Influence of Various Model Compounds on the Rheological Properties of Zein-Based Gels.

The controlled release of a compound entrapped in a biocompatible formulation is a sought-after goal in modern pharmaceutical technology. Zein is a hydrophobic protein which has several advantageous properties that make it suitable for use as a biocompatible and degradable material under physiological conditions. It is, therefore, proposed for different biomedical and pharmaceutical applications. In particular, due to its gelling properties, it can be used to form a polymeric network able to preserve biomolecules from harsh environments. The current study was designed to investigate the influence of different probes on the rheological properties of gels made up of zein, in order to characterize the systems as a function of the polymer concentration. Four model compounds characterized by different physico-chemical properties were entrapped in zein gels, and different behaviors (viscoelastic or pronounced solid-like characteristics) of the systems were observed. Zein-based gels showed various release profiles of the encapsulated compounds, suggesting that there are different interaction rates between the probes and the polymeric matrix.

Lipid- and polymer-based formulations containing TNF-α inhibitors for the treatment of inflammatory bowel diseases.

Monoclonal antibodies inhibiting tumor necrosis factor-alpha (iTNF-α) have revolutionized the therapeutic regimen of inflammatory bowel disease, but their main drawback is the parenteral route of administration they require. An alternative approach lies in the delivery of these molecules to the area involved in the inflammatory process by means of innovative formulations able to promote their localization in affected tissues while also decreasing the number of administrations required. This review describes the advantages deriving from the use of lipid- and polymer-based systems containing iTNF-α, focusing on their physicochemical and technological properties and discussing the preclinical results obtained in vivo using rodent models of colitis.

DIFUCOSIN: DIclofenac sodium salt loaded FUCOidan-SericIN nanoparticles for the management of chronic inflammatory diseases.

The current investigation emphasizes the use of fucoidan and sericin as dual-role biomaterials for obtaining novel nanohybrid systems for the delivery of diclofenac sodium (DS) and the potential treatment of chronic inflammatory diseases. The innovative formulations containing 4 mg/ml of fucoidan and 3 mg/ml of sericin showed an average diameter of about 200 nm, a low polydispersity index (0.17) and a negative surface charge. The hybrid nanosystems demonstrated high stability at various pHs and temperatures, as well as in both saline and glucose solutions. The Rose Bengal assay evidenced that fucoidan is the primary modulator of relative surface hydrophobicity with a two-fold increase of this parameter when compared to sericin nanoparticles. The interaction between the drug and the nanohybrids was confirmed through FT-IR analysis. Moreover, the release profile of DS from the colloidal systems showed a prolonged and constant drug leakage over time both at pH 5 and 7. The DS-loaded nanohybrids (DIFUCOSIN) induced a significant decrease of IL-6 and IL-1ß with respect to the active compound in human chondrocytes evidencing a synergistic action of the individual components of nanosystems and the drug and demonstrating the potential application of the proposed nanomedicine for the treatment of inflammation.

Rutin-loaded zein gel as a green biocompatible formulation for wound healing application.

Wound healing is a challenging clinical problem and efficient wound management is essential to prevent infection. This is best done by utilizing biocompatible materials in order to complete the healing in a rapid manner, with functional and esthetic outcomes. In this context, the zein protein fulfills the criteria of the ideal wound dressing which include non-toxicity and non-inflammatory stimulation. Zein gels containing rutin were prepared without any chemical refinement or addition of gelling agents in order to obtain a natural formulation characterized by antioxidant and anti-inflammatory properties to be proposed for the treatment of burns and sores. In vitro scratch assay showed that the proposed gel formulations promoted cell migration and a rapid gap closure within 24 h (~90 %). In addition, the in vivo activities of rutin-loaded zein gel showed a greater therapeutic efficacy in Wistar rats, with a decrease of the wound area of about 90 % at day 10 with respect to the free form of the bioactive and to DuoDERM®. The evaluation of various markers (TNF-α, IL-1ß, IL-6, IL-10) confirmed the anti-inflammatory effect of the proposed formulation. The results illustrate the feasibility of exploiting the peculiar features of rutin-loaded zein gels for wound-healing purposes.

Characterization and Preliminary In Vitro Antioxidant Activity of a New Multidrug Formulation Based on the Co-Encapsulation of Rutin and the α-Acylamino-ß-Lactone NAAA Inhibitor URB894 within PLGA Nanoparticles.

