Polymers for DNA Vaccine Delivery - American Chemical Society

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Polymers for DNA Vaccine Delivery Mingming Zhang, Yanhang Hong, Wenjuan Chen, and Chun Wang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00418 • Publication Date (Web): 30 Sep 2016 Downloaded from http://pubs.acs.org on October 4, 2016

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ACS Biomaterials Science & Engineering

Polymers for DNA Vaccine Delivery Mingming Zhang1, Yanhang Hong1, Wenjuan Chen1, Chun Wang2,* 1

Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, 236 Baidi Road, Nankai District, Tianjin, 300192, China 2

Department of Biomedical Engineering, University of Minnesota, 7-105 Hasselmo Hall, 312 Church Street S. E., Minneapolis, Minnesota, 55455, USA * Corresponding author: [email protected]

Abstract DNA vaccine is a third generation vaccine type based on concepts and techniques of molecular biology. It can closely mimic live infections and induce both antibody and cell mediated immune responses, and thus, has much potential for treating chronic viral infection and cancer. How to transport DNA vaccine to the right target cells in lymphoid tissues and organs? How to achieve high and robust gene transfection efficiency while simultaneously induce DC maturation and antigen presentation? These questions pose significant challenges and addressing them may require serious efforts in developing better biomaterials as carriers. This review is dedicated to the discussion of polymers as nano-scale carriers for DNA vaccine. We summarize recent advances in polymer science and engineering to overcome multi-level hurdles for DNA vaccine delivery, and conclude with thoughts on challenges and opportunities that may shape the future of polymers in DNA vaccination.

Keywords Polymers; DNA vaccine; Gene delivery; Immunotherapy

ACS Paragon Plus Environment

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1. Introduction Ever since Edward Jenner demonstrated in 1798 that inoculation with pus from cowpox lesions conferred protection against smallpox infection, the development and application of vaccines have seen tremendous progress. Traditional subunit protein based vaccines have been shown to elicit robust immunity against many microbes and prevent human infections1, but they have limited impact on diseases such as cancer and chronic viral infections such as HIV2. A key reason is that subunit vaccines are only adequate in eliciting humoral immune response3, whereas controlling cancer and chronic viral infections relies more on T cell mediated immune response4. Live attenuated viruses and bacteria are able to produce cell-mediated immune responses, yet they may be dangerous to administer into patients with compromised immune systems5-6. DNA vaccine is a third generation vaccine type developed in the early 1990s based on concepts and techniques of molecular biology. It works by incorporating the gene of a protein antigen in a recombinant eukaryotic expression vector (such as a plasmid) and introducing it into the body to produce the exogenous antigen, which then elicits antigen-specific immune responses that control diseases7. Compared with traditional vaccines, DNA vaccine can closely mimic live infections and induce both antibody and cell mediated immune responses, and thus, has much potential for treating chronic viral infection and cancer. In addition, DNA vaccine has multiple advantages, for examples, stable gene expression in transfected host cells providing sufficient supply of antigen, easy design of genetic sequences using recombinant techniques, possibility of expressing specific antigen and immunoregulatory proteins, scalable manufacturing, easy


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to store and transport without cold-chain, and demonstrated safety in animal and human clinical experiments6, 8-10. DNA vaccine often uses plasmid as vector that contains one or more exogenous DNA antigens and regulatory components to drive expression in a eukaryotic host. The typical process of DNA vaccination begins by introducing recombinant DNA vaccine into the body through intradermal, subcutaneous, or intramuscular injection. Professional antigen-presenting cells (APCs) such as dendritic cells (DCs) and macrophages can be transfected directly to express the encoded protein antigen endogenously. APCs can also acquire and process exogenous antigens shed by other transfected cells (bystanders). After phenotypic maturation, the APCs migrate to the draining lymph nodes and the spleen, where epitopes are presented by major histocompatibility complex (MHC) class I/II molecules, and along with the necessary co-stimulatory signals and cytokine environment, enable the activation of naïve CD8 (to become cytotoxic T lymphocytes, or CTLs) and CD4 helper T cells (to stimulate antibody production by B cells)11. The first clinical investigation on DNA vaccine was for treating HIV infection6, 12. Since then, DNA vaccine has been evaluated extensively for a wide range of diseases including various cancers (breast8, 13, prostate14-15, cervical16-18, colorectal19-20, hepatocellular21-23, melanoma24-26) and infections (influenza27-29, malaria30, hepatitis B, human papillomavirus (HPV31-34, HIV6, 35-36). Despite potential technological and commercial value, there has not been any FDA approved DNA vaccine for human use in the US. Poor immunogenicity of DNA vaccine is one of the most frequently cited problems. Recent years have seen development of several approaches to enhance the immunogenicity of naked DNA vaccine. These include using more potent promoter,


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codon optimization, addition of adjuvants, electroporation, intradermal delivery, and various prime-boost schemes14. Despite these efforts, naked DNA as vaccine has serious limitations. The in vivo half-life of naked DNA is minutes due to degradation by nucleases and removal by the reticular endothelial system (RES)16. For protein expression, naked DNA has to overcome multiple cellular and subcellular barriers including the plasma membrane17. Naked DNA also lacks the ability of facilitating specific uptake by DCs, the most important APCs37. How to transport DNA vaccine to the right target cells in lymphoid tissues and organs, achieve high and robust gene transfection efficiency while simultaneously induce DC maturation and antigen presentation, present significant challenges and require serious efforts in developing better biomaterials as carriers. Polymers are extensively investigated in drug and gene delivery. In comparison with small molecule, polymers have certain advantages and unique features as carriers for DNA vaccine. First, long chains of macromolecules afford multivalency and cooperativity in binding to DNA, which effectively collapses into compact, stable nanoparticles resistant to degradation in biological medium. Second, polymers are easily rendered multifunctional through chemical and biological derivatization that provide reactive groups and ligands for mediating cellular targeting and transport. Third, polymer carriers are highly tunable for incorporating dynamic structural transitions (such as degradation and disassembly) in response to biological stimuli (such as pH and redox state), in order to modulate transport and environment-specific release of DNA. Finally, in some sense, polymers are the ultimate bio-inspired gene carriers: they resemble viral


