Macrophage-targeted delivery systems for nucleic acid therapy of inflammatory diseases.

COREL-07131; No of Pages 16 Journal of Controlled Release xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel

Review

Macrophage-targeted delivery systems for nucleic acid therapy of inflammatory diseases Amit Singh, Meghna Talekar, Ankita Raikar, Mansoor Amiji ⁎ Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, Boston 02115, USA

a r t i c l e

i n f o

Article history: Received 25 January 2014 Accepted 21 March 2014 Available online xxxx Keywords: Inflammatory diseases Nucleic acid therapy Macrophage-targeting Nanoparticles

a b s t r a c t Inflammation is an immune response that marks several pathophysiological conditions in our body. Though adaptive immune cells play a major role in the progression of the disease, components of innate immune system, mainly monocytes and macrophages play the central role in onset of inflammation. Tissue-associated macrophages are widely distributed in the body showing tremendous anatomical and functional diversity and are actively involved in maintaining the homeostasis. They exhibit different phenotypes depending on their residing tissue microenvironment and the two major functional phenotypes are classically activated M1 phenotype showing pro-inflammatory characteristics and alternatively activated M2 phenotype demonstrating anti-inflammatory nature. Several cytokines, chemokines and other regulatory mediators delicately govern the balance of the two phenotypes in a tissue. This balance, however, is subverted during infection, injury or autoimmune response leading to increased population of M1 phenotype and subsequent chronic inflammatory disease states. This review underlines the role of macrophages in inflammatory diseases with an insight into potential molecular targets for nucleic acid therapy. Finally, some recent nanotechnology-based approaches to devise macrophagespecific targeted therapy have been highlighted. © 2014 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . Inflammatory diseases and therapeutic targets . . . 2.1. Inflammatory bowel disease (IBD) . . . . . 2.2. Rheumatoid arthritis (RA) . . . . . . . . . 2.3. Atherosclerosis . . . . . . . . . . . . . . 2.4. Diabetes and insulin resistance . . . . . . . 2.5. Cancer . . . . . . . . . . . . . . . . . . 3. Nucleic acid therapy . . . . . . . . . . . . . . . 3.1. Challenges in nucleic acid delivery . . . . . 3.2. Strategies in nucleic acid delivery . . . . . 3.3. Safety considerations in delivery vector design 3.4. Targeting macrophages . . . . . . . . . . 4. Nucleic acid therapy of inflammatory diseases . . . 4.1. IBD therapy . . . . . . . . . . . . . . . 4.2. RA therapy . . . . . . . . . . . . . . . . 4.3. Atherosclerosis therapy . . . . . . . . . . 4.4. Therapy against diabetes . . . . . . . . . 4.5. Therapy against cancer . . . . . . . . . . 5. Conclusions . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . .

⁎ Corresponding author. Tel.: +1 617 373 3137; fax: +1 617 373 8886. E-mail address: [email protected] (M. Amiji).

http://dx.doi.org/10.1016/j.jconrel.2014.04.021 0168-3659/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: A. Singh, et al., Macrophage-targeted delivery systems for nucleic acid therapy of inflammatory diseases, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.04.021

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

2

A. Singh et al. / Journal of Controlled Release xxx (2014) xxx–xxx

1. Introduction Inflammation is a stimuli-activated adaptive response of the body that initiates a coordinated cascade of complex regulatory network upon tissue injury, microbial infection or autoimmune response [1]. The pathological implication of inflammatory response of the body is very well understood especially during microbial infection though its physiological function is yet to be characterized. Macrophages, the bone marrow derived leucocytes, are widely distributed cell population in the body that exhibit different phenotypic characteristics depending on their location and function (Fig. 1) [2]. They serve an important role in maintaining homeostasis in the body and are also key regulators of the inflammatory process. They along with dendritic cells are key members of the innate immune system responsible for recognizing “nonself” through membrane bound or intracellular pattern-recognition receptors (PRRs) and initiating the production of inflammatory cytokines and chemokines and subsequent involvement of adaptive immune system. Macrophages are derived from monocyte precursor under tissue specific differentiation and infiltrate the site of infection or injury to produce inflammatory mediators including chemokines, cytokines vasoactive amines, eicosanoids and other products of proteolytic cascades. They can be classically polarized to pro-inflammatory M1 phenotype and function as an effector of TH1 and TH17 mediated immune response upon stimulation by lipopolysaccharide (LPS), toll like receptor (TLR) activation, granulocyte monocyte colony stimulating factor (GM-CSF) or interferon-γ (IFN-γ) produced from pathogenic invasion. The M1 phenotype macrophages produce abundant amounts of tumor necrosis factor (TNF) and interleukins (IL-12, IL-23) that attract tissue specific TH1 and TH17 to the site of infection, thereby increasing the inflammatory

response. The clearance of parasite infection however can also result in non-specific tissue damage and enhanced inflammatory response that can cause inflammatory diseases. Instead, macrophages can be alternatively polarized to anti-inflammatory M2 phenotype by TH2 specific cytokine, IL-4 and result in higher expression of IL-10 and IL-1RA and lower the production of IL-12. M2 polarized macrophages have immunosuppressive function and are involved in pathogen clearance, alleviating inflammation, tissue repair and remodeling as well as tumor progression. The two phenotypes of macrophages show diversity in functionspecific surface markers as well as secreted chemokine profile that differentiates the two populations [3]. Macrophages therefore play a pivotal role in maintaining homeostasis and regulating the inflammatory response of the body and thus, their phenotypic balance is an important parameter in defining the initiation, progression and resolution of inflammatory conditions (Fig. 1). In the event of infection or injury in body tissues, circulating monocytes are recruited and transformed primarily to pro-inflammatory M1 polarized macrophage phenotype under the influence of local tissue environment. The M1 polarization of macrophages is regulated by nuclear factor (NF)-κb, activator protein 1 (AP1), interferon regulatory factor (IRF) and mineralocorticoid receptor (MR) [4]. The pro-inflammatory macrophages exhibit enhanced endocytic activity and antigen presentation to initiate immunogenic response against the microbial infection. They also secrete inflammatory mediators such as TNF, nitric oxide, reactive oxygen intermediates, hydrolytic enzymes and cytokines such as IL-1 that in turn activate the anti-microbial defense mechanism involving a highly oxidative environment that results in killing the invading organisms. Furthermore, M1 polarized macrophages produce TH1 attracting chemokines such as C-X-C motif chemokine CXCL9 and CXCL10. The inflammatory process that commences due to the

Fig. 1. Tissue macrophages perform important homeostatic functions. Mononuclear phagocytes are generated from committed hematopoietic stem cells located in the bone marrow. Macrophage precursors are released into the circulation as monocytes and quickly migrate into nearly all tissues of the body, where they differentiate into mature macrophages. Various populations of mature tissue macrophages are strategically located throughout the body and perform important immune surveillance activities, including phagocytosis, antigen presentation and immunosuppression. Reprinted with permission from NPG [2].



production of reactive oxygen and nitrogen species along with the presence of TH1 and TH17 cells results in significant levels of collateral damage of healthy tissues. In the regular course of infection or injury, the inflammatory process is controlled and the M1 phenotype macrophages undergo apoptosis or switch to their anti-inflammatory M2 phenotype, thereby mitigating the process of inflammation. However, if the inflammatory response of macrophages is not controlled, it becomes pathogenic resulting in significant levels of non-specific tissue damage and causing inflammatory and autoimmune diseases [5]. Macrophage targeted therapy therefore is extremely relevant in improving the prognosis of inflammatory diseases and is the mainstay of this review with special focus on inflammatory bowel disease (IBD), rheumatoid arthritis (RA), atherosclerosis, diabetes and cancer. Several surface receptors generally expressed on macrophages are also expressed on other mononuclear phagocytes since they all originate from the same myeloid progenitors. CD68 is a common surface marker of macrophages and is constitutively expressed in all macrophages alike. Disease-specific activated macrophages however exhibit elevated levels of several other markers on their surface that have been characterized and are used for population analysis during the course of therapy. It is also important to realize that surface marker expression and overexpression in monocytes and macrophages can be a strong function of their local microenvironment. The cell surface receptor profile of blood monocytes for example shows a marked difference from that of intestinal macrophages [6]. CD40 and CD40 ligand (CD154) overexpression in macrophages and T-cells has been closely associated with all inflammatory conditions and their inhibition aid in containment of inflammation. The CD40 receptor has been reported to be severely elevated in CD68+ macrophages in inflammatory bowel disease (IBD) compared to normal small bowel and colon [7]. Similarly, an increased subpopulation of CD16+ (Fc-γRIII) and CD56+ blood monocytes has been implicated in Crohn's disease [8]. Rugtveit et al. studied the specimens from IBD patients and normal controls to reveal that while normal control samples exhibited selective CD86high (B7.2) subpopulation, patients suffering from IBD show CD80high (B7.1) and CD86high (B7.2) subpopulation along with CD14high (LPS coreceptor) macrophages [9]. TLRs similarly are overexpressed in the majority of macrophages involved in inflammation and recognize microbial products as well as endogenous ligands such as heat-shock proteins (HSPs) and fibronectin [10]. Tumor associated macrophages (TAMs) on the other hand express a host of cell markers on their surface apart from CD68 such as matrix metalloproteinases (MMPs), human leukocyte antigen (HLA-DR), vascular endothelial growth factor (VEGF), etc. Heusinkveld et al. have comprehensively reviewed different organ specific cell markers in TAMs that are overexpressed in various cases of cancer [11]. 2. Inflammatory diseases and therapeutic targets 2.1. Inflammatory bowel disease (IBD) Inflammatory bowel disease is a general medical condition used to describe chronic or recurring immune reaction and inflammatory condition of the digestive tract and majorly includes ulcerative colitis (UC) and Crohn's disease (CD). IBD is generally marked by an increased autoimmune response of the body to food, altered or normal microflora of the gastrointestinal tract and any other material. However, several other factors such as diet, consumption of tobacco, occupation and exposure to infection have also been found to be contributing to the etiology of the disease [12]. T-cells are believed to be the major contributor of UC and CD but recent evidences suggest that innate immune components play a major role at least in CD, where they initiate the inflammatory process as well as adaptive immune response [13]. Macrophages are an integral part of the normal intestinal tissues and their location is well established in lamina propria and Peyer's patch where they function as immune effector cells against any pathogenic attack. Macrophages in lamina

3

propria are more prominent in the subepithelial region demonstrating a larger, round morphology and actively produce acid phosphatases and nonspecific esterases while in the deeper lamina propria, they are smaller with irregular morphology [14]. Similarly, macrophages in Peyer's patch also demonstrate significantly varying morphology and surface markers characteristics with distinct phenotype presence in different regions. Macrophages in lamina propria for example are CD68+, major histocompatibility complex II+ (MHCII+), RFD1+, RFD7+ and lysozyme+ but RFD9− and S100−. Alternatively, macrophages in the dome region of Peyer's patch are CD68+, major histocompatibility complex II+ (MHCII+), RFD1+ and S100+ but lysozyme−, RFD9− and RFD7− [15]. Immune response in IBD is a complex cascade of events involving a coordinated activation of immune cells through an array of cytokines and chemokines and is predominated by activation of CD4 + cells. Onset of CD is associated with cytokine profile involved in TH1-mediated activation of immune response with IFN-γ and IL-2 being the major contributors. UC on the other hand, is marked by TH2 linked cytokine profile involving transforming growth factor-β (TGF-β), IL-5 and IL-13 but without any change in expression profile of IL-4 (alternate TH2 cytokine). Recent studies have revealed a distinct lineage of T helper cells, called TH17 cells that are effectively inhibited by IFN-γ and IL-4 (key activators of TH1 and TH2 lineage respectively) when in naïve form. However, in absence of these inhibitory factors, IL-23 activates them to committed TH17 cells that are resistant to suppression by TH1 and TH2 cytokines [17]. Macrophages along with dendritic cells are major contributors of enhanced expression of IL-23 [18], thereby mediating inflammatory tissue destruction by production of IL-17. Therefore, enhanced level of cytokines such as IL-12, IL-18 and IL-23 due to increased population of M1 macrophage phenotype push toward T-cell differentiation into TH1 and TH17 mediating a chronic inflammatory immune response. This along with reduced population of anti-inflammatory macrophage M2 result in the altered physiological condition from tolerance to active immune state resulting in IBD and therefore present as attractive targeting strategy for intervention against anticytokine therapy, T-cell population modulation and switch in macrophage polarization [19]. Genomic abnormalities are alternate focal points in understanding development of IBD and devising modalities for potential therapeutic targets. Genome wide screening with microsatellite markers has revealed 10 genomic sites that have been implicated in altering the course of both forms of IBD [20]. Caspase recruitment domain family, member 15 (CARD15) (aka nucleotide-binding oligomerization domain 2; NOD2) was the first IBD specific gene located on chromosome 16 to be identified and is therefore popularly called IBD-1. Interestingly, this gene is most prominently expressed in macrophages, suggesting another alternative mechanism by which macrophages contribute to development of IBD. More than 60 mutations have been reported in NOD2 gene out of which 3 have been directly implicated in affecting the development of CD [21]. Although the exact mechanism by which CARD15 influences the inflammatory response is not entirely clear, several indications suggest that the mutated gene results in a defective clearance of infecting microorganisms from the intestines [22]. Mutation in the gene has also been linked to the altered activation of NF-κB and TLR, though contrasting reports are available on how it effects the receptor activation [19]. Carnitine/organic cation transporter 3 (OCTN3) (also known as IBD-5) [23] and disks, large homolog 5 (DLG5) [24] are two new gene mutations that have been connected to the progression and outcome of IBD. Mutations in gene encoding IL10R subunit protein have recently been confirmed in patients suffering from hyperinflammatory immune response in intestines [25]. Table 1 shows the reported genetic mutations common/specific to CD and UC [16]. Therefore, there is a pool of gene defects that can be potentially targeted using nucleic acidmediated intervention against IBD by exploiting the advantages offered by nanodelivery systems (Fig. 2).


