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Cited by 108Advanced Drug Delivery SystemsNanoparticle-Based Drug DeliveryImmunotherapy and Immune Responses

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Alginic Acid-Coated Chitosan Nanoparticles Loaded with Legumain DNA Vaccine: Effect against Breast Cancer in Mice

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Liu, Ze, Lv, Dan, Liu, Shu, Gong, Junbo, Wang, Da, Xiong, Min, Chen, Xiaoniao, Xiang, Rong, Tan, Xiaoyue·2013·PLoS ONE
DOI 10.1371/journal.pone.0060190PMC PMC3618226
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Abstract

Legumain-based DNA vaccines have potential to protect against breast cancer. However, the lack of a safe and efficient oral delivery system restricts its clinical application. Here, we constructed alginic acid-coated chitosan nanoparticles (A.C.NPs) as an oral delivery carrier for a legumain DNA vaccine. First, we tested its characteristic in acidic environments in vitro. DNA agarose electrophoresis data show that A.C.NPs protected DNA better from degradation in acidic solution (pH 1.5) than did chitosan nanoparticles (C.NPs). Furthermore, size distribution analysis showed that A.C.NPs tended to aggregate and form micrometer scale complexes in pH<2.7, while dispersing into nanoparticles with an increase in pH. Mice were intragastrically administrated A.C.NPs carrying EGFP plasmids and EGFP expression was detected in the intestinal Peyer's patches. Full-length legumain plasmids were loaded into different delivery carriers, including C.NPs, attenuated Salmonella typhimurium and A.C.NPs. A.C.NPs loaded with empty plasmids served as a control. Oral vaccination was performed in the murine orthotopic 4T1 breast cancer model. Our data indicate that tumor volume was significantly smaller in groups using A.C.NPs or attenuated Salmonella typhimurium as carriers. Furthermore, splenocytes co-cultured them with 4T1 cells pre-stimulated with CoCl2, which influenced the translocation of legumain from cytoplasm to plasma membrane, showed a 4.7 and 2.3 folds increase in active cytotoxic T lymphocytes (CD3(+)/CD8(+)/CD25(+)) when treated with A.C.NPs carriers compared with PBS C.NPs. Our study suggests that C.NPs coated with alginic acid may be a safe and efficient tool for oral delivery of a DNA vaccine. Moreover, a legumain DNA vaccine delivered orally with A.C.NPs can effectively improve autoimmune response and protect against breast cancer in mice.

