PH-responsive nanoparticle vaccines for dual-delivery of antigens and immunostimulatory oligonucleotides

ACS Nano

Volumn 7, Issue 5, 2013, Pages 3912-3925

  Wilson, John T ^a   Keller, Salka ^a   Manganiello, Matthew J ^a   Cheng, Connie ^a   Lee, Chen Chang ^a   Opara, Chinonso ^a   Convertine, Anthony ^a   Stayton, Patrick S ^a  

^a University of Washington   (United States)

Author keywords

antigen cross presentation; CpG adjuvant; endosomal escape; micelle; pH responsive polymer; vaccine

Indexed keywords

AMPHIPHILIC DIBLOCK COPOLYMERS; CPG ADJUVANT; ENDOSOMAL ESCAPES; HUMORAL IMMUNE RESPONSE; MICELLAR NANOPARTICLES; PH-RESPONSIVE POLYMER; POLYMER NANOPARTICLES; POLYMERIC NANOPARTICLES;

ANTIGENS; BLOCK COPOLYMERS; IMMUNE SYSTEM; IMMUNIZATION; MAMMALS; MICELLES; NANOPARTICLES; OLIGONUCLEOTIDES; POLYMERS; PROTEINS; RNA; VACCINES;

T-CELLS;

ANTIBODY; ANTIGEN; DRUG CARRIER; HLA ANTIGEN; IMMUNOLOGICAL ADJUVANT; NANOPARTICLE; OLIGONUCLEOTIDE; VACCINE;

ANIMAL; ANTIGEN PRESENTATION; ARTICLE; CD4+ T LYMPHOCYTE; CD8+ T LYMPHOCYTE; CHEMISTRY; CPG ISLAND; CYTOSOL; DRUG EFFECT; ENDOSOME; FEMALE; GENETICS; IMMUNOLOGY; METABOLISM; MOUSE; PH; TRANSPORT AT THE CELLULAR LEVEL;

ADJUVANTS, IMMUNOLOGIC; ANIMALS; ANTIBODIES; ANTIGEN PRESENTATION; ANTIGENS; BIOLOGICAL TRANSPORT; CD4-POSITIVE T-LYMPHOCYTES; CD8-POSITIVE T-LYMPHOCYTES; CPG ISLANDS; CYTOSOL; DRUG CARRIERS; ENDOSOMES; FEMALE; HLA ANTIGENS; HYDROGEN-ION CONCENTRATION; MICE; NANOPARTICLES; OLIGONUCLEOTIDES; VACCINES;

EID: 84878276759     PISSN: 19360851     EISSN: 1936086X     Source Type: Journal    
DOI: 10.1021/nn305466z     Document Type: Article

References (82)

