gute Fragestellung, holpriger Antwortversuch. Alles funzt in Fließgleichgewichten mit Kipppunkten, wartet nicht, bis ein Wecker klingelt. Es beginnt stets eine Autopoesis, die in ein neues System führt.
Structure and Function of Cationic and Ionizable Lipids for Nucleic Acid Deliver
"Saturation status of the lipid tails also affects the overall property of the cationic or ionizable lipids that, in turn, determines the endosomal escape properties [118]. Cationic/ionizable lipids can have various dynamic structural phases, including the micellar, lamellar, cubic and inverted hexagonal phase [119]. The type of structure can be predicted by the packing parameter (P), which represents the ratio of the area occupied by the hydrophobic tails versus the hydrophilic head [120–122]. When P value exceeds 1, the area occupied by the hydrocarbon lipid tails is much larger than the head group, the lipid tends to adopt the inverted hexagonal phase, which is in favor of endosomal membrane destabilization, which is crucial for LNP gene delivery [112, 123]. The study utilized lipids of the same alkyl chain length (C18) modified by a systematic addition of double bonds and the effect of double bonds addition against bi-layer transition temperature demonstrated that the lipid tails containing 1 or 2 double bonds had much lower phase transition temperature for HII phase transition to get the ‘cone’ shape character [66]. Lower phase transition temperature and the ‘cone’ shape structure can facilitate efficient membrane fusion and thereby endosomal escape."
"Notably, ≈75% of the most efficacious LNPs were formulated with ionizable lipids containing branched, unsaturated tails. This is consistent with previous observations from our group and others.[9, 50, 54] Indeed, the COVID-19 mRNA vaccine lipids—Moderna's SM-102 and Acuitas’ ALC-0315—both include branched tails,[7, 55] and ongoing clinical trials are thought to be conducted using similarly branched lipids.[56] Regarding mechanism, the efficaciousness of branched tail lipids is likely facilitated by their enhanced cone-like structure that, when ionized and paired with anionic phospholipids in the endosomal membrane, more effectively disrupts the endosome for intracellular RNA release.[56-58]"
Tear of lipid membranes by nanoparticles
"Health concerns associated with the advent of nanotechnologies have risen sharply when it was found that particles of nanoscopic dimensions reach the cell lumina. Plasma and organelle lipid membranes, which are exposed to both the incoming and the engulfed nanoparticles, are the primary targets of possible disruptions."
Ist eigentlich ziemlich eindeutig und auch logisch: Du unterbrichst den eigentlichen PtdIns-Zyklus (sprich den endozytisch-phagozytisch-lysischen Regelkreislauf) durch die Protonierung innerhalb des sauren Milieus der Endsome und damit erfolgt die Penetration der Endosome.
Nö, damit erfolgt eine Änderung des Milieus - daraus folgt eine Erweiterung der Poren (passive Penetration) oder Aktivierung von aktiven/Desaktivierung von Ausschleusung (aktive Penetration)… nirgendwo hockt ein Minion mit Schaufelchen, sondern überall entstehen/zerfallen Gradienten…
"Acridine orange assay by confocal imaging and flow cytometer
Lysosomal pH changes were tested by acridine orange staining. To analyze acridine orange by flow cytometry, Raw264.7 cells were seeded at 4e5 cells/well and then incubated with 400 ng/mL LNP for 6 h. For positive control, 6.8 mM LLOMe was cocultured with cells for 1 h. Then, cell pellets were harvested and stained by 1 ug/mL acridine orange for 10 min. At the end of staining, add 1 mL/tube PBS immediately. Then cells were washed by centrifugation at 4℃, 300 g, 5 min and finally analyzed by Guava easyCyte flow cytometer (Luminex). To record acridine orange images, cells were seeded to 8-well μ-Slide chambers (Raw264.7 cells: 1.5E5 cells/well; A549GFP1-10 cells: 1.5E4 cells/well) and then treated with LNP for 6 h. As for the positive control group, Raw264.7 cells were incubated with 6.8 mM LLOMe for 1 h and A549GFP1-10 cells were treated with 68 μM LLOMe for 2 h. Next, cells were labeled by 10 μg/mL acridine orange for 10 min and then imaged by LSM980 confocal microscope (Zeiss).
