Poly(l-lactide-co-glycolide) nanospheres conjugated with a nuclear localization signal for delivery of plasmid DNA

Abstract

Polymeric nanospheres fabricated from biodegradable poly(lactide-co-glycolide) (PLGA) have been extensively investigated for applications in gene delivery. In this study, we show that the covalent conjugation of a nuclear localization signal (NLS, SV40 peptide) on PLGA nanospheres enhances the gene transfection efficiency. NLS conjugated PLGA copolymer was prepared by using a coupling reaction between maleimide-terminated PLGA copolymer and NLS in the presence of Imject maleimide conjugation buffer. PLGA nanospheres encapsulating plasmid (pDNA) were prepared by using a double emulsion-solvent evaporation method. The kinetics of in vitro release of pDNA from PLGA nanospheres was determined with UV in phosphate buffered saline (PBS). Gene transfection efficiency in human dermal fibroblasts was tested in vitro using nanospheres encapsulating the luciferase gene. The conjugation of the NLS peptide to the PLGA nanospheres could improve the nuclear localization and/or cellular uptake of PLGA nanosphere/pDNA constructs and thereby improve the transfection efficiency of a PLGA nanosphere gene delivery system. The pDNA was released from PLGA nanospheres over nine days. NLS conjugation enhanced the gene transfection efficiency in vitro by 1.2 approximately 3.2-fold over 13 days. PLGA/pDNA nanospheres appeared to be superior to PEI/pDNA complexes for the long-term expression of pDNA. Furthermore, the level of the sustained gene expression of the PLGA nanospheres was enhanced by the conjugation of NLS to the PLGA nanospheres. This study showed that the NLS conjugation enhanced the gene transfection efficiency of the PLGA nanosphere gene delivery system in vitro and that the enhanced gene expression was sustained for at least 13 days.

Chitosan-Modified PLGA Nanoparticles with Versatile Surface for Improved Drug Delivery

“…Drug delivery carriers with a versatile surface can improve the effectiveness of the systems. Recent studies have shown that polymeric material PLGA is a suitable vehicle that can incorporate various chemotherapeutic agents and deliver them to tumors (1,3). One of the only few drawbacks related to PLGA-based drug delivery system is the difficulty to have the targeting effects, either the passive or active ones (4), largely caused by the barren PLGA polymeric surfaces.…”

Controlling and Monitoring Intracellular Delivery of Anticancer Polymer Nanomedicines

“…[178] In another example, covalent conjugation of an NLS derived from the SV40 virus on PLGA nanospheres was demonstrated to enhance the gene transfection efficiency of the encapsulated pDNA. [179] While decoration of polymers or polymer nanoparticles with NLSs provides a powerful strategy to enhance nuclear delivery, in vivo the positively charged nature of these peptides may lead to nonspecific cellular uptake in the blood stream. [175] One way to address this drawback is to mask the NLS such that it is negatively charged in circulation but upon internalization undergoes charge reversal.…”

“…[147] Flierl et al used another approach to overcome the size limitations. In this work, an MTS peptide-peptide nucleic acid (PNA) conjugate was annealed to an oligonucleotide with a complementary PNA sequence and the complex was Linear polymer [177] α,β-Importins Nuclear localization signals (NLSs) [167,168,174,175] CGYGPKKKRKVGG Micelles [18] DRQIKIWFQNRRM-KWKK Nanoparticles [178] PKKRKV Nanoparticles [179] CVKRKKKP Linear polymers [63,79] Cytoplasmic NFkB Linear polymers [180,181] (11 of 26) 1700022 transferred into the cytosol using a PEI carrier. While the carrier allowed endosomal escape, the MTS targeting peptide directed the oligonucleotide to the mitochondria.…”

The Pharmacology of Regenerative Medicine

“…Systems with reduced levels of toxicity and improved transgene expression are being developed through increased understanding of the role of chemical constituents of the delivery vehicle (Liu and Reineke, 2010). Peptidic sequences have been conjugated to various nonviral gene delivery systems to improve cellular uptake , subcellular transport (Kwon et al, 2008;Moseley et al, 2010), and nuclear localization (Jeon et al, 2007;Moore et al, 2009). Based on safety and toxicity profiles, it is conceivable that these approaches may supplant viral technologies for gene delivery (Li and Huang, 2007).…”