Full length articleUnraveling the role of Intralipid in suppressing off-target delivery and augmenting the therapeutic effects of anticancer nanomedicines
Graphical abstract
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.
Section snippets
Materials
RPMI-1640 medium, Dulbecco's modified Eagle medium (DMEM), CDDP, isoflurane, and all solvents were purchased from Wako Pure Chemical (Osaka, Japan). Pyropheophorbide-a was from Frontier Scientific (Inochem, Ltd., UK) and poly(styrene maleic anhydrate) (SMA) was from Sigma-Aldrich (St. Louis, MO, USA). Fetal calf serum was obtained from GIBCO (Grand Island, NY). Latex Beads-Rabbit IgG-FITC Complex was from Cayman Chemical (Ann Arbor, MI, USA). Lipi-Red and Hoechst 33342 were from Dojindo
Intralipid uptake by Kupffer cells
We first confirmed Intralipid uptake by Kupffer cells by using Oil Red O staining. Lipid droplets inside Kupffer cells were clearly visible after Intralipid addition (Fig. 1A). Quantification of results showed that Intralipid was taken up by Kupffer cells over time; uptake reached a plateau at 8 h and remained constant up to 24 h (Fig. 1B). We also found lipid droplets in liver tissues of S180 tumor-bearing mice treated with Intralipid for 1–24 h by the same Oil Red O staining method
Discussion
Reduced liver toxicity and increased bioavailability of nanomedicines are reportedly the two major advantages of Intralipid pretreatment to improve the therapeutic outcome of nanomedicines. Here in this study, we confirmed that Intralipid blocked the capture of nanomedicines by the liver both in vivo and in vitro (Figs. 2 and 3), which led to reduced liver toxicity (Fig. 4). More important, we also found significantly increased tumor accumulation of nanomedicines, which resulted in improved
Conclusions
In this study, we validated the beneficial effect of Intralipid pretreatment as a strategy to temporarily suppress the RES to induce improved EPR effect-based delivery of nanomedicines. Reduced liver uptake of nanomedicines and a simultaneous increase in tumor accumulation of nanomedicines were clearly results of pretreatment with Intralipid. The consequences were not only hepatoprotection but also, and more important, significantly enhanced therapeutic effects of nanomedicines, which were
Funding
This work was partly supported by Grants-in-Aid for Scientific Research, on Scientific Research (C) (JSPS KAKENHI grant numbers 16K08217 and 19k07743) to J. Fang; partly by a Bilateral Joint Research Project between the Japan Society for the Promotion of Science (JSPS) and the Czech Academy of Sciences (CAS) (29400001) to H. Maeda and J. Fang; and partly by the Czech Science Foundation (grant 19-01417S) to T. Etrych.
CRediT authorship contribution statement
Rayhanul Islam: Methodology, Software, Data curation, Writing – original draft, Visualization, Investigation, Validation, Writing – review & editing. Shanghui Gao: Methodology. Waliul Islam: Methodology. Vladimír Šubr: Methodology. Jian-Rong Zhou: Methodology. Kazumi Yokomizo: Methodology. Tomáš Etrych: Funding acquisition, Writing – review & editing. Hiroshi Maeda: Conceptualization, Supervision, Validation, Funding acquisition, Writing – review & editing. Jun Fang: Conceptualization,
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The authors thank Dr. Tsukasa Takeuchi of Shimadzu Corporation for kindly providing the in vivo fluorescence imaging probe o-SWCNT-PEG, and Ms. Hitomi Miyahara of Summit Pharmaceuticals International for her technical assistance in tumor blood vessel imaging. We also thank Ms. Judith Gandy for editing the manuscript.
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