Alginate Nanoformulation: Influence of Process and Selected Variables

Nanocarriers are defined as structures and devices that are constructed using nanomaterials which add functionality to the encapsulants. Being small in size and having a customized surface, improved solubility and multi-functionality, it is envisaged that nanoparticles will continue to create new biomedical applications owing to their stability, solubility, and bioavailability, as well as controlled release of drugs. The type and physiochemical as well as morphological attributes of nanoparticles influence their interaction with living cells and determine the route of administration, clearance, as well as related toxic effects. Over the past decades, biodegradable polymers such as polysaccharides have drowned a great deal of attention in pharmaceutical industry with respect to designing of drug delivery systems. On this note, biodegradable polymeric nanocarrier is deemed to control the release of the drug, stabilize labile molecules from degradation and site-specific drug targeting, with the main aim of reducing the dosing frequency and prolonging the therapeutic outcomes. Thus, it is essential to select the appropriate biopolymer material, e.g., sodium alginate to formulate nanoparticles for controlled drug delivery. Alginate has attracted considerable interest in pharmaceutical and biomedical applications as a matrix material of nanocarriers due to its inherent biological properties, including good biocompatibility and biodegradability. Various techniques have been adopted to synthesize alginate nanoparticles in order to introduce more rational, coherent, efficient and cost-effective properties. This review highlights the most used and recent manufacturing techniques of alginate-based nanoparticulate delivery system, including emulsification/gelation complexation, layer-by-layer, spray drying, electrospray and electrospinning methods. Besides, the effects of the main processing and formulation parameters on alginate nanoparticles are also summarized.


Introduction
Nanomaterials have recently received much attention as drug carriers. In particular, nanoparticle-based nanoparticles [1,2], lipids [3,4], magnetic nanoparticles [5,6] and liposomes [7] have been widely studied in drug delivery systems. Among all, polymeric nanoparticles have been widely investigated due to their unique physicochemical properties [8]. The natural and synthetic polymers are versatile materials and are preferred for many applications, including the pharmaceutical industry [9].
Naturally-derived polymers are superior to synthetic ones due to their biodegradability, biocompatibility and biological activity. Hydrogel-based natural polymers such as alginate, collagen and gelatin can be used to deliver hydrophilic drugs due to their ability to absorb large amounts of water while maintaining their structures. The water absorption ability is related to the hydrophilic groups and gelatin can be used to deliver hydrophilic drugs due to their ability to absorb large amounts of water while maintaining their structures. The water absorption ability is related to the hydrophilic groups such as -OH, -COOH, -CONH -SO3H and -CONH2 that are contained in the hydrogel matrices [10]. Besides, the chelating, biocompatible, immunogenic and mucoadhesive properties of alginate make it as an attractive polymer in drug and cell delivery systems [11,12]. Alginates are considered among the most biosynthesized polymers, where 70% of annual alginate production is allocated to pharmaceutical and biomedical applications and the remaining is used in the food industry [13,14].
After alginate evolution in the 1980s and their spread as microparticles for encapsulation purposes, several studies were carried out to synthesize nano-sized alginate particles [15,16]. Gelling properties of alginate and its remarkable processing ease have grown its importance in drug delivery, cell immobilization, food industry and research perspectives [17,18]. Furthermore, it is considered as an environmentally friendly polymer that can undergo recycling and degradation. Alginate nanomaterials represent a fast-developing field, particularly for the pharmaceutical and food industry as well as academia. The gelling property makes alginate polymer as one of the most frequently used in drug delivery [19].
The physicochemical properties of alginate, such as viscosity, thermo-stability, sol-gel transformation, pH-responsivity, as well as drug release can gain better insight into their potential applications. Many factors could impact the properties of alginate nanoparticles, such as alginate, surfactant and crosslinker concentrations, stirring time and speed as well as pH value [20]. In this review, different aspects related to the formulation and processing parameters using various techniques for preparation of alginate nanoparticles were discussed with regard to the size, encapsulation efficiency, zeta potential and drug release profile.

Alginate Polymer
Over the last decades, researchers were extensively utilizing natural polymers, especially in the pharmaceutical [21] and food industry [22,23] due to their advantages such as biocompatibility, biodegradability and low cost [11]. Alginate is an anionic polymer that typically obtained from brown marine algae. It is an unbranched polysaccharide copolymer consisting of alternating of dmannuronate (M) and l-guluronic (G) blocks linked together by 1,4-glycosidic linkages ( Figure 1A). Divalent cations, such as Ba 2+ and Ca 2+ , can quickly form so-called egg-box complexes with G block to create alginate hydrogel through gelation phenomenon ( Figure 1B) [24]. Hydrogelling ability of alginate has broadened its applications in biomedical and pharmaceutical research to encapsulate proteins and drugs for controlled release and targeted delivery. Hydrogel alginate matrix is a pH responsive where it shrinks at low pH, thus, the payload is preserved for extended period of time. Conversely, it swells and releases the encapsulated drug at higher pH values, offering a great carrier for oral delivery. The small size and large surface area of nanoparticles increase the dissolution rate and solubility of poorly soluble drugs. Nanoparticles can enhance the targetability of the encapsulant to specific sites of the body/tissue/cells, whether paracellularly or transcellularly [48]. The relationship between the rate of dissolution and particles size as well as surface area is explained by Noyes-Whitney Equation (1) as follows [49]: where dc/dt = dissolution rate, D is the diffusion coefficient of the substance, A is the surface area of exposed solid, Cs and C represent the concentration of the dissolved substance at a given time t and the solubility concentration of the substance, respectively, and h is the thickness of the diffusion layer. Also, nanoparticles enhance the solubility, dissolution rate and bioavailability of poorly soluble drugs by promoting the interaction propensity with the medium, owing to their large surface area [50]. Several researchers had developed and characterized nanoparticles-based natural polymers [51], lipids [52], polysaccharides [53] and synthetic biodegradable polymers over the last ten years [54]. Due to the unique physicochemical properties of alginate polymer among various natural polysaccharides, the ability to encapsulate foods, drugs and proteins into alginate nanoparticles has attracted the interest of many researchers [14]. Alginate-based nanocarrier seems to have all optimal requirements to be a successful drug delivery system due to its biodegradability, biocompatibility, protection effect of oral drugs against harsh gastrointestinal environment, controllable release, water solubility (avoiding the effect of noxious solvents during processing), availability and low cost [55]. Scanning electron microscopy images of alginate nanoparticles prepared by: (a) emulsification/ internal gelation, 1% w/w alginate, CaCO 3 :alginate mass ratios 0.1:1, pH 6 medium chain triglyceride (MCT) oil [40]; (b) nanospray dryer, spray cap 7 µm, flow rate 7 mL/min, drying gas flow of 110 L/min with relative flow rate 100%, inlet drying gas temperature 120 • C and outlet temperature 35 • C [41]; (c) polyelectrolyte complexation, nisin-loaded nanoparticles alginate 250 mg/mL and chitosan 250 mg/mL [42]; (d) evaporation method, zein-to-propylene glycol, alginate mass ratio 20:1 [43]; (e) layer-by-layer, paclitaxel, poly (lactic-co-glycolic acid) (PLGA) 10 mg/mL, alginate 5 mg/mL and chitosan 5 mg/mL [44]; (f) emulsification/external gelation, alginate 0.03% w/v and CaCl 2 18 mM [45]; (g) electrospray DNA plasmid loaded alginate nanoparticles, flow rate 0.1 mL/h, voltage 12.5 kV, alginate 1% w/v, Tween 20 1% v/v, CaCl 2 1.5% w/v, collector distance 4 cm and nozzle size 30 G [46]; (h) nanofiber produced by electrospinning, alginate 1.74% w/w, voltage 12 kV, needle 27 G, flow rate 0.6 mL/h and distance 12 cm [47].
The small size and large surface area of nanoparticles increase the dissolution rate and solubility of poorly soluble drugs. Nanoparticles can enhance the targetability of the encapsulant to specific sites of the body/tissue/cells, whether paracellularly or transcellularly [48]. The relationship between the rate of dissolution and particles size as well as surface area is explained by Noyes-Whitney Equation (1) as follows [49]: where dc/dt = dissolution rate, D is the diffusion coefficient of the substance, A is the surface area of exposed solid, Cs and C represent the concentration of the dissolved substance at a given time t and the solubility concentration of the substance, respectively, and h is the thickness of the diffusion layer. Also, nanoparticles enhance the solubility, dissolution rate and bioavailability of poorly soluble drugs by promoting the interaction propensity with the medium, owing to their large surface area [50]. Several researchers had developed and characterized nanoparticles-based natural polymers [51], lipids [52], polysaccharides [53] and synthetic biodegradable polymers over the last ten years [54]. Due to the unique physicochemical properties of alginate polymer among various natural polysaccharides, the ability to encapsulate foods, drugs and proteins into alginate nanoparticles has attracted the interest of many researchers [14]. Alginate-based nanocarrier seems to have all optimal requirements to be a successful drug delivery system due to its biodegradability, biocompatibility, protection effect of oral drugs against harsh gastrointestinal environment, controllable release, water solubility (avoiding the effect of noxious solvents during processing), availability and low cost [55]. Various studies Pharmaceuticals 2020, 13, 335 5 of 34 have focused on enhancing the low intestinal penetration, gastrointestinal degradation and low bioavailability of orally administered insulin utilizing alginate nanoparticles as an oral drug delivery system [56,57]. On top of that, the application of alginate nanoparticles in cancer treatment has gained wide attention due to the ability to deliver anti-cancer therapeutics in sufficient manner at target site, promoting the bioavailability as well as reducing drug dosage and its side effects to the normal tissues [58,59]. Alginate nanoparticles have been also used for targeted antibiotic delivery applications without inducing resistant strains of bacteria [60,61]. The applications and fields that utilized alginate nanoparticles grew proportionally due to their useful properties and simple synthesis methods.

