Not New: A long research history of Lipid Nanoparticles and PEGs targeting the brain
Otherwise known as 'Stays in the arm'
Magic Appearance:
Lipid nanoparticles and PEG used in the vaccine are not new – they have a long history in the lab, especially as tools to break through the protective Blood Brain Barrier. This post highlights a cluster of biolab tools frequently used together for gene transfection and targeting the brain, which materialized together in the Spike + shots.
Enjoy reading and Happy Holidays.
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Summary of this post:
LNPs and PEG are in the Covid shots.
LNPs and PEG target the brain and have long been developed and used for these purposes, as well as for gene transfection.
Included in this biolab tool-set are cationic Cell Penetrating Peptides and Transferrin Receptor ligands. Spike targets the Transferrin Receptor and has a cationic multi-Arginine repeat motif that magically appeared.
Described below by Ansari et al. (2020), Cell Penetrating Peptides are inactivated by proteases and thus “should be stabilized via the incorporation of … lipid-based nanocarriers … to avoid inactivation by proteases”.
The multi-arginine repeat in Spike (a classic cell penetrating peptide that I propose is inspired by HIV Tat) has been discussed as the Furin Cleavage Site. Furin is a protease. This motif is also targeted by the protease TMPRSS2. As Ansari mentions, a goal of using CPPs and LNPs / PEG is to avoid cleavage by proteases. According to lab history, this could be questioned as being not only a 'cleavage site’, but a ‘hopefully-not-cleaved before steering its cargo into the nucleus’ site.
[Background info not covered in this post due to streamlining]: Research articles describing brain-targeting LNP / PEG technologies traditionally have the end-goal of getting a cargo past the BBB into the brain. What happens after to the brain cells and the long-term effects are very rarely questioned by the authors, and remains unknown. Meanwhile, current status in neuroscience holds that provoking neuroinflammation or BBB disturbance can trigger ongoing neurodegenerative processes.
Bottom of page: links to Wright’s post discussing specific history of the development of these biolab tools; links to my other posts on the subject including Syncytia-building, in which my ideas of the Arginine-rich motif contributing to sudden death via syncytial disturbance in brain stem / heart, destruction of eggs and pregnancies, and building of giant clots via syncytia are discussed.
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Magic Disappearance
Pause for a moment and see how many search engine results you get for “stays in the arm”. This content has magically disappeared from the web.
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Stays in the arm:
2008 Wuhan:
doi: 10.1007/s12272-001-1191-8
“The objective of this study is to investigate the pharmacokinetics and biodistribution of free breviscapine (BVP) and coated BVP-loaded poly (D, L-lactic acid) nanoparticles (BVP-PLA-NPs) in rats after i.v. administration. … NPs were mainly distributed in liver, spleen, heart and brain. ... Coated BVP-PLA-NPs could effectively avoid the capture by the reticuloendothelial system and prolong the half-life of BVP. Moreover, these NPs could penetrate BBB and enhance the accumulation of BVP in brain.”
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LNPs + PEG in vaccine:
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Assessment report COVID-19 Vaccine Moderna, Common name: COVID-19 mRNA Vaccine (nucleoside-modified).
European Medicines Agency, Committee for Medicinal Products for Human Use. 11 March 2021 EMA/15689/2021 Corr.1*1. Procedure No. EMEA/H/C/005791/0000
Description of the product and Pharmaceutical Development
“The finished product … contains an mRNA active substance … that encodes for the pre-fusion stabilised spike glycoprotein of 2019-novel Coronavirus (SARS-CoV-2) encapsulated into lipid nanoparticles (LNP) …. The LNP are composed of four lipids which act as protectants and carriers of the mRNA. These are: heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102, a custommanufactured, ionisable lipid), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol.
PEG2000 DMG… is a novel excipient, not previously used in an approved finished product within EU.”
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LNPs + PEG target the brain
Brain-targeting technologies use Lipid Nanoparticles and PEG; — bonus — gene delivery.
Transferrin Receptor, mentioned below, is also part of this system; Spike targets Transferrin Receptor.
Notice ‘cationic’ being repeated, and recall the cationic multi-Arginine motif that appeared in Spike.
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Cationic albumin-conjugated pegylated nanoparticles as novel drug carrier for brain delivery
Lu W, Zhang Y, Tan YZ, Hu KL, Jiang XG, Fu SK.J Control Release. 2005 Oct 20;107(3):428-48. doi: 10.1016/j.jconrel.2005.03.027.
