Preclinical Validation of Tumor-Penetrating and Interfering Peptides against Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in adults. The disease is characterized by the accumulation of tumoral B cells resulting from a defect of apoptosis. We have in vitro and in vivo preclinically validated a tumor-penetrating peptide (named TT1) coupled to an interfering peptide (IP) that dissociates the interaction between the serine/threonine protein phosphatase 2A (PP2A) from its physiological inhibitor, the oncoprotein SET. This TT1-IP peptide has an antitumoral effect on CLL, as shown by the increased survival of mice bearing xenograft models of CLL, compared to control mice. The peptide did not show toxicity, as indicated by the mouse body weight and the biochemical parameters, such as renal and hepatic enzymes. In addition, the peptide-induced apoptosis in vitro of primary tumoral B cells isolated from CLL patients but not of those isolated from healthy patients. Finally, the peptide had approximately 5 h half-life in human serum and showed pharmacokinetic parameters compatible with clinical development as a therapeutic peptide against CLL.

Novel Anthracycline Utorubicin for Cancer Therapy.

Novel anticancer compounds and their precision delivery systems are actively developed to create potent and well-tolerated anticancer therapeutics. Here, we report the synthesis of a novel anthracycline, Utorubicin (UTO), and its preclinical development as an anticancer payload for nanocarriers. Free UTO was significantly more toxic to cultured tumor cell lines than the clinically used anthracycline, doxorubicin. Nanoformulated UTO, encapsulated in polymeric nanovesicles (polymersomes, PS), reduced the viability of cultured malignant cells and this effect was potentiated by functionalization with a tumor-penetrating peptide (TPP). Systemic peptide-guided PS showed preferential accumulation in triple-negative breast tumor xenografts implanted in mice. At the same systemic UTO dose, the highest UTO accumulation in tumor tissue was seen for the TPP-targeted PS, followed by nontargeted PS, and free doxorubicin. Our study suggests potential applications for UTO in the treatment of malignant diseases and encourages further preclinical and clinical studies on UTO as a nanocarrier payload for precision cancer therapy.

Targeting Pro-Tumoral Macrophages in Early Primary and Metastatic Breast Tumors with the CD206-Binding mUNO Peptide.

M2-like tumor-associated macrophages (M2 TAMs) play important roles in the resistance of tumors to immunotherapies. Selective depletion or reprogramming of M2 TAMs may sensitize the nonresponsive tumors for immune-mediated eradication. However, precision delivery of payloads to M2 TAMs, while sparing healthy tissues, has remained an unresolved challenge. Here, we studied the application of a short linear peptide (CSPGAK, "mUNO") for the delivery of molecular and nanoscale cargoes in M2 TAMs in vitro and the relevance of the peptide for in vivo targeting of early-stage primary breast tumors and metastatic lung foci. First, we performed in silico modeling and found that mUNO interacts with mouse CD206 via a binding site between lectin domains CTLD1 and CTLD2, the same site previously demonstrated to be involved in mUNO binding to human CD206. Second, we showed that cultured M2 macrophages take up fluorescein-labeled (FAM) polymersomes conjugated with mUNO using the sulfhydryl group of its N-terminal cysteine. Pulse/chase studies of FAM-mUNO in M2 macrophages suggested that the peptide avoided lysosomal entrapment and escaped from early endosomes. Third, our in vivo studies with FAM-mUNO demonstrated that intraperitoneal administration results in better pharmacokinetics and higher blood bioavailability than can be achieved with intravenous administration. Intraperitoneal FAM-mUNO, but not FAM-control, showed a robust accumulation in M2-skewed macrophages in mouse models of early primary breast tumor and lung metastasis. This targeting was specific, as no uptake was observed in nonmalignant control organs, including the liver, or other cell types in the tumor, including M1 macrophages. Collectively, our studies support the application of the CD206-binding mUNO peptide for delivery of molecular and nanoscale cargoes to M2 macrophages and manifest the relevance of this mode of targeting primary and metastatic breast tumors.

Paclitaxel-Loaded Cationic Fluid Lipid Nanodiscs and Liposomes with Brush-Conformation PEG Chains Penetrate Breast Tumors and Trigger Caspase-3 Activation.

