Amazing porn model Belle Delphine nudes on sexe-libre.org. Watch free video collection of Belle Delphine nede leaked
Rare Muslim porn and سكس on sexsaoy.com. Tons of Arab porn clips.
XNXX and Xvideos porn clips free on xnxxarabsex.com. Best XnXX porn tube channels, categorized sex videos, homemade and amateur porn.
Exlusive russian porn russiainporn.com. Get uniqe porn clips from Russia
Find out on sexjk.com best collection of Arabain and Hijab سكس
Received Date: 29/08/2016; Accepted Date: 15/09/2016; Published Date: 25/09/2016
Visit for more related articles at Research & Reviews: Journal of Material Sciences
Physically complexed Polyelectrolyte (PE) Complexes (PECs) have been widely used for different purposes. Mixing solutions of oppositely charged PEs leads to spontaneous formation of PEC dispersions (complex coacervates) of interpolymer complexes driven by electrostatic interactions. Assembling of poly(γ-glutamic acid) (γ-PGA) with chitosan (Ch) in PECs provides an easy and flexible technology for the delivery of biomolecules in tissue engineering. γ-PGA is an anionic polymer, recently explored but with interesting biological properties, namely nonimmunogenicity and in vivo biodegradability into glutamatic acid units. Its combination with cationic polymers, namely Ch, is being increasingly investigated. Here the formation of PECs, namely different architectures, as well as detailed features of biomedical applications of Ch/γ-PGA PECs as delivery systems and mediators of tissue regeneration is reviewed. Besides overviewing the literature on Ch/γ-PGA PECs applications, the main challenges to the use of Ch/γ-PGA PECs and the most promising applications of these structures in regenerative medicine are covered.
Polyelectrolyte complexes, Poly(γ- glutamic acid) , Chitosan, Regenerative medicine, Delivery system
Development of more advanced and functional biomaterials has been an ongoing challenge in the last decades towards the benefit of millions of patients worldwide that need to be treated with biomaterial-based therapies. The ability of individual polymers to respond to changes in pH, temperature, electric or magnetic fields, together with the possibility of combining different polymers, is a strategy largely pursued in the field [1-4].
Polyelectrolyte (PE) Complexes (PECs) have been particularly under focus, especially as delivery systems of biomolecules [2-5]. Systems that can respond to a dynamic pH environment are of particular interest for applications within different locations in the body that exhibits substantial pH changes during either healthy or diseased state . PECs have the ability to improve intrinsic properties shown by each PE (e.g. cytocompatibility and hydrophilicity [6-10]), enhance bulk properties (e.g. mechanical properties [11,12]) and act as delivery systems with increased capability (e.g. pH-responsiveness [13-17], higher loading efficiency [13,18] and effectiveness [9,18]).
Chitosan (Ch) has been one of the most studied PEs. Ch’s chemical structure resembles human polysaccharides that are present in the extracellular matrix of numerous tissues as proteoglycans. Ch is the only naturally occurring cationic Glycosaminoglycan (GAG) analogue [1,19-21], prone to molecular recognition by living cells or tissues. It is an efficient delivery vehicle for nucleic acids, binding strongly with negatively charged biomolecules, and useful for gene therapy [22-25]. Plus, as a cationic polymer, Ch can be combined with anionic polymers.
Poly(γ-glutamic acid (γ-PGA) is one of the most promisor anionic PEs, highly due to its non-immunogenic behavior [26-28] and biodegradability into glutamic acid residues [29,30]. Glutamate is mostly known as major excitatory neurotransmitter in the central nervous system, acting as a synaptic transmitter, and influencing neuronal excitability, synaptic structure and function, neuronal migration during development, and neuronal viability [31-35]. It is also present in other tissues and organs, specifically in non-neuronal tissues like bone, pancreas, skin and cartilage [31,33-36], and having significant roles in their normal function, although yet not fully understood. γ-PGA, being a poly(amino acid) built by glutamic acid monomers [29,30,37,38], may participate in similar processes.
Ch/γ-PGA PECs appear to be promising with an increasing number of references in the literature, but there is a lack of systematized information. Therefore, this review aims to overview the process of PEC formation within different architectures, as well as the current status of the Ch/γ-PGA PECs and their applications in the field of drug delivery and regenerative medicine. Within this scope, we expect to identify the main challenges of Ch/γ-PGA PECs application for tissue regeneration.