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capsid proteins and nuclear histones in eukaryotic cells, but with structural diversity far exceeding that of natural proteins. This review is dedicated to the discussion of polymers as nano-scale carriers for DNA vaccine. After an analysis of multi-level hurdles for DNA vaccine delivery, we summarize recent advances in polymer science and engineering to overcome these hurdles. We conclude with thoughts on challenges and opportunities that may shape the future of polymers in DNA vaccination. 2. Overcoming hurdles Multiple hurdles must be overcome for DNA vaccine to succeed in generating robust, antigen-specific immune responses in vivo. After DNA is condensed or packaged by polymer and introduced into the body, it must overcome those barriers generic to all gene delivery applications and be able to transfect DCs. Unfortunately, DCs are particularly resistant to the entry of exogenous DNA into the cell nuclei and thus, are very difficult to be transfected by DNA encoding foreign antigens. For this reason, current research on DNA vaccine delivery has focused on overcoming barriers to intracellular delivery of DNA and achieving high transfection efficiency in APCs. In addition, DCs need to process and present the DNA-encoded protein antigen, express high levels of co-stimulatory molecules, and produce immunostimulatory cytokines, so as to prime naïve T and B cells. Here we describe these multitudes of barriers to DNA vaccine delivery and outline general strategies to overcome them. 2.1 Gene transfection 2.1.1 Extracellular delivery


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The lymphatic system, including lymph nodes (LNs), spleen, and mucosaassociated lymphoid tissue (MALT), where T and B lymphocytes reside, is the main locale for immune activation and response toward foreign antigen. Extracellular hurdles that a DNA vaccine carrier may face depend on which type of lymphatic organ and tissue it is designed to target, and this will dictate the choice of route of vaccination. An overview of these target tissues and organs, the hurdles they pose, and potential polymerbased delivery approaches to overcome them, is given in Figure 1. 2.1.1.1 Lymph nodes (LNs) LNs are specialized peripheral lymphatic organs distributed widely throughout the body. Lymphocytes are preserved in distinct layers inside the LNs, where they are stimulated by antigens and can generate antigen-specific immune responses. LNs are the final destination of DNA vaccine administered to the skin and muscle through microneedles, intradermal injection, subcutaneous injection, intramuscular injection, electroporation, gene gun, etc. During the course of vaccination, the majority of the DNA vaccine carriers may be taken up by cells at the site of administration (such as dermal fibroblasts and myocytes), which could lead to expression of the DNA-encoded antigen. If the expressed antigen is released from these cells, it may be captured by DCs at the injection site and reach the draining LNs. Alternatively, DNA vaccine carriers may transfect directly Langerhans cells in the epidermis, or drain directly into the LNs and transfect resident DCs there.


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Figure 1. Extracellular hurdles of DNA vaccine delivery and polymer-based strategies to overcome them. Abbreviations: NALT (nasal associated lymphoid tissue); BALT (bronchus associated lymphoid tissue); GALT (gut associated lymphoid tissue); i.n. (intranasal administration); p.o. (oral administration); i.m. (intramuscular administration); i.v. (intravenous administration); i.d. (intradermal administration); s.c. (subcutaneous administration); APCs (antigen-presenting cells); RES: reticulo-endothelial system.


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Engineering the physico-chemical properties of DNA vaccine carriers and selecting the appropriate route of vaccination may enhance the capture of DNA vaccine by cells and facilitate lymphatic drainage and immune activation. Particle size, surface charge and colloidal stability of the DNA vaccine carriers can influence the transport of DNA and the ultimate type of immune responses. In general, particles of 9-10 nm in diameter will rapidly drain to the LNs through lymph ducts, whereas particles smaller than 6 nm will enter directly into the blood circulation. Particles larger than 500 nm may be trapped in the extracellular matrix of the injection site, captured through phagocytosis or macropinocytosis by peripheral DCs or monocytes from the blood, which bias toward eliciting humoral immunity. On the other hand, particles of 20-200 nm in size are expected to drain into the LNs and to be captured by resident DCs and macrophages, which bias toward eliciting Th1 type immune response38. DNA vaccine carriers with neutral or negatively charged surface are easier to drain into the LNs, whereas positively charged carriers may interact electrostatically with negatively charged extracellular matrix that impedes lymphatic drainage39. Moreover, modification of carriers with certain moieties may actively engage APCs and enhance lymphatic retention. Studies have indicated that polymers conjugated with specific ligands for APC surface receptors, such as Fc receptors, CD40, C-type lectins (DC-SIGN, DEC-205, mannose receptor, etc), can help increase lymphatic drainage40-42. Currently the most widely investigated strategy to improve polymer/DNA vaccine delivery to APCs is the use of mannosylated polymers, which has been shown to promote the generation of immune responses43-45. 2.1.1.2 Spleen


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The spleen is the body’s largest lymphatic organ and is structurally separate from the lymph ducts. While the LNs are responsible for immune responses toward antigens that drain through the lymph, it is the spleen that responds to antigens coming through the blood circulation. Therefore, the spleen is the major destination of DNA vaccine carriers administered via intravenous injection. The carriers are expected to travel through the blood circulation to reach the spleen and transfect DCs or macrophages in the spleen. During this process, the carriers should avoid interacting with blood components (such as plasma proteins and red blood cells), because it might cause aggregation or dissociation of polyplexes, depending on particle size and surface charge. The carriers also need to avoid opsonization by IgG, complement proteins as well as proteins of the blood coagulation cascade. Otherwise, it could lead to rapid clearance by the mononuclear phagocytotic system (MPS) and even activation of the innate immune responses, resulting in acute inflammation and causing severe acute renal and hepatic toxicity46. However, it should be noted that complement activation by polymer carriers with certain surface chemistry could be exploited to enhance the immune responses against antigens47. Nonetheless, in addition to particle size and surface charge, maintaining colloidal stability of DNA vaccine carriers is key to retention in the spleen and cellular uptake. For example, covalent conjugation with biocompatible hydrophilic polymer (such as PEG)44,48-50 and chemical crosslinking51-52 are effective approaches to stabilize polymer carriers and prolong their circulation during systemic administration. Besides spleen targeting, it may be desirable to target DNA vaccines directly to numerous types of circulating lymphocytes by incorporating cell-specific ligands to the carriers. 2.1.1.3 Mucosa-associated lymphoid tissue (MALT)