1. Black, M.; Trent, A.; Tirrell, M.; Olive, C. Advances in the Design and Delivery of Peptide Subunit Vaccines with a Focus on Toll-like Receptor Agonists Expert Rev. Vaccines 2010, 9, 157-173
2. Perrie, Y.; Mohammed, A. R.; Kirby, D. J.; McNeil, S. E.; Bramwell, V. W. Vaccine Adjuvant Systems: Enhancing the Efficacy of Sub-Unit Protein Antigens Int. J. Pharm. 2008, 364, 272-280
3. Moon, J. J.; Huang, B.; Irvine, D. J. Engineering Nano- and Microparticles To Tune Immunity Adv. Mater. 2012, 24, 3724-3746
4. Ahmed, S. S.; Plotkin, S. A.; Black, S.; Coffman, R. L. Assessing the Safety of Adjuvanted Vaccines Sci. Transl. Med. 2011, 3, 93rv2
5. Hubbell, J. A.; Thomas, S. N.; Swartz, M. A. Materials Engineering for Immunomodulation Nature 2009, 462, 449-460
6. De Temmerman, M.-L.; Rejman, J.; Demeester, J.; Irvine, D. J.; Gander, B.; De Smedt, S. C. Particulate Vaccines: On the Quest for Optimal Delivery and Immune Response Drug Discovery Today 2011, 16, 569-582
7. Foged, C.; Hansen, J.; Agger, E. M. License to Kill: Formulation Requirements for Optimal Priming of CD8(+) CTL Responses with Particulate Vaccine Delivery Systems Eur. J. Pharm. Sci. 2012, 45, 482-491
8. Yewdell, J. W. Designing CD8+ T Cell Vaccines: It's Not Rocket Science (Yet) Curr. Opin. Immunol. 2010, 22, 402-410
9. Amigorena, S.; Savina, A. Intracellular Mechanisms of Antigen Cross Presentation in Dendritic Cells Curr. Opin. Immunol. 2010, 22, 109-117
10. Zhang, N.; Bevan, M. J. CD8(+) T Cells: Foot Soldiers of the Immune System Immunity 2011, 35, 161-168
11. Liu, M. A. Immunologic Basis of Vaccine Vectors Immunity 2010, 33, 504-515
12. Moon, J. J.; Suh, H.; Bershteyn, A.; Stephan, M. T.; Liu, H.; Huang, B.; Sohail, M.; Luo, S.; Ho, U. S.; Khant, H. et al. Interbilayer-Crosslinked Multilamellar Vesicles as Synthetic Vaccines for Potent Humoral and Cellular Immune Responses Nat. Mater. 2011, 10, 243-251
13. Reddy, S. T.; van der Vlies, A. J.; Simeoni, E.; Angeli, V.; Randolph, G. J.; O'Neil, C. P.; Lee, L. K.; Swartz, M. A.; Hubbell, J. A. Exploiting Lymphatic Transport and Complement Activation in Nanoparticle Vaccines Nat. Biotechnol. 2007, 25, 1159-1164
14. Nembrini, C.; Stano, A.; Dane, K. Y.; Ballester, M.; van der Vlies, A. J.; Marsland, B. J.; Swartz, M. A.; Hubbell, J. A. Nanoparticle Conjugation of Antigen Enhances Cytotoxic T-Cell Responses in Pulmonary Vaccination Proc. Natl. Acad. Sci. U.S.A. 2011, 108, E989-E997
15. Kwon, Y. J.; James, E.; Shastri, N.; Fréchet, J. M. J. In Vivo Targeting of Dendritic Cells for Activation of Cellular Immunity Using Vaccine Carriers Based on pH-Responsive Microparticles Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 18264-18268
16. Kasturi, S. P.; Skountzou, I.; Albrecht, R. A.; Koutsonanos, D.; Hua, T.; Nakaya, H. I.; Ravindran, R.; Stewart, S.; Alam, M.; Kwissa, M. et al. Programming the Magnitude and Persistence of Antibody Responses with Innate Immunity Nature 2011, 470, 543-547
17. Lee, I.-H.; Kwon, H.-K.; An, S.; Kim, D.; Kim, S.; Yu, M. K.; Lee, J.-H.; Lee, T.-S.; Im, S.-H.; Jon, S. Imageable Antigen-Presenting Gold Nanoparticle Vaccines for Effective Cancer Immunotherapy in Vivo Angew. Chem., Int. Ed. 2012, 51, 8800-8805
18. Scott, E. A.; Stano, A.; Gillard, M.; Maio-Liu, A. C.; Swartz, M. A.; Hubbell, J. A. Dendritic Cell Activation and T Cell Priming with Adjuvant- and Antigen-Loaded Oxidation-Sensitive Polymersomes Biomaterials 2012, 33, 6211-6219
