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. 2024 May 18;15(1):4247.
doi: 10.1038/s41467-024-48442-7.

Enhancing in vivo cell and tissue targeting by modulation of polymer nanoparticles and macrophage decoys

Alexandra S Piotrowski-Daspit #  1   2   3 , Laura G Bracaglia #  4   5 , David A Eaton  6 , Owen Richfield  6 , Thomas C Binns  6   7 , Claire Albert  6 , Jared Gould  6 , Ryland D Mortlock  6 , Marie E Egan  8   9 , Jordan S Pober  9   10 , W Mark Saltzman  11   12   13   14
Affiliations

Enhancing in vivo cell and tissue targeting by modulation of polymer nanoparticles and macrophage decoys

Alexandra S Piotrowski-Daspit et al. Nat Commun. .

Abstract

The in vivo efficacy of polymeric nanoparticles (NPs) is dependent on their pharmacokinetics, including time in circulation and tissue tropism. Here we explore the structure-function relationships guiding physiological fate of a library of poly(amine-co-ester) (PACE) NPs with different compositions and surface properties. We find that circulation half-life as well as tissue and cell-type tropism is dependent on polymer chemistry, vehicle characteristics, dosing, and strategic co-administration of distribution modifiers, suggesting that physiological fate can be optimized by adjusting these parameters. Our high-throughput quantitative microscopy-based platform to measure the concentration of nanomedicines in the blood combined with detailed biodistribution assessments and pharmacokinetic modeling provides valuable insight into the dynamic in vivo behavior of these polymer NPs. Our results suggest that PACE NPs-and perhaps other NPs-can be designed with tunable properties to achieve desired tissue tropism for the in vivo delivery of nucleic acid therapeutics. These findings can guide the rational design of more effective nucleic acid delivery vehicles for in vivo applications.

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Conflict of interest statement

