain (RTA) and a B-chain (RTB) linked by a disulfide bridge. The RTB binds to cell membranes promoting cytosolic entry of the RTA moiety which acts by cleavage of the N-glycosidic bond of adenosine4324 nucleoside leading to inhibition of protein synthesis. Gelonin has similar activity to RTA, but lacks the equivalent of RTB and therefore is non-toxic to intact cells. When PAAs were incubated with B16F10 melanoma cells in vitro in combination with RTA or gelonin (at non-toxic concentrations of toxin) it was found that tyramine-modified ISA1 could restore toxin cytotoxicity, whereas the amphoteric, but prevailingly anionic tyramine-modified ISA23 did not. The ability of ISA1 to promote intracellular delivery of non-permeant toxins was confirmed by comparing in this respect ISA1 with its random and block copolymers with ISA23. It was found that only ISA1 and the block copolymer ISA23:ISA1 having a 2:1 molar ratio were able to promote intracellular delivery. These findings were further confirmed by studying the covalent conjugates of the same PAAs with the membrane disrupting peptide melittin. In a parallel study, the ability of ISA23 to establish interaction with model membrane vesicles was investigated using EPR in conjunction with SANS. Besides pH, also the type of counterion determined the gyration radius of ISA23, as well some important biological properties, such as toxicity and hemolytic properties of both ISA1 and ISA23. For EPR, 16-DSE was dissolved in the vesicle membrane to measure its dynamics and polarity, whereas a spin-labeled ISA23 (Table 6, No. 47) analogue was used to give a measure of the polymer flexibility. No interaction was found adding ISA23 to the external vesicle surface. This observation conflicts with the reported ability of ISA23 to lyse the membrane of red blood cells (RBC) at pH ≤ 5, but is in agreement with previous studies showing no effect on membrane permeability when this PAA was added to an incubation medium containing isolated lysosomal vesicles, whereas destabilized the lysosomal membrane if internalized into the lysosomal compartment. All the above studies point to the conclusion that linear PAAs can be designed to exhibit minimal non-specific toxicity, display pH-dependent membrane lysis, and deliver genes and toxins in vitro. A related study was aimed at measuring PAA cellular uptake using ISA1-Oregon Green conjugate (ISA1-OG) [and as a reference ISA23-Oregon Green conjugate (ISA23-OG)] in B16F10 cells in vitro and, by subcellular fractionation, quantitate intracellular trafficking of 125I-labelled ISA1-tyrosine in liver cells after intravenous administration to rats. ISA1-OG displayed ∼60-fold greater B16F10 cell uptake than ISA23-OG. Passage of ISA1 along the liver cell endocytic pathway caused a transient decrease in vesicle buoyant density. Increasing ISA1 dose from 10 mg/kg to 100 mg/kg increased both radioactivity and N-acetylglucosamine levels in the cytosolic fraction (5- to 10-fold) at 1 h. Moreover, internalized ISA1 provoked N-acetylglucosamine release from an isolated vesicular fraction in a dose-dependent manner. These results provide direct evidence, for the first time, of PAA permeabilization of endocytic vesicular membranes in vivo, and they have important implications for potential efficacy/toxicity of such polymeric vectors. It is worth mentioning that one of the important observations in this study is that PAA endosomolytic activity is probably due to physical PAA–membrane interaction, rather than to the proton sponge effect as hypothesized for PEI and other traditional polycations. Most of these latest studies have been recently reviewed and discussed elsewhere.
PAAs as DNA Carriers and Transfection Promoters
Triggered by the basic investigations reported in the previous paragraph, several papers on the use of PAAs other than ISA1 and ISA23 as DNA condensing agents and transfection promoters have been published in recent years. For the sake of clarity, these papers will be grouped in two categories dealing, respectively, with PAAs or PAA-PEG copolymers whose polymer chain contains only carbon, oxygen, and nitrogen (“traditional” PAAs) and PAAs with reducible SS bonds in the polymer chain.