Polyelectrolyte (PE) is the term used to classify polymeric macromolecules with charged or chargeable groups when dissolved in polar solvents (predominantly water) [39,40]. When a PE dissociates, it gives rise to a macroion and counterions in aqueous solution . The macroion can be named polycation (positively charged PE) or polyanion (negatively charged PE) . Table 1 highlights some of the most studied PEs, either from natural or synthetic origin.
|Polycation||Ch, Col, Gelatin, PLL, Arginine, Starch.||Poly(ethylene imine), Poly(allylamine hydrochloride), PDADMAC, Poly(vinyl pyrrolidone), Poly(amidoamine), Poly(N-isopropyl acrylamide), Poly(2-(dimethylamino)ethyl methacrylate), Poly(2-(diisopropylamino)ethyl methacrylate).|
|Polyanion||HA, chondroitin sulfate, PGA, Cellulose sulfate, Alginate, Pectin, Heparin, Mucin, DNA, Poly(aspartic acid), Dextran sulfate, CMC, GG.||Poly(acrylic acid), Poly(styrene sulfonate), Poly(vinyl sulfate), Poly(methacrylic acid).|
*PEC: Polyelectrolyte Complex; Ch: Chitosan; Col: Collagen; PLL: Poly(L-lysine); PDADMAC: Poly(diallyldimethylammonium chloride); HA: Hyaluronic Acid; PGA: Poly(glutamic acid); DNA: Deoxyribonucleic Acid; CMC: Carboxymethylcellulose; GG: Gellan Gum.
In particular, Ch is a polysaccharide and a linear copolymer of glucosamine and N-acetylglucosamine units connected through a ß-1,4-linkage [41-51]. On the other hand, γ-PGA is a poly(amino acid) built by D- and L-glutamic acid residues linked by peptide bonds [29,37]. Both PEs are represented in Figure 1.
C. Rouget was the first to identify Ch in 1859 [52,53]. Ch is mainly obtained by N-deacetylation of chitin under alkaline conditions [20,54-56]. Chitin is abundant in Nature (primary structural component of the exoskeleton of shrimps, crabs and lobster and squid pens as an equivalent to collagen, and present in lesser amounts in cell walls of some fungi and yeast and in plants as the equivalent of cellulose) [20,54,56-58]. When the degree of acetylation (DA, molar fraction of N-acetylated units) is lower than about 50%, the polymer is termed Ch . Ch’s molecular weight (Mw) and DA are its main structural parameters influencing the overall behavior of the polymer as a biomaterial [1,59]. Ch is soluble in nearly all diluted aqueous acidic solutions and insoluble in water, concentrated acid, alkali, alcohol, and acetone and in common organic solvents. The dissolution occurs by protonation of the primary amine (-NH2) groups of the D-glucosamine molecular unit [20,54,56,59,60]. These amine groups become protonated, and the polymer behaves as a PE [61,62]. As a PE, Ch has been extensively screened for its ability to form complexes with other polymers, particularly through electrostatic interactions [1,20,50,57,63].
Ch has been extensively described for a large range of biomedical applications [1,20,53,55,64-66], in numerous structures and forms : 2D films [67-70], PEM films [71-74] or nanostructured surfaces ; small-scaled particles like nanoparticles [76-80], microparticles [81-83] or PEM micro- [84-86] or nanocapsules [87-89]; and 3D porous structures such as sponges [90-95], fiber meshes [96-101], or gels [102-106], either alone or in combination with other molecules. Parameters such as biodegradability and biocompatibility are particularly important. Ch can be degraded enzymatically, through chemoenzymatic means, recombinant approaches and physical means like electromagnetic radiation and sonication [57,60,107]. In humans, In vivo degradation of Ch is thought to be primarily due to the activity of lysozymes (present in articular cartilage, liver, plasma, saliva, tears and milk ) and bacterial chitinolytic enzymes (e.g. chitosanase) that have been identified in human tissues of the gastrointestinal tract and lung [54,107,109,110]. These enzymes hydrolyze both glucosamine and acetylated residues, leading to polymer erosion into a suitable size for renal clearance [19,50,58,107]. Ch has additionally been regarded as a non-toxic and a biologically compatible polymer [1,55,56,111]. Ch and its derivatives have also been tested successfully against numerous cell types . Moreover, it is approved by the Center for Devices and Radiological Health (CDRH) of the Food and Drug Administration (FDA) as a wound dressing material (topical intended use) . Ch has also been shown to possess mucoadhesive [112,113], chemoattractive [114-117], analgesic , antibacterial and antifungal [119-121], and hemostatic properties [122,123] (among others).
