Nanocarrier Systems for Transdermal Drug Delivery

The nanomedicine which is the application of technologies on the scale of 1 to 500 nm to diagnose and treat diseases, it has become a very relevant topic nowadays. During the last century, there has been a lot of new research and patents regarding nanomedicine in health sciences [1]. The objective of nanomedicine is to diagnose and preserve the health without side effects with noninvasive treatments. To reach these goals, nanomedicine offers a lot of new tools and capabilities. The manipulation that nanomedicine provides to the drugs and other materials in the nanometer scale can change the basic properties and bioactivity of materials. The solubility, increment in surface area, control release and site-targeted delivery are some characteristics that nanotechnology can manipulate on drug delivery systems.


Introduction
The nanomedicine which is the application of technologies on the scale of 1 to 500 nm to diagnose and treat diseases, it has become a very relevant topic nowadays. During the last century, there has been a lot of new research and patents regarding nanomedicine in health sciences [1]. The objective of nanomedicine is to diagnose and preserve the health without side effects with noninvasive treatments. To reach these goals, nanomedicine offers a lot of new tools and capabilities. The manipulation that nanomedicine provides to the drugs and other materials in the nanometer scale can change the basic properties and bioactivity of materials. The solubility, increment in surface area, control release and site-targeted delivery are some characteristics that nanotechnology can manipulate on drug delivery systems. Nanotechnology applied to health sciences contains new devices used in surgery, new chips for better diagnostics, new materials for substituting body structures and some structures capable to carry drugs through the body for treatment of a lot of diseases. These structures can be made of a lot of different materials and they are very different in structure and chemical nature. All these nanostructures are called nanocarriers and they can be administrated into the organisms by topical and transdermal routes [2]. Nanocarriers are a powerful weapon against a lot of illnesses since they are so small to be detected by immune system and they can deliver the drug in the target organ. For that reason, drug doses using nanocarriers and side effects decrease a lot.
The idea for using these tiny systems is not as new as we think but the use of nanocarriers in pharmaceutical products is not frequent, since the technology is expensive for certain types of nanoparticles and because nanocarriers need to be evaluated for demonstrating they do not have toxic effects. Nowadays the controversy of biological effects due to nanostructures Many agents are applied to the skin either deliberately or accidentally, with either beneficial or deleterious outcomes. The main interest in dermal absorption assessment is related to: a) Local effects in dermatology (e.g., corticosteroids for dermatitis); b) transport through the skin seeking a systemic effect (e.g., nicotine patches, hormonal drug patches, etc.) [31]; c) surface effects (e.g., sunscreens, cosmetics, and anti-infectives) [32,33]; d) targeting of deeper tissues (e.g., nonsteroidal anti-inflammatory agents) [34][35][36][37]; and e) unwanted absorption (e.g., solvents in the workplace, pesticides or allergens) [38,39].

Stratum corneum
The stratum corneum is the heterogeneous outermost layer of the epidermis and is approximately 10-20 µm thick. The stratum corneum consists of about 15 to 25 layers of flattened, stacked, hexagonal, and cornified cells embedded in an intercellular matrix of lipids. These lipid domains form a continuous structure so they are considered to play a crucial role in the maintenance of the skin barrier that helps avoid transepidermal water loss. Each cell is approximately 40 µm in diameter and 0.5 µm thick [40].
The barrier function is further facilitated by the continuous desquamation of this horny layer with a total turnover of the stratum corneum occurring once every 2-3 weeks. The stratum corneum functions as a barrier are to prevent the loss of internal body components, particularly water, to the external environment. The cells of the stratum corneum originate in the viable epidermis and undergo many morphological changes before desquamation. Thus, the epidermis consists of several cell strata at varying levels of differentiation.
The origins of the cells of the epidermis lie in the basal lamina between the dermis and viable epidermis. In this layer there are melanocytes, Langerhans cells, Merkel cells, and two major keratinic cell types: the first functioning as stem cells having the capacity to divide and produce new cells; the second serving to anchor the epidermis to the basement membrane [46]. The basement membrane is 50-70 nm thick and consists of two layers, the lamina densa and lamina lucida, which comprise mainly proteins, such as type IV collagen, aminin, nidogen and fibronectin. Type IV collagen is responsible for the mechanical stability of the basement membrane, whereas laminin and fibronectin are involved with the attachment between the basement membrane and the basal keratinocytes. The cells of the basal lamina are attached to the basement membrane by hemidesmosomes, which are found on the ventral surface of basal keratinocytes [47]. Hemidesmosomes appear to comprise three distinct protein groups: two of which are bullous pemphigoid antigens (BPAG1 and BPAG2), and the other epithelial cellspecific integrins [48][49][50]. BPAG1 is associated with the organization of the cytoskeletal structure and forms a link between the hemidesmosome structure and the keratin intermediate filaments. The integrins are transmembrane receptors that mediate attachment between the cell and the extracellular matrix. Human epidermal basal cells contain integrins α2β1, α3β1 and α6β4. Integrin α6β4 and BPAG2 appear to be the major hemidesmosomal protein contributors to the anchoring of the keratinocyte, spanning from the keratin intermediate filament, through the lamina lucida, to the lamina densa of the basement membrane [51]. In the lamina densa, these membrane-spanning proteins interact with the protein laminin-5 which, in turn, is linked to collagen VII, the major constituent of the anchoring fibrils within the dermal matrix. It has also been suggested that both BPAG2 and integrin α6β4 mediate in the signal transductions required for hemidesmosome formation and cell differentiation and proliferation. Integrin α3β1 is associated with actin and may be linked with laminin-5. Epidermal wounding results in an up-regulation of these proteins that appears to be involved with cell motility and spreading. The importance of maintaining a secure link between the basal lamina cells and the basement membrane is obvious, and the absence of this connection results in chronic blistering diseases such as pemphigus and epidermolysis bullosa.

