*Review* **Nutraceuticals and Food-Grade Lipid Nanoparticles: From Natural Sources to a Circular Bioeconomy Approach**

**Cristina Blanco-Llamero 1,2, Joel Fonseca 1, Alessandra Durazzo 3, Massimo Lucarini 3, Antonello Santini 4,\*, Francisco J. Señoráns 2 and Eliana B. Souto 1,5,\***


**Abstract:** Nutraceuticals have gained increasing attention over the last years due to their potential value as therapeutic compounds formulated from natural sources. For instance, there is a wide range of literature about the cardioprotective properties of omega-3 lipids and the antioxidant value of some phenolic compounds, which are related to antitumoral activity. However, the value of nutraceuticals can be limited by their instability under gastric pH and intestinal fluids, their low solubility and absorption. That is why encapsulation is a crucial step in nutraceutical design. In fact, pharmaceutical nanotechnology improves nutraceutical stability and bioavailability through the design and production of efficient nanoparticles (NPs). Lipid nanoparticles protect the bioactive compounds from light and external damage, including the gastric and intestinal conditions, providing a retarded delivery in the target area and guaranteeing the expected therapeutic effect of the nutraceutical. This review will focus on the key aspects of the encapsulation of bioactive compounds into lipid nanoparticles, exploring the pharmaceutical production methods available for the synthesis of NPs containing nutraceuticals. Moreover, the most common nutraceuticals will be discussed, considering the bioactive compounds, their natural source and the described biological properties.

**Keywords:** nutraceuticals; lipids; solid lipid nanoparticles; nanostructured lipid carriers; food-grade ingredients

**1. Introduction**

In recent decades, nanoparticles have increasingly become a subject of interest for researchers, mainly due to the use of materials and production methods that are easy to scale and safe, especially the ones that avoid solvents. These properties make these systems especially attractive for the synthesis of nutraceuticals. A nutraceutical is formed by nutrient compounds, each of which has a pharmaceutical and standardized value, with physiological benefits for human health, performance and well-being, and which is obtained from natural sources. In fact, the description of bioactive compounds includes lowering the risk of developing specific medical diseases, and these compounds include, for instance, bioactive peptides, polyphenols, omega-3 PUFA, probiotics, carotenoids, etc. However, nutraceuticals derived from natural sources are quickly oxidized and unstable, which limits their utilization. On the other hand, the bioactive ingredients present in nutraceuticals are often unable to achieve their potential outcomes due to limited aqueous solubility, leading to a poor bioavailability profile and interaction with gastro-intestinal fluids. Therefore,

**Citation:** Blanco-Llamero, C.; Fonseca, J.; Durazzo, A.; Lucarini, M.; Santini, A.; Señoráns, F.J.; Souto, E.B. Nutraceuticals and Food-Grade Lipid Nanoparticles: From Natural Sources to a Circular Bioeconomy Approach. *Foods* **2022**, *11*, 2318. https:// doi.org/10.3390/foods11152318

Academic Editor: Aimin Shi

Received: 12 June 2022 Accepted: 1 August 2022 Published: 3 August 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

their encapsulation in order to increase their bioavailability and adsorption is instrumental to improve their therapeutic potential. Recent progress in the field of nutraceutical delivery has incorporated nanotechnology to overcome the drawbacks accompanying nutraceuticals. Nano-based carrier systems provide several benefits, including an undesirable taste, odor and color masking, providing a pH-triggered controlled release, improved stability, improved shelf life, preservation of volatile ingredients, protection against gastric conditions and pH before reaching the target and protection for the ingredients from different environmental parameters, including oxygen, heat, water and light.

Nanotechnology can improve the bioavailability of nutraceuticals by their encapsulation using nanocarriers for proper delivery to their target, or by the transformation of these compounds into a nanoparticle form. This comprehensive review focuses on issues associated with nutraceuticals and nano-scale formulation approaches, describing recent nanodelivery systems used to encapsulate different nutraceuticals or bioactive compounds with biological value and therapeutic properties. In addition, the advantages and disadvantages of the most common encapsulation techniques and nanodelivery systems used to encapsulate nutraceuticals will be explored, taking into account the main challenges related to their stability.

#### **2. Sources of Nutraceuticals with Biological Value**

Bioactive compounds obtained from natural sources are a widespread and heterogeneous group with highly different biological properties described to be potential alternative therapeutic tools useful in both the prevention or treatment of some diseases. Their therapeutic value has already been proven effective by numerous clinical trials. Most common bioactive compounds can be classified as bioactive lipids (omega-3 fatty acids, oleic acid), carotenoids, bioactive peptides and phenolic compounds, among others [1,2]. Table 1 shows the different more common bioactive compounds, including their natural sources and their proven biological value.




