將抗原以奈米粒子的形式包覆傳遞，可增加其被免疫細胞胞吞的機會，以提升疫苗的免疫效力。本研究以具佐劑特性的幾丁聚醣及聚麩胺酸為奈米粒子之材料，透過於弱酸中帶NH3+基團的幾丁聚醣與本身帶COO基團的聚麩胺酸，將抗原包覆並形成離子性奈米粒子，藉由改變材料的組成比例，調控粒徑大小及粒子表面帶電荷。為探討粒子表面電荷性對抗原免疫效力之影響，本研究分別製備出粒徑約為200奈米，表面帶有相反電荷的兩組奈米粒子(35.9 ± 3.2 及36.9 ± 4.6 mV)。經細胞實驗證實，此兩組幾丁聚醣/聚麩胺酸奈米微粒並沒有明顯的細胞毒性，且不論是表面帶正電或負電荷的奈米粒子，皆能促進巨噬細胞(RAW 264.7)吞噬抗原(Ovalbumin; OVA)的能力。以皮下注射方式將包覆OVA之奈米粒子打入Sprague Dawley大鼠體內，帶正電荷的粒子，在第二週即可產生較單純施打OVA的組別明顯更高的IgG抗體量，且持續長達十六週。然而，帶負電荷粒子的組別，對刺激抗體的產生卻完全沒有幫助。為評估微針經皮傳輸是否能更進一步促進免疫效力，利用包覆正電荷粒子的快溶型玻尿酸微針進行免疫試驗，結果顯示，將奈米粒子包覆在微針中恐喪失OVA之抗原性，推測是與微針製程中抗原本身嚴重聚集或失活有關。

Using nanocarriers to deliver antigens may enhance the immune responses to antigens by stimulating endocytosis into immune cells. Because of their adjuvant properties, we utilized ionic-gelation method to prepare a polyelectrolyte complex (PEC) to encapsulate antigens and for antigen delivery. This PEC comprises chitosan with NH3+ group as the cationic polyelectrolyte and poly-γ-glutamic acid (γ-PGA) with COO as the anionic polyelectrolyte. The DLS result indicated that the particle size and the zeta potential value of the prepared nanoparticles can be controlled by changing their constituted compositions. In this study, 2 groups of 200 nm-sized nanoparticle with opposite surface charge (35.9 ± 3.2 and 36.9 ± 4.6 mV) were fabricated for the effect of surface charges of the nanoparticle to the immune responses. The in vitro cellular experiments showed that the chitosan/γ-PGA nanoparticles caused little cytotoxicity to macrophages (Raw 264.7) with increasing concentration and time, and both positive charged and negative charged nanoparticles promoted the endocytosis of Raw 264.7 cells for antigens (Ovalbumin , OVA). We applied OVA-loaded nanoparticles to Sprague Dawley Rats by subcutaneous injection. Compared to free OVA, combination with positive charged nanoparticles can induce a much higher OVA specific antibody response at week 2 and can last until week 16. However, the negative charged nanoparticles did not induce notable immune responses.

[1] Wiechelman KJ, Braun RD, Fitzpatrick JD. Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation. Analytical biochemistry 1988;175:231-237.
[2] Kenyon NJ, Bratt JM, Lee J, Luo J, Franzi LM, Zeki AA, et al. Self-assembling nanoparticles containing dexamethasone as a novel therapy in allergic airways inflammation. PloS one 2013;8:e77730.
[3] Luo L, Qin T, Huang Y, Zheng S, Bo R, Liu Z, et al. Exploring the immunopotentiation of Chinese yam polysaccharide poly (lactic-co-glycolic acid) nanoparticles in an ovalbumin vaccine formulation in vivo. Drug delivery 2017;24:1099-1111.
[4] Park JW, Lee IC, Shin NR, Jeon CM, Kwon OK, Ko JW, et al. Copper oxide nanoparticles aggravate airway inflammation and mucus production in asthmatic mice via MAPK signaling. Nanotoxicology 2016;10:445-452.
