Paul Ehrlich proposed the concept of the immune system as a useful strategy against cancer, in the beginning of the Twentieth century (Ehrlich, 1909). Some decades after, Thomas and Burnet postulated the immunosurveillance theory based on Ehrlich's hypothesis. Cancer arousal was suggested to be caused by the lack of efficiency of the immune system or the modification in antigen expression of tumor cells, leading to its evasion (Burnet, 1957; Thomas, 1982). Thomas and Burnet also claimed that antitumor immune response generally happens at an early stage of the cancer development (Burnet, 1957; Thomas, 1982). Therefore, once the tumor has grown, it escaped the immunosurveillance barrier and started developing additional mechanisms to evade from the immune system (Ahmad et al., 2004). Nevertheless, Strutman's later studies showed that cancer susceptibility of immune-competent mice was similar to that observed in mice with major immunodeficiency, setting against the immunosurveillance hypothesis (Shankaran et al., 2001; Dunn et al., 2002). In the beginning of this century, the immunosurveillance hypothesis was revised, as several studies have shown that the immune system may not only destroy tumor cells but also shape their phenotypes, leading to reduction of immunogenicity (Shankaran et al., 2001; Dunn et al., 2002; Schreiber et al., 2011).

Currently, there is increasing evidence that tumor cells can be recognized and destroyed by the immune system, as developing tumor cells often co-express tumor antigens and ligands for activating receptors (Schreiber et al., 2011). Therefore, it is important to describe which immune components display major roles in tumor rejection. It is also important to clarify the appropriate time and efficient type of action (Swann and Smyth, 2007).

As reviewed by Schreiber et al. (2011), cancer immunoediting can be divided in three different phases: “elimination,” “equilibrium” and “escape.”

In the first stage—“elimination”—both innate and adaptive immunities act combined to identify the formation of tumor cells and to destroy them, resembling the immunosurveillance theory. Although many mechanisms are still poorly known, it has been reported that cytokines, “danger signals” and DCs have important roles in this phase (Sims et al., 2010; Vesely et al., 2011). It has also been suggested that the required components for an effective “elimination” depend on specific characteristics of the tumor cells, such as its origin or anatomical location (Sims et al., 2010). If the “elimination” stage is well succeeded, tumor cells are destroyed, constituting an endpoint for cancer immunoediting (Schreiber et al., 2011; Vesely et al., 2011).

The next stage—“equilibrium”—is described as a period of tumor latency. In other words, when a tumor cell survives the elimination phase, the adaptive immune response can control tumor cell growth and shape its immunogenicity. “Equilibrium” is believed to be the longest phase of cancer immunoediting process. It seems to allow cancer cells to reside in patients' body even decades before it restarts to grow and become clinically evident (Schreiber et al., 2011; Vesely et al., 2011).

The third phase—“escape”—occurs when tumor cells have developed the ability to evade the mechanisms of recognition of the immune system and/or their elimination. Tumor cells are thought to progress from “equilibrium” phase to “escape” through several mechanisms and/or pathways. For instance, an alteration in immune system response, which may be triggered by cancer-induced immunosuppression or a change in tumor cells induced by immunoediting, or even immune system deterioration (Schreiber et al., 2011; Vesely et al., 2011).

Cellular immunity has been shown to play a major role in the control of tumor generation. Even though, recent findings revealed that tumors often manage to evade it through several different mechanisms. It has been reported that there is a reduction or even loss of MHCI molecules, mostly associated to gene mutations or impairment of MHCI-dependent antigen processing (Garrido and Algarra, 2001; Ahmad et al., 2004; Vesely et al., 2011). In addition, an antigenic drift in cancer cells has lately been observed and appears to be related with the mutation, loss or down-expression of TAAs in tumor cells (Uyttenhove et al., 1997; Ahmad et al., 2004). Similarly, the lack or reduction of the expression of co-stimulatory patterns by tumor cells direct T lymphocytes to an anergy state. These mechanisms altogether seem to reduce and difficult the detection of cancer cells by CTLs and NK cells, which consequently leads to tumor growth (Ahmad et al., 2004).

Alterations in apoptotic receptor signaling seem to help tumor cells to evade the immune system. Molecules such as phosphatidylinositol 3-kinase (PI3K), protein kinase B and Fas ligand (FasL) have modified expression and might be implicated in this process (Davidson et al., 1998; Osaki et al., 2004).

Tumor eradication is also dependent on the manipulation of immunosuppressive properties of tumor microenvironment, where inducing and suppressing cytokine imbalance impairs DC activation and maturation, compromising immune cell effector properties and supporting tumor growth. Tumor cells can indeed secrete immunosuppressive molecules, including vascular endothelial growth factor (VEGF), IL-10 and transforming growth factor-β (TGF-β) (Fortis et al., 1996; Tsushima et al., 1996; Oyama et al., 1998). VEGF appears to be responsible for down-regulation of NF-κB expression, which interferes in DC maturation and differentiation, limiting the immune response against tumor cells (Oyama et al., 1998). On the other hand, TGF-β1 is an immune suppressive cytokine involved in the conversion of CD4+T cells into immunosuppressive T regulatory (Treg) cells that are mainly produced by DCs and tumor cells (Zou, 2005).

These immunosuppressive molecules are interesting targets to achieve tumor growth inhibition and might be a very useful tool for cancer immunotherapy. The use of nanoparticles (NPs) containing small interfering RNA (siRNA) to knock-down TGF-β in the tumor microenvironment has resulted in increased levels of CD8+ T cells and lower number of Treg cells, leading to tumor growth inhibition by 52% (Xu et al., 2014). A similar strategy using polyethylenimine-capped silica NPs carrying VEGF siRNA has been designed as a highly effective approach for lung cancer growth suppression and metastasis (Chen et al., 2014b). High levels of indoleamine 2,3-dioxygenase have also been found in tumor microenvironment, reducing tryptophan pool levels, which drive T lymphocytes to be arrested at G1 phase of the cell cycle (Ahmad et al., 2004).