A biodegradable and biocompatible polymeric matrix made up of poly(d,l-lactide-co-glycolide) (PLGA) was used for the simultaneous delivery of rutin and the (S)-N-(2-oxo-3-oxetanyl)biphenyl-4-carboxamide derivative (URB894). The goal was to exploit the well-known radical scavenging properties of rutin and the antioxidant features recently reported for the molecules belonging to the class of N-acylethanolamine-hydrolyzing acid amidase (NAAA) inhibitors, such as URB894. The use of the compounds, both as single agents or in association promoted the development of negatively-charged nanosystems characterized by a narrow size distribution and an average diameter of ~200 nm when 0.2-0.6 mg/mL of rutin or URB894 were used. The obtained multidrug carriers evidenced an entrapment efficiency of ~50% and 40% when 0.4 and 0.6 mg/mL of rutin and URB894 were associated during the sample preparation, respectively. The multidrug formulation evidenced an improved in vitro dose-dependent protective effect against H2O2-related oxidative stress with respect to that of the nanosystems containing the active compounds as a single agent, confirming the rationale of using the co-encapsulation approach to obtain a novel antioxidant nanomedicine.

Gliadin Nanoparticles Containing Doxorubicin Hydrochloride: Characterization and Cytotoxicity.

Doxorubicin hydrochloride (DOX) is a well-known antitumor drug used as first line treatment for many types of malignancies. Despite its clinical relevance, the administration of the compound is negatively affected by dose-dependent off-target toxicity phenomena. Nanotechnology has helped to overcome these important limitations by improving the therapeutic index of the bioactive and promoting the translation of novel nanomedicines into clinical practice. Herein, nanoparticles made up of wheat gliadin and stabilized by polyoxyethylene (2) oleyl ether were investigated for the first time as carriers of DOX. The encapsulation of the compound did not significantly affect the physico-chemical features of the gliadin nanoparticles (GNPs), which evidenced a mean diameter of ~180 nm, a polydispersity index < 0.2 and a negative surface charge. The nanosystems demonstrated great stability regarding temperature (25−50 °C) and were able to retain high amounts of drug, allowing its prolonged and sustained release for up to a week. In vitro viability assay performed against breast cancer cells demonstrated that the nanoencapsulation of DOX modulated the cytotoxicity of the bioactive as a function of the incubation time with respect to the free form of the drug. The results demonstrate the potential use of GNPs as carriers of hydrophilic antitumor compounds.

Strategies of stabilization of zein nanoparticles containing doxorubicin hydrochloride.

Hybrid nanoparticles made up of zein and various stabilizers were developed and characterized. In detail, a zein concentration of 2 mg/ml was blended with various amounts of different phospholipids or PEG-derivatives in order to obtain formulations with suitable physico-chemical properties for drug delivery purposes. Doxorubicin hydrochloride (DOX) was used as a model of a hydrophilic compound and its entrapment efficiency, release profile and cytotoxic activity were investigated. Photon correlation spectroscopy showed that the best formulations were obtained using DMPG, DOTAP and DSPE-mPEG2000 as stabilizers of zein nanoparticles, which were characterized by an average diameter of ~100 nm, a narrow size distribution and a significant time- and temperature-dependent stability. The interaction between protein and stabilizers was confirmed through FT-IR analysis, while TEM analysis showed the presence of a shell-like structure around the zein core. The release profiles of the drug from the zein/DSPE-mPEG2000 nanosystems, evaluated at two pHs (5.5 and 7.4), showed a prolonged and constant leakage of the drug. The encapsulation of DOX within zein/DSPE-mPEG2000 nanosystems did not compromise its biological efficacy, demonstrating the potential application of these hybrid nanoparticles as drug carriers.

Ascorbic acid-loaded gliadin nanoparticles as a novel nutraceutical formulation.