Alginate Nanoparticles Preparation Methods
Alginate nanoparticles are utilized as carriers to improve the bioavailability of drugs. Various techniques have been proposed to synthesize alginate nanosystem. Selection of the preparation method is highly related to the nature of encapsulant, as well as the pre-determined attributes that nanoparticles should meet. The following sections review the most common fabrication methods of alginate nanoparticles and their effective variables.

Emulsification/Gelation
Emulsification/gelation is defined as a gelation process of the emulsion droplets that consist of an alginate solution dispersed in an oil phase to fabricate nanospheres [40]. Generally, this is a simple and low-cost technique compared to the nozzle-based methods [62,63]. This technique consists of two main stages: preparation of alginate-in-oil (w/o) emulsion, followed by gelation of the alginate emulsion droplets in aid of a covalent or ionic crosslinker [16]. Gelation of alginate takes place through two conventional ways: external and internal gelation [64]. For external gelation, crosslinker such as CaCl 2 diffuses from the outer phase into the inner core of alginate emulsion droplets to react immediately with carboxylic groups of α-L-guluronic acid. Typically, after gelation of alginate emulsion droplets by adding the crosslinker, the emulsion is demixed (Figure 3) [16,19]. Emulsification/external gelation outputs micro/nanospheres consisting of a soft core and a rigid outer matrix [65]. Internal gelation technique on the other hand, depends on the release of cations from the inner core of alginate emulsion droplets [66]. Basically, a water-insoluble calcium salt, such as CaCO 3 , is mixed with alginate before emulsification ( Figure 3). Gelation of alginate is initiated by increasing the solubility of calcium source and/or lowering the pH of the emulsion from 7.5 to 6.5, where calcium ions begin to migrate from the inner droplets to the outer part as shown in Equations (2) and (3) [67,68]. Internal gelation produces symmetrical micro/nanospheres with large pores and low matrix density compared to that prepared by external gelation [60,61].
Paques et al., have prepared alginate nanospheres via w/o emulsification followed by internal gelation using CaCO 3 micro/nano particle. The concentration of alginate solution was at 1% w/w and CaCO 3 to alginate weight ratio at 0.1:1. Glucono delta-lactone (GDL) as an acidifier, together with CaCO 3 were dispersed in alginate solution. The final mixture was subsequently emulsified in the continuous phase of the medium chain triglyceride (MCT) oil. The results showed that gelation time and size of alginate spheres reduced proportionally to CaCO 3 size from micro to nano-sized, while higher alginate concentration has resulted in delaying of the gelation time. The mass ratios at 0.1:1 and 1.98:1 of CaCO 3 :alginate and GDL:CaCO 3 , respectively, with pH value around 6 were considered as the optimum values in gel properties [40].
Another study has fabricated doxorubicin-loaded alginate nanospheres via w/o emulsification/external gelation intended for breast cancer therapy. The formulation composed of an aqueous phase (doxorubicin dissolved in alginate solution) mixed with an organic phase (cyclohexane/dodecylamine) by stirring at 1200 rpm. The synthesized nanospheres exhibited particle  In the Spadari et al. study, miltefosine (antifungal drug) has been encapsulated in alginate nanospheres by emulsification/external gelation method. Miltefosine was dissolved in an aqueous alginate solution (1% w/w) followed by emulsification with sunflower oil (3% v/v) containing Span 80 as a surfactant. After sonication of the mixture in an ice bath, CaCl2 and Poloxamer 407 were added dropwise to the emulsion as a crosslinker and surfactant, respectively. The mixture was sonicated for 5 min, accompanied by stirring for 30 min. Subsequently, the system was centrifuged at 3000× g for 5 min, discarding the supernatant followed by oil residues removal through complexation with isopropanol. In order to obtain a homogeneous fine powder of alginate nanospheres, 500 µL of trehalose (10% w/v) was applied prior to freeze-drying for 24 h. The dried nanospheres had a zeta potential of −39.7 ± 5.2 mV with a mean particle size of 279.1 ± 56.7 nm, a polydispersity index of 0.42 ± 0.15 and an encapsulation efficiency of 81.70 ± 6.64%. The study concluded that emulsification/external gelation method had efficiently encapsulated miltefosine in alginate matrix with an enhanced antifungal effect for treatment of Galleria mellonella infection and reduced drug toxicity [70].

Emulsification-Solvent Displacement Technique
Emulsification-solvent displacement method was first described in 1997 when Quintanar et al., proposed a new way to prepare concentrated pseudolatex nanoparticles using acceptable solvents [71]. This process involves emulsifying an organic solution of the polymer and drug (water-saturated) in an aqueous phase of a stabilizer (solvent-saturated) through conventional stirrers, accompanied by direct displacement of solvents under rapid evaporation using vacuum and/or temperature (e.g., rotary evaporator). Compared to conventional solvent evaporation method, emulsion homogenization step is avoided in emulsification-solvent displacement approach to produce nanoparticles. This method depends on the aggregation of the polymer and drug through a rapid diffusion of the solvent from the internal into the external phase [72].
In the study of Dai et al., zein nanoparticles were synthesized through Pickering emulsions of zein and propylene glycol alginate (PGA) via solvent evaporation method. Firstly, PGA was mixed with ethanolic zein solution at various mass ratios of 1:1, 5:1, 10:1, 20:1 and 40:1, then ethanol was evaporated by rotary evaporator followed by freeze drying for 3 days to obtain solid zein-PGA nanoparticles. The In the Spadari et al. study, miltefosine (antifungal drug) has been encapsulated in alginate nanospheres by emulsification/external gelation method. Miltefosine was dissolved in an aqueous alginate solution (1% w/w) followed by emulsification with sunflower oil (3% v/v) containing Span 80 as a surfactant. After sonication of the mixture in an ice bath, CaCl 2 and Poloxamer 407 were added dropwise to the emulsion as a crosslinker and surfactant, respectively. The mixture was sonicated for 5 min, accompanied by stirring for 30 min. Subsequently, the system was centrifuged at 3000× g for 5 min, discarding the supernatant followed by oil residues removal through complexation with isopropanol. In order to obtain a homogeneous fine powder of alginate nanospheres, 500 µL of trehalose (10% w/v) was applied prior to freeze-drying for 24 h. The dried nanospheres had a zeta potential of −39.7 ± 5.2 mV with a mean particle size of 279.1 ± 56.7 nm, a polydispersity index of 0.42 ± 0.15 and an encapsulation efficiency of 81.70 ± 6.64%. The study concluded that emulsification/external gelation method had efficiently encapsulated miltefosine in alginate matrix with an enhanced antifungal effect for treatment of Galleria mellonella infection and reduced drug toxicity [70].

