Chitosan/Poly(γ-glutamic acid) Polyelectrolyte Complexes: From Self- Assembly to Application in Biomolecules Delivery and Regenerative Medicine

PEC of different polymers can be proposed, such as Poly(Styrenesulfonate) (PSS) and Poly(Allylamine Hydrochloride) (PAH), two synthetic polymers initially studied by Decher et al. [213] and one of the most widely studied PE pairs, namely to further analyze PEM formation [304-307]. Nevertheless, studies with natural polymers have been preferred for biomolecules delivery and regenerative medicine, since biological recognition allows the PEs to become bioactive and biodegradable [19,58,308,309]. Within natural polymers, focus has been given to Ch, collagen, hyaluronic acid and chondroitin sulfate [221,231,310,311], mostly to act as ECM or potential ECM analogues. For example, the work of Thierry et al. [74] with Ch and hyaluronic acid as PE counterparts to release nitric-oxidedonor sodium nitroprusside: loaded PEMs reduced platelet adhesion and enhanced thromboresistance, while promoted the antiinflammatory and wound healing properties of both PEs. Moreover, with the long-lasting delivery of matrix-bound recombinant human bone morphogenetic protein 2 (rhBMP-2) [310], PEMs could further assist tissue-engineered constructs or metallic biomaterial surfaces, to promote tissue formation or implant integration within the native tissue.

PE Self-Assembly into Patterned Surfaces

As aforementioned, electrostatic interactions can be used to direct the patterning of surfaces through selective electrostatic attachment [233]. This can be achieved by generating charged surface patterns at different substrates based on different lithographic methods, followed by adsorption from solution of molecules carrying the opposite charge [41,233,312-314]. As illustrated in Figure 3, charged gold particles can selectively attach to regions presenting the opposite charge, when stirred in suspension with a surface patterned with charged Self-Assembled Monolayers (SAMs). Electroplating gold into cavities of patterned photoresist, releasing the gold into ethanol, and modifying the surfaces with charged alkanethiols, can create charged gold particles with welldefined shapes; various sizes and shapes can be tailored by altering the photoresist pattern [233].

Micropatterning with PEMs can be used to build biosensors, which can be used for immunoassays, drug screening, and Tissue Engineering (TE) [299]. On the other hand, research on PE micropatterning alone has been directed to produce nanostructured coatings with complex patterns or guide area-selective patterned assembly, with uses mostly found within flexible transistors, sensing elements, integrated circuits, antennas, as well as biomedical implants and other devices [41].

PE Self-Assembly into Small-Scaled Particles

PEs can also spontaneously self-assemble in the form of small-scaled particles, namely at the nanometer scale with a size ranging from 1 to 1000 nm [315-317], by direct mixture of polycation and polyanion aqueous solutions [172,255,318,319].

The process of PEC formation of nanoparticles can also be influenced by several parameters. In what concern PE counterparts, PE type, Mw and charge density, have to be defined first. Then, media parameters such as concentration of PE solutions, mixing ratio of charged units, salt concentration, pH, temperature and volume of all solutions, must be selected. Finally, while preparing the nanoparticles, the order of PE addition (polyanion to polycation, or vice-versa) as well as questions related to the mixing procedure (mixing type, protocol and device) should be taken into consideration (although typically PE solutions are mixed with a magnetic stirrer at a fixed velocity) [240,318,319]. Environmental conditions, such as temperature and humidity of the surrounding air, are also probable influences in the assembly [256,267,270].

PEC formation into nanoparticles is usually determined by a fast kinetics (values as low as 5 μs have been described) [255,318], including the initial diffusion stage of mutual entanglement between polymers under release of counterions, formation of primary particles, and the further stage of their aggregation and rearrangement of the already formed aggregates [238,255,318]. This process is illustrated in Figure 5.

Figure 5: Simplified schematic representation of the process involved in the formation of PEC nanoparticles (a) Representation of the PEs used in the assembly: Ch as a polycation and γ-PGA as a polyanion. (b) Formation of primary complexes comprising a few self-assembled PEs. (c) Formation of secondary complex particles, with a typical core/shell structure, in which the shell contains an excess of one of the PEs. Figure adapted from [320].

