Table of contents
Vaccines are important tools to prevent infections. Over the last two centuries, vaccination has been one of the most successful medical interventions in reduction of infectious diseases (1). Infections are responsible for almost one-third of all deaths worldwide (2). Currently vaccines are available in three forms: live-attenuated, inactivated and subunit (3). Classical vaccines were mostly based on live-attenuated and inactivated pathogen. Due to the complex nature and safety issues of these classic vaccines, subunit vaccines are preferred over these classical vaccines. However, disadvantages of subunit vaccines are that they are based on a purified protein, peptide or gene fragment of the pathogen and are poor immunogens compared to the traditional vaccines (4).
Almost all subunit vaccines are administered by intramuscular or subcutaneous injection, but alternative routes of administration such as intradermal and subcutaneous are widely explored. Injections can be painful and require syringes, needles, trained personnel and carry the risk of transmitting various bio hazardous pathogens (5). Novel vaccination strategies are still needed which should result in safer injections but at the same time still being cost-effective and capable of evoking an immune response in patients (6)(7).
The skin is an attractive target for vaccination as it acts as a major barrier for the entry of environmental pathogens, while at the same time being easily accessible for vaccine administration (8)(9). The skin has a great possibility for painless drug delivery. First of all, skin tissue contains more antigen presenting cells (APCs) than muscle and subcutaneous tissue. The skin has an advanced immune system with APCs, such as Langerhans cells (LCs) and dermal dendritic cells (dDCs) (10). Targeting the skin immune system can be carried out with transcutaneous and intradermal immunization. Transcutaneous immunization refers to the needle-free topical application of a vaccine with or without an adjuvant. Transcutaneous immunization has several benefits such as inducing a strong humoral and cellular response. Intradermal immunization is the delivery of molecules into the dermis. DCs in the dermis are able to capture and process foreign antigen resulting in a strong cellular and humoral immune response.
In addition, the skin has a large surface area for drug application (11). These two methods allow for the use of smaller quantities of antigen (12). Furthermore the transdermal route bypasses the first-pass effect of the liver, which also suggests that the skin is a very attractive place for vaccination.
1.2 Nanotechnology in vaccination
Nanotechnology is an exhilarating area for the development of drug delivery systems using nanoparticles (23). In the past micro particles were often used. However, the size of these micro particles limits their ability to permeate skin and/or most other membranes. So is seen in the past that these particles are too large to cross the intestinal mucosal barrier of the gastrointestinal (GI) tract after the drug has been delivered orally. Nanoparticles on the other hand have an advantage over micro particles due their nano-sizes. Nanoparticles are particles from different materials with dimensions ranging from 1-1000 nm, to which drugs or antigens can be attached, adsorbed, encapsulated or entrapped. They demonstrate unique properties and functions due to their nano-scale size (26).The size of these particles resemble natural pathogens and are therefore easily internalized by cells. Due to their size, nanoparticles can enter living cells by pinocytosis and presentation of antigens is better recognized by the immune system(27). In the past years the application of nanoparticles has increased rapidly which may lead to a new generation of improved vaccines also called ‘nanovaccinology’ (24). Nanoparticles can be produced in various sizes, surface charges, shapes and compositions (25).
However, the use of nanoparticles encounters also some problems. Intravenously administered nanoparticles were rapidly cleared from the body by phagocytic cells. The problem of phagocytic removal of nanoparticles has been solved by surface modification of nanoparticles (30). The surface modification protects nanoparticles from being phagocytosed and removed from the blood vascular system after intravenous injections. Currently, a wide variety of biomolecules, vaccines and drugs can be delivered into the body using nano-particulate carriers and a number of routes of delivery.
Nanoparticles can be used as either a delivery system and/or as an adjuvant to enhance the immune response.Nanoparticles can act as a delivery system by delivering antigens to the cells of the immune system. They can protect the antigen and then release it at the target location. Nanoparticles have the ability to deliver a range of molecules to varying areas of the body and for sustained and controlled periods of time (31). Other advantages of nanoparticle delivery systems, includes improved drug solubility, prolonged systemic circulation, real-time read on the in vivo efficacy of a therapeutic agent and etc. (32). For a nanoparticle based delivery system, the key objectives are the size of the particles, the shape of the particles, the loading capacity and the surface properties as well as the charge of drugs or the active ingredients to accomplish highest efficacy (33).
Currently, the subunit vaccines are preferred over the traditional vaccines, but their poor immunogenicity requires the addition of an adjuvant in order to get an improved immune response. An adjuvant is a compound that can be added to the vaccine formulation in order to enhance the immunostimulatory effects on the antigen presenting cells (19). Another promising approach to increase the immunogenicity of subunit vaccines is the formulation of antigens into nanoparticles. Nanoparticles facilitate the uptake by antigen presenting cells, such as dendritic cells, due to their similarity in size to pathogens (34). Furthermore, nanoparticle formulations can protect the antigen from enzymatic breakdown, allow sustained antigen release over time and offer the possibility of co-encapsulation of adjuvants (35).
Recently, skin vaccination gets more and more attention. The skin is a primary barrier for nanoparticles. Although nanoparticles are very small, they still cannot permeate the stratum corneum (SC). If this was not the case the skin would be vulnerable for pathogens. Therefore other ways have to be explored to deliver nanoparticles into the skin. Therefore in this review, we aim to investigate the different types of nanoparticles that are currently known and their usage as delivery vehicles and/or immune enhancers in vaccination. We also want to explore the interaction of nanoparticles with the antigen, with APCs and the human bio-system. Finally we will look into the use of skin vaccination in combination with nanoparticles.
2. Different types of nanoparticles as delivery vehicle
There are several classes of nanoparticles and each class of nanoparticles consists of several types of particles. Most of these nanoparticles can act as delivery vehicles. As carriers, nanoparticles have been found to be able to enhance the cellular uptake of DNA, lengthen the circulation time, target specific cells and regulate immune response. Here, we will discuss the most important and frequent used nanoparticles as antigen delivery vehicle (Figure 1).
Figure 1: Schematic representation of different nanoparticle delivery systems. (A) Virus-likeparticle, (B) Liposome, (C) ISCOM, (D) Polymeric nanoparticle, (E) Non-degradable nanoparticle.
2.1 Polymeric nanoparticles
Polymeric nanoparticles are colloidal carriers that vary in size from 10 to 1000 nm (37). They can be divided into two categories: nano-spheres and nano-capsules. A nano-spheres is a polymeric matrix in which the drug is uniformly dispersed. Nano-capsules are vesicular systems in which the drug is constricted to a cavity surrounded by a polymer membrane, whereas Polymeric nanoparticles are prepared using synthetic polymers such as poly (d,l-lactide-co-glycolide) (PLG) and polystyrene. Poly (lactic-co-glycolic acid) (PLGA), PLG and poly lactic acid (PLA) are the most extensively investigated nanoparticles due to their excellent biocompatibility and biodegradability. These polymers have already been approved for human use (38).
2.1.1 Poly (lactic-co-glycolic acid)
The degradation time of PLGA can vary from several months to several years. The degradation period depends on factors such as the molecular weight and copolymer ratio (39)(40). PLGA nanoparticles are negatively charged which can be shifted to neutral or positive charges by surface modification, for example by PEGylation of the PLGA polymer or chitosan coating (41)(42). One of the major pitfalls of PLGA-based nanoparticles is the poor loading capacity. They show high encapsulation efficiencies (98%) while the drug loading is around the 1% (which means that nanoparticles contain 1 mg active ingredient per 100 mg polymers of nanoparticles) (43)(44). This low drug loading constitutes a major problem for some drugs in combination with PLGA-based nanoparticles. PLGA has been used as a delivery system to carry antigens from several pathogens such as Chlamydia, hepatitis B, Bacillus anthracis and Plasmodium vivax malaria with mono-phosphoryl lipid A as adjuvant (43)(45)(46)(47). PLGA polymers can encapsulate one antigen or a combination of antigens and adjuvants in the same particle (48). Antigens and adjuvants must be delivered by the same particle to be internalized simultaneously. A study by Diwan et al. has showed that PLGA nanoparticles containing very low doses of antigens and adjuvants are able to induce strong T cell responses (49). These studies show that PLGA nanoparticles can act as efficient delivery vehicles. Another major advantage of PLGA over other polymers is that PLGA is approved by the food and drug administration (FDA) and European medicines agency (EMA) in various drug delivery systems, leading PLGA-based nanoparticles in a good position for clinical trials.