Emulsification-Solvent Displacement Technique
Emulsification-solvent displacement method was first described in 1997 when Quintanar et al., proposed a new way to prepare concentrated pseudolatex nanoparticles using acceptable solvents [71]. This process involves emulsifying an organic solution of the polymer and drug (water-saturated) in an aqueous phase of a stabilizer (solvent-saturated) through conventional stirrers, accompanied by direct displacement of solvents under rapid evaporation using vacuum and/or temperature (e.g., rotary evaporator). Compared to conventional solvent evaporation method, emulsion homogenization step is avoided in emulsification-solvent displacement approach to produce nanoparticles. This method depends on the aggregation of the polymer and drug through a rapid diffusion of the solvent from the internal into the external phase [72].
In the study of Dai et al., zein nanoparticles were synthesized through Pickering emulsions of zein and propylene glycol alginate (PGA) via solvent evaporation method. Firstly, PGA was mixed with Pharmaceuticals 2020, 13, 335 7 of 34 ethanolic zein solution at various mass ratios of 1:1, 5:1, 10:1, 20:1 and 40:1, then ethanol was evaporated by rotary evaporator followed by freeze drying for 3 days to obtain solid zein-PGA nanoparticles. The results showed that the nanoparticle size was significantly reduced at 10:1 mass ratio, while higher PGA concentration enhanced the sample stability as a function of strong electrostatic repulsion between particles, promoting the production of stable Pickering emulsions [43].

Solvent Evaporation Technique
In this technique, the polymer and the hydrophobic drug are dissolved in a volatile organic solvent [8]. The emulsion (o/w) is prepared by adding organic solution such as ethyl acetate, dichloromethane or chloroform (oil phase) to the aqueous solution of surfactant under ultra-sonication or rapid homogenization [73,74]. The organic solvent is then evaporated by means of continuous stirring or high temperature under reduced pressure. The nanoparticles are then collected through ultracentrifugation to remove the excess solvent, while free drug and surfactant are washed away with distilled water ( Figure 4A). Double emulsion (w/o/w) technique on the other hand, is commonly used for encapsulation of hydrophilic molecules such as proteins, peptides and antigens (vaccines), where aqueous drug phase is added to the oil phase of polymer and volatile solvent under continuous stirring to prepare stable emulsion (w/o). The resulted emulsion is then transferred under the same condition to the aqueous phase of surfactant to produce a double emulsion (w/o/w) ( Figure 4B) [8]. Under the same condition, nanoparticles are hardened after solvent removal, while the physical and chemical properties of the synthesized nanoparticle can be tuned by tailoring the emulsion properties, such as surfactant concentration/type, o/w phase ratio, polymer concentration and evaporation rate as well as processing parameters, including agitation rate and time, geometry of homogenizing tip, and shape and volume of homogenizer vessel [75,76]. results showed that the nanoparticle size was significantly reduced at 10:1 mass ratio, while higher PGA concentration enhanced the sample stability as a function of strong electrostatic repulsion between particles, promoting the production of stable Pickering emulsions [43].

Solvent Evaporation Technique
In this technique, the polymer and the hydrophobic drug are dissolved in a volatile organic solvent [8]. The emulsion (o/w) is prepared by adding organic solution such as ethyl acetate, dichloromethane or chloroform (oil phase) to the aqueous solution of surfactant under ultra-sonication or rapid homogenization [73,74]. The organic solvent is then evaporated by means of continuous stirring or high temperature under reduced pressure. The nanoparticles are then collected through ultracentrifugation to remove the excess solvent, while free drug and surfactant are washed away with distilled water ( Figure 4A). Double emulsion (w/o/w) technique on the other hand, is commonly used for encapsulation of hydrophilic molecules such as proteins, peptides and antigens (vaccines), where aqueous drug phase is added to the oil phase of polymer and volatile solvent under continuous stirring to prepare stable emulsion (w/o). The resulted emulsion is then transferred under the same condition to the aqueous phase of surfactant to produce a double emulsion (w/o/w) ( Figure 4B) [8]. Under the same condition, nanoparticles are hardened after solvent removal, while the physical and chemical properties of the synthesized nanoparticle can be tuned by tailoring the emulsion properties, such as surfactant concentration/type, o/w phase ratio, polymer concentration and evaporation rate as well as processing parameters, including agitation rate and time, geometry of homogenizing tip, and shape and volume of homogenizer vessel [75,76].   In the study of K.S et al., zidovudine nanoparticles were prepared from alginate and stearic acid polyethylene glycol via two-step emulsion solvent evaporation technique. This study aimed to confer enhancement of the drug loading capacity, the drug release efficacy and the biocompatibility. The organic phase consisted of a chloroform as a solvent, polyethylene glycol (0.2% w/v) and stearic acid (100 mg in 25 mL), while zidovudine was dissolved into the alginate solution (1 mg/mL) to form the aqueous solution. The first emulsion was prepared by dissolving polyethylene glycol (0.2% w/v) and stearic acid (0.4% w/v) in chloroform at 55 • C. Zidovudine of different strengths (100 mg, 150 mg and 200 mg) was dissolved in alginate (0.1% w/v) solution at 55 • C to form a double emulsion system, stirred for 2 h in a fume hood to evaporate the organic solvent and solidify the nanoparticles. Then, nanoparticles suspension was centrifuged (20,000 rpm), washed and dried by freeze-drying technique. The optimized formulation produced particles size of 407.67 ± 19.18 nm, zeta potential of −42.53 mV and encapsulation efficiency of 83.18 ± 22%. The drug release was examined in different dissolution medium at pH 1.2 and 7.4. Initially, zidovudine nanoparticles exhibited a burst release around 36% and 20% at pH 1 and 7.4, respectively within 2 h, followed by a prolonged release of 95% and 48% within 28 h [77].

Complexation
The complexation method produces two types of nanoparticles with reference to the medium used, where alginate nanoaggregates and nanocapsules are synthesized in an aqueous solution and on the interface of oil droplet, respectively. In general, complexation of alginate takes place using divalent ions like Ca 2+ (available in calcium chloride), which act as a crosslinker and/or by incorporating of oppositely charged polyelectrolytes such as chitosan [78] and polymethacrylate [79]. The production of alginate nanoaggregates is attained by pre-gelation state, in which alginate solution is mixed with calcium chloride in aqueous continuous phase. In addition, complexation of alginate is also feasible by polyelectrolyte complex through mixing alginate and polycationic polymer such as chitosan and polymethacrylate to obtain polyelectrolyte complex coated-alginate nanoparticles ( Figure 5) [80,81].
Commonly, nanocapsule is synthesized through forming a shell on template droplets by polymer deposition on their interface with subsequent solvent removal. The crosslinker is added to stabilize the polymer shell by physical or covalent intermolecular forces ( Figure 5). Briefly, this method consists of an organic solvent mixed with a drug to be encapsulated to form the interior phase of nanocapsule. This mixture is slowly added to an alginate solution that contains surfactant such as Tween 80, where oil-in water (o/w) emulsion is prepared by means of sonication [82]. In addition, chitosan can be synergistically included together with the crosslinker to promote the encapsulation efficiency and reduce the porosity of the alginate nanocapsules [83]. Table 1 summarizes recent studies of alginate produced by means of complexation method. divalent ions like Ca 2+ (available in calcium chloride), which act as a crosslinker and/or by incorporating of oppositely charged polyelectrolytes such as chitosan [78] and polymethacrylate [79]. The production of alginate nanoaggregates is attained by pre-gelation state, in which alginate solution is mixed with calcium chloride in aqueous continuous phase. In addition, complexation of alginate is also feasible by polyelectrolyte complex through mixing alginate and polycationic polymer such as chitosan and polymethacrylate to obtain polyelectrolyte complex coated-alginate nanoparticles ( Figure 5) [80,81]. Commonly, nanocapsule is synthesized through forming a shell on template droplets by polymer deposition on their interface with subsequent solvent removal. The crosslinker is added to