Monoclonal antibodies are the main cancer immunotherapy used currently in clinic to treat solid tumors and lymphomas (Krishnamachari et al., 2011). For example, trastuzumab has been used to treat HER2⁺ breast cancer and adenocarcinoma, whilst alemtuzumab has been applied in chronic lymphocytic leukemia treatment (Lee et al., 2013).

The mechanism of action of mAbs is related to their ability to interfere with both growth factor ligands and receptors or pro-apoptotic targets, inducing apoptosis of cancer cells. Besides, mAbs may activate components of the immune system through Fc-region-based mechanisms. This leads to antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) responses by macrophages and NK cells (Krishnamachari et al., 2011).

The use of mAbs in clinic has been increasing in the last decades. The first generation of mAbs used in cancer therapy was originated from mouse. Their origin often resulted in limited half-life, decreasing mAbs efficacy. Further progresses conducted to the development of chimeric mAbs, with enhanced properties, and then humanized mAbs. Nowadays, fully human mAbs are already available (Lee et al., 2013). Several novel mAbs for different cancer types are presently in clinical trials, as reviewed by Lee et al. (2013). For example, ganitumab—for pancreatic cancer –and necitumumab—for non-small cell lung cancer—are now in phase III of clinical trials.

This approach is based on the transfer of mature tumor-reactive T lymphocytes to act against tumor cells. Unlike cancer vaccines, this strategy is independent from an immune response elicited by an exogenous antigen. Instead, it relies on the delivery of a great amount of ex vivo-expanded cells (Gajewski, 2012; Kirkwood et al., 2012; Helmy et al., 2013).

Adoptive T-cell therapy with tumor-infiltrating lymphocytes (TILs) has been proposed. In a successful study, autologous TILs—T cells with potent antitumor activity found within tumors—were harvested, activated ex vivo and reinfused in patients. The total remission was reported in more than 20% of the treated patients (Rosenberg et al., 2011).

Complementary research has been made to improve T-cell adoptive therapies. Genetically engineered T cells are under study, in order to manipulate the properties of the administered T-cell population, such as proliferation and migration characteristics (Liu and Rosenberg, 2001; Hinrichs et al., 2011). Also, T cells have been genetically modified to have antitumor specificity by introducing a T-cell receptor for a particular tumor, as previously described in a review by Helmy et al. (2013).

These vaccines use isolated CD14+ monocytes or CD34+ DC precursors from an individual. After being isolated, these cells are then cultured and differentiated in immature DCs (Romani et al., 1994; Chapuis et al., 1997). The following process is TAA-loading of DCs, which consists in adding proteins, peptides or tumor lysates to its culture medium or through transfection. Additional maturation stimuli, such as CD40L or pro-inflammatory cytokines, may be used to ensure DCs will be able to induce a strong cellular immune response. Finally, loaded mature DCs are administered back into the patient by intravenous (i.v.), subcutaneous (s.c.), intradermal (i.d.), intratumoral (i.t.) or intralymphatic (i.l.) route (Hamdy et al., 2011).

The use of a tumor cell to stimulate DCs seems to induce a better immune response, but it is limited by a possible induction of autoimmune diseases, due to the lack of antigen specificity among the undefined antigen found at cancer cell surface.

Whichever the type of antigen used to pulse DCs, although it has been reported that this approach is safe and able to induce CTL immunity, the clinical observed goal is low, possibly due to the in vivo general complex interactions between immune cells (Rosenberg et al., 2004).

DC therapy involves the isolation, culture and stimulation of patient's monocytes and macrophages ex vivo using TAAs (Cho et al., 2011). When administered back to the patient, antigen-loaded DCs will bypass the in vivo uptake of tumor antigens. DCs are already activated and therefore they are able to migrate to the secondary lymph nodes wherein they will trigger T cells. However, the relative short half-life of TAA-MHC complexes on DC membrane surface, and the low percentage (3–5%) of DCs that can migrate to the lymph nodes and contact with T cells can contribute to the low rate of success of these vaccines (De Vries et al., 2003; Hamdy et al., 2011). Also, being produced specifically for a particular patient, ex vivo DC-based vaccines are a highly complex, laborious, time-consuming and expensive approach. Futhermore, the vaccine quality might depend on the clinic where it is produced, once there are several variable parameters in the process, such as dose of DCs and posology (Hamdy et al., 2011). The type of DCs stimulated, antigen loading method and DC maturation level are also important aspects to be characterized to better understand the adjuvant role of DCs.

To overcome the lack of clinical efficacy of ex vivo DC-based cancer vaccines, it is extremely recommended to develop an alternative way to target antigens directly to DCs in vivo, which can be achieved using peptide-based vaccines. These are mainly based on MHCI peptides, which are simple to produce and administer, and guarantee DC activation and expansion for prolonged periods of time (Figure 1) (Cheong et al., 2010; Silva et al., 2013). However, the cytoplasmic delivery of the antigen is limited by low membrane permeability and frequent destruction after intracellular entry, being their immunogenicity considerably lower than the traditional vaccines. Hence, their association to potent adjuvants, as particulate vaccine delivery systems, or immunomodulatory molecules is being widely investigated (Al-Hanbali et al., 2006; Hillaireau and Couvreur, 2009; Sharp et al., 2009; Shahar et al., 2010; Smith et al., 2012).

Numerous studies have demonstrated that these delivery platforms could increase the uptake of antigens and adjuvants by DCs, leading to better immune responses (Diwan et al., 2002; Schlosser et al., 2008; Florindo et al., 2009a). In vivo DC-targeted vaccines are able to deliver, within the same platform, both antigens and additional stimuli (i.e., adjuvants) to the same cell in its natural environment, enhancing and maximizing the outcome (Kazzaz et al., 2006). Particulate delivery systems range from micro and nanoparticles, liposomes, to virus-like particles (VLPs). Unlike ex vivo DC vaccines, the clinical intervention is limited to vaccine administration, sparing time in fastidious cycles of blood withdrawal and in vitro cell culture. Also, it offers on-shelf products, which can be produced at large scale with cost reduction and increased quality.

