Zein Microparticles and Nanoparticles as Drug Delivery Systems

Zein is a natural, biocompatible, and biodegradable polymer widely used in the pharmaceutical, biomedical, and packaging fields because of its low water vapor permeability, antibacterial activity, and hydrophobicity. It is a vegetal protein extracted from renewable resources (it is the major storage protein from corn). There has been growing attention to producing zein-based drug delivery systems in the recent years. Being a hydrophobic biopolymer, it is used in the controlled and targeted delivery of active principles. This review examines the present-day landscape of zein-based microparticles and nanoparticles, focusing on the different techniques used to obtain particles, the optimization of process parameters, advantages, disadvantages, and final applications.


Introduction
Zein is the major storage protein of corn first identified in the nineteenth century. It constitutes 44-79% of the endosperm protein depending on the variety of corn and the technique used for its extraction [1]. Being a natural material, it can be easily extracted from corn and is extensively available in nature [2]. Zein belongs to the category of prolamins and, from the biological point of view, it is a mixture of four components (α, β, γ, and δ) with different peptide chains, molecular size, and solubility [3]. Without any doubt, α-zein is the most abundant constituent of the mixture, accounting for about 70-80% of the whole zein, followed by γ-zein, which constitutes 10-20% [4,5]. It is the main component of commercially available zein, which is available in two forms, yellow and white zein. The former contains a high concentration of xanthophyll pigments and has a purity of about 90%. In contrast, white zein is obtained by decolorization of the yellow protein, has a negligible amount of xanthophylls, and has a purity higher than 96% [6]. Zein is a hydrophobic protein: it is poorly soluble in water alone and is soluble in aqueous ethanol, aqueous acetone, and some organic solvents [7].
The hydrophobicity, biodegradability, and biocompatibility of zein have been exploited in various fields, such as food coating [8] and packaging [9], adhesives [10], coatings [11], in the textile sector [12], in the biomedical sector and tissue engineering [13], and in the pharmaceutical industry [14]. Being a versatile polymer, zein is processed to obtain different shapes, such as particles [15], films [16], membranes [17], and scaffolds [18], which have been used for different applications. Zein films obtained by solvent casting, extrusion, or compression molding are generally used in the biomedical field, but because of the poor ability of zein to act as a water barrier, plasticizers or crosslinkers are necessary, or, in some cases, hybrid films are used incorporating other biopolymers [19,20]. Zein nanofibers produced by electrospinning are used to obtain scaffolds for tissue engineering [21] or food packaging applications [22].
Recently, zein has been utilized as a carrier for oral drug delivery systems [23] because it guarantees enhanced bioavailability, preparing sustained-release dosage forms, and targeting/protecting drugs [24]. Different reviews have been published on this topic, generally focused on the different morphologies obtained [13,24,25]; in some cases, the use of zein in combination with other substances has been deepened [7]. From the results reported in Table 1, it is clear that zein + API powders are obtained almost in all cases in the form of nanoparticles using the LAS method. In some cases, particle size distributions are broad, but in other cases, monodisperse NPs are obtained. An exemplificative SEM image of monodisperse nanoparticles obtained using liquid antisolvent precipitation is reported in Figure 1. The particles are constituted by resveratrol, a polyphenol with antioxidant, antiaging, and anticancer properties, loaded into zein. The nanoparticles obtained are constituted by a homogeneous population of spherical particles with a smooth surface [95].  The dimensions of the particles are determined either with the aid of software based on image analysis or, more correctly, through dynamic light scattering (DLS). An exemplificative distribution obtained using a DLS particle size analyzer is reported in Figure 2. The LAS technique has been used to process different materials with anti-inflammatory, chemotherapeutic, and antioxidant properties. In some cases, the in vitro cytotoxicity of the processed NPs against cancer cells was evaluated with encouraging results [44,46,62,68,70,85,93,99,102]. Moreover, in vivo studies on rats [74,92,95] or on human volunteers [97] were performed in some cases. When a comparative investigation of different carriers was attempted, zein showed a greater ability to retain the active compound effectively. Indeed, Gagliardi et al. [99] coprecipitated rutin, a polyphenolic bioflavonoid characterized by peculiar antioxidant properties, using poly(lactic-co-glycolic acid) (PLGA) or zein as the carrier. They observed that PLGA nanoparticles showed a poorer ability to retain rutin with respect to zein nanosystems.
