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.

The animal experiments were conducted in accordance with the Laboratory Animal&#8212;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.&#160;The mice were euthanized by an intraperitoneal injection of 100 mg/kg of 1% sodium pentobarbital.
1. Preparation of the I-OVA NE
<ol>
	<li>Admix 1 mg of monophosphoryl lipid A (MPLA) with 100 &#181;L of DMSO, vortex for 5 min, and allow it to stand for 4 h at room temperature (RT) to dissolve completely.</li>
	<li>Add Tween 80 and I-OVA quantitatively and mix. 
	NOTE: Tween 80 and I-OVA were mixed at a mass ratio of 25:119.</li>
	<li>Add squalene into the mixture prepared in step 1.2 (7:3, Smix: squalene).</li>
	<li>Add 100 &#181;L of MPLA solution (10 mg/mL) to the mixture prepared in step 1.3.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>Blend 50 mg of mucin protein with 100 mL of water for injection to prepare a 0.05% mucin solution.</li>
	<li>Dilute 4 mg/mL I-OVA NE 200-fold with 0.05 mg/mL mucin protein or deionized water.</li>
	<li>Observe the particle sizes, zeta potential, polydispersity index (PDI), and electrophoretic mobility at 25 &#176;C using a nanoanalyzer19.</li>
	<li>Dilute 10 &#181;L of I-OVA NE 200-fold with 2 mL of deionized water.</li>
	<li>Place 5 &#181;L of prediluted I-OVA NE vaccine (step 2.4) on a carbon-coated copper grid, and cover it with 10 &#181;L of 1% phosphotungstic acid for 3 min.</li>
	<li>Remove the excess phosphotungstic acid with filter paper.</li>
	<li>Obtain images using TEM. Place a 10 &#181;L 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 &#181;L of phosphotungstic acid (1%, pH 7.4). Examine all the samples by TEM at a voltage of 120 kV.</li>
	<li>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&#183;m-1); scan range: 450 nm x 450 nm; tapping mode: imaging mode; and scanning method: point-by-point scanning at RT.</li>
</ol>
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.
<ol>
	<li>Revive human BEAS-2B epithelial cells following steps 3.1.1-3.1.4 and culture them in a complete growth medium at 37 &#176;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.
	<ol>
		<li>Turn on the water bath and adjust the temperature to 37 &#176;C. Remove the cell vials frozen in liquid nitrogen and thaw quickly in a 37 &#176;C water bath.</li>
		<li>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.</li>
		<li>Remove the supernatant, add 2 mL of the complete growth medium to resuspend the cells, and centrifuge at 129 x g for 5 min.</li>
		<li>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 &#176;C in a 5% CO2 incubator.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Mix and aspirate the samples into a 15 mL sterile centrifuge tube and centrifuge at 129 x g for 5 min.</li>
	<li>Remove the supernatant and resuspend the cells in 1 mL of RPMI-1640 complete medium. Use 20 &#181;L for cell counting, and dilute the cells to 1 x 105 cells/mL.</li>
	<li>Plate the BEAS-2B cells at a density of 1 x 104 cells/well in 96-well plates in 100 &#181;L of RPMI-1640 complete medium and preincubate the plates for 24 h at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Discard the supernatant, and add 100 &#181;L of I-OVA NE, 100 &#181;L of I-OVA+BNE (physically mixed), and 100 &#181;L 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 &#176;C. 
	NOTE: Add 100 &#181;L of complete growth medium to the negative control groups and 100 &#181;L of cell suspension (1 x 105/mL) to the positive control groups.</li>
	<li>Remove the medium and add 90 &#181;L of complete growth medium and 10 &#181;L of CCK-8 solution to each well of the plate.</li>
	<li>Incubate the plate for 2 h at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Measure the absorbance of each well at 450 nm using an enzyme-labeled plate reader.</li>
	<li>Calculate the survival ratio of BEAS-2B cells as indicated in the following equation: 
	(OD450 sample&#8722; OD450 negative control/OD450 positive control&#8722; OD450 negative control) &#215; 100%</li>
	<li>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.</li>
	<li>Use 10 &#181;L pipette tips to perform nasal immunization of the mice with 10 &#181;L/nostril of I-OVA, I-OVA+BNE, and IOVA NE at 4 mg/mL for all the&#160;3 days. Use BNE and PBS as the experimental control. 
	NOTE: Provide thermal support till the animal recovers from anesthesia.</li>
	<li>Euthanize all the mice by an intraperitoneal injection of 100 mg/kg 1% sodium pentobarbital on day 4.</li>
	<li>Cut approximately 3 mm thick samples of nasal tissues with scissors, and remove the lung tissues.</li>
	<li>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 &#181;m.</li>
	<li>Stain the sections with hematoxylin and eosin (H&#38;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.</li>
</ol>
4. In vitro cellular uptake
<ol>
	<li>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 &#176;C in a 5% CO2 incubator.</li>
	<li>Add 100 &#181;L of FITC-labeled I-OVA NE (purity: 98.3%, produced by the company, 4 mg/mL) or I-OVA (4 mg/mL) to 900 &#181;L of cell suspension and place at 37 &#176;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.</li>
	<li>Wash three times with 0.1 M PBS (1 mL/well) for 30 min at 37 &#176;C after the treatment.</li>