Alginate as a Coating Material for Nanocarrier (Layer-by-Layer Approach)
Layer-by-layer is a bottom-up coating technique, in which the film is formed through construction layers of micro/nanometric thicknesses to produce core-shell system. This method involves electrostatic interaction between oppositely charged polyelectrolytes, where alternating adsorption of multi-layers cationic and anionic polymers occurs on flat substrates. In addition, this technique enables drug to be embedded between the layers, and hence promoting encapsulation efficiency and drug release control propensity with the reference of the physiochemical properties of the polymeric carrier used [92]. Layer-by-layer process can be influenced by various factors, such as pH of the medium, saturation adsorption time, polyelectrolyte concentration, adsorption temperature and salt concentration of polyelectrolyte solutions as well as the type of the matrix and core used [93].
In the study of Liu et al., nanoliposomes were coated with alginate and chitosan via layer-by-layer method to improve liposomal stability and prevent leakage of the payload [94]. In order to form the first layer, anionic nanoliposomes were added into chitosan solution (0.6% w/v) and then incubated for 1 h under gentle stirring. The subsequent layer was deposited by dropping chitosan-nanoliposomes into alginate solution (0.5% w/v) using the same procedure. The size, polydispersity index and zeta potential of the prepared nanoliposomes were around 89 nm, 0.26 and −6.3 mV, respectively. On the other hand, the final formulation of coated nanoliposomes exhibited larger mean size at 330 nm, polydispersity index at 0.37 and zeta potential at −15.8 mV in pH 5.5. The enzymatic digestion stability test demonstrated that coated nanoliposomes have conferred lipolytic degradation resistance and delayed release of encapsulant in simulated gastrointestinal conditions compared to coat-free nanoliposomes.
In order to control doxorubicin release for antitumor activity, Chai et al., have prepared alternative multilayer of chitosan (cationic polymer) and alginate (anionic polymer) on the surface of doxorubicin-loaded PLGA nanoparticles. The influences of temperature, polyelectrolyte polymers concentration and NaCl concentration on the multilayer growth were investigated. Doxorubicin PLGA nanoparticles were prepared via double water in-oil-in-water (w/o/w) emulsion-solvent evaporation method. Subsequently, chitosan and alginate were alternately deposited on the nanoparticle surface to form multilayers of polyelectrolyte. It was found that, increasing polyelectrolyte or NaCl concentration as well as the adsorption temperature, the coat weight of the multilayer film was intensified. The initial burst release was significantly reduced as a result of employing layer-by-layer technique, while lowering the pH of dissolution medium conferred an increase of doxorubicin release [95].
Wang et al., have modified paclitaxel-PLGA nanoparticles prepared via solvent evaporation method by depositing chitosan and alginate layer-by-layer to improve the drug burst release tendency. After chloroform removal (volatile solvent) from w/o single emulsion, chitosan solution (0.5% w/v) was added dropwise into the emulsion under continues stirring to adsorb the first coat layer on the surface of nanoparticles by electrostatic force. In order to form a second coat layer, alginate solution (5 mg/mL) was added to PLGA-chitosan nanoparticles solution and ultrasound sonicated for 10 min. Three types of nanoparticles (PLGA, PLGA/chitosan and PLGA/chitosan/alginate) were separately prepared and characterized with regard to mean size, zeta potential, encapsulation efficiency and loading capacity. The particle size, encapsulation efficiency and loading capacity were increased proportionally after depositing each layer. The values of zeta potential of the above-mentioned nanoparticles were −2.72 ± 0.17 mV, +17.36 ± 0.84 mV and −10.62 ± 0.38 mV, respectively. The drug release study was carried out in PBS buffer (pH 7.4), and paclitaxel nanoparticles exhibited low initial burst release and prolonged release properties [44]. Khan et al., have investigated the influence of alginate/chitosan coat complex on the sustained release and bio-accessibility of resveratrol-zein nanoparticles [96]. The average particle size and zeta potential of initial nanoparticles were increased from 16.9 to~72 nm and from +15.01 to +43.01 mV, respectively, while the effect on the encapsulation efficiency was negated. Besides, the release of resveratrol was sustained under simulated gastrointestinal condition, and its bio-accessibility was improved significantly.

