Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

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-OVA_257-264 (I-OVA) was prepared by a low-energy emulsification method. The combination of IKVAV and OVA_257-264 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 vaccines, which have shown advantages in the treatment of metastatic cancers because of the lack of the significant toxicity associated with radiotherapy and chemotherapy^4,5. Since the mid-1990s, preclinical and clinical trials for tumor treatment have been conducted mainly using antigen peptide vaccines, but few vaccines exhibit an adequate therapeutic effect on cancer patients⁶. Furthermore, cancer vaccines with peptide epitopes have poor immunogenicity and insufficient delivery efficiency, which may be due to the rapid degradation of extracellular peptides that diffuse rapidly from the site of administration, which leads to insufficient antigen uptake by immune cells⁷. Therefore, it is necessary to overcome these obstacles with vaccine delivery technology.

OVA_257-264, the MHC class I-binding 257-264 epitope expressed as a fusion protein, is a frequently used model epitope⁸. In addition, OVA_257-264 is crucial to the adaptive immune response against tumors, which depends on the cytotoxic T lymphocyte (CTL) response. It is mediated by antigen-specific CD8⁺ T cells in the tumor, which are induced by the OVA_257-264 peptide. It is characterized by insufficient granzyme B, which is released by cytotoxic T cells, leading to the apoptosis of target cells⁸. However, free OVA_257-264 peptide administration may induce little CTL activity because the uptake of these antigens occurs in nonspecific cells rather than antigen-presenting cells (APCs). The deficiency of appropriate immune stimulation results in CTL activity⁵. Therefore, the induction of efficacious CTL activity demands considerable advancement.

Owing to the barrier provided by epithelial cells and the continuous secretion of mucus, vaccine antigens are rapidly removed from the nasal mucous^9,10. Developing an efficient vaccine vector that can pass through the mucosal tissue is crucial because the antigen-presenting cells are situated under the mucosal epithelium⁹. Intranasal injection of vaccines theoretically induces mucosal immunity to fight mucosal infection¹¹. In addition, nasal delivery is an effective and safe administration method for vaccines due to its convenience, the avoidance of intestinal administration, and improved patient compliancy⁷. Therefore, nasal delivery is a good means of administration for the novel peptide epitope nanovaccine.

Several synthetic biomaterials have been devised to combine epitopes of cell-tissue and cell-cell interactions. Certain bioactive proteins, such as Ile-Lys-Val-Ala-Val (IKVAV), have been introduced as part of the structure of the hydrogel to confer bioactivity¹². This peptide likely contributes to cell attachment, migration, and outgrowth¹³ and binds integrins α_3β1 and α_6β1 to interact with different cancer cell types. IKVAV is a cell adhesion peptide derived from the laminin basement membrane protein α₁ chain that was originally used to model the neural microenvironment and cause the neuronal differentiation¹⁴. Therefore, finding an efficient delivery vehicle for this novel vaccine is important for disease control.

Recently reported emulsion systems, such as W805EC and MF59, have also been compounded for the nasal cavity delivery of inactivated influenza vaccine or recombinant hepatitis B surface antigen and illustrated to trigger both mucosal and systemic immunity¹⁵. Nanoemulsions (NEs) have the advantages of easy administration and convenient co-formation with effective adjuvants compared with particulate mucosal delivery systems¹⁶. Nanoemulsion vaccines have been reported to alter the allergic phenotype in a sustained manner different from traditional desensitization, which results in long-term suppressive effects¹⁷. Others reported that nanoemulsions combined with Mtb-specific immunodominant antigens could induce potent mucosal cell responses and confer significant protection¹⁸. Therefore, a novel intranasal self-assembled nanovaccine with the synthetic peptide IKVAV-OVA_257-264 (I-OVA, the peptide consisting of IKVAV bound to OVA_257-264) was designed. It is important to assess this novel nanovaccine systematically.

The purpose of this protocol is to systematically evaluate the physicochemical characteristics, toxicity, and stability of the nanovaccine, detect whether antigen uptake and protective and therapeutic effects are enhanced using technical means, and elaborate on the main experimental contents. In this study, we established a series of protocols to study the physicochemical characteristics and stability, determine the magnitude of toxicity of the I-OVA NE to BEAS-2B cells by CCK-8, and observe the antigen-presenting ability of BEAS-2B cells to the vaccine using confocal microscopy, evaluate the release profiles of this novel nanovaccine in vivo and in vitro, and detect the protective and therapeutic effect of this vaccine by using an E.G7-OVA tumor-bearing mouse model.

