key: cord-022142-d4yxgv83
authors: David, Ayelet; Golani-Armon, Adi
title: Polymer-Based DNA Delivery Systems for Cancer Immunotherapy
date: 2016-05-28
journal: Nanomedicine
DOI: 10.1007/978-1-4939-3634-2_10
sha: 
doc_id: 22142
cord_uid: d4yxgv83

The use of gene delivery systems for the expression of antigenic proteins is an established means for activating a patient’s own immune system against the cancer they carry. Since tumor cells are poor antigen-presenting cells, cross-presentation of tumor antigens by dendritic cells (DCs) is essential for the generation of tumor-specific cytotoxic T-lymphocyte responses. A number of polymer-based nanomedicines have been developed to deliver genes into DCs, primarily by incorporating tumor-specific, antigen-encoding plasmid DNA with polycationic molecules to facilitate DNA loading and intracellular trafficking. Direct in vivo targeting of plasmid DNA to DC surface receptors can induce high transfection efficiency and long-term gene expression, essential for antigen loading onto major histocompatibility complex molecules and stimulation of T-cell responses. This chapter summarizes the physicochemical properties and biological information on polymer-based non-viral vectors used for targeting DCs, and discusses the main challenges for successful in vivo gene transfer into DCs.

Cancer has long represented a major burden on health and longevity [ 1 ] . The high prevalence of the disease has made it the basis of a major research focus, with the disease being investigated in many contexts. Intensive research has yielded a large body of information that has served to uncover biological, biochemical and pathophysiological aspects of the disease, as well as their underlying mechanisms. Based on the information gathered by the efforts of numerous researchers, novel nanosized medicines (nanomedicines), that can be effectively localized to tumors and actively taken up by cancer cells have been developed, and are expected to replace traditional chemotherapy and possibly irradiation due to increased effi ciencies and attenuated adverse side-effects [ 2 ] .

Unfortunately, recent years have seen only minor improvements in cancer morbidity and mortality rates , with the most commonly used cancer therapies still being conventional chemotherapy, despite their serious side-effects. This has encouraged researchers to seek answers in the fi eld of immunotherapy [ 3 ] . Whereas exogenous anti-cancer agents have failed to generate strong and effective responses against tumors and induce regression, the endogenous activity of the immune system was hoped to elicit specifi c anti-tumor immunity that would result in tumor regression and elimination. In particularly, dendritic cell (DC) vaccinations were expected to induce immunological memory that would enable recognition and destruction of residual tumor cells that evaded earlier treatment, thereby avoiding recurrence of the disease [ 4 ] . Previous studies employing mouse models indicated that the establishment of anti-tumor immunity required the presentation of tumorassociated antigens (TAA) by DCs [ 5 , 6 ] . Two unique properties make DCs the most potent antigen-presenting cells (APCs), namely their ability to present exogenous antigens on major histocompatibility complex (MHC)-I molecules to prime CD8 + T-cells (i.e., cross-presentation), and their capability to initiate, activate and modulate the various arms of the immune system in a coordinated manner. Indeed, in vivo targeting of tumor antigens into DCs was shown to elicit strong TAAspecifi c DC4 + and CD8 + immune responses [ 7 ] . Therefore, DCs loaded with TAA or tumor antigen-encoding plasmid DNA may facilitate the development of new immunotherapies for cancer treatment. This chapter highlights the repertoire of non-viral, nanosized polymeric DNA delivery systems (polyplexes) available to achieve effi cient gene transfer into DCs for immunotherapeutic applications in cancer therapy. The physicochemical characteristics and surface properties of polyplexes required for effi cient gene transfer and gene expression in DCs are discussed. The infl uence of the ligand valency on the targeting of DCs via dendritic cell surface receptors is also described.

The fi eld of immunotherapy aims at activating a patient's own immune system against a host disease. Immunotherapy includes the investigation of different mechanisms leading to activation of the immune response, and the development of methods to control such responses in a desired manner so as to combat disease, either as a single strategy or in combination with other treatments. In the specifi c case of cancer immunotherapy, it is speculated that the in vivo activity of endogenous immune cells would be far more sensitive and specifi c than would any exogenous treatment [ 8 ] . Anti-cancer vaccines have, thus been designed to induce both tumorspecifi c effector T cells and tumor-specifi c memory T-cells [ 4 ] . Such anti-cancer vaccination, it is hoped, will induce regression of established tumors, as well as prevent the onset of secondary tumors and metastases, possibly by addressing the issue of minimal residual disease (MRD). In this case, sparse cancerous cells that evaded irradiation or chemotherapy will be detected and destroyed by the immune system cells that, unlike the temporary exogenous treatment, are present in the body at all times. A major requirement for an effi cient anti-cancer vaccination is the generation of a cytotoxic T-lymphocyte (CTL) response, but manipulation of the other arms of the immune response, mainly CD4 + T helper cells, is critical for a robust and long lasting CTL response [ 9 -11 ] . CTL response generation depends on antigen (Ag) presentation on MHC t o naïve DC8 + T cells. The poor Ag presentation activity demonstrated by tumor cells [ 12 ] highlights the need for professional APCs to generate the desired CTL response. Among the several types of APCs in the body, DCs are the most suitable for this purpose.

In 2011, Ralph Steinman was awarded the Nobel Prize for his 1973 discovery of DCs. DCs are a family of professional APCs. As the most powerful APCs, they are equipped with specialized machinery that enables them to regulate the initiation of a primary immune response [ 13 -15 ] . Originally derived from bone marrow, DCs are seeded in all tissues, where they sample their environment in an attempt to detect tissue damage, pathogen entry, infl ammation and malignantly transformed cells by taking up particles and molecules, processing them into short peptides and presenting the resulting peptides on MHC molecules. DCs use different routes to capture Ags, including phagocytosis, micro-and macropinocytosis, and receptormediated endocytosis [ 9 , 15 , 16 ] . Importantly, receptor-mediated endocytosis can be exploited for the targeting of TAAs into DCs.