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The MALT is a diffuse system of small clusters of lymphoid tissue found in sites in the epithelium and sub-mucosa (lamina propria) of the gastrointestinal tract, respiratory tract, salivary glands, genitourinary tract, and includes those containing germinal centers such as the thyroid, Peyer’s patches of the small intestine, the appendix, etc. Thus, the MALT can be subdivided into GALT (gut-associated lymphoid tissue), BALT (bronchus-associated lymphoid tissue), NALT (nasal-associated lymphoid tissue), LALT (larynx-associated lymphoid tissue), and so on. The MALT contains a plethora of immune cells and is the main location where mucosal immunity is induced. For DNA vaccine delivered through intranasal, oral, buccal/sublingual, pulmonary, and vaginal routes, the MALT is the destination. To illustrate the hurdles to DNA vaccine delivery posed by the MALT, consider the intranasal route. DNA vaccine carriers must be able to penetrate mucus and reach the NALT through epithelial cells or M cells (microfold epithelial cells). M cells are single layer of epithelial cells above the NALT and are responsible for sampling antigens in the lumen for presentation to B and T cells in the mucosal tissue53-55. M cells can translocate particles as large as several microns, and thus, the size of the DNA vaccine carriers (too large or too small) may not be a significant constraint for cellular uptake. However, quick turnover of mucus in the nasal cavity may remove the vaccine carriers. Several approaches have been proposed to improve intranasal vaccine delivery through engineering carrier properties. One, surface modification with PEGylated polymers may enable better diffusion through the nasal mucosa and make them available for acquisition by APCs or epithelial cells48. Two, using mucoadhesive polymers as carriers will increase the retention time on mucosal tissue surface. Third, modification of carrier surface with


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M cell specific ligands was shown to target DNA vaccine to mucosal tissue and respiratory epithelium with improved immune responses56. Furthermore, it may be worthwhile borrowing from certain protein-based vaccine strategies to incorporate heparin-binding hemagglutinin adhesion (HBHA) protein to promote binding between DNA vaccine carriers and epithelial cells57. Similar to intranasal delivery, DNA vaccine delivered orally by swallowing – once reaching the intestinal wall – must overcome tissue barriers to reach the GALT. A difference, however, is that orally delivered DNA vaccine has to survive the hostile environment of the gastrointestinal tract, which includes the extreme acidic pH in the stomach (pH 2), intestinally secreted nucleases for digesting nucleic acid in food, bile salts, and gut microbiota. It is thus imperative to use highly stable polymers to protect DNA vaccine from hydrolytic and enzymatic degradation. Co-delivery of agonists of tolllike receptors 2 and 4 can also enhance the absorption of DNA vaccine carriers by the intestinal mucosa58-59. 2.1.2 Cellular uptake DNA vaccine carriers arriving at the lymphoid tissues of the skin, muscle, LNs, spleen, and MALT, will need to be captured and internalized by immune cells 60. Given that there are already a large number of studies elucidating the uptake mechanism of particles by various cell types61-62, here we only discuss those factors that influence the uptake of DNA vaccine carriers by APCs (Figure 2). APCs capture particulates through receptor-mediated or non-receptor-mediated endocytosis. Receptor-mediated endocytosis can be mediated by clathrin, caveolin, or be independent of clathrin and caveolin63, and is triggered by a variety of molecular entities


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such as serum-transport proteins, transferrin, hormones, growth factors, antigens and viruses64. Targeting receptor-mediated endocytosis of APCs by using carriers bearing APC-specific ligands (such as mannose, anti-DEC205 antibodies, Fc receptor antibodies) has been demonstrated to improve cell specificity and transfection efficiency of DNA vaccine2, 65. Moreover, particle size of carriers also influences the pathway of uptake and can lead to different immune responses63. Studies have revealed that inert particles of 40100 nm in size are prone to be internalized by DED205+ DCs, whereas 1-µm size particles are preferred by F4/80+ macrophages. Particles with size similar to viruses (20200 nm) are particularly attractive to DCs66.


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⑩ T cell activation

① Receptor-mediated endocytosis

② Non receptor-mediated endocytosis

•Co-delivery of DNA with molecular adjuvants

• Conjugation cell-specific targeting moieties

•Particle size & shape

•“Smart” polymers for subcellular targeted delivery of immunomodulatory agents

•Surface charge

①

•Conjugation to cell penetrating peptides

•Co-delivery of gene-based adjuvants

②

•Co-delivery of antiapoptotic agents macropinocytosis Secret antigen

Transfection of bystander cells

phagocytosis

③

③ Endosomal escape

⑥ Transcription

• pH-sensitive “protonsponge” polymers

④

⑦ ⑧

⑤

⑥ Antigen processing & presentation

• pH-sensitive membrane lytic polymers and peptides

CD8+ T cell

④ DNA dissociation

CD4+ T cell

•Promote cross-presentation via bystander cells ⑨

• Backbone or side chain degradable polymers • pH or redox or enzyme sensitive polymers

•Recombinant DNA approaches to promote MHC I ⑦ & II ⑧ pathways

⑤ Nucleus entry

•Co-delivery of antiapoptotic signals

• Conjugation to nuclear localization signals • Manipulation of cell cycle

Plasmid

Intracellular antigen

Proteasome

MHC class I

Endoplasmic Reticulum

Costimulator

Nanoparticle

Extracellular antigen

Peptide

MHC class II

T cell receptor

Costimulator receptor

Figure 2. Cellular and subcellular hurdles to DNA vaccine delivery and polymer-based strategies to overcome them. Non-receptor-mediated endocytosis includes macropinocytois and phagocytosis, and particle size is a significant factor in the pathway of uptake. Carriers with particle size of 0.5-5 µm can be taken up by APCs through both macropinocytosis and phagocytosis, whereas particles larger than 5 µm are mainly taken up through phagocytosis. Both pathways eventually lead to acidic subcellular vesicles and fusion


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with lysosome61 that expose DNA vaccine to damage and destruction. It has been reported that DNA vaccine carriers conjugated to certain cell-penetrating peptides, such as MPG, could enhance cellular uptake, potentially through non-receptor-mediated pathways, and result in better immune responses67. Regardless of whether receptors are involved, surface charge and shape of vaccine particles have much impact on cellular uptake. Interestingly, both cationic and anionic carriers can be taken up by phagocytic cells68. Electrostatic interaction between cationic carriers and glycoproteins in the cell membrane favors cellular uptake69, but could also cause cytotoxicity associated with membrane damage and apoptosis, which leads to inflammation70. How to strike a balance between enhancing cellular uptake and avoiding cytotoxicity is crucial in designing the proper cationic DNA vaccine carriers. Equally important is the shape of carriers. Optimal phagocytosis requires a particle shape with high length-normalized curvature71. Cellular uptake of spherical particles is higher than particles of other shapes, because nonspherical particles with odd geometries do not engage cells that facilitate the formation of actin filaments requisite for phagocytosis72. 2.1.3 Intracellular delivery and antigen expression Polymer/DNA vaccine complexes internalized by APCs must traverse several intracellular barriers to transfect cells that express antigen (Figure 2). First, DNA vaccine carriers must either avoid or escape from the acidic phagosome. Several polymer designs are known to be effective in mediating endosomal escape via different mechanisms. These include PEI and poly(amidoamine)s that may cause leakage in endo-lysosomes due to the “proton sponge” effect73, and membrane-lytic polymers (such as poly(propyl acrylic acid) and fusogenic peptides), whose pH-sensitive conformational changes can