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, vortex for 5 min, and allow it to stand for 4 h at room temperature (RT) to dissolve completely.
2. Add Tween 80 and I-OVA quantitatively and mix.
   NOTE: Tween 80 and I-OVA were mixed at a mass ratio of 25:1¹⁹.
3. Add squalene into the mixture prepared in step 1.2 (7:3, Smix: squalene).
4. Add 100 µL of MPLA solution (10 mg/mL) to the mixture prepared in step 1.3.
5. Prepare the nanoemulsion vaccine using low-energy emulsification methods¹⁹: add the mixed solution to water droplets at approximately 70% of the total volume and stir gently to obtain a transparent and easily flowing mixture.
   NOTE: The BNE control (blank emulsion) was prepared by the same method, replacing water with I-OVA.

2. Physicochemical characterization and stability

NOTE: Assess the droplet size distribution, zeta potential, and other physicochemical data of the I-OVA NE vaccine following steps 2.1-2.3; perform morphological characterization of the I-OVA NE vaccine following steps 2.4-2.7; and examine the 3D structure of the I-OVA NE vaccine following steps 2.8-2.9.

1. Blend 50 mg of mucin protein with 100 mL of water for injection to prepare a 0.05% mucin solution.
2. Dilute 4 mg/mL I-OVA NE 200-fold with 0.05 mg/mL mucin protein or deionized water.
3. Observe the particle sizes, zeta potential, polydispersity index (PDI), and electrophoretic mobility at 25 °C using a nanoanalyzer¹⁹.
4. Dilute 10 µL of I-OVA NE 200-fold with 2 mL of deionized water.
5. Place 5 µL of prediluted I-OVA NE vaccine (step 2.4) on a carbon-coated copper grid, and cover it with 10 µL of 1% phosphotungstic acid for 3 min.
6. Remove the excess phosphotungstic acid with filter paper.
7. Obtain images using TEM. Place a 10 µL sample diluted 50 times on a 100-mesh carbon copper grid and allow it to stand at room temperature (RT) for 5 min before adding 10 µL of phosphotungstic acid (1%, pH 7.4). Examine all the samples by TEM at a voltage of 120 kV.
8. Characterize the molecular morphology of the I-OVA NEusing a high-resolution atomic force microscope. Obtain the color images of I-OVA NE under the following conditions: for tungsten probes (force constant: 0.06 N·m^-1); scan range: 450 nm x 450 nm; tapping mode: imaging mode; and scanning method: point-by-point scanning at RT.

3. In vitro and in vivo toxicity assays

NOTE: The in vitro toxicity of the I-OVA NE vaccine was assessed following steps 3.1-3.9, and the in vivo toxicity of the I-OVA NE vaccine was assessed following steps 3.10-3.13.

1. Revive human BEAS-2B epithelial cells following steps 3.1.1-3.1.4 and culture them in a complete growth medium at 37 °C in a 5% CO₂ incubator.
   NOTE: To prepare the complete growth medium, add fetal bovine serum (FBS) and penicillin/streptomycin to the RPMI-1640 medium at final concentrations of 10% and 1%, respectively.
   1. Turn on the water bath and adjust the temperature to 37 °C. Remove the cell vials frozen in liquid nitrogen and thaw quickly in a 37 °C water bath.
   2. After thawing, quickly pipette the cells into a 15 mL sterile centrifuge tube, add 2 mL of the complete growth medium, and centrifuge at 129 x g for 5 min.
   3. Remove the supernatant, add 2 mL of the complete growth medium to resuspend the cells, and centrifuge at 129 x g for 5 min.
   4. Remove the supernatant, add 6 mL of complete growth medium to resuspend the cells, and transfer the cells to a T25 culture flask to culture the cells at 37 °C in a 5% CO₂ incubator.
2. When the cell density reaches 80%-90%, discard the culture medium and wash the cells twice with 2 mL of PBS. Add 1 mL of 0.25% trypsin to digest the cells for 1-2 min. When rounding of the cells is observed, immediately add 4 mL of the complete growth medium to neutralize the trypsin.
3. Mix and aspirate the samples into a 15 mL sterile centrifuge tube and centrifuge at 129 x g for 5 min.
4. Remove the supernatant and resuspend the cells in 1 mL of RPMI-1640 complete medium. Use 20 µL for cell counting, and dilute the cells to 1 x 10⁵ cells/mL.
5. Plate the BEAS-2B cells at a density of 1 x 10⁴ cells/well in 96-well plates in 100 µL of RPMI-1640 complete medium and preincubate the plates for 24 h at 37 °C in a 5% CO₂ incubator.
6. Discard the supernatant, and add 100 µL of I-OVA NE, 100 µL of I-OVA+BNE (physically mixed), and 100 µL of I-OVA prediluted with a complete growth medium at various final concentrations (0.5 mg/mL, 1 mg/mL, 2 mg/mL, 4 mg/mL, 8 mg/mL), with BNE as a control. Incubate for 24 h at 37 °C.
   NOTE: Add 100 µL of complete growth medium to the negative control groups and 100 µL of cell suspension (1 x 10⁵/mL) to the positive control groups.
7. Remove the medium and add 90 µL of complete growth medium and 10 µL of CCK-8 solution to each well of the plate.
8. Incubate the plate for 2 h at 37 °C in a 5% CO₂ incubator.
9. Measure the absorbance of each well at 450 nm using an enzyme-labeled plate reader.
10. Calculate the survival ratio of BEAS-2B cells as indicated in the following equation:
    (OD450 _sample− OD450 _{negative control}/OD450 _{positive control}− OD450 _{negative control}) × 100%
11. Randomly divide 6-week-old C57BL/6 mice into five groups (n = 5 in each group) and anesthetize them with 4% isoflurane for induction. Maintain anesthesia with 2% isoflurane. Use neomycin sulfate ointment on the eyes of the mice to prevent dryness.
12. Use 10 µL pipette tips to perform nasal immunization of the mice with 10 µL/nostril of I-OVA, I-OVA+BNE, and IOVA NE at 4 mg/mL for all the 3 days. Use BNE and PBS as the experimental control.
    NOTE: Provide thermal support till the animal recovers from anesthesia.
13. Euthanize all the mice by an intraperitoneal injection of 100 mg/kg 1% sodium pentobarbital on day 4.
14. Cut approximately 3 mm thick samples of nasal tissues with scissors, and remove the lung tissues.
15. Fix the nasal tissues and the whole lung tissues in 4% paraformaldehyde for 24 h. Dehydrate the tissues through a serial alcohol and xylene gradient and then embed in paraffin.Slice the finished wax blocks on a paraffin slicer at a thickness of 4 µm.
16. Stain the sections with hematoxylin and eosin (H&E). Then, observe the mucosal toxicity, including hyperemia, edema, neutrophil infiltration, and structural damage in the nasal mucosa and lung tissue, under a microscope (100x and 200x)⁷.