On the other hand, γ-PGA was firstly detected by Ivanovics and Bruckner  in 1937, as the sole constituent of the Bacillus anthracis surface capsule, and released into the medium upon bacterial cell lysis. After its discovery, γ-PGA was identified in the growth medium of several Bacillus strains as an extracellular product of their fermentation [29,125]. γ-PGA can be characterized by its Mw distribution, the ratio of D- to L-glutamic acid monomers and the carboxyl-group (a or γ) engaged in the peptide bond . γ-PGA is soluble in water ; in acidic solutions, the carboxyl groups of γ-PGA become deprotonated [126,127] and the polymer can also be explored as a PE. As a PE, γ-PGA has been object of some studies regarding its ability to form complexes with other polymers, namely through electrostatic interactions [8,128-132].
In the case of γ-PGA, its potential for biomedical applications has been less explored than for Ch [29,30,133,134]. Still, γ-PGA has been processed in the form of 2D films [135,136], or PEM films [71,131,137]; small-scaled particles like nanoparticles [128,138-142], microparticles [143,144] or PEM nanocapsules ; and 3D porous structures such as sponges [7,8,11,12,145,146], fiber meshes [147-151], or gels [152-158], particularly in combination with other molecules. γ-PGA can be chemically degraded by prolonged exposure to an extreme pH value at high temperatures, physically by ultrasonic irradiation, or enzymatically . In γ-PGA producing Bacilli, endo-γ-glutamylpeptidase and exo-γ-glutamyl-peptidase are the two types of enzymes that can degrade γ-PGA. In mammals, the existence of γ-PGA has also been reported ; being assumed that this polymer can be biochemically degraded into glutamic acid residues In vivo . γ-PGA non-immunogenicity feature has been demonstrated by a lack of immune response after repeated polymer injections [26-28], reinforcing a fact already discovered when γ-PGA was shown to mask the dangerous pathogen Bacillus anthracis from immune surveillance of the host [26,160-162]. Afterwards, γ-PGA and its derivatives have been tested successfully under different shapes and against some cell lines [6-8,139,145,148,155,163], and primary cells [164,165]. Moreover, since γ-PGA is edible, it is generally regarded as safe [26,30,160,166].
Self-assembly is a spontaneous association of multiple components into larger entities [167-171]. Its concept can be enlightened as follows: the ‘assembly’ connotes ‘to put together or build’ and the ‘self’ implies ‘without outside help or on its own’. The ‘self’ that drives the assembly is(are) the interaction(s) between the assembling objects . This process can occur at any scale, ranging from nano- and micrometer scale to macroscopic dimensions [173,174]. Self-assembly is the most ubiquitous process in Nature [171,175-178], being present in many biological systems. Classical examples include the hierarchical self-assembled rod-like structure of tobacco mosaic virus [179-181], phospholipids forming the membranes of cells and organelles [127,182-184], deoxyribonucleic acid (DNA) self-complementary double-helix annealing [127,185], and protein aggregation forming the Extracellular Matrix (ECM) of connective tissues, namely fibronectin and collagen [186-189]. The assembling objects behind self-assembly are active building units bearing recognition information, and thus capable of recognizing each other. If the assembly happens in multiple stages, the building block can be different in each stage [168,190].
The self-assembly process relies on the action of non-covalent interactions (or weak, and also reversible, covalent interactions) between the building blocks, underlying their assembly into larger and thermodynamically stable structures. A balance between attractive and repulsive forces should take place in order to obtain stable self-assembled structures [168,191,192]. The most described forces behind self-assembly comprehend van der Waals forces (including hydrogen bonding), hydrophobic interactions, electrostatic forces, magnetic interactions, aromatic stacking, metal-ligand bonds and/or entropic effects (Figure 2) [169,171,172,191,193,194].
