Last Fifteen Years of Nanotechnology Application with Our Contribute

1. Introduction

Currently, nanotechnology is the most promising science, engineering, and technology conducted at the nanoscale and is used in several sectors. The interest in nanomaterials is strongly increased during the two last decades and can be easily evaluated by considering the number of studies present in literature. From a survey conducted using the Scopus database (https://www.scopus.com/, accessed on 06 November 2024), the number of experimental studies intended as full articles, conference papers and letters, found using keyword “nanoparticles”, published in the last fifteen years (2009-2024) was 764,279 (Figure 1, light blue line).

That found summing the results obtained by using as keywords “antimicrobial AND nanoparticles” and then “antibacterial AND nanoparticles” was 82,286 (Figure 1 and Figure 2, blue lines).

That found summing the results obtained by using “anticancer AND nanoparticles”, “antitumor AND nanoparticles” and then “antitumour AND nanoparticles” resulted 42,390 (Figure 1 and Figure 2, red lines), that obtained using “biomedical AND nanoparticles” was 24,056 (Figure 1 and Figure 2, purple lines), that using “environment AND nanoparticles” was 42,845 (Figure 1 and Figure 2, green lines) and finally that obtained using “nanomedicine” was 21,555 (Figure 1 and Figure 2, pink lines). As observable in Figure 1, the number of studies published on NPs (764,279) is very large and the sum of publications dealing more specifically with definite applications of NPs, considered in Figure 1 and Figure 2 (213,132) does not reach it, thus leaving a great clean space between the light blue line of NPs and all other lines grouped below. This establishes how the number of NPs applications missing in the graph is high. Particularly, among the applications of NPs randomly chosen and considered in the graphs of Figure 1 and Figure 2, those dealing with human health and medicine (170,287) are almost 4-fold more numerous than those dealing with environment (42,845). However, several sectors of both these areas have not been reported, including those of food, food packaging, diagnosis, imaging, sensors, electric and electronic components, thermal and electric conductors, fertilizers, pesticides, soil improvers and so on. They would account for 225,129 other publications, for a total of only 57% of publications found on nanomaterials. During the period reported in Figure 1 and Figure 2, our group contributed to the field of applicative nanotechnology with several experimental and review articles, which we hope could have relevantly enhanced the knowledge of the scientific community in the field. In this new work, an extensive overview concerning the main types of NPs and their applications in the medical and food sector developed so far, with particular attention to those regarding the enhancement of bioavailability, target delivery and reduction of possible toxicity of bioactive molecules, and those used for ameliorating the traditional food packaging, have been provided. Then, we have reviewed all our experimental works on nanosized materials, both in the form of nanoparticles (NPs), dendrimers, micelles and liposomes developed in the years 2009-2024 [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51]. Additionally, a list of review articles composed by us along the same years and concerning the same topics, particularly useful because collecting the most relevant advances made by other eminent scientists in nanotechnology has been reported [52,53,54,55,56,57,58,59,60,61,62]. Anyway, paradoxically, although the application of nanotechnology translates in a great improvement of properties of pristine materials, there may be a still not predictable risk, for humans, animals and the environment associated with an extensive application of NPs. Unfortunately, despite nanotoxicology is a science in large expansion, such risks are not yet fully disclosed. On this state, the final part of this work discusses the pending issue relating to the possible toxic effect of NPs and their impact on customers’ acceptance, in a scenario of limited knowledge.

2. Among the Nanotechnology Applications

2.1. Application of Nanomaterials to Natural and Synthetic Bioactive Molecules

Before passing on reviewing our fifteen years of working in the field of nanotechnology, an exhaustive overview about its employment in the medicine and food sectors has been provided in the following sections, with particular attention to the use of nanotechnology to improve the characteristics, often not favorable, of bioactive principles (APs).

Nanotechnology, the different techniques of nano-formulation, and nanomaterials are strongly implied in the current methods used to address the drawbacks concerning bioactive molecules bioavailability [63]. The solubility, delivery, and cell uptake of APs can be strongly improved by using NPs, as well as their protection from early degradation and fast metabolism. In this context, although the existence of several critical challenges, including reproducibility, proper characterization, and biological evaluation via proper assays, are still associated with their use, the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have approved several nanomedicines, which are now commercially available[63]. Rigorous studies besides stringent guidelines are warranted, for effective and safe nanomedicine development and use [63]. Moreover, by the nanotechnological manipulation of bioactive molecules is possible to prepare food products enriched with them and therefore with improved health properties without interfering with the sensory and qualities of the original food. It is foreseen that the market for nanotechnological items produced in the food and beverage sectors as health promoters will be incessantly increasing[64]. The nanomanipulation of APs, regardless of their natural or synthetic origin, can allow them to more easily bypass the physiological barriers that commonly limit their oral delivery. It is forecast that synthetic nanomedicines will have nonpareil advantages in drug delivery, as well as in clinical practice in the future [65]. Several factors could in fact affect oral absorption of APs, including poor aqueous solubility and therefore a slow dissolution rate in gastrointestinal (GIT) fluids, instability in the acidic environment of the stomach, the presence of degrading enzymes in GIT, the presence of food, biological barriers, and finally, first-pass metabolism in the liver[66]. Moreover, also when systemic circle and/or cells are reached, other issues could consist in the tendency of APs to bind irreversibly to blood proteins. Furthermore, APs could tie permanently to cellular DNA and proteins or form weakly soluble complexes with calcium and magnesium ions, which greatly reduce transcellular absorption[67], thus reducing their health effects. The lack of pathogenesis-targeting effects in neurodegenerative diseases such as Parkinson disease (PD), Alzheimer disease (AD), the varies form of sclerosis and dementia is principally due to the limiting effects of the blood–brain barrier (BBB), which keeps out of the brain about 99% of all “foreign substances”[68]. Nanotechnology, when correctly applied to drugs suffering from the above-mentioned drawbacks can enhance their efficacy and in vivo stability, while reducing their toxicity, thus aligning the excellent results commonly observed in vitro with those found in vivo, which are usually much less satisfactory[54]. Carrying bioactive compounds in NPs favors their distribution in specific brain areas, thus providing more valuable benefits in neuro-regenerative treatments, while minimizing their accumulation in the systemic circulation, as well as the related toxic side effects[69]. Specifically, by loading neurotrophin in NPs, its distribution in specific brain areas has been favoured, thus providing more valuable benefits in different types of neuro-regenerative treatments [69]. Moreover, NPs formulation of APs protects them from early degradation and rapid metabolism. Several are the natural APs extracted by fruits, seeds and vegetables, which are endowed with several health-promoting properties. These activities of natural APs have been extensively improved by engineering using nanotechnology, thus ameliorating their very poor solubility, as well as the many pharmacokinetic drawbacks associated with their pristine form [54]. Several studies have reported on the development of appropriate nanomaterial-based devices used to enhance the solubility of strong antioxidants polyphenols, their hydrophilic–lipophilic balance (HLB), GIT absorbability and/or to protect them from early oxidation and/or metabolism [11,70,71,72,73,74,75]. It is the case of the insoluble ellagic acid (EA), which was gifted with high water solubility using cyclodextrins [73,74,75], pectin[11], and polyester-based dendrimers[11]. In a paper, the effects of phospholipid composition on pharmacokinetics and biodistribution of epirubicin-loaded liposomes were examined, proving a significantly prolonged circulating time, reduced clearance, and reduced heart toxicity[76].

