Lipid and Polymeric Nanocapsules

Abstract

In recent years, innovative drug nanocarriers have been developed to enhance stability, bioavailability, and provide sustained release. In this chapter, systems based on natural macromolecules, lipids, or polymeric/polyelectrolyte nanocapsules and their principal chemical and functional characteristics are described. Nano-vesicular systems are especially relevant in different fields. Particularly, a promising potential is offered by systems based on colloidal nanocapsules, that exhibit a typical core-shell structure in which the drug can be confined into the cavity or in the polymeric coating that surrounds it. Both the cavity and the active substance can be lipophilic or hydrophilic and in solid or liquid form depending on the materials and methods used, making these nanocapsules attractive carriers for drug delivery. In addition, a compilation of different methods and materials employed in the preparation of these nanosystems and a recent review of applications of lipid and polymeric nanocapsules have been made, focussing on the encapsulation of drugs.

1. Introduction

Nanostructures appear as either nanofibers, nanocompounds, nanomembranes, nanoparticles, or nanotubes, and have applications in different fields, such as medicine, cosmetics, environment, and nutrition. They can be used in biomedicine for diagnosis or the prevention and treatment of different diseases. They are employed as drug, protein, nucleic acid, and peptide carriers, or as biosensors, as well as for medical imaging [1, 2]. The incorporation of active principles in nano- or micrometric scale devices is called encapsulation. The encapsulated material is covered with a different type of material, which can be a polymer, a lipid, or a macromolecule. In general, encapsulation provides an increase in the stability of the encapsulated material, it also preserves its chemical and therapeutic properties, and it enlarges its average life, since it protects it from the effects of pH, heat, light, oxygen, humidity, and even from enzymatic degradation by nucleases and proteases, for instance. Besides, encapsulation offers the possibility to modify the physicochemical properties of the encapsulated material to facilitate manipulation, it reduces the loss of volatile compounds, it can mask unpleasant flavor and odor, improve bioavailability, and help the controlled release of active substances following a certain stimulus (pH, T, P, etc.) [3].

Regarding medical applications, encapsulated systems offer great possibilities of improving the safety and efficiency of countless drugs. They are capable of traveling across biological barriers, such as the skin, the gastrointestinal or respiratory mucous membranes, and even the blood-brain barrier (BBB). They can reach the target organ, tissue, or cell group where the drug has to act; they can even reach intracellular compartments. The distribution of the active principle, and therefore, its concentration in the target, is influenced by the size and the properties of the nanoparticles. This dependence permits minimization of side effects and an increase in the therapeutic power of the released molecule of interest, for example, in cancer treatment. Administration in the form of nanoparticles allows to orally dispense antitumor drugs, as well as biotechnologically originated molecules (peptides, proteins, plasmids, etc.), which are very sensitive to physicochemical and enzymatic degradation and cannot cross the mucous membranes [3].

Organic nanocarriers include nanoparticles such as solid lipid nanocarriers, liposomes, dendrimers, polymeric nanocarriers, micelles, and viral nanocarriers. Nanoparticles are defined as solid vesicles under 1000 nm, usually between 100 and 500 nm, formed by natural macromolecules, lipids, or synthetic polymers. For therapeutic application they must have a size under 200 nm since the width of the microcapillaries of the body is 200 nm [1]. The active principle is incorporated inside the nanoparticle, which can be a nanocapsule or a nanosphere. Nanocapsules are kind of reservoirs, they are vesicular systems, that is to say, traditional hollow shell structures constituted by a polymeric or lipid membrane and an internal core where the molecules of the drug are dissolved or dispersed. As to nanospheres, they are spherical matrixial systems, and the drug is homogeneously dispersed in the solid polymeric matrix [2, 4]. Core-shell nanoparticles offer great versatility and, depending on their composition, permit the encapsulation of a huge variety of molecules in solid, liquid, and semi-solid state. The nanocapsule shells can be prepared from several materials, such as polymers, lipids, phospholipids, and silica [5]. Different methods are used to build the core-shell structure, the polymers and methods employed are chosen according to the properties of the compound to be encapsulated and its application [6].

Some criteria to be considered before nanoencapsulation are clear definition of the desired objective, assurance that the active principle does not degrade during the fabrication process and that it disperses homogeneously inside the nanocapsule, choice of a suitable polymer or macromolecule, and cost/performance optimization. The encapsulating material must be biodegradable/bioerodible and inert with respect to the encapsulated material both in production and storage, offer the highest protection of the active principle, and be processable in suitable solvents for biomedical application. There is a real influence of the nature of the material and the production methodology on the physicochemical properties of the prepared systems. The main characteristics of nanoparticles are their large specific surface area, their homogenous dispersion in fluids, the capacity to encapsulate small molecules, and an adequate release rate [3].

In this chapter, systems based on natural macromolecules, lipids, or polymeric/polyelectrolyte nanocapsules and their principal chemical and functional characteristics are described. In addition, a compilation of different methods and materials employed in the preparation of nanocapsules and a recent review of applications of lipid and polymeric nanocapsules have been made, focussing on the encapsulation of drugs.

2. Common materials

As it has been mentioned above, polymeric nanoparticles and lipid-based systems are included in the research of efficient drug-delivery systems. Lipid polymer hybrid nanoparticles (LPNs) are being explored as well, combining the advantages and properties of polymers and lipids [7]. In this chapter, mainly polymeric and lipid nanocapsules are dealt with.

2.1 Polymeric nanocapsules

Polymeric nanoparticles are colloidal and are prepared from degradable/nondegradable hydrophilic/hydrophobic natural/synthetic polymers. Natural polymers, such as polysaccharides (hyaluronic acid, starch, maltodextrins, chitosan, cyclodextrins, alginate, carrageenan, gums, and agar) or proteins (gliadin, vicilin, legumin, casein, gelatines, and albumin) are not usually used alone due to the variability of their purity. Nanocapsules based on saccharides (glyco nanocapsules) are very interesting for bioapplications. The synthetic polymers usually used are poly(lactic-co-glycolic) acid (PLGA), poly(lactic acid) (PLA), poly(ε-caprolactone) (PCL), polyanionic cellulose (PAC), poly(D,L-glycolide) (PLG), polyethylene glycol (PEG), and poly-cyanoacrylate (PCA) [1, 7, 8, 9, 10]. Polymeric nanoparticles made from natural or synthetic polymers are easy to modify superficially and are, in general, stable. Their features can be tuned to achieve better bioavailability and a controlled drug release in specific locations. Biodegradable polymers have been widely used for the preparation of systems to control drug release, which can stabilize certain labile molecules, such as proteins, peptides, or DNA, and can also be used for site-specific drug targeting. The preparation of biodegradable polymeric nanoparticles for application in tissue engineering is also pursued [1]. As they are biodegradable, they can remain for days or even weeks and release the drug in the target during that time. PLA and PLGA have proved to be effective with intracellular drugs. PLGA is usually combined with PEG, as PEGylation increases solubility and stability in water, reduces intramolecular aggregation, decreases immunogenicity, and prolongs the permanence of the drug in the systemic circulation [1, 7]. As an example, a recent review (in press) shows a representation of some modifications on the surface of polymeric nanocapsules (polymer-coating; PEG-coating; layer-by-layer; polymersomes; and inner portion—hollow-core) [5].

Chitosan, alginate, and cellulose are the natural polymers most widely used in medicine due to their geometry, their large specific surface, their mechanic and barrier properties, their low toxicity, and their biodegradability and biocompatibility [11, 12]. Chitosan is a biodegradable cationic polymer that can be obtained from crustaceans, insects, mollusks, and fungi. Usually, it is obtained for industrial use from crustacean exoskeletons, mainly from the waste products of the fishing industry. The properties that make it interesting for use are its molecular weight, deacetylation degree, solubility, biocompatibility, and bioadhesion. It presents antimicrobial activity against fungi, viruses, and bacteria. The application of nanospheres and nanocapsules of chitosan in cartilage and bone regenerative medicine is currently being studied due to the aforementioned properties [13]. The polysaccharide alginate is used in therapeutics because of its biocompatibility, low immunogenicity, and ability to gelation. Moreover, it is a pH-sensitive polymer that can be used to prevent drug release in an acid medium, such as gastric juice, when it is necessary for the active to be released in an alkaline medium (e.g., in oral administration of intestinal targeted drug delivery) [6]. In Ref. [14], nanospheres with alginate and chitosan loaded with the insecticide captan hydrochloride are obtained. Cellulose can be extracted from different natural sources. It is highly abundant, and the development of cellulose-based systems for drug release applicable to cancer therapy has increased enormously in the last decade [11].

