Article | Published:

Radiation-enhanced delivery of plasmid DNA to tumors utilizing a novel PEI polyplex

Cancer Gene Therapyvolume 25pages196206 (2018) | Download Citation


The excitement surrounding the potential of gene therapy has been tempered due to the challenges that have thus far limited its successful implementation in the clinic such as issues regarding stability, transfection efficiency, and toxicity. In this study, low molecular weight linear polyethyleneimine (2.5 kDa) was modified by conjugation to a lipid, lithocholic acid, and complexed with a natural polysaccharide, dermatan sulfate (DS), to mask extra cationic charges of the modified polymer. In vitro examination revealed that these modifications improved complex stability with plasmid DNA (pDNA) and transfection efficiency. This novel ternary polyplex (pDNA/3E/DS) was used to investigate if tumor-targeted radiotherapy led to enhanced accumulation and retention of gene therapy vectors in vivo in tumor-bearing mice. Imaging of biodistribution revealed that tumor irradiation led to increased accumulation and retention as well as decreased off-target tissue buildup of pDNA in not only pDNA/3E/DS, but also in associated PEI-based polyplexes and commercial DNA delivery vehicles. The DS-containing complexes developed in this study displayed the greatest increase in tumor-specific pDNA delivery. These findings demonstrate a step forward in nucleic acid vehicle design as well as a promising approach to overall cancer gene therapy through utilization of radiotherapy as a tool for enhanced delivery.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    Baum C, Kustikova O, Modlich U, Li Z, Fehse B. Mutagenesis and oncogenesis by chromosomal insertion of gene transfer vectors. Hum Gene Ther. 2006;17:253–63.

  2. 2.

    Bessis N, GarciaCozar FJ, Boissier MC. Immune responses to gene therapy vectors: influence on vector function and effector mechanisms. Gene Ther. 2004;11:S10–7.

  3. 3.

    Waehler R, Russell SJ, Curiel DT. Engineering targeted viral vectors for gene therapy. Nat Rev Genet. 2007;8:573–87.

  4. 4.

    Nagasaki T, Shinkai S. The concept of molecular machinery is useful for design of stimuli-responsive gene delivery systems in the mammalian cell. J Incul Phenom Macrocycl Chem. 2007;58:205–19.

  5. 5.

    Bouard D, Alazard-Dany D, Cosset FL. Viral vectors: from virology to transgene expression. Br J Pharmacol. 2009;157:153–65.

  6. 6.

    Hatakeyama H, Akita H, Harashima H. A multifunctional envelope type nano device (MEND) for gene delivery to tumours based on the EPR effect: a strategy for overcoming the PEG dilemma. Adv Drug Deliv Rev. 2011;63:152–60.

  7. 7.

    Ito T, Iida-Tanaka N, Niidome T, Kawano T, Kubo K, Yoshikawa K, et al. Hyaluronic acid and its derivative as a multi-functional gene expression enhancer: protection from non-specific interactions, adhesion to targeted cells, and transcriptional activation. J Control Release. 2006;112:382–8.

  8. 8.

    Wang Y, Xu Z, Zhang R, Li W, Yang L, Hu Q. A facile approach to construct hyaluronic acid shielding polyplexes with improved stability and reduced cytotoxicity. Colloids Surf B Biointerfaces.2011;84:259–66.

  9. 9.

    Fan Y, Yao J, Du R, Hou L, Zhou J, Lu Y, et al. Ternary complexes with core-shell bilayer for double level targeted gene delivery: in vitro and in vivo evaluation. Pharm Res. 2013;30:1215–27.

  10. 10.

    Sun X, Ma P, Cao X, Ning L, Tian Y, Ren C. Positive hyaluronan/PEI/DNA complexes as a target-specific intracellular delivery to malignant breast cancer. Drug Deliv. 2009;16:357–62.

  11. 11.

    He Y, Cheng G, Xie L, Nie Y, He B, Gu Z. Polyethyleneimine/DNA polyplexes with reduction-sensitive hyaluronic acid derivatives shielding for targeted gene delivery. Biomaterials. 2013;34:1235–45.

  12. 12.

    Bahadur KCR, Thapa B, Xu P. Design of serum compatible tetrary complexes for gene delivery. Macromol Biosci. 2012;12:637–46.

  13. 13.