Figure 2: Forces involved in self-assembly. (a) Van der Waals forces; (b) hydrogen bonds between a H-bond donor and a H-bond acceptor; (c) electrostatic interactions between oppositely, or likely, charged species; (d) magnetic interactions in response to a magnetic field (H); (e) aromatic stacking with face-to-face or edge-to-face interactions; (f) metal-ligand binding between a soluble metal acceptor centre (M) and organic ligand donors (X and Y); and (g) entropic effects, namely hydrophobic effects (separation of hydrophobic parts of amphiphilic objects from water molecules), confinement effects (increase of steric repulsion) or depletion forces (mediated by smaller particles or solvent molecules). Figure adapted from [171,190,191,195-202].
Electrostatic forces offer a type of bond that is low demanding in terms of its directionality and the distance between oppositely charged functional groups (it has the least steric demand of all chemical bonds [195-203]), in addition to the possibility of forming multi-centre bonds . Furthermore, the magnitude and length scale of these interactions can be regulated, namely by choosing the solvent (e.g., dielectric constant) and/or the concentration and chemical nature (e.g., size and valence) of the surrounding charged counterparts . Electrostatic self-assembly is an easy, reliable, cheap and versatile example of the known self-assembly processes; ergo, subject of numerous studies up until now [41,173,191,203,205-209].
Numerous building blocks – amphiphiles (lipids and surfactants) [210,211], polymers [212-214], copolymers [215-217], functional molecules/ macromolecules (poly(amino acids) , peptides , proteins [220,221] and DNA [222,223]), nanoparticles (from biological origin – charged viruses  – to inorganic – magnetic, gold or silver [225-228]) – have been assembled by electrostatic interactions [199,207,209]. Although not complete, this list demonstrates the diversity of building blocks that can be used. Different self-assembled shapes have been obtained, as a result of the geometries of all interactions elicited by that object : two-dimensional (2D) films, small-scaled particles or three-dimensional (3D) structures [168,199,207,209,229]. Examples of such outlines are represented in Figure 3.
Figure 3: Self-assembled entities obtained from oppositely charged self-assembling objects. 2D films: cationic poly(L-lysine) (PLL) and anionic hyaluronic acid (HA) alternate deposition onto a planar substrate; and planar and curved patterned gold substrates (NMe3+-terminatedor, CO2 --terminated self-assembled monolayers (SAMs)), coated with PO3H--terminated gold disks. Small-scaled particles: nanoparticles composed by the block copolymer poly(ethylene oxide)210-b-poly(tert-butyl methacrylate)97 (PEO-b-PMA) and oppositely charged surfactant hexadecyltrimethylammonium bromide (HTAB); and hollow layer-by-layer (LbL) capsules containing cationic polymer poly(diallyldimethylammonium chloride) (PDADMAC) and anionic silicon dioxide nanoparticles (SiO2). 3D structures: freeze-dried scaffolds composed of cationic chitosan (Ch), and anionic poly(γ-glutamic acid) (γ-PGA) and carboxymethylcellulose (CMC); and microfibers containing cationic Ch and anionic gellan gum (GG). Figure adapted from [212,230-235].
PECs in Solution
Molecules aggregate with each other in solution . In general, mixtures of two neutral polymers (or PEs with same/similar charge density) in solution present thermodynamic incompatibility, with phase separation occurring when the polymer concentra tion increases and the different phases formed become enriched in one of the components. However, when PEs present opposite charges, electrostatic interactions are established between them. When phase separation occurs, a phase becomes enriched in the two polymers while the other is composed by solvent only . Hence, phase separation results in macroscopic phase separation, or in polymeric complexes .
Bungenberg de Jong [236,238] was the first to suggest PEC formation in solution, between gelatin and gum arabic, by observing the latest aforementioned phenomenon. Such phase separation occurrence was named "complex coacervation", and the polymer condensed liquid phase "complex coacervate" . Therefore, complex coacervation is the spontaneous process that results in the aggregation of macromolecules or formation of colloidal particles under the action of electrostatic attractive forces [239,240]. Fuoss [241-243], Michaels [40,208,244,245], Kabanov [246-248], Tsuchida [236,249,250] and Dautzenberg [215,251-253], among others , were pioneers in the study of this method with PEs in solution, and at the colloidal level. Mixing solutions of polycations and polyanions lead to spontaneous formation of PEC dispersions (complex coacervates) of interpolymer complexes [236,254,255].