Spray Drying
Spray drying is one of the common techniques of micro and nanoparticle production where a liquid is atomized to droplets and dried using a hot gas [97]. In 1872, Samuel Percy was the first person to patent a spray drying technique that has been developed concerning its safety and productivity [98]. The production of nanoparticles using spray drying process is based on the removal of moisture from sprayed wet droplets by using a heated atmosphere. The working principle of a spray dryer includes four basic steps: emulsifying, dissolving or dispersing the drug in solvent (i), atomization of the solution into a spray using specific nozzle (ii), drying the sprayed droplets by drying gas (iii), and collecting the product (iv) [99]. The drying gas is introduced via an air dispenser from top of the chamber. At a constant flow rate and appropriate temperature, the feed solution is atomized into the drying gas chamber, in which the wet fine droplets are dried by moisture vaporization. Dry particles are collected through electrostatic particle collector that confers the particle surface charged and deflects by an electric filed. The collector consists of a rounded stainless still tube linked to a high voltage supplier (anode) and a grounded star electrode (cathode) inside the tube. Finally, the exhausted gas is passed through outlet filter that traps free particles from the gas ( Figure 6) [100].
Alfatama et al., prepared three types of nanoparticles for oral delivery of insulin via spray drying technique. Simple alginate, alginate-C18 and alginate-stearic acid were used to obtain spherical nanoparticles with particle size of 513 nm, 522 nm, 619 nm, insulin encapsulation efficiency of 44.4%, 44.9%, 76.7% and polydispersity index of 0.54, 0.74, 0.33, respectively. The processing parameters used were as follows: air flow rate 2-2.5 m/s, solution feed rate 4 mL/min and atomizing air pressure 6 bar. The inlet temperature and outlet temperature were 60 • C and 23 • C, respectively [57]. Shehata and Ibrahima, on the other hand, have encapsulated metformin hydrochloride into nanoparticles consisting of alginate/gelatin with 1:3 ratio using nano-spray dryer. The prepared nanoparticles exhibited a mean diameter around 850 nm, polydispersity index 0.14, yield 81% and encapsulation efficiency 90%, employing nano-spray parameters: spray cap 7 µm, flow rate 7 mL/min, flow of drying gas 110 L/min with relative flow rate 100%, inlet drying gas temperature 120 • C, outlet temperature 35 • C, and actuator 60 kHz to form droplets through vibration membrane. The obtained dried nanoparticles manifested good flowability with an angle of repose around 31 • due to their spherical shaped, however the reduced yield was attributed to the nanoparticles powder that stuck to the scraper and the electrode in the collection chamber [41]. from top of the chamber. At a constant flow rate and appropriate temperature, the feed solution is atomized into the drying gas chamber, in which the wet fine droplets are dried by moisture vaporization. Dry particles are collected through electrostatic particle collector that confers the particle surface charged and deflects by an electric filed. The collector consists of a rounded stainless still tube linked to a high voltage supplier (anode) and a grounded star electrode (cathode) inside the tube. Finally, the exhausted gas is passed through outlet filter that traps free particles from the gas ( Figure 6) [100]. Alfatama et al., prepared three types of nanoparticles for oral delivery of insulin via spray drying technique. Simple alginate, alginate-C18 and alginate-stearic acid were used to obtain spherical nanoparticles with particle size of 513 nm, 522 nm, 619 nm, insulin encapsulation efficiency of 44.4%, 44.9%, 76.7% and polydispersity index of 0.54, 0.74, 0.33, respectively. The processing parameters used were as follows: air flow rate 2-2.5 m/s, solution feed rate 4 mL/min and atomizing air pressure 6 bar. The inlet temperature and outlet temperature were 60 °C and 23 °C, respectively [57]. Shehata and Ibrahima, on the other hand, have encapsulated metformin hydrochloride into nanoparticles consisting of alginate/gelatin with 1:3 ratio using nano-spray dryer. The prepared nanoparticles exhibited a mean diameter around 850 nm, polydispersity index 0.14, yield 81% and encapsulation efficiency 90%, employing nano-spray parameters: spray cap 7 µm, flow rate 7 mL/min, flow of drying gas 110 L/min with relative flow rate 100%, inlet drying gas temperature 120 °C, outlet In another study by A. El-Missiry et al., ellagic acid nanoparticles were synthesized using calcium-alginate via nano-spray drying and followed by ionotropic gelation with calcium ions. Alginate and ellagic acid were used at mass ratio 1:1 with alginate concentration at 0.025% w/v. The spray drying condition used: inlet temperature 120 • C and an air flow rate 135 L/min. The obtained nanoparticles were weighted and added into calcium chloride solution (0.1% w/v) as a crosslinker. The results showed that the nanoparticles mean size was around 670 nm [101].
De Cicco et al., have encapsulated gentamicin sulfate into alginate pectin nanoparticles via nano-spray dryer technique after adjusting feed rate and nozzle spray mesh size. The process conditions and parameters used: inlet temperature 90 • C, air flow 100 L/min, feed rate 9.5 mL/min, relative spray rate 100% and nozzle size 4.0 µm, 5.5 µm or 7.0 µm. Modulating nozzle size has directly impacted the particles size and particles size distribution proportionally, while the feed rate has affected the particles size distribution only in symmetrical manner. Apparently, the above-mentioned parameters had no significant effect on the encapsulation efficiency [102]. In the pharmaceutical industry, spray drying technique plays a significant role in manufacturing of drug powders and other therapeutic products, and this technique, in particular, can produce a high drug encapsulation efficiency and operate under sterile conditions [103,104]. It has also become an attractive technique in other industries such as cosmetics [105], food [106], and photoluminescence [107].

Electrospray
Electrospray technique exhibits unique advantages to develop micro-and nanoparticles due to the friendly single step approach, the ability to control the particle size, the low amount of solvents and the yield control [108]. This technique is booming in the research and industry fields because of its ability to produce monodisperse droplets from nano-size to hundreds of micrometers, depending on the processing parameters [109,110]. Several researchers have successfully employed this technique to encapsulate macromolecular bioactive agents such as cells [111], proteins [112], nucleic acids [46] and others. Firstly, a liquid is pumped slowly through a thin metal needle using a syringe pump by a constant flow rate. Then, high voltage is applied on the needle to increase the acceleration of exited liquid away from the needle and overcome the surface tension of droplets. Finally, the droplets at the tip of the needle are converted into fine nano/micro-sized spray and form a cone called the Taylor cone (Figure 7) [113]. The size of the final droplet can be controlled by modulating the processing parameters, such as the flow rate, voltage, needle size, and the distance between the needle and the surface of the collector, and formulation parameters, such as materials concentration, crosslinkers, and surfactants. This method offers many advantages, such as using small input materials to get high yield of nanoparticles and thus avoiding waste of expensive substances, a single continuous one-step approach, and low cost, while the disadvantage is associated with time consuming [114]. Tsai and Ting have synthesized alginate and alginate/chitosan bilayer nanoparticles via electrospray technique. Central Composite Design-Response Surface Methodology (CCD-RSM) was applied to investigate the influence of flow rate, applied voltage, needle distance and alginate concentration on the particle diameters. The results reported that the size of alginate nanoparticles was significantly influenced by the applied voltage and the concentration of alginate solution, whereas the effects of the distance between the needle to the surface of the receiving medium and flow rate were negated. The optimum parameters produced alginate nanoparticles with size 279.17 ± 16.33 nm and zeta potential −57.3 ± 0.15 mV via modulating the flow rate, collector distance, applied voltage and alginate concentration are 0.6 mL/h, 20 cm, 27 kV and 3% w/v, respectively. Finally, chitosan/alginate bilayer nanocarrier were synthesized using chitosan as a core and alginate as a shell. Electrospray nozzle comprising two concentric stainless-steel needles (coaxial needle) was utilized to produce nanocarrier with dual layer, in which, the outer needle diameter 0.96 mm (for shell), while the inner needle diameter 0.52 mm (for core). The processing parameters were as follows: concentration of chitosan and alginate solutions at 2% w/w and 3% w/w, respectively, the distance of collector 20 cm, and voltage 27 kV. Taylor cone structure consisted of two solutions was determined experimentally to be at ratio 1:11 of chitosan solution (core) and alginate solution (shell). The final results showed that chitosan/alginate nanocarrier were produced by electrospray technique with mean size particles 112.1 ± 35.2 nm, polydispersity 0.42 ± 0.06 and zeta potential +21.82 ± 2.23 mV [115].
In another study, B. Alallam et al., have formulated alginate nanoparticles as a carrier for plasmid DNA by means of electrospray technique [46]. The study reported that the particle size of the nanoparticles reduced significantly from 1500 nm to 420 nm as a function of voltage rise from 9 kV to 12 kV. This can be advocated that higher voltage brings about stronger coulombic forces which intensify repulsion between adjacent droplets, producing smaller droplets size. Furthermore, the effect of flow rate of atomized solution was also investigated and reported a decrease in particle size from 533 nm to 463 nm upon acceleration the flow rate from 0.1 mL/h to 0.5 mL/h. In order to find the optimum ratio between flow rate and applied voltage, the interaction effect was examined using Minitab software. The suggested parameters from the design were the highest voltage (12.5 kV) with the lowest flow rate of 0.1 mL/h. This can be explained by the uneven spread of the emulsion at the tip of the needle, caused by high flow rate, resulted in uncontrolled atomization and formation of large particles [116]. Tsai and Ting have synthesized alginate and alginate/chitosan bilayer nanoparticles via electrospray technique. Central Composite Design-Response Surface Methodology (CCD-RSM) was applied to investigate the influence of flow rate, applied voltage, needle distance and alginate concentration on the particle diameters. The results reported that the size of alginate nanoparticles was significantly influenced by the applied voltage and the concentration of alginate solution, whereas the effects of the distance between the needle to the surface of the receiving medium and flow rate were negated. The optimum parameters produced alginate nanoparticles with size 279.17 ± 16.33 nm and zeta potential −57.3 ± 0.15 mV via modulating the flow rate, collector distance, applied voltage and alginate concentration are 0.6 mL/h, 20 cm, 27 kV and 3% w/v, respectively. Finally, chitosan/alginate bilayer nanocarrier were synthesized using chitosan as a core and alginate as a shell. Electrospray nozzle comprising two concentric stainless-steel needles (coaxial needle) was utilized to produce nanocarrier with dual layer, in which, the outer needle diameter 0.96 mm (for shell), while the inner needle diameter 0.52 mm (for core). The processing parameters were as follows: concentration of chitosan and alginate solutions at 2% w/w and 3% w/w, respectively, the distance of collector 20 cm, and voltage 27 kV. Taylor cone structure consisted of two solutions was determined experimentally to be at ratio 1:11 of chitosan solution (core) and alginate solution (shell). The final results showed that chitosan/alginate nanocarrier were produced by electrospray technique with mean size particles 112.1 ± 35.2 nm, polydispersity 0.42 ± 0.06 and zeta potential +21.82 ± 2.23 mV [115].
In another study, B. Alallam et al., have formulated alginate nanoparticles as a carrier for plasmid DNA by means of electrospray technique [46]. The study reported that the particle size of the nanoparticles reduced significantly from 1500 nm to 420 nm as a function of voltage rise from 9 kV to 12 kV. This can be advocated that higher voltage brings about stronger coulombic forces which intensify repulsion between adjacent droplets, producing smaller droplets size. Furthermore, the effect of flow rate of atomized solution was also investigated and reported a decrease in particle size from 533 nm to 463 nm upon acceleration the flow rate from 0.1 mL/h to 0.5 mL/h. In order to find the optimum ratio between flow rate and applied voltage, the interaction effect was examined using Minitab software. The suggested parameters from the design were the highest voltage (12.5 kV) with the lowest flow rate of 0.1 mL/h. This can be explained by the uneven spread of the emulsion at the tip of the needle, caused by high flow rate, resulted in uncontrolled atomization and formation of large particles [116].