Omega-3 fatty acids are long-chain polyunsaturated fatty acids, and their main bioactive forms are: docosahexaenoic acid, DHA (22:6 Ω-3), eicosapentaenoic acid, EPA (20:5 Ω-3) and α-linolenic acid, ALA (18:3 Ω-3). In fact, ALA is the precursor of the bioactive fatty acids EPA and DHA; nevertheless, its conversion rate in the human body has been described as low; thus, the bioactive forms of EPA and DHA must be obtained from natural sources. The traditional sources of omega-3 fatty acids, including EPA and DHA, have been fish and krill oil. This approach, however, supports the overexploiting of the ocean and abusive fishing. Therefore, new alternative biomasses are needed to obtain EPA and DHA. Microalgae are a promising source of bioactive compounds, especially omega-3 fatty acids, as they are the only nonanimal source of the bioactive form of omega-3. One of the main advantages of microalgae as omega-3 producers is their ability to grow in wastewater without competing with terrestrial plants for arable lands. Microalgae have a grow rate 2–3 times higher than that of terrestrial plants, and they are able to accumulate a wide range of bioactive compounds in their cells depending on the microalgae species. Among them, *Nannochloropsis gaditana*, *Isochrysis galbana*, *Tetraselmis chuii* and *suecia* and *Phaedodactylum tricornutum* are the main species producing EPA and DHA [1,3,4,45–48]. Omega-3 lipids have been proven to be related to the prevention and treatment of some world-recognized diseases, such as cardiometabolic disease and age-related macular degeneration, with promising results [5–11,49].

Carotenoids are a widespread group constituted by more than 400 different types of substances, which have been described in natural sources such as vegetables, egg, fish, algae and microalgae. The most common carotenoids included in nutraceuticals are lutein, zeaxanthin, fucoxanthin, astaxanthin and beta-carotene, which have been proven to be related to different biological values in clinical trials [20,21,32–34]. For instance, lutein and zeaxanthin have been shown to be alternative and effective treatments for early AMD after supplementation, and they have been proven to influence neurological and visual development during pregnancy. Lycopene has been shown to be able to reduce the risk of prostate cancer due to its antioxidant activity and ability to realize the induction of the apoptosis, the inhibition of cellular growth, the decrease in IGF-1 and IGF-BP-3, the induction of phase II enzymes and the modulation of androgenic metabolism [2,20–24,33,50–52].

It is interesting to highlight the wide biological properties that this group of bioactive peptides shows depending on the peptide studied, including antihypertensive, antioxidant, antiproliferative, anti-inflammatory, apiaceous, hypocholesteremia, antithrombotic and mineral chelator functions [40–44,53]. On the other hand, phenolic compounds, including flavonoids (anthocyanins, flavanols, catechins, gallo catechins, etc.), phenolic acids (caffeic acid, vanillin acid, etc.), lignans and stilbenes, show mainly antioxidant and antitumoral activities related to the reduced risk of cardiovascular disease due to the inhibition of LDL oxidation, an antihypertensive effect, anti-inflammatory effect and regulation of the immune response, platelet antiaggregant. Isoflavones found in soy, on the other hand, are associated with an antiestrogenic effect due to their interaction with 17-b-estradiol receptors [35–39,54–56].

#### **3. Challenges Encountered in Nutraceutical Stability**

Due to the complex nature of the different available bioactive compounds and the heterogenicity of them all (including molecular weight, charge, thermosensitivity or polarity), there are important challenges related to the properties of each one that must be considered. First of all, the low solubility of most of the bioactive compounds described should be highlighted, including, for instance, bioactive lipids or carotenoids. Due to this fact, optimal formulations must be designed to encapsulate these compounds. Indeed, if the final purpose of using a nutraceutical is its inclusion in the food matrix, the dosage of the bioactives is crucial, whereas micro/nanoemulsions must be produced to make them dispersible in the often-aqueous food matrix and resistant enough to pass through the rest of the food process. Additionally, the nutraceutical must be delivered in the target area at a controlled velocity, which will depend on the nanoparticle's constituents. Regarding

bioactive lipids, their low solubility, the crystallinity of some at room temperature and their thermosensitivity, which forbids the use of high temperatures in the process, should be highlighted. Additionally, the formulation should protect the bioactive from the stomach pH and digestion, maintaining the biological properties after these processes. This is also a crucial point for bioactive peptides, which should be protected from high temperatures, physiology pH and organic solvents, which may damage their structure and make them lose their biological value. Additionally, as some of them are bitter, they benefit from an adequate vehiculation system masking their flavor. Regarding phenolic compounds and phytochemicals, their solubility is affected by their polarity, functional groups, molecular weight, if it is oxidized or reduced, or if it is complexed to another molecule. Therefore, each compound should be studied deeply before choosing one production technique or another to preserve their biological value and to load it efficiently [57–62].

