Apparent Cytotoxicity and Intrinsic Cytotoxicity of Lipid Nanomaterials Contained in a COVID-19 mRNA Vaccine

Abstract

The medicinal preparation called Comirnaty by Pfizer-BioNTech is an aqueous dispersion of lipid nanomaterials, intended to constitute, after thawing and dilution, the finished product for intramuscular injection. In the present study, we examine some evident chemical-physical criticalities of the preparation, particularly regarding the apparent and the intrinsic pKa (acid dissociation constant) of its main excipient, the ionizable cationic lipid ALC-0315. The very high value of its intrinsic pKa causes, after internalization and endosomal escape of LNPs, a sudden increase of its cationic charge concentration and consequently the formation of pro-inflammatory cytokines and ROS (reactive oxygen species), that can disrupt the mitochondrial membrane and release its content, cause RNA mistranslation, polymerization of proteins and DNA, DNA mutations, destruction of the nuclear membrane and consequent release of its content. Additionally, the apparently low pKa value (6.09) of ALC-0315 associated with other lipids in the LNP, is not suitable for intramuscular application. Its value is too low to enable a proper transfection of host cells, despite what is stated by EMA (European Medicines Agency) in its Assessment report dated 19 February 2021, in flagrant contradiction with the same bibliographic source therein cited. Furthermore, the exceptional penetrability, mobility, chemical reactivity and systemic accumulation of uncontrollable cationic lipid nanoparticles, with high cytotoxicity levels, shed in unpredictable biological locations, even far from the site of inoculation, are all factors that can lead to an unprecedented medical disaster. Meanwhile, further immediate studies and verifications are recommended, taking into consideration, in accordance with the precautionary principle, the immediate suspension of vaccinations with the COVID-19 mRNA- LNP-based vaccines.

Full text

International Journal of Vaccine Theory, Practice, and Research 3(1)
https://doi.org/10.56098/ijvtpr.v3i1.84
October 16, 2023 | Page 957
Apparent Cytotoxicity and Intrinsic Cytotoxicity of Lipid
Nanomaterials Contained in a COVID-19 mRNA Vaccine
Gabriele Segalla, PhD
Pure Chemistry (Organic Biological Chemistry), specialist in chemistry of micro-emulsions and colloidal systems, CEO
& Chief Scientist of Multichem R&D Italy, email: gabriele.segalla@gmail.com
ORCID: https://orcid.org/0000-0002-5969-3732
Abstract
The medicinal preparation called Comirnaty by Pfizer-BioNTech is an aqueous dispersion of lipid nanomaterials,
intended to constitute, after thawing and dilution, the finished product for intramuscular injection. In the
present study, we examine some evident chemical-physical criticalities of the preparation, particularly regarding
the apparent and the intrinsic pKa (acid dissociation constant) of its main excipient, the ionizable cationic lipid ALC-
0315. The very high value of its intrinsic pKa causes, after internalization and endosomal escape of LNPs, a
sudden increase of its cationic charge concentration and consequently the formation of pro-inflammatory
cytokines and ROS (reactive oxygen species), that can disrupt the mitochondrial membrane and release its
content, cause RNA mistranslation, polymerization of proteins and DNA, DNA mutations, destruction of the
nuclear membrane and consequent release of its content. Additionally, the apparently low pKa value (6.09) of
ALC-0315 associated with other lipids in the LNP, is not suitable for intramuscular application. Its value is too
low to enable a proper transfection of host cells, despite what is stated by EMA (European Medicines Agency)
in its Assessment report dated 19 February 2021, in flagrant contradiction with the same bibliographic source
therein cited. Furthermore, the exceptional penetrability, mobility, chemical reactivity and systemic accumulation
of uncontrollable cationic lipid nanoparticles, with high cytotoxicity levels, shed in unpredictable biological
locations, even far from the site of inoculation, are all factors that can lead to an unprecedented medical disaster.
Meanwhile, further immediate studies and verifications are recommended, taking into consideration, in
accordance with the precautionary principle, the immediate suspension of vaccinations with the COVID-19
mRNA- LNP-based vaccines.
Keywords: mRNA vaccine, LNP, lipid nanoparticles, ROS, reactive oxygen species, pKa, apparent pKa, intrinsic pKa
INTRODUCTION
Lipid nanoparticles (LNPs) in the two COVID-19 mRNA-LNP-based vaccines (Comirnaty
by Pfizer/BioNTech and Spikevax by Moderna Therapeutics) are formed by four different
types of lipids: an ionizable cationic lipid whose positive charge binds to the negatively
charged backbone of the mRNA, a polyethylene glycol (PEG)-linked lipid that helps
prolonging the half-life of the composition, a phospholipid to facilitate the formation of a