Other authors compared the performance in terms of drug release of differently obtained systems based on the use of zein. Indeed, Chuacharoen and Sabliov [69] compared The dimensions of the particles are determined either with the aid of software based on image analysis or, more correctly, through dynamic light scattering (DLS). An exemplificative distribution obtained using a DLS particle size analyzer is reported in Figure 2.  The dimensions of the particles are determined either with the aid of software based on image analysis or, more correctly, through dynamic light scattering (DLS). An exemplificative distribution obtained using a DLS particle size analyzer is reported in Figure 2. The LAS technique has been used to process different materials with anti-inflammatory, chemotherapeutic, and antioxidant properties. In some cases, the in vitro cytotoxicity of the processed NPs against cancer cells was evaluated with encouraging results [44,46,62,68,70,85,93,99,102]. Moreover, in vivo studies on rats [74,92,95] or on human volunteers [97] were performed in some cases. When a comparative investigation of different carriers was attempted, zein showed a greater ability to retain the active compound effectively. Indeed, Gagliardi et al. [99] coprecipitated rutin, a polyphenolic bioflavonoid characterized by peculiar antioxidant properties, using poly(lactic-co-glycolic acid) (PLGA) or zein as the carrier. They observed that PLGA nanoparticles showed a poorer ability to retain rutin with respect to zein nanosystems.
Other authors compared the performance in terms of drug release of differently obtained systems based on the use of zein. Indeed, Chuacharoen and Sabliov [69] compared zein NPs covalently linked to folic acid (FA) (through the reaction between the carboxyl The LAS technique has been used to process different materials with antiinflammatory, chemotherapeutic, and antioxidant properties. In some cases, the in vitro cytotoxicity of the processed NPs against cancer cells was evaluated with encouraging results [44,46,62,68,70,85,93,99,102]. Moreover, in vivo studies on rats [74,92,95] or on human volunteers [97] were performed in some cases. When a comparative investigation of different carriers was attempted, zein showed a greater ability to retain the active compound effectively. Indeed, Gagliardi et al. [99] coprecipitated rutin, a polyphenolic bioflavonoid characterized by peculiar antioxidant properties, using poly(lactic-co-glycolic acid) (PLGA) or zein as the carrier. They observed that PLGA nanoparticles showed a poorer ability to retain rutin with respect to zein nanosystems.
Other authors compared the performance in terms of drug release of differently obtained systems based on the use of zein. Indeed, Chuacharoen and Sabliov [69] compared zein NPs covalently linked to folic acid (FA) (through the reaction between the carboxyl group of FA and the primary amino group of zein) and zein NPs with physically entrapped FA. In the former case, the particles were able to sustain the release of the active principle and target cancer cells overexpressing folate-binding receptors. In contrast, in the latter case, zein NPs controlled the release of the bioactive substance without targeting cancer cells. The proposed release mechanisms are sketched in Figure 3. entrapped FA. In the former case, the particles were able to sustain the release of the active principle and target cancer cells overexpressing folate-binding receptors. In contrast, in the latter case, zein NPs controlled the release of the bioactive substance without targeting cancer cells. The proposed release mechanisms are sketched in Figure 3. Contado et al. [98] coated resveratrol-loaded zein-pectin nanoparticles with Eudragit S100 to avoid the degradation of the dissolved drug in the gastrointestinal tract; they obtained nanoparticles with a mean diameter (MD) of 250 nm with targeted delivery in the colon tract. Indeed, it is well-known that Eudragit polymers are pH-sensitive polymers and, therefore, a specific Eudragit can be chosen depending on the type of desired drug release [103].
