The Strange World of "Nano"

Do the LNPs have quantum properties?

Introduction

I want to talk more about the LNPs as nanoparticles. We must understand the nature of nanoparticles (NPs) to understand these shots.

Definition

The ISO issued guidelines defining a nanoparticle as an object with 3 dimensions below 100 nm. It doesn’t have to be a sphere, they can be elliptoid etc. They deemed under 100 nm to be the most appropriate cut off for nanoparticles, as it is within this range that most of the special properties characterized by being "nano" are exhibited.

The LPNs in the vax are about 60-80nm (Process 2) but the accepted range was considered to be 40-180nm.

There are many kinds of nanoparticles Structural properties of nanoparticles

1. carbon based, like carbon nanotubes

2. inorganic based, like metals (gold, silver, etc) and metal oxides

3. organic based, from organic matter like polymer NP; LNPs are nanostructured lipid carriers

4. Composite-based NMs include multiphase materials which have one phase on the nanoscale dimension

5. Bio-based NMs are mainly comprised of biomaterials like nanobacteria and enzymes

we will of course, just examine the organic nanoparticles, and specifically the LNPs

Special Properties of Being “Nano”

NPs are more chemically active and toxic due to its greater surface area and small size. They behave differently in different biological environments. Not all NPs are the same and the toxicity and behaviours differ depending on various factors.

Hmmm, based on these properties, can we explain some of the weird phenomenon we saw with these LNPs? The glowing is from NPs optical effects? The magnetism phenomenon might be from metal contamination in the LNPs at nanoscale? Metallic NP can be supraparamagnetic. Just some thoughts.

What we need to know about nanotechnology

How to teach nanotechnology to biologists/health care professionals? Educating biologists on nanotechnology

Since we are mainly going to talk about the LNPs, we will be focusing on the size-dependent and surface-related properties and a little on how they are designed and engineered.

Manufacturing

Essentially, the manufacturing of these LNPs is that you end up with an oil in water emulsion, or a liquid in liquid presentation.

You have all the lipids dissolved in ethanol (the organic phase) and the mRNA plus buffer (aqueous phase) and you mix them at high speeds with various flow rates and the SELF ASSEMBLE into LNPs. They are held together with electrostatic forces, that is they are not chemically bound together into an LNP. So like making a salad dressing.

The figure below shows how they are made in detail, using a herringbone mixer, instead of a T-mixer which is also used. Pilkington et al

Pilkington is a big name in LNPs BTW

Because it is basically an emulsion or more accurately a dispersion, the LNPs get bigger over time (called the Oswalt effect) and merge together. The PEG lipid is supposed to slow that process. Does the lipid part separate out from the buffer/water part? Who knows, but maybe given enough time, I think this is possible. We do know that if you shake them vigorously for 5 minutes (a vortex) a clear collapse of the nanoparticle structure was observed. See Japanese stability study of LNPs

How many LNPs in a dose?

Ay, there is the rub. How to measure these tiny particles? For the LNPs they range in size from 40-180nm. I think the manufacturers really had no clue on how to measure how many NP there were in a dose.

Here are the results of a FOIA to the UK regulator asking that very question.

MHRA FOIA on number of LNPs per dose

Huh. Not needed to know. Its because NO ONE actually knows. In August 2022, no one KNEW HOW to measure how many individual particles there were in a dose. They were all guesses. Because it is very hard to measure teeny tiny LNPs. One group is working on it, and they came up with a number of 10 to the 16th. What is that? A quadrillion? or 1 000 000 000 000 000 000 000 000, per mL. So in 30microL we would have a third of that. Whew. See look for yourself. And this scientist thinks that about 2/3rds are empty LNPs.

Payload distribution and capacity of LNPs

But they are all guesses.

Nanostructure

What do these LNPs look like? What are the nanostructure and what does it tell us? Scientists are still trying to find out, after jabbing billions with these synthetic LNPs. Here are some of the issues regarding the actual structure of the LNP and what biophysical attributes they have.

The multiplicity of biophysical attributes must be controlled on a consistent basis to get a pure, high quality product. Many of these properties are yet to be defined. Like payload quantity. How many mRNAs per LNP? On average, one method measures a mean of 2.8 per LNP. Another method of measurement closer to 5 per LNP. Plus you have various manufacturers of the lipids themselves, and various sites that make the mRNA/LNP product.

