The field of therapeutic nanomedicines is still attempting to fully characterize and understand the immune responses triggered by intravenous administration of nanoparticles. The most persistent problems the field faces are unintended interactions with the body's milieu, insufficient tumor delivery, and off-target accumulation of nanomedicines in the Mononuclear Phagocytic System (MPS) (a.k.a reticuloendothelial system), which includes the liver, spleen, and other major organs [[1], [2], [3], [4]]. The innate immune system governs the clearance and accumulation of nanomedicines in the MPS and initiates rapid responses to repeat injections of nanomedicines [[5], [6], [7], [8], [9], [10]]. Even with the most promising “stealth” or “targeted” formulations approximately 99% of an injected dose of nanomedicines will accumulate in the MPS and other off-target tissues [11]. Since many chemotherapeutic nanomedicines require repeat injections, this off-target accumulation can lead to altered pharmacokinetics and toxicities that can compromise the therapeutic benefits [[12], [13], [14]]. However, if a reduction in drug exposure in non-tumor tissues could be achieved, it would allow for more aggressive dosing regimens leading to greater therapeutic efficacy. Indeed, the most well-established chemotherapeutic nanomedicine, Doxil® (liposomal encapsulated doxorubicin), has been successful by exploiting a similar strategy. Doxorubicin administered as a free drug is extremely toxic and can cause fatal cardiotoxicity [15]. Doxil® effectively minimizes this cardiotoxicity by reducing off-target doxorubicin accumulation in cardiomyocytes and increasing tumor delivery of doxorubicin [16]. Nonetheless, current research predominantly focuses on “targeting” tumor tissues and “hiding” from the immune system. We suggest that it may be more productive to focus on exploiting the innate immune system to reduce off-target accumulations.

One such immune response that is of particular interest is a poorly understood phenomenon that happens when complexes of nucleic acids and cationic lipids (i.e., lipoplexes) are repeatedly administered (∼every 24 h). Curiously, studies have reported that neither organ accumulation nor expression of the gene therapy vector increases with repeated administrations of lipoplexes, and actually decreases in some cases [9,10,17,18]. This phenomenon was dubbed the “refractory” response, initiating approximately 12 h after administration and has been reported to last for an average of approximately 2–3 weeks [9,10,17,[19], [20], [21], [22]]. It is understood that this response cannot be due to the complement/antibody driven Accelerated Blood Clearance (ABC) phenomenon which requires several days to elicit after the initial injection [23]. Additionally, the ABC effect involves an adaptive immune response to PEGylation [23,24], and not all the nanoparticles in the reported studies were PEGylated. Furthermore, an ABC event would be expected to result in increased hepatic accumulation of the gene therapy vector, which was not observed [6,7,25]. Since neither expression nor accumulation of the gene therapy vectors in MPS organs significantly increases with repeated administration, it suggests that the refractory response is an immunological reaction to lipoplexes that directly limits tissue deposition of subsequently administered particles.

When considering the similarities between the structure of viruses and lipoplexes, it is understood why lipoplexes would cause immune reactions. Viruses are on the nanoscale, often possess lipid membranes, have surfaces coated with polysaccharides, and contain DNA/RNA. Similarly, lipoplexes consist of lipid particles that are on the nanoscale, usually contain polyethylene glycol (PEG), which has a similar structure to polysaccharides, and are complexed/conjugated to nucleic acids. Considering these similarities, we propose that the refractory response is an anti-viral response that is triggered when lipoplexes lead to activation of the innate immune system. Recent studies in the field of viral invasion have revealed a novel anti-viral response that provides resistance to viral spread and re-infection [[26], [27], [28], [29], [30], [31], [32], [33], [34], [35]]. This response is rapidly elicited upon sensing of virus-like materials to induce interferons including type III interferons (IFN-λ) produced by epithelial cells, leading to the induction of interferon stimulated genes and a broad anti-viral state within the tissue [28,[36], [37], [38], [39], [40], [41], [42]]. Additionally, IFN-λ enhances epithelial/endothelial barrier (GI tract, Blood Brain Barrier, Skin) functions, “tightens” cellular junctions, and limits viral invasion and infection of the underlying tissues [27,29,31,35,39,43]. We hypothesize that administration of lipoplexes leads to an anti-viral response that systemically tightens healthy epithelium and limits tissue deposition of a subsequently administered particle.

It could be beneficial to tighten the body's epithelial barriers and limit systemic exposure to potentially toxic particles such as chemotherapeutic nanomedicines. However, chemotherapeutic nanomedicines also need to be delivered to the tumor to be efficacious. In this context, it is well established that the tumor microenvironment is highly dysregulated especially with regards to epithelial structure and immune responses [[44], [45], [46], [47]]. In fact, avoiding immune detection and promoting inflammation are two of the most prominent hallmarks and enabling characteristics of cancers [44]. Due to the dysfunction of the tumor microenvironment, it is likely that tumor tissues will respond differently to a lipoplex injection as compared to healthy tissues. Therefore, if the tumor possesses a compromised ability to properly respond to immunological signals such as IFN-λ, then the tumor microenvironment may remain relatively unaffected during a systemic tightening event. Under such conditions, a dose of nanoparticles administered to a tumor-bearing subject would potentially exhibit reduced particle deposition in healthy tissues while accumulation in the tumor remains unimpeded. This is consistent with our studies on the repeat administration of lipoplexes in tumor-bearing mice [18,48].

In this study we conducted experiments to characterize the refractory response and determine the organ/tumor accumulation of intravenously injected dextran/Doxil® in mice that had been pretreated with saline, lipoplexes, liposome, or IFN-λ. We also demonstrate that the refractory response is unique to virus-like particles that contain nucleic acids. Our results clearly illustrate that an IFN-λ response is initiated when lipoplexes are intravenously administered while a liposome treatment does not elicit the production of IFN-λ. Additionally, our data demonstrate that pretreatment with either a lipoplex or IFN-λ leads to a significant decrease in organ dextran/Doxil® deposition coupled with an increase in plasma and tumor accumulation.