Unraveling the role of Intralipid in suppressing off-target delivery and augmenting the therapeutic effects of anticancer nanomedicines

Abstract

Intralipid, a clinically used lipid emulsion, was reportedly utilized as one strategy to suppress off-target delivery of anticancer nanomedicines; Intralipid also effectively improved drug delivery to tumors and produced better therapeutic effects. However, the mechanisms involved—the why and how—in Intralipid's facilitation of delivery of nanomedicines to tumors have not yet been reported in detail. In this study, we investigated Intralipid and discovered the beneficial effects of Intralipid pretreatment when using three anticancer nanomedicines, including the clinically approved drug doxorubicin (Doxil). Intralipid pretreatment induced a 40% reduction in liver uptake of a polymeric nanoprobe used in photodynamic therapy as well as a 1.5-fold-increased nanomedicine accumulation in tumors. This increased accumulation consequently led to significantly better therapeutic effects, and this finding was validated by using Doxil. As an interesting result, Intralipid pretreatment significantly prolonged the plasma half-life of nanomedicines in normal healthy mice but not in tumor-bearing mice, which suggests that tumors become an alternative route of nanomedicine delivery when liver delivery is suppressed. Also, we found markedly increased tumor blood flow, as measured by fluorescence angiography, and significantly lower blood viscosity after Intralipid pretreatment. All our results together indicate that Intralipid treatment not only suppressed off-target nanomedicine delivery by the reticuloendothelial system, but more important, it enhanced nanomedicine delivery to tumors by improving tumor blood flow, which is key to satisfactory drug delivery via the enhanced permeability and retention effect. Significantly better therapeutic outcomes were thus achieved by the strategy of combining utilization of nanomedicines and Intralipid pretreatment.

Statement of significance

Off-target delivery to organs such as the liver and obstructed tumor blood flow as is often seen in advanced cancers are major barriers to the therapeutic efficacy of anticancer nanomedicines. Intralipid has been shown effective for suppressing nanomedicine accumulation in the liver, resulting in improved anticancer effects. Unraveling the mechanisms involved in this process will be greatly helpful for the clinical application of anticancer nanomedicines. We reported here that Intralipid could also significantly increase tumor delivery of nanomedicine, which is beneficial for improving tumor blood flow and lowering blood viscosity. To our knowledge, this is the first study to investigate the role of Intralipid in this regard. This knowledge provides a solid rationale for the use of Intralipid in combination with anticancer nanomedicines.

Introduction

The enhanced permeability and retention (EPR) effect serves as the foundation of tumor-targeted delivery of anticancer nanomedicines [1,2]. The EPR effect is a unique phenomenon occurring in tumor tissues that is related to tumor-associated physiological and anatomical abnormalities, including large fenestrations, increased permeability, and defective lymphatic function in tumor vasculature, that allow the entry of macromolecules into tumor tissues and their retention in those tissues [1]. EPR effect-based anticancer nanomedicines have shown superior tumor-targeting efficacy and therapeutic activity compared with low-molecular-weight counterparts [3], [4], [5], [6], [7]. Nonetheless, delivery of anticancer nanomedicines to advanced solid tumors is challenging, and clinical use of these agents is still not very popular because of various problems [2]. Enhancement of the EPR effect by improving its heterogeneity and by utilizing vascular mediators in tumor tissues is urgently needed, and some solutions have recently been reported [2].

The reticuloendothelial system (RES) associated with the liver and spleen is also a key factor that affects nanomedicine biodistribution and bioavailability by sequestering nanoparticles from the circulation. Kupffer cells in the liver are mainly responsible for endocytic uptake of nanomedicines. Tsoi et al. showed that about 84% of Kupffer cells residing in the liver take up injected nanoparticles [8]. This unwanted off-target delivery greatly hampers the delivery of nanomedicines to tumors. In addition, accumulation of nanomedicines in the liver may induce liver injury and adverse side effects [9]. Thus, suppressing off-target delivery of nanomedicines to achieve satisfactory anticancer efficacy of nanomedicines is challenging.