4


Table 1 Polymorphisms and mutations associated with IBD. Reprinted with permission from NPG [16]. Pathway of site affected

Genes affected in CD

Paneth cells Bacterial sensing Innate mucosal defense Authophagy Immune cell recruitment Antigen presentation IL-23/Th17 T cell regulation B cell regulation Immune tolerance

ITN1, NOD2, ATG16L1 TLR4, TLR9, CD14, MAL NOD2, ITLN1, CARD8, NLRP3, ILI8RAP ATG16L1, IRGM, NOD2, LRRK2 CCL11-CCL2-CCL7-CCL8, CCR6 ERAP2, LNPEP, DENND1B STAT3 NDFIP1, TAGAO, IL2RA IL5, IKZF1, BACH2 IL27, SBNO2, NOD2

2.2. Rheumatoid arthritis (RA) Rheumatoid arthritis is another autoimmune disease where macrophages play the primary role in chronic inflammation of synovium while other cells from innate and adaptive immune system such as fibroblasts, dendritic cells, neutrophils, T and B cells contribute to the secondary dysfunction. The synovial membrane primarily consists of type A (macrophages) and type B (fibroblasts) cells where in normal condition, the type B cells outnumber type B cells but in RA, the macrophage population is severely increased [26]. Increased infiltration of monocytes through chemotaxis, reduced emigration, increased local proliferation and reduced apoptosis are some of the contributing factors in the increase of the macrophage population during RA [27]. The increased population of macrophages mounts an autoimmune response through production of several cytokines such as TNF-α, granulocytemacrophage colony-stimulating factor (GM-CSF), IL-1, IL-6, IL-8, IL-10, IL-13, IL-15, IL-18, migration inhibitory factor (MIF) and chemokines such as macrophage inflammatory protein 1 (MIP-1), monocyte chemoattractant protein 1 (MCP-1) and fractalkine. An increased level

Genes affected in UC

Genes affected in CD and UC XBP1

SLC11A1, FCGR2A/B PARK7, DAP IL8RA-IL8RB

CARD9, REL CUL2 MST1

IL21 IL2, TNFRSF9, PIM3, IL7R, TNFSF8, IFNG, IL21 IL7R, IRF5 IL1R/IL1R2

IL23R, JAK2, TYK2, ICOSLG, TNFSF15 TNFSF8, IL12B, IL23R, PRDM1, ICOSLG IL10, CREM

of macrophage derived proteins such as matrix metalloproteinases (MMPs) and leucocyte elastase has also been reported [28]. Similarly, increased population of osteoclasts, giant multinucleated cells of monocyte lineage, mediate bone desorption in the articular region and further aggravate the disease pathogenesis. Macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) modulate the osteoclast formation from its precursor cells. TNF and IL-7 promotes M-CSF production from synovial fibroblasts and T-cells respectively while RANKL levels are increased by cytokines such as TNF, IL-1β, IL-6 and IL-17 as well as non-cytokines such as prostaglandin E2 [10]. Fig. 3 comprehensively summarizes the interconnection and crosstalk of various cytokines, chemokines and molecular events in the pathogenesis of RA. TNF-α plays a major role in RA since it not only promotes the expression of pro-inflammatory cytokines but also induces macrophages to produce reactive oxygen species (ROS) that increase inflammation in the joints. Besides, macrophages overproduce nitric oxide in the joints that stimulates synovial cells to produce more TNF-α and further promote inflammation and bone destruction [29]. IL-1 similarly has a

Fig. 2. Novel therapeutic targets in inflammatory bowel disease (IBD). Potential therapies in IBD encompass interventions at a variety of pathways in the inflammatory cascade. These include altering luminal factors, enhancing intestinal repair, augmenting the intestinal innate immune barrier function, inhibiting cell adhesion and blocking cytokine activity. GM-CSF, granulocyte-macrophage colony-stimulating factor; ICAM1, intercellular adhesion molecule 1; IFN, interferon; IL, interleukin; Mφ, macrophage cell; PMN, peripheral blood mononuclear cell; SAM, selective adhesion molecule. Reprinted with permission from NPG [19].



5

Fig. 3. An overview of the cytokine-mediated regulation of synovial interactions. The component cells of the inflamed rheumatoid synovial membrane are depicted in innate and adaptive predominant compartments of the inflammatory response. Pivotal cytokine pathways are depicted in which activation of dendritic cells (DCs), T cells, B cells and macrophages underpins the dysregulated expression of cytokines that in turn drive activation of effector cells, including neutrophils, mast cells, endothelial cells and synovial fibroblasts. The clinical manifestations of such effects are highlighted. Only key cytokines are shown in each domain for relative simplicity; the main text contains more detailed description of the precise role of additional cytokines in these processes. Bidirectional arrows represent a relationship between cells that is influenced by the cytokines listed. APRIL, a proliferation-inducing ligand; BAFF, B-cell activating factor; bFGF, basic fibroblast growth factor; CCL21, CC-chemokine ligand 21; CXCL13, CXC-chemokine ligand 13; FcγR, Fc receptor for IgG; IFN, interferon; IL, interleukin; LTβ, lymphotoxin-β; M-CSF, macrophage colony-stimulating factor; PAR2, protease activated receptor 2; RANKL, receptor activator of nuclear factor-κB (RANK) ligand; TGFβ, transforming growth factor-β; TH, T helper; TLR, Toll-like receptor; TNF, tumor-necrosis factor; VEGF, vascular endothelial growth factor. Reprinted with permission from NPG [10].

major influence in joint inflammation and articular destruction since it inhibits proteoglycan synthesis, promotes its degradation and stimulates production of MMP-1 and MMP-3 [28,30]. IL-15 produced by macrophages as well as T cells and IL-17 produced by T-helper cells regulate the production of TNF-α and IL-1 by macrophages and thus indirectly influence the disease progression [28]. A detailed account of other pro-inflammatory cytokines produced by macrophages and their subsequent contribution to disease pathogenesis is out of the scope of this review but has been comprehensively summarized in a recent review and is highly recommended to the reader [10]. However, it is important to consider the balance in the macrophage phenotypes M1 and M2 in RA and the role of anti-inflammatory cytokines in pathogenesis. IL-4 is the primary cytokine that shows anti-inflammatory effect by inhibiting the production of IL-1β, TNF-α and TNF-α receptors [31]. IL-10 is another anti-inflammatory cytokine that inhibits the production of GM-CSF and IL-1 receptor antagonist. Studies have conclusively demonstrated a deficiency in the expression of anti-inflammatory cytokines in RA and therefore a reduced local concentration of IL-10, IL-11 and IL-1RA that is ineffective in eliciting an anti-inflammatory effect. T-cell derived cytokines such as IL-2 and IL-4 are also significantly lower in concentration, which impairs the regulatory TH2 cell activation and skews the T-cell differentiation in favor of TH1 or TH17 population. Concentration of other cytokines such as TNFR1 and type II IL-1R that sequester TNF and IL-1, is also diminished in the synovial fluid and thus makes it inefficient in performing the regulatory functions [32]. Gene therapy to replenish the concentration of the anti-inflammatory cytokines presents as a viable strategy to reduce inflammation and initiate wound healing and tissue remodeling, which has been previously attempted but without substantial benefits [33]. However, the emergence of improved delivery vehicles with target-specific delivery capability holds tremendous promise in improving the efficacy profile of gene therapy approach. Alternatively, selective downregulation of the pro-inflammatory cytokines such as TNF-α, IL-1

or other modulators promoting chronic inflammatory response may provide a more beneficial outcome of the disease. 2.3. Atherosclerosis Atherosclerosis is a lipid disorder and a chronic inflammatory disease, which is initiated with migration and deposition of cholesterolrich apolipoprotein B-containing lipoproteins in the arterial blood vessels. In the event of hypercholesterolemia, the low-density lipoproteins (LDL) tend to sequester in the walls of arteries, more so at the inner corners and branching arches where the blood flow is disturbed and they are prone to undergo chemical modifications such as oxidation, enzymatic and non-enzymatic cleavage and aggregation. The oxidized LDL is pro-inflammatory in nature leading to activation of the endothelium and initiating an inflammatory process that largely involves recruitment of circulating monocytes into the endothelial space. The activation of endothelium initiates a leucocyte adhesion cascade where endothelial cells express cell adhesion molecules such as intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM1) that leads to binding of monocytes on the surface [34]. The firm adhesion of the circulating monocytes on to the endothelial surface is mediated by chemokine–chemokine receptor recognition, leading to their infiltration through the intima (Fig. 4). The infiltrated monocytes differentiate into mononuclear phagocytic cells that along with originally resident macrophages of the intima actively ingest and present the normal and modified lipid on their surface to initiate a T-cell mediated immune response. Though lipid clearance and presentation is a normal event in containing hypercholesterolemia, lack of a suitable negative feedback mechanism for uptake and abundance of lipids in the vicinity leads to a significantly higher accumulation of lipids in the macrophages, causing the formation of foam cells and promoting the disease progression. Though the contribution of several cell types such as endothelial cells, dendritic cells, lymphocytes, mast cells, eosinophils and smooth


6


Fig. 4. Mechanisms regulating monocyte recruitment and accumulation in plaques. Hyperlipidemia increases the number of GR1 + LY6Chi monocytes, which constitute 80% of the monocytes recruited to mouse atherosclerotic plaques, with the remainder being the GR1-LY6Clow patrolling monocytes. These monocyte subsets use different chemokine–chemokine receptor pairs to infiltrate the intima, which is facilitated by endothelial adhesion molecules, including selectins, intercellular adhesion molecule 1 (ICAM1) and vascular adhesion molecule 1 (VCAM1). The recruited monocytes differentiate into macrophages or dendritic cells (DCs) in the intima, where they interact with atherogenic lipoproteins. Macrophages avidly take up native and modified (for example, oxidized) low-density lipoprotein (LDL) via macropinocytosis or scavenger receptor-mediated pathways (including via scavenger receptor A1 (SR-A1) and CD36), which results in the formation of the foam cells that are a hallmark of the atherosclerotic plaque. These foam cells secrete pro-inflammatory cytokines (including interleukin-1 (IL-1), IL-6, and tumor necrosis factor (TNF)) and chemokines (such as CC-chemokine ligand 2 (CCL2), CCL5 and CXC-chemokine ligand 1 (CXCL1)), as well as macrophage retention factors (such as netrin 1 and semaphorin 3E) that amplify the inflammatory response. CX3CL1, CX3C-chemokine ligand 1; CX3CR1, CX3C-chemokine receptor 1; LFA1, lymphocyte function-associated antigen 1; PSGL1, P-selectin glycoprotein ligand 1; VLA4, very late antigen 4. Reprinted with permission from NPG [34].

muscle cells is well acknowledged in the onset of chronic inflammatory response, it's the formation of foam cells that has been found critical in dysregulation of lipid metabolism, resulting in development and progression of atherosclerotic plaques. Macrophages express several receptors on their surface such as LDL receptor and scavenger family of receptors such as scavenger receptor A1 (SR-A1), SR-A2, CD36, SR-B1, lectin like oxidized LDL receptor 1 (LOX1), etc. that facilitate modified lipid uptake and promote foam cell formation. The cholesteryl esters of internalized lipoproteins are hydrolyzed to free cholesterol and fatty acid and the free cholesterol is then transported to the endoplasmic reticulum (ER) where it is re-esterified by acetyl Co-A acetyltransferase 1 (ACAT1) in macrophages to cholesteryl fatty acid ester thus giving them foamy characteristics [35]. In the later stages of atherosclerotic plaques, altered lipid metabolism and ER stress increase apoptosis in macrophages, which is poorly managed due to impaired lipid pathway and inability of surrounding macrophages to perform efficient clearance. The lipids and other cellular content of dying macrophages form the prothrombotic necrotic core, a characteristic of unstable plaques that are prone to rupture and can lead to eventual myocardial infarction and stroke. Development of atherosclerotic plaques involves various cell types and key molecular level recognition elements that present as excellent therapeutic targets. High accumulation of lipids in the macrophages is counteracted by expression of surface receptors that promote cholesterol and other lipid efflux from the cells. Macrophages, for example, express ATP-binding cassette subfamily members ABCA1, ABCG1 and SR-B1 that mediate lipid efflux to lipid poor ApoA1 or HDL particles [36]. Genes encoding the expression of these receptors are upregulated by liver X receptors (LXRs) in response to increased levels of cholesterol and other sterols [37]. Deficiency in expression of ABCA1 and upregulation of ABCG1 expression does not significantly change the course of plaque formation but knocking down both receptors promotes atherosclerosis in mice [38] and thus is a promising target for nucleic acid therapy. Similarly, regulatory receptors of lipid metabolism pathways are

looked upon as an alternative therapeutic target for controlling the plaque formation. The homeostasis of lipid levels is strictly regulated by transcription factors such as sterol regulatory element-binding protein (SREBPs) and peroxisomes proliferator-activated receptors (PPARs) [39]. The intracellular lipid accumulation and subsequent foam cell formation has been linked to defective suppression in the expression of SREBP-dependent gene expression as well as failed activation of LXR-mediated pathways. PPARs on the other hand are expressed in 3 isoforms (α, γ and δ) in macrophages and play a crucial role in maintaining glucose and lipid homeostasis. In vivo studies on murine models have revealed that PPARα and δ lead to decreased incidence of atherosclerosis while PPARγ promotes it. Expression of these receptors not only affects the disease progression directly but also contributes to other related processes that influence the outcome of the disease. PPARα for example induces the expression of Niemann–Pick C1 (NPC1) and NPC2 that are key regulators of cholesterol trafficking while PPARα and γ induce the expression of ABCA1. Development of atherosclerosis therefore is a complex phenomenon of closely-knit crosstalk of cytokines and chemokines with macrophages being the key cells dictating the disease onset and progression. 2.4. Diabetes and insulin resistance Insulin resistance is a medical condition where insulin becomes ineffective in blood glucose level leading to hyperglycemia and is generally associated with obesity, aging and physical inactivity among many other potential causes. Islet cells of pancreas increase their insulin secretion to counter insulin resistance to a certain degree but in the event of their failure, insulin deficiency and type-2 diabetes (T2D) develops. Obesity is generally associated with chronic low-grade systemic inflammation with excessive adipocyte necrosis where adipose tissue macrophages (ATMs) contribute to the production of pro-inflammatory cytokines that results in insulin resistance and eventually T2D (Fig. 5). Macrophages tend to accumulate in the adipose tissue over time and