The main drawback of the LAS process is the presence of a high amount of ethanol; this can limit the use of the LAS technique because of the high costs related to the removal of the solvent itself and the residual solvent which can be contained in the precipitated powders.

Supercritical Antisolvent Process
An innovative process that has been used to coprecipitate a carrier with active principles is supercritical antisolvent precipitation (SAS) [104,105]. In this process, a liquid antisolvent is substituted with carbon dioxide in supercritical conditions. Therefore, this process is based on two prerequisites: the carrier (zein) and the drug have to be soluble in an organic solvent but insoluble in the mixture formed by this organic solvent and supercritical carbon dioxide (scCO2); the organic solvent and scCO2 have to be miscible under the process conditions.
Concerning the liquid antisolvent process, in the SAS process, the peculiarities of scCO2 can be exploited [106]. As a consequence, the size of the obtained particles can be easily controlled at the micro-and nanoscale by varying the process parameters, such as pressure, temperature, total concentration of the liquid solution, and ratio between the zein and the drug; moreover, complex post-process operations to separate the solvent and the antisolvent are not necessary, considering that in correspondence of the ambient conditions of temperature and pressure carbon dioxide is in the gaseous state whereas the Contado et al. [98] coated resveratrol-loaded zein-pectin nanoparticles with Eudragit S100 to avoid the degradation of the dissolved drug in the gastrointestinal tract; they obtained nanoparticles with a mean diameter (MD) of 250 nm with targeted delivery in the colon tract. Indeed, it is well-known that Eudragit polymers are pH-sensitive polymers and, therefore, a specific Eudragit can be chosen depending on the type of desired drug release [103].
The main drawback of the LAS process is the presence of a high amount of ethanol; this can limit the use of the LAS technique because of the high costs related to the removal of the solvent itself and the residual solvent which can be contained in the precipitated powders.

Supercritical Antisolvent Process
An innovative process that has been used to coprecipitate a carrier with active principles is supercritical antisolvent precipitation (SAS) [104,105]. In this process, a liquid antisolvent is substituted with carbon dioxide in supercritical conditions. Therefore, this process is based on two prerequisites: -the carrier (zein) and the drug have to be soluble in an organic solvent but insoluble in the mixture formed by this organic solvent and supercritical carbon dioxide (scCO 2 ); -the organic solvent and scCO 2 have to be miscible under the process conditions.
Concerning the liquid antisolvent process, in the SAS process, the peculiarities of scCO 2 can be exploited [106]. As a consequence, the size of the obtained particles can be easily controlled at the micro-and nanoscale by varying the process parameters, such as pressure, temperature, total concentration of the liquid solution, and ratio between the zein and the drug; moreover, complex post-process operations to separate the solvent and the antisolvent are not necessary, considering that in correspondence of the ambient conditions of temperature and pressure carbon dioxide is in the gaseous state whereas the organic solvent is a liquid. It was previously demonstrated that the use of different carriers in the SAS process leads to different drug releases: hydrophilic polymers such as polyvinylpyrrolidone can be used to obtain an increase in the drug dissolution rate, whereas hydrophobic carriers, such as zein, are the right choice to obtain a prolonged release of the active principle [107].
