SELF-ASSEMBLY OF BIOHYBRID POLYMERS
7.4 SELF-ASSEMBLY DRIVEN BY ELECTROSTATIC INTERACTIONS
Polyelectrolytes belong to a class of water-soluble macromolecules that bear high amounts of charges. Such materials are divided into either weak (annealed) or strong (quenched) polycations and polyanions. Strong polyelectrolytes are permanently charged, regardless of the pH in aqueous media. For weak polyelectrolytes, the situation is different: the charge density, and hence the chain conformation and the solubility depend on the solution pH [111]. Other than cationic or anionic polyelectrolytes, polyampholytes contain both positive and negative charges, which can be randomly distributed or arranged block-wise along the polymeric backbone [112]. In the case of polyzwitterions, each monomer unit carries at least one positive and one negative charge [113]. Two oppositely charged polyelectrolytes do co-react in solution to form interpolyelectrolyte complexes (IPECs) [114].
Interpolyelectrolyte complex is a specific term for the complex formation between two polyelectrolytes of opposite charge [115]; block ionomer complex (BIC) is another term specifically used for the complex formation between ionic/non-ionic block copolymers with one charged and one neutral segment and polyelectrolytes of opposite charge [116]. Polyion complex (PIC) [117], polyplex [18], or complex coacervate (CC), introduced for the electrostatic co-assembly of oppositely charged synthetic polyelectrolytes [119] are definitions found as well in the literature.
IPECs have attracted attention due to the large number of emerging and poten- tial applications. Currently, various synthetic IPECs did find commercial applica- tions [113]. Some examples include synthetic ionic polymers used in the field of water treatment [120], in the oil industry [121], and nanotechnology [122]. Other approaches focus on IPEC systems based on biomolecules (DNA, protein, polysac- charides), which are very attractive because of their numerous potential applications notably for medical purposes (targeted drug delivery, imaging) [118, 123–126]. This
k k part of the chapter will be restricted to IPEC systems based on biomolecules, named
as bio-IPECs. First, the factors influencing the complex formation will be briefly described. Later on, relevant but not exhaustive examples of bio-IPEC will be given.
As already mentioned, the mixing of oppositely charged polyelectrolytes in aqueous solution immediately induces IPECs formation. Such macromolec- ular co-assembly processes proceed with high rates, which are typical of diffusion-controlled reaction (millisecond range) [127]. As a result of colli- sions between oppositely charged macromolecules, contacts (referred to as ion-ion or salt bonds) are formed between their monomer units. Any low molecular weight counterions, which are initially located near the polymeric backbone of the involved macromolecules, are released into the solution, which causes the entropy of the system to increase. This co-assembly process is thus predominantly driven by a gain in entropy [128]. This effect was notably observed by Sato et al. in their investigation of interaction between DNA and a cationic comb-type copolymer (PLL-g-Dex). Double-stranded (ds) DNA has a higher density and a larger total number of anionic charges than single-strand (ss) DNA, and therefore has larger counterion condensation. These counterions are released upon IPEC formation with the cationic polymers, leading to the formation of a more stable IPEC with dsDNA than with ssDNA [129].
Hydrogen bonding might as well play an important role in complexation, notably stabilization (Figure 7.14). For example, the results obtained by Mascottiet al. in the study of the IPEC formation between DNA strands and oligoarginines, show that H-bonding of arginine with the phosphate backbone of the nucleic acid contributes to the increased stability of these complexes [130]. Another example is the study of IPEC based on polymethacrylate (Eudragit, E100) and sodium carboxymethylcellu- lose (NaCMC) achieved by Ngwulukaet al. In this study, computational modeling revealed that the formation of the IPEC was due to electrostatic interaction, hydrogen bonding, and van der Waals interactions [131]. Thus the hydrophilic weight frac- tion of the polymer is another important parameter to be taken in consideration. This parameter influences both the solubility of the polymer in aqueous media and its com- plex formation via hydrophobic or ionic association, hydrogen bonding, and van der Waals interaction [110, 131].