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two-layer structure, and cholesterol having a function of membrane fluidity
modulator/stabilizer (Figure 1).
Figure 1. “Structures of the lipid constituents of the LNPs of the COVID-19 mRNA vaccines” reprinted from Figure 8, page
16989 from the article by Tenchov, R., Bird, R., Curtze, A. E., & Zhou, Q., entitled “Lipid nanoparticles ─ from liposomes to
mRNA vaccine delivery, a landscape of research diversity and advancement” published in ACS Nano 2021, 15, 11, 16982–17015,
15(11), 16982–17015, https://pubs.acs.org/doi/full/10.1021/acsnano.1c04996. Copyright © by the authors 2021 and licensed
under CC-BY 4.0.
These nanoparticles have the primary purpose of encapsulating the mRNA, protecting it
from enzymatic degradation and assisting its penetration into the cells of the host
organism, after intramuscular injection (Nance & Meier, 2021).
The messenger RNA (mRNA BNT162b2) of the medicinal product Comirnaty by
Pfizer/BioNTech, which is expected to encode the viral Spike protein inside the host cell,
is encapsulated in lipid nanoparticles formed by the two functional lipids ALC-0315 ((4-
hydroxybutyl (azanediyl) bis (hexane-6,1-diyl) bis (2-hexyldecanoate)) and ALC-0159 (2
([polyethylene glycol]-2000)-N,N-ditetradecylacetamide), and the two structural lipids
DSPC (1,2-Distearoyl-snglycero-3-phosphocholine) and cholesterol.
In this narrative review, the purpose is to provide a detailed and documented account of
the currently existing scientific evidences proving the toxicity and hazardousness of
cationic lipid nanomaterials contained in mRNA vaccines, with particular attention to
Comirnaty by Pfizer/BioNTech, and the serious proven contradictions, omissions and
non-compliances by both the manufacturers and the regulatory bodies responsible for the
scientific evaluation, supervision and safety monitoring of medicinal products.
REGULATORY NON-COMPLIANCES AND ABSENCE OF
TOXICOLOGICAL STUDIES
ALC-0315 and ALC-0159 are classified by EMA as novel excipients, never previously used in a
medicinal product in Europe and not registered in the EU Pharmacopoeia (EMA/707383, p. 23).

