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ADR-12332; No of Pages 11 Advanced Drug Delivery Reviews xxx (2012) xxx–xxx Contents lists available at SciVerse ScienceDirect Advanced Drug Delivery Reviews journal homepage: www.elsevier.com/locate/addr Immunotoxicity derived from manipulating leukocytes with lipid-based nanoparticles☆ Dan Peer ⁎ Laboratory of Nanomedicine, Department of Cell Research and Immunology, George S. Wise Faculty of Life Science, Tel Aviv University, Tel Aviv 69978, Israel Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel Aviv 69978, Israel a r t i c l e i n f o Article history: Received 14 May 2012 Accepted 14 June 2012 Available online xxxx Keywords: Lipid-based nanoparticles Liposomes Immune response Leukocytes RNAi T cells Antigen-presenting cells a b s t r a c t Lipid-based nanoparticles (LNPs) such as liposomes, micelles, and hybrid systems (e.g. lipid-polymer) are prominent delivery vehicles that already made an impact on the lives of millions around the globe. A common denominator of all these LNP‐based platforms is to deliver drugs into specific tissues or cells in a pathological setting with minimal adverse effects on bystander cells. All these platforms must be compatible to the physiological environment and prevent undesirable interactions with the immune system. Avoiding immune stimulation or suppression is an important consideration when developing new strategies in drug and gene delivery, whereas in adjuvants for vaccine therapies, immune activation is desired. Therefore, profound understanding of how LNPs elicit immune responses is essential for the optimization of these systems for various biomedical applications. Herein, I describe general concepts of the immune system and the interaction of subsets of leukocytes with LNPs. Finally, I detail the different immune toxicities reported and propose ways to manipulate leukocytes’ functions using LNPs. © 2012 Elsevier B.V. All rights reserved. Contents 1. 2. 3. 4. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immune recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Pattern recognition receptors (PRRs) . . . . . . . . . . . . . . . . . 2.2. The adaptive immune arm . . . . . . . . . . . . . . . . . . . . . . Lipid-based nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Structural and physicochemical properties of LNPs determine the type of 3.1.1. LNPs size distribution . . . . . . . . . . . . . . . . . . . . 3.1.2. Formulation additives and contaminations . . . . . . . . . . 3.1.3. LNPs surface charge . . . . . . . . . . . . . . . . . . . . . 3.1.4. Aggregation of LNPs. . . . . . . . . . . . . . . . . . . . . 3.1.5. Route of administration . . . . . . . . . . . . . . . . . . . 3.2. LNPs may stimulate or suppress the immune system. . . . . . . . . . Modulating the immune response by LNPs as a potential therapeutic strategy . 4.1. Immunostimulation by LNPs . . . . . . . . . . . . . . . . . . . . . 4.2. Modulating the immune response with RNAi-LNPs strategies . . . . . . 4.2.1. Targeted delivery into pan leukocytes . . . . . . . . . . . . 4.2.2. Targeting subsets of leukocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . immune response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Abbreviations: mAb, monoclonal antibody; BD, biodistribution; DOTAP, N-[1-(2,3-dioleoyloxy)-propyl]-N,N,N- trimethylammonium methylsulfate; DOTMA, 3b-[N-(N′,N ′-dimethy lainin(ethyl) carbamoyl; dsRNA, double stranded ribonucleic acid; EPR, enhanced permeability and retention; I.V, intravenous; miRNA, micro ribonucleic acid; MPS, mononuclear phagocytic system; MW, molecular weight; NLR, Nod‐like receptors; NP, nanoparticle; nt, nucleotide; PEG, polyethylene glycol; PEI, polyethyleneimine; PC, phosphatidylcholine; PK, pharmacokinetics; RES, reticuloendothelial system; RISC, RNA-induced silencing complex; RNA, ribonucleic acid; shRNA, short hairpin ribonucleic acid; siRNA, small interfering RNA; SNALP, stable lipid-nucleic acid particle; T1/2, half time; TH, T helper lymphocytes; TLR, Toll-like receptor. ☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Nanotoxicity: from bench to bedsite.” ⁎ Tel.: +972 3640 7925; fax: +972 3640 5926. E-mail address: [email protected]. 0169-409X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2012.06.013 Please cite this article as: D. Peer, Immunotoxicity derived from manipulating leukocytes with lipid-based nanoparticles, Adv. Drug Deliv. Rev. (2012), doi:10.1016/j.addr.2012.06.013 2 D. Peer / Advanced Drug Delivery Reviews xxx (2012) xxx–xxx 5. Conclusions and future prospective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction Profound understanding of the immune system and the interaction of various materials with subsets of immune cells (leukocytes) is important for optimizing novel drug delivery systems. The immune system guards the body from internal and external intruders by holding the host protection mechanism for self-defense against infections and malignancies. This is a fine-tuning mechanism that allows leukocytes to respond to cellular and extracellular stimuli, identify pathogens, and respond to different types of materials with various size, geometry, surface curvature and charge [1–5]. Here, I will describe the general mechanisms by which leukocytes interact with lipid-based nanoparticles (LNPs) and detail immuno-toxicities caused by these nanomaterials. Finally, I will suggest ways to manipulate leukocytes’ function using LNPs loaded with various types of drugs. 2. Immune recognition The immune system can be divided into two divisions: the innate arm and the adaptive arm. The innate immune arm constitutes the first host defense line, mainly composed of the phagocytic cells and circulating macrophages (derived from blood monocytes) [6] and of the complement system (which is often considered as the link between the innate and the adaptive arms). The “complement cascade” is a complex multi-component system composed of 26 proteins, which combine with antibodies or cell surfaces, is constitutive and nonspecific but must be activated in order to function [7,8]. Macrophages mediate crucial innate immune responses via the clearance mechanism: caspase-1-dependent processing and secretion of interleukin (IL)-1β and IL-18. The second arm of the immune system, the adaptive immune arm, is able to response in a highly specific manner against molecular determinants on pathogens and this process may proceed over weeks [9]. The immune response against a potential threat (internal or external) can be divided into stimulation or suppression of the immune system. Immune stimulating via activation of the innate or adaptive immune arms can be caused by nanoparticles (NPs) made from different types of materials, having various sizes, shapes and surface charges in a variety of molecular recognition mechanisms [1,3,8,10,11]. It is possible to utilize the type of response (either stimulation or suppression) for therapeutic purposes [1,12]. When provoking a stimulation response the impact of the therapeutic efficacy might be effected, such as in the case of cancer treatment and vaccine efficacy. In contrast, robust immune stimulation might cause an undesirable response, for example, when lipid-based nanoparticles (LNPs) interact with specific cell surface receptors on subsets of leukocytes [1,10,13,14] and can induce cytokine storm, interferon response and/or lymphocyte activation causing severe adverse effects that may diminish the therapeutic effect. Correspondingly, a suppression response can be desired and as such may enhance the therapeutic benefits of treatments for allergies and autoimmune diseases as well as prevent rejection of transplanted organs [1,3–5] or may be inadvertent and lower the body's defense against infection and cancerous cells. 