PECs in 2D: PE Self-Assembly into Multilayers (PEMs)
The technique known by layer-By-layer (LbL) self-assembly is considered the most well established bottom-up assembly methods of thin film deposition , and widely used for 2D PEC formation. With this method, PEs can be self-assembled by sequential adsorption of multiple thin polyion films from aqueous solutions onto charged solid substrates, forming PE Multilayers (PEMs) (Figure 3) [170,203,213,256].
Multilayer formation was first suggested by Kirkland  and Iler  in 1965-1966 [41,209,259]. They described for the first time the formation of layers of charged particles by adsorption from solution, by alternating deposition of positively and negatively charged colloid particles, such as silica and alumina. However, it was Iler  that suggested a way to prove the existence of a multilayered structure by use of reflected light after each layer addition. Still, Kirkland patented the system in 1970-1984 [260,261]. In 1980 , 1982 , 1984  and 1990  other researchers started suggesting multilayered film assembly based on electrical charge reversal after each deposition step. In 1991, Decher and Hong [210,266] analyzed the thickness of multilayered films built by anionic and cationic bipolar amphiphiles and suggested the use of PEs for PEM formation from sufficiently concentrated PE aqueous solutions. Layer numbers of 35 and 39 were achieved, with corresponding 170 and 151 nm of thickness, calculated through small angle X-ray scattering.
Regardless, only in 1992, PEM formation through electrostatic interaction exclusively between PEs was reported . PEM films of 100 consecutively alternating layers were assembled [203,213]. Through this new technique, a solid substrate with a positively charged planar surface (silanized aminopropylsilanized fused quartz or silicon single crystal substrates) was immersed in a solution containing an anionic PE (poly(styrene sulfonate)) and a monolayer of the polyanion was adsorbed . Since the adsorption was carried out at relatively high concentrations of PE, a large number of ionic residues remained exposed to the interface with the solution and thus the surface charge was effectively reversed. After rinsing in pure water, the substrate was immersed in the solution containing a cationic PE (poly-4-vinylbenzyl-(N,N-diethyl-N-methyl-)-ammonium iodide). Again, a monolayer was adsorbed and the original surface charge was restored. By repeating both steps in a cyclic fashion, alternating multilayer assemblies of both polymer pairs were obtained. A linear increase of film thickness with layer number was observed. It was technically possible to calculate a PEM thickness of 42.5 nm of 39 deposited layers composed by the PE pair of poly(styrene sulfonate) and poly(allylamine hydrochloride) . This process was designed by layer-by-layer (LbL) deposition by Decher and colleagues [203,213]. Since then, surface modification via the LbL deposition technique through electrostatic interactions gained motion. An adaptation of this technique is represented in Figure 4.
Figure 4: (a) Molecular units of both polymers used in the polyelectrolyte multilayer (PEM) buildup: Ch (left), as a polycation, and γ-PGA (right), as a polyanion. (b) Simplified molecular picture of the first two deposition steps, illustrating film deposition starting with a positively charged surface. Counterions are included. Polyion conformation and layer intermingling are omitted for clarity. (c) Schematics of the layer-by-layer (LbL) deposition process, by horizontal immersion, using gold-coated silicon wafers as substrates, coated with a Ch film, obtained by spin-coating. Steps 1 and 3 represent the adsorption of the γ-PGA and Ch, respectively. Steps 2 and 4 are the intermediate rinsing with buffer solution. With such cyclic depositions, the simplest multilayer film architecture can be obtained. Figure reprinted with permission from .
