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Intelligent Biosynthetic Nanobiomaterials (IBNs) for Hyperthermic Gene Delivery

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Abstract

Purpose

Intelligent biosynthetic nanobiomaterials (IBNs) were constructed as recombinant diblock copolymers, notated as K8-ELP(1–60), containing a cationic oligolysine (VGK8G) and a thermosensitive elastin-like polypeptide (ELP) block with 60 repetitive pentapeptide units [(VPGXG)60; X is Val, Ala and Gly in a 5:2:3 ratio].

Methods

K8-ELP(1–60) was synthesized by recursive directional ligation for DNA oligomerization. Purity and molecular weight of K8-ELP(1–60) were confirmed by SDS-PAGE and mass spectrometry. DNA polyplexes were prepared from K8-ELP(1–60) and pGL3-Control (pGL3–C) plasmid DNA (pDNA) and stability was evaluated by gel retardation, DLS, and DNA displacement with heparin. Thermal transition profiles were studied by measuring the turbidity change at 350 nm and the polyplexes were used to transfect MCF-7 cells with a concomitant cytotoxicity assay.

Results

SDS-PAGE and MALDI-TOF studies showed highly pure copolymers at the desired molecular weight. K8-ELP(1-60) condensed pDNA at a cation to anion (N/P) ratio above 0.25 with a tight distribution of particle size ranging from 115.5–32.4 nm with increasing N/P ratio. Thermal transition temperatures of K8-ELP(1-60)/pDNA and K8-ELP(1-60) alone were 44.9 and 71.5°C, respectively. K8-ELP(1-60)/pDNA complexes successfully transduced MCF-7 cells with qualitative expression of enhanced green fluorescent protein (EGFP) and minimal cytotoxicity compared to branched poly(ethyleneimine) controls.

Conclusions

K8-ELP(1-60) was successfully designed and purified through recombinant means with efficient and stable condensation of pDNA at N/P ratios > 0.25 and polyplex particle size < 115 nm. MCF-7 cells successfully expressed EGFP with minimal cytotoxicity compared to positive controls; moreover, polyplexes retained sharp, thermotransitive kinetics within a narrow Tt range at clinically relevant hyperthermic temperatures, where the decrease of Tt was due to the increased hydrophobicity upon charge neutralization.

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Abbreviations

ELP:

elastin-like polypeptide

ELP(1–60):

ELP with 60 repetitive pentapeptide units [(VPGXG)60, X is Val, Ala and Gly in a 5:2:3 ratio]

IBN:

intelligent biosynthetic nanobiomaterial

ITC:

inverse transition cycling

K8 :

oligolysine (VGK8G)

RDL:

recursive directional ligation

Tt (°C):

phase transition temperature

References

  1. P. Factor. Gene therapy for acute diseases. Mol. Ther. 4:515–524 (2001).

    Article  PubMed  CAS  Google Scholar 

  2. S. Yla-Herttuala, and K. Alitalo. Gene transfer as a tool to induce therapeutic vascular growth. Nat. Med. 9:694–701 (2003).

    Article  PubMed  Google Scholar 

  3. J. T. Santoso, D. C. Tang, S. B. Lane, J. Hung, D. J. Reed, C. Y. Muller, D. P. Carbone, J. A. Lucci 3rd, D. S. Miller, and J. M. Mathis. Adenovirus-based p53 gene therapy in ovarian cancer. Gynecol. Oncol. 59:171–178 (1995).

    Article  PubMed  CAS  Google Scholar 

  4. F. Siddiqui, E. J. Ehrhart, B. Charles, L. Chubb, C. Y. Li, X. Zhang, S. M. Larue, P. R. Avery, M. W. Dewhirst, and R. L. Ullrich. Anti-angiogenic effects of interleukin-12 delivered by a novel hyperthermia induced gene construct. Int. J. Hyperthermia 22:587–606 (2006).

    Article  PubMed  CAS  Google Scholar 

  5. J. Zabner, A. J. Fasbender, T. Moninger, K. A. Poellinger, and M. J. Welsh. Cellular and molecular barriers to gene transfer by a cationic lipid. J. Biol. Chem. 270:18997–19007 (1995).

