TITULO IX : DE LAS MEDIDAS DE SEGURIDAD, INFRACCIONES
DE LAS MEDIDAS DE SEGURIDAD, INFRACCIONES Y SANCIONES
It is vital to assess the viability of the cells when these are stimulated with a certain substance. It is important to observe elevated signal from the receptors, and elevated cytokine expression, however, the potential adjuvant cannot be too toxic because the cells would be terminated which is not desired, some cell death is not a bad outcome though as it sends signals and stimulates the immune response. The viability testing was performed with Alamar Blue assay for Zr- MOFs, and by FACS for Al-MOFs.
Figure 6-10 Viability of dendritic cells after exposure to Al-MOFs and alum for 24h, measured by FACS based on Annexin V/PI staining. Results were averaged based on 3 independent experiments, courtesy of Dr Kubajewska. Error bars are SEM (standard mean of error).
Based on the results obtained (Figure 6-10) for the viability screening, almost all the MOFs, except MIL-101-NH2 can be regarded as non-toxic and do not
compromise the safety of the living system - the cells. MIL-101-NH2 decreases
viability to70 % and when compared with different tested MOFs, may show higher toxicity, due to the presence of amine group. Alum shows the viability at the level of 80%.
Similar experiments were performed to measure the viability of macrophages when stimulated with Zr-MOFs. After the 24-hour simulation was completed, the aliquots of cells suspension containing secreted cytokines were harvested. Please refer to Chapter 3 Section 3.5.2. for a detailed description of the procedure.
131 Figure 6-11 Viability of macrophages (THP-1 cells) after 24-hour stimulation with Zr-MOFs and alums AlOOH and ALPO4, measured by Alamar Blue assay. Error bars refer to SEM (standard error of mean), results from 2 independent experiments, each in duplicate.
The viability results show that the stimulation with Zr-MOFs did not compromise the cell viability, as opposed to the alum samples (AlO(OH) and ALPO4), which
decreased the number of live cells down to 84-85%. Control wells, stimulated with PBS solution or LPS (100 ng/ml) show that LPS decreases the viability slightly, PBS stimulation does not harm the cells. The results obtained, viability testing on dendritic cells and macrophages, show that Zr-MOF and Al-MOF, apart from MIL-101-NH2, are non-toxic to the cells and could be potentially used as
adjuvants. The obtained results show that generally MOFs do not compromise cells’ viability. This property of MOFs combined with the data showing the induction of higher immune responses based on the levels of IL-6 and TNF- cytokines and the expression of cell receptors on the dendritic cells responsible for the stimulation of T cells, suggests that MOFs might be strong candidates for novel adjuvants in vaccines. Recently, some attempts were made to synthesise biocompatible MOFs with lower toxicity, especially the ones containing Fe in their framework30-32, the MOFs containing Cr and Co cations in their framework show the highest toxicity33, 34, but still can be used as drug carriers for anti-cancer drugs as the latter are very toxic anyway.
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6.4.Conclusions
The concept of providing a more potent adjuvant to replace alum was the foundation of this research project. It is strongly believed that Al (Aluminium) may be the key to triggering the immune responses3, and with the new class of MOF materials allowing to play around with variety of their structures and linkers with an exact composition, showed a promising route to follow as MOFs are seen as potential candidates for variety of applications, with some in medicine35-37. There have been a lot of vaccines invented over the years for variety of diseases, with the idea to immunize the organism with the pathogen’s subunit and induce specific immune response against it. Likewise, the idea that links the immune system with the cancer prevention emerged, and there were some trials made to design a vaccine for cervical cancer that led to Gardasil invention38.
In this chapter, a series of Al- and Zr-containing MOFs were synthesised and their immunogencity explored in the in vitro studies. Experiments were performed to probe the maturation of macrophages and dendritic cells, looking at the levels of cytokines secreted by the macrophages and surface protein expression by the dendritic cells. The results show that MOFs containing Al-metal in their framework were more potent in provoking cytokine secretion than a commercially used alum or MOFs with a Zr-metal, and leading to increased concentrations of TNF-α and IL-6. The cell viability levels were also higher in the case of Al- MOFs, than those measured for alum, and it may be concluded that alum led to decreased viability. Upon stimulation with Al-MOFs, higher percentages of dendritic cells (DCs) expressed signal from CD40, CD86 and CD1a receptors and thus are expected to be able to activate T-cells more effectively than alum39. The results obtained with Zr-MOFs showed the levels of cytokines secreted were only slightly higher than these obtained after stimulation with alum. The viability studies showed that Zr-MOFs are non-toxic to the cells and the viability levels are sustained at nearly 100%.
Overall, the data collected indicated that Al-containing MOFs could be potent vaccine adjuvants. To prove this, further studies must be performed to link different parameters of the substances e.g. shape of grains, size of particles, zeta- potential, distances between Metal-carbon chemistry of the compounds to their
133 immunogenic properties. This would allow to build an adjuvant model and predict the potentially best match for an adjuvant. Such modelling was performed by Dr Williams showing promising results for LDH (layered double hydroxides) to link their chemistry to their performance as adjuvants16. The in vivo results were promising. However, in case of MOFs, in order to prove the model, some more in vitro studies are needed and ultimately some in vivo, also taking into consideration further MOF structures.
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