In Figure 4, a typical SAS plant is sketched. It comprises a carbon dioxide tank, a liquid solution burette, two pumps (one for CO 2 and one for the liquid solution), a precipitation vessel, and a separator. The precipitation vessel is the heart of the plant and is equipped with an injector mounted in the top part of the chamber and a filter at the bottom from which the particles can be recovered after depressurization. polyvinylpyrrolidone can be used to obtain an increase in the drug dissolution rate, whereas hydrophobic carriers, such as zein, are the right choice to obtain a prolonged release of the active principle [107]. In Figure 4, a typical SAS plant is sketched. It comprises a carbon dioxide tank, a liquid solution burette, two pumps (one for CO2 and one for the liquid solution), a precipitation vessel, and a separator. The precipitation vessel is the heart of the plant and is equipped with an injector mounted in the top part of the chamber and a filter at the bottom from which the particles can be recovered after depressurization. In Table 2, a list of the active ingredients coprecipitated with zein using the SAS process and the operating conditions chosen by the different research groups, and the main results obtained are reported. Table 2. Zein-based particles obtained using the SAS process. AC, acetone; β-car, β-carotene; c, concentration in the liquid solution; δ-toc, δ-tocopherol; DCM, dichloromethane; DIC, diclofenac sodium; DMSO, dimethyl sulfoxide; DR, dissolution rate; EE, encapsulation efficiency; EtOH, ethanol; HCPT, 10-hydroxycamptothecin; MP, microparticles; NP, nanoparticles; P, operating pressure; Rib, riboflavin; T, operating temperature.  In Table 2, a list of the active ingredients coprecipitated with zein using the SAS process and the operating conditions chosen by the different research groups, and the main results obtained are reported. Table 2. Zein-based particles obtained using the SAS process. AC, acetone; β-car, β-carotene; c, concentration in the liquid solution; δ-toc, δ-tocopherol; DCM, dichloromethane; DIC, diclofenac sodium; DMSO, dimethyl sulfoxide; DR, dissolution rate; EE, encapsulation efficiency; EtOH, ethanol; HCPT, 10-hydroxycamptothecin; MP, microparticles; NP, nanoparticles; P, operating pressure; Rib, riboflavin; T, operating temperature.  It can be noted that using the SAS process, zein, in general, precipitates in the form of microparticles rather than nanoparticles and that the organic solvents most frequently used are DMSO or aqueous ethanol. The pressure ranges from 8.0 to 16.0 MPa, the temperature-from 30 to 55 • C. The zein/API weight ratio can vary in a wide range, but typically ratios in the range of 5:1-20:1 are preferred by different authors. The selected active principles belong to different categories, such as vitamins [109,113], antibiotics [108], anti-inflammatory drugs [111], anticancer drugs [112], or antihistamine drugs [110]. When the dissolution rate of the active principle coprecipitated with zein was compared with the dissolution rate of the pure API, a clear effect of prolonging the release was observed. For example, in the case of amoxicillin [108], complete API dissolution was achieved in almost 3 days and, therefore, the formulation can be used for "long-term antibiotic therapy". In the case of 10-hydroxycamptothecin (HCPT) nanocrystals coprecipitated with zein microparticles [112], there was an initial burst corresponding to the fast release of the 50% of the drug in the first 10 h (attributed to the immediate dissolution and release of the HCPT located near the surface of the particles), followed by slow dissolution of the API corresponding to the release of the 70% of the drug in 82 h (because of the drug entrapped into the zein microspheres). A bimodal release was also observed in the case of antihistamines [110]: an immediate release of a small amount of the drug (22%) useful to rapidly relieve the symptoms associated with allergy, followed by a prolonged release of the remaining drug (that was completely dissolved in 36 h), which reduces the number of administrations throughout the day.

Active
The SAS process has also been used to combine zein with other substances to deliver active principles. For example, Liu et al. [116] prepared nanospheres constituted by zein decorated with folic acid to obtain targeted delivery of HCPT. Indeed, HCPT is a promising natural anticancer ingredient characterized by poor aqueous solubility and in vitro and in vivo instability. The authors added folic acid to obtain a sustainable and targeted delivery system, enhancing the intracellular uptake of HCPT within cancerous cells. Compared to zein nanoparticles, folic acid/zein nanoparticles were smaller (in the range of 350-820 nm) and had a higher stability; moreover, folic acid/zein conjugates have the potential to selectively target tumor cells, with an associated reduction in nonspecific toxicity in the normal cells.
