{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,6]],"date-time":"2026-03-06T04:09:40Z","timestamp":1772770180891,"version":"3.50.1"},"reference-count":66,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2021,8,31]],"date-time":"2021-08-31T00:00:00Z","timestamp":1630368000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["UIDB\/00709\/2020"],"award-info":[{"award-number":["UIDB\/00709\/2020"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["2020.10201.BD"],"award-info":[{"award-number":["2020.10201.BD"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["IF\/01459\/2015"],"award-info":[{"award-number":["IF\/01459\/2015"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>In current times, DNA vaccines are seen as a promising approach to treat and prevent diseases, such as virus infections and cancer. Aiming at the production of a functional and effective plasmid DNA (pDNA) delivery system, four chitosan polymers, differing in the molecular weight, were studied using the design of experiments (DoE) tool. These gene delivery systems were formulated by ionotropic gelation and exploring the chitosan and TPP concentrations as DoE inputs to maximize the nanoparticle positive charge and minimize their size and polydispersity index (PDI) as DoE outputs. The obtained linear and quadratic models were statistically significant (p-value &lt; 0.05) and non-significant lack of fit, with suitable coefficient of determination and the respective optimal points successfully validated. Furthermore, morphology, stability and cytotoxicity assays were performed to evaluate the endurance of these systems over time and their further potential for future in vitro studies. The subsequent optimization process was successful achieved for the delivery systems based on the four chitosan polymers, in which the smallest particle size was obtained for the carrier containing the 5 kDa chitosan (~82 nm), while the nanosystem prepared with the high molecular weight (HMW) chitosan displayed the highest zeta potential (~+26.8 mV). Delivery systems were stable in the formulation buffer after a month and did not exhibit toxicity for the cells. In this sense, DoE revealed to be a powerful tool to explore and tailor the characteristics of chitosan\/pDNA nanosystems significantly contributing to unraveling an optimum carrier for advancing the DNA vaccines delivery field.<\/jats:p>","DOI":"10.3390\/pharmaceutics13091369","type":"journal-article","created":{"date-parts":[[2021,8,31]],"date-time":"2021-08-31T08:42:32Z","timestamp":1630399352000},"page":"1369","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":24,"title":["Design of Experiments to Achieve an Efficient Chitosan-Based DNA Vaccine Delivery System"],"prefix":"10.3390","volume":"13","author":[{"given":"Carlos","family":"Rodolfo","sequence":"first","affiliation":[{"name":"CICS-UBI\u2014Health Science Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilh\u00e3, Portugal"}]},{"given":"Dalinda","family":"Eus\u00e9bio","sequence":"additional","affiliation":[{"name":"CICS-UBI\u2014Health Science Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilh\u00e3, Portugal"}]},{"given":"Cathy","family":"Ventura","sequence":"additional","affiliation":[{"name":"CICS-UBI\u2014Health Science Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilh\u00e3, Portugal"}]},{"given":"Renato","family":"Nunes","sequence":"additional","affiliation":[{"name":"CICS-UBI\u2014Health Science Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilh\u00e3, Portugal"}]},{"given":"Helena F.","family":"Florindo","sequence":"additional","affiliation":[{"name":"Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisbon, Portugal"}]},{"given":"Diana","family":"Costa","sequence":"additional","affiliation":[{"name":"CICS-UBI\u2014Health Science Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilh\u00e3, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9155-7581","authenticated-orcid":false,"given":"\u00c2ngela","family":"Sousa","sequence":"additional","affiliation":[{"name":"CICS-UBI\u2014Health Science Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilh\u00e3, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2021,8,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e1","DOI":"10.1017\/erm.2017.2","article-title":"Molecular approaches for HPV genotyping and HPV-DNA physical status","volume":"19","author":"Tsakogiannis","year":"2017","journal-title":"Expert Rev. Mol. Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"546","DOI":"10.1002\/btpr.2603","article-title":"HPV-16 targeted DNA vaccine expression: The role of purification","volume":"34","author":"Almeida","year":"2018","journal-title":"Biotechnol. Prog."