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alter cell membrane structure in ways that leaky holes can form74-76. Second, the release of antigen-encoding plasmid DNA from its carrier must be accomplished along the way of transit to the cell nucleus. Various polymer chemistries have been tested for promoting intracellular release of cargos. These include endosomal pH triggered degradation of polymer backbone or side chain77-84, pH triggered polymer solubilization85, reduction of disulfide linkages86 and enzymatic degradation of polymer carriers87. Finally, the entry into the cell nucleus often relies on cell division to breach the nucleic membrane88. In nondividing cells, plasmid DNA larger than 2000 base-pairs may require coupling to nuclear localization signals (NLS) to be transported into the nucleus. Some studies showed that NLS coupling was helpful in enhancing gene transfection efficiency89-92, but others suggested that NLS didn’t seem to have much positive enhancement93-95. At this point the jury is still out there in terms of the necessity and benefit of the NLS. Nevertheless, nuclear entry is perhaps the most challenging intracellular hurdle for DNA vaccine delivery, because the target cells, primarily immature DCs, may not be actively dividing. Furthermore, as DCs undergo phenotypic maturation, intracellular transport processes of DNA vaccine are surely affected, but how and to what degree, remain unknown. 2.2 Immune activation 2.2.1 Antigen processing and presentation DCs use two main pathways for antigen presentation that are mediated by either MHC I or MHC II (Figure 2). When DCs are transfected directly by DNA vaccine, antigens are produced in the cytoplasm and mainly processed via the MHC I pathway. During antigen synthesis, some of them are labeled by ubiquitin. Ubiquitinylated antigens


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are digested into polypeptides by proteases and translocated to the ER (endoplasmic reticulum) by TAP (Transporter Associate with antigen Processing), where they are further cut into 8-10 residue peptides, loaded onto MHC I and transported to the cell membrane for engaging CD8+ T cells. If DNA vaccine happens to transfect bystander cells (i.e. nonphagocytic somatic cells), expressed antigen may be released and captured by DCs (Figure 2). These DCs may process the acquired antigen either by crosspresentation or by MHC II mediated pathway. Cross-presented antigen will be processed and presented by MHC I on the cell surface as described above. For the MHC II pathway, the antigen is localized in the lysosome and hydrolyzed into fragments of 20-25 residues, and after fusing with MHC II containing vesicles, the peptides are further processed to 12-15 mers, loaded onto MHC II molecules, shipped to the cell surface for stimulating CD4+ T cells. Thus, whether the direct target of DNA vaccine transfection is APCs or bystander cells may influence the outcome of antigen processing and presentation. 2.2.2 T cell activation The display of MHC-epitope complex on the surface of APCs, often called Signal 1, is responsible for antigen recognition by B and T cells. Also required are Signal 2– DC surface bound co-stimulatory molecules (such as CD80, CD86, CD40), and Signal 3 – secreted cytokines that stimulate T cell differentiation and expansion (such as IL-12 and Type I IFN). These signals are provided by phenotypically mature DCs and are crucial in eliciting immune responses (Figure 2). Molecular adjuvants based on pathogenassociated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) are potent stimuli of DC maturation via pattern recognition receptors (PRRs) such as TLRs. It is recognized that in addition to antigen-encoding DNA, one must


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deliver molecular adjuvants, preferably through the same carriers, to DCs. Considering that different TLRs are found in different subcellular locations, proper engineering of the polymer carriers is necessary to deliver DNA to the nucleus and adjuvants to TLRs in other organelles96-97. An alternative method is to deliver a single recombinant DNA that encodes both the antigen and the adjuvant (such as GM-CSF)98-102. Another hurdle to T cell activation is that activated DCs have limited life span and will undergo apoptosis to avoid “over-activating” the immune system. Therefore, combined delivery of DNA antigen with apoptosis inhibitors (such as Bcl-xL and Bcl-2) to prolong the survival of DCs and their engagement with T cells will likely boost the effectiveness of DNA vaccine103. 3. Polymer carriers for DNA vaccine delivery Table 1 is a comprehensive list of natural and synthetic polymers used as DNA vaccine carriers along with their intended disease targets and, if tested in vivo, routes of administration and animal species involved. Major classes of these polymers are discussed in detail as follows. 3.1 Natural polymers 3.1.1 Chitosan Chitosan is a natural linear polysaccharide, obtained through deacetylation of chitin found in exoskeleton of Crustaceans. As a copolymer of glucosamine and acetyl glucosamine, chitosan contains large numbers of amino groups that can interact with DNA electrostatically to form polyplexes. It is a widely investigated polymer carrier for gene delivery104.


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Chitosan is well known for its mucoadhesive property. Positively charged chitosan/DNA complexes can bind to negatively charged sialic acid residues in mucins found at the surface of nasal mucosa and delay the clearance of polyplexes. Chitosan can also open up tight junctions between epithelial cells and increase transport across the epithelium. Therefore, chitosan has long been regarded as an ideal DNA vaccine carrier for intranasal administration. For example, as reported in 2003, L. Illum et al tested chitosan for intranasal DNA vaccination against Respiratory Syncytial Virus (RSV) and demonstrated protective cytotoxic T lymphocyte (CTL) response in mice105. More recently, M. Fan and X. Yang et al combined chitosan/DNA complexes with anionic liposome into hybrid nanoparticles. It was reasoned that DNA binding to these nanoparticles was depended on pH. Stable binding was expected at intranasal pH of 5.56.5, but electrostatic repulsion within the complexes favored DNA release at cytoplasmic pH of 7.2-7.6 after cellular internalization, thus promoting gene transfection efficiency. Intranasal immunization with an anti-caries DNA vaccine formulated with the hybrid nanoparticles led to absorption by the nasal mucosa, the secretion of IgA, and a longlasting (12-week) immune response in vivo106. It should be noted that the molecular weight (MW) of chitosan significantly affects transfection efficiency of DNA vaccine. L. Zong et al compared chitosan with high (173 kDa) and low (5 kDa) molecular weights in delivering DNA vaccine to treat atherosclerosis. They found that the low MW chitosan had higher in vitro transfection efficiency than the high MW polymer. Intranasal vaccination with the low MW chitosan DNA vaccine induced systemic immune responses and delayed sclerotic process through modulating plasma lipoprotein in a high-cholesterol rabbit model107. However, low MW


