**1. Introduction**

Fortification of edible products (e.g., food, food constituents, or supplements) with nutrients or non-nutrient bioactive components can help to balance the total nutrient profile of a diet and supplement nutrients lost in processing and thus to correct or prevent insufficient nutrient intake and associated deficiencies [1]. Compounds of natural origin, such as curcumin (CUR) occurring in turmeric, ω-3-fatty acid in fish oil, vitamins from fruits, when encapsulated in an appropriate nanocarrier, will be released after consumption of the food in the target organ and utilized according to its nutritional property [2].

Basic types of preparations/materials influencing human health or condition can be classified as follows: (i) drug products, (ii) homeopathics, (iii) dietary supplements (DISs), (iv) medical devices, (v) cosmetics, and (vi) biocidal products. Dietary (food) supplements are products that look similar

to medicines (can be sold in pharmacies) but are a special category of foods. They contain vitamins, minerals, amino acids, essential fatty acids, natural products, probiotics, etc., as active ingredients. The purpose of a DIS is to keep the human body functioning properly by delivering compounds that are needed by the human body but could not be received sufficiently from a regular diet. According to the manufacturers, DISs have beneficial effects on health conditions. They are manufactured in the form of pills, capsules, tablets, or liquids [3,4]. DISs are regulated by many guidelines, regulations and directives, for example, by the European Commission directives 2002/46/EC and 2006/37/EC, European regulations 1924/2006, 1137/2008, 1170/2009, 1161/2011, 119/2014, 2015/414, 2017/1203 [4], and by a number of documents published by the U.S. Food and Drug Administration (FDA) [5], to ensure the quality and safety of these products, to protect consumers against potential health risks from such products, and to ensure that they are not provided with misleading information. In addition, DISs are regulated by the European Food Safety Authority (EFSA), national legislation (e.g., on food) and regulations (e.g., requirements for food supplements and food enrichment), etc. For example, in the EU market, approx. 30 approved nutrition claims (meaning that specific requirements are to be met) can be found, and a product can be marketed as a DIS only if it meets the so-called health claims, which is any statement about a relationship between food and health. The European Commission approves various health claims, which have to be easily understood by consumers, based on scientific evidence. The EFSA is responsible for evaluating the scientific evidence supporting health claims, the types of which are as follows: (i) 'Function Health Claims' (relating to the growth, development and functions of the body, or referring to psychological and behavioral functions, or on slimming or weight-control), (ii) 'Risk Reduction Claims' (on reducing a risk factor in the development of a disease), and (iii) Health 'Claims referring to children's development' [6].

It is important to note that DISs are not food additives, which are special excipients added to foods for modifications of their flavor, color, or longevity [7].

Foods for special medical purposes (FSMPs), based on the definition of the EFSA, "are designed to feed patients who, because of a particular disease, disorder, or medical condition, have nutritional needs that cannot be met by consuming standard foodstuffs. Specifically, according to EU legislation, they are intended for patients with a limited, impaired, or disturbed capacity to take, digest, absorb, metabolize, or excrete ordinary foods, or certain nutrients or metabolites; or with other medically nutrient requirements whose dietary managemen<sup>t</sup> cannot be achieved by modification of the normal diet alone" [8]. It means that FSMPs are foods that are intended for nourishment at: (i) certain groups of people whose digestive process or metabolism is impaired, (ii) certain groups of persons in a particular physiological state, which therefore, may have specific benefits from controlled consumption of certain substances in food, or (iii) healthy infants and young children. Therefore, the following categories of FSMPs can be distinguished: (i) food for infant and follow-on nourishment and nutrition of small children, (ii) food for cereal and other non-cereal food for infant and young children, (iii) low-energy foods designed to reduce body weight, (iv) food without phenylalanine, (v) gluten-free foods, (vi) foods for people with disorders of carbohydrate metabolism (diabetics), (vii) low lactose or lactose-free foods, (viii) foods with low protein content, (ix) foods intended for athletes and for persons with increased physical performance. FSMPs are advised to be used only under medical supervision and have to be provided with labels with information about their intended use. The European Commission also issued several documents for the regulation of FSMPs, e.g., Commission Directive 1999/21/EC, Commission Regulations No. 953/2009, 609/2013, 2016/128 [9], and so-called "medical foods" are regulated also by FDA [10].

