Solid lipid nanoparticles (SLN)

C H A P T E R

1 Solid lipid nanoparticles (SLN): prediction of toxicity, metabolism, fate and physicochemical properties J.R. Campos1, P. Severino2,3,4, A. Santini5, A.M. Silva6,7, Ranjita Shegokar8, S.B. Souto9, E.B. Souto1,10 1

Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra (FFUC), P
olo das Ci^encias da Sa
ude, Coimbra, Portugal; 2Universidade Tiradentes (Unit), Aracaju, Sergipe, Brazil; 3 Instituto de Tecnologia e Pesquisa, Laborat
orio de Nanotecnologia e Nanomedicina (LNMed), Aracaju, Sergipe, Brazil; 4Tiradentes Institute, Dorchester, United States; 5Department of Pharmacy, University of Napoli “Federico II”, Napoli, Italy; 6School of Biology and Environment, University of Tr
as-os-Montes e Alto Douro (UTAD), Vila Real, Portugal; 7Centre for Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of Tr
as-os-Montes e Alto Douro (UTAD), Vila Real, Portugal; 8Capnomed GmbH, Zimmern, Germany; 9Department of Endocrinology, S. Jo~ao Hospital, Alameda Prof. Hern^ani Monteiro, Porto, Portugal; 10CEB - Centre of Biological Engineering, University of Minho, Braga, Portugal

1. Introduction

formulations [2]. Various controlled drug delivery systems like polymer-based controlledrelease systems, hydrogels, as well as nanoand microparticles have been introduced in recent years in order to improve solubility, stability and bioavailability of poorly water-soluble drugs. In this context, lipid nanoparticles offer attractive and ideal properties for drug or gene delivery. These particles (either composed of solid lipids only in SLNs, or of a blend of solid and liquid lipids in NLCs) stabilized with surfactants have the advantages of other colloidal

Solid lipid nanoparticles (SLNs) gained greater attention as a drug delivery system when in 1991 M€ uller developed them [1]. This promising drug carrier system is at the interface in the preexisting lipid systems (emulsions and liposomes) and polymeric nanoparticle systems. Lipid nanoparticles, known as SLNs or nanostructured lipid carriers (NLCs), have specific features of structure and composition, showing benefits in comparison to conventional

Nanopharmaceuticals https://doi.org/10.1016/B978-0-12-817778-5.00001-4

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© 2020 Elsevier Inc. All rights reserved.

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1. Solid lipid nanoparticles (SLN)

particles (polymeric nanoparticles, fat emulsions, and liposomes) by overcoming their limitations [3,4]. Taking account their polymeric and lipid raw materials, several modifications of drug delivery systems have been proposed to increase the bioavailability of loaded drugs. SLNs are made of a solid lipid matrix and a surfactant layer and they can load poorly watersoluble drugs, delivering them at defined rates and with improved bioavailability [5]. These colloidal drug delivery systems protect the drug against chemical degradation and modify its release profile since the drug is entrapped in the solid lipid matrix [6,7]. These nanoparticles of spherical shape have a mean size of 40e1000 nm [8,9]. The lipid matrix is composed of a solid lipid (or a mixture of solid and liquid lipids) in a 0.1%e30% (w/w) concentration dispersed in aqueous medium, and their stability is ensured by the presence of a surfactant in a 0.5%e5% (w/w) concentration [8] and can be used for lipophilic or hydrophilic drugs [9]. Triglycerides (tristearin), sterols (cholesterol), partial glycids (glyceryl monostearate), fatty acids (stearic acid), as well as waxes (cetylpalmitate) are especially used as lipids in the SLNs. In these systems emulsifiers and polymers are used as stabilizers in order to avoid aggregation of the particles. Examples of stabilizers are bile salts (e.g., taurodeoxycholate), lecithins, and copolymers of polyoxyethylene and polyoxypropylene (Poloxamer) [10]. It is clear that lipid nanocarriers are ideal for sensitive bioactive compounds. SLNs exhibit biocompatibility, matrix with lipophilic nature protecting active compounds of chemical degradation, drug targeting, controlled release profile, and high drug payload [9,12]. Moreover, they are suitable for industrial production mainly because they are easy to scale up, are stable under sterilization conditions, and they have the advantage of being non-toxic or of very low toxicity, because of their composition in Generally