2.1.1. NPs-Mediated Controlled Release of APs

The controlled and targeted release of APs from NPs has been recognized as a pivotal step for realizing their effective administration. A controlled and targeted delivery of APs allows to reach their higher concentration at the desired site, thus permitting to reduce the overall dosage and the systemic toxicity affecting the pristine AP. Many are the parameters, which can be optimized to control the specific release of APs, including pH, temperature, ultrasound or magnetic fields applications, light incidence, type and physicochemical features of NPs, chemical structure, physicochemical features[77].

APs-loaded stimuli-sensitive nano-capsules possessing an oil core were shown to improve the effects following the oral administration of pristine APs, while the dose and the administration frequency, thus ameliorating the patient compliance, was reduced [77].

Rhodamine-loaded poly-alkylene glycol (PAG)-NPs were applied to SH-SY5Y NB cells or prostate cancer DU145 cells and were visualized by fluorescence. PAG-NPs were visualized in the cytoplasm, suggesting that they have been internalized via endocytosis, overcoming, without damage, the phospholipidic barrier of the cell membrane, which represents an impediment for hydrophilic compounds to enter the cells [76].

2.1.2. Main NPs Developed to Nano Formulate Natural and Synthetic APs

To provide readers several information using a tool as much as possible reader-friendly, the details on this topic have been organized in Table 1, that summarizes the most used engineered NPs for biomedical uses and/or in food sector.

The conventional techniques to prepare NSs consist of the bottom-up and top-down methods. Figure A1, in Appendix A provides a schematic representation of both techniques and additional information has been included in the Figure A1 caption [62].

β-carotene was formulated as nanomaterial by precipitation from pressurized ethyl acetate-on-water emulsions for application as natural colorant[78]. Quercetin was instead subjected to high pressure homogenization (HPH), achieving an NS of amorphous NPs[79]. Using spray drying (SD) technique and maltodextrin as encapsulating agent, water-re-dispersible powders loaded with the products derived from acai fruit were prepared. They demonstrated improved nutritional values, extended shelf life, and radicals scavenging activity[80]. A stable aqueous NPs (150 nm) suspension of α-tocopherol, with improved solubility and bioavailability was obtained by supercritical assisted process [81]. To improve the performances of the single approaches, combination methods were born by merging the top-down and bottom-up techniques. They include Nanoedge™ Technique (Baxter Healthcare) [52,121], H 69 Technology, H 42 Technology, H 96 Technology[52] and Combination Technology (CT)

CT, which is suitable for scaling up[122], was used to formulate hesperidin. In vitro studies have established its antioxidant activity and when assumed with the diet, it has proven to be a valid vase protector. NPs were characterized by improved solubility and long-term stability. They were suitable both for oral administration and topical application [52]. Hesperidin nanocrystals are in the Platinum Rare cosmetic product (La Prairie, Volketswil, Switzerland). Furthermore, rutin and apigenin were processed with the CT technology. Rutin NPs of about 600 nm, suitable both for oral and topical administration and apigenin NPs of 275 nm were obtained. Rutin nanocrystals can be found in a cosmetic product launched by Juvena, St. Margrethen, Switzerland [52].

Nanoedge-like techniques were employed to formulate all-trans retinoic acid (ATRA) in 155-nm-sized particles, suitable for oral administration, in 30′ operation time [52].

H 69 Technology (SmartCrystal^® technology group) was approached to formulate resveratrol (RES) in particles of 150 nm, suitable for oral administration [62].

H 42 Technology is like H 69 [121]. When H 42 was used for formulating RES, particles of 200 nm eligible for oral administration were obtained [52,121]. Differently, through NEs technology, APs are encapsulated in small droplets mixing an aqueous phase (w) with an oil one (o) and obtaining water in oil (w/o), oil in water (o/w), or bi-continuous colloidal dispersions. The colloids are stabilized using specific additives, such as generally-regarded-as-safe (GRAS) pharmaceutical surfactants, co-surfactants, and emulsifiers (5–10%). Oils utilized in NEs encompass Captex 355, Captex 8000, Witepsol, Myritol 318, Isopropyl myristate, Capryol 90, Sefsol-218, triacetin, isopropyl myristate, castor oil, olive oil, etc. In bi-continuous colloidal dispersions, microdomains of oil and water are inter-dispersed in the system.

