Controlling Nanoparticle Uptake in Innate Immune Cells with Heparosan Polysaccharides

Abstract

We used heparosan (HEP) polysaccharides for controlling nanoparticle delivery to innate immune cells. Our results show that HEP-coated nanoparticles were endocytosed in a time-dependent manner by innate immune cells via both clathrin-mediated and macropinocytosis pathways. Upon endocytosis, we observed HEP-coated nanoparticles in intracellular vesicles and the cytoplasm, demonstrating the potential for nanoparticle escape from intracellular vesicles. Competition with other glycosaminoglycan types inhibited the endocytosis of HEP-coated nanoparticles only partially. We further found that nanoparticle uptake into innate immune cells can be controlled by more than 3 orders of magnitude via systematically varying the HEP surface density. Our results suggest a substantial potential for HEP-coated nanoparticles to target innate immune cells for efficient intracellular delivery, including into the cytoplasm. This HEP nanoparticle surface engineering technology may be broadly used to develop efficient nanoscale devices for drug and gene delivery as well as possibly for gene editing and immuno-engineering applications.

Keywords: Heparosan; antigen-presenting cells; endocytosis; immuno-engineering; nanoparticles; surface modification.

Figure 1:. The cellular uptake of heparosan (HEP) modified gold nanoparticles (AuNPs) is time-dependent.

(A) Representative brightfield light micrographs of HEP-AuNPs internalization in RAW 264.7 macrophages at 0 h, 1 h, 3 h, and 9 h. Scale bar: 50 μm. (B) ICP-MS results of 55-nm HEP-AuNPs uptake in RAW 264.7 macrophages over time. The data points indicate mean values and standard deviation (n=3–4). (C) Real-time confocal laser scanning microscopy (CLSM) imaging of HEP-AuNP internalization in live RAW 264.7 macrophages. Scale bars: 20 μm. (D) A representative individual cell image was selected from panel C. The right panel shows the AuNPs channel. Scale bars: 10 μm. (E) Transmission electron micrographs of 55-nm HEP-AuNP internalization in RAW 264.7 after 3 h, 6 h, and 24 h incubation. The insert at the bottom right corner of each micrograph shows a higher magnification view of the selected field of view sections. Scale bars: 500 nm.

Figure 2:. HEP-coated nanoparticles enter innate immune cells through endocytosis.

(A) Schematic representation of the uptake pathway study: (i) non-specific endocytosis inhibition to determine whether nanoparticle cellular uptake is energy-dependent. (ii-iv) Specific endocytosis inhibitors for studying (ii) caveolae-mediated endocytosis, (iii) clathrin-mediated endocytosis, and (iv) macropinocytosis. (B-D) ICP-MS quantification of the nanoparticle cellular uptake in RAW 264.7 macrophages at 4°C (B), in the presence of ATPase inhibitor sodium azide (C), or chemical endocytosis inhibitors of caveolae-mediated endocytosis, clathrin-mediated endocytosis, and macropinocytosis (D). AuNPs modified with 13-kDa HEP (at 0.2 nM) were used as control without inhibitors at 37°C. Bars indicate mean ± SD (n=3–4); statistical tests used one-way ANOVA (p<0.0001 (****); p<0.0021 (**); p<0.0332 (*).

Figure 3:. HEP-coated nanoparticles enter cells primarily through clathrin-mediated endocytosis and macropinocytosis.

(A) Schematic representation of HEP-AuNPs uptake through clathrin-mediated endocytosis or macropinocytosis. (B-C) ICP-MS was used to quantify the nanoparticle cellular uptake in RAW 264.7 macrophages upon inhibition with different concentrations of chlorpromazine (B; clathrin-mediated endocytosis) and cytochalasin D (C; macropinocytosis). Bars indicate mean values ± SD (n=3–4). The statistical analysis of groups with competitors showed p<0.0001 compared to the no-competitor group using one-way ANOVA. (D) Confocal laser scanning micrographs of nanoparticle uptake in the presence of endocytosis inhibitors chlorpromazine or cytochalasin D along with non-inhibition control. Scale bar: 20 μm.

Figure 4:. Evaluation of structural HEP analog polymers as competitors for HEP-coated nanoparticle uptake.

(A) Schematic illustration of the experimental design. (B) ICP-MS was used to quantify the cellular uptake of HEP-AuNPs in the presence of HEP structural analogs: 0.1 mg/mL 1,000-kDa HA, 160-kDa HA, 169-kDa HEP, heparin sulfate (HS), or heparin, and 1 mg/mL chondroitin sulfate A (CS A) or chondroitin sulfate C (CS C). The bars indicate mean values ± SD (n=3–4). Statistical tests were performed using one-way ANOVA (p<0.0001 (****); p<0.0021 (**); p<0.0332 (*); n.s. indicates no statistically significant differences). (C-E) Representative brightfield light micrographs of HEP-AuNPs cell uptake in the presence of competitors. The inserted bar graphs represent the quantitative ICP-MS results. The bars indicate mean values ± SD (n=3–4). Scale bar: 50 𝜇m. (F-G) ICP-MS was used to quantify the CS A competition efficiency to reduce HEP-AuNPs cellular uptake over time (F; 1 mg/mL CS A was used) and various CS A concentrations (G). The graphs indicate mean values ± SD (n=3–4).

Figure 5:. Nanoparticle surface coating with HEP promotes multivalent interactions with innate immune cells.

(A) Schematic representation of the surface coating process. (i) The HEP polymers were added to the AuNPs with theoretical surface coating densities ranging from 0 to 14 HEP/nm². (ii) Backfilling of the nanoparticle surface was achieved by adding a constant saturating amount of PEG (adding the equivalent of 7 PEG/nm²) to generate HEP/PEG-AuNPs. (B) The uptake efficiency was measured as a function of surface HEP density by ICP-MS. The data points indicate mean values ± SD (n=3–4). (C-E) Representative brightfield light micrographs of HEP/PEG-AuNPs in cells. The dark spots within cells indicate nanoparticle accumulation. The inserted bar graphs display the quantitative ICP-MS results of nanoparticle cell uptake. The data points indicate mean values ± SD (n=3–4). Scale bar: 50 𝜇m.