Pharmaceutical Research

, Volume 27, Issue 5, pp 841–854 | Cite as

Release of Plasmid DNA-Encoding IL-10 from PLGA Microparticles Facilitates Long-Term Reversal of Neuropathic Pain Following a Single Intrathecal Administration

  • Ryan Gene Soderquist
  • Evan M. Sloane
  • Lisa C. Loram
  • Jacqueline A. Harrison
  • Ellen C. Dengler
  • Scott M. Johnson
  • Luke D. Amer
  • Courtney S. Young
  • Makenzie T. Lewis
  • Stephen Poole
  • Matthew G. Frank
  • Linda R. Watkins
  • Erin D. Milligan
  • Melissa J. MahoneyEmail author
Research Paper



Interleukin-10 (IL-10) is an anti-inflammatory molecule that has achieved interest as a therapeutic for neuropathic pain. In this work, the potential of plasmid DNA-encoding IL-10 (pDNA-IL-10) slowly released from biodegradable microparticles to provide long-term pain relief in an animal model of neuropathic pain was investigated.


PLGA microparticles encapsulating pDNA-IL-10 were developed and assessed both in vitro and in vivo.


In vitro, pDNA containing microparticles activated macrophages, enhanced the production of nitric oxide, and increased the production of IL-10 protein relative to levels achieved with unencapsulated pDNA-IL-10. In vivo, intrathecally administered microparticles embedded in meningeal tissue, induced phagocytic cell recruitment to the cerebrospinal fluid, and relieved neuropathic pain for greater than 74 days following a single intrathecal administration, a feat not achieved with unencapsulated pDNA. Therapeutic effects of microparticle-delivered pDNA-IL-10 were blocked in the presence of IL-10-neutralizing antibody, and elevated levels of plasmid-derived IL-10 were detected in tissues for a prolonged time period post-injection (>28 days), demonstrating that therapeutic effects are dependent on IL-10 protein production.


These studies demonstrate that microparticle encapsulation significantly enhances the potency of intrathecally administered pDNA, which may be extended to treat other disorders that require intrathecal gene therapy.

Key words

interleukin-10 microparticle pDNA PLGA neuropathic pain 



anti-rat interleukin-10 IgG neutralizing antibody


chronic constriction injury


cerebrospinal Fluid


threshold cycle


glyceraldehyde-6-phosphate dehydrogenase




limulus amebocyte lysate




major histocompatibility complex-II


nitric oxide


plasmid DNA


poly (lactic-co-glycolic-acid)



Funding for this work was provided by NIH grant DA018156, and E. Dengler was supported by NSF Grant DGE-0549500. The authors would like to acknowledge the expert assistance of Dr. Travis Hughes and Dr. Leslie Leinwand at the University of Colorado for consultations on plasmid DNA growth and characterization. We would also like to thank Avigen Inc. (Alameda, CA) for the purification of Anti-IL-10-IgG and Control-IgG antibodies used in this work and Jenny L. Wilkerson at the University of New Mexico for her assistance with surgical procedures.