NEs can be achieved by high and low energy methods, such as high-pressure homogenization, ultrasonication, phase inversion temperature and emulsion inversion point, as well as recently developed approaches such as bubble bursting methods[82]. High drug loading (DL%) is possible, and solutions are isotropic, transparent, and kinetically stable, even if NEs stability is lower than that of micro-emulsions, due to the very small droplets initially obtained, which tend to re-aggregate along time with the formation and growth of undesired great crystals[123,124,125]. Using NEs-based delivery systems, herbal drugs, whole plant extracts or their constituents, as well as food-related APs, unstable in highly acidic pH and/or undergoing liver metabolism if administered as free, were formulated. NE techniques were considered to reduce possible side effects due to the accumulation of some APs in the non-targeted areas. For this characteristic, NEs are authorized also for paediatric and geriatric oral the administration [123].

Self-emulsifying drug delivery systems (SEDDSs) represent a particular type of NE. SEDDSs are generally suitable for orally delivering LBACs and include SNEDDSs and SMEDDSs based on their droplets size[52]. They can be taken orally by either solubilizing them in water and drinking the obtained NE or by ingesting capsules filled with gelatin as schematized in Figure A2 in Appendix A.

Many formulation parameters, including surfactants concentrations, the oil/surfactant ratio, the polarity of the emulsion, the droplets’ size and charge, the physicochemical properties of APs, such as pKa, log P, molecular structure, MW, presence, and quantity of ionizable groups, have remarkable effects on the performances of SEDDSs.

The group of Hu manufactured a self-double-emulsifying drug delivery system (SDEDDS) loaded with epigallocatechin-3-gallate (EGCG), having improved photo-stability in respect of free EGCG[90].

NE techniques were used to nano-formulate turmeric, curcumin (diferuloylmethane), and di-benzoyl-methane (a structural analogue of curcumin). Curcumin, also used as GRAS food supplement, possesses antiseptic, analgesic, antimalarial, and insect-repellent activities. Triacylglycerol was chosen as the oil phase and Tween-20 as an emulsifier to formulate curcumin in NE, achieving NPs with reduced toxicity, improved bioavailability and bioactivity and strong anti-inflammatory properties[83]. Turmeric is instead commonly used to treat biliary disorders, jaundice, anorexia, cough, diabetic ulcers, liver disorders, rheumatism, inflammation, sinusitis, menstrual disorders, haematuria, and haemorrhage[52]. Furthermore, tannins, stilbenes, and flavonoids, possessing at least in vitro antioxidant effects, have been encapsulated in NEs[84]. Differently from in vitro results, the in vivo antioxidant activity shown by EGCG was very poor. However, it was significantly increased by formulating it in small NPs using NE technique[85]. Bioactive lipids and carotenoids were converted in NEs achieving respectively, more stability against autoxidation and increased bio-accessibility[52]. NEs were successful also in saving lactic acid bacteria from degradation and in restoring the proper microbiota in diverse intestinal diseases conditions[126].

Pomegranate peel ethyl acetate extracts containing several polyphenols, including high levels of ellagic acid (EA) were beat together with pomegranate seed oil, achieving polyphenols-loaded NEs, suitable for topical applications. The NE possessed the capability to avoid or delay UV radiation damage, thus being suitable as anti-photo-aging cosmetic [52,86]. Lemongrass essential oil (LEO), often found in soaps and other personal care products as flavour, is traditionally used to treat digestive problems and high blood pressure, as a tool in aromatherapy to relieve stress, anxiety, and depression, or like an antimicrobial. Unfortunately, LEO is prone to autooxidation and easy degradation, by which it loses activity, and provides smelly or even harmful compounds, responsible for allergic reactions and skin irritation.

To address such drawbacks, NE formulations of LEO were prepared with reduced undesired sensory impact, while enhancing its antimicrobial activity. Edible carnauba wax and LEO NEs were developed, achieving a coat packaging for protecting plums, which proved to inhibit the growth of food-borne Salmonella spp. and E. coli [87].

By using APs-loaded solid nanoparticles delivery systems (SNDSs), highly soluble bioactive nanomaterials were obtained. Further details are reported in the previous Table 1 [91], [92]. The SNDSs’ digestibility in the GIT or in others body districts controls the release of APs in that precise body area, thus realizing a targeted release. In this regard, materials of carrier agents should be selected based on their physicochemical features, which should be appropriate to permit SNDSs degradation where desired [91]. Starch-based NPs are digested at an oral level by the activity of amylase, while polysaccharide- and protein/polysaccharide-based NPs are assimilated in the small intestine, due to variations of pH and salt concentrations[93]. According to these degradative processes, the APs release happens in the oral cavity from starch-based NPs, while in the small intestine from polysaccharide and protein/polysaccharide-based NPs. On the contrary, lipid-based NPs will release APs in the small intestine simultaneously with the digestion of triglycerides[91].

Particles sizes of SNDSs can positively influence the transport of APs through enterocytes by transcellular endocytosis, while their surface charge could be responsible for the formation of hydrogen bonds with the mucosal surfaces, contributing to momentary retention[127]. On the other hand, the presence of surface cell-penetrating ligands could enhance transmembrane transport efficiency[91], thus influencing positively the effectiveness and bioactivity of the transported APs. Additionally, NPs equipped with a lipid phase can access the bloodstream via mesenteric lymph and thoracic ducts, avoiding hepatic first-pass metabolism, thus extending the half-life of APs-loaded SNDSs.

APs-loaded SNDSs were prepared in the form of micelles (MICs) using some of the polymers reported in Table 1 (row 4). They are present in many therapeutic devices approved by the Food and Drug Administration (FDA) or in clinical trials Phases II-IV [94].

Different APs-loaded SNDSs were prepared also in form of hydrogels using different polymers or co-polymers, including PCL-b-PEG-b-PCL (10 nm), PLGA-b-PEG-b-PLGA (77–84 nm), PLA-b-PEG (<200 nm), Pluronics® (<60 nm), PGA-b-PAE (100–200 nm), PLL-b-DOCA-b-mPEG (<200 nm), PEG-b-Pasp (22 to 60 nm), PLH-b-PEG (112 nm), PEI-g-PVP (142 nm), PDMAEMA-PCL (<150 nm), PEG-b-PLL-b-PLLeu (100–125 nm), PIHCA-Tween80 (<320 nm), sodium alginate-HPMC, PEO-b-PHB-b-PEO, OncoGelTM, PAH/Chitosan and are already approved and marketed as medical treatments for different diseases [94]. Interestingly, regarding dendrimers characterized by a core-shell structure, when APs were physically encapsulated, the resulting AP-enriched dendrimers were characterized by having a bioactive functional core and a dendrimer shell. On the contrary, when APs were covalently bound on the surface of dendrimers, the dendrimer formulations were typified by a dendrimer core and a bioactive shell. The drug-loaded dendrimers showed a favourable drug release profile protracted in time and improved biological activities.