PAA No. 3 of Table 5 and its PEG copolymers have first been the object of independent studies as DNA delivery systems with remarkable success.[208-210] Later on, an extensive study was performed on AGMA1. AGMA1 easily entered HT-29 cells, gave stable complexes with DNA and showed good transfection efficiency suggesting the ability to transport in the cytoplasm a DNA payload. AGMA1 probably established non-disruptive membrane interactions allowing membrane crossing of AGMA1 together with its payload without exerting membranolytic activity. Further investigation on three AGMA1 samples, AGMA5, AGMA10, and AGMA20 of 5100, 10,100, and 20,500, respectively, as DNA non-viral carriers were performed. All samples condensed DNA in spherical, positively charged nanoparticles and protected it against enzymatic degradation. AGMA10 and AGMA20 polyplexes had average diameters lower than 100 nm. AGMA5 polyplexes were larger. All polyplexes showed negligible cytotoxicity and were internalized in cells. AGMA10 and AGMA20 effectively promoted transfection, whereas AGMA5 was ineffective, suggesting a MW dependence of the transfection efficiency. Fluorescein isothiocyanate (FITC)-labeled AGMA10 was also prepared and its intracellular trafficking, as well as that of its DNA polyplex, studied. FITC-AGMA10 concentrated in the perinuclear region, but did not enter the nucleus, whereas DNA/FITC-AGMA10 polyplex largely localized inside the nucleus. DNA/AGMA10 polyplex intravenously administered to mice promoted gene expression in liver, but not in other organs and proved exempt from detectable toxic side effects.[89, 92]
The transfection and intracellular trafficking of three PAAs with pendant primary amine groups obtained from mono-protected diamines followed by deprotection have been described (Table 4, Nos. 35–40).[55-57] These PAAs exhibited good DNA-binding capacities and even higher transfection efficiencies than commercial PEI of molecular weight 25,000, being in the meantime less cytotoxic. Their favorable performance was attributed to efficient cell uptake and intracellular trafficking. In a different study, branched PAAs were developed by polyaddition of 1-(2-aminoethyl)piperazine with N,N′-methylenebisacrylamide and in a water/N,N-dimethylformamide mixture. By increasing the branching degree, the polymers became more compact and their DNA condensation ability increased, whereas their cytotoxicity decreased. Correspondingly, their efficiency as transfection promoters improved by more than three orders of magnitude.
A differently conceived PAA-based carrier consisted of PAA-grafted carbon multiwalled nanotubes. The PAA chains were attached to chemically oxidized nanotubes by amide bonds. The adducts, besides giving stable suspensions, showed lower cytotoxicity and comparable or even higher transfection efficiency than both PEI of molecular weight 25,000 and the parent PAA.
“Inverted” PAAs obtained by sequential polycondensation of diacids with polyamines are also considered as DNA carriers, possibly susceptible of considerable development.
PAAs with SS Bonds in the Main Chain (SSPAAs)
Reduction-sensitive biodegradable polymers and conjugates soon appeared highly promising functional biomaterials with enormous potential in formulating sophisticated drug and gene delivery systems. In fact, the SS bond is reductively cleaved after cell internalization or, if administered orally, after reaching the lower portions of the gastrointestinal tract. As regards PAAs, this technique, besides obviously facilitating the selective intracellular release of a drug, protein or plasmid payload, greatly increased biocompatibility even in the presence of structural features apt to enhance transport efficiency, but in the meantime normally imparting toxicity, such as hydrophobic side substituents and high density of positive charges. In particular, the effects of variation in charge density and hydrophobicity on the gene delivery properties of SSPAAs with aminobutyl side chains were investigated by varying the degree of acetylation and benzoylation of the side amine groups. Hydrophobic benzoyl groups imparted higher transfection efficiencies. In a parallel study, a SSPAA was modified by introducing long-chain alkyl groups, thus apparently enhancing its ability to condense DNA into compact nanoparticles with positive surface charges. Bioreducible copolymeric PAAs with tunable charge densities were prepared by polyaddition of N,N′-bisacryloylcystamine with variable ratios of 4-amino-1-butanol and ethylenediamine or triethylenetetramine and optimized as silencing RNA (siRNA) vectors. It was found that whereas 20–30% ethylenediamine or triethylenetetramine units was needed to encapsulate siRNA into small and stable polyplexes, higher proportions of these units did not significantly improve their performance in this respect, but resulted in higher cytotoxicity and hemolytic activity, probably owing to the increased cationic charge. siRNA transfecting properties were also revealed by bioreducible PAAs with ω-aminohexyl pendants. Interestingly, bioreducible PAAs with polyamine moieties inserted in the main chain proved to condense DNA forming nanoparticles and efficiently in vitro transfect the murine capillary endothelial cells forming the blood brain barrier.