Taking all these aspects into account, the main key points that must be considered in the formulation of NPs including nutraceuticals are shown below (Figure 1).

**Figure 1.** Diagram representing the key points to consider in nanoparticle formulation for loading unstable bioactive compounds.

The main principles of different nutraceuticals to be considered in the selection of wall materials and nanodelivery system production procedures are described below:


Oleic acid or PUFAs are commonly used in the food and pharmaceutical industries. However, their susceptibility to oxidation may cause damage to encapsulated compounds through production of free radicals; thus, their use should be limited to highly stable compounds, avoiding carotenoids or omega-3 lipids. Emulsifiers have been used for stabilization of the lipid dispersions by reduction in interfacial tension between the lipid phase and the aqueous phase during the production of the particles, leading to fine nanocarriers. It has been found that utilization of a mixture of emulsifiers can be more efficient in preventing particle aggregation. A combination of tween 80 and lecithin caused smaller particles with a lower PI and higher stability due to an increased zeta potential in comparison to their use separately. Additionally, compared to ionic surfactants, tween 80 has low toxicity and is approved for use in specific food products and is GRAS [64,65].

• Bioactive thermosensitivity: the method used for NP production should be selected based on the bioactive compound properties, thermosensitivity and water solubility, which are the main factors affecting its selection. Homogenization at high pressure is the most used method in food industry; however, the high temperatures employed in this technique have led to the development of cold homogenization, which could be more adequate for carotenoids, bioactive lipids and peptides [65–67].

#### **4. Nanoparticles Used for the Loading of Nutraceuticals: Food-Grade Nanosystems**

Overcoming the main drawbacks related to some bioactive compounds, such as low water solubility, poor chemical and oxidation stability associated with thermosensitivity and photosensitive compounds, gastric degradation and thus poor bioavailability, must be the focus. Many of these challenges, such as the low water solubility, gastric degradation and the poor bioavailability, can be overcome by encapsulating these compounds into nanoparticles as efficient delivery vehicles contributing to the industry's economy. A circular bioeconomy has become a model for commercial production that enhances reuse, recycle and recovery with a smaller environmental footprint in nutraceutical industries. Nutraceutical industry costs are mainly related to the challenges mentioned above, requiring large numbers of bioactive ingredients to achieve an adequate in vivo effect, so many natural biomasses are still unexploited [68,69]. Nutraceuticals' production using lipid nanoparticles as carriers allows for their obtention in one stage of production with higher encapsulation efficiencies, as was reported in previous works, providing economic advantages, decreasing the number of process steps and the dosage of nutrients by increasing their bioavailability [66,70–73]. NPs overcoming the main challenges related to nutraceuticals have been widely reported in the scientific literature. Recent works on NP nutraceutical release showed an increased water solubility and in vitro bioavailability of curcumin [72]. Improved oxidative stability has also been described in NP omega-3 works [74]. Thus, the encapsulation of bioactive compounds in nanocarriers allows for the incorporation of natural biomasses into the circular bioeconomy.

Interestingly, lipid NPs' structure can overcome the abovementioned poor bioavailability by different causing factors. The main critical steps in the oral absorption of nutrients could be the rate of dissolution and the rate of nutrient penetration across the bio membrane. An essential prerequisite for the absorption of a bioactive compound is its ability to exist in a stable aqueous solution. This fact depends on its aqueous solubility and dissolution rate. Lipid-based nanoparticles improve the bioavailability due to their increased surface area. Additionally, by decreasing the particle size, the thickness of the diffusion layer is decreased, leading to faster transport and faster dissolution [57,63,64].

Going deeper, lipid NPs promote enhanced gastrointestinal (GI) absorption due to induced permeability changes caused by the surfactant, and due to the increased residence time in the stomach and upper small intestine owing to their lipidic nature and their adhesion to the intestinal underlying epithelium. The bioactive compound is protected from the harsh gastric conditions as it is encapsulated in the NPs, promoting their stability. On the other hand, NPs have low stability in acidic environments, which make them

degradable by gastric lipases [75]. Thus, lipid NPs are transformed by lipase and colipase into micelles (consisting of bioactive and lipid monoglycerides), stimulating bile flow to form mixed micelles. Mixed micelles are absorbed by chylomicron formation into lymphatic vessels, avoiding the first-pass effect and enhancing drug bioavailability alongside the fat absorption process. Through systemic and lymphatic transport, they increase the concentration of nutrients in the systemic circulation. On the other hand, nanoparticulate systems were reported to improve oral drug bioavailability by intracellular uptake by M cells of Peyer's patches. Indeed, transient opening of tight junctions (gaps between two adjacent intestinal epithelial cells) has been reported due to the effect of highly lipophilic surfactants, improving paracellular absorption [63].