A solid substrate with a positively charged planar surface is immersed in the solution containing the anionic PE and a monolayer of the polyanion is adsorbed (step 1 or step A). Since the adsorption is carried out at relatively high concentrations of PE, a number of ionic groups remain exposed to the interface with the solution and thus the surface charge is reversed. After rinsing in a buffered solution (or in pure water) the substrate is immersed in the solution containing the cationic PE. Again a monolayer is adsorbed but now the original surface charge is restored (step 3 or step B). These four steps are the basic buildup sequence for the simplest film architecture, (A/B)n, since by repeating the steps in a cyclic fashion (A, B, A, B, …), alternating multilayer assemblies of both polymers are obtained . In more detail, LbL film-deposition requires a solid support bearing a minimal surface charge to initiate the assembly process. If not inherent, the substrate surface charge can be created by chemical or physical modifications. Cleaned hydrophilic glass, quartz, silicon wafers, mica, and gold-coated supports are the most frequently used materials. They are typically chosen according to their convenience for particular analytical methods [209,256,267]. The structure and properties of each layer of PE are typically independent on the substrate used after a few deposition cycles, being then governed by the choice of the respective polyanion/polycation pair and by the deposition conditions . Self-repulsion of the already deposited layers should also take place, to adjust molecules conformation and consequent flexibility of the underlying layers, and promote further PE deposition [170,267]. Layer intermingling, imposed by intrinsic charge compensation , is also frequent [170,267].
Several important parameters influence PEMs buildup and need optimization for each PEM. They include: pre-treatment of the substrate, nature of the PEs, concentration and temperature of the solutions, solvent, salt type and ionic strength, pH, dipping time, rinsing time, agitation during adsorption or rinsing, drying step, and environmental conditions such as temperature and humidity of the surrounding air [209,256,267,269,270].
The main advantages of PEM formation through LbL self-assembly technique derives from: i) its simplicity, since no complex equipment is required for the buildup (a suitable substrate, container(s) compatible with the materials used and a substrate handling tool ); ii) each adsorption step is relatively quick (typically 10-20 min/adsorption step, but can be between 1 min and 1 h due to PE adsorption kinetics [221,231,256,269,271,272]); iii) low costs [170,273,274]; iv) compatible with industrial production methods [231,275]; v) easy build-up [231,256,267]; vi) the possibility of introducing different molecules [203,209,256,259,276], into the multilayer in a programmed sequence, adding new functionalities [170,231,275,276]; vii) the possibility of controlling PEC architecture at the nanometer-level, by selecting the substrate and building blocks, layering sequence, experimental conditions and influencing parameters [203,231,256]; viii) the lack of need for auxiliary molecules, such as catalysts or initi ators, to initiate the reaction (generally performed in aqueous solution), which represents the main advantage over covalently crosslinked networks, favoring biocompatibility thus avoiding purification before administration .
The main limitations of PEMs include: i) fuzzy nano-assemblies, derived from the intermingled structures fabricated from LbL technique [170,203]; ii) interpolymer orientation or alignment of polymer chains in a given layer may be difficult to control [208,269,277-279]; iii) long dipping times in the presence of biological materials can be prone to denaturation or photobleaching; or iv) eventual contamination by previous solutions of the layering scheme .
PEMs can be built not only on planar substrates, but also on substrates with different shapes, like protein aggregates, enzyme crystals, colloidal particles, cylindrical systems and even living cells [170,209,270,273,274]. Additionally, when using two building blocks, PEM preparation only requires the two PEs carrying complementary charges [1,203,256]. An exact positional matching of the charged groups is also not required . Regardless, technical advances of this technique, which include the use of automated dipping robots [269,280,281], hydrodynamic LbL (with moving substrates [269,282], or solutions [283-285]), spin-assisted LbL [286-288], sprayassisted LbL assembly [289-292], ink-jet assisted LbL [293,294], among others, are assisting in overcoming PEMs limitations, while increasing its use [41,209]. Combination of the bottom-up LbL technique with top-down lithographic techniques [295-298] are another valuable possibility .