0 0 0 tissue. This interaction may lead to signal cascades upon activation of pattern recognition receptors (PRRs) [15–17]. PRRs are proteins expressed by cells of the innate immune arm to identify pathogen-associated molecular patterns (PAMPs), which are associated with microbial pathogens or cellular stress. Other types of receptors are the damage-associated molecular patterns (DAMPs), which are associated with cell components released during cell damage (Fig. 1). On the bases of their function, PRRs may be divided into signaling PRRs or endocytic PRRs. Signaling PRRs include the large families of membrane-bound Toll-like receptors (TLRs) [18–20] that can recognize nucleic acids (single and double stranded), parts of bacteria's well such as lipopolysaccharides (LPS), several types of charged phospholipids and more [13,21]. In addition, cytoplasmic NOD-like receptors (NLRs) for nucleotide oligomerization domain receptors are cytoplasmic proteins that may have a variety of functions in regulation of inflammation and apoptotic responses [15,22]. Endocytic PRRs promote the attachment, engulfment and destruction of microorganisms by phagocytes, without relaying an intracellular signal. These types of PRRs recognize carbohydrates and include mannose receptors of macrophages, glucan receptors present on all phagocytes and scavenger receptors that recognize charged ligands and are found on all phagocytes and mediate removal of apoptotic cells [23,24]. If the NPs indeed interact similar to some pathogens (such as in the case of several cationic lipids [13,21]) then it is highly possible that a similar cascade that may activate PAMPs and DAMPs by a pathogen can occur by interaction between cationic NPs in the PRRs (Fig. 1). This highly possible activation is described below. Upon interaction of the NPs with endothelial cells or cells in a particular tissue, PAMPs and DAMPs bind to Toll-like receptors (TLRs) expressed by innate immune cells, such as dendritic cells (DCs) [17]. This binding promotes DC maturation and migration to lymph nodes. In the lymph nodes, the mature DCs release cytokines (proinflammatory), such as interleukin-12 (IL-12), which induces T helper 1 (TH1) cell differentiation, and IL-6, which works together with transforming growth factor-β (TGF-β) to promote the differentiation of TH17 cells. Then, T cells that undergone differentiation leave the lymph nodes and migrate through the bloodstream to tissues, where they are further activated by encounter with self‐antigens presented by antigen-presenting cells. Then, in the already inflamed tissues, TH17 cells are further activated by a panel of cytokines such as IL-1 and IL-23, but also by IL-17 and IL-21 produced early in the immune response by subsets of T cells (γδ T cells) and other innate lymphoid cells (ILCs). Granulocyte–macrophage colony-stimulating factor (GM-CSF) that is produced by both TH1 and TH17 cells activates CD11b + myeloid cells, DCs and macrophages. Cytokines produced by TH1 and TH17 cells activate macrophages, DCs and monocytes, promoting the release of proinflammatory mediators, such as IL-1β, tumor necrosis factor (TNF), matrix metalloproteinases (MMPs) and reactive oxygen species (ROS), which all mediate tissue damage. IL-17 induces the production of chemokines, especially CXC-chemokine ligand 2 (CXCL2; also known MIP2) and IL-8 (also known as CXCL8), which recruit neutrophils to the site of inflammation (Fig. 1). 2.1. Pattern recognition receptors (PRRs) 2.2. The adaptive immune arm Immune stimulation often initiates when NPs interact with cells of the innate immune arm such as dendritic cells and macrophages in a similar manner to a pathogen infection or being sensed as a damaged The second defense line, the adaptive arm, involves antigen-mediated T- and B-lymphocytes carrying antigen-specific Please cite this article as: D. Peer, Immunotoxicity derived from manipulating leukocytes with lipid-based nanoparticles, Adv. Drug Deliv. Rev. (2012), doi:10.1016/j.addr.2012.06.013 D. Peer / Advanced Drug Delivery Reviews xxx (2012) xxx–xxx surface receptors (e.g. TH cells and cytotoxic T lymphocytes; CTLs). TH cells are composed of several subsets of T cells such as TH1 [25], TH2 [26], TH17 [25,27], Tregs [27], TH9 [28] and the newly identified TH22 cells [29]. These subsets of T cells are defined according to their 3 production of lineage-indicating cytokines and functions. For example, TH1 cells mediate a cellular immune response, which is mainly a proinflammatory response by producing cytokines such as interleukin (IL)-2, interferon (IFN) γ and tumor necrosis factor (TNF)-α. TH2 Fig. 1. Effector cells signaling cascades upon interaction of cationic LNPs with leukocytes. (A) Due to LNP interaction with a specific tissue, which might be similar to a pathogen interaction, pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPS) bind to Toll-like receptors (TLRs) expressed by innate immune cells, such as dendritic cells (DCs). This promotes DC maturation and migration to the lymph nodes. (B) In the lymph nodes, mature DCs release proinflammatory cytokines, including interleukin-12 (IL-12), which induces T helper 1 (TH1) cell differentiation, and IL-6, which works together with transforming growth factor-β (TGF-β) to promote the differentiation of TH17 cells. Differentiated autoreactive T cells exit the lymph nodes and migrate through the blood to tissues, where they are further activated by encounter with self-antigens presented by antigen-presenting cells. (C) In the inflamed tissues, TH17 cells are further activated by IL-1 and IL-23, but also by IL-17 and IL-21 produced early in the immune response by γδ T cells and other innate lymphoid cells (ILCs). Granulocyte–macrophage colony-stimulating factor (GM-CSF) that is produced by both TH1 and TH17 cells activates CD11b+ myeloid cells. DCs and macrophages and appears to be essential for the development of autoimmunity. Cytokines produced by TH1 and TH17 cells activate macrophages, DCs and other cells, promoting