Immunostimulatory effect of chitosan conjugated green copper oxide nanoparticles in tumor immunotherapy

doi:10.1016/j.cyto.2019.154958

Cytokine

Volume 127, March 2020, 154958

https://doi.org/10.1016/j.cyto.2019.154958 Get rights and content

Highlights

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CuONPs@CS was able to activate the macrophages that killed cancer cells.
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Activated macrophages stimulated Th1 & Th2 cells.
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Specific antigen conjugated CuONPs@CS functioned as an antigen delivery vehicle.
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Antigen conjugated CuONPs@CS triggered cytotoxic T lymphocytes.

Abstract

Current study demonstrates the immunogenic role of biopolymer coated green synthesized copper oxide nanoparticles by the induction of cellular immunity through the activation immune cells. Alongside humoral immunity response was triggered by the surface coated NPs through IgG response which indicate the adjuvanic role of the nano conjugate. Th1 (Type 1 and Type 2 helper T cells) and Th2 cells were activated after the treatment with nano conjugate and act as an immunostimulant which would inhibit the proliferation of breast cancer (MCF-7) and cervical cancer (HeLa) cells in in vitro. Solid tumor induced by 4 T1 cells were also inhibited in in vivo Balb/C mice model. Secretion of pro-inflammatory cytokines and the increase in CD + 4 populations indicate the activation of immune cells in the current study. Immunotherapy by the help of metal nano conjugate can be an effective tool to eradicate the cancer cells from the system.

Introduction

Metal-based nanoparticles or nano-metalloids are promising tools in several biomedical applications, in particular, in drug and gene delivery [1], [2]. These nano-metalloids open a new avenue by acting as a convenient tool for the delivery of antigen to antigen-presenting cell (APC) and nano-vaccine scaffolds [3], [4]. As surface chemistry of the engineered nano-metalloids affects the immune response [5], enhancing the pre-existing adjuvanticity of these nanometalloids remains the main aim [6]. These nano-sized particles (NPs) loaded with antigen freely migrate to lymph node and stimulate the immune cells influencing the adaptive immune response [7]. Prolonged retention of NPs facilitates antigen uptake by antigen presenting cell (APC) [8], [9]. Another mechanism of the adjuvanticity of the positively charged nano-metalloid NPs is adsorption of negatively charged proteins enhancing their immunogenicity [10]. Even positively charged chitosan polymers induces inflammasome response [11], [12], [13] while some other NPs modulate Th1 and Th2 balance [14], [15].

Cancer immunotherapy is an effective treatment route because in this process natural defense mechanism of our system boosted up against cancer antigen. Tumor-associated antigens (TAAs) can be recognized by both T cells and B cells as well as TAAs is the main key regulator of immune reactions. To inhibit tumorigenesis, TAAs targeting using antigens is more common and effective method.

Immunomodulating potential of NPs can arise several side effects which can be neutralized by designing engineered NPs in medicine. Immuno imbalance occurs mainly due to oxidative damage which is responsible for ROS generation by enhancing the inflammatory response in vitro and in vivo model [16], [17], [18]. The shift of Th1/Th2 balance could modulate the immune cells where NF-κB is a key regulator for proinflammatory gene expression by producing cytokines such as TNF-α, IL-1β, IL-6, IL-8 [19], [20], [21].

Shape and surface modification of NPs enhances their cell penetration and permeation, we have studied how a spherical, bio-engineered nano-metalloid NPs influences the cellular internalization, transport through the receptor and modulation of ‘Th subsets’ anti-tumor therapy. Here, we have synthesized a novel chitosan coated green copper oxide nano NPs (CuONPs) and assessed their anti-tumor functions.

Section snippets

Chemicals and reagents

Anti CD4⁺ Ab was purchased from Pertec (Germany Code No-05-8401). Anti IgG, IgG1 and IgG2 were obtained from Imgenex, India. Chitosan, Copper Sulphate, Histopaque 1077, propidium Iodied, were purchased from Sigma (St. Louis, MO, USA). DMEM, RPMI 1640, fetal bovine serum (FBS), penicillin, streptomycin, sodium carbonate (Na₂CO₃), sucrose, and ethylene diamine tetraacetate (EDTA) were purchased from Himedia, India. Tris-Hcl, Tris buffer, KH₂PO₄, K₂HPO₄, alcohol and other chemicals were procured