PEM films have been mainly studied as potential coatings for implantable biomaterials, namely to inhibit coagulation, inflammation or infections (choosing anti-thrombogenic, anti-inflammatory or anti-bacterial PEs, for instance), non-fouling properties (to prevent immune rejection or to enhance the efficiency of injected delivery vehicles), increase protein adsorption and/or promote integration with native tissue (having adhesive proteins or ECM components as building blocks, among other possibilities), cell/ biomolecules encapsulation and preservation of their functionality (parameters such as wall thickness and permeation can be modified according to the needs of the system), localized delivery of different molecules in response to different stimuli (namely pH-responsiveness), and modification of substrates to influence cellular processes as adhesion, proliferation, differentiation and ECM synthesis/degradation (tuning PEMs biodegradability, mechanical properties and bioactivity, either inherent to the PEs or induced by added molecules) [42,45,299-301]. Thin-film biosensors (incorporating able molecules within PEM structure – enzymes, antibodies, proteins, nucleic acids, inorganic colloids or organic molecules) are also important fields of application .
Ch/γ-PGA PEMs have been proposed as model surfaces to use as delivery systems (Table 2). For example, the chemokine stromal-derived factor-1 (SDF-1) was incorporated into Ch/γ-PGA PEMs using different approaches. Ch/γ-PGA PEMs with SDF-1 were able to increase the recruitment of human bone marrow-derived mesenchymal stem/stromal cell (hMSC) In vitro due to a sustained SDF-1 release from the PEMs structure. This highlighted Ch/SDF-1/γ-PGA PEMs as potential hMSC attractants, clinically relevant for different clinical settings, from tissue regeneration to immunomodulation . This strategy inspired the use of Ch/γ-PGA PEMs as protein reservoir to other molecules. For example, the pro-inflammatory cytokine interferon-γ (IFN-γ) was incor porated into Ch/γ-PGA PEMs and IFN-γ-PEMs were capable of counteracting human M2 macrophage phenotype while decreasing human cancer cell invasion, pointing the potential of this therapeutic strategy to target macrophages at the tumor site and hamper their cooperation in cancer cell-related activities such as invasion .
Table 2. Ch/γ-PGA PECs in regenerative medicine: 2D PEMs.
|Ch||γ-PGA||Molecule incorporated||In vitro||In vivo||Main achievements||Ref.|
|Mw (KDa)||DA (%)||Mw (KDa)|
|200||-||1230||-||-||-||First PEM formation with organic solvents|||
|650, 1200 and 1400||-||600 and 1230||-||-||-||Successful PEM formation with organic solvents, particularly with higher Mw of γ-PGA|||
|324||10||10-50||-||NIH3T3 cell line||-||No cytotoxicity|||
|324||10||10-50||SDF-1||hMSCs||-||Chemoattractor-delivery system for SDF-1|||
|366||14||10-50||IFN-γ||Human monocytes||-||IFN-γ delivery system able to modulate macrophage phenotype (M2 into M1) and counteract cancer cell invasion|||
*2D: Two-Dimensional; PEM: Polyelectrolyte Multilayer; Mw: Molecular Weight; DA: Degree of Acetylation; NIH3T3: Mouse Embryonic Fibroblast; SDF-1: Stromal-Derived Factor-1; hMSCs: Human Bone Marrow-Derived Mesenchymal Stem/Stromal Cells; IFN-γ: Interferon-γ.
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.  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.  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) , 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 . 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 .
Micropatterning with PEMs can be used to build biosensors, which can be used for immunoassays, drug screening, and Tissue Engineering (TE) . 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 .
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 .
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 . They can have spherical, rod-like, or toroid shapes, and a loose gel-like up to compact internal structure .
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 . 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 . However, the use of PEC nanoparticles present some advantages over PEM films as: i) absence of substrate restrictions ; ii) high surface/volume ratio : small enough to pass through biological barriers , internalize into target cells  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 . The interaction with surfaces can also be improved by adding molecules specifically to that effect. In the work of Hadju et al. , 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 . An increased protection from immune surveillance was also shown by Moon and colleagues using Ch/γ-PGA PEC nanoparticles : 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.