7

Fig. 5. Development of inflammation in type 2 diabetes. Excessive levels of nutrients, including glucose and free fatty acids, will stress the pancreatic islets and insulin-sensitive tissues such as adipose tissue (and the liver and muscle, not shown), leading to the local production and release of cytokines and chemokines. These factors include interleukin-1β (IL-1β), tumor necrosis factor (TNF), CC-chemokine ligand 2 (CCL2), CCL3 and CXC-chemokine ligand 8 (CXCL8). Furthermore, production of IL-1 receptor antagonist (IL-1RA) by β-cells is decreased. As a result, immune cells will be recruited and contribute to tissue inflammation. The release of cytokines and chemokines from the adipose tissues into the circulation promotes inflammation in other tissues, including the islets. Reprinted with permission from NPG [44].

produce cytokines such as TNF and IL-6 along with chemokines like CCchemokine ligand 2 (CCL-2; also known as MCP1). Besides, a marked increase in circulation level of acute-phase proteins such as C-reactive protein (CRP) and sialic acid as well as IL-1β, IL-1RA has been observed in patients with obesity and T2D. Similarly, monocytes isolated from T1D patients show an inflammatory phenotype and secret proinflammatory cytokines [40] while those isolated from T2D patients are involved in TH17 cells activation [41]. In lean individuals, M2 macrophage phenotype is induced by PPARγ signaling and they maintain adequate adipose metabolism, insulin sensitivity and glucose tolerance. However, under obesity, these macrophages switch to M1 phenotype, with NOD-like receptor proteins (NLRPs) inflammasome acting as a molecular switch sensing the obesity-induced danger signals [42]. IL-1β has been mainly implicated as the driver of the disease and two processes tightly regulate its level. Its production in inactive form can be induced by pro-inflammatory signal through toll-like receptors (TLRs) or by cytokines from M1 macrophages. However, its activation, maturation and secretion require caspase-1 activity, which in turn is activated by inflammasomes [43]. Type-2 diabetes development is marked by monocyte recruitment by expressing molecules including ICAM-1, MCP-1, osteopontin, macrophage migration inhibitory factor (MIF) and M-CSF in adipose tissue, islet cells of pancreas, liver and muscle. The recruited monocytes differentiate to M1 phenotype under the influence of pro-inflammatory signal from adipose tissue and existing ATMs, and they in turn secrete more cytokines and chemokines to recruit more monocytes, thus unleashing a feed-forward loop to enrich macrophage population and leading to chronic inflammation and insulin resistance. Though macrophages form the majority of infiltrated innate immune cells in adipose tissue, there is compelling evidence of accumulation of mast cells during obesity. Mast cell deficient obese mice (Kitw-sh/w-sh) or obese mice treated with ketotifen, which blocks mast cell function has shown better insulin resistance profile compared to normal or untreated mice respectively [45]. Similarly, components of the adaptive immune system including CD4+ and CD8+ T-cells, TH1 and TH2 helper T-cells and

forkhead box P3 (FOXP3) regulatory T-cells (Tregs) also populate the adipose tissue simultaneously and contribute to the fate of the disease progression based on their population balance. CD8+ T-cells and TH1 cells promote the insulin resistance phenotype while Tregs and TH2 cells tend to counter this effect [46]. Tregs cell population specifically shows a drastic decrease in obese mice leading to significantly lower levels of anti-inflammatory cytokine IL-10 and higher inflammation, suggesting that induction of Tregs population could be a potential strategy for treatment [47]. Interestingly, IL-1β is a cytokine common to both T1D as well as T2D and plays a central role in inflammatory process though T1D is driven by autoimmune response while T2D shows characteristics of metabolic stress such as hyperglycemia and elevated concentration of free fatty acids [48]. Presence of human islet amyloid polypeptide (IAPP) as inducer of IL-1β production by macrophages has recently been confirmed in T2D [49]. Insulitis, the inflammation of pancreatic islets, a new concept in T2D development has long been established and accepted as a mechanism for T1D [50]. These new developments indicate that though not clearly established, there is a significant overlap in T1D and T2D development and thus therapies targeting the keys of common players could prove beneficial for either disease. Hypoxia and neovascularization have also been reported in adipose tissue of obese mice and human, which induces expression of pro-inflammatory and proangiogenic genes in the macrophage and thus show a connection between adipose tissue expansion and macrophage-mediated inflammation [51]. Inflammation and stress induced kinases such as IκB kinaseβ (IKKβ) and JUN N-terminal kinase (JNK) are also activated in T2D, which in turn activate pro-inflammatory cytokines and transcription factors such as NF-κB to promote the disease progression. NF-κB especially is activated in the majority of vital organs such as adipose tissue, liver, muscles, and hypothalamus to affect development of diabetes directly or indirectly [44]. Diabetes as an inflammatory disease is therefore a complex interplay of cytokines, chemokines, transcription factors and other regulatory molecules that are delicately managed at the genetic and molecular levels. The role of macrophages is central in


8


the onset and progression of the disease and thus devising macrophagetargeted therapy holds tremendous promise in improving the prognosis of disease. 2.5. Cancer Cancer is a complex physiological condition of cells characterized by six hallmark properties namely self-sufficiency in growth signals, insensitivity to negative growth signals, resistance to apoptosis, incessant cell replication capability, angiogenic competence and invasiveness [52]. It is also clearly evident that manifestation of malignancy by transformed epithelial cells involves a sophisticated support from stromal cells composed of fibroblasts, adipocytes, blood and infiltrated hematopoietic cells. Tumor-associated macrophages (TAMs) are the major component of the infiltrated cells in stromal compartment as well as the majority of tumors that demonstrate M2 phenotypes. The majority of tumors produce cytokine colony stimulating factor-1 (CSF-1) that recruits, proliferates and prolongs the life of TAMs in tumors and there are compelling evidences suggesting that lack of CSF-1 production impedes tumor growth and almost completely obliterates its metastatic potential. On the other hand, overexpression of CSF-1 supports tumor progression and dissemination [53]. Besides, cytokines such as VEGF and plateletderived growth factor (PDGF) as well as chemokines CCL-5, 7, 8 and 12 have also been implicated in the recruitment of monocytes [54]. TAMs in turn produce growth and angiogenic factors as well as proteases that promote tumor cell proliferation, angiogenesis, degradation of extracellular matrix and invasiveness. Further, most common tumors produce pro-inflammatory cytokines such as TNF, IL-1 and IL-6, growth factors and chemokines but lack regulatory factors for macrophagemediated sustained immune response. Inflammation therefore is a vital physiological component of several common tumors which is mediated majorly by TAMs in coordination with dendritic cells and other lymphocytes [55]. TNF is a major regulator of inflammation and as with other inflammatory diseases, its chronic production leads to tissue remodeling and development of stromal component that is necessary for malignancy. Elevated levels of TNF along with IL-1, IL-6 and M-CSF have been successfully established in ovarian, breast, prostate, bladder and colorectal cancers, lymphomas and leukemia, suggesting its role in development of inflammation in tumors [55]. In ovarian cancer, TNF has been found to be involved in stimulation of chemokine monocyte chemotactic protein-1 (MCP-1)/chemokine ligand-2 (CCL-2) production, which in turn is responsible for promoting macrophage and lymphocyte infiltration. Infiltrated macrophages further produce MCP-1 themselves to generate an amplification loop resulting in further recruitment of monocytes. TAMs in the tumor milieu preferentially migrate to the necrotic, avascular region in response to the cytokines and chemokines (VEGF, CXCL-12 and its receptor CXCR-4) that are regulated and expressed by hypoxia inducible factor-1 (HIF-1) [56]. NF-κB is another master regulator of inflammation and its expression is promoted by cytokines such as IL-1 and TNF expressed by TAMs and other stromal cells, by tumor microenvironment or by genetic abnormalities [57]. TAMs also mediate immune suppression in tumor by production of IL-10 along with chemokines that draw TH2 and Treg (e.g. CCL-17 and CCL22) and induce T-cell anergy (e.g. CCL-18) [54]. Besides mediating tumor growth, inflammation and immunosuppression, TAMs play a major role in angiogenesis and induction of metastasis. TAMs in the avascular region of the tumor express VEGF along with other hypoxia-inducible chemokines that promote neoangiogenesis. Besides, they produce CXCL-12 (a strong attractant of endothelial cells), its receptor CXCR-4 and other angiogenic chemokines such that CCL-2, CXCL-1, 8 and 13 and CCL5 [54]. Simultaneously, TAMs also modulate lymphangiogenesis through production of factors such as VEGF-C and VEGF-D that regulate intra and peritumoral lymphatics [58]. Malignancy is the most devastating aspect of neoplastic diseases and TAMs have been implicated to play a crucial

role in induction of dissemination of the cancer cells through blood or lymphatic vessels. Their impact on invasiveness of cancer cells is multidimensional, influencing not only the neoplastic cells but also the tumor microenvironment. TNF-α is the major established contributor to invasive behavior of malignant cells by promoting the production of MMPs in the macrophages. TAMs also produce a host of matrix proteins and other proteases such as serine proteases, MMPs and cathepsins that modify ECM composition, interrupt cell–cell interaction and facilitate basal membrane disruption. At another level, macrophages contribute to epithelial-to-mesenchymal transition (EMT), the preliminary step in induction of metastasis, by regulation of TGF-β signaling and activation of β-catenin in the tumor [59]. IL-1 is another cytokine that initiates angiogenesis and potentiates the metastatic capability of the tumors. Voronov et al. demonstrated that administration of IL-1 in IL-1β knockout mice restores angiogenesis and metastatic potential while IL-1 receptor antagonist counters the effect [60]. The multi-faceted control of TAMs in moderating tumor growth, immune suppression, angiogenesis, tissue remodeling and metastasis by a complex crosstalk with neoplastic cells has been summarized in Fig. 6 and they are therefore looked upon as a promising target for therapeutic intervention. 3. Nucleic acid therapy Nucleic acid therapy classically included insertion of gene of interest as DNA duplexes into the deficient somatic cells to enhance the expression of the product and achieve the desired effect. Advances in molecular biology methods, synthetic biology and bioengineering have however extended the repertoire of nucleic acid candidates for therapeutic application to include antisense oligonucleotides, messenger RNA, small interfering RNA (siRNA), small hairpin RNA (shRNA) and microRNA (miRNA) [61]. The following section will discuss some challenges associated with nucleic acid delivery, strategies to overcome these challenges and methods to selectively target macrophage therapy for inflammatory diseases. 3.1. Challenges in nucleic acid delivery Bioavailability is the biggest challenge to nucleic acid therapy primarily due to poor half-life of DNA or RNA duplexes in body fluids. Any therapeutically active moiety has to encounter several physiological barriers before it can reach its target site and show its desired effect. Naked unprotected nucleic acids rapidly degrade in biological fluids due to the presence of extra and intra-cellular nucleases, thereby showing poor therapeutic outcome. Plasmid DNA (pDNA) for example degrades rapidly within 5 min of intravenous injection in a mouse [62]. The uptake of nucleic acid by cells is further impeded because of their polar nature and high molecular weight. The limited amount of nucleic acid that are taken up by the cells are further trapped into the endosomes where they are subjected to low pH and enzymes that limits their release to reach their target in the cytoplasm or nucleus. Nucleic acids that target penetration into the nucleus have to further translocate from cytoplasm to nucleus where selectivity of nuclear membrane and the nuclear pore complexes limit their permeation and thus affect the efficacy [63]. The size restrictive permeation through the nuclear membrane allows only particles smaller than 40 kDa or sizes less than 25 nm, thereby limiting the entry of large size plasmids but allowing smaller antisense oligonucleotides [64,65]. Exogenous DNA/RNA have also proven to be immunogenic and can potentially mount systemic and local immune response mediated by TLRs [66]. The majority of these limitations can be circumvented by the use of a suitable viral or non-viral delivery vector to protect the nucleic acid payload to interact directly with the in vivo bio-milieu. Delivery vectors not only protect the structural integrity of the cargo but can also aid in cellular uptake, endosomal release, nuclear penetration using homing peptides and prevent immunological effects [67]. To this end, non-viral vectors have gained popularity in recent



Circulating Monocytes CSF VEGF PDGF MCP-1/CCL-2 CCL-5, 7, 8, 12

9

Immunosuppression

MCP-1/CCL-2

Monocytes Amplification Recruitment loop

IL-3 M-CSF

CCL-17, 18, 22

Differentiation

CSF IL-4, 10, 13 TGF-β

IL-10 TGF-β

TAMs (M2 polarization) CSF VEGF, PDGF FGF MMPs TGF-β ECM proteins

Tumor Growth

TNF-α IL-1β MMPs Proteases

Metastasis

Fig. 6. Schematic elucidating the factors involved in monocyte recruitment and differentiation in tumors and subsequent role of TAM in moderating tumor growth, immunosuppression, angiogenesis and metastasis.