Palazzo et al. [117] used a supercritical-based process named supercritical assisted injection in a liquid antisolvent (SAILA) to entrap luteolin in zein microparticles. The process is based on the continuous injection of an expanded liquid constituted by an organic solvent, zein, API, and scCO 2 in an aqueous solution, which has the role of the antisolvent. Therefore, unlike the SAS process in which scCO 2 is the antisolvent, in the SAILA process, scCO 2 is a co-solute. The authors identified the best operating conditions at a pressure of 10.0 MPa, a temperature of 40 • C, and a zein/luteolin ratio of 20:1. In correspondence with these conditions, they obtained microparticles with an MD of 1.20 µm and an entrapment efficiency of 82%.
The main advantage of the techniques based on the use of scCO 2 lies in controlling the size of the particles as the operating conditions vary. In general, the particle size increases with increasing concentration of the liquid solution and decreasing pressure. Another significant advantage lies in the absence or presence of traces below the permitted limits of the solvent residue in the powders. On the other hand, the investment costs for the construction of plants and the operating costs of the processes themselves are higher than those of the LAS process due to high operating pressures.

Coacervation
The coacervation process involves no harsh solvents or high temperatures. The process consists of the separation of solutions into colloidal systems with two liquid phases: one, called coacervate, is rich in polymer and another phase is without the polymer. Coacervation can be simple when it involves the use of a single polymer or complex when two natural biopolymers of opposite charges are involved.
Simple coacervation has been used to prepare zein microspheres conjugated with different drugs, such as heparin [118], gitoxin [119], chemotherapeutic agents [120], and antigens for the preparation of vaccines [121,122] or DNA [123]. For example, Wang et al. [118] prepared a drug-eluting coating film containing zein + heparin microspheres with slow API release; indeed, about 55% of the entrapped heparin was released after 20 days. The film constituted by microparticles showed adequate anticoagulation and improved hemocompatibility. Susuki et al. [120] conjugated zein microspheres with some antitumor drugs, such as mitomycin C, daunomycin hydrochloride, and peplomycin sulfate, obtaining sustained API release systems to be used in selective cancer chemotherapy by oral or intratumoral administration. Regier et al. [123] prepared zein/DNA nanoparticles with an MD ranging from 158 to 397 nm depending on the zein/DNA ratio. The authors demonstrated a sustained plasmid release for at least 7 days, with a minimal initial burst. Zein/DNA nanospheres showed robust biocompatibility. They can be fine-tuned for specific applications including oral gene delivery, intramuscular delivery, and in the fabrication of tissue engineering scaffolds.
Complex coacervation has been used, for example, to prepare zein and chitosan coacervates, considering the zein/chitosan ratio, solid/liquid ratio, and pH. Indeed, Li et al. [123] studied the morphology and encapsulation efficiency of curcumin in the correspondence of different coacervation conditions. The in vitro release study showed that the stronger the zein-chitosan interaction was, the less the amount of curcumin released from the nanoparticles was. The API at the optimized operating conditions has a slight burst effect followed by a slow release.
The main advantages of coacervation are the rapidity of the process and the absence of solvents. In contrast, the drawbacks are the potential toxicity of the crosslinkers and the difficulty of controlling the coagulation step.

Other Techniques
Zein has also been processed using other techniques. In the emulsification and solvent evaporation method, an oily phase constituted by zein + API + organic solvent is emulsified into an aqueous phase in which a surfactant is dissolved. The system is continuously stirred to reduce the droplet size that forms the emulsion. Then, the organic solvent is evaporated under vacuum, and microparticles and nanoparticles precipitate in the second step. In Figure 5, a schematic representation of the process is reported. specific applications including oral gene delivery, intramuscular delivery, and in rication of tissue engineering scaffolds.
Complex coacervation has been used, for example, to prepare zein and chito acervates, considering the zein/chitosan ratio, solid/liquid ratio, and pH. Indeed, [123] studied the morphology and encapsulation efficiency of curcumin in th spondence of different coacervation conditions. The in vitro release study showed stronger the zein-chitosan interaction was, the less the amount of curcumin releas the nanoparticles was. The API at the optimized operating conditions has a slig effect followed by a slow release.
The main advantages of coacervation are the rapidity of the process and the of solvents. In contrast, the drawbacks are the potential toxicity of the crosslinkers difficulty of controlling the coagulation step.