	<li>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 &#176;C.</li>
	<li>Preincubate the samples with DAPI (4&#39;,6-diamidino-2-phenylindole) at a final concentration of 10 &#181;g/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 &#176;C after treatment.</li>
	<li>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.</li>
</ol>
5. In vivo release
<ol>
	<li>Anesthetize nude mice with 4% isoflurane gas and maintain anesthesia at 2% isoflurane. Immunize nude mice intranasally with 10 &#181;L of PE-labeled I-OVA at 4 mg/mL or PE-labeled I-OVA NE at 4 mg/mL in each nostril.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Click on the Living Image software to initiate the sequence and click Initialize to initialize the IVIS system</li>
	<li>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.</li>
	<li>Click Imaging Wizard and then choose Fluorescence in the dialog box that appears.</li>
	<li>Adjust the following parameters: Exposure Time: auto sec, Binning: 8, F/Stop: 2, Field of View: D.</li>
	<li>Select the 620 nm excitation filter and the 670 nm emission filter. Click on Acquire Sequence to acquire the image.</li>
	<li>Process the live images of all the mice and the radiance data with Living Image software.
	<ol>
		<li>After acquiring the picture, click on ROI Tools in the Tool Palette, select Circle, and make an ROI circle.</li>
		<li>Click on Measure ROIs to obtain a quantitative value for the ROI area.</li>
	</ol>
	</li>
</ol>
6. In vivo antitumor efficacy
<ol>
	<li>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%.</li>
	<li>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.</li>
	<li>Evaluate the preventive protective effect on mice as described in steps 6.3.1-6.3.4..
	<ol>
		<li>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.</li>
		<li>Intranasally immunize the mice with 10 &#181;L 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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>On day 0, inject the C57BL/6 mice subcutaneously into the right back with E.G7-OVA cells (5 x 105 cells/mouse).</li>
		<li>At 0, 7, and 14 days after injection, immunize all the mice intranasally immunized with 10 &#181;L of I-OVA, BNE+I-OVA, I-OVA NE (all at concentrations of 1 mg/mL), BNE, or PBS three times in each nostril.</li>
		<li>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,&#160;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 = &#960;/6 x L x W2 
		where L represents the tumor length, and W represents the tumor width (length unit: mm, volume unit: mm3).</li>
	</ol>
	</li>
</ol>
7. Statistical analysis
<ol>
	<li>Analyze the differences in data between the different groups using appropriate statistical software with one-way ANOVA, Tukey&#39;s multiple comparisons, or a Student&#39;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 &#177; SD. The significance of P values P &#60; 0.05, P &#60; 0.01, and P &#60; 0.001 is represented using *, **, and ***, respectively, on the plots.</li>
</ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispersity+effects+in+polymer+self-assemblies:+A+matter+of+hierarchical+control.">Dispersity effects in polymer self-assemblies: A matter of hierarchical control.</a> Chemical Society Reviews. 46 (14), 4119-4134 (2017).</li><li>Pei, M., Li, H., Zhu, Y., Lu, J., Zhang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+evidence+of+oncofetal+antigen+and+TLR-9+agonist+co-delivery+by+alginate+nanovaccines+for+liver+cancer+immunotherapy.">In vitro evidence of oncofetal antigen and TLR-9 agonist co-delivery by alginate nanovaccines for liver cancer immunotherapy.</a> Biomaterials Science. 10 (11), 2865-2876 (2022).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Titanium+dioxide+nanoparticles+induced+reactive+oxygen+species+(ROS)+related+changes+of+metabolomics+signatures+in+human+normal+bronchial+epithelial+(BEAS-2B)+cells.">Titanium dioxide nanoparticles induced reactive oxygen species (ROS) related changes of metabolomics signatures in human normal bronchial epithelial (BEAS-2B) cells.</a> Toxicology and Applied Pharmacology. 444, 116020 (2022).</li><li>Kumar, V., Sharma, N., Maitra, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+toxicity+assessment+of+nanoparticles.">In vitro and in vivo toxicity assessment of nanoparticles.</a> International Nano Letters. 7 (4), 243-256 (2017).</li><li>Tong, Y. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+immunopotentiator,+ophiopogonin+D,+encapsulated+in+a+nanoemulsion+as+a+robust+adjuvant+to+improve+vaccine+efficacy.">An immunopotentiator, ophiopogonin D, encapsulated in a nanoemulsion as a robust adjuvant to improve vaccine efficacy.</a> Acta Biomaterialia. 77, 255-267 (2018).</li><li>Elliott, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscopy:+Principles+and+modern+practices.">Confocal microscopy: Principles and modern practices.</a> Current Protocols in Cytometry. 92 (1), 68 (2020).</li><li>Huang, Y., Zou, Y., Lin, L., Zheng, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ginsenoside+Rg1+activates+dendritic+cells+and+acts+as+a+vaccine+adjuvant+inducing+protective+cellular+responses+against+lymphomas.">Ginsenoside Rg1 activates dendritic cells and acts as a vaccine adjuvant inducing protective cellular responses against lymphomas.</a> DNA and Cell Biology. 36 (12), 1168-1177 (2017).</li></ol>