Liposomes consist of self-assembled lipid bilayer membranes with size ranging from 90 to 150 nm, which are formed by phospholipids and cholesterol that enclose an aqueous core (Figure 3A). Phospholipids are composed by hydrophilic heads and hydrophobic long tails. Thus, as previously described in several reviews, their structure allows hydrophilic molecules to be incorporated within the inner compartments, while the hydrophobic compounds will be entrapped within the hydrophobic bilayer (Sahoo and Labhasetwar, 2003; Aslan et al., 2013; Sharma et al., 2013).

The potential use of liposomes as delivery systems is based on the fact that they provide a slow and sustained release, improving the accumulation of the entrapped molecules. Also, on their ability to decrease cytotoxicity of incorporated molecules, since they modulate the biodistribution and pharmacokinetics (Khan et al., 2008; Sharma et al., 2013). Having in consideration their biocompatibility, the biodegradability and ability to cross lipid bilayers and cell membranes, liposomes have been proposed as delivery platforms for vaccines, anticancer drugs and gene therapy (Ewert et al., 2005). However, one of the major drawbacks of conventional liposomes is the short circulation time, being rapidly removed by mononuclear phagocytes of the reticular endothelial system (RES). Stealth liposomes, or long-circulating liposomes, have been developed to overcome this problem. They consist in liposomes that are sterically stabilized, presenting thus a prolonged half-life (Frank, 1993; Krishnamachari et al., 2011).

Regarding the success attained by liposomal platforms in the clinic and advanced-stage clinical trials, several liposomal-based delivery systems are nowadays offered as an anticancer strategy, such as liposomal doxorubicin, cytarabine and cisplatin (Abraham et al., 2005; Huwyler et al., 2008; Aslan et al., 2013). The use of liposomes for doxorubicin delivery prevents the damage of heart and renal healthy tissues that is usually induced by the extreme toxicity of the drug (Abraham et al., 2005). Moreover, doxorubicin has already been formulated in active targeted liposomes for breast cancer therapy, using engineered peptide ligands (Sharma et al., 2013). Other attractive approach is the use of liposomes as carriers for antisense oligonucleotides, as siRNA, in cancer therapy (Tari et al., 1996).

Van Broekhoven et al. (2004) have reported a DC-targeting vaccine, based on a liposomal formulation, as an outstanding platform to induce a highly effective immunity against tumor cells (Van Broekhoven et al., 2004). Preclinical studies of liposome-DNA complexes have also been described, constituting an effective strategy to elicit anti-tumor immunity (U'ren et al., 2006).

The phase I clinical trial of a liposomal cancer vaccine for breast, ovarian and prostate cancer has already been reported. It has been proved that this peptide vaccine, which is intended to elicit multi-functional T-cell responses, is safe and immunogenic (Berinstein et al., 2012).

Additionally, liposomes have been studied as carriers for alternative bioorganic and biodegradable contrast agents, as glycogen and poly-L-Lysine. With these liposomes, it was possible to develop an in vivo multi-color magnetic resonance imaging for lymph node mapping (Chan et al., 2014).

Polymeric NPs are submicron-sized polymeric colloidal particles with excellent features as vehicle for the delivery of drugs, biomolecules and genes (Panyam and Labhasetwar, 2003; Mahapatro and Singh, 2011).

Polymer properties such as biocompatibility, low toxicity and biodegradability have highlighted polymeric NPs as an interesting delivery strategy. The chemical structure of the polymers is easily modified, allowing the development of multifunctional engineered systems. Nanoparticle size, shape and surface properties can also be tailored, as well as the degradation kinetics and mechanical properties (Albertsson, 2002).

Polymeric NPs are usually highly stable and can easily entrap and/or adsorb both hydrophilic and hydrophobic molecules with good efficacy (Gelperina et al., 2005). The drug entrapment protects molecules from degradation (Singh and Lillard, 2009). Additionally, as nano-sized polymeric particles, these carriers are easily transported through extra and intracellular barriers. As a result, entrapped agents may be delivered site-specifically, for instance in inflamed areas or tumors, after crossing the endothelium (Prokop and Davidson, 2008; Singh and Lillard, 2009).

Two different types of polymeric NPs are usually considered: nanospheres and nanocapsules (Figure 3B). Nanospheres consist in a polymeric matrix in which the drug or cargo is homogenously dispersed, whereas nanocapsules are vesicular systems formed by a polymer wall that surrounds a core containing the cargo (Singh and Lillard, 2009).

Several methods have been used to produce polymeric nanoparticles. Some of the most studied are spray-drying, salting out, nanoprecipitation and emulsion-based methods, The latter, in particular, lies on an emulsification process with the removal of organic solvents used for polymer dissolution, by extraction or evaporation. The emulsified organic drops containing the polymer originate nanoparticles, when the organic solvent is eliminated (Lassalle and Ferreira, 2007).

Nevertheless, it is important to bear in mind that the chosen method will influence the characteristics of the obtained NPs, such as the size and the surface. Besides, it is crucial to have a great knowledge about the different experimental variables, in order to achieve the intended formulation characteristics (Gorner et al., 1999; Lassalle and Ferreira, 2007).