Ascorbic acid (AA) is one of the foremost antioxidants. Unfortunately, its sensitivity to different external stimuli such as light, heat and oxygen are concrete limitations for its use. Various approaches have been investigated in order to circumvent this problem and enhance the stability of the active compound, besides promoting its use for different applications. In this investigation, AA was encapsulated in a vegetal protein-based matrix made up of gliadin, the prolamin obtained from wheat kernels, with the aim of proposing a novel nutraceutical formulation. The nanosystems were characterized by an average diameter of < 200 nm and a negative surface charge of âˆ¼ -40 mV. The samples were not destabilized after incubation at different temperatures (up to 70 °C) or after the pasteurization procedure. Suitable stability was also observed in NaCl 100 mM, as well as after cryodesiccation when 10 % w/v of mannose was used. The gliadin nanoparticles showed the ability to retain high amounts of AA, promoting its prolonged release in PBS and under simulated gastrointestinal conditions. The nanosystems enhanced the antioxidant features of the compound as compared to its free form and preserved its chemical stability following UV exposition. The results demonstrate the potential application of the investigated nanoparticles as a novel nutraceutical formulation or as food fortificants.

α-Acylamino-ß-lactone N-Acylethanolamine-hydrolyzing Acid Amidase Inhibitors Encapsulated in PLGA Nanoparticles: Improvement of the Physical Stability and Protection of Human Cells from Hydrogen Peroxide-Induced Oxidative Stress.

N-Acylethanolamine acid amidase (NAAA) is an N-terminal cysteine hydrolase that preferentially catalyzes the hydrolysis of endogenous lipid mediators such as palmitoylethanolamide, which has been shown to exhibit neuroprotective and antinociceptive properties by engaging peroxisome proliferator-activated receptor-α. A few potent NAAA inhibitors have been developed, including α-acylamino-ß-lactone derivatives, which are very strong and effective, but they have limited chemical and plasmatic stability, compromising their use as systemic agents. In the present study, as an example of a molecule belonging to the chemical class of N-(2-oxo-3-oxetanyl)amide NAAA inhibitors, URB866 was entrapped in poly(lactic-co-glycolic acid) nanoparticles in order to increase its physical stability. The data show a monomodal pattern and a significant time- and temperature-dependent stability of the molecule-loaded nanoparticles, which also demonstrated a greater ability to effectively retain the compound. The nanoparticles improved the photostability of URB866 with respect to that of the free molecule and displayed a better antioxidant profile on various cell lines at the molecule concentration of 25 µM. Overall, these results prove that the use of polymeric nanoparticles could be a useful strategy for overcoming the instability of α-acylamino-ß-lactone NAAA inhibitors, allowing the maintenance of their characteristics and activity for a longer time.

Influence of the Dispersion Medium and Cryoprotectants on the Physico-Chemical Features of Gliadin- and Zein-Based Nanoparticles.

The evaluation of the physico-chemical features of nanocarriers is fundamental because the modulation of these parameters can influence their biological and in vivo fate. This work investigated the feasibility of saline, 5% w/v glucose and phosphate-buffered saline solution, as polar media for the development of nanoparticles made up of two vegetal proteins, zein from corn and gliadin from wheat, respectively. The physico-chemical features of the various systems were evaluated using dynamic and multiple light scattering techniques, and the results demonstrate that the 5% w/v glucose solution is a feasible medium to be used for their development. Moreover, the best formulations were characterized by the aforementioned techniques following the freeze-drying procedure. The aggregation of the zein nanoparticles prepared in water or glucose solution was prevented by using various cryoprotectants. Mannose confirmed its crucial role in the cryopreservation of the gliadin nanosystems prepared in both water and glucose solution. Sucrose and glucose emerged as additional useful excipients when they were added to gliadin nanoparticles prepared in a 5% glucose solution. Specifically, their protective effect was in the following order: mannose > sucrose > glucose. The results obtained when using specific aqueous media and cryoprotectants permitted us to develop stable zein or gliadin nanoparticles as suspension or freeze-dried formulations.

Application of Biocompatible Drug Delivery Nanosystems for the Treatment of Naturally Occurring Cancer in Dogs.

BACKGROUND: Cancer is a common disease in dogs, with a growing incidence related to the age of the animal. Nanotechnology is being employed in the veterinary field in the same manner as in human therapy. AIM: This review focuses on the application of biocompatible nanocarriers for the treatment of canine cancer, paying attention to the experimental studies performed on dogs with spontaneously occurring cancer. METHODS: The most important experimental investigations based on the use of lipid and non-lipid nanosystems proposed for the treatment of canine cancer, such as liposomes and polymeric nanoparticles containing doxorubicin, paclitaxel and cisplatin, are described and their in vivo fate and antitumor features discussed. CONCLUSIONS: Dogs affected by spontaneous cancers are useful models for evaluating the efficacy of drug delivery systems containing antitumor compounds.