2.1.2 Poly (lactic acid)
PLA nanoparticles are anionic and have also been used as a delivery system, but to a lesser extent than PLGA nanoparticles. PLA nanoparticle applications are limited due to its weak hydrophilicity, excessively long degradation time, and low drug loading of polar drugs. However modification of PLA with PEGylation has shown some promising results regarding sustained release and hydrophilicity (50). The encapsulation efficiency of antigens is not well reported. The encapsulation efficiency for the antigen quercitrin is approximately 40% (51). PLA nanoparticles have already been used to deliver drugs such as zidovudine and antigens such as HIV-1 p24 and gp120 proteins (52)(53).
2.1.3 Poly (d,l-lactide-co-glycolide)
PLG nanoparticles have almost the same characteristics as PLGA nanoparticles but are less often used compared to PLGA particles. However several studies have shown that PLG nanoparticles are excellent delivery vehicles. A study by Elamanchili et al. showed that PLG nanoparticles loaded with MPL and a cancer associated antigen were efficiently taken up by DCs (54). This study showed that PLG is also an efficient delivery vehicle. Glutamic acid-PGA (g-PGA) nanoparticles are also sometimes used as delivery vehicles. g-PGA are comprised of amphiphilic polymers. These particles self-assemble into Nano-micelles with a hydrophilic outer shell and a hydrophobic inner core (55). In contrast to PLGA nanoparticles, g-PGA nanoparticles are generally used to encapsulate hydrophobic antigens.
2.1.4 Natural polymers
Natural polymer nanoparticles are also used as delivery vehicles. This group of polymers consists of pullulan, alginate, inulin and chitosan (56)(57)(58)(59). However, chitosan is the most intensively studied polymer nanoparticle for antigen delivery. Chitosan nanoparticles are a promising delivery vehicle due to their biodegradability, biocompatibility, non-toxicity, immune modulating properties and their ability to be easily modified in different shapes and sizes (60)(61). Chitosan particles have a size in the range of 150-450 nm and are specifically adapted to carry large amounts of antigens. Oliveira and coworkers described that chitosan nanoparticles loaded with plasmid DNA were able to form complexes electrostatically with DNA and condense it into positively charged nanostructures (62). These particles have been used in various vaccines against several diseases such as Newcastle disease vaccines.
There are also some chitosan Nano gels available on the market. Nano gels are favorable due to their flexible mesh size, large surface area for multivalent conjugation, high water content and high antigen loading capacity (63). In addition, polymer nanoparticles can be used as delivery vehicles and immune enhancers at the same time.
2.2 Inorganic nanoparticles
Currently there are many inorganic nanoparticles extensively studied and used in vaccines. Inorganic nanoparticles are mostly used for their rigid structure and controllable synthesis. One side point of inorganic nanoparticles is the fact that they are non-biodegradable. Several materials are being examined such as silica, gold, carbon and polystyrene for their usage as vaccine delivery system.
2.2.1 Gold nanoparticles
One of the earliest used materials for vaccine delivery are gold nanoparticles (AuNPs). These nanoparticles can be produced into different shapes such as spherical, rod and cubic nanoparticles. AuNPs are characterized by their size with a size range of 2-150 nm. The size of AuNPs can be modulated to optimize the delivery to the immune system and can be modified with groups for immune modulation (64). It has been reported that AuNPs can be surface-modified with carbohydrates and in addition can become cationic, neutral or anionic (65)(66). The loading efficiency of the antigen is not well investigated but numbers between 40% and 60% are reported (67). AuNPs have been used for the antigen delivery of several viruses such as influenza and foot and mouth disease (68)(69). The group of Safari et al. explored AuNPs as carriers for a synthetic Streptococcus pneumoniae type 14 conjugate vaccine (70). This study showed that AuNPs carriers elicited a humoral and cellular immune response. Another study by Chen et al. investigated the potential of AuNPs in BALB/c mice to act as a size-dependent carrier for a synthetic peptide resembling foot-and-mouth disease virus (FMDV) (69). This study showed that AuNPs could induce an antibody response.
2.2.2 Silica nanoparticles
One of the most promising inorganic materials for vaccination is silica. Silica (SiO2), an oxide of silicon with the chemical formula SiO2, is the most common element found in nature and is widely distributed in dusts, sands, planetoids, and planets. Silica based nanoparticles are biocompatible and can be used as nanocarriers for vaccine delivery. These carriers are also frequently used for different applications such as tumor targeting and real-time multimodel imaging. One of the most frequent used inorganic nanoparticles are the porous silica based nanoparticles in particularly the mesoporous silica nanoparticle (MSNs). This nanoparticle is a form of amorphous silica. They are characterized by the size of their pores, which range from 2’50 nm. MSNs with sizes ranging from 50-200 nm are extensively studied as nanocarriers and adjuvants for the delivery of antigens (71). MSNs are currently extensively investigated because these particles show enhanced stability and no leakage issues compared to other nanoparticles. MSNs have a higher loading capacity, because of their larger specific surface area, improved performance in delivery and controlled release due to the specific porous structure. In addition, MSNs can be varied in their size, pore size, morphology and structure. It is reported that MSNs can be made in all kind of shapes such as rods, spheres and worm like structures (72). The surface of these particles can also be chemically modified to act as efficient delivery systems. Due to its flexible psychical and chemical properties through which adsorption and release of biomolecules can be tuned, MSNs are frequently used (73).
Hollow mesoporous silica particles (HMSNs) are also being extensively investigated due to their high loading capacity for antigens. HMSNs show also multifunctional surface modification, controlled release capability, and good thermal stability (74).These properties indicate that they can be used as carriers for therapeutic compounds in vitro and in vivo. These particles are already used for intracellular delivery of bovine serum albumin (BSA) and goat Immunoglobulin G (IgG) (75). In addition, HMSNs have been approved by the FDA as a new inorganic material.
2.2.3 Calcium phosphate nanoparticle
Another well-known type of inorganic nanoparticles is the calcium phosphate nanoparticle (CaP). These nanoparticles can be generated by mixing calcium chloride, dibasic sodium phosphate and sodium citrate under specific conditions (76). These particles are approximately 50-100 nm in size (77). Calcium phosphate has been used for over 30 years as delivery vehicles to mammalian cells. This vector has proved advantages over other delivery vehicles such as viruses and dendrimers in terms of superior biocompatibility (78). CaP-nanoparticles are excellent for usage in vaccines due to their exquisite biocompatibility, biodegradability and non-toxicity. Their surface can be modified by substituting cations like Mg2+ and strontium which affects the physical and chemical properties of CaP-nanoparticles (79)(80). It is reported that the maximum amount of 50 ??g of pDNA per mg of nanoparticles could be loaded and that the entrapment efficiency was found to be more than 95%. The capabilities of CaP-nanoparticles as a delivery vehicle is already explored in animals (77)(81). For example, CaP-nanoparticles as an antigen/protein delivery vehicle was explored in a fish model Labeo rohita H (81). The S-layer protein (Aeromonas hydrophila) was adsorbed on CaP nanoparticles and elicited both innate and adaptive immune parameters, which persisted up to 63 days of post immunization through parenteral immunization and gave cross protections. However, clinical application of calcium phosphate based delivery vehicles is hampered by poor understanding of the key factors underlying its action.