the release of proinflammatory mediators, such as IL-1β, tumor necrosis factor (TNF), matrix metalloproteinases (MMPs) and reactive oxygen species (ROS), which mediate tissue damage. IL-17 induces the production of chemokines, especially CXC-chemokine ligand 2 (CXCL2; also known MIP2) and IL-8 (also known as CXCL8), which recruit neutrophils to the site of inflammation. Please cite this article as: D. Peer, Immunotoxicity derived from manipulating leukocytes with lipid-based nanoparticles, Adv. Drug Deliv. Rev. (2012), doi:10.1016/j.addr.2012.06.013 4 D. Peer / Advanced Drug Delivery Reviews xxx (2012) xxx–xxx cells secret anti-inflammatory cytokines such as IL-4, IL-5, IL-10 and IL-13 and also support the production of circulating antibodies. Another set of newly discovered subgroup is the TH17 group that is responsible for regulation of the inflammatory response and these subsets of T cells secret mainly IL-17, IL-6 and transforming growth factor (TGF)‐β [30]. T regulatory cells (Tregs) are CD4 +CD25 +FOXP3 + cells and are involved in shutting down immune responses after they have successfully tackled invading organisms, and also in regulating immune responses that may potentially attack one's own tissues [27]. The TH1–TH2–TH17 cell paradigm now includes a fourth subset of IL-9 producing effector T cells, TH9 cells [28,31], raising questions about the plasticity of T helper cell subsets. TH9 cells are generated under the presence of IL-4 and TGF-β1, but the costimulatory signals that induce TH9 cell differentiation and the transcriptional regulation of these cells are still not known. Newly identified TH22 cells [29] are a subset of human TH cells that infiltrates the epidermis in individuals with inflammatory skin disorders and are characterized by the secretion of IL-22 and TNF-α, but not IFN-γ, IL-4, or IL-17. These cells might have a role in skin diseases and thus might be important to study when applying topically nanoparticles in the context of skin disorders. At least two types of signals are required for the activation of T lymphocytes to proliferate and differentiate into functional effectors cells: A specific signal of T cells recognition by T cell receptors peptide fragments bound to the major histocompatibility complex (MHC) class I and class II molecules and an interaction signal of receptors with costimulatory molecules on antigen-presenting cell (APC) surface (e.g., B7 family and inducible costimulators ligands (ICOS)). The extra signals activate the priming and differentiation of cytotoxic T lymphocytes (CTLs) and Th cells. Th cells deliver antigen‐specific B cells resulting in antibody production [6,27,32]. 3. Lipid-based nanoparticles Liposomes, micelles, solid lipid-base nanoparticles (SLNPs) and polymersomes are nano-scale lipid-based particles. Liposomes, the most veterans delivery vehicles, were described more than 40 years ago by Bangham et al. [33–35] and were the first to be approved by the FDA to carry several types of chemotherapeutics (see Table 1) [36,37]. Liposomes are spherical, self-closed structures formed by one or several concentric lipid bilayers with aqueous phases inside and between the lipid bilayers. The properties of liposomes are highly attributable to their physicochemical properties such as size, surface charge, composition, rigidity of bilayer and preparation methods [38]. These integrated liposome features enable the encapsulation, embedding or association with a wide range of molecules (i.e., small molecules drugs, antigens, proteins and nucleotides) as well as enhance the delivery of therapeutic payloads into specific tissues and cells. Liposomes also improve in vitro and in vivo stability and reduce adverse effects [39]. Several challenges exist with these carriers, including rapid clearance, serum instability (dependent on the specific formulation), and nonspecific uptake by the mononuclear phagocytic system (MPS), whose function is to remove foreign materials from the circulation. To overcome these limitations, poly (ethylene glycol) (PEG) was introduced for coating lipid-based carriers to provide a hydrophilic layer resulting in increased circulation time [40,41]. To further enhance the selectivity to cell surface markers, targeting moieties were attached to the liposomal surface via a PEG spacer arm, which reduced steric hindrances [42]. Instances of selective targeting using lipid-based carriers include anti-CD19 and anti-CD20 modified long-circulating liposomes that deliver doxorubicin to B cell lymphoma [43] and anti-Her2 liposomes that show effective targeting to breast cancer cells overexpressing erbB2 receptor [44]. Other carriers, including folate-targeted liposomes, have been also proposed as vehicles for boron neutron capture therapy [45]. Transferrin (Tf)-mediated liposome delivery is an emerging strategy to target tumors overexpressing the Tf receptors; Tf-coated liposomes entrapping doxorubicin have been developed to target C6 glioma cells [46] and solid tumors [47]. Additional approaches include the combination of fusogenic peptides, long-circulating agents such as PEG, targeting moieties, and pH sensitive lipids that will cause liposome disruption in the endosome [48,49]. The length of shelf life is one of the challenges facing drug-containing liposomes. Existing liposomal formulations can be stored as suspensions (with a limited shelf life) or as lyophilized powder. In the latter, the lyophilized formulation includes up to 30% v/v sugars as cryo-protectants to maintain the carriers’ nano-scale dimensions upon rehydration. High sugar content in the nanocarrier system may limit the number of patients who can receive these nanocarriers, particularly those patients who suffer from diabetes. Recent attempts to address these challenges involve the covalent immobilization of high molecular weight hyaluronan (HA) on unilamellar liposomes. This serves to cryo-protect nano-scale liposomes in the process of lyophilization and rehydration [50]. In addition, the HA coatings improve circulation time and enhance targeting to HA receptor-expressing tumors (CD44 and RHAMM) [51–56]. In addition, HA was also used as a scaffold and a linker for monoclonal antibody binding to nanoliposomes’ surface directing these particles into subsets of leukocytes in vivo while delivering RNAi payloads [14,57,58]. 3.1. Structural and physicochemical properties of LNPs determine the type of immune response Physicochemical properties of LNPs such as size, geometry, lipid composition, charge, surface characteristics, bilayer packing and defects, as well as LNPs quantity, dose and way of administration, can all act as immunological adjuvant and trigger a robust immune response [11] (see also Table 2). When developing an LNP formulation for clinical application, the prevention of unwanted adverse immune effects can be ruled out by taking into consideration several crucial parameters. 