    Article  PubMed  CAS  Google Scholar 

  6. P. C. Bell, M. Bergsma, I. P. Dolbnya, W. Bras, M. C. Stuart, A. E. Rowan, M. C. Feiters, and J. B. Engberts. Transfection mediated by gemini surfactants: engineered escape from the endosomal compartment. J. Am. Chem. Soc. 125:1551–1558 (2003).

    Article  PubMed  CAS  Google Scholar 

  7. J. Y. Legendre, and F. C. Szoka Jr. Delivery of plasmid DNA into mammalian cell lines using pH-sensitive liposomes: comparison with cationic liposomes. Pharm. Res. 9:1235–1242 (1992).

    Article  PubMed  CAS  Google Scholar 

  8. X. Gao, and L. Huang. Cationic liposome-mediated gene transfer. Gene Ther. 2:710–722 (1995).

    PubMed  CAS  Google Scholar 

  9. O. Boussif, F. Lezoualc’h, M. A. Zanta, M. D. Mergny, D. Scherman, B. Demeneix and J. P. Behr. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. U. S. A. 92:7297–7301 (1995).

    Article  PubMed  CAS  Google Scholar 

  10. Z. Megeed, M. Haider, D. Li, B. W. O’Malley Jr, J. Cappello, H. Ghandehari. In vitro and in vivo evaluation of recombinant silk-elastinlike hydrogels for cancer gene therapy. J. Control. Release 94:433–445 (2004).

    Article  PubMed  CAS  Google Scholar 

  11. K. Itaka, K. Yamauchi, A. Harada, K. Nakamura, H. Kawaguchi, and K. Kataoka. Polyion complex micelles from plasmid DNA and poly(ethylene glycol)-poly(l-lysine) block copolymer as serum-tolerable polyplex system: physicochemical properties of micelles relevant to gene transfection efficiency. Biomaterials 24:4495–4506 (2003).

    Article  PubMed  CAS  Google Scholar 

  12. D. Fischer, T. Bieber, Y. Li, H. P. Elsasser and T. Kissel. A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: effect of molecular weight on transfection efficiency and cytotoxicity. Pharm. Res. 16:1273–1279 (1999).

    Article  PubMed  CAS  Google Scholar 

  13. P. van de Wetering, E. E. Moret, N. M. Schuurmans-Nieuwenbroek, M. J. van Steenbergen, and W. E. Hennink. Structure-activity relationships of water-soluble cationic methacrylate/methacrylamide polymers for nonviral gene delivery. Bioconjug. Chem. 10:589–597 (1999).

    Article  PubMed  CAS  Google Scholar 

  14. D. E. Meyer, and A. Chilkoti. Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system. Biomacromolecules 3:357–367 (2002).

    Article  PubMed  CAS  Google Scholar 

  15. D. W. Lim, K. Trabbic-Carlson, J. A. Mackay, and A. Chilkoti. Improved non-chromatographic purification of a recombinant protein by cationic elastin-like polypeptides. Biomacromolecules 8(5):1417–1424 (2007).

    Article  CAS  Google Scholar 

  16. C. Plank, M. X. Tang, A. R. Wolfe, and F. C. Szoka Jr. Branched cationic peptides for gene delivery: role of type and number of cationic residues in formation and in vitro activity of DNA polyplexes. Hum. Gene Ther. 10:319–332 (1999).

    Article  PubMed  CAS  Google Scholar 

  17. D. Fischer, H. Dautzenberg, K. Kunath, and T. Kissel. Poly(diallyldimethylammonium chlorides) and their N-methyl-N-vinylacetamide copolymer-based DNA-polyplexes: role of molecular weight and charge density in complex formation, stability, and in vitro activity. Int. J. Pharm. 280:253–269 (2004).

    Article  PubMed  CAS  Google Scholar 

  18. D. T. McPherson, J. Xu, and D. W. Urry. Product purification by reversible phase transition following Escherichia coli expression of genes encoding up to 251 repeats of the elastomeric pentapeptide GVGVP. Protein Expr. Purif. 7:51–57 (1996).

    Article  PubMed  CAS  Google Scholar 

  19. D. E. Meyer, and A. Chilkoti. Purification of recombinant proteins by fusion with thermally-responsive polypeptides. Nat. Biotechnol. 17:1112–1115 (1999).