Recently, the gene delivery properties of new bioreducible branched PAAs were investigated in comparison with their linear analogs. The branched PAAs were prepared from N,N′-bisacryloylpiperazine and cystamine or ethylenediamine using unbalanced monomer mixtures to avoid crosslinking. Their linear counterparts were obtained by substituting N,N′-dimethylcystamine or N,N′-dimethylethylenediamine for cystamine and ethylenediamine, respectively. All PAAs were terminated with 4-aminobutanol or 2-aminoethanol. All PAAs formed polyplexes with plasmid DNA with sizes around 200 nm and positive zeta potentials. Remarkably, little to negligible cytotoxicity was observed in all cases. Branched N,N′-bisacryloylcystamine-based PAAs showed higher gene expression in DNA transfection tests with COS-7 cells than their linear analogues and up to two times higher than linear PEI used as reference polymer. Moreover, the transfection efficiencies of branched PAAs were generally enhanced by the presence of serum.[213, 214]
An original modification of disulfide-containing PAAs was recently described. It consisted of introducing phenylboronic acid moieties either by grafting 4-carboxyphenylboronic acid on a PAA sample with aminobutyl side chains, or incorporating 2-aminomethylphenylboronic acid units in the PAA chain by copolymerization. The ratio of phenylboronic-substituted units versus residual aminobutyl units was 30:70 for both PAAs. Compared with non-boronate benzoylated PAA samples (see above), both polymers were approximately as effective as gene delivery vectors, but more highly cytotoxic, possibly due to increased membrane disruptive interactions.
PEG-ylated bioreducible PAAs prepared by polyaddition of N,N′-bisacryloyl cystamine with a mixture of 4-amino-1-butanol and mono-tert-butoxycarbonyl-diamino PEG were evaluated as gene delivery vectors in comparison with their de-protected analogs carrying ω-prim-amine groups. These PAA-PEG copolymers proved to condense DNA into nanoscaled polyplexes (< 250 nm) with a stability in buffer suspension significantly higher than their non-PEG-ylated counterparts. The PEG-ylated polyplexes, however, remarkably biocompatible were less effective as transfection promoters, possibly because the PEG substituent biased the endosomal escape of the polyplexes. The cellular uptake and intracellular trafficking of bioreducible PAA–gene complexes in cells of the retinal pigment epithelium, considered good targets for ocular gene therapy, was studied in a few recent papers. The polyplexes exhibited a cationic surface, attached to cell surface proteoglycans of the cells, and were subsequently internalized via a phagocytosis-like mechanism.[109, 216, 217]
The introduction of disulfide linkages for achieving controlled degradation has been employed also for “inverted” PAA-PEG block copolymers. In particular, a single disulfide moiety was incorporated between the two blocks. This technique was used to establish a two-phase process for releasing active substances inside cells and tested by analyzing the complexation behavior of the system with plasmid DNA, before and after reductive degradation of the block copolymer. SSPAAs have also been used for localized gene delivery. Multilayered films composed of bioreducible cationic micelles, obtained from amphiphilic disulfide-containing PAAs and DNA, were prepared and employed as transfection promoters. Films with 10 bilayers prepared from PAAs with 28% of alkyl side chains showed the fastest release of DNA in the presence of 2.5 mM glutathione and the highest transfection efficiency toward 293T cells cultured on the film surface. SSPAAs obtained from N,N′-cystamine bisacrylamide, 4-amino-1-butanol, and ethylene diamine are promising carriers also as delivery systems of siRNA. In particular, the effects of the percentages of butanolic side chains and the density of basic sites in the main chain on siRNA complexation, cellular uptake, gene silencing, and toxicity were investigated. More than 80% knockdown efficiency, combined with low cytotoxicity, was found for polyplexes formed with polymers containing 25% or 50% ethylenediamine (EDA).
