Polyelectrolyte complexes: mechanisms, critical experimental aspects, and applications

Abstract

The polyelectrolyte complexes (PECs) are versatile formulations formed by electrostatic interactions between oppositely charged biopolymers. PECs have been investigated widely by the researchers to explore the virtues of this formulation viz. high biocompatibility, excellent biodegradability, low toxicity, cost-effective, environment-friendly, and energy-efficient production. The prime object of the present review is to present the prominent features of PECs including mechanism of PEC formation, structural models of PECs, interactions involved in PEC formation, steps involved in PEC fabrication, factors affecting the formation of PECs and applications of PECs. The patents pertaining to PECs have briefly been tabulated as well.

Keywords:

• Biomedical applications
• drug delivery
• electrostatic interactions
• polyelectrolyte complex
• polyions

Introduction

Coulomb’s (electrostatic) interactions between charged microdomains of two oppositely charged polyionic components result in the formation of polyelectrolyte complexes (PECs) (Dautzenberg and Kriz 2003, Schatz et al. 2004a, Wu and Delair 2015). PEC formation, in general, results in the formation of optically homogeneous and stable nanodispersion with dimensions in a colloidal size range (Mao et al. 2006, Sun et al. 2008). The avoidance of the chemical cross-linking agents reduces the possible toxicity and other undesirable effects of the involved reagents (Lankalapalli and Kolapalli 2009).

PECs can be formed with different number of structures and properties, which grants to them, a wide range of applications in different technological and scientific fields viz. medicine, pharmacy, biotechnology and tissue engineering, biomaterials and biomedicals, cosmetics, and so on (Koetz and Kosmella 2007). As PECs are biodegradable, biocompatible, and non-toxic, they find utility as a subject of extreme interest in pharmaceutical and biomedical research (Boeris et al. 2007).

In the present review, we highlight the key aspects of PECs like structural models of PECs, preparation methodologies, factors affecting the formation and stability of PECs, and characterization of PECs. We also discuss potential applications of PECs, especially in pharmaceutical and biomedical fields. As a framework, we start by introducing the basic mechanism of PEC formation and interactions involved therein. Few patents pertinent to PECs have been tabulated briefly.

Mechanism of PEC formation

The PECs are formed as a consequence of strong electrostatic (Coulomb’s) interactions between charged counterparts of at least two oppositely charged polyelectrolytes (Table 1) (Dautzenberg and Kriz 2003; Schatz et al. 2004a, Wu and Delair 2015). Quantitatively, PECs are categorized as either stoichiometric (S-PECs; PECs prepared by polymers in equimolar ratio), or non-stoichiometric (N-PECs; one polymer in excess compared to another, so that the resulting complex becomes more hydrophilic and soluble) (Robertis et al. 2015).

Table 1. List of natural and synthetic polymers having PEC-forming potential.

Polymer category	Cationic	Anionic
Natural	Chitosan, poly(l-lysine), dextran, starch, cellulose	Alginate, carrageenan, carboxymethyl cellulose, gelatin, gellan gum, gum kondagogu, hyaluronic acid, pectin, xanthan gum, xyloglucan, γ-poly(glutamic acid), maleic starch, polybetaine, heparin, chodroitin sulfate, poly-(l-glutamic acid)
Synthetic	Poly(vinylbenzyl trialkyl ammonium), poly(4-vinyl-N-alkyl-pyridimiun), poly(acryloyl-oxyalkyl-trialkyl ammonium), poly(acryamidoalkyl-trialkyl ammonium), poly(diallydimethyl-ammonium), N,N,N-trimethyl chitosan	Dextran sulfate, cross-linked-poly(acrylic acid), Eudragit (poly(acrylic or methacrylic acid), polyalkylenoxide-maleic acid, poly-(l-aspartic acid), sodium cellulose sulfate, poly(styrenesulfonic acid), poly(vinylsulfonic acid), poly(itaconic acid)

The worldwide consensus among researchers is that the PEC formation is an entropy-driven phenomenon. The contributing force for the formation of PECs in aqueous solutions is the release of low molecular weight counterions (which were previously associated with the charged groups on the polymer chains) that results in a gain in entropy by the system (Michaels and Miekka 1961, Pergushov et al. 2012, Philipp et al. 1989). Kabanov and Zezin (1984) first investigated the kinetics of complex formation and stop-flow measurements have demonstrated that this complex formation process takes place in less than 5 ms.