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Of the two, the most important functional
lipid is ALC-0315, instrumental to the
formation of spheroidal lipid nanoparticles.
ALC-0315 is an ionizable cationic amino-
lipid consisting of a tertiary amine with a
hydroxy-butyl and two exilic groups
esterified with 2-hexyldecanoic acid
(Segalla, 2023).
Thanks to its particular tertiary aminic
structure, ALC-0315 tends to be
protonated in a neutral or moderately low pH environment, thus giving rise to the formation
of cationic nanoparticles, i.e. having a predominant positive surface charge. This positive charge
is critical as it is what allows the formation of nano-complexes with negatively charged
genetic materials such as mRNA (Figure 2).
Experimental data, however, have shown that cytotoxic and genotoxic effects are enhanced
if nanoparticles have a positive charge (Kanasty et al., 2012; Fröhlich, 2012; Barone et al.,
2017). As admitted even by BioNTech (co-owner, together with Pfizer, of the Comirnaty
vaccine) in its patent RNA Formulation for Immunotherapy dated November 26, 2019, the
elevated toxicity attributed to positively charged liposomes and lipoplexes makes them problematic
and unsuitable for use in pharmaceuticals. The reference is to formulations of RNA
encapsulated in cationic lipid nanoparticles — i.e. very similar to those used in Comirnaty
— and called, in this context, “lipoplexes”:
Unfortunately, for positively charged liposomes and lipoplexes elevated toxicity has been reported,
which can be a problem for the application of such preparations as pharmaceutical products
(patent US 10,485,884 B2)
Nevertheless, EMA, in its Assessment report dated 19 February 2021, surprisingly asserts:
No genotoxicity nor carcinogenicity studies have been provided. The components of the vaccine
formulation are lipids and RNA that are not expected to have genotoxic potential. (EMA/707383,
2021, p. 55)
As per guidance, no genotoxicity nor carcinogenicity studies were performed. The components of
the vaccine (lipids and mRNA) are not expected to have genotoxic potential. This is acceptable to
the CHMP. 1 (EMA/707383, 2021, p. 56).
REACTIVE OXYGEN SPECIES (ROS) FORMATION AND LIPID
NANOPARTICLE TOXICITY
In stark contrast to what EMA asserts, nanoparticles consisting of monovalent cationic lipids have
been shown to be significantly efficient in inducing cell death through the production of reactive
oxygen species (ROS) (Yun et al., 2016). There is overwhelming evidence that overproduction of ROS
is the main cause of nanoparticle biotoxicity. By concentrating mainly in lysosomes, mitochondria,
and the nucleus of the cell, and generating ROS at those sites, positively charged nanoparticles can
cause devastating consequences. Numerous studies irrefutably confirm that nucleotides components
1 CHMP: European Committee for Medicinal Products for Human Use.
Figure 2. Molecular structure of cationic lipid ALC-0315.

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of cellular DNA and RNA constitute a significantly vulnerable target to the aggression of ROS
generated by nanomaterials (Imlay et al., 1988; Maki et al., 1992; Demple et al., 1994). The major
challenges for the excessive use of cationic nanomaterials are their dose-dependent toxicity,
hepatotoxicity, and pulmonary inflammation by promoting the release of reactive oxygen species
and increasing intracellular calcium levels (Ozpolat et al., 2014; Lee et al., 2013; Zhang et al., 2014).
Moreover, cationic liposomes can interact with negatively charged cellular constituents, such as
opsonins and serum proteins, resulting in hemolysis, i.e. the rupture or destruction of red blood
cells (Buyens et al., 2012). In addition, LNPs can induce activation of the immune system resulting
in complement activation-related pseudoallergy (CARPA), an acute immunological response that can
lead to anaphylactic-like shock (Szebeni et al., 2014).
INTRINSIC pKa OF THE AMINO-LIPID ALC-0315
The amount of an ionizable compound being protonated (i.e. positively charged) in an aqueous
solution, at a certain pH, is defined by the value of its acid dissociation constant (pKa)2.
The pKa value of a ionizable compound defines the pH at which its functional ionizable groups are
50% in ionized and 50% in a de-ionized form. Since ALC-0315 contains a protonatable tertiary
amine head group with an intrinsic pKa of 9.6 (Zhang et al., 2022), this implies that, at a pH value of
9.6, there would be 50% of the molecules in the protonated form (R3NH+) and 50% in the neutral
form (R3N), according to the equation:
R3N + H+
R3NH+ (1)
which, for ALC-0315, can be simplified and schematized as follows:
+ H+ (2)
ALC-0315
neutral
ALC-0315
protonated
The neutral, non-protonated form of ALC-0315 (represented in green) will express the least toxicity,
while its fully protonated form (represented in red) will express the maximum toxicity due to the
disrupting interactions of its cationic charges with anionic parts of endosomal, lysosomal and
mitochondrial membranes (Figure 3).
2 Acid dissociation constant (pKa): the pH at which molecules are half dissociated. pKa is of utmost importance for
understanding drug absorption and biodistribution in the systemic circulation. pKa measurements enable the proportion
of the molecules in the ionized (charged) or deionized state to be determined (P. Patel et al., 2021).

