Title : Overcoming the PEG dilemma via tris-Based surface engineering for acidic microenvironment-responsive tumor-specific uptake and Intracellular delivery
Abstract:
Background: The clinical success of cancer nanomedicines depends on their ability to overcome the “PEG dilemma.” While modification with polyethylene glycol (PEG) is the gold standard for achieving prolonged systemic circulation and enhanced tumor accumulation via the enhanced permeation and retention (EPR) effect, the steric hindrance of PEG chains often significantly suppresses cellular uptake and endosomal escape after the nanoparticles reach the tumor tissue. To address this limitation, we previously developed a pathological environment-responsive strategy using SAPSp-modified liposomes (SAPSp-lipo). These nanoparticles undergo charge inversion from negative to positive in response to the mildly acidic tumor microenvironment (pH ~6.5), thereby promoting efficient cellular internalization. However, the relatively hydrophobic surface of SAPSp-lipo results in rapid clearance from the bloodstream. In this study, we hypothesized that novel Tris-lipid derivatives—Tris-succinic acid (TSA) and Tris-glutaric acid (TGA)—could serve as superior alternatives to PEG by providing a sufficient hydration layer for stealth properties while preserving the pH-responsive functionality of the SAPSp moiety.
Methods: Liposomes were prepared by a simple hydration method, followed by modification with 5 mol% SAPSp and additional incorporation of TSA or TGA at varying molar ratios (10–50 mol%). Surface hydration properties were quantitatively evaluated using the Fixed Aqueous Layer Thickness (FALT) method. Systemic circulation and tumor-targeting efficiency were assessed in B16-F1 tumor-bearing mice using DiR-labeled liposomes after intravenous administration. pH-dependent cellular uptake was quantified by fluorescence-activated cell sorting (FACS). Furthermore, intracellular distribution and endosomal escape behavior were visualized using confocal laser scanning microscopy (CLSM) under different pH conditions.
Results: FALT analysis revealed that TGA modification significantly increased the thickness of the aqueous layer compared with unmodified SAPSp-lipo, indicating the formation of an effective hydration barrier. In vivo pharmacokinetic studies demonstrated that liposomes modified with 30 mol% TGA (TGA-SAPSp-lipo) exhibited markedly improved blood circulation and tumor accumulation compared with SAPSp-lipo. FACS analysis showed that PEG modification severely hindered cellular uptake at pH 6.5 due to steric hindrance, whereas TGA-SAPSp-lipo retained pH-responsive charge inversion and achieved high cellular uptake. CLSM observations further confirmed efficient endosomal escape of TGA-SAPSp-lipo into the cytoplasm under acidic conditions, demonstrating that the Tris-based surface layer does not interfere with the pH sensitivity of SAPSp.
Conclusion: We successfully developed a multifunctional carrier that bridges the gap between systemic stability and intracellular efficacy. By employing TGA as a novel surface modifier, we overcame the PEG dilemma, achieving both prolonged blood circulation and acidic microenvironment-responsive tumor-specific uptake and intracellular delivery. This platform provides a promising strategy for the efficient delivery of a wide range of therapeutic agents to solid tumors.