Recognized as Safe (GRAS) excipients [4,6]. SLNs are biodegradable (fulfilling the requirements of preclinical safety) and are also stable in blood, with prolonged lifetime in the bloodstream [13e15]. In addition, compared to liposomes, SLNs exhibit high encapsulation efficiency, stability against light and oxygen, do not need organic solvents for their preparation, and have high drug-loading capacity (mainly for lipophilic compounds) [12,15,16]. On the other hand, SLNs have also limitations, mainly attributed to the risk of polymorphic transitions (from a to b0 , and from b0 to b) which causes stability challenges during administration or storage, resulting in drug expulsion from the particles and eventual particle size increase [6,10,17]. These disadvantages are related to the crystallization behavior and lipid matrix’s polymorphic transitions, which depend on the type of lipids used for the production of SLNs [6]. There are various methods described in the literature to produce SLNs based on solidified emulsion technologies: high shear homogenization and ultrasound, high pressure (hot and cold) homogenization, oil/water (O/ W) and water/oil/water (W/O/W) microemulsions, as well as solvent evaporation [10]. These techniques interfere with various characteristics of the particles, mainly in morphology. According to the literature, the most commonly applied methods are those that use high pressure homogenizers (HPH). M€ uller and Luck developed the HPH technique (European Patent No. 0605497) for obtaining nanoemulsions for largescale parenteral nutrition [10]. There are diverse kinds of equipment with various sizes, prices as well as different capacities. The different equipments work by pulling the liquid in high pressure (100e2000 bar) through a narrow piston (nanometer scale), which is accelerated over a small distance at a high speed (over 1000 km/h). This fluid is subjected to high stress, disrupting the macroscopic oil droplets by cavitation forces and thus generating the nanodroplets [10]. In

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2. Toxicity profiling

the hot process, the hot preemulsion is passed through the hot homogenizer to obtain nanoemulsions, which are then cooled down in order to solidify and crystallize the hot inner liquid phase to obtain SLNs. In the cold process, the drug is firstly ground milled in a mortar mill with the solid lipid at room temperature, and then the obtained powder is dispersed in an aqueous surfactant solution, which is then subjected to HPH. The influence of the type of homogenizer, pressure, and number of cycles employed, and the temperature used to obtain the ideal particle size, have been intensively studied. Depending on the type of lipid, it is possible to use lipid concentrations above 40% and obtain the particle size distribution in a low range (polydispersity index <0.2) [18]. To obtain SLNs from microemulsions, Gasco and collaborators developed a technique that has been modified by numerous researchers. In this technique, SLNs are produced by diluting a hot oil-in-water (O/W) microemulsion in high volume of coldwater (0e4o C). The internal phase of this microemulsion is composed of low-melting lipids and, when in contact with the cold water, they suffer crystallization and form SLNs [18e20]. The type of lipids used to make the microemulsion, the preparation parameters (stirring and temperature), rate of microemulsion’s addition in the cold water, volume of water, and the technique used to remove the excess water, all affect the characteristics of obtained nanoparticles [18]. This technique is therefore difficult to scale up. Marengo has developed a device that processes 100 mL of microemulsion and can produce SLNs of mean size below 100 nm [20]. The biomedical applications of lipid nanoparticles are manyfold. Indeed, they can be used as drug and gene carriers, and as contrast agents for imaging analysis [21]. In recent years, different administration routes (e.g., oral, parenteral, dermal, pulmonary, rectal, ocular) have

been explored for drug delivery using lipid nanoparticles. Components of lipid nanoparticles (lipids and surfactants) determine the product quality, its physicochemical properties, as well as the administration route [4] (Table 1.1). In this chapter, the concept behind the SLNs and their physicochemical properties, pharmacokinetics, and biopharmaceutics and their toxicological testing are discussed.