19. Andrews, C. D.; Huh, M.-S.; Patton, K.; Higgins, D.; Van Nest, G.; Ott, G.; Lee, K.-D. Encapsulating Immunostimulatory CpG Oligonucleotides in Listeriolysin O-Liposomes Promotes a Th1-Type Response and CTL Activity Mol. Pharmaceutics 2012, 9, 1118-1125
20. Beaudette, T. T.; Bachelder, E. M.; Cohen, J. A.; Obermeyer, A. C.; Broaders, K. E.; Fréchet, J. M. J.; Kang, E.-S.; Mende, I.; Tseng, W. W.; Davidson, M. G. et al. In Vivo Studies on the Effect of Co-encapsulation of CpG DNA and Antigen in Acid-Degradable Microparticle Vaccines Mol. Pharmaceutics 2009, 6, 1160-1169
21. Schlosser, E.; Mueller, M.; Fischer, S.; Basta, S.; Busch, D. H.; Gander, B.; Groettrup, M. TLR Ligands and Antigen Need To Be Coencapsulated Into the Same Biodegradable Microsphere for the Generation of Potent Cytotoxic T Lymphocyte Responses Vaccine 2008, 26, 1626-1637
22. Gruenberg, J.; van der Goot, F. G. Mechanisms of Pathogen Entry through the Endosomal Compartments Nat. Rev. Mol. Cell Biol. 2006, 7, 495-504
23. Cho, Y. W.; Kim, J.-D.; Park, K. Polycation Gene Delivery Systems: Escape from Endosomes to Cytosol J. Pharm. Pharmacol. 2003, 55, 721-734
24. Flanary, S.; Hoffman, A. S.; Stayton, P. S. Antigen Delivery with Poly(propylacrylic acid) Conjugation Enhances MHC-1 Presentation and T-Cell Activation Bioconjugate Chem. 2009, 20, 241-248
25. Foster, S.; Duvall, C. L.; Crownover, E. F.; Hoffman, A. S.; Stayton, P. S. Intracellular Delivery of a Protein Antigen with an Endosomal-Releasing Polymer Enhances CD8 T-Cell Production and Prophylactic Vaccine Efficacy Bioconjugate Chem. 2010, 21, 2205-2212
26. Convertine, A. J.; Benoit, D. S. W.; Duvall, C. L.; Hoffman, A. S.; Stayton, P. S. Development of a Novel Endosomolytic Diblock Copolymer for siRNA Delivery J. Controlled Release 2009, 133, 221-229
27. Convertine, A. J.; Diab, C.; Prieve, M.; Paschal, A.; Hoffman, A. S.; Johnson, P. H.; Stayton, P. S. pH-Responsive Polymeric Micelle Carriers for siRNA Drugs Biomacromolecules 2010, 11, 2904-2910
28. Bode, C.; Zhao, G.; Steinhagen, F.; Kinjo, T.; Klinman, D. M. CpG DNA as a Vaccine Adjuvant Expert Rev. Vaccines 2011, 10, 499-511
29. Krieg, A. M. Therapeutic Potential of Toll-like Receptor 9 Activation Nat. Rev. Drug Discovery 2006, 5, 471-484
30. Szablan, Z.; Toy, A. A.; Terrenoire, A.; Davis, T. P.; Stenzel, M. H.; Müller, A. H. E.; Barner-Kowollik, C. Living Free-Radical Polymerization of Sterically Hindered Monomers: Improving the Understanding of 1,1-Disubstituted Monomer Systems J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 3692-3710
31. Szablan, Z.; Toy, A. A.; Davis, T. P.; Hao, X.; Stenzel, M. H.; Barner-Kowollik, C. Reversible Addition Fragmentation Chain Transfer Polymerization of Sterically Hindered Monomers: Toward Well-Defined Rod/Coil Architectures J. Polym. Sci., Part A: Polym. Chem. 2004, 42, 2432-2443
32. Bioley, G.; Lassus, A.; Bussat, P.; Terrettaz, J.; Tranquart, F.; Corthésy, B. Gas-Filled Microbubble-Mediated Delivery of Antigen and the Induction of Immune Responses Biomaterials 2012, 33, 5935-5946
33. Fothergill, L. A.; Fothergill, J. E. Thiol and Disulphide Contents of Hen Ovalbumin. C-Terminal Sequence and Location of Disulphide Bond Biochem. J. 1970, 116, 555-561
34. Schafer, F. Q.; Buettner, G. R. Redox Environment of the Cell As Viewed through the Redox State of the Glutathione Disulfide/Glutathione Couple Free Radical Biol. Med. 2001, 30, 1191-1212