Proteins, such as albumin, which are biocompatible and able to be tuned, are also used as polymeric shells for nanocapsules. Albumin is water-soluble and biodegradable. Apart from controlling drug release rate, albumin can reduce the nanosystem immunogenicity, and it can be useful as a drug targeting vector [6].

In Ref. [15] several polymeric systems are described, containing hybrid lecithin/chitosan nanoparticles, PCL nanocapsules stabilized with the non-ionic surfactant polysorbate 80, and polymeric PCL nanocapsules stabilized with a polysaccharide-based surfactant, that is, sodium caproyl hyaluronate. These three systems present interesting physicochemical and structural properties from the biopharmaceutical viewpoint for nasal and nose-to-brain delivery—biocompatibility, drug release, mucoadhesion, and permeation across the nasal mucous membrane. All three systems improved the transport of the hypolipidemic drug simvastatin through the epithelial barrier of the nasal cavity, compared to the traditional formulation.

According to reference [7], polymeric nanoparticles present several disadvantages, such as toxicity, presence of organic solvent residues, inadequate encapsulation of hydrophilic drugs, losses, difficulty of large-scale production, and storage and sterilization issues. Besides, the organism may receive them as strange particles. To avoid this, lipids can be employed. Their instability and the consequent reduction of their average life hinder their clinical applications. Nevertheless, their core-shell structure presents countless advantages, especially for drug delivery. In the case of oily core nanocapsules, the pharmaceutical industry opts for lyophilization, especially if there are thermolabile compounds. Definitely, they result in promising structures as they offer a high capability of drug encapsulation, protection from degradation, and biocompatibility; they hardly irritate tissues and certain polymers have been observed to actively interact with biological fluids [4, 5].

2.2 Lipid-based formulations

Delivery systems with lipid-based formulations are mainly of three types—liquid, solid, and lipid as colloidal carriers (liposomes). The liquid formulations are emulsions or micro-emulsions, self-emulsifying or self-nanoemulsified drug-delivery systems, and solid in oil suspensions. The solid lipid-based systems include solid-state micro-emulsions, solid self-emulsifying drug-delivery systems for dry emulsions, microspheres, nanoparticles, and suppositories. The incorporation of the drug to the matrix or shell-core of the solid lipid nanoparticle relies on the composition and the preparation mode of the formulations [7].

The different types of lipid-based nanocarriers are solid lipid nanoparticles (SLNs), liposomes, lipid-drug conjugates, lipid nanocapsules (LNCs), and nanostructured lipid carriers. Nanocarriers fabricated using lipid biomolecules show low in vivo toxicity and are subject to parenteral, oral, transdermal, intranasal, and ocular administration.

SLNs are stable in biological fluids and offer a good therapeutic alternative—their size is between 80 and 100 nm, they are more efficient than polymeric nanoparticles, and have the advantage of being able to be prepared from non-toxic physiological lipids, habitually used as excipients. In Ref. [7] the authors present various advantages compared to other colloidal carriers, such as controlled drug release and targeted therapy; and they protect encapsulated compounds from degradation. Their nature is very versatile and they can be applied in chemotherapy. The solid matrix forms an o/w emulsion, it is formed by well-tolerated lipids, and it allows for the incorporation of hydrophilic and/or hydrophobic drugs. The amount of encapsulated active compounds ranges from 1 to 5% for hydrophilic compounds and reaches 80% for lipophilic ones [16]. The lipids used as the coating can increase bioavailability, they help drug release and protect from water permeability. SLNs are most useful for the oral administration of drugs and vitamins that can solubilize in a lipid medium [17]. They have a lower cost than synthetic polymers, than PLGA for instance, and besides, can bind PEG as ligand [7].

LNCs are hybrid structures between polymeric nanoparticles and liposomes. Their size is small (between 20 and 100 nm), they are very stable, biodegradable and biocompatible, easy to manufacture, they can accommodate one or two drugs together, which can be released in a sustained manner. They show an oily liquid core surrounded by a solid or semisolid hydrophobic shell made of solid lipids and emulsifying agents. They are prepared through micro-emulsification or high-pressure homogenization. The principal components are oils, a lipophilic surfactant, and a non-ionic surfactant. Usually, fatty alcohol or acids; steroids or waxes; mono, di, or triglycerides; and phospholipids are employed [2, 18]. Triglycerides used as excipients, such as caprylic acid (Labrafat^®), lauric acid, palmitic acid, oleic acid, and behenic acid, present different chain lengths; mixed glycerides and polar oils like sorbitan trioleate (span 85), and oleic acid, are also used as emulsifying agents. Vegetable oils obtained from castor, soybean, olive, argan, eucalyptus, orange, and sesame are also being tested. Lecithin obtained from egg, soybean, rapeseed, sunflower, and lysolecithin is used as a lipophilic surfactant and is available as Lipoid^® and Phospholipon^® brands. Phospholipon^® is a mixture of nature hydrogenated lecithin and phospholipids. On the other hand, ethanol, glycerol, propylene glycol, and PEG, as well as water-soluble and insoluble (non-ionic) surfactants are used as cosolvents to improve solubility. Water-soluble surfactants have HLB numbers of 12 or more and are, for example, alkyl ether ethoxylate, cremophor RH40 and RH60 (ethoxylated hydrogenated castor oil); and water-insoluble ones have values of HLB from 8 to 12 and can adsorb on the oil-water interface, such as polyoxyethylene and sorbitan trioleate (Tween-85). Finally, anti-oxidants like α-tocopherol, β-carotene, propyl gallate, and butylated hydroxytoluene are added [7, 18]. Among hybrid lipid-polymer nanoparticles (LPNs) are polymer-core lipid shell nanoparticles, formed by a polymer inside the core that is surrounded by one or more membrane-like lipids. The space between the polymer and the lipid is filled with water or aqueous buffer. The core polymer delays drug delivery and favors lipid stability. It is possible to encapsulate lipophilic drugs easily. In the case of highly water-soluble drugs, lipid-polymer complexes can be used [7]. The anti-cancer drug salidroside is incorporated in polymer-core lipid shell NPs formed by PLGA-PEG-PLGA triblock and the lipids lecithin and cholesterol, with high encapsulation efficiency, negative charge, and 150 nm size [19].

There is a type of hollow-core/shell lipid-polymer-lipid hybrid nanoparticles where polymeric NPs and PEGylated lipoplexes are combined. They contain a layer of positively charged lipid, which forms the inner hollow core, a middle layer of PLGA, which is hydrophobic, external to the PEG, and a neuter lipid layer between them. These systems are not simple LPNs, they present the features of PEGylated lipoplexes and PLGA nanoparticles. The positively charged hollow core can accommodate anionic drugs more efficiently than a polymer alone, the polymeric layer of the medium will allow sustained release, and the PEG-lipid layer avoids the particle being recognized by a macrophage, increases stability, and slows down polymer degradation and drug release. The combination of si-RNA and small drug molecules in the hydrophobic layer of PLGA is very useful for the treatment of different diseases, including multidrug-resistant cancers [7, 20].