[5] Hsiao YP, Shen CC, Huang CH, Lin YC, Jan T-R. Iron oxide nanoparticles attenuate T helper 17 cell responses in vitro and in vivo. International immunopharmacology 2018;58:32-39.
[6] Yang Y-SS, Atukorale PU, Moynihan KD, Bekdemir A, Rakhra K, Tang L, et al. High-throughput quantitation of inorganic nanoparticle biodistribution at the single-cell level using mass cytometry. Nature communications 2017;8:14069.
[7] Omlor AJ, Le DD, Schlicker J, Hannig M, Ewen R, Heck S, et al. Local effects on airway inflammation and systemic uptake of 5 nm PEGylated and citrated gold nanoparticles in asthmatic mice. Small 2017;13:1603070.
[8] Li X, Hufnagel S, Xu H, Valdes SA, Thakkar SG, Cui Z, et al. Aluminum (oxy) hydroxide nanosticks synthesized in bicontinuous reverse microemulsion have potent vaccine adjuvant activity. ACS applied materials & interfaces 2017;9:22893-22901.
[9] Fröhlich E. The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles. International journal of nanomedicine 2012;7:5577.
[10] Kumar R, Ray PC, Datta D, Bansal GP, Angov E, Kumar N. Nanovaccines for malaria using Plasmodium falciparum antigen Pfs25 attached gold nanoparticles. Vaccine 2015;33:5064-5071.
[11] Lundquist CM, Loo C, Meraz IM, Cerda JDL, Liu X, Serda RE. Characterization of free and porous silicon-encapsulated superparamagnetic iron oxide nanoparticles as platforms for the development of theranostic vaccines. Medical Sciences 2014;2:51-69.
[12] Tu J, Du G, Nejadnik MR, Mönkäre J, van der Maaden K, Bomans PH, et al. Mesoporous silica nanoparticle-coated microneedle arrays for intradermal antigen delivery. Pharmaceutical research 2017;34:1693-1706.
[13] Mody KT, Mahony D, Cavallaro AS, Stahr F, Qiao SZ, Mahony TJ, et al. Freeze-drying of ovalbumin loaded mesoporous silica nanoparticle vaccine formulation increases antigen stability under ambient conditions. International journal of pharmaceutics 2014;465:325-332.
[14] Kim KM, Kim HM, Lee WJ, Lee CW, Kim Ti, Lee JK, et al. Surface treatment of silica nanoparticles for stable and charge-controlled colloidal silica. International journal of nanomedicine 2014;9:29.
[15] Han H, Park YH, Park HJ, Lee K, Um K, Park JW, et al. Toxic and adjuvant effects of silica nanoparticles on ovalbumin-induced allergic airway inflammation in mice. Respiratory research 2016;17:60.
[16] Fine JH, Bondy GS, Coady L, Pearce B, Ross N, Tayabali AF, et al. Immunomodulation by gastrointestinal carbon black nanoparticle exposure in ovalbumin T cell receptor transgenic mice. Nanotoxicology 2016;10:1422-1430.
[17] Weilhammer D, Dunkle AD, Blanchette CD, Fischer NO, Corzett M, Lehmann D, et al. Enhancement of antigen-specific CD4+ and CD8+ T cell responses using a self-assembled biologic nanolipoprotein particle vaccine. Vaccine 2017;35:1475-1481.
[18] Swaminathan G, Thoryk EA, Cox KS, Meschino S, Dubey SA, Vora KA, et al. A novel lipid nanoparticle adjuvant significantly enhances B cell and T cell responses to sub-unit vaccine antigens. Vaccine 2016;34:110-119.
[19] Courant T, Bayon E, Reynaud-Dougier HL, Villiers C, Menneteau M, Marche PN, et al. Tailoring nanostructured lipid carriers for the delivery of protein antigens: Physicochemical properties versus immunogenicity studies. Biomaterials 2017;136:29-42.