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Figure 3. Representation of charged and uncharged forms of ALC-0315.
The value of the pKa of an ionizable compound is of utmost importance for understanding its
absorption and biodistribution, and, as we will see shortly, also for consistently estimating its
cytotoxicity. Measurements of the pKa make it possible to calculate the exact proportion of ionized
(positively charged) and deionized (neutral) molecules of ALC-0315, at a certain pH.
The relationship between the ratio of the protonated form to the non-protonated form and the pKa
of the ionizable molecule is regulated by the Henderson-Hasselbach equation, which, in the case of a
tertiary amine, can be written as follows:
Log [R3N]/[ R3NH+] = pH - pKa (3)
or
[R3N]/[ R3NH+] = 10 (pH – pKa) (4)
where R3N is the deprotonated form (neutral) and R3NH+ is the protonated form (cationic) of
ALC-0315.
The Henderson-Hasselbach equation clearly shows that there is a strict correlation between the pH
of the aqueous medium, the pKa of the ionizable substance and the relative concentrations of its
cationic and neutral forms. This provides as well a good assessment of its theoretical cytotoxicity
(according to the principle that, within the same number of moles, the more protonated a species is,
the greater the resulting cytotoxicity). The resulting pKa determines also the ionization behavior and
surface charge of the ionized nanoparticles, which substantially influence their stability, potency 3,
and toxicity (Alabi et al., 2013).
At pH values below pKa, the predominant chemical species is the protonated form (cationic, more
cytotoxic) of the amino-lipid, while at pH values above pKa, the predominant chemical species is
the basic de-protonated (neutral, less cytotoxic) form of the amino-lipid.
3 Potency is an expression of the activity of a drug, in terms of the concentration or amount needed to produce a
defined effect (Neubig et al., 2003)

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Applying equation 4 above, at a physiological pH of 7.4, we will have:
[R3N]/[ R3NH+] = 10 (7.4 – 9.6) = 0.006309573
which simply indicates that, in a physiological environment, 99.37% of the molecules of ALC-0315
are protonated, thus expressing their maximum cytotoxicity.
APPARENT pKa OF LIPID NANOPARTICLES
To reduce cytotoxicity of cationic liposomes and nanomaterials in general, a relatively effective
solution has been the modulation of their acid dissociation constant (pKa), whereby values of 7 or
lower were demonstrated to be of importance in RNA encapsulation and in vivo activity. When the
amino-lipid is inserted, together with other structural lipids, within the structure of a lipid
nanoparticle, its pKa can undergo a lowering of 2-3 units, due to the forces of interaction with
anionic species or with polar portions of the other contiguous lipids.
The new lower value achieved for the particle is called apparent pKa (or surface pKa), to distinguish it
from the original intrinsic pKa, i.e. the pKa value of the amino-lipid measured before the formation of
the lipid nanoparticle. 4
The apparent pKa of a lipo-
nanoparticle is then defined
as the pH at which 50% of
the ionizable lipids,
associated to that LNP, is
protonated.
For example, the lipid
nanoparticle used to
encapsulate Onpattro (a drug
for the treatment of
hereditary amyloidosis and
administered via intravenous
infusion) contains the FDA-
approved amino-lipid DLin-
MC3-DMA (intrinsic pKa
9.4), whose LNP apparent
pKa gets thus lowered to
6.44. The apparent pKa of
LNPs formed by amino-lipid
SM-102 and used to
Figure 4. Structure and pKa values of amino-lipids: 1) DLin-MC3-DMA
(Onpratto/ Patisiran by Alnylam): apparent pKa 6.44, intrinsic pKa 9.4 2),
SM-102 (Spikevax by Moderna): apparent pKa 6.75, intrinsic pKa 8.9 3) Alc-
0315 (Comirnaty by Pfizer BioNTech): apparent pKa 6.09, intrinsic pKa 9.6.
encapsulate the Moderna vaccine Spikevax, from the intrinsic value 8.9, gets down to 6.75. The
apparent pKa of ALC-0315-based LNPs is reduced from 9.6 to 6.09 (P. Patel et al., 2021; Zhang et
al., 2022; and see Figure 4).
4 The apparent pKa of LNPs is dependent not only on the pKa of individual lipid but also on the molar ratio of all the
lipids. Each lipid has a distinct pKa which can be changed by modifying its headgroup and the hydrophobic tail.
Therefore, one strategy to adjust the apparent pKa of LNPs is to chemically modify the lipid. Another strategy is to use
a mixture of two or more lipids with different pKa values and adjust their ratio to achieve the desirable apparent pKa (P.
Patel et al., 2021).