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.

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 controllingphysical and chemical properties such as the measurement, shape, and electric charge. Hence, nanovaccines have become important in a wide range of applications21. These properties are major decisive factors regarding uptake and toxicity, and nanovaccines can be rendered nontoxic only by manipulation22. Therefore, this protocol for the study of physicochemical characteristics, including shape, size, and charge, is vital. TEM is a highly precise instrument that has been widely used in the scientific community for many years23. It has become an essential tool to understand the properties of nanostructured materials and to manipulate their behaviors. In addition, atomic force microscopy (AFM) has emerged as a powerful technique for the nanomechanical characterization of biological samples24. It can provide high-resolution 3D and nanoscale information while also analyzing surface details at the atomic level. In addition to determining changes in particle size distribution, DLS can measure both size and charge to provide information about the aggregation state of nanoparticles in solution25.
It has been reported that high zeta potential values are essential for the good stability of colloidal suspensions26. In this study, we used these protocols to assess the physicochemical characteristics of nanovaccines with TEM, AFM, and DLS. Moreover, the surface of the nasal mucosa contains a large amount of mucus, which provides lubrication, moisture, and a chemical protective barrier. This maybe due to the interaction between the mucus andsome antigens or delivery systems, resulting in the accumulation and &#34;capture&#34; of antigens or delivery systems and their subsequent removal, enormously reducing the delivery efficiency7. It is well known that the stability of nanovaccines is vital for nasal administration. Therefore, we used Nano ZS to determine a series of stability parameters, including the particle size, polydispersity indexes, zeta potentials, and electrophoresis mobility, after treatment with 0.5% mucin protein.
The in vivo histocompatibility and in vitro cell viability assays showed that this novel nanovaccine was nontoxic in the range of the tested concentrations27. Human normal bronchial epithelial (BEAS-2B) cells are standard cell lines used to study the human respiratory tract28. Due to its lower cost, rapidity, and minimal ethical concerns, in vitro toxicity assessment is an important method. In this study, the in vitro cell viability assay was determined by a CCK8 assay. In addition, in vivo toxicity assessment is usually performed in animal models such as mice and rats. Histopathological examination is often performed on tissues exposed to nanoparticles, such as the heart, eye, brain, liver, kidney, lung, and spleen29. Therefore, we used these methods to assess&#160;the toxicity of this novel nanovaccine in vitro and in vivo.
Antigen uptake and prolongation are prerequisites for antigen submission to trigger a subsequent immune response30. It is necessary that the cellular uptake of BEAS-2B and the release profiles of the novel nanovaccine in vivo and in vitro&#160;be determined. CLSM is the most common commercial implementation of the relevant technology, which can be found in the great majority ofimaging laboratories and has a wide range of applications. These instruments are widely used and relatively easy to use; however, they are usually not optimal for quantitative data collection.
In our protocol, cellular uptake was detected by CLSM because of the clear resolution, optical sectioning capability, and versatility with 3D imaging31. In addition, IVIS was used to obtain the in vivo release profiles of the novel nanomaterial because it can provide an imaging chamber with exterior light excluded for quantitative bioluminescence and fluorescence imaging in vivo and in vitro. Therefore, in this protocol, we used these methods to determine the cellular uptake and release profiles of novel nanovaccines in vivo and in vitro.
It is also important to assess the tumor efficacy of the novel nanovaccine. In our study, the E.G7 tumor-bearing model was used to determine the therapeutic and protective effects of the vaccine. EL4 cells are derived from the T lymphocytes of C57BL/6 mice with high-grade malignancy. E.G7 cells are derived from EL4 lymphoma cells transfected by electroporation32. In our study, this nanovaccine induced protective immunity in E.G7-OVA tumor-bearing mice. In summary, it is necessary to establish a series of protocols to study the physicochemical characteristics, stability, toxicity, release profiles, cellular uptake, and antitumor effects of nanovaccines in vitro and in vivo. These protocols will provide useful results for the novel nasal nanovaccine.