3.1.1. LNPs size distribution Size distribution has been suggested as the leading parameter that determines the potential to induce cytokine expression [5,59–61]. For example, particles that were prepared at a narrow size range (e.g., 20, 40, 49, 67, 93, 101, and 123 nm in diameter) were shown to determine whether antigens loaded into NPs enhance cellular type I interferon (IFN) γ response or antibody type II (IL-4) cytokine response. After a single immunization in mice with 40 and 49 nm beads containing ovalbumin (OVA) antigen, IFNγ response was significantly lower than other particles sizes tested. In contrast, IL-4 response to OVA was higher after immunization with OVA conjugated to larger beads (93, 101, and 123 nm) [60]. Table 1 FDA approved liposomal drugs on the market (many more are under clinical evaluation). Compound Commercial name ™ Indications Daunorubicin Doxorubicin DaunoXome Kaposi's sarcoma Mycet Combinational therapy of recurrent breast cancer Doxorubicin Doxil/ Refractory Kaposi's sarcoma; recurrent breast (PEG-liposomes) Caelyx cancer; ovarian cancer, multiple myeloma Amphotericin B AmBisome Fungal infections Cytarabine DepoCyt Lymphomatous meningitis Vincristine Onco TCS Relapsed aggressive non-Hodgkin's lymphoma (NHL) Please cite this article as: D. Peer, Immunotoxicity derived from manipulating leukocytes with lipid-based nanoparticles, Adv. Drug Deliv. Rev. (2012), doi:10.1016/j.addr.2012.06.013 D. Peer / Advanced Drug Delivery Reviews xxx (2012) xxx–xxx 3.1.2. Formulation additives and contaminations Several studies demonstrated that induction of cytokines can be triggered not only by the liposomes but also by surfactants or bacterial endotoxins present in the liposome formulation [75,76]. Schöler et al. showed that cytotoxicity was strongly influenced by the surfactant used being marketed with cetylpyridinium chloride (CPC) coated solid lipid nanoparticles (SLN) at a concentration of 0.001% and above. All other SLN formulations, containing Poloxamine 908 (P908), Poloxamer 407 (P407), Poloxamer 188 (P188), Solutol HS15 (HS15), Tween 80 (T80), Lipoid S75 (S75), sodium cholate (SC), or sodium dodecylsulfate (SDS), used at the same concentrations, modestly reduced the cell viability [75]. Therefore, it is important to quantify the presence of byproducts and endotoxins contaminants before administration of any formulation into a vertebrate. 3.1.3. LNPs surface charge Among non-viral delivery systems for oligonucleotides (e.g., plasmid DNA, DNA and RNA anti-sense, siRNAs and other oligonucleotides), cationic lipids have been widely used as part of the formulation [49,63,77]. Cationic lipids are lipid-bilayer‐forming vesicles with a positive surface charge. Common cationic lipids that were extensively studied included 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and N-(1-(2,3Dioleoyloxy)propyl)-N,N,N-trimethyl-ammoniummethyl sulphate (DOTMA), 3b-[N-(N′,N′-dimethy lainin(ethyl) carbamoyl cholesterol (DC-Chol), and dimethyldioctadecylammonium bromide (DDAB) but the variety of cationic lipids and lipid-like novel materials is enormous [78,79]. Cationic liposomes showed a superior adjuvant effect compared to anionic or neutral liposomes as demonstrated in vivo in animal models [64,66]. Since cationic liposomes can activate the complement system and cause rapid clearance by macrophages of the mononuclear phagocytic system (MPS) [63], these cationic particles can be used to enhance and modulate the immune response in a desirable direction and represent an efficient tool when designing tailor-made adjuvant for a specific disease target. The use of cationic liposomes in vivo elicited dose-dependent toxicity, as demonstrated by the multivalence cationic liposome, LipofectAMINE2000 a comparison with the monovalence cationic lipids, such as DOTAP [62,65]. While it was found that DOTAP-based cationic liposomes also caused severe damage to the mitochondria [67], the specific mechanism by which this cytotoxicity occur is still unknown and might involve different cellular pathways depending on the particular cell type. In addition, mice treated with positively charged LNP‐containing DOTAP showed increased liver enzyme release and mild body weight loss compared to mice treated with neutral or negatively charged LNPs [1,13]. Intravenous administration of cationic LNPs induced type I IFN response and elevated mRNA levels of interferon responsive genes 15–25 fold higher than neutral and negatively charged NPs in mouse splenocytes. Treatment with cationic LNPs provoked a dramatic Table 2 Different types of immune responses generated when LNPs interact with subsets of leukocytes. Type of an immune response Liposome feature Parameter examined Induction of various types of cytokines Proinflammatory response inducing TH1 cytokine expression Complement activation Liposome size Diameter > 100 nm [59–61] Liposome surface's charged Liposome aggregation Induced a robust high IgG1, Route of IgG2a and IFN-γ response administration Lymphocyte activation Liposomes’ surface charge Cationic lipids: DOTAP DOTMA PEG References [13,62–67] [11,68–70] Intralymphatic [71,72] injection Negatively charged [73,74] PS-containing liposomes 5 proinflammatory response by inducing TH1 cytokine expression (IL-2, IFNγ and TNFα) 10- to 75-fold higher than treatment with control particles (neutral or negatively charged particles). Using TLR4 knockout mice, it was shown that the induction of cytokines and IFN response to cationic liposomes decreased dramatically [13], strengthening the hypothesis suggested by Ruysschaert several years ago that cationic liposomes can agonize TLR4 [80]. PEGylated liposomes, which are mildly negatively charged under physiological conditions [81], have been extensively used as drug delivery carriers. Their advantage in improve circulation time of the entrapped therapeutic payload is well known [82,83]. However, it was observed that when PEGylated liposomes were injected into mice, rats and rhesus monkeys repeatedly, they lost this long circulation feature and was accumulated in the liver, a phenomenon which was termed accelerated blood clearance (ABC) [84,85]. Ishida et al. suggested a possible mechanism of action for the ABC phenomenon. The empty PEGylated liposomes elicited PEG-specific IgM response, which are produced in the spleen in response to the first injection, selectively bind to the PEG on a second injected liposome dose and subsequently activate the complement system. An opsonization of liposomes by C3 fragment occurred, which lead to an enhance uptake of the liposomes by specialized macrophages in the liver known as the Kupffer cells [86]. The surface charge of the LNPs affects also the tissue specificity of the particles uptake. Macrophages seem to preferentially take up negatively charged LNPs [73,81,87]. Different malignant cell lines have different uptake patterns with respect to positive, neutral, or negative charges, and in vivo uptake patterns can be further different [87]. Anionic liposomes interact with a limited fraction of dendritic cells in vitro, whereas cationic liposomes interact with a high percentage of the dendritic cells, probably by means of electrostatic binding to the negatively charged surface heparane sulfate proteoglycans resulting in intracellular localization [88]. 