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The apparent pKa of nanoparticles
can be measured by different
techniques. TNS5 fluorescence
titration is considered the most
accurate method: amino lipid pKa
values are determined for each LNP
by measuring the fluorescence of
TNS during titration at different pH
values (Jayaraman et al., 2012; P.
Patelet al., 2021). An apparent pKa
between 6 and 7 has been generally
estimated as the optimum range for
the development of efficient
nanoparticles for RNA delivery.
Nanoparticles with lower pKa values
have insufficient ionic charges and
polarity at neutral pH, thereby
leading to aggregation of the
nanoparticles and consequent
instability of the whole composition.
On the other hand, nanoparticles
with a higher pKa exibit more
positive charges at physiological pH,
which results in higher stability but, unfortunately, also in higher toxicity (Figure 5).
When the pH of the preparation medium is below the apparent pKa of the lipid nanoparticles, the
amino groups are protonated and bear a positive charge that interacts with negatively charged RNA
to form stable cationic nanoparticles.
OPTIMAL AND NON-OPTIMAL PKA RANGE OF LNPS FOR
INTRAMUSCOLAR ADMINISTRATION OF MRNA VACCINES
According to the EMA Assessment Report, dated 19 February 2021 (EMA, 2021, page 42), in the
physiological environment, where the pH is 7.4, these lipid nanoparticles, thanks to the contribution
of the primary driver ALC-0315, have a neutral charge:
The potency of the RNA vaccine is further optimized by encapsulation of the RNA into lipid
nanoparticles (LNPs), which protects the RNA from degradation by RNAses and enable
transfection of host cells after intramuscular (i.m.) delivery. The functional and ionizable lipid, ALC-
0315, is identified as the primary driver of delivery as it allows the LNPs to have a neutral charge in
a physiological environment to facilitate internalization; the endosomal environment exhibits a
positive charge and therefore triggers the translocation of RNA into the cytosol (Midoux & Pichon,
2015; Hassett et al, 2019; S. Patel et al, 2019; and see Figure 6).
5 TNS: 2-(p-toluidino)-6-napthalene sulfonic acid.
Figure 5. The Effects of pKa on the Instability, Potency, and
Toxicity of Nanoparticles.
Reprinted from Trends in Pharmacological Sciences, Vol.
42(6), 448–460, Patel et al., 2021, The Importance of Apparent
pKa in the Development of Nanoparticles Encapsulating siRNA and
mRNA, Page No. 458, Copyright © 2021, with permission
from Elsevier Ltd.