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chitosan/DNA complexes may be unstable in vivo due to weaker binding between the polymers108. Chitosan has been modified with mannose for targeting mannose receptor of APCs. W. Kong et al prepared microspheres of mannosylated chitosan encapsulating PEI/DNA complexes to enhance the uptake by DCs. As a result, CTL response was achieved at reduced, safer vaccine dose109. L. Zong et al used mannosylated chitosan to carry a DNA vaccine targeting gastrin-release peptide and demonstrated enhanced uptake by macrophages via C-type lectin binding. Compared with unmodified chitosan and naked DNA vaccine, mannosylated chitosan was a more effective carrier in suppressing the growth of prostate tumor in mice110. J. Singh et al synthesized mannosylated phenylalanine grafted chitosan to complex with DNA encoding HBV antigen. Enhanced cellular uptake and transfection were demonstrated in vitro using macrophages (RAW 264.7) and DCs (DC 2.4). Intradermal vaccination of the polyplexes stimulated T cell proliferation and maturation as well as humoral immune response in mice43. In addition to mannose, other chemical modifications have been made to chitosan for APC targeting. For example, S. Heuking et al conjugated a TLR7 agonist to chitosangraft-PEG. In vitro experiments showed that conjugated polyplexes stimulated human THP-1 macrophages to secrete IL-8, which plays a role in leukocyte recruitment and chemotaxis to the mucosal site of infection111. K. Kaur et al evaluated biotinylated chitosan nanoparticles decorated with streptavidin-antibody fusion protein to target DEC205+ DCs. Intranasal vaccination resulted in DC maturation and elevated levels of mucosal IgA and systemic IgG112.


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Coupling chitosan with other polymers through chemical or physical means presents new opportunities. J. Gao et al conjugated chitosan with PEI chemically and showed transfection of DCs and modest suppression of B16 melanoma in animals113. Chitosan nanoparticles coated with alginic acid were prepared by X. Tan et al for oral delivery of DNA vaccine. Alginic acid coating improved stability of polyplexes under gastric pH (1.5). Tumor suppression and CTL response were observed in a mouse breast cancer model114. In summary, much promise is seen with chitosan, in particular for mucosal delivery of DNA vaccine115. Modification of chitosan to reduce solubility at gastric pH may expand the usage of this polymer in oral DNA vaccination116. 3.1.2 Other polysaccharides Besides chitosan, other natural polysaccharides have also found use in DNA vaccination. For example, X. Zeng et al demonstrated that Astragalus polysaccharides (APS), an adjuvant of Hepatitis B virus (HBV) DNA vaccine (pcDS2), could enhance the humoral and cellular immune responses with increased IgG level as well as T cell proliferation. Compared to naked DNA, the addition of APS enhanced IFN-γ expression byCD8+ T cells, and induced CD4+ T cells to produce IL-4, IL-2 and IFN-γ. Moreover, APS also induced robust CTL, stimulated DC maturation, and decreased regulatory T cell level117. C. Letellier et al used agarose hydrogel for encapsulation and sustained release of DNA vaccine against Herpesvirus. Antibody response was demonstrated in cattle after intradermal implantation of the vaccine without the need of using osmotic pump118. 3.2 Synthetic polymers 3.2.1 Polypeptides


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As one of the earliest synthetic cationic gene carriers, poly-L-lysine (PLL) has already been explored for DNA vaccine delivery57,129. More recently, M. Plebanski et al condensed a plasmid encoding chicken egg ovalbumin (OVA) with PLL-coated polystyrene nanoparticles. The obtained complexes could induce high levels of CD8+ T cells and OVA-specific antibodies in C57BL/6 mice as well as significant inhibition of tumor growth after challenge with the OVA expressing EG7 tumor cells119. On the other hand, the anionic poly (γ-glutamic acids) (γ-PGA) was used by H. Sasaki et al to coat the surface of PEI/DNA complexes. The negatively charged γ-PGA could reduce the high liver toxicity of the cationic PEI. After intravenous injection, the γ-PGA-coated complexes showed selectively high transgene expression in the marginal zone of the spleen, where DCs and macrophages are abundant, and delivered a therapeutic DNA vaccine that significantly inhibited the growth and metastasis of B16-F10 melanoma cells120. In the last few years, K. Nakano and K. Kataoka et al introduced a simple but elegant carrier system based on polyplexes of DNA with mixture of a homopolymer of cationic polyaspartamide P[Asp(DET)] and a block copolymer PEG-b-P[Asp(DET)]121122

. The ratio between the homopolymer and block copolymer was optimized for DNA

complexation, stability against nonspecific clearance, and transfection efficiency of cells. The polyplexes containing DNA encoding a tumor antigen SART3, adjuvants CD40L and GM-CSF, was administered i.p. and shown to prolong survival of animals bearing CT26 colon carcinoma through suppression of tumor growth and metastasis121. Subsequently, these researchers compared the performance of the vaccine given through different routes (Figure 3). It was found that s.c. injected polyplexes were localized in the


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inguinal LN and the skin, whereas i.p. injected polyplexes were found in the mesenteric LNs, the liver and spleen, and positive gene transfection was detected from both routes. In contrast, i.v. injected polyplexes and electroporation did not produce detectable gene expression, perhaps due to dilution in the blood and limited area of impact. It was the DNA vaccine injected subcutaneously that achieved the most positive immunological and therapeutic responses as measured by tumor growth suppression, animal survival, activation of CTL and NK cells, and tumor infiltration of DCs and CD8/CD4 T cells122.

A

B

C

D

Figure 3. The transgene expression and anti-tumor efficiency of SART3/CD40L/GMCSF gene-loaded homopolymer P[Asp(DET)]/block copolymer PEG-b-P[Asp(DET)]mixed polyplexes in a mouse tumor model. (A) GM-CSF mRNA expression in the indicated tissues after s.c. and i.p. administrations of the polyplexes. (B) Tumor volumes for four weeks. (C) The number of CD11c+ DCs in groin LN, spleen, and tumor tissues. (D) Infiltration of CD4+ and CD8a+ T cells into tumor tissues after s.c. administration of polyplexes. Reprinted with permission from reference 122. Copyright 2015 Elsevier.