Dermis
The dermis is about 0.1-0.5 cm thick and consists of collagenous (70 %) and elastin fibres. In the dermis, glycosaminoglycans or acid mucopolysaccharides, are covalently linked to peptide chains to form proteoglycans, the ground substance that promotes the elasticity of the skin. The main cells present are the fibroblasts, which produce the connective tissue components of collagen, laminin, fibronectin and vitronectin; mast cells, which are involved in the immune and inflammatory responses; and melanocytes involved in the production of the pigment melanin [51]. Nerves, blood vessels and lymphatic vessels are also present in the dermis.
Contained within the dermis is an extensive vascular network providing for the skin nutrition, repair, and immune responses for the rest of the body, heat exchange, immune response, and thermal regulation. Skin blood vessels derive from those in the subcutaneous tissues (hypodermis), with an arterial network supplying the papillary layer, the hair follicles, the sweat and apocrine glands, the subcutaneous area, as well as the dermis itself. These arteries feed into arterioles, capillaries, venules, and, thence, into veins. Of particular importance in this vascular network is the presence of arteriovenous anastomoses at all levels in the skin. These arteriovenous anastomoses, which allow a direct shunting of up to 60% of the skin blood flow between the arteries and veins, thereby avoiding the fine capillary network, are critical to the skin's functions of heat regulation and blood vessel control. Blood flow changes are most evident in the skin in relation to various physiological responses and include psychological effects, such as shock (''draining of color from the skin'') and embarrassment (''blushing''), temperature effects, and physiological responses to exercise, hemorrhage, and alcohol consumption.
The lymphatic system is an important component of the skin in regulating its interstitial pressure, mobilization of defense mechanisms, and in waste removal. It exists as a dense, flat meshwork in the papillary layers of the dermis and extends into the deeper regions of the dermis. Also present in the dermis are a number of different types of nerve fibers supplying the skin, including those for pressure, pain, and temperature [52]. Epidermal appendages such as hair follicles and sweat glands are embedded in the dermis [53].

Hypodermis
The deepest layer of the skin is the subcutaneous tissue or hypodermis. The hypodermis acts as a heat insulator, a shock absorber, and an energy storage region. This layer is a network of fat cells arranged in lobules and linked to the dermis by interconnecting collagen and elastin fibers. As well as fat cells (possibly 50% of the body's fat); the other main cells in the hypodermis are fibroblasts and macrophages. One of the major roles of the hypodermis is to carry the vascular and neural systems for the skin. It also anchors the skin to underlying muscle. Fibroblasts and adipocytes can be stimulated by the accumulation of interstitial and lymphatic fluid within the skin and subcutaneous tissue [54]. The total thickness of skin is about 2-3 mm, but the thickness of the stratum corneum is only about 10-15 µm.

Skin appendages
There are four skin appendages: the hair follicles with their associated sebaceous glands, eccrine and apocrine sweat glands, and the nails, but these occupy only about 0.1 % of the total human skin surface.
The pilosebaceous follicles have about 10 to 20 % of the resident flora and cannot be decontaminated by scrubbing. The hair follicles are distributed across the entire skin surface with the exception of the soles of the feet, the palms of the hand and the lips. A smooth muscle, the erector pilorum, attaches the follicle to the dermal tissue and enables hair to stand up in response to fear. Each follicle is associated with a sebaceous gland that varies in size from 200 to 2000 µm in diameter. The sebum secreted by this gland consisting of triglycerides, free fatty acids, and waxes, protects and lubricates the skin as well as maintaining a pH of about 5. Sebaceous glands are absent on the palms, soles and nail beds. Sweat glands or eccrine glands respond to temperature via parasympathetic nerves, except on palms, soles and axillae, where they respond to emotional stimuli via sympathetic nerves [51]. The eccrine glands are epidermal structures that are simple, coiled tubes arising from a coiled ball, of approximately 100 µm in diameter, located in the lower dermis. It secretes a dilute salt solution with a pH of about 5, this secretion being stimulated by temperaturecontrolling determinants, such as exercise and high environmental temperature, as well as emotional stress through the autonomic (sympathetic) nervous system. These glands have a total surface area of about 1/10,000 of the total body surface. The apocrine glands are limited to specific body regions and are also coiled tubes. These glands are about ten times the size of the eccrine ducts, extend as low as the subcutaneous tissues and are paired with hair follicles.
Nail function is considered as protection. Nail plate consists of layers of flattened keratinized cells fused into a dense but elastic mass. The cells of the nail plate originate in the nail matrix and grow distally at a rate of about 0.1 mm/day. In the keratinization process the cells undergo shape and other changes, similar to those experienced by the epidermal cells forming the stratum corneum. This is not surprising because the nail matrix basement membrane shows many biochemical similarities to the epidermal basement membrane [55,56]. Thus, the major components are highly folded keratin proteins with small amounts of lipid (0.1-1.0%). The principal plasticizer of the nail plate is water, which is normally present at a concentration of 7-12 %.