    Chen CJ, Zhao ZX, Wang JC, Zhao EY, Gao LY, Zhou SF, et al. A comparative study of three ternary complexes prepared in different mixing orders of siRNA/redox-responsive hyperbranched poly (amido amine)/hyaluronic acid. Int J Nanomedicine. 2012;7:3837–49.

  14. 14.

    Xu P, Quick GK, Yeo Y. Gene delivery through the use of a hyaluronate-associated intracellularly degradable crosslinked polyethyleneimine. Biomaterials. 2009;30:5834–43.

  15. 15.

    Hamada K, Yoshihara C, Ito T, Tani K, Tagawa M, Sakuragawa N, et al. Antitumor effect of chondroitin sulfate-coated ternary granulocyte macrophage-colony-stimulating factor plasmid complex for ovarian cancer. J Gene Med. 2012;14:120–7.

  16. 16.

    Pathak A, Kumar P, Chuttani K, Jain S, Mishra AK, Vyas SP, et al. Gene expression, biodistribution, and pharmacoscintigraphic evaluation of chondroitin sulfate-PEI nanoconstructs mediated tumor gene therapy. ACS Nano. 2009;3:1493–505.

  17. 17.

    Ibrahim BM, Park S, Han B, Yeo Y. A strategy to deliver genes to cystic fibrosis lungs: a battle with environment. J Control Release. 2011;155:289–95.

  18. 18.

    Han S, Mahato RI, Kim SW. Water-soluble lipopolymer for gene delivery. Bioconjug Chem. 2001;12:337–45.

  19. 19.

    Furgeson DY, Cohen RN, Mahato RI, Kim SW. Novel water insoluble lipoparticulates for gene delivery. Pharm Res. 2002;19:382–90.

  20. 20.

    Bajaj A, Kondaiah P, Bhattacharya S. Synthesis and gene transfection efficacies of PEI-cholesterol-based lipopolymers. Bioconjug Chem. 2008;19:1640–51.

  21. 21.

    Wang DA, Narang AS, Kotb M, Gaber AO, Miller DD, Kim SW, et al. Novel branched poly(ethylenimine)-cholesterol water-soluble lipopolymers for gene delivery. Biomacromolecules. 2002;3:1197–207.

  22. 22.

    Falamarzian A, Aliabadi HM, Molavi O, Seubert JM, Lai R, Uludag H, et al. Effective down-regulation of signal transducer and activator of transcription 3 (STAT3) by polyplexes of siRNA and lipid-substituted polyethyleneimine for sensitization of breast tumor cells to conventional chemotherapy. J Biomed Mater Res A. 2014;102:3216–28.

  23. 23.

    Neamnark A, Suwantong O, Bahadur RK, Hsu CY, Supaphol P, Uludag H. Aliphatic lipid substitution on 2 kDa polyethylenimine improves plasmid delivery and transgene expression. Mol Pharm. 2009;6:1798–815.

  24. 24.

    Aliabadi HM, Landry B, Bahadur RK, Neamnark A, Suwantong O, Uludag H. Impact of lipid substitution on assembly and delivery of siRNA by cationic polymers. Macromol Biosci. 2011;11:662–72.

  25. 25.

    Incani V, Tunis E, Clements BA, Olson C, Kucharski C, Lavasanifar A, et al. Palmitic acid substitution on cationic polymers for effective delivery of plasmid DNA to bone marrow stromal cells. J Biomed Mater Res A. 2007;81:493–504.

  26. 26.

    Aliabadi HM, Landry B, Mahdipoor P, Hsu CY, Uludag H. Effective down-regulation of breast cancer resistance protein (BCRP) by siRNA delivery using lipid-substituted aliphatic polymers. Eur J Pharm Biopharm. 2012;81:33–42.

  27. 27.

    Chae SY, Kim HJ, Lee MS, Jang YL, Lee Y, Lee SH, et al. Energy-independent intracellular gene delivery mediated by polymeric biomimetics of cell-penetrating peptides. Macromol Biosci. 2011;11:1169–74.

  28. 28.

    Goldberg AA, Beach A, Davies GF, Harkness TA, Leblanc A, Titorenko VI. Lithocholic bile acid selectively kills neuroblastoma cells, while sparing normal neuronal cells. Oncotarget. 2011;2:761–82.

  29. 29.

    Chae SY, Jin CH, Shin JH, Son S, Kim TH, Lee S, et al. Biochemical, pharmaceutical and therapeutic properties of long-acting lithocholic acid derivatized exendin-4 analogs. J Control Release. 2010;142:206–13.

  30. 30.