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Gene delivery targeted to the brain using an Angiopep-conjugated polyethyleneglycol-modified polyamidoamine dendrimer
Ke W, Shao K, Huang R, Han L, Liu Y, Li J, Kuang Y, Ye L, Lou J, Jiang C.Biomaterials. 2009 Dec;30(36):6976-85. doi: 10.1016/j.biomaterials.2009.08.049.
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Transport of PEGylated-PLA nanoparticles across a blood brain barrier model, entry into neuronal cells and in vivo brain bioavailability
Rabanel JM, Piec PA, Landri S, Patten SA, Ramassamy C. J Control Release. 2020 Dec 10;328:679-695. doi: 10.1016/j.jconrel.2020.09.04
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Brain delivery property and accelerated blood clearance of cationic albumin conjugated pegylated nanoparticle
Lu W, Wan J, She Z, Jiang X.J Control Release. 2007 Mar 12;118(1):38-53. doi: 10.1016/j.jconrel.2006.11.015.
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Targeting the brain with PEG-PLGA nanoparticles modified with phage-displayed peptides
Li J, Feng L, Fan L, et al.Biomaterials. 2011;32(21):4943-4950. doi:10.1016/j.biomaterials.2011.03.031
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Preparation, characterization and transfection efficiency of cationic PEGylated PLA nanoparticles as gene delivery systems.
Chen J, Tian B, Yin X, Zhang Y, Hu D, Hu Z, Liu M, Pan Y, Zhao J, Li H, Hou C, Wang J, Zhang Y.J Biotechnol. 2007 Jun 15;130(2):107-13. doi: 10.1016/j.jbiotec.2007.02.007.
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Transferrin + LNPs / PEG to brain
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Transferrin /Receptor is included in this system of brain-targeting tools:
Nanoparticulate systems for brain delivery of drugs
Jörg Kreuter. Advanced Drug Delivery Reviews. Volume 47, Issue 1, 23 March 2001, Pages 65-81 https://doi.org/10.1016/S0169-409X(00)00122-8
“…A number of attempts have been made to overcome the above barrier including osmotic opening of the tight junctions [5], use of prodrugs or carrier systems such as antibodies [6], liposomes [7], [8], [9], and nanoparticles…. The colloidal carriers… may take advantage of the biochemical transport systems that are also present in the BBB: … carrier-mediated transport systems exist that enable the entry or the elimination of a variety of compounds… for instance, the LDL-receptor and the *transferrin* transcytosis systems may be employed in the delivery of drugs by the above particulate colloidal drug delivery systems. …
… One of the possibilities to deliver drugs to the brain is the employment of nanoparticles.”
*Spike targets Transferrin Receptor: See my other posts, such as ‘Not New. Spike —> Transferrin Receptor, B-Cells, Tumors’.
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*Transferrin*-conjugated nanoparticles of poly(lactide)-D-alpha-tocopheryl polyethylene glycol succinate diblock copolymer for targeted drug delivery across the blood-brain barrier.
Gan CW, Feng SS. Biomaterials. 2010 Oct;31(30):7748-57. doi: 10.1016/j.biomaterials.2010.06.053.
“We developed in this research a nanoparticle system for targeted drug delivery across the blood-brain barrier (BBB), which consists of the transferrin (Tf) conjugated nanoparticles of poly(lactide)-D-alpha-Tocopheryl polyethylene glycol succinate (PLA-TPGS) diblock copolymer. … The Tf-conjugated PLA-TPGS NPs formulation demonstrated great advantages over the other two NPs formulations … IC50 data showed that the Tf-conjugated PLA-TPGS NPs formulation of Docetaxel could be 23.4%, 16.9% and 229% more efficient than the PLGA NPs, the PLA-TPGS NPs formulations and Taxotere… ”
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Brain tumor-targeted therapy by systemic delivery of siRNA with Transferrin receptor-mediated core-shell nanoparticles.
Wei L, Guo XY, Yang T, Yu MZ, Chen DW, Wang JC.Int J Pharm. 2016 Aug 20;510(1):394-405. doi: 10.1016/j.ijpharm.2016.06.127.
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HIV Tat + LNPs / PEG to brain
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HIV-1 Tat (poly-Arginine, cationic) is included in this system of brain-targeting tools:
Benefit of a Short Chain Peptide as a Targeting Ligand of Nanocarriers for a Brain-Driven Purpose
Yu-Chen Lo and Wen-Jen Lin. Pharmaceutics. 2021 Aug; 13(8): 1249. doi: 10.3390/pharmaceutics13081249
“In this study, a peptide-conjugated nano-delivery system was explored for the purpose of glioma therapy. … An in vitro cocultured BBB model illustrated the internalization of peptide-conjugated NPs … indicating both BBB-crossing and glioma-penetrating abilities. IVIS (In Vivo Imaging System) images revealed that T7-conjugated NPs specifically accumulated in the brain … The benefit of T7-peptide as a targeting ligand for NPs across the BBB with accumulation in the brain was elucidated.