A functional food or functional ingredient is any food or food component providing health benefits beyond basic nutrition, and natural bioactive compounds as functional ingredients showing beneficial effects for health become increasingly popular in the diet [11]. Therefore, functional foods are similar to traditional conventional foods but have more advantageous properties in relation to healthy physical condition. Nutraceuticals are based on both food and herbal or other natural products and are used in the form of pharmaceutical formulations, i.e., tablets, capsules, drops, or liquids, and have physiological benefits. The main focus of all these products is to improve health and reduce the risk of disease. In contrast to drugs, in all these cases, the active substance or a mixture of active compounds is present in low concentration [12].

Nanotechnology is a rapidly growing field that ensures the development of materials with new dimensions, novel properties, and a wider range of applications. U.S. National Nanotechnology Initiative defines nanoparticles (NPs) in the range of 1–100 nm [13]. According to the Recommendation on the definition of a nanomaterial adopted by the European Commission, the term "nanomaterial" means "a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range 1–100 nm. In specific cases and where warranted by concerns for the environment, health, safety or competitiveness the number size distribution threshold of 50% may be replaced by a threshold between 1 and 50%" [14]. However, in pharmacy, particles of 10–500 nm have been used, rarely up to 700 nm. From the aspect of passage through vessels, the inside diameter of which is in the range from 25 mm (aorta) to 5 μm (capillaries), the ideal size of NPs should be <300 nm to ensure efficient transport for targeted distribution of drugs [15–20].

NPs can be prepared from both inorganic and organic materials [21–23], and currently, especially encapsulation to various biodegradable nature-based biopolymers is more and more frequently used [19,20,24,25]. NPs can be generated by either top-down methods (dispergation, fluidization, homogenization processes, or emulsifying technologies) or bottom-up methods (precipitation/condensation processes, evaporation techniques, various controlled sol-gel syntheses) [21,26]. NPs produced using mechanical approaches are usually, in the range 100–1000 nm; to produce NPs of size 10–100 nm, chemical and bottom-up methods are used [21,27]. "Green" synthesis of NPs or innovative biotechnological approaches related to the synthesis of NPs are summarized by Singh and Shukla et al. [22,23].

The physical, chemical, and biological properties of nanoscale materials are significantly, different from those of bulk materials and single atoms or molecules; therefore, different properties of active pharmaceutical ingredients have been modified in such a way [14,28–33]. In biomedical branches, NPs can be used for nanodiagnostics, as nanomaterials for tissue engineering, as drug carriers for specific delivery/targeted biodistribution or controlled release, and as agents/drugs for prevention/treatment of diseases. Therefore, application of nanotechnology can be considered as an excellent tool for modification of parameters of bioactive agents. Modification of properties using nanosystems/nanoformulations helps to enhance the bioavailability of active substances and change the route of administration when needed. Therefore, smaller amounts of substances can be used, which allows decreasing dose-dependent toxicity and various side effects. In addition, many formulations also protect bioactive molecules from degradation [19,20,24,25,28,29,31,34–42]. The enhancement of bioavailability could be achieved by the improved solubility of bioactive compounds under gastrointestinal (GI) conditions, their protection from the chemical conditions in the GI tract, and controlled release within the GI tract, or by an improved transfer through the intestinal wall, and the particle size, surface properties, and physical state of the nanomaterials used in food supplements are crucial characteristics affecting their final nutritional value [43]. Recent findings and advancements related to lipid nanoscale cargos for the protection and delivery of food bioactive ingredients and nutraceuticals were overviewed by Akhavan et al. [44]. Nanoemulsion (NE) compositions, types of active ingredients, applications in different types of food systems, toxicological and safety aspects, and future directions were summarized by Kumar and Sarkar [45]. For encapsulating drugs/nutraceuticals and fortification of food products, especially beverages with water insoluble nutraceuticals, nanostructured lipid carriers (NLCs) could be successfully applied [46]. Micro- and nano bio-based delivery systems (DESs) for food applications were discussed also by Simoes et al. [47].

The above-mentioned nanoformulations can be found in many drug classes, and so it is not surprising that supplements and FSMPs have also started being formulated in the nanoscale, especially with the aim to improve bioavailability, protect active ingredients against degradation, or reduce side effects. Therefore, this contribution summarizes the current state of the research focused on nanoformulated human and veterinary DISs and FSMPs.