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Figures

Figure 1 Illustration of A.C.NPs-legumain synthesis.
Figure 1 Illustration of A.C.NPs-legumain synthesis.CC BY · PubMed Central
Figure 2 Characteristics of A.C.NPs-legumain and C.NPs-legumain. (A)Representative images of A.C.NPs- legumain (left) and C.NPs-legumain (right) observed by TEM. Scale bar = 100 nm. (B& C) Size distributions in purified water (pH7.0) and the mean value of zeta-potentials of A.C.NPs-legumain and C.NPs-legumain at 37°C. (D) FTIR spectra of uncoated chitosan particles and alginate acid-coated chitosan particles.
Figure 2 Characteristics of A.C.NPs-legumain and C.NPs-legumain. (A)Representative images of A.C.NPs- legumain (left) and C.NPs-legumain (right) observed by TEM. Scale bar = 100 nm. (B& C) Size distributions in purified water (pH7.0) and the mean value of zeta-potentials of A.C.NPs-legumain and C.NPs-legumain at 37°C. (D) FTIR spectra of uncoated chitosan particles and alginate acid-coated chitosan particles.CC BY · PubMed Central
Figure 3 Characteristics of C.NPs-legumain and A.C.NPs- legumain in an acidic environment. (A) Nanoparticles were treated in different acidity levels (pH 1.8∼12) for 2 hours. C.NPs-legumain and A.C.NPs-legumain particle diameter and zeta potential measurements at 37°C. (B) Representative images of A.C.NPs at pH 1.5 (left, scale bar = 1µm) and pH 7.0 (right, scale bar = 100 nm). (C) FTIR spectra of A.C.NPs-legumain at pH 1.5 and pH 7.0.
Figure 3 Characteristics of C.NPs-legumain and A.C.NPs- legumain in an acidic environment. (A) Nanoparticles were treated in different acidity levels (pH 1.8∼12) for 2 hours. C.NPs-legumain and A.C.NPs-legumain particle diameter and zeta potential measurements at 37°C. (B) Representative images of A.C.NPs at pH 1.5 (left, scale bar = 1µm) and pH 7.0 (right, scale bar = 100 nm). (C) FTIR spectra of A.C.NPs-legumain at pH 1.5 and pH 7.0.CC BY · PubMed Central
Figure 4 A.C.NPs protect DNA against degradation at low pH. Naked, full-length legumain DNA plasmids, C.NPs-legumain, and A.C.NPs-legumain were each incubated with artificial gastric fluid (pH 1.5) for 0, 0.5, 1, 2 or 4 hours. Naked plasmid DNA was incubated in solution of pH 7.0 for the same time points to serve as a positive control. (A) A representative image of the agarose gel electrophoresis. A lane is observed in the A.C.NPs even after incubation at pH 1.5 for 4 h. (B) Graphical representation of relative OD values. Data are presented as mean ± SD of three independent experiments (**P<0.01).
Figure 4 A.C.NPs protect DNA against degradation at low pH. Naked, full-length legumain DNA plasmids, C.NPs-legumain, and A.C.NPs-legumain were each incubated with artificial gastric fluid (pH 1.5) for 0, 0.5, 1, 2 or 4 hours. Naked plasmid DNA was incubated in solution of pH 7.0 for the same time points to serve as a positive control. (A) A representative image of the agarose gel electrophoresis. A lane is observed in the A.C.NPs even after incubation at pH 1.5 for 4 h. (B) Graphical representation of relative OD values. Data are presented as mean ± SD of three independent experiments (**P<0.01).CC BY · PubMed Central
Figure 5 A.C.NPs loaded with DNA pass through the acidic gastric barrier and are taken up by macrophages and dendritic cells in the intestinal Peyer’s patches. Naked EGFP DNA plasmids, C.NPs-EGFP, and A.C.NPs-EGFP were separately given to BALB/c mice (n = 5) via intragastric gavage at a daily dose equivalent to 30 µg plasmid DNA per mouse for three consecutive days. Peyer’s patches were isolated and analyzed by flow cytometry. PE-conjugated F4/80 and APC-conjugated CD11c antibodies were used to stain (A) the macrophages and (B) dendritic cells, respectively. Histograms of the percentage of EGFP-positive (C) macrophages and (D) dendritic cells. (E) Histograms of the ratio of F4/80- or CD11c- positive cells to total EGFP-positive cells. Data are presented as mean ± SD of three independent experiments (**P<0.01; n = 5).
Figure 5 A.C.NPs loaded with DNA pass through the acidic gastric barrier and are taken up by macrophages and dendritic cells in the intestinal Peyer’s patches. Naked EGFP DNA plasmids, C.NPs-EGFP, and A.C.NPs-EGFP were separately given to BALB/c mice (n = 5) via intragastric gavage at a daily dose equivalent to 30 µg plasmid DNA per mouse for three consecutive days. Peyer’s patches were isolated and analyzed by flow cytometry. PE-conjugated F4/80 and APC-conjugated CD11c antibodies were used to stain (A) the macrophages and (B) dendritic cells, respectively. Histograms of the percentage of EGFP-positive (C) macrophages and (D) dendritic cells. (E) Histograms of the ratio of F4/80- or CD11c- positive cells to total EGFP-positive cells. Data are presented as mean ± SD of three independent experiments (**P<0.01; n = 5).CC BY · PubMed Central
Figure 6 Oral vaccination using A.C.NPs-legumain inhibits tumor growth and increases survival in orthotopic 4T1 breast cancer model. (A) Illustration of the experimental design. (B) Tumor growth curves and (C) ratio of tumor weight vs. body weight in different groups. Data are presented as mean ± SD (*P<0.05, **P<0.01). (D) 50 female BALB/c mice were randomly divided into 5 groups (n = 10). Tumor challenge and oral vaccination were performed as described. A Kaplan-meier survival curve is shown.
Figure 6 Oral vaccination using A.C.NPs-legumain inhibits tumor growth and increases survival in orthotopic 4T1 breast cancer model. (A) Illustration of the experimental design. (B) Tumor growth curves and (C) ratio of tumor weight vs. body weight in different groups. Data are presented as mean ± SD (*P<0.05, **P<0.01). (D) 50 female BALB/c mice were randomly divided into 5 groups (n = 10). Tumor challenge and oral vaccination were performed as described. A Kaplan-meier survival curve is shown.CC BY · PubMed Central
Figure 7 Oral vaccination via A.C.NPs-legumain induces autoimmune CTLs that target legumain. Activated CTLs were accessed by flow cytometry. (A) Representative confocal microscopy images of immnofluorescence staining of 4T1 cells treated with or without CoCl2 for 6 h. E-cadherin was used as a cell membrane marker. Significant translocation expression of legumain to the membrane after CoCl2 stimulation was observed. (B) Representative flow cytometry histogram of splenocytes co-cultured with 4T1 cells treated with or without CoCl2. (C) Histogram of the percentages of CD8 and CD25 double positive cells. Data are presented as mean ± SD of five independent experiments (*P<0.01).
Figure 7 Oral vaccination via A.C.NPs-legumain induces autoimmune CTLs that target legumain. Activated CTLs were accessed by flow cytometry. (A) Representative confocal microscopy images of immnofluorescence staining of 4T1 cells treated with or without CoCl2 for 6 h. E-cadherin was used as a cell membrane marker. Significant translocation expression of legumain to the membrane after CoCl2 stimulation was observed. (B) Representative flow cytometry histogram of splenocytes co-cultured with 4T1 cells treated with or without CoCl2. (C) Histogram of the percentages of CD8 and CD25 double positive cells. Data are presented as mean ± SD of five independent experiments (*P<0.01).CC BY · PubMed Central
Figure 8 Oral vaccination via A.C.NPs-legumain enhances the autoimmune response of CTLs compared with other treatment. Animals (n = 5) were grouped and treated as described. Upon sacrifice, splenocytes were isolated and co-cultured with 4T1 cells, which had been pretreated with CoCl2 for 24 h. The percentage of CTLs activity was measured via flow cytometry. (A) Representative histograms of different groups. (B) The percentages of CD8 and CD25 double positive cells. Data are presented as mean ± SD (**P<0.01).
Figure 8 Oral vaccination via A.C.NPs-legumain enhances the autoimmune response of CTLs compared with other treatment. Animals (n = 5) were grouped and treated as described. Upon sacrifice, splenocytes were isolated and co-cultured with 4T1 cells, which had been pretreated with CoCl2 for 24 h. The percentage of CTLs activity was measured via flow cytometry. (A) Representative histograms of different groups. (B) The percentages of CD8 and CD25 double positive cells. Data are presented as mean ± SD (**P<0.01).CC BY · PubMed Central
Figure 9 Diagram shows the protective effect of A.C.NPs on DNA against enzymatic and acidic degradation. (A) Schematic of A.C.NPs-legumain preparation. (B) Schematic representation of A.C.NPs-legumain passing through the acidic gastrointestinal track and taken up by antigen-presenting cells in the intestinal Peyer’s patches.
Figure 9 Diagram shows the protective effect of A.C.NPs on DNA against enzymatic and acidic degradation. (A) Schematic of A.C.NPs-legumain preparation. (B) Schematic representation of A.C.NPs-legumain passing through the acidic gastrointestinal track and taken up by antigen-presenting cells in the intestinal Peyer’s patches.CC BY · PubMed Central