2. Toxicity profiling SLNs are known to be stable in aqueous dispersion, allows the encapsulation of hydrophilic and lipophilic drugs, are adaptable to several administration routes, can modify the release profile and avoid their adverse effects (protecting the drug from undesirable interactions or directing it to its target) [30]. However, their toxicological profile has to be very well characterized in vitro before any pre-clinical and clinical studies [31]. There is a relationship between the size of the particles and their toxicity, as the lower the size, the higher the surface area and the higher the reactivity. A prerequisite to be marketable is the GRAS status of the excipients of SLNs [32], but additional nanotoxicological studies are needed to allow the understanding of the effect of nanoparticles in the body [14,33,34]. Nanotoxicology helps in identifying the SLN formulations to be selected for preclinical studies by evaluating their safety, tolerance, and cytotoxicity. Preclinical toxicological studies allow to determine the concentration of substances that cause toxic effects, and allow identification of target organs predisposed to these effects. Preclinical safety tests require the appropriate selection of the animal species, age, physiological status, the administration route, dosage form as well as the treatment regime. Preclinical

TABLE 1.1 Commonly used lipids in the composition of SLNs/NLCs. Name

Chemical structure

Therapeutic application

Surfactant

System

Drug

Dihexadecyl phosphate

SLNs

e

Pulmonary delivery

[22]

PEGs

SLNs

e

Oral delivery

[23]

Glyceryl monostearate

Mixture of Tween 80 and Span 80

SLNs

e

e

[24]

Stearic acid

Omega-3 PUFA

SLNs

Resveratrol

Oral delivery

[25]

Cystamine

SLNs

e

e

[26]

Tween 80

NLCs

e

e

[27]

Pluronic F-68

NLCs

Simvastatin

Oral delivery

[28]

D-a-tocopheryl polyethylene glycol succinate (TPGS)

NLCs

Rapamycin

Ocular delivery

[29]

Tristearin

Cholesterol

Vitamin E O R

O n O

O O

O

References

3. Physicochemical properties

toxicological evaluation of a new compound can provide information about acute, subacute, subchronic, and chronic toxicity. Before any preclinical study, cytotoxicity assessment is carried out in cell culture, also allowing the study of the interaction between nanoparticles and cells. The use of primary cells (isolated directly from the animals) provides more realistic toxicological results. On the other hand, in vivo studies evaluate the organism as a whole. Therefore, in vivo studies are relevant to determine the location and concentration of the drug in the tissues, and systemic toxicity [35]. Systems with large quantities of surfactants (e.g., nanoemulsions) have higher risk of exhibiting cytotoxicity, since these agents interact with cell membranes composed of phospholipids. Likewise, their production requires the use of organic solvents, which may also increase the risk of toxicological events if poorly removed. Although precise determination of the toxicity can only be quantified in vivo, there are several in vitro toxicological tests that provide preliminary information [10,14,36]. In vitro studies have shown that SLNs are acceptable at concentrations <1 mg/ mL (total lipids), and with particle diameter >500 nm can be less tolerated, which can be explained by their aggregation. It was also shown that the stabilized formulations composed of several surfactants are less biocompatible in comparison to those based on one surfactant only. For polysorbate 80 and poloxamer 188, two surfactants mostly used in SLNs formulations, enough evidence has been found to determine their safety [14]. It is clear that the knowledge of the toxicological profile of any material and the biocompatibility of the drug delivery systems are crucial for the implementation of drug therapies, but the information of

EEð%Þ ¼

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nanoparticle-based drug therapies available is still very limited [8].

3. Physicochemical properties The characterization of SLNs usually requires the determination of the particle size and zeta potential, morphological evaluation, determination of the loading capacity and encapsulation efficiency, kinetics of drug release, as well as the nanoparticles over time and storage temperature.