Physical characterization of CuONPs@CS

The CuONPs@CS conjugate was characterized by FT-IR technique with characteristic peak near 1218 cm⁻¹ and 1155 cm⁻¹ assign the peak of alkyl group and O single bond H stretching frequency of CS, indicating successful conjugation of CS with green CuONPs (Fig. 1A). Peak around 1588 cm⁻¹ assign to the NH2 group from the CS. The NH2 group was slightly shifted in case of conjugate compared to the green CuONPs which indicated successful conjugation with CS. To detect the surface morphology and average size of the

Discussion

Nanoparticles are able to produce oxidative stress and to induce inflammation as reflected in the production of several cytokines [33], [34], [17]. NPs with unique physico-chemical property can modulate the tumor microenvironment by the macrophage activation that may constitute an effective way for cancer immunotherapy. In our present study, we observed that the spherical CuONPs@CS NPs of about 62 ± 10 nm diameter activated macrophages and executed efficient anti-tumor immunity perhaps due to

Conclusion

CS conjugated green CuONPs induced Th1 cells by producing several cytokines that activated the macrophages to eradicate the cancer cells. Alteration in redox status deciphered the ROS generation which interplay between depleted level of GSH and intracellular signaling or co-stimulatory molecules, i.e., adjuvant (CuONPs@CS). We improved the immunostimulatory role of CuONPs@CS and simultaneously evaluated its anti-tumerogenic response via instigation of CD4 + T cells. We designed an effective

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

We are Thankful to Department of Science and Technology (DST) for providing financial assistance to Miss. Aditi Dey through the INSPIRE Fellowship scheme [Grant no. DST/INSPIRE Fellowship/2015/IF 150762]. We are thankful to the Central Lab of Vidyasagar University (USIC) for instrumental support.

References (48)

N.G. Bastus et al.
Peptides conjugated to gold nanoparticles induce macrophage activation
Mol. Immunol.
(2009)
B. Slutter et al.
Mechanistic study of the adjuvant effect of biodegradable nanoparticles in mucosal vaccination
J. Control. Release.
(2009)
C.L. Bueter et al.
Chitosan but not chitin activates the inflammasome by a mechanism dependent upon phagocytosis
J. Biol. Chem.
(2011)
H. Liu et al.
Comparative study of respiratory tract immune toxicity induced by three sterilization nanoparticles: silver, zinc oxide and titanium dioxide
J. Hazard. Mater.
(2013)
A. Srinivas et al.
Acute inhalation toxicity of ceriumoxide nanoparticles in rats
Toxicol. Lett.
(2011)
A. Dey et al.
Azadirachta indica leaves mediated green synthesized copper oxide nanoparticles induce apoptosis through activation of TNF-α and caspases signaling pathway against cancer cells
J. Saudi Chem. Soc.
(2019)
A. Dey et al.
Biodistribution and toxickinetic variances of chemical and green Copper oxide nanoparticles in vitro and in vivo
J. Trace Elem. Med Biol.
(2019)
S. Goswami et al.
Neem leaf glycoprotein matures myeloid derived dendritic cells and optimizes anti-tumor T cell functions
Vaccine.
(2010)
O.H. Lowry et al.
Protein measurement with the folin phenol reagent
J. Biol. Chem
(1951)
H. Liu et al.
Comparative study of respiratory tract immune toxicity induced by three sterilisation nanoparticles: silver, zinc oxide and titanium dioxide
J. Hazard. Mater.
(2013)

T. Biedermann et al.

TH1 and TH2 lymphocyte development and regulation of TH cell-mediated immune responses of the skin

J. Investig. Dermatol. Symp. Proc

(2004)

E.E. Connor et al.

Gold nanoparticles are taken up by human cells but do not cause acute cytotoxicity

Small.

(2005)

D. Pissuwan et al.

The forthcoming applications of gold nanoparticles in drug and gene delivery systems

J. Control Release.

(2009)

K. Kostarelos et al.

Cellular uptake of functionalised carbon nanotubes is independent of functional group and cell type

Nat. Nanotechnol.

(2007)

M. Kovacsovics-Bankowski et al.

A phagosome-to-cytosol pathway for exogenous antigens presented on MHC class I molecules

Science

(1995)

T. Fifis et al.

Size-dependent immunogenicity: therapeutic and protective properties of nano-vaccines against tumors

J. Immunol.

(2004)

L. Melero et al.

Therapeutic vaccines for cancer: an overview of clinical trials

Nat. Rev. Clin. Oncol.

(2014)

A.E. Gregory et al.

Vaccine delivery using nanoparticles

Send Front. Cell. Infect. Microbiol.

(2013)

Z.J. Deng et al.