It is finally worth mentioning that PAA-PEG nanoparticulated constructs crosslinked with SS bonds, to which DNA end-capped with SH group had been grafted, were also employed for a purpose widely different from transfection, that is, as environmentally safe and extremely sensitive tracers for detecting sources of water pollution.
Comments on PAAs as DNA Carriers and Transfection Promoters
By considering the remarkable wealth of positive results by many authors on polyplex forming and transfection promoting ability of PAAs, it can be concluded that these are virtually general properties of this class of polymers, probably related to the fundamentals of their structure. Going deeper into details, favorable elements are, in the order, the basicity, the presence of some proportions of hydrophobic substituents, and of other structural elements enhancing cell membrane interaction and cell internalization followed by endosomal escape, probably including branched molecular architectures and presence of guanidine side substituents owing to the well-known chaotropic properties of this group.[221, 222] The presence of SS linkages in the polymer chain with the consequent selective degradation inside cells is a structural feature reducing cytotoxicity to acceptable levels even in the presence of other features acting in the opposite sense, such as the first two listed above as favorable to transfection efficiency. However, its positive effect goes farther, in that intracellular degradation triggers the release of the DNA loading in the right place and at the right moment.
Linear SSPAAs were also studied as carriers for intracellular protein delivery. To this purpose, a SSPAA bearing citraconic acid side moieties was prepared. This polymer gave nanosized complexes with proteins of opposite charge by electrostatic interaction. After cell internalization, the release of the protein payload was triggered by a dual mechanism, the reduction of the disulfide bond, and the inversion of the protein–polymer interaction at the endosomal pH, due to the charge-reversal of the citraconic side group. A series of functionalized PAAs were synthesized, two of which contained disulfide bonds in the main chain. They self-assembled into cationic, and essentially non-toxic nanocomplexes with oppositely charged proteins, as for instance β-galactosidase. The results indicated that these PAAs were highly potent and non-toxic intracellular protein carriers. Bioreducible SSPAAs containing multiple disulfide linkages in the polymer backbone were used to form nanocomplexes by self-assembly with human insulin, used as a negatively charged model protein at neutral pH. They were analyzed upon adsorption on model membranes. Imidazole pendants were also introduced in cationic SSPAAs for the same purpose, by employing histamine as comonomer.[112, 113] Moreover, two PAAs, one of which containing SS bonds in the main chain, both capable of forming self-assembled cationic nanocomplexes with oppositely charged proteins such as albumin, were recently prepared and the uptake of the resultant albumin-PAA nanoparticles by human-derived intestinal mucus secreting cells was studied. Both types of nanoparticles acted as intracellular protein transporters, but the SSPAA based nanoparticles were more effective owing to their mucoadhesive properties. The delivery of the hypoxia-inducible vascular endothelial growth factor (RTP-VEGF) plasmid assisted by a soluble SS PAA obtained by polyaddition of excess 1,2-diaminoethane with bisacryloylcystamine was also studied.
Comments on PAAs as Protein Carriers
The results of the above papers on PAAs as protein carriers are in agreement with the previously mentioned results of basic research in the same field[82-84, 95, 96, 204-207] and, not unexpectedly, point to a similar conclusion. By a proper design of the acid/base properties and the hydrophobic/hydrophilic balance, there is little doubt that a number of PAAs may display intracellular protein transport ability. As in the case of transfection, the presence of SS bonds in the main chain is advisable or even determinant, and for the same reasons outlined there.