The authors declare the following competing interests: A.S.P., M.E.E. and W.M.S. are co-founders of and, at the time of preparing this manuscript, consultants for Xanadu Bio, Inc., W.M.S. is a member of the Board of Directors of Xanadu Bio, Inc., A.S.P., L.G.B., C.A., J.S.P., and W.M.S. are inventors on patent applications related to the work described here. The remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Effects of PACE composition and NP characteristics on blood concentration and biodistribution.
a Schematic of tunable PACE family polymers from monomer building blocks to formulated NPs, NA loading: nucleic acid encapsulation into NPs. b Blood NP concentration over time following systemic IV injection of dye-loaded PACE (blue), PACE-COOH (green), and PACE-PEG NPs (purple) of various sizes (see Supplementary Table 1) using quantitative microscopy (n = 3 mice per group per time point; error bars represent standard deviation (SD)). Representative end-point (c) IVIS analysis of PACE NP uptake in various organs (heart, lungs, liver, spleen, kidneys, and bone), CTL: untreated control. End-point analyses of (d) whole organ fluorescence quantification of PACE NP uptake in various organs (n = 3 mice per group per organ; error bars represent SD), (e) %NP+ cells in homogenized organs by flow cytometry (n = 3 mice per group per organ; error bars represent SD), (f) %NP+ cells in homogenized liver populations (bulk, F4/80+, and CD31+) by flow cytometry (n = 3 mice per group per population; error bars represent SD), and (g) %NP+ cells in homogenized lung populations (bulk, CD45+, and EpCAM+) by flow cytometry (n = 3 mice per group per population; error bars represent SEM). The effects of PACE NP characteristics on biodistribution as measured by (h) cumulative extrahepatic %NP+ cells and (i) cumulative number of NP+ extrahepatic organs, both as a function of time in circulation. PACE NPs are represented according to chemistry (PACE (blue), PACE-COOH (green), and PACE-PEG NPs (purple)) and size (see Supplementary Table 1).
Fig. 2
Fig. 2. Effects of PACE-PEG dosing on blood concentration and biodistribution.
a Blood NP concentration over time following systemic IV injection of dye-loaded PACE-PEG NPs at various doses (0.1 mg: pink, 0.5 mg: orange, 2.0 mg: green, 2.5 mg: turquoise, and 3.5 mg: indigo per animal corresponding to approximately 30 billion, 160 billion, 650 billion, 800 billion, and 1 trillion NPs, respectively) using quantitative microscopy (n = 3 mice per group per time point; error bars represent standard deviation (SD)). b Circulation half-life of PACE-PEG NPs as a function of dose. Representative end-point (c) IVIS analysis of PACE-PEG NP uptake in various organs (heart, lungs, liver, spleen, kidneys, and bone), CTL: untreated control. End-point analysis of (d) whole organ fluorescence quantification of PACE-PEG NP uptake in various organs (n = 3 mice per group per organ; error bars represent SD). End-point analyses of (e) %NP+ cells and (f) Mean fluorescence intensity (MFI) in arbitrary units (arb. units) of homogenized organs by flow cytometry (n = 3 mice per group per organ; error bars represent SD). End-point analysis of (g) %NP+ cells and (h) MFI in homogenized liver populations (bulk, F4/80+, and CD31+) by flow cytometry (n = 3 mice per group per population; error bars represent SD). End-point analyses of (i) %NP+ cells and (j) MFI in homogenized lung populations (bulk, CD45+, and EpCAM+) by flow cytometry (n = 3 mice per group per population; error bars represent SD). k End-point analyses of %NP + F4/80− cells in the liver as a function of dose (n = 3 mice per dose; error bars represent SD). l End-point analysis of the ratio of %NP + F4/80− cells to %NP + F4/80+ cells in the liver as a function of dose (n = 3 mice per dose; error bars represent SD).
Fig. 3
Fig. 3. Schematic of PBPK model describing PACE NP biodistribution in blood and various organs.
The other tissues compartment is defined as all tissues separate from the ones displayed here.
Fig. 4
Fig. 4. PBPK Modeling of PACE-PEG NP Biodistribution.
a Model prediction of NP blood concentration for various NP doses (0.1 mg: pink, 0.5 mg: orange, 2.0 mg: green, 2.5 mg: turquoise, and 3.5 mg: indigo per animal corresponding to approximately 30 billion, 160 billion, 650 billion, 800 billion, and 1 trillion NPs, respectively) over time with experimental datapoints overlaid (n = 3 mice per group per time point; error bars represent standard deviation (SD)). Solid lines indicate the mean of the model output, shaded area indicates the +/−  standard error of the mean (SEM). Colors correspond to dosages simulated in the model, compared to corresponding data. b Model prediction of NP tissue concentration over time in the heart. c Model prediction of NP tissue concentration over time in the liver. Solid lines indicate the mean of the model output, shaded area indicates +/−  SEM. d Model prediction of NP tissue concentration over time in the spleen. Solid lines indicate the mean of the model output, shaded area indicates +/−  SEM. e Model prediction of NP tissue concentration over time in the kidneys. Solid lines indicate the mean of the model output, shaded area indicates +/−  SEM. f Model prediction of NP tissue concentration over time in the lungs. Solid lines indicate the mean of the model output, shaded area indicates +/−  SEM. g Model prediction of NP tissue concentration over time in the bone marrow. Solid lines indicate the mean of the model output, shaded area indicates +/−  SEM. h Model prediction of NP tissue concentration over time in the brain and other tissues (combined). Solid lines indicate the mean of the model output, shaded area indicates +/−  SEM.