35. Hirosue, S.; Kourtis, I. C.; van der Vlies, A. J.; Hubbell, J. A.; Swartz, M. A. Antigen Delivery to Dendritic Cells by Poly(propylene sulfide) Nanoparticles with Disulfide Conjugated Peptides: Cross-Presentation and T Cell Activation Vaccine 2010, 28, 7897-7906
36. Wendorf, J.; Singh, M.; Chesko, J.; Kazzaz, J.; Soewanan, E.; Ugozzoli, M.; O'Hagan, D. A Practical Approach to the Use of Nanoparticles for Vaccine Delivery J. Pharm. Sci. 2006, 95, 2738-2750
37. Demento, S. L.; Siefert, A. L.; Bandyopadhyay, A.; Sharp, F. A.; Fahmy, T. M. Pathogen-Associated Molecular Patterns on Biomaterials: A Paradigm for Engineering New Vaccines Trends Biotechnol. 2011, 29, 294-306
38. Krishnamachari, Y.; Salem, A. K. Innovative Strategies for Co-delivering Antigens and CpG Oligonucleotides Adv. Drug Delivery Rev. 2009, 61, 205-217
39. Shen, Z.; Reznikoff, G.; Dranoff, G.; Rock, K. L. Cloned Dendritic Cells Can Present Exogenous Antigens on Both MHC Class I and Class II Molecules J. Immunol. 1997, 158, 2723-2730
40. West, M. A.; Bretscher, M. S.; Watts, C. Distinct Endocytotic Pathways in Epidermal Growth Factor-Stimulated Human Carcinoma A431 Cells J. Cell Biol. 1989, 109, 2731-2739
41. Aoki, T. Tyrosine Phosphorylation of Caveolin-1 in the Endothelium Exp. Cell Res. 1999, 253, 629-636
42. Wang, L. H.; Rothberg, K. G.; Anderson, R. G. Mis-assembly of Clathrin Lattices on Endosomes Reveals a Regulatory Switch for Coated Pit Formation J. Cell Biol. 1993, 123, 1107-1117
43. Sallusto, F.; Cella, M.; Danieli, C.; Lanzavecchia, A. Dendritic Cells Use Macropinocytosis and the Mannose Receptor To Concentrate Macromolecules in the Major Histocompatibility Complex Class II Compartment: Downregulation by Cytokines and Bacterial Products J. Exp. Med. 1995, 182, 389-400
44. Sandgren, K. J.; Wilkinson, J.; Miranda-Saksena, M.; McInerney, G. M.; Byth-Wilson, K.; Robinson, P. J.; Cunningham, A. L. A Differential Role for Macropinocytosis in Mediating Entry of the Two Forms of Vaccinia Virus Into Dendritic Cells PLoS Pathog. 2010, 6, e1000866
45. Burgdorf, S.; Kurts, C. Endocytosis Mechanisms and the Cell Biology of Antigen Presentation Curr. Opin. Immunol. 2008, 20, 89-95
46. Akinc, A.; Langer, R. Measuring the pH Environment of DNA Delivered Using Nonviral Vectors: Implications for Lysosomal Trafficking Biotechnol. Bioeng. 2002, 78, 503-508
47. Accapezzato, D. Chloroquine Enhances Human CD8+ T Cell Responses Against Soluble Antigens in Vivo J. Exp. Med. 2005, 202, 817-828
48. Martin, R. M.; Brady, J. L.; Lew, A. M. The Need for IgG2c Specific Antiserum When Isotyping Antibodies from C57BL/6 and NOD Mice J. Immunol. Methods 1998, 212, 187-192
49. Maubant, S.; Banissi, C.; Beck, S.; Chauvat, A.; Carpentier, A. F. Adjuvant Properties of Cytosine-Phosphate-Guanosine Oligodeoxynucleotide in Combination with Various Polycations in an Ovalbumin-Vaccine Model Nucleic Acid Ther. 2011, 21, 231-240
50. Lührs, P.; Schmidt, W.; Kutil, R.; Buschle, M.; Wagner, S. N.; Stingl, G.; Schneeberger, A. Induction of Specific Immune Responses by Polycation-Based Vaccines J. Immunol. 2002, 169, 5217-5226
51. Matzelle, M. M.; Babensee, J. E. Humoral Immune Responses to Model Antigen Co-delivered with Biomaterials Used in Tissue Engineering Biomaterials 2004, 25, 295-304
52. Wegmann, F.; Gartlan, K. H.; Harandi, A. M.; Brinckmann, S. A.; Coccia, M.; Hillson, W. R.; Kok, W. L.; Cole, S.; Ho, L.-P.; Lambe, T. et al. Polyethyleneimine Is a Potent Mucosal Adjuvant for Viral Glycoprotein Antigens Nat. Biotechnol. 2012, 30, 883-888