Immature DCs in peripheral tissues are characterized by high Ag capture activity, low surface MHC levels and co-stimulatory molecules, as well as a tendency to induce tolerance to self-antigens. Upon recognition of "danger" signals, DCs undergo a maturation process characterized by a reduction of Ag capture activity and an increase of MHC and co-stimulatory molecule expression on the cell surface [ 17 ] . Mature DCs migrate to adjacent lymph nodes, where they present antigenic peptides to naïve T-cells [ 4 ] . Ag presentation, co-stimulatory molecules expression, and the secretion of an appropriate set of cytokines and chemokines by DCs facilitate the differentiation of naïve CD4 + and CD8 + T-cells into effector T-helper or T-cytotoxic cells, respectively [ 18 , 19 ] . DCs can, thus, simultaneously activate both a CTL response to directly kill pathogens or affected cells and a T-helper response to recruit and enhance the activity of other immune cells in the right context. This unique ability of DCs to activate the various arms of the immune system to achieve a powerful and effi cient immune response makes them the most promising candidates for immunotherapeutic manipulations .

Three distinct pathways are used by different cell types for the presentation of antigens, namely MHC class I and class II antigen presentation, and cross-presentation.

This pathway is present in all nucleated cells, and is used to present endogenous antigens derived from the nucleus or the cytoplasm. MHC class I antigen presentation can, therefore, report intracellular bacterial or viral infections, as well as malignant transformation in the presenting cell. The resulting intracellular products are degraded in the cytoplasm by the proteasome, translocated into the endoplasmic reticulum (ER) via the transporter associated with antigen presentation (TAP), and loaded on newly formed MHC class I molecules. The MHC class I/peptide complexes are then exported along the constitutive secretory pathway to the cell membrane, where they are presented to naïve CD8 + T-cells. The interaction between MHC class I/peptide complexes on DCs and T-cell receptors (TCR) on CD8 + T-cells induce the differentiation of naïve CD8 + T-cells into cytotoxic T-cells that are capable of killing the infected or transformed cells ( Fig. 10 .1 ) [ 14 , 18 , 20 ] . 

This pathway is restricted to APCs, including macrophages (MΦs), DCs and B lymphocytes, and is used to present exogenous antigens. The exogenous antigens are taken up into these cells by endocytosis, where they are found in early endosomes, a mildly acidic cell compartment containing a small amount of proteases. Early endosomes develop into late endosomes that are more acidic and present higher proteolytic activity, which process the captured Ag into short peptides suitable for MHC presentation [ 21 ] . The α and β chains of the MHC class II molecule are assembled in the ER and are associated with the Invariant chain (Ii) that 

The generation of a CTL response against virally infected or transformed cells is possible through the MHC class I pathway described above. However, other kinds of threats can be addressed by the immune system APCs through the MHC class II pathway, but this pathway exclusively generates a T-helper response. As it is obvious that not every viral infection or malignant transformation involves APCs, and that many pathogens can impair antigen presentation by their host cells, an alternative mechanism must exist that would enable APCs to generate a cytotoxic T-cell response against the various exogenous threats they encounter [ 22 ] . Such a pathway indeed exists and is a unique property of DCs. In addition to the MHC class I and MHC class II antigen presentation pathways, DCs can present exogenous antigens on MHC class I molecules to CD8 + T-cells. This pathway is termed cross-presentation, implying that it involves mechanisms from both the MHC class I and the MHC class II pathways [ 23 ] . Although fi rst described more than three decades ago, the details of this pathway remain only poorly understood, with two main theories attempting to explain the mechanisms leading to MHC class I loading of endocytosed exogenous antigens. The cytosolic track suggests that the antigen is translocated from the endosome into the cytosol, where it enters the cytosolic processing pathway. The endocytic track, on the other hand, suggest that MHC I molecules are recycled from the cell membrane to the endosome, where they are loaded with antigenic peptides processed by endosomal proteases [ 21 , 24 ] .

As mentioned earlier, anti-cancer immunity requires the generation of a specifi c anti-tumor CTL response that, in turn, requires proper antigen presentation. As part of the many techniques tumor cells use to avoid or weaken the immune system, they can substantially reduce the expression of MHC class I/peptide complexes on their cell surface. Since tumor cells are, thus, poor Ag-presenting cells, cross-presentation of tumor antigens by DCs is essential for the generation of tumor-specifi c CTL responses [ 4 ] . In addition, given the unique ability of DCs to initiate and orchestrate the various arms of the immune system, including recruitment and activation of macrophages, natural killer (NK) cells, T-helper cells and B-cells, a strong and comprehensive anti-cancer immune response can hopefully be achieved by inducing such cells to present an appropriate tumor antigen [ 10 ] .

DC immunization requires that an appropriate antigen be presented to DCs in a proper manner and in the right immune context so as to allow Ag uptake, processing and presentation on MHC molecules, parallel to DC maturation and T-cell priming. The fi rst method developed for DC immunization involved the use of whole tumor cells (WTC) or tumor cell lysates, either alone, mixed with an adjuvant, or genetically modifi ed to express an adjuvant. The major advantage of this method is that no Ag identifi cation is required, and multiple Ags are being delivered simultaneously. Nevertheless, all WTC vaccine methods showed limited efficiency in clinical trials, probably due to insuffi cient interaction between the tumor antigens and the DCs [ 25 ] . Pulsing DCs with antigenic proteins or peptides corresponds to another approach. Protein-pulsed DCs are capable of presenting Ags on both MHC class I and MHC class II molecules, with a long half-life of MHC class I presentation and no HLA restriction (i.e., no need for patient selection), but they require an appropriate processing of the protein by DCs, which may be diffi cult to achieve. Peptide-pulsed DCs, on the other hand, require no processing. Still, they show limited MHC class II presentation, a short half-life of MHC class I presentation, and a HLA restriction. Specifi c or total tumor mRNA can be processed and presented on both MHC class I and class II molecules. This, however, requires mRNA extraction from a tumor sample and is, thus, patient-specifi c [ 15 , 26 ] . Lately, the delivery of tumor Ag-encoding plasmid DNA has emerged as a promising method for DC immunization [ 19 , 27 ] .