Other Techniques
Zein has also been processed using other techniques. In the emulsification a vent evaporation method, an oily phase constituted by zein + API + organic so emulsified into an aqueous phase in which a surfactant is dissolved. The system is uously stirred to reduce the droplet size that forms the emulsion. Then, the organic is evaporated under vacuum, and microparticles and nanoparticles precipitate in ond step. In Figure 5, a schematic representation of the process is reported. For example, aceclofenac sodium, a nonsteroidal anti-inflammatory drug use sively in treating rheumatoid arthritis and osteoarthritis, was processed with th nique [124]. The disperse phase was prepared by dissolving the drug and zein alcohol. This solution was added to the continuous phase (sesame oil) containin Span 80 as an emulsifying agent. MPs with an MD in the range of 136-174 μm w tained; the EE varied from 11.6 to 26.1% depending on the zein/drug ratio. In vitro studies were attempted simulating gastric and intestinal fluids: a sustained relea 72 h was detected. Biocompatibility of the zein microspheres was evaluated thr vitro cytotoxicity studies using fibroblast cells from the explant tissue. Karthikey [125] used the same method to prepare zein microspheres charged with aceclofen formin, and promethazine. The three active principles were chosen as examples of phobic, hydrophilic, and amphiphilic drugs. The average particle size of the d zein/API microspheres was found to be 68-136 μm depending on the drug. The around 20-25% depending on the zein/API ratio. The higher the ratio, the higher A novel approach proposed by some authors is the preparation of zein mic tures by electrospinning and spray-drying (SD). For example, Coelho et al. [126] i rated vitamin B12, the most chemically complex and the largest molecule among vitamins, into zein. The microparticles obtained by electrospinning had a size of For example, aceclofenac sodium, a nonsteroidal anti-inflammatory drug used extensively in treating rheumatoid arthritis and osteoarthritis, was processed with this technique [124]. The disperse phase was prepared by dissolving the drug and zein in 90% alcohol. This solution was added to the continuous phase (sesame oil) containing 0.5% Span 80 as an emulsifying agent. MPs with an MD in the range of 136-174 µm were obtained; the EE varied from 11.6 to 26.1% depending on the zein/drug ratio. In vitro release studies were attempted simulating gastric and intestinal fluids: a sustained release up to 72 h was detected. Biocompatibility of the zein microspheres was evaluated through in vitro cytotoxicity studies using fibroblast cells from the explant tissue. Karthikeyan et al. [125] used the same method to prepare zein microspheres charged with aceclofenac, metformin, and promethazine. The three active principles were chosen as examples of hydrophobic, hydrophilic, and amphiphilic drugs. The average particle size of the different zein/API microspheres was found to be 68-136 µm depending on the drug. The EE was around 20-25% depending on the zein/API ratio. The higher the ratio, the higher the EE.
A novel approach proposed by some authors is the preparation of zein microstructures by electrospinning and spray-drying (SD). For example, Coelho et al. [126] incorporated vitamin B12, the most chemically complex and the largest molecule among all the vitamins, into zein. The microparticles obtained by electrospinning had a size of around 3 µm and an EE of 91%. In contrast, wrinkled coprecipitated microparticles were obtained by SD with an average size of 6.4 µm and an EE of 95%. In vitro release tests revealed that a controlled release profile characterized the API and that vitamin B12-loaded zein microstructures produced by electrospinning have a slower release profile than the structures obtained by spray-drying. Mahalakshmi et al. [127] encapsulated β-carotene in zein at the microlevel using SD (with an MD in the range of 1.4-2.5 µm) and at the nanolevel using electrospraying (with an MD of 600-900 nm). Electrospraying proved to have higher encapsulation efficacy than SD. In vitro simulated gastrointestinal stability studies showed that the release of encapsulated β-carotene from NPs is faster than from MPs due to the larger surface area interacting with the release medium. SD was also used by Sousa et al. [128] to develop microspheres of PLGA and zein for amoxicillin and indomethacin delivery. In the case of amoxicillin, they obtained MPs with a mean diameter in the range of 9.4-38.3 µm depending on the PLGA/zein ratio with an EE up to 51%; in the case of indomethacin, the mean diameter varied in the range of 5.6-38.5 µm, and the EE was higher (up to 99%). In vitro release studies revealed a sustained-release pattern for all the formulations. De Sousa et al. [129] obtained tetracycline-loaded microparticles made of PLGA and zein using SD for teeth preservation therapy (chronic periodontitis). They obtained a product with a different EE depending on the PLGA/zein ratio. In vitro drug release studies showed a sustained release of tetracycline over 700 h in water (30 days), which is a period that would guarantee the treatment of a long course of periodontitis. The antimicrobial activity against Staphylococcus aureus was also evaluated.