1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), a neutral phospholipid‐forming liposome, was used to deliver siRNA payloads in vivo into tumor cells [77,89–92] 10- and 30-fold more effectively than cationic, DOTAP-based liposomes and naked siRNA, respectively [93]. DOPC-based nano-scaled liposomes (~ 100 nm in diameter) did not induce any detectable toxicity and were found to be safe in orthotopic mouse models, making them highly attractive for further therapeutic development [94]. Phagocytic cells fervently take up anionic liposomes in an unclear mechanism. When entrapping nucleic acids such as siRNAs or DNA the loading efficiency into these liposomes is quite low, probably due to the negative charge of these molecules [1,87,95]. Clearly, a complete understanding of the best liposomal design for delivery of therapeutic substances is still evolving. It is possible that with nucleic acid delivery the use of a neutral lipid, such as DOPC, which will enter the cells via a mechanism that will not involve endosomes, will allow a balance between efficient uptake of the therapeutic payload into a liposome at preparation, uptake of the liposome into a particular cell type and the release of the payload from the liposome inside the cell cytoplasm, as was demonstrated for multiple targets using siRNAs entrapped in DOPC‐based particles [91,92,96]. 3.1.4. Aggregation of LNPs Aggregation might cause complement activation even in negligible quantities. It was shown that Doxil® (pegylated liposomes entrapping doxorubicin) or Danuxome® (daunorubicin entrapped in nanoliposomes) activate the complement, despite the fact that both drugs are located within the liposome particles, apparently shielded from plasma, and aggregates are present within these formulations [68]. It is thus recommended that all new liposomalbased formulation candidates undergo adequate investigations into their interactions with the immune system prior to their use in Please cite this article as: D. Peer, Immunotoxicity derived from manipulating leukocytes with lipid-based nanoparticles, Adv. Drug Deliv. Rev. (2012), doi:10.1016/j.addr.2012.06.013 6 D. Peer / Advanced Drug Delivery Reviews xxx (2012) xxx–xxx clinical trials [97,98]. Recent excellent reviews and commentaries on LNPs that activate the complement can be found elsewhere and are beyond the scope of this report [8,11,70,99]. 3.1.5. Route of administration Routes of administrations such as subcutaneous (S.C.), intradermal (I.D.), intramuscular (I.M.) and intralymphatic (I.L.) and their influence on the immune response were studied by Mohanan et al. [71]. The researchers used the ovalbumin (OVA) in three different particulate antigen-delivery systems: dimethyldioctadecylammonium bromide (DDA) liposomes, N-trimethyl chitosan (TMC) NPs, and poly lactic-co-glycolide (PLGA) microspheres for evaluating the influence of the administration routes on the immune response. Whether the vaccine was administrated S.C., I.D., I.M., or I.L., a minor effect was observed, inducing an antibody response of IgG1 subclass, associated with TH2-type (anti-inflammatory) response. In contrast, the administration route strongly affected both kinetic and magnitude of the IgG2a subclass that is associated with TH1-type (proinflammatory) response: A single I.L. administration of all the tested delivery systems induced a robust IgG2a response. The I.D. and I.M. routes generated intermediate IgG2a titers. A thorough investigation into the rough of administration of different LNPs (made from various lipids) and characterized based on their size, geometry and surface charge will be a substantial contribution to this field and will add important knowledge correlating the administration route to structural properties of the LNPs. 3.2. LNPs may stimulate or suppress the immune system Despite advantages of increased stability, facilitated delivery and protection of its cargo, following systemic administration, LNPs may trigger the innate immune system, especially the complement cascade and macrophage clearance mechanisms [11] (Fig. 2). As other circulating particles, liposomes, for example, are often first taken up by phagocytic cells (such as blood monocytes and macrophages of the liver, spleen and bone marrow) [100]. There might be undesirable interactions between the liposomes and the immune system, such as immunostimulation or immunosuppression. Liposomes have been well documented as agonists of TLRs and can be also internalized into macrophages by scavenger receptors, but there is lack of immunological knowledge about the interaction of liposomes and other LNPs with NLRs and specifically with the inflammasome. Liposomes can also activate complement or induce an ‘educational’ event with the adaptive immune system. Interaction of LNPs with TH and B-lymphocytes is less characterized and has a tremendous potential to explore new avenues in the adaptive immune system. The inadvertent recognition of liposomes as foreign entities by leukocytes may result in a multilevel immune response against the liposomes and eventually lead to toxicity in the host and/or lack of therapeutic benefit [1,100]. A harmful activation of the complement cascade may occur in some types of particles. This event may lead to hypersensitivity reactions and anaphylaxis [4]. Szebeni et al. showed that an intravenous injection of LNPs could cause acute hypersensitivity reactions (HSRs) in up to 45% of patients, with hemodynamic, respiratory and cutaneous manifestations. The phenomenon can be explained with activation of the complement system on the surface of lipid particles, leading to anaphylatoxin (C5a and C3a) liberation and subsequent release reactions of mast cells, basophiles and possibly other inflammatory cells in the blood [99]. LNPs decorated with polyethylene glycol (PEG) and entrapping doxorubicin (Doxil™) also activate the complement system. The reported frequency of HSRs to Doxil™ is up to 25% of all the treated patients. Unlike IgE-mediated (type I) allergy, these reactions occur mostly at the first exposure to the formulation without prior sensitization [68]. An additional example is a harmful activation of the complement system at tumor sites, which may stimulate tumor- associated immune cells and promote their conversion into a tumor-supportive phenotype, thereby stimulating cancer progression [4,70,101]. 