DNA vaccines are DCs that were genetically modifi ed to express TAAs. DNA vaccines are advantageous for several reasons. They enable the presentation of multiple epitopes of full-length TAAs on MHC class I and class II molecules, and since processing occurs within the cell, they are not HLA-restricted. In addition, effi cient gene transfer allows for a continuous supply of peptides in the modifi ed DC [ 10 ] . Genetic modifi cation of DCs can be achieved either by ex vivo gene delivery methods, including use of a gene gun, electroporation, ultrasound and microinjection, or by in vivo approaches, including naked DNA delivery, and viral or synthetic vectors [ 8 , 27 ] . Ex vivo delivery offers many advantages. The extracted cells are cultured and undergo maturation under controlled conditions and in the absence of inhibitory signals provided by the tumor cells, such that maturation status can be determined before re-administration. Since transfection occurs in culture, high specifi city is achieved as only the selected cells are transfected. Nevertheless, ex vivo gene delivery carries substantial limitations. The process is laborious, costly and time-consuming [ 12 ] . Patients must undergo cytopheresis, followed by culturing and maturation of the acquired cells, steps that must be performed for each patient separately. Reproducibility is very low and different quality control methods are used. Thus, only a limited number of patients would be expected to benefi t from this approach. Hence, ex vivo gene delivery is not likely to become widely marketable. Moreover, transfected mature DCs can show poor distribution from the injection site and ex vivo maturation can impair DC traffi cking to lymph nodes, a critical requirement for cross-priming [ 28 , 29 ] . In contrast, in vivo gene delivery may be expected to become "off the shelf" therapy. As a single product suitable for all patients, in vivo gene delivery can be produced on a large scale with lower costs. Simple and uniform manufacturing and product control procedures will enable reproducibility and control over product quality. In addition, DCs can be targeted at different sites and in their natural environment, thereby not impairing their natural course of maturation and activation [ 25 , 29 ] . For these reasons, in vivo gene delivery is considered by many to be the best strategy for Ag delivery into DCs [ 8 , 10 , 19 ].

In vivo gene carriers must meet important requirements. First, they should be able to incorporate their plasmid DNA cargo into the core of the nanoparticle and be stable enough to carry such cargo in the circulation and protect it from degradation. Second, they should be able to selectively target the desired cell type and be properly internalized. Third, they must facilitate escape from the endosome, cytoplasm traffi cking, nuclear transport and DNA unpacking [ 30 ] . To date, the most effi cient gene delivery systems are viral vectors. Using small amounts of DNA, viral vectors can induce high transfection effi ciency and stable, long-term gene expression.

Unfortunately, viral vectors possess some serious safety issues, including toxicity, immunogenicity and oncogenicity, with numerous clinical trials having been terminated because of this [ 15 , 31 , 32 ] . Restricted gene size is another major limitation of viral vectors. Synthetic vectors correspond to cationic lipids or cationic polymers, respectively termed lipoplexes or polyplexes, which electrostatically bind the negatively charged DNA. The main drawback of such vectors is their low transfection effi ciency, as compared to viral vectors. Yet, synthetic vectors are simple, safe, easy to manufacture on a large scale and can carry plasmids of unrestricted size. Moreover, they can be easily modifi ed to possess desirable properties, including targetability, serum stability, reactivity to external signals and an ability to stimulate an immune response. In fact, cationic polymers themselves may provide immune stimulation and mimic nuclear localization signals (NLS), facilitating nuclear transport of their cargo [ 9 , 30 ] .

Various polymeric carriers for DNA and RNA delivery have been developed in the last decade as alternatives to viral vectors. Cationic polymers with free primary, secondary and/or tetiary amines were the earliest DNA carriers investigated as transfection reagents in mammalian cells. Such polymers can condense large genes into smaller structures, protect DNA from enzymatic degradation, and can mask the negative charges of DNA, a prerequisite for successful transfection of most types of cells. Some cationic polymers, such as poly-( L -lysine) (PLL), L -polyethylenimine ( L -PEI) and chitosan, are linear, while others, like B-PEI and polyamidoamine (PAMAM) dendrimers, are highly branched chains [ 33 ] . Branched cationic polymers, such as PEI and PAMAM, exhibit high transfection effi ciency in mammalian tissues. Micro-and nanoparticulate systems which adsorb or encapsulate oligonucleotides or genes, based on, for example, poly(lactide-co-glycolide) (PLGA), polycyanoacrylate, polyorthoesters, gelatin, alginate or chitosan, are also under investigation as sustained release matrices for genetic drugs. Some of the most important polymer-based systems utilized for DNA delivery into APCs are discussed below.