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1186\/s12929-016-0293-9","article-title":"Perspectives for therapeutic HPV vaccine development","volume":"23","author":"Yang","year":"2016","journal-title":"J. Biomed. Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"158","DOI":"10.1016\/j.tim.2017.07.007","article-title":"The HPV E6\/E7 Oncogenes: Key Factors for Viral Carcinogenesis and Therapeutic Targets","volume":"26","author":"Bossler","year":"2018","journal-title":"Trends Microbiol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"3116","DOI":"10.3389\/fmicb.2019.03116","article-title":"Human Papillomavirus E6 and E7: The Cervical Cancer Hallmarks and Targets for Therapy","volume":"10","author":"Pal","year":"2020","journal-title":"Front. Microbiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"196","DOI":"10.1016\/j.ygyno.2017.04.004","article-title":"HPV vaccines\u2014A review of the first decade","volume":"146","author":"Harper","year":"2017","journal-title":"Gynecol. Oncol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"971","DOI":"10.1089\/hum.2017.197","article-title":"Therapeutic DNA Vaccines for Human Papillomavirus and Associated Diseases","volume":"29","author":"Cheng","year":"2018","journal-title":"Hum. Gene Ther."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2044","DOI":"10.1016\/j.drudis.2019.07.011","article-title":"Cervical cancer and HPV infection: Ongoing therapeutic research to counteract the action of E6 and E7 oncoproteins","volume":"24","author":"Almeida","year":"2019","journal-title":"Drug Discov. Today"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1056\/NEJMoa2035389","article-title":"Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine","volume":"384","author":"Baden","year":"2021","journal-title":"N. Engl. J. Med."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Valente, J.F.A., Pereira, P., Sousa, A., Queiroz, J.A., and Sousa, F. (2021). Effect of plasmid DNA size on chitosan or polyethyleneimine polyplexes formulation. Polymers, 13.","DOI":"10.3390\/polym13050793"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Faria, R., Viv\u00e9s, E., Boisguerin, P., Sousa, A., and Costa, D. (2021). Development of peptide-based nanoparticles for mitochondrial plasmid dna delivery. Polymers, 13.","DOI":"10.3390\/polym13111836"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"110610","DOI":"10.1016\/j.colsurfb.2019.110610","article-title":"Cancer gene therapy mediated by RALA\/plasmid DNA vectors: Nitrogen to phosphate groups ratio (N\/P) as a tool for tunable transfection efficiency and apoptosis","volume":"185","author":"Neves","year":"2020","journal-title":"Colloids Surf. B Biointerfaces"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Serra, A.S., Eus\u00e9bio, D., Neves, A.R., Albuquerque, T., Bhatt, H., Biswas, S., Costa, D., and Sousa, \u00c2. (2021). Synthesis and Characterization of Mannosylated Formulations to Deliver a Minicircle DNA Vaccine. Pharmaceutics, 13.","DOI":"10.3390\/pharmaceutics13050673"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Xing, L., Fan, Y.T., Zhou, T.J., Gong, J.H., Cui, L.H., Cho, K.H., Choi, Y.J., Jiang, H.L., and Cho, C.S. (2018). Chemical modification of Chitosan for efficient vaccine delivery. Molecules, 23.","DOI":"10.3390\/molecules23020229"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1016\/j.addr.2009.08.004","article-title":"Chitosan-based formulations for delivery of DNA and siRNA","volume":"62","author":"Mao","year":"2010","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"280","DOI":"10.1016\/j.jconrel.2018.05.035","article-title":"Lipid nanoparticles with minimum burst release of TNF-\u03b1 siRNA show strong activity against rheumatoid arthritis unresponsive to methotrexate","volume":"283","author":"Aldayel","year":"2018","journal-title":"J. Control. Release"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Bai, K., Hong, B., Huang, W., and He, J. (2020). Selenium-nanoparticles-loaded chitosan\/chitooligosaccharide microparticles and their antioxidant potential: A chemical and in vivo investigation. Pharmaceutics, 12.","DOI":"10.3390\/pharmaceutics12010043"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Mohammed, M.A., Syeda, J.T.M., Wasan, K.M., and Wasan, E.K. (2017). An overview of chitosan nanoparticles and its application in non-parenteral drug delivery. Pharmaceutics, 9.","DOI":"10.3390\/pharmaceutics9040053"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Eus\u00e9bio, D., Neves, A.R., Costa, D., Biswas, S., Alves, G., Cui, Z., and Sousa, \u00c2. (2021). Methods to improve the immunogenicity of plasmid DNA vaccines. Drug Discov. Today, in press.","DOI":"10.1016\/j.drudis.2021.06.008"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1016\/j.ejpb.2015.11.003","article-title":"Mucoadhesive