[0:00]Thank you so much. So my name is Stephanie Andrade, and today I will speak a little bit of the work that I have developed during my PhD, that is based on the production and characterization of transferrin-modified nanoparticles for Alzheimer's disease therapy with natural compounds.
[0:19]So, as you may know, Alzheimer's disease is the most common form of dementia, accounting with around 70% of the cases in the world.
[0:30]Alzheimer's disease is a progressive and irreversible disease that affects cognitive functions like memory.
[0:37]The age is the major risk factor of the disease, and with the increase of the life expectancy, it is predictable that in 2050, the number of patients with Alzheimer will triplicate.
[0:52]Unfortunately, the disease remains incurable since no medications are able to prevent or cure the disease.
[1:00]Some months ago, was approved the first medication that targets the pathogenesis of the disease.
[1:06]However, Adulm is under a lot of controversy because the clinical benefit of the medication were not proved.
[1:17]So, what is behind the Alzheimer's disease?
[1:21]The mechanism of the disease, why why the disease appears is still unknown.
[1:26]However, the brain of Alzheimer's patients is characterized by the presence of plaques composed of amyloid beta peptide.
[1:35]This peptide is present in the brain of all of us, in the monomeric form that is not toxic.
[1:46]However, by a mechanism that is still not well understood, monomers start to aggregate into oligomers, then protofibrils and then fibrils.
[1:55]So, there is several evidence that establish a direct relationship between the degree of the disease and the amount of aggregates.
[2:06]Therefore, strategies that can inhibit the aggregation of the peptide, or disaggregate fibrils are becoming very relevant.
[2:17]So, in this context, natural compounds have arose a lot of a lot of interest due to their neuroprotective effects and also because they are presumably safe for human intake.
[2:32]Among those natural compounds that have been described for Alzheimer's disease, caffeic acid is a natural compound known to to have antioxidant and anti-inflammatory properties, and our group recently discovered that caffeic acid is able to interact with amyloid beta, inhibiting the aggregation of the peptide and also disrupting performing fibrils.
[2:56]However, like many other compounds, caffeic acid has some limitations that hinders its clinical application, like its low bioavailability and also the lack of specificity to the blood-brain barrier.
[3:11]The BBB, it's a barrier that impairs the passage of toxic substances from the blood to the brain, however, it also induces the passage of therapeutic compounds.
[3:24]So, to overcome the limitation of caffeic acid and other molecules, nanocarriers have been developed for drug delivery.
[3:35]Nanoparticles are able to protect compounds from degradation, masking their limiting properties without modifying them.
[3:47]They also allow for controlled and sustained drug release, and have the ability to interact with specific receptors, therefore being able to direct our nanoparticles to a specific site.
[4:02]They are also recognized to be biocompatible and biodegradable.
[4:06]From the a lot of kind of particles that that are being described, liposomes are the are one of of kind of particles that have been widely used.
[4:19]So liposomes are spherical vesicles composed of of phospholipids.
[4:26]Conventional liposomes were the first generation to be to be developed.
[4:31]They are composed by neutral and anionic phospholipids, however, they are quickly cleared from our immune system.
[4:40]It is why appeared the second generation by adding polymers like PEG, it allows to increase the circulation time of the liposomes in the in the body.
[4:54]So the main goals of these studies was to produce liposomes containing our natural compounds caffeic acid by several techniques, aiming to find the best technique, so the formulation with the best properties.
[5:10]Then, we we modified the the surface of the liposomes with transferrin to direct our liposomes to the blood-brain barrier because the the BBB have the the transferrin receptors overexpressed.
[5:27]This way, we are able to direct our liposomes to the BBB.
[5:31]We then evaluate the anti-immunogenic properties of our nano system.
[5:39]So, our liposomes are composed of neutral phospholipids. We also added cholesterol and PEG to increase the stability of our nano system, and as I told you, we added transferrin to to the surface of the liposomes.
[5:55]So four techniques were used to produce the liposomes.
[6:00]We characterized our nanoparticles according to their size, the potential, encapsulation efficiency, conjugation efficiency, stability at storage conditions, and release profile of caffeic acid.
[6:14]We then evaluated the ability of our of our system to inhibit the aggregation of A-beta and disrupt the mature preformed fibrils.
[6:26]So, here are the the characterization of our liposomes produced by the four techniques.
[6:33]We can see here, the size is one of the most important characteristic because nanoparticles larger than 200 nanometers cannot cross the BBB.
[6:46]So, as we can see here, all techniques, liposomes have all sizes lower than 200, which is good for brain delivery.
[7:00]Then we have here the the polydispersity index.
[7:05]All of our formulation have values lower than 0.3, which suggests that the liposomes the formulation have a monodisperse monodisperse population in terms of size.
[7:16]We have then the zeta potential of all formulation that is close to zero, it is expected due to the nature of our liposomes, and then we have here the encapsulation efficiency.
[7:30]We can see here that from the four techniques uh tested, the reverse-phase evaporation was the one that presented higher values, 23% of encapsulation.
[7:44]Therefore, we select this technique in the further experiments.
[7:48]So, we then modified the surface of the liposomes with transferrin.
[7:54]We can see that transferrin did not affect the properties of our of our liposomes, in terms of size, PDIs, zeta potential, and encapsulation efficiency.