Moreover, the ratio of polyelectrolyte chain lengths and nature of the substituent (inducing screening of charges) or counterions (uni- or multivalent) can be significant in the formation of IPEC [132]. Investigations of the binding interactions between native DNA and dendrimers (Astramol) of five generations (G1, G2, G3, G4, and G5) have been carried out by Kabanovet al. All dendrimers interacting with DNA at an equal concentration of amine and phosphate groups did form neutral water-insoluble IPECs. However, G4 and G5 added to the DNA solution in excess formed positively charged water-soluble IPECs, contrary to G1 and G2 [133].
Environmental factors such as the medium also play a role in the complex forma- tion process, especially pH and ionic strength (salt and polyelectrolyte concentration).
The presence of salt (e.g., NaCl) drastically affects the characteristics of IPECs [135, 136]. At low salt levels, structure formation leads to the formation of rather small par- ticles. Increased ionic strength can induce the formation of aggregated IPEC species
k k
Polyplex Size Decreasing
Increasing Charge Density or Hydroxyl Concentration
Electrostatic and Hydrogen Bond Interactions Present in Polyplex
OH
OH
OH
HO HO HO HO
Maximum Transfection Efficiency
Figure 7.14 IPEC formed between a cationic polymer and condensed anionic plasmid DNA.
Various factors including hydrogen bonding, impact the IPEC stability. Reprinted with the permission from [134]. (See color insert for color representation of this figure).
and even cause macroscopic phase separation, leading to a precipitate of an insoluble nearly stoichiometric IPEC while the prevailing supernatant solution contains either nonstoichiometric (Z≠1) IPECs or, in some cases, a pure host (excess) polyelec- trolyte [111]. For example, Sunget al. observed that the polycation (PEG grafted PEI)/DNA complexes, in excess of the polycation, had an effective diameters of 500 nm in 10 mM NaCl, whereas, in 150 mM NaCl, complexes having size greater than 900 nm were observed [137]. At still higher salt concentrations, a threshold ionic concentration is reached, which induces dissociation of the IPEC. Therefore no com- plex formation was observed [138, 139]. In addition to the salt concentration, in the case of weak polyelectrolytes, the solution pH also strongly influences the amount of charges, and therefore also the extent of formation of interpolyelectrolyte com- plexes [131]. This factor was used by Elschneret al. to produce a pH-responsive IPEC based on a well soluble cellulose-based polyzwitterion with weak ionic groups. Their studies showed the formation of pH-responsive IPECs in the presence of polydial- lyldimethylammonium chloride. These particles are switchable in a physiologically relevant range of pH [140].
Also of prime importance is the way that the complex formation itself is con- ducted, that is, in mixing parameters such as the charge-to-charge stoichiometry of the components, the addition rate, order of addition of the components (kinetic versus thermodynamic) [127], and temperature [141]. The charge-to-charge stoichiometry
k k corresponds to the overall ratio of positive to negative charges of the oppositely
charged polyelectrolytes involved. Stoichiometric IPECs are hydrophobic due to the mutual screening of electrostatic interaction and precipitate from aqueous solution, although a certain swelling due to the incorporation of water molecules cannot be excluded [135]. If nonstoichiometric mixtures are prepared, overcharging effects due to an excess of either the polycation or the polyanion occur. The solubility and sta- bility of the IPEC then depends as well on the molecular weight of the respective polyelectrolyte added.
IPECs structure formation thus offers unique control over both properties and morphology of the resulting structures. Moreover, bio-IPECs based on biological molecules (DNA/RNA, protein, saccharide) reveal very interesting properties that include biocompatibility, swellability, stimuli responsiveness, and physicochemical stability [142]. Thus bio-IPECs belong to a very interesting and promising class of materials for several applications, notably in bio-medicine, pharmacy, and the food industry, among others. One of the most important application of bio-IPECs is as gene and/or drug delivery vectors for therapeutics [118, 125, 143, 144].
In the following discussion are given relevant examples of bio-IPECs.
7.4.1 DNA/Polymer Bio-IPECs
Gene therapy has emerged as a promising approach for the treatment or prevention of acquired and genetic diseases. The formation of bio-IPECs with DNA is encouraging for future gene therapy developments. DNA, an anionic polymer, can be complexed by cationic polymers, water-soluble or amphiphilic copolymers (depending on the hydrophilic weight fraction). The interaction between DNA and polycation can form various self-assembled structures, from spherical micelles to vesicles [145, 146]. The common polycationic polymers of natural origin are poly(L-lysine) [147, 148], and chitosan [149, 150].