Structural versatility has been already reported as a general property of PAAs. Two specific elements, however, deserve to be underlined here. First of all, as already mentioned, PAAs can be assembled in a modular fashion by simply employing the right amines and bisacrylamides, either singly or in combination with other monomers of the same category. Secondly, nearly all PAAs carry in the polymer chain a tert-amine group, only rarely a sec-amine group, flanked by one or two carbonyl group in β-position. The pKa values of these groups are without exceptions in the 7.25–8.25 (frequently in the 7.4–7.8) range. PAAs deriving from sec-diamines carry a second amine group per unit whose pKa is usually in the range 3.25–7.5. Therefore, the omnipresent tert-amine group in the repeating unit or, in the case of diamine-deriving units, at least one of them at the pH of extracellular fluids (∼7.5) is in proximity of the mid-flex point, hence the maximum slope of its titration curve. Reminding that PAAs behave as regards ionization as if each repeating unit were an isolated molecule, most PAAs change abruptly, albeit predictably, their positive charge density passing from the body fluids to intracellular endosomal compartments (pH ∼5.5). This brings about conformational changes and allows displaying their latent endosomolytic activity.
BIOTECHNOLOGICAL APPLICATIONS OF CROSSLINKED PAAs AND PAA BLOCK AND GRAFT COPOLYMERS
Heparin Absorbing Resins
Heparin is a natural mucopolysaccharide containing carboxyl- and sulfonic groups commonly used in clinics as anticoagulant agent. It is a polyanion with a high density of negative charges. In some cases, the anticoagulant activity of heparin must be inhibited when no longer needed. Traditionally, this is achieved by administering salmin, a polymer pertaining to a family of highly basic natural polypeptides indicated with the generic term of protamine. Several PAAs proved capable of neutralizing the anticoagulant activity of heparin in solution much as salmin does (but with vastly inferior toxicity) undoubtedly through the formation of PAA–heparin polyelectrolyte complexes. Not unexpectedly, crosslinked PAAs display the same heparin-complexing ability as their linear counterparts, but, being insoluble, act as heparin removing resins from aqueous media, including biological fluids such as plasma or blood.[151, 225-229] Since heparin is commonly administered to hemodialyzed patients to minimize blood coagulation and this can result in morbidity in patients at risk of bleeding, heparin-absorbing PAA resins have been proposed to achieve regional deheparinization in hemodialysis and extracorporeal circuits in general. These resins displayed remarkable heparin-absorbing capacity, even when incubated with very dilute solutions of heparin. One of the best-performing resins was a hybrid PAA-N-vinylpyrrolidinone copolymer prepared by applying the above reported V-PAA radical polymerization method in the presence of N-vinylpyrrolidone. These resins absorbed heparin to an extent ranging from 30 to 100 wt % (calculated on dry resin) according to their PAA content and apparently did not adversely affect any normal blood parameter. In particular, they were not hemolytic and did not alter recalcification time, prothrombin time, partial thromboplastin time, and fibrinogen content. The retentive power of a typical resin at various pH values after heparin loading was investigated by eluting with M/15 phosphate buffers at pH 4.8, 7.4, and 8.5. No heparin was released. The resin was then eluted with carbonate buffers. All heparin was desorbed between pH 10.8 and 11.4 and the remarkably sharp elution peak was centered at pH 11.0.
Non-physiological materials usually induce thrombus formation when placed in contact with blood. The development of permanently non-thrombogenic materials for cardivascular prosteses was a long-coveted, but never completely achieved target in the second half of the twentieth century. One of the earliest and most promising approaches was to ionically adsorb heparin on polymeric materials, a process hindering thrombus formation as long as heparin was present. The PAA property of giving stable complexes with heparin both in solution and in the hydrogel state, coupled with the presence in them of reactive end-functions amenable to coupling reactions, was soon exploited in this direction. Two ways were explored, the surface-grafting of PAAs on commercial polymers and the preparation of PAA block—an graft copolymers with medical grade commodity plastics. The preparation of these modified materials has been reported above in the “PAA-based block and graft copolymers” and “Surface-grafting of PAAs onto organic and inorganic materials” sections.
The previously mentioned polyurethane-PAA block copolymers nicknamed PUPA were extensively studied. PUPA samples containing 5–30 wt % PAA showed approximately the same mechanical properties and hydrolytic stability of parent polyurethane, even if their tensile strength was somewhat lower and the elongation at break higher. PUPA samples were capable of adsorbing, and probably to some extent also absorbing relatively large amounts of heparin (0.002–0.7 mg/cm2) and stably retained it.[128-136] No heparin was stably retained by the corresponding native polyurethane. PUPA samples proved an excellent coating for poly(vinylchloride), virgin polyurethanes, and other materials. Segmented polyurethanes containing quaternary ammonium groups behave in a similar way to PAA-polyurethane block and graft copolymers, but unlike the latter were liable to display hemolytic properties.