The mechanism of PEC formation can be viewed, at least in its initial stage, as the net effect of two counteracting processes (Philipp et al. 1989), namely:

1. Electrostatic charge compensation leads to the ordering of two oppositely charged polyions to a complex molecule and this occurs in connection with cooperative effects inducing conformational changes favorable for mutual charge compensation;

2. A “chaotic” aggregation of polyanions and polycations with only partial mutual charge compensation and a considerable number of ionic sites still charge-compensated by low molecular weight counterions.

The basic mechanism of PEC formation, as illustrated in Figure 1, involves three major steps:

1. Primary complex formation;

2. New bond formation within intracomplexes;

3. Intercomplex aggregation.

Figure 1. Schematic representation of the aggregation of PECs. Figure depicts the process of PEC formation with three major steps involved, namely Primary complex formation; new bond formation process within intracomplexes; intercomplex aggregation process (modified after Tsuchida 1994).

The first step proceeds with the establishment of secondary binding forces such as Coulomb’s interactions immediately after mixing oppositely charged polyelectrolyte solutions. This reaction is very rapid. The second step proceeds within the order of an hour and involves the formation of new bonds and/or the correction of the distortions of the polymer chains to define new conformation of the polymer chains. The third step involves the aggregation of secondary complexes, mainly through hydrophobic interactions. The final aggregates of the PECs are insoluble in ordinary solvents, and the molar ratio of the polymer components in the aggregates is almost unity (Tsuchida 1994).

PECs were proposed to consist of two models (Figure 2), on the basis of supermolecular order of polymeric chains in a PEC, namely Ladder-like model with an ordered chain packing and Scrambled egg model with disordered packing (Pergushov et al. 2012). From all the experimental evidences available today, it has been concluded that the above-mentioned models represent the limiting cases, with the reality being existing in between these two models, but the closer to the latter than to the former model (Philipp et al. 1989).

Figure 2. Mechanism of PEC formation. On the basis of supermolecular order of polymeric chains, PECs were proposed to consist of two models, namely Ladder-like model with an ordered chain packing and Scrambled egg model with disordered packing (Modified after Purgushov et al. 2012).

Structural models of PECs

The three different types of structures can result, while in solution, from polycation–polyanion interactions, namely (i) water soluble, (ii) colloidally stable, and (iii) two phase systems (Koetz and Kosmella 2007).

Water-soluble aggregates can be formed at molecular scale when polyelectrolytes with weak ionic groups and large differences in molar mass are mixed in a non-stoichiometric ratio (i.e., the ratio of cationic to anionic functional groups is either >1 or <1 but = 1). Such structures consist of long host molecules sequentially complexed with shorter guest polyions of opposite charge. The concentration of soluble salts and the charge ratio of two polyelectrolytes affect the stability of water-soluble PECs, that as coexist in solution with insoluble and colloidally stable PECs, or aggregates and precipitates (Koetz and Kosmella 2007, Zezin and Kabanov 1982).

Complex formation between high or similar molecular weight polyelectrolytes produces highly aggregated and macroscopically heterogeneous systems. The complexes obtained from polyelectrolytes having significantly different molar masses, mixed in non-stoichiometric ratios, produces water-soluble aggregates at the molecular scale. However, at low or moderate ionic strengths, in extremely diluted solutions and non-stoichiometric conditions, the aggregation process can be stopped at a colloidal level resulting in colloidally stable PECs (Delair 2011; Gucht et al. 2011).

The mixing of highly concentrated solutions of high and similar molecular weight polyelectrolytes near stoichiometric ratio forms two-phase systems, consisting of a liquid phase and a PECs-rich phase. The rheological properties of PECs-rich phase depends on the properties of polyelectrolytes and salt concentrations and are similar to the either soft-solids or otherwise have liquid-like properties, being called in this case as complex coacervates (Gucht et al. 2011).

Interactions involved in PEC formation

The interactions established during complex formation between polyelectrolytes includes Coulomb’s interactions, hydrogen bonding, hydrophobic interactions, van der Waals interactions, and dipole interactions (Delair 2011, Koetz and Kosmella 2007, Philipp et al. 1989, Robertis et al. 2015).

