Periodic Reporting for period 1 - NMR4Nanos (Development and application of NMR-based tools to inorganic nanocarriers for effective vaccine delivery)
Okres sprawozdawczy: 2017-09-04 do 2019-09-03
The period between 2011-2020 has been designated by WHO the Decade of Vaccines . In addition to the eradication of poliomyelitis, the reduction of child mortality and other goals of prime interest, special focus is given to the development and introduction of new and improved vaccine technologies.
Since the first vaccines that contained attenuated or dead microorganisms a series of discoveries led to the concept of third generation vaccines that are safe and can induce long term protection against infections. These new insights, which also enabled development of the principles for therapeutic vaccines against cancer, were the following:
- Part of the microorganism is sufficient to generate an adequate immune response, moreover, a DNA sequence encoding the production of a protein specific to the given microorganism can also lead to the activation of the immune system. The benefit of this solution is the fast, cheap and safe production of the antigen in the form of a DNA sequence in comparison to using the whole pathogen.
- Some cancer antigens have been identified that allow for the prevention or immunotherapy of specific tumours.
- Specific auxiliary substances called adjuvants are needed to enhance the reaction of the body to antigens. Without adjuvants, antigens can be degraded and eliminated from the body or the immune response against the antigen is too weak. This is especially applicable to cancer antigens, which are recognized by the body as endogenous (like the body itself) and as result the production of killer cells against them is reduced.
- Adjuvants can be, for example, short DNA fragments of the bacterial or viral genome that are recognized as dangerous intruders. One promising type of adjuvants are CpG ODNs (OligoDeoxyNucleotides with CpG motifs) that are particularly suitable inducing strong immune responses against tumor cells and thus are used as adjuvants in cancer vaccine development.
- Small vehicles such as microparticles and even smaller nanoparticles have an intrinsic additional adjuvant effect due to their size, which is similar to the size of pathogens. This effect is independent of the material of the particles. Furthermore, small carriers can bind and transport several adjuvants and antigens on their surface presenting them together to immune cells.
- Immune cells recognize the nanoparticles as intruders, engulf them and take them up into specific organelles. As a result, the nanocarriers quickly accumulate in the immune cells and deliver the antigen and the adjuvant to their target cell, where they can launch the immune response.
Objectives
Based on this framework, our project aimed at the preparation of new type of nanoparticles that can readily bind CpG ODN adjuvants on their surface and can potentially become potent adjuvant nanocarriers for cancer vaccination. The scientific objectives were:
1. elaboration of the new nanocarriers
2. application of nuclear magnetic resonance techniques to confirm the binding of oligonucleotides at the surface of the nanocarriers and to characterize the interaction
3. preliminary evaluation of the new nanocarriers in biological assays that would tell us, if our product has an added value in comparison to the CpG ODN adjuvant alone.
Since the first vaccines that contained attenuated or dead microorganisms a series of discoveries led to the concept of third generation vaccines that are safe and can induce long term protection against infections. These new insights, which also enabled development of the principles for therapeutic vaccines against cancer, were the following:
- Part of the microorganism is sufficient to generate an adequate immune response, moreover, a DNA sequence encoding the production of a protein specific to the given microorganism can also lead to the activation of the immune system. The benefit of this solution is the fast, cheap and safe production of the antigen in the form of a DNA sequence in comparison to using the whole pathogen.
- Some cancer antigens have been identified that allow for the prevention or immunotherapy of specific tumours.
- Specific auxiliary substances called adjuvants are needed to enhance the reaction of the body to antigens. Without adjuvants, antigens can be degraded and eliminated from the body or the immune response against the antigen is too weak. This is especially applicable to cancer antigens, which are recognized by the body as endogenous (like the body itself) and as result the production of killer cells against them is reduced.
- Adjuvants can be, for example, short DNA fragments of the bacterial or viral genome that are recognized as dangerous intruders. One promising type of adjuvants are CpG ODNs (OligoDeoxyNucleotides with CpG motifs) that are particularly suitable inducing strong immune responses against tumor cells and thus are used as adjuvants in cancer vaccine development.
- Small vehicles such as microparticles and even smaller nanoparticles have an intrinsic additional adjuvant effect due to their size, which is similar to the size of pathogens. This effect is independent of the material of the particles. Furthermore, small carriers can bind and transport several adjuvants and antigens on their surface presenting them together to immune cells.
- Immune cells recognize the nanoparticles as intruders, engulf them and take them up into specific organelles. As a result, the nanocarriers quickly accumulate in the immune cells and deliver the antigen and the adjuvant to their target cell, where they can launch the immune response.
Objectives
Based on this framework, our project aimed at the preparation of new type of nanoparticles that can readily bind CpG ODN adjuvants on their surface and can potentially become potent adjuvant nanocarriers for cancer vaccination. The scientific objectives were:
1. elaboration of the new nanocarriers
2. application of nuclear magnetic resonance techniques to confirm the binding of oligonucleotides at the surface of the nanocarriers and to characterize the interaction
3. preliminary evaluation of the new nanocarriers in biological assays that would tell us, if our product has an added value in comparison to the CpG ODN adjuvant alone.