4. In vitro cellular uptake

1. Plate BEAS-2B cells at a density of 5 x 10⁵ cells/well in 12-well plates with coverslips in 2 mL of the complete growth medium and preincubate the plates overnight at 37 °C in a 5% CO₂ incubator.
2. Add 100 µL of FITC-labeled I-OVA NE (purity: 98.3%, produced by the company, 4 mg/mL) or I-OVA (4 mg/mL) to 900 µL of cell suspension and place at 37 °C for 90 min.
   NOTE: Prepare FITC-labeled I-OVA NE as described in step 4.1. Add 1 mL of complete growth medium to the control groups.
3. Wash three times with 0.1 M PBS (1 mL/well) for 30 min at 37 °C after the treatment.
4. Fix these samples with 4% paraformaldehyde for 20 min in the dark. After fixation, remove the paraformaldehyde and wash 3 times with 0.1 M PBS (1 mL/well) for 30 min at 37 °C.
5. Preincubate the samples with DAPI (4',6-diamidino-2-phenylindole) at a final concentration of 10 µg/mL for 10 min in the dark, and then wash 5 times with 0.1 M PBS (1 mL/well) for 30 min at 37 °C after treatment.
6. Obtain the cellular uptake by CLSM withthe following parameter settings: Frame size: 512 px x 512 px, Scan speed: 8; Line step: 1; Averaging: 2.

5. In vivo release

1. Anesthetize nude mice with 4% isoflurane gas and maintain anesthesia at 2% isoflurane. Immunize nude mice intranasally with 10 µL of PE-labeled I-OVA at 4 mg/mL or PE-labeled I-OVA NE at 4 mg/mL in each nostril.
2. At 0 h, 0.5 h, 1.5 h, 3, 6 h, 9 h, 12 h, and 24 h, capture all the mice under anesthesia by the IVIS system.
3. Perform a background scan immediately before intranasal administration to provide a threshold value (Min = 1.06 x 10⁶) to adjust the images gathered at all time points.
4. Click on the Living Image software to initiate the sequence and click Initialize to initialize the IVIS system
5. When it is initialized, the temperature status light in the IVIS acquisition control panel is red. When the temperature status light changes to green, imaging can be performed.
6. Click Imaging Wizard and then choose Fluorescence in the dialog box that appears.
7. Adjust the following parameters: Exposure Time: auto sec, Binning: 8, F/Stop: 2, Field of View: D.
8. Select the 620 nm excitation filter and the 670 nm emission filter. Click on Acquire Sequence to acquire the image.
9. Process the live images of all the mice and the radiance data with Living Image software.
   1. After acquiring the picture, click on ROI Tools in the Tool Palette, select Circle, and make an ROI circle.
   2. Click on Measure ROIs to obtain a quantitative value for the ROI area.