Electrospinning
Electrospinning is a simple technique that produces nanofibers via electric fields to extract ultrafine fiber from polymer solution or melt [117]. This technique has attracted high attention in regenerative medicine, tissue engineering and industrial fields due to the ease of controlling the formed fiber properties such as the shape, size, and porosity. In addition, the nanofiber has been also applied in drug delivery system such as protein, antibiotics, DNA, RNA, living cells and growth factors, because it offers a high surface-to-volume ratio and ability to control drug loading and release profiles [118,119]. Electrospinning setup normally comprises of a syringe pump, high voltage power supply and collector that separated at a defined distance. In general, electrospinning process is considered as a sister technology with electrospray technique (Figure 8) [120]. The major difference between both techniques is the polymer concentration, in which high concentration can be used in electrospinning to obtain a more stable jet as well as elongation takes place by whipping instability mechanism [121]. The essential parameters that affect diameter of the electrospun and morphology of nanofibers are: needle diameter, distance between needle and collector, flow rate, solvent volatility and applied voltage [122].

Electrospinning
Electrospinning is a simple technique that produces nanofibers via electric fields to extract ultrafine fiber from polymer solution or melt [117]. This technique has attracted high attention in regenerative medicine, tissue engineering and industrial fields due to the ease of controlling the formed fiber properties such as the shape, size, and porosity. In addition, the nanofiber has been also applied in drug delivery system such as protein, antibiotics, DNA, RNA, living cells and growth factors, because it offers a high surface-to-volume ratio and ability to control drug loading and release profiles [118,119]. Electrospinning setup normally comprises of a syringe pump, high voltage power supply and collector that separated at a defined distance. In general, electrospinning process is considered as a sister technology with electrospray technique (Figure 8) [120]. The major difference between both techniques is the polymer concentration, in which high concentration can be used in electrospinning to obtain a more stable jet as well as elongation takes place by whipping instability mechanism [121]. The essential parameters that affect diameter of the electrospun and morphology of nanofibers are: needle diameter, distance between needle and collector, flow rate, solvent volatility and applied voltage [122]. Table 2 represents examples of previous research studies of alginate nanofibers synthesized via electrospinning method.

Limitations of Alginate Nanofabrication
Despite the rapid progression and verities of techniques to synthesize alginate nanoparticles, it is still less striking compared to synthetic polymers due to extensive molecular weight variations and source-related properties [128,129]. Alginate purity determines the biocompatibility of the prepared nanoparticles, thus the presence of hazardous contaminants such as endotoxins, polyphenolic compounds, proteins and heavy metals, may cause an immune response in the site of administration [130]. In the market, alginates are available in different grades, purities, and qualities, based on the manufacturer and source [131]. Moreover, the difference in M/G blocks ratios of alginate has been correlated to innate immune system stimulation as a result of the presence of cytokines in response to high M block (β-d-mannuronic acid) content [132].
On the other hand, many studies have reported a poor encapsulation efficiency and high burst drug release of alginate nanoparticles [86,90,133]. This can be attributed to the hydrophilicity and high porosity of alginate nanoparticles leading to instability and swelling at biological fluids as well as leakage of entrapped drugs during the preparation process [18,134]. Different chemical modification approaches have been implemented to overcome these limitations, improving the mechanical strength of alginate nanoparticles and to confer a hydrophobic character of alginate [135,136]. However, this usually involves chemical reagents such as aldehydes, which may require purification prior to clinical uses [137]. Moreover, the use of organic solvents for alginate derivation may influence the physiochemical and biological characteristics of the encapsulant [138]. In addition, some of the preparation methods of alginate nanoparticles including emulsification-solvent displacement and solvent evaporation are mediated by organic solvents [128].

The Influence of Alginate Concentration
Yasmin et al., have highlighted the effect of alginate concentration on the size, size distribution and encapsulation efficiency of nanospheres prepared by means of emulsification/external gelation method. The results revealed that alginate concentration has a significant effect on the mean diameter of nanospheres, in which alginate concentration 3% w/v and 5% w/v resulted in nanospheres with size range 700 nm to 900 nm, respectively. Further, high alginate concentration 5% w/v was associated with improved encapsulation efficiency of bovine serum albumin loaded-nanospheres. The results also showed that high alginate concentration is effective in reducing the initial burst release from 86 ± 3.62% to 74 ± 1.53%, while the effect of alginate concentration on the total protein release was negated [139]. Similar results were reported by Sarei et al., whereby direct proportional relationship between alginate concentration and size, encapsulation efficiency and polydispersity index of the prepared nanospheres was found, while a slow drug release was attainable. This phenomenon was observed and explained extensively, advocating the use of high alginate concentration produces high solution viscosity and low shear stress that lead to larger emulsion droplets [140]. In another study, Mokhtari et al., have analyzed the influence of alginate concentration on nanoparticle's attributes prepared by emulsification/internal gelation technique. They have reported an increase in particle size from 512 nm to 4303 nm and encapsulation efficiency from 5.26% to 7.62%, when alginate concentration was raised from 0.5% w/v to 1% w/v, respectively [141]. This may be explicated by higher alginate concentration induced more forces in alginate droplets that may resist droplets breakdown and hinder the diffusion of drug from alginate droplets to the oil phase during preparation process [142]. Besides, nanocarriers, being relatively large in size and hydrophilic in nature, possess small specific surface area and are less prone to burst or premature release [143].
Novitasari et al., have synthesized alginate-chitosan nanoparticles by means of ionic gelation technique using calcium ions. The influence of the processing parameters was assessed using full factorial design to predict the effect of each factor and their interactions on the responses. Based on the results, high alginate concentration led to an increase in particles size and polydispersity index and improved encapsulation efficiency of the nanoparticles. With reference to the interaction effect among all factors (alginate, calcium chloride and chitosan concentration), the lowest particles size (200 nm) and polydispersity index (0.27) with encapsulation efficiency around 35% were attainable by reducing both alginate and chitosan concentrations while maintaining high value of calcium chloride concentration [144]. Similar findings were also reported by Zimet et al., when Nisaplin ® loaded alginate nanoparticles have been prepared by means of ionic gelation/complexation method [145]. The results showed an increase in particle size from 86 nm to 204 nm, zeta potential from −33.2 mV to −38.7 mV and a decrease in encapsulation efficiency from 35.6% to 30.5% when alginate concentration was increased from 0.03% w/v to 0.07% w/v, respectively. Encapsulation efficiency was directly proportional to Nisaplin ® : alginate mass ratio of the nanoparticles.
Mansourpour et al., have applied factorial models to examine the influence of alginate concentration on the size and polydispersity index of nanoparticles produced by ionotropic gelation. Higher alginate concentration resulted in particles with larger size and higher polydispersity index, in which the effect of alginate concentration was driven by other independent factors namely, CaCl 2 and cationic β-cyclodextrin concentrations [146]. Furthermore, Rahaiee et al., have investigated the effects of alginate and chitosan concentrations to optimize the characteristics of the prepared nanoparticle via ionic gelation method. The influence of alginate concentration on the size and encapsulation efficiency of the nanoparticles was dominated compared to other factors. Simultaneous rise of alginate and chitosan concentration rendered encapsulation efficiency to drop, but this was reversible by mere reducing chitosan concentration. This can be attributed to the competition of the drug and chitosan on the binding sites onto alginate moieties. On the other hand, the nanoparticle size was reducible by decreasing the concentration of alginate [133]. Another study by Govindaraju et al., indicated the role of alginate on curcumin loaded alginate nanoparticles synthesized by ionotropic gelation method [147]. They have reported that high alginate concentration was associated with a significantly increased zeta potential from +150 ± 1.15 mV to +200 ± 2.15 mV and a slightly changed size and encapsulation efficiency of the prepared nanoparticle. The positive value of zeta potential was elucidated by its relationship with the absorbed amount of Ca 2+ after alginate neutralization by CaCl 2 .
Based on the abovementioned studies, it can be concluded that alginate concentration has the main influence on the size, polydispersity index and encapsulation efficiency of nanoparticles prepared via ionic gelation method. This influence can be advocated by the extent of interaction between the functional groups of alginate chains (COO − ) and calcium ions. Higher alginate concentration produces particles with larger size and wider polydispersity index as a result of increasing the number of carboxylate groups and alginate chain layers which can intensify around calcium cations. This in turn creates additional space for more drug to be entrapped, and thus improves the encapsulation efficiency, as summarized in Table 3 [45,148].