2.3 Liposomes and virosomes
Liposomes are capable to act as delivery systems. It was first reported by Gregoriadis et al. that liposomes induced immune responses of encapsulated or associated antigens (82). Since then, liposomes were extensively studied for their use in human vaccines. Liposomes are spherical vesicles, which are composed of a phospholipid bilayer membrane. They are formed by biodegradable phospholipids. Liposomes resemble cells but with nothing in the core. The size of liposomes is usually between 90 and 150 nm.
Drug delivery with liposomes has certain benefits as liposomes are made of natural mammalian membranes (83). A general key advantage of liposomes and virosomes is their adaptability and plasticity. The composition and preparation of liposomes can be altered for the usage purpose. Liposome properties that can be achieved by altering the composition and preparation method are lipid composition, charge, size distribution, entrapment and location of antigens or adjuvants. This feature was investigated by Guan et al. in mice (84). In this study the effect of the liposome formulation on the type of immune response was tested. This revealed that antigens being encapsulated or surface adsorbed produced a strong cytotoxic T lymphocyte response (CTL), while an antibody response was only achieved with the surface adsorbed formulation. In the study of Fox et al., anionic liposomes were used to deliver toll like receptor ligands 4 and 7 synergistically in mice (85). It was seen that the combination of liposome formulation and ligand enhanced and resulted in a specific Th1 immune response, which make liposomes efficient antigen deliverers.
For vaccine delivery, the antigen can be presented in different ways to the antigen presenting cells using liposomes. Antigens can either be captured in the empty core of liposomes for delivery (86). They can also be buried within the lipid bilayer or adsorbed on the surface of liposomes. There are some human clinical trials ongoing with liposomes as delivery systems against diseases such as malaria, HIV, hepatitis A, influenza and cancer (87)(88)(89)(90). Stimuvax, a liposome formulation, is being investigated for the treatment of small lung cancer. A phase 2 trial showed that Stimuvax increased survival rates for patients with small lung cancer (91). Another liposomal based delivery system, called NeuGcGM3 vaccine, is investigated for the treatment of melanoma (92). This study showed enhanced immunogenicity in patients with the liposome-based vaccine compared to traditional vaccines in phase I trial. Despite these ongoing trials there are currently no liposome based vaccines on the market (87).
Furthermore, liposomes can become virosomes by incorporating viral envelope glycoproteins in their core. The first virosomes were prepared by the group of Ameida et al. (93). They removed the surface haemagglutinin and neuraminidase projections of influenza virus from the viral envelope. Then, they purified and relocated this on the surface of unilamellar liposomes. The structures obtained were examined and found to resemble the original influenza virus. To be more specific, virosomes are liposomes by combining natural or synthetic phospholipids with virus phospholipids, viral spike glycoproteins and other viral proteins.
One of the most important groups of virosomes are the immunopotentiating reconstituted influenza virosomes (IRIVs). These virosomes are small unilamellar vesicles (SUV) with spike projections of the influenza surface glycoproteins haemagglutinin (HA) and neuraminidase. The fusogenic properties of HA make them so interesting for vaccine delivery. Some virosome-based vaccines have already reached the market. The first virosome based vaccine in humans was called Inflexal, an influenza vaccine. This vaccine showed excellent results after vaccination in immunocompromised and healthy volunteers (94). Another well-known virosome based vaccine is called Epaxel, an inactivated hepatitis A vaccine. Epaxel was tested in Thai children with HIV infection. The prevalence of hepatitis A antibodies was 100% after vaccination which resulted in concluding that Epaxal is an effective vaccine for children with HIV (95). Another vaccine using virosomes is called Invivac. All these virosome based vaccines have shown that they possess excellent immunogenicity and tolerability (47)(48)(96).
Currently, several vaccines are undergoing pre-clinical or clinical studies such as a virosome based vaccine containing surface HIV-1 gp41-derived P1 lipid conjugated peptides and IRIVs with potential for delivery of peptides derived from Plasmodium falciparum-antigens (97)(98).
2.4 Immunostimulating complex
Immunostimulating complexes are also called ISCOMs. These complexes can be used as delivery vehicles with potent adjuvant activity. ISCOMs are made of the saponin adjuvant Quil A, cholesterol, phospholipids and protein antigen. Quil A is extracted from the bark of the South American Quillaja Saponaria Molina tree. The saponin and cholesterol molecules interact to form a subunit, ring-like micelle which, in the presence of a phospholipid, creats a cage-like structure. These cage like structures are approximately 40 nm in size and are spherical shaped (99). Vaccines using ISCOM have been shown to induce humoral as well as cellular immune responses.
ISCOMs are able to generate a broad range of immune responses such as APC activation and CD4- and CD8- T cell responses (100)(101)(102). This feature makes ISCOMs perfect for the use in vaccines that are directed against chronic diseases and cancer. In addition, ISCOMs can result in an induced major histocompatibility complex II (MHC II) expression on APCs and cytokine induction (103)(104). However, the type of immune response after ISCOM vaccination is highly dependent on the type of antigen and the route of administration. ISCOMs have several advantages above other colloidal delivery systems such as liposomes. ISCOMs are more immunogenic due to their in-built adjuvant (105). Quil A exerts it in adjuvant activity in ISCOMs while at the same time the stability is increased and toxicity is decreased. Another major advantage is the fact that ISCOMs require less antigen and adjuvant to induce an immune response with vaccination compared to traditional vaccines (106). This makes them especially applicable for vaccines that require antigens that have limited availability or are expensive to manufacture. Furthermore, ISCOMs display specific advantages for humoral responses such as the speed and the longevity of the antibody response.
Due to these advantages over other delivery systems many vaccines with ISCOMs are currently investigated. To date many studies have already showed the ability of ISCOM vaccines to induce antigen specific humoral and cellular immune responses in animal models (107). There are some vaccines which have been approved for veterinary use (108). These successes in animals resulted in undergoing clinical trials in humans. ISCOM vaccines for humans have been tested with various antigens and resulted in highly humoral and cellular immune responses. Currently, ISCOMs with different antigens have been used such as influenza, herpes simplex virus, HIV, and Newcastle disease (109)(110)(111)(112). In all these vaccines, the studies have shown a good safety and tolerability profile in humans (113). In addition, induction of both humoral and cellular immune responses were showed (114). Although there are currently registered ISCOMs vaccines for veterinary applications, the ISCOMs vaccines need further clinical investigation for novel human vaccines and further cellular or humoral immune responses should also be required to demonstrate efficacy in humans for future usages.
2.5 Virus like particles
Virus like particles (VLP) are self-assembling non infective viruses, formed by self-assembly of biocompatible capsid proteins (115). These naturally occurring bionanomaterials can be produced from several viruses. VLPs sizes can range from 20nm to 800 nm (116). VLPs, like their parental viruses, can have different conformations such as enveloped or non-enveloped and spherical or filamentous.
VLPs contain functional viral proteins, which are responsible for cell penetration by the virus. This leads to efficient cell entry. Most important application of VLPs is in vaccinology, whereby they provide delivery systems that combine good safety profiles with strong immunogenicity (117). VLPs are ideal for nanovaccine purposes as they contain the power of a viral structure which is naturally optimized for interaction with the immune system, but avoid the infectious system (118). These complexes stimulate an immune response by delivering an antigen that mimics viral properties. They stimulate both cellular and humoral immunity when used in vaccines even without an adjuvant (119)(120). VLPs are also convenient to chemical and genetic modifications which makes VLPs ideal carriers for antigens (121). Furthermore, based on the size and number of copies of protein needed to assemble particles, VLPs can be designed to carry multiple epitopes for the detection of viral agents (122). Traditionally, vaccines against viral diseases have been prepared from attenuated or inactivated viruses. VLPs lacking the viral genome, but are able to penetrate cells and tissues, are a much safer alternative.