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3.2.2 Degradable polyesters 3.2.2.1 PLGA Perhaps the most popular biodegradable polymer, PLGA is well-known for its safety and track-record in FDA approved product applications123. Earlier studies showed that PLGA microspheres could encapsulate DNA plasmid and target APCs passively to induce CTL-mediated immune response124-125. Such microsphere DNA vaccines could be given orally and parenterally through injection, which induced mucosal and systemic antibody responses and antitumor responses126-130. This delivery system is also effective in antiviral applications such as in the case of delivering prophylactic DNA vaccine against foot-and-mouth disease virus (FMDV)131. PLGA alone does not carry any positive charges. Simple encapsulation of DNA into PLGA may lead to DNA degradation due to acidic polymer degradation products. Transfection efficiency of pure PLGA/DNA microspheres is low132. A popular and successful strategy is to combine PLGA with a cationic molecule, through physical mixing, adsorption, or chemical conjugation, in order to improve DNA loading, stability, and transfection efficiency. Numerous cationic surfactants and polymers have been used in this context, such as dimethyldioctyldecyl ammonium bromide (DDA) through mixing 133

, cetyl trimethylammonium bromide (CTAB) through surface adsorption 134-136, PEI

through blending137-138or chemical conjugation139-140, chitosan through blending141, glycol chitosan by coating of PLGA nanoparticles142, dendrimers through emulsified blending143, and cationic poly(β-aminoesters) (PBAE) through layer-by-layer coating onto PLGA microneedles144. These binary polymer systems have been extensively examined for


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DNA vaccine delivery in vitro as well as in numerous animal disease models with promising results. 3.2.2.2 Other polyesters PBAE and poly(ortho esters) (POE) are two types of acid-labile biodegradable polymers investigated for DNA vaccine delivery. There is much tunability in both types of polymers through adjusting monomer type and ratio145-146, polymerization pathway147, introducing targeting ligand45, control of hydrophilicity and degradation rate, and so on. Similar to PLGA, these polymers can be readily processed into various physical forms including microparticles148, nanoparticles149, and microneedle arrays150. C. Wang et al and S. Little et al were the first to demonstrate that acid-labile POE148 and PBAE/PLGA151 microspheres could be used to carry DNA vaccine to APCs and induce robust immune responses in healthy and tumor-bearing animals. The rationale was based on accelerated degradation of microspheres and release of DNA vaccine in response to the acidic environment of the phagosomes of APCs, potentially synchronizing DNA delivery with the maturation of transfected cells. Subsequently, a variety of modifications to these polymers have been explored, including physical blending with PEI152, layered deposition with PLGA and adjuvant poly(I:C) 150, and changes in composition of polymer backbone146. An interesting recent development is a library of mannosylated PBAE by B. A. Pfeifer et al that produced optimized polymer composition for targeted delivery of APCs153 and strong humoral immune response in vivo without exogenous adjuvant44. Furthermore, these novel synthetic polyesters have been used to construct hybrid delivery systems by coating the surface of bacterial cells with promising results in animal models154-155.


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3.2.3 PEI Well-known for its “proton sponge” mechanism of endosomal escape, PEI (particularly the branched polymer of 25 kDa) has long been the “gold standard” of polymeric gene carriers115, 156. Interestingly, PEI alone was found to have some immunostimulatory effect, as demonstrated in animals after intratracheal157, subcutaneous158, and intranasal delivery159 of antigen-encoding DNA. At least part of this effect is attributed to PEI induced cytotoxicity, as demonstrated by C. Wang et al that moderate cell death due to exposure to PEI might be exploited for promoting DC crosspresentation of antigen produced by transfected bystander cells, leading to activation of CD8+ T cells. Such intrinsic immunostimulatory property combined with high transfection efficiency have made PEI an attractive DNA vaccine carrier160. Multiple molecular designs of PEI, including the hyperbranched structures, have been explored for DNA vaccine delivery. J. F. S. Mann et al synthesized dPEI (a nearly fully hydrolyzed linear PEI with 11% additional free protonatable nitrogen atoms) with reduced toxicity compared to regular PEI. They used dPEI to deliverDNA vaccine through the pulmonary route and detected mucosal and systemic immune responses against influenza challenge161. E. V. Grant et al examined several linear PEI with different structures and found that physical properties of the PEI/DNA complexes correlated with immunogenicity of the vaccine. Interestingly, CD8+ T cell responses inversely correlated with particle size of the polyplexes but positively correlated with surface charge. Further, their study showed that i.v. injection of polyplexes was more effective than i.m. injection in inducing gene expression in secondary lymphoid organs and CD8+ T cell responses162. Yet a separated study on poly(propylenimine) (PPI)


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dendrimer with a structure analogous to PEI showed effectiveness in i.m. delivery of DNA vaccine163. Direct comparison among studies like these is difficult, given the diverse chemical structure of the polymers, type of vaccine, and choices of animal models. PEI has been extensively modified with small molecule ligands and other polymers through covalent conjugation, aiming to achieve cell specific targeting, reduce toxicity, and enhance transfection. E. R. Tőke et al have shown that mannose-PEI conjugate formed nanoparticles of 50-240 nm in size with plasmid DNA. These virus-like particles were able to enter Langerhans cells (LCs) through receptor-mediated endocytosis and be transported to the draining LNs by the LCs164. In fact, PEIm is the only polymer carrier used in a DNA vaccine product (DermaVir) being clinically tested165. A. David et al synthesized multivalent mannose ligands and modified PEI through a PEG spacer. Subcutaneous injection of the polyplexes achieved targeted cellular uptake and higher transfection of CD11c+ DCs in the inguinal LNs than nontargeted polyplexes44. Similar strategy was employed by the same group in PEI modification with a DC targeting peptide (DC3) 166. Another study incorporated mannose and deoxycholic acid dual modified PEI into polyelectrolyte multilayer assembly films for cutaneous delivery of DNA vaccine167. Finally, modification of low MW PEI (600 Da) with cationic cell-penetrating Tat peptide was also effective in intracellular delivery of DNA vaccine and eliciting humoral and cellular immune responses after s.c. injection168. More recently, cyclodextrin-modified PEI/DNA polyplexes were used to coat live attenuated bacteria for delivering a DNA tumor antigen VEGFR2 orally and achieved antitumor effect169.


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3.2.4 Vinyl polymers Vinyl polymers can be easily synthesized by various radical polymerization methods, achieving broad diversity of well-defined architectures, compositions and functionalities. Therefore, they are perfect model polymers for structure-function relationship studies. In addition, practical application of vinyl polymers as DNA vaccine carriers may also be explored. Varieties of vinyl polymers tested for DNA vaccine delivery range from corecrosslinked particles to linear water-soluble polymers. Back in 2004, J. M. J. Frechet et al. prepared acid-labile crosslinked acrylamide-based microspheres by free radical polymerization, which were used for condense DNA. The encapsulated DNA can transfect and activate RAW264.7 macrophages, leading to secretion of IL-6 with a response 40-fold higher than the naked plasmid170. A more recently example is microgel particles whose vinyl polymer side-chains hydrolyze to convert positive charges to zwitterions, thus facilitating the delivery of DNA to macrophages171. Core-shell cationic nanoparticles consisting of vinyl polymers were prepared by A. Caputo et al. using emulsion polymerization. The inner core was polymethylmethacrylate (PMMA) and the shell was poly(2-(dimethyloctyl) ammonium ethyl methacrylate bromine) and PEG. HIV DNA vaccine was condensed into the nanoparticles via electrostatic interactions. Intramuscular immunizations of the complexes followed by protein boosts in BALB/c mice induced significant antigen-specific humoral and cellular responses, and greatly increased Th1-type T cell responses and CTLs against HIV 172. T. H. Young et al prepared five kinds of PMMA particles with different size and surface charge. The ones with positively charged surface and larger size (460±160 nm) showed stronger immune