4. Modulating the immune response by LNPs as a potential therapeutic strategy Nano-scale liposomes that can modulate an immune response can be divided into two categories: liposomes encapsulating an antigen type I, which are designed to elicit an immune response, and a type II polymer coated liposomes, which are designed to prevent an immune recognition [4]. One example of immunosuppressive liposomes is the liposomal alendronate (LA). LA is a bisphosphonate drug used for osteoporosis and other types of bone diseases. It has been shown that partial systemic inactivation and transient depletion of monocytes and macrophages is caused by liposomal bisphosphonates (BP), which reduce neointimal hyperplasia and restenosis in animal models [102]. The anti-inflammatory effect resulting from inhibition of tissue macrophages by LA has been documented in experimental arthritis [103], delayed graft rejection [104] and tumor angiogenesis models [105]. Haber et al. demonstrated that a high dose of LA (10 mg/kg) administrated either intraperitoneal (I.P.) or intravenous (I.V.) exhibited a significant depletion of the circulating monocytes in the pathological conditions of restenosis and endometriosis. The mode of liposome administration (I.P. or I.V.) determines whether systemic or local inhibition of monocytes or macrophages will occur [100]. An additional, elegant example for liposomes, which induce an anti-inflammatory effect, is demonstrated by the phosphatidylserine (PS)-presenting liposomes strategy [81]. Following myocardial infarction (MI) resident and recruitment of macrophages remove necrotic and apoptotic cells, secreted cytokines, and modulated angiogenesis at the infarct site [106]. In a rat model of acute MI, targeting of PS-presenting liposomes to infarct macrophages after i.v. injection Fig. 2. The interaction of different LNPs with subsets of leukocytes can suppress or activate the immune response. The first line of defense by the innate immune arm includes different pattern recognition receptors such as membrane‐bound Toll-like receptors (TLRs), cytoplasmic NOD-like receptors (NLRs) and scavenger receptors on innate immune cells such as monocytes, macrophages and dendritic cells. The second line of defense includes the adaptive immune arm with several important T helper subsets such as TH1, TH2, TH17, Tregs, TH9 and TH22 cells. Each subset of leukocytes can interact differently with different types of nanoparticles made from different materials, sizes, geometry, and surface charges. Adapted with permission from ref. [5]. Please cite this article as: D. Peer, Immunotoxicity derived from manipulating leukocytes with lipid-based nanoparticles, Adv. Drug Deliv. Rev. (2012), doi:10.1016/j.addr.2012.06.013 D. Peer / Advanced Drug Delivery Reviews xxx (2012) xxx–xxx demonstrated improved infract repair by mimicking the antiinflammatory effects of apoptotic cells. Following PS-liposome uptake by macrophages in vitro and in vivo, the cells secreted high levels of anti-inflammatory cytokines (e.g., TGF-β and IL-10) and upregulated the expression of mannose-CD206, concomitant with downregulation of proinflammatory markers such as TNF-α and the costimulatory molecule CD86. The treatment promoted angiogenesis, preservation of small scars and prevented ventricular dilatation and remodeling. The strategy of resolving inflammation in this case was to mimic anti-inflammatory effects of apoptotic cell clearance with macrophages by the use of natural, well-defined materials [107]. One important note is that strongly negatively charged particles may also increase cytotoxicity and promote cell death; thus, prior to translating this strategy into clinical practice several important investigations need to be done in order to understand the level of the anti-inflammatory response and the depletion of important innate immune players such as monocytes and macrophages. 4.1. Immunostimulation by LNPs In order to induce a robust immune response, it is necessary to develop vaccine strategies involving the generation of cytotoxic (CD8 +) T lymphocytes (CTL) to possess a mechanism by which administrated exogenous antigens can be presented not only on MHC class II, but also using MHC class I molecules on antigen‐presenting cells [32,108,109]. A drug delivery system based on the oligomannosecoated liposomes (OML) has been developed and tested in an attempt to increase the effect of generating CTLs against the delivered antigens. The OML‐based vaccine could reject transplanted tumor cells, prevent progression of encephalitis and vertical transmission and reduce offspring mortality of Neospora caninum as shown in the studies by Ikehara et al. [110–112]. OML-based vaccine produces strong adjuvanicity for CTLs, as liposomes coated by oligomannose are exclusively taken up by F4/80 + intraperitoneal mononuclear cells and gathered at extranodal lymphoid tissues [113]. The OML-based vaccine mechanism of action is based on the immune-surveillance system for detecting pathogens invading the abnormal cavity in either a mannose dependent or mannose independent manner. Taken together, a line of clearance process for OML may associate with strong adjuvanticity that induced CTLs [114]. Another strategy built on induction of CTL and antibody response was detailed by the groups of Barenholz and Kedar [115]. This strategy is based on a novel polycationic sphingolipid (CCS)/cholesterol liposomal formulation that markedly enhanced the hepatitis A virus (HAV)-specific antibody response at the intestinal interface, particularly when delivered intrarectally or intranasally, to Balb/c mice at low HAV doses. Although the specific mechanism is not discussed, no dramatic adverse effects were observed and this strategy is now under clinical investigation. An additional formulation that is now under clinical evaluation is the CAF01 [116]. This is a cationic liposomal formulation that incorporates synthetic mycobacterial cord factor and primes protective TH1, TH17, and antibody responses in animal models of bacterial, viral, and parasitic infections. Using CAF01 as a backbone, a recent study demonstrated that incorporating the TLR3 ligand polyinosinic/ polycytidylic acid, poly(I:C), primes CD8 + T cells specific to the SIINFEKL epitope of the model antigen ovalbumin. The CAF01/ poly(I:C) can induce CD8 + T cells that efficiently lyse target cells and significantly reduce tumor growth in two different mouse tumor models [117]. Adjuvant utilizing liposomes is not always effective, as demonstrated by Amin et al. The impact of chitosan-coated liposomes (CCL) as a nasal vaccine vehicle for eliciting viral specific humoral mucosal and cellular immune response was studied. CCL interacted with nasal associated lymphoid tissue failed to increase the residence 7 time of the particles in the nasal cavity, nor induced mucosal and systemic responses as efficiently as non-coated liposomes [118]. 