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  1. Borges O, Cordeiro-da-Silva A, Romeijn SG, Amidi M, de Sousa A, <etal>et al</etal>.. (2006) Uptake studies in rat Peyer’s patches, cytotoxicity and release studies of alginate coated chitosan nanoparticles for mucosal vaccination. J Control Release 114: 348–358. Epub 2006 Jun 2016. · DOI

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Metadata

Authors
Liu, Z., Lv, D., Liu, S., Gong, J., Wang, D., Xiong, M., Chen, X., Xiang, R., & Tan, X.
Affiliations
  • Nankai University (CN)
  • Tianjin University (CN)
Corresponding author
Tan, Xiaoyue
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2013-04-05
Volume
8
Issue
4
Pages
e60190
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0060190
PMC
PMC3618226
OpenAlex
W1993169857
Cited by
108
References
37
Citations by year
2026: 1 · 2025: 4 · 2024: 2 · 2023: 8 · 2022: 8 · 2021: 13 · 2020: 9 · 2019: 7 · 2018: 9 · 2017: 7 · 2016: 13 · 2015: 9 · 2014: 11 · 2013: 7
FWCI
8.02
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Figshare (submittedVersion, cc-by)
Funders
  • National Natural Science Foundation of China (81273331, 2013CB967201, 973 Program, 81270774)
  • National Key Research and Development Program of China (2013CB967201)
Topics
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmaceutical Science › Advanced Drug Delivery Systems
  • Physical Sciences › Materials Science › Biomaterials › Nanoparticle-Based Drug Delivery
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
Keywords
legumain, DNA vaccine, breast cancer, chitosan nanoparticles, oral vaccine delivery, attenuated Salmonella typhimurium, 4T1 breast cancer, cytotoxic T lymphocytes, Peyer's patches, nanoparticle aggregation
MeSH terms
Asparaginyl Endopeptidase, Alginates, Animals, Antigen-Presenting Cells, Biological Transport, Cysteine Endopeptidases, Drug Carriers, Female, Gastric Mucosa, Hexuronic Acids, Hydrogen-Ion Concentration, Macrophages, Mammary Neoplasms, Experimental, Mice, Inbred BALB C, Peyer's Patches, T-Lymphocytes, Vaccines, DNA, Cancer Vaccines, Glucuronic Acid, Chitosan, Cell Proliferation, Mice, Nanoparticles

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