A large number of polymers from different origins have already been described as useful materials for polymeric NP production and used in preclinical studies. Polymers can be from natural origin, as chitosan, or synthesized, as polylactic acid and poly-lactic-co-glycolic acid (PLGA) (Krishnamachari et al., 2011; Mizrahy and Peer, 2012). Particulate adjuvants, such as PLGA and PCL NPs, have generated a lot of interest due to their biodegradability, biocompatibility and mechanical strength. (Danhier et al., 2012) has nicely reviewed the main properties and applications of PLGA-based nanocarriers. These NPs can also act as adjuvants, maintaining the antigenicity and immunogenicity of encapsulated proteins. In fact, PLGA, used for decades in humans, is the most studied polymer for vaccine formulation and it was shown to increase antibody and cellular responses to antigen-loaded PLGA NP (Johansen et al., 2000; Shen et al., 2006; Chen et al., 2014a). PCL has a great potential for developing antigen controlled release matrices by its low degradation rate, hydrophobicity, good drug permeability, in vitro stability and low toxicity. The adjuvant effect of PCL NPs to induce immune responses against an infectious disease was previously confirmed by several studies (Benoit et al., 1999; Florindo et al., 2008, 2009b; Labet and Thielemans, 2009). If the encapsulated antigen fails to induce DC activation, these NPs can be modified with maturation signals at their surface for direct ligand-receptor interaction, as mannose receptor is overexpressed at DCs and macrophage cell surface. Chitosan NPs, for instance, are an interesting strategy for gene delivery, namely small interfering RNA (siRNA). As chitosan is positively charged, electrostatic interactions occur with negatively charged siRNA, and thus the biomolecule is safely carried to its in vivo target (Aslan et al., 2013).

Nanocarriers produced using polypeptide-based polyanionic, zwitteronic and polycationic polymers (e.g., polyglutamic acid, polyarginine) have also been described (Christian et al., 2009). These are endosomolytic polymers and have been used to promote the cytosolic delivery of these biomolecules. Although clinical trials with peptide-based cancer nanovaccines have shown little success, more recent research has been developed to improve them, using novel polymeric NPs systems.

It has been reported that PLGA NPs loaded with melanoma antigens can elicit effective anti-tumor activity by CTLs in vivo (Zhang et al., 2011; Ma et al., 2012). DC-targeting chitosan NPs, carrying IL-12, were also used in a preclinical study. The administration of this nanovaccine in an animal model resulted in suppression of tumor growth and increased induction of apoptosis (Kim et al., 2006).

Regarding immune cell tracking, biodegradable PLGA NPs have been used in a combined multimodal imaging strategy for a DC-targeting nanovaccine. Superparamagnetic iron oxide particles and a fluorescently labeled antigens were incorporated within the same nanosystem, allowing not only the analysis and quantification of NPs uptake, but also the subcellular tracking of NPs (Cruz et al., 2011).

Polymeric micelles are self-assembled spherical nanocarriers formed by amphiphilic block copolymers in aqueous medium (Figure 3C). A hydrophobic core and a hydrophilic surface compose these structures, and their size ranges from 10 to 100 nm (Torchilin, 2001; Jhaveri and Torchilin, 2014).

Polymer micelles have been investigated as delivery systems for poorly water-soluble/hydrophobic drugs due to the hydrophobic core. It has been shown that micelles can enhance the bioavailability of hydrophobic molecules, which is reassured because they protect the drug from in vivo degradation (Torchilin, 2001; Jhaveri and Torchilin, 2014). Other advantages of polymeric micelles are the low toxicity, the prolonged circulation time and good levels of accumulation in tumor areas (Ganta et al., 2008). In an experiment with nude mice xenograft model, PLGA-PEG polymeric micelles have shown increased tumoral uptake (Yoo and Park, 2004).

Novel pH-responsive polymer micelles formed by an N-(2-hydroxypropyl) methacrylamide corona and a propylacrylic acid (PAA)/dimethylaminoethyl methacrylate (DMAEMA)/butyl methacrylate (BMA) core have already been investigated for antigen trafficking modulation in DCs. The results showed that this nanosystem facilitates the antigen delivery to DCs in the lymph nodes and enhances CD8+ T cell responses, being thus a potential carrier for cancer vaccines (Keller et al., 2014). Also, micelles formed by DMAEMA and pyridyl disulfide ethyl methacrylate (PDSEMA), carrying both CpG ODN and protein antigens, have shown to elicit and increase cellular and humoral immune response by modulating and stimulating antigen cross-presentation, as summarized by Wilson et al. (2013).

Dendrimers consist in hyperbranched spherical nanocarriers formed by a central core, branching monomers and functionalized peripheral groups. Dendrimers can be produced by convergent or divergent polymerization of branching units, resulting in a structure with a hydrophilic surface and a hydrophobic central core (Figure 3D) (Lee et al., 2005). Their main physicochemical features are low viscosity, hyperbranched molecular topology, marcromolecular size, high density of chemical functionality and multiple end groups that can be chemically functionalized (Lee et al., 2005). Also, the depolymerization of dendrimers can be tailored in order to control the release profile of the loaded agents, as described in a review by Wong et al. (2012). Besides vaccines, therapeutic and targeting carriers, dendrimers have also been reported as diagnostic tools due to their ability to protect imaging agents, decreasing its toxicity and enhancing specificity (Yang et al., 2009).

Nowadays, the most described family of dendrimers is the well-studied polyamidoamine (PAMAM). Poly(propyleneimine) and peptide dendrimers, such as poly(L-glutamic acid) dendrimers, have also been studied (Nanjwade et al., 2009). Linear poly(glutamic acid) is a poly(amino acid) polymer with considerable potential for antigen delivery to DCs, and adjuvant properties for DC maturation, able to induce CTLs (Yoshikawa et al., 2008). Additionally, it has been shown to be safe for use in clinic (Chipman et al., 2006) providing the necessary safety profile for human use. These glycopeptide dendrimers have shown promise for antitumor and antiviral prophylactic or therapeutic vaccines, as well as antiviral agents (Niederhafner et al., 2008). Several formulations have reached clinical trials as vaccines against breast (Gilewski et al., 2007), prostate (Slovin et al., 2003), and small cell lung cancers (Krug et al., 2004) with encouraging results. Even though, further investigation must be done in order to guarantee the long-term safety, before they become clinically available (Aslan et al., 2013).