Colocalization imaging of LNPs and endo-lysosomes
Cells were seeded to 8-well μ-Slide chambers (Raw264.7 cells: 1.5e5 cells/well; A549GFP1-10 cells: 1.8e4 cells/well) and then treated with cKK-E12 LNPs formulated with 0.3 mol % of 18:1 TopFluor PE-Alexa Fluor 594 for 0.5, 1 and 6 h. Endo-lysosomes were labeled by 200 nM LysoTracker DeepRed (L12492, Invitrogen) for 30 min and nuclei were stained by Hoechst33342 (R37650, Invitrogen). Fluorescent images were acquired by LSM980 microscopy (Zeiss) and colocalization of LNPs and endo-lysosomes was analyzed by Pearson’s coefficient in ImageJ."
Sauber nachgewiesen in dem erwähnten Preprint zu endosomale Disruption/ Schäden.
2) "nirgendwo hockt ein Minion mit Schaufelchen, sondern überall entstehen/zerfallen Gradienten…"
Die gängigste These ist endosomal/lysomale Disruption. Ebenfalls aus dem pre-print. Zudem: Wieso "Nö"? Wo genau widersprach ich diesem Faktum, welches sich durch die Protonierung der ionisierbaren tertiären Amine logischerweise ergibt?:
"Once protonated in the acidic endosome, ionizable lipids form ion pairs with the anionic lipids in the endosomal membrane, leading to the formation of an inverse hexagonal phase that disrupts the endosomal membrane and releases the RNA cargo into the cytosol31–33. Different ionizable lipids have different endosomal disruption capabilities as a function of lipid structural properties which influence their ability to form the inverse hexagonal phase34,35. We hypothesized that these differences also correlate with inflammatory responses elicited by LNPs. We propose that LNPs formulated with less potent ionizable lipids induce less endosomal escape of RNA, lower RNA expression, and less inflammation, and more potent ionizable lipids induce higher RNA expression but more severe inflammatory responses (Fig. 2A). This is part of our central hypothesis that some aspect of endosomal escape itself induces inflammation and follows from our results in the previous section showing that lipid components of LNPs, rather than RNA cargo, drive inflammation (Fig. 1K)." (Lesen hilft also!)
Wie das ganze physisch, mathematisch funktioniert, mag ich getrost Ihnen überlassen, Herr Umann. Dies widerspricht jedoch in keinster Weise meiner Kernaussage, dass eine Disruption und zuvor Penetration der Zellmembran stattfindet. Niemand sagte also etwas von Minions. Und dann hätte ich gerne mal ein paar Papers zum Gradientenverhältnis, wenn Sie schon nicht auf meine Argumente eingehen und scheinbar die Papers getrost ignorierten.
Statt mir also Worte in den Mund zu legen, die ich nicht gebrauchte, vielleicht doch mal das ein oder andere Paper lesen?
Lysosome toxicities induced by nanoparticle exposure and related mechanisms
Nanoparticles distribute the acidity of lysosomal.
Nanoparticles disrupt lysosomal function by the regulation of ion channel proteins.
Und nicht vergessen: Wir reden hier nicht über rezeptorvermittelte Aufnahme, sondern über einen endozytoseÄHNLICHEN (nicht direkt vergleichbaren) Prozess, da einige ionisierbare Lipide auch zwischen der Doppelmembran "hängen" bleiben könnten, wenn die gesamt LNP-formulierung beginnt, instabil zu werden...:
Ionizable lipids penetrate phospholipid bilayers with high phase transition temperatures: perspectives from free energy calculations
"Also, the ionizable lipids should become positive again upon exposure to the typical acidic environment of the endosomes, thus, destabilizing the endosomal membrane and promoting the cytosolic delivery of the genetic cargo. Finally, in the cytosol, the neutral pH would favor the release of the NA from the ionizable lipids, thus, enabling their free interaction with the cell machinery."
(...)
