Nanoparticle uptake by circulating leukocytes: A major barrier to tumor delivery
Graphical abstract
Introduction
The use of nanoparticles as drug delivery vehicles offers the potential for reduced toxicity, greater efficacy, and enhanced retention at the target site. While this approach could potentially be used for delivery to any tissue, the predominant focus of current nanoparticle delivery is to achieve selective drug accumulation in tumors [1]. It is well known that nanoparticle-mediated delivery to tumors via intravenous administration is aided by the abnormal vasculature associated with rapidly growing tumors, especially in animal models [2, 3]. In spite of this advantage, the amount of the injected dose that accumulates in tumors remains very low (≈ 1%), even in animal models [4]. It follows that the vast majority of the injected dose (> 90%) accumulates in tissues other than the tumor, primarily in the lungs, liver and spleen, and the potential for toxicity of both the drug cargo and the delivery vehicle needs to be considered [[5], [6], [7], [8], [9], [10], [11]]. Furthermore, the resident macrophages in these organs are the major components of the reticuloendothelial system (RES) responsible for nanoparticle clearance that is thought to play the predominant role in limiting particle deposition in the tumor [12, 13]. Accordingly, strategies that reduce uptake by resident immune cells in the lung, liver, and spleen have been shown to prolong circulation times and improve delivery to the tumor [[13], [14], [15], [16]]. More recently, it has been suggested that this uptake by immune cells could be exploited for immuno-oncology, and usher in a new era for cancer nanomedicine [17].
It should be recognized that intravenous administration introduces nanoparticles into the vasculature where delivery systems immediately come in contact with serum proteins and blood cells. The effects of serum proteins on the stability of delivery systems is well studied, and the tendency of researchers to optimize formulations for in vitro transfection under conditions of low/no serum undoubtedly contributes to the inability of many formulations to perform well in vivo [[18], [19], [20], [21], [22], [23]]. In addition to the effects of serum proteins, intravenously-injected nanoparticles interact with circulating blood cells, and fusogenic particle formulations have been shown to cause aggregation of red blood cells that promotes rapid accumulation in the lung [[24], [25], [26], [27]]. Some researchers have attempted to exploit interactions with red and white blood cells (“hitchhiking”) to achieve prolonged circulation and facilitate delivery to sites of inflammation and cancer [[28], [29], [30], [31]]. However, other studies have demonstrated that some formulations are avidly taken up by circulating leukocytes, and shown that monocytes ultimately migrate to the RES tissues and differentiate into resident macrophages [[32], [33], [34], [35], [36], [37]]. Therefore, initial interactions with circulating cells can potentially lead to the eventual accumulation of nanoparticles in the lung, liver, and spleen. Regardless of the specific interactions with various cell types after intravenous administration, uptake by non-target cells ultimately sequesters nanoparticles and affects drug delivery to the target tissue (e.g., tumor). Although targeting ligands can promote the retention of deposited nanoparticles in the target tissue, non-specific uptake by blood and/or immune cells nullifies targeting by preventing the ligand from gaining access to its receptor on the target tissue. It follows that strategies that reduce uptake/clearance by non-target tissues not only reduce toxicity in those tissues, but also increase the potential for systemically-administered nanoparticles to passively deposit in the target tissue.
Although interactions between delivery systems and circulating cells is well documented, strategies attempting to extend circulation times and increase delivery to the tumor predominantly focus on preventing uptake by resident macrophages in the lung, liver, and spleen. Furthermore, it is common practice to measure bioavailability by harvesting a blood sample and quantifying levels of radio-labelled nanoparticles under the assumption that circulating nanoparticles have the potential to distribute to target tissues. While blood levels are the traditional measure of “bioavailability” used for conventional small molecule pharmaceuticals, this approach ignores the potential for particles to be sequestered by circulating blood cells and thereby unavailable for diffusion into target tissues despite being present in the blood.
Our previous work has demonstrated that the serum stability of lipoplexes can be greatly enhanced by including high levels of cholesterol, consistent with earlier work with lipid-based delivery systems [23, [38], [39], [40], [41], [42]]. We also documented that lipoplexes can be formulated to promote the formation of cholesterol domains, which have been shown to enhance serum stability and transfection rates in vitro and in vivo [40, [43], [44], [45], [46]]. More recently, we have substituted sphingosine for synthetic cationic lipids, and greatly reduced the toxicity and immunogenicity of the delivery system [5, 6, 47]. This optimized formulation was employed in the current study to investigate the uptake of lipoplexes by blood cells, and the concomitant effects on circulation times and tissue distribution. In addition, we use flow cytometry to quantify uptake by specific blood cells and probe the relationships among plasma levels, leukocyte uptake, half-life, tissue distribution, and tumor deposition.
Section snippets
Materials
Cholesterol, N-(1-(2, 3-dioleoyloxy) propyl)-N, N, N-trimethylammonium chloride (DOTAP), diarachidoyl-sn-glycero-3-phosphocholine (DAPC), egg phosphatidylcholine, sphingosine, rhodamine-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl), and PEG750-ceramide were purchased from Avanti Polar Lipids (Alabaster, AL). PicoGreen® was obtained from Thermo Fisher Scientific
Results
Early studies with conventional, multilamellar liposomes demonstrated that leukocytes are capable of internalizing lipid-based particles [52, 53]. Uptake by leukocytes is part of the innate immune system which has evolved to provide efficient surveillance for foreign pathogens [54]. It follows that the introduction of nanoparticles via intravenous injection mimics the infection process, and thus might trigger sequestration by leukocytes. In addition, previous studies have demonstrated that
Discussion
Researchers have been attempting to improve delivery to tumors for decades [[14], [15], [16]]. It was recognized in early studies with liposomes that resident macrophages in the lung, liver, and spleen are part of the endogenous system of clearing foreign particulates, and studies have predominantly focused on reducing uptake in these organs [15, 16, 57, 58]. Although some studies have demonstrated the ability of monocytes and neutrophils to phagocytose nanoparticles [[29], [30], [31], [32], 35
Acknowledgments
This work was supported by grants from the ALSAM Foundation (TJA and MAN) and the National Institutes of Health (RO1EB016378 to TJA and R01NS094758/P01AG032958 to MAN). In addition, the flow cytometry facility is supported by the University of Colorado Cancer Center via Grant P30CA046934 from NIH.
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2022, Journal of Pharmaceutical SciencesCitation Excerpt :However, studies have established that ABC also occurs in immunocompromised animals that are incapable of producing antibodies, demonstrating that the innate immune response is involved in accelerated clearance.24 Previous work has documented the extensive uptake of naked lipoplexes by circulating immune cells,48,54,65 and our recent work has demonstrated that a lactose coating provides an equivalent reduction in leukocyte uptake and superior tumor accumulation to that observed with PEGylated lipoplexes.21 We have previously shown that lactosylated lipoplexes elicit less of a cytokine response as compared to PEG, but the results in Fig. 1 clearly indicate that ABC is also observed when lipoplexes are coated with lactose.
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