vs. mucopenetrating particulate drug delivery","volume":"98","author":"Netsomboon","year":"2016","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1016\/j.jconrel.2018.10.020","article-title":"Intranasal immunization with aluminum salt-adjuvanted dry powder vaccine","volume":"292","author":"Thakkar","year":"2018","journal-title":"J. Control. Release"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"15101","DOI":"10.1088\/0957-4484\/22\/1\/015101","article-title":"Formulation of chitosan-TPP-pDNA nanocapsules for gene therapy Applications","volume":"22","author":"Gaspar","year":"2011","journal-title":"Nanotechnology"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1016\/j.colsurfb.2011.09.042","article-title":"Formation mechanism of monodisperse, low molecular weight chitosan nanoparticles by ionic gelation technique","volume":"90","author":"Fan","year":"2012","journal-title":"Colloids Surf. B Biointerfaces"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2856","DOI":"10.1021\/mp300162j","article-title":"Insight on the formation of chitosan nanoparticles through ionotropic gelation with tripolyphosphate","volume":"9","author":"Koukaras","year":"2012","journal-title":"Mol. Pharm."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Yan, J., Guan, Z.Y., Zhu, W.F., Zhong, L.Y., Qiu, Z.Q., Yue, P.F., Wu, W.T., Liu, J., and Huang, X. (2020). Preparation of puerarin chitosan oral nanoparticles by ionic gelation method and its related kinetics. Pharmaceutics, 12.","DOI":"10.3390\/pharmaceutics12030216"},{"key":"ref_26","unstructured":"Eriksson, L., Johansson, E., Kettaneh-Wold, N., Wikstr\u00c4om, C., and Wold, S. (2000). Design of Experiments: Principles and Applications, Umetrics AB, Ume\u00e5 Learnways AB."},{"key":"ref_27","unstructured":"Myers, J., Well, A., and Lorch, R. (2010). Research Design and Statistical Analysis, Routledge. [3rd ed.]."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Valente, J.F.A., Dias, J.R., Sousa, A., and Alves, N. (2019). Composite central face design-an approach to achieve efficient alginate microcarriers. Polymers, 11.","DOI":"10.3390\/polym11121949"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1016\/j.ijpharm.2017.04.055","article-title":"Optimization of critical quality attributes in continuous twin-screw wet granulation via design space validated with pilot scale experimental data","volume":"525","author":"Liu","year":"2017","journal-title":"Int. J. Pharm."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"341","DOI":"10.3389\/fbioe.2021.657201","article-title":"Toward QbD Process Understanding on DNA Vaccine Purification Using Design of Experiment","volume":"9","author":"Hocharoen","year":"2021","journal-title":"Front. Bioeng. Biotechnol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"818","DOI":"10.1080\/15226514.2020.1859985","article-title":"Bisphenol A removal by the Chlorophyta Picocystis sp.: Optimization and kinetic study","volume":"23","author":"Leboulanger","year":"2021","journal-title":"Int. J. Phytoremed."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"488","DOI":"10.1016\/j.carbpol.2016.05.092","article-title":"Preparation of chitosan-TPP sub-micron particles: Critical evaluation and derived recommendations","volume":"151","year":"2016","journal-title":"Carbohydr. Polym."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1430","DOI":"10.1016\/j.carbpol.2011.01.044","article-title":"The effect of prolonged storage at different temperatures on the particle size distribution of tripolyphosphate (TPP)-chitosan nanoparticles","volume":"84","author":"Morris","year":"2011","journal-title":"Carbohydr. Polym."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"3747","DOI":"10.1021\/bm301207a","article-title":"Stability of chitosan nanoparticles cross-linked with tripolyphosphate","volume":"13","author":"Jonassen","year":"2012","journal-title":"Biomacromolecules"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"113157","DOI":"10.1016\/j.molliq.2020.113157","article-title":"Polymer-peptide ternary systems as a tool to improve the properties of plasmid DNA vectors in gene delivery","volume":"309","author":"Albuquerque","year":"2020","journal-title":"J. Mol. Liq."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"975","DOI":"10.1016\/j.biomaterials.2009.09.102","article-title":"Molecular design of chitosan gene delivery systems with an optimized balance between polyplex stability and polyplex unpacking","volume":"31","author":"Strand","year":"2010","journal-title":"Biomaterials"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1016\/j.ijpharm.2007.11.052","article-title":"Preparation, characterization and antibacterial activities of chitosan, N-trimethyl chitosan (TMC) and N-diethylmethyl chitosan (DEMC) nanoparticles loaded with insulin using both the ionotropic gelation and polyelectrolyte complexation methods","volume":"355","author":"Sadeghi","year":"2008","journal-title":"Int. J. Pharm."