RETRACTED: Molecular targeting of FATP4 transporter for oral delivery of therapeutic peptide

Abstract

Low oral bioavailability of peptide drugs has limited their application to parenteral administration, which suffers from poor patient compliance. Here, we show that molecular targeting of the FATP4 transporter is an effective approach to specifically transport long-chain fatty acid (LCFA)-conjugated peptides across the enterocytic membrane and, thus, enables oral delivery of drug peptides. We packaged LCFA-conjugated exendin-4 (LCFA-Ex4) into liposomes and coated with chitosan nanoparticles to form an orally deliverable Ex4 (OraEx4). OraEx4 protected LCFA-Ex4 from damage by the gastric fluid and released LCFA-Ex4 in the intestinal cavity, where LCFA-Ex4 was transported across the enterocyte membrane by the FAPT4 transporter. OraEx4 had a high bioavailability of 24.8% with respect to subcutaneous injection and exhibited a substantial hypoglycemic effect in murine models of diabetes mellitus. Thus, molecular targeting of the FATP4 transporter enhances oral absorption of therapeutic peptides and provides a platform for oral peptide drug development.

Fig. 1. FATP4 transporter mediates uptake of LCFA-Ex4 in epithelial cells.

(A) Schematic view of chemical structure of LCFA-Ex4 (upper panel) and FATP4-mediated transport of LCFA-Ex4 across intestinal epithelial membrane (bottom panel). (B) Glucose tolerance test in C57BL/6 mice to evaluate bioequivalence between free Ex4 and LCFA-Ex4 after subcutaneous injection (n = 6). (C) AUC (area under the curve) of blood glucose levels in glucose tolerance test. (D) Flow cytometry analysis on FATP4 expression in Caco-2 and HCC827 cells. (E) Uptake of Alexa Fluor 594–labeled LCFA-Ex4 (Alexa 594–LCFA-Ex4) in the FATP4-expressing Caco-2 cells and non–FATP4-expressing HCC827 cells. (F) Microscopic analysis on uptake of Alexa 594–LCFA-Ex4 conjugates [short-chain fatty acid (SCFA), C6; medium-chain fatty acid (MCFA), C10; long-chain fatty acid (LCFA), C16] in Caco-2 cells. (G) Competitive binding to the FATP4 transporter by Alexa 594–LCFA-Ex4 in the presence of increasing concentrations (500, 5000, or 10,000 times) of palmitic acid (C16). (H) Uptake of LCFA-Ex4 in FATP4 knockdown Caco-2 cells. FATP4-specific small interfering RNA (siRNA) oligos were used to knock down FATP4 expression, and a scrambled siRNA served as a control. Inset: Western blot analysis on FATP4 expression. Black bars: normalized FATP4 expression levels; white bars: normalized fluorescence intensity (n = 3). (I) Microscopic analysis on intracellular trafficking of Alexa 594–LCFA-Ex4. Nucleus is in blue, the ER track is in green, and Alexa 594–LCFA-Ex4 is in red. Data are presented as means ± SEM. *P < 0.05, **P < 0.01.

Fig. 2. Characterization of OraEx4 on zeta potential, size, and in vitro release.

(A) Schematic view of OraEx4 synthesis, including loading of LCFA-Ex4 into DOPA liposome (Lipo) and sequential coating with chitosan nanoparticle (ChiNP). Chito, chitosan; TPP, sodium triphosphate; DOPA, 1,2-dioleoyl-sn-glycero-3-phosphate; Chole, cholesterol; LCFA,16-hydroxyhexadecanoic acid; Ex4, exendin-4. (B) Zeta potential and size of ChiNP, Lipo, and ChiNP-Lipo. (C) Transmission electron microscopy (TEM) images of ChiNP-Lipo with increasing ChiNP/Lipo ratio. (D) Fourier-transform infrared (FT-IR) spectra of ChiNP, Lipo, and ChiNP-Lipo under different pH environments. (E) Changes in hydrodynamic radius of ChiNP-Lipo under different pH conditions. (F) Release profiles of LCFA-Ex4 from OraEx4 and conventional liposome (T-Lipo). (G) TEM images of OraEx4 at different pH. Data are presented as means ± SEM.

Fig. 3. Biodistribution and lymphatic transport of OraEx4.

(A) Western blot analysis of FATP4 expression in jejunum, ileum, duodenum, and colon and correlation with uptake of LCFA-Ex4 (n = 3). (B) Quantification of fluorescence in major organs at different time points after oral administration of Alexa Fluor 594–labeled OraEx4. All data are normalized to tissue weight (n = 3). (C) LCSM images of Alexa 594–LCFA-Ex4 in different intestinal sections after oral treatment. Representative images are shown. (D) IVIS (In vivo imaging system) images of LCFA-Ex4 in mesenteric lymph node (MLN) from mice treated with OraEx4. (E) Quantitative analysis on fluorescence intensity of LCFA-Ex4 in MLN from (D). (F) Intravital microscopic images of MLN from mice after treatment with OraEx4. (G) Hematoxylin and eosin (H&E) staining of MLN from mice treated with nanogold-conjugated OraEx4 with or without phloretin. Nanogold was stained with silver enhancer. Data are presented as means ± SEM, **P < 0.01.

Fig. 4. In vivo efficacy evaluation of OraEx4.

(A) IPGTT after C57BL/6 mice were treated with oral administration of Ex4 solution (4 mg/kg), OraEx4 (4 mg/kg), or OraEx4 (4 mg/kg) together with phloretin (50 mg/kg; n = 6). (B) AUC of blood glucose levels in (A). (C) Blood glucose levels after a single treatment of OraEx4 (4 or 8 mg/kg) in db/db mice (n = 5). (D) Pharmacokinetic profiles of Oral-Ex4 (4 and 8 mg/kg), compared with subcutaneous (SC) injection of LCFA-Ex4 (n = 5). (E) Long-term blood glucose regulation by daily gavage of OraEx4 (8 mg/kg; n = 5). (F) Body weight changes in db/db mice during treatment with OraEx4 for 12 days (n = 5). (G) IPGTT after treatment with a single dose of OraEx4 (8 mg/kg) in db/db mice (n = 5). (H) Insulin levels before and after OraEx4 treatment in db/db mice (n = 5). (I) Immunofluorescence images on insulin secretion from islets in C57BL/6 and db/db mice. (J) H&E staining of pancreatic islets from posttreatment db/db mice. Images show representative islets from five mice per group. Data are presented as means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5. Analysis on safety profiles after C57BL/6 mice were treated with OraEx4.

Serum samples were collected from mice (n = 5) after daily treatment with indicated agents for 10 days, and levels of enzymes and other biomarkers indicating (A) hepatic function, (B) renal function, and (C) other organs were analyzed. Changes in (D) white blood cells and (E) red blood cell and platelets were also measured. (F) H&E staining of tissue blocks from major organs. Images are representative of five mice per group. Data are presented as means ± SEM.