Skin functions
Many of the functions of the skin can be classified as essential to survival of the body bulk of mammals and humans in a relatively hostile environment. In a general context, these functions can be classified as a protective, maintaining homeostasis or sensing. The importance of the protective and homeostatic role allows the survival of humans in an environment of variable temperature; water content (humidity and bathing); and the presence of environmental dangers, such as chemicals, bacteria, allergens, fungi and radiation. In a second context, the skin is a major organ for maintaining the homeostasis of the body, especially in terms of its composition, heat regulation, blood pressure control, and excretory roles. It has been argued that the basal metabolic rate of animals differing in size should be scaled to the surface area of the body to maintain a constant temperature through the skin's thermoregulatory control [57]. Third, the skin is a major sensory organ in terms of sensing environmental influences, such as heat, pressure, pain, allergen, and microorganism entry. Finally, the skin is an organ that is in a continual state of regeneration and repair. To fulfill each of these functions, the skin must be tough, robust, and flexible, with effective communication between each of its intrinsic components mentioned above.
The stratum corneum also functions as a barrier to prevent the loss of internal body components, particularly water, to the external environment. The epidermis plays a role in temperature, pressure, and pain regulation.
Appendage functions are following: hair follicle and sebaceous gland fulfill with protect (hair) and lubricate (sebum), eccrine and apocrine glands have the functions of cooling and vestigial secondary sex gland, respectively; and nails has the function of to protect.The hypodermis acts as a heat insulator, a shock absorber and an energy storage region. One of the major roles of the hypodermis is to carry the vascular and neural systems for the skin.

Routes of drug penetration through the skin
The determination of penetration pathways of topically applied substances into the skin is the subject of several investigations. The permeation of drugs through the skin includes the diffusion through the intact epidermis y through the skin appendages. These skin appendages are hair follicles and sweat glands which form shunt pathways through the intact epidermis, occupying only 0.1% of the total human skin [58]. It is known drug permeation through the skin is usually limited by the stratum corneum. Two pathways through the intact barrier may be identified, the intercellular and transcellular route ( Figure  2): a. The intercellular lipid route is between the corneocytes.
Interlamellar regions in the stratum corneum, including linker regions, contain less ordered lipids and more flexible hydrophobic chains. This is the reason of the non-planar spaces between crystalline lipid lamellae and their adjacent cells outer membrane. Fluid lipids in skin barrier are crucially important for transepidermal diffusion of the lipidic and amphiphilic molecules, occupying those spaces for the insertion and migration through intercellular lipid layers of such molecules [59,60]. The hydrophilic molecules diffuse predominantly "laterally" along surfaces of the less abundant, water filled inter-lamellar spaces or through such volumes; polar molecules can also use the free space between a lamella and a corneocyte outer membrane to the same end [61].
b. The transcellular route contemplates the crossing through the corneocytes and the intervening lipids [24].
Intracellular macromolecular matrix within the stratum corneum abounds in keratin, which does not contribute directly to the skin diffusive barrier but supports mechanical stability and thus intactness of the stratum corneum. Transcellular diffusion is practically unimportant for transdermal drug transport [62]. The narrow aqueous transepidermal pathways have been observed using confocal laser scanning microscopy (CLSM). Here regions of poor cellular and intercellular lipid packing coincide with wrinkles on skin surface and are simultaneously the sites of lowest skin resistance to the transport of hydrophilic entities. This lowest resistance pathway leads between clusters of corneocytes at the locations where such cellular groups show no lateral overlap.
The better sealed and more transport resistant is the intra-cluster/inter-corneocyte pathway [63]. Hydrophilic conduits have openings between ≥5 µm (skin appendages) and ≤10 nm (narrow inter-corneocyte pores). So sweat ducts (≥50 µm), pilosebaceous units (5-70 µm), and sebaceous glands (5-15 µm) represent the largest width/lowest resistance end of the range. Junctions of corneocytes-clusters and cluster boundaries fall within the range [64]. It was determined that the maximally open hydrophilic conduits across skin are approximately 20-30 nm wide, including pore penetrant/opener thickness [63]. Another studies revealed the width of the negatively charged hydrophilic transepidermal pores expanded by electroosmosis to be around of 22-48 nm [65]. Lipophilic cutaneous barrier is governed by molecular weight and distribution coefficient rather than molecular size [66]. The relative height of cutaneous lipophilic barrier consequently decreases with lipophilicity of permeant, but molecules heavier than 400-500 Da are so large permeants to find sufficiently wide defects in the intercellular lipidic matrix to start diffusing through the lipidic parts of cutaneous barrier [64,66,67].
The contribution to transdermal drug transport can increases with the pathways widening or multiplication, for example such that is caused by exposing the stratum corneum to a strong electrical (electroporation/iontophoresis), mechanical (sonoporation/sonophoresis), thermal stimulus, or suitable skin penetrants [59].
Recently, follicular penetration has become a major focus of interest due to the drug targeting to the hair follicle is of great interest in the treatment of skin diseases. However due to follicular orifices only occupying 0.1% of the total skin surface area, it was assumed as a non important route. But a variety of studies shown the hair follicles as could be a way to trough the skin [68][69][70][71][72][73]. Such follicular pathway also has been proposed for topical administration of nanoparticles and microparticles and it has been investigated in porcine skin, because in recent studies the results have confirmed the in vitro penetration into the porcine hair follicles might be considered similar to those on humans in vivo. After topical application of dye sodium fluorescein onto porcine skin mounted in Franz diffusion cells with the acceptor compartment beneath the dermis, the fluorescence was detected on the surface, within the horny layer, and in most of the follicles confirming the similarity in the penetration between porcine and human skin [72]. So nanoparticles have been studied in porcine skin revealing in the surface images that polystyrene nanoparticles accumulated preferentially in the follicular openings, this distribution was increased in a time-dependent manner, and the follicular localization was favored by the smaller particle size [74]. In other investigations, it has been shown by differential stripping the influence of size microparticles in the skin penetration. It can act as efficient drug carriers or can be utilized as follicle blockers to stop the penetration of topically applied substances [73].
It has already been postulated that certain molecules can hydrogen bond to groups present on the surfaces of follicular pores [75]. However, more studies have to be made in order to identify all the molecular properties that influence drug penetration into hair follicles.
Nowadays, there are currently a number of methods available for quantifying drugs localized within the skin or various layers of the skin. To date, a direct, non-invasive quantification of the amount of topically applied substance penetrated into the follicles had not been possible. Therefore, stripping techniques, tape stripping and cyanoacrylate skin surface biopsy have been used to remove the part of the stratum corneum containing dye topically applied [76]. Thus, the "differential stripping" has been shown as a new method that can be used to study the penetration of topically applied substances into the follicular infundibula non-invasively and selectively [20,26,30,76,77].