  1. 1.
    Miller G. The dark side of glia. Science. 2005;308:778–81.CrossRefPubMedGoogle Scholar
  2. 2.
    Suter MR, Wen YR, Decosterd I, Ji RR. Do glial cells control pain? Neuron Glia Biol. 2007;3:255–68.CrossRefPubMedGoogle Scholar
  3. 3.
    Watkins LR, Hutchinson MR, Ledeboer A, Wieseler-Frank J, Milligan ED, Maier SF. Glia as the “bad guys”: Implications for improving clinical pain control and the clinical utility of opioids. Brain Behav Immun. 2007;21:131–46.CrossRefPubMedGoogle Scholar
  4. 4.
    Reeve AJ, Patel S, Fox A, Walker K, Urban L. Intrathecally administered endotoxin or cytokines produce allodynia, hyperalgesia and changes in spinal cord neuronal responses to nociceptive stimuli in the rat. Eur J Pain. 2000;4:247–57.CrossRefPubMedGoogle Scholar
  5. 5.
    Sawada M, Suzumura A, Hosoya H, Marunouchi T, Nagatsu T. Interleukin-10 inhibits both production of cytokines and expression of cytokine receptors in microglia. J Neurochem. 1999;72:1466–71.CrossRefPubMedGoogle Scholar
  6. 6.
    Pahan K, Khan M, Singh I. Interleukin-10 and interleukin-13 inhibit proinflammatory cytokine-induced ceramide production through the activation of phosphatidylinositol 3-kinase. J Neurochem. 2000;75:576–82.CrossRefPubMedGoogle Scholar
  7. 7.
    Milligan ED, Langer SJ, Sloane EM, He L, Wieseler-Frank J, O’Connor K, et al. Controlling pathological pain by adenovirally driven spinal production of the anti-inflammatory cytokine, interleukin-10. Eur J Neurosci. 2005;21:2136–48.CrossRefPubMedGoogle Scholar
  8. 8.
    Milligan ED, Sloane EM, Langer SJ, Cruz PE, Chacur M, Spataro L, et al. Controlling neuropathic pain by adeno-associated virus driven production of the anti-inflammatory cytokine, interleukin-10. Mol Pain. 2005;1:1–9.CrossRefGoogle Scholar
  9. 9.
    Milligan ED, Sloane EM, Langer SJ, Hughes TS, Jekich BM, Frank MG, et al. Repeated intrathecal injections of plasmid DNA encoding interleukin-10 produce prolonged reversal of neuropathic pain. Pain. 2006;126:294–308.CrossRefPubMedGoogle Scholar
  10. 10.
    Sloane E, Ledeboer A, Seibert W, Coats B, van Strien M, Maier K, et al. Anti-inflammatory cytokine gene therapy decreases sensory and motor dysfunction in experimental Multiple Sclerosis. Brain Behav Immun. 2009;23:92–100.CrossRefPubMedGoogle Scholar
  11. 11.
    Sloane EM, Soderquist RG, Maier SF, Mahoney MJ, Watkins LR, Milligan ED. Long term control of neuropathic pain in a non-viral gene therapy paradigm. Gene Ther. 2009;16:470–5.CrossRefPubMedGoogle Scholar
  12. 12.
    Ledeboer A, Jekich BM, Sloane EM, Mahoney JH, Langer SJ, Milligan ED, et al. Intrathecal interleukin-10 gene therapy attenuates paclitaxel-induced mechanical allodynia and proinflammatory cytokine expression in dorsal root ganglia in rats. Brain Behav Immun. 2007;21:686–98.CrossRefPubMedGoogle Scholar
  13. 13.
    Gomez-Nicola D, Valle-Argos B, Suardiaz M, Taylor JS, Nieto-Sampedro M. Role of IL-15 in spinal cord and sciatic nerve after chronic constriction injury: regulation of macrophage and T-cell infiltration. J Neurochem. 2008;107:1741–52.CrossRefPubMedGoogle Scholar
  14. 14.
    Dijkstra CD, Dopp EA, Joling P, Kraal G. The heterogeneity of mononuclear phagocytes in lympoid organs - distinct macrophage subpopulations in the rat recognized by monoclonal antibody-ED1, antibody-ED2 and antibody-ED3. Immunology. 1985;54:589–99.PubMedGoogle Scholar
  15. 15.