After the mixture of the PEs, the dispersion mostly consists of three components: (i) small soluble primary complexes comprising a few polycations and polyanions, (ii) dispersed colloidal particles of aggregated primary complexes (secondary complexes), and (iii) larger insoluble precipitated particles. The former typically exhibit sizes of a few nanometers, the second exhibit sizes of 20-500 nm, and the latter exhibit sizes of 0.1-1 mm. The secondary complex particles (case ii) are the ones with most interest to a clinical setting. They typically form a core/shell structure, according to which a 1:1 charge stoichiometry prevails in the particle core, and in the shell the excess PE component is located, giving the particle the defined charge sign [254,319-321]. Even if the stoichiometry of the charged units is 1:1, the major component may be bound in excess [255]. They can have spherical, rod-like, or toroid shapes, and a loose gel-like up to compact internal structure [319].

The attachment of a PEC colloidal particle is mainly caused by the strong electrostatic interactions between the PE assembling units, whereby the gain in entropy due to the release of low molecular counterions has a major role [255,316,322-324]. Moreover, as for any colloidal particle, a decrease in interfacial energy is critical for its formation [324]. Repulsive forces (Figure 2) are also commonly present and may assist in the stabilization of PEC structures. Other attractive forces may additionally take part in the assembly process [255,318].

As for PEMs, PEC formation into nanoparticles is a simple and versatile process, which uses mild conditions, and allows the incorporation of other molecules (drugs, genes, proteins, vaccines, or diagnostic agents) within their structure [47,318,319,325-327]. Control over size, shape and surface chemistry is also a possibility [319]. However, the use of PEC nanoparticles present some advantages over PEM films as: i) absence of substrate restrictions [328]; ii) high surface/volume ratio [321]: small enough to pass through biological barriers [327], internalize into target cells [4] and influence a number of cellular processes [329-331]. Current limitations of PEC nanoparticles include difficulties in the reproducibility of the preparation protocol, size and shape uniformity, conservation of colloidal stability after binding of compounds and interaction with surfaces [240,321].

A higher control over all parameters influencing PEC assembly into nanoparticles is the most described strategy to overcome all limitations [238,240,332-335]. Post-treatment of the dispersions can be applied as well. A known example comprehends consecutive centrifugation, separation and re-dispersion steps of the coacervate phase, which can increase the monomodality of the complexes [214,319,336]: the initial raw dispersion can be centrifuged, the supernatant discarded and the formed coacervate phase of the serum dissolved again to the original volume. The latter is able to increase the amount of secondary complex particles and reduce the polydispersity of the dispersion. Other authors [323,337] filtered the raw dispersions, aiming at the removal of larger aggregate particles and collection of smaller particles towards a lower dispersion of particle sizes [319]. The interaction with surfaces can also be improved by adding molecules specifically to that effect. In the work of Hadju et al. [338], folic acid was covalently attached to γ-PGA, Ch/γ-PGA PEC nanoparticles were prepared, and were then able to target folate receptors overexpressed in tumor cells. Loaded with the magnetic resonance imaging (MRI) metal, gadolinium, the nanoparticles showed an increased efficiency as MRI contrast agents. A resume of the main applications of Ch/γ-PGA nanoparticles is described in Table 3. As small-scaled particles, Ch/γ-PGA PEC nanoparticles have already been studied as a diclofenac-delivery system and were shown to reduce human macrophage activation In vitro [339]. An increased protection from immune surveillance was also shown by Moon and colleagues using Ch/γ-PGA PEC nanoparticles [340]: intranasal administration of recombinant influenza hemagglutinin antigen, or inactivated virus, loaded into Ch/γ-PGA nanoparticles protected mice against the highly pathogenic influenza A H5N1 virus. The safety of γ-PGA was highlighted in addition to its potential to be used as a vaccine adjuvant, not only against influenza, but also against other viruses.

Table 3. Ch/γ-PGA PECs in regenerative medicine: small-scaled particles.

PE Self-Assembly into PEM Nanocapsules

PEM nanocapsules is another spherical architecture at the nanoscale that has been actively studied in the field [3,129,230,341-343]. Colloid-templated electrostatic LbL self-assembly starts with the selection of a colloidal particle template such as silica or polysterene latex particles. Anionic PEs can subsequently adsorb to the charge particles and initiate the LbL self-assembly process, as thoroughly described in section 3.2. Hollow nanocapsules can be obtained after removal of the core particles through their dissolution or decomposition [230].

The process of PEM nanocapsule formation combines the advantages of both PEM films along with the benefits of a spherical shape at the nano-scale, leading to increased control over their size and membrane thickness, in comparison to PEC nanoparticles alone [5,129,344].