The biocompatibility of all the PAA-modified materials was evaluated.[134, 136] Heparinization significantly improved their blood compatibility, especially as regards clotting formation. They showed a definite potential for the fabrication of thromboresistant medical devices addressed to short- and medium-term applications involving contact with blood.
Blood biocompatible PAA-polyamide block-copolymer were also synthesized by reacting amine end-capped polyamides with N,N-methylenebisacrylamide in m-cresol solution. The block copolymers absorbed heparin. After heparinization, they showed significant improvement in blood compatibility. PAA-polymethylmethacrylate block copolymers were also prepared and studied for the same purpose. After heparinization, the resultant materials showed acquired non-thrombogenic properties. PAAs could not be used directly in the making of blood-contacting materials due to their poor mechanical strength. Characterization studies indicated that the PAAs have been suitably incorporated into the MMA matrix. The relative hydrophilic nature of the synthesized copolymers was established from the measurement of water contact angle. Two mixed PAA-PVP hydrogels were also obtained from piperazine, cyclohexylamine, and N,N′-methylenebisacrylamide by copolymerizing N-vinylpyrrolidone with the terminal double bonds. After heparinization, these materials exhibited non-thrombogenic and non-hemolytic properties. The maximum achievable duration of the thromboresistance in vivo of all the above reported heparinizable materials was apparently never determined, but could be hardly expected to be indefinite. However, the discovery of permanently thromboresistant synthetic materials does not seem anymore a priority biomedical target, possibly owing to the improved methods of controlling blood coagulation presently available coupled by the widespread use of fast eliminable low molecular weight heparin, or simply because of the diffuse feeling that reaching it is virtually impossible.
Release of Bioactive Substances from Tailored PAA-Based Hydrogels
Horseradish peroxidase (HRP)-mediated crosslinking of PAA copolymers was recently applied in the preparation of in situ forming degradable hydrogels. In particular, PAA copolymers containing different amounts of tyramine residues were synthesized. The gelation time varied with the amount of tyramine residue, the HRP, and H2O2 concentration. These hydrogels completely degraded under physiological conditions within ten weeks. They proved suitable for the sustained release of model low molecular weight substances and proteins.
A novel application of PAA-based copolymers as injectable pH- and temperature-sensitive hydrogels has been proposed. A series of poly(amidoamine)-poly(ethyleneglycol)-poly(amidoamine) (PAA-PEG-PAA) triblock copolymers were designed and prepared to examine the factors affecting their sol–gel transition behavior. The PAA-PEG-PAA copolymers underwent sol–gel transitions in solution (10–15 wt %) in response to changes in both the pH and temperature. Adjusting the molecular weights of the PEG and PAA blocks and changing the PAA-PEG-PAA concentration allowed to control the sol–gel phase transition behavior. These copolymers demonstrated stronger bioadhesive properties than chitosan and poly(acrylic acid) in an aqueous solution. The in vitro release of flubiprofen, as a model drug, from these hydrogels was tested and found to be controllable.[232, 233]
Recently, a new pH sensitive, biocompatible, and biodegradable polymer hydrogel obtained from the combination of Shellac (a natural polymer secreted by lac insect) and PAA by a photopolymerization process has been reported. This new material has been proposed for the controlled release of colon specific therapeutic agents.