More specifically, with organic solid nanoparticles (OSNPs), organic nano carriers are intended, whose classification is based on their physicochemical nature, production method, properties, free energy, interactions type, and typology, etc. [128]. To date, the most adopted OSNPs for APs encapsulation are those reported in Table 1. Anyway, inorganic metal oxide-based and clay-based NPs are also extensively used.

The main typologies of lipid-based nanoparticles (LNPs) have been reported both in Table 1 and in Figure A3 in Appendix A[129].

SLNPs are emerging products of lipid nanotechnology [130,131], are commercially available NPs and are suitable for delivering LAPs. The lipids used to obtain SLNPs include triglycerides (tristearin), diglycerides (glycerol bahenate), monoglycerides (glycerol monostearate), fatty acids (stearic acid), steroid molecules (cholesterol), and waxes (cetyl palmitate) [52].

LPs have demonstrated remarkable therapeutic benefits in clinical applications, even if their approval is still limited by all stages necessary for the liposomal development and for the production process that encompasses manufacturing methods, regulatory approval by the competent authorities and intellectual property[132]. Anyway, due to intensive research in the development of liposomal formulations for clinical use, a few liposomes have entered the market as commercialized liposomal products [133]. The main liposomal products approved and marketed have been reported in Figure 3 [133], while the main APs contained in such clinical approved liposomal formulation have been reported in Table 1[96].

Anyway, even if not marketed util now, several other or similar APs have been already formulated in several other liposome carriers and are currently in clinical trial Phase I-III. Those that are in Phase III clinical trials include APs such as amikacin, tecemotide, T4 endonuclease V, prostaglandin E-1 (PGE-1), doxorubicin, and cisplatin. Those in Phase II encompass platinum analogue cis-(trans-R,R-1,2-diaminocyclohexane) bis (neo-decanoato), platinum (II) semi-synthetic doxorubicin analogue, annamycin, cisplatin, lurtotecan, potent topoisomerase I inhibitor, irinotecan’s active metabolite, paclitaxel and ATRA. Finally, those in Phase I include APs such as mitoxantrone, antisense oligodeoxynucleotide growth factor receptor bound protein 2 (Grb-2), vinorelbine tartrate, topotecan, PLK1 siRNA, PKN3 siRNA, doxorubicin, CEBPA siRNA, docetaxel, cisplatin, doxorubicin, p53 gene, and vinorelbine[96]. Furthermore, LNPs have been used to entrap essential oils (EOs), ferulic acid and tocopherol achieving loaded lipid NPs, which showed the capability of reaching different types of cells and improved antioxidant activity[134]. Micelles (MICs) are tiny spherical lipid particles made using both hydrophilic and hydrophobic copolymers like those reported for SDDSs. Usually, PEG-PLGA micelles are normal micelles (n-MICs), while PLC-P2VP micelles are inverse micelles (i-MICs). MICs-based delivery systems allow the intravenous administration of HAPs, without using solubilizing adjuvants which can cause undesired toxic symptoms[97]. The release of APs from MICs can be voluntarily provoked at the target site by local stimuli, as variation in pH, temperature, or the application of ultrasounds or light. Drug-loaded MICs found applications especially in the treatment of cancer disease, and the selection of stimuli-sensitive polymers used in MICs preparation is based on the specific conditions found in the tumor microenvironment[97]. The current clinical trends in using stimuli-responsive MICs to treat cancer have been reported and discussed in a relevant work by Wang et al [97]. Stimuli include pH, ROS, hypoxia, enzymes, thermic and magnetic stimuli[97].

Anyway, many factors including MIC intrinsic stability, APs diffusion rate, their partition coefficient, the copolymers biodegradation rate, APs concentration within the MICs, their MW, physicochemical features, and location within the MICs can also influence their release[52].

Fatty acid-based micelles were used to solubilize and transport plant oxylipins, phytoprostanes, and phytofurans, which were derived by the non-enzymatic oxidation of linolenic acid [91].

Niosomes (NIOs) are vesicles osmotically active and stable, representing an alternative option to LPs specifically used for ameliorating oral bioavailability of APs with limited absorption in GIT. They can act as reservoir systems capable to providing controlled and sustained delivery of encapsulated APs. NIOs made with non-ionic surfactants demonstrated low levels of toxicity for cells, due to their uncharged structure [98]. NIOs, as MICs and LPs, have been employed to formulate APs such as those reported in Table 1, clinically applied to treat different forms of cancer including breast, lung, colorectal, prostate and skin cancer [98]. By mixing Span 60 and Tween 60, with 15% PEG 400 as a solvent, a dermal delivery system consisting of EA-loaded NIOs was prepared. It exhibited very high EE% and high efficacy in delivering EA to human epidermis and dermis[135].

ONPs consist mainly of cyclodextrins (CDs) which are commonly used as host molecules for encapsulating and delivery LAPs by the monomolecular inclusion complex technique[104,105]. CDs are cyclic oligosaccharides of different dimensions obtained through enzymatic degradation of amylose by the enzyme cyclodextrin glucosyl transferase[52]. They have a truncated cone structure and can accommodate hydrophobic molecules inside their hydrophobic interior cavity. CDs’ outer side, due to the presence of several OH groups, forms a hydrophilic layer, which confers CDs high water solubility (Figure A4 in Appendix A).

Low doses of CDs are well tolerated by humans, but high doses may cause some adverse effects such as diarrhoea and soft stools. β-CDs are currently mostly used as devices for drug delivery and loading several non-polar APs[103] and found applications as carriers in the food, pharmaceutical, and cosmetic industries. Different methods are available to prepare the inclusion complexes (ICPXs) of LAPs using CDs[104,105].