Poly( L -lysine) (PLL) was the fi rst cationic polymer developed for gene delivery. PLL is a linear polypeptide presenting L -lysine residues in repeat units ( Fig. 10.3c ). This fi rst generation cationic polymer bears ɛ-amino groups that electrostatically bind the negatively charged nucleic acids, and interact with the negatively charged cell membrane. The large number of active functional amine groups allows for easy modifi cation with targeting ligands [ 34 ] . However, the transfection effi ciency of PLL is very low because it cannot mediate escape from the endosomal compartment and release into the cytosol. To overcome this barrier, PLL is usually used in combination with chloroquine, although use of this agent is limited due to its cytotoxicity [ 35 ] . Another disadvantage of PLL polyplexes is that transfection effi ciency is signifi cantly infl uenced by serum, probably due to the rapid binding to negativelycharged serum components [ 36 ] . Furthermore, in vivo applications of PLL polyplexes are complicated by a high level of cytotoxicity [ 37 ] and lack of in vivo stability [ 38 , 39 ] . Because of low transfection effi ciency, imidazole groups (pKa ~ 6.5) were introduced into PLL to improve buffer capacity, thereby, enhancing transfection effi ciency [ 40 ] . PLL with high imidazole content mediated high gene transfection effi ciency, with gene expression levels close to those of PEI (discussed below), and exhibited low cytotoxicity. The PEGylation of PLL can also improve its cytotoxicity profi le [ 36 ] . Finally, whereas it has been found that PLL/DNA complexes can be taken up by DCs, PLL/DNA did not alter DC phenotype through surface marker expression [ 41 ] . Complexation of plasmid DNA encoding for chicken egg ovalbumin (OVA) with PLL-coated polystyrene (PS) particles induced high levels of CD8+ T-cells as well as OVA-specifi c antibodies in C57BL/6 mice, and further inhibited tumor growth after challenge with OVA expressing tumor cells [ 42 ] . PLL-based microspheres displaying mannan or mannoside-modifi ed surfaces (for targeting the mannose receptor, discussed below) were readily phagocytosed by both DCs and macrophages, however neither surface-assembled mannan-nor mannoside-modifi ed microspheres could stimulate DC maturation [ 43 ] . Thus, despite the promising results shown in early studies, PLL is not likely to fi nd clinical applications [ 30 ] .

Poly(ethylenimine) (PEI) is a second generation and one of the most useful polycations for gene delivery [ 44 ] . Linear PEI has only secondary amino groups that are almost all protonated under physiological conditions. Branched PEI presents not only primary and secondary amines but also tertiary amines. As such, only about two-thirds of the amino groups in branched PEI are protonable under physiological conditions. The transfection effi ciency of PEIs depends on the molecule weight, the PEI nitrogen/DNA phosphate charge ratio (N/P) and the cell type. As every third position on the polymer backbone is occupied by a protonable amino group (Fig.  10.3a ) , its cationic charge density is very high, allowing for condensation of the negatively charged DNA. Under physiological conditions, only about 20 % of the PEI nitrogen atoms are protonated, leaving the other 80 % to facilitate the important step of endosomal escape by the so-called " proton sponge effect " [ 30 , 45 , 46 ] . Thus, PEI is the most popular and most effective polymeric transfection reagent cited to date [ 47 ] . However, the positive charge of PEI/DNA polyplexes cause some serious problems, including adsorption to cells and negatively charged blood components, recognition by the immune system components, resulting in rapid clearance from the circulation, and cytotoxicity to non-target cells [ 44 , 48 ] . To overcome these limitations, PEI has been conjugated to hydrophilic polymers (i.e., poly(ethylene glycol) (PEG) [ 49 ] , or hyaluronic acid (HA)) of different molecular weights [ 50 ] . PEG is wildly used in drug and gene delivery systems to shield charged, immunogenic or toxic segments, resulting in less toxic "stealth" particles that can evade the immune system and, thus, avoid rapid clearance from the circulation [ 51 , 52 ] . The conjugation of PEG to PEI was previously shown to increase polyplex solubility and serum stability and reduce cytotoxicity by shielding the high positive charge of PEI [ 49 ] . PEGylated PEI, however, showed lower transfection effi ciency, as compared to PEI/DNA complexes, and also lacked cell-specifi city [ 53 ] . Ligation of PEI/ DNA complexes to molecules targeting DC uptake receptors can signifi cantly increase transfection effi ciency.

Approaches for targeting DC receptors have generally involved either natural receptor ligands or the use of antibodies raised against specifi c receptors. Targeting DC cell surface receptors may also provide cell activation signals [ 4 , 54 ] . Different DC receptors have been used to facilitate targeted delivery of gene and drug carriers into DCs, including β-integrins (CD11b and CD11c) [ 55 ] , CD40 [ 56 ] and the Fc receptor [ 57 ] , but the most studied proteins for this purpose are members of the C-type lectin receptor (CLR) family [ 58 ] . CLR is a family of receptors sharing structural homology in their carbohydrate recognition domain (CRD), where specifi c sugar residues are bound in a calcium-dependent manner. One of the CLRs, the mannose receptor (MR, CD206), is the most widely used receptor for targeting DCs, using both mannose and mannan [ 59 ] . While several studies demonstrated that mannosylated-PEI conjugates are effective in gene delivery via MR [ 60 -62 ] , their transfection potential for primary human and mouse DC was found to be rather low [ 60 ] . This might be due to the low affi nity of carbohydrate ligands to their receptors, which is usually in the low millimolar range [ 63 ] , thus, limiting in vivo transfection effi ciency. The eight adjacent CRDs in MR may help to increase the binding affi nity and specifi city of polyplexes containing mannosylated glycans in a multivalent display [ 59 , 64 ] . We have recently described the design of multivalent mannosylated PEI/DNA complexes bearing mono-and trivalent mannose as a ligand for targeting MR-positive DCs [ 65 ] . Complexes bearing mono-and trivalent mannose (Man-PEG-b-PEI/DNA and Man 3 -PEG-b-PEI/DNA, respectively) were safe and demonstrated signifi cantly higher in vitro transfection effi ciency in DCs. The mannosylated complexes were injected into the tail base of C57/BL6 mice, and 24 h post-injection, the percentages of GFP positive cells in the entire cell population and in the CD11c + cells taken from the inguinal lymph nodes were measured. When examining the entire cells population extracted from the lymph node, only PEI/DNA complexes showed detectable GFP activity, but no signifi cant change was observed between the treatments (Fig. 10.4a ) . However, Man 3 -PEGb -PEI/DNA was signifi cantly more effi cient in transfecting CD11c+ DCs collected from inguinal lymph nodes, as compared to polyplexes prepared with PEG-b-PEI/DNA or PEI/DNA (Fig. 10.4b ) .