The low toxicity, ability to incorporate different molecules, increase load efficiency and stability, control load release and the small size and shape of PEC nanoparticles are giving added value as contrast agents in biomedical imaging, signaling molecules that enhance diagnostic ability, carriers for drug/gene/protein/polynucleotide delivery, and themselves as therapeutic elements [316,319,327,345]. PEM nanocapsules are promising candidates for more complex tasks of storage, encapsulation and release [5]. Main reasons comprise the ability to readily tailor their properties, namely their size, membrane thickness, composition, surface functionality, permeability, and colloidal stability [5,129,344]. Moreover, the stepwise formation of the PEM nanocapsules allows the introduction of multiple functionalities [344], similarly as previously described for the PEM films.

PECs in 3D Hydrogels

During complexation, PEs can either coacervate, or form a more or less compact hydrogel: macromolecular networks swollen in water or biological fluids [1]. These hydrophilic polymer networks have a high affinity for water, thus high water content [48,346], but are prevented from dissolving due to their physically crosslinked network. As such, water or biological fluids infiltrate the polymer chains of the polymer network, leading to swelling and the formation of a hydrogel [1,346].

After the pioneering work of Wichterle and Lim in 1960 about crosslinked 2-Hydroxyl Methacrylate (HEMA) hydrogels [347], and knowledge of their hydrophilic character and potential to be biocompatible, hydrogels are a thriving research field [2,348,349]. Hydrogels can assume different shapes and reach macroscopic dimensions, depending of the processing methodology employed [1,349,350]. In particular, the formation of 3D PEC hydrogels combines the features of PEs in aqueous solutions with processing methodologies at the macroscale, namely self-assembled gels [351-353], porous scaffolds [7,10,354], microfibers [212,355], or nanofibers [356,357].

The spontaneous gelation into 3D porous PEC scaffolds is one of the most common methodologies actually studied in the field. Main steps of the processing methodology are represented in Figure 6.

Figure 6: Simplified schematic representation of the process involved in the formation of 3D porous PEC scaffolds. Representation of the PEs used in the assembly: Ch as a polycation and γ-PGA as a polyanion. (b) Figure adapted from [2,71,349].

Formation of 3D porous PEC scaffolds typically starts by blending PE solution with the powder of its PE counterpart to obtain homogeneous dispersion of the latter component. Dissolution and pH adjustment is followed. Spontaneously, charges are mixed at the pH-range of interaction and gel formation occurs [7,10,349,354]. Water-filled ‘voids’ or ‘macropores’ can also be formed [349]. Subsequently, the mixture can be cast into shape-forming molds [358], followed by freeze-drying [7,10,354]. pH-responsive 3D porous scaffolds are ergo obtained, structures able to respond to perturbations in the environmental pH through swelling/deswelling, as water is either absorbed or expelled from the hydrogel network [2].

Factors common to all PEC self-assemblies also apply to 3D porous PEC scaffolds, with the main consequence being the degree of PE interaction. Within these structures, global charge densities, PE concentration and relative proportion in the PEC, pH, temperature, ionic strength and order of mixing are the main studied parameters, together with parameters specific of each particular PE [1,349]. Moreover, physical hydrogels such as these are typically reversible and it is possible to dissolve them by changing environmental conditions such as pH and the ionic strength of solution [2,349].

PECs in 3D present some advantages as: i) no need to remove residues of toxic crosslinkers; ii) reversible sol-gel transitions with high sensitivity under mild conditions; iii) convenient incorporation of multiple properties to introduce heterogeneity for hosting hydrophobic drugs [48]. However, we cannot exclude that hydrogels in general tend to possess low mechanical strength, posing significant difficulties in handling, and their sterilization can also be challenging [349].