Most studies assert that by encapsulation in CDs, significant improvements were observed in polyphenols, such as flavonoids or other APs from plants, including those reported in Table 1 [52,62,106,107,108]. The improvements concerned mainly ameliorated water solubility, water dispersibility, stability, antioxidant and anti-inflammatory activity, drug loading (DL%), controlled release, oral bioavailability, while possible bitter taste perception and degradation were reduced.

Polysaccharides NPs (PNPs) are instead synthesized from natural polyelectrolytes or non-electrolytes hydrophilic polysaccharides such as alginate, chitosan, hyaluronic acid, pectin, and cellulose derivatives (hydroxyethyl cellulose and carboxymethylcellulose) and proper cross linkers or other substances inducing polymer–polymer interactions, as schematized in Figure A5 in Appendix A.

APs were either physically entrapped during NPs formation, covalently attached to the precursor materials, or absorbed into NPs after their preparation. PNPs can be freeze-dried (FD) in the presence of a suitable cryoprotectant or spray-dried (SD) into a microparticulate powder. PNPs have a high affinity to mucosal layers of the cells present in the respiratory tract and GIT, thus being capable of long residence time in these districts. Moreover, their biodegradability, biocompatibility, mucoadhesive features, and tunable properties make them attractive as carriers for formulating colon-targeted drug delivery systems[136]. PNPs, mainly those made using cationic chitosan, anionic alginate or combinations of alginate and chitosan allowed the administration of several APs, also food-derived, for treating diseases in several body compartments such as nasal, oral, ocular, and dermal with enhanced circulation time[62]. Anionic hyaluronic acid-based NPs are particularly efficient for targeting delivery of anticancer drugs, due to hyaluronic acid affinity for hyaluronan receptors, which are highly expressed in tumour cells. Finally, neutral PNPs, made of dextran, maltodextrin, pullulan, pectin, were used to prepare delivery systems able to escape the reticuloendothelial system, thus possessing long systemic residence time, circulation permanence, and higher efficiency[62]. Maltodextrin, that is digested like glucose; is massively used by the bodybuilding industry to increase the intake of carbohydrates in the diet without resorting to sugar[62]. Some plant extracts and APs encapsulated in PNPs have been listed in Table 1 [109,110,111,112,113,114,115,116,117,118,119,120].

Protein-based NPs (ProNPs) can be prepared through proteins precipitation methods including de-solvation, coacervation, emulsification, nanoprecipitation, SD, NP albumin-bound technology, self-assembly, electro-spraying, salting out, and crosslinking[62]. Specifically, proteins are dissolved in a suitable solvent and a non-solvent is added. Also, by changing the physicochemical parameters of the protein solution (pH, salinity, or temperature) precipitation of the pristine protein can be caused[99]. De-solvating agents are often added to promote the dehydration of the system. The stability of the pristine protein is increased using chemical, ionic, thermal, and enzymatic crosslinking agents, among which 8% glutaraldehyde aqueous solution or calcium phosphate are the most used. Figure A6 in Appendix A, shows a casein protein complex stabilized with calcium phosphate [62].

An innovative method was introduced, which allows to produce crosslinked and sterilized ProNPs in a one-step procedure is based on γ-irradiation of ProNPs in phosphate buffer (pH = 7.2), in the absence and/or presence of ethanol and methanol at 30% and 40% (v/v). The results showed that by controlling the irradiation dose, it was possible to modulate the crosslinking density and the particle size[100]. Moreover, to enhance their circulation residence time, ProNPs have been surface modified with PEG [52].

Several food-related APs have been formulated using ProNPs, some of which have been included in Table 1, such as EGCG, GA, and probiotics microorganisms[52,101].

Concerning organo-synthetic biodegradable polymer nanoparticles (OBP-NPs) they are already described when SNPs made with biodegradable polymers were discussed. They can load different APs, either by physical interactions or by covalently binding by utilizing their several chemical functions. Depending on the hydrophilic/hydrophobic balance (HLB) of polymers or copolymers, NPs characterized by various shapes and morphologies can be prepared.

They are also suitable for oral administration of nutraceuticals and phytochemicals and for producing food-grade smart nanocomposites for food-packaging (FP), able to preserve food quality, looks, and taste along with storage.

In the food industry, a topical ointment was prepared with PEG and 5% pomegranate rind extract, with excellent release profile and skin-permeation capability of EA and anti-inflammatory effects in a mouse model of contact dermatitis[137,138]. NPs (150–300 nm) made of PLGA, chitosan, and PEG, were loaded with EA (up to 100 μM), achieving EA-loaded PLGA-chitosan-PEG NPs, that were able to potentiate apoptosis-mediated cell death in HepG2 human hepatoma cells[139]. PLGA-based NPs stabilized by PEG were used to encapsulate anthocyanins, obtaining anthocyanins-loaded biodegradable NPs that showed an EE% of 60%, improved stability, extended life, and a biphasic release profile in vitro. In vivo, they proved anti-inflammatory and anti-neurodegenerative capacities, preventing memory losses in estrogen-deficient rats, and showed a neuroprotective power against Alzheimer’s dementia[140,141,142]. Finally, anthocyanins formulated as NPs significantly upregulated endogenous antioxidant genes, thus helping in the prevention of oxidative stress (OS), with consequent attenuation of the clinical symptoms of the Alzheimer’s dementia and reduction of DNA damage to a higher extent than the native non-conjugated AP[140].

The oral administration of EA-loaded PLC-NPs (EA-NPs), which proved to have high EE% and DL%, produced an EA plasma concentration 3.6-fold higher than that produced administering free EA[143].

Several structurally different eco-friendly soybean-oil-based cationic polyurethanes (PURs) were prepared to develop edible food coatings with antimicrobial properties toward a panel of bacterial pathogens including Listeria monocytogenes NADC 2045, Salmonella typhimurium ATCC 13311, and S. minnesota R613. Tested against the same strains of wild-type, the PURs-based NPs exhibited better antibacterial activity on the Gram-positive L. monocytogenes than on the Gram-negative S. minnesota and excellent activity against S. Minnesota R613 [144].

