The application of reconstituted high-density lipoproteins (rHDL) as a drug-carrier has during the past decade been established as a promising approach for effective receptor-mediated drug delivery, and its ability to target tumors has recently been confirmed in a clinical trial. The rHDL mimics the endogenous HDL, which is known to be highly dynamic and undergo extensive enzyme-mediated remodulations. Hence, to reveal the physiological rHDL stability, a thorough characterization of the dynamics of rHDL in biologically relevant environments is needed. We employ a size-exclusion chromatography (SEC) method to evaluate the dynamics of discoidal rHDL in fetal bovine serum (FBS), where we track both the rHDL lipids (by the fluorescence from lipidconjugated fluorophores) and apoA-I (by human apoA-I ELISA). We show by using lipoprotein depleted FBS and isolated lipoproteins that rHDL lipids can be transferred to endogenous lipoproteins via direct interactions in a nonenzymatic process, resulting in rHDL compositional-and size-remodeling. This type of dynamics could lead to misinterpretations of fluorescencebased rHDL uptake studies due to desorption of labile lipophilic fluorophores or off-target side effects due to desorption of incorporated drugs. Importantly, we show how the degree of rHDL remodeling can be controlled by the compositional design of the rHDL. Understanding the correlation between the molecular properties of the rHDL constituents and their collective dynamics is essential for improving the rHDL-based drug delivery platform. Taken together, our work highlights the need to carefully consider the compositional design of rHDL and test its stability in a biological relevant environment, when developing rHDL for drug delivery purposes.
Most
tumor-targeted drug delivery systems must overcome a large
variety of physiological barriers before reaching the tumor site and
diffuse through the tight network of tumor cells. Many studies focus
on optimizing the first part, the accumulation of drug carriers at
the tumor site, ignoring the penetration efficiency, i.e., a measure
of the ability of a drug delivery system to overcome tumor surface
adherence and uptake. We used three-dimensional (3D) tumor spheroids
in combination with light-sheet fluorescence microscopy in a head-to-head
comparison of a variety of commonly used lipid-based nanoparticles,
including liposomes, PEGylated liposomes, lipoplexes, and reconstituted
high-density lipoproteins (rHDL). Whilst PEGylation of liposomes only
had minor effects on the penetration efficiency, we show that lipoplexes
are mainly associated with the periphery of tumor spheroids, possibly
due to their positive surface charge, leading to fusion with the cells
at the spheroid surface or aggregation. Surprisingly, the rHDL showed
significantly higher penetration efficiency and high accumulation
inside the spheroid. While these findings indeed could be relevant
when designing novel drug delivery systems based on lipid-based nanoparticles,
we stress that the used platform and the detailed image analysis are
a versatile tool for in vitro studies of the penetration efficiency
of nanoparticles in tumors.
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