One challenge of targeting the MR, as holds true for all of the other abovementioned receptors, is that they are not exclusively expressed on DCs, and are also on several other cell types [ 57 ] . MR, for example, is also expressed on monocytes, subsets of endothelial cells and tumor-associated macrophages [ 9 ] . Furthermore, the synthesis of carbohydrate ligands and analogs, especially multivalent or complex carbohydrates, often requires many time-consuming, low yielding steps [ 66 ] . The stereochemistry of such carbohydrate ligands is diffi cult to control, and the products are diffi cult to purify. A frequently used alternative for carbohydrate ligands are antibodies raised against DC receptors (i.e., integrin CD11c/CD18 [ 55 ] , Fc receptors [ 67 ] , DEC-205 [ 7 ] , DC-SIGN [ 68 ] and MR [ 69 ] ), but these, even when humanized, may still elicit adverse immune responses that decrease the effi ciency of treatment and induce auto-immune side-effects [ 70 ] .

The use of peptide ligands remains, nonetheless, a promising method to target DC receptors. Such ligands are easily synthesized, do not possess immunogenicity, and show high selectivity to DCs (Table 10 .1 ). Accordingly, peptide ligands that could serve as DC-targeting moieties have been sought.

A DC3-nona-arginine fusion peptide (DC3-9dR) that binds nucleic acids by electrostatic interactions was previously exploited for the specifi c delivery of siRNA to DCs in vitro and in vivo [ 78 ] and was also used to silence immunosuppressive molecules in DCs so as to induce strong human T-cell immune responses [ 79 ] . The DC3 targeting peptide (Table 10 .1 ) was recently studied by our group to mediate the specifi c delivery of PEGylated-PEI/DNA polyplexes into DCs [ 80 ] . Polyplexes show signifi cant transfection effi ciency in DCs when decorated with DC3 peptide but not in endothelial cells. The transfection effi ciency observed was higher than that of PEI/DNA, signifying the potential use of DC3-bearing polyplexes for immunotherapy via DCs .

Polyamidoamine ( PAMAM ) dendrimers are hyper-branched, symmetrical, fl exible and monodisperse polymeric molecules (Fig. 10.3d ) . Szoka et al. fi rstly investigated PAMAM cascade polymers as non-viral gene delivery vectors [ 81 ] . These polymers have an ammonia initiator core and amido-amine repeat units of different A. David and A. Golani-Armon generations (G2-G10). Their stepwise synthesis results in products that are uniform in terms of structure and size [ 82 ] and bear a well-defi ned number of primary and tertiary amines. The surface primary amines are protonated, resulting in an extremely positively charged surface that enables DNA binding in stable complexes, and interaction with the cell membrane. The tertiary amines in the interior of the polyplexes are protonable, endowing the dendrimers with pH buffering capacity that enables endosomal escape of the polyplexes following cellular uptake [ 83 ] . All these properties, in addition to their non-immunogenic nature, make PAMAM dendrimers an alternative to the highly effi cient and highly immunogenic viral vectors. However, a major drawback of PAMAM dendrimers is that low generation dendrimers (G5 or lower) show poor transfection effi ciencies, while dendrimers of high generation (G6 and higher) are effi cient transfection agents, yet possess serious cytotoxicity [ 82 ] . In addition, synthesis of high generation dendrimers is a high cost, labor-consuming process that last several days, making them less likely to be designed for large scale production [ 83 ] . Mannosylated G4-PAMAM dendrimers conjugated to OVA specifi cally targeted DCs and induced cross-presentation in vivo [ 84 ] . Moreover, pre-immunization with mannosylated PAMAM-OVA leads to delayed onset of B16-OVA melanoma development, slower kinetics of tumor growth and increased survival of OVAimmunized mice. G4-PAMAM dendrimers bearing DC-SIGN ligand in multivalent presentation also achieved effi cient DC targeting properties, however, did not affect DCs maturation [ 20 ] . It has been postulated that mannosylated dendrimers, as opposed to DC-SIGN-modifi ed dendrimers, may trigger not only DC-SIGN, but also other mannose-specifi c CLRs that contributes to DC maturation and activation. G5-PAMAM dendrimers conjugated to MHC class II-targeting peptide (PADRE ,  Table 10 .1 ) and surface-loaded DNA have been shown to effectively transfect murine and human APCs in vitro [ 85 ] . When applied subcutaneously, this conjugate preferentially transfected DCs in draining lymph nodes, promoted generation of high affi nity T-cells, and elicited rejection of established B16 tumors.

Chitosan is a linear polysaccharide (Fig. 10 .3e ) obtained by deacetylation of its parent polymer chitin, a compound that is widely distributed in nature. Chitosan is composed of randomly distributed ß-(1-4)-linked D -glucosamines (deacetylated units) and N-acetyl-D -glucosamines (acetylated units) and has an apparent pKa value of 6.5 [ 86 ] . Chitosan is a biodegradable and biocompatible polymer, with low or no immunogenicity and antibacterial activity [ 86 , 87 ] . It was previously shown to be non-toxic in both test animals [ 88 ] and humans [ 89 ] . Due to these properties, chitosan is attractive for drug and gene delivery. The high density of positive charges along the polymeric chain contributes to the condensation of the negatively charged DNA, and more frequently, siRNA molecules, into compact structures, thus protecting them from degradation by blood nucleases, promoting their cellular uptake [ 90 ] . Chitosan also possess adhesive properties by interacting with glycoproteins in the mucus [ 91 ] , and is thus used in muco-adhesive drug delivery systems as an adsorption enhancer [ 92 ] . DNA plasmid incorporated into chitosan nanoparticles was able to induce DCs maturation and increase IFN-γ secretion from T cells after pulmonary mucosal immunization [ 93 ] . However, unless modifi ed, chitosan has low solubility at physiological pH, which signifi cantly limits its use in many applications [ 87 , 92 ] . Chemical modifi cations of chitosan have been performed to improve its solubility at physiological pH, including PEGylation or quaternization of the amine groups of chitosan. However, the transfection efficiency of chitosan-based derivatives reported so far is generally not superior to that of PEI [ 94 ] .