#### **2. Types of Formulations and Used Materials**

Nutraceuticals' functionality in food products can be stabilized and enhanced using bio-based nanoscaled DESs that help to improve their bioavailability and protect valuable nutraceuticals at food processing or digestion, see Figure 1, where individual most frequently applied nanoformulations are mentioned. Selected nanoformulations are discussed below in the following subchapters. Advances in nutraceutical DESs with focus on the formulation design for the enhancement of nutraceuticals' bioavailability with the purpose to ensure effective preservation or maximization of their bioactivity and safety inside the human body were summarized by Goncalves et al. [48]. A review of recent research developments related to nanocarrier-based delivery of nutraceuticals for cancer prevention and treatment was presented by Arora and Jaglan [49]. Recent findings related to advances made in the nanoencapsulation of lipophilic and hydrophilic vitamins, safety issues, and health risks regarding the consumption of these products, which would result in widespread utilization of nanoencapsulated vitamins in the food and beverage products in the future, were summarized by Katouzian and Jafari [50]. The intelligent DESs for bioactive compounds in foods designed to improve their low solubility, poor stability, and low permeability in the GI tract and improving their oral bioavailability were discussed by Chai et al. [51] from the aspect of physicochemical and physiological conditions, absorption mechanisms, obstacles, and responsive strategies.

**Figure 1.** Most frequently used nanoformulation types of dietary supplements and foods for special medical purposes.

Gleeson et al. [52] looked into the potential of certain delivery strategies for the improvement of the oral bioavailability of different types of nutraceuticals, such as fatty acids, bioactive peptides, micronutrients, and phytochemicals, and emphasized that nutraceutical and pharmaceutical industries could leverage approaches to oral delivery formulations, which would result in synergies for nutraceutical and pharmaceutical molecules. For example, microfluidization could be considered as an efficient emulsification technique resulting in fish oil encapsulated powder producing emulsions at the nanoscale range (*d*43 of 210–280 nm) with the lowest unencapsulated oil at the surface of particles [53]. At investigating the effect of excipient emulsions with different surface-weighted mean droplet diameters *d*32 = 0.15 μm (small), 0.40 μm (medium), and 22.3 μm (large) on the bioaccessibility of carotenoids from tomatoes using a simulated GI tract, it was found that the bioaccessibility of carotenoids decreased with an increase of initial droplet size, which could be attributed to more efficient extraction of carotenoids from tomato tissues by smaller droplets that were digested faster. This caused faster mixed micelle formation and, consequently, enhanced solubilization of carotenoids in intestinal fluids. Moreover, when tomatoes were boiled with emulsions, the bioaccessibility of carotenoids was higher than when they were boiled alone and subsequently added to emulsions [54]. Electrospinning and electrospraying technologies constitute useful and modern techniques used for the encapsulation and controlled release of bioactive compounds, including drugs and health-promoting agents. Both electrospinning, mostly used for fibres, and electrospraying, mostly used for particles, are voltage-driven fabrication technologies enabling tight control of fibres and particles in the micro-, submicro- and nanoscale dimensions suitable for a wide range (polymers, proteins, inorganic) of materials. Both processes are able to replace traditional techniques, e.g., spray-drying or lyophilisation, as they propose several benefits such as (i) production of dry products in a single step, (ii) room temperature operation (suitable for labile components, e.g., antioxidants, omega-3 oils, living cells, etc.), (iii) enabling to produce single-phase or multi-component fibres and particles, and (iv) high effectivity of encapsulation [53,55–60], as mentioned below.

#### *2.1. Liposomes and Nanoscale Emulsions*

Nanoliposomes, or nanometric bilayer phospholipid vesicles, have a very promising potential for the nutraceutical industry, because they can encapsulate simultaneously lipophilic and hydrophilic materials, ensuring a synergistic effect, and can protect sensitive bioactive compounds, enhance their bioavailability, ensure sustained-release, and improve storage stability. The unique properties of nanoliposomes predestine them to be used in DISs for effective disease prevention and health promotion [61].

Nanophytosome is one of the newest lipid-based nanocarriers enabling the delivery of botanical based nutraceuticals, which could be potentially used in food products for designing novel functional foods and beverages [62]. Phytosomes-phosphatidylcholine (PC)—rutin complexes prepared by the encapsulation of rutin with PC using rutin:PC molar ratio 1:3 were found to provide the highest physical and chemical stability (during 30 days of storage) with fine particle sizes (<100 nm) and the encapsulation efficiency (EE) of 99%, and due to the ability of masking undesirable features of rutin, they may be applied in fortification of food products with water insoluble nutraceuticals [63].