    Polfliet MMJ, Goede PH, van Kesteren-Hendrikx EML, van Rooijen N, Dijkstra CD, van den Berg TK. A method for the selective depletion of perivascular and meningeal macrophages in the central nervous system. J Neuroimmun. 2001;116:188–95.CrossRefGoogle Scholar
  16. 16.
    Duffield JS. The inflammatory macrophage: a story of Jekyll and Hyde. Clin Sci. 2003;104:27–38.CrossRefPubMedGoogle Scholar
  17. 17.
    de Vos AF, van Meurs M, Brok HP, Boven LA, Hintzen RQ, van der Valk P, et al. Transfer of central nervous system autoantigens and presentation in secondary lymphoid organs. J Immunol. 2002;169:5415–23.PubMedGoogle Scholar
  18. 18.
    Honda H, Kimura H, Rostami A. Demonstration and phenotypic characterization of resident macrophages in rat skeletal-muscle. Immunology. 1990;70:272–7.PubMedGoogle Scholar
  19. 19.
    Zhang J, Shi XQ, Echeverry S, Mogil JS, De Koninck Y, Rivest S. Expression of CCR2 in both resident and bone marrow-derived microglia plays a critical role in neuropathic pain. J Neurosci. 2007;27:12396–406.CrossRefPubMedGoogle Scholar
  20. 20.
    El Khoury J, Luster AD. Mechanisms of microglia accumulation in Alzheimer’s disease: therapeutic implications. Trends Pharmacol Sci. 2008;29:626–32.CrossRefPubMedGoogle Scholar
  21. 21.
    Sloane E, Langer S, Jekich B, Mahoney J, Hughes T, Frank M, et al. Immunological priming potentiates non-viral anti-inflammatory gene therapy treatment of neuropathic pain. Gene Ther. 2009;16:1210–22.CrossRefPubMedGoogle Scholar
  22. 22.
    Hedley ML. Formulations containing poly(lactide-co-glycolide) and plasmid DNA expression vectors. Exp Opin Biol Ther. 2003;3:903–10.CrossRefGoogle Scholar
  23. 23.
    Remaut K, Sanders NN, De Geest BG, Braeckmans K, Demeester J, De Smedt SC. Nucleic acid delivery: Where material sciences and bio-sciences meet. Mater Sci Eng R-Rep. 2007;58:117–61.CrossRefGoogle Scholar
  24. 24.
    Yoshida M, Babensee JE. Molecular aspects of microparticle phagocytosis by dendritic cells. J Biomater Sci-Polym Ed. 2006;17:893–907.CrossRefPubMedGoogle Scholar
  25. 25.
    Kanangat S, Nair S, Babu JS, Rouse BT. Expression of cytokine messenger-mRNA in murine splenic dendritic cells and better induction of T-cell-derived cytokines by dendritic cells than by macrophages during in-vitro costimulation assay using specific antigens. J Leukoc Biol. 1995;57:310–6.PubMedGoogle Scholar
  26. 26.
    Kelleher P, Knight SC. IL-12 increases CD80 expression and the stimulatory capacity of bone marrow-derived dendritic cells. Int Immunol. 1998;10:749–55.CrossRefPubMedGoogle Scholar
  27. 27.
    Ding TT, Sun J, Zhang P. Immune evaluation of biomaterials in TNF-alpha and IL-1 beta at mRNA level. Springer 2007, pp 2233–2236.Google Scholar
  28. 28.
    Prior S, Gander B, Blarer N, Merkle HP, Subira ML, Irache JM, et al. In vitro phagocytosis and monocyte-macrophage activation with poly(lactide) and poly(lactide-co-glycolide) microspheres. Eur J Pharm Sci. 2002;15:197–207.CrossRefPubMedGoogle Scholar
  29. 29.
    Parsa S, Wang Y, Fuller J, Langer R, Pfeifer BA. A comparison between polymeric microsphere and bacterial vectors for macrophage P388D1 gene delivery. Pharm Res. 2008;25:1202–8.CrossRefPubMedGoogle Scholar
  30. 30.
    Mert T, Gunay I, Ocal I, Guzel AI, Inal TC, Sencar L, et al. Macrophage depletion delays progression of neuropathic pain in diabetic animals. Naunyn-Schmiedebergs Arch Pharmacol. 2009;379:445–52.CrossRefPubMedGoogle Scholar
  31. 31.