Various DC-targeted chitosan-based DNA delivery formulations has been designed to overcome the major obstacles facing the clinical development of chitosan, namely, the lack of cell-specifi city and low transfection effi ciency. Mannosylated chitosan/DNA complexes were more effi cient in transfecting DCs, as compared to water-soluble chitosan/DNA, and induced better INF-γ production from DCs [ 95 ] . Mannosylated-chitosan-entrapping PEI/HBV-DNA complexes induced signifi cantly enhanced serum antibody production and CTL levels after intramuscular immunization [ 96 ] . Biotinylated chitosan nanoparticles were modifi ed with bifunctional fusion protein (bfFp) vectors for achieving DC-selective targeting. bfFp is a recombinant fusion protein consisting of truncated core-streptavidin fused to an anti-DEC-205 single chain antibody (scFv). Intranasal administration of plasmid DNA-loaded bfFp/chitosan nanoparticles, along with anti-CD40 DC maturation stimuli, enhanced the amount of mucosal IgAs, as well as systemic IgGs, against nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) [ 97 ] . Finally, to improve low transfection effi ciency, chitosan-linked-PEI/DNA complexes have been designed, and showed high transfection effi ciency and low cytotoxicity towards DCs [ 98 ] . Vaccination with DCs transfected with chitosan-linked-PEI/DNA encoding gp100 (melanoma-associated antigen) slightly improved resistance to the B16BL6 melanoma challenge .

Polymeric particulates have been shown to be effi cient in delivering plasmid DNA into APCs (reviewed in [ 99 -101 ] ). A key advantage of particulate vectors relative to other non-viral gene delivery systems is their superior in vivo stability. The principal types of polymers studied in this context include those made of poly(lactide) (PLA; reviewed in [ 99 ] ), poly(lactide-co-glycolide) (PLGA; reviewed in [ 99 , 100 , 102 ] ), polyorthoesters [ 103 ] , polystyrene (PS) [ 104 , 105 ] and poly(ε-caprolactone) [ 106 ] . Of these, PLGA has been studied most extensively in terms of its capacity to stimulate APCs.

Poly(lactide-co-glycolide) (Fig. 10.3b ) is an FDA-and European Medicine Agencyapproved polymer for drug delivery systems for parenteral administration [ 107 ] . This biocompatible and biodegradable polymer slowly degrades in vivo by hydrolysis, and its byproducts (lactic and glycolic acid) are easily metabolized and excreted. DCs and macrophages appear to have high affi nity for PLGA particles, as a high level of internalization of antigen-loaded particles has been demonstrated in both in vitro and in vivo settings [ 108 , 109 ] . PLGA microspheres carrying protein antigens or antigen-encoding plasmid DNA are capable of eliciting potent antigenspecifi c immune responses [ 100 , 110 , 111 ] . PLGA also affected expression maturation markers and cytokine production in DCs [ 112 ] . This capacity appears to be driven by unique physical features of PLGA particles (i.e., surface charge, shape and the rate of polymer degradation [ 100 ] ). The mechanisms leading to maturation induction in DCs, however, remain unclear. With respect to their stability in PLGA particles, antigen-encoding plasmid DNA offers advantages over protein-based immuno-modulators, which can lose their biological activity in response to small changes in their tertiary and quaternary structures during formulation.

DNA delivery into DCs can be improved by using ligand-decorated PLGA particles. Mannose-grafted PLGA nanoparticles lead to a signifi cant enhancement of OVA accumulation in the infl amed colon compared to the healthy one, underlining the benefi t of active targeting of macrophages and dendritic cells in diseased tissues [ 113 ] . Mannan-decorated OVA-loaded PLGA nanoparticles simultaneously enhanced antigen-specifi c CD4+ and CD8+ T-cell responses in vaccinated mice [ 114 ] . Modifi cation of PLGA micro-particles with P-D2 peptide (Table 10 .1 ) signifi cantly improved DC antigen presentation in vitro, and increased the rate and extent of microsphere translocation by DCs and macrophages in vivo [ 110 ] .

PLGA has been also utilized in combination with other cationic polymers for gene expression in DCs, with [ 115 ] or without targeting ligands [ 116 , 117 ] . PLGA scaffolds encapsulating PEI/DNA and granulocyte-macrophage colony-stimulating factor (GM-CSF) led to a signifi cant increase in gene expression, and high levels of expression that persisted for a period of time [ 116 ] . Yet, since PLGA nanoparticles are hydrophobic in nature, they tend to form aggregates that reduce the effi ciency of the system [ 108 ] . In addition, PLGA particles are opsonized by the immune system components, and degraded before reaching their destination. Another drawback of PLGA for delivery of genetic vaccines stems from their sustained release property [ 102 ] . PLGA particles release their cargo very slowly, over days or even weeks, but in vivo they may be exocytosed from the cell or degraded in the lysosome over much shorter periods of time, before suffi cient amounts of cargo are released [ 35 ] . Moreover, most DCs die within 7 days after activation and migration to draining lymph nodes [ 118 ] , hence even fast degrading PLGA systems, which fully release the encapsulated DNA within few weeks, cannot meet with the rapid release kinetic criteria and thus fail to induce high levels of target gene expression [ 108 ] . Finally, hydrolysis of PLGA leads to low pH within the particle and thus to DNA degradation [ 102 ] . For these reasons, despite the promising results obtained with animal models [ 114 ] and clinical trials [ 119 , 120 ] , PLGA has been of limited use in this sense [ 121 ] .