The Influence of Surfactant
Mokhtari et al., highlighted the effect of variant concentrations of Tween 80 as a surfactant with hydrophile-lipophile balance (HLB) of 4.3 used to aid the encapsulation of peppermint phenolic extract in alginate nanospheres via emulsification/internal gelation method. The results indicated that higher surfactant content produced nanospheres with lower particles size and higher encapsulation efficiency as a result of decreasing the surface tension to promote breakdown extent of alginate droplets [141]. Another study by Elgegren et al., indicated the role of surfactant on sacha inchi (Plukenetia volubilis L.) oil-loaded alginate nanoparticles synthesized by emulsification/external gelation with complexation process. They have reported that high Poloxamer 407 concentration was associated with decreased both size and polydispersity index of the prepared nanoparticles [149]. Moreover, incorporation of Polysorbate 80 (10% v/v, 20% v/v, and 40% v/v) has rendered a rise in particle size, while the encapsulation efficiency peaked at medium concentration of the surfactant [147]. Similar results were also reported by Scolari et al., in which rifampicin-ascorbic acid-loaded alginate nanoparticles coated with chitosan were fabricated by ionic gelation/complexation method. The results reported a significant increase in particle size when the Tween 80 concentration increased from 0.25% w/v to 0.5% w/v [150].
Within a single assembled system, Baghbani et al., have investigated the effect of surfactant (Tween 20) and co-surfactant (Span 60/Poloxamer 188) on the nanoparticles attributes prepared by emulsification/external gelation method. Mere use of Tween 20 at low concentration (0.1% v/v) was undesirable as it did not inhibit the separation of the emulsion phases, while at higher surfactant concentration (0.3% v/v), smaller particle size and polydispersity index, improved encapsulation efficiency and delayed manner of drug release were achieved. Besides, the effect of co-surfactant inclusion (Span 60 or Poloxamer 188) at three different concentrations (0%, 0.15% w/v and 0.3% w/v) coupled with 0.3% v/v Tween 20 on the particle size, entrapment efficiency, and drug release kinetics of doxorubicin-loaded alginate nanodroplets was also investigated. The particle size was decreased and then increased upon using higher strength of Span 60 or Poloxamer 188 in the formulation from 0% to 0.15% w/v. Using high Span 60 concentration impacted the encapsulation efficiency and cumulative drug release negatively at a fixed concentration of Tween 20, while the effect was negated upon varying the amounts of Poloxamer 188. This can be explained by the hydrophobic nature of Poloxamer 188 that covers the oil nanodroplets more efficiently and reduces drug release by retarding the degradation of alginate through neutralizing the acidity produced as a result of its degradation [148].
The large size and low encapsulation efficiency of nanoparticles associated with low concentration of surfactant can be explained by the production of unstable droplets and high propensity of coalescence, as a results of irregular surface tension due to incomplete covering of the particle surfaces. On the other hand, high levels of surfactant lead to similar effects as above in addition to higher polydispersity index. This can be advocated by higher interaction tendency between polymers or hydrophilic chains of different particles [150,151]. In addition, reduced encapsulation efficiency upon increasing the amount of surfactant was also observed, it may be attributed to the reduction in the spaces within polymeric chains (as a result of generating reverse micelles inside it) as well as losing the entrapped drug during preparation as a function of low surface tension of droplets. Table 4 represents recent examples of the effect of surfactant on the nanoparticle's attributes [141].

The Influence of CaCl 2 Concentration
Mokhtari et al., evaluated the effect of CaCl 2 concentration on the size and encapsulation efficiency of peppermint phenolic extract-loaded nanospheres prepared by means of emulsification/internal gelation. The results reported a decrease in the mean particle size and enhancement of encapsulation efficiency as a function of increasing the molarity of CaCl 2 [141]. For the development of metronidazole loaded chitosan-alginate nanoparticles, Sabbagh et al., applied a full factorial design to achieve the optimum concentration of CaCl 2 and other factors related to ionotropic pre-gelation process. The study reported that high concentration of CaCl 2 led to formation of smaller particle size and improved loading efficiency as well as reduced zeta potential [152]. Similar findings were also observed by Ahdyani et al., in which timolol maleate-loaded chitosan-alginate nanoparticles were prepared via ionic gelation technique. The results indicated reduction in particle size when CaCl 2 concentration was increased from 0.05% w/v to 0.25% w/v (Table 5) [144]. The crosslinking process has a significant impact on the physiochemical properties of alginate hydrogel, in which the carboxylic groups of alginate backbone interact preferentially with calcium ions to form a stable three-dimensional network [146,153]. The higher cross-linking concentration induces shorter polymer chain and more network density of gel matrix, resulting in smaller size and size distribution as well as improved drug release extent of alginate particles [154]. Besides, the decrease in calcium cationic ions improves zeta potential of particle surface to be more negative, conferring better stability, drug loss retardation, and high encapsulation efficiency [80,155].