|Ch/γ-PGA PECs in small-scaled particles|
|Ch||γ-PGA||Molecule incorporated||In vitro||In vivo||Main achievements||Ref.|
|Mw (KDa)||DA (%)||Mw (KDa)|
|20||15||-||Lansoprazole||-||-||First description of Ch/γ-PGA microparticle preparation|||
|400||≤10||1230||Antimicrobial peptides LL-37 and NO||-||-||First description of Ch/γ-PGA microparticles as multifunctional carriers for LL-37 and NO|||
|50||15||160||-||Caco-2 cell line||-||Intestinaldelivery systems|||
|80||15||60||Insulin||Caco-2 cell line||Diabetic male Wistar rats. Oral intake.||Oral insulin delivery|||
|15||-||>2000||rHA antigen or inactivated virus||-||Female BALB/c mice, seronegative for influenza A virus. Intranasal vaccination||Intranasal mucosal adjuvant for influenza A vaccine|||
|320||12||1200||-||-||-||First PEC nanoparticle preparation with pH adjustment before self-assembly|||
|320||12||1200||Gadolinium||HeLa cell line||CD1 female nude mice. MRI imaging in tumor-bearing mice.||Injectable MRI-targeting contrast agents for early cancer detection|||
|Low||-||-||Doxorubicin||HN-5a cell line||-||No cytotoxicity|||
|324||11||10-50||-||Primary human macrophages||-||No cytotoxicity|||
|324||10||10-50||Diclofenac||Primary human macrophages||-||Suppress local inflammation|||
|60||15||100||DTPA||-||Wistar rats. Study absorption enhancements, after in situ intestinal closed-loop technique||Injectable treatment to improve insulin absorption|||
|80||15||20||siRNA||HT1080 cell line||-||Cell transfection|||
|600||20||1000||FITC-labeled dextran||-||-||First PEM nanocapsule preparation. Delivery systems in acidic environments|||
|10||10||200-500||Insulin||L929 cell line||-||PEM nanocapsules. Cytocompatible. Glucose-triggered insulin delivery|||
*NO: Nitric Oxide; Caco-2: Human Epithelial Colorectal Adenocarcinoma; rHA: Recombinant Influenza Hemagglutinin; HeLa: Human Cervical Carcinoma; MRI: Magnetic Resonance Imaging; HN-5a: Human Oral Squamous Cell Carcinoma; DTPA: DiethyleneTriaminePentaacetic Acid; siRNA: Small InterferingRibonucleicAcid; HT1080: HumanFibrosarcoma; FITC: fluoresceinIsothiocyanate; L929: Mouse Fibroblast.
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 .
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 . 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 , 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 . 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 , 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.
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 . Subsequently, the mixture can be cast into shape-forming molds , 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 .
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 . 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 .
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 . 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 .
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 .
Table 4. Ch/γ-PGA PECs in regenerative medicine: 3D structures.
|Ch/γ-PGA PECs in 3D|
|Ch||γ-PGA||Molecule incorporated||In vitro||In vivo||Main achievements||Ref.|
|Mw (KDa)||DA (%)||Mw (KDa)|
|400||10||2000||-||ROS cells||-||First Ch/γ-PGA 3D PECs. Cytocompatible|||
|400||15||-||-||NHDF cell line||-||No cytotoxicity|||
|30 and 300||3||1250||-||NIH3T3 cell line||-||No cytotoxicity. Promotion of cell proliferation|||
|300||3||1250||-||-||ICR male mice. Observation of wound closure.||Wound dressing material, with adequate moisture and increased healing capacity|||
|300||2-3||1250||-||-||Male Wistar rat. Evaluation of socket healing following tooth extraction||New bone formation in the alveolar socket following tooth extraction|||
*3D: Three-Dimensional; ROS: Rat Osteosarcoma; NHDF: Human Normal Human Dermal Fibroblasts.
As PEC hydrogels, Ch and γ-PGA have been shown to be cytocompatible towards fibroblasts and promote cell proliferation , improve wound closure rate and wound healing In vivo in mice  and promote an earlier and increased bone tissue formation in the alveolar socket following tooth extraction in rats . Interestingly, Chang and colleagues  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 , as glutamic acid-rich sequences are involved in the nucleation of hydroxiapatite by bone sialoproteins .
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  and enhance type II collagen deposition in an injury model of intervertebral disc . 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  to recruit hMSCs, IFN-γ  to polarize macrophages, diclofenac  to control inflammation, gadolinium  to detect carcinogenic features; recombinant antigens or inactivated viruses  for vaccines; small interfering ribonucleic acid (siRNA)  to increase cell uptake and biological effect; insulin  or Diethylene Triamine Pentaacid Acid (DTPA)  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.
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).