… The attachment of a variety of high-affinity targeting ligands to the surface of nanocarriers has been applied to enhance the active targeting ability of nanodelivery systems [7]. The targeting ligands can be endogenous substances (e.g., transferrin, folic acid), enzymes, engineered antibodies, and macromolecules like proteins and carbohydrates [8]. *The targeted delivery is ensured by a high specificity of the ligand for its receptor, which is overexpressed on tumor cells or tumor vasculature, but has no or low-expression on normal cells [9]*.” …
*Not high-specificity in terms of cells: Transferrin receptors are expressed in many tissues throughout the body including every region of brain and the hematopoetic system.
“T7-peptide (HAIYPRH) … exhibits a high affinity for the human transferrin receptor (TfR)… The binding of Tf with TfR promotes the transportation of T7-peptide as well… The best characterized receptor-mediated transcytosis systems for brain targeting include the TfR and low-density-lipoprotein receptor (LDLR), which are highly expressed on brain endothelial cells [13]. On the basis of the expression level of TfR on brain endothelial cells and glioma cancer cells being observably higher than that in other normal cells, T7-peptide is considered as an ideal targeting ligand of TfR and is applied in a variety of drug delivery systems in cancer therapy [14,15,16].
Cell penetrating peptides (CPPs) are a family of short chain peptides, generally comprising 5–30 amino acids, with the ability to spontaneously enter across the cell membrane [17]. They have been extensively shown to transport a wide variety of biologically active cargos, including proteins, plasmid DNA, RNA, oligonucleotides, liposomes and anti-cancer drugs, into the cells [18]. For example, *Tat-peptide* conjugated drug or vaccine was successfully delivered by lipid-based nanocarriers for SARS-CoV-2 therapy [19,20]. Polyarginine exhibits the highest level of cellular uptake among CPPs, thus offering a higher potential for therapeutics [21,22,23,24,25]. Studies on a series of arginine-based peptides (from R3 to R12) have shown that the minimal sequence necessary for cellular uptake is six arginines, and R6 to R9 are optimally translocated through the cell membrane [26]. Arginine-rich CPPs are widely employed as delivery vehicles for a large variety of cargos. However, with a high transduction efficiency, cytotoxic effects of polyarginine peptides have also been reported [27]. Some studies have put emphasis on how to mask or shield the CPPs while in the circulation in order to minimize their exposure to normal cells [28].” …
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Lipid-based nano delivery of Tat-peptide conjugated drug or vaccine-promising therapeutic strategy for SARS-CoV-2 treatment.
Ansari M.A., Almatroudi A., Alzohairy M.A., AlYahya S., Alomary M.N., Al-Dossary H.A., Alghamdi S. Expert Opin. Drug Deliv. 2020;17:1671–1674. doi: 10.1080/17425247.2020.1813712.
Detail:
“… Most importantly, the nano delivery of repurposed antiviral drugs can be further enhanced by conjugating to cell-penetrating peptides (CPPs). The discovery of CPPs, a short cationic peptide with a high content of basic amino acid residues [10], readily facilitate intracellular intake and delivery of a variety of nanosize, small and large molecules into cytoplasm or nucleus *without causing toxicity* and damaging the cell membranes integrity [11,12].”
* Good we cleared that up. (See my post ‘Cells melt together into Syncytia following Spike exposure: what happens to ovaries and eggs?’)
“The ‘cargo’ of these molecules is either due to electrostatic forces between the positively charged TAT-peptide and negatively charges of phospholipids membrane or nonelectrostatic hydrogen or hydrophobic interactions [12,13]. CPPs deliver the cargo into the cell either through macropinocytosis, caveolae-mediated endocytosis or clathrin-independent endocytosis mechanism. It has also been studied that HIV1 TAT-peptides directly penetrate the membranes by generating nanoscale pores [13].”
Bird: This demonstrates that it may be irrelevant whether a tissue expresses ACE2 etc. or not when considering what will be targeted by Spike and thus potentially destroyed by the immune system – any cell with a membrane is susceptible to cell-penetrating peptides. This is a question to investigate further.