Several VLP based vaccines are already on the market. VLP based vaccines were one of the first nanoparticle based vaccines to reach the market. A prophylactic hepatitis B vaccine was the first recombinant protein based VLP vaccine for humans approved by the FDA (USA) in 1986 (123). It is based on a recombinant HBV surface antigen (HBsAg), which upon production in yeast or mammalian cells forms 22- nm spherical VLPs that are adsorbed on an aluminum hydroxide gel (124). Most commercialized VLP vaccines are based on self-assembly of proteins derived from the target virus. However, VLPs can also act as delivery platform where a target antigen from a virus unrelated to the VLP used is modularized on the surface of a VLP (125)(126). These modularized VLPs exploit known benefits of VLPs to target disease.
Currently, two VLP vaccines have been approved for use in humans. VLP based vaccines that are already on the market are against HPV which have been approved for use in the United States, Gardasil?? in 2006 and Cervarix?? in 2009 (127) (128). These are a quadrivalent (types 6, 11, 16 and 18) HPV vaccine and a bivalent (types 16 and 18) vaccine and are approved for human use (129). Both vaccines are made of non-infectious, DNA free virus like particles and combined with an adjuvant. These vaccines showed to induce high levels of serum antibodies in almost all vaccinated patients. Many VLP based vaccines are in the clinical or pre-clinical trials phase. Approximately 88 new VLP based vaccines are under clinical evaluation whereas most of them are against viral infections. One of these clinical trials is performed with a vaccine against HIV. In this study they investigated the immunity of VLP made of yeast Ty protein and decorated with HIV p24 protein (130). This therapeutic anti-HIV vaccination with p17/p24: Ty- VLP elicited antibodies against p24. These studies show that VLP nanoparticles are excellent vaccine delivery vehicles. In nanovaccinology VLP nanoparticles are clearly the safest to use for vaccination in healthy human. This is also supported by several studies and evidence. In addition with so many VLP based vaccines undergoing clinical trials, an increase in the number of approved VLP-based vaccines can be expected (131).
2.6 Self-assembled proteins
Particles similar to the VLPs are the self-assembled proteins. These types of nanoparticles are especially important in driving higher-order molecular structure in modern vaccine delivery. There are several examples of self-assembled proteins, but the most important one is the cage like structure of ferritin. Ferritin is a protein that can self-assemble into a spherical form of approximately 10 nm. Kanekiyo et al reported the use of influenza virus haemagglutinin (HA) in self-assembling proteins (132). They genetically fused HA to ferritin, which resulted in the assembling of the genetically engineered protein into an octahedrally symmetric particle. This assembled protein also contained 8 reconstructed HA spikes to give a higher immune response compared to a classical inactivated influenza vaccine.
Another example of a self-assembling protein is the major vault protein (MVP). Vaults are ubiquitous, ribonucleoprotein particles found in eukaryotic cells (133). Native vaults are composed of multiple copies of three protein species and several copies of a small untranslated RNA. The most abundant protein is the 97 kDa major vault protein. This protein consists of 96 units of MVP and these units assemble into a barrel shaped vault protein (134). This protein is approximately 40 nm wide and 70 nm long. The cavities of these vault proteins are large enough to encapsulate hundreds of proteins. Furthermore, the protein shell of the vault offers some protection from external proteases and vaults appear to be biodegradable. Two additional properties of the vault particle that may make it an ideal structure to use as a therapeutic delivery system are its monodispersity and regularity. Because vaults are the size of small microbes, a vault particle containing an immunogenic protein could be easily phagocytosed by dendritic cells (135). Therefore, vault nanoparticles are straightforward to produce and make vaults viable vaccine delivery vehicles. One of the major issues facing the field of nanotechnology is cellular compatibility. To overcome this problem, naturally occurring vault Nano capsules as drug delivery devices could potentially have therapeutic applications. In many diseases such as Chlamydia and cancer vault proteins are already used for vaccine delivery. A phase I clinical trial investigated the effectiveness of CCL21 delivery using vault proteins for antitumor activity (136).This study showed that the vault Nano capsule can efficiently deliver CCL21 to sustain antitumor activity and inhibit lung cancer growth. Vault nanoparticles are better delivery vehicles compared to liposomes as the stability and targeting of liposomes can be problematic and the controlled release of encapsulated drugs has not been easy to regulate.
3. Nanoparticles act as adjuvants
3.1 Classic adjuvants
Adjuvants have an immunostimulatory effect and are therefore used in poorly immunogenic vaccines. Emulsions were first used in the late 30s as adjuvants. One of the first adjuvants was called Freunds Complete Adjuvant (FCA), an oil-in-water emulsion (140). Although this adjuvant was highly immunogenic, this emulsion had strong toxic side effects. This led to the development of a less toxic version called FIA and eventually the aluminum based adjuvants were used from the mid-60s (141). Currently, the most widely used immune adjuvants are the aluminum salts. Aluminium salts can trigger the inflammasome mechanism in the cells by acting as a danger signal and thereby enhancing the immune response (142). Aluminum salts have been successfully and extensively used since 1926 (143). These adjuvants are highly immunogenic and have a history of safe administrations (144). But there are some constraints to these adjuvants. First of all, aluminum salts are unable to stimulate cellular immune responses (145). Secondly, their adverse local reactions and lastly their degradation upon freeze-drying limits the use of these adjuvants (143)(146). These limitations have stimulated the search for new adjuvants to solve the problems of traditional vaccine adjuvants. This led to the use of nanoparticles as adjuvants in vaccines. However, only few studies have investigated the use of nanoparticles as adjuvants in vaccine delivery.
3.2 Inorganic nanoparticles
It was reported that MSNs can be used as adjuvants, however this depends on the binding capacity of the antigens, protein conformation, concentration of the nanoparticles, surface charge of nanoparticles and proteins and the uptake of protein loaded nanoparticles by APCs. There are some studies that report the use of MSNs as immune enhancers. The first MSN that was used as an adjuvant were SBA-15 nanoparticles (147). This study showed that SBA-15 elicited a significantly stronger immune response compared to the traditional aluminum salt adjuvant. This was also confirmed by the group of Carvalho et al.(148). HMSNs have also been investigated for adjuvant use. Guo et al. showed that HMSNs were used as an adjuvant for the Porcine Circovirus type 2. This study revealed that HMSNs induced a strong cellular and humoral immune response (74).
Calcium phosphate nanoparticles can also be used as adjuvant. When used as an adjuvant the calcium phosphate nanoparticles are smaller than 1000 nm in diameter, but a precise size is not defined (76). CaP, a calcium phosphate adjuvant, has been developed by BioSante Pharmaceuticals. This adjuvant has already been tested in phase I clinical studies. This study showed that CaP was safe and non-toxic in healthy volunteers. Preclinical studies showed that vaccines containing CaP as an adjuvant resulted in a higher immune response compared to those vaccines with classical adjuvants. There are some vaccines using calcium phosphate nanoparticles as an adjuvant in pre-clinical studies, such as HBV, flu and HSV-2 (149).