years due to the simplicity and versatility of the approach, lack of possible viral pathogenicity and immunogenicity. 3.2. Strategies in nucleic acid delivery Nucleic acid therapy is an extremely challenging field and several factors have to be taken into account to ensure that the therapeutic moiety reaches it target site of action. Route of administration is one such important criterion that defines the bioavailability, accessibility of the target site and physiological challenges that the delivery vehicle has to overcome. Common approaches to route of administration can be broadly classified into oral, local and systemic delivery, though the boundaries of such classification are largely vague. Choice of delivery vehicle and route of administration is largely governed by the target site of action. Oral route for example is preferred for delivering the nucleic acid payload across intestinal mucosa of the GI tract for therapy of IBD though alternative systemic and intranasal delivery has also shown significant efficacy [67]. However, oral delivery of nucleic acid could be highly challenging due to the extreme physiological conditions in the stomach and intestines and barrier of penetration through the mucosal layer for uptake. Such physiological challenges restrict the application of conventional delivery vehicles but also provide the opportunity to design smart delivery systems that can exploit physiological barrier to improve delivery capability [68,69]. The less accessible regions of the body such as head, neck and eye are treated by localized delivery though systemic administration is more common for the delivery of nucleic acid. The circulatory system filters the particles based on their size such that particles smaller than 10 nm in size are filtered by the renal system within a few hours after being injected whereas particles larger than 200 nm are retained in the spleen. Thus, an optimal delivery system should have a size ranging from 50 to 200 nm. Due to the aforementioned limitations of the viral delivery systems, novel non-viral substitutes to improve pharmacokinetics, pharmacodynamics and efficacy of nucleic acids are always desired. The advent of nanotechnology with our better understanding of material science, organic synthesis and disease pathophysiology has led to several biocompatible and biodegradable materials that can be used to administer nucleic acid. Commonly used non-viral delivery systems include lipoplexes, polyplexes, polymer systems, lipid delivery systems and

hybrids. One of the challenges to systemic therapy is recognition of the nucleic acid loaded particles by reticuloendothelial system (RES) that leads to rapid clearance of the delivery vehicle as well as the payload. Application of polyethylene glycol as surface coating polymer however not only aids evasion from the RES recognition but also provides long circulating property to the delivery system, thus improving the residence time in the body and the probability to reach the target site [70]. Particle shape, size and charge are other important parameters that should be considered in designing the delivery system as these parameters not only govern the stability of the system in the body but also impact their uptake behavior [71,72]. 3.3. Safety considerations in delivery vector design Safety certainly is the most prime consideration when designing a delivery system for therapeutic application. More importantly, material safety for macrophage-targeted therapy becomes even more important since the macrophages are among the first cells of the body to encounter the administered delivery system and are capable of mounting immunological response against it upon stimulation. Nucleic acid delivery has classically been performed with the use of viral vectors and the subsequent sections of the review will highlight that the majority of gene therapy approaches for treatment of inflammatory diseases have relied on the use of these vectors. Viral vectors, though a popular choice for nucleic acid therapy, have always remained controversial due to the immune response that they can potentially elicit. The incident in 1999 leading to the death of a patient in gene therapy clinical trials using adenoviral vectors at the University of Pennsylvania emphasized the need to understand the interaction of the delivery vector with the host body prior to its application in human subjects. Even when such studies are conducted, it is important to consider inter-patient variability due to the influence of genetic and epigenetic effects on the immune system of an individual. For e.g. another patient in the same clinical trial in 1999 received a similar therapeutic dose but did not show any undesired side effects. Thomas et al. comprehensively summarized the application of viral vectors in gene therapy along with potential challenges and associated problems [73]. Non-viral vectors therefore have been actively sought for nucleic acid delivery and nanodelivery systems have been looked upon as an


10


attractive alternative. Advances in material science have led to the development of several synthetic and natural polymers, lipid and inorganic materials whose properties can be tailored to achieve biocompatibility and low to negligible cytotoxicity. Cationic polymer and lipid systems have been popularly used for condensation of nucleic acid for delivery to cells through electrostatic interaction with negatively charged cell surface. However, a higher charge density also accounts for material-related cytotoxicity and endosomal entrapment of the cargo; thus surface charge is another important consideration in the design of the delivery system [74]. Clearance of the materials used for vector design is another criterion that has not been studied in depth for the majority of the delivery systems used for nucleic acid therapy. Materials that cannot be naturally degraded or cleared from the body may not exhibit any short-term cytotoxicity but their accumulation over the course of therapy may lead to toxicity that should be taken into consideration. Degradation and clearance of polymers usually depend on the molecular weight (Mw). Chitosan, a natural polysaccharide, has been extensively used for charge-based nucleic acid complexation and shows Mw dependent cytotoxicity profile and clearance [75]. Similarly, choice and composition of lipids for lipid-based delivery system and surface functionality are other key parameters for material safety [74]. 3.4. Targeting macrophages Macrophages and monocytes play a central role in the autoimmune response and regulating the molecular cascade that leads to inflammatory disease. Apart from resident macrophages in the area of disease onset, circulating monocytes and macrophages are continuously recruited to meet the demands of inflammatory response and expression of chemokines, cytokines and cell adhesion molecules. Thus, considerable emphasis has been laid upon targeted therapies against them to treat inflammatory diseases. One of the most exploited approaches is to facilitate phagocytosis of loaded micro or nanodelivery vehicle by them, which is then passively targeted to the site of inflammation due to mounting immune response. The active targeting approach however has become more popular where the surface of the delivery vehicle is decorated with a ligand that selectively interacts with their target receptors. Several earlier studies have targeted the overexpressed cell adhesion molecules on the surface of vascular endothelial cells (ICAM-1, ICAM-2, VCAM-1, MAdCAM-1, etc.) to selectively deliver the payload at the site of inflammation [76]. However, some recent reports have used ligands to selectively target receptors expressed on the surface of the macrophages and monocytes. Folate receptor (FR), commonly overexpressed on the surface of cancer cells, has been reported on the surface of activated monocytes and macrophages residing in the synovial fluid of rheumatoid arthritis [77] and subsequently those in the atherosclerotic plaques [78,79]. Since then, FR has been actively pursued to target nanocarriers to the site of inflammation for improved delivery and efficacy in arthritis and atherosclerosis [80–83]. CD36, a member of scavenger receptor class B, is expressed on the surface of macrophages, monocytes, platelets and endothelial cells and play a central role in foam cell formation by oxidized LDL uptake during development of atherosclerosis and has recently been implicated in the development of insulin resistance as well [84]. Hexarelin, a growth hormone releasing peptide, binds to CD36 and not only facilitates macrophage targeting but also impedes foam cell formation [85]. In yet another approach, anti-CD36 antibody was anchored on the surface of MRI contrast agent containing lipid-based nanoparticles for imaging of atherosclerotic plaques [86]. Similarly, macrophages express several lectin-like receptors on their surface that aid in recognition of ligands commonly present on the surface of microbial pathogens and have recently been exploited for delivery of nanoparticles [87,88]. A much more indigenous approach to macrophage targeting was adapted recently where the glucan shell of baker's yeast was used as a delivery vector to successfully ferry the cargo to the macrophages. The

β1,3-D-glucan shells (2–4 μm) loaded with siRNA were successfully taken up by macrophages through dectin-1 receptor or other glucan receptor-mediated pathways upon oral delivery [89,90]. Several other macrophage and monocyte specific receptors have also been targeted using ligands such as peptides, antibody, antibody fragments, polysaccharides, etc. [91]. However, targeted therapy has evidently been underexploited against inflammatory diseases even though the approach has clearly shown promising results. 4. Nucleic acid therapy of inflammatory diseases 4.1. IBD therapy IBD is a disease of the gastrointestinal tract and therefore oral drug delivery approach with reduced systemic absorption is the most ideal way to present the therapeutic molecules at the site of action. To this end, several strategies have been adopted to design nanocarriers for specific delivery and absorption of drug through intestinal mucosa while reducing systemic availability by promotion of first pass metabolism. The drug delivery strategy for IBD has been reviewed in detail elsewhere [92] and this review will therefore focus on elucidating novel macrophage-targeted nucleic acid therapy to modulate disease prognosis. Macrophage population skewed in favor of M1 phenotype resulting in increased pro-inflammatory cytokines is a mainstay for all the inflammatory diseases including IBD and one of the earliest therapeutic strategies has been to restore the cytokine homeostasis at the site of inflammation. IL-10 gene therapy has been a popular choice because IL-10 cytokine has shown inflammation reduction in animal models but has failed to live up to the promise in clinical trials [93]. Conventional gene transfer approaches for treatment of IBD has been extensively reviewed elsewhere [94]. Bhavsar et al. developed a nanoparticles-in-microspheres oral system (NiMOS) for delivery of IL-10 gene in trinitrobenzenesulfonic acid (TNBS)-induced acute colitis Balb/c mice model. Their result indicated that IL-10 gene expression could successfully suppress the expression of pro-inflammatory cytokines including IFN-γ, TNF-α, IL-1α, IL-1β and IL-12 and demonstrated a favorable clinical score [95]. NiMOS technology was further employed for successful delivery of siRNA in dextran sulfate sodium (DSS) induced acute colitis mice model to silence TNF-α and subsequently downregulate the expression of IL-1β, IFN-γ and MCP-1 and reduce myeloperoxidase activity in vivo [96]. Likewise, this system was used for delivery of dual siRNA combination to silence TNF-α and cyclin-D1 independently and simultaneously to demonstrate the versatility of the delivery vehicle in oral therapy of IBD [96]. More recently, polyethylenimine (PEI)-nucleic acid complexes have been attempted for IBD therapy. PEI has high positive charge, which enables efficient electrostatic complexation with negatively charged nucleic acid and the complex thus formed can be loaded into a secondary delivery container to be ferried to the target of interest. Laroui et al. loaded CD98 siRNA/PEI complex into the polylactic acid nanoparticles and delivered it to the DSS-induced colitis model to demonstrate efficient delivery and gene silencing in vivo [97]. The same group previously used TNF-α siRNA loaded in same system to successful delivery in vivo [98]. Xiao et al. developed a mannosylated cationic polymer system to specifically target the mannose receptor on the surface of macrophages for delivery of TNF-α siRNA. Their study indicates that the nanoparticles were efficiently internalized inside macrophages, mediated silencing of TNF-α expression and therefore showed a promising anti-inflammatory effect in vitro as well as ex vivo [99]. Polymeric particles therefore have proved to be an efficient delivery system for nucleic acid delivery for treatment of inflammatory diseases [100]. Interestingly, though lipid-based nanoparticles have been extensively studied as delivery systems for nucleic acid therapy of several target diseases; there is a dearth of reports on their application in IBD therapy and targeting macrophages in particular. In one of the earlier attempts, Peer et al. used hyaluronan-stabilized liposomes targeted



against β7-integrin receptor overexpressed in circulating leukocytes [101]. Protamine was used to condense the cyclin D1 siRNA in order to achieve high loading efficiency (80%) without significantly increasing the size of the nanoparticles. Nanodelivery vector enabled delivery of ~ 4000 siRNA/particle compared to integrin targeted antibodyprotamine fusion protein delivery system, which could carry only 5 siRNA molecules per fusion protein [102]. Most importantly, the stabilized liposomal nanoparticles showed very high targeting efficiency, increased receptor-mediated uptake and efficient gene silencing in vivo. Similarly, Wilson et al. developed unique ROS responsive delivery system with poly (1,4-phenyleneacetone dimethylene thioketal) (PPADT) loaded with TNF-α siRNA complexed to 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) lipid. The thioketal group confers a ROS responsive behavior to the delivery system, which was administered orally to release its payload in response to the level of ROS at the site of inflammation in the intestines. Interestingly, the delivery vehicle is resistant to acidic, basic or high-protease environment, thus protecting the payload from the harsh conditions in the gastrointestinal tract. In vivo studies on DSS-induced colitis mice model revealed that the PPADT particles could successfully silence TNF-α expression along with other key proinflammatory markers like IL-6, IL-1 and IFN-γ and thus improve prognosis of the disease significantly [103]. In a more recent report, Attarwala et al. reported a water-in-oil-in-water (W-O-W) and nanoparticle-inemulsion (NiE) systems for delivery of IL-10 gene in vitro to J774.A1 macrophage cells. The NiE delivery system outperformed W-O-W and other controls with effective transfection efficiency, higher expression of IL-10 and subsequent downregulation of TNF-α and IL-1β pro-inflammatory cytokines in lipopolysaccharide stimulated macrophages [104]. 4.2. RA therapy In inflammatory conditions such as arthritis, pro-inflammatory cytokines are essential for disease progression. Thus, antibody and gene therapies directed against pro-inflammatory cytokines would be a rational therapeutic approach in RA. Several studies have investigated the use of adenoviral vectors for the treatment of RA [105–107]. However, only a moderate impact on severity and incidence of established arthritis has been observed with this approach [108]. Adenoviral vectors are also known to induce an immune response and hence alternative delivery strategies are continually being explored. Non-viral vectors are potential alternatives to the use of viral vectors for delivery of nuclei acid [109]. However, non-viral vectors are often associated with poor or low transfection efficiency. In order to overcome this issue, researchers have exploited various receptors on macrophages (mannose, scavengers, tuftsin, etc.) for improving transfection. Jain et al. recently evaluated macrophage-targeted tuftsin modified alginate nanoparticles for the delivery of murine interleukin-10 (mIL-10) [110]. These nanoparticles showed rapid internalization using the targeted particles with a significant effect on the levels of pro-inflammatory cytokines. Likewise, Fernandes et al. designed a chitosan based folate receptor targeted system for the delivery of interleukin-1 receptor antagonist (IL-1Ra) gene encoding plasmid DNA in rats with adjuvant-induced arthritis [111]. The targeted nanoparticles provided significant downregulation of expression of pro-inflammatory cytokines such as TNF-α and IL-1β, thus indicating the potential of non-viral gene delivery platforms for successful targeting of macrophages. Similar to antibody and naked plasmids based delivery, siRNA mediated knockdown of pro-inflammatory cytokines at an mRNA level is an alternative therapeutic strategy for inflammatory conditions. Some of the earlier work on siRNA mediated RA therapy showed the feasibility of direct intra-articular injection of TNF-α specific siRNA along with electroporation for reducing joint inflammation in mice [112]. Although these studies showed some degree of clinical efficacy, repeated intra-articular electrotransfer of siRNA was required for sustained local and systemic inflammatory response. Previously, neutral and anionic liposomal systems indicated their feasibility in delivering drugs to macrophages [113,114]