NP size appears to influence the cellular uptake mechanism and the endocytic pathway of NPs, dictating their ultimate intracellular fate and thus overall biological effect. NPs may be assimilated by receptor-mediated endocytosis, clathrin-dependent and/or caveolae-mediated, and phagocytosis, or through a receptor independent mechanism—macropinocytosis. Particulate systems with a larger diameter (>0.5 μm) tend to be assimilated through macropinocytosis and/or phagocytosis by some specific cells, as macrophages and Langerhans cells in the skin. Smaller particles usually enter the cell through endocytosis. NPs with size <150 nm are generally taken by cells via classic receptor-mediated endocytosis (clathrin-dependent) or endocytosis caveolae-mediated if ranging from 50 to 80 nm (Pelkmans and Helenius, 2002). These NPs with size equivalent to viruses are usually able to initiate a virus-like immune response with activation of CTL and Th1. On the other hand, larger particles normally generate a similar immune response to that induced by bacteria, with Th2 activation and antibody production (Xiang et al., 2006).

Besides size, it has also been reported that particle shape may influence cellular uptake and biodistribution. Although it has been suggested non-spherical particles may be valuable for their increased blood circulation time, due to reduced phagocytosis by unspecific cells, they also demonstrated decreased cellular uptake, when compared to spherical NPs. According to Gratton et al., rod-shaped NPs show the highest uptake performance, followed by spheres, cylinders and finally cubical NPs (Gratton et al., 2008).

NP surface charge also seems to play an important role in their particle internalization and thus will also determine the nature of the induced immune response (Xiang et al., 2006). As cell membrane charge is negative, positively charged molecules/systems will show high affinity to it. After cellular uptake, it has been observed that negatively charged or neutral NPs tend to localize within lysosomes, whilst positively charged NPs showed ability to escape from these. Cationic NPs were found in the perinuclear area and have been reported as effective for uptake by macrophages and DCs (Thiele et al., 2003; Yue et al., 2011). On the other hand, the interaction of those delivery systems with cell depends on multiple factors and some studies have reported the presence of neutral NP at endoplasmic reticulum, suggesting their ability to escape degradation at lysossomal/endossomal compartment (Zhou et al., 2014).

Passive targeting results from the transport of nano-based systems across the abnormal leaky vasculature of tumors, into the tumor interstitium or cells, by their movement within fluids—convection—or by passive diffusion. Whereas convection is observed for larger molecules, compounds with low molecular weight cross the membranes by diffusion, depending only on the concentration gradient (Iyer et al., 2006; Danhier et al., 2010).

As blood vessels architecture and its regulation are compromised, caused by unpaired angiogenesis, nanocarriers tend to accumulate selectively in tumor interstitium due to the “Enhanced Permeability and Retention (EPR) effect.” The increased size of gaps in endothelial cells creates pores ranging from 10 to 1000 nm, which along with the poor lymphatic drainage, contributes to the EPR effect, that was first described by Matsumura and Maeda (1986); Yuan et al. (1995); Danhier et al. (2010). This effect has become very important for the design of targeted nanocarriers for cancer therapies. It has been reported that NP levels of accumulation in tumor interstitium are 10–50-fold higher than in normal tissues, leading to improved therapeutic efficacy and less side effects (Iyer et al., 2006; Danhier et al., 2010).

Nanotechnology-based strategies have been explored as platforms for drug delivery, cancer vaccination and/or diagnosis, due to their capacity for overcoming biological barriers and to modulate payloads' intracellular trafficking. These nanoparticulate systems present a good potential for site-selective delivery by binding recognition ligands to NP surface, which can enhance NP endocytosis, influencing their intracellular trafficking and thus inducing prolonged effects (Danhier et al., 2010).

Surface functionalization of nano-based systems (Figure 4) has been used to improve tissue and cell surface antigen targeting, thus moderating non-specific distribution and prolonging the blood circulation time of nano-based systems (Alexis et al., 2008).

PEGylation is a widespread strategy to improve the half-life time of nanocarriers, through steric stabilization and “stealth” properties. It relies on the introduction of poly(ethylene glycol) (PEG) molecules by conjugation, grafting or adsorption onto the surface of nanosystems (Figure 5). The terminal groups of PEG chains also present very suitable moieties to attach functional ligands and attain active-targeted carriers (Freichels et al., 2012). The conjugation of antibody fragments to PEG ends, using disulfide bonds, may consist in an interesting strategy to develop platforms for active targeting (Brocchini et al., 2008). D-α-tocopheryl polyethylene glycol succinate (TPGS) has been reported as an alternative to PEG (Pan and Feng, 2008).

Active-targeted nanosystems are based on the design of nanocarriers with bioactive ligands placed onto their surface or periphery. They will be recognized by overexpressed molecular patterns at the tissues/cells intended to target, facilitating NP recognition and subsequent receptor-mediated endocytosis (Figure 6) (Cheng et al., 2007; Kumar et al., 2009; Danhier et al., 2010; Aslan et al., 2013; Nicolas et al., 2013; Wang et al., 2013a; Gao et al., 2014). Surface modifications represent an outstanding tool for cell targeting allowing a specific contact of nanoparticulate systems with critical immune cells, as evidenced in Stephan et al. (2010). For example, the ligand DEC-205 is highly expressed by CD8+DCs, cells particularly efficient at “cross-presenting” exogenous antigens on MHCI, constituting a highly relevant pathway for the development of a cytolytic immune response. Moreover, recent studies have indicated that the triggering of CD40 on APCs can lead to CD8 T-cell effectors, without the need of common stimulation by MHCII-related Th cells via CD40 ligands (Vonderheide et al., 2013). Mannose receptors at DCs are also associated to ligand internalization and further processing and presentation by immune cells, leading to a more extensive immune response (Lu et al., 2007; Carrillo-Conde et al., 2011; Silva et al., 2013).

These ligands, such as peptides, antibodies and antibody fragments, carbohydrates and even vitamins, may be either attached before the nanocarrier production or afterwards. Liking ligands prior to nanocarrier production may be advantageous, so that the conjugation yield of the ligand to the polymer can be assessed and controlled. Nanocarriers can be thus produced with a well-characterized (co)polymer and the density of ligands on their surface can be tailored. Physicochemical properties of the polymers must be evaluated after ligand conjugation, because the hydrophilic/hydrophobic balance may be altered, particularly if macromolecules are linked (Betancourt et al., 2009; Sperling and Parak, 2010; Nicolas et al., 2013).