3.1 Encapsulation parameters Determination of the amount of drug associated with the nanoparticles is a crucial parameter for the characterization of SLNs. This task is however difficult because of the small size of the particles, which compromises the separation of the free drug fraction from the associated fraction. Ultracentrifugation is the most commonly used separation procedure, after which the non-loaded drug is quantified in the supernatant. From the difference between total drug and the drug found in the supernatant, the drug concentration associated with nanostructures is calculated [37,38]. Ultrafiltration can be coupled to the process of ultracentrifugation. In this approach, a membrane (100 kDa) is used to separate the aqueous phase from the nanoparticles. Although the free drug concentration in this technique is determined in the ultrafiltrate, the drug fraction associated with the nanostructures is also found by the difference between the total and free concentrations [39]. Encapsulation efficiency (EE%) and loading capacity (LC%) are determined using the following equations [40]:

Amount ðmgÞ of loaded drug determined experimentaly  100 Theoretical amount of drug ðmgÞ in formulation

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1. Solid lipid nanoparticles (SLN)

LCð%Þ ¼

Amount ðmgÞ of loaded drug determined experimentaly  100 Theoretical amount of lipid ðmgÞ in formulation

3.2 Particle size

3.3 Zeta potential

Size and polydispersity index are parameters that indicate the stability of the nanoparticles. An optimized SLN formulation should exhibit a mean particle size less than 1 mm, together with a small polydispersity index. Several parameters affect the particle size and polydispersity, e.g., composition of the formulation (mixture of surfactants, lipid structural properties, and also incorporated drug), methods and conditions of the production (time, temperature, stirring velocity, pressure, etc) [41]. There is a relationship between the proportion of surfactant/lipid and the particle size, i.e., the greater the concentration of surfactant, the smaller the particle size [42]. The temperature used in the HPH technique is another parameter that interferes with the particle size. In the production of SLNs and NLCs, the lipid phase is heated up to a temperature higher than the melting point of the solid lipid. The final step is the cooling down of the systems so that the lipid recrystallizes to solidify the lipid droplets [43]. Homogenization at low temperatures (i.e., below the melting point of the solid lipid) favors the increase of particle size, but temperatures near the melting point improve the homogenization and lower particle sizes can be obtained. Similarly, the hot HPH procedure produces particles with lower mean size and polydispersity than the cold procedure [41]. Generally, the particle size increases when lipids of long fatty acid chains are employed. The use of mixtures of short- and long-chain fatty acids may therefore reduce the mean particle size and polydispersity index of SLNs/NLCs [4,44].

Zeta potential is another parameter used to evaluate the long-term stability of the particles and its assessment is instrumental for the physicochemical characterization of the nanoparticles' dispersion. When determining the zeta potential, it is possible to understand the interactions between the particle and the drug. This parameter shows the surface electrical charge of the particles, which is modified by changes in the interface with the dispersing medium since there is a dissociation of functional groups on the particle' surface and adsorption of ionic species of the aqueous dispersion medium [39]. The measurement of this parameter allows to clarify the stability of the formulation during its storage time. The higher the zeta potential, the higher the electrostatic repulsion between the particles, resulting in reduction of the risk of particles' aggregation. According to literature, a zeta potential higher than the 30 mV modulus guarantees the stability of SLNs [45]. However, there are SLN formulations with zeta potential below |30 mV| that remain stable over time due to the stereochemical stability offered by surfactants [46]. Changing a formulation by varying the concentration of its components, allows understanding which of those whether contribute to the stereochemical stability or to the electrostatic stability [47]. Differential scanning calorimetry, based on the study of the peaks of SLN formulation recorded on the thermogram, allows assessing if the drug is loaded within the particles and eventually its contribution to the surface electrical charge of SLNs [48].

3. Physicochemical properties

3.4 Particle morphology To learn about the shape and size of nanoparticles, scanning (SEM) and transmission electron microscopy (TEM) are commonly used [50]. Atomic force microscopy (ATM) is another technique that gives information with high resolution in three dimensions at the nanometer scale (even surface details at the atomic level), and is also used to understand the surface morphology of nanoparticles [39].