53. Amorij, J.-P.; Kersten, G. F. A.; Saluja, V.; Tonnis, W. F.; Hinrichs, W. L. J.; Slütter, B.; Bal, S. M.; Bouwstra, J. A.; Huckriede, A.; Jiskoot, W. Towards Tailored Vaccine Delivery: Needs, Challenges and Perspectives J. Controlled Release 2012, 161, 363-376
54. Reed, S. G.; Bertholet, S.; Coler, R. N.; Friede, M. New Horizons in Adjuvants for Vaccine Development Trends Immunol. 2009, 30, 23-32
55. Vollmer, J.; Krieg, A. M. Immunotherapeutic Applications of CpG Oligodeoxynucleotide TLR9 Agonists Adv. Drug Delivery Rev. 2009, 61, 195-204
56. Sablan, B. P.; Kim, D. J.; Barzaga, N. G.; Chow, W. C.; Cho, M.; Ahn, S. H.; Hwang, S. G.; Lee, J. H.; Namini, H.; Heyward, W. L. Demonstration of Safety and Enhanced Seroprotection Against Hepatitis B with Investigational HBsAg-1018 ISS Vaccine Compared to a Licensed Hepatitis B Vaccine Vaccine 2012, 30, 2689-2696
57. Nierkens, S.; den Brok, M. H.; Sutmuller, R. P. M.; Grauer, O. M.; Bennink, E.; Morgan, M. E.; Figdor, C. G.; Ruers, T. J. M.; Adema, G. J. In Vivo Colocalization of Antigen and CpG within Dendritic Cells Is Associated with the Efficacy of Cancer Immunotherapy Cancer Res. 2008, 68, 5390-5396
58. Cho, H. J.; Takabayashi, K.; Cheng, P. M.; Nguyen, M. D.; Corr, M.; Tuck, S.; Raz, E. Immunostimulatory DNA-Based Vaccines Induce Cytotoxic Lymphocyte Activity by a T-Helper Cell-Independent Mechanism Nat. Biotechnol. 2000, 18, 509-514
59. Lee, J.; Sohn, J. W.; Zhang, Y.; Leong, K. W.; Pisetsky, D.; Sullenger, B. A. Nucleic Acid-Binding Polymers as Anti-inflammatory Agents Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 14055-14060
60. Chen, H. C.; Sun, B.; Tran, K. K.; Shen, H. Effects of Particle Size on Toll-like Receptor 9-Mediated Cytokine Profiles Biomaterials 2011, 32, 1731-1737
61. Honda, K.; Ohba, Y.; Yanai, H.; Negishi, H.; Mizutani, T.; Takaoka, A.; Taya, C.; Taniguchi, T. Spatiotemporal Regulation of MyD88-IRF-7 Signalling for Robust Type-I Interferon Induction Nature 2005, 434, 1035-1040
62. Paludan, C.; Schmid, D.; Landthaler, M.; Vockerodt, M.; Kube, D.; Tuschl, T.; Münz, C. Endogenous MHC Class II Processing of a Viral Nuclear Antigen After Autophagy Science 2005, 307, 593-596
63. Schmid, D.; Pypaert, M.; Münz, C. Antigen-Loading Compartments for Major Histocompatibility Complex Class II Molecules Continuously Receive Input from Autophagosomes Immunity 2007, 26, 79-92
64. Dengjel, J.; Schoor, O.; Fischer, R.; Reich, M.; Kraus, M.; Müller, M.; Kreymborg, K.; Altenberend, F.; Brandenburg, J.; Kalbacher, H. et al. Autophagy Promotes MHC Class II Presentation of Peptides from Intracellular Source Proteins Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 7922-7927
65. Lee, H. K.; Lund, J. M.; Ramanathan, B.; Mizushima, N.; Iwasaki, A. Autophagy-Dependent Viral Recognition by Plasmacytoid Dendritic Cells Science 2007, 315, 1398-1401
66. Bertin, S.; Pierrefite-Carle, V. Autophagy and Toll-like Receptors: A New Link in Cancer Cells Autophagy 2008, 4, 1086-1089
67. Combadiere, B.; Liard, C. Transcutaneous and Intradermal Vaccination Hum. Vaccines 2011, 7, 811-827
68. Romani, N.; Flacher, V.; Tripp, C. H.; Sparber, F.; Ebner, S.; Stoitzner, P. Targeting Skin Dendritic Cells To Improve Intradermal Vaccination Curr. Top. Microbiol. Immunol. 2012, 351, 113-138