Co-Encapsulation of Paclitaxel and JQ1 in Zein Nanoparticles as Potential Innovative Nanomedicine.

The manuscript describes the development of zein nanoparticles containing paclitaxel (PTX) and the bromo-and extra-terminal domain inhibitor (S)-tertbutyl2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno(3,2-f)(1,2,4)triazolo(4,3-a)(1,4)diazepin-6-yl)acetate (JQ1) together with their cytotoxicity on triple-negative breast cancer cells. The rationale of this association is that of exploiting different types of cancer cells as targets in order to obtain increased pharmacological activity with respect to that exerted by the single agents. Zein, a protein found in the endosperm of corn, was used as a biomaterial to obtain multidrug carriers characterized by mean sizes of ˂200 nm, a low polydispersity index (0.1-0.2) and a negative surface charge. An entrapment efficiency of ~35% of both the drugs was obtained when 0.3 mg/mL of the active compounds were used during the nanoprecipitation procedure. No adverse phenomena such as sedimentation, macro-aggregation or flocculation occurred when the nanosystems were heated to 37 °C. The multidrug nanoformulation demonstrated significant in vitro cytototoxic activity against MDA-MB-157 and MDA-MB-231 cancer cells by MTT-test and adhesion assay which was stronger than that of the compounds encapsulated as single agents. The results evidence the potential application of zein nanoparticles containing PTX and JQ1 as a novel nanomedicine.

Oleic acid-based vesicular nanocarriers for topical delivery of the natural drug thymoquinone: Improvement of anti-inflammatory activity.

The topical administration of a drug compound remains the first choice for the treatment of many local skin ailments. Many skin diseases can be treated by applying the active formulation directly to the skin, but unfortunately some drugs are unable to overcome the stratum corneum and exert their pharmacological action. An example is thymoquinone, a naturally derived drug obtained from Nigella sativa L. and potentially effective in the treatment of inflammatory and oxidative skin conditions. Since its physico-chemical properties are not suitable for overcoming the stratum corneum, we wanted to circumvent the problem by proposing new lipid-based nanovesicles called "oleoethosomes", made up of naturally derived ingredients, for its delivery. Among several formulations of oleoethosomes, the sample made up of 2% (w/w) oleic acid:PL90G 1:1 (molar ratio), and ethanol 15% showed the best physico-chemical characteristics and above all it showed the capacity to contain a suitable amount of thymoquinone (2 mg/ml). The formulation was tested in vitro on stratum corneum and viable epidermis membranes confirming its ability to induce the passage of thymoquinone through the human stratum corneum and to act as a permeation enhancer. In fact, it showed thymoquinone permeation values of 22.63 ± 1.49% regarding the applied drug amount. Oleoethosomes were compared with oleosomes, another kind of naturally derived nanosystems but free of ethanol. The experimental data confirmed that ethanol was an important component that enhanced the activity of the oleoethosomes when tested on the skin of healthy volunteers. The thymoquinone-loaded oleoethosome treatment demonstrated a significantly greater extent of anti-inflammatory activity than the treatment with thymoquinone-loaded oleosomes or the conventional dosage form of the drug. These in vivo results confirmed the synergic effect between oleic acid and ethanol on the lipid and protein compartments of the outermost skin layer, thus promoting a greater penetration capacity.

Identification of Exosomal microRNAs and Their Targets in Papillary Thyroid Cancer Cells.

The release of molecules in exosomal cargoes is involved in tumor development and progression. We compared the profiles of exosomal microRNAs released by two thyroid cancer cell lines (TPC-1 and K1) with that of non-tumorigenic thyroid cells (Nthy-ori-3-1), and we explored the network of miRNA-target interaction. After extraction and characterization of exosomes, expression levels of microRNAs were investigated using custom TaqMan Advanced array cards, and compared with those expressed in the total cell extracts. The functional enrichment and network-based analysis of the miRNAs' targets was also performed. Five microRNAs (miR-21-5p, miR-31-5p, miR-221-3p, miR-222-3p, and let-7i-3p) were significantly deregulated in the exosomes of tumor cells vs. non-tumorigenic cells, and three of them (miR-31-5p, miR-222-3p, and let-7i-3p) in the more aggressive K1 compared to TPC-1 cells. The network analysis of the five miRNAs identified some genes as targets of more than one miRNAs. These findings permitted the identification of exosomal microRNAs secreted by aggressive PTC cells, and indicated that their main targets are regulators of the tumor microenvironment. A deeper analysis of the functional role of the targets of exosomal miRNAs will provide further information on novel targets of molecular treatments for these neoplasms.