    Qiagen. Endofree plasmid purification handbook. Qiagen Inc; 2006, pp. 38–41.Google Scholar
  32. 32.
    Tinsley-Bown AM, Fretwell R, Dowsett AB, Davis SL, Farrar GH. Formulation of poly(D, L-lactic-co-glycolic acid) microparticles for rapid plasmid DNA delivery. J Control Rel. 2000;66:229–41.CrossRefGoogle Scholar
  33. 33.
    Milligan ED, Soderquist RG, Malone SM, Mahoney JH, Hughes TS, Langer SJ, et al. Intrathecal polymer-based interleukin-10 gene delivery for neuropathic pain. Neuron Glia Biol. 2006;2:293–308.CrossRefPubMedGoogle Scholar
  34. 34.
    Hartmann B, Ahmadi S, Heppenstall P, Lewin G, Schott C, Borchardt T, et al. The AMPA receptor subunits GluR-A and GluR-B reciprocally modulate spinal synaptic plasticity and inflammatory pain. Neuron. 2004;44:637–50.CrossRefPubMedGoogle Scholar
  35. 35.
    Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction. Anal Biochem. 1987;162:156–9.CrossRefPubMedGoogle Scholar
  36. 36.
    Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method. Methods. 2001;25:402–8.CrossRefPubMedGoogle Scholar
  37. 37.
    Yeo Y, Park KN. Control of encapsulation efficiency and initial burst in polymeric microparticle systems. Arch Pharm Res. 2004;27:1–12.CrossRefPubMedGoogle Scholar
  38. 38.
    Ito H, Koide N, Morikawa A, Hassan F, Islam S, Tumurkhuu G, et al. Augmentation of lipopolysaccharide-induced nitric oxide production by alpha-galactosylceramide in mouse peritoneal cells. J Endotox Res. 2005;11:213–9.Google Scholar
  39. 39.
    Tumer C, Bilgin HM, Obay BD, Diken H, Atmaca M, Kelle M. Effect of nitric oxide on phagocytic activity of lipopolysaccharide-induced macrophages: possible role of exogenous L-arginine. Cell Biol Int. 2007;31:565–9.CrossRefPubMedGoogle Scholar
  40. 40.
    Lichtman SN, Wang J, Lemasters JJ. LPS receptor CD14 participates in release of TNF-alpha in RAW 264.7 and peritoneal cells but not in Kupffer cells. Am J Physiol-Gastrointest Liver Physiol. 1998;38:G39–46.Google Scholar
  41. 41.
    Bergman I, Burckart GJ, Pohl CR, Venkataramanan R, Barmada MA, Griffin JA, et al. Pharmacokinetics of IgG and IgM anti-ganglioside antibodies in rats and monkeys after intrathecal administration. J Pharmacol Exp Ther. 1998;284:111–5.PubMedGoogle Scholar
  42. 42.
    Gretzer C, Emanuelsson L, Liljensten E, Thomsen P. The inflammatory cell influx and cytokines changes during transition from acute inflammation to fibrous repair around implanted materials. J Biomater Sci-Polym Ed. 2006;17:669–87.CrossRefPubMedGoogle Scholar
  43. 43.
    Eldridge JH, Hammond CJ, Meulbroek JA, Staas JK, Gilley RM, Tice TR. Controlled vaccine release in the gut-associated lymphoid-tissues. 1. Orally-administered biodegradable microspheres target the peyers patches. Elsevier Science Bv, 1990, pp. 205–214.Google Scholar
  44. 44.
    Champion JA, Walker A, Mitragotri S. Role of particle size in phagocytosis of polymeric microspheres. Pharma Res. 2008;25:1815–21.CrossRefGoogle Scholar
  45. 45.
    Shakweh M, Besnard M, Nicolas V, Fattal E. Poly (lactide-co-glycolide) particles of different physicochemical properties and their uptake by Peyer’s patches in mice. Eur J Pharm Biopharm. 2005;61:1–13.CrossRefPubMedGoogle Scholar
  46. 46.
    Foged C, Brodin B, Frokjaer S, Sundblad A. Particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model. Int J Pharm. 2005;298:315–22.CrossRefPubMedGoogle Scholar
  47. 47.