69. Mohanan, D.; Slütter, B.; Henriksen-Lacey, M.; Jiskoot, W.; Bouwstra, J. A.; Perrie, Y.; Kündig, T. M.; Gander, B.; Johansen, P. Administration Routes Affect the Quality of Immune Responses: A Cross-Sectional Evaluation of Particulate Antigen-Delivery Systems J. Controlled Release 2010, 147, 342-349
70. Eby, J. K.; Dane, K. Y.; O'Neil, C. P.; Hirosue, S.; Swartz, M. A.; Hubbell, J. A. Polymer Micelles with Pyridyl Disulfide-Coupled Antigen Travel through Lymphatics and Show Enhanced Cellular Responses Following Immunization Acta Biomater. 2012, 8, 3210-3217
71. Sheng, K.-C.; Kalkanidis, M.; Pouniotis, D. S.; Esparon, S.; Tang, C. K.; Apostolopoulos, V.; Pietersz, G. A. Delivery of Antigen Using a Novel Mannosylated Dendrimer Potentiates Immunogenicity in Vitro and in Vivo Eur. J. Immunol. 2008, 38, 424-436
72. Kar, U. K.; Jiang, J.; Champion, C. I.; Salehi, S.; Srivastava, M.; Sharma, S.; Rabizadeh, S.; Niazi, K.; Kickhoefer, V.; Rome, L. H. et al. Vault Nanocapsules as Adjuvants Favor Cell-Mediated over Antibody-Mediated Immune Responses Following Immunization of Mice PLoS One 2012, 7, e38553
73. Bulmus, V.; Woodward, M.; Lin, L.; Murthy, N.; Stayton, P.; Hoffman, A. A New pH-Responsive and Glutathione-Reactive, Endosomal Membrane-Disruptive Polymeric Carrier for Intracellular Delivery of Biomolecular Drugs J. Controlled Release 2003, 93, 105-120
74. Murthy, N.; Campbell, J.; Fausto, N.; Hoffman, A. S.; Stayton, P. S. Bioinspired pH-Responsive Polymers for the Intracellular Delivery of Biomolecular Drugs Bioconjugate Chem. 2003, 14, 412-419
75. Broaders, K. E.; Cohen, J. A.; Beaudette, T. T.; Bachelder, E. M.; Fréchet, J. M. J. Acetalated Dextran Is a Chemically and Biologically Tunable Material for Particulate Immunotherapy Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 5497-5502
76. Karttunen, J.; Sanderson, S.; Shastri, N. Detection of Rare Antigen-Presenting Cells by the LacZ T-Cell Activation Assay Suggests an Expression Cloning Strategy for T-Cell Antigens Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 620-624
77. Belizaire, R.; Unanue, E. R. Targeting Proteins to Distinct Subcellular Compartments Reveals Unique Requirements for MHC Class I and II Presentation Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 17463-17468
78. Schwarz, K.; de Giuli, R.; Schmidtke, G.; Kostka, S.; van den Broek, M.; Kim, K. B.; Crews, C. M.; Kraft, R.; Groettrup, M. The Selective Proteasome Inhibitors Lactacystin and Epoxomicin Can Be Used To Either Up- or Down-Regulate Antigen Presentation at Nontoxic Doses J. Immunol. 2000, 164, 6147-6157
79. Gerelchuluun, T.; Lee, Y.-H.; Lee, Y.-R.; Im, S.-A.; Song, S.; Park, J. S.; Han, K.; Kim, K.; Lee, C.-K. Dendritic Cells Process Antigens Encapsulated in a Biodegradable Polymer, Poly(d, l -lactide- co -glycolide), via an Alternate Class I MHC Processing Pathway Arch. Pharmacol. Res. 2007, 30, 1440-1446
80. Murthy, N.; Robichaud, J. R.; Tirrell, D. A.; Stayton, P. S.; Hoffman, A. S. The Design and Synthesis of Polymers for Eukaryotic Membrane Disruption J. Controlled Release 1999, 61, 137-143
81. De Koker, S.; De Geest, B. G.; Singh, S. K.; De Rycke, R.; Naessens, T.; van Kooyk, Y.; Demeester, J.; De Smedt, S. C.; Grooten, J. Polyelectrolyte Microcapsules as Antigen Delivery Vehicles to Dendritic Cells: Uptake, Processing, and Cross-Presentation of Encapsulated Antigens Angew. Chem., Int. Ed. 2009, 48, 8485-8489
82. Brito, L. A.; Singh, M. Acceptable Levels of Endotoxin in Vaccine Formulations During Preclinical Research J. Pharm. Sci. 2010, 100, 34-37