With the aim of ameliorating their performances, several EOs and their constituents have been subjected to modifications by nanotechnology and converted into NPs formulations for improving their antimicrobial activity, thus allowing their exploitation to extend food shelf-life and to minimize the growth of foodborne pathogens.

More in Deep about Nanotechnology Applications to Nutraceuticals (Nuts) and Phytochemicals (Phys): in Vivo Experimental Advances

To exploit phytochemicals as health enhancers, researchers extensively engineered nanomaterials and resorted to nanotechnology and nanostructures with dimensions of nano meters (nm). Formulation of Phys using NPs has allowed their controlled and targeted release, which is essential for effective administration [70]. Controlled nano delivery translates in a higher concentration at the target, thus allowing a reduction in the overall administered dose and consequently systemic toxicity [1]. Both internal and external factors, such as pH, temperature, ultrasound or magnetic field application, light incidence, and the type and physicochemical features of NPs, as well as the chemical structure and the physicochemical features of the bioactive compounds themselves, can control their specific release [1].

Stimuli-sensitive nano-capsules containing a bioactive derivative of paclitaxel and possessing an oil core showed the capability to improve the anticancer effects of the encapsulated compound taken by oral administration, thanks to targeted delivery and controlled long-term release[77]. The improved effects allowed a decrease in the dosage and the administration frequency, thus improving patient compliance[77]. Starting from biocompatible pH-dependent polyelectrolytes, nontoxic nanocarriers with high permeability were designed[77].

The layer-by-layer self-assembly of pH-sensitive building blocks proved to be a promising approach to obtaining Phys-based biomaterials with customized properties, which were successfully applied as stimuli-responsive nanocarriers[145]. The encapsulation of bioactive compounds contained in food in properly functionalized NPs permitted increased cellular uptake and slower drug release, thus improving their bioactivity and contributing to sustained therapy[52].

According to a not recent but relevant research paper, only up to the year 2019, while liposomes were the most studied NPs for nano-manipulating Phys, nano-emulsions (NEs) were little considered as nanotechnological approach, while nano-suspensions (NSs) were not even reported [146]. Additionally, NSs and NEs are usually produced using regarded as safe (GRAS) ingredients like liposomes, so that they should be considered among the less toxic and the most suitable tactics for developing Phys-loaded NPs finalized to clinical application. Anyway, due to this statistic, a relevant Review dedicated to these too little considered nanotechnologies has been published in 2023. Such work could be of interest to readers particularly attracted by the topic [54].

The poor solubility, permeability, and negative pharmacokinetics of a series of nutraceuticals (Nuts) were enhanced by developing different nanosized delivery systems[147]. Table 2 and Table 3 summarize some examples of Nuts nanotechnologically formulated using different NPs and methods, associated to their activity as demonstrated by in vivo experiments and/or structural characteristics.

In addition to enhance bioavailability of Phys and Nuts, using different techniques, nanotechnology was and is used in the food sector to prepare NPs finalized to act as colour additives, flavourings, and preservatives, as well as to prepare improved food packaging, with the aim to enhance food shelf-life, taste, and appearance [70].

More in Deep about Nanotechnology Applications in Food-Packaging (FP) Industry

In order to improve the mandatory properties of traditional materials for FP, which have been listed in in studies by Kuswandy, 2017, Kuswandi and Moradi, 2019 and more recently reviewed by Alfei et al in 2020 [61,192], nanotechnology is nowadays intensively studied, also for application in the FP industry. It has been demonstrated that the nanoencapsulation of bioactive natural compounds, by using particles with diameters ranging from 1 to 100 nm, leads to a remarkable increase of their solubility and stability, as well as to a decrease of their inactivation rate, thus offering the possibility of preparing better performant food packaging exploitable as preservative agents in comparison to conventional ones[193]. The inclusion in food packaging of NPs with intrinsic antioxidant, antimicrobial and antifungal properties or capable to release antioxidant, antimicrobial, preservative APs, flavours or enzymes, nutraceuticals and/or phytochemicals previously entrapped, can allow further improvements, including longer shelf life and higher food overall quality[194].

Collectively, the use of nanomaterials has improved FP both in physical and in biochemical characteristics [192,195].

Table 4 reports the main advanced FP types achieved using functional nanomaterials [61].

Note that both natural and synthetic polymers have been employed in the past to produce conventional FPs. Natural biopolymers include lipids-based, polysaccharides-based and proteins-based polymers like those previously discussed but obtained by the action of living organisms. They are completely degradable, while synthetic ones comprehend both petroleum-based plastics and eco-friendly bio-based polymers, that can be in turn degradable and not degradable materials. Degradable bio-based polymers and natural degradable biopolymers allow to reduce the levels of environmental pollution, but lack of suitable mechanical properties. Resorting to nanotechnology, both eco-friendly bio-based and petroleum-based nanocomposites have been prepared, which allow the development of nanomaterial-based physically improved FP.

3. Our Dealing with Nanotechnology Applications: Last 15 Years Studies

The following Table 5 collects the main details about the nanosized materials developed in our laboratories in the years 2009-2024, while additional twelve review articles we have published, concerning the most relevant nanomaterials for specific applications such as improvement of solubility and bioavailability of natural and/or synthetic APs, food sectors, food packaging, etc., developed by other eminent scientist in the same years [52,53,54,55,56,57,58,59,60,61,62].

4. Nanotoxicology

Nowadays, it is universally recognized that the application of nanotechnology can allow to achieve many nonpareil advantages, regardless the sector of application. Concerning APs, their nanomanipulation has permitted to efficiently administer not soluble and not bioavailable bioactive compounds and to prepare unconventional food-related therapeutics. The protective action of nanoencapsulations of unstable APs has provided bioactive NPs capable to undergo the strong conditions of processes necessary to enrich food with them, thus achieving functional foods (FFs), food supplements (FSs), as well as manufacturing active food packaging (FP) or preservative additives.