11

and inflammatory sites [115]. However due to the inability of these systems to deliver DNA, cationic liposomes have been investigated. Cationic liposomes have been used for the delivery of genes in cystic fibrosis [116] or endotoxic shock [117]. Fellowes et al. showed that a significant therapeutic effect was obtained in mice with CIA when ACHx/DCChol:DOPE liposomes containing IL-10 expression plasmid were used for in vivo gene delivery. The delivery system showed marked and prolonged (up to 30 days post injection) amelioration of inflammation in arthritic joints [118]. This concept of cationic liposomal-based delivery systems was also extended to the delivery of siRNA in RA. Anti-TNF siRNA formulated with RPR209120/dioleoylphosphatidylethanolamine (DOPE) cationic liposome showed decreases in local and systemic inflammation [119]. Likewise, anti-IL1-β; IL-6, IL-18 siRNA lipoplexes abrogated joint swelling and destruction of cartilage and bone [120]. Further, combination of the three siRNA's reduced all pathological features of CIA including improving overall anti-inflammatory parameters compared to siTNF lipoplex treatment indicating the possibility of a multi-pronged approach for the treatment of RA. Cationic lipid based nanoparticles have also been used for the systemic delivery of TNF-α specific siRNA, however the application of this route of delivery has been hindered due to systemic aggregation and clearance by liver [121]. I.p. injection has been sought as an alternative to systemic delivery [122,123], which enables therapeutic targeting to blood-free macrophage rich environment. This strategy has been previously investigated for murine sepsis [122] and antiviral host immunity in herpes simplex viral infections [121]. For the treatment of RA, Howard et al., reported development of chitosan/siRNA nanoparticles containing unmodified anti-TNA-α siRNA, which mediated ~ 66% TNF-α knockdown in primary peritoneal macrophage in vitro. In vivo downregulation of TNF-α-induced inflammatory responses and arrested joint swelling in CIA mice. As these nanoparticles have shown a decrease in local and systemic inflammation they provide a novel strategy for arthritis treatment [124]. Similar to siRNA-based therapy, microRNA (miRNA) based therapy of RA is a prospective therapeutic strategy. Aberrant expression of several miRNA's (miR-155, miR-146a, miR-9) has been observed in RA [125] with overexpression of miR-146a being characterized with chronic inflammation of the synovial tissue which leads to irreversible joint damage. Likewise miR-155 overexpression in synovial fibroblasts has also been associated with downregulation of MMP-1 and MMP-3, two molecules considered markers of the destructive and inflammatory synovial fibroblasts. miRNA based nanoparticles have been successfully explored for cancer therapy and thus provide promising avenues for RA therapy. Like siRNA and miRNA, ribozymes are also being explored as a potential therapeutic strategy in RA. Ribozyme also known as ribonucleic acid enzyme is an RNA molecule that has enzymatic properties to catalyze specific RNA cleavage, which reduces gene expression. Macrophage production of the potent pro-inflammatory molecule, TNF-α has been implicated in the pathogenesis of inflammation in arthritic joints. Kisich et al. previously investigated the use of anti-TNF-α ribozyme to selectively inhibit the secretion of TNF-α. However, as ribozymes are not normally expressed in mammalian cells, they have to be incorporated in delivery systems to enable them to enter the cytoplasm or the nucleus to achieve gene silencing. In order to deliver the ribozymes Kisich et al. developed cationic lipid mediated delivery systems to deliver the RNA molecules to cells in vitro and in vivo to achieve suppression of TNF-α production [126]. Following intraperitoneal administration, the ribozymes accumulated in peritoneal macrophages and TNF-α release in response to LPS was reduced. Thus combination of nuclei acid therapy with nanoparticles holds a promising approach for the treatment of RA. 4.3. Atherosclerosis therapy In atherosclerosis, macrophage dominant maladaptive inflammatory response develops as a reaction to subendothelial retention and


12


modification of apolipoprotein B containing lipoprotein [127]. In all stages of atherosclerotic lesions, classically M1 subsets of macrophage secrete inflammatory cytokines and other molecules, which contribute to lesion progression. In advanced lesions these macrophages are ineffectively cleared by apoptosis or phagocytosis, which leads to plaque necrosis, plaque disruption and acute thrombosis. On the other hand, induction of macrophage apoptosis would result in lesion cellularity and promotion of plaque necrosis. Several targeted nanoparticle approaches have been explored for imaging of macrophages in atherosclerosis [128, 129]. Unique approaches are being investigated to non-invasively detect apoptosis in smooth muscle cells and macrophages. Marrache et al., reported the construction of a synthetic, biodegradable HDL-NP for detection of vulnerable plaques [130]. The system comprised an HDL resembling hydrophobic core of PLGA and cholesteryl oleate and PLGA-b-PEG-QD for optical imaging. The core was surrounded by 1,2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG-COOH lipid layer embedded with cholesterol apoA-I mimetic 4F peptide with a FAEKFKEAVKDYFAKFWD sequence and a triphenyl phosphonium (TPP) cation containing stearyl-TPP ligand for targeting the collapse of mitochondrial membrane potential observed during apoptosis. This delivery system indicated promising detection ability and therapeutic potential of TPP-HDL-apoA-I-QD NP's. Recently, a few papers have examined the role of miRNA-based therapy in atherosclerosis [131,132]. However, only a few reports using nanoparticle-mediated delivery of miRNA have been reported in cardiovascular therapy. As nanoparticle based approach for the delivery of miRNA and antimir's has been extensively developed for other inflammatory conditions and cancer this is a viable option to be explored in animal models of atherosclerosis in the near future. Chistiakov et al. have recently published a detailed account of miR's potential for therapy of cardiovascular disease and is highly recommended to the reader [133]. siRNA based therapeutic approach has also been investigated for the treatment of atherosclerosis. Previously Aouadi et al. reported the engineering of β1,3-D-Glucan-encapsulated siRNA particles as efficient oral delivery vehicles for targeting macrophages [89]. Likewise Peer et al. investigated targeted stabilized nanoparticles loaded with si-cyclinD1 functionalized with b7 integrin for targeting specific leukocyte subsets [101]. Tian et al. stably transfected macrophages with siRNA to inhibit the expression of allograft inflammatory factor 1 (AIF1). AIF-1 is a inflammation-responsive cytoplasmic protein and studies have indicated its increased levels in activated macrophages. siRNA therapy successfully reduced AIF-1 protein expression by 79% thereby reducing macrophage migration, proliferation and signal transduction capability in response to atherogenic stimuli [134]. Leuschner et al. extended this concept of cell specific siRNA delivery to target CCR2 mRNA expression. The authors reported the preparation of si-CCR2 nanoparticle for efficient degradation CCR2 mRNA that encodes for the synthesis of the chemokine receptor CCR2 that is responsible for migration of inflammatory monocyte substrate [135]. In atherosclerotic plaque this downregulation resulted in a decrease in infarct size after coronary artery occlusion, prolonged normoglycemia in diabetic mice after pancreatic islet transplantation and reduced tumor volumes and tumor-associated macrophages. Thus nanoparticle based nuclei delivery approach needs to be investigated further to gain a better insight in the management of atherosclerosis. 4.4. Therapy against diabetes Insulin resistance refers to a decreased capacity of circulating insulin to regulate nutrient metabolism, which predisposes people to Type 2 diabetes. It was previously observed that high doses of anti-inflammatory agents such as sodium salicylate and aspirin could be used to inhibit nuclear factor kappa B (NF-κB) and its upstream activator IkB kinase β (IKKβ). However, this approach did not only provide an antiinflammatory effect but helped to improve insulin sensitivity in vivo [136]. In contrast, blocking pro-inflammatory cytokines using antibodies

or antagonists provided conflicting outcomes for overcoming insulin resistance in humans [137,138]. It was presumed that poor penetration into certain tissues where these pro-inflammatory cytokines are produced could be an explanation for lack of therapy. Lately it was observed that adipose tissue inflammation and infiltration by macrophages is often associated with resistance and type 2 diabetes. Aouadi et al., recently reported the development of siRNA-Endo-Porter complexes encapsulated in glucan nanocomplexes (GeRP's), which were specifically targeted to macrophages [139,140]. It was observed that following i.p. administration in obese mice, gene silencing was specifically observed in epididymal ATM, whereas macrophages within lungs, spleen, kidney, heart, skeletal muscle, subcutaneous adipose tissue and liver were not targeted. The down-regulation in TNF-a and osteopontin also caused a significant improvement in glucose tolerance indicating the importance of ATM cytokines in exacerbating whole body glucose tolerance. The dearth of reports on macrophage-targeted nucleic acid therapy of diabetes despite the acknowledgment of several potential targets shows that the field has been highly under-explored and such studies in future can tremendously benefit the clinical outcome of the disease. 4.5. Therapy against cancer It has been well established that TAMs promote inflammation, tumor growth and development, angiogenesis, remodeling of ECM and metastasis and thus they can be excellent targets for anti-cancer therapy. Various anti-macrophage therapies such as receptor antagonists, inhibitors and antibodies have been undertaken to disrupt the activity of different cytokines and chemokines that play key roles in augmentation of tumors but this review will focus exclusively on nucleic acid therapy. One of the earlier attempts have relied on the use of engineered macrophages to express factors that neutralize or negate the tumorigenic behavior. There are substantial evidences indicating that macrophages tend to accumulate in the hypoxic avascular region of the tumor. Carta et al. designed a hypoxia responsive construct using HRE3x-Tk promoter and IFN-γ gene that could be successfully transfected in murine macrophages. Exposure of the transfected macrophages to a hypoxic environment confirmed marked increase in IFN-γ mRNA level, subsequent protein level and other related downstream molecular responses [141]. IFN-γ is a strong stimulator of macrophage polarization and induces proinflammatory cytotoxic M2 phenotype, which can potentially restore the otherwise suppressed immune response within the tumor. Satoh et al. transfected macrophages with murine IL-12 recombinant adenoviral vector, which upon intratumoral injection in orthotopic 178-2 BMA mice showed increased expression of MHC classes I and II as well as F4/80 antigen. Tumor analysis further revealed enhanced infiltration of CD4+ and CD8+ T cells and NK cells, thereby increasing the cumulative immune response and significant reduction in tumor burden [142]. More recently, a similar approach was adopted for macrophage transfection with Glipr1 gene in adenoviral vector leading to higher expression of CD40, CD80, MHC class II molecules, IL-12 and IL-6 in vitro and increased suppression of prostate tumor growth and lung cancer metastasis in vivo [143]. Gene transfection using engineered macrophages to boost immune response in the tumor therefore appears to be a promising approach for clinical translation. Monocyte recruitment and infiltration is another key process to tumor-associated inflammation and is regulated primarily by expression of CSF-1. Aharinejad et al. hypothesized that blocking CSF-1 or its receptor using antisense technology and siRNA interference would inhibit the recruitment process, resulting in poor infiltration and thus the tumor growth. Treatment of MCF-7 mammary carcinoma xenograft mouse model either with mouse CSF-1 antisense oligonucleotide or siRNA interfering with CSF-1 expression or c-fms gene indeed showed growth suppression. Further analysis revealed a reduced macrophage infiltration, MMP-12, MMP-2 and VEGF-A expression and endothelial cell proliferation in treated mice [144]. Macrophage polarization is another key aspect of anti-cancer therapy and existing evidences confirm



that tumor infiltration macrophages are M2 polarized, equipped with immunosuppressive, tissue repair and remodeling capabilities. Studies have indicated that activation of toll-like receptors (TLR) such as TLR9 promote cytotoxic M1 population of macrophages, thereby reinstating the immune response against tumor cells. TLR9 receptors are localized in the endosomal compartment of B cells and dendritic cells, where they are activated by unmethylated CpG dinucleotide motifs commonly prevalent in bacterial and viral genomes but largely suppressed or methylated in vertebrate genomes [145]. Though not targeted specifically for macrophage therapy, the use of CpG oligodendronucleotides mediates restoration of M1 macrophage phenotype and adaptive immune response in tumors and has shown anti-tumor effect alone or in combination with other chemotherapeutic drugs [146]. More recently, a combination of CpG activation of TLR9 and siRNA mediated silencing of signal transducer and activator of transcription 3 (Stat3) demonstrated augmentation of CD8+ T cells and dendritic cells and improved antitumor response in vivo [147,148]. Similarly, CCR-2 inhibiting siRNA encapsulated in lipid nanoparticles showed reduction in the TAM population in lymphoma EL4 cells bearing tumors [135]. Despite these positive outcomes from nucleic acid therapy, the application of nanoparticle system to selectively target and improve delivery to TAMs has not yet been explored. However, given the advances in nucleic acid therapy using advanced drug delivery vectors, this direction of research certainly holds promise in improving the existing cancer therapy alternatives. Table 2 summarizes the nucleic acid therapy approaches for macrophage specific therapeutic intervention against IBD, RA, atherosclerosis and cancer. 5. Conclusions Inflammation is a natural adaptive process in the body to cope with pathogen infection or tissue injury that is very tightly regulated by the components of the innate and adaptive immune systems. Macrophages particularly are ubiquitously distributed throughout the body and play a vital role in containment of any physical or metabolic insults to the body and reinstating homeostasis. The phenotypic status of the resident macrophages of a tissue is essentially governed by the metabolic status in the local environment. In the event of injury or infection, they are the

13

first line of immune cells along with dendritic cells to get activated, phagocytose anything “foreign” and function as antigen presenting cell to initiate a cascade of immune responses. Inflammation is an integral part of this process, which is delicately controlled by a complex and versatile crosstalk between structural cells and the immune effector cells, involving several cytokines and chemokines. The autoimmune response or chronic infection however subverts this balance resulting in chronic inflammatory process and disease condition involving pro-inflammatory M1 macrophages and subsequent activation of TH1 and TH17 mediated immune response. Inflammatory bowel disease, rheumatoid arthritis, atherosclerosis and diabetes are four major conditions that result from activation of a chronic inflammatory response. All inflammatory diseases also show a markedly decreased population of anti-inflammatory M2 phenotype of macrophages that exhibit immunosuppressive, tissue repair and remodeling functions. Alternatively, in cancer, TAMs exhibiting a M2 phenotype are predominant while the cytotoxic pro-inflammatory M1 polarization is diminished resulting in immunosuppression and tumor progression. Macrophages thus play a central role in regulating the inflammation process and are therefore prime target for therapeutic intervention. Increase of pro-inflammatory and decrease of anti-inflammatory markers are characteristics of IDs and this altered physiological profile is closely related to differential gene expression. Besides, IDs themselves show a close connection to genetic level dysfunction. Recent studies have further demonstrated the successful repolarization of macrophage phenotype by microRNA intervention, which could be key to reversing the pro-inflammatory population in IDs and immunosuppressive population in cancer [149]. Nucleic acid therapy therefore is a promising approach for treatment of inflammatory diseases. While gene therapy has been applied to increasing the expression of regulators responsible for decreasing inflammatory condition, antisense therapy has been exploited to selectively interfere with the expression of molecular targets that promote inflammation. Nucleic acid therapy though is challenging in in vivo settings, the advent of novel nanodelivery systems and ability to guide the therapeutic molecule to a specific target have led to dramatic advances in controlling the fate of a diseased condition. Macrophage-targeted nucleic acid therapy using novel nanodelivery systems therefore certainly shows tremendous promise.