Moreover, bioactive compound release from lipid particles occurs by diffusion and simultaneous degradation of lipid particles in the body. Controlled release from NPs can lead to a prolonged half-life and slows down the enzymatic attack in systematic circulation. The degradation rate, and therefore the kinetics of compound release, depend on the type of lipid used. The shorter the fatty acids of triacylglycerols, the faster the degradation rate. Surfactants including lecithin or sodium cholate can play an important role in accelerating the degradation rate by inducing attachment of the lipase/colipase to the nanoparticle [65,76].

Other main drawbacks must be studied to achieve an adequate formulation. Therefore, in order to produce lipid NPs with thermosensitive compounds, including omega-3 FA, carotenoids and flavonolignans, the selection of a low-melting-point lipid, such as glyceryl monooleate, glycerin, Monosteol ™ or Softisan ™, and a production procedure avoiding high temperatures, is a crucial step. It is also important to avoid the use of organic solvents that could damage the stability of the bioactive compounds, and to work with photosensitive compounds, opaque lipids must be selected as formulation components. Lipid components, solids and liquids (in the case of NLCs), will be selected based on these points, and based on their miscibility with the bioactive compounds.

Once the lipid components are selected, the surfactant type should be chosen as a function of the former. The lipid and surfactant content in the formulation will be the main factors affecting the chemical properties of the nanoparticles developed. Higher numbers of solid lipids (SL) will lead to a larger particle size, although it will also depend on the surfactant content [70,77]. However, to increase the NPs' load, it is necessary to increase the SL rate, and thus, compromise conditions must be selected for each case. Frequently, lipid NPs are produced through the combined use of Ultra-Turrax with sonication to reduce particle size. Nevertheless, the addition of the sonication step can also increase the polydispersity index and particle size distribution. On the other hand, when the target administration is intravenous or ocular, the rheology of the NPs produced must be studied to explore the NPs' behavior under different conditions. Ideally, the viscosity must decrease with the stress applied, whereas the loss and modulus storage must increase, with the storage always being higher than the loss (G > G"), to achieve an appropriate rheological behavior upon administration [25,58–60,62,70,77–85]. The lipid-based nanosystems solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) will be described, as well as the component types and the production procedures available. Table 2 lists the latest works on the production of NPs encapsulating nutraceuticals, highlighting the ones focused on lipid nanoparticles. Recent studies have also investigated the use of resistant starch or soy-protein-based polymeric nanoparticles for the encapsulation of ferulic acid and curcumin, respectively, showing higher stability of the nutraceutical loaded [60,84].



Lipid nanoparticles are similar to polymeric ones in terms of the solid matrix structure that they share. However, lipid nanoparticles appear to avoid the problems of toxicity of some polymers and solvents used in their production. Lipid nanoparticles are based on biodegradable lipids and emulsifiers, either lipophilic or hydrophilic. Among lipids, those mostly used were tristearin and tripalmitin, whereas amongs<sup>t</sup> the surfactant agents, soy lecithin and polysorbate 80 should be emphasized as lipophilic agents and poloxamer 188 and tween 80 as hydrophilic ones [31,54,70,80–83].

The other advantage of these kinds of systems is their ability to encapsulate either lipophilic or hydrophilic molecules, and the possibility of covering them with polymers to modify their characteristics, such as the use of polyethylene glycol or chitosan to modulate the permanence of the molecules in the mucous membranes of the organism. Indeed, lipid nanoparticles are versatile systems that can be administrated by different pathways, such as intravenous, oral, cutaneous, pulmonary, ocular and transdermal pathways. They also have limitations though, since during storage, the purest lipids tend to crystalize in a perfect crystal structure that may lead to the drug or bioactive being loaded. That is why a new generation of lipid nanoparticles appeared to solve this problem, the nanolipid carriers (NLCs), whose structure is just imperfect enough to avoid the expulsion of the loaded drug, which is achieved by formulations including not only solid lipids but liquid and solid lipids in combination, which increases the encapsulation efficiency and minimizes the expulsion of the bioactive compounds inside during the storage [25,58–60,62,70,77–86].