The strong intermolecular interactions existing in between polycation and polyanion, responsible for effective complex formation are referred to as Coulomb’s interactions. In this case, PEC formation process depends on the nature of the polymer in excess. Sæther et al. (2008) proposed a core-shell model of PECs based on electrostatic interactions between chitosan and alginate (Figure 3). In excess of either chitosan or alginate, the zeta potential has a large positive or negative value and small, non-aggregating particles are formed. This non-aggregating behavior is suggested to arise due to an excess of the major component, which forms a stabilizing shell around the particles. At the crossover from a negative to positive zeta potential, which occurs at K∼1, the results indicated that there is an increment in the average size of PECs. These findings probably reflect the formation of flocks and aggregates. Similar findings were observed near net charge neutrality for chitosan and dextran sulfate (Schatz et al. 2004a). The capability to build larger aggregates or agglomerates therefore mainly occurs close to K∼1. Figure 4 represents the preparation methodology and structural model of PECs suggesting core-shell topography of the fabricated PECs (Sæther et al. 2008).

Figure 3. Schematic illustration of a core-shell model for polyelectrolyte complexes of alginate (polyanion) and chitosan (polycation) at net charge ratios less than one, equal to one and larger than one (reproduced from Sæther et al. 2008, with kind permission of the copyright holder, Amsterdam).

Figure 4. Preparation methodology and structural model of PECs based on the arrangement of polymer chains involved in PEC formation, suggesting core-shell topography of the fabricated PECs (modified after Lu et al. 2012).

Preparation of PECs

The preparation protocol for the fabrication of PECs, in general, follows the below mentioned steps (Schatz et al. 2004b).

Preparation of polyelectrolyte solution

The two oppositely charged polyelectrolytes are dispersed in a suitable dispersion medium (usually deionized water) at various concentrations and in defined stoichiometric ratios, so as to obtain the solutions of both polyelectrolytes with required molar concentrations. A polysalt (like sodium chloride) can be added to obtain required ionic strength. Finally pH is adjusted to a desired value. All prepared solutions are filtered through 0.22 μ millipore membrane filter before use.

PEC formation

The PECs are generally prepared at room temperature using either polycation or polyanion as the starting solution. The complex formation process, in general, can be completed using either the slow addition of polyanionic solution dropwise at a predetermined flow rate to polycationic solution of same ionic strength or the rapid one-shot addition of one polyelectrolyte solution to oppositely charged polyelectrolyte solution of similar ionic strength under a constant speed magnetic stirring.

Particle refinement

Particle refinement is done by centrifugation process and is necessary in order to avoid physisorption of free polymers onto the surface of already formed PECs. The final product is either re-suspended in a minimum volume of deionized water for immediate characterization or is lyophilized for better stability.

Factors affecting the formation and stability of PEC particles: critical experimental aspects

The mixing of two oppositely charged polyelectrolytes usually leads to the separation of a polymer-rich phase (milky phase) from a polymer-depleted phase (clear phase) (Muller 2012). Figure 5 enlists various factors affecting the formation, stability of PECs.

Figure 5. Illustrative representation of various factors affecting the formation and stability of PECs.

Charge-to-charge stoichiometry

While investigating polyelectrolyte complexation, what needs to be studied is the net ratio of positive to negative charges of the oppositely charged polyelectrolytes involved in fabricating PECs. This ratio is referred as charge ratio and denoted as Z, with the subscripts (+/− or −/+). The charge-to-charge stoichiometry of the formed PECs is denoted as Φ. S-PECs (Φ = 1) are sufficiently hydrophobic due to the mutual screening of the charges and precipitate from the aqueous solution. If the N-PECs (Φ = 1) are prepared, overcharging effects due to either excess polycation or polyanion can be observed (Pergushov et al. 2012).

Charge density

Dautzenberg et al. (1982) reported the effect of charge density on PEC properties using cationic and anionic copolymers of acrylamide. They observed that for equal charge density of the polyelectrolyte components, the symplex particles adopt a compact structure, whereas in systems with strongly deviating charge density of the polyelectrolyte components a loose fluctuating structure prevails. Claesson et al. (2009) recently reported the controversial findings with a cationic methacryloxyethyl trimethylammonium (METAC) and anionic poly(styrenesulfonate) (PSS). They observed that with decreasing charge density it was possible to obtain soluble PECs with decreasing hydrodynamic radii of around 20 nm and increasing stability, even for 1:1 stoichiometry.