3.3 Immunostimulating complexes
ISCOMs have also been developed to act as immune enhancers. These complexes are made of the same material but minus the antigen and these are called ISCOMATRIX (150). The antigen is added in a later stadium in formulating the vaccine. A major benefit in using ISCOM as an immune enhancer rather than a Nano carrier, is the general use of antigens. ISCOMATRIX works similar as ISCOMs but in this material there is no need for specific hydrophobic regions for antigens to encapsulate. A study by Windon et al. showed that ISCOMATRIX’ can induce pro-inflammatory cytokines (IL-1, IL-6, IL-8 and IFN-??) production without an antigen (151). These inflammatory cytokines are important for the induction of an immune response. Other studies suggested that vaccines containing ISCOM activate the innate immune response in combination with IL-12 (150)(152). It is also observed that ISCOMATRIX’ vaccines induce the up-regulation of MHC class II and I expression on APCs and activate DCs much better compared to other colloidal structures (153). ISCOMATRIX’ vaccines are able to produce broad immune responses due to antigen presentation by both MHC class I and class II pathways (150). This suggests that ISCOMATRIX’ particles are excellent immune enhancers and suitable to use as adjuvants.
Liposomes can be modified to act as adjuvants in vaccines. Positively charged compounds is a general method used to alter liposome properties for adjuvant use. Positive charged liposomes are also called cationic liposomes and they are frequently used as cell transfection reagents. The high surface density of positive charges increases liposome adsorption on negatively charged cell surfaces. Cationic liposomes penetrate into cells through specific mechanisms and activate different cellular pathways depending on cell type, cationic lipid nature, but also on formulation types and liposome size (154)(155). In DNA vaccine studies, liposomes combined with 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) modified-cationic liposome and a cationic polymer (usually protamine) condensed DNA are called liposome-polycation-DNA nanoparticles (LPD). These nanoparticles are used as adjuvants in vaccine studies. The size of these LPD nanoparticles is around 150 nm and they contain condensed DNA inside the liposome.
3.5 Self-assembling proteins
Besides the delivery function, self-assembling proteins can also act at the same time as immune enhancers. Self-assembling proteins are most of the time used as an immune enhancer and delivery system in one, but never as an immune enhancer alone. To act at the same time as an immune enhancer the self-assembling nanocarriers are usually modified. These recombinant vaults can be modified in different ways for example by addition of a cell penetration peptide (TAT) or adding a cysteine rich peptide on the N terminus to increase stability (183)(184). Recombinant vaults can also be produced to contain a bacterial antigen and induce adaptive immune responses and protective immunity following immunization (185). Self-assembling proteins are already used for delivery and immune enhancing purposes in several studies. A study by Kar et al. tried to characterize the immunity produced in response to OVA within vault nanoparticles (184). This study showed that vault nanocapsules induced strong anti-OVA CD8+ and CD4+ T cell memory responses and modest antibody production.
These examples show that nanoparticles are not only excellent delivery systems but are also promising adjuvants. Based on these studies we can say that the safety, tolerability and adjuvanticity of nanoparticles in humans seems to be well established. All the different classes of nanoparticles can be used as delivery vehicles or immune enhancers. Some classes of nanoparticles are simultaneously used as delivery vehicles and immune enhancers at the same time. These nanoparticles require no extra adjuvant. These findings indicate that the use of nanoparticles as immune enhancers will surely increase and may eventually replace old adjuvants such as aluminum salts.
4. Nanoparticle interaction with antigen
4.1 Delivery system
When nanoparticles act as a delivery system, the association of nanoparticle and antigen is very important. Inorganic nanoparticles are often used as delivery systems and are thoroughly investigated for this purpose. The attachment of antigen on the nanoparticle can be achieved in several ways such as encapsulation, adsorption or conjugation (Figure 2). Among these 3 attachments methods adsorption is the weakest interaction. The association between nanoparticle and antigen in this method is very weak. This often results in a rapid dissociation of antigen from nanoparticle in vivo. Adsorption of antigens is solely based on electrostatic and hydrophobic interactions (186). Generally, encapsulation and conjugation are categorized as the stronger antigen attachment methods. In the encapsulation method, the antigen is mixed with the nanoparticle precursor during synthesis (76). This procedure results in the encapsulation of the antigen in the nanoparticle. The antigen is released in vivo when the particle has been decomposed. In the chemical conjugation method, the antigen is chemically cross-linked to the surface of nanoparticles (187). This nanoparticle with antigen is taken as a whole up by APCs and is then released in the cell.
4.2 Immune enhancers
When nanoparticles act as immune enhancers, the interaction between nanoparticle and antigen is not necessary. Therefore, the formulation of a vaccine with an immune enhancer and antigen is possible through simple mixing of these two. The mixing of nanoparticle and adjuvant could occur prior to injection. This is already investigated for soft material adjuvants such as emulsions. MF59 and AS03 have been shown to act as immune potentiators even when they are injected independently of the antigen (181)(188). For other nanoparticles this approach is not that common and is extensively investigated. Hard material nanoparticle immune enhancers showed that they act as size-dependent immune enhancers without association with the antigen. This effect was also observed in a number of other studies which showed that hard material nanoparticles induced the immune response with and without the antigen (189)(190).
4.3 Polymeric nanoparticles
In polymeric formulations the antigen can be either entrapped or adsorbed to the surface of the particles. It has been shown that PLG and PLGA nanoparticles entrap antigens either for delivery to certain cells or sustained antigen release due to their slow biodegradation rate (46)(60). PLGA generally encapsulates hydrophilic antigens while g-PGA encapsulates hydrophobic antigens due to its hydrophobic inner core. Joshi et al. fabricated and characterized PLGA particles with different sizes co-loaded with a model antigen ovalbumin (OVA) and CpG oligodeoxynucleotides (CpG ODN) (191).
PLGA particles showed a size-dependent release of encapsulated molecules. Particle internalization
by dendritic cells (DCs) increases with a decrease in particle size (71). Mice vaccinated with 300 nm sized particles generated the highest fraction of OVA specific of cytotoxic T lymphocytes (CTLs) on days 14 and 21, demonstrating that 300 nm sized particles can rapidly stimulate cell-mediated immunity (71).
4.4 Inorganic nanoparticles
Inorganic nanoparticles mostly encapsulate or adsorb the antigen of interest. When silica nanoparticles act as delivery systems they mainly encapsulate or adsorb the antigen (71). Functionalization is one of the most widely used strategies to alter the adsorption and release of antigens from MSNs. It is seen in several studies that the adsorption and release efficiency is dependent on the type of functional group associated with the nanoparticle. There are several functional groups investigated while one of the most important is the positively charged groups such as amino, triethanolamine and PEI. Calcium phosphate nanoparticles deliver their antigen by encapsulation. A study reported that biocompatible DNA/gold nanoparticle complexes with a protective calcium phosphate (CaP) coating were prepared by incubating DNA/gold nanoparticle complex coated by hyaluronic acid in simulated body fluid (192). These particles turned out to have high compatibility with cells and resistance against enzymatic degradation. CaP nanoparticles release their encapsulated antigen by a low pH (192)(193). This example of CaP capsule including a DNA complex is promising as a sustained-release system of DNA complexes for gene therapy.
Liposomes have different ways of association with the antigen, depending on the chemical properties of the compound. Water-soluble compounds (proteins, peptides, nucleic acids, carbohydrates, haptens) are entrapped within the aqueous inner space, whereas lipophilic compounds (lipopeptides, antigens, adjuvants, linker molecules) are intercalated into the lipid bilayer (194). In addition, antigens can be attached to the liposome surface either by adsorption or stable chemical linking (195). Self-assembled proteins can bind the antigens in several ways. One-way is the genetically fusion of the antigen with a minimal interaction domain of the vault protein. It is also reported that vault nanoparticles can encapsulate the antigen (185). Virus like particles can associate with different methods to the antigen. It is known that antigens can be encapsulated, conjugated, self-assemble in VLPs or adsorbed at the surface of VLPs (196)(197)(198)(199). VLPs release their antigen by disassembly after endocytosis by APCs.