[20] Xie S, Pan B, Shi B, Zhang Z, Zhang X, Wang M, et al. Solid lipid nanoparticle suspension enhanced the therapeutic efficacy of praziquantel against tapeworm. International journal of nanomedicine 2011;6:2367.
[21] Luo Z, Shi S, Jin L, Xu L, Yu J, Chen H, et al. Cationic micelle based vaccine induced potent humoral immune response through enhancing antigen uptake and formation of germinal center. Colloids and Surfaces B: Biointerfaces 2015;135:556-564.
[22] Salman HH, Gamazo C, de Smidt PC, Russell-Jones G, Irache JM. Evaluation of bioadhesive capacity and immunoadjuvant properties of vitamin B12-Gantrez nanoparticles. Pharmaceutical research 2008;25:2859-2868.
[23] Rietscher R, Czaplewska JA, Majdanski TC, Gottschaldt M, Schubert US, Schneider M, et al. Impact of PEG and PEG-b-PAGE modified PLGA on nanoparticle formation, protein loading and release. International journal of pharmaceutics 2016;500:187-195.
[24] Orozco VH, Palacio J, Sierra J, López BL. Increased covalent conjugation of a model antigen to poly (lactic acid)-g-maleic anhydride nanoparticles compared to bare poly (lactic acid) nanoparticles. Colloid and Polymer Science 2013;291:2775-2781.
[25] Sharpe LA, Vela Ramirez JE, Haddadin OM, Ross KA, Narasimhan B, Peppas NA. pH-Responsive Microencapsulation Systems for the Oral Delivery of Polyanhydride Nanoparticles. Biomacromolecules 2018;19:793-802.
[26] Etorki AM, Gao M, Sadeghi R, Maldonado‐Mejia LF, Kokini JL. Effects of Desolvating Agent Types, Ratios, and Temperature on Size and Nanostructure of Nanoparticles from α‐Lactalbumin and Ovalbumin. Journal of food science 2016;81.
[27] Chang TZ, Stadmiller SS, Staskevicius E, Champion JA. Effects of ovalbumin protein nanoparticle vaccine size and coating on dendritic cell processing. Biomaterials science 2017;5:223-233.
[28] Dombu C, Carpentier R, Betbeder D. Influence of surface charge and inner composition of nanoparticles on intracellular delivery of proteins in airway epithelial cells. Biomaterials 2012;33:9117-9126.
[29] Kulkarni AD, Vanjari YH, Sancheti KH, Patel HM, Belgamwar VS, Surana SJ, et al. Polyelectrolyte complexes: mechanisms, critical experimental aspects, and applications. Artificial cells, nanomedicine, and biotechnology 2016;44:1615-1625.
[30] Cole H, Bryan D, Lancaster L, Mawas F, Vllasaliu D. Chitosan nanoparticle antigen uptake in epithelial monolayers can predict mucosal but not systemic in vivo immune response by oral delivery. Carbohydrate polymers 2018;190:248-254.
[31] Li N, Peng LH, Chen X, Zhang TY, Shao GF, Liang WQ, et al. Antigen-loaded nanocarriers enhance the migration of stimulated Langerhans cells to draining lymph nodes and induce effective transcutaneous immunization. Nanomedicine: Nanotechnology, Biology and Medicine 2014;10:215-223.
[32] Howe SE, Sowa G, Konjufca V. Systemic and Mucosal Antibody Responses to Soluble and Nanoparticle-Conjugated Antigens Administered Intranasally. Antibodies 2016;5:20.
[33] Kulig D, Zimoch-Korzycka A, Jarmoluk A, Marycz K. Study on alginate–chitosan complex formed with different polymers ratio. Polymers 2016;8:167.
[34] Trefalt G, Borkovec M. Overview of DLVO theory. Laboratory of Colloid and Surface Chemistry, University of Genebra 2014.
[35] Hotze EM, Phenrat T, Lowry GV. Nanoparticle Aggregation: Challenges to Understanding Transport and Reactivity in the Environment All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Journal of environmental quality 2010;39:1909-1924.