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1007\/s10856-018-6160-3","article-title":"Chitosan\/hyaluronic acid\/plasmid-DNA nanoparticles encoding interleukin-1 receptor antagonist attenuate inflammation in synoviocytes induced by interleukin-1 beta","volume":"29","author":"Deng","year":"2018","journal-title":"J. Mater. Sci. Mater. Med."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/j.foodchem.2017.01.081","article-title":"Preparation of astaxanthin-loaded DNA\/chitosan nanoparticles for improved cellular uptake and antioxidation capability","volume":"227","author":"Wang","year":"2017","journal-title":"Food Chem."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1037","DOI":"10.1016\/j.ijbiomac.2019.01.008","article-title":"Chitosan-DNA polyelectrolyte complex: Influence of chitosan characteristics and mechanism of complex formation","volume":"126","author":"Rosselgong","year":"2019","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1186\/s12951-018-0337-2","article-title":"Chitosan-DNA nanoparticles enhanced the immunogenicity of multivalent DNA vaccination on mice against Trueperella pyogenes infection","volume":"16","author":"Huang","year":"2018","journal-title":"J. Nanobiotechnol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1016\/j.colsurfa.2013.12.058","article-title":"Factorial design as tool in chitosan nanoparticles development by ionic gelation technique","volume":"445","author":"Milioli","year":"2014","journal-title":"Colloids Surf. A Physicochem. Eng. Asp."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1441","DOI":"10.1038\/sj.gt.3302312","article-title":"Improved chitosan-mediated gene delivery based on easily dissociated chitosan polyplexes of highly defined chitosan oligomers","volume":"11","author":"Issa","year":"2004","journal-title":"Gene Ther."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1016\/S0168-3659(98)00097-2","article-title":"Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery","volume":"56","author":"MacLaughlin","year":"1998","journal-title":"J. Control. Release"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Bruinsmann, F.A., Pigana, S., Aguirre, T., Souto, G.D., Pereira, G.G., Bianchera, A., Fasiolo, L.T., Colombo, G., Marques, M., and Pohlmann, A.R. (2019). Chitosan-coated nanoparticles: Effect of chitosan molecular weight on nasal transmucosal delivery. Pharmaceutics, 11.","DOI":"10.20944\/preprints201902.0034.v1"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"119038","DOI":"10.1016\/j.ijpharm.2020.119038","article-title":"Formulation of simvastatin chitosan nanoparticles for controlled delivery in bone regeneration: Optimization using Box-Behnken design, stability and in vivo study","volume":"577","author":"Delan","year":"2020","journal-title":"Int. J. Pharm."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"110417","DOI":"10.1016\/j.colsurfb.2019.110417","article-title":"Optimization of peptide-plasmid DNA vectors formulation for gene delivery in cancer therapy exploring design of experiments","volume":"183","author":"Sousa","year":"2019","journal-title":"Colloids Surf. B Biointerfaces"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"112488","DOI":"10.1016\/j.molliq.2020.112488","article-title":"Design of experiments to select triphenylphosphonium-polyplexes with suitable physicochemical properties for mitochondrial gene therapy","volume":"302","author":"Sousa","year":"2020","journal-title":"J. Mol. Liq."},{"key":"ref_49","first-page":"148763","article-title":"Hydrodynamic delivery of chitosan-folate-DNA nanoparticles in rats with adjuvant-induced arthritis","volume":"2011","author":"Shi","year":"2010","journal-title":"J. Biomed. Biotechnol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1016\/j.jconrel.2005.05.004","article-title":"Transfection efficiency of chitosan vectors: Effect of polymer molecular weight and degree of deacetylation","volume":"106","author":"Huang","year":"2005","journal-title":"J. Control. Release"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"215","DOI":"10.3109\/10717544.2010.544688","article-title":"Plasmid DNA-loaded chitosan\/TPP nanoparticles for topical gene delivery","volume":"18","year":"2011","journal-title":"Drug Deliv."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"2337","DOI":"10.1038\/s41467-020-15889-3","article-title":"Size and surface charge characterization of nanoparticles with a salt gradient","volume":"11","author":"Rasmussen","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"339","DOI":"10.1186\/s11671-018-2728-6","article-title":"Insight into Cellular Uptake and Intracellular Trafficking of Nanoparticles","volume":"13","author":"Foroozandeh","year":"2018","journal-title":"Nanoscale Res. Lett."