The strategy of attaching ligand molecules after nanocarrier production is usually applied, when antibodies, proteins and polypeptides are chosen as targeting agents. As some organic solvents are generally used in the preparation of nanosystems, this method is preferred to avoid denaturation of the secondary structure of the ligands. Also, since they are bulky molecules, they will disturb the hydrophilic/hydrophobic balance which can difficult the method of nanocarrier production (Nicolas et al., 2013). The drawbacks of this approach are related with subsequent purification of the formulation and its characterization. The processes frequently used for purification, such as centrifugation, filtration and dialysis, may degrade or alter the nanosystems. Additionally, it is usually difficult to prove that the ligand is covalently linked to the surface of the nanocarrier and not only adsorbed (Nicolas et al., 2013).

Ligation strategies for functionalization. Several pathways have been developed to attach ligands onto nanosystems surface, such as the carbodiimide strategy, the Michael addition pathway, the biotin–streptavidin approach and the Copper-catalized ligation method (Betancourt et al., 2009; Nicolas et al., 2013). The native terminal groups of some polymers or specific moieties, introduced through chemical modifications, are generally used to apply these schemes of functionalization. For instance, carboxylic acid terminals in aliphatic polyesters and poly(ethylene glycol) (Betancourt et al., 2009).

The most used scheme is based on the carbodiimide chemistry. It relies on the coupling of a molecule containing a terminal amine group with another with an N-hydroxysuccinimide (NHS) ester end or an end group that can be easily esterified to NHS moiety (Betancourt et al., 2009; Nicolas et al., 2013). The Michael addition pathway is based on the thiol-maleimide coupling. Maleimide-polymers are used to produce nanocarriers, which are then decorated with thiol-containing targeting agents (Betancourt et al., 2009; Nicolas et al., 2013). However, the presence of native thiol groups in some molecules, as proteins and peptides, is usually low (or absent in some cases) and many are hard to access. To overcome this, disulfide bonds can be reduced in thiol groups or heterobifunctional cross-linking agents may be used (Nicolas et al., 2013). The biotin–streptavidin approach utilizes a strong non-covalent biological interaction between biotin and avidin (Betancourt et al., 2009; Nicolas et al., 2013). Still, for this strategy, a targeting agent is usually chemically bound to avidin, which is a bulky glycoprotein that may then obstruct the interaction ligand-receptor, essential for targeting (Betancourt et al., 2009). The Copper-catalized ligation is a highly efficient method, based on a cycloaddition reaction that fits in the “click chemistry” class of reactions. The chemical reaction is developed in mild conditions and with little or absent byproducts. The major disadvantage of this approach is the elimination of the Cu-based catalyst used for the reaction (Nicolas et al., 2013).

Functionalization of nanosystems for immune cell targeting. Extensive research has been made regarding cell surface receptors in immune cells, the so-called PRRs. PRRs recognize pathogen-associated molecular patterns (PAMPS) and are involved in several stages of the immune response, from its initiation and proliferation, to its execution (Kumar et al., 2009). Different types of molecules may act as PAMPs, known as “danger signals,” for instance lipids, lipoproteins, proteins, carbohydrates and nucleic acids. The recognition of PAMPs by PRRs triggers immune responses by activating multifactorial signaling pathways. This leads to the induction of inflammatory responses mediated by several cytokines and chemokines (Kumar et al., 2009).

Several classes of PRRs have been reviewed, such as TLRs, retinoic-acid inducible gene (RIG)-like receptors (RLRs), nucleotide oligomerization domain (NOD)-like receptors (NLRs), DNA receptors (cytosolic sensors for DNA), scavenger receptors, and C-type lectin receptors (CLRs) (Kumar et al., 2009; Carrillo-Conde et al., 2011; Shen et al., 2013; Silva et al., 2013). In mammals, the most studied PRR class is the TLRs class. TLRs are predominantly expressed by APCs, as DCs, but they are also found on cells of the adaptive immune system, such as in αβT cells, regulatory T cells, and γδT cells, as well as NKT cells (Wesch et al., 2011). Through TLR activation, both the innate and the adaptive immune responses can be engaged, either by direct activation of TLRs with their ligands on T and B cells, or by indirect mechanisms involving TLR-activated DCs (Silva et al., 2013). C-type lectin receptors (CLRs) belong to another class of PRRs expressed by APCs. This receptor family is characterized by the presence of domains that bind to carbohydrates (Van Kooyk, 2008). CLRs are specific receptors particularly engaged in the internalization of antigens. CLRs enable the intracellular uptake and processing of antigens, as well as influence their cytosolic fate and the loading on MHC class I and II (Unger and Van Kooyk, 2011).

Regarding the involvement of PRRs in several strategic immune pathways, the design of nano-based systems for immune cell targeting can be extremely interesting. Not only because a more specific delivery can be achieved, but also because the cellular internalization of the targeted nanosystem can be modulated and potentiated. Additionally, the attachment of PRRs ligands on the surface of nanocarriers may boost their immunogenicity, which can be an outstanding strategy for the development of vaccines, since it allows the incorporation of an antigen and the “danger signal” in the same platform (Silva et al., 2013).

Fluorescence imaging is an optical imaging method based on the excitation/emission of molecules (Cai and Chen, 2007). The use of fluorescent molecular probes—as fluorescent dyes and fluorescent proteins—has been widely applied in the labeling of biomolecules, cells and tissues. Although these probes are already used in vivo, for instance in retinal angiography and visualization of arteries, they are unsuitable for real-time imaging assays, regarding their low photostability and sensitivity at the cellular and molecular levels (Santra and Malhotra, 2011). The application of fluorophores in real-time in vivo imaging has also been limited by the high absorption of optical signal by tissues and body fluids in the UV and visible wavelength. The light scattering caused by tissues that attenuate the optical signal and the tissue auto-fluorescence that influences the background signal is also a limitation (Santra and Malhotra, 2011). Additionally, some fluorescent probes may be toxic for cells and body (Li et al., 2013). Several NP-based strategies have been proposed to overcome the limitations of fluorescent dyes for real-time in vivo imaging (Supplementary Material) (Santra and Malhotra, 2011; Wang et al., 2013b).