3. Preparation methods

The production of polymeric nanocapsules has been increasing in the last decade, mainly in relation to the great potential of their applications in the fields of Biology, Medicine, and Pharmaceutics. The characteristics and usability of these nanosystems depend strongly on the production method chosen and the process variables, as well as on the formulation materials used [5]. On these grounds, several methods and processing techniques have been developed in the last two decades to obtain nanocapsules with the desired properties and biological performance according to their purpose. Generally, there are three classical methods for the preparation of polymeric nanocapsules—nanoprecipitation, emulsion template method, and layer-by-layer method [6, 21]. Regardless of the method used, the production of core-shell structures requires a non-solvent/continuous phase (water) and a solvent/dispersed phase (organic solvent that can be removed later), plus one or more polymers and surfactants to contribute to structure and stability, respectively. The nanocapsules are obtained as colloidal dispersions, or in powder if some drying method is added.

3.1 Nanoprecipitation

Nanoprecipitation, also named interfacial deposition method or solvent displacement, was the first method described [22], and it has been widely used in the last two decades, principally because it is fast, and it has extensive applicability, being able to be used with many types of materials, and allowing various drugs to be encapsulated. In addition, it is a low-cost and simple operation technique since it does not require any special equipment [5]. The preparation of nanocapsules using this method involves both an organic and an aqueous phase. Typically, the organic phase, which consists of oil, polymer, and the active compound dissolved in an organic solvent, is added slowly and with moderate stirring to the aqueous phase (in most cases water and a selected surfactant). Hence, the formation of nanocapsules results from a combination of the spontaneous emulsification of oily droplets and the simultaneous precipitation of polymer onto the water-oil interface during the diffusion of phases [23]. Finally, the colloidal aqueous suspension is obtained by eliminating the organic solvent via evaporation or through a drying process [6, 24]. The characteristics of nanocapsules formed by nanoprecipitation are mainly influenced by several process variables, such as nature, concentration, and compatibility of the components [21], volume ratio between organic and aqueous phase, and the selected method for the injection of the organic phase. In fact, there is some evidence that varying the organic phase injection rate, the aqueous phase agitation rate, and adding the organic phase through a thin needle, leads to a significant decrease in the average size of the nanocapsules, compared with the technique consisting in just pouring one phase over the other [25]. This is probably due to the increase of the contact surface between the phases [26].

3.2 Emulsification-based methods

There are different ways to obtain nanocapsules using nanoemulsions as a template, including emulsion-diffusion, emulsion-coacervation, and double emulsification. All of them involve the emulsification of the organic or the aqueous phase in the other using a low or high-energy homogenization technique, causing the surfactant to self-assemble at the interface. The nature of the rest of the materials used in the continuous or in the dispersed phase will depend on the desired characteristics of the nanocapsules to be formulated.

3.2.1 Emulsion-diffusion/evaporation method

Emulsion-diffusion is a technique that involves the emulsification of an organic phase onto an aqueous phase and the subsequent elimination of the organic solvent by diffusion into the external phase or driven by evaporation, which results in the formation of nanocapsules [6, 27]. The formation of a conventional oil-in-water emulsion within a partially water-soluble solvent via diffusion requires a second aqueous phase (also named dilution phase) that promotes the solvent to diffuse into the external phase causing polymer precipitation and interfacial phenomena, forming a core-shell structure. The homogenization of both phases can be attained through low or high-energy shaking (by means of magnetic or mechanical stirring, ultra turrax, ultrasound, high-pressure homogenizers, etc.), being the latter a better option to obtain smaller nanocapsules [5, 28]. The basis of this method, which differentiates it from nanoprecipitation, is mainly the use of an organic phase partially miscible in water and a polymer partially miscible in both phases. Some other factors that can affect the final characteristics of the nanocapsules obtained by this technique are the amount of dilution phase, the surfactant and polymer concentrations, the oil-to-polymer ratio, and the drop size of the primary emulsion [21]. The advantages of this method include better reproducibility, control of particle size, and therefore better scaling response; but enough energy must be spent to remove large amounts of water, and it is a recommended method only for particles with an oily core [5, 23, 29].

3.2.2 Double emulsification method

Multiple emulsion systems are capable of encapsulating both hydrophilic and lipophilic molecules simultaneously and can be obtained through the double emulsification method [20, 30, 31]. Depending on the established phase sequence, double emulsions can be water-in-oil-in-water (w/o/w) or oil-in-water-in-oil (o/w/o). The preparation of nanocapsules using double emulsification involves a two-step emulsification process and the use of two stabilizers or surfactants. In fact, the key to ensuring good interphase stability and improving drug encapsulation and particle size is the correct selection and concentration of surfactants. As an example, based on w/o/w, a low hydrophilic-lipophilic balance (HLB) surfactant is needed to stabilize the w/o interface. In contrast, to stabilize the oil-in-water interface, a high HLB surfactant is required. Finally, particle hardening, or in other words, polymer shell formation, has been reported to be achieved by polymer precipitation, solvent diffusion, coacervation, or a combination of these strategies [5, 32].

3.2.3 Emulsion-coacervation method

Like the methods described in Sections 3.2.1 and 3.2.2, the emulsion-coacervation method uses the emulsion as a template, the difference is that the formation and stabilization of the polymer shell can be achieved by physical coacervation, chemical crosslinking, photopolymerization, sonochemical techniques, in situ polymerization, atom transfer radical polymerization (ATRP), and addition-fragmentation chain transfer (RAFT) [5, 33, 34]. Therefore, the materials commonly used for the fabrication of nanocapsules are usually monomers, polymers possessing cross-linking function groups or polyelectrolytes [35]. The general procedure involves the formation of the nanoemulsion firstly, coacervation phase stabilization, and finally, monomer/polymer crosslinking. Thus, some factors that are also necessary for the fabrication of nanocapsules by the emulsion-coacervation method include the addition of coacervation or crosslinking agents and the modification of certain variables, such as pH and temperature [36]. The main advantage of this method is that it permits to obtain a rigid nanocapsule shell structure, which can help to minimize the leakage of the payloads to the external phase. However, the final product may contain monomer residues that did not react [33].

3.3 Layer-by-layer method (LBL)

The layer-by-layer method has a great potential to develop multi-compartmental delivery devices since nanocapsules with multiple polymeric layers around the core are obtained. This method requires a colloidal core, which can be the solid form of the active substance, inorganic particles, biological cells, or an oil-in-water nanoemulsion prepared using any of the methods described in Section 3.2. The mechanism of this nanocapsules formation is the irreversible electrostatic attraction—a sequential adsorption of oppositely charged polyelectrolytes is achieved [5, 37]. The sequential deposition of polycations and polyanions on the inorganic core can be followed by the sacrifice of the template core, resulting in a hollow nanocapsule where the payload can be trapped [6, 38, 39]. The main advantages of the LBL technique are the possibility to simultaneously encapsulate different drugs at different positions, and the possibility to control release properties by modulating the composition and the thickness or number of layers of the polymeric shell [24, 40, 41]. On the other hand, this method bears some difficulties, such as the separation of the polyelectrolyte and the remaining counterions in each deposition cycle. Otherwise, aggregates of these may form. This high number of assembly steps is quite complex and time-consuming. In addition, larger nanocapsules are obtained compared to other methods, due to the number of polymeric layers deposited [21].

3.4 Comparative analysis for the selection of nanocapsules production method

Table 1 summarizes the main advantages and disadvantages of the processing methods described in Section 3 in terms of water volume consumption, additional purification steps, contaminant generation, time consumption, and others. Overall, nanoprecipitation represents a simple, fast, low-cost, and versatile method, but its simplicity can lead to some repeatability and scale-up problems. In contrast, emulsion template methods need agitation and solvent removal equipment and thus the technique becomes more expensive but with better control of particle size and scale response. Finally, the LBL technique offers many possibilities, as several active ingredients of different nature can be encapsulated simultaneously, and the release mechanism can be controlled according to the nature and number of polyelectrolyte layers; however, it becomes a more complex and time-consuming technique and larger particle sizes can be obtained compared to the other methods. The selection of the nanocapsule production method should mainly consider the desired characteristics of the final nanocarrier, the formulation materials, and the availability of laboratory equipment [5].

