Surface functionalization of nanoparticles with diverse ligands is crucial for efficient drug delivery, overcoming physiological barriers and enhancing targeting. However, mutual interference between ligands, such as steric hindrance and electrostatic interactions, compromises their function and reduces efficacy. Strategies to address this challenge include optimizing ligand ratios and densities, using enzyme-responsive linkers, and employing pH-sensitive molecular actuators. While these methods improve targeting, they often fail to fully realize the potential of multiple ligands. This research introduces ligand-switchable poly(lactic-co-glycolic acid) (PLGA) nanoparticles (Pep/Gal-PNPs) to minimize ligand interference and improve oral insulin therapy. The pH-responsive Pep mimics viral spike proteins, undergoing conformational changes to avoid mutual interference between Pep and the liver-targeting Galactose (Gal). Oral delivery of Pep/Gal-PNPs is hypothesized to utilize the pH changes along the gastrointestinal tract to sequentially expose ligands for efficient absorption and liver targeting. This work seeks to demonstrate the efficacy of this approach using oral insulin delivery as a proof of concept.

Existing research on multifunctional nanoparticles highlights the challenge of ligand interference. Studies show that covalently attaching multiple ligands, like RGD and transferrin, can enhance drug delivery but is limited by steric hindrance. Optimizing ligand ratios and lengths, using enzyme-responsive linkers, and employing pH-sensitive actuators are promising strategies but haven't fully solved the problem of realizing the complete functionality of multiple ligands. This study builds upon these previous efforts by introducing a novel approach that directly addresses the issue of ligand interference by mimicking the dynamic surface features of viruses, allowing sequential activation of ligands along the delivery route.

The researchers synthesized a pH-responsive cell-penetrating peptide (Pep) and conjugated it with PLGA to create PLGA-Pep polymers. They also synthesized PLGA-PEG-Gal polymers, using PEG as a linker to hold the Galactose (Gal) moiety. PLGA nanoparticles (PNPs) were then fabricated using a double emulsion solvent evaporation method, incorporating both PLGA-Pep and PLGA-PEG-Gal polymers. The nanoparticles’ characteristics (size, zeta potential, morphology) were analyzed at various pH levels using techniques such as DLS, zeta potential measurements, Cryo-TEM, and AFM to confirm the pH-responsive conformational changes of Pep. In vitro studies were conducted to evaluate the nanoparticles’ stability in simulated gastric and intestinal fluids, insulin release kinetics, mucus penetration, cellular uptake by Caco-2 cells (investigating the endocytosis mechanisms), and transcytosis efficiency across Caco-2 cell monolayers. The integrity of nanoparticles post-transcytosis was confirmed using FRET and Cryo-TEM. The interaction of the nanoparticles with hepatocytes (LO2 cells) was studied, investigating cellular uptake and the activation of insulin signaling pathways. In vivo studies in diabetic rats involved assessing intestinal absorption using two-photon microscopy and CLSM, liver accumulation and selectivity using IVIS and CLSM (with immunofluorescence staining for ASGPR), and in vivo ligand switching using FRET. Therapeutic efficacy was evaluated by monitoring blood glucose levels, serum insulin levels, hepatic glycogen content (using ELISA and PAS staining), and liver function tests (ALT, AST). Toxicity was assessed by monitoring body weight and histology.

The synthesized Pep exhibited a pH-dependent conformational change, extending in acidic environments and folding at physiological pH, as confirmed by CD, FRET, and AFM. Pep/Gal-PNPs showed a size reduction at physiological pH due to Pep folding, exposing the Gal ligand. The nanoparticles were stable in simulated gastrointestinal fluids and exhibited sustained insulin release. Pep/Gal-PNPs demonstrated significantly enhanced mucus penetration and Caco-2 cell uptake at acidic pH compared to PNPs and CPP/Gal-PNPs. Cellular uptake was mainly through clathrin-mediated endocytosis at acidic pH and macropinocytosis at physiological pH. Pep/Gal-PNPs exhibited efficient transcytosis across Caco-2 monolayers at acidic pH, maintaining structural integrity after exiting the cells, and retained their pH sensitivity. In vivo, Pep/Gal-PNPs showed superior intestinal absorption at simulated intestinal pH compared to CPP/Gal-PNPs and PNPs, as evidenced by two-photon microscopy and CLSM. Pep/Gal-PNPs exhibited significantly greater liver accumulation than CPP/Gal-PNPs and PNPs and specifically targeted hepatocytes via Gal-ASGPR interaction. In vivo FRET confirmed the pH-dependent conformational changes of Pep, exhibiting a low FRET in the intestine and a high FRET in the liver. Confocal laser endomicroscopy visualized the sequential transport of Pep/Gal-PNPs from the intestine to the liver. Insulin-loaded Pep/Gal-PNPs elicited a sustained and significant hypoglycemic effect in diabetic rats, with significantly higher insulin levels in the liver compared to subcutaneous insulin and other nanoparticle formulations. They also significantly increased hepatic glycogen production, restoring levels similar to healthy rats. Importantly, Pep/Gal-PNPs showed no significant toxicity in vivo.

This study successfully demonstrated the efficacy of ligand-switchable nanoparticles for targeted drug delivery. The pH-responsive conformational change of the Pep ligand enabled sequential activation of functions, facilitating intestinal absorption at acidic pH and liver targeting at physiological pH. The design closely mimics viral surface features, offering a safer alternative to virus-based delivery systems. The superior performance of Pep/Gal-PNPs compared to CPP/Gal-PNPs highlights the importance of the switchable ligand system in maximizing both intestinal absorption and hepatic targeting. The sustained hypoglycemic effect and enhanced hepatic glycogen production observed in diabetic rats indicate the potential of this technology for improved oral insulin therapy, mimicking the natural insulin gradient for better glucose homeostasis. This approach may minimize the side effects associated with conventional insulin delivery methods, such as hypoglycemia.

This research presents a novel ligand-switchable nanoparticle platform (Pep/Gal-PNPs) for targeted drug delivery, showcasing its effectiveness for oral insulin therapy. The unique design, mimicking viral surface mechanisms, allows sequential ligand activation to overcome physiological barriers and achieve highly efficient liver targeting. The resultant sustained hypoglycemic effect and restored hepatic glycogen levels in diabetic rats represent a significant advancement in diabetes management. Future research could explore applications of this platform for delivering other biomacromolecules and therapeutic agents to various target organs.

While this study demonstrates significant promise, some limitations exist. The study used a rat model, and further investigation in larger animal models and human clinical trials is necessary. The long-term effects of Pep/Gal-PNPs and potential long-term toxicity need further evaluation. The synthesis and characterization methods might require optimization for large-scale production. Although the in vivo findings were extensively investigated, further in-depth mechanistic studies could enhance understanding and potentially improve the delivery system further.