ABSTRACT
Trastuzumab (Herceptin(®) ) is a monoclonal antibody (mAb) for specific ablation of HER2-overexpressing malignant breast cancer cells. Intensification of antiproliferative activity of trastuzumab through construction of immunotoxins and nano-immunoconjugates is a promising approach for treatment of cancer. In this study, trastuzumab was directly conjugated to diphtheria toxin (DT). Also, conjugates of trastuzumab and multiwalled carbon nanotubes (MWCNT) were constructed by covalent immobilization of trastuzumab onto MWCNTs. Then, antiproliferative activity of the fusion constructs against HER2-overexpressing SK-BR-3 and also HER2-negative MCF-7 cancer cell lines were examined. Cells treated with trastuzumab-MWCNT conjugates were irradiated with near-infrared (NIR) light. Efficient absorption of NIR radiation and its conversion to heat by MWCNTs can be resulted to thermal ablation of cancerous cells. Our results strongly showed that both trastuzumab-MWCNT and trastuzumab-DT conjugates were significantly efficient in the specific killing of SK-BR-3 cells. Targeting of MWCNTs to cancerous cells using trastuzumab followed by exposure of cells to NIR radiation was more efficient in repression of cell proliferation than treatment for cancer cells with trastuzumab-DT. Our results also showed that conjugation linkers can significantly affect the cytotoxicity of MWCNT-immunoconjugates. In conclusion, our data demonstrated that trastuzumab-MWCNT is a promising nano-immunoconjugate for killing of HER2-overexpressing cancerous cells.
Subject(s)
Antibodies, Monoclonal, Humanized/chemistry , Antibodies, Monoclonal, Humanized/pharmacology , Diphtheria Toxin/chemistry , Nanotubes, Carbon/chemistry , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/radiation effects , Cell Survival/drug effects , Cell Survival/radiation effects , Drug Carriers/chemistry , Female , Humans , Infrared Rays , MCF-7 Cells , Temperature , TrastuzumabABSTRACT
Bacterial lipopolysaccharide (LPS) has T-helper 1 (Th1) immunostimulatory activities but because of toxicity and pyrogenicity cannot be used as an adjuvant. Brucella abortus LPS has less toxicity and no pyrogenic properties in comparison to other bacterial LPS. In the current study, the immunostimulatory properties of B. abortus LPS were evaluated for its adjuvant activity. Tuberculin purified protein derivative (PPD) from Mycobacterium tuberculosis was extracted and after anion-exchange chromatography on Q-sepharose column, two fractions (17 and 23), which dominantly contained 30- and 70-kDa antigens, were collected for immunological studies. BALB/c mice were immunized with four different antigen preparations (BCG, PPD, 17th and 23rd PPD fractions) along with complete Freund's adjuvant or B. abortus LPS. The T-cell immune response of mice was assessed by measurement of Th1-type cytokine (IFN-γ) and Th2-type cytokines (IL-5 and IL-10) levels. Also, the humoral immunity was evaluated by measuring the specific IgG levels. Our results showed that immunization of mice with 17th PPD fraction along with B. abortus LPS can induce a Th1-type cytokine response characterized with a high IFN-γ/IL-5 ratio, while immunization with PPD or 23rd PPD fraction along with the same adjuvant resulted to a mixed Th1/Th2-type cytokine response.
Subject(s)
Bacterial Proteins/immunology , Brucella abortus/immunology , Lipopolysaccharides/immunology , Tuberculin/immunology , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/standards , Animals , Antigens, Bacterial/administration & dosage , Antigens, Bacterial/immunology , BCG Vaccine/administration & dosage , BCG Vaccine/immunology , Bacterial Proteins/administration & dosage , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Immunization/methods , Immunization/standards , Interferon-gamma/immunology , Interferon-gamma/metabolism , Interleukin-10/immunology , Interleukin-10/metabolism , Interleukin-5/immunology , Interleukin-5/metabolism , Lipopolysaccharides/administration & dosage , Male , Mice , Mice, Inbred BALB C , Mycobacterium tuberculosis/immunology , Reproducibility of Results , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tuberculin/administration & dosageABSTRACT
Tuberculosis (TB) represents one of the leading killers among all infectious disease. Protection against TB depends on the activation of T-helper type I (Th1) immune response. Carbon nanotubes (CNTs) have attracted considerable attention because of their potential applications as new nanovehicle. In the current study, tuberculin purified protein derivative (PPD) was conjugated to carboxylated single-walled carbon nanotubes (SWCNTs). Cytotoxicity of the carboxylated SWCNT and SWCNT-PPD conjugate was analyzed with MTT assay and by reactive oxygen species (ROS) and nitric oxide (NO) generation. Male BALB/c mice were immunized with BCG, PPD, SWCNT-PPD conjugate and PPD in complete Freund's adjuvant (CFA). Induction of cellular immune response was analyzed by measuring the levels of Th1 cytokines (IFN-gamma and IL-12) and Th2 cytokines (IL-10 and IL-5). Immunization with non-conjugated PPD or PPD in Freund's adjuvant induced a Th2 cytokine response while immunization with BCG resulted to a mixed Th1/Th2 cytokine response. In contrast, PPD in conjugation with SWCNT generated preferentially a Th1-type cytokine response in the absence of potential cytotoxic effects.