4.6 Immunostimulating complexes
In ISCOM vaccines the antigen is incorporated into the ISCOM structure through hydrophobic interactions. ISCOM complexes can trap the antigen by apolair/hydrophobic interactions formed by the matrix (34) for delivery purposes. However this method is limiting the amount and type of antigen that could be incorporated (105)(107). Therefore, the ISCOMATRIX’ is more often used for the association of antigens. ISCOMs and ISCOMATRIX’ have glucuronic acid on their surface, which gives these nanostructures a negative surface charge. Efforts have also been made to develop cationic ISCOMATRIX’ particles to associate more different types of antigens. Lendemans et al. developed positively charged ISCOM particles but it turned out to be unsuitable to produce cationic equivalents (200). In a different approach, fusion proteins have been produced with the addition of hydrophobic peptide tags from viral or bacterial membranes. This also resulted in the incorporation of hydrophilic proteins into ISCOM vaccines (201). Another antigen association technique uses chelation which is the
combination of a metal ion with a chemical compound to form a ringed structure. The antigen has to be modified by addition of a poly-histidine tag to either the N- or C-terminal. If the poly-histidine tagged protein is mixed with the chelating ISCOMATRIX’ in the presence of copper ions, the protein becomes chelated to the ISCOMATRIX’ and can act as delivery system/immune enhancer (202).
Figure 2: different ways of antigen- nanoparticle interaction. The formulation of nanoparticle and antigen for drug delivery can be prepared through conjugation, encapsulation and adsorption (attachment). For immune enhancing effect, the formulation of nanoparticle and antigen can be achieved by simple mixing.
5. Nanoparticle interaction with antigen presenting cells
5.1 Targeting APCs
As nanoparticles have become very important tools in the delivery of antigens it is also very important on how to deliver these antigens more efficiently to APCs. When antigens are presented to APCs they induce the maturation of these APCs and subsequently are cross presented for activation of a potent immune response (203). Vaccines are usually designed to target the APCs as they efficiently take up and process antigens. A good understanding of DC and macrophage uptake mechanism for nanoparticles is essential for efficient nanoparticle vaccines. Several studies have reported that size, shape and charge are important in the antigen uptake by APCs.
5.2 Effects of particle size
Nanoparticles are in size comparable to pathogens and are therefore recognized and processed by APCs for the induction of an immune response (204)(205). DCs have a preference for smaller nanoparticles in size ranging from 20-200 nm (204). In contrast, macrophages prefer larger particles from 500 nm-5000 nm. In vitro studies using polymeric nanoparticles ranging from 0.04 ??m to 15 ??m were performed to identify the optimal size for DC uptake. For example, Jung et al. (69) studied the effect of particle size on the immune responses induced by tetanus toxoid adsorbed onto particulates prepared from sulphobutylated poly (vinyl alcohol)-graft-PLGA and showed that small particles of 100 and 500 nm induced significantly higher antibody titters than larger ones (>1000 nm) after oral or intranasal administration. The optimum size for DC uptake was found to be smaller than 500 nm (206). It is also found that 300 nm sized PLGA particles showed a higher activation and higher internalization of DCs in comparison with particles from another size (191). However, several studies report different optimal PLGA sizes for vaccination usage. Virus like particles and self-assembling proteins such as vaults are also efficiently taken up by DCs due to their size comparable to microbes (135)(207). VLPs can enter the cell via the endocytotic pathway where they dissemble and release their contents (207). In addition, the C-terminus of MVP can be engineered to target cell surface receptors on DCs for delivery of the encapsulated antigen (134). ISCOMs and ISCOMATRIXTM are directly targeted to APCs. They are more efficiently taken up by DCs and macrophages via endocytosis due to their particulate nature (208). It is thought that Saponin mediates the targeting of the mannose receptor DEC-205 on DCs which results in a higher uptake of antigens and more efficient presentation to T cells of antigens (209)(210).
5.3 Effects of particle surface charge
Besides particle size, surface charge also plays an important role on their interaction with cells and in the activation of an immune response. Cationic nanoparticles induce higher extent of internalization, apparently as a result of the ionic interactions established between positively charged particles and negatively charged cell membranes (206)(211). Nonetheless, this is not the case for nanoparticles below 100 nm in size. Furthermore, positively charged nanoparticles are able to escape from lysosomes after being internalized and exhibit perinuclear localization, whereas the negatively and neutrally charged nanoparticles prefer to co-localize with lysosomes. However, cationic particles could induce hemolysis and platelet aggregation, due to strong interactions with anionic cell membranes (212). Some nanoparticles can be modified to be taken up by APCs. PLGA nanoparticles are anionic particles, which can be shifted to neutral or positive charges by surface modification, for example by PEGylation. For liposomes there are contra-dictionary results reported for efficient uptake by APCs. Liposomes IRIVs present antigens in the context of MHC-I and MHC-II and induce B- and T-cell responses (213)(214). The group of Nakanishi showed that cationic liposomes are much more potent inducing an immune response compared to anionic and neutral liposomes (215). In contrast the group of Yotsumoto reported that anionic liposomes could act as effective adjuvants to induce an immune response (216).
5.4 Effects of particle shape
Particle shape plays an equal important role with size and charge in the interaction between APCs and nanoparticles. In vitro assays demonstrated that MSNs with a long rod shape were taken up in larger amounts and showed faster internalization rate than spherical shape like particles (217). Niikura et al. reported that spherical AuNPs were more effective in inducing an antibody response compared to cubes and rods (218). From some nanoparticles the precise mechanism of uptake by APCs is not known. For example, the precise mechanism of oil-in-water emulsions taken up by DCs is not fully understood. It is thought that the adjuvant itself promotes antigen engulfment and presentation. By better understanding of the precise mechanism of antigen uptake by DCs more potent immune responses can be elicited using vaccines.
6. Nanoparticle-biosystem interactions
6.1 Nanoparticle application in humans
Nanoparticle-assisted drug delivery has been emerging as an active research area in recent years. The in vivo bio distribution of nanoparticle and its following mechanisms of biodegradation and/or excretion determine the feasibility and applicability of such a nano-delivery platform in the practical clinical translation. In addition, the toxicity profile of nanoparticles are important for their application in humans. Toxicity is dependent on the physicochemical properties of nanoparticles (219)(220). Besides targeting the lymph nodes for efficient activation of the immune response, the design of nanoparticle vaccines also needs to consider nanoparticle clearance from the body. Toxic effects may occur when nanomaterial’s are not degraded or excreted from the body. Using nanoparticles in vaccines as delivery systems or immune enhancers requires the thorough understanding of its interactions with the bio system to ensure safe and efficacious vaccines.
6.2 Lymph nodes
Besides APCs, lymph nodes are also a target for vaccine delivery. Distribution of nanoparticles to the lymph node are affected by size (221)(222). Lymph nodes contain cells of the immune system such as T and B cells. These cells are important for eliciting a potent immune response by antigen delivery of well-armed peripheral APCs (223). Distribution of nanoparticles to the lymph node is mainly affected by size. Nanoparticles with a size between 10 and 100 nm can penetrate the extracellular matrix easily. From there they travel to the lymph node, where they are taken up by DCs for immune response activation (224). Particles larger than 100 nm are mostly scavenged by local APCs (225).
6.3 Liver and kidney
Biliary clearance through the liver allows excretion of nanoparticles larger than 200 nm (226)(227). Beside size, surface charge and shape of nanoparticles are also very important for the excretion through the liver. An increased surface charge led to an increased distribution of nanoparticles in the liver (228). Another study reported that short rod nanoparticles were mostly found in the liver and long rods in the spleen. In addition it was reported that short rods were excreted at a faster rate than longer ones (229). Size, surface charge and shape of nanoparticles are also very important for the excretion through the kidney. The kidney can excrete nanoparticles smaller than 8 nm through renal clearance (227)(230). Surface charge also plays an important role in the clearance of nanoparticles by the kidney. It is reported that anionic charged nanoparticles are easily removed by the kidney (231).