"Indeed, the direct siRNA visualization method optimized for lipoplexes was not applicable to LNPs (formulated with a biodegradable derivative of MC3). This was probably due to the lower amount of siRNA loaded on LNPs compared with larger lipoplexes. In agreement with the previous study, the authors estimated that only 3.5% of LNP‐administered siRNA was released in the cytosol. The escape took place in a narrow time window, and at a slightly earlier stage compared to lipoplex‐delivered siRNA: a lower RAB7 expression of and no RAB9 were found on the endosomal membrane. Noteworthy, the recruitment of Gal8 was recently validated as a quantitative method for the detection of endosomal disruption triggered by cationic polymers, 69 and sensors based on Gal8‐split luciferase fusion proteins were developed for high throughput screening purposes. 70 Although not specifically tested on ionizable LNPs, these sensors might provide a formidable tool for the screening of new cytosol‐targeting nanoparticles."
Da sind wir dann auch direkt im PtdIns-Zyklus und Feedbackschleifen.
The endosomal-lysosomal system: from acidification and cargo sorting to neurodegeneration
"Compared to a cytoplasmic pH (of about 7.0), the endosomal and lysosomal lumen pH is maintained in a range of 6.5 to 4.5, due to the activity of the ATP-dependent proton pumps present in the membrane of both endosomes and lysosomes [7]. Molecular trafficking and sorting along the endocytic pathway is regulated by the Rab family of small GTPases, which are critically important membrane association proteins. The Rabs function as molecular switches that alternate between two conformational states: the activated GTP-bound form and the GDP-bound inactivated form [8]. Different Rab proteins have corresponding host organelles; therefore, they are often regarded as markers of different endosomal compartments."
"The majority of the particles are endocytosed by macropinocytosis; however, clathrin-mediated endocytosis is a prerequisite. Endocytosed LNPs are transferred to early endosomes, which mature into late endosomes and eventually into lysosomes (21). For efficient delivery, the nucleic acid payloads must be released into the cytosol before the maturation of late endosomes to lysosomes where the majority of the foreign materials are degraded enzymatically. The release of the payload prior to lysosomal maturation is a crucial stage for efficient delivery and is known as endosomal escape. This process is inefficient and is considered a bottleneck in this field (17, 18)."
Und die Mehrheit der Papers geht eben exakt von diesem Prozess aus. Was sich dann auch 1:1 damit deckt, dass NF-kB beispielsweise getriggert wird, durch diverse Signalwege der MAPk, mTORC1 etc, etc, etc.... (Bereits von kationischen Liposomen ohne den Fettschwanzmod der ALC-0315 und SM-102 bekannt gewesen:
Cationic liposomal lipids: From gene carriers to cell signaling
Es ging mir also zunächst ausschließlich um die Protonierung als AUSLÖSER zur Disruption. Dass dazwischen sich logischerweise das Säureverhältnis ändern wird, erschien mir nicht mal mehr weiter erwähnenswert, da dies eine streng logische Reaktion ist.
I'm so pleased they gained mastery of the mechanisms and addressed all safety concerns before administering it into billions of arms.
The "experts" complicit in this crime probably spent longer choosing their next smartphone than they did on how LNPs work.
"... More than 5.55 billion people worldwide have received a dose of a Covid-19 vaccine, equal to about 72.3 percent of the world population."
https://www.nytimes.com/interactive/2021/world/covid-vaccinations-tracker.html
gute Fragestellung, holpriger Antwortversuch. Alles funzt in Fließgleichgewichten mit Kipppunkten, wartet nicht, bis ein Wecker klingelt. Es beginnt stets eine Autopoesis, die in ein neues System führt.
Structure and Function of Cationic and Ionizable Lipids for Nucleic Acid Deliver
"Saturation status of the lipid tails also affects the overall property of the cationic or ionizable lipids that, in turn, determines the endosomal escape properties [118]. Cationic/ionizable lipids can have various dynamic structural phases, including the micellar, lamellar, cubic and inverted hexagonal phase [119]. The type of structure can be predicted by the packing parameter (P), which represents the ratio of the area occupied by the hydrophobic tails versus the hydrophilic head [120–122]. When P value exceeds 1, the area occupied by the hydrocarbon lipid tails is much larger than the head group, the lipid tends to adopt the inverted hexagonal phase, which is in favor of endosomal membrane destabilization, which is crucial for LNP gene delivery [112, 123]. The study utilized lipids of the same alkyl chain length (C18) modified by a systematic addition of double bonds and the effect of double bonds addition against bi-layer transition temperature demonstrated that the lipid tails containing 1 or 2 double bonds had much lower phase transition temperature for HII phase transition to get the ‘cone’ shape character [66]. Lower phase transition temperature and the ‘cone’ shape structure can facilitate efficient membrane fusion and thereby endosomal escape."
