ABSTRACT
The ability to selectively kill cancerous cell populations while leaving healthy cells unaffected is a key goal in anticancer therapeutics. The use of nanoporous silica-based materials as drug-delivery vehicles has recently proven successful, yet production of these materials requires costly and toxic chemicals. Here we use diatom microalgae-derived nanoporous biosilica to deliver chemotherapeutic drugs to cancer cells. The diatom Thalassiosira pseudonana is genetically engineered to display an IgG-binding domain of protein G on the biosilica surface, enabling attachment of cell-targeting antibodies. Neuroblastoma and B-lymphoma cells are selectively targeted and killed by biosilica displaying specific antibodies sorbed with drug-loaded nanoparticles. Treatment with the same biosilica leads to tumour growth regression in a subcutaneous mouse xenograft model of neuroblastoma. These data indicate that genetically engineered biosilica frustules may be used as versatile 'backpacks' for the targeted delivery of poorly water-soluble anticancer drugs to tumour sites.
Subject(s)
Antineoplastic Agents/administration & dosage , Antineoplastic Agents/therapeutic use , Camptothecin/administration & dosage , Camptothecin/therapeutic use , Diatoms/metabolism , Animals , Antibodies , Cell Line, Tumor , Cloning, Molecular , Diatoms/genetics , Drug Delivery Systems , Gene Expression Regulation , Genetic Engineering , Immunoglobulin G , Liposomes , Lymphoma, B-Cell/drug therapy , Mice , Micelles , Nanoparticles , Neoplasms, Experimental/drug therapy , Neuroblastoma/drug therapy , Particle Size , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Silicon Dioxide/metabolism , Transplantation, HeterologousABSTRACT
The biological formation of inorganic materials (biomineralization) often occurs in specialized intracellular vesicles. Prominent examples are diatoms, a group of single-celled eukaryotic microalgae that produce their SiO2 (silica)-based cell walls within intracellular silica deposition vesicles (SDVs). SDVs contain protein-based organic matrices that control silica formation, resulting in species specifically nanopatterned biosilica, an organic-inorganic composite material. So far no information is available regarding the molecular mechanisms of SDV biogenesis. Here we have investigated by fluorescence microscopy and subcellular membrane fractionation the intracellular transport of silaffin Sil3. Silaffins are a group of phosphoproteins constituting the main components of the organic matrix of diatom biosilica. We demonstrate that the N-terminal signal peptide of Sil3 mediates import into a specific subregion of the endoplasmic reticulum. Additional segments from the mature part of Sil3 are required to reach post-endoplasmic reticulum compartments. Further transport of Sil3 and incorporation into the biosilica (silica targeting) require protein segments that contain a high density of modified lysine residues and phosphoserines. Silica targeting of Sil3 is not dependent on a particular peptide sequence, yet a lysine-rich 12-14-amino acid peptide motif (pentalysine cluster), which is conserved in all silaffins, strongly promotes silica targeting. The results of the present work provide the first insight into the molecular mechanisms for biogenesis of mineral-forming vesicles from an eukaryotic organism.
Subject(s)
Cell Wall/metabolism , Diatoms/metabolism , Oligopeptides/metabolism , Peptides/metabolism , Silicon Dioxide/metabolism , Amino Acid Sequence , Base Sequence , Blotting, Western , Cell Wall/ultrastructure , Cytoplasmic Vesicles/metabolism , Diatoms/genetics , Diatoms/ultrastructure , Endoplasmic Reticulum/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Microscopy, Confocal , Microscopy, Electron, Scanning , Microscopy, Fluorescence , Models, Biological , Molecular Sequence Data , Oligopeptides/genetics , Peptides/genetics , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Sorting Signals/genetics , Protein TransportABSTRACT
The formation of SiO(2)-based cell walls by diatoms (a large group of unicellular microalgae) is a well established model system for the study of molecular mechanisms of biological mineral morphogenesis (biomineralization). Diatom biomineralization involves highly phosphorylated proteins (silaffins and silacidins), analogous to other biomineralization systems, which also depend on diverse sets of phosphoproteins (e.g. mammalian teeth and bone, mollusk shells, and sponge silica). The phosphate moieties on biomineralization proteins play an essential role in mineral formation, yet the kinases catalyzing the phosphorylation of these proteins have remained poorly characterized. Recent functional genomics studies on the diatom Thalassiosira pseudonana have revealed >100 proteins potentially involved in diatom silica formation. Here we have characterized the biochemical properties and biological function of one of these proteins, tpSTK1. Multiple tpSTK1-like proteins are encoded in diatom genomes, all of which exhibit low but significant sequence similarity to kinases from other organisms. We show that tpSTK1 has serine/threonine kinase activity capable of phosphorylating silaffins but not silacidins. Cell biological and biochemical analysis demonstrated that tpSTK1 is an abundant component of the lumen of the endoplasmic reticulum. The present study provides the first molecular structure of a kinase that appears to catalyze phosphorylation of biomineral forming proteins in vivo.