With 3D porous PEC scaffolds, the control of all parameters influencing their formation, working in consonance to reach the final PEC structure, remains an additional difficult task. However, preparing PECs under reproducible conditions is one strategy that is being attempted. For instance, and as aforementioned in the description of the processing methodology, PE solutions can be mixed at a pH value where complexation does not occur in order to obtain a homogeneous mixture. Only then the pH of the solution can be adjusted to the intended value, thereby promoting the electrostatic interaction [1]. The latter strategy has been found within PEC formation at the macroscale [7,10,354]. Still, physical hydrogels can be heterogeneous, due to clusters of molecular entanglements, or hydrophobically- or ionically-associated domains, which can create inhomogeneities. Free chain ends or chain loops also are also included in the transient network defects of physical gels such as these [349].

pH-sensitive hydrogels based on PEs have additionally been designed to possess precise responsiveness by exploiting the wide pH range of different sites of the human body in order to achieve controlled site-specific biomolecules delivery. PECs pH-responsive hydrogels can be applied in the biomedical field as delivery systems, cell encapsulation, tissue regeneration and wound dressing (within specific applications with low mechanical demands) (Table 4) [1,2,48,348,349]. Hydrogel swelling and release profiles can be modulated by tailoring processing parameters [1].

Table 4. Ch/γ-PGA PECs in regenerative medicine: 3D structures.

As PEC hydrogels, Ch and γ-PGA have been shown to be cytocompatible towards fibroblasts and promote cell proliferation [10], improve wound closure rate and wound healing In vivo in mice [12] and promote an earlier and increased bone tissue formation in the alveolar socket following tooth extraction in rats [11]. Interestingly, Chang and colleagues [11] observed a decrease of inflammatory cells presence in the sockets of PEC-treated groups in their histological findings. Moreover, PEC hydrogels led to a larger presence of mineralized tissue, exhibiting significantly earlier as well as greater amounts of new bone formation than treatment with Ch alone. This fact was also attributed to the presence of γ-PGA in the treatment performed. Another possible explanation to this outcome is the possible activation of the glutamatergic signaling, which is functional in bone and results in increased bone formation [35,359]. It is expected that γ-PGA might be consumed/degraded, thus generating a glutamate pool able to activate glutamate signaling pathway [359], as glutamic acid-rich sequences are involved in the nucleation of hydroxiapatite by bone sialoproteins [360].

Conclusion

In conclusion, Ch/γ-PGA PECs can be useful for numerous applications. Their cytocompatibility, their immunomodulatory capacity [302,303,339], their stimuli responsiveness [128,129], the ability to control their hydrophilicity and swelling ratio [8,129], together with their versatility to be used as vehicles for drugs, genes, vaccines, enzymes and/or other biomaterials [9,129,144,340] and furthermore promote new tissue formation [11,12] are strong aspects that Ch/γ-PGA PECs have a role in restoring tissue homeostasis.

In truth, PEMs can be successfully produced with many PEs, either biostable or degradable/biodegradable, synthetically produced or biologically derived. But Ch/γ-PGA PECs importance in the regenerative medicine field can have a particular impact. For example, these PECs appear to play a significant role in cartilage tissue, as γ-PGA revealed to promote chondrogenic ECM by hMSCs [164] and enhance type II collagen deposition in an injury model of intervertebral disc [361]. Moreover, γ-PGA-based PECs in proper forms like films, particles, capsules and scaffolds can be used as carriers of different biomolecules to several purposes: SDF-1 [302] to recruit hMSCs, IFN-γ [303] to polarize macrophages, diclofenac [339] to control inflammation, gadolinium [338] to detect carcinogenic features; recombinant antigens or inactivated viruses [340] for vaccines; small interfering ribonucleic acid (siRNA) [9] to increase cell uptake and biological effect; insulin [13] or Diethylene Triamine Pentaacid Acid (DTPA) [341] to increase insulin bodily levels. The non-immunogenic feature of γ-PGA has been opening new perspectives on their use as immunoadjuvant in vaccines and is expected to increase in the field of immunotherapy.

Still, the control of all parameters influencing PEC formation, working in consonance to reach the final PEC structure, together with a high-throughput method of fabricating different structures remain the main drawbacks to the widespread use of PECs. Preparing PECs in a high-throughput manner, under reproducible conditions is one the main challenges to be pursuit in the near future. The control of all the different parameters that are known to influence PEC formation is essential to keep reproducibility among different PEC batches and will require a synergy between different expertise and knowledge fields, from chemistry to robotics, to physics, to nanotechnology, to biomaterials engineering, to fundamental biology, among others. Only through the combination of the different areas, a real effective and standardized method of producing Ch/γ-PGA PEC can be reached and widespread their use within tissue engineering and regenerative medicine approaches.

Acknowledgments

This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the project "Institute for Research and Innovation in Health Sciences" (POCI-01-0145-FEDER-007274). Raquel Gonçalves is also grateful to FCT for her starting grant (IF/00638/2014).

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