Method	Advantages	Disadvantages	References
Nanoprecipitation	Fast and low-cost method No require any special equipment Many types of materials and drugs	Oil core: lipophilic active substance nature Reproducibility and scale-up problems due to its manual operation	[5, 21, 42]
Emulsion-diffusion	Better reproducibility, particle size control, and scaling response. Different ways of homogenization, including low or high-energy shaking	High water volume consumption (dilution) Enough energy to remove large amounts of water Only recommended for particles with an oily core	[5, 23, 28, 29]
Double emulsification	Encapsulation of hydrophilic and lipophilic molecules simultaneously Preparation of o/w or w/o emulsions	Contaminant generation (use of two surfactants) Additional purification steps	[20, 30, 31]
Emulsion-coacervation	Rigid nanocapsule shell structure Good release control and minimization of the “burst” effect	The final product may contain monomer residues	[21, 33]
Layer-by-layer	Many colloidal core possibilities (solid drug, inorganic particles, biological cells, or an o/w nanoemulsion) Simultaneous encapsulation of different drugs Control of the release properties by modulating the composition and the thickness or the number of layers of the polymer shell	Contaminant generation (remaining polyelectrolyte and counterions in each deposition cycle) Additional purification steps Complex and time-consuming technique Larger nanocapsules compared to other methods	[6, 21, 24, 38, 39, 40, 41]

Table 1.

Comparative analysis of the advantages and disadvantages of nanocapsule production methods.

4. Physicochemical characterization of nanocapsules

Next to the technical challenge of fabricating nano-vesicular systems, there is an inevitable need both for monitoring the whole process and characterizing the properties of the nanocapsules produced [43]. A variety of characterization techniques can be found in the literature. Some of them are important techniques required to be done in any colloidal system, while the choice of others depends on the specific area of application of the systems. In this sense, the physicochemical characterization involves techniques to study or determine particle size, morphology, and dynamic stability of the nanocapsule suspension, as well as to know their effectiveness as drug entrapment and release systems.

4.1 Average size and size distribution

The average size and size distribution of submicron particles are usually measured by dynamic light scattering (DLS), which is based on the equivalent sphere principle when an incident beam interacts with the sample particles. Well-prepared nanocapsule systems should be in the nanometer range and with a narrow particle size distribution. Therefore, numerous studies have paid attention to the effect of both the type and the concentration of the constituents, as well as the fabrication process variables, on the size and polydispersity index (PI) of the sample [44]. The disadvantage of DLS is that there are some parameters that may influence data, such as viscosity, pH, and temperature of the suspension medium, as well as concentration, colloidal instability, or the presence of aggregates [23]. On the other hand, microscopic methods are also used to determine nanocapsules’ mean size, but they require the imaging of a large number of particles, and the measurement may be affected due to the sample dry state required for the analysis [24]. So, it is generally recommended to use at least two methods to determine the particle size and size distribution [23].

4.2 Morphology

Different microscopy techniques can be used not only to observe the nanocapsule morphology and structure, but also to determine the average size, elemental composition, and state of aggregation. Scanning electron microscopy (SEM) or transmission electron icroscopy (TEM) are the most common techniques, and their choice depends on the size of the studied system and the established purposes [23, 45]. The principle of SEM is to scan the sample with a high-energy electron beam, and image formation is achieved by collecting low-energy secondary electrons or backscattered electrons that are released from the sample surface. For this reason, SEM images present a three-dimensional appearance and are useful to appreciate the structure, shape, and surface defects of the sample [45, 46].

Compared to SEM, TEM needs a higher voltage, resulting in higher resolution (0.2 nm). Since electrons can pass through the sample, the internal structure, whether crystalline or amorphous, can be observed [44]. Both techniques are expensive and require high vacuum, the main difference laying in the preparation of the sample and the information obtained from it. SEM requires sample conductivity, which is usually achieved by coating the sample with a thin layer of gold or platinum. In contrast, for TEM analysis the sample must be thin enough to be electron-transparent [45]. It is worth mentioning that a qualitative or semi-quantitative elemental chemical analysis can be performed by electron microscopy, coupling energy-dispersive X-ray spectroscopy (XEDS) [24]. In addition, the use of scanning transmission electron microscopy (STEM), a technique that combines both principles, has been reported for the characterization of micro- and nanocapsules [47].

Another useful tool for simultaneously determining particle shape, surface structure, and even some mechanical properties is atomic force microscopy (AFM). AFM images are obtained by measuring the displacement of the AFM tip during a raster scanning over the immobilized sample. The result is a high-resolution 3D profile of the surface under study. The principal advantage of AFM over electron microscopy is that it permits the imaging of almost any type of surface and biomolecules under different physicochemical conditions, during biological processes, or even the study of the mechanical properties of delivery systems at the nanoscale [45, 48].

4.3 Stability of nanocapsule suspensions

An important property of colloidal systems is that they remain stable over time and under certain conditions of interest. Some physicochemical instability phenomena of nanocapsules are aggregation, precipitation, creaming, nanocapsule chemical degradation, and consequently, the reduction of drug content within the nanocapsule. All these phenomena can occur during the production process or after storage. Among the main reasons for them are inadequate steric or electrostatic stabilization, and the combination with external agents, such as oxygen, temperature, and ultraviolet radiation. In this way, the stability of nanocapsules is generally evaluated in terms of drug content, variations of zeta potential values, and average particle size as a function of time, pH, or temperature, using DLS or Zeta Potential techniques [23]. In some cases, the visual inspection of the colloidal suspension is also useful. On the other hand, some degradation products can be evaluated by chromatography or spectrometry [49].

4.3.1 Zeta potential

The zeta potential is the electric potential at the interfacial double layer between the dispersed particle and the liquid layer surrounding it, and can be determined by electrophoretic light scattering, where the particle migrates toward the electrode of opposite charge with a velocity that is proportional to the magnitude of the zeta potential [23]. Regarding stability, it is important to ensure that the zeta potential values of the nanosystems are greater than ±30 mV, as this guarantees the strong electrostatic repulsion forces that prevent the occurrence of aggregation phenomena among the particles [50]. In addition to evaluating the stability of nanodevices, zeta potential measurements can also confirm the coating or adsorption of a specific material on the nanocapsules surface, which is useful specifically in the layer-by-layer technique described in Section 3.3 [24].

4.4 Encapsulation efficiency and in vitro drug release

Drugs can be loaded onto nanosystems by incorporating them during the nanocapsule production, or either after the formation of the nanocapsules, incubating the carrier with the concentrated drug solution [51]. In both cases, the drug can be physically loaded onto the polymeric matrix or the oily core, or it can be adsorbed on the surface, in the function of the affinity and the physicochemical characteristics of both the drug and the components of the nanocapsules [52]. The total content of a drug in the nanocapsule suspension can be determined after dissolving or extracting the drug from the carrier, or calculated from the difference between the total and free drug concentrations after the separation of nanocapsules by centrifugation or ultrafiltration [23]. The determination of loaded or released drugs can be carried out by means of high-performance liquid chromatography (HPLC), fluorescence spectroscopy, UV-Vis spectroscopy, or another analytical technique.

Nanocapsule erosion or swelling can lead to drug release. In vitro drug delivery depends upon the localization of the drug within the particle, the physicochemical properties of both the drug and the nanocapsule constituents, size, morphology, and cross-linking, and also on release conditions, such as pH, temperature, polarity, and the presence of enzymes or an adjuvant. Regarding the release of the active principle, the process is governed by solubility, diffusion, and polymer biodegradation. In the case of nanospheres, where the drug is evenly distributed, drug release occurs through diffusion or matrix erosion. If diffusion is faster than erosion, the release mechanism is said to be controlled by diffusion. If the drug is weakly bound to the surface, a rapid initial release or “burst” will take place. If the drug has been incorporated into the polymeric matrix (nanocapsule), it will present a relatively small “burst” effect and a sustained release profile instead. In this case, the release will be controlled by drug dissolution and diffusion through the polymeric membrane. To avoid the “burst” effect, compounds can be added to the matrix that reduces the drug solubility, or, as in the case of chitosan, ethylene oxide-propylene oxide block copolymer (PEO-PPO) can be added, which increases release rate because it diminishes drug-matrix interaction [1].