Molecular weight

Recently, Hu et al. (2012) reported the effect of chitosan (CHT) molecular weight on the particle size of carboxymethylpachyman (CMP)/CHT PEC nanoparticles. Increase in the molecular weight of CHT from 12,000 to 46,000 g/mole resulted in increased particle size of PECs from 135 to 279 nm. In the conclusion, it was underlined that longer chains of positively charged CHT molecules can complex with a large number of negatively charged CMP molecules.

Polyelectrolyte concentration

Schatz et al. (2004a) reported the increase in size of CHT/DS biopolyelectrolyte complex particles with increasing DS concentration, but only at high mixing ratios n⁺/n⁻. Muller et al. (2011) suggested that, based on the model of aggregation of primary PEC to secondary PEC particles due to short range dispersive interactions, increasing polyelectrolyte concentration results in reduced electrostatic repulsion between like-charged primary PEC particles and thus, in their elevated dispersive attraction. Furthermore, an increase in polyelectrolyte concentration might also result in a large number of primary PEC particles per volume. Both could lead to larger secondary PEC particles. Exceeding certain polyelectrolyte concentration values can also lead to precipitation.

pH

Polyelectrolyte complexation between chitosan and dextran sulfate sodium prepared at low and high pH values have been investigated by Fukuda and Kikuchi (1977), who suggested that the complexes prepared at different pH conditions were different in their molecular structures. Muller et al. (2011) studied the influence of pH combination on the particle size of PEC system consisting of weak polyelectrolytes poly(ethyleneimine) (PEI) and poly(acrylic acid) (PAC) at molar mixing ratio n⁻/n⁺ = 1.5 and observed a decrease in particle size from ∼400 nm to ∼160 nm with decreasing values of pH (10, 8.5, 7, and 4) of the PEI solution at a constant pH of PAC solution. Highly charged particles of PEI/PAC at lower pH may have a lower tendency of coagulation due to mutual electrostatic repulsion, compared to the lower charged particles formed at higher pH due to electrostatic attraction.

Ionic strength

The ionic strength of the polyelectrolytes has great impact on the final size of the PECs. An increase in ionic strength induces a decrease in the average diameter, which could be related to the increase in chain flexibility (Delair 2011). The complexation can occur only at pH values in the vicinity of the pK_a interval of the two polyelectrolytes. During complexation, polyelectrolytes can either coacervate or form a more or less compact hydrogel. Nevertheless, if ionic strength is too strong, precipitation can occur (Berger et al. 2004).

Mode of mixing polyelectrolyte solutions

Mixing of polycation and polyanion solutions to produce PECs is expected to be dependent on the mixing type, mixing protocol, and the device used. Ankerfors et al. (2010) reported the influence of mixing procedure on the complexation of poly(allylamine) (PAH) and PAC by comparing jet mixing with the frequently used colloid titration. They obtained small PECs for both low molecular weight polyelectrolytes at short mixing times, whereas for high molecular weight polyelectrolytes PEC size first decreased with decreasing mixing time until a minimum and then increased again. This behavior was attributed to the diffusion-controlled formation of pre-complexes occurring sufficiently quickly so that stable complexes were formed. However, for larger polyelectrolytes non-equilibrium pre-complexes prone to aggregation were formed. Comparing the two mixing procedures, jet mixing gave smaller PECs, allowing mixing time to control PEC size, whereas colloid titration gave larger PECs. Sæther et al. (2008) observed decrease in particle size of chitosan-alginate PECs with increasing mixing speed using an Ultra-Turrax procedure.

Order of addition

A very sensitive experimental variable was investigated by several researchers, the order of polyelectrolytes addition, while polyelectrolyte solutions are mixed slowly with one another (Mende et al. 2004). Schatz et al. (2004a) compared the ordinary slow dropwise mixing with rapid one-shot mixing for the preparation of CHT-DS PECs. Rapid one-shot mixing procedure gave PECs with small diameter and higher stability compared to the PECs prepared by a slow dropwise mixing process. While adding the titrant solution dropwise to the starting solution, the order of mixing was essential and the polymer in default has to be added to the one in excess in order to avoid the aggregate formation. In contrast, the one-shot addition of the titrant solution was insensitive to the order of addition, confirming that the particle formation process is kinetically controlled (Schatz et al. 2004a).