    Article  PubMed  CAS  Google Scholar 

  20. J. Cappello, J. Crissman, M. Dorman, M. Mikolajczak, G. Textor, M. Marquet, and F. Ferrari. Genetic engineering of structural protein polymers. Biotechnol. Prog. 6:198–202 (1990).

    Article  PubMed  CAS  Google Scholar 

  21. D. W. Urry, T. M. Parker, M. C. Reid, and D. C. Gowda. Biocompatibility of the bioelastic materials, poly(GVGVP) and its γ-irradiation cross-linked matrix: summary of generic biological test results. J. Bioact. Compat. Poly. 6:263–282 (1991).

    Article  CAS  Google Scholar 

  22. A. C. Rincon, I. T. Molina-Martinez, B. de Las Heras, M. Alonso, C. Bailez, J. C. Rodriguez-Cabello, and R. Herrero-Vanrell. Biocompatibility of elastin-like polymer poly(VPAVG) microparticles: in vitro and in vivo studies. J. Biomed. Mater. Res. A 78:343–351 (2006).

    PubMed  CAS  Google Scholar 

  23. D. W. Urry. Physical chemistry of biological free energy transduction as demonstrated by elastic protein-based polymers. J. Phys. Chem. B 101:11007–11028 (1997).

    Article  CAS  Google Scholar 

  24. A. Girotti, J. Reguera, F. J. Arias, M. Alonso, A. M. Testera, and J. C. Rodríguez-Cabello. Influence of the molecular weight on the inverse temperature transition of a model genetically engineered elastin-like pH-responsive polymer. Macromolecules 37:3396–3400 (2004).

    Article  CAS  Google Scholar 

  25. M. R. Dreher, D. Raucher, N. Balu, O. Michael Colvin, S. M. Ludeman and A. Chilkoti. Evaluation of an elastin-like polypeptide-doxorubicin conjugate for cancer therapy. J. Control. Release 91:31–43 (2003).

    Article  PubMed  CAS  Google Scholar 

  26. D.Y. Furgeson, M. R. Dreher, and A. Chilkoti. Structural optimization of a “smart” doxorubicin–polypeptide conjugate for thermally targeted delivery to solid tumors. J. Control. Release 110:362–369 (2006).

    Article  PubMed  CAS  Google Scholar 

  27. W. Liu, M. R. Dreher, D. Y. Furgeson, K. V. Peixoto, H. Yuan, M. R. Zalutsky, and A. Chilkoti. Tumor accumulation, degradation and pharmacokinetics of elastin-like polypeptides in nude mice. J. Control. Release 116:170–178 (2006).

    Article  PubMed  CAS  Google Scholar 

  28. D. Raucher, and A. Chilkoti. Enhanced uptake of a thermally responsive polypeptide by tumor cells in response to its hyperthermia-mediated phase transition. Cancer Res. 61:7163–7170 (2001).

    PubMed  CAS  Google Scholar 

  29. G. L. Bidwell 3rd, and D. Raucher. Application of thermally responsive polypeptides directed against c-Myc transcriptional function for cancer therapy. Mol. Cancer. Ther. 4:1076–1085 (2005).

    Article  PubMed  CAS  Google Scholar 

  30. I. Massodi, G. L. Bidwell 3rd, and D. Raucher. Evaluation of cell penetrating peptides fused to elastin-like polypeptide for drug delivery. J. Control. Release 108:396–408 (2005).

    Article  PubMed  CAS  Google Scholar 

  31. R. Langer, and D. A. Tirrell. Designing materials for biology and medicine. Nature 428:487–492 (2004).

    Article  PubMed  CAS  Google Scholar 

  32. A. Aris, J. X. Feliu, A. Knight, C. Coutelle, and A. Villaverde. Exploiting viral cell-targeting abilities in a single polypeptide, non-infectious, recombinant vehicle for integrin-mediated DNA delivery and gene expression. Biotechnol. Bioeng. 68:689–696 (2000).

    Article  PubMed  CAS  Google Scholar 

  33. L.K. Medina-Kauwe, M. Maguire, N. Kasahara, and L. Kedes. Nonviral gene delivery to human breast cancer cells by targeted Ad5 penton proteins. Gene Ther. 8:1753–1761 (2001).

    Article  PubMed  CAS  Google Scholar 

  34. A. Hatefi, Z. Megeed, and H. Ghandehari. Recombinant polymer–protein fusion: a promising approach towards efficient and targeted gene delivery. J. Gene Med. 8:468–476 (2006).