The delivery of antigens into DCs carries tremendous potential for immune modulation, and specifi cally, for cancer immunotherapy manipulations. Various polymeric nanomedicines for DNA delivery, ranging from linear homopolymers to block copolymers, branched polymers, as well as combinations of different types of polymers, selected to reduce cytotoxicity and facilitate endocytosis of particles into DCs, are now been routinely examined for their ability to modulate DCs and macrophages. Ultimately, DNA delivery systems that can also induce DC maturation and activation would be advantageous. The main challenges for successful in vivo gene transfer into DCs are the cytotoxicity associated with many cationic polymer gene carriers, the lack of cell specifi city and relatively low transfection effi ciency, when compared to viral vectors. Together with the fact that DCs compose only 1-3 % of cells in peripheral tissues, such gene systems will have limited success unless targeted. Given the number of targeting molecules, immune-modulatory agents, chemokines, growth factors and antigens that can be considered for DC-specifi c delivery, large numbers of potentially useful formulations for DC manipulation are available. Targeting DCs in vivo with tumor antigen-encoding plasmid DNA can elicit effective and long-lasting tumor antigen-specifi c immunity, with minimal inconvenience to the patient. Furthermore, DNA delivery formulations that are stable for extended periods of time and which can enhance antigen presentation on both MHC-I and MHC-II molecules are preferable. With respect to clinical translation, effi cacious non-viral gene delivery into DCs will depend on the combination of intelligent material design, the appropriate tumor specifi c antigen-encoding DNA and immuno-stimulatory molecules to promote DC maturation and activation.

Cancer statistics

Nanoparticle therapeutics: an emerging treatment modality for cancer

Gene therapy for cancer treatment: past, present and future

Cancer immunotherapy via dendritic cells

Type I interferon is selectively required by dendritic cells for immune rejection of tumors

Host type I IFN signals are required for antitumor CD8+ T cell responses through CD8{alpha}+ dendritic cells

In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination

Gene carriers and transfection systems used in the recombination of dendritic cells for effective cancer immunotherapy

Targeting tumor antigens to dendritic cells using particulate carriers

Engineering dendritic cells to enhance cancer immunotherapy

Combining immunotherapy and targeted therapies in cancer treatment

Dendritic cell based PSMA immunotherapy for prostate cancer using a CD40-targeted adenovirus vector

Immunobiology of dendritic cells

Taking dendritic cells into medicine

Presentation of tumour antigens by dendritic cells and challenges faced

Intracellular events regulating cross-presentation

Spinning molecular immunology into successful immunotherapy

Towards a systems understanding of MHC class I and MHC class II antigen presentation

Dendritic cell-based cancer gene therapy

Multivalent glycopeptide dendrimers for the targeted delivery of antigens to dendritic cells

Into the intracellular logistics of cross-presentation

Cross-priming in health and disease

A modular and combinatorial view of the antigen crosspresentation pathway in dendritic cells

Regulation of antigen transport into the cytosol for crosspresentation by ubiquitination of the mannose receptor

Cellular vaccine approaches

Whole tumor antigen vaccines

Improvement of different vaccine delivery systems for cancer therapy

Third generation dendritic cell vaccines for tumor immunotherapy

Dendritic-cell immunotherapy: from ex vivo loading to in vivo targeting

Design and development of polymers for gene delivery

RNA-based vaccines

Mesenchymal stem cells: a promising targeteddelivery vehicle in cancer gene therapy

Cationic polymer based gene delivery systems

Biodegradable nanoparticles of mPEG-PLGA-PLL triblock copolymers as novel non-viral vectors for improving siRNA delivery and gene silencing

Polymers for intracellular delivery of nucleic acids

Synthesis of biodegradable multi-block copolymers of poly( L -lysine) and poly(ethylene glycol) as a non-viral gene carrier

Novel vectors for gene delivery formed by self-assembly of DNA with poly( L -lysine) grafted with hydrophilic polymers

Stabilization of poly-L -lysine/ DNA polyplexes for in vivo gene delivery to the liver

Steric stabilization of poly-L -lysine/DNA complexes by the covalent attachment of semitelechelic poly

Polymer-based gene delivery with low cytotoxicity by a unique balance of side-chain termini

Role of DNA topology in uptake of polyplex molecules by dendritic cells

Poly-L -lysine-coated nanoparticles: a potent delivery system to enhance DNA vaccine effi cacy

Mannose-based molecular patterns on stealth microspheres for receptor-specifi c targeting of human antigen-presenting cells

Revisit complexation between DNA and polyethylenimine-effect of length of free polycationic chains on gene transfection

The proton sponge: a trick to enter cells the viruses did not exploit

Polycation gene delivery systems: escape from endosomes to cytosol

Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo

Polyethylenimine-mediated gene delivery to the lung and therapeutic applications

Biodegradable poly(ethylenimine) for plasmid DNA delivery

Design of magnetic polyplexes taken up effi ciently by dendritic cell for enhanced DNA vaccine delivery

Polyethylenimine-graft-poly(ethylene glycol) copolymers: infl uence of copolymer block structure on DNA complexation and biological activities as gene delivery system

The decrease of PAMAM dendrimerinduced cytotoxicity by PEGylation via attenuation of oxidative stress

PEGylation signifi cantly affects cellular uptake and intracellular traffi cking of non-viral gene delivery particles

Recent developments in cancer vaccines

Rapid monoclonal antibody generation via dendritic cell targeting in vivo

Delivery of antigen to CD40 induces protective immune responses against tumors

Targeting antigens to dendritic cells in vivo

Distinct pathways of antigen uptake and intracellular routing in CD4 and CD8 T cell activation

The mannose receptor and other macrophage lectins

Mannose polyethylenimine conjugates for targeted DNA delivery into dendritic cells

Mannosylated biodegradable polyethyleneimine for targeted DNA delivery to dendritic cells

Enhanced immune response against HIV-1 induced by a heterologous DNA prime-adenovirus boost vaccination using mannosylated polyethyleneimine as DNA vaccine adjuvant

Biological roles of oligosaccharides: all of the theories are correct

Macrophage receptors and immune recognition

Mannosylated polyion complexes for in vivo gene delivery into CD11c(+) dendritic cells

Peptide ligands for a sugar-binding protein isolated from a random peptide library

Antigen-antibody immune complexes empower dendritic cells to effi ciently prime specifi c CD8+ CTL responses in vivo

Effective induction of naive and recall T-cell responses by targeting antigen to human dendritic cells via a humanized anti-DC-SIGN antibody