Analysis of serum microRNA in exosomal vehicles of papillary thyroid cancer.

PURPOSE: In this study, we investigated the profile of microRNAs (miRNAs) contained in exosomes secreted in the serum of patients with papillary thyroid cancer (PTC). METHODS: Exosome were isolated by adding ExoQuick Exosome Precipitation Solution. Dynamic light scattering (DLS) and western blotting analysis were used to ensure the quality of exosomes. The expression levels of miRNAs were investigated using custom-designed TaqMan Advanced miRNA Array Cards in the screening cohort and using specific TaqMan Advanced MicroRNA Assays in the validation cohort. RESULTS: We identified miR24-3p, miR146a-5p, miR181a-5p and miR382-5p with different expression levels in two different series of 56 and 58 PTC patients as compared with healthy controls. Significant differences in the expression of three PTC exosomal miRNAs, depending on the presence of lymph node metastasis, were detected in only one PTC series. When comparing the expression levels of some PTC-specific exosomal miRNAs with those of the same miRNAs circulating free of any encapsulation, we found a significant correlation for only miR24-3p, suggesting that only select miRNAs are secreted in exosomes. CONCLUSIONS: Our findings demonstrate that four miRNAs are differently secreted in the exosomes of PTC patients, whereas no conclusive results were found to characterize PTCs with lymph node metastasis, suggesting caution in the use of circulating exosomal miRNA expression levels as lymph node metastasis biomarkers. Further investigation into the mechanisms governing miRNA secretion in tumor cells are required.

Real-World Safety Profile of Biologics Used in Rheumatology: A Six-Year Observational Pharmacovigilance Study in the Calabria Region.

Background: The introduction of biological agents into the clinical armamentarium has modified the management of moderate-severe inflammatory arthritis (IA). However, these drugs can lead to serious adverse events (SAEs) and unpredictable adverse events (AEs) that are difficult to detect in pre-marketing clinical trials. This pharmacovigilance project aimed to study the AEs associated with biologics use in rheumatology. Methods: The current investigation is a multicenter, prospective, observational cohort study based on the Calabria Biologics Pharmacovigilance Program. Patients treated with one biologic agent from January 2016 to January 2022 were enrolled. Results: Overall, 729 (86.3%) of a total of 872 patients did not develop AEs or SAEs, whereas 143 (16.4%) patients experienced at least one AE, of which 16 (1.8%) had at least one SAE. The most common AEs were administration site conditions followed by gastrointestinal, nervous system and skin disorders. We reported a total of 173 switches and 156 swaps. Switches mainly occurred for inefficacy (136; 77.7%), whereas only 39 (22.3%) were due to the onset of an AE. Primary/secondary failure was the most frequent reason for swaps (124, 79%), while AEs onset led to 33 (21%) swaps. Conclusions: This study supports the validity of our program in monitoring and detecting AEs in the rheumatological area, confirming the positive beneficial/risk ratio of biologics.

Recent Advances of Taxol-Loaded Biocompatible Nanocarriers Embedded in Natural Polymer-Based Hydrogels.

The discovery of paclitaxel (PTX) has been a milestone in anti-cancer therapy and has promoted the development and marketing of various formulations that have revolutionized the therapeutic approach towards several malignancies. Despite its peculiar anti-cancer activity, the physico-chemical properties of PTX compromise the administration of the compound in polar media. Because of this, since the development of the first Food and Drug Administration (FDA)-approved formulation (Taxol®), consistent efforts have been made to obtain suitable delivery systems able to preserve/increase PTX efficacy and to overcome the side effects correlated to the presence of some excipients. The exploitation of natural polymers as potential materials for drug delivery purposes has favored the modulation of the bioavailability and the pharmacokinetic profiles of the drug, and in this regard, several formulations have been developed that allow the controlled release of the active compound. In this mini-review, the recent advances concerning the design and applications of natural polymer-based hydrogels containing PTX-loaded biocompatible nanocarriers are discussed. The technological features of these formulations as well as the therapeutic outcome achieved following their administration will be described, demonstrating their potential role as innovative systems to be used in anti-tumor therapy.