“CPPs has several advantages over other delivery and translocation approaches as it is inexpensive, easy to manufacture and usually nontoxic [14]. They have a higher capability to translocate into a wide range of cell types, higher rate of cellular permeability and uptake, more comfortable to pass other biological barriers. Additionally, they have a large cargo capacity and reduced cell toxicity with no immunological response [14,15]. Apart from the several advantages of CPPs, these peptides have also limitations, such as low cell specificity, uptake into intracellular endosomes and inactivation of CPPs by proteases* could be the most significant drawbacks of the first generation of CPPs [16]. Hence, to the therapeutic application, these CPPs should be stabilized via the incorporation of multifunctional organic polymeric or lipid-based nanocarriers to improve selectivity, efficiency, and capacity of cargo transport to avoid inactivation by proteases* [17].
Bird: The multi-arginine repeat in Spike (a classic cell penetrating peptide that I propose is inspired by HIV Tat) has been discussed as the Furin Cleavage Site. Furin is a protease. In this text we see that a goal of using CPPs and LNPs / PEG is to avoid cleavage by proteases. In my view according to lab history, this could be questioned as being not a 'cleavage site’, but a ‘hopefully-not-cleaved before steering its cargo into the nucleus’ site. This multi-arginine motif in Spike is also a target for protease TMPRSS2: see e.g. Weatherbee et al. 2020. ‘Expression of SARS-CoV-2 receptor ACE2 and the protease TMPRSS2 suggests susceptibility of the human embryo in the first trimester.’ DOI: 10.1098/rsob.200162
“It has been reported that CPP-based drug delivery systems have reached clinical trials for cancer diagnosis and therapy, where they showed enhanced efficacy [18]. It is, therefore, reasonable to expect similar progress in the development of nanoformulation-based CPP-conjugated nano delivery of repurposed antiviral drugs for the treatment of COVID-19. The HIV-1 TAT-peptide47-57 (GRKKRRQRRRP), a short cationic (8 positive charges) and high content of essential amino acid (2 lysine and 6 arginine), rapidly became a popular and powerful research tool to enhance the transport and delivery of proteins, DNA/RNA, viruses, drugs and nanoparticles inside the cells [12,15]. CPPs might be promising immune enhancers when incorporated into appropriate nanoformulation-based nanocarriers systems. According to Milken Institute, ~123 vaccines, ~21 repurposed antiviral drugs, ~58 antibodies, ~15 cell-based, 6 RNA-based and more than 80 such as immune enhancers, immune-modulating, antimalarial, antiparasitic and anti-inflammatory are at different stages of clinical trials for the treatment of COVID-19 infections [19]. *Unluckily*, none of the ongoing clinical trials and treatments is based on nanocarriers and Tat-peptide conjugated-nanoformulation strategies despite the many advantages in nanocarriers and Tat-peptide drug delivery systems. Therefore, we suggest that the efficacy of antiviral activity of repurposed drug or vaccine against COVID-19 can be improved and enhanced by conjugating it to the Tat-peptides by utilizing nanoformulation-based nanocarriers delivery systems.”
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Not New
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Charles Wright Substack: Was Merck’s HIV-producing “vaccine” technology a forerunner to vaccines producing viruses today?
Charles Wright looks into the history of these tools - a succinct synopsis of what we have faced in this pandemic and the shots can be found in the excerpt / image from his post below:
Wright: “On December 3, 1996, patent #5,580,859 was issued to [RM] for a Delivery of exogenous DNA sequences in a mammal. The December 3, 1996 screenshot below is from [RM's] curriculum vitae.”
Regarding RM’s CV points (especially point 10, polyfunctional cationic cytofectins), see my posts about the cationic multi-Arginine repeat motif in Spike, which I have been questioning as a gene transfection (cytofection) tool. Charles Wright’s post brings this together by discussing not only the gene transfection history of RM et al.’s work, but their focus on trying to create an HIV vaccine that would deliver e.g. 3 HIV peptides into the nucleus, attached to an Adeno delivery vector (see my post on the history of Adeno virus vaccines.) In addition to the gene transfection potential of multi-Arginine cationic motifs, I have pointed to their known ability to trigger Syncytia, and suggested that Syncytia should be investigated as a contributor to sudden death via e.g. disruption in brain stem / heart and building of massive blood clots (extracellular matrix / netosis / white blood cells?).
History of Adenovirus vectors with gene transfection:
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Multi-arginine cationic cytofectins developed from HIV-1 Tat:
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Transferrin Receptor and Spike: Targeting Transferrin Receptor (mitochondrial dysfunction, cell death, immunosuppression) has a long history in the lab.
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General background description of cell transfection:
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Thank you for reading.
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