3.5 Differential scanning calorimetry The physical and chemical changes of a sample can be measured as a function of its temperature; differential scanning calorimetry (DSC) quantifies the loss or heat gain resulting from these changes. There are two types of DSC instruments: (1) the power compensate DSC, which is made of two separate ovens; and (2) the heat-flux DSC, which has only an oven that heats up the reference and sample pans. The sample and reference receive the heat through the sample pan, which is placed in a disc that is the main source of heat. Both differential heat flow and sample temperature are monitored, and the calorimetric sensitivity is maintained by the software linearization of the cell calibration [52]. The endothermic processes, i.e., those that absorb heat, include fusion (melting), boiling, sublimation, vaporization, desolvation, as well as solid-solid transitions. The crucial exothermic process, i.e., the one that releases energy, is crystallization. These analyzes can be used to identify materials, investigate their purity, polymorphism or solvation, analyze quantitative and qualitative degradation, aging, glass transition temperature, as well as their affinity to other substances. DSC is useful to obtain information about the degree of crystallinity of the lipid matrix and polymorphic behaviour. In

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DSC analysis, a melting point depression is usually observed when transforming the bulk lipid into lipid nanoparticles [48,52].

3.6 Stability of formulations and release profile The stability of the loaded drug within lipid nanoparticles is dependent on the chemical composition of the lipid matrix (e.g., type of lipid, surfactant) and production procedure. Upon storage, triglycerides undergo polymorphic changes that may result in drug expulsion from the lipid nanoparticles [53]. Surfactants also play a relevant role on the crystallization behavior of the lipid nanoparticles, i.e., whether the recrystallization of the particles occurs onto their surface or within the lipid core [54]. The long-term stability of lipid nanoparticles over storage is therefore dependent on the lipid and surfactant composition of the particles and of the production procedure [55]. The presence of imperfections in the lipid matrix offers higher capacity to accommodate drug molecules, improving the loading capacity of the particles [19,56]. To achieve a modified release profile of the loaded drug, the particles should however retain the drug during storage until their administration. To increase the encapsulation efficiency, the use of mixed lipids (having fatty acids with different liquid and solid chain lengths) is usually recommended. These blends create small liquid reservoirs inside the particles e as happens in the NLCs e delaying the polymorphic changes over storage [18, 53, 57]. The encapsulation efficiency of lipophilic drugs is also higher in SLNs and NLCs than hydrophilic drugs. To load these latter in lipid matrices, other strategies are needed such as the development of insoluble conjugates between the

8

1. Solid lipid nanoparticles (SLN)

lipid and the drug by a covalent bonding [58, 59]. Hydrophilic drugs might show higher risk to be partitioned to the aqueous phase during the production of SLNs and create a drugenriched shell model. Indeed, upon cooling of the dispersions, the lipid solidifies first, crystallizes and forms the cores in which the hydrophilic drug will be precipitating onto their surface. This type of SLNs (drug-enriched shell model) does not exhibit a modified release profile but rather a fast release attributed to the presence of drug onto the surface of the SLNs. For drugs that solidify first (e.g., of melting point higher than that of the solid lipid) or for lipophilic drugs, a drug enriched core model can be produced, which exhibits a modified release profile [17]. Typical methods used for assessment of the in vitro release are the dialysis and the static or dynamic Franz diffusion methods. The assays can be designed so that isotonicity and pH value can be adjusted to mimic the intended administration route, and can also include the effect of protein adsorption, plasma compatibility, whole blood compatibility and sterilisation. It is known that in a physiological environment, proteins can bind the surfaces of nanoparticles, forming a nanoparticleeprotein complex that influences the biological response. Nanoparticles can be incubated with bulk serum, plasma and also with solutions of individual proteins, in order to evaluate which physical properties are responsible for the protein binding onto their surface [60], such as the type of polymer used to stabilize the particles [61,62]. Freeze-drying may also be used to enhance the long-term stability of SLNs and NLCs. Lipid nanoparticles can be transformed in a dry product to improve their physicochemical stability [63].