PAA Hydrogels and as Scaffolds for Cell Culturing and Tissue Engineering Applications
As stated above, PAAs can be easily obtained in crosslinked form either directly, by introducing multifunctional monomers in the polymerizing system, or in two steps consisting in preparing first α,ω-divinyl-terminated PAA oligomers with unbalanced monomer mixtures, and then triggering a vinyl polymerization by radical initiators. All crosslinked PAAs in aqueous media absorb high amounts of water, ranging from 100 to 1000% of their own dry weight, or even more, depending from the crosslinking degree and the crosslinking method adopted, and give rise to hydrogels. A comprehensive structural characterization of crosslinked insoluble PAA networks can be performed by high-resolution magic angle spinning (HRMAS) NMR spectroscopy. The interaction of water with PAA hydrogels in the absence and presence of inorganic ions was carefully studied by NMR techniques.[235-237]
The potential of crosslinked PAAs as scaffolds for cell culturing and tissue regeneration started only recently. In a first paper, amphoteric PAA hydrogels were obtained from 2,2-bisacrylamidoacetic acid, 2-methylpiperazine, and primary bis-amines as crosslinking agents. In some instances, mono-acrylamides as modifiers were added. These hydrogels were essentially crosslinked versions of ISA23. Hybrid PAA/albumin hydrogels were also prepared. Cytotoxicity tests demonstrated that all the amphoteric PAA hydrogels considered were cytobiocompatible both as free bases and salts. Pure PAA hydrogels completely dissolved within two weeks in Dulbecco medium at pH 7.4 and 37°C, but hybrid PAA/albumin hydrogels did not dissolve within eight months, suggesting a way to tune the degradation time in vitro. The degradation products of all samples turned to be completely non-cytotoxic. Shortly after, two new hydrogels (PAA-AG1 and PAA-AG2) were prepared by polyaddition of 2,2-bisacrylamidoacetic acid with 4-aminobutylguanidine (agmatine) employing two different crosslinkers, namely 1,10-decanediamine for PAA-AG1 and PAA-NH2, that is a PAA containing pendant NH2 groups, for PAA-AG2. These hydrogels were differently crosslinked versions of AGMA1 (Table 6, No. 28). Both PAA-AG1 and PAA-AG2 proved non-cytotoxic and adhesive to cell membranes. Compared with PAA-AG1, PAA-AG2 exhibited improved mechanical strength. The dissolution times of PAA-AG1 and PAA-AG2 under the conditions reported for ISA23 hydrogels were approximately 10 and 40 days, respectively. As in the previous case, the degradation products were completely non-cytotoxic. These data were later substantially confirmed.
In a parallel study, nanometric hydrogel layers based on ISA23 and AGMA1 crosslinked with 1,2-diaminoethane supported on transparent substrates for cell culture were prepared by in situ polymerization carried out on glass substrates purposely modified with γ-aminopropyltriethoxysilane, followed by swelling in water, which invariably led to spontaneous delamination of the external bulk of the hydrogel. AGMA1 hydrogel layers exhibited a level of cell adhesion toward epithelial cells (MDCK) comparable to that of commercial plastic substrates. On the contrary, ISA23-hydrogels showed a vastly inferior cell adhesion, thus demonstrating that epithelial cell adhesion was probably due to the side guanidine groups of the former hydrogel coupled with its prevailingly cationic character at pH 7.4. Slightly later, it was found that the treatment of plastic or glass wells for cell culture with a solution of linear AGMA1 rendered them good substrates for neuronal cell culturing, promoting Schwann and Dorsal Root Ganglion neurons cell adhesion and/or proliferation to the same extent as poly-l-lysine, but with a vastly inferior toxicity. These results provided the rationale for proposing tubular scaffolds made of AGMA1 hydrogels as guides for peripheral nerve regeneration in vivo. The first reported AGMA1 hydrogels prepared with polyamines as crosslinking agents were mechanically too weak to be inserted in animals, but hydrogels prepared by radical polymerization of vinyl-α,ω-terminated AGMA1 oligomers showed improved mechanical properties and proved amenable to be glued to living tissues by commercial fibrin glue. Rats with severed sciatic nerve were implanted with AGMA1 tubes of 1.2 mm internal lumen. The animals were analyzed at 30, 90, and 180 days post-surgery. The tubing made nerve regeneration remarkably easier. Good surgical outcomes were achieved with no inflammation or neuroma signs. Moreover, nerve regeneration was morphologically sound and the quality of functional recovery satisfactory. At the end of the experiment (90 days) the PAA tubing had faded away.