At the preparation of liposomes starting from multilamellar large vesicles with a diameter range 2.9–5.7 μm using an ultrasound-assisted approach based on the thin-film hydration method, unilamellar vesicles with diameter sizes ranging from 40 nm to 51 nm were achieved, showing the EE of 56% for cobalamin, 76% for α-tocopherol, and 57% for ergocalciferol. The nanovesicles and their content were kept intact for >10 days when incubated at simulated conditions of extracellular environment thanks to the used lipid composition [64]. The investigation of the encapsulation and preservation of quercetin (Q) with cyclodextrins (CDs), conventional liposomes composed of three different types of phospholipids (unsaturated egg Lipoid E80, unsaturated soybean Lipoid S100, and saturated soybean Phospholipon 90H), and drug-in-CD-in-liposomes showed that the application of Lipoid E80-liposomes resulted in a better protection of Q against UV irradiation, and its photostability was additionally improved when encapsulated in drug-in-CD-in-liposomes (sulfobutylether β-CD/Q inclusion complex in Lipoid E80 liposomes) [65].

Compared to CUR liposomes, the Pluronic® modified CUR liposomes showed a slower release rate and lower cumulative release percentage for CUR, enhanced pH stability and thermal stability, and pronouncedly improved absorption in simulated GI tract in vitro, suggesting that both types

of liposomes could be used as carriers of CUR in nutraceuticals and functional foods. The best bioaccessibility was observed for CUR liposomes modified with Pluronic® F-127 [66].

Stimuli-sensitive (smart) nano DESs for nutraceuticals of both a nutritional and pharmaceutical value are of grea<sup>t</sup> importance for the formulation of novel functional foods. Because the best effect on the human health was observed when the weight ratio of ω-6/ω-3 polyunsaturated fatty acids (PUFAs) is in the range between 1:1 and 5:1, Semenova et al. [67] focused their attention on the molecular design of DESs on the basis of nanoscale complexes formed between a covalent conjugate (sodium caseinate (SCas) + maltodextrin; dextrose equivalent = 2) and combinations of polyunsaturated lipids that are mutually complementary in the content of ω-6 and ω-3 PUFAs: α-linolenic acid (α-LNA) + α-linoleic acid (α-LLA); liposomes of soy PC + α-LNA, and micelles of soy lyso-PC + α-LNA. The researchers concluded that thanks to the EE of all these lipid combinations by the conjugate, lipids were highly protected against oxidation, and their high solubility in an aqueous medium was reached. Dey et al. [68] designed ω-3 PUFA enriched biocompatible NE with sesame protein isolate (SPI) as a natural surfactant. NE with 0.5% (*w*/*v*) SPI and Tween 20 and Span 80 used in 1:1 ratio having the hydrodynamic droplet size of 89.68 ± 2.38 nm effectively enhanced the shelf-life stability of NEs, and the fatty acid release from NE droplets was ≥90% during 120 min of simulated two-step in vitro digestion.

The short-chain triglyceride-based NE encapsulating vitamin E did not physically withstand temperatures exceeding 25 ◦C, while with long-chain triglyceride-based NEs, good vitamin E retention even at 40 ◦C was observed, and the retention was increased when the NEs were stored in the dark [69]. Vitamin D NEs with small droplet diameters (*d* < 200 nm) fabricated by spontaneous emulsification using medium chain triglycerides (MCT) and Tween 80 at surfactant-to-oil ratio ≥1 at high stirring speeds (800 rpm) were found to be relatively stable at ambient temperatures and unstable at heating (T > 80 ◦C), but the application of a cosurfactant (sodium dodecyl sulfate) could improve their thermal stability [70]. The investigation of the effect of excipient NEs formulated from long or medium chain triglycerides (LCT or MCT) on β-carotene (β-Car) bioaccessibility from commercial DISs (tablets or soft gels) studied using an in vitro GI tract model showed that the application of LCT NEs enhanced β-Car bioaccessibility from tablets and soft gels by 20% and 5%, respectively, while the effect of MCTs was minor. This could be connected with the fact that large carotenoid molecules could be incorporated only into large mixed micelles formed by LCT digestion, and thus, excipient NEs could be applied to improve nutraceutical bioavailability from DISs [71]. NEs prepared using three LCT oils (flaxseed, olive and corn oil) increased the bioaccessibility of astaxanthin (AST) compared to the control due to the formation of mixed micelles that solubilized the hydrophobic carotenoids. The final amount of free fatty acids released affected lipid digestion and AST bioaccessibility, which decreased in the following order: olive oil > flaxseed oil > corn oil, and free fatty acids unsaturation and chain length affected lipid digestion and micelle formation [72]. Saxena et al. [73] increased the bioavailability of the model bioactive compound α-tocopherol as a food supplement using edible (coconut) oil NEs. The prepared NEs were found stable and biocompatible, and the contribution of kinetic-controlled release was found to be approx. 70%, while that of diffusion-controlled release was approx. 30%, suggesting the potential of the use of edible oil NEs in food and beverages.