4.2. Modulating the immune response with RNAi-LNPs strategies Gene silencing by RNA interference (RNAi) was discovered by Fire and Mello in 1998 [119]. This discovery has rapidly emerged as a promising new strategy for drug target validation, and it is currently evaluated in clinical trials as a potential therapy for diseases caused by undruggable therapeutic targets [120]. The use of RNAi for therapeutics holds a great promise to change the therapeutic modality in many diseases; however, it is a daunting task to deliver small interfering RNA (siRNA) molecules into specific cell types in a safe manner. A naked siRNA has a limited ability to cross the cell membrane due to its 13 KDa molecular weight and the polyanionic nature (~ 40 negative phosphate residues) [121]. Unmodified, naked siRNA is unstable in blood and serum, as it is rapidly degraded by endo- and exonucleases, with a very short half-life in vivo. In addition, unless chemically modified, siRNA can induce immune responses and may lead to off-target effects. In vertebrates, an immune response can be induced by dsRNA, which is part of a defense mechanism against viral infection. dsRNA can be sensed in the cytoplasmic and endosomal compartments by RNA-dependent kinases (PKRs) and induce [122] an interferon (IFN) response that will end up in production of proinflammatory cytokines, by activating NF-kappa-B dependent pathway. Other known mechanisms, which may cause NF-kappa-B activation and IFN production, are helicase retinoid-acid-inducible gene I (RIG-I) and melanoma differentiationassociated gene 5 (MDA-5). Studies have found that both chemically synthesized siRNAs and siRNAs generated from in vitro transcription with various lengths can lead to the activation of PKR. The dsRNA length increased the IFN induction levels. For example, Rossi et al. demonstrated that a 5′ triphosphate on T7 polymerase-transcribed RNA molecules is a potent inducer of IFN response [123]. Non-self RNA can also be recognized on the cell surface by TLRs. Recognition of RNA by TLRs activates cellular signaling pathways that lead to the activation of NF-κB and production of proinflammatory cytokines. Kleinman et al. showed that siRNA against VEGF, a pro-angiogenic factor that is involved in age-related macular degeneration in the eye, can antagonize TLR3 on the cell surface of fibroblasts. The observed effect of decreased blood vessel growth was demonstrated regardless of the sequence, which meant that sequence-independent RNAi triggered TLR3 and subsequent local inflammation that lead to a therapeutic benefit was not part of a specific silencing of VEGF target [124]. In order to avoid or reduce unfavorable immune response, siRNAs need to be optimized in a way that evades triggering TLRs responses as well as intracellular responses such as with the inflammasome. For example, avoid GU-rich sequences, optimizing the length and/or reducing the dosing of siRNA and perform an experimental screening of multiple siRNAs against the same target in order to identify the optimal candidate with minimal immune response. There are several exceptional reviews on the chemical modifications of RNAi payloads [125,126], which are beyond the scope of this report. It is widely known that efficient intracellular RNAi delivery to specific target cells following systemic administration is the challenge for widespread use of RNAi in the clinic [121]. Among many studied delivery systems, only few seem to hold more promise. One of the reasons is the toxicity and immunogenicity caused by the interactions of the particles with leukocytes. As with other therapeutics delivered via systemic strategy, this approach can be divided into two major categories: passive and active (targeted) delivery. Passive delivery utilizes the inherited propensity of liposomes and other LNPs to accumulate in cancerous or highly inflamed tissues due to the enhanced permeability and retention (EPR) effect. In this effect an increased permeability of blood vessels in solid tumors and highly inflamed tissues with dysfunctional lymphatic Please cite this article as: D. Peer, Immunotoxicity derived from manipulating leukocytes with lipid-based nanoparticles, Adv. Drug Deliv. Rev. (2012), doi:10.1016/j.addr.2012.06.013 8 D. Peer / Advanced Drug Delivery Reviews xxx (2012) xxx–xxx drainage is occurring, which retain the accumulated liposomes/LNPs in close proximity to the tumor or the inflamed tissue [127]. Different studies have shown that the efficacy of extravasation into tumors is higher with smaller particles (b200 nm) [128–131]. Liposomes and NPs also tend to accumulate in organs of the mononuclear phagocytic system (MPS). The MPS, part of the immune system, consists of phagocytic cells located in reticular connective tissue, primarily monocytes, dendritic cells and macrophages. These cells accumulate in the spleen, liver and lymph nodes and take up foreign particles such as bacteria, viruses, and parasites with different sizes and surface charges [132,133]. Active siRNA delivery is based on specific antibodies and ligands that direct the nanocarriers to specific target cells and tissues in order to achieve maximal therapeutic effect, decrease the amount of RNAi payload required and avoid nonspecific silencing and toxicity in bystander cells [128]. Tremendous progress has been made in systemic siRNA delivery and those have been reviewed extensively elsewhere by us and by others [1,95,134–136]. Below we will only detail example of modulating the immune response by passive and by active delivery strategies targeted to leukocytes (see Table 3). 4.2.1. Targeted delivery into pan leukocytes The systemic delivery of RNAi for specific gene silencing to target leukocytes is an unconquered and extremely challenge task that holds a great potential for treatment of leukocyte-implicated diseases such as inflammation, blood cancers (lymphoma, leukemia, myeloma), and leukocyte-tropic viral infections (HIV, ebola, dengue). Since leukocytes are resistant to conventional transfection reagents and are dispersed throughout the body, it becomes a major challenge to achieve successful delivery of RNAi payloads selectively in leukocytes. Several strategies are under different research stages. For example, PEGylated liposomes are being used for delivery and targeting of siRNA in vitro with ligands covalently attached to the liposomal surface that bind to receptors expressed on leukocytes [139]. Unfortunately, these PEGylated particles induced a significant increase in serum cytokine levels in an unclear mechanism, thus reducing the therapeutic potential concealed with these particles. Nevertheless, cytokine induction might be considered as a therapeutic advantage, when an immune activation could strengthen the therapeutic effect. Table 3 Immune modulation by selected delivery platforms. Type of immune modulation Target cells LNP Dimensions of the LNPs (nm in technology diameter) References Anti-inflammatory (depletion of the circulating monocytes) Anti-inflammatory Macrophages, monocytes, dendritic cells ~180 Liposomal aldronate P [100] Infarct macrophages 1200 [81] ~1053 PS‐ presenting liposomes⁎ OML⁎ [111,114] 2300 CCL⁎ [118] ~100 PEGylated [86] liposomes⁎ [137] P-selectin coated liposomes⁎⁎ I-tsNP⁎⁎ [138] CTL adjuvanicity Cytotoxic T lymphocytes Failure in inducing Lymphoid tissue mucosal and systemic adjuvant responses Cytokine Leukocytes induction Inflammation T cells Gut inflammation ⁎ ⁎⁎ Gut lymphocytes Passive delivery. Active targeted delivery. 