Nanoparticleinduced unfolding of fibrinogen promotes Mac-1 receptor activation and inflammation

Nat. Nanotechnol.

(2011)

C.L. Bueter et al.

Spectrum and mechanisms of inflammasome activation by chitosan

J. Immunol.

(2014)

D. Fong et al.

Chitosan immunomodulatory properties: perspectives on the impact of structural properties and dosage

Future Sci. OA.

(2018)

M. Zhu et al.

Exosomes as extrapulmonary signaling conveyors for nanoparticle-induced systemic immune activation

Small.

(2012)

C.C. Shen et al.

Iron oxide nanoparticles suppressed T helper 1 cell-mediated immunity in a murine model of delayed-type hypersensitivity

Int. J. Nanomed.

(2012)

T. Morishige et al.

Suppression of nanosilica particle-induced inflammation by surface modification of the particles

Arch. Toxicol.

(2012)

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Citation Excerpt :
In another study, Dey et al. found that the chitosan conjugated copper oxide NPs (CuONPs@CS) could activate macrophages and stimulate both Th1 and Th2 cells, which leads to inhibition of tumor cell proliferation. The CuONPs@CS nanovaccine also increased pro-inflammatory cytokines, including IL-12, IFN-γ, and TNF-α, and reduced IL-10 levels as an anti-inflammatory cytokine [43]. The oxide form of FeNPs (IONPs) is another metallic carrier widely used in developing nanovaccines.
Cancer immunotherapy is proposed to eradicate tumors by stimulating host anti-tumor immunity through utilizing various therapeutic approaches. Cancer vaccines have become a promising approach for cancer immunotherapy among the proposed platforms, either alone or in combination with other therapeutic agents. Due to the suboptimal efficacy of cancer vaccines in clinical trials and the advent of nanotechnology in the biomedicine field, scientists developed nanoplatforms, such as various nanoparticles (NPs), cell-derived components, and nanocomplexes, to deliver vaccine components to target cells and tissues, thereby supporting their anti-tumor efficacy and minimizing adverse side effects. To increase the therapeutic effects of nanovaccines in cancer therapy, dendritic cell (DC) targeting through the modulation of the structure of the vaccines, such as using DC-specific ligands, has attracted extensive interest. Here, we reviewed the various forms of nanovaccines in cancer therapy and their therapeutic effects; we highlighted the properties and functions of DCs as the main antigen-presenting cells in immune responses and focused on targeting DCs in developing nanovaccines.

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Immunostimulatory effect of chitosan conjugated green copper oxide nanoparticles in tumor immunotherapy

Highlights

Abstract

Introduction

Section snippets

Chemicals and reagents

Physical characterization of CuONPs@CS

Discussion

Conclusion

Declaration of Competing Interest

Acknowledgement

Mol. Immunol.

J. Control. Release.

J. Biol. Chem.

J. Hazard. Mater.

Toxicol. Lett.

J. Saudi Chem. Soc.

J. Trace Elem. Med Biol.

Vaccine.

J. Biol. Chem

J. Hazard. Mater.

J. Investig. Dermatol. Symp. Proc

Gold nanoparticles are taken up by human cells but do not cause acute cytotoxicity

Small.

The forthcoming applications of gold nanoparticles in drug and gene delivery systems

J. Control Release.

Cellular uptake of functionalised carbon nanotubes is independent of functional group and cell type

Nat. Nanotechnol.

A phagosome-to-cytosol pathway for exogenous antigens presented on MHC class I molecules

Science

Size-dependent immunogenicity: therapeutic and protective properties of nano-vaccines against tumors

J. Immunol.

Therapeutic vaccines for cancer: an overview of clinical trials

Nat. Rev. Clin. Oncol.

Vaccine delivery using nanoparticles

Send Front. Cell. Infect. Microbiol.

Nanoparticleinduced unfolding of fibrinogen promotes Mac-1 receptor activation and inflammation

Nat. Nanotechnol.

Spectrum and mechanisms of inflammasome activation by chitosan

J. Immunol.

Chitosan immunomodulatory properties: perspectives on the impact of structural properties and dosage

Future Sci. OA.

Exosomes as extrapulmonary signaling conveyors for nanoparticle-induced systemic immune activation

Small.

Iron oxide nanoparticles suppressed T helper 1 cell-mediated immunity in a murine model of delayed-type hypersensitivity

Int. J. Nanomed.

Suppression of nanosilica particle-induced inflammation by surface modification of the particles

Arch. Toxicol.