Nanoprecipitation was successfully used by de Souza Tavares et al. [130] who nanoencapsulated ellagic acid, a compound with antioxidant and antimicrobial activities, into zein nanoparticles. They obtained spherical, non-aggregated, smooth-surface particles under 370 nm in diameter with relevant inhibitory and bactericide activity against S. aureus and P. aeruginosa. The obtained system can represent a suitable alternative to prevent and treat infectious attributed to Gram-positive and Gram-negative bacteria; moreover, it was demonstrated that the antioxidant effect was preserved for 24 h, such as required in skin repairing and topical treatments. Weissmueller et al. [131] used a flash nanoprecipitation process to encapsulate some APIs, such as vitamin E acetate and anticholera quorumsensing modulator CAI-1 ((S)-3-hydroxytridecan-4-one). They obtained particles with a diameter less than 100 nm with high loading for both active principles. The stability of the obtained particles in the simulated intestinal fluid was demonstrated for 24 h. Zein nanoparticles incorporated with digoxin, a drug used to treat heart failure, were obtained by nanoprecipitation and then charged in an alginate film to prepare a buccal drug delivery system [132]. Digoxin was successfully encapsulated into zein nanoparticles with an EE of 91% and a mean size of 87 nm. It was also demonstrated that the mucoadhesive film incorporated with zein + API nanoparticles presented a controlled swelling profile and mechanical properties compatible with the application as a drug delivery system through the buccal mucosa.
Another process to reduce the use of organic solvents is acidification of a strong alkaline solution containing zein and an API, as proposed by Yuan et al. [133], who prepared pH-driven zein/tea saponin composite NPs containing curcumin. The process is based on the principle that the solubility of dissolved zein + API decreases during the acidification process and forms a sphere spontaneously to avoid the polar environment. The obtained spherical NP had an EE equal to 84% and high bioaccessibility with respect to free curcumin. Sabra et al. [134] synthesized amphiphilic protein copolymers via a carbodiimide coupling reaction for the tumor-targeted delivery of rapamycin and wogonin, two anticancer drugs. The nanoplatform was composed of a hydrophobic zein core to encapsulate drugs with high EE, a hydrophilic lactoferrin corona to enhance tumor targeting and prolong systemic circulation of nanocarriers, and glutaraldehyde crosslinking to reduce the particle size and improve micellar stability. The particle size of the micelles was around 260-290 nm and the EE was higher in the case of wogonin than for rapamycin. In vitro release profiles revealed that wogonin release from micelles was biphasic, characterized by initial fast release of about 64% of the drug during the first 6 h followed by the second phase of very slow release with about 67.59% of WOG released after 24 h. On the contrary, rapamycin showed a very slow release (<20% drug release after 72 h) without a considerable initial burst effect. Moreover, this combined nanodelivery system maximized synergistic cytotoxicity of the two drugs in terms of tumor inhibition in MCF-7 breast cancer cells.