Advantages and disadvantages of transdermal drug delivery
Transdermal drug delivery systems offer several important advantages over more traditional approaches, in addition to the benefits of avoiding the hepatic first-pass effect, and higher patient compliance, the additional advantages and the disadvantages [78][79][80] that transdermal drug delivery offers can be summarized as follows in Table 1.

Nanocarrier systems
Nanocarriers have demonstrated increased drug absorption, penetration, half-life, bioavailability, stability, etc. Nanocarriers are so small to be detected by immune system and they can deliver the drug in the target organ using lower drug doses in order to reduce side effects. Nanocarriers can be administrated into the organisms by all the routes; one of them is the dermal route. The nanocarriers most used and investigated for topical/transdermal drug delivery in the pharmaceutical field are shown in Figure 3 and Table 2.

Nanoparticles
Nanoparticles are smaller than 1,000 nm. Nowadays, it is possible to insert many types of materials such as drugs, proteins, peptides, DNA, etc. into the nanoparticles. They are constructed from materials designed to resist pH, temperature, enzymatic attack, or other problems [81]. Nanoparticles can be classified as nanospheres or nanocapsules (See Figure  4). Nanospheres are solid-core structures and nanocapsules are hollow-core structures. Nanoparticles can be composed of polymers, lipids, polysaccharides and proteins [82,83]. Nanoparticles preparation techniques are based on their physicochemical properties. They are made by emulsification-diffusion by solvent displacement, emulsificationpolymerization, in situ-polymerization, gelation, nanoprecipitation, solvent evaporation/extraction, inverse salting out, dispersion polymerization and other derived from these one.

Nanoemulsions
Nanoemulsions are isotropic dispersed systems of two non miscible liquids, normally consisting of an oily system dispersed in an aqueous system (o/w nanoemulsion), or an aqueous system dispersed in an oily system but forming droplets or other oily phases of nanometric sizes (100 nm). They can be stable (methastable) for long times due to the extremely small sizes and the use of adequate surfactants. Nanoemulsions can use hydrophobic and hydrophilic drugs because it is possible to make both w/o or o/w nanoemulsions [84]. They are non-toxic and non-irritant systems and they can be used for skin or mucous membranes, parenteral and non parenteral administration in general and they have been used in the cosmetic field. Nanoemulsions can be prepared by three methods mainly: high-pressure homogenization, microfluidization and phase inversion temperature. Transdermal delivery using nanoemulsions has been reduced due to the stability problems inherent to this dosage form.

Liposomes
Liposomes are hollow lipid bilayer structures ( Figure 5) that can transport hydrophilic drugs inside the core and hydrophobic drugs between the bilayer [85]. They are structures made of cholesterol and phospholipids. They can have different properties depending on the excipients included and the process of their elaboration. The nature of liposomes makes them one of the best alternatives for drug delivery because they are non-toxic and remain inside the bloodstream for a long time. Liposomes can be surface-charged as neutral, negative or positive, depending on the functional groups and pH medium. Liposomes can encapsulate both lipophilic and hydrophilic drugs in a stable manner, depending on the polymer added to the surface [86]. There are small unilamellar vesicles (25 nm to 100nm), medium-sized unilamellar vesicles (100 nm and 500nm), large unilamellar vesicles, giant unilamellar vesicles, oligolamellar vesicles, large multilamellar vesicles and multivesicular vesicles (500 nm to microns). The thickness of the membrane measures approximately 5 to 6 nm. These shapes and sizes depend of the preparation technique, the lipids used and process variables. Depending on these parameters, the behavior both in vivo and in vitro can change and opsonization processes, leakage profiles, disposition in the body and shelf life are different due to the type of liposome [86].
Liposomes preparation techniques follow three basic steps with particular features depending on safety, potential scale up and simplicity: 1) Lipid must be hydrated, 2) Liposomes have to be sized and 3) Nonencapsulated drug has to be removed. The degree of transdermal drug penetration is affected by the lamellarity, lipid composition, charge on the liposomal surface, mode of application and the total lipid concentrations [87].

Figure 5.
Liposomes are spherical vesicles that comprise one or more lipid bilayer structures enclosing an aqueous core. They protect encapsulated drugs from degradation. Liposomes can also be functionalized to improve cell targeting and solubility