},{"key":"ref_54","doi-asserted-by":"crossref","unstructured":"Manzanares, D., and Ce\u00f1a, V. (2020). Endocytosis: The nanoparticle and submicron nanocompounds gateway into the cell. Pharmaceutics, 12.","DOI":"10.3390\/pharmaceutics12040371"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"105270","DOI":"10.1016\/j.ejps.2020.105270","article-title":"Design and characterization of chitosan\/citrate films as carrier for oral macromolecule delivery","volume":"146","author":"Ibrahim","year":"2020","journal-title":"Eur. J. Pharm. Sci."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"2215","DOI":"10.1016\/j.ijbiomac.2020.07.245","article-title":"Effect of chitosan molecular weight on zein-chitosan nanocomplexes: Formation, characterization, and the delivery of quercetagetin","volume":"164","author":"Chen","year":"2020","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"1877","DOI":"10.1039\/C9SM02387A","article-title":"Viability of: Lactobacillus rhamnosus GG microencapsulated in alginate\/chitosan hydrogel particles during storage and simulated gastrointestinal digestion: Role of chitosan molecular weight","volume":"16","author":"Qi","year":"2020","journal-title":"Soft Matter"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"115689","DOI":"10.1016\/j.carbpol.2019.115689","article-title":"Effect of molecular weight of chitosan and its oligosaccharides on antitumor activities of chitosan-selenium nanoparticles","volume":"231","author":"Song","year":"2020","journal-title":"Carbohydr. Polym."},{"key":"ref_59","first-page":"955","article-title":"Synthesis, characterization and cytotoxic evaluation of graphene oxide nanosheets: In vitro liver cancer model","volume":"18","author":"Loutfy","year":"2017","journal-title":"Asian Pac. J. Cancer Prev."},{"key":"ref_60","doi-asserted-by":"crossref","unstructured":"Tomaz, A.F., de Carvalho, S.M.S., Barbosa, R.C., Silva, S.M.L., Gutierrez, M.A.S., de Lima, A.G.B., and Fook, M.V.L. (2018). Ionically crosslinked chitosan membranes used as drug carriers for cancer therapy application. Materials, 11.","DOI":"10.3390\/ma11102051"},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"1388","DOI":"10.1016\/j.ijbiomac.2020.08.048","article-title":"Sustainable antimicrobial modified chitosan and its nanoparticles hydrogels: Synthesis and characterization","volume":"162","author":"Ahmed","year":"2020","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"344","DOI":"10.1016\/j.jcis.2004.08.186","article-title":"Physicochemical characterization of chitosan nanoparticles: Electrokinetic and stability behavior","volume":"283","author":"Carvalho","year":"2005","journal-title":"J. Colloid Interface Sci."},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1002\/bit.26475","article-title":"Metabolic reduction of resazurin; location within the cell for cytotoxicity assays","volume":"115","author":"Chen","year":"2018","journal-title":"Biotechnol. Bioeng."},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1111\/imm.12059","article-title":"Chemokine programming dendritic cell antigen response: Part II\u2014programming antigen presentation to T lymphocytes by partially maintaining immature dendritic cell phenotype","volume":"139","author":"Park","year":"2013","journal-title":"Immunology"},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"3141","DOI":"10.1021\/acs.molpharmaceut.6b00338","article-title":"Incorporation of a nuclear localization signal in pH responsive LAH4-L1 peptide enhances transfection and nuclear uptake of plasmid DNA","volume":"13","author":"Xu","year":"2016","journal-title":"Mol. Pharm."},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"181","DOI":"10.18433\/J3TP4T","article-title":"Preparation of chitosan-plasmid DNA nanoparticles encoding interleukin-12 and their expression in CT-26 colon carcinoma cells","volume":"14","author":"Valizadeh","year":"2011","journal-title":"J. Pharm. Pharm. Sci."}],"container-title":["Pharmaceutics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4923\/13\/9\/1369\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:53:33Z","timestamp":1760165613000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4923\/13\/9\/1369"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,8,31]]},"references-count":66,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2021,9]]}},"alternative-id":["pharmaceutics13091369"],"URL":"https:\/\/doi.org\/10.3390\/pharmaceutics13091369","relation":{},"ISSN":["1999-4923"],"issn-type":[{"value":"1999-4923","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,8,31]]}}}