Several in vivo and in vitro studies revealed that mesoporous silica nanoparticles don’t show any signs of toxicity when used. MSNs can be processed which results in excretion by the urine. It has been shown that high positively charged MSNs are excreted quickly from the liver while less charged MSNs are sequestered in the liver (232). This work suggests that charge-dependent adsorption of serum proteins greatly facilitates the hepatobiliary excretion of silica nanoparticles, and that nanoparticle residence time in vivo can be regulated by modification of surface charge. Calcium phosphate particles are naturally occurring in the body, therefore there are less issues around the safety of these materials in the body (233)(234). AuNPs have also been suggested to have some cytotoxicity due to their cationic polarity and surface modifications (212)(235). Anionic AuNPs show no signs of toxicity. ISCOMs have shown to be very successful when used in vaccines in the clinic. However the actual use of ISCOMs in human has been restrained. Some saponins (component of the ISCOM matrix) can be toxic at elevated levels (236). As self-assembling proteins are naturally occurring the vault particle may be useful as a therapeutic delivery system. These nanoparticles are small enough to prevent them from being trapped in the kidney and/or liver if administered intravenously (225). This is often seen with structures above 200 nm. In addition particles less than 200 nm can freely access the draining lymph nodes when injected intradermally.
6.5 Biodegradable nanoparticles
Generally speaking, biodegradable polymers are safer to use in humans than non-biodegradable nanomaterial’s. PLGA is one of the most successfully used biodegradable polymers. PLGA undergoes hydrolysis in the body to produce monomers such as lactic acid and glycolic acid. Lactic acid and glycolic acids are normally found in the body and participate in a number of physiological and biochemical pathways (237). Lactic acid is a natural intermediate/by product of anaerobic respiration, which is converted into glucose by the liver during the Kreb cycle (44). The product glucose is then used as an energy source in the body. Therefore there is minimal systemic toxicity associated with the use of PLGA for the drug delivery. The use of PLA nanoparticles is therefore safe and does not result in any major toxicity.
Although, there is some information available on the effect of nanoparticles on the bio system and the resulting toxicity, this is still not enough to make clear conclusions about the use of nanoparticles in humans. More research has to be done on the interaction of nanoparticles and the bio system and the resulting toxicity.
7. Skin vaccination using nanoparticles
7.1 Skin barrier
Two main functions of the skin are to act as an effective barrier against the invasion of pathogens as well as to prevent excessive water loss from the body. The skin consists of several layers, the epidermis, dermis and the hypodermis containing subcutaneous fat tissue (13) (Figure 3). The outmost layer of the skin is the epidermis. Although all the skin layers are actively engaged in the host defense, the epidermis is especially important in preventing loss of water and other components of the body to the environment and in protecting the body from unwanted environmental influences (14).
The most important barrier of the skin is the stratum corneum (SC), which is the outermost layer of the epidermis, and consists of corneocytes (dead cells), which is approximately 15’20 ??m thick (15). The physical barrier mainly consists of the stratum corneum (SC) and the corneocytes, although the cell’cell junctions and associated cytoskeletal proteins in the below layers provide further physical barriers (16). The SC is a tough barrier due to the overlapping alignment of the flat cornified cell layers, which increases the intracellular diffusion path length. Human SC contains 10 to 25 corneocyte layers that are parallel oriented to the skin surface and are embedded in a lipid matrix (17). The lipid matrix composes the intercellular space of the SC. This lipid matrix gives the SC the extra rigid structure in combination with the corneocytes (18). Furthermore the poor permeability of the corneocytes and the unusual lipid composition and organization also enhance the barrier function of the stratum corneum (19). This layer results in a practically impermeable barrier which limits the passage of molecules, especially molecules larger than 500 Da (20).
Figure 3: microanatomy of the skin. The skin with its different layers.
Various pharmaceutical approaches and devices have been developed to enable vaccine formulations to overcome the penetration barrier of the SC. Targeting the skin immune system is possible either by transcutaneous vaccination or by intradermal vaccination. Together, this is called cutaneous vaccination. In this section, the techniques, technologies, and devices used for the enhancement of cutaneous vaccination are reviewed.
7.2 Different routes of delivery with different delivery technologies
Electroporation is a technique to increase the permeability of the skin by applying single or multiple short duration pulses (238). The voltage of these pulses can be varied. With high-voltage pulses (75-100 V), micro channels or local transport regions are created though lipid bilayer membranes including the SC (239). Zhao et al. reported that electroporation induced Ag-specific immune responses (240). However, this method requires power-supply equipment, thus they may not be an optimal ease of self-administration. In addition, disrupting SC as skin barriers may lead to secondary infection.
Iontophoresis is a method to enhance the transportation of ionic or charged molecules through a biological membrane by passing direct or periodic electric current through an electrolyte solution with an appropriate electrode polarity. This technique is especially used for transdermal drug delivery. Several groups reported the penetration of peptides and proteins such as insulin, calcitonin, or botulinum toxin through the SC (241). In addition, it is reported that the combination of iontophoresis and electroporation results in even more effective drug penetration across the SC (242). So iontophoresis seems to be a promising devise for drug delivery. However, problems about lack of convenience and risk of secondary infections remain because this method requires power-supply equipment and may break cutaneous barrier.
Sonophoresis is a technique to disrupt the structure of the SC by low frequency ultrasound. This leads to increased permeability for drugs to penetrate the skin. Sonophoresis uses cavitation and oscillations to increase skin permeability. Cavitation is the formation of gaseous cavities in an ultrasound-coupling medium upon exposure to ultrasound. This also involves the rapid growth and collapse of a bubble (transient cavitation) or slow oscillatory motion of a bubble (stable cavitation) in the ultrasound field. Oscillations and collapse of cavitation bubbles disorder the lipid bilayers of the SC, which results in enhanced transport (243). The group of Tezel et al. showed that using sonophoresis at a low frequency ultrasound, enhanced the Ag-specific immune response (244). This suggests that sonophoresis may act as a physical adjuvant. Like electrophoresis and iontophoresis this method also requires power-supply equipment and disrupt cutaneous barrier.
7.2.4 Jet injectors
Jet injectors are devices that use pressure to deliver substances into the skin (245). One of the first jet injectors was the multiple-use nozzle jet injector (246). With this injector many patients could be vaccinated through the same fluid and nozzle. However, these devices are no longer used due to contamination. Most recent developed jet injectors are the disposable syringe jet injectors. The group of Simon et al. reported a clinical study of the immunogenicity of inactivated influenza virus delivered by the LectraJet M3?? RA disposable syringe jet injector (247). These jet injectors induce the immune response and administration is simple compared to other methods of drug delivery.
7.2.5 Patch formulations
Patch formulations are one of the commonly used systems for transcutaneous immunization. Several groups have reported that TCI using gauze patches or adherent patches induced Ag-specific immune responses (248). The gauze patch needs to be saturated with Ag solution just before application to the skin. Eetchart et al. investigated the use of a patch formulation in a vaccine, called ROUVAX??, with live-attenuated measles in a phase I/II clinical trial (249). This trial showed that the vaccine is safe, poorly reactogenic and induces measles specific antibodies. However, there are some disadvantages with the use of patch formulations. The SC has to be removed before patch application with skin preparation system (SPS) or cyanoacrylate skin surface stripping (CSSS) procedures for improvement of Ag penetration into skin. These methods may carry a risk of increasing sensitivity to secondary infection due to disrupting the cutaneous barrier.