6. In vivo antitumor efficacy

1. Refresh and culture the mouse lymphoma E.G7-OVA cells from EL4 with a complete growth medium following steps 2.1.1-2.1.4.
   NOTE: To prepare E.G7-OVA cell complete growth medium, mix RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate and supplemented with 0.05 mM 2-mercaptoethanol and 0.4 mg/mL G418, 90%, and fetal bovine serum, 10%.
2. Subculture the cells at a ratio of 1:2 when the cells reach a density between 1 x 10⁶ cells/mL and 1 x 10⁷ cells/mL.
3. Evaluate the preventive protective effect on mice as described in steps 6.3.1-6.3.4..
   1. Randomly divide 6-week-old C57BL/6 mice into five groups (n = 8 in each group). Anesthetize nude mice with 4% isoflurane gas and maintain anesthesia with 2% isoflurane. Partially shave the back hair and neomycin sulfate ointment on the eyes of the mice to prevent dryness.
   2. Intranasally immunize the mice with 10 µL of 1 mg/mL I-OVA, BNE+I-OVA, or I-OVA NE in each nostril, BNE (diluted four times with PBS), or a PBS control 3 times with a 7-day interval between each immunization.
   3. Inoculate all the mice hypodermically into the right back with 5 x 10⁵ E. G7-OVA cells on the 7th day after the final immunization.
   4. At 0, 6, 9, 12, 15, and 18 days after the final immunization, monitor the tumor volumes by measuring two axes of the tumor using digital calipers, and record the 30-day (after the final immunization) survival of the mice.
4. Evaluate the therapeutic protective effect on mice after inoculating E.G7-OVA cells as described in steps 6.4.1-6.4.3. For the grouping and the handling of mice refer to step 6.3.1.
   1. On day 0, inject the C57BL/6 mice subcutaneously into the right back with E.G7-OVA cells (5 x 10⁵ cells/mouse).
   2. At 0, 7, and 14 days after injection, immunize all the mice intranasally immunized with 10 µL of I-OVA, BNE+I-OVA, I-OVA NE (all at concentrations of 1 mg/mL), BNE, or PBS three times in each nostril.
   3. At 0, 6, 9, 12, 15, and 18 days after injection, monitor the tumor volume and record the 30-day survival of the mice.
      NOTE: If the tumor volume exceeds 3,000 mm³, the mice should be euthanized for humane reasons, and these mice will be considered dead in the survival curve. Tumor volume is calculated by a modified ellipsoidal formula as indicated in the following equation:
      Volume = π/6 x L x W²
      where L represents the tumor length, and W represents the tumor width (length unit: mm, volume unit: mm³).

7. Statistical analysis

1. Analyze the differences in data between the different groups using appropriate statistical software with one-way ANOVA, Tukey's multiple comparisons, or a Student's t-test. Use the Kaplan-Meier method to estimate the survival outcomes and compare the groups with log-rank statistics. Express all the results as mean ± SD. The significance of P values P < 0.05, P < 0.01, and P < 0.001 is represented using *, **, and ***, respectively, on the plots.

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 cooperatively^16,20. Desirable attributes can be obtained by controllingphys...

Materials

Name	Company	Catalog Number	Comments
96-well plates	Corning Incorporated, USA	CLS3922
Bio-Rad 6.0 microplate reader	Bio-Rad Laboratories Incorporated Limited Co., CA, USA	Bio-Rad 6.0
CCK-8 kits	Dojindo, Japan	CK04
Centrifuge 5810 R	Eppendorf, Germany	5811000398
DAPI	Sigma-Aldrich, St. Louis, USA	D9542
fetal bovine serum (FBS)	Hyclone (Life Technology, USA)	SH30088.03
FITC-labeled I-OVA	Shanghai Botai Biotechnology Co., Ltd.	NA
HF 90/240 Incubator	Heal Force, Switzerland	NA
HPLC	Shanghai Botai Biotechology Co., Ltd.	E2695
Inverted Microscope	Nikon,Japan	DSZ5000X
IPC-208	Chong Qing University, China	NA
IVIS system	Caliper Life Science Limited Company	NA
JEM-1230 TEM	JEOL Limited Company of Japan	1230 TEM
Malvern NANO ZS	Malvern Instruments Ltd., UK	NA
MPLA	Invivogen Lit. Co.	tlrl-mpla
Neomycin Sulfate Ointment	Shanghai CP General Pharmaceutical Co. , Ltd.	H31022262
OVA257–264	Shanghai Botai Biotechnology Co., Ltd.	NA
RPMI 1640 medium	Hyclone (Life Technology, USA)	SH30809.01
Synthetic peptide (I-OVA) conjugation of IKVAV-PA	Shanghai Botai Biotechnology Co., Ltd.	NA
Zeiss LSM800 laser scanning confocal fluorescence microscope	Zeiss, Germany	Zeiss LSM800