Many nanocomposites are still at the lab stage, but several products have been already approved by EFSA and by the Member States and the European Commission[199]. Nanotechnology application to APs, food and beverages has had an exponential growing over the past 15 years, and due to the obtained advantages, the presence in the market of APs and foods nanotechnologically manipulated is destined to increase further. Anyway, a major problem remains to be solved, which concerns the poor knowledge about the possible risky effects on health of humans, animals and environment, which can derive from ingestion and massive exposure to NPs, and from the possible migration of NPs from FP into foodstuff [199]. In this context, the amplification of the development of nanomaterial-based food-related products is a topic debated incessantly among researchers with contrasting opinions, thus spreading concern and prejudice both among producers in various sectors and among consumers[200].

The Possible Migration of NPs From FP to Food and Toxicity of Ingesting Them

The migration rate of NPs from nanocomposites used to manufacture innovative food packaging (FP) into food or food simulants has been measured using European and U.S. (FDA) standard migration tests. Anyway, numerous issues have emerged that complicated the determination and interpretation of NPs migration studies and related results [199]. Commonly, “migration into foodstuff” indicates the process by which the constituents initially present in the package, possibly including NPs, are liberated into the food or beverage packaged [61]. Limits of safety and the list of authorized substances for manufacturing polymeric food-contact nanomaterials have been established by the European Regulation (European Commission, 2011) [199]. Data of migration are available mainly for inorganic NPs, thus evidencing a very restricted knowledge on the question. They mainly include nano-clay, titanium nitride, nano-silver, silanated silicon dioxide, titanium oxide, zinc oxide, and iron oxide NPs. Collectively, concerning these NPs, the reported findings have established that the migration of NPs from FP would be low and slow[61]. Additionally, the migration rate of a system increases when NPs size and polymer dynamic viscosity decrease[201].

The practice to insert NPs in the FP materials is still at its infancy, therefore only a few studies are available in the literature and current information on their possible migration and on their toxicity upon exposure are limited. Further fundamental studies on toxicity, ecotoxicity, migration tendency and on risk of the intake of nanocomposite materials are needed, to authorize massive application of nanomaterials in the FP field[202]. What is the actual behaviour of NPs once inserted in packaging and in contact with packaged foodstuff? What are the eventual mechanisms involved in the migration and how can the diffusion process change the size and morphology of nanomaterials?

With the aim at answering to these questions, a standardized food model (SFM) for evaluating the toxicity and fate of NPs migrated in food matrix after ingestion was proposed for the first time by Zhang et al, in the year 2019 and its efficacy was assessed by examining the impact of food matrix on the toxicity of TiO₂NPs[203]. SFM is an oil-in-water emulsion usable both in wet and in dried forms. Using a simulated GIT model was observed that all the SFM food components were well digested and that the potential toxicity of TiO₂NPs was reduced in the presence of the SFM, by underling the importance of food matrix effects on the actual toxicity of NPs[203]. Table 6 collects the results achieved by evaluations made on some NPs, while Table 7 reports detailed migration results expressed as wt/volume or as wt/wt of numerous inorganic NPs from different food contact materials (FCMs) into food or foods simulants[199].

The overall migration of nanoclay/starch nanoparticles into vegetables was in conformity with European directives, thus establishing that these materials are suitable for utilization in FP sector [61]. Similarly, the migration of PLA/laurate LDH-C12-modified NPs used to reduce gas permeability of a packaging in modified atmosphere for conservation of meat, resulted largely below the total legislative migration limits established for all materials [204]. Experimental results and theoretical considerations about the migration of two types of CNTs/LDPE/PS NPs, in different food simulants, established that such NPs do not migrate from the polymer matrix into food[205]. Even if a commercially available FP improved with AgNPs intended to package chicken meatballs, under common domestic storage conditions, demonstrated no significant antibacterial effects, evidenced a migration rate encouragingly slow[206]. AgNPs did not migrate from AgNPs/PEF into chicken breast or distilled water, after 168 and 72 hours respectively[207]. Inductively coupled plasma mass spectrometry (ICP-MS) analytical technique evidenced that, migration of AgNPs from AgNPs/PE packaging films into an acidic food simulant (3% acetic acid) was promoted by the presence of organic additives, as Irganox 1076, Irgafos 168, Chimassorb 944, Tinuvin 622, UV-531 and UV-P, and by Ag oxidation endorsed by high humidity and temperature treatment[208].

The evaluation of the degree of migration of NPs from active/smart/mechanically improved FP into foods and into the environment should be associate with an all-round knowledge of the possible toxic outcomes that the NPs eventually could have on humans and animals when ingested, as well as on the environment. NPs are not normally eaten and metabolized by humans and animal species and, paradoxically, even if by adding NPs in FP, new advantageous opportunities can be achieved such as ingesting higher-quality and more safe aliments, new risks to human health and the environment can occur[199]. In this regard, the overall risks potentially associated to the intake of food containing NPs is still unclear. Currently, we know that smaller particles are usually absorbed easier and faster and are more promptly distributed into the organs. Here, NPs can damage cells and tissues by reactive oxygen species (ROS) generation or by other type of direct or indirect toxicity[209]. As represented in Figure 4, the toxicological effects of NPs and their mechanisms have been evaluated in vitro and in vivo [210]. For evaluations in vitro, human and/or rodent cell lines from intestine, liver, lung and skin, and plants cells, have been used. The cytotoxicity tests comprised the lactate dehydrogenase (LHD) release assay, live-dead assay, cell counting, Alamar blue assay, neutral red uptake, the evaluation of protein content and, trypan blue dye exclusion test. Specific mechanisms are generally studied by observing the changes in different biomarkers, such as ROS production, glutathione (GSH) levels, inflammation response, DNA damage and cell death. On the contrary, for assessing potential genotoxicity Comet assay, Ames test and micronucleus essay have been adopted.