Determining the drug release mechanism from the particle system can give valuable information about the interactions between the drug and the nanocapsule. Drug release kinetics from nanocapsules may be obtained using ultracentrifugation, centrifugal ultrafiltration, dialysis techniques, or side-by-side diffusion cells with an artificial or biological membrane [23, 52]. Furthermore, the kinetic data can be fitted to mathematical models to determine the predominant release mechanism, which is very convenient in the design and evaluation of the utility of nanocapsules as drug-delivery systems in pharmaceutical applications [24].

5. Applications of nanocapsules in therapeutics

Nanotechnology has revolutionized cancer diagnosis and therapy. Protein engineering and materials science have contributed to the development of new nanosystems for drug delivery. The major features of nanoparticles for their application in drug delivery are particle size and size distribution. These determine the capacity to reach the target, drug distribution and toxicity, and influence charge and drug release, as well as the stability of nanoparticles. Smaller nanoparticles present a larger surface/volume ratio; in this case, the drug, which is closer to the surface, is expected to be released at a higher speed. Because of their small size, these nanoparticles can cross the sore endothelium, the intestinal epithelium, for example, in tumors, and enter microcapillaries of 5–6 microns diameter, and it also enables them to be selectively captured by cells and cause drug accumulation in certain places. On the other hand, the smaller the particle size, the more the risk of aggregation during the storage, transport, and manipulation. With respect to bigger particles, they allow for a larger drug per particle encapsulation, which derives in a slower release. Therefore, particle size control results in a regulation of the drug release rate. Moreover, size also affects polymeric degradation. In the case of cancer treatment, the aforementioned properties are fundamental; due to their small size, nanoparticles can access tumors and concentrate there through the EPR effect (enhanced permeability and retention). To the moment, many nanotechnological systems have been developed and tested as anticancer drug carriers, however, difficulty arises from the fact that these drugs do not differentiate healthy from tumoral cells. For that reason, it is necessary to investigate strategies that permit systems to reach the tumor specifically [1, 53].

As to the surface properties of nanoparticles, hydrophobicity influences their destiny, as it determines the level of blood components (such as opsonin) that will join them. It is essential to minimize opsonization to prolong the circulation of nanoparticles in blood. With this goal, nanoparticles can be coated with hydrophilic/surfactant and/or biodegradable polymers, such as PEG, polyethylene oxide, poloxamer, poloxamine, and polysorbate 80 (Tween 80) [1]. Targeted delivery can be active or passive. In the first case, the active principle or the nanosystem must conjugate to a specific ligand from the cell or tissue, whereas when the delivery is passive, the drug is released in the target organ. Nanocarriers concentrate preferably in tumors, inflammatory sites, and at antigen sampling sites due to the EPR effect of the vasculature. Anti-neoplastic, anti-viral drugs, and several other drugs are unable to cross the BBB. Nanoparticles with Tween 80 and those formulated with hyper-osmotic mannitol, which breaks the strong unions present, have been proved to be able to cross the BBB and provide a sustained release of drugs for the treatment of brain tumors. Once the target is reached, nanoparticles from biodegradable hydrophobic polymers become like a reservoir and start releasing the active compound in a continuous way. This type of system is usually employed to improve bioavailability and sustained release, and even to solubilize drugs for systemic delivery, and the systems adapt to protect bioactives from enzymatic degradation by nucleases and proteases, for instance [1].

5.1 Drug-delivery systems

Next, some most recent examples are gathered where polymeric and lipid nanocapsules are used to carry a great variety of drugs. In Table 2 some recent relevant studies are compiled.

Pharmacological group	Drug/compound	Type of nanocapsule	References
Antimicrobial and antibiotics	Fluoxetine	Polymeric	[54]
	Carvacrol	Polymeric	[55]
	Amoxicillin	Polymeric	[56]
	Florfenicol	Polymeric	[57]
	Tea tree oil	Polymeric	[58]
	Fusidic acid	Lipid	[59]
	Albendazole	Lipid	[60]
Anticancer drugs	Curcumin	Polymeric	[61, 62, 63]
		Hybrid lipid	[64]
		Liquid lipid	[65, 66]
	Docetaxel	Polymeric	[67, 68]
		Lipid	[69]
	Perillyl alcohol	Polymeric	[70]
	Tamoxifen	Polymeric	[71, 72]
	Aprepitant	Polymeric	[73]
	5-fluoroacil	Polymeric	[74, 75]
	Doxorubicin	Polymeric	[76]
	Paclitaxel	Polymeric	[77, 78]
		Lipid	[79]
	Oleic acid	Hybrid	[80]
	Simvastatin	Hybrid	[81]
	Thymoquinone	Polymeric	[82]
	Gemcitabine	Lipid	[83]
	Phloretin	Lipid	[84]
	Itraconazole	Lipid	[85, 86]
	Regorafenib	Lipid	[87]
	Sorafenib	Lipid	[88]
	Imatinib	Lipid	[89]
	Ifosfamide	Lipid	[90]
	Vinorelbine	Lipid	[91]
Anti-leishmaniasis	Glucantime	Polymeric	[92]
Anti-hyperglycaemic	Chrysin	Polimeric	[93]
Inmunological	Imiquimod	Hybrid	[94]
Anti-inflammatory	Diflunisal	Hybrid	[24]
	Celecoxib	Polymeric	[95]
	Nicotine	Lipid	[96]
Antidiabetic	Anthocyanin	Polymeric	[97]
Anesthetics	Prilocaine, Lidocaine	Lipid	[98]
Ophthalmic	Astragaloside	Lipid	[99]

Table 2.

Recent studies based on lipid and polymeric nanocapsules used as drug carriers.

5.1.1 Antimicrobial and antibiotics

Several reviews exist about systems developed for the treatment of infections. Specifically, in Ref. [100], the most important works concerning the use of lipid-based formulations from 2000 to 2020 are compiled. Infections caused by bacteria resistant to antimicrobial drugs available for use in humans have increased exponentially. This revision highlights the importance of the development of nanotechnology in lipid systems as an innovative tool for infection treatment. Chitosan nanocapsules, and lecithin-polysorbate 80, containing dapsone, have resulted useful; also, lipid nanocapsules with carvacrol and cinnamaldehyde. Another review about the incorporation of natural substances with antimicrobial activity in polymeric nanoparticles highlights specifically the antifungal activity against Candida species of Glycyrrhiza glabra L., which is included in mucoadhesive nanoparticles constituted by PLA, PLGA, and alginate. Nanocapsules were also prepared from polymyxin B cross-linked with sodium alginate and solid lipid nanoparticles with Ginkgo biloba L., and their antimicrobial activity against Pseudomonas aeruginosa was studied [101]. For the delivery of fluoxetine, starch nanocapsules with core-shell morphology were prepared and joined to polyurethane. The system presented antibacterial activity against Staphylococcus aureus [54]. The incorporation of antimicrobial peptides appears as a promising alternative for infection treatment, as well as carvacrol loaded onto nanocapsules formed by PCL [55]. PCL nanocapsules loaded with amoxicillin trihydrate were prepared to investigate the gastric stability of this drug, as well as its therapeutic activity against H. pilori [56]. Eudragit^® polymers (polymethacrylate-based copolymers) are easy to handle and are used to prepare formulations for oral administration. Antibiotic florfenicol was encapsulated in Eudragit^® nanocapsules [57].