    Henry JA, Burugapalli K, Neuenschwander P, Pandit A. Structural variants of biodegradable polyesterurethane in vivo evoke a cellular and angiogenic response that is dictated by architecture. Acta Biomater. 2009;5:29–42.CrossRefPubMedGoogle Scholar
  48. 48.
    Zhang SL, Li J, Lykotrafitis G, Bao G, Suresh S. Size-dependent endocytosis of nanoparticles. Adv Mater. 2009;21:419–424.Google Scholar
  49. 49.
    Coonrod A, Li FQ, Horwitz M. On the mechanism of DNA transfection: efficient gene transfer without viruses. Gene Ther. 1997;4:1313–21.CrossRefPubMedGoogle Scholar
  50. 50.
    Greitz D, Hannerz J. A proposed model of cerebrospinal fluid circulation: observations with radionuclide cisternography. Am J Neuroradiol. 1996;17:431–8.PubMedGoogle Scholar
  51. 51.
    Stacey KJ, Sweet MJ, Hume DA. Macrophages ingest and are activated by bacterial DNA. J Immunol. 1996;157:2116–22.PubMedGoogle Scholar
  52. 52.
    Fukuhara Y, Naoi T, Ogawa Y, Nishikawa M, Takakura Y. Plasmid DNA uptake and subsequent cellular activation characteristics in human monocyte-derived cells in primary culture. J Pharm Sci. 2007;96:1576–84.CrossRefPubMedGoogle Scholar
  53. 53.
    Pratten MK, Lloyd JB. Pinocytosis and phagocytosis - the effect of size of a particulate substrate on its mode of capture by rat peritoneal macrophages culture in vitro. Biochim Biophys Acta. 1986;881:307–13.PubMedGoogle Scholar
  54. 54.
    Cooper PH, Mayer P, Baggiolini M. Stimulation of phagocytosis in bone marrow-derived mouse macrophages by bacterial lipopolysaccharide - correlation with biochemical and functional parameters. J Immunol. 1984;133:913–22.PubMedGoogle Scholar
  55. 55.
    Collins HL, Bancroft GJ. Cytokine enhancement of complement-dependent phagocytosis by macrophages - synergy of tumor-necrosis-factor-alpha and granulocyte-macrophage colony-stimulating factor for phagocytosis of cryptococcus-neoformans. Eur J Immunol. 1992;22:1447–54.CrossRefPubMedGoogle Scholar
  56. 56.
    Hasegawa T, Hirota K, Tomoda K, Ito F, Inagawa H, Kochi C, et al. Phagocytic activity of alveolar macrophages toward polystyrene latex microspheres and PLGA microspheres loaded with anti-tuberculosis agent. Colloids Surf B-Biointerfaces. 2007;60:221–8.CrossRefPubMedGoogle Scholar
  57. 57.
    Anderson JM, Shive MS. Biodegradation and biocompatibility of PLA and PLGA microspheres. Adv Drug Deliv Rev. 1997;28:5–24.CrossRefPubMedGoogle Scholar
  58. 58.
    Samuvel DJ, Sundararaj KP, Nareika A, Lopes-Virella MF, Huang Y. Lactate boosts TLR4 signaling and NF-kappa B pathway-mediated gene transcription in macrophages via monocarboxylate transporters and MD-2 Up-regulation. J Immunol. 2009;182:2476–84.CrossRefPubMedGoogle Scholar
  59. 59.
    Zolnik BS, Burgess DJ. Evaluation of in vivo-in vitro release of dexamethasone from PLGA microspheres. J Control Release. 2008;127:137–45.CrossRefPubMedGoogle Scholar
  60. 60.
    Tousignant JD, Zhao HM, Yew NS, Cheng SH, Eastman SJ, Scheule RK. DNA sequences in cationic lipid: pDNA-mediated systemic toxicities. Hum Gene Ther. 2003;14:203–14.CrossRefPubMedGoogle Scholar
  61. 61.
    Yasuda K, Wagner H, Takakura Y. Role of immunostimulatory DNA and TLR9 in gene therapy. Crit Rev Ther Drug Carr Syst. 2006;23:89–109.Google Scholar
  62. 62.
    Zielhuis SW, Nijsen JFW, Seppenwoolde JH, Bakker CJG, Krijger GC, Dullens HFJ, et al. Long-term toxicity of holmium-loaded poly(L-lactic acid) microspheres in rats. Biomaterials. 2007;28:4591–9.CrossRefPubMedGoogle Scholar