    Article  PubMed  CAS  Google Scholar 

  35. M. Haider, V. Leung, F. Ferrari, J. Crissman, J. Powell, J. Cappello, and H. Ghandehari. Molecular engineering of silk-elastinlike polymers for matrix-mediated gene delivery: biosynthesis and characterization. Mol. Pharm. 2:139–150 (2005).

    Article  PubMed  CAS  Google Scholar 

  36. A. Hatefi, J. Cappello, and H. Ghandehari. Adenoviral gene delivery to solid tumors by recombinant silk-elastinlike protein polymers. Pharm. Res. 24:773–779 (2007).

    Article  PubMed  CAS  Google Scholar 

  37. A. Zintchenko, M. Ogris, and E. Wagner. Temperature dependent gene expression induced by PNIPAM_based copolymers: potential of hyperthermia in gene transfer. Bioconjug. Chem. 17:766–772 (2006).

    Article  PubMed  CAS  Google Scholar 

  38. H. S. Bisht, D. S. Manickam, Y. You, and D. Oupicky. Temperature-controlled properties of DNA complexes with poly(ethylenimine)-graft-poly(N-isopropylacrylamide). Biomacromolecules 7:1169–1178 (2006).

    Article  PubMed  CAS  Google Scholar 

  39. P.H. Hirel, M. J. Schmitter, P. Dessen, G. Fayat, and S. Blanquet. Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. Proc. Natl. Acad. Sci. U. S. A. 86:8247–8251 (1989).

    Article  PubMed  CAS  Google Scholar 

  40. D. W. Urry. Free energy transduction in polypeptides and proteins based on inverse temperature transitions. Prog. Biophys. Mol. Biol. 57:23–57 (1992).

    Article  PubMed  CAS  Google Scholar 

  41. A.M. Ponce, Z. Vujaskovic, F. Yuan, D. Needham and M. W. Dewhirst. Hyperthermia mediated liposomal drug delivery. Int. J. Hyperthermia 22:205–213 (2006).

    Article  PubMed  CAS  Google Scholar 

  42. M. Neu, J. Sitterberg, U. Bakowsky, and T. Kissel. Stabilized nanocarriers for plasmids based upon cross-linked poly(ethylene imine). Biomacromolecules 7:3428–3438 (2006).

    Article  PubMed  CAS  Google Scholar 

  43. D. Putnam, C. A. Gentry, D. W. Pack, and R. Langer. Polymer-based gene delivery with low cytotoxicity by a unique balance of side-chain termini. Proc. Natl. Acad. Sci. U. S. A. 98:1200–1205 (2001).

    Article  PubMed  CAS  Google Scholar 

  44. K. L. Kiick, E. Saxon, D. A. Tirrell, and C. R. Bertozzi. Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation. Proc. Natl. Acad. Sci. U.S.A. 99:19–24 (2002).

    Article  PubMed  CAS  Google Scholar 

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ACKNOWLEDGEMENTS

This work was funded by University of Wisconsin-Madison start-up funds to DYF. The ELP(1–30) gene was donated by Prof. Ashutosh Chilkoti (Duke University). We thank Prof. Glen Kwon (University of Wisconsin—Madison) for use of the NICOMP DLS; Prof. Maureen Barr (University of Wisconsin—Madison) for microscope access; and the UW Biotechnology Center for performing MALDI-TOF mass spectrum analysis. We express our thanks to Ms. Tracy P. Williamson for her critical review of the manuscript.

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Authors and Affiliations

  1. Division of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin—Madison, Madison, Wisconsin, 53705-2222, USA

    Tze-Haw Howard Chen, Younsoo Bae & Darin Y. Furgeson

  2. Department of Biomedical Engineering, University of Wisconsin—Madison, Madison, Wisconsin, 53705-2222, USA

    Darin Y. Furgeson

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  1. Tze-Haw Howard Chen
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Correspondence to Darin Y. Furgeson.

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Chen, TH.H., Bae, Y. & Furgeson, D.Y. Intelligent Biosynthetic Nanobiomaterials (IBNs) for Hyperthermic Gene Delivery. Pharm Res 25, 683–691 (2008). https://doi.org/10.1007/s11095-007-9382-5

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