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responses than other particles. They stimulated the highest level of TNF-α production by transfected macrophages cell line J774A.1 in vitro without inducing significant IL-6 production, demonstrating potential advantage of using PMMA particles as adjuvants for DNA vaccination 173. Water-soluble block copolymers PEG-b-poly(dimethylaminoethyl methacrylate) (PDMAEMA) were synthesized by A. Dong et al using atom transfer radical polymerization (ATRP) and used to condense HIV DNA vaccine into polyplexes with size of 150 nm. After intranasal administration in mice, the polyplexes enhanced the priming effect of the vaccine and stimulated cytokine secretion by mouse macrophages48. Homopolymers of 2-aminoethyl methacrylate (PAEM) bearing primary amines on the side chains were synthesized by C. Wang et al using ATRP. The effect of polymer chain length on DC transfection, DC maturation, and CD8+ T cell activation by a model OVA DNA vaccine was investigated174. Subsequently, PAEM homopolymer was compared with PEG-b-PAEM block copolymer with equivalent cationic segment in terms of DNA complexation capacity and distribution in the skin after s.c. injection. Both polyplexes formed depots at the injection site that persisted for days, allowing engagement and transfection of APCs and dermal fibroblasts. PEGylated polyplexes showed higher colloidal stability and broader tissue distribution than non-PEGylated polyplexes49. A potential weakness of vinyl polymers as practically useful DNA vaccine carriers is that their carbon-carbon backbone chains are not biodegradable. The possibility of synthesizing biodegradable vinyl polymers with similarly exquisite structural control remains to be explored. 3.2.5 Dendrimers


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Cationic dendrimers represent another family of polycations that have been extensively investigated as gene carriers. Compared to linear polycations, dendrimers typically have higher cationic charge density, more globular shape, and higher DNA binding and gene transfection efficiency. The chemical structure of dendrimers can be easily tuned and adapted for many gene delivery applications175. Recent examples of using dendrimers for DNA vaccine delivery include genenration-4 poly(propyl ether imine) (PETIM) dendrimer for stimulating production of neutralizing antibody in mice against rabies176, generation-4 polyamidoamine (PAMAM) dendrimer modified with lysine for DNA vaccination against S. japonica infection177, and generation-5 PAMAM dendrimer-MHC-II-peptide conjugate as a universal APC-targeted plartform showing efficacy in vivo animal against established tumors231.

4. Concluding remarks Published studies on polymer mediated DNA vaccine delivery have largely focused on a few major types of polymers that include chitosan, degradable polyesters, synthetic polypeptides, PEI, nondegradable vinyl polymers, PAMAM dendrimers, and some nonionic block copolymers. Much progress has been made toward developing effective polymer carriers that show promise in enhancing the efficacy of DNA vaccine both in vitro and in vivo. Studies in the past have established several unequivocal benefits of using polymers as DNA vaccine carriers in comparison to naked DNA or lipid-based carriers. One, polymers can protect DNA from degradation by complexation or encapsulation, thus prolonging the in vivo lifetime of the DNA. Two, compact, nanoscale polyplexes can be internalized by phagocytic APCs through various pathways much


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efficiently than naked DNA. Three, polymers can form depots after local vaccination and provide sustained supply of antigen, resulting in more robust immune responses. Four, conjugation of polymer carriers to specific targeting ligands can lead to enhanced uptake by APCs. Five, composition, architecture, size, surface charge of polymer carriers can be tuned to have profound influence on transfection efficiency of APCs and immune responses. Despite such progress, clinical translation of polymer mediated DNA vaccination has been slow. To our knowledge, currently there is only one DNA vaccine formulation in clinical trial that involves a polymer as delivery system (DermaVir, mannosylated PEI)115. The paucity of viable polymer candidate for clinically relevant DNA vaccination suggests that there is much to be learned about the fundamental science of polymer based DNA vaccine delivery, before robust, practical technologies can emerge. Two outstanding problems of current polymer carriers are apparent. The first is the lack of sufficient transfection efficiency of APCs in vivo. DCs are notoriously difficult to transfect, both because of its intrinsic resistance to foreign DNA and its rarity in the body. Localized delivery to DC-rich tissues and organs has achieved some success, but the appealing idea of cell-specific targeting through systemic delivery remains elusive. Without sufficiently high level of antigen expression in the right type of APCs, there is the risk of inducing anergy or even immune tolerance. The second problem is the lack of intrinsic immunogenicity of DNA vaccine. While the use of adjuvants along with DNA vaccination is widely practiced, such approaches have yet to live up to their full potential in jump-starting the immune system, largely due to poor choices of adjuvant and suboptimal delivery.


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It is our opinion that future research effort needs to focus on increasing our understanding of structure-function relationship in polymer mediated DNA vaccine delivery, in light of searching for solutions to overcome multitudes of hurdles in delivery. One aspect of structure-function relationship is the interaction between polymer carriers and immune cells. A potentially fruitful approach is to synthesize structurally welldefined polymer carriers using tools such as controlled polymerization and click reactions, and characterize systematically the uptake and intracellular trafficking dynamics in DCs. This approach may identify rate-limiting processes in antigen expression and presentation and reveal critical structural features of polymers that have impact on these processes. It is important to note that biological state of the target cells may have unexpected influence on antigen expression and presentation. For example, cell death by apoptosis may reduce antigen expression level and limit the duration of DC engagement with naïve T cells necessary for immune activation. However, cell death by necrosis may facilitate cross-presentation by releasing endogenous danger signals that promote immune activation. Furthermore, autophagy induced by polymer-mediated transfection either augments or reduces antigen expression through currently unknown mechanisms. Finally, phenotypic maturation of DCs confers profound changes in several aspects of cell behavior, such as endocytosis, fusion of intracellular vesicles, and transcriptional activity, all of which are expected to alter polymer mediated DNA delivery to DCs. Understanding the dynamics of DC maturation and gene delivery will provide guidance to designing polymers for synergistic co-delivery of molecular adjuvants and DNA vaccine.