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 chemotherapy4,5. 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.
OVA257-264, the MHC class I-binding 257-264 epitope expressed as a fusion protein, is a frequently used model epitope8. In addition, OVA257-264 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 OVA257-264 peptide. It is characterized by insufficient granzyme B, which is released by cytotoxic T cells, leading to the apoptosis of target cells8. However, free OVA257-264 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 mucous9,10. 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 &#945;3&#946;1 and &#945;6&#946;1 to interact with different cancer cell types. IKVAV is a cell adhesion peptide derived from the laminin basement membrane protein &#945;1 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-OVA257-264 (I-OVA, the peptide consisting of IKVAV bound to OVA257-264) 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.

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

preparation, characteristics, toxicity, and efficacy evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo

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.

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 microphotographs stained with H&#38;E illustrate that I-OVA NE had no obvious mucosal toxicity, including tissue damage, bleeding, or inflammatory cell infiltration (Figure 2B). Efficient uptake of the antigen by BEAS-2B cells in the nasal cavity is a prerequisite for antigen presentation to elicit subsequent immune responses (Figure 3). The IVIS system helps to elucidate the sustained-release effect of I-OVA NE in vitro and suggests that this nanovaccine can delay rapid release, prolong the time in the nasal area, and improve the uptake of peptides in cells (Figure 4). The preventive protective models and the therapeutic protective models directly reflect the protective effect of the I-OVA NE vaccine and the ability of the I-OVA NE vaccine to inhibit tumor growth and prolong the median survival time of mice (Figure 5). The above experimental results have been published by Yang et al.7.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64299/64299fig01.jpg" /> 
Figure 1: Physical characteristics and stability of I-OVA NE. (A) Transmission electron micrograph (TEM), scale bar = 100 nm. (B) Atomic force microscopy (AFM) micrograph. The X and Y axes both have a total length of 450 nm. (C) Size diameter and distribution. (D) Zeta potential and distribution of I-OVA NE analyses performed using Nano ZS. (E) Particle sizes, (F) polydispersity indexes, (G) zeta potentials, and (H) electrophoresis mobility of I-OVA NE in mucin stability analyses performed using Nano ZS. This figure has been adapted with permission from Yang et al.7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64299/64299fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64299/64299fig02.jpg" /> 
Figure 2: In vitro and in vivo toxicity of I-OVA NE. (A) Relative viability of BEAS-2B cells in culture exposed to different peptide concentrations of I-OVA, BNE+I-OVA, and I-OVA NE for 24 h. BNE was used as a control. The data are expressed as the mean &#177; SD (n = 3). (B) Microscopic examination of pathological sections of the nasal mucosa and lung tissues fixed 5 h after the challenge. Images were captured at 100x and 200x magnification. This figure has been adapted with permission from Yang et al.7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64299/64299fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64299/64299fig03.jpg" /> 
Figure 3: Cellular uptake of the I-OVA NE. In vitro confocal fluorescence imaging of BEAS-2B cells treated for 1 h with I-OVA or I-OVA NE. PBS was used as a control, I-OVA was labeled with FITC (green fluorescence), and the nuclei were stained with DAPI (blue fluorescence) (scale bar = 50 &#181;m). This figure has been adapted with permission from Yang et al.7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64299/64299fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64299/64299fig04.jpg" /> 
Figure 4: In vitro release of I-OVA NE. (A) In vivo fluorescence imaging of PE-labeled I-OVA in the mouse nasal cavity. Relative fluorescence intensity recorded at 0 h, 0.5 h, 1.5 h, 3 h, 6 h, 9 h, 12 h, and 24 h after nasal administration of I-OVA or I-OVA NE. (B) Quantitation of fluorescence intensity. The data are expressed as the mean &#177; SD (n = 5). *: P &#60; 0.05; **: P &#60;0.01; and ***: P &#60; 0.001. This figure has been adapted with permission from Yang et al.7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64299/64299fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64299/64299fig05.jpg" /> 
Figure 5: In vivo anti-tumor efficacy of I-OVA NE. (A) Average tumor growth curves of the vaccinated mice in the preventive protective models. (B) Percent survival rate of the vaccinated mice in the preventive protective models. (C) Average tumor growth curves of the vaccinated mice in the therapeutic protective models. (D) Percent survival rate of the vaccinated mice in the therapeutic protective models. *: P &#60; 0.05; **: P &#60; 0.01; and ***: P &#60; 0.001. This figure has been adapted with permission from Yang et al.7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64299/64299fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo at JoVE.com