In vivo evaluations used mainly rodents and macro-toxic or histopathological effects, following the sub-chronic/chronic exposure to different kind of NPs were assessed[210]. Unfortunately, the results from both in vitro and in vivo analyses are often contrasting and questionable. As an example, findings on nano organoclay have established that their toxicological evaluation, case by case, should be performed [210]. In addition, since the average amount of clay added as reinforcement of polymers should be around 5 wt%, it is important that the concentrations tested would be pertinent with a real oral exposure scenario. Collectively, more toxicity data from studies on an increasing number of nanocomposites are necessary for a more reliable evaluation of nanotoxicology and exposure estimation[211].

Table 8 collects the results from in vitro studies we have reviewed. Toxicity depended mainly on concentrations, type of cation of metal NPs and especially morphology of NPs, (nano wires, nano roads, nano spheres etc.). Similarly, Table 9 reports the results from in vivo evaluations of different NPs with different morphologies on different animal models.

Although the potential toxicity by ingestion of Ag NPs is still debated, it has been found that the cytotoxicity reported in Table 8 (first row) can be nullified by the addition of carboxymethylcellulose (CMC) to the colloidal solutions of Ag NPs, while the genotoxic effects of Ag NP dispersions was observed at concentration of 12.4 ppm [212]. One of the most accredited mechanisms by which SPM iron oxide NPs, also referred to as USPIO or SPION (USPIO = ultrasmall superparamagnetic iron oxide; SPION = superparamagnetic iron oxide nanoparticles) can induce cytotoxicity consists in causing an aberrant increasing of ROS. By crossing mitochondrial membrane, the free iron in the form of ferrous ions (Fe^2 +) can react with hydrogen peroxide and oxygen produced by the mitochondria to produce highly reactive hydroxyl radicals and ferric ions (Fe^3 +) via the Fenton reaction. Hydroxyl radicals generated could indirectly damage DNA, causes peroxidation of proteins and lipids and generates inflammation [214]. Physiological doses of SiO₂NPs tested in an in vitro model, to assess their effects on gastrointestinal function and health, evidenced damage to the brush border membrane and both acute and chronic adverse effects in gastrointestinal tract (GIT) cells[224]. Furthermore, the results of an in vitro study have demonstrated that ZnO₂ NPs may cause a decrement in the transport of Fe and glucose and affect the microvilli of the intestinal cells [225]. Anyway, the toxicity of ZnO₂ NPs decreased when a surface modification by a silica coating was performed [226]and proposed as a possible solution to broaden the applications of ZnO₂ NPs as antibacterial agent in FP.

Although both in vitro and in vivo studies have established that TiO₂ NPs accumulate in the tissues of mammals and are eliminated very slowly, the results both on their accumulation and toxicity are conflicting. More reliable in vivo toxicokinetic data are needed to provide conclusions concerning the risk of TiO₂NPs oral exposure[199].

Some in vivo studies on SiO₂ NPs showed that they may cause cytotoxicity and ROS generation and may accumulate in liver causing hepatotoxicity evidenced by alterations in morphometry, biochemistry, hematology, liver tissues and the expression of drug-metabolizing enzyme genes[241]. On the contrary, other studies, performed by administrating SiO₂NPs, as well as Fe NPs to both female and male rats over a 13-week period, reported no accumulation or toxicity[199].

Antimicrobial ZnO₂ NPs and ZnO₂ NPs can reach several organs through ingestion, inhalation, and parenteral routes. Their oral administration induced neurotoxicity and proinflammatory response in rats and immune-toxicity in different ages of BALB/c mice[247,248].

5. Conclusions

The biomedical revolution is governed by nanotechnology, which aids resolve several issues associated with the most part of natural and-or synthetic APs, thus limiting their effectiveness, as well as their actual applicability in vivo. Target treatment administration and maximized therapeutic effectiveness can be achieved, while side effects can be reduced by formulating APs using nanomaterials. Applying nanotechnology, vaccines, anticancer drugs, antimicrobial agents and other nanomedicines can be engineered, while wearable equipment, diagnostic and imaging equipment can be realized. Combining standard medicines with nanoscale technologies, the blood brain barrier (BBB) can be crossed intact and nanodrugs can circulate inside the brain. This technology offers enormous potential markets and benefits to whole classes of current drugs. It is possible to develop tailored mechanisms for medication administration, new diagnostic methods, and nanoscale medical devices with high residence time. Technological advancements in the fields of nanoscience and nanotechnology have led to a remarkable innovation also in the food sector that could bring wide-ranging benefits to the whole food chain. Such innovations include the development of new tastes, textures, mouth sensations and consistencies of food products. The reduction in the amount of fat and certain additives, such as salt, an enhancement in the absorption and bioavailability of nutrients and supplements, the preservation of food quality and freshness represent other innovative challenges which are progressively solved by nanotechnological studies. The research for novel nanomaterial-based packaging solutions, allowing better traceability and security of food products in the supply chain, is incessantly in rapid expansion. Food packaging (FP) applications currently represent the largest portion of the nanofood market, following the nanosized and/or nano-encapsulated ingredients and additives for healthy food production. Although still new and scarce in Europe and other regions, a discrete amount of nanomaterial-improved food-contact materials (FCMs) and more functional food products containing nanosized ingredients and additives are already available in some countries. It is anyway rational to imagine that such products will be available on the global market in increasing numbers and variety in the coming years. Their expansion will depend mainly on the price, quality and, above all, acceptance by consumers. Although nanotechnology applications for the food, healthy food and biomedicine sectors have undoubtedly unlocked up enormous opportunities for innovation and new developments, they have also opened new happenstances for ensuring safety and evidencing all the potential risks of this new technology, without highlighting only the benefits. Reliable strategies to better know the risks for human, animals and the environment, which can derive from a massive exposition to nanomaterials and to regulate them in a globally harmonized manner are needed. Unfortunately, food laws in different countries may not conform to each other and this fact is a challenge to the regulatory authorities. In this regard, it would desirable that in due course, such issues could be addressed through the development of frameworks relating to key international trade agreements, such as those administered by the World Trade Organization. We hope that this new review could provide much-needed insights into the various aspects and issues relating to the new and exciting developments that nanotechnologies are offering to the food, medicine and related sectors.