[36] Bhattacharjee S. DLS and zeta potential–What they are and what they are not? Journal of Controlled Release 2016;235:337-351.
[37] Kouchakzadeh H, Shojaosadati SA, Maghsoudi A, Farahani EV. Optimization of PEGylation conditions for BSA nanoparticles using response surface methodology. Aaps Pharmscitech 2010;11:1206-1211.
[38] Almeida TCA, Larentis AL, Ferraz HC. Evaluation of the stability of concentrated emulsions for lemon beverages using sequential experimental designs. PloS one 2015;10:e0118690.
[39] Philipp B. Poylelectrolyte complexs-recent developments and open problems. Prog Poh,m Sei 1989;14:91-172.
[40] Tsuchida E. Formation of polyelectrolyte complexes and their structures. Journal of Macromolecular Science—Pure and Applied Chemistry 1994;31:1-15.
[41] Pergushov DV, Muller AH, Schacher FH. Micellar interpolyelectrolyte complexes. Chemical Society reviews 2012;41:6888-6901.
[42] Koetz J, and Sabine Kosmella. Polyelectrolytes and nanoparticles. Springer Science & Business Media 2007.
[43] Ernst JD, Stendahl O. Phagocytosis of bacteria and bacterial pathogenicity: Cambridge University Press; 2006.
[44] Couzinet S, Cejas E, Schittny J, Deplazes P, Weber R, Zimmerli S. Phagocytic uptake of Encephalitozoon cuniculi by nonprofessional phagocytes. Infection and immunity 2000;68:6939-6945.
[45] Nel AE, Mädler L, Velegol D, Xia T, Hoek EM, Somasundaran P, et al. Understanding biophysicochemical interactions at the nano–bio interface. Nature materials 2009;8:543.
[46] Van Hong Nguyen B-JL. Protein corona: A new approach for nanomedicine design. International journal of nanomedicine 2017;12:3137.
[47] Oh N, Park J-H. Endocytosis and exocytosis of nanoparticles in mammalian cells. International journal of nanomedicine 2014;9:51.
[48] Gan Q, Wang T, Cochrane C, McCarron P. Modulation of surface charge, particle size and morphological properties of chitosan–TPP nanoparticles intended for gene delivery. Colloids and Surfaces B: Biointerfaces 2005;44:65-73.
[49] Alkilany AM, Murphy CJ. Toxicity and cellular uptake of gold nanoparticles: what we have learned so far? Journal of nanoparticle research 2010;12:2313-2333.
[50] Pegoraro C, MacNeil S, Battaglia G. Transdermal drug delivery: from micro to nano. Nanoscale 2012;4:1881-1894.
[51] MacNeil S. Biomaterials for tissue engineering of skin. Materials Today 2008;11:26-35.
[52] Cevc G, Vierl U. Spatial distribution of cutaneous microvasculature and local drug clearance after drug application on the skin. Journal of controlled release : official journal of the Controlled Release Society 2007;118:18-26.
[53] Loan Honeywell-Nguyen P, Wouter Groenink HW, Bouwstra JA. Elastic vesicles as a tool for dermal and transdermal delivery. Journal of liposome research 2006;16:273-280.
[54] Medzhitov R. Recognition of microorganisms and activation of the immune response. Nature 2007;449:819-826.
[55] Matzinger P. The danger model: a renewed sense of self. Science 2002;296:301-305.
[56] Pancer Z, Cooper MD. The evolution of adaptive immunity. Annual review of immunology 2006;24:497-518.
[57] Harty JT, Tvinnereim AR, White DW. CD8+ T cell effector mechanisms in resistance to infection. Annual review of immunology 2000;18:275-308.
[58] Abbas AK, Murphy KM, Sher A. Functional diversity of helper T lymphocytes. Nature 1996;383:787.
[59] Pulendran B, Ahmed R. From Innate Immunity to Immunological Memory: Springer Science & Business Media; 2006.