Antigenic targeting of the human mannose receptor induces tumor immunity

Strengths and weaknesses of immunotherapy for advanced non-small-cell lung cancer: a meta-analysis of 12 randomized controlled trials

Interaction of ICAM-1 with beta 2-integrin CD11c/CD18: characterization of a peptide ligand that mimics a putative binding site on domain D4 of ICAM-1

Development of a dendritic cell-targeting lipopeptide as an immunoadjuvant that inhibits tumor growth without inducing local infl ammation

Peptides identifi ed through phage display direct immunogenic antigen to dendritic cells

Red-cell ICAM-4 is a ligand for the monocyte/macrophage integrin CD11c/CD18: characterization of the binding sites on ICAM-4

Targeted delivery of vaccine to dendritic cells by chitosan nanoparticles conjugated with a targeting peptide ligand selected by phage display technique

A novel peptide carrier for effi cient targeting of antigens and nucleic acids to dendritic cells

Prototype Alzheimer's disease vaccine using the immunodominant B cell epitope from beta-amyloid and promiscuous T cell epitope pan HLA DR-binding peptide

Enhanced induction of HIV-specifi c cytotoxic T lymphocytes by dendritic cell-targeted delivery of SOCS-1 siRNA

Targeted delivery of small interfering RNA to human dendritic cells to suppress dengue virus infection and associated proinfl ammatory cytokine production

DC3-decorated polyplexes for targeted gene delivery into dendritic cells

Polyamidoamine cascade polymers mediate effi cient transfection of cells in culture

Cellsurface glycosaminoglycans inhibit intranuclear uptake but promote post-nuclear processes of polyamidoamine dendrimer-pDNA transfection

Disulfi de cross-linked low generation dendrimers with high gene transfection effi cacy, low cytotoxicity, and low cost

Delivery of antigen using a novel mannosylated dendrimer potentiates immunogenicity in vitro and in vivo

Peptide-conjugated PAMAM dendrimer as a universal DNA vaccine platform to target antigen-presenting cells

Chitosan-a versatile semi-synthetic polymer in biomedical applications

Biocompatibility, cellular uptake and biodistribution of the polymeric amphiphilic nanoparticles as oral drug carriers

Preparation of chitosan nanoparticles using methacrylic acid

Chitosan as a nasal delivery system: the effect of chitosan solutions on in vitro and in vivo mucociliary transport rates in human turbinates and volunteers

Chitosanhyaluronic acid nanoparticles for gene silencing: the role of hyaluronic acid on the nanoparticles' formation and activity

Preparation and effi cacy of a live newcastle disease virus vaccine encapsulated in chitosan nanoparticles

Tetraphenylporphyrin tethered chitosan based carriers for photochemical transfection

Pulmonary delivery of chitosan-DNA nanoparticles enhances the immunogenicity of a DNA vaccine encoding HLA-A*0201-restricted T-cell epitopes of Mycobacterium tuberculosis

Chitosan and its use as a pharmaceutical excipient

Receptor-mediated gene delivery into antigen presenting cells using mannosylated chitosan/DNA nanoparticles

Controlled release of PEI/DNA complexes from mannose-bearing chitosan microspheres as a potent delivery system to enhance immune response to HBV DNA vaccine

Dendritic cell targeted chitosan nanoparticles for nasal DNA immunization against SARS CoV nucleocapsid protein

Gene-carried chitosan-linked polyethylenimine induced high gene transfection effi ciency on dendritic cells

Microparticles for the delivery of DNA vaccines

Activation of antigen-presenting cells by DNA delivery vectors

DNA-loaded biodegradable microparticles as vaccine delivery systems and their interaction with dendritic cells

Microencapsulation of DNA using poly( DL -lactide-co-glycolide): stability issues and release characteristics

Enhancement of poly(orthoester) microspheres for DNA vaccine delivery by blending with poly(ethylenimine)

Gene delivery to dendritic cells facilitated by a tumor necrosis factor alpha-competing peptide

Polymeric microspheres as stabilizing anchors for oligonucleotide delivery to dendritic cells

Development of functionalized nanoparticles for vaccine delivery to dendritic cells: a mechanistic approach

PLGA-based nanoparticles: an overview of biomedical applications

Hydrophilic poly( DL -lactide-co-glycolide) microspheres for the delivery of DNA to human-derived macrophages and dendritic cells

Uptake of poly( D , L -lactic-coglycolic acid) microspheres by antigen-presenting cells in vivo

Microparticle surface modifi cations targeting dendritic cells for non-activating applications

Poly-(lacticco-glycolic-acid)-based particulate vaccines: particle uptake by dendritic cells is a key parameter for immune activation

Composition and surface charge of DNAloaded microparticles determine maturation and cytokine secretion in human dendritic cells

Drug delivery to infl amed colon by nanoparticles: comparison of different strategies

Activation of antigen-specifi c T cell-responses by mannan-decorated PLGA nanoparticles

Uptake characteristics of NGR-coupled stealth PEI/pDNA nanoparticles loaded with PLGA-PEG-PLGA tri-block copolymer for targeted delivery to human monocyte-derived dendritic cells

Sustained GM-CSF and PEI condensed pDNA presentation increases the level and duration of gene expression in dendritic cells

Enhancement of gene transfection into human dendritic cells using cationic PLGA nanospheres with a synthesized nuclear localization signal

Genetic tagging shows increased frequency and longevity of antigen-presenting, skin-derived dendritic cells in vivo

Experimental studies and preliminary clinical trial of vinorelbine-loaded polymeric bioresorbable implants for the local treatment of solid tumors

An eightmonth clinical study of LA-2575 30.0 mg: a new 4-month, subcutaneous delivery system for leuprolide acetate in the treatment of prostate cancer

Polymers for gene delivery across length scales

Acknowledgments This work was supported by a grant from the Israeli National Nanotechnology Initiative (INNI), Focal Technology Area (FTA) program, Nanomedicine for Personalized Theranostics.