An innovative approach is the application of micro needle arrays. The micro needle technique disrupt the barrier properties of the SC, which results in an enhanced transdermal drug delivery (250). The first micro needle arrays introduced were solid micro needles, to pre-treat the skin. Currently, a variety of types, including hollow, solid, coated and dissolvable microneedles, have been developed. Micro needles are needle-like structures with diameters in the size order of microns and lengths up to 1 mm. Micro needles are needles that pierce the SC while being short enough to avoid pain feelings. They penetrate the skin and go into the viable epidermis (251). Contact with nerves, blood vessels and fibers are avoided, because these lay primarily in the dermal layer. Due to these characteristics, micro needles would provide a pain-free delivery of vaccines (252).
As mentioned before, there are several types of micro needles. Most used are the solid micro needles. These solid micro needles can either be used to pre-treat the skin for pore formation through which drugs can transport or to deliver vaccines directly (253). In the case of the coated micro needles, the needles are coated with a vaccine that is suitable for coating. When the needles are applied to the skin, the desired dose of the vaccine is released into the tissue (254). However, the dose is limited by the amount that can be applied to the micro needles. Dissolving micro needles are polymeric micro needles that are dissolved completely in the skin. These micro needles are made of compounds that will completely dissolve in the skin after penetration of the SC. These needles are used for quick release of drugs in vivo (255). Hollow micro needles enable pressure-driven flow of a liquid formulation into the skin (256). With this delivery method the pressure and flow rate can be modulated for a rapid bolus injection, a slow infusion or a time-varying delivery rate (257).
7.3 Delivery of nanoparticles with transcutaneous methods
7.3.1 Micro needles for transcutaneous delivery
From the transcutaneous delivery routes, micro needles seem to be the most promising one for vaccination. Micro needles are very promising for the transcutaneous delivery of nanoparticles because they enable them to pass the SC via micro pores. Micro needles can be used to pre-treat the skin and then apply the nanoparticles so they can penetrate the skin. Another possibility is to coat the micro needles with nanoparticles, to pierce through the skin and release the nanoparticles in the skin layer. This is a simple and minimally invasive method to deliver both existing and novel vaccine candidates, including nanoparticles to humans. It is logically to assume that the micro channels created by SC are large enough for nanoparticles to penetrate the intra-epidermal layer. However, the movement of nanoparticles trough these micro channels within the skin tissue is a complex process that needs a lot of further investigation. Despite this fact there are some examples known of vaccination with a combination of micro needles and nanoparticles.
7.3.2 Nanoparticle delivery using micro needles
A study by Kumar et al. investigated micro needle-mediated transcutaneous immunization with plasmid DNA coated on cationic PLGA nanoparticles (258). In this study the skin was pretreated with solid micro needles before applying cationic nanoparticles coated with plasmid DNA onto the skin. This resulted in a significantly enhanced immune response. Cutaneous immunization with plasmid DNA-coated positively charged nanoparticles elicited a stronger immune response compared to plasmid DNA-coated negatively charged nanoparticles or by intramuscular immunization with plasmid DNA alone.
Another study by Lee et al. used nanostructured lipid carriers to formulate a lipophilic drug into hydrophilic polymeric micro needles (259). They found that, in a skin permeation study using a Franz diffusion cell with minipig dorsal skin, approximately 70% of NR was localized in the skin after 24-hour application of micro needles with nanostructured lipid carriers. This suggests that micro needles are an excellent strategy for the penetration of nanoparticles through the skin.
A nanomaterial-strengthened dissolving micro needle patch for transdermal delivery is reported by the group of Yan et al (260). This patch comprises thousands of micro needles, which are composed of dissolving polymers, nanomaterial’s, and drug/biomolecules in their interior. These Nano composite-strengthened micro needle arrays were also tested in vivo where they showed significantly higher antibody responses compared to subcutaneous injection.
These studies suggest that nanomaterial’s in combination with micro needles could be useful in vaccines for the penetration through skin and the delivery of antigen to APCs. Several other groups are also investigating the potential of micro needles in combination with nanoparticles to penetrate the human skin. This also indicates that this combination may be highly useful and is the most interesting for drug delivery and especially vaccines. In addition they show rapid dissolution rates and may therefore be potentially useful in clinical applications.
7.4 Delivery of nanoparticles using intradermal vaccination
7.4.1 Nanoparticles for intradermal delivery
It is already established that nanoparticles are efficiently delivered using subcutaneous vaccination (see paragraph 2: Different types of nanoparticles as delivery vehicle). It is important to investigate whether nanoparticles are also efficient delivery systems in combination with intradermal vaccination techniques as intradermal vaccination is important for the future of vaccination. Some currently licensed vaccines such as Hepatitis B or Rabies vaccines, the protective immunity by the ID vaccination route is comparable or superior to standard intramuscular or subcutaneous routes, but allows consequent reduction of antigenic doses (generally 1/5th of the dose used SC) (261). To date, some studies are performed using nanoparticles for intradermal vaccination.
7.4.2 Progress in nanoparticle mediated intradermal vaccination
Especially the last three years progress is made in the field of intradermal vaccination using nanoparticles. The groups of Cui et al. were one of the first to report the use of nanoparticles in combination with intradermal vaccination. In that time subcutaneous vaccination was the most used method. Cui et al. showed that a combination of a novel cationic nanoparticle-based DNA delivery system with ID jet injection led to enhanced antibody production in mice (262).The group of Arias et al. used HIV-gp140-adsorbed nanoparticles for intradermal vaccination. Immunogenicity studies in mice showed that intradermal vaccination with HIV-gp140 antigen-adsorbed nanoparticles induced high levels of specific IgG (263). Another study by Verheul et al. reported the use of stabilized trim ethyl chitosan (TMC) and hyaluronic acid (HA) nanoparticles in combination with intradermal vaccination. This study also resulted in an efficient delivery of the antigen and elicited immune responses (264). In addition, it is reported that intradermal vaccination using HIV-1 p24 PLA nanoparticles induced both humoral and cellular immunity while subcutaneous vaccination in combination with nanoparticles only induced a humoral response (265).
Although there are not many studies reporting the use of nanoparticles in intradermal vaccination, we were able to show that intradermal vaccination results in an efficient delivery of nanoparticles. It is even reported that intradermal vaccination is preferred over subcutaneous vaccination due to higher immune responses. These studies also showed that nanoparticles were able to elicit an immune response and can be used for intradermal vaccination purposes.
Novel nanoparticle based delivery systems and adjuvants are being evaluated in a number of vaccines. These vaccines are against a variety of diseases such as hepatitis, influenza, HIV and cancer in which a humoral and/or cellular response is required. Clinical studies of several nanoparticle immunopotentiators and antigen delivery systems have shown CTL responses for ISCOMs while Th1 responses have been detected for liposomal MF59 adjuvant. In addition, cellular immune responses have also been generated using VLPs, liposomes/virosomes and non-degradable nanoparticles. There is no clear line in which of the nanoparticles is the best to use in vaccines. Despite this fact there are some differences noted in the use of nanoparticles as some of classes of nanoparticles are only used as immune enhancer, delivery vehicle or as both. In addition, some nanoparticle properties showed a better interaction with APCs to induce an immune response such as size, charge and shape. Nanoparticles may show some concern regarding toxicity. This is due to their ability to cross biological membranes and the slow biodegradability of some materials. However it seems reasonable to suggest that in the case of vaccines the infrequent and low-level exposure to nanoparticles will not result in adverse health problems, which indicates that they could be safely used in humans.
The use of lower doses of antigens would be advantageous not only for minimizing the potential side effects often associated with the use of adjuvants but also from an economical point of view.
However, the application and use of nanoparticles in vaccines and drug delivery is still at an early stage of development. There are still some challenges to overcome such as a lack of understanding of how the physical properties of nanoparticles affect their bio distribution and targeting, and how these properties influence the interaction of nanoparticles and the bio system at all levels. Therefore, there is more research needed in the use of nanoparticles in humans. Only then, novel vaccine system for unmet needs such as needle-free delivery will become practical in the near future.
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