4. Administration routes and drug bioavailability Pharmaceutical nanotechnology comes up as a strategy to improve the bioavailability of poorlywater soluble drugs, enhancing their therapeutic effectiveness and reducing the risk of adverse reactions [64e67]. SLNs have been extensively exploited as an interesting approach to improve the drug’s bioavailability in particular those of class II and IV of the biopharmaceutical classification system (BCS) [64, 67]. Indeed, due to their lipid composition, they may act as absorption enhancers [67]. On the other hand, surfactants surrounding the particles besides ensuring their steric stability in aqueous dispersion, they induce specific surface-chemical properties and may also modulate the biopharmaceutical profile. For the selection of the best surfactant, several parameters have to be taken into account e.g., hydrophiliclipophilic balance (HLB) values, their effect on the lipid polymorphism and on the particle size. The HLB values for the stabilization of oil-inwater dispersions vary between 8e18 [68]. The right choice of the surfactant minimizes the risk of production of particles’ aggregates which may compromise the stability of the dispersion in vitro and its performance in vivo [45].

4.1 Topical and dermal routes The administration of drugs through the skin may contribute to increase the drug’s bioavailability as it overcomes the first-pass metabolism. This administration route reduces the in inter/intra-patient variation and increases patient compliance. However, it is also associated with interactions of drug and/or excipients with skin that may cause irritation [69]. The

4. Administration routes and drug bioavailability

loading of drugs within lipid nanoparticles can minimize the risk of skin irritation and allergenic reactions, by preventing direct contact between the drug and the skin and by controlling the drug release through the skin [14,70e72]. Besides, as they are composed of biocompatible and physiological lipids of GRAS status, SLNs and NLCs exhibit low risk of acute and/or chronic toxicity [10]. Most of the surfactants used in the production of SLNs and NLCs are already used in topical pharmaceutical or cosmetic formulations. To further reduce the risk of irritation, the surfactants should be non-ionic, while polyethoxylated surfactants should also be avoided [14,42,49,73e75]. There is evidence that most of the PEG-free surfactants have shown to stabilize lipid nanoparticles without the need of cosurfactants. In addition, when compared to the PEG-containing counterparts, the PEG-free surfactants have been shown to require less concentration of surfactant to obtain small and uniform particle size [76]. The methods used to evaluate the skin irritation include typical in vitro test methods based on the reconstructed human epidermis (RhE) and also in vivo animal tests. In vivo experiments are more useful but their cost, tight regulation, and ethical issues towards the promotion of reduction, reuse, and recycling imply the need to develop improved in vitro tests [77]. New strategies to decrease skin irritation are based on the use of controlled release systems, i.e., creating a gradual drug delivery that prevents the accumulation of high concentrations of drug in the skin, which are usually responsible for this skin irritation, and also increase drug deposition in the pilosebaceous unit, which reduces the dose frequency and risk of adverse events [78]. Lipid nanoparticles have additional advantages as they can prevent and even reduce skin irritation by the reinforcement and repair of the stratum

9

corneum lipid film. Indeed, these nanoparticles create a protective lipid film onto the skin upon their topical application, promoting skin hydration [76].

4.2 Ocular delivery For the treatment of eye diseases, direct ocular instillation is the most accepted approach by patients. Lipid nanoparticles have also gained interest as drug carriers for this administration route, due to their biocompatibility with the ocular tissues, mucoadhesiveness and modified-release profile [79e81]. Conventional ophthalmic solutions have low precorneal retention time. Lipid nanoparticles increase the retention time of ocular drugs, improving their bioavailability [82]. To be suitable for ocular instillation, lipid nanoparticles should be of small particle size to avoid blurred vision and discomfort [83]. To avoid damage of the corneal tissues, inflammation and immunologic reactions, the formulations also need to exhibit sterility, isotonicity and pH between 7e9. Toxicity of the formulations that could alter the corneal epithelial integrity or disrupt the tissue, resulting in deficient drug delivery into eye (which is not their aim) also need to be considered [84]. The Draize rabbit eye test is routinely used to evaluate eye irritation. This test was developed with the aim to predict human eye irritation of pharmaceutic and cosmetic products. Its drawbacks are the ethical issues associated with the use of animals, its costs and the number of variables of the test [85].