Fluorescent-labeled NPs are more stable in the body and increase the detection sensitivity and photostability. In the same platforms, a great number of probe molecules can be incorporated, in opposite to a single conventional molecule. Also, in NPs, fluorescent dyes can be protected from quenching and degradation (Santra and Malhotra, 2011; Wang et al., 2013b).

The most extensively studied nanosystems for fluorescence imaging are quantum dots (QDs) (Cai and Chen, 2007), inorganic fluorescent NPs that can be based on metallic or semiconductor materials, such as CdSe and CdTe (Ballou et al., 2004). As reviewed by Cai and Chen, in ideal conditions, QDs can have better properties than organic fluorescence probes. These include high resistance to degradation and photobleaching, high quantum yields, high molar extinction coefficients, continuous absorption spectra covering from UV to near-infrared, long fluorescence lifetimes (>10 ns), narrow emission spectra and very long effective Stokes shifts (Cai and Chen, 2007). QDs have been used for innumerous applications, from cell tracking (Voura et al., 2004) to mapping of sentinel lymph nodes (Ballou et al., 2007). QDs can be used to identify several ligands in the same experiment, using multiple colors and intensities to detect different structures (Ballou et al., 2004). The potential use of DC-targeting QDs as both fluorescent NPs for in vivo and in vitro imaging, and antigen-delivery system has also been investigated. In this study, it was proved that QDs display promising properties for combined priming and immunoimaging of DC (Sen et al., 2008). Functionalization and modifications of the surface of QDs with PEG chains and ligands for active targeting, such as peptides and antibodies, have been under research to improve the application of these nanosystems in the biomedical field (Ballou et al., 2007; Cai and Chen, 2007). QD conjugates are already commercially available for immunospecific labeling (Ballou et al., 2004). Thus, the development of multifunctional nanoplatforms holds a great promise for the future of biomedicine, since it will be possible to combine simultaneously both diagnosis and therapy in the same nanostructure (Kim et al., 2008a).

Several other groups have suggested the use of silica-based NPs (siNPs) as an interesting strategy to perform imaging assays using fluorescence (Santra et al., 2005; Kim et al., 2008a; Wang et al., 2013b). siNPs have been used for high sensitive and specific in situ labeling and tracking of cell surface receptors (He et al., 2004, 2007). Relying on the affinity of antigen-antibody or ligand-receptor interactions, NPs were functionalized with antibodies and ligands and applied as an immunediagnostic method (He et al., 2002). siNPs have also been used as a non-invasive tool for intracellular labeling, tracking and sensing in living cells, contributing with novel information about dynamic biological processes of subcellular structures, such as lysosomes and endosomes (Shi et al., 2010). Finally, siNPs were applied to better understand the biodistribution and fate of NPs, in vivo (Wang et al., 2013b).

The key role of immune cells in the development of future immunotherapeutic approaches against chronic pathologies, mainly cancer diseases, has fostered the design and optimization of different real-time imaging techniques, avoiding the classic ex vivo histologic analysis (Kircher et al., 2011; Ahrens and Bulte, 2013; Liu and Li, 2014). In fact, most of the information obtained for immune cell tracking has arisen from optical and confocal microscopy and flow cytometry. Two-photon microscopy allowed the observation of different immune cells in their biological environment at real time (Progatzky et al., 2013). However, despite being a powerful tool to observe these highly motile cells and characterize their interaction with native environment, this imaging technique is unsuitable for detection of deeper events due to tissue opacity (Dzhagalov et al., 2012).

Bioluminescence imaging techniques, on the other hand, enable deeper tissue penetrations while tracking immune cells in vivo. Even though, it is one of the most commonly used techniques for immune cell tracking in vivo, allowing whole-body non-invasive tomography. This technique is only useful for preclinical studies in small animals, due to the limits related to the attenuation of light in tissues (Kircher et al., 2011).

All near-infrared (NIR) multiphoton microscopy methods are potential techniques for deep tissue imaging but further studies are needed to better characterize the capabilities of these NIR-excitation techniques and background reduction (Joshi et al., 2013).

Magnetic resonance imaging (MRI), ultrasound, positron emission tomography (PET) (Yaghoubi et al., 2009), single photon emission tomography (SPECT) and X-ray computed tomography (CT) are the imaging techniques approved for medical applications (Bernsen et al., 2014). PET and SPECT are high-sensitivity and low-resolution techniques, while MRI and CT provide high-resolution images (Liu and Li, 2014). However, the use of radionuclide-based techniques, as PET and SPECT, has brought questions regarding their safety (Laskey et al., 2010). In addition, their combination with additional methods is fundamental to obtain an anatomical image. Therefore, the combination of these different imaging modalities constitutes a multimodality imaging method that has been explored in preclinical and clinical development, including SPECT/CT and MRI/PET (Naumova et al., 2014).

Among these techniques, MRI is the most versatile and sensitive method allowing the study of immune cell morphology and function (Ahrens and Bulte, 2013). In fact, innovative and safer techniques are emerging from the use of different biocompatible cell labeling probes and MRI to obtain high-resolution images without using ionizing radiation (Sosnovik and Nahrendorf, 2012; Thu et al., 2012). The signal used for MRI arises from the water protons (1H) or different fluorinated molecules (e.g., ¹⁹F) under a static magnetic field and after pulsed by a radio-frequency radiation, which alters the equilibrium of their nuclei. The MRI signal will then result from a transient voltage determined by the properties of labeled tissue (Ahrens and Bulte, 2013).