Besides, it was possible to demonstrate the antimicrobial activity of chitosan against Escherichia coli through the assembly of bacteria cell membranes. This last finding represents an advance in delivery systems based on chitosan nanocapsules since it can enhance the effects of carried antibiotics [102]. As a strategy to increase the solubility of capsaicin, a major component of chili peppers known for its numerous therapeutic activities, nanocapsules of chitosan in the form of high-payload submicron capsaicin-chitosan colloidal particle complex were prepared. Besides achieving 75% of the loaded compound, capsaicin antimicrobial activity remained intact [103]. Chitosan-PCL core-shell nanocapsules were obtained and loaded with tea tree oil. These nanosystems presented activity against Cutibacterium acnes, which makes them useful for topical acne treatment [58]. Also, lipid-core nanocapsules coated with chitosan to test the antimicrobial in vitro activity of fusidic acid against Gram-positive bacteria were prepared [59]. The side effects of bedaquiline, a very effective drug against tuberculosis, can be very dangerous. The result of its encapsulation in lipid nanoparticles and chitosan-based nanoparticles suggests that it is possible to achieve a high concentration of the drug in the place of the infection, reducing the dose and therefore, side effects [104]. Encapsulation of the anthelmintic drugs mebendazole, albendazole, and their main metabolite in lipid nanoparticles, showed efficacy for Cystic echinococcosis treatment [60]. On the other hand, bacterial biofilms often impede the diffusion and accumulation of antimicrobial compounds, which is why the development of systems able to cross the biofilm is paramount. It has been observed that polymeric nanoparticles can access and change the properties of the biofilm microenvironment due to their size and specific structure. In this way, they interact with bacteria and/or release the encapsulated drugs. Thus, they are systems with a projection in anti-biofilm therapy [105].

5.1.2 Anticancer drugs

Curcumin has been described, among many other applications, to possess anticancer properties against different tumor types, including colorectal cancer. Nevertheless, it presents an inconveniently low bioavailability and a short average life, as well as a limited absorption and quick metabolism. The use of non-toxic nanocapsules prepared from the biodegradable polymer polyallyhydrocarbon proved useful for drugs with low bioavailability, including curcumin. The efficacy of these systems was confirmed with the use of mice as models [61]. In the same way, curcumin was encapsulated in systems prepared from chitosan and carboxymethyl cellulose which presented good stability [62] In Ref. [63], nanocapsules with different polymeric coatings (P80, PEG, chitosan, and Eudragit^®) were prepared and compared as curcumin carriers. They evaluated the release of the active, cytotoxicity, and in this case, antimalaria activity was tested instead of antitumoral potential. The highest activity observed was that of nanocapsules prepared from chitosan. On the other hand, a curcumin-loaded nanostructure of hybrid lipid capsules of three different sizes has been shown to present 2.5 times the anti-cancerous efficacy of free curcumin in breast cancer cells and breast cancer stem-like cells [63, 64]. Also, liquid lipid nanocapsules coated with human serum albumin to carry curcumin were proposed. To strengthen the protecting role of the protein layer, this was cross-linked [65]. Moreover, liquid lipid nanocapsules were obtained from olive oil emulsification, where nanocapsules were coated by a protective shell composed of bovine serum albumin and hyaluronic acid [66]. The so-called nanocurcumin was also formulated, consisting of the anti-cancerous compound incorporated into a polymeric nanoparticle, which enhances its solubility. Due precisely to its solubility, its gelifying capacity and its ability to form complexes, pectin has also been widely used for the preparation of diverse nanomaterials, including nanocapsules. It presents medical uses as a coagulant, anti-diarrheic, anti-ulcerous, and anti-cancerous for colon cancer [106].

The anticancer drug docetaxel has been carried in multiple types of nanocapsules. As a novelty, nanocapsules were prepared that consisted of a polymeric shell coating an oily core, targeted to Tn-expressing carcinomas. Chitosan was PEGylated and modified with a monoclonal antibody that recognizes the antigen Tn, which is highly specific for carcinomas. Internalization of nanoparticles and reduction of cellular viability were proved. The release is pH-dependent, being faster in acid pH, which favors intracellular release [67]. Also for docetaxel delivery, nanocapsules were formulated from hyaluronic acid through self-emulsification in absence of organic solvents. They were studied in vitro with lung cancer cells and an effective release of the drug was observed [68]. In addition, the system composed of docetaxel and thymoquinone co-encapsulated in PEGylated lipid nanocapsules was explored. Cytotoxicity was improved and enhanced antitumor efficacy and apoptotic effects were observed. Reduced oxidative stress and toxicity to liver and kidney tissues occurred [69]. Chitosan-coated PLC nanocapsules resulted effective for the oral administration of perillyl alcohol, an essential oil with chemo-preventive activity for anticancer therapy. These nanocapsules present the mucoadhesive properties of the oil [70]. Chitosan and gellan gum were combined in the preparation of natural nanocapsules for tamoxifen delivery [71]. Tamoxifen delivery in biocompatible nanocapsules made from a PLA core and a 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] shell was studied for breast cancer treatment. Cell proliferation results indicated cytotoxicity of nanocapsules in MCF-7 cells, as compared to free tamoxifen [72].

The drug aprepitant is a selective neurokinin 1 antagonist with low solubility in water, clinically used for the prevention of vomits and sickness provoked by chemotherapy. Chitosan-PEG-coated cyclodextrin nanoparticles and nanocapsules were designed and evaluated in vitro and in vivo [73]. New topic formulations of the antitumoral drug 5-fluorouracil were studied using sodium alginate and hyaluronic acid-containing AS1411 aptamer-functionalized polymeric nanocapsules. It was proved that nanoencapsulation improves drug permeability, and the nanoparticles prepared showed favorable biosafety and good antitumor effects for skin cancer treatment [74]. To improve the efficiency of the antitumoral treatment, hybrid nanocapsules obtained from the interfacial condensation between chitosan and poly(N-vinyl pyrrolidone-alt-itaconic anhydride), containing both magnetic nanoparticles and 5-fluorouracil, were developed. Their nanometric size and their spherical shape were confirmed by SEM [75].

Polysaccharide-based nanocapsules prepared from furcellaran and chitosan via LBL deposition using electrostatic interaction were studied. To achieve targeted delivery, the surface was modified with a peptide. Doxorubicin was encapsulated with excellent drug loading properties, and release and stability proved to be influenced by pH. This system showed compatibility with eukaryotic organisms and good anticancer effects [76]. Paclitaxel, like most anticancer drugs, is low water-soluble and presents high toxicity at therapeutic doses. Nanoencapsulation seems a good strategy to overcome these difficulties. In Ref. [77] three kinds of nanocapsules using derivates of PEG dimethacrylates as crosslinking agents were obtained. It was possible to prove that the nanocapsule system provides an effective and universal strategy for lung targeting, esterase triggering, and synergy therapy. In another study, paclitaxel was loaded onto chitosan-poly(isobutyl cyanoacrylate) core-shell nanocapsules designed for oral drug delivery. The nanocapsules thus prepared had low polydispersity, spherical shape, and good mucoadhesive properties [78]. Another strategy for the encapsulation of paclitaxel and the reduction of its toxicity is the preparation of lipid and biosurfactant-based core-shell-type nanocapsules. In one such study, Acconon® was the lipid, and stearic-acid-valine conjugate the biosurfactant [79].

Health properties are attributed to the low water-soluble components of garlic oil diallyl disulfide (DADS) and diallyl trisulphide (DATS). Among these is anticancer activity. Both compounds were encapsulated in oil-core nanocapsules of hyaluronic acid. It was proved that encapsulation inhibits the membrane lysis of red blood cells (chiefly provoked by DADS), that the shell acts as a limiting barrier for the sulfur oxidation of the compounds, and that they preserve their biological and anticancer properties after encapsulation. Oleic acid was carried in this same type of nanocapsules and it was concluded that in presence of amphiphilic derivates of hyaluronic acid as a shell of the nanocapsules it is not necessary to include low molecular weight (co)surfactants [80].