Table 2 Macrophage-specific nucleic acid therapeutic approach against inflammatory diseases. Disease

Target

Therapeutic molecule

Delivery vector

Reference

IBD

IL-10 TNF-α TNF-α, Cyclin-D1 CD98, TNF-α TNF-α Cyclin-D1 TNF-α IL-10 IL-10 IL-1Ra IL-10 TNF IL-1β, IL-6, IL-18 TNF-α TNF-α – AIF-1 CCR-2 TNF-α, Osteopontin CCR-2 IFN-γ IL-12 Glipr1 CSF-1 TLR9 TLR9, Stat3 CCR-2

Gene siRNA siRNA siRNA siRNA siRNA siRNA Gene Gene Gene Gene siRNA siRNA siRNA Ribozyme HDL siRNA siRNA siRNA siRNA Gene Gene Gene Antisense nucleotide/siRNA CpG oligonucleotide CpG oligonucleotide, siRNA siRNA

Polymer Polymer Polymer Polymer Polymer, mannose-targeted Liposome, β7-integrin targeted Lipid Polymer Polymer Polymer, folate targeted Liposomes Liposome Lipid Polymer Lipid Polymer–lipid Lipid Lipid Glucan particles Lipid Adenovirus Adenovirus Adenovirus Lipid – – Lipid

[95] [96] [96] [97,98] [99] [101,102] [103] [104] [110] [111] [118] [119] [120] [124] [126] [130] [134] [135] [140] [135] [141] [142] [143] [144] [146] [147,148] [135]

RA

Atherosclerosis

Diabetes Cancer


14


References [1] R. Medzhitov, Origin and physiological roles of inflammation, Nature 454 (2008) 428–435. [2] P.J. Murray, T.A. Wynn, Protective and pathogenic functions of macrophage subsets, Nat. Rev. Immunol. 11 (2011) 723–737. [3] D.M. Mosser, J.P. Edwards, Exploring the full spectrum of macrophage activation, Nat. Rev. Immunol. 8 (2008) 958–969. [4] T. Lawrence, G. Natoli, Transcriptional regulation of macrophage polarization: enabling diversity with identity, Nat. Rev. Immunol. 11 (2011) 750–761. [5] T.A. Wynn, A. Chawla, J.W. Pollard, Macrophage biology in development, homeostasis and disease, Nature 496 (2013) 445–455. [6] L.E. Smythies, M. Sellers, R.H. Clements, M. Mosteller-Barnum, G. Meng, W.H. Benjamin, J.M. Orenstein, P.D. Smith, Human intestinal macrophages display profound inflammatory anergy despite avid phagocytic and bacteriocidal activity, J. Clin. Invest. 115 (2005) 66–75. [7] H.S. Carlsen, T. Yamanaka, H. Scott, J. Rugtveit, P. Brandtzaeg, The proportion of CD40+ mucosal macrophages is increased in inflammatory bowel disease whereas CD40 ligand (CD154) + T cells are relatively decreased, suggesting differential modulation of these costimulatory molecules in human gut lamina propria, Inflamm. Bowel Dis. 12 (2006) 1013–1024. [8] O. Grip, A. Bredberg, S. Lindgren, G. Henriksson, Increased subpopulations of CD16(+) and CD56(+) blood monocytes in patients with active Crohn's disease, Inflamm. Bowel Dis. 13 (2007) 566–572. [9] J. Rugtveit, A. Bakka, P. Brandtzaeg, Differential distribution of B7.1 (CD80) and B7. 2 (CD86) costimulatory molecules on mucosal macrophage subsets in human inflammatory bowel disease (IBD), Clin. Exp. Immunol. 110 (1997) 104–113. [10] I.B. McInnes, G. Schett, Cytokines in the pathogenesis of rheumatoid arthritis, Nat. Rev. Immunol. 7 (2007) 429–442. [11] M. Heusinkveld, S.H. van der Burg, Identification and manipulation of tumor associated macrophages in human cancers, J. Transl. Med. 9 (2011) 216. [12] S.B. Hanauer, Inflammatory bowel disease: epidemiology, pathogenesis, and therapeutic opportunities, Inflamm. Bowel Dis. 12 (Suppl. 1) (2006) S3–S9. [13] D.K. Podolsky, Inflammatory bowel disease, N. Engl. J. Med. 347 (2002) 417–429. [14] Y.R. Mahida, S. Patel, P. Gionchetti, D. Vaux, D.P. Jewell, Macrophage subpopulations in lamina propria of normal and inflamed colon and terminal ileum, Gut 30 (1989) 826–834. [15] Y.R. Mahida, The key role of macrophages in the immunopathogenesis of inflammatory bowel disease, Inflamm. Bowel Dis. 6 (2000) 21–33. [16] E.M. Brown, M. Sadarangani, B.B. Finlay, The role of the immune system in governing host-microbe interactions in the intestine, Nat. Immunol. 14 (2013) 660–667. [17] L.E. Harrington, R.D. Hatton, P.R. Mangan, H. Turner, T.L. Murphy, K.M. Murphy, C.T. Weaver, Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages, Nat. Immunol. 6 (2005) 1123–1132. [18] Y. Iwakura, H. Ishigame, The IL-23/IL-17 axis in inflammation, J. Clin. Invest. 116 (2006) 1218–1222. [19] J.R. Korzenik, D.K. Podolsky, Evolving knowledge and therapy of inflammatory bowel disease, Nat. Rev. Drug Discov. 5 (2006) 197–209. [20] J. Satsangi, J. Morecroft, N.B. Shah, E. Nimmo, Genetics of inflammatory bowel disease: scientific and clinical implications, Best Pract. Res. Clin. Gastroenterol. 17 (2003) 3–18. [21] Y. Ogura, D.K. Bonen, N. Inohara, D.L. Nicolae, F.F. Chen, R. Ramos, H. Britton, T. Moran, R. Karaliuskas, R.H. Duerr, J.P. Achkar, S.R. Brant, T.M. Bayless, B.S. Kirschner, S.B. Hanauer, G. Nunez, J.H. Cho, A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease, Nature 411 (2001) 603–606. [22] T. Hisamatsu, M. Suzuki, H.C. Reinecker, W.J. Nadeau, B.A. McCormick, D.K. Podolsky, CARD15/NOD2 functions as an antibacterial factor in human intestinal epithelial cells, Gastroenterology 124 (2003) 993–1000. [23] A.M. Lamhonwah, J. Skaug, S.W. Scherer, I. Tein, A third human carnitine/organic cation transporter (OCTN3) as a candidate for the 5q31 Crohn's disease locus (IBD5), Biochem. Biophys. Res. Commun. 301 (2003) 98–101. [24] M. Stoll, B. Corneliussen, C.M. Costello, G.H. Waetzig, B. Mellgard, W.A. Koch, P. Rosenstiel, M. Albrecht, P.J. Croucher, D. Seegert, S. Nikolaus, J. Hampe, T. Lengauer, S. Pierrou, U.R. Foelsch, C.G. Mathew, M. Lagerstrom-Fermer, S. Schreiber, Genetic variation in DLG5 is associated with inflammatory bowel disease, Nat. Genet. 36 (2004) 476–480. [25] E.O. Glocker, D. Kotlarz, K. Boztug, E.M. Gertz, A.A. Schaffer, F. Noyan, M. Perro, J. Diestelhorst, A. Allroth, D. Murugan, N. Hatscher, D. Pfeifer, K.W. Sykora, M. Sauer, H. Kreipe, M. Lacher, R. Nustede, C. Woellner, U. Baumann, U. Salzer, S. Koletzko, N. Shah, A.W. Segal, A. Sauerbrey, S. Buderus, S.B. Snapper, B. Grimbacher, C. Klein, Inflammatory bowel disease and mutations affecting the interleukin-10 receptor, N. Engl. J. Med. 361 (2009) 2033–2045. [26] N.A. Athanasou, Synovial macrophages, Ann. Rheum. Dis. 54 (1995) 392–394. [27] A. Gierut, H. Perlman, R.M. Pope, Innate immunity and rheumatoid arthritis, Rheum. Dis. Clin. N. Am. 36 (2010) 271–296. [28] N. Maruotti, F.P. Cantatore, E. Crivellato, A. Vacca, D. Ribatti, Macrophages in rheumatoid arthritis, Histol. Histopathol. 22 (2007) 581–586. [29] I.B. McInnes, B.P. Leung, M. Field, X.Q. Wei, F.P. Huang, R.D. Sturrock, A. Kinninmonth, J. Weidner, R. Mumford, F.Y. Liew, Production of nitric oxide in the synovial membrane of rheumatoid and osteoarthritis patients, J. Exp. Med. 184 (1996) 1519–1524. [30] H. von den Hoff, M. de Koning, J. van Kampen, J. van der Korst, Interleukin-1 reversibly inhibits the synthesis of biglycan and decorin in intact articular cartilage in culture, J. Rheumatol. 22 (1995) 1520–1526.

[31] J.B. Allen, H.L. Wong, G.L. Costa, M.J. Bienkowski, S.M. Wahl, Suppression of monocyte function and differential regulation of IL-1 and IL-1ra by IL-4 contribute to resolution of experimental arthritis, J. Immunol. 151 (1993) 4344–4351. [32] M. Feldmann, F.M. Brennan, R.N. Maini, Role of cytokines in rheumatoid arthritis, Annu. Rev. Immunol. 14 (1996) 397–440. [33] B. Bresnihan, The safety and efficacy of interleukin-1 receptor antagonist in the treatment of rheumatoid arthritis, Semin. Arthritis Rheum. 30 (2001) 17–20. [34] K.J. Moore, F.J. Sheedy, E.A. Fisher, Macrophages in atherosclerosis: a dynamic balance, Nat. Rev. Immunol. 13 (2013) 709–721. [35] F.R. Maxfield, I. Tabas, Role of cholesterol and lipid organization in disease, Nature 438 (2005) 612–621. [36] L. Yvan-Charvet, N. Wang, A.R. Tall, Role of HDL, ABCA1, and ABCG1 transporters in cholesterol efflux and immune responses, Arterioscler. Thromb. Vasc. Biol. 30 (2010) 139–143. [37] A.C. Calkin, P. Tontonoz, Transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR, Nat. Rev. Mol. Cell Biol. 13 (2012) 213–224. [38] L. Yvan-Charvet, M. Ranalletta, N. Wang, S. Han, N. Terasaka, R. Li, C. Welch, A.R. Tall, Combined deficiency of ABCA1 and ABCG1 promotes foam cell accumulation and accelerates atherosclerosis in mice, J. Clin. Invest. 117 (2007) 3900–3908. [39] Y. Yuan, P. Li, J. Ye, Lipid homeostasis and the formation of macrophage-derived foam cells in atherosclerosis, Protein Cell 3 (2012) 173–181. [40] S. Devaraj, N. Glaser, S. Griffen, J. Wang-Polagruto, E. Miguelino, I. Jialal, Increased monocytic activity and biomarkers of inflammation in patients with type 1 diabetes, Diabetes 55 (2006) 774–779. [41] M. Jagannathan-Bogdan, M.E. McDonnell, H. Shin, Q. Rehman, H. Hasturk, C.M. Apovian, B.S. Nikolajczyk, Elevated proinflammatory cytokine production by a skewed T cell compartment requires monocytes and promotes inflammation in type 2 diabetes, J. Immunol. 186 (2011) 1162–1172. [42] B. Vandanmagsar, Y.H. Youm, A. Ravussin, J.E. Galgani, K. Stadler, R.L. Mynatt, E. Ravussin, J.M. Stephens, V.D. Dixit, The NLRP3 inflammasome instigates obesityinduced inflammation and insulin resistance, Nat. Med. 17 (2011) 179–188. [43] J.H. Pedra, S.L. Cassel, F.S. Sutterwala, Sensing pathogens and danger signals by the inflammasome, Curr. Opin. Immunol. 21 (2009) 10–16. [44] M.Y. Donath, S.E. Shoelson, Type 2 diabetes as an inflammatory disease, Nat. Rev. Immunol. 11 (2011) 98–107. [45] J. Liu, A. Divoux, J. Sun, J. Zhang, K. Clement, J.N. Glickman, G.K. Sukhova, P.J. Wolters, J. Du, C.Z. Gorgun, A. Doria, P. Libby, R.S. Blumberg, B.B. Kahn, G.S. Hotamisligil, G.P. Shi, Genetic deficiency and pharmacological stabilization of mast cells reduce diet-induced obesity and diabetes in mice, Nat. Med. 15 (2009) 940–945. [46] Y. Ilan, R. Maron, A.M. Tukpah, T.U. Maioli, G. Murugaiyan, K. Yang, H.Y. Wu, H.L. Weiner, Induction of regulatory T cells decreases adipose inflammation and alleviates insulin resistance in ob/ob mice, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 9765–9770. [47] M. Feuerer, L. Herrero, D. Cipolletta, A. Naaz, J. Wong, A. Nayer, J. Lee, A.B.. Goldfine, C. Benoist, S. Shoelson, D. Mathis, Lean, but not obese, fat is enriched for a unique population of regulatory T cells that affect metabolic parameters, Nat. Med. 15 (2009) 930–939. [48] M.Y. Donath, J. Storling, K. Maedler, T. Mandrup-Poulsen, Inflammatory mediators and islet beta-cell failure: a link between type 1 and type 2 diabetes, J. Mol. Med. 81 (2003) 455–470. [49] S.L. Masters, A. Dunne, S.L. Subramanian, R.L. Hull, G.M. Tannahill, F.A. Sharp, C. Becker, L. Franchi, E. Yoshihara, Z. Chen, N. Mullooly, L.A. Mielke, J. Harris, R.C. Coll, K.H. Mills, K.H. Mok, P. Newsholme, G. Nunez, J. Yodoi, S.E. Kahn, E.C. Lavelle, L.A. O'Neill, Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1beta in type 2 diabetes, Nat. Immunol. 11 (2010) 897–904. [50] M.Y. Donath, D.M. Schumann, M. Faulenbach, H. Ellingsgaard, A. Perren, J.A. Ehses, Islet inflammation in type 2 diabetes: from metabolic stress to therapy, Diabetes Care 31 (Suppl. 2) (2008) S161–S164. [51] B. Burke, A. Giannoudis, K.P. Corke, D. Gill, M. Wells, L. Ziegler-Heitbrock, C.E. Lewis, Hypoxia-induced gene expression in human macrophages: implications for ischemic tissues and hypoxia-regulated gene therapy, Am. J. Pathol. 163 (2003) 1233–1243. [52] D. Hanahan, R.A. Weinberg, The hallmarks of cancer, Cell 100 (2000) 57–70. [53] J. Condeelis, J.W. Pollard, Macrophages: obligate partners for tumor cell migration, invasion, and metastasis, Cell 124 (2006) 263–266. [54] G. Solinas, G. Germano, A. Mantovani, P. Allavena, Tumor-associated macrophages (TAM) as major players of the cancer-related inflammation, J. Leukoc. Biol. 86 (2009) 1065–1073. [55] F. Balkwill, A. Mantovani, Inflammation and cancer: back to Virchow? Lancet 357 (2001) 539–545. [56] H.J. Knowles, A.L. Harris, Macrophages and the hypoxic tumour microenvironment, Front. Biosci. 12 (2007) 4298–4314. [57] A. Mantovani, P. Allavena, A. Sica, F. Balkwill, Cancer-related inflammation, Nature 454 (2008) 436–444. [58] R.C. Ji, Lymphatic endothelial cells, tumor lymphangiogenesis and metastasis: new insights into intratumoral and peritumoral lymphatics, Cancer Metastasis Rev. 25 (2006) 677–694. [59] A.K. Bonde, V. Tischler, S. Kumar, A. Soltermann, R.A. Schwendener, Intratumoral macrophages contribute to epithelial-mesenchymal transition in solid tumors, BMC Cancer 12 (2012) 35. [60] E. Voronov, D.S. Shouval, Y. Krelin, E. Cagnano, D. Benharroch, Y. Iwakura, C.A. Dinarello, R.N. Apte, IL-1 is required for tumor invasiveness and angiogenesis, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 2645–2650.