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Figure 6. EMA Assessment Report on Comirnaty by Pfizer/ BioNTech, dated 19 February 2021, page 42.
From such statement, it is clear that EMA, by even calling it the primary driver, has undoubtedly
understood that ALC-0315 is an instrumental and determinant factor in facilitating and optimizing the
internalization of LNPs into the endosomal environment and the consequent translocation of
mRNA into the cytosol.
However, it seems that the author of the EMA assessment report has scrupulously omitted to
assess, if not willingly ignored, what is reported in one of the scientific references cited in support
of such a claim, namely, the 2019 article by Hassett et al., with the eloquent title: Optimization of Lipid
Nanoparticles for Intramuscular Administration of mRNA Vaccines.
Unexpectedly, all 19 authors of this paper are either current or previous employees of Moderna
Therapeutics and own stock options and/or shares in the company, the main Pfizer's competitor for
what regards mRNA COVID-19 vaccines.
Figure 7. Extract of scientific article by Moderna Therapeutics (2021) on optimization of LNPs for intramuscular and
intravenous administration.

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But what is even more unexpected is found on page 3 of this paper, with regard to the optimal value of
pKa (rightly defined as a strong determinant) that lipids for intramuscular administration should have:
[…] One strong determinant of immunogenicity was the lipid pKa, with a range of 6.6–6.9 being optimal for
IM [intramuscular] immunogenicity […]. This differs from the optimal pKa range for IV [intravenous] delivery
of siRNAs and mRNAs, which has been reported as 6.2–6.6. (Figure 7).
The scientific article by Hassett et al.,
cited by EMA in support of the
claimed ideal role played by ALC-
0315 in the intramuscular
administration of the Comirnaty
vaccine, clearly and thoroughly
explains that the optimal lipid pKa
range for intramuscular applications
should be between 6.6 and 6.9.
Such range is considered optimal, as
LNPs with those values of pKa
produce an efficient immune
response after intramuscular
administration of mRNAs (P. Patel
et al., 2021). It is therefore clear that
ALC-0315 is NOT suitable for
intramuscular delivery, since its
apparent pKa (6.09) is much lower
than the optimum (Figure 8),
making the RNA vaccine
composition oo unstable and
ineffective for intramuscular
Figure 8. Lipid pKa of ALC-0315 with regards to instability, potency and
toxicity. Modified from Trends in Pharmacological Sciences, Vol. 42(6),
448–460, Patel et al., 2021, The Importance of Apparent pKa in the Development of
Nanoparticles Encapsulating siRNA and mRNA, page No. 458, copyright ©
2021, with permission from Elsevier Ltd.
application. According to Hassett et al., it would not be suitable either for intravenous delivery, this
later one requiring apparent pKa values between 6.2 and 6.6. The optimal range indicated by Hassett
et al. is in sharp contradiction with the technical information that EMA highlights in its official
assessment report on the medicinal product Comirnaty, with regard to its primary driver ALC-0315.
In the light of what has been so far exposed and what will be hereafter presented, asserting that
Comirnaty LNPs enable transfection of host cells after intramuscular (i.m.) delivery and that ALC-0315
allows the LNPs to have a neutral charge in a physiological environment to facilitate internalization, is
scientifically unacceptable and potentially misleading, as it promotes the idea that the composition
of the LNP-based vaccine Comirnaty has been somehow "optimized" for intramuscular inoculation.
Such statement is disavowed by the very reference which it is based on.
In the final analysis, it is evident (Figure 8), that the apparent pKa value of ALC-0315 is too low to
be defined optimal, and that a so low value makes the entire structure of the LNP unstable, inducing
the formation of aggregates and particulates, which may inhibit the transfection and inevitably
influence, not only the efficacy of the product, but also both the biodistribution and the
bioaccumulation of lipid nanoparticles in unexpected tissues and organs. Bioaccumulation can lead
to blockage of small blood and lymphatic vessels, while an abnormal biodistribution means that cell