  63. 63.
    Koval M, Preiter K, Adles C, Stahl PD, Steinberg TH. Size of IgG-opsonized particles determines macrophage response during internalization. Exp Cell Res. 1998;242:265–73.CrossRefPubMedGoogle Scholar
  64. 64.
    Grosse S, Tremeau-Bravard A, Aron Y, Briand P, Fajac I. Intracellular rate-limiting steps of gene transfer using glycosylated polylysines in cystic fibrosis airway epithelial cells. Gene Ther. 2002;9:1000–7.CrossRefPubMedGoogle Scholar
  65. 65.
    Wang DQ, Robinson DR, Kwon GS, Samuel J. Encapsulation of plasmid DNA in biodegradable poly(D, L-lactic-co-glycolic acid) microspheres as a novel approach for immunogene delivery. J Control Release. 1999;57:9–18.CrossRefPubMedGoogle Scholar
  66. 66.
    Cohen H, Levy RJ, Gao J, Fishbein I, Kousaev V, Sosnowski S, et al. Sustained delivery and expression of DNA encapsulated in polymeric nanoparticles. Gene Ther. 2000;7:1896–905.CrossRefPubMedGoogle Scholar
  67. 67.
    Houk BE, Martin R, Hochhaus G, Hughes JA. Pharmacokinetics of plasmid DNA in the rat. Pharm Res. 2001;18:67–74.CrossRefPubMedGoogle Scholar
  68. 68.
    Martinez FO, Sica A, Mantovani A, Locati M. Macrophage activation and polarization. Front Biosci. 2008;13:453–61.CrossRefPubMedGoogle Scholar
  69. 69.
    Ma J, Chen T, Mandelin J, Ceponis A, Miller NE, Hukkanen M, et al. Regulation of macrophage activation. Cell Mol Life Sci. 2003;60:2334–46.CrossRefPubMedGoogle Scholar
  70. 70.
    Gordon S. Alternative activation of macrophages. Nat Rev Immunol. 2003;3:23–35.CrossRefPubMedGoogle Scholar
  71. 71.
    Polfliet MMJ, Fabriek BO, Daniels WP, Dijkstra CD, van den Berg TK. The rat macrophage scavenger receptor CD163: Expression, regulation and role in inflammatory mediator production. Immunobiology. 2006;211:419–25.CrossRefPubMedGoogle Scholar
  72. 72.
    Pinheiro ED, Antunes OAC, Fortunak JMD. A survey of the syntheses of active pharmaceutical ingredients for antiretroviral drug combinations critical to access in emerging nations. Antivir Res. 2008;79:143–65.CrossRefGoogle Scholar
  73. 73.
    de Lissovoy G, Matza LS, Green H, Werner M, Edgar T. Cost-effectiveness of intrathecal baclofen therapy for the treatment of severe spasticity associated with cerebral palsy. J Child Neurol. 2007;22:49–59.CrossRefPubMedGoogle Scholar
  74. 74.
    Plassat R, Verbe BP, Menei P, Menegalli D, Mathe JF, Richard I. Treatment of spasticity with intrathecal baclofen administration: long-term follow-up, review of 40 patients. Spinal Cord. 2004;42:686–93.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Ryan Gene Soderquist
    • 1
  • Evan M. Sloane
    • 2
  • Lisa C. Loram
    • 2
  • Jacqueline A. Harrison
    • 2
  • Ellen C. Dengler
    • 3
  • Scott M. Johnson
    • 1
  • Luke D. Amer
    • 1
  • Courtney S. Young
    • 1
  • Makenzie T. Lewis
    • 2
  • Stephen Poole
    • 4
  • Matthew G. Frank
    • 2
  • Linda R. Watkins
    • 2
  • Erin D. Milligan
    • 3
  • Melissa J. Mahoney
    • 1
    Email author
  1. 1.Department of Chemical & Biological EngineeringUniversity of Colorado at BoulderBoulderUSA
  2. 2.Department of Psychology and Neuroscience, & the Center for NeuroscienceUniversity of Colorado at BoulderBoulderUSA
  3. 3.Department of NeurosciencesUniversity of New Mexico School of MedicineAlbuquerqueUSA
  4. 4.Division of EndocrinologyNational Institute for Biological Standards and ControlHertsUK

Personalised recommendations