[60] Sproul TW, Cheng PC, Dykstra ML, Pierce SK. A role for MHC class II antigen processing in B cell development. International reviews of Immunology 2000;19:139-155.
[61] Wen ZS, Xu YL, Zou XT, Xu ZR. Chitosan nanoparticles act as an adjuvant to promote both Th1 and Th2 immune responses induced by ovalbumin in mice. Marine drugs 2011;9:1038-1055.
[62] Zhao Jh, Zhang QB, Liu B, Piao XH, Yan YL, Hu XG, et al. enhanced immunization via dissolving microneedle array-based delivery system incorporating subunit vaccine and saponin adjuvant. International journal of nanomedicine 2017;12:4763.
[63] Chu LY, Prausnitz MR. Separable arrowhead microneedles. Journal of controlled release : official journal of the Controlled Release Society 2011;149:242-249.
[64] Kim YC, Park JH, Prausnitz MR. Microneedles for drug and vaccine delivery. Advanced drug delivery reviews 2012;64:1547-1568.
[65] Lee JW, Park JH, Prausnitz MR. Dissolving microneedles for transdermal drug delivery. Biomaterials 2008;29:2113-2124.
[66] Kolli CS, Banga AK. Characterization of solid maltose microneedles and their use for transdermal delivery. Pharmaceutical research 2008;25:104-113.
[67] Park JH, Allen MG, Prausnitz MR. Polymer microneedles for controlled-release drug delivery. Pharmaceutical research 2006;23:1008-1019.
[68] Balmayor ER, Azevedo HS, Reis RL. Controlled delivery systems: from pharmaceuticals to cells and genes. Pharmaceutical research 2011;28:1241-1258.
[69] Hiraishi Y, Nakagawa T, Quan YS, Kamiyama F, Hirobe S, Okada N, et al. Performance and characteristics evaluation of a sodium hyaluronate-based microneedle patch for a transcutaneous drug delivery system. Int J Pharm 2013;441:570-579.
[70] Heffernan MJ, Zaharoff DA, Fallon JK, Schlom J, Greiner JW. In vivo efficacy of a chitosan/IL-12 adjuvant system for protein-based vaccines. Biomaterials 2011;32:926-932.
[71] Cetin EO, Gundogdu E, Kirilmaz L. Novel microparticle drug delivery systems based on chitosan and Eudragit® RSPM to enhance diltiazem hydrochloride release property. Pharmaceutical development and technology 2012;17:741-746.
[72] Sasaki S, Okuda K. The use of conventional immunologic adjuvants in DNA vaccine preparations. Methods Mol Med 2000;29:241-249.
[73] Lim C, Lee DW, Israelachvili JN, Jho Y, Hwang DS. Contact time-and pH-dependent adhesion and cohesion of low molecular weight chitosan coated surfaces. Carbohydrate polymers 2015;117:887-894.
[74] Lee DW, Lim C, Israelachvili JN, Hwang DS. Strong adhesion and cohesion of chitosan in aqueous solutions. Langmuir 2013;29:14222-14229.
[75] Manocha B, Margaritis A. Controlled release of doxorubicin from doxorubicin/γ-polyglutamic acid ionic complex. Journal of Nanomaterials 2010;2010:12.
[76] Liao Z-X, Peng S-F, Ho Y-C, Mi F-L, Maiti B, Sung H-W. Mechanistic study of transfection of chitosan/DNA complexes coated by anionic poly (γ-glutamic acid). Biomaterials 2012;33:3306-3315.
[77] Scheibner KA, Lutz MA, Boodoo S, Fenton MJ, Powell JD, Horton MR. Hyaluronan fragments act as an endogenous danger signal by engaging TLR2. The Journal of Immunology 2006;177:1272-1281.
[78] Sæther HV, Holme HK, Maurstad G, Smidsrød O, Stokke BT. Polyelectrolyte complex formation using alginate and chitosan. Carbohydrate Polymers 2008;74:813-821.