The weak point of the AGMA1 hydrogels, however prepared, consisted of mechanical properties that, though barely sufficient to perform animal experiments, were still unfit to be proposed for human use. A strategy for obtaining PAA hydrogels combining mechanical strength with ability to promote neuronal, in particular Schwann and DRG cell adhesion and proliferation, was to capitalize on structure-forming properties exhibited by some linear PAAs, which could be expected to be shared by their crosslinked analogs. In particular, the PAA No. 32 of Table 5 was highly crystalline and during polymerization in water showed a strong tendency to precipitate by crystallization. Indeed, moderately crosslinked PAA samples maintained to a considerable extent the same structure-forming properties in aqueous media of their linear counterpart. They formed hydrogels that, notwithstanding their high swelling degree, contained crystalline domains acting as effective reinforcing agents. Consequently, their mechanical properties were remarkably superior to those of all other PAA hydrogels described so far. Interestingly, this property vanished by increasing the crosslinking degree, probably because the length of the linear segments connecting the crosslink points became too short to allow extensive crystalline structuring in a tight network. In vitro experiments on the structured hydrogels showed that Schwann and Dorsal Root Ganglion neurons adhered and grew satisfactorily on them, warranting potential for in vivo applications.
A parallel study dealt with the direct micro fabrication of topographical clues for the guided growth of neural PC12 cells along patterns sculptured by E-beam microlithography on the prevailingly anionic, non-adhesive ISA23 hydrogels deposited on a solid surface. The cells grew only along the sculptured patterns and a neural network of single cells connected by neuritis extending along microchannels was obtained. The reason why the cells grew only along the channels neglecting the rest of the surface was not fully explained, but it might be supposed that the electron beam induced de-carboxylation of the 1,1-bisacrylamidoacetic acid moieties of the hydrogel, locally reversing from anionic to cationic its prevailing charge.
PAAs are a polymer family endowed with a rarely equalled combination of relevant properties making them eligible to a variety of applications mainly, but not exclusively, in the biomedical field. The fundamentals of their structure are tert-amine groups and amide groups regularly arranged along the the polymer chain, of which they constitute integral parts. The carbonyl groups, in β-position to the amine groups, significantly lower both the basicity and the toxicity of PAAs (indeed, some PAAs are nearly as biocompatible as dextran), provided strongly basic- or long-chain hydrophobic side substituents are absent. Notwithstandingly, most PAAs are able to interact with polyanions yielding polyplexes. In dilute aqueous solution, at pH ≥ 7.5 and T ≥ 30°C, PAAs degrade also in the absence of specific enzymes, whereas crosslinked PAAs under the same conditions are apparently much more stable even if ultimately dissolve both in vitro and, more rapidly, in vivo. Their degradation rate is largely tuneable according to needs. Both linear and crosslinked PAAs are remarkably more stable in slightly acidic media.
In addition to the above general properties, PAAs are endowed with a structural versatility enabling to finely tune their acid–base properties, to introduce additional functions for the sake of specific properties, to prepare PAA block copolymers with other PAAs or with most other polymer families, to surface-graft PAA chains on a number of organic or inorganic materials. Last but not least, the PAA preparation is usually simple and environmentally friendly.
PAAs have been and are being successfully considered for a number of disparate applications, such as selective absorbers of inorganic water pollutants, as active componens of sensors, as matrices for immobilizing NLO probes, as hydrogel scaffolds for cell culturing and peripheral nerve regeneration, as selective heparin absorbers, as polymer-drug conjugates, as antiviral and antimalarial agents, as protein intracellular carriers, as nucleic acids complexing agents and transfection promoters. Possibly, from a practical standpoint, PAAs have somewhat resented of having been developed mostly in the Academy without having been sponsored ab initio by a strong research-oriented Company. Nevertheless, it may be expected that continuing the present trend their impact will steadily increase in the next years.
Paolo Ferruti took his degrees at the University of Pavia and then was summoned by Giulio Natta at the Polytechnic of Milan. In 1968, he worked with Melvin Calvin in Berkeley at the Lawrence Radiation Laboratory of the University of California. In 1976, he became Full Professor at the University of Naples and then commuted to Bologna, Brescia and finally Milan. He authored more than 400 papers and 50 patents. Functional polymers for technical and biotechnological applications are his chief interest. His main scientific achievement has been the discovery of poly(amidoamine)s.