One most interesting aspect of the LNPs is their tendency to form BLEBS. If filled with DNA, they don’t’ do that as much as they do with RNA. Are blebs beneficial? Help with transfection? Some beginning works seems to suggest this. This article is by Brader from Moderna who has been studying LNPs for quite some time. Why do they form? No one knows, but it appears dependent on pH. Encapsulation rate of LNPs

I reviewed the nanostructure of the LNPs in more detail here.

The LNPs re about the same size as a virus and the same size as a lipoprotein particle in your blood called a chylomicron. So yes, the LNPs resemble a viral system with a lipid envelope and blebs. Quite something. As I said earlier, the LNPs are stealth synthetic biology.

Properties of Nanoparticles (focus on LNPs)

What are the particular properties of LNPs that make them perhaps less than safe?

1. size

2. surface charge

3. surface modifications

4. lipid composition

5. hydrophobicity

One of the most defining properties of a nanoparticle is the size to volume ratio. This is critical to understand.

Size/Volume Ratio

I found this engaging explanation of the size/volume ratio properties of nanoparticles using M&Ms as an example useful Valentines day nanotechnology

1. dispersed nanomaterials have a very large surface area and high particle number per mass unit,

2. the fraction of atoms at the surface in nanomaterials is increased,

3. the atoms situated at the surface in nanomaterials have fewer direct neighbors

It is all about the SURFACE of the LNPs. The surface of the LNPs are highly reactive compared to the bulk materials. They have strong attractive forces and can agglomerate, which is the reason for needing the pegylated lipid (about 1.5%) to slow down that agglomeration in the dispersion (ie in the vials).

Here is a visual representation of this. Nanotechnology & Nanoscience

Surface charge/Surface Hydrophobicity/Surface Modification/Lipid Composition

Here is the best graphical representation regarding how important the surface of a nanoparticle really is, and here specifically for the LNPs Impact of the physicochemical properties of the LNPs

Surface to Volume Ratio

1. transportation

2. stability of the LNPs

3. loading capacity or the number of LNPs you can encapsulate per particle (but difficult to measure!)

4. penetration of the cell membrane (ie transfection)

Hydrophobic interactions

1. formation of the biocorona. SOOOO important. In biological fluids the surface of the LNPs are coated with proteins such as albumin and complement as well as lipoproteins, particularly ApoE forming a BIOCORONA. Then they have a biological identity.

Surface Charge

1. electrostatic interference with say blood

2. circulation time (and the role of PEG shedding)!!

3. involved in the corona formation

4. stability

Lipid Composition

1. tissue tropism; not all LNPs transfect all cell types

2. organ and cell targeting; well that is still in progress but they types and proportion of lipids forming the surface can affect targeting

3. cellular uptake and membrane permeability; how efficient at transfections?

4. subcellular distribution; this is about how the LNPs are released from the endosome/lysosome to get into the cytoplasm and then the ribosomes. This is the rate limiting step of the LNPs BTW

   Most of what is out there in the literature talks about the lipid composition which is important but it is not the only thing. What you find is that IF you change the molar ratio a bit then these other aspects of the LNP also changes. A very complex entity are these LNPs.

Surface Modification

1. Blood stability: again this is about the amount an type of pegylated lipids used, and the formation of the biocorona

2. retention time: see above

3. delivery efficacy

4. designing the surface means adding antibodies, or say anti-inflammatory lipids to counter the toxicity of the LNPs?

DID EVERYONE SEE THIS BRAND NEW PAPER FROM UPENN? HOW THEY REDESIGNED THE SURFACE OF THE LNPs?

Enabling non-viral DNA delivery using lipid nanoparticles co-loaded with endogenous anti-inflammatory lipids

Here they can transfect plasmid DNA in large quantities without immediate overwhelming cytokine storm. Seriously? Well, this is UPenn after all. Home of the noble prize winner Weissman.

SUMMARY

If there is nothing else you learn from this substack entry but the following, I will have achieved my goal.

THE LNPS ARE NOT CARRIERS FOR THE MRNA. THE LNPS AND MRNA ARE A MORE THAN THEIR PARTS; THEY ARE A NANOPARTICLE WITH SPECIFIC PHYSICAL AND CHEMICAL PROPERTIES. AND IN BIOLOGICAL FLUIDS HAVE A BIOLOGICAL IDENTITY

1. nanoparticles have specific physicochemical properties

2. it is all about the surface with nanoparticles and LNPs are no exception

3. they are easy to make and complicated to characterize

4. scientists still don’t have a frigging clue on how to measure or analyze them

5. they are stealth, shape shifting, unknowable almost sentient particles

Thanks for reading. And pray the rosary