Nanoparticle elasticity directs tumor uptake

Abstract

To date, the role of elasticity in drug delivery remains elusive due to the inability to measure microscale mechanics and alter rheology without affecting chemistry. Herein, we describe the in vitro cellular uptake and in vivo tumor uptake of nanolipogels (NLGs). NLGs are composed of identical lipid bilayers encapsulating an alginate core, with tunable elasticity. The elasticity of NLGs was evaluated by atomic force microscopy, which demonstrated that they exhibit Young's moduli ranging from 45 ± 9 to 19,000 ± 5 kPa. Neoplastic and non-neoplastic cells exhibited significantly greater uptake of soft NLGs (Young's modulus <1.6 MPa) relative to their elastic counterparts (Young's modulus >13.8 MPa). In an orthotopic breast tumor model, soft NLGs accumulated significantly more in tumors, whereas elastic NLGs preferentially accumulated in the liver. Our findings demonstrate that particle elasticity directs tumor accumulation, suggesting that it may be a design parameter to enhance tumor delivery efficiency.

Fig. 1

Synthesis and characterization of NLP and NLGs with varying elasticity. a Schematic illustration of nanoliposome–hydrogel complex system. NLP represents nanoliposome encapsulating PBS. Uncrosslinked NLG represents nanoliposome encapsulating uncrosslinked alginate (0 mM CaCl₂). Crosslinked NLG represents nanolipogel encapsulating 1–5 mM CaCl₂ crosslinked alginate. b The internal structure of NLP-45KPa, NLG-1.6MPa, and NLG-19MPa characterized by TEM. Scale bars represent 100 nm. c The Young’s moduli of synthesized NLP and NLGs characterized by AFM. The encapsulation efficiencies of FITC-dextran (d) and siRNA (e) in synthesized NLP and NLGs. f Sustained release profiles of synthesized FITC-dextran encapsulating NLP and NLGs. *P < 0.05, **P < 0.01, ***P < 0.001. The mean values and error bars are defined as mean and S.D., respectively

Fig. 2

Particle elasticity regulates in vitro cellular uptake. Relative cellular uptake of synthesized NLP and NLGs by MDA-MB-231 (a), MCF7 (b), and MCF10A (c) cells. Relative cytotoxicity of engineered NLP and NLGs in MDA-MB-231 (d), MCF7 (e), and MCF10A (f) cells. The concentration (g) and serum (h) dependence of particle elasticity-mediated cellular uptake in MDA-MB-231 cells. i Fluorescent microscope images of MDA-MB-231 cellular uptake of synthesized NLP and NLGs with varying elasticity. The scale bars represent 50 μm. ***P < 0.001. The mean values and error bars are defined as mean and S.D., respectively

Fig. 3

Cell internalization pathway shift by varying particle elasticity. Soft NLP-45KPa (a) enters the cell via two pathways: fusion (predominant) and endocytosis (inferior). Hard NLG-19MPa (b) enters cell via only clathrin-mediated endocytosis

Fig. 4

Particle elasticity mediates cellular uptake via different internalization pathways. Relative cellular uptake of NLP and NLGs in MDA-MB-231 (a), MCF7 (b), and MCF10A (c) cells in the presence of small molecules inhibiting clathrin (Chlorpromazine), caveolae (Filipin) and both endocytic pathways (Dynasore). Representative fluorescent microscope images of cellular uptake of soft NLP-45KPa (d) and hard NLG-19MPa (e) in response to endocytosis inhibitors. The scale bars represent 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001. The mean values and error bars are defined as mean and S.D., respectively

Fig. 5

Particle elasticity regulates in vivo tumor uptake. a In vivo NIR fluorescent images of mice at post-6h and -48h after i.v. administration of DiR-labeled NLP-45KPa, NLG-1.6MPa, NLG-13.8MPa, and NLG-19MPa (n = 10 for each group). b In vivo tumor accumulation of synthesized NLP-45KPa and NLGs was quantified by fluorescent intensity. c Ex vivo NIR fluorescent images of excised tumors at post 48 h. d The biodistribution of NLP-45KPa and NLGs in organs (liver, spleen, lung, kidney, heart, and brain) and tumors was quantified by their fluorescent intensity at post-48h (n = 10 for each group). e Uptake of NLP-45KPa and NLGs by mouse leukocytes was quantified by flow cytometry (n = 8–10 for each group). ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001. The mean values and error bars are defined as mean and S.D., respectively