100–150 ~100 Huang et al. demonstrated another delivery platform to silence genes in circulating cells with the P-selectin coated liposomes. P-selectin can be absorbed onto the surface of a blood-compatible microrenathane tube. P-selectin-coated surface could successfully captured P-selectin receptor-positive stem cells from physiological shear flow in vitro and from the bloodstream in vivo, although previous studies have shown that P-selectin-based cell capture is also associated with inflammation of the liver or lung in vivo [137]. 4.2.2. Targeting subsets of leukocytes An elegant approach for the delivery of RNAi molecules into dendritic cells that is based on liposomes decorated with monoclonal antibody against DEC-205, a dendritic cell specific protein and CD40-siRNA was reported. After i.v. administration to mice, a functional silencing of CD40 was demonstrated [140]. This approach can be used to target CD40 siRNA specifically to dendritic cells in vivo and consequently induce antigen-specific immune suppression and may be feasible for siRNA-based clinical therapy. Leukocyte integrins mediate the adhesive interaction between endothelial cells and leukocytes, one of the crucial steps in the migration of cells to the inflamed site. Antibodies such as efalizumab (Raptiva) and natalizumab (Tysabri) were developed in order to block these adhesive interactions and are examples of FDA approved, humanized, integrin-blocking antibodies for treatment of autoimmune diseases. The potential use of integrins as receptor targets for RNAi delivery to lymphocytes is based on the fact that two of the family members, β2 and β7 integrins, are expressed exclusively on leukocytes and thus enable the selective targeting. Moreover, these integrins are constitutively internalized and recycled. Thus, integrin recycling supports the internalization of bound antibodies, a prerequisite for siRNA-mediated activation of the RNAi pathway. The most unique feature of integrins is their ability to bind ligands via a conformational change from a low affinity conformation in resting cells to a high affinity conformation upon activation. A proof-of-concept for utilizing leukocyte integrins as receptor targets for RNAi delivery was demonstrated first by the use of scFv against the high affinity form of the integrin lymphocyte function-associated antigen I (LFA-I), which was fused to protamine, a highly positively charged protein that nucleates DNA in the sperm. This fusion protein selectively delivered siRNAs into activated lymphocytes, both in vitro and in vivo, but not to resting lymphocytes [138]. Based on this strategy, a sophisticated method was devised termed the integrin‐targeted and stabilized nanoparticles (I-tsNPs) [14,57,58,141]. Using this approach, it was demonstrated that a single i.v. injection of I-tsNP-encapsulated siRNA (2.5 mg/kg) targeted against the DNA repair-associated gene Ku70 was determined to be sufficient to reduce target gene expression by approximately 80% in β7 integrin-positive cells of gut lymphocytes [14]. The same platform was used for validation of the role of the cell cycle regulator, cyclin D1, as a potential anti-inflammatory target. Cyclin D1 was found to be upregulated in T lymphocytes at sites of inflammation such as in inflammatory bowel diseases. An i.v. injection of β7 I-tsNP-entrapping cyclin D1-siRNAs in colitis-induced mice reduced cyclin D1 mRNA and protein levels and caused a remarkable reduction in intestinal inflammation [14] without activating lymphocytes, inducing an interferon response and provoking a proinflammatory cytokine secretion with minimal off-target effects and toxicities. This strategy is now widely exploited in several different types of blood cancer, gut inflammation and HIV infection. 5. Conclusions and future prospective Understanding the complicated nature of the immune system with its unique arms can open the door for utilizing LNPs to manipulate the immune response. In a recent study [16], it was demonstrated that hydrophobicity can dictate the immune response. A small library of gold NPs was synthesized and characterized. These gold NPs had increased hydrophobic carbon chains on their surface and had almost Please cite this article as: D. Peer, Immunotoxicity derived from manipulating leukocytes with lipid-based nanoparticles, Adv. Drug Deliv. Rev. (2012), doi:10.1016/j.addr.2012.06.013 D. Peer / Advanced Drug Delivery Reviews xxx (2012) xxx–xxx a linear behavior (both in vitro and in vivo) when interacted with subsets of leukocytes. LNPs are one of the most veteran delivery vehicles used already in clinical practice for many years and several new classes of LNPs including hybrid systems (lipid-polymers) are under clinical evaluation with therapeutic and imaging payloads. Adequate and comprehensive understanding of the specific interaction between LNPs with T cells, B cells, DCs, macrophages and monocytes at the cellular and molecular level, and appropriate assays to probe immune suppression or stimulation such as cytokine induction, interferon response, lymphocyte activation, coagulation cascades and complement activation, will aid in designing safer vehicles for therapeutic and imaging strategies that will benefit not only the patients, but also the entire pharmaceutical and biotech industry. Those might experience shorter development path for new chemical entities that are entrapped within LNPs. Acknowledgement Dan Peer wishes to thank Ms. Varda Wexler for her help with the graphics and illustrations and the Peer laboratory members for helpful discussions. This work was supported in part by grants from the Marie Curie IRG-FP7 of the European Union, Lewis Family Trust, Israel Science Foundation (Award #181/10), The MAGNET program of the Israeli OCS, the Kenneth Rainin Foundation, the Israeli Centers of Research Excellence (I-CORE), Gene Regulation in Complex Human Disease, Center No 41/11 and by the FTA: Nanomedicine for Personalized Theranostics awarded to D.P. References [1] M. Goldsmith, S. Mizrahy, D. Peer, Grand challenges in modulating the immune response with RNAi nanomedicines, Nanomedicine (Lond.) 6 (10) (2011) 1771–1785. [2] S. Mizrahy, D. Peer, Polysaccharides as building blocks for nanotherapeutics, Chem. Soc. Rev. 41 (7) (2012) 2623–2640. [3] M.A. Dobrovolskaia, S.E. McNeil, Immunological properties of engineered nanomaterials, Nat. 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