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It is vitally important to consider the investigation of structure-function relationship of DNA vaccine delivery in the context of in vivo environment. Clearly, different route of DNA vaccine delivery involves different target tissue sites that vary in anatomical features, cell types and distribution of various cell types, demanding completely different sets of carrier design principles. Thus, polymers that perform well in one delivery route (such as subcutaneous injection) may be completely ineffective in another (such as systemic injection). Ideally, tracking the fate of polymer DNA vaccine carriers should be performed on live animals in real-time. Further, somewhat unconventional routes of delivery, such as intranodal injection and buccal/sublingual vaccination, may be worth exploring.

Acknowledgment C. Wang acknowledges funding from the NIH/NCI (grant R01CA129189).


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Animal Formulation

Disease

Administration

Ref. model

Natural polymers Chitosan

Chitosan

Chitosan/anionic liposomes Mannosylated chitosan Chitosan-g-PEG Biotinylated chitosan Chitosan-linked PEI Chitosan/PEI Alginic acid-coated Chitosan nanoparticle GNPs-chitosan N-acetylated chitosan N-trimethyl chitosan Albumin-based chitosan

RSV Streptococcus pneumoniae Atherosclerosis Coxsackievirus B3 infection Tuberculosis Toxoplasma gondii Vibrio parahaemolytucus Chlamydia trachomatis Anatid herpesvirus Newcastle disease Viral myocarditis Influenza virus HPV White spot syndrome virus Tumor antigen Leptospirosis Caries HBV Tumor antigen

i.n., pulmonary, p.o., i.m.

Mouse, rabbit, black seabream , duck, chicken, shrimp

i.n.

Mouse

i.m., i.d. i.n.

Mouse, guinea pig

i.n. i.m.

Mouse Mouse

p.o., i.m.

Mouse

i.m. p.o. i.n. p.o.

Mouse Mouse Mouse Mouse

106 43, 109 110, 195 111 112 113 196 114, 197 198 199 200 201

HBV Bovine herpesvirus 1 Melanoma

i.v. s.c. s.c.

Mouse Cattle Mouse

117 118 202

HIV

i.m., i.d., s.c.

Mouse

203

Colorectal cancer

i.p., s.c., i.m.

Mouse

121-

FMDV Model antigen SARS Melanoma Reporter system Breast cancer HBV HBV Model antigen Modal antigen HBV

105, 107, 178194

Other polysaccharides Astragalus polysaccharides Agarose hydrogel Spermine dextran Synthetic polymers Peptides and polypeptides Peptide-based nanofibrous hydrogel P[Asp(DET)]/PEG-b-


33


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P[Asp(DET)] Cell penetrating peptides (MPG, CPP-PEI1800-Man) γ-PGA PEI/γ-PGA PLL-coated PS nanoparticle Cell-penetrating peptides (CPPs) Th1-type epitope peptides

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Electroporation

122

HPV

12, 67

s.c., t.c

Mouse

Melanoma Melanoma Malaria Model antigen

i.v., i.p.

Mouse

i.d.

Mouse

120 204 119

HBV

-

-

205

Schistosomiasis

s.c.

Mouse

206

i.p., p.o, i.m., i.d., i.n., intranodal

Mouse， sheep, chicken, nonhuma n primate

96, 126127, 129130, 207214

i.m.

Mouse

Degradable polyesters ---- PLGA

PLGA

PLGA/DDA

Rotavirus HIV Model antigen FMDV HBV Allergic response Tubercolosis Newcastle disease Fungal infection Alphavirus-based measles virus Mycobacterium tuberculosis

PLGA-CTAB

HIV HBV FMDV

i.m.

Mouse， pig

PLGA/PEI PLGA-PEI

Listeria monocytogenes HIV Model antigen

i.m., pulmonary

Mouse

i.n., gene gun, i.m,

Mouse, chicken

i.m. t.c. -

Mouse Mouse -

i.d. -

Mouse -

i.m., i.d., t.c.

Mouse

s.c.

Mouse

PLGA-Chitosan PLGA/Chitosan PLGA-PEG/NGR-PEI PLGA-dendron PLGA microneedles PLGA/PBAE

FMDV Model antigen Newcastle disease Model antigen Anthrax Model antigen Model antigen

133 134, 136, 215216 137138, 140, 217 141142, 218 219 143 144 220

Degradable polyesters ---- POE and PBAE Poly(ortho ester) (POE) POE-PEI Poly(beta-amino esters) (PBAE) Mannosylated PBAE

Model tumor antigen Reporter system HIV Modal antigen Model antigen

148 152 146, 150 45

PEI PEI (branched PEI, dPEI, linear PEI)

Model antigen Influenza Model antigen HIV Renal carcinoma

s.c., i.n., pulmonary, i.v., i.m.

Mouse

Mannosylated PEI (mannose-PEI, mannosePEG-b-PEI)

Model antigen HPV

s.c., t.c., i.m.

Mouse, rabbit


159, 161162, 221223 44, 164, 167,

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Mouse Mouse Mouse

Malaria HPV

s.c. t.c. i.m. i.p., i.m., s.c., i.d. s.c.

Mouse

224 166 168 225 163 226227 228 229

Reporter system

-

-

170

DC-targeting PEI CPP-PEI Pluronic P123-PEI PPI dendrimer

Reporter system HPV HBV HBV

SPIONs-PEI

Malaria

SPIONs/PEI/HA PEI-Tat

Mouse

Vinyl polymers Acrylamide microspheres Hydrolytically degradable polyamine PDMAEMA-PEG-PMMA PMMA

Reporter system

-

-

230

HIV HPV

i.m. Gene gun

Mouse Mouse

PEGylated PDMAEMA

HIV

i.n.

Mouse

PAEM PEG-b-PAEM

Model antigen

i.d., i.m.

Mouse

PDMAEMA-PEG/MMA

HBV Tat

i.m.

Mouse

172 173 48, 231 174, 232 233234

Rabies

i.m.

Mouse

176

S. japonicum infection Model tumor antigens

i.m. s.c.

Mouse Mouse

177 235

Dendrimers Poly(propyl ether imine) (PETIM) (G4) (G4) PAMAM-lys Peptide-PAMAM (G5)

i.m., intramuscular; i.p., intraperitoneal; i.v., intravenous; i.d., intradermal; i.n., intranasal; s.c., subcutaneous; t.c., transcutaneous

Table 1


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TOC graphic DNA

Delivery

Complexes Polymer

C MH

Peptide

Others

PLGA

T cell

t o ry ula e l tim Cos olecu m

APC Cy

Chitosan

TCR

tok

ine

s

PEI T cell differentiation and expansion


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Polymers for DNA Vaccine Delivery - American Chemical Society

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