Freeze-drying of ovalbumin loaded mesoporous silica nanoparticle vaccine formulation increases antigen stability under ambient conditions

Mody, Karishma T.; Mahony, Timothy J.; Cavallaro, Antonino S.; Stahr, Frances; Qiao, Shi Zhang; Mahony, Timothy J.; Mitter, Neena

doi:10.1016/j.ijpharm.2014.01.037

Cited by 21 publications

(21 citation statements)

References 35 publications

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“…In our laboratory, we have previously described the use of OVA loaded amino functionalised MSNs and BVDV E2 loaded on hollow mesoporous silica nanoparticles (HMSA) (23)(24)(25). Though, we were able to get low levels of detectable antibody and cell-mediated responses, the major limitations were very low protein loading efficiency (60e80 mg/mg of particles), small entrance size (2e3.5 nm) and possibly only surface presentation of proteins on these silica nanocarriers.…”

Section: Introductionmentioning

confidence: 99%

Silica vesicles as nanocarriers and adjuvants for generating both antibody and T-cell mediated immune resposes to Bovine Viral Diarrhoea Virus E2 protein

et al. 2014

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Section: Introductionmentioning

confidence: 99%

Silica vesicles as nanocarriers and adjuvants for generating both antibody and T-cell mediated immune resposes to Bovine Viral Diarrhoea Virus E2 protein

et al. 2014

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“…After washing, cells were treated with trypan blue staining as indicated above. For a long time, trypan blue dye has been generally used at 0.2% concentration in independent studies on different cell types [4][5][6][7]. The dye penetrates into membranes of dead cells since dead cells have damaged membranes, whereas the dye is excluded from live cells with intact cell membrane so that dead cells are seen as blue under light microscope (Figure 4a) [8].…”

Section: Resultsmentioning

confidence: 99%

Development of a Direct Trypan Blue Exclusion Method to Detect Cell Viability of Adherent Cells into ELISA Plates

Uzuner¹

2018

Celal Bayar Üniversitesi Fen Bilimleri Dergisi

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Cell viability detection is important in cell culture applications including measurement of cell proliferation i.e for understanding cytotoxic effects of compounds on cells. There are some cell viability methods based on fluorescence or non-fluorescence detection. More simplified evaluation for cell viability, such as trypan blue staining, can be preferred before performing fluorescence assays. This appears advantageous when to have a large number of cell samples in ELISA plates after treatments with different concentrations of drug candidates. Thus, further fluorescence assays can include less concentrations rather than experiencing all used along 96well plates. For this, trypan blue exclusion method is an option. Traditionally, treated cells are harvested by centrifugation and incubated with trypan blue within tubes followed by transferring the mixture into a hemacytometer with two chambers and assessed under the microscope. Nevertheless, using a hemacytometer limits practicability of this method when analyzing various cell samples into 96-well plates at the same time. This study was aimed to adapt trypan blue method to in situ staining of adherent cells cultured on ELISA plates. For this, cells were fixed with different fixatives after trypan blue incubation to maintain cells in impenetrable meshwork, and paraformaldehyde was the most effective fixative. This modified protocol was validated by testing the effect of dimethylsulfoxide-a cytotoxic agent-on cells, and expectedly found that cell viability reduced with higher concentrations of dimethylsulfoxide suggesting that in situ detection of cell viability by trypan blue can be a useful tool for preliminary detection of cells cultured on ELISA plates before performing automatized experiments with such flow cytometer and/or microplate reader.

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“…Previously we have shown successful freeze-drying of ovalbumin adsorbed mesoporous silica nanoparticles using 5% trehalose and 1% PEG8000 [ 21 ]. Therefore the first combination that was trialled with Opti-E2 bound HMSA was 5% trehalose with either 1%, 0.5% or 0.1% PEG8000.…”

Section: Resultsmentioning

confidence: 99%

“…The freeze-dried formulation included the excipients 5% trehalose and 1% glycine which are not known to be immunogenic. We have previously used 5% trehalose and 1% PEG8000 [ 21 ] with ovalbumin protein to generate freeze-dried nanovaccine and did not see increased cell-mediated responses. Once the reconstituted vaccine is injected the excipients should be rapidly desorbed from the injection site and be metabolised or cleared from the system of the animal and therefore not have any non-specific stimulatory effect on the immune system.…”

Section: Resultsmentioning

confidence: 99%

Immunisation of Sheep with Bovine Viral Diarrhoea Virus, E2 Protein Using a Freeze-Dried Hollow Silica Mesoporous Nanoparticle Formulation

et al. 2015

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Bovine viral diarrhoea virus 1 (BVDV-1) is arguably the most important viral disease of cattle. It is associated with reproductive, respiratory and chronic diseases in cattle across the world. In this study we have investigated the capacity of the major immunological determinant of BVDV-1, the E2 protein combined with hollow type mesoporous silica nanoparticles with surface amino functionalisation (HMSA), to stimulate immune responses in sheep. The current work also investigated the immunogenicity of the E2 nanoformulation before and after freeze-drying processes. The optimal excipient formulation for freeze-drying of the E2 nanoformulation was determined to be 5% trehalose and 1% glycine. This excipient formulation preserved both the E2 protein integrity and HMSA particle structure. Sheep were immunised three times at three week intervals by subcutaneous injection with 500 μg E2 adsorbed to 6.2 mg HMSA as either a non-freeze-dried or freeze-dried nanoformulation. The capacity of both nanovaccine formulations to generate humoral (antibody) and cell-mediated responses in sheep were compared to the responses in sheep immunisation with Opti-E2 (500 μg) together with the conventional adjuvant Quil-A (1 mg), a saponin from the Molina tree (Quillaja saponira). The level of the antibody responses detected to both the non-freeze-dried and freeze-dried Opti-E2/HMSA nanoformulations were similar to those obtained for Opti-E2 plus Quil-A, demonstrating the E2 nanoformulations were immunogenic in a large animal, and freeze-drying did not affect the immunogenicity of the E2 antigen. Importantly, it was demonstrated that the long term cell-mediated immune responses were detectable up to four months after immunisation. The cell-mediated immune responses were consistently high in all sheep immunised with the freeze-dried Opti-E2/HMSA nanovaccine formulation (>2,290 SFU/million cells) compared to the non-freeze-dried nanovaccine formulation (213–500 SFU/million cells). This study is the first to demonstrate that a freeze-dried silica mesoporous nanovaccine formulation gives balanced immune responses in a production animal.

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Freeze-drying of ovalbumin loaded mesoporous silica nanoparticle vaccine formulation increases antigen stability under ambient conditions

Cited by 21 publications

References 35 publications

Silica vesicles as nanocarriers and adjuvants for generating both antibody and T-cell mediated immune resposes to Bovine Viral Diarrhoea Virus E2 protein

Silica vesicles as nanocarriers and adjuvants for generating both antibody and T-cell mediated immune resposes to Bovine Viral Diarrhoea Virus E2 protein

Development of a Direct Trypan Blue Exclusion Method to Detect Cell Viability of Adherent Cells into ELISA Plates

Immunisation of Sheep with Bovine Viral Diarrhoea Virus, E2 Protein Using a Freeze-Dried Hollow Silica Mesoporous Nanoparticle Formulation

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