Chitosan-coated PCL nanocapsules loaded with simvastatin resulted to be a promising strategy for simvastatin administration within a nose-to-brain approach for brain tumors therapy. Lipid-core nanocapsules coated with chitosan of different molecular weights were prepared by a novel one-pot technique. All formulations presented adequate particle sizes, positive surface charge, narrow droplet size distribution, and high encapsulation efficiency. The nanocapsules allowed for controlled drug release and displayed mucoadhesive properties dependent on the molecular weight of the coating chitosan [81]. Polymeric nanocapsules of Eudragit® were successfully prepared to load thymoquinone. They were conjugated with anisamide as ligand for sigma receptors overexpressed by colon cancer cells [82]. In a bibliographical review, the use of nanocapsules is summarized, among other nanocarriers, for immunotherapy against cancer. As an example, gemcitabine encapsulated in PEGylated lipid nanocapsules is reported. These nanostructures enter macrophages and tumoral cells [107]. Also, lipid nanocapsules (100 nm) loaded with lauroyl-modified gemcitabine efficiently target monocytic myeloid-derived suppressor cells in melanoma patients. The size and charge of nanocapsules can be modulated to reach immunosuppressive cells [83]. The antitumoral effect and the safety of nanocapsules made from a multifunctional component based on Lecigel^® phospholipids loaded with the anticancer drug phloretin were tested. This drug is little soluble in an aqueous medium, and therefore, its dermatologic formulations are limited. With the prepared hydrogel, capacity to get through the skin layers was proved, as well as the drug reservoir role in the corneum stratum. Hence, it is presented as an innovative formulation to be applied in melanoma therapy [84]. Itraconazole is an antifungal drug to which are attributed potential anti-cancerous effects with few side effects. Lipid nanocapsules were proposed for the combined therapy with miltefosine and itraconazole, and an increase in the chemotherapeutic efficacy was observed. These nanocapsules were prepared from Labrafil^® (oleoyl polyoxyl-6 glycerides), Labrafac^® (caprylic-capric acid triglycerides), Transcutol^® (diethylene glycol monoethyl ether), and Lipoid^® (soybean lecithin, phosphatidylcholine, and phosphatidyl ethanolamine). Lipid systems were also prepared to study their efficacy as topic formulations with fungal and non-fungal effects [85, 86].

In Ref. [87], the possibility to supply intravenously lipid nanocapsules of 50 nm approx. was investigated, and it was checked whether this is viable for the treatment of different cancer types. Six different kinds of drugs were employed; encapsulation efficacy was good and in vivo experiments showed that the combination of SN38 and regorafenib in lipid nanocapsules is useful for the treatment of colorectal cancer. Also, sorafenib, a tyrosine kinase inhibitor, was encapsulated in lipid nanocapsules against glioblastoma and the results suggest that they can be used to improve chemotherapy and radiotherapy efficacy [88]. There also exist studies on imatinib, another tyrosine kinase inhibitor, delivered in lipid nanocapsules against melanoma [89]. The therapeutic efficacy against osteosarcoma was increased by ifosfamide-loaded-lipid-core nanocapsules, with significantly higher cytotoxicity of the drug than free ifosfamide at the same concentration. The apoptosis of cancer cells was increased by the prepared system by increasing the expression levels of caspase-3 and caspase-9 in MG63 cells [90]. A hydrophilic antimitotic agent for breast cancer, vinorelbine bitartrate, was incorporated in the lipid aqueous core of nanocapsules protected with a lipid shell. The release mechanism resulted to be Fickian diffusion, and hemocompatibility studies were also carried out to ensure safety in the case of intravenous administration [91].

5.1.3 Genes

The new systems for oncotherapy under development focus on selectivity so that they reach tumor cells without affecting the healthy ones. One strategy is to incorporate genes in non-viral carriers via surface modification to increase selectivity and affinity for the target cell’s receptors. Nanocapsules with ter-polymers, to deliver DNA into tumoral cells, were developed. The surface of the nanocapsules was activated with folic acid so as to enable interaction with the folate receptors overexpressed [108]. Folate-decorated reductive-responsive carboxymethylcellulose-based nanocapsules were also prepared for targeted delivery and controlled release of hydrophobic drugs. In this case, the shell was cross-linked by disulfide bonds formed from hydrosulfuryl groups on the thiolated carboxymethylcellulose. These systems could become potential hydrophobic drug carriers for cancer therapy [109]. In addition, the first experiment with DNA-derived nanocapsules designed to reach podocytes, which damage plays a central role in the pathogenesis of idiopathic nephrotic syndrome, as well as in the progression of many chronic glomerular diseases, has been developed. These nanocapsules were composed of chitosan and plasmids, and their size, the number of plasmid layers, and the presence of the solid template were investigated in particular as the main parameters impacting the biological assay [110]. Besides, nanocapsules obtained from chitosan with hyaluronic acid for genes delivery into the lung epithelium were described. The nanocapsules were introduced in mannitol microspheres to facilitate administration in the lungs. This was seen as a good strategy for the delivery of genetic material into the lung [111]. In a different study, spherical nanocapsules were obtained from chitosan and loaded with capsaicin for cystic fibrosis treatment, and wtCFTR-mRNA was linked to the surface. They happened to be highly stable in the cell culture transfection medium [112]. Also, polyarginine was encapsulated in glyceryl-monooleate-based liquid droplets together with the immunomodulator chemokine CCL2 and two RNAi sequences. It was concluded that polymeric shells confer multifunctionality to the nanocapsules due to their versatility, which permits control of the mechanism of the therapeutic action [107]. An analogous of the nucleotide GMP was encapsulated in lipid nanocapsules for the treatment of neurodegenerative retinal degenerations. The nanocapsules were prepared from Labrafac™ lipophile, Kolliphor^®, and phospholipids, and remained stable for 6 days in phosphate buffer and in vitreous components, allowing for a sustained release [113].

5.2 Stimuli-responsive systems

Stimuli-responsive drug-delivery systems are of interest because they can restrict drug delivery to the target. Nevertheless, it is difficult for most systems to reach all the cells of the tumoral tissue, due to the natural tumoral barrier. There are nanosystems that can be pre-programmed to alter their structure and release the encapsulated molecule more effectively. In this case, molecular sensors are incorporated which respond to physiological or biological stimuli, such as changes in pH, redox potential, or enzymes. Drug release can occur via passive systemic targeting or active receptor targeting. Plasmids of DNA, si-RNA, and other therapeutic nucleic acids can be carried [1]. The development of pH-sensitive drug-delivery systems for the selective release of anticancer drugs is promising, given that healthy tissue has a pH of 7.4, the average extracellular pH in tumoral tissue is 6.8, and the pH of intracell components such as endosomes and lysosomes varies from 4.5 to 6.5. The cause of this difference is that the high metabolic rate required for tumor growth provokes hypoxia in the tumoral region. Therefore, the specificity of delivery systems for low pH levels is a suitable strategy to improve chemotherapy effectiveness, on one side, and reduce cytotoxicity levels, on the other. Most systems proposed for this kind of strategy are organic, but they still present some inconveniences such as low biocompatibility, complex manufacture, and limited drug release indexes [53]. In the case of melanoma therapy, lipid nanocapsules prepared from N-vinylpyrrolidone and vinyl imidazole showed pH-responsive ability and improved drug entrance into the tumoral cells. The copolymers were inserted into the surface of the nanocapsules, and they particularly changed from neutral charge at physiological pH to positive charge in acid conditions [114]. Besides, polyurea/polyurethane nanocapsules displaying pH-synchronized amphoteric properties were proposed. Such properties facilitate their accumulation and their selectivity for acidic tissues, such as tumoral tissues [115]. Stimuli-responsive multi-layered nanocapsules were also prepared with Eudragit^®, chitosan, sodium alginate, and poly-L-arginine. They were loaded with curcumin and delivery was studied under similar pH conditions to those of the gastrointestinal tract. These nanocapsules were observed to shield the compound from being released in the stomach and allow it to be released in the intestine [116]. A LBL nanocapsule was obtained from hyaluronic acid functionalized with anionic azobenzene co-assembled with cationic poly diallyl dimethylammonium chloride. It is a novel UV-induced (365 nm) decomposable nanocapsule. Its size enables it to cross biological barriers, permits a prolonged circulation in the blood, and improves accumulation in the tumor. Later, it can be eliminated after UV-induced dissociation. Similarly, a nanocapsule was prepared with the anionic alginate-azo and cationic chitosan, and the anticancer drug doxorubicin was loaded onto these nanocapsules [117, 118].