A. Singh et al. / Journal of Controlled Release xxx (2014) xxx–xxx [61] J.B. Opalinska, A.M. Gewirtz, Nucleic-acid therapeutics: basic principles and recent applications, Nat. Rev. Drug Discov. 1 (2002) 503–514. [62] M. Harada-Shiba, K. Yamauchi, A. Harada, I. Takamisawa, K. Shimokado, K. Kataoka, Polyion complex micelles as vectors in gene therapy–pharmacokinetics and in vivo gene transfer, Gene Ther. 9 (2002) 407–414. [63] T. Bieber, W. Meissner, S. Kostin, A. Niemann, H.P. Elsasser, Intracellular route and transcriptional competence of polyethylenimine-DNA complexes, J. Control. Release 82 (2002) 441–454. [64] J.P. Leonetti, N. Mechti, G. Degols, C. Gagnor, B. Lebleu, Intracellular distribution of microinjected antisense oligonucleotides, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 2702–2706. [65] C.M. Roth, S. Sundaram, Engineering synthetic vectors for improved DNA delivery: insights from intracellular pathways, Annu. Rev. Biomed. Eng. 6 (2004) 397–426. [66] K. Kariko, P. Bhuyan, J. Capodici, D. Weissman, Small interfering RNAs mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3, J. Immunol. 172 (2004) 6545–6549. [67] M. Elsabahy, A. Nazarali, M. Foldvari, Non-viral nucleic acid delivery: key challenges and future directions, Curr. Drug Deliv. 8 (2011) 235–244. [68] S. Ganta, H. Devalapally, A. Shahiwala, M. Amiji, A review of stimuli-responsive nanocarriers for drug and gene delivery, J. Control. Release 126 (2008) 187–204. [69] A.K. Iyer, A. Singh, S. Ganta, M.M. Amiji, Role of integrated cancer nanomedicine in overcoming drug resistance, Adv. Drug Deliv. Rev. 65 (2013) 1784–1802. [70] S.M. Moghimi, A.C. Hunter, J.C. Murray, Long-circulating and target-specific nanoparticles: theory to practice, Pharmacol. Rev. 53 (2001) 283–318. [71] M. Caldorera-Moore, N. Guimard, L. Shi, K. Roy, Designer nanoparticles: incorporating size, shape and triggered release into nanoscale drug carriers, Expert Opin. Drug Deliv. 7 (2010) 479–495. [72] E. Frohlich, The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles, Int. J. Nanomedicine 7 (2012) 5577–5591. [73] C.E. Thomas, A. Ehrhardt, M.A. Kay, Progress and problems with the use of viral vectors for gene therapy, Nat. Rev. Genet. 4 (2003) 346–358. [74] F. Chellat, Y. Merhi, A. Moreau, L. Yahia, Therapeutic potential of nanoparticulate systems for macrophage targeting, Biomaterials 26 (2005) 7260–7275. [75] T. Kean, M. Thanou, Biodegradation, biodistribution and toxicity of chitosan, Adv. Drug Deliv. Rev. 62 (2010) 3–11. [76] H. Ulbrich, E.E. Eriksson, L. Lindbom, Leukocyte and endothelial cell adhesion molecules as targets for therapeutic interventions in inflammatory disease, Trends Pharmacol. Sci. 24 (2003) 640–647. [77] N. Nakashima-Matsushita, T. Homma, S. Yu, T. Matsuda, N. Sunahara, T. Nakamura, M. Tsukano, M. Ratnam, T. Matsuyama, Selective expression of folate receptor beta and its possible role in methotrexate transport in synovial macrophages from patients with rheumatoid arthritis, Arthritis Rheum. 42 (1999) 1609–1616. [78] F. Antohe, L. Radulescu, E. Puchianu, M.D. Kennedy, P.S. Low, M. Simionescu, Increased uptake of folate conjugates by activated macrophages in experimental hyperlipemia, Cell Tissue Res. 320 (2005) 277–285. [79] W. Ayala-Lopez, W. Xia, B. Varghese, P.S. Low, Imaging of atherosclerosis in apoliprotein e knockout mice: targeting of a folate-conjugated radiopharmaceutical to activated macrophages, J. Nucl. Med. 51 (2010) 768–774. [80] U. Bilthariya, N. Jain, V. Rajoriya, A.K. Jain, Folate-conjugated albumin nanoparticles for rheumatoid arthritis-targeted delivery of etoricoxib, Drug Dev. Ind. Pharm. (2013), http://dx.doi.org/10.3109/03639045.2013.850705. [81] T.P. Thomas, S.N. Goonewardena, I.J. Majoros, A. Kotlyar, Z. Cao, P.R. Leroueil, J.R. Baker Jr., Folate-targeted nanoparticles show efficacy in the treatment of inflammatory arthritis, Arthritis Rheum. 63 (2011) 2671–2680. [82] J.W. van der Heijden, R. Oerlemans, B.A. Dijkmans, H. Qi, C.J. van der Laken, W.F. Lems, A.L. Jackman, M.C. Kraan, P.P. Tak, M. Ratnam, G. Jansen, Folate receptor beta as a potential delivery route for novel folate antagonists to macrophages in the synovial tissue of rheumatoid arthritis patients, Arthritis Rheum. 60 (2009) 12–21. [83] Y. Furusho, M. Miyata, T. Matsuyama, T. Nagai, H. Li, Y. Akasaki, N. Hamada, T. Miyauchi, Y. Ikeda, T. Shirasawa, K. Ide, C. Tei, Novel therapy for atherosclerosis using recombinant immunotoxin against folate receptor beta-expressing macrophages, J. Am. Heart Assoc. 1 (2012) e003079. [84] J.F. Glatz, Y. Angin, L.K. Steinbusch, R.W. Schwenk, J.J. Luiken, CD36 as a target to prevent cardiac lipotoxicity and insulin resistance, Prostaglandins Leukot. Essent. Fat. Acids 88 (2013) 71–77. [85] R. Avallone, A. Demers, A. Rodrigue-Way, K. Bujold, D. Harb, S. Anghel, W. Wahli, S. Marleau, H. Ong, A. Tremblay, A growth hormone-releasing peptide that binds scavenger receptor CD36 and ghrelin receptor up-regulates sterol transporters and cholesterol efflux in macrophages through a peroxisome proliferatoractivated receptor gamma-dependent pathway, Mol. Endocrinol. 20 (2006) 3165–3178. [86] M.J. Lipinski, J.C. Frias, V. Amirbekian, K.C. Briley-Saebo, V. Mani, D. Samber, A. Abbate, J.G. Aguinaldo, D. Massey, V. Fuster, G.W. Vetrovec, Z.A. Fayad, Macrophage-specific lipid-based nanoparticles improve cardiac magnetic resonance detection and characterization of human atherosclerosis, J. Am. Coll. Cardiol. Img. 2 (2009) 637–647. [87] Y. Chao, P.P. Karmali, D. Simberg, Role of carbohydrate receptors in the macrophage uptake of dextran-coated iron oxide nanoparticles, Adv. Exp. Med. Biol. 733 (2012) 115–123. [88] Y. Chao, P.P. Karmali, R. Mukthavaram, S. Kesari, V.L. Kouznetsova, I.F. Tsigelny, D. Simberg, Direct recognition of superparamagnetic nanocrystals by macrophage scavenger receptor SR-AI, ACS Nano 7 (2013) 4289–4298. [89] M. Aouadi, G.J. Tesz, S.M. Nicoloro, M. Wang, M. Chouinard, E. Soto, G.R. Ostroff, M. P. Czech, Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation, Nature 458 (2009) 1180–1184.

15

[90] M. Aouadi, M. Tencerova, P. Vangala, J.C. Yawe, S.M. Nicoloro, S.U. Amano, J.L. Cohen, M.P. Czech, Gene silencing in adipose tissue macrophages regulates whole-body metabolism in obese mice, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 8278–8283. [91] C. Kelly, C. Jefferies, S.A. Cryan, Targeted liposomal drug delivery to monocytes and macrophages, J. Drug Deliv. 2011 (2011) 727241. [92] F. Kesisoglou, E.M. Zimmermann, Novel drug delivery strategies for the treatment of inflammatory bowel disease, Expert Opin. Drug Deliv. 2 (2005) 451–463. [93] H. Braat, M.P. Peppelenbosch, D.W. Hommes, Interleukin-10-based therapy for inflammatory bowel disease, Expert. Opin. Biol. Ther. 3 (2003) 725–731. [94] S. Wirtz, M.F. Neurath, Gene transfer approaches for the treatment of inflammatory bowel disease, Gene Ther. 10 (2003) 854–860. [95] M.D. Bhavsar, M.M. Amiji, Oral IL-10 gene delivery in a microsphere-based formulation for local transfection and therapeutic efficacy in inflammatory bowel disease, Gene Ther. 15 (2008) 1200–1209. [96] C. Kriegel, M.M. Amiji, Dual TNF-alpha/cyclin D1 gene silencing with an oral polymeric microparticle system as a novel strategy for the treatment of inflammatory bowel disease, Clin. Transl. Gastroenterol. 2 (2011) e2. [97] H. Laroui, D. Geem, B. Xiao, E. Viennois, P. Rakhya, T. Denning, D. Merlin, Targeting intestinal inflammation with CD98 siRNA/PEI-loaded nanoparticles, Mol. Ther. 22 (2014) 69–80. [98] H. Laroui, A.L. Theiss, Y. Yan, G. Dalmasso, H.T. Nguyen, S.V. Sitaraman, D. Merlin, Functional TNFalpha gene silencing mediated by polyethyleneimine/TNFalpha siRNA nanocomplexes in inflamed colon, Biomaterials 32 (2011) 1218–1228. [99] B. Xiao, H. Laroui, S. Ayyadurai, E. Viennois, M.A. Charania, Y. Zhang, D. Merlin, Mannosylated bioreducible nanoparticle-mediated macrophage-specific TNFalpha RNA interference for IBD therapy, Biomaterials 34 (2013) 7471–7482. [100] M.D. Bhavsar, M.M. Amiji, Polymeric nano- and microparticle technologies for oral gene delivery, Expert Opin. Drug Deliv. 4 (2007) 197–213. [101] D. Peer, E.J. Park, Y. Morishita, C.V. Carman, M. Shimaoka, Systemic leukocytedirected siRNA delivery revealing cyclin D1 as an anti-inflammatory target, Science 319 (2008) 627–630. [102] D. Peer, P. Zhu, C.V. Carman, J. Lieberman, M. Shimaoka, Selective gene silencing in activated leukocytes by targeting siRNAs to the integrin lymphocyte functionassociated antigen-1, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 4095–4100. [103] D.S. Wilson, G. Dalmasso, L. Wang, S.V. Sitaraman, D. Merlin, N. Murthy, Orally delivered thioketal nanoparticles loaded with TNF-alpha-siRNA target inflammation and inhibit gene expression in the intestines, Nat. Mater. 9 (2010) 923–928. [104] H. Attarwala, M. Amiji, Multi-compartmental nanoparticles-in-emulsion formulation for macrophage-specific anti-inflammatory gene delivery, Pharm. Res. 29 (2012) 1637–1649. [105] N. Bessis, M.C. Boissier, P. Ferrara, T. Blankenstein, D. Fradelizi, C. Fournier, Attenuation of collagen-induced arthritis in mice by treatment with vector cells engineered to secrete interleukin-13, Eur. J. Immunol. 26 (1996) 2399–2403. [106] F. Apparailly, C. Verwaerde, C. Jacquet, C. Auriault, J. Sany, C. Jorgensen, Adenovirus-mediated transfer of viral IL-10 gene inhibits murine collageninduced arthritis, J. Immunol. 160 (1998) 5213–5220. [107] J.D. Whalen, E.L. Lechman, C.A. Carlos, K. Weiss, I. Kovesdi, J.C. Glorioso, P.D. Robbins, C.H. Evans, Adenoviral transfer of the viral IL-10 gene periarticularly to mouse paws suppresses development of collagen-induced arthritis in both injected and uninjected paws, J. Immunol. 162 (1999) 3625–3632. [108] Y. Ma, S. Thornton, L.E. Duwel, G.P. Boivin, E.H. Giannini, J.M. Leiden, J.A. Bluestone, R. Hirsch, Inhibition of collagen-induced arthritis in mice by viral IL-10 gene transfer, J. Immunol. 161 (1998) 1516–1524. [109] M. Jafari, M. Soltani, S. Naahidi, D.N. Karunaratne, P. Chen, Nonviral approach for targeted nucleic acid delivery, Curr. Med. Chem. 19 (2012) 197–208. [110] S. Jain, M. Amiji, Tuftsin-modified alginate nanoparticles as a noncondensing macrophage-targeted DNA delivery system, Biomacromolecules 13 (2012) 1074–1085. [111] J.C. Fernandes, H. Wang, C. Jreyssaty, M. Benderdour, P. Lavigne, X. Qiu, F.M. Winnik, X. Zhang, K. Dai, Q. Shi, Bone-protective effects of nonviral gene therapy with folate-chitosan DNA nanoparticle containing interleukin-1 receptor antagonist gene in rats with adjuvant-induced arthritis, Mol. Ther. 16 (2008) 1243–1251. [112] R.M. Schiffelers, J. Xu, G. Storm, M.C. Woodle, P.V. Scaria, Effects of treatment with small interfering RNA on joint inflammation in mice with collagen-induced arthritis, Arthritis Rheum. 52 (2005) 1314–1318. [113] N. Van Rooijen, A. Sanders, Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications, J. Immunol. Methods 174 (1994) 83–93. [114] C.D. Muller, F. Schuber, Neo-mannosylated liposomes: synthesis and interaction with mouse Kupffer cells and resident peritoneal macrophages, Biochim. Biophys. Acta 986 (1989) 97–105. [115] W.J. Oyen, O.C. Boerman, G. Storm, L. van Bloois, E.B. Koenders, R.A. Claessens, R.M. Perenboom, D.J. Crommelin, J.W. van der Meer, F.H. Corstens, Detecting infection and inflammation with technetium-99 m-labeled Stealth liposomes, J. Nucl. Med. 37 (1996) 1392–1397. [116] N.J. Caplen, E.W. Alton, P.G. Middleton, J.R. Dorin, B.J. Stevenson, X. Gao, S.R. Durham, P.K. Jeffery, M.E. Hodson, C. Coutelle, et al., Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis, Nat. Med. 1 (1995) 39–46. [117] M.A. Rogy, T. Auffenberg, N.J. Espat, R. Philip, D. Remick, G.K. Wollenberg, E.M. Copeland III, L.L. Moldawer, Human tumor necrosis factor receptor (p55) and interleukin 10 gene transfer in the mouse reduces mortality to lethal endotoxemia and also attenuates local inflammatory responses, J. Exp. Med. 181 (1995) 2289–2293. [118] R. Fellowes, C.J. Etheridge, S. Coade, R.G. Cooper, L. Stewart, A.D. Miller, P. Woo, Amelioration of established collagen induced arthritis by systemic IL-10 gene delivery, Gene Ther. 7 (2000) 967–977.