Recently, bio-IPECs with potential for application have been evidenced by Nardin et al. [104]. In their work, they showed that the inhibition of amyloidβ(1-42) fibril genesis could occur if Aβ42proteins interact with a polyanionic polymer, like short DNA strands. Bio-IPECs might be assembled through interaction between the protein and either synthetic polyions or nucleic acid strands through electrostatic attraction between monomers or nucleic acids and amino acids of opposite charges. Moreover, the incubation of the amyloid fibers with synthetic nucleotide sequences induced their disassembly. If the resulting complexes are less toxic than the prefibrillar oligomers or fibers, this strategy could be of particular relevance for future drug developments against amyloid-based diseases [151].
7.4.2 DNA/Copolymer Bio-IPECs
An elegant strategy, to improve the efficiency of gene delivery, is to use a diblock copolymer. Kakizawa et al. proposed a diblock copolymer composed of a neutral block (polyethylene glycol, PEG) and a ionic block (polyamino acid) [152]. The ionic block interacts with the phosphate groups of DNA or RNA, and thus ensures the
k k formation of the complex. The PEG block was used to confer colloidal stability to
the nanocarriers and to delay phagocytosis by prolonging the blood circulation time.
A similar strategy has been developed for protein cell targeting. However, the pro- teins can be neutral or charged (either positively or negatively), and several proteins are unstable in serum (parenteral route) or in the gastric and intestinal media (oral route). A solution, recently proposed by Novoa-Carballal et al. is the use of sul- fated glycosaminoglycan (GAG) [139] (or hyaluronic acid (HA) [138])), negatively charged polysaccharides [139], as the ionic block of a PEG-based diblock copolymer (Figure 7.15) [153, 154].The main advantage of the GAGs approach is that it allows formation of complexes of neutral charge, small size, and colloidal stability at physi- ological (and even higher) ionic strength. All these properties contribute to prolonged blood circulation times of the formed IPECs, and thus more effective delivery to the targeted site. We can note here that PEG is the most common neutral block used for biological application of bio-IPECs due to its biocompatibility and enzymatic resistance. Furthermore, the application of various polypeptide-b-PEG copolymers as drug delivery nanocarriers (DNA, proteins, or charged anticancer drugs) has recently reached the phase III clinical trials development stage [155].
Spontaneous complexation
Phosphate buffer, pH 7.4
Poly-L-lysine (PLL)
Glycosaminoglycan (GAG)
Polyethylene glycol (PEG)
Core:
Interpolyelectrolyte complex (IPEC) PEG shell: stealth effect
Figure 7.15 Schematic presentation of the complexation process between GAG-b-PEG and polycationic protein (poly-L-lysine (PLL)). Reprinted with the permission from [139]. (See color insert for color representation of this figure).
k k The majority of bio-IPECs strategies employed for drug delivery are very similar
to those used for gene or protein delivery. For example, nanoparticles based on polysaccharide-based diblock copolymers have been developed using dextran [156]
or chitosan-based copolymers [124] for the design of colon targeted ibuprofen tablets. The ternary bio-IPECS (insulin-poly(methylaminophosphazene)-dextran sulphate developed by Burovaet al. for oral delivery of insulin is an elegant approach as well [141]. As for gene delivery, the majority of bio-IPECs structures for drug delivery assemble into a variety of structures, from spherical micelles to vesicles [114, 146].
DNA copolymers are as well investigated to induce IPECs by, for instance, the cou- pling of thec-mybantisense nucleic acid sequence with poly(ethylene glycol) (PEG) through an acid cleavable phosphoramidate linkage [157]. The resulting biocompat- ible diblock was complexed with a cationic peptide through electrostatic interaction in order to give rise to stable micellar aggregates. The resulting complex is com- posed of an inner polyelectrolyte core surrounded by a PEG chain corona. Coating of the nanocarriers with the hydrophilic, inert, and biocompatible polyethylene gly- col (PEG) decreases the rate of opsonisation, leading to reduced clearance by the mononuclear phagocytic system (MPS) and prolonged circulation time but increased delivery efficiency [158, 159]. Indeed the transport of nucleic acids to cells was greatly improved in comparison to the pristine ODN sequence [157].