A saponin coated NE with mean droplet diameter 277 nm encapsulating vitamin E was found to be more stable to droplet coalescence at thermal processing (30–90 ◦C), long-term storage, and mechanical stress than a conventional emulsion with mean droplet diameter 1.285 μm. At application of both emulsion formulations to male Wistar rats, droplet flocculation and coalescence during in vivo digestion was observed, however, the higher in vivo oral bioavailability of vitamin E encapsulated in the NE was reflected in a 3-fold increase in the area under the curve (AUC) compared to the conventional emulsion [74]. The lowest particle diameters (*d*32) of vitamin E NEs fabricated using natural surfactants, quillaja saponin, and lecithin and high-pressure homogenization were 0.13 μm for lecithin and 0.12 μm for quillaja saponin at vitamin E to orange oil ratio 50:50%. At pH 7, both systems were stable in the temperature range 3–90 ◦C but unstable at pH 2 or in the presence of NaCl

(>100 mM NaCl for lecithin and ≥400 mM NaCl for quillaja saponin) [75]. The encapsulation of CUR in saponin-coated CUR NEs fabricated using a simple pH-driven loading method improved CUR solubility and bioavailability, and its in vitro bioaccessibility was approx. 3.3-fold higher compared to free CUR. In an in vivo study, oral administration of these NPs to Sprague Dawley rats resulted in approx. 8.9-fold higher in vivo bioavailability than that estimated with free CUR [76].

Zheng et al. [77] subjected CUR loaded oil-in-water (O/W) NEs prepared using the conventional oil-loading method, the heat-driven method, and the pH-driven method and three commercial CUR supplements (Nature Made, Full Spectrum, and CurcuWin) to a simulated GI tract model consisting of mouth, stomach, and small intestine phases and found that the three tested NEs showed similar CUR bioaccessibility (74–79%) with the highest absolute amount of CUR in the mixed micelle phase of the NE fabricated by the pH-driven method. The concentration of CUR in mixed micelles decreased as follows: CurcuWin ≈ pH-driven method > heat-driven method > conventional method >> full spectrum > nature made, and CUR encapsulated in small lipid particles had an improved absorption in GI tract.

Cholecalciferol (vitamin D3) minitablets and an optimized bile salt/lipase alginate-glycerin film provided unique oral components for inclusion in a bioactive association platform (BAP) capsule designed to deliver the active nutraceutical ingredient from the formulation framework resulting in the enhanced in vitro and in vivo performance of cholecalciferol. The in vivo experiment showed that cholecalciferol bioavailability from the BAP was 3.2-fold greater than that of the conventional product, and improved and maintained serum levels of 25-hydroxyvitamin D3 were observed as well, suggesting that BAP could be considered as an ideal oral vehicle for enhanced delivery of cholecalciferol [78].

β-Car enriched O/W emulsions, in which chlorogenic acid-lactoferrin-polydextrose conjugate was used as an emulsifier to stabilize lipid droplets, showed improved stability to droplet aggregation under simulated GI tract conditions, resulting in increased β-Car bioaccessibility, suggesting that the ternary conjugate-stabilized emulsions could be used as protectors and carriers of hydrophobic drugs, supplements, and nutraceuticals [79]. On the other hand, excipient NEs had much less effect on the bioaccessibility of phenolic compounds, probably due to their smaller and more polar molecules, which could be more easily solubilized in aqueous intestinal fluids [80].

Among NEs prepared using soy protein isolate (132 nm), whey protein concentrate (190 nm), maltodextrin (266 nm), and gum arabic (468 nm), the soy protein isolate NE showing the smallest droplet size provided the highest protection of vitamin D (85%) at 4 wt % concentration, pH 7, and 25 ◦C [81].
