Intracellular GRP78-Directed Delivery of Rapamycin by Biomolecular Condensates of Hydra-Elastin-like Polypeptides

Apr 9, 2026Biomacromolecules

Using Protein-Based Droplets to Deliver Rapamycin Inside Cells Targeting GRP78

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Abstract

The novel GRP78-targeted carrier (L-5FV) showed significant cellular association and inhibition of p-rpS6 in a breast cancer cell line.

L-5FV is engineered to enhance the solubility and cellular uptake of rapamycin by utilizing FKBP12 and targeting GRP78.
The carrier forms a biomolecular condensate depot at body temperature, facilitating controlled drug release.
Both targeted and untargeted formulations exhibited phase separation and demonstrated nanomolar affinity for FKBP12 and rapamycin.
Cellular internalization and mTORC1 inhibition were confirmed in BT-474 breast cancer cells in a dose- and time-dependent manner.
L-5FV showed a more significant effect on inhibiting p-rpS6 compared to untargeted formulations.

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Rapamycin (Rapa) is a potent inhibitor of the mammalian target of rapamycin complex 1 (mTORC1) with possible applications in multiple diseases; however, it and its analogues exhibit low solubility, variable bioavailability, and dose-limiting side effects. To engineer a long-release carrier, we employ Rapa's cognate receptor (FKBP12) to modulate its solubility, rate of release, and cellular uptake. To target its internalization into cancer cells under stress with an unfolded protein response (UPR), we use an L-peptide that binds cell-surface glucose-regulated protein 78 (GRP78). Herein, the L-peptide was fused to five FKBP domains linked by an elastin-like polypeptide (ELP) selected to form a biomolecular condensate depot at body temperature. This novel GRP78-targeted carrier (L-5FV) was characterized by UV-vis spectrophotometry, dynamic light scattering (DLS), surface plasmon resonance (SPR), and dialysis under sink conditions to assess its thermosensitivity, particle assembly, binding kinetics to both Rapa and GRP78, and drug release, respectively. Functional delivery of cellular internalization and mTORC1 inhibition were confirmed using fluorescence microscopy and Western blot in dose- and time-dependent manners in a breast cancer cell line, BT-474. Both targeted and untargeted formulations are phase-separated at physiological temperatures and exhibit nanomolar affinity for FKBP12 and Rapa. Notably, L-5FV demonstrated a more significant cellular association and inhibition of p-rpS6, a mechanistic target of mTORC1 activity. This report provides insight into how to construct long-release, molecularly targeted drug carriers with applications in UPR-active cancers.

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Intracellular GRP78-Directed Delivery of Rapamycin by Biomolecular Condensates of Hydra-Elastin-like Polypeptides

Abstract

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