Novel approaches are required to address the urgent need to develop lipid-based carriers of paclitaxel (PTX) and other hydrophobic drugs for cancer chemotherapy. Carriers based on cationic liposomes (CLs) with fluid (i.e., chain-melted) membranes (e.g., EndoTAG-1) have shown promise in preclinical and late-stage clinical studies. Recent work found that the addition of a cone-shaped poly(ethylene glycol)-lipid (PEG-lipid) to PTX-loaded CLs (CLsPTX) promotes a transition to sterically stabilized, higher-curvature (smaller) nanoparticles consisting of a mixture of PEGylated CLsPTX and PTX-containing fluid lipid nanodiscs (nanodiscsPTX). These CLsPTX and nanodiscsPTX show significantly improved uptake and cytotoxicity in cultured human cancer cells at PEG coverage in the brush regime (10 mol % PEG-lipid). Here, we studied the PTX loading, in vivo circulation half-life, and biodistribution of systemically administered CLsPTX and nanodiscsPTX and assessed their ability to induce apoptosis in triple-negative breast-cancer-bearing immunocompetent mice. We focused on fluid rather than solid lipid nanodiscs because of the significantly higher solubility of PTX in fluid membranes. At 5 and 10 mol % of a PEG-lipid (PEG5K-lipid, molecular weight of PEG 5000 g/mol), the mixture of PEGylated CLsPTX and nanodiscsPTX was able to incorporate up to 2.5 mol % PTX without crystallization for at least 20 h. Remarkably, compared to preparations containing 2 and 5 mol % PEG5K-lipid (with the PEG chains in the mushroom regime), the particles at 10 mol % (with PEG chains in the brush regime) showed significantly higher blood half-life, tumor penetration, and proapoptotic activity. Our study suggests that increasing the PEG coverage of CL-based drug nanoformulations can improve their pharmacokinetics and therapeutic efficacy.

Novel Anthracycline Utorubicin for Cancer Therapy.

Novel anticancer compounds and their precision delivery systems are actively developed to create potent and well-tolerated anticancer therapeutics. Here, we report the synthesis of a novel anthracycline, Utorubicin (UTO), and its preclinical development as an anticancer payload for nanocarriers. Free UTO was significantly more toxic to cultured tumor cell lines than the clinically used anthracycline, doxorubicin. Nanoformulated UTO, encapsulated in polymeric nanovesicles (polymersomes, PS), reduced the viability of cultured malignant cells and this effect was potentiated by functionalization with a tumor-penetrating peptide (TPP). Systemic peptide-guided PS showed preferential accumulation in triple-negative breast tumor xenografts implanted in mice. At the same systemic UTO dose, the highest UTO accumulation in tumor tissue was seen for the TPP-targeted PS, followed by nontargeted PS, and free doxorubicin. Our study suggests potential applications for UTO in the treatment of malignant diseases and encourages further preclinical and clinical studies on UTO as a nanocarrier payload for precision cancer therapy.

Application of polymersomes engineered to target p32 protein for detection of small breast tumors in mice.

Triple negative breast cancer (TNBC) is the deadliest form of breast cancer and its successful treatment critically depends on early diagnosis and therapy. The multi-compartment protein p32 is overexpressed and present at cell surfaces in a variety of tumors, including TNBC, specifically in the malignant cells and endothelial cells, and in macrophages localized in hypoxic areas of the tumor. Herein we used polyethylene glycol-polycaprolactone polymersomes that were affinity targeted with the p32-binding tumor penetrating peptide LinTT1 (AKRGARSTA) for imaging of TNBC lesions. A tyrosine residue was added to the peptide to allow for 124I labeling and PET imaging. In a TNBC model in mice, systemic LinTT1-targeted polymersomes accumulated in early tumor lesions more than twice as efficiently as untargeted polymersomes with up to 20% ID/cc at 24 h after administration. The PET-imaging was very sensitive, allowing detection of tumors as small as ∼20 mm3. Confocal imaging of tumor tissue sections revealed a high degree of vascular exit and stromal penetration of LinTT1-targeted polymersomes and co-localization with tumor-associated macrophages. Our studies show that systemic LinTT1-targeted polymersomes can be potentially used for precision-guided tumor imaging and treatment of TNBC.