16


[119] M. Khoury, P. Louis-Plence, V. Escriou, D. Noel, C. Largeau, C. Cantos, D. Scherman, C. Jorgensen, F. Apparailly, Efficient new cationic liposome formulation for systemic delivery of small interfering RNA silencing tumor necrosis factor α in experimental arthritis, Arthritis Rheum. 54 (2006) 1867–1877. [120] M. Khoury, V. Escriou, G. Courties, A. Galy, R. Yao, C. Largeau, D. Scherman, C. Jorgensen, F. Apparailly, Efficient suppression of murine arthritis by combined anticytokine small interfering RNA lipoplexes, Arthritis Rheum. 58 (2008) 2356–2367. [121] K.A. Howard, P.R. Dash, M.L. Read, K. Ward, L.M. Tomkins, O. Nazarova, K. Ulbrich, L.W. Seymour, Influence of hydrophilicity of cationic polymers on the biophysical properties of polyelectrolyte complexes formed by self-assembly with DNA, Biochim. Biophys. Acta 1475 (2000) 245–255. [122] D.R. Sorensen, M. Leirdal, M. Sioud, Gene silencing by systemic delivery of synthetic siRNAs in adult mice, J. Mol. Biol. 327 (2003) 761–766. [123] P. Lundberg, P.V. Welander, C.K. Edwards III, N. van Rooijen, E. Cantin, Tumor necrosis factor (TNF) protects resistant C57BL/6 mice against herpes simplex virusinduced encephalitis independently of signaling via TNF receptor 1 or 2, J. Virol. 81 (2007) 1451–1460. [124] K.A. Howard, S.R. Paludan, M.A. Behlke, F. Besenbacher, B. Deleuran, J. Kjems, Chitosan/siRNA nanoparticle-mediated TNF-alpha knockdown in peritoneal macrophages for anti-inflammatory treatment in a murine arthritis model, Mol. Ther. 17 (2009) 162–168. [125] E. Sonkoly, A. Pivarcsi, microRNAs in inflammation, Int. Rev. Immunol. 28 (2009) 535–561. [126] K.O. Kisich, R.W. Malone, P.A. Feldstein, K.L. Erickson, Specific inhibition of macrophage TNF-alpha expression by in vivo ribozyme treatment, J. Immunol. 163 (1999) 2008–2016. [127] T. Seimon, I. Tabas, Mechanisms and consequences of macrophage apoptosis in atherosclerosis, J. Lipid Res. 50 (2009) S382–S387. [128] V. Amirbekian, M.J. Lipinski, K.C. Briley-Saebo, S. Amirbekian, J.G.S. Aguinaldo, D.B. Weinreb, E. Vucic, J.C. Frias, F. Hyafil, V. Mani, E.A. Fisher, Z.A. Fayad, Detecting and assessing macrophages in vivo to evaluate atherosclerosis noninvasively using molecular MRI, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 961–966. [129] J. Sanz, Z.A. Fayad, Imaging of atherosclerotic cardiovascular disease, Nature 451 (2008) 953–957. [130] S. Marrache, S. Dhar, Biodegradable synthetic high-density lipoprotein nanoparticles for atherosclerosis, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 9445–9450. [131] J. Madrigal-Matute, N. Rotllan, J. Aranda, C. Fernández-Hernando, MicroRNAs and atherosclerosis, Curr. Atheroscler Rep. 15 (2013) 1–8. [132] C. Urbich, A. Kuehbacher, S. Dimmeler, Role of microRNAs in vascular diseases, inflammation, and angiogenesis, Cardiovasc. Res. 79 (2008) 581–588. [133] D.A. Chistiakov, I.A. Sobenin, A.N. Orekhov, Strategies to deliver microRNAs as potential therapeutics in the treatment of cardiovascular pathology, Drug Deliv. 19 (2012) 392–405. [134] Y. Tian, S.E. Kelemen, M.V. Autieri, Inhibition of AIF-1 expression by constitutive siRNA expression reduces macrophage migration, proliferation, and signal transduction initiated by atherogenic stimuli, Am. J. Physiol. Cell Physiol. 290 (2006) C1083–C1091. [135] F. Leuschner, P. Dutta, R. Gorbatov, T.I. Novobrantseva, J.S. Donahoe, G. Courties, K. M. Lee, J.I. Kim, J.F. Markmann, B. Marinelli, P. Panizzi, W.W. Lee, Y. Iwamoto, S. Milstein, H. Epstein-Barash, W. Cantley, J. Wong, V. Cortez-Retamozo, A. Newton, K. Love, P. Libby, M.J. Pittet, F.K. Swirski, V. Koteliansky, R. Langer, R. Weissleder, D.G. Anderson, M. Nahrendorf, Therapeutic siRNA silencing in inflammatory monocytes in mice, Nat. Biotechnol. 29 (2011) 1005–1010.

[136] M. Yuan, N. Konstantopoulos, J. Lee, L. Hansen, Z.-W. Li, M. Karin, S.E. Shoelson, Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikkβ, Science 293 (2001) 1673–1677. [137] C.M. Larsen, M. Faulenbach, A. Vaag, A. Vølund, J.A. Ehses, B. Seifert, T. MandrupPoulsen, M.Y. Donath, Interleukin-1-receptor antagonist in type 2 diabetes mellitus, N. Engl. J. Med. 356 (2007) 1517–1526. [138] E.J.P. van Asseldonk, R. Stienstra, T.B. Koenen, L.A.B.. Joosten, M.G. Netea, C.J. Tack, Treatment with anakinra improves disposition index but not insulin sensitivity in nondiabetic subjects with the metabolic syndrome: a randomized, doubleblind, placebo-controlled study, J. Clin. Endocrinol. Metab. 96 (2011) 2119–2126. [139] G.J. Tesz, M. Aouadi, M. Prot, S.M. Nicoloro, E. Boutet, S.U. Amano, A. Goller, M. Wang, C.A. Guo, W.E. Salomon, J.V. Virbasius, R.A. Baum, M.J. O'Connor Jr., E. Soto, G.R. Ostroff, M.P. Czech, Glucan particles for selective delivery of siRNA to phagocytic cells in mice, Biochem. J. 436 (2011) 351–362. [140] M. Aouadi, M. Tencerova, P. Vangala, J.C. Yawe, S.M. Nicoloro, S.U. Amano, J.L. Cohen, M.P. Czech, Gene silencing in adipose tissue macrophages regulates whole-body metabolism in obese mice, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 8278–8283. [141] L. Carta, S. Pastorino, G. Melillo, M.C. Bosco, S. Massazza, L. Varesio, Engineering of macrophages to produce IFN-gamma in response to hypoxia, J. Immunol. 166 (2001) 5374–5380. [142] T. Satoh, T. Saika, S. Ebara, N. Kusaka, T.L. Timme, G. Yang, J. Wang, V. Mouraviev, G. Cao, M.A. Fattah el, T.C. Thompson, Macrophages transduced with an adenoviral vector expressing interleukin 12 suppress tumor growth and metastasis in a preclinical metastatic prostate cancer model, Cancer Res. 63 (2003) 7853–7860. [143] K. Tabata, S. Kurosaka, M. Watanabe, K. Edamura, T. Satoh, G. Yang, E. Abdelfattah, J. Wang, A. Goltsov, D. Floryk, T.C. Thompson, Tumor growth and metastasis suppression by Glipr1 gene-modified macrophages in a metastatic prostate cancer model, Gene Ther. 18 (2011) 969–978. [144] S. Aharinejad, P. Paulus, M. Sioud, M. Hofmann, K. Zins, R. Schafer, E.R. Stanley, D. Abraham, Colony-stimulating factor-1 blockade by antisense oligonucleotides and small interfering RNAs suppresses growth of human mammary tumor xenografts in mice, Cancer Res. 64 (2004) 5378–5384. [145] A.M. Krieg, Therapeutic potential of Toll-like receptor 9 activation, Nat. Rev. Drug Discov. 5 (2006) 471–484. [146] C. Manegold, D. Gravenor, D. Woytowitz, J. Mezger, V. Hirsh, G. Albert, M. AlAdhami, D. Readett, A.M. Krieg, C.G. Leichman, Randomized phase II trial of a toll-like receptor 9 agonist oligodeoxynucleotide, PF-3512676, in combination with first-line taxane plus platinum chemotherapy for advanced-stage nonsmall-cell lung cancer, J. Clin. Oncol. 26 (2008) 3979–3986. [147] A. Herrmann, M. Kortylewski, M. Kujawski, C. Zhang, K. Reckamp, B. Armstrong, L. Wang, C. Kowolik, J. Deng, R. Figlin, H. Yu, Targeting Stat3 in the myeloid compartment drastically improves the in vivo antitumor functions of adoptively transferred T cells, Cancer Res. 70 (2010) 7455–7464. [148] M. Kortylewski, P. Swiderski, A. Herrmann, L. Wang, C. Kowolik, M. Kujawski, H. Lee, A. Scuto, Y. Liu, C. Yang, J. Deng, H.S. Soifer, A. Raubitschek, S. Forman, J.J. Rossi, D.M. Pardoll, R. Jove, H. Yu, In vivo delivery of siRNA to immune cells by conjugation to a TLR9 agonist enhances antitumor immune responses, Nat. Biotechnol. 27 (2009) 925–932. [149] J.W. Graff, A.M. Dickson, G. Clay, A.P. McCaffrey, M.E. Wilson, Identifying functional microRNAs in macrophages with polarized phenotypes, J. Biol. Chem. 287 (2012) 21816–21825


Advancing polymeric delivery systems amidst a nucleic acid therapy renaissance.

Nucleic acid aptamer-mediated drug delivery for targeted cancer therapy.

Gold nanoparticles for nucleic acid delivery.

Nucleic acid detection systems for enteroviruses.

Nucleic acid based logical systems.

Stimuli-responsive liposomes for the delivery of nucleic acid therapeutics.

Electroporation-enhanced delivery of nucleic acid vaccines.

Dendrimers as Nanocarriers for Nucleic Acid and Drug Delivery in Cancer Therapy.

Sequence-defined shuttles for targeted nucleic acid and protein delivery.

Nanodelivery systems for nucleic acid therapeutics in drug resistant tumors.

A nucleic acid-based medication for allergic skin diseases.

Independent blood-brain barrier transport systems for nucleic acid precursors.

Nucleic acid delivery systems based on poly(galactosyl ureaethyl methacrylate-b-dimethylamino ethyl methacrylate) diblock copolymers.

Nucleic acid-scavenging electrospun nanofibrous meshes for suppressing inflammatory responses.

Fungal diseases: could nanostructured drug delivery systems be a novel paradigm for therapy?

Immune sensing of nucleic acids in inflammatory skin diseases.

New polymer of lactic-co-glycolic acid-modified polyethylenimine for nucleic acid delivery.

Systems biology in inflammatory bowel diseases: ready for prime time.

Applications of nucleic acid probes in veterinary infectious diseases.

Phytosome-hyaluronic acid systems for ocular delivery of L-carnosine.

Drug Delivery Systems for Imaging and Therapy of Parkinson's Disease.

Nucleic acid-mediated intracellular protein delivery by lipid-like nanoparticles.

Microbial Nucleic Acid Sensing in Oral and Systemic Diseases.

HDL as a drug and nucleic acid delivery vehicle.