Dendrimers
Dendrimers are monodisperse populations that are structurally and chemically uniform (Figure 6). They allow conjugation with numerous functional groups due to the nature of their branches. The amount of branches increases exponentially and dendrimers growth is typically about 1 nm per generation [88]. The dendrimers classification is based on the number of generations. After the creation of a core, the stepwise synthesis is called first generation; after that, every stepwise addition of monomers creates the next generation. This approach allows an iterative synthesis, providing the ability to control both molecular weight and architecture.
The kind of polymer chosen to construct the dendrimer by polimerization is very important with regard to the final architecture and features. In addition, the use of branched monomers has the peculiarity of providing tailored loci for site-specific molecular recognition and encapsulation. Notably, 3D and fractal architecture, as well as the peripheral functional groups, provide dendrimers with important characteristic physical and chemical properties. In comparison with linear polymers, dendritic structures have "dendritic voids" that give these molecules important and useful features. These spaces inside dendrimers can mimic the molecular recognition performed by natural proteins.
Furthermore, dendrimers have a high surface-charge density due to ionizable groups that help them to attach drugs by electrostatic forces, regardless of the stoichimetry. This dendrimer-drug association provides drugs with better solubility, increasing their transport through biological membranes and sometimes increasing drug stability. The number of molecules that can be incorporated into dendrimers is related to the number of surface functional groups; therefore, later-generation dendrimers are more easily incorporated into dendritic structure. However, not all the functional groups are available for interaction due to steric volume, molecule rotation or stereochemistry effects. Dendrimers can have positive and negative charges, which allows them to complex different types of drugs [89].

Advantages and limitations of using nanocarriers for transdermal drug delivery
As it is has been mentioned before, the search for new strategies able to enhance the topical and transdermal penetration of drugs has become essential [61]. Different carrier systems have been proposed in an attempt to favour the transport of drugs through the skin, enabling drug retention and in some cases allowing a controlled release. Skin penetration is essential to a number of current concerns, for example, contamination by microorganisms and chemicals, drug delivery to skin (dermatological treatments) and through skin (transdermal treatments), and skin care and protection (cosmetics) [103][104][105][106].
Follicular penetration has become a major focus of interest due to the drug targeting to the hair follicle is of great interest in the topical treatment of skin diseases. However due to follicular orifices only occupying ∼0.1% of the total skin surface area, it was assumed as a non important route. But recently, a variety of studies have shown that the hair follicles represent a important way to trough the skin and some techniques have been used to test the penetration of drugs loaded nanoparticles/microsparticles [70,72,73,[107][108][109]. Confocal laser scanning microscopy which permits optical sectioning of thick tissues and cells and their subsequent computerized three-dimensional reconstruction, has been used to study the entry of drugs through the skin [110]. It was visualized in the fresh human scalp skin on-line the diffusion processes of a model fluorophore into the hair follicle at different depths [72]. Such follicular pathway also has been proposed for topical administration of nanoparticles and microparticles using porcine skin. Recent studies have confirmed that the in vitro penetration into the porcine hair follicles might be considered similar to those on humans in vivo [73]. Studies in porcine skin revealed in the surface images that polystyrene nanoparticles accumulated preferentially in the follicular openings, this distribution was increased in a time-dependent manner, and the follicular localization was favored by the smaller particle size [76]. In other investigations, it has been shown by differential stripping the influence of size microparticles in the skin penetration. It can act as efficient drug carriers or can be utilized as follicle blockers to stop the penetration of topically applied substances [75]. An alternative technique is multiphoton microscopy (MPM) especially two-photon excitation microscopy has been widely used in imaging biological specimens treated with nanoparticles [111][112][113][114]. The near-infrared light used in the two-photon microscope can penetrate deeper in highly scattering tissues such as in vivo human skin than confocal microscopes operated with ultraviolet excitation [115]. Futhermore, this technique provides both cellular and extracellular structural information, with subcellular resolution helpful for clinical dermatological diagnosis, both ex vivo and in vivo. In addition, it can be used to characterize stratum corneum structures, visualize and quantify transcutaneous drug delivery, detect skin cancers, explore collagen structural transitions, and watch laser-skin interactions [116,117]. A common method used for quantifying drugs localized within the skin or various layers of the skin are the tape stripping and cyanoacrylate skin surface biopsy techniques, which have been used to remove the part of the stratum corneum containing dye topically applied. Thus, the "differential stripping" has been shown as a new method that can be used to study the penetration of topically applied substances into the follicular infundibula non-invasively and selectively [118]. It has been reported in a previous in vitro permeation studies using tape stripping, that triclosan-loaded nanoparticles penetrated into the skin and their retention favoured a local effect. Moreover, polymeric nanoparticles are expected to be able to form a depot in the hair follicles, providing a targeted controlled drug delivery [33,119].
In general, the principal advantages of microparticles and nanoparticles over conventional formulations such as creams, solutions, ointments, lotions, gels, and foams, is that the second ones have different absorption characteristics and aesthetic properties, and they also have some major limitations, such as poor penetration and uncontrolled drug release. Furthermore, tolerability and safety end points, such as irritation, dryness, erythema, itching, stinging and burning will be key factors in determining its usefulness. It happens because using a traditional system, drug delivery is sometimes rapid and topical or plasmatic concentrations can result in toxic effects. However, for the case of nanoparticles a smaller amount of the drug is necessary and due to the targeted nature of delivery [120]. Topical or transdermal drug deliveries have many advantages over the other routes: fewer side effects, increased patient compliance, controlled release, and the lack of a hepatic first pass [121].