[79] Hu Y, Yang T, Hu X. Novel polysaccharides-based nanoparticle carriers prepared by polyelectrolyte complexation for protein drug delivery. Polymer bulletin 2012;68:1183-1199.
[80] Schatz C, Domard A, Viton C, Pichot C, Delair T. Versatile and efficient formation of colloids of biopolymer-based polyelectrolyte complexes. Biomacromolecules 2004;5:1882-1892.
[81] Müller M, Keßler B, Fröhlich J, Poeschla S, Torger B. Polyelectrolyte complex nanoparticles of poly (ethyleneimine) and poly (acrylic acid): preparation and applications. Polymers 2011;3:762-778.
[82] Peng S-F, Yang M-J, Su C-J, Chen H-L, Lee P-W, Wei M-C, et al. Effects of incorporation of poly (γ-glutamic acid) in chitosan/DNA complex nanoparticles on cellular uptake and transfection efficiency. Biomaterials 2009;30:1797-1808.
[83] Sonaje K, Chen Y-J, Chen H-L, Wey S-P, Juang J-H, Nguyen H-N, et al. Enteric-coated capsules filled with freeze-dried chitosan/poly (γ-glutamic acid) nanoparticles for oral insulin delivery. Biomaterials 2010;31:3384-3394.
[84] Wu C, Wu T, Fang Z, Zheng J, Xu S, Chen S, et al. Formation, characterization and release kinetics of chitosan/γ-PGA encapsulated nisin nanoparticles. RSC Advances 2016;6:46686-46695.
[85] Lin YH, Mi FL, Chen CT, Chang WC, Peng SF, Liang HF, et al. Preparation and characterization of nanoparticles shelled with chitosan for oral insulin delivery. Biomacromolecules 2007;8:146-152.
[86] Akagi T, Watanabe K, Kim H, Akashi M. Stabilization of polyion complex nanoparticles composed of poly (amino acid) using hydrophobic interactions. Langmuir 2009;26:2406-2413.
[87] Lin Y-H, Chung CK, Chen CT, Liang HF, Chen SC, Sung HW. Preparation of nanoparticles composed of chitosan/poly-γ-glutamic acid and evaluation of their permeability through Caco-2 cells. Biomacromolecules 2005;6:1104-1112.
[88] Domingos RF, Baalousha MA, Ju-Nam Y, Reid MM, Tufenkji N, Lead JR, et al. Characterizing manufactured nanoparticles in the environment: multimethod determination of particle sizes. Environmental science & technology 2009;43:7277-7284.
[89] Berne B, Pecora R. Dynamical Light Scattering with Reference to Physics, Chemistry and Biology. Wiley/Interscience, New York; 1976.
[90] Sawyer L, Grubb DT, Meyers GF. Polymer microscopy: Springer Science & Business Media; 2008.
[91] Wischke C, Borchert HH. Fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) as a model protein drug: opportunities and drawbacks. Die Pharmazie-An International Journal of Pharmaceutical Sciences 2006;61:770-4.
[92] Verheul RJ, Slütter B, Bal SM, Bouwstra JA, Jiskoot W, Hennink WE. Covalently stabilized trimethyl chitosan-hyaluronic acid nanoparticles for nasal and intradermal vaccination. Journal of controlled release 2011;156:46-52.
[93] Verheul RJ, van der Wal S, Hennink WE. Tailorable thiolated trimethyl chitosans for covalently stabilized nanoparticles. Biomacromolecules 2010;11:1965-1971.
[94] Yoshikawa T, Okada N, Oda A, Matsuo K, Matsuo K, Mukai Y, et al. Development of amphiphilic γ-PGA-nanoparticle based tumor vaccine: potential of the nanoparticulate cytosolic protein delivery carrier. Biochemical and biophysical research communications 2008;366:408-413.
[95] prasanth Koppolu B, Smith SG, Ravindranathan S, Jayanthi S, Kumar TKS, Zaharoff DA. Controlling chitosan-based encapsulation for protein and vaccine delivery. Biomaterials 2014;35:4382-4389.
