Enhancing subcutaneous injection and target tissue accumulation of nanoparticles via co-administration with macropinocytosis inhibitory nanoparticles (MiNP)

Abstract

A significant barrier to the application of nanoparticles for precision medicine is the mononuclear phagocyte system (MPS), a diverse population of phagocytic cells primarily located within the liver, spleen and lymph nodes. The majority of nanoparticles are indiscriminately cleared by the MPS via macropinocytosis before reaching their intended targets, resulting in side effects and decreased efficacy. Here, we demonstrate that the biodistribution and desired tissue accumulation of targeted nanoparticles can be significantly enhanced by co-injection with polymeric micelles containing the actin depolymerizing agent latrunculin A. These macropinocytosis inhibitory nanoparticles (MiNP) were found to selectively inhibit non-specific uptake of a second "effector" nanoparticle in vitro without impeding receptor-mediated endocytosis. In tumor bearing mice, co-injection with MiNP in a single multi-nanoparticle formulation significantly increased the accumulation of folate-receptor targeted nanoparticles within tumors. Furthermore, subcutaneous co-administration with MiNP allowed effector nanoparticles to achieve serum levels that rivaled a standard intravenous injection. This effect was only observed if the effector nanoparticles were injected within 24 h following MiNP administration, indicating a temporary avoidance of MPS cells. Co-injection with MiNP therefore allows reversible evasion of the MPS for targeted nanoparticles and presents a previously unexplored method of modulating and improving nanoparticle biodistribution following subcutaneous administration.

Figure 1:. Schematic of tumor bearing mice being co-administered with latrunculin A-loaded macropinocytosis inhibitory nanoparticles (MiNP).

MiNP were developed and evaluated for their effect on the accumulation of a targeted “effector” nanoparticle via subcutaneous and intravenous injection. As MiNP interferes with macropinocytosis but not receptor-mediated endocytosis, pre- and/or co-injection of MiNP with an effector nanoparticle displaying targeting ligands allows enhanced uptake by cells expressing the target receptor. As an example, MiNP are shown enhancing the targeting of receptors highly expressed within tumor microenvironments by interfering with off-target mononuclear phagocyte system (MPS) clearance.

Figure 2:. LatA retains its endocytic inhibition properties and does not change the size of PEG-b-PPS micelles when encapsulated.

a) Cryogenic transmission electron microscopy (CryoTEM) of MiNP visually confirms retention of micellar structures. b) MiNP with ((+)MiNP) and without ((−)MiNP) loaded LatA were characterized via small angle x-ray scattering (SAXS) and fitted with a micelle model fit using SASView. c) Free LatA and (+)MiNP significantly inhibited macropinocytosis by RAW264.7 macrophages as compared to clathrin-mediated endocytosis inhibitor chlorpromazine. Cells were treated with each inhibitor for 2 h followed by 30 min of incubation with pHrodo dextran prior to analysis by flow cytometry. Data are shown as a percentile scale of endocytosis inhibition. On this scale, 0% represents standard cell uptake with no inhibitor, while 100% represents complete inhibition with no uptake of dye. N=3 p<0.001. d) In comparison, uptake of transferrin conjugated pHrodo dextran by macrophages via receptor-mediated endocytosis (RME) was significantly inhibited by chlorpromazine compared to (+)MiNP. N=3 p<0.001. e) Loading within (+)MiNP significantly decreased the toxicity of LatA. Macrophages were incubated with various doses of free LatA or (+)MiNP for 4 h and assessed by flow cytometry for viability via the Zombie Aqua live/dead assay. N=3 p<0.05.

Figure 3.. Latrunculin A loaded MC ((+)MiNP) co-injection and −4 h pre-injection lead to similar effector particle biodistributions.

a) Timeline showing the injection times for the co-injection and −4 h injection methods, which were evaluated for both subcutaneous (SC) and intravenous (IV) administration. “(+/−)MiNP” indicated an injection of either (+)MiNP or (−)MiNP, and effector micelle injections are indicated by “E-MC”. All mice were sacrificed at 24 h post E-MC injection. Comparisons of cell uptake in spleen and liver for the different SC (b, c) and IV (d, e) injection methods are shown. In all cases, mice were injected with 100 μL 7μM LatA (+)MiNP or (−)MiNP and E-MC were labelled with DiR for flow cytometric quantification of cellular uptake within the spleen and liver. Data are reported as fold increase median fluorescence intensity of the E-MC over an untreated control. N=5 p<0.0001. To assess the transience of the MiNP effect, mice were injected SC (f) or IV (g) with (+/−)MiNP and E-MC according to the co-injection method, and serum levels of E-MC were evaluated by fluorescence spectroscopy. Mice were then rested for 72 hours and injected again with only E-MC to determine whether the inhibitory effect remained. N=3 for 2 h and 4 h timepoints and N=6 for 24 h timepoints, *p<0.05.

Figure 4.. (+)MiNP treatment increases the accumulation of folate-targeted E-MC (E-MC(FA)) in B16F10 tumors following SC injection.

a) Timeline of injection protocol assessing the tumor-targeting co-injection method. (+/−)MiNP indicates an injection of either (+)MiNP or (−)MiNP. Mice were sacrificed 24 h after the co-injection for analysis by flow cytometry. Results are shown for IV (b, c, d) and SC (e, f, g) injections of 3 co-injection modalities: (−)MiNP treatment/E-MC, (+)MiNP treatment/E-MC, and (+)MiNP/E-MC(FA). Fluorescent E-MC and E-MC(FA) uptake by 3 different cell subsets were quantified: non-immune cells (b, e), dendritic cells (c, f), and macrophages (d,g) for 4 different organs. Data are reported as fold increase median fluorescence intensity of E-MC or E-MC(FA) over a PBS baseline control. N=4–10 p<0.05. Significance was determined within each organ by separate unpaired student’s t-tests.