Nanocarrier Size range Preparation methods Characteristics References
Polymeric nanoparticles [97] Aquasomes 60-300 nm -Self-assembling of hydroxyapatite by coprecipitation method The particle core is composed of noncrystalline calcium phosphate or ceramic diamond, and it is covered by a polyhydroxyl oligomeric film. [98] Pharmacosomes <200 nm -Hand-shaking method -Ether-injection method Pure drug vesicles formed by amphiphilic drugs. [99] Colloidosomes  It has been reported that nanoencapsulation of drugs (nano-medicines) increases their efficacy, specificity, tolerability and therapeutic index [122][123][124]. These nano-formulations are reported to be superior to traditional medicine with respect to controlled release, targeted delivery and therapeutic impact. The targeting capabilities of nanomedicines are influenced by particle size, surface charge, surface modification, and hydrophobicity. Of these, nanoparticle size distribution is an important factor in determining the interaction with the cell membrane and their penetration across physiological barriers, being dependent on the tissue, target site and circulation [125]. Example of this are the nanostructured lipid carriers (NLC) by their structure (lipid nanoparticles with solid matrix) increase in loading capacity, physical and chemical long-term stability, triggered release and potentially supersaturated topical formulations with respect to solid lipid nanoparticles (SLN). Other advantages of NLC include improvement in stabilisation of incorporated compounds, controlled release, occlusivity, film formation on skin including in vivo effects on the skin. Lipid nanoparticles have been observed as a good option for transdermal delivery because they can be prepared in different sizes and it is possible to modify surface polarity in order to improve skin penetration [126,127]. From the upper skin, nanoparticles can reach deeper skin regions because they exhibit mechanical flexion [128].
Additionaly, transdermal nanocarriers are able to reach target organs because they can be attached to antibodies, antigens, vitamins and other molecules to be more specific. Nanoparticles can travel largely undetected by the immune system depending of the nanocarriers size of the antigen added as well as its composition. So, by hiding functional groups or protecting these groups with other molecules, drugs can be released specifically in the target organ. Consecuently, nanoparticles can even travel from the skin to lymph nodes, representing a promising tool for immunomodulation [129]. One of the first strategies for transdermal delivery were the liposomes. The nature of liposomes makes them one of the best alternatives for drug delivery because they are non-toxic and remain inside the bloodstream for a long time [130][131][132][133]. Nevertheless, some factors affect the degree of transdermal drug penetration such as the lamellarity, the lipid composition, the charge on the liposomal surface, the mode of application and the total lipid concentrations [89,134]. For that reason, flexible vesicles called transfersomes or transformable liposomes have been compared with those rigid vesicles to enhance penetration [135][136][137][138][139][140][141][142]. The lipids present in the liposome bilayer can interact with lipids present in the stratum corneum changing the structure of the upper skin. This change is beneficial for the penetration of lipophilic drugs into the stratum corneum [143]. Some liposomes may have a deformable structure and pass through the stratum or may accumulate in the channel-like regions in the stratum corneum, depending upon their composition [144,145]. In order to obtain transformable liposomes more flexible, they are prepared using surfactants or alcohol (ethosomes) in the lipid bilayer, to be able to deform them when a pressure is applied in the transdermal route.
Some limitations for nanocarriers are the important tests and regulations that should be carried out to ensure an adequate characterization, analytical evaluation, toxicological and pharmacological assessment, which is necessary to determine the efficacy of using these nanostructures in therapies and diagnosis because of their tiny size, their high surface energy, their composition, their architecture, their attached molecules, etc. Those things are frequently reviewed for the dendrimers. One of the main advantages is that they have multivalency and it is possible to get control of the functional groups on the surface [146,147]. Due to their form and size (1-10 nm), these molecules can carry drugs, imaging agents, and can interact with lipids present in membranes, because it was reported a better permeation in cell cultures and intestinal membranes. They also increased the permeation of lipophilic drugs instead of hydrophilic drugs. The main problems with this kind of transdermal carrier are poor biodegradation and inherent cytotoxicity [148]. To obtain dendrimers less toxic, dendrimers have been linked to peptides. Dendrimers-peptides are formed from amino acids linked via peptide-amide bonds to the branches of dendrimers in the core or on the surface to get down the toxicity. Then, they are bio-transformed to produce amino-acid derivatives. Besides, the synthesis of these structures is less expensive and purification does not present any difficulty [149,150].
It is suggested in future research to elucidate the interactions between nanocarriers and other molecules as well as interactions between nanocarriers and biological entities. The toxicology of nanostructures is also a current concern. Materials behave very differently when they are diminished to nanosizes. Traditional laws do not work at this "meso-scale" in the same way as they function at the macro-scale. On the macro scale, bulk properties in a material predominate over surface properties. At the micro-scale, surface properties tend to dominate. At the meso-scale, both types of properties play significant roles [151,152]. Furthermore, the effects of metabolized/altered nanostructures on the biological system are difficult to predict. Regulatory agencies are taking action to assess new Nanotechnologybased products.
In addition, the fabrication of nanocarriers scaling up from the lab at the industrial production is difficult and the materials used to prepare nanocarriers are very expensive in the majority of the cases.