    Appelbe OK, Zhang Q, Pelizzari CA, Weichselbaum RR, Kron SJ. Image-guided radiotherapy targets macromolecules through altering the tumor microenvironment. Mol Pharm. 2016;13:3457–67.

  31. 31.

    Filonov GS, Piatkevich KD, Ting LM, Zhang J, Kim K, Verkhusha VV. Bright and stable near-infrared fluorescent protein for in vivo imaging. Nat Biotechnol. 2011;29:757–61.

  32. 32.

    Moding EJ, Clark DP, Qi Y, Li Y, Ma Y, Ghaghada K, et al. Dual-energy micro-computed tomography imaging of radiation-induced vascular changes in primary mouse sarcomas. Int J Radiat Oncol Biol Phys. 2013;85:1353–9.

  33. 33.

    Ma CM, Coffey CW, DeWerd LA, Liu C, Nath R, Seltzer SM, et al. AAPM protocol for 40-300 kV x-ray beam dosimetry in radiotherapy and radiobiology. Med Phys. 2001;28:868–93.

  34. 34.

    Inoue Y, Izawa K, Kiryu S, Tojo A, Ohtomo K. Diet and abdominal autofluorescence detected by in vivo fluorescence imaging of living mice. Mol Imaging. 2008;7:21–7.

  35. 35.

    Neu M, Germershaus O, Mao S, Voigt KH, Behe M, Kissel T. Crosslinked nanocarriers based upon poly(ethylene imine) for systemic plasmid delivery: in vitro characterization and in vivo studies in mice. J Control Release. 2007;118:370–80.

  36. 36.

    Doh KO, Yeo Y. Application of polysaccharides for surface modification of nanomedicines. Ther Deliv. 2012;3:1447–56.

  37. 37.

    Alshamsan A, Haddadi A, Incani V, Samuel J, Lavasanifar A, Uludag H. Formulation and delivery of siRNA by oleic acid and stearic acid modified polyethylenimine. Mol Pharm. 2009;6:121–33.

  38. 38.

    Moon HH, Joo MK, Mok H, Lee M, Hwang KC, Kim SW, et al. MSC-based VEGF gene therapy in rat myocardial infarction model using facial amphipathic bile acid-conjugated polyethyleneimine. Biomaterials. 2014;35:1744–54.

  39. 39.

    Uchida S, Itaka K, Chen Q, Osada K, Miyata K, Ishii T, et al. Combination of chondroitin sulfate and polyplex micelles from Poly(ethylene glycol)-poly{N'-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} block copolymer for prolonged in vivo gene transfection with reduced toxicity. J Control Release. 2011;155:296–302.

Download references


This work was supported by NIH R01s CA199663 to SJK and EB017791 to YY as well as NSF DMR-1056997 to YY.

Author contributions

OKA, BKK, YY, and SJK initiated the project. OKA led in vivo experimental design, data acquisition and analysis, and manuscript preparation. BKK designed and prepared all novel PEI polyplexes and performed in vitro data acquisition and analysis. NR and JW aided in data acquisition. YY and SJK participated in experimental design, data analysis, and manuscript preparation.

Author information


  1. Ludwig Center for Metastasis Research, The University of Chicago, 5758 South Maryland Avenue, MC 9006, Chicago, IL, 60637, USA

    • Oliver K. Appelbe
    • , Nick Rymut
    •  & Stephen J. Kron
  2. Department of Molecular Genetics and Cellular Biology, The University of Chicago, 929 East 57th Street, GCIS W519, Chicago, IL, 60637, USA

    • Oliver K. Appelbe
    • , Nick Rymut
    •  & Stephen J. Kron
  3. Department of Industrial and Physical Pharmacy, Purdue University, 575 Stadium Mall Dr., West Lafayette, IN, 47907, USA

    • Bieong-Kil Kim
    • , Jianping Wang
    •  & Yoon Yeo
  4. Department of Pharmaceutics, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, China

    • Jianping Wang
  5. Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, 47907, USA

    • Yoon Yeo


  1. Search for Oliver K. Appelbe in:

  2. Search for Bieong-Kil Kim in:

  3. Search for Nick Rymut in:

  4. Search for Jianping Wang in:

  5. Search for Stephen J. Kron in:

  6. Search for Yoon Yeo in:

Conflict of interest

The authors declare that thay have no competing interests.

Corresponding author

Correspondence to Stephen J. Kron.

Electronic supplementary material

About this article

Publication history