5.3 Theragnostics and diagnostics

The field of theragnostics has been rapidly amplified in the last years, thanks to nanotechnology. As it has been commented above, nanomaterials can provide useful action following a great variety of stimuli, be they internal (enzymes, redox potential, pH, and temperature) or external (light, heat, magnetic fields, and ultrasounds-US). In Ref. [119], an interesting review of US-responsive theragnostic nanomaterials under the following categories can be found: microbubbles, micelles, liposomes (conventional and echogenic), niosomes, nanoemulsions, polymeric nanoparticles, chitosan nanocapsules, dendrimers, hydrogels, nanogels, gold nanoparticles, titania nanostructures, carbon nanostructures, mesoporous silica nanoparticles, and fuel-free nano/micro-motors. Theragnostic nanomaterials in service can produce an imaging signal and/or a therapeutic effect, which frequently involves cell death. It is much interesting to combine the ability for theragnostics of the nanocarriers designed with the clinical imaging ultrasound technique. High-intensity-focused ultrasound appears as a promising and minimally invasive therapeutic modality against various solid tumors. Although it has received considerable attention in the biomedical field, both the accuracy and efficacy of this technique are currently unsatisfactory. A nanometer-sized organic/inorganic hybrid enhancement agent for photoacoustic imaging-guided high-intensity-focused ultrasound therapy was designed and fabricated by concurrently encapsulating both Cu2-xS nanodots and perfluorooctyl bromide into a PLGA nanocapsule. These nanocapsules assumed a unique core/satellite/shell sandwich structure and combined the merits of small and uniform particle size, favorable biosafety, and multifunctional theragnostic ability into one system [120]. A biocompatible theragnostic platform consisting of luminescent upconversion nanocapsules encapsulated with cellulose acetate, a biocompatible polymer, was developed. This theragnostic platform is able to simultaneously perform diagnosis and drug delivery. On the one hand, the luminescence properties of the nanocapsules were observed to remain stable even after encapsulation. On the other, the chemotherapeutic drug doxorubicin was successfully loaded onto the nanocapsules [121]. Also, a new theragnostic nanoplatforms based on nanocapsules and PLGA, which were chemically modified so that they could incorporate several imaging moieties was produced. The nanocapsules can be endowed with a magnetic resonance imaging reporter, two fluorescence imaging probes (blue/NIR), and a positron emission tomography (PET) reporter. In vitro toxicity was not observed in any of the two different types of human endothelial cells with concentrations up to 100 μg mL⁻¹. Versatile in vitro/in vivo multimodal imaging ability was observed, as well as excellent biosafety and over 1% wt protein loading. In the same way, nanocapsules fabricated from biodegradable and photoluminescent polyester with PLGA were reported. Superparamagnetic iron oxide nanoparticles (SPIONs) were incorporated into the polymeric shell so as to transform the system into a magnetic resonance/photoluminescence dual-modal imaging theragnostic platform [122, 123]. Polydopamine is a polymer with adhesive properties; nanoparticles were prepared from it where cisplatin prodrug has been loaded via supramolecular interaction between β-cyclodextrin and adamantyl groups [124]. The nanoparticles exhibited photoacoustic imaging capacity for in vivo monitoring of the drug in the tumor site, and the chemo-photothermal therapy of the system showed a powerful anticancer activity against osteosarcoma cells in vitro. This is an innovative strategy for the preparation of multifunctional nanotheragnostics for combined anticancer therapy [125]. Polymeric Pluronic-F127-chitosan nanocapsules were obtained and explored as theragnostic agents. IR780 iodide, a near-infrared fluorescent dye that can be applied as a photosensitizer in photodynamic and photothermal therapies, was loaded for single-wavelength NIR laser imaging-assisted dual-modal phototherapy. Besides, the nanocapsules were functionalized with folic acid so as to activate their targeting capacity against folate receptor-expressing ovarian cancer cells [126]. The same compound was encapsulated in PEG-PLA nanocapsules and demonstrated potential as a multifunctional theragnostic agent for breast cancer treatment, with increased cellular uptake and photodynamic activity, and more reliable tracking in cell-image studies [127]. Magnetic lipid nanocapsules that show higher structural stability and better theragnostic properties than traditional lipid-based nanocarriers were reported as therapeutic nanocarriers displaying drug-delivery capacity. These nanocapsules are 16 times more efficient than free drugs and their diagnostic imaging capability was also demonstrated [128]. Increasing attention is being payed to multilayer nanocarriers loaded with optically activated payloads, since they are expected to provide new mechanisms of energy transfer in health-oriented applications, at the same time as they look promising for energy storage and environmental protection. The combination of a careful selection of optical components for efficient Förster resonance energy transfer and surface engineering of the nanocarriers allowed to synthesize and characterize novel theragnostic nanosystems for deep-seated tumors diagnosis and therapy [129]. Recently, a review has been published where the most interesting advances in nanocarriers applications, including polymeric nanocapsules as tools for Alzheimer’s diagnosis and treatment, are compiled [130].

5.4 Biosensors

Biosensors measure biological, chemical, and physical signals related to health. They are used to track diseases and thus improve health. A multilayer system via LBL deposition of biopolymers, based on electrostatic interaction and intended to be applied in diabetes detection was developed. They obtained a new glucose-responsive system using poly(lysine) derivatives and alginate as polycation and polyanion, respectively [131]. Also, chitosan-based nanocapsules were demonstrated as E. coli bacterial quorum sensing reporter strain [132].

5.5 Others

Next are collected the most recent studies of polymeric and lipid nanocapsules as drug carriers not included in the previous groups:

Regarding polymeric nanocapsules, systems prepared from PLA and PLGA were loaded with glucantime, active against leishmaniasis, and the anti-hyperglycaemic flavonoid chrysin, respectively [92, 93]. Another example is that of the chitosan nanocapsules prepared with the novel excipient Compritol^® for transdermal delivery of imiquimod, a modifier of the immunological response [94]. Natural polymers k-carrageenan and chitosan were deposited onto olive oil nanoemulsion droplets via LBL self-assembly. The anti-inflammatory drug diflunisal was used as a lipophilic drug model in the nanocapsules thus prepared and was introduced into the oily core [24]. Also, a new system was developed with optimized hyaluronan nanocapsules for intra-articular delivery of the anti-inflammatory celecoxib [95]. In addition, Eudragit^® nanocapsules loaded with nicotine as an adjuvant were studied for the anti-inflammatory therapy of the central nervous system. In this case, some polymer toxicity was observed [96]. An alternative to insulin therapy for diabetes is the use of nanocapsules loaded with anthocyanin. A study explored the potential use of purple sweet potato extract, with high levels of anthocyanin, loaded onto carboxymethyl cellulose and alginate nanocapsules [97].

On the other hand, there are several examples of lipid nanocapsules for the delivery of diverse drugs; lipid spherical nanocapsules with negative zeta potential were prepared with Labrafac^® lipophile WL 1349 and Lipoid^®, and incorporated in a gel for topical administration. The local anesthetics prilocaine and lidocaine were successfully encapsulated [98]. Phospholipid nanocapsules of three sizes were obtained and loaded with astragaloside to treat age-related macular degeneration. Ocular penetration was corroborated through pharmacokinetic studies [99].

6. Conclusion

The number of publications reporting new strategies for the obtention of nanocapsules for biomedical applications considerably rises every year. New materials are being included for the preparation of nanocapsules, and their applications as stimuli-responsive systems, biosensors, and for theragnostic uses are being explored. The nanocapsule production method depends on the desired characteristics, the formulation materials, and the availability of laboratory equipment. Once the nanocapsules are fabricated, the protocol of characterization of the prepared systems is followed. In general, the nanoparticles described are spherical, with suitable sizes, size distributions, and zeta potentials according to their application, specifically, under 300 nm and with zeta potentials around ±30 mV. On the other hand, the activity of the nanoparticles loaded with the corresponding drug is tested in vitro in many of the works, although it is crucial to test that activity in vivo so as to access clinical study. For this reason, more and more in vivo studies appear, especially in the case of antitumoral therapy, which refers to the activity of the encapsulated drug and the ability of nanocapsules to release it in the desired target, avoiding side effects in healthy cells of the body.