[96] Bal SM, Hortensius S, Ding Z, Jiskoot W, Bouwstra JA. Co-encapsulation of antigen and Toll-like receptor ligand in cationic liposomes affects the quality of the immune response in mice after intradermal vaccination. Vaccine 2011;29:1045-1052.
[97] Pitaksuteepong T, Davies NM, Tucker IG, Rades T. Factors influencing the entrapment of hydrophilic compounds in nanocapsules prepared by interfacial polymerisation of water-in-oil microemulsions. European Journal of Pharmaceutics and Biopharmaceutics 2002;53:335-342.
[98] Koppolu B, Zaharoff DA. The effect of antigen encapsulation in chitosan particles on uptake, activation and presentation by antigen presenting cells. Biomaterials 2013;34:2359-2369.
[99] Du G, Hathout RM, Nasr M, Nejadnik MR, Tu J, Koning RI, et al. Intradermal vaccination with hollow microneedles: A comparative study of various protein antigen and adjuvant encapsulated nanoparticles. Journal of Controlled Release 2017;266:109-118.
[100] Chen MC, Lai KY, Ling MH, Lin CW. Enhancing immunogenicity of antigens through sustained intradermal delivery using chitosan microneedles with a patch-dissolvable design. Acta biomaterialia 2018;65:66-75.
[101] Chiu YH, Chen MC, Wan SW. Sodium hyaluronate/chitosan composite microneedles as a single-dose intradermal immunization system. Biomacromolecules 2018;19:2278-2285.
[102] Xiao B, Ma P, Ma L, Chen Q, Si X, Walter L, et al. Effects of tripolyphosphate on cellular uptake and RNA interference efficiency of chitosan-based nanoparticles in Raw 264.7 macrophages. Journal of colloid and interface science 2017;490:520-528.
[103] Jia J, Zhang W, Liu Q, Yang T, Wang L, Ma G. Adjuvanticity regulation by biodegradable polymeric nano/microparticle size. Molecular pharmaceutics 2016;14:14-22.
[104] Laroui H, Theiss AL, Yan Y, Dalmasso G, Nguyen HT, Sitaraman SV, et al. Functional TNFα gene silencing mediated by polyethyleneimine/TNFα siRNA nanocomplexes in inflamed colon. Biomaterials 2011;32:1218-1228.
[105] Almalik A, Karimi S, Ouasti S, Donno R, Wandrey C, Day PJ, et al. Hyaluronic acid (HA) presentation as a tool to modulate and control the receptor-mediated uptake of HA-coated nanoparticles. Biomaterials 2013;34:5369-5380.
[106] Liu Q, Zhang C, Zheng X, Shao X, Zhang X, Zhang Q, et al. Preparation and evaluation of antigen/N-trimethylaminoethylmethacrylate chitosan conjugates for nasal immunization. Vaccine 2014;32:2582-2590.
[107] Zhou JC, Yang ZL, Dong W, Tang RJ, Sun LD, Yan CH. Bioimaging and toxicity assessments of near-infrared upconversion luminescent NaYF4:Yb,Tm nanocrystals. Biomaterials 2011;32:9059-9067.
[108] Berridge MV, Herst PM, Tan AS. Tetrazolium dyes as tools in cell biology: New insights into their cellular reduction. Biotechnol Annu Rev 2005;11:127-152.
[109] Jonassen H, Kjøniksen AL, Hiorth M. Stability of chitosan nanoparticles cross-linked with tripolyphosphate. Biomacromolecules 2012;13:3747-3756.
[110] Sedar AW. Electron microscopic demonstration of polysaccharides associated with acid-secreting cells of the stomach after “inert dehydration”. Journal of ultrastructure research 1969;28:112-124.
[111] Cui Z, Mumper RJ. Chitosan-based nanoparticles for topical genetic immunization. Journal of Controlled Release 2001;75:409-419.
[112] He C, Hu Y, Yin L, Tang C, Yin C. Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials 2010;31:3657-3666.