https://link.springer.com/article/10.1007/s11095-022-03460-2
Branched-Tail Lipid Nanoparticles for Intravenous mRNA Delivery to Lung Immune, Endothelial, and Alveolar Cells in Mice
https://advanced.onlinelibrary.wiley.com/doi/full/10.1002/adhm.202400225
"Notably, ≈75% of the most efficacious LNPs were formulated with ionizable lipids containing branched, unsaturated tails. This is consistent with previous observations from our group and others.[9, 50, 54] Indeed, the COVID-19 mRNA vaccine lipids—Moderna's SM-102 and Acuitas’ ALC-0315—both include branched tails,[7, 55] and ongoing clinical trials are thought to be conducted using similarly branched lipids.[56] Regarding mechanism, the efficaciousness of branched tail lipids is likely facilitated by their enhanced cone-like structure that, when ionized and paired with anionic phospholipids in the endosomal membrane, more effectively disrupts the endosome for intracellular RNA release.[56-58]"
Tear of lipid membranes by nanoparticles
"Health concerns associated with the advent of nanotechnologies have risen sharply when it was found that particles of nanoscopic dimensions reach the cell lumina. Plasma and organelle lipid membranes, which are exposed to both the incoming and the engulfed nanoparticles, are the primary targets of possible disruptions."
https://pubs.rsc.org/en/content/articlelanding/2022/sm/d2sm00179a
Lipid Nanoparticle-Associated Inflammation is Triggered by Sensing of Endosomal Damage: Engineering Endosomal Escape Without Side Effects
https://www.biorxiv.org/content/10.1101/2024.04.16.589801v1.full
Ist eigentlich ziemlich eindeutig und auch logisch: Du unterbrichst den eigentlichen PtdIns-Zyklus (sprich den endozytisch-phagozytisch-lysischen Regelkreislauf) durch die Protonierung innerhalb des sauren Milieus der Endsome und damit erfolgt die Penetration der Endosome.
Nö, damit erfolgt eine Änderung des Milieus - daraus folgt eine Erweiterung der Poren (passive Penetration) oder Aktivierung von aktiven/Desaktivierung von Ausschleusung (aktive Penetration)… nirgendwo hockt ein Minion mit Schaufelchen, sondern überall entstehen/zerfallen Gradienten…
Und da wären wir bei den 7 sich widersprechenden Theorien ohne Messmethoden eine wirklich zu belegen.
Da kann man sich gemütlich bis zum St. Nimmerleinstag über Theorien streiten, wie in den Geisteswissenschaften.
ansonsten ging’s über große Zahlen und Kombinatorik (die Habil meines Doktorvaters)
einerseits jep, oder man schließt eine nach der anderen als unzutreffend aus
Sobald man dafür Messmethoden entwickelt hat.
1) Zu "Messmethoden":
"Acridine orange assay by confocal imaging and flow cytometer
Lysosomal pH changes were tested by acridine orange staining. To analyze acridine orange by flow cytometry, Raw264.7 cells were seeded at 4e5 cells/well and then incubated with 400 ng/mL LNP for 6 h. For positive control, 6.8 mM LLOMe was cocultured with cells for 1 h. Then, cell pellets were harvested and stained by 1 ug/mL acridine orange for 10 min. At the end of staining, add 1 mL/tube PBS immediately. Then cells were washed by centrifugation at 4℃, 300 g, 5 min and finally analyzed by Guava easyCyte flow cytometer (Luminex). To record acridine orange images, cells were seeded to 8-well μ-Slide chambers (Raw264.7 cells: 1.5E5 cells/well; A549GFP1-10 cells: 1.5E4 cells/well) and then treated with LNP for 6 h. As for the positive control group, Raw264.7 cells were incubated with 6.8 mM LLOMe for 1 h and A549GFP1-10 cells were treated with 68 μM LLOMe for 2 h. Next, cells were labeled by 10 μg/mL acridine orange for 10 min and then imaged by LSM980 confocal microscope (Zeiss).