Applications of nanocarrier systems in topical/transdermal delivery
Nanocarriers as drug delivery systems were first intended for use in parenteral or oral routes of administration and as such still continue to be the focus of many studies [153]. However skin application of these nanocarriers, and especially for liposomes, polymeric and lipidic nanoparticles, also makes sense when considering surface effects (film formation and occlusive effects), local effects in the skin (drug delivery in the epidermis and dermis) and systemic effects (deeper drug permeation and transdermal delivery). In potential uses apart from those concerned with surface effects the nanocarrier has to overcome the SC barrier in order to deliver the drug more or less deeply into skin layers. Recent advances in the study of penetration mechanisms deal with the control of the intercellular penetration route by the crystalline state of lipids, and the penetration through skin appendages (the follicular pathway) that appears to contribute much more than was previously thought. Applications dependent on skin penetration that have received special attention include transdermal delivery of nano-and microparticles by hair follicles, especially for nanoparticles which penetrate hair follicles very efficiently targeting the skin immune system in order to develop new vaccination strategies, and problems relating to skin diseases [154,155].
Options for topical and transdermal delivery are the solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) [156]. Some drugs such as triptolide, triamcinolone acetonide acetate, cyclosporin A have been used to be entrapped in SLN [157][158][159]. SLN can be admixed to an already commercially available and established topical formulation, e.g. a cosmetic day cream. Admixing the SLN leads to an increase in occlusivity while still maintaining the 'light character' of the day cream and avoiding the glossiness of more occlusive night creams. This phenomena is explained in Figure 7. However, having a highly occlusive night cream already, addition of SLN will have little or no effect [156].
Lipid nanoparticles are other options to load arthemeter and econazole nitrate [160,161]. Celecoxib [162]. It was compared the permeability of coenzima Q 10 incorporated in NLC and in an emulsion with the same lipid contain. The occlusion effect of the cream was also investigated. The result showed a higher permeability of the molecule and a higher occlusive effect for the NLC than for the emulsion as it could be observed in Figures 8 and 9 [163]. Figure 7. The occlusion factor of lipid nanoparticles depends on various factors: at identical lipid content, reducing the particle size leads to an increase in particle number, the film becomes denser (left) and therefore the occlusion factor increases. At a given particle size, increasing the lipid concentration increases particle number and density of the film (right) which also leads to a higher occlusion factor.
Three vitamin derivatives including vitamin C (ascorbyl tetraisoplamitate), vitamin E (tocopherol acetate) and vitamin A (retinyl palmitate) were also loaded in PLGA nanospheres, for skin whitening and anti-wrinkles/aging applications due vitamin C suppresses the blemishes because it limits the activity of tyrosinase, which promotes melanin production. Furthermore, it increases collagen formation to reduce wrinkles, and prevents cell oxidation by eliminating active oxygen. As to vitamin E and A, they also act as antioxidant and collagen promoter, respectively. They were able to reach the target areas in a stable form, sustain the pharmacological effect for a long time and be effective to reduce the wrinkles and produce a whitening effect [171]. In a sense, the idea of nanoparticle design for drug delivery systems in cosmetics applications is important.  . Tape stripping test: summation of coenzyme Q10 found in the tapes related to the applied amount (with permission from authors) [163].
As it was described before, the follicles are deep invaginations inside skin where the SC is thinner, the vascularisation is denser, and there are several targets of interest along a follicle structure from both cosmetic and pharmaceutical viewpoints [172]. Here, minoxidil which is an antihypertensive has been introduced in a block of copolymer poly (-caprolactone)block-poly(ethyleneglycol) to treat the alopecia areata disorder , by widening blood vessels and opening potassium channels, it allows more oxygen, blood, and nutrients to the follicle. This disorder is an inflammatory condition, often reversible hair loss affecting mainly children and young adults. Clinically, round hairless patches appear on the scalp while hair follicles remain intact. This skin disorder is related with the distal part of the human hair follicle immune system, especially with the interacting intraepithelial T cells. The cause of this condition is diverse and seems to involve T cell-mediated immunologic changes, neuropeptides, genetic disposition to autoimmunity, and distress [30,173,174]. As the infundibulum of the hair follicle is surrounded by an extensive capillary network and the permeability of its epithelium allows the transport of molecules or particles to the blood circulatory system. There is a high density of immune cells in and around the infundibulum epithelium which could be targets also for hair follicle immune system and topical vaccination.
The sebaceous glands associated with hair follicles provide another potential target for delivering drugs against acne, androgenetic alopecia and other sebaceous gland dysfunctions. Different nanoparticles formulations have been prepared in order to treat acne vulgaris, which is an inflammatory disease of the pilosebaceous units, most densely concentrated on the face and torso [175]. Pathogenesis is multifactorial, but Propionibacterium acnes a Gram-positive bacterium plays a central role in the promotion of inflammation in acne. The most commonly formulations are prepared with different topical antimicrobials, either alone or in combination with other drugs. It is expected that an agent able to inhibit P. acnes growth and to suppress the inflammatory response will provide significant benefits to patients with acne vulgaris [30,176,177]. For that reason triclosan has been used in several systems. It was reported the characterization of triclosan loaded polymeric nanoparticles. They showed a good encapsulation efficiency and also a good physical stability representing an alternative as a treatment of acne [33]. Triclosan loaded nanoparticles made of chitosan and cyclodextrins were prepared using a very simple ionic gelation technique. This new approach permits to enhance the entrapment of hydrophobic drugs by forming molecular inclusion complexes with cyclodextrins in aqueous media. Such a device could be of interest for conferring protection to some specific drug molecules through the complexation followed by entrapment in the polymer matrix [109]. Another drug used to treat this disorder is tretinoin (all-trans-retinoic acid) which is the active form of a metabolic product of Vitamin A, also called retinoic acid. Tretinoin-loaded nanocapsules improved tretinoin photostability, independently on the type of oily phase used (capric/caprylic triglycerides and sunflower seed oil) in this study, and represent a potential system to be incorporated in novel topical or systemic dosage forms containing tretinoin [178].
