pDNA bioparticles: comparative heterogeneity, surface, binding, and activity analyses

https://doi.org/10.1016/j.bbrc.2004.04.188Get rights and content

Abstract

New applications for nucleic acid-bound micro/nanoparticles are emerging in drug delivery, biocatalysis, diagnostics, and toxicology. Bioactivity of viral or liposomal based technologies is limited by heterogeneity, partitioning, aggregation, and protein binding in physiological fluids, underlying immunotoxicity, and poor in vitro and cell-culture corollaries. Here we have systematically investigated novel pDNA bioparticles formed through complexation to model non-viral/non-lipid materials, peptides, aminoglycans, and small molecules (polybrene, chitosan, butirosin, protamine, Lys10, RGDS, bupivacaine, and chlorpromazine). On the basis of characterization by heterogeneity, kinetics, partitioning in physiological fluid and serum protein-binding, surface, size and electrophoretic behavior, transfection, and immunotoxicity, notably protamine, and chitosan DNA particles gave a long lifetime (12–18 h), low protein-binding (<10 μg/ml), good transfection activity (102–104 RLU/mg cell protein), and low immunotoxicity. Our results support further evaluation of these materials as potential alternatives to viral or liposomal approaches, in combination with pDNA as binding, expression or therapeutic agents.

Section snippets

Materials and methods

Reagents. All model compounds were obtained from Sigma/Aldrich (St Louis, MO). 1:1 wt:wt DOTAP/cholesterol and rhodamine labeled plasmid DNA were generous gifts from Megabios (Burlingame, CA). Luciferase and green fluorescent protein plasmid were obtained from Aldevron (Fargo, ND). Gold microparticles were obtained from PowderJect Vaccines (Madison, WI).

Controlled complex formation. Compounds were added to plasmid at approximately 1 ml/min while the plasmid was stirring within a 12 ml glass vial

Model compounds

Chemical features of the model compounds, lipid or tributyrin controls are shown in Table 1. All compounds’ transfection and toxicity in combination with DNA had been previously described [3], [4], [5], [6], [7], [8]. All agents contained cationic–NH-groups permitting stabilizing charge–charge interactions with the anionic phosphodiester backbone on the DNA, except tributyrin whose charge was mediated through combination with RGDS peptide at the vesicles’ surface. In addition, intercalation of

Discussion

In brief summary we have characterized the DNA-binding, charge and size heterogeneity, surface, sedimentation, protein-binding, transfection activity, and immunotoxicity of a range of non-lipid biomaterials which produce micro/nanoparticles when complexed to plasmid DNA. Unlike viral or liposomal DNA particles [1], [2], our results especially with chitosan and protamine materials suggest that these materials will be less limited from the standpoint of aggregation, sedimentation, and

Acknowledgements

We gratefully acknowledge J. Koe, G. Koe, and S. Venkatesh (Megabios Corp., Burlingame, CA) for the provision of liposome, rhodamine labeled plasmid DNA, and assistance with the sedimentation assays, B. Lemos (Geron Corp.) for technical assistance with mouse intra-peritoneal assays, S. Ghivizzani and T. Oligino (University of Pittsburg Center for Biotechnology and Bioengineering) for assistance with the rabbit knee model white blood cell assays, and J. Haynes (PowderJect Vaccines, Madison, WI)

References (28)

  • C.M Wiethoff et al.

    The structural organization of cationic lipid-DNA complexes

    J. Biol. Chem.

    (2002)
  • A Hawtrey et al.

    Low concentrations of chlorpromazine and related phenothiazines stimulate gene transfer in HeLa cells via receptor-mediated endocytosis

    Drug Deliv.

    (2002)
  • W.F Anderson et al.

    Protamine sulfate as an effective alternative to polybrene in retroviral-mediated gene-transfer: implications for human gene therapy

    J. Virol. Methods

    (1989)
  • H Hanenburg et al.

    Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells

    Nat. Med.

    (1996)
  • Cited by (18)

    • Substrate-mediated delivery of gene complex nanoparticles via polydopamine coating for enhancing competitiveness of endothelial cells

      2016, Colloids and Surfaces B: Biointerfaces
      Citation Excerpt :

      Protamine is a cationic small and high arginine content protein [36]. Protamine sulfate (PrS) has been used as a non-viral carrier to condense DNA into complex nanoparticles for accelerating delivery of gene into target cells [51,52]. In addition, PrS shows other particular properties including weak anticoagulation and antibacterial activity [14,53,54], readily available properties when PrS is applied in coatings for cardiovascular devices.

    • In vitro design and characterization of the nonviral gene delivery vector iopamidol, protamine, ethiodized oil reagent

      2011, Journal of Vascular and Interventional Radiology
      Citation Excerpt :

      And although the VIPER conditions are less efficient gene carriers than adenovirus, the nonviral vector is within 1.5 orders of magnitude of viral activity and an order of magnitude of FuGENE activity (Fig 4a), a cationic lipid commonly used for nonviral in vitro transfection that is too toxic for clinical applications. In addition, unlike adenovirus, the immunogenicity and toxicity of which limits iterative gene therapy treatments (34), P:D complexes, as in VIPER, are significantly less toxic and less immunogenic (35). With an iterative approach to gene delivery, the transfection efficiency of this nonviral system could be within a range suitable for clinical translation.

    • Characterization and performance of nucleic acid nanoparticles combined with protamine and gold

      2009, Biomaterials
      Citation Excerpt :

      Evidence for DNA attachment to gold is shown in Fig. 1. Particles were prepared with DNA attached to gold [11,19], and the supernatant or particle-associated fraction (eluted from the particles) was run by gel electrophoresis (panel A). In comparison to internal load standards, all five batches appeared to have 25–50 ng of DNA loaded per 0.5 mg of gold eluted.

    • Chitosan-based nanoformulation as carriers of small molecules for tissue regeneration

      2020, Functional Chitosan: Drug Delivery and Biomedical Applications
    View all citing articles on Scopus
    View full text