Mixing ratio

Dautzenberg (1997) studied the effect of mixing ratio (X) on the molecular weight and particle size of PEC particles between poly(diallyldimethylammonium chloride) (PDADMAC) and poly(styrenesulfonate) (PSS). A slight decrease in molecular weight and a significant decrease in particle size from 400 nm (X = 0.1) to 200 nm (X = 1.0) was found with increasing mixing ratio in a saltless system. It was qualitatively interpreted as an increase in structural density when X approaches 1:1 stoichiometry because mutual charge-compensated chains might be more compactly coiled. In a salted system, the presence of salt (Cs = 0.01 M) increased the particle size from 50 nm (X = 0.1) to about 120 nm (X = 1.0). It was interpreted by a lower aggregation number of the salted system.

Salt concentration

The influence of salt concentration on the properties of PECs can be studied either during complexation or after complexation.

Dautzenberg (1997) prepared PDADMAC/PSS PEC particles at a polyelectrolyte concentration of 0.00025 M (PSS) and 0.0005 M (PDADMAC) in the presence of no salt, 0.01 M and 0.1 NaCl. For no salt, the PEC particle size dropped from around 300 nm at mixing ratio X = 0.1 to about 130 nm at X = 0.95. For 0.01 M NaCl, the size was constant at about 40 nm at X = 0.1–0.9. For 0.1 M NaCl, the PEC size was 50 nm at X = 0.1, which was slightly higher than for 0.01 M, and increased to about 120 nm at X = 0.95. In this investigation, increasing salt concentration favored coagulation of primary PECs to larger secondary PECs, while on the other hand the polyelectrolyte shells of the finally formed PEC aggregates shrink to a smaller one.

Dautzenberg and Kriz (2003) evaluated the influence of addition of salts (LiCl, NaCl, and KCl) on the size of PECs between PDADMAC and acrylamide complexed by polymethacrylate. A constant mixing ratio of X = 0.6 was used. Using salt addition, after complexation, there was an increase in PEC size from around 70 nm to at C_NaCl = 0 up to a sharp increase up to 370 nm at around C_NaCl = ∼0.5 M and decrease to again around 80 nm at C_NaCl > 0.5 M. The other salts also followed the similar trends, which were attributed in terms of salt-induced aggregation of small soluble complexes to larger aggregates at salt concentrations of 0–0.5 M, whereas the sharp drop in particle size of PECs for C_salt > 0.5 M was attributed to the dissolution of PEC aggregates to small soluble complexes or partly to the polyelectrolyte components.

Characterization of PECs

The key parameters to be examined and the techniques used for the characterization of PECs are briefly discussed in this section. The PEC characterization mainly includes the physicochemical, morphological analysis of PEC particles in dispersion, and solid state characterization of the lyophilized complexes.

Physicochemical characterization

Static or dynamic light scattering (SLS/DLS) is the most widely used technique for the estimation of size or size distribution (polydispersity index) of PEC particles. Stokes–Einstein equation is employed to determine the mean hydrodynamic diameter of the PEC particles in these techniques (Delair 2011). SLS measures time-average intensities (mean square fluctuations). SLS is also commonly utilized to determine the weight–average molecular weight and hydrodynamic diameter of particles in the sub-micron and supra-micro ranges. DLS (also known as photon correlation spectroscopy or quasi-elastic light scattering) measures the time-dependent fluctuations of the intensity of scattered light caused by particle movement. This fluctuation is due to the fact that the small molecules in solutions undergoing Brownian motion, and so the distance between the scatterers in the solution is constantly changing with time. SLS/DLS covers a range from a few nanometers to about 3 μm (Pardeshi et al. 2012). However, SLS/DLS is not able to detect larger microparticles. One can draw the predictions regarding storage stability of colloidal PEC particles from zeta potential measurements using SLS or DLS (Pardeshi et al. 2012).

Morphological characterization

The morphological analysis of the colloidal PEC particles can be done using scanning or transmission electron microscopy (SEM/TEM) or using scanning force microscopy (SFM). It has been noted, by various investigators, that PECs are generally polydisperse quasi-spherical particles, sometimes affected by the vacuum drying during SEM analysis (Delair 2011, Wu and Delair 2015).