Colocalization imaging of LNPs and endo-lysosomes
Cells were seeded to 8-well μ-Slide chambers (Raw264.7 cells: 1.5e5 cells/well; A549GFP1-10 cells: 1.8e4 cells/well) and then treated with cKK-E12 LNPs formulated with 0.3 mol % of 18:1 TopFluor PE-Alexa Fluor 594 for 0.5, 1 and 6 h. Endo-lysosomes were labeled by 200 nM LysoTracker DeepRed (L12492, Invitrogen) for 30 min and nuclei were stained by Hoechst33342 (R37650, Invitrogen). Fluorescent images were acquired by LSM980 microscopy (Zeiss) and colocalization of LNPs and endo-lysosomes was analyzed by Pearson’s coefficient in ImageJ."
(https://www.biorxiv.org/content/10.1101/2024.04.16.589801v1.full)
Sauber nachgewiesen in dem erwähnten Preprint zu endosomale Disruption/ Schäden.
2) "nirgendwo hockt ein Minion mit Schaufelchen, sondern überall entstehen/zerfallen Gradienten…"
Die gängigste These ist endosomal/lysomale Disruption. Ebenfalls aus dem pre-print. Zudem: Wieso "Nö"? Wo genau widersprach ich diesem Faktum, welches sich durch die Protonierung der ionisierbaren tertiären Amine logischerweise ergibt?:
"Once protonated in the acidic endosome, ionizable lipids form ion pairs with the anionic lipids in the endosomal membrane, leading to the formation of an inverse hexagonal phase that disrupts the endosomal membrane and releases the RNA cargo into the cytosol31–33. Different ionizable lipids have different endosomal disruption capabilities as a function of lipid structural properties which influence their ability to form the inverse hexagonal phase34,35. We hypothesized that these differences also correlate with inflammatory responses elicited by LNPs. We propose that LNPs formulated with less potent ionizable lipids induce less endosomal escape of RNA, lower RNA expression, and less inflammation, and more potent ionizable lipids induce higher RNA expression but more severe inflammatory responses (Fig. 2A). This is part of our central hypothesis that some aspect of endosomal escape itself induces inflammation and follows from our results in the previous section showing that lipid components of LNPs, rather than RNA cargo, drive inflammation (Fig. 1K)." (Lesen hilft also!)
Wie das ganze physisch, mathematisch funktioniert, mag ich getrost Ihnen überlassen, Herr Umann. Dies widerspricht jedoch in keinster Weise meiner Kernaussage, dass eine Disruption und zuvor Penetration der Zellmembran stattfindet. Niemand sagte also etwas von Minions. Und dann hätte ich gerne mal ein paar Papers zum Gradientenverhältnis, wenn Sie schon nicht auf meine Argumente eingehen und scheinbar die Papers getrost ignorierten.
Statt mir also Worte in den Mund zu legen, die ich nicht gebrauchte, vielleicht doch mal das ein oder andere Paper lesen?
Lysosome toxicities induced by nanoparticle exposure and related mechanisms
Nanoparticles distribute the acidity of lysosomal.
Nanoparticles disrupt lysosomal function by the regulation of ion channel proteins.
https://www.sciencedirect.com/science/article/pii/S0147651324012910
Wenn Sie den PtdIns-Zyklus begreifen, erschließt sich der Rest automatisch und logisch:
https://genervter.substack.com/p/die-phospholipide-doppelschicht-und
Und nicht vergessen: Wir reden hier nicht über rezeptorvermittelte Aufnahme, sondern über einen endozytoseÄHNLICHEN (nicht direkt vergleichbaren) Prozess, da einige ionisierbare Lipide auch zwischen der Doppelmembran "hängen" bleiben könnten, wenn die gesamt LNP-formulierung beginnt, instabil zu werden...:
Ionizable lipids penetrate phospholipid bilayers with high phase transition temperatures: perspectives from free energy calculations
https://www.sciencedirect.com/science/article/pii/S0009308423000166
"Also, the ionizable lipids should become positive again upon exposure to the typical acidic environment of the endosomes, thus, destabilizing the endosomal membrane and promoting the cytosolic delivery of the genetic cargo. Finally, in the cytosol, the neutral pH would favor the release of the NA from the ionizable lipids, thus, enabling their free interaction with the cell machinery."
