RESUMO
The pH (low) insertion peptides (pHLIPs) target acidity at the surfaces of cancer cells and show utility in a wide range of applications, including tumor imaging and intracellular delivery of therapeutic agents. Here we report pHLIP constructs that significantly improve the targeted delivery of agents into tumor cells. The investigated constructs include pHLIP bundles (conjugates consisting of two or four pHLIP peptides linked by polyethylene glycol) and Var3 pHLIPs containing either the nonstandard amino acid, γ-carboxyglutamic acid, or a glycine-leucine-leucine motif. The performance of the constructs in vitro and in vivo was compared with previous pHLIP variants. A wide range of experiments was performed on nine constructs including (i) biophysical measurements using steady-state and kinetic fluorescence, circular dichroism, and oriented circular dichroism to study the pH-dependent insertion of pHLIP variants across the membrane lipid bilayer; (ii) cell viability assays to gauge the pH-dependent potency of peptide-toxin constructs by assessing the intracellular delivery of the polar, cell-impermeable cargo molecule amanitin at physiological and low pH (pH 7.4 and 6.0, respectively); and (iii) tumor targeting and biodistribution measurements using fluorophore-peptide conjugates in a breast cancer mouse model. The main principles of the design of pHLIP variants for a range of medical applications are discussed.
Assuntos
Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Proteínas de Membrana/química , Peptídeos/administração & dosagem , Amanitinas/química , Animais , Antineoplásicos/química , Dicroísmo Circular , Feminino , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas/química , Lipossomos/química , Proteínas de Membrana/genética , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/tratamento farmacológico , Peptídeos/química , Peptídeos/farmacocinética , Polietilenoglicóis/química , Distribuição TecidualRESUMO
pH (low) insertion peptides (pHLIP peptides) target acidic extracellular environments in vivo due to pH-dependent cellular membrane insertion. Two variants (Var3 and Var7) and wild-type (WT) pHLIP peptides have shown promise for in vivo imaging of breast cancer. Two positron emitting radionuclides ((64)Cu and (18)F) were used to label the NOTA- and NO2A-derivatized Var3, Var7, and WT peptides for in vivo biodistribution studies in 4T1 orthotopic tumor-bearing BALB/c mice. All of the constructs were radiolabeled with (64)Cu or [(18)F]-AlF in good yield. The in vivo biodistribution of the 12 constructs in 4T1 orthotopic allografted female BALB/c mice indicated that NO2A-cysVar3, radiolabeled with either (18)F (4T1 uptake; 8.9 ± 1.7%ID/g at 4 h p.i.) or (64)Cu (4T1 uptake; 8.2 ± 0.9%ID/g at 4 h p.i. and 19.2 ± 1.8% ID/g at 24 h p.i.), shows the most promise for clinical translation. Additional studies to investigate other tumor models (melanoma, prostate, and brain tumor models) indicated the universality of tumor targeting of these tracers. From this study, future clinical translation will focus on (18)F- or (64)Cu-labeled NO2A-cysVar3.
Assuntos
Radioisótopos de Cobre , Espaço Extracelular/química , Radioisótopos de Flúor , Proteínas de Membrana , Tomografia por Emissão de Pósitrons/métodos , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/farmacocinética , Camundongos , Camundongos Endogâmicos BALB C , Traçadores Radioativos , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
Acidity is a biomarker of cancer that is not subject to the blunting clonal selection effects that reduce the efficacy of other biomarker technologies, such as antibody targeting. The pH (low) insertion peptides (pHLIP®s) provide new opportunities for targeting acidic tissues. Through the physical mechanism of membrane-associated folding, pHLIPs are triggered by the acidic microenvironment to insert and span the membranes of tumor cells. The pHLIP platform can be applied to imaging acidic tissues, delivering cell-permeable and impermeable molecules to the cytoplasm, and promoting the cellular uptake of nanoparticles. Since acidosis is a hallmark of tumor development, progression, and aggressiveness, the pHLIP technology may prove useful in targeting cancer cells and metastases for tumor diagnosis, imaging, and therapy.