Solid state characterization

The FTIR spectroscopy is used to assess the complex formation process. The formation of the new complex was evidenced by the appearance of new bands, change in intensity of already existing bands or the shift of bands from their original position (Delair 2011).

The alterations in the thermal properties, through DSC and TGA analysis, are also a mean of characterization of PEC particles as revealed by the Sarmento et al. (2006b). The thermal stability and the effect of process parameters like pH on the properties of PEC particles can be studied using DSC/TGA analysis (Martins et al. 2011a, 2011b).

The X-ray powder diffraction (XRD) analysis can be used to estimate the nature and degree of crystallinity of the PEC particles, as a consequence of change in pH or electrostatic interactions among the charged functional groups of participating polymers in PECs (Martins et al. 2011b).

The qualitative structure of the PEC particles can be evaluated using nuclear magnetic resonance (NMR) spectroscopy. The selectivity offered by the chemical shifts compliments the sensitivity to molecular mobility so as to provide information on the physicochemical status of the PEC particles (Martins et al. 2011a).

Complex stoichiometry

The PEC formation process depends on charge neutralization, and the end point of titration can be determined by using various techniques, namely viscosimetry, potentiometry, or conductometry. These methods cannot give an idea regarding the composition of the PECs that can be obtained using elemental analysis, solid state NMR spectroscopy, and X-ray photocorrelation spectroscopy (Delair 2011).

Applications of PECs

Applications of PECs in drug delivery

The PEC particles find wide applications in various disease conditions, including cancer (Anitha et al. 2011, Martins et al. 2013, Wang et al. 2014), HIV/AIDS (Costalat et al. 2014, Polexe et al. 2013), gastric ulcer (Volodko et al. 2013), Staphylococci infections (Lankalapalli et al. 2014), and fungal infections (Lefnaoui and Moulai-Mostefa 2014). The particulars of polycations and polyanions used, therapeutic agents loaded into, and the diseases targeted using these PECs are presented in Table 2.

Table 2. Applications of PECs as drug delivery carriers.

Polycation	Polyanion	Drug	Formulation	Target disease	Reference
Chitosan	Dextran sulfate	Curcumin	PEC nanoparticles	Cancer	Anitha et al. (2011)
TMC	Alginate	Curcumin	PEC	Cancer	Martins et al. (2013)
Chitosan	Poly(l-malic acid-co-d,l-lactic acid)	Doxorubicin	PEC nanoparticles	Cancer	Wang et al. (2014)
Chitosan	Dextran sulfate	Tenofovir	PEC nanoparticles	HIV/AIDS	Polexe et al. (2013)
Chitosan	Dextran sulfate, heparin	Adenosine 5′-monophosphate  monohydrate	PEC nanoparticles	HIV/AIDS	Costalat et al. (2014)
Chitosan	Carrageenan	NS	PEC	Gastric ulcer	Volodko et al. (2013)
Chitosan	Neem gum, Hupu gum	Vancomycin	PEC nanoparticles	Staphylococci infections	Lankalapalli et al. (2014)
Eudragit RL 30D	Carboxymethyl kappa carrageenan	Miconazole	Bioadhesive matrix  tablets	Fungal infections	Lefnaoui and  Moulai-Mostefa (2014)

NS, not specified; TMC, N,N,N-trimethyl chitosan.

Applications of PECs in gene delivery

The design of biocompatible PECs is a promising strategy for in vivo delivery of non-viral vectors loaded with therapeutic genes to find applications in gene therapy. The condensation of DNA by polycations, leading to the formation of so-called polyplexes, received considerable attention for its potential in gene delivery applications, where the development of safe and effective non-viral vectors remain a central challenge (Amaduzzi et al. 2014). The selected examples of PECs utilized as gene delivery carriers are presented in Table 3.

Table 3. Selected examples of PECs in gene delivery applications.

