title: pH-responsive polymeric sirna carriers sensitize multidrug resistant ovarian cancer cells to doxorubicin via knockdown of polo-like kinase 1.
abstract: Small interfering RNA (siRNA)-based therapies have great potential for the treatment of debilitating diseases such as cancer, but an effective delivery strategy for siRNA is elusive. Here, pH-responsive complexes were developed for the delivery of siRNA in order to sensitize drug-resistant ovarian cancer cells (NCI/ADR-RES) to doxorubicin. The electrostatic complexes consisted of a cationic micelle used as a nucleating core, siRNA, and a pH-responsive endosomolytic polymer. Cationic micelles were formed from diblock copolymers of dimethylaminoethyl methacrylate (pDMAEMA) and butyl methacrylate (pDbB). The hydrophobic butyl core mediated micelle formation while the positively charged pDMAEMA corona enabled siRNA condensation. To enhance cytosolic delivery through endosomal release, a pH-responsive copolymer of poly(styrene-alt-maleic anhydride) (pSMA) was electrostatically complexed with the positively charged siRNA/micelle to form a ternary complex. Complexes exhibited size (30-105 nm) and charge (slightly positive) properties important for endocytosis and were found to be noncytotoxic and mediate uptake in >70% of ovarian cancer cells after 1 h of incubation. The pH-responsive ternary complexes were used to deliver siRNA against polo-like kinase 1 (plk1), a gene upregulated in many cancers and responsible for cell cycle progression, to ovarian cancer cell lines. Treatment resulted in approximately 50% reduction of plk1 gene expression in the drug-resistant NCI/ADR-RES ovarian cancer cell model and in the drug-sensitive parental cell line, OVCAR8. This knockdown functionally sensitized NCI/ADR-RES cells to doxorubicin at levels similar to OVCAR8. Sensitization occurred through a p53 signaling pathway, as indicated by caspase 3/7 upregulation following plk1 knockdown and doxorubicin treatment, and this effect could be abrogated using a p53 inhibitor. To demonstrate the potential for dual delivery from this polymer system, micelle cores were subsequently loaded with doxorubicin and utilized in ternary complexes to achieve cell sensitization through simultaneous siRNA and drug delivery from a single carrier. These results show knockdown of plk1 results in sensitization of multidrug resistant cells to doxorubicin, and this combination of gene silencing and small molecule drug delivery may prove useful to achieve potent therapeutic effects.
authors.affiliation: Department of Bioengineering, University of Washington, Seattle Washington 98195, USA.
authors.forename: Danielle S W
authors.initials: DS
authors.lastname: Benoit
authors.forename: Scott M
authors.initials: SM
authors.lastname: Henry
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Shubin
authors.forename: Allan S
authors.initials: AS
authors.lastname: Hoffman
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Stayton
mesh_terms: D018797:Cell Cycle Proteins / Q000235:genetics*; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000737:chemistry / Q000627:therapeutic use*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects* / Q000235:genetics; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008689:Methacrylates / Q000737:chemistry; D008823:Micelles; D008954:Models, Biological; D009757:Nylons / Q000737:chemistry; D010051:Ovarian Neoplasms / Q000188:drug therapy; D011108:Polymers / Q000008:administration & dosage / Q000138:chemical synthesis / Q000737:chemistry*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D034741:RNA, Small Interfering / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000097869:Polo-Like Kinase 1
chemical_list: D018797:Cell Cycle Proteins; D004317:Doxorubicin; D008689:Methacrylates; D008823:Micelles; D009757:Nylons; D011108:Polymers; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D000097869:Polo-Like Kinase 1; C407037:poly(2-(dimethylamino)ethyl methacrylate); C016284:butyl methacrylate