Transepithelial transport of Fc-targeted nanoparticles by the neonatal fc receptor for oral delivery

Abstract

Nanoparticles are poised to have a tremendous impact on the treatment of many diseases, but their broad application is limited because currently they can only be administered by parenteral methods. Oral administration of nanoparticles is preferred but remains a challenge because transport across the intestinal epithelium is limited. We show that nanoparticles targeted to the neonatal Fc receptor (FcRn), which mediates the transport of immunoglobulin G antibodies across epithelial barriers, are efficiently transported across the intestinal epithelium using both in vitro and in vivo models. In mice, orally administered FcRn-targeted nanoparticles crossed the intestinal epithelium and reached systemic circulation with a mean absorption efficiency of 13.7%*hour compared with only 1.2%*hour for nontargeted nanoparticles. In addition, targeted nanoparticles containing insulin as a model nanoparticle-based therapy for diabetes were orally administered at a clinically relevant insulin dose of 1.1 U/kg and elicited a prolonged hypoglycemic response in wild-type mice. This effect was abolished in FcRn knockout mice, indicating that the enhanced nanoparticle transport was specifically due to FcRn. FcRn-targeted nanoparticles may have a major impact on the treatment of many diseases by enabling drugs currently limited by low bioavailability to be efficiently delivered though oral administration.

Fig. 1. Schematic of Fc-targeted nanoparticle transport across the intestinal epithelium by the FcRn through a transcytosis pathway

(1) IgG Fc on the NP surface binds to the FcRn on the apical side of absorptive epithelial cells under acidic conditions in the intestine. (2) NP-Fc are then trafficked across the epithelial cell through the FcRn transcytosis pathway in acidic endosomes. (3) Upon exocytosis on the basolateral side of the cell, the physiological pH causes IgG Fc to dissociate from the FcRn, and NP-Fc are free to diffuse through the intestinal lamina propria to the capillaries or lacteal and enter systemic circulation.

Fig. 2. Nanoparticle assembly, characterization, and in vitro transepithelial transport

(A) Schematic of NP-Fc assembly. NPs consist of a biodegradable PLA core for drug encapsulation and a PEG surface coating for particle stability and to reduce phagocytic uptake. NPs were formed using the nanoprecipitation self-assembly method (25) and surface-modified with IgG Fc for FcRn targeting. (B) Dynamic light scattering measurements for non-targeted NPs and NP-Fc. Data are means ± SD (n=3). (C) IgG Fc ligand density on the NP surface with (Fc-SH) and without (Fc) thiol modification of the IgG Fc. Data are means ± SD (n=3). (D) In vitro transepithelial transport of non-targeted NPs, NP-Fc, and NP-Fc with an excess of human IgG Fc as a blocking agent for FcRn. Data are expressed as mean basolateral ³H disintegrations per minute (DPM) as a percentage of the initial amount of ³H (± SEM; n = 4 wells per group). *P < 0.05, two-tailed Student’s t-test.

Fig. 3. FcRn expression and nanoparticle intestinal uptake in mice

(A) Western blot of mouse FcRn (mFcRn) in mouse intestinal tissue. (B) Quantification of Western blot band intensity. The relative band intensity was calculated as the ratio of mFcRn to B-Actin band intensity from the Western Blot. (C) Immunohistochemistry on sections of mouse duodenum. mFcRn appears brown. The negative control was tissue stained with polyclonal IgG. (D) Fluorescently labeled NPs (red) were administered to fasted wild-type mice by oral gavage and the intestines were collected for sectioning and imaging 1.5 h after administration. The panels are confocal fluorescence images of 12-μm sections of mouse duodenum. Cell nuclei were stained with DAPI (blue). Images are representative for n=3 mice.

Fig. 4. Nanoparticle absorption and biodistribution in mice

(A) Biodistribution of ¹⁴C-labeled non-targeted NPs and NP-Fc after oral administration to fasted wild-type mice. Data are mean % initial dose (ID) per gram of tissue ± SEM (n=5 mice per time point). (B) Release of ¹⁴C from ¹⁴C-labeled NPs in PBS at 37°C. Data are means ± SD for n=4 release experiments. (C) Total absorbed ¹⁴C over time for non-targeted NPs and NP-Fc after administration by oral gavage. Data are mean %ID measured in all of the organs added together ± SEM (n=5 mice per time point). **P < 0.01 for comparison of non-targeted NPs and NP-Fc at respective time point, two-tailed Student’s t-test.

Fig. 5. Encapsulation of insulin and oral delivery of Fc-targeted insulin nanoparticles to mice

(A) Release of insulin from insNP into PBS. Data are means ± SD (n=3 per time point). (B) Blood glucose response of fasted wild-type mice to insulin encapsulated and released from NPs in (A) before administration. Fasted wild-type mice received free insulin (3.3 U/kg) administered by tail-vein injection. Data are means ± SEM (n=3 mice per group). (C) Blood glucose response of fasted wild-type mice to free insulin solution, NP-Fc containing no insulin, non-targeted insNP, and insNP-Fc, each administered by oral gavage. Data are means ± SEM (n=6 mice per group). *P < 0.05 for comparison of non-targeted insNP and insNP-Fc at corresponding time points, two-tailed Student’s t-test. (D) Blood glucose response of fasted wild-type mice to insNP-Fc, insNP-Fc administered concurrently with excess of IgG Fc, and insNP with chicken IgY Fc fragments, each administered by oral gavage. Data are means ± SEM (n=5 mice per group). **P<0.01 for comparison between insNP-Fc with insNP-Fc + free IgG Fc at the 15 and 19 h timepoints and between insNP-IgG Fc and insNP-IgY Fc at the 10, 15, and 19 h timepoints using a two-tailed Student’s t-test. (E) Blood glucose response to equivalent insulin doses (3.3 U/kg) administered by tail-vein injection into fasted wild-type and FcRn KO mice. Data are means ± SEM (n=3 mice per group). (F) Fasted FcRn KO mice blood glucose response to free insulin solution, NP-Fc containing no insulin, non-targeted insNP, or insNP-Fc, each administered by oral gavage. Data are means ± SEM (n=5 mice per group). (G) Fasted wild-type and FcRn KO mice were dosed by oral gavage with non-targeted insNP and insNP-Fc at two different doses. *P<0.05 for comparison between insNP-Fc at 1.1 U/kg and each of the other groups at corresponding timepoints, two-tailed Student’s t-test.