Polycation	Polyanion	Formulation	Therapeutic outcomes	Reference
Chitosan	pDNA	Nanocomplex	Nanocomplexes exhibited high pDNA transfection efficiencies in HEK293 cells in vitro	Cifani et al. (2015)
Chitosan	pDNA	Nanocomplexes	ELISA assay revealed an efficient expression of PDGF-BB and FGF-2 with specific antibodies in mice sera	Jean et al. (2009)
Polyethylenimine	pDNA	Nanoparticles	Nanoparticles exhibited high transfection efficiency with the gene encoding human urokinase plasminogen activator (Hu-uPA)	Zaitsev et al. (2004)
Chitosan	DNA	Chitoplexes	High binding affinity of chitosan to DNA was obtained at pH 5.4 At high charge ratio, the spherical nanocomplexes were formed, without free DNA	Liu et al. (2005)
PEI-PEG-PCP	siRNA	Polyplexes	Polyplexes exhibited high VEGF inhibition efficiency in human prostate carcinoma (PC-3) cells	Kim et al. (2009)
PEI	siRNA	Polyplexes	High uptake of siRNA by HeLa cells at higher concentration of copolymer used to modify the surface of polyplexes	Bui et al. (2012)
PEI-DA	siRNA	Polyplexes	The polyplexes showed high stability against heparin exchange reaction and improved gene silencing efficacy	Lee et al. (2012)
PFNBr	siRNA	Nanocomplexes	Nanocomplexes showed enhanced cellular uptake in PANC-1 cells	Jiang et al. (2013)

pDNA, plasmid DNA; PDGFBB, platelet-derived growth factor-bb; FGF-2, fibroblast growth factor-2; VEGF, vascular endothelial growth factor; PEI-PEG-PCP, prostate cancer-binding peptide (PCP) conjugated with polyethylenimine (PEI) via poly(ethylene glycol) (PEG) linker; PEI-DA, polyethylenimine modified by deoxycholic acid; PFNBr, brush-like conjugated polyelectrolyte nanoparticles.

Applications of PECs in protein and peptide delivery

Several laboratories have reported the possible applications of PECs in the delivery of therapeutic proteins or peptides. The non-covalent association between positively charged polymer and negatively charged protein or peptide to yield a PEC is based on the principle of electrostatic or hydrophobic interactions in between participating polymers and protein/peptide (Cheng et al. 2010). Table 4 shows the selected examples of PECs investigated for protein and peptide delivery.

Table 4. Applications of PECs in protein and peptide delivery.

Polycation	Polyanion	Therapeutic protein/peptide	Formulation	Reference
Chitosan	Dextran sulfate and alginate	Insulin	PEC nanoparticles	Sarmento et al. (2006a)
Chitosan, polyethylenimine, poly-l-lysine	Dextran sulfate	VEGF	PEC nanoparticles	Huang et al. (2007)
APEG	PMMA	BSA	PEC nanoparticles	Sajeesh and Sharma (2007)
Chitosan, polyethylenimine, poly-l-lysine	Dextran sulfate	Repifermin®	PEC nanoparticles	Huang and Berkland (2008)
Chitosan	Alginate	bFGF	Scaffolds	Ho et al. (2009)
Chitosan	Hyaluronate	Insulin or vancomycin	Nasal insert	Luppi et al. (2009)
Chitosan	Alginate	Ovalbumin	Nanocomplex	Cegnar et al. (2011)
Chitosan	Poly(γ-glutamic acid)	SDF-1	Multilayer films	Goncalves et al. (2012)
Chitosan	NaCS and PPS	Lactoferrin	PEC capsules	Wu et al. (2013)

VEGF, vascular endothelial growth factor; PMMA, poly(methacrylic acid); APEG, bis-(2-aminopropyl)poly(ethylene glycol); bFGF, basic fibroblast growth factor; SDF-1: stromal-derived factor 1; BSA, bovine serum albumin; NaCS, sodium cellulose sulfate, PPS, sodium polyphosphate.

Applications of PECs in vaccine delivery

Micro- or nanoparticulate formulations provide a right mode for the delivery of antigenic molecules. In addition to the improved protection, prolonged residence at the desired site, and facilitated transport of the antigen, particulate formulations could also offer more effective antigen recognition by immune cells (Csaba et al. 2009, Xiang et al. 2006).

Recently, Verheul et al. (2011) developed covalently stabilized polyelectrolyte nanocomplexes composed of thiolated trimethyl chitosan (TMC-SH) and thiolated hyaluronic acid (HA-SH), with or without PEG coating, as a potential nasal and intradermal vaccine delivery system. The PEC particles were loaded with ovalbumin (OVA) as a model antigen. OVA-loaded stabilized TMC-S-S-HA particles demonstrated superior adjuvanticity and immunogenicity compared to non-stabilized particles, after both nasal and intradermal vaccination. PEGylation abolished the beneficial effects of stabilization after nasal vaccination while having similar immunogenicity as stabilized particles after intradermal vaccination. Finally, the authors concluded that stabilization of TMC-HA PEC particles greatly enhance the immunogenicity of OVA after nasal and intradermal vaccination.

