A PLZ4-targeted nanoplatform changes shape at bladder tumors and uses MMP9 to unlock a doxorubicin prodrug, testing a two-stage drug-delivery strategy in cells and mouse models.
Paper: Transformable peptide-drug conjugate nanoparticle for bladder cancer therapy. Image Credit: Inkoly / Shutterstock
*Important notice: This news reports on an unedited version of an accepted paper and is awaiting final editing. Therefore, the paper should not be regarded as conclusive or treated as established information.
A paper recently published in the Journal of Nanobiotechnology reported a transformable peptide nanoparticle (NP) platform for bladder cancer (BC) therapy that combines the doxorubicin prodrug leucine-doxorubicin (Leu-DOX) with the PLZ4 targeting ligand and a KLVFF β-sheet-forming sequence.
The work is preclinical: the platform was tested in cell studies and in immunodeficient mice bearing bladder tumors, including tumors derived from two patients, rather than in patients themselves.
Importance of Prodrugs
BC, the tenth most diagnosed cancer globally and a potentially lethal disease, is a major health concern with a high cost of treatment. For BC management, DOX is commonly used in neoadjuvant and adjuvant chemotherapy (NAC).
DOX is associated with severe side effects, including bone marrow aplasia, gastrointestinal disturbances, stomatitis, cumulative cardiotoxicity, acute nausea and vomiting, alopecia, and neurologic disturbances such as dizziness and hallucinations.
Developing prodrugs that activate selectively within tumor cells or the tumor microenvironment (TME) offers one way to improve DOX's therapeutic effect.
Tumor-associated enzyme-activated prodrugs (TAEAPs) can be activated selectively inside the TME, where overexpressed enzymes trigger drug release. This approach can reduce exposure in healthy tissues and improve chemotherapy selectivity.
Enzymes such as metalloproteinases (MMPs), prostate-specific antigen, and cathepsin proteases can trigger the release of active compounds from TAEAPs within the TME. MMPs are well suited for this purpose because their activity is linked with tumorigenesis and disease progression.
Role of NPs
Nanoparticle-based drug delivery systems offer another route to reducing side effects and improving anti-tumor efficacy. Peptides have gained attention as drug-delivery carriers because they can self-assemble, are easy to modify, and are highly biodegradable and biocompatible.
Researchers have studied amphiphilic peptides rich in aromatic groups for their self-assembling properties. The authors' group studied KLVFF and its reverse sequence, FFVLK, along with tumor-targeting ligands, to build receptor-mediated transformable NPs.
The Transformable Peptide-drug Conjugate NP
In this work, the researchers developed BC cell surface receptor-mediated, transformable peptide NPs from a monomer that combines the PLZ4 targeting ligand, the β-sheet-forming KLVFF sequence, and the Leu-DOX prodrug. The design aims to improve drug delivery and reduce toxicity.
Leu-DOX Preparation: Doxorubicin hydrochloride and Fmoc-Leu-OH were reacted in anhydrous dimethylformamide (DMF) with N,N-diisopropylethylamine (DIPEA) and HATU. The reaction mixture was stirred at 0 °C, precipitated into icy anhydrous diethyl ether, washed, centrifuged, and vacuum-dried.
The intermediate Fmoc-Leu-DOX was treated with 30% 4-methylpiperidine in DMF to remove the Fmoc group. Ten percent lactate was added to quench the reaction. The product was precipitated again, washed, centrifuged, and vacuum-dried to yield solid Leu-DOX.
NP Preparation: Standard solid-phase peptide synthesis techniques were used to prepare the monomers. DMSO solutions of the monomers were prepared at a DOX-equivalent concentration of 20 mg/mL. The solutions were sonicated to dissolve the monomers, then diluted 100-fold into PBS under sonication. Brief vortexing and filtration through a 0.22 μm filter produced the NPs.
Effectiveness of the NP Platform
In the cell and mouse experiments, BC cell receptor-mediated transformable peptide NPs carrying Leu-DOX showed MMP9-dependent cytotoxicity in cultured BC cells and stronger tumor control than free DOX and control formulations in mice. Several measured toxicity markers were lower than those seen with free DOX.
The transformable peptides self-assembled into NPs that targeted αvβ3 integrin on BC cells through PLZ4. Binding to αvβ3 integrin triggered a structural change from nanoparticles to nanofibrils on the cancer cell surface, followed by gradual uptake.
After intravenous administration in mice, the transformable peptide NPs accumulated in bladder tumors.
Whole-body and organ biodistribution imaging used pheophorbide a (Pa), a near-infrared tracer incorporated into the nanoparticle formulation.
At 36 h, the PLZ4-TP-DOX formulation produced a strong Pa signal in the bladder and relatively weak signals in the liver and kidneys.
Non-targeting and non-transformable controls showed weak bladder signals and stronger liver and kidney signals.
Separate measurements of intrinsic DOX fluorescence in bladder sections found a higher signal in the tumor region and a higher tumor-to-normal bladder-wall ratio with PLZ4-TP-DOX than with free DOX.
The authors attribute tumor accumulation to the EPR effect, which reflects increased permeability and retention, and PLZ4 binding to αvβ3 on BC cells. They also propose that αvβ3-bearing tumor-associated exosomes may provide another route for nanofibril formation and uptake within the TME.
The resulting nanofibrils showed several distributions: some remained attached to the cell surface, some were taken into cells, likely via endocytosis, and some remained in the TME.
Laboratory release assays showed that active MMP9 released 75.0% of conjugated Leu-DOX by 12 h and 83.75% by 48 h. Heat-inactivated MMP9 and the MMP inhibitor GM6001 kept release near baseline. Over time, T24 bladder cancer cells converted released Leu-DOX into active DOX, consistent with the authors' proposed two-step activation mechanism.
In T24 cells, intact PLZ4-TP-DOX was far less cytotoxic than free DOX. Adding MMP9 increased cytotoxicity, and an MMP9 inhibitor reversed that gain, supporting enzyme-dependent drug release.
The platform was designed to exploit differences in MMP9 activity between tumor and normal tissues, with lower activity outside tumors expected to limit prodrug release and reduce drug exposure in healthy organs.
In mice, the PLZ4-TP-DOX group had blood chemistry and blood cell counts close to the PBS control, while free DOX caused abnormal liver and kidney markers and pancytopenia. Cardiac troponin rose about tenfold with free DOX and about twofold with PLZ4-TP-DOX.
In the T24 orthotopic model, all five mice given PLZ4-TP-DOX survived to day 25, while mice in the PBS, free DOX, and Leu-DOX groups died by days 15, 18, and 22, respectively.
In the patient-derived orthotopic model, all seven PLZ4-TP-DOX-treated mice survived the 120-day follow-up, and H&E staining showed no remaining tumor.
Comparisons with the non-targeting and non-transformable nanoparticle controls also indicated that both PLZ4 targeting and transformability contributed to the treatment effect.
The findings support further preclinical work on targeted therapies for BC. The platform has not been tested in people, so its safety, dosing, and antitumor activity in patients remain unknown.
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