Brij-stabilized zein nanoparticles as potential drug carriers.

The current study was designed to provide a preliminary physico-chemical characterization of zein nanosystems prepared with various Brij surfactants (for the first time to the best of our knowledge) as a function of various external stimuli such as temperature, pH, serum incubation and the freeze-drying process. The results demonstrate that when Brijs are characterized by unsaturation (C18), considerable stabilization of the colloidal structure is promoted while the length of the polyethylene glycol fraction does not significantly modulate the physico-chemical properties of the nanosystems. Specifically, dynamic light scattering and nanoparticle tracking analysis demonstrated that the use of 0.2 % w/v of Brij O10 promoted the formation of stable zein nanosystems with mean sizes of ∼150 nm and a narrow size distribution, preserving their structures at various pHs and temperatures. The use of mannitol as cryoprotectant resulted in a formulation that can easily be re-suspended in water after the freeze-drying process. This nanoformulation demonstrated that it efficiently retained different amounts of both hydrophilic and lipophilic compounds and showed a prolonged release of the entrapped molecules. In addition, the nanosystems showed a favorable degree of in vitro safety on various cell lines when a concentration <50 µg/mL of protein was used, demonstrating the potential application of Brij O10-stabilized zein nanoparticles as innovative nanocarriers of several active compounds.

Development of polyoxyethylene (2) oleyl ether-gliadin nanoparticles: Characterization and in vitro cytotoxicity.

Natural polymers have been widely investigated as materials for the delivery of active compounds as a consequence of their biocompatibility, low-cost and the opportunity they furnish to obtain micro- and nanostructures. In this investigation, commercial wheat gliadin was used as raw material with the aim of obtaining a vegetal protein-based nanoformulation to be used for various applications. The influence of non-ionic and anionic surfactants on the physico-chemical properties of gliadin nanoparticles was evaluated in order to propose a suitable candidate able to stabilize the colloidal structure. The use of Super Refined polyoxyethylene (2) oleyl ether gave the best results, promoting the formation of spherical-shaped nanosystems with a narrow size distribution. The oleyl ether-based emulsifier prevented the destabilization of the colloidal systems when pH- and temperature-dependent stress was applied. A freeze-dried formulation was obtained when mannose was used as a cryoprotectant. Polyoxyethylene (2) oleyl ether-stabilized nanosystems were shown to retain and release both hydrophilic and lipophilic model compounds in a controlled manner. The cytotoxicity of the surfactant-free and polyoxyethylene (2) oleyl ether-stabilized gliadin based nanosystems was assessed on human cells, both normal and tumoural, in order to investigate the concentrations of particles that can be used during in vitro experiments. Polyoxyethylene (2) oleyl ether-stabilized gliadin-based nanosystems are promising carriers for the delivery of several active compounds.

Zein- vs PLGA-based nanoparticles containing rutin: A comparative investigation.

In the last few decades, several natural and synthetic polymers have been used as starting material for the development of innovative polymeric nanoparticles able to encapsulate biologically active substances and to modulate their biopharmaceutical features and/or therapeutic efficacy. This investigation focused on the comparison of the physico-chemical properties of nanosystems made up of two of the most successfully used biodegradable biomaterials, namely poly(lactic-co-glycolic acid) (PLGA) and zein, belonging to the synthetic and natural family of polymers, respectively. Rutin, a polyphenolic bioflavonoid characterized by peculiar antioxidant properties, was chosen as the model drug to be encapsulated in the polymeric systems. The results demonstrated a greater ability of zein-based nanosystems to effectively retain the active compound with respect to the PLGA particles. The integration of rutin in the protein matrix favored a controlled drug leakage, and was influenced by the surfactant used to stabilize the formulation. Moreover, rutin-loaded zein nanoparticles showed significant in vitro antioxidant activity, evidencing a synergistic action between the intrinsic antioxidant activity of the protein and the pharmacological properties of the active compound. Finally, the intracellular localization of the zein nanosystems was demonstrated through confocal laser scanning microscopy.