[8:06]And we can see here that we obtain a conjugation efficiency of 21%.
[8:14]This corresponds to about 80% of transferring molecules per liposome.
[8:21]This number allows according to the literature allows nanoparticles to cross the BBB.
[8:26]Then we performed FTIR to confirm the functionalization of our nanoparticles.
[8:33]Here in blue, we have the spectrum of transferrin.
[8:36]We can see that is characterized by two main two main bands, we can see here the value corresponding to amine 1 and amine 2.
[8:44]We can see that the two peaks are present in the in the transferring modified liposomes.
[8:52]In contrast, when we have just our liposomes without functionalization, we don't have the first peak, which confirms that these two peaks are due to the conjugation of transferrin.
[9:07]We also evaluate the stability of our systems, and in terms of size, PDI, and zeta potential, for two months and we did not observe variations suggesting that our nano system without and with encapsulation without and with modification of the surface, all formulations remained stable.
[9:31]We also evaluate the release of caffeic acid from our liposomes, and we can see here in the pink data that the release during five days was very sustained in contrast to free caffeic acid that was completely released after 24 hours.
[9:51]So, this is in agreement with the structure of caffeic acid that is a nerophobic compound.
[9:59]So it is expected to be um retained in the bilayer of the liposome.
[10:04]So we expected that in vivo, due to the degradation of the liposome, the molecules remaining in the bilayer will be further released.
[10:13]So, we then evaluated the activity of our nano system.
[10:18]We first evaluate its ability to inhibit the aggregation of A-beta peptide.
[10:24]We used a molecule that is called the the that emits fluorescence when it binds to fibrils.
[10:34]So we can see here in the orange data that is corresponding to untreated A-beta, that it starts with a very low fluorescence, which means that we don't have fibrils in our samples.
[10:48]However, it it then starts to increase the fluorescence, which is due to the formation of fibrils.
[11:00]Then we have the stabilization of the fluorescence that is due to when all peptide already aggregate.
[11:06]And then we can observe here a decrease in the fluorescence.
[11:10]Rather than being related to the disaggregation because the interaction between peptides are very strong, this is due to the intense aggregation of the peptide that masks the fluorescence of the the.
[11:27]We can see here that when we added caffeic acid to A-beta monomers, no fluorescence was observed, that means that there is no formation of fibrils, which which confirms the ability of our compound to inhibit the aggregation.
[11:50]We then when we added our unloaded liposomes, we can see here a very high increase in the fluorescence of the.
[12:00]Which mean that our liposomes in has decelerated the the formation of fibers.
[12:07]However, when we encapsulate our compound in the liposomes, we can see that caffeic acid the presence of the compound significantly reduced the the fluorescent of the.
[12:22]Implying that the compound that was released in this short time, two hours, was enough to contradict this negative effect of the liposomes.
[12:32]Of course, as I told you, by the release, we expect that then in vivo, the molecules that are in the liposomes will have more effect in the amyloid beta aggregation.
[12:47]Here we perform transmission electron microscopy to visualize our fibers.
[12:54]We can see here that untreated A-beta have long fibers and thin.
[13:00]Here in the presence of caffeic acid, we can observe just small fibers and a lot of small structures.
[13:10]And here in the presence of unloaded liposomes and the natural compound loaded liposomes.
[13:17]No difference were observed in the structure of of our fibers.
[13:23]Then we also evaluate the the ability of our nano system to disaggregate preformed fibrils.
[13:32]So, orange data.
[13:35]We can see the fluorescence of our fibers that was normalized.
[13:40]And when we added our caffeic acid, there is a immediate decrease in the fluorescence, which means that when we added our natural compounds, there there is an uh disaggregation of fibers.
[13:56]We can see here after one hour of incubation, there is only 22% of fibers.
[14:03]However, when we put our unloaded liposomes, there is a small increase in the fluorescence.
[14:10]This may be due to the presence of some pre-fibrillar species that have finished to aggregate.
[14:20]When we put amyloid fibers incubated with our complete nano system.
[14:27]So we can see here at the beginning of the experiment, we don't have variation of on the fluorescence, however, after one hour, we can see here a reduction of about 30% in the content of amyloid fibers.
[14:42]Which which suggests that our nano system is able to disrupt preformed fibers.
[14:49]We then obtained a microscopic image that confirmed the presence of the fibers.
[14:58]Here in the presence of the natural compounds, we can see a lot of small structures, in addition, of course, of some fibers.
[15:05]In this image, we can see here the presence of fibers.
[15:09]Here too, and these structures are our liposomes.
[15:15]So, to conclude, we were able to to demonstrate that reverse phase evaporation was the technique most appropriate to produce liposomes and encapsulate caffeic acid.
[15:29]Our nano system was stable for one month at storage condition and allowed the sustained and slow release of caffeic acid.
[15:40]We also in terms of activity, we also revealed that our natural compound is able to inhibit in some degree the formation of fiber and also disrupt preformed fibers.
[15:54]Of course, in vivo studies with transgenic animals models of Alzheimer are now planned to validate the therapeutic efficacy of our nano system.
[16:07]I would like to thank all all the person involved in this work and also uh the the support that I received.
[16:15]And thank you all.