(...)
"Indeed, the direct siRNA visualization method optimized for lipoplexes was not applicable to LNPs (formulated with a biodegradable derivative of MC3). This was probably due to the lower amount of siRNA loaded on LNPs compared with larger lipoplexes. In agreement with the previous study, the authors estimated that only 3.5% of LNP‐administered siRNA was released in the cytosol. The escape took place in a narrow time window, and at a slightly earlier stage compared to lipoplex‐delivered siRNA: a lower RAB7 expression of and no RAB9 were found on the endosomal membrane. Noteworthy, the recruitment of Gal8 was recently validated as a quantitative method for the detection of endosomal disruption triggered by cationic polymers, 69 and sensors based on Gal8‐split luciferase fusion proteins were developed for high throughput screening purposes. 70 Although not specifically tested on ionizable LNPs, these sensors might provide a formidable tool for the screening of new cytosol‐targeting nanoparticles."
Da sind wir dann auch direkt im PtdIns-Zyklus und Feedbackschleifen.
https://pmc.ncbi.nlm.nih.gov/articles/PMC7995196/
The endosomal-lysosomal system: from acidification and cargo sorting to neurodegeneration
"Compared to a cytoplasmic pH (of about 7.0), the endosomal and lysosomal lumen pH is maintained in a range of 6.5 to 4.5, due to the activity of the ATP-dependent proton pumps present in the membrane of both endosomes and lysosomes [7]. Molecular trafficking and sorting along the endocytic pathway is regulated by the Rab family of small GTPases, which are critically important membrane association proteins. The Rabs function as molecular switches that alternate between two conformational states: the activated GTP-bound form and the GDP-bound inactivated form [8]. Different Rab proteins have corresponding host organelles; therefore, they are often regarded as markers of different endosomal compartments."
https://pmc.ncbi.nlm.nih.gov/articles/PMC4596472/
"The majority of the particles are endocytosed by macropinocytosis; however, clathrin-mediated endocytosis is a prerequisite. Endocytosed LNPs are transferred to early endosomes, which mature into late endosomes and eventually into lysosomes (21). For efficient delivery, the nucleic acid payloads must be released into the cytosol before the maturation of late endosomes to lysosomes where the majority of the foreign materials are degraded enzymatically. The release of the payload prior to lysosomal maturation is a crucial stage for efficient delivery and is known as endosomal escape. This process is inefficient and is considered a bottleneck in this field (17, 18)."
https://pmc.ncbi.nlm.nih.gov/articles/PMC10945858/
Linkage between endosomal escape of LNP-mRNA and loading into EVs for transport to other cells
https://www.nature.com/articles/s41467-019-12275-6
Und die Mehrheit der Papers geht eben exakt von diesem Prozess aus. Was sich dann auch 1:1 damit deckt, dass NF-kB beispielsweise getriggert wird, durch diverse Signalwege der MAPk, mTORC1 etc, etc, etc.... (Bereits von kationischen Liposomen ohne den Fettschwanzmod der ALC-0315 und SM-102 bekannt gewesen:
Cationic liposomal lipids: From gene carriers to cell signaling
https://www.sciencedirect.com/science/article/abs/pii/S0163782708000271
Cationic lipids activate intracellular signaling pathways
https://www.sciencedirect.com/science/article/abs/pii/S0169409X12001901 )
Es ging mir also zunächst ausschließlich um die Protonierung als AUSLÖSER zur Disruption. Dass dazwischen sich logischerweise das Säureverhältnis ändern wird, erschien mir nicht mal mehr weiter erwähnenswert, da dies eine streng logische Reaktion ist.