In some cases, zein was not used as the carrier coprecipitated with the active principle but as the coating material. For example, Vozza et al. [135,136] used ionotropic gelation to encapsulate selenoamino acids (selenomethionine, methylselenocysteine, and selenocysteine) with antioxidant and anticancer properties into chitosan nanoparticles; the NPs were, then, coated with zein. At the best operating conditions in terms of the chitosan/zein ratio, they obtained particles with an MD of 271 nm and an encapsulation efficiency (EE) of 81% in the case of methylselenocysteine, particles with an MD of 377 nm in the case of selenomethionine and an EE of 80%, whereas in the case of selenocysteine, the MD was 262 nm and the EE was 79%. The analyses of the particles showed no cytotoxicity in Caco-2 cell lines and a sustained release of the APIs. Farris et al. [137] prepared chitosan/zein nano-in-microparticles constituted by a core of chitosan/DNA nanoparticles prepared by ionic gelation and further encapsulated in zein microparticles obtained using a water-in-oil emulsion. Well-defined micrometric particles were obtained, as reported in the FESEM image in Figure 6. Analyses such as DNA release profiles, site-specific degradation of the outer zein matrix, and in vivo transfection demonstrate that the formulated particles can improve oral gene delivery through enhanced protection and controlled release of the DNA cargo.
Polymers 2022, 14, x FOR PEER REVIEW rapamycin showed a very slow release (<20% drug release after 72 h) without a c able initial burst effect. Moreover, this combined nanodelivery system maximize gistic cytotoxicity of the two drugs in terms of tumor inhibition in MCF-7 breas cells.
In some cases, zein was not used as the carrier coprecipitated with the active p but as the coating material. For example, Vozza et al. [135,136] used ionotropic ge encapsulate selenoamino acids (selenomethionine, methylselenocysteine, and se teine) with antioxidant and anticancer properties into chitosan nanoparticles; were, then, coated with zein. At the best operating conditions in terms of the chito ratio, they obtained particles with an MD of 271 nm and an encapsulation efficien of 81% in the case of methylselenocysteine, particles with an MD of 377 nm in the selenomethionine and an EE of 80%, whereas in the case of selenocysteine, the M 262 nm and the EE was 79%. The analyses of the particles showed no cytotoxicity 2 cell lines and a sustained release of the APIs. Farris et al. [137] prepared chito nano-in-microparticles constituted by a core of chitosan/DNA nanoparticles prep ionic gelation and further encapsulated in zein microparticles obtained using a w oil emulsion. Well-defined micrometric particles were obtained, as reported in the image in Figure 6. Analyses such as DNA release profiles, site-specific degradatio outer zein matrix, and in vivo transfection demonstrate that the formulated parti improve oral gene delivery through enhanced protection and controlled releas DNA cargo.

Conclusions
This review focused on the use of zein particles in the pharmaceutical field. Z versatile polymer, which can be used alone, in blends with other polymers, and core systems constituting both the shell or the core polymer to obtain sustained drug delivery systems. Depending on the process and the operating conditions precipitated in the form of microparticles or nanoparticles. The liquid antisolvent generally generates powders in the nanometric range, whereas supercritical ant precipitation gives microparticles. The traditional and largely employed LAS met the advantage of simplicity and low costs. Conversely, the SAS method is chara by lower amounts of organic solvents and high costs; for this reason, it has been lently applied only at a laboratory scale. Other processes, such as coacervation, e cation, and solvent evaporation, or electrospinning, have also been used to obt tained-release powders.

Conclusions
This review focused on the use of zein particles in the pharmaceutical field. Zein is a versatile polymer, which can be used alone, in blends with other polymers, and in shellcore systems constituting both the shell or the core polymer to obtain sustained-release drug delivery systems. Depending on the process and the operating conditions, zein is precipitated in the form of microparticles or nanoparticles. The liquid antisolvent process generally generates powders in the nanometric range, whereas supercritical antisolvent precipitation gives microparticles. The traditional and largely employed LAS method has the advantage of simplicity and low costs. Conversely, the SAS method is characterized by lower amounts of organic solvents and high costs; for this reason, it has been prevalently applied only at a laboratory scale. Other processes, such as coacervation, emulsification, and solvent evaporation, or electrospinning, have also been used to obtain sustainedrelease powders.