This non-invasive and safe imaging technique has been expected to track immune cells in vivo, enabling the characterization of their biodistribution and fate. MRI also seems suitable for the detection/quantification of surface markers and secreted factors resultant from biological processes occurred in vivo at a particular disease stage (Lu et al., 2013; Naumova et al., 2014). The rapid evolution in this field, advanced by the potential efficacy of next-generation cellular-based therapeutic approaches (e.g., immunotherapy and stem cell-based therapy), will certainly make this method a crucial tool to follow detailed biological and immunological processes in vivo.

The successful application of these in vivo cell-tracking tools can potentially optimize image-guided diagnostics and the overall efficacy of different therapeutic options. Particularly, those based on the modulation of endogenous cells support the selection of a specific treatment, the choice of the best administration route and also the use of a correct dose for each patient (Ahrens and Bulte, 2013).

Different exogenous cell-labeling probes have been explored but superparamagnetic iron oxide (SPIO) nanoparticles and perfluorocarbon (PFC) nanoemulsions seem to be the most promising for those advanced MRI-based techniques (Supplementary Material). Moreover, these are the unique in vivo MRI cell-labeling techniques approved for human clinical trials, and thus will be further discussed (Ahrens and Bulte, 2013).

Nano-based systems for MRI real-time tracking of immune cells. Different nanosystems (Supplementary Material) have been developed for MRI-based in vivo cell tracking, but the negative contrast agents based on SPIO and PFC constitute the most explored ways to control MRI signal and consequent detection (Hawrylak et al., 1993; Bulte and Kraitchman, 2004). SPIO contrast agents are small particles composed by ferrous and ferric oxides, usually coated by dextran. Even though, these ionic NPs have been modified by other biodegradable polymer (e.g., chitosan, PEG, siloxanes, polyaniline, glyceryl monooleate) and labeled with targeting moieties to potentiate their delivery to certain tissues (Supplementary Material) (Shubayev et al., 2009; Dilnawaz et al., 2010). These MRI-based contrast agents strongly perturb the magnetic field of the region in which they are embedded. The water molecules will sense that alteration in the magnetic field and the resultant loss of signal will lead to a dark image (Ahrens and Bulte, 2013). On the other hand, fluorinated-based probes directly label targeted cells and thus the MRI signal is dependent on the number of fluorine atoms and labeled cells, which can be observed in their biological environment (Srinivas et al., 2012).

The labeling of cells using these nano-based systems can be performed ex vivo or in vivo, through their direct administration in the body. The labeling of immune cells ex vivo with SPIO NPs has been explored to track and clarify migratory patterns of diverse immune cells, as NK (Daldrup-Link et al., 2005), cells from T lineage (Kircher et al., 2011), and DCs (De Vries et al., 2005; Rohani et al., 2011) used during immunotherapeutic cancer approaches. Innovative immunotheranostic strategies under development combine these metal ion-based NP with targeted nanoparticulate cancer vaccines. One interesting study has shown multifunctional iron oxide NPs formulated in order to deliver carcinoembryonic antigens to DCs and be detected by MRI (Cho et al., 2011). Alternatively, some SPIO NPs have been developed to label DCs membranes by modifying their surface with CD11c antibodies, promoting receptor-mediated endocytosis (Ahrens et al., 2003; Yu et al., 2012). Despite being a promising approach against cancer disease, their clinical translation is still unclear.

The ex vivo labeling of a DC-based cancer vaccine by SPIO NP was used in the first clinical trial that involved the cell tracking by MRI techniques, where it was possible to detect the target lymph node only in half of the patients with melanoma (De Vries et al., 2005).

T cells have been sorted and cultured with SPIO NPs, mostly coated by transfection agents, as poly-L-lysine or protamine sulfate, to promote their capture due to the non-phagocytic nature of these immune cells (Arbab et al., 2005; Thorek and Tsourkas, 2008; Thu et al., 2012). These intracellular labeling was also attempted through the use cell-penetrating peptides and HIV-TAT (Torchilin, 2008).

The in vivo labeling of immune cells by SPIO NPs is often used to track monocytes and macrophages to characterize inflammatory events, due to their phagocytic behavior (Settles et al., 2011). The in vivo labeling can be achieved by the intravenous administration of SPIO NPs, or alternatively after their direct injection into tumor tissue. Both options were successfully used to label immune cells and track their migration pattern toward lymph nodes, which allows for example the definition of tumor specific stage (Harisinghani et al., 2003).

It is important to emphasize that the cell labeling strategy must not alter the function and normal phenotype of immune cells, which could limit the efficacy of cellular-based therapies. The SPIO NPs are known as safe systems due to their biodegradability nature and usual rapid metabolization in vivo (Yu et al., 2012). Therefore, the SPIO-based cell labeling is mostly suitable for short-term studies. On the other hand, false positives may be detected after the accumulation of the detection agent in macrophages after the destruction of labeled cells (Ahrens et al., 2003; Thorek and Tsourkas, 2008). This disadvantage is in fact common to different imaging reagent-labeled techniques.

The ¹⁹F MRI is a highly sensitive technique that allows the direct quantification of labeled immune cells, as T cells and phagocytic cells, either in vivo or ex vivo (Srinivas et al., 2009; Helfer et al., 2010). Unlike SPIO NPs, this labeling method usually does not detect false positives and, once is not metabolized in vivo, constitutes a suitable approach for long-term studies (Janjic and Ahrens, 2009; Srinivas et al., 2012).

The droplet surface of these PFC colloidal systems has been changed with charged entities to potentiate their efficient delivery at intracellular level. Therefore, the safety of these labeling systems is increased, which has been shown using different immune cells, as DCs and T cells (Ahrens et al., 2005; Srinivas et al., 2009; Helfer et al., 2010; Ahrens and Bulte, 2013).

Recent studies have shown the promising combination of ¹⁹F labeling techniques with fluorescence or NIR probes, as well as with nuclear magnetic resonance (NMR) (Patel et al., 2013). Even though, the use of these colloidal system for cell tracking is considerably recent and further studies are urged in order to confirm these indications.