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

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

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

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; ...

preparation-characteristics-toxicity-efficacy-evaluation-nasal-self

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

1.6K Views.  Third Military Medical University of Chinese PLA. Nieuw nano-emulsievaccin heeft grote voordelen bij ziektetactiektherapie. Deze set-effecten worden geminimaliseerd door eigenschappen zoals uniek tumor chopisme, de identificatie van specifieke tactieken en de lage systemische toxiciteit. We verwachten dat het protocol technische en theoretische aanwijzingen zal bieden voor de toekomstige ontwikkeling van nieuwe T-cel ectype peptide mucosale vaccins.Begin met het mengen van een milligram monofosforollipide A met 100 microliter DMSO. Vo...

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

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

Cancer Research

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Medicine

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Bioengineering

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1&#945;) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1&#945; would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1&#945; systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1&#945;-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1&#945;-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; in a syngeneic mouse model of HNSCC.

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Synthesis and Characterization of mRNA-Loaded Poly(Beta Aminoesters) Nanoparticles for Vaccination Purposes

Vaccination has been one of the major successes of modern society and is indispensable in controlling and preventing disease. Traditional vaccines were composed of entire or fractions of the infectious agent. However, challenges remain, and new vaccine technologies are mandatory. In this context, the use of mRNA for immunizing purposes has shown an enhanced performance, as demonstrated by the speedy approval of two mRNA vaccines preventing SARS-CoV-2 infection. Beyond success in preventing viral infections, mRNA vaccines can also be used for therapeutic cancer applications.
Nevertheless, the instability of mRNA and its fast clearance from the body due to the presence of nucleases makes its naked delivery not possible. In this context, nanomedicines, and specifically polymeric nanoparticles, are critical mRNA delivery systems. Thus, the aim of this article is to describe the protocol for the formulation and test of an mRNA vaccine candidate based on the proprietary polymeric nanoparticles. The synthesis and chemical characterization of the poly(beta aminoesters) polymers used, their complexation with mRNA to form nanoparticles, and their lyophilization methodology will be discussed here. This is a crucial step for decreasing storage and distribution costs. Finally, the required tests to demonstrate their capacity to in vitro transfect and mature model dendritic cells will be indicated. This protocol will benefit the scientific community working on vaccination because of its high versatility that enables these vaccines to prevent or cure a wide variety of diseases.

Synthesis and Characterization of mRNA-Loaded PolyBeta Aminoesters Nanoparticles for Vaccination Purposes

Here, a simple protocol is presented for producing mRNA nanoparticles based on poly(beta aminoester) polymers, easy to be tailored by changing the encapsulated mRNA. The workflow for synthesizing the polymers, the nanoparticles, and their in vitro essential characterization are also described. A proof-of-concept regarding immunization is also added.

Vaccination has been one of the major successes of modern society and is indispensable in controlling and preventing disease. Traditional vaccines were ...