To avoid off-target delivery or cellular uptake of nanomedicines by the RES, attempts have been made to deplete macrophages, for example, such as by induction of macrophage apoptosis by liposomal clodronate administration, or direct blockade of resident macrophages of the RES with an array of inorganic and organic compounds [10]. However, adverse effects including infection and problems related to reduced innate immunity became a serious issue [10].

Intralipid, an FDA-approved lipid emulsion for hyperalimentation that comprises soybean oil (20%), egg-yolk phospholipids (1.2%), and glycerin (2.25%) in water, has been used to temporarily block the RES [11]. Intralipid pretreatment, which markedly inhibited the RES, reduced drug-induced toxicities in the liver, spleen, bone marrow, and kidneys [12]. More important, pretreatment with Intralipid significantly improved the therapeutic effects of anticancer nanomedicines [13]. This strategy was validated in a more recent study, in which increased drug delivery to tumors was clear when Intralipid pretreatment was combined with use of an anticancer nanomedicine [13]. A rationale behind the enhanced delivery of nanomedicines by Intralipid pretreatment was the prolonged plasma half-life of nanomedicines, i.e. increased bioavailability of these agents in the circulation [11,12]. This explanation seems logical because the prolonged plasma half-life of nanomedicines is the basic principle of EPR effect-based tumor drug delivery [2]. However, reasons for and mechanisms related to the increased accumulation in tumors of nanomedicines after Intralipid pretreatment, i.e.—the why and how Intralipid drives nanomedicines into tumors—have not been completely explored.

A noteworthy report concerned the prolonged plasma half-life of nanomedicines in normal healthy mice pretreated with Intralipid [12]; tumor-bearing mice were assumed to manifest the same results. Nevertheless, in our experience, data obtained with normal healthy mice may not necessarily correlate with those obtained with tumor-bearing mice. In non-tumor-bearing normal mice, when the RES is blocked, nanomedicines remain too long in the circulation. In contrast, in tumor-bearing mice, nanomedicines can use an alternative route to tumors when the RES is blocked. We believe that this mechanism may lead to the Intralipid-induced increase in accumulation of nanomedicines in tumors. Here, we therefore designed experiments to investigate this issue in detail.

In addition, when we reviewed previous literature on Intralipid, we found another interesting effect, which Kessler et al. reported [14]: Intralipid infusion significantly reduced blood viscosity in neonates by interfering with the binding of fibrinogen and other larger proteins with the red blood cell (RBC) surface, thereby increasing the negative charge on the RBC surface. The negative surface charge of the vascular endothelial luminal surface repelled contact with the RBC surface, so clot formation was disrupted and blood flow improved. Upregulation of fibrinogen and elevated levels of thrombin have also been reported in cancer patients [15,16]. Fibrinogen-mediated clot formation is partly responsible for blood vessel occlusion and reduced tumor blood flow, which are major barriers to delivery of nanomedicines to tumors [2]. Thus, a reasonable expectation is that Intralipid may augment delivery of nanomedicines to tumors by restoring and improving tumor blood flow.

The present study therefore aimed to explore and verify the beneficial effects of Intralipid pretreatment, with a focus not only on reduced off-target delivery but also on augmented delivery of nanomedicines to tumors. In this study we used several polymeric nanodrugs, including a polymeric photosensitizer used in photodynamic therapy (PDT): N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-conjugated pyropheophorbide-a (P-PyF) [17], which was synthesized at the Institute of Macromolecular Chemistry of the Czech Academy of Sciences. We also used a complex of the cisplatin (CDDP) ion with poly(styrene maleic anhydrate) (SMA-CDDP) [18] developed in our laboratory. In addition, we used a clinically approved anticancer nanomedicine, Doxil [19]. Thus, in our investigations here we clarified the increased tumor blood flow and lowered blood viscosity after Intralipid infusion, which may play a role in enhancing EPR effect-based drug delivery and result in greater therapeutic efficacy as well as reduced side effects.