The Influence of Crosslinking Time
The effect of varying crosslinking times on the properties of bovine serum albumin-loaded alginate nanospheres, prepared via emulsification/external gelation method was assessed [139]. The results demonstrated that prolonging gelation time from 1 min to 10 min led to a slight reduction of the particle size and initial burst release profiles, while encapsulation efficiency was improved (43 ± 7.50% to 51 ± 8.09%), however, further lengthening of the processing time exhibited a counteractive effect. This can be attributed to the completion of alginate gelation within 10 min, whereas a shorter stirring time was insufficient [156,157]. On the other hand, longer crosslinking time resulted in diffusing more Ca 2+ ions into alginate nanospheres that led to an increase in viscosity of the alginate phase, increased porosity, more junction zones and, hence a leakage of drug molecules from the alginate droplets to the medium [158,159].The effect of the complexation time was examined at 90 min, 120 min, and 180 min with respect to the particle size and entrapment efficiency to produce streptomycin-loaded alginate-chitosan nanoparticles via ionotropic complexation method. It was found that the lowest particles size (374 nm) and the highest encapsulation efficiency (93.32%) were achieved at complexation duration of 90 min [160]. However, the effect of complexation time on the encapsulation efficiency of trans-cinnamaldehyde nanoparticles prepared by combination of ionic gelation and complexation techniques, according to Loquercio study, was negated, while the mean particle size (294.78 nm) was reducible by increasing the crosslinking duration from 45 min to 90 min [20].
Another study was conducted to evaluate the particle size of insulin nanoparticles synthesized via emulsification/internal gelation. Similar trend of results was observed where the reduction of particle size (442 nm to 317 nm) was enabled by applying ultrasonication for 15 min, after which the size started to expand [161]. To enable a marked reduction of particle size, it was envisaged that ultrasound sonication brought about emulsion droplet breakdown by means of cavitation phenomena, yet lengthening this process time rendered the coalescence of emulsion droplets as a result of elevating the medium temperature and hence larger particle size [162].
The short stirring duration may not be adequate to generate intensive electrolyte interactions that compact the polymer chains on the nanoparticles surface [163]. This may lead to the production of larger particles with exposed pores that could result in the loss of the encapsulant during preparation [18,164]. On the other hand, longer stirring time might cause aggregation of polymer molecules on the surface of nanoparticles and reduction of free spaces within alginate matrix [157].

The Influence of Stirring Rate
J. Emami et al., have evaluated the effect of stirring rate from 500 rpm to 2000 rpm on the mean particle size, encapsulation efficiency and drug release profiles of glipizide-loaded alginate-chitosan nanoparticles using ionotropic controlled gelation technique. The results showed a negative influence on average particles size and encapsulation efficiency of alginate nanoparticles, while drug release was remarkably prolonged by increasing the stirring rate [165]. Similar findings were reported by other researchers in which the nanoparticle diameter decreased from 627 nm to 236 nm as a function of intensifying stirring rate from 500 rpm to 1000 rpm [166].
In another study, Samprasit et al., have prepared mangostin-loaded chitosan-alginate nanoparticles via ionotropic gelation method. It was found that an increase of stirring speed from 1000 rpm to 1400 rpm has resulted in a significant decrease in nanoparticle mean size [167]. Mohamed and Laraba-Djebari have also prepared calcium alginate nanoparticles as vaccine delivery by means of ionic gelation method. The concentration of alginate, CaCl 2 and the time of homogenization remained constant, while the homogenization rate varied from 500 to 1500 rpm. The results indicated that higher homogenization extent conferred a significant decrease in the size of the nanoparticles from <1000 to 85-300 nm [168].
From the previous studies, it can be concluded that stirring speed impacted the particle size significantly. This phenomenon can be evidently observed by transfer of mechanical energy of different stirring rates, in which the breaking energy is increased upon rising the stirring speed, resulting in smaller particles size [169,170]. The variability in the drug release profiles can be indicated by the variance in the particle size [171,172]. Smaller particles have a faster drug release tendency due to their large surface-to-volume area, as well as shorter diffusion pathways that transfer the payload to the outer dissolution medium [148].

The Influence of pH
In general, the complex of anionic and cationic ions occurs based on the electrostatic interactions between charged molecules [173]. The strength of the polyelectrolyte complex is influenced by the pH value of the solution [174]. In order to find the optimum pH value that offers the highest number of ionized or protonated groups required for interaction, the pH range should be investigated [175]. The pKa value of alginate is between 3.4 and 4.4 (depending on its source). At pH > pKa, the carboxylate groups of alginates are ionized (Alg-COO − ) and electrostatically linked to calcium ions and cationic polymers [92]. Furthermore, the negatively charged carboxylate groups are predominant at weak acid medium around pH = 5 [85]. While, amino groups (NH 2 ) of chitosan are protonated (NH 3 + ) at pH below its pKa (6.5), it allows chitosan to coacervate with anionic polymers such as alginate [176]. As shown in Table 6, alginate-chitosan nanoparticles with small size and high encapsulation efficiency were produced at halfway between pKa of these species (pH~4.8). Besides, intensifying of electrostatic interactions is represented by zeta potential values, in which high negative values indicate an absence of free cationic groups on the surface of nanoparticles, that correlated to high encapsulation efficiency and drug loading propensity [161].  Table 7 represents recent examples of nanoparticles prepared from alginate/chitosan blends via different approaches. It can be evidently noticed that higher amounts of chitosan produce larger particles as well as lower encapsulation efficiency. This can be explained by the higher affinity of amino groups of chitosan to M residues than G residues of alginate [133,163,178]. Hence, reducing the alginate:chitosan ratio may lead to a competition between amino groups and cationic Ca 2+ ions on the G block binding sites after saturation of the binding sites of M residues of alginate. This may bring about particles aggregation which could have a negative impact on the encapsulation efficiency and loading capacity, as well as an increase in particles size and zeta potential. On the other hand, decreasing the alginate:chitosan ratio can minimize the electrostatic attraction between them, suppresses encapsulation ability and drug release control as well as increase the particle size [133,179].

Comparison of Alginate Nanoparticles' Synthesis Methods
Hydrophobic and hydrophilic molecules such as anti-cancer drugs (doxorubicin [89], crocin [86], curcumin, resveratrol [88], exemestane [87]) were successfully loaded into alginate nanoparticles via complexation method. Emulsification/gelation method on the other hand, is preferable to encapsulate hydrophobic substances due to the presence of two phases. This method enables synthesis of relatively small size nanoparticles as low as 39.2 nm [148]. In addition, nozzle-based methods such as spray drying [106], electrospray [182] and electrospinning [183] are able to produce nanoparticles of reduced polydispersity index and improved encapsulation efficiency due to the fact of single-step method where drug loss is minimized. Table 8 summarizes the characterization and formulation/processing factors that can be modulated to obtain an optimal formulation of alginate nanoparticles for specific drug delivery system.  [126,127]

Conclusions
Alginate possesses great biocompatibility and approved as food additive by the US-FDA, thus it is a preferred candidate among pharmaceutical excipients for designing of advanced drug delivery system for oral delivery. The high interest of the scientific community in alginate synchronized with the revolution in therapeutics-driven by nanomedicine. The present spotlight review highlighted the most recent studies of alginate as a platform to develop nanoparticles intended for oral administration. The most elegant attributes are mucoadhesive and mucopenetration that promote the passage of payloads through the gastrointestinal epithelium. This could be availed to improve local and systemic delivery, enhance oral bioavailability, and control drug release. Even the research of alginate nanoparticles is less profuse and has been limited to a few cargo substances compared to microparticles, and various techniques have been adopted to design different types of nanocarriers. The selection of the polymers and formulation technique mainly relies on the pharmaceutical excipients and application goal, such as improving payload delivery and enhancing sustained release and site-targeting. Generally, emulsification/gelation is a low-cost technique for producing small particles in large quantity, involving preparation of alginate-in-oil (w/o), followed by gelation process. Polyelectrolyte complexation method on the other hand, is the simplest and most common technique to prepare alginate nanoparticles, where oppositely charged polyelectrolyte complex takes place. Moreover, layer-by-layer technique mainly used in drug targeting delivery and controlled drug release systems. In addition, nozzle-associated methods are based on extruding the polymer solution from the nozzle tip, including nano-spray dryer, electrospray and electrospinning. Careful selection of recipe and processing parameters is essential to formulate nanocarrier with tuned attributes. Additionally, future perspectives in polymeric nanoparticles should concentrate on studies of employing new and most performing techniques to develop advanced delivery systems, thereby expanding the applications of polymeric nanostructure in pharmaceutical field. To conclude, alginate endures a great potential and its extensive implementation in the advancement of innovative nanocarrier delivery systems with translation possibility is a matter of time.