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated with antigens. Ophiopogonin D (OP-D), a purified component extracted from the plant Ophiopogon japonicus, has been found to be useful as a vaccine adjuvant. The problems of the low solubility and toxicity of OP-D can be effectively overcome by using a low-energy emulsification method to prepare nanoemulsion ophiopogonin D (NOD). In this article, a series of in vitro protocols for cellular activity evaluation are examined. The cytotoxic effects of L929 were determined using a cell counting kit-8 assay. Then, the secreted cytokine levels and corresponding immune cell numbers after the stimulation and culture of splenocytes from immunized mice were detected by ELISA and ELISpot methods. In addition, the antigen uptake ability in bone marrow-derived dendritic cells (BMDCs), which were isolated from C57BL/6 mice and matured after incubation with GM-CSF plus IL-4, was observed by laser scanning confocal microscopy (CLSM). Importantly, macrophage activation was confirmed by measuring the levels of IL-1&#946;, IL-6, and tumor necrosis factor alpha (TNF-&#945;) cytokines by ELISA kits after coculturing peritoneal macrophages (PMs) from blank mice with the adjuvant for 24 h. It is hoped that this protocol will provide other researchers with direct and effective experimental approaches to evaluate the cellular response efficacies of novel vaccine adjuvants.

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

The protocol presents detailed methods for evaluating whether the nanoemulsion ophiopogonin D adjuvant promotes effective cellular immune responses.

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated ...

Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus (MRSA) Infection

Nanoemulsion adjuvant vaccines have attracted extensive attention because of their small particle size, high thermal stability, and ability to induce validly immune responses. However, establishing a series of comprehensive protocols to evaluate the immune response of a novel nanoemulsion adjuvant vaccine is vital. Therefore, this article features a rigorous procedure to determine the physicochemical characteristics of a vaccine (by transmission electron microscopy [TEM], atomic force microscopy [AFM], and dynamic light scattering [DLS]), the stability of the vaccine antigen and system (by a high-speed centrifuge test, a thermodynamic stability test, SDS-PAGE, and western blot), and the specific immune response (IgG1, IgG2a, and IgG2b). Using this approach, researchers can evaluate accurately&#160;the protective effect of a novel nanoemulsion adjuvant vaccine in a lethal MRSA252 mouse model. With these protocols, the most promising nanoemulsion vaccine adjuvant in terms of effective adjuvant potential can be identified. In addition, the methods can help optimize novel vaccines for future development.

Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus MRSA Infection

The present protocol prepares and evaluates the physical properties, immune response, and in vivo protective effect of a novel nanoemulsion adjuvant vaccine.

Nanoemulsion adjuvant vaccines have attracted extensive attention because of their small particle size, high thermal stability, and ability to induce ...

Evaluating Immunogenicity of Nanoemulsion&#45;Encapsulated Retinoic Acid in Mice

Cationic Nanoemulsion-Encapsulated Retinoic Acid as an Adjuvant to Promote OVA&#45;Specific Systemic and Mucosal Responses

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen uptake and then promotes antigen-specific immune responses. In recent years, retinoic acid (RA) has received increasing attention due to its effect in activating the mucosal immune response; however, in order to use RA as a mucosal adjuvant, it is necessary to solve the problem of its dissolution, loading, and delivery. Here, we describe a cationic nanoemulsion-encapsulated retinoic acid (CNE-RA) delivery system composed of the cationic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOTAP), retinoic acid, squalene as the oil phase, polysorbate 80 as surfactant, and sorbitan trioleate 85 as co-surfactant. Its physical and chemical properties were characterized using dynamic light scattering and a spectrophotometer. Immunization of mice with the mixture of antigen (ovalbumin, OVA) and CNE-RA significantly elevated the levels of anti-OVA secretory immunoglobulin A (sIgA) in vaginal lavage fluid and the small intestinal lavage fluid of mice compared with OVA alone. This protocol describes a detailed method for the preparation, characterization, and evaluation of the adjuvant effect of CNE-RA.

Author Spotlight: Development and Evaluation of a Cationic Nanoemulsion&#45;Encapsulated Retinoic Acid System for Mucosal Vaccination

In this protocol, we developed a cationic nanoemulsion-encapsulated retinoic acid (RA) to be used as an adjuvant to promote antigen-specific systemic and mucosal responses. By adding the FDA-approved RA to the nanoemulsion, antigen-specific sIgA was promoted in the vagina and small intestine after intramuscular injection of the nanoemulsion.

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen ...

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

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