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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present detailed methods for the preparation and evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo.

Abstract

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; in particular, some MHC I-restricted epitopes can induce effective cytotoxic T lymphocyte activity to clear tumor cells. Additionally, nasal administration is an effective and safe delivery technique for tumor vaccines due to its convenience and improved patient compliance. However, epitope peptides are unsuitable for nasal delivery because of their poor immunogenicity and lack of delivery efficiency. Nanoemulsions (NEs) are thermodynamically stable systems that can be loaded with antigens and delivered directly to the nasal mucosal surface. Ile-Lys-Val-Ala-Val (IKVAV) is the core pentapeptide of laminin, an integrin-binding peptide expressed by human respiratory epithelial cells. In this study, an intranasal self-assembled epitope peptide NE tumor vaccine containing the synthetic peptide IKVAV-OVA257-264 (I-OVA) was prepared by a low-energy emulsification method. The combination of IKVAV and OVA257-264 can enhance antigen uptake by nasal mucosal epithelial cells. Here, we establish a protocol to study the physicochemical characteristics by transmission electron microscopy (TEM), atomic force microscopy (AFM), and dynamic light scattering (DLS); stability in the presence of mucin protein; toxicity by examining the cell viability of BEAS-2B cells and the nasal and lung tissues of C57BL/6 mice; cellular uptake by confocal laser scanning microscopy (CLSM); release profiles by imaging small animals in vivo; and the protective and therapeutic effect of the vaccine by using an E.G7 tumor-bearing model. We anticipate that the protocol will provide technical and theoretical clues for the future development of novel T cell epitope peptide mucosal vaccines.

Introduction

As one of the most critical public health innovations, vaccines play a key role in fighting the global burden of human disease1. For example, at present, more than 120 candidate vaccines for COVID-19 diseases are being tested, some of which have been approved in many countries2. Recent reports state that cancer vaccines have effectively improved the progress of clinical cancer treatments because they direct the immune system of cancer patients to recognize antigens as foreign to the body3. Moreover, multiple T cell epitopes located inside or outside tumor cells can be used to design peptide vaccin....

Protocol

The animal experiments were conducted in accordance with the Laboratory Animal—Guideline for ethical review of animal welfare (GB/T 35892-2018) and were approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University. The mice were euthanized by an intraperitoneal injection of 100 mg/kg of 1% sodium pentobarbital.

1. Preparation of the I-OVA NE

  1. Admix 1 mg of monophosphoryl lipid A (MPLA) with 100 µL of DMSO, v.......

Representative Results

According to the protocol, we completed the preparation and in vitro and in vivo experimental evaluation of the nasal tumor nanovaccine delivery. TEM, AFM, and DLS are effective means for the assessment of the basic characteristics of the surface zeta potential and the particle size of the nanovaccine (Figure 1). BEAS-2B epithelial cells are a useful screening model for the in vitro toxicity testing of nasal vaccines (Figure 2A). The m.......

Discussion

Nanovaccines functionalized with immunocyte membranes have great advantages in disease-targeted therapy, and the side effects are minimized by properties such as unique tumor tropism, the identification of specific targets, prolonged circulation, enhanced intercellular interactions, and low systemic toxicity. They can also be easily integrated with other treatment modules to treat cancers cooperatively16,20. Desirable attributes can be obtained by controllingphys.......

Acknowledgements

This study was supported by No. 31670938, 32070924, 32000651 of the National Natural Science Foundation Program of China, No. 2014jcyjA0107 and No. 2019jcyjA-msxmx0159 of the Natural Science Foundation Project Program of Chongqing, No. 2020XBK24 and No. 2020XBK26 of the Army Medical University Special projects, and No. 202090031021 and No. 202090031035 of National Innovation and Entrepreneurship Program for college students.

....

Materials

NameCompanyCatalog NumberComments
96-well platesCorning Incorporated, USACLS3922
Bio-Rad 6.0 microplate readerBio-Rad Laboratories Incorporated Limited Co., CA, USA Bio-Rad 6.0
CCK-8 kitsDojindo, JapanCK04
Centrifuge 5810 REppendorf, Germany 5811000398
DAPISigma-Aldrich, St. Louis, USAD9542
fetal bovine serum (FBS)Hyclone (Life Technology, USA)SH30088.03
FITC-labeled I-OVAShanghai Botai
Biotechnology Co., Ltd.
NA
HF 90/240 IncubatorHeal Force, SwitzerlandNA
HPLC Shanghai Botai Biotechology Co., Ltd.E2695
Inverted MicroscopeNikon,JapanDSZ5000X
IPC-208Chong Qing University, ChinaNA
IVIS system Caliper Life Science Limited CompanyNA
JEM-1230 TEMJEOL Limited Company of Japan1230 TEM
Malvern NANO ZSMalvern Instruments Ltd., UKNA
MPLA Invivogen
Lit. Co.
tlrl-mpla
Neomycin Sulfate OintmentShanghai CP General Pharmaceutical Co. , Ltd.H31022262
OVA257–264Shanghai Botai
Biotechnology Co., Ltd.
NA
RPMI 1640 mediumHyclone (Life Technology, USA)SH30809.01
Synthetic peptide (I-OVA) conjugation of IKVAV-PAShanghai Botai
Biotechnology Co., Ltd.
NA
Zeiss LSM800 laser scanning confocal fluorescence microscopeZeiss, GermanyZeiss LSM800

References

  1. Sung, H., et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians. 71 (3), 209-249 (2021).
  2. Mohammed, I., et al.

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