Applications of PECs in tissue engineering

The biodegradable or biocompatible materials have been used as support matrices for delivery of bioactive species to target specific tissues and to promote three dimensional tissue reconstructions (Liu et al. 2007). There are a number of reports explaining the potential of PECs in various areas of tissue engineering like bone tissue engineering (Coimbra et al. 2011a), cartilage tissue engineering (Whu et al. 2013), cardiac tissue engineering (Ceccaldi et al. 2014), and dental tissue engineering (Chang et al. 2014, Coimbra et al. 2011b).

Diagnostic and imaging applications of PECs

Huang et al. (2008) developed PECs capable of MRI contrast enhancement using gadolinium (Gd). PECs were 300 nm in diameter, produced by complex coacervation of chitosan with dextran sulfate. Gd was incorporated by two different mechanisms, namely First, diethylenetriamine pentaacetic acid (DTPA) with chelated Gd was grafted to chitosan before complexation with dextran sulfate. Second, Gd was incorporated into PECs by mixing them with gadolinium chloride (GdCl₃), which resulted in ionic trapping of Gd ions within the PECs. In this way, three contrast enhanced PECs were produced: (i) Gd-DTPA grafted to chitosan, then complexed with dextran sulfate; (ii) ionically trapped Gd (Gd³⁺) in preformed PECs; and (iii) a combination including Gd-DTPA grafted to chitosan and ionically trapped Gd. To their conclusions, PECs combining grafted Gd-DTPA and ionically trapped Gd produced significant contrast enhancement in vivo. Particles were observed to accumulate in the rat kidneys. PECs modified using these techniques offer MRI contrast enhancement of a widely used drug and gene delivery vehicles as a method of quantifying particle biodistribution and pharmacokinetics.

Patents pertaining to PECs

The limitation of large-scale commercial production of PECs restricted their transfer from the laboratory to the commercial market. Only few patents have been transferred into the commercial marketed products. Some of the patents relevant to PECs are briefly outlined in Table 5.

Table 5. Patents pertinent to PECs.

Payload	Therapeutic applications	Year	Patent No.	References
Heparin	For hemophilia	1984	4471112	Johnson (1984)
Protein, nucleic acid, antigen	Nanoparticulate system for drug, protein, antigen or  nucleic acid delivery	2003	US 2003/0170313A1	Prokop (2003)
RNA, DNA, peptides, hormones, antigen	For flavor, fragrance delivery	2006	WO2006/064331	Laue and Kauper (2006)
Not specified	Coating of medical devices, kidney hemodialysis,  and dental applications	2007	WO2007/108775A1	Ying and Wan (2007)
Vitamins, enzymes, proteins peptides	As an absorption enhancer and drug carrier	2008	US2008/0254078A1	Laue and Kauper (2008)
Not specified	For dental application and kidney hemodialysis	2010	US2010/0111917A1	Ying and Wan (2010)
Protein, DNA, and peptides	For protein delivery and treatment of lysosomal disease	2012	WO2012/085888A2	Giannotti et al. (2012)
Silver, triclosan	Antimicrobial and antifungal	2012	US2012/8092854B2	Toreki and Moore (2012)
Not specified	For water disinfection and antimicrobial	2013	US2013/0273174A1	Scheuing et al. (2013)
Radio opaque contrast media	For magnetic resonance imaging	2013	US2013/0302255A1	Borbely et al. (2013)

Conclusions and future perspectives

PECs, being stable, biodegradable, ad biocompatible formulations, are capable of merging the unique properties of different polymers without losing their inherent characteristics. The particle formation process can be considered as an environment friendly, energy-efficient production process utilizing only aqueous solvents and can be carried out at ambient temperature conditions. Because of their versatility and above mentioned merits, PECs find utility as a subject of keen interest for the researchers.

A pharmaceutical formulation if, along with academic researchers, well accepted by the pharmaceutical industries for commercialization and clinical applications, would be considered as an effective therapeutic formulation. Since, PECs have already been proven their potential in pharmaceutical, biomedical, and other allied fields; they must engage a sizeable place in the pharmaceutical market.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.