Fig. 5
Fig. 5. Effects of decoy pre-administration on PACE NP blood concentration and biodistribution.
a Schematic of decoys to deplete or occupy macrophages in the liver and spleen. b Schematic of decoy pre-administration scheme. c Blood NP concentration over time following intravenous injection of dye-loaded PACE NPs alone (gray) or with decoy pre-administration (intralipid: pink, PLGA NPs: cyan, anti-RBC Ab: red, or clodronate liposomes: mint green) using quantitative microscopy (n = 3 mice per group per time point; error bars represent standard error of the mean (SEM)). Representative end-point (d) IVIS analysis of PACE NP uptake in organs (heart, lungs, liver, spleen, kidneys, and bone) alone or following decoy-pre-administration, CTL: untreated control. End-point analysis of (e) whole organ fluorescence quantification of PACE NP uptake in organs (n = 3 mice per group per organ; error bars represent SEM) alone or following decoy pre-administration. End-point analyses of (f) %NP+ cells in homogenized organs by flow cytometry (n = 3 mice per group per organ; error bars represent SEM), (g) %NP+ cells in homogenized liver populations (bulk, F4/80+, and CD31+) by flow cytometry (n = 3 mice per group per population; error bars represent SEM), and (h) %NP+ cells in homogenized lung populations (bulk, CD45+, and EpCAM+) by flow cytometry (n = 3 mice per group per population; error bars represent SEM). End-point analyses of (i) Mean fluorescence intensity (MFI) in arbitrary units (arb. units) of homogenized organs by flow cytometry (n = 3 mice per group per organ; error bars represent SEM), (j) MFI in homogenized liver populations (bulk, F4/80+, and CD31+) by flow cytometry (n = 3 mice per group per population; error bars represent SEM), and (k) MFI cells in homogenized lung populations (bulk, CD45+, and EpCAM+) by flow cytometry (n = 3 mice per group per population; error bars represent SEM).
Fig. 6
Fig. 6. Effects of decoy pre-administration on PACE NP distribution in the liver.
Representative epifluorescence liver images from three independent experiments of (a) untreated control animals, (b) PACE NP-treated animals, (c) intralipid and PACE NP-treated animals, (d) PLGA NP and PACE NP-treated animals, (e) anti-RBC Ab and PACE NP-treated animals, and (f) clodronate liposome and PACE NP-treated animals. Nuclei: blue, F4/80+ macrophages: green, PACE NPs: red. Scale bars, 100 μm.
Fig. 7
Fig. 7. PLGA decoy pre-administration significantly enhances PCSK9 knockdown by PACE siRNA NPs in hepatocytes.
a Schematic of PLGA NP decoy pre-administration scheme with PCSK9 siRNA-loaded PACE NPs and in vivo assessment workflow. b Schematic of liver and blood end-point analyses to assess PCSK9 activity. c %PCSK9 mRNA knockdown as measured by quantitative RT-PCR in hepatocytes of PACE PCSK9 siRNA NP-treated mice and PACE PCSK9 siRNA NP-treated mice following PLGA NP pre-administration (n = 6 mice per group per population; error bars represent standard error of the mean (SEM)). An unpaired two-tailed t test was used for statistical analysis; p = 0.000000024, t = 15.601510, df = 10. d Cholesterol levels in untreated control, PACE PCSK9 siRNA NP-treated mice, and PACE PCSK9 siRNA NP-treated mice following PLGA NP pre-administration (n = 3 mice per group per population; error bars represent SEM). An ordinary one-way ANOVA was used for statistical analysis (F = 11.85, R2 = 0.7980, p = 0.0082) with Holm-Šídák’s post-test for multiple comparisons; for untreated control (CTL) vs. PLGA + PACE NPs, p = 0.0084. e Representative epifluorescence liver image from three independent experiments of dye-loaded PLGA NP-treated animals 24 h post-IV administration. Nuclei are shown in blue, F4/80+ macrophages are shown in green, and PLGA NPs are shown in red. Scale bar, 100 μm.
Fig. 8
Fig. 8. CD4-targeting PACE NPs accumulate selectively in CD4+ T-cells in vitro in human blood and in vivo in rats.
a Schematic of PACE-PEG-maleimide NP monobody and antibody conjugation process. b %NP + CD4+ PBMCs from human blood following in vitro incubation with either isotype control (yellow) or CD4-targeting (pink) DiD-loaded PACE-PEG-maleimide NPs for 5 min, 12 min, 30 min, and 1 hr (n = 3 blood samples per group per time point; error bars represent standard error of the mean (SEM)). c %NP+ PBMCs from human blood following in vitro incubation with either isotype control or CD4-targeting DiD-loaded PACE-PEG-maleimide NPs for 5 min, 12 min, 30 min, and 1 hr (n = 3 blood samples per group per time point; error bars represent SEM). d Schematic of in vivo CD4+ T-cell PACE-PEG-maleimide NP targeting experiment with clodronate liposome decoy pre-treatment in rats. e %NP + CD4+ PBMCs from rat blood two hours after intravenous delivery in vivo of either isotype control or CD4-targeting DiD-loaded PACE-PEG-maleimide NPs (n = 3 rats per group; error bars represent SEM). f %NP+ PBMCs from rat blood two hours after intravenous delivery in vivo of either isotype control or CD4-targeting DiD-loaded PACE-PEG-maleimide NPs (n = 3 rats per group; error bars represent SEM).
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