4.3 Oral administration Oral delivery is painless and is easy for selfadministration. This administration route has high patient compliance and is appropriate for outpatients. All these advantages make it the most accepted drug administration route [86].

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1. Solid lipid nanoparticles (SLN)

However, the gastrointestinal tract has chemical and enzymatic barriers that limit the effectiveness of oral drug delivery, and also show low permeability for several drugs [87]. By optimizing the formulations, it is possible to ameliorate their efficiency and bioavailability, promoting the therapeutic potency and reducing side effects. Lipid nanoparticles may be used as absorption enhancers through the gastrointestinal tract to improve the oral bioavailability of several drugs [88]. SLNs developed for oral administration have been shown to enhance and control drug delivery, mainly due to the specific characteristics of the surface modification, increased permeation of the gastrointestinal tract, as well as resistance against degradation. The solid state of the matrix can protect chemically unstable drugs and promote the drug residence onto the site of absorption. SLNs have shown low cytotoxicity against mammalian cells and high tolerance in vivo. They can be further formulated in classical dosage forms e.g., capsules, tablets or pellets for oral administration [89, 90]. To improve the formulation stability, SLN conversion into powders for further processing into solid dosage forms have also been proposed. Shegokar et al. investigated the possibility of enhancing the long-term stability of nanoparticles with freeze-drying studies, converting SLNs in a dry product and observing that have good size distribution, which also proves that SLNs are a suitable drug delivery system [63]. There is still a demand to design and develop new SLNs to obtain a viable release profile, which depends on the drugs selected for these formulations as different drugs have different physicochemical characteristics and also different interactions with nanoparticles. The release profile from solid dosage forms needs to be critically controlled; a burst drug release can be linked to toxicity problems whereas a slow drug release that can cause

inefficient activity in treating the diseases [91]. Many oral SLN tests in cell-based and animal studies are described in the literature, however, their clinical trials are still limited due to their cost, as well as the unknown side effects (they have to be investigated first). From the commercial point of view, to be viable the nanoparticles have to show 5-fold-improved oral bioavailability or other convincing benefits [91].

4.4 Parenteral administration Until now, typical SLN components have not yet been used in parenteral formulations, except medium-chain triglycerides (MCTs) and polysorbate 80 [92]. Usually, the final suspension for injection contains solid particles, which means that its safety profile will have to be confirmed. It is known that the parenteral dispersion should not show any solid particles visible to the naked eye. The admissible limits of solid particles of size below 50, 25, and 10 mm are not yet defined, and are still open to discussion, and there is no available regulation of particles with size below 1 mm. SLNs have shown intensity with a diameter below 1 mm and, in some cases, below 200 nm, but it is necessary to take into account that they have a tendency to aggregate over time (especially when exposed to high ionic strength environment or proteins) [14,93,94]. The key parameters to be evaluated for parenteral application include understanding of plasma drug profile, fate of nanoparticles, and acute and chronic dose toxicity. Some studies have been published on bare and surface-coated nanoparticles for parenteral application, lab to large scale, targeting, toxicity studies, solid product conversion, phagocytic uptake studies, cytotoxicity studies, as well as organ distribution studies. For example, Shegokar et al. evaluated the potential of lipid nanoparticles for active delivery of an antiretroviral

5. Conclusions

drug to lymphatic tissues. SLNs were developed taking account of various physicochemical parameters, e.g., appearance, particle size, polydispersity index, as well as zeta potential. Authors investigated the targeting potential of these SLNs through ex vivo cellular uptake studies, showing an enhanced uptake in comparison to pure drug, and the lymphatic drug levels and organ distribution studies also showed the efficiency of these nanoparticles for prolonged residence. The study demonstrated that these lipid carriers can be used for effective and targeted drug delivery, enhance the therapeutic safety and decrease collateral effects [95,96].

