Oral delivery of protein drugs based on nanoparticulate delivery system requires permeation of the nanoparticles through the mucus layer and subsequent absorption via epithelial cells. However, overcoming these two barriers requires very different or even contradictory surface properties of the nanocarriers, which greatly limits the oral bioavailability of macromolecular drugs. Here we report a simple zwitterions-based nanoparticle (NP) delivery platform, which showed a great potency in simultaneously overcoming both the mucus and epithelium barriers. The dense and hydrophilic coating of zwitterions endows the NPs with excellent mucus penetrating ability. Moreover, the zwitterions-based NPs also possessed excellent affinity with epithelial cells, which significantly improved (4.5-fold) the cellular uptake of DLPC NPs, compared to PEGylated NPs. Our results also indicated that this affinity was due to the interaction between zwitterions and the cell surface transporter PEPT1. Moreover, the developed NPs loaded with insulin could induce a prominent hypoglycemic response in diabetic rats following oral administration. These results suggest that zwitterions-based NPs might provide a new perspective for oral delivery of protein therapeutics.

Nanoparticles (NPs) for oral delivery of peptide/protein drugs are largely limited due to the coexistence of intestinal mucus and epithelial barriers. Sequentially overcoming these two barriers is intractable for a single nanovehicle due to the requirements of different or even contradictory surface properties of NPs. To solve this dilemma, a mucus-penetrating virus-inspired biomimetic NP with charge reversal ability (P-R8-Pho NPs) was developed by densely coating poly(lactic- co-glycolic acid) NPs with cationic octa-arginine (R8) peptide and specific anionic phosphoserine (Pho). The small size (81.81 nm) and viruslike neutral charged surface (-2.39 mV) of the biomimetic NPs achieved rapid mucus penetration, which was almost equal to that of the conventional PEGylated mucus-penetrating nanoparticles. The hydrolysis of surface-anchored anionic Pho was achieved by intestinal alkaline phosphatase, which led to the turnover of ζ potential to positive (+7.37 mV). This timely charge reversal behavior also exposed cationic R8 peptide and induced efficient cell-penetrating peptide (CPP)-mediated cellular uptake and transepithelial transport on Caco-2/E12 cocultured cell model. What's more, P-R8-Pho NPs showed excellent stability in simulated gastrointestinal conditions and enhanced absorption in intestine in vivo. Finally, oral administration of insulin-loaded P-R8-Pho NPs enabled to induce a preferable hypoglycemic effect and a 1.9-fold higher oral bioavailability was achieved compared with single CPP-modified P-R8 NPs on diabetic rats. The combinative application of biomimetic mucus-penetrating strategy and enzyme-responsive charge reversal strategy in a single nanovehicle could sequentially overcome mucus and epithelial barriers, thus showing great potential for the oral peptide/protein delivery.

Although nanoparticles (NPs) have been demonstrated as promising tools for improving oral absorption of biotherapeutics, most of them still have very limited oral bioavailability. Lyso-endosomal degradation in epithelial cells is one of the important but often-neglected physiological barriers, limiting the transport of cargoes across the intestinal epithelium. We herein reported a solid lipid nanoparticle (SLN) platform with a unique feature of endosomal escape for oral protein drug delivery. The SLNs consisted of a solid-lipid shell, which contained an endosomal escape agent (GLFEAIEGFIENGWEGMIDGWYG, HA2), and an aqueous core that is loaded with insulin (INS HA2-O-SLNs). SLNs without and with the HA2 peptide in the aqueous core (INS SLNs and INS HA2-W-SLNs, respectively) were used as the control groups. Our study showed that INS HA2-O-SLNs effectively facilitated the escape of the loaded insulin from the acidic endosomes, which preserved the biological activity of insulin to a greater extent during the intracellular transport. The spatial location of the HA2 peptide was demonstrated to determine the endosomal escape efficiency. As demonstrated in the intracellular trafficking of SLNs, INS HA2-O-SLNs displayed much less distribution in late endosomes and lysosomes. Meanwhile, insulin in INS HA2-O-SLNs exhibited the highest transepithelial permeation efficiency, with 2.19 and 1.72 folds higher accumulated amount in the basolateral side as compared to that in INS SLNs and INS HA2-W-SLNs. In addition, insulin from INS HA2-O-SLNs exhibited the highest insulin permeation in various regions of small intestines. INS HA2-O-SLNs generated an excellent hypoglycemic response following oral administration in diabetic rats. Thus, such functional SLNs demonstrated a great potency for oral delivery of peptide/protein drugs.

Ligand-modified nanoparticles (NPs) are an effective tool to increase the endocytosis efficiency of drugs, but these functionalized NPs face the drawback of "easy uptake hard transcytosis" in the oral delivery of proteins and peptides. Adversely, the resulting deficiency in transcytosis has not attracted much attention. Herein, NPs modified with the low-density lipoprotein receptor (LDLR) ligand NH-C6-[cMPRLRGC]c-NH, i.e., peptide-22 (P22NPs) were fabricated to investigate strategies related to the enhancement of transcytosis. By systematically studying the intracellular trafficking of NPs, it was found that reduced transcytosis might be associated with the entrapment of P22NPs in endosomes or lysosomes and limited basolateral exocytosis. On this basis, the prevention of the endolysosomal entrapment of NPs and the acceleration of basolateral exocytosis should be considered as strategies to enhance the transcytosis of NPs. By screening chemicals that could help the endosomal/lysosomal escape of chemicals related to LDLR-mediated transcytosis, it was shown that hemagglutinin-2 (HA) and metformin had higher abilities to enhance the exocytosis of P22NPs. The transcytosis efficiencies of insulin loaded in P22NPs were also investigated, and a 3.2-fold increase in transcytosis was observed in comparison with free insulin. The transcytosis efficiencies of insulin could be further increased by the addition of metformin or HA (3.6-fold or 4.1-fold higher than that of free insulin). Inspiringly, the simultaneous addition of the abovementioned two chemicals led to the highest transcytosis efficiency of insulin, which was up to 5.1-fold higher than that of free insulin. These results demonstrated that endolysosomal entrapment and basolateral exocytosis are two of the most important limiting steps for the "easy uptake hard transcytosis" of orally administered ligand-modified NPs. Moreover, our work provides a new point of view for the design of novel oral drug delivery systems.

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