The nanocarriers were prepared in four different sizes between 20 to 100 nanometer (nm) in diameter. We carried out an in-depth analysis of their structure and followed their changes in time to find the optimal age of the particles for the binding of CpG ODN adjuvants. As smaller particles can bind more oligonucleotides on their surface, we have chosen the smallest particle size that stayed stable to investigate the binging of CpG ODN adjuvant. This was found to be 50 nm in diameter.
After preparation of the particles in ethanol, we transferred the nanocarriers into water and studied their stability in aqueous solution. We found that the transfer into water changed the crystallinity of the nanocarriers and thus increased the number of surface defects enabling increased binding of oligonucleotides. This was highly beneficial for improving capacity of adjuvant binding. The particles transferred into water could bind larger amount of oligonucleotide than those in ethanol. As a result, our new nanocarrier could bind higher amount of CpG ODN adjuvants than similar nanocarriers described earlier by other researchers. This was due to the improved preparation method leading to different crystallinity and smaller particle size.
We studied the details of the surface modification of the particles on the molecular level using multiple physicochemical techniques. Nuclear magnetic resonance spectroscopy was proven particularly useful in detecting and characterizing the binding of oligonucleotides to the surface of the nanocarriers. It enabled us to directly observe the binding event to the nanoparticle surface and determine the nature of the interaction.
In order to evaluate the toxicity and the immunological properties of the functionalized nanocarriers, they were prepared under sterile conditions. In the first round, they were added to sensitive liver cells to study their eventual toxicity, however, no adverse effects were observed. The toxicity of the nanocarriers were further tested on specific immune cells, whereby we found that the cells have taken up the nanocarriers in large quantities, but no toxic effects were observed with these cells either. Finally, in order to evaluate the effectiveness of the developed nanoplatform, we studied to what extent the functionalized nanocarriers induced maturation of specific immune cells, which is required for the desired immune activation in fighting cancer. We found that the nanocarriers modified with the CpG ODN adjuvants on their surface induced higher degree of maturation of the immune cells than the adjuvant monotherapy alone. Thus, they were more effective in inducing immune responses required against tumor cells. In summary, the developed nanocarriers could be useful as vaccine adjuvant carriers as they can improve the effect of adjuvants and deliver them safely to immune cells.
After preparation of the particles in ethanol, we transferred the nanocarriers into water and studied their stability in aqueous solution. We found that the transfer into water changed the crystallinity of the nanocarriers and thus increased the number of surface defects enabling increased binding of oligonucleotides. This was highly beneficial for improving capacity of adjuvant binding. The particles transferred into water could bind larger amount of oligonucleotide than those in ethanol. As a result, our new nanocarrier could bind higher amount of CpG ODN adjuvants than similar nanocarriers described earlier by other researchers. This was due to the improved preparation method leading to different crystallinity and smaller particle size.
We studied the details of the surface modification of the particles on the molecular level using multiple physicochemical techniques. Nuclear magnetic resonance spectroscopy was proven particularly useful in detecting and characterizing the binding of oligonucleotides to the surface of the nanocarriers. It enabled us to directly observe the binding event to the nanoparticle surface and determine the nature of the interaction.
In order to evaluate the toxicity and the immunological properties of the functionalized nanocarriers, they were prepared under sterile conditions. In the first round, they were added to sensitive liver cells to study their eventual toxicity, however, no adverse effects were observed. The toxicity of the nanocarriers were further tested on specific immune cells, whereby we found that the cells have taken up the nanocarriers in large quantities, but no toxic effects were observed with these cells either. Finally, in order to evaluate the effectiveness of the developed nanoplatform, we studied to what extent the functionalized nanocarriers induced maturation of specific immune cells, which is required for the desired immune activation in fighting cancer. We found that the nanocarriers modified with the CpG ODN adjuvants on their surface induced higher degree of maturation of the immune cells than the adjuvant monotherapy alone. Thus, they were more effective in inducing immune responses required against tumor cells. In summary, the developed nanocarriers could be useful as vaccine adjuvant carriers as they can improve the effect of adjuvants and deliver them safely to immune cells.
First of all, the success of this project provided a ‘proof of concept’ that the developed material is suitable as a nanocarrier in vaccines. Second, we have shown the usefulness of nuclear magnetic resonance techniques to study the details of the binding of biomolecules to inorganic nanoparticles, thus providing a more detailed insight into the nature of interactions between the components of the nanovaccine platform at the molecular level.
The results of this project were shared with other scientists through presentations in meetings, publications in scientific journals and were also presented to potential investors. Through dedicated dissemination of the results of the project, we have contributed to the increase of overall knowledge in the field and hope to have incited interest that can bring about further investments into the development of similar therapeutic products in the future.
The results of this project were shared with other scientists through presentations in meetings, publications in scientific journals and were also presented to potential investors. Through dedicated dissemination of the results of the project, we have contributed to the increase of overall knowledge in the field and hope to have incited interest that can bring about further investments into the development of similar therapeutic products in the future.