4.5 Nasal and pulmonary delivery Lipid nanoparticles are also a new approach for controlled drug delivery to lungs. There are various studies of SLN formulations with the aim of administration by inhalation. Many that have been developed for delivery of antifungal and antimicrobial drugs have proved efficacy in vivo. Nasal administration is commonly used to deliver drugs to the central nervous system (CNS) in a noninvasive way, thereby providing higher patients' compliance. SLNs for intranasal administration are gaining more attention lately but there are still few reports about their safety; adverse effects have been observed in laboratory animals, probably due to encapsulated drugs, inappropriate SLN doses used for these studies, or faulty selection of excipients [14].

5. Conclusions SLNs and NLCs are the most studied lipidbased drug delivery systems, having potential to deliver drugs and also nutrients for several administration routes due to their biocompatibility, low toxicity, high-loading capacity, slow release rate, and high stability. They are in development as drug carriers for administration by

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various routes, including dermal, ocular, and oral, with the dermal route the safest. Lipid nanoparticles are composed of biocompatible and biodegradable lipids, with a melting point above 40 C to ensure solid status at room and also at body temperatures. SLN production is based on the incorporation of the drug in the melted lipid and then mixed with the aqueous surfactant solution, and they can be made by high energy techniques (e.g., ultrasound methods and supercritical fluid technologies) or low-energy techniques (e.g., solvent emulsification evaporation, coacervation, microemulsion technique, and phase-inversion temperature method). Lipid nanoparticles protect the drug against chemical degradation and achieve controlled drug release, once the drug is entrapped in a biocompatible lipid core surrounded by a surfactant at the outer surface. Melt temperature and crystallinity index as well as the selection of excipients and SLN dose are their most important features. They need to be evaluated for final product stability, their thermal behavior during storage, as well as their safety. Nanoparticles have physicochemical properties that give them exceptional biological activity, with their toxicological profile dependent on these properties, mainly particle size and size distribution, as well as zeta potential. This is crucial in toxicological studies because it allows assessing their toxic effects, identifying routes of exposure as well as predicting the risks of their synthesis or use. The potential toxicity and the biocompatibility of drug delivery formulations are crucial for the implementation of drug therapies. Although the toxicity mechanisms of nanoparticles have been well studied, the available information about nanoparticlebased drug therapies is still very limited. In summary, the development of SLN and NLC formulations continues to grow, with many patents created worldwide. Toxicological testing documents that lipid nanoparticles are safe drug carriers for the various administration routes.

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1. Solid lipid nanoparticles (SLN)

Abbreviations 3 Rs 7AAD ATM AUC BCS BHT CNS DMSO DNA DPPH DSC EE% FBS GRAS HIV HLB HPH HPLC MCTs TEM SEM NLCs O/W ORAC PBS PEG RhE SLNs SRE W/O/W W/W

Replace, reduce, and refine 7-amino-actinomycin D Atomic force microscopy Area under the curve Biopharmaceutical classification system Butylhydroxytoluene Central nervous system Dimethyl sulfoxide Deoxyribonucleic acid 2,2-DIPHENYL-1-PICRYLHYDRAZYL Differential Scanning Calorimetry Encapsulation efficiency Fetal bovine serum Generally recognized as safe Human immunodeficiency virus Hydrophilic-lipophilic balance High-pressure homogenization High-performance liquid chromatography Medium-chain triglycerides Transmission electron microscopy Scanning electron microscopy Nanostructured lipid carriers Oil/water Oxygen radical absorbance capacity Phosphate-buffered saline Polyethylene glycol Reconstructed human epidermis Solid lipid nanoparticles Transcription factor e the GATA factor Water/oil/water weight for weight

Acknowledgments The authors acknowledge the financial support received from Portuguese Science and Technology Foundation (FCT/MCT) and from European Funds (PRODER/COMPETE) under the project references M-ERA-NET/0004/2015-PAIRED and UID/QUI/50006/2013, cofinanced by FEDER, under the Partnership Agreement PT2020.

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