Formulations of nanoparticles are often used in combination with penetration chemical and physical enhancers to modify the physical state of the stratum corneum, affecting the degree of transdermal drug penetration. DNA has been entrapped in nanoparticle of polysaccharide such as chitosan/poly--glutamic acid and in a multifunctional core-shell polymeric nanoparticle of PLGA core and a positively-charged glycol chitosan (GC) shell. Another drugs used in the preparation of nanoparticles made of propyl-starch derivatives are flufenamic acid, testosterone and caffeine [179,180]. Insulin is a protein which has also been introduced in chitosan nanoparticles [83]. Poly (lactide-co-glycolide) polymer has been used to prepare biodegradables nanoparticles containing dexamethasone phosphate and 5-Fluorouracil [181,182]. Chlorhexidine loaded polymeric nanoparticle are used to treat cutaneous infections [183].
Inflammatory skin diseases account for a large proportion of all skin disorders and constitute a major health problem worldwide. Psoriasis, atopic dermatitis, poison ivy, and eczema are another skin disorders. Contact dermatitis, atopic dermatitis, and psoriasis represent the most prevalent inflammatory skin disorders and share a common efferent Tlymphocyte mediated response. Oxidative stress and inflammation have recently been linked to cutaneous damage in T-lymphocyte mediated skin diseases, particularly in contact dermatitis [184]. Poison ivy and atopic dermatitis may also present with bullous and vesicular changes [185]. Lipid nanoparticles have been investigated to improve the treatments of skin diseases such as atopic eczema, psoriasis, skin mycosis and inflammations. Apart from the treatment of skin diseases by topical application, e.g. gastrointestinal side effects of non-steroidal anti-inflammatory drugs can be decreased by topical antirheumatic therapy. Drugs under investigations for dermal application using lipid nanoparticles at the present are for instance glucocorticoids, retinoids, non-steroidal antiinflammatory drugs, COX-2 inhibitors and antimycotics. It was showed that it is possible to enhance the percutaneous absorption with lipid nanoparticles. These carriers may even allow drug targeting to the skin or even to its substructures. Thus they might have the potential to improve the benefit/risk ratio of topical drug therapy [186].
Perioral dermatitis is commonly seen in women aged 20-35 years. It presents as red papules that form superficial plaques around the perioral area, nasolabial folds and/or lower eyelids. It is minimally itchy [187,188]. Topical corticosteroids are the first-line therapy of acute exacerbations of atopic dermatitis and contact dermatitis. Prednicarbate is superior to the halogenated glucocorticoids because of an improved benefit/risk ratio. However, at the present the separation of desired anti-inflammatory effects and undesired antiproliferative effects is still not satisfying. Therefore, lipid nanoparticles were investigated as a delivery system for prednicarbate. The report show an improved extent of prednicarbate uptake by human skin in vitro, if applied as SLN dispersion or cream containing prednicarbate-loaded SLN. The authors found that a prednicarbate targeting to the epidermis [189]. This is particular relevant because prednicarbate in the dermis is responsible for the induction of irreversible skin atrophy while the inflammatory process is most pronounced within the epidermis [186]. Therefore, a better benefit/risk ratio is expected for the application of pretnicarbate in SLN containing topical formulations.
Tretinoin, a metabolite of vitamin A is used for topical treatment of various proliferateive and inflammatory skin diseases such as psoriasis, acne (as mentioned before), photo aging, epidermotropic T-cell lymphomas and epithelial skin cancer. One of the major disadvantages associated with the topical application of tretinoin is local skin irritation such as erythrema, peeling and burning as well as increased sensitivity to sunlight. To overcome these problems tretinoin was incorporated into SLN [190]. In vitro permeation studies through rat skin indicated that SLN-based tretinoin gel has a permeation profile comparable to that of the market tretinoin cream. Furthermore, Draize patch test showed that SLN-based tretinoin gel resulted in remarkably less erythremic episodes compared to the currently marketed tretinoin cream (Figure 10). Therefore, also for formulations containing tretinoinloaded SLN a better benefit/risk ratio is expected. Liposomes which were one of the first strategies for transdermal delivery are being successfully used in cancer therapy [139,141]. However to date, many liquid-type nanocosmetics carriers, such as liposomes, are structurally unstable. Specifically, when passing through the skin, they adhere to the inside walls of the skin cells causing the collapse of phospholipid association bodies and the leak of their encapsulated ingredients. As a result, their ability to transport active ingredients to deep skin is not likely good. Some authors report the use of flexible vesicles called transfersomes or transformable liposomes in comparison with rigid vesicles to enhance penetration [148,191]. The application of transformable liposomes more flexible, which are prepared using surfactants or alcohol (ethosomes) in the lipid bilayer, to be able to deform them when a pressure is applied in the transdermal route has been increased.
In some researches, dendrimers are used for transdermal drug delivery. They show promising results in the delivery of drugs such as tamsulosin [192], indomethacin [193], ketoprofen and diflunisal [194 ] and 5-fluorouracil [195]. The main problems with this kind of transdermal carrier are poor biodegradation and inherent cytotoxicity [158]. In order to get down the toxicity dendrimers have been linked to peptides (dendrimers-peptides) from amino acids linked via peptide-amide bonds to the branches of dendrimers in the core or on the surface [159,160].
Nowadays transdermal delivery using nanoemulsions it is not so used as nanoparticles or liposomes delivery due to the stability problems inherent to this dosage form. Nevertheless, Gamma Tocopherol, Caffeine, Plasmid DNA, Aspirin, Methyl Salicylate, Insulin, Nimesulide have been included in nanoemulsion. The use of these nanocarriers to deliver analgesics, corticosteroids, anti cancer agents, etc. is very important since these drugs are able to act immediately because they do not need to cross extra barriers. The drug is bioavailable easily and faster [204][205][206][207][208][209][210].

Conclusions
Nanocarriers have shown many advantages for topical and trasdermal delivery of drugs. It could be shown already for various drugs that topical/trandermal formulations containing nanoparticles can enhance the penetration into the skin increasing treatment efficiency, target the epidermis or follicles, reducing side effects. Furthermore, an increased activities as well as prolonged activities have been reported. These delivery systems can deliver both hydrophilic and lipophilic molecules. Advances with regard to materials, fabrication methods and techniques facilitate the development of new and better nanocarriers. Nonetheless, future researches must ensure the benefit and evaluate the risk ratio for many drugs included in nanocarriers.