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death and inflammation caused by the COVID-19 mRNA vaccine could occur in organs not
foreseen by its biological destiny, such as the brain, placenta, and testes (Parry, P.I., et al., 2023;
Zhou, Y., et al., 2018; Wick, P., et al., 2010).
Other significant evidences relating to the unsuitability of the medicinal product Comirnaty for
intramuscular application, as well as its instability and inefficacy due to the formation of LNP
aggregations and agglomerations caused by the addition of destabilizing ionic compounds (PBS pH
buffer), have already been described in detail (Segalla, 2023).
PREDOMINANCE OF THE CYTOTOXIC PROTONATED LIPID FORM
DURING THE ENDOCYTOTIC PROCESSES
In the acidic environment of endosomes, the amino groups are protonated and their positive charge
promotes interaction with anionic endosome lipids, inducing destabilization of the endosome
membrane and promoting the release of mRNA into the cytosol (Wan et al., 2014; Draz et al., 2014;
Tam et al., 2013), as also briefly mentioned in the aforementioned EMA report:
[…] In general, following endocytosis of LNPs, the mRNA is released from the endosome into the
host cell cytosol (Sahay et al, 2010; Maruggi et al, 2019).
The RNA-loaded nanoparticle, once penetrated by endocytosis into the cell, undergoes various
passages through early endosomes, late endosomes and lysosomes, along a decreasing pH gradient
(Figure 9), until its cationic charge causes the rupture of the endosomal membrane and the
consequent release of RNA inside the cytosol (P. Patel, et al., 2021).
Figure 9. Delivery of RNAs into the cytoplasm through endosomal escape with ionizable nanoparticles. Once
nanoparticles are taken up by the cells, the charges on the nanoparticle increase as the pH decreases below the pKa
during endosomal maturation (pH 7–5.5). The charges on nanoparticles decrease in the cytosol and weaken the binding
interaction with RNAs. Finally, the nanoparticles dissociate to release the RNAs and produce the desired activity.
Reprinted from Trends in Pharmacological Sciences, Vol. 42(6), 448–460, Patel et al., 2021, The Importance of Apparent pKa in the
Development of Nanoparticles Encapsulating siRNA and mRNA, Page No. 451, Copyright © 2021, with permission from
Elsevier Ltd.

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The pH thus decreases along the endosomal-lysosomal system: early endosomes have a pH between
6 and 7, late endosomes have a pH in the range of 5 and 6, and in lysosomes the pH can decrease to
4.5 (Hu et al., 2015).
Applying equation (4) to Comirnaty LNP composition, we can calculate the ratio [R3N]/[R3NH+]
and, consequently, the percentage of the protonated species (i.e. the most cytotoxic), at various pKa
and pH conditions, as follows:
- Before endocytosis: physiological pH 7.4, ALC-0315/LNP apparent pKa 6.09
- In early endosomes: pH 6.5, ALC-0315/LNP apparent pKa 6.09.
- In late endosomes: pH 5.5, ALC-0315/LNP apparent pKa 6.09.
- In lysosomes: pH 4.5, ALC-0315/LNP apparent pKa 6.09.
- In cytosol, after endosomal escape and LNP disassembling: pH 7.4, ALC-0315 intrinsic pKa
9.6.
The results are shown in Table 1 and Figure 10.
Table 1.
Another issue that cannot be overlooked is undoubtedly the enormous difference between the
apparent pKa and intrinsic pKa values of the lipid ionizable species, particularly for the Pfizer
preparation. Considering that these values are logarithmic values, a difference of 3.51 between the
intrinsic pKa and the apparent pKa of ALC-0315 means that its intrinsic tendency to protonation
(i.e. its base strength) is 3,236 times higher than the one expressed by its apparent LNP pKa. Similarly
for SM-102, a difference between the two pKa values of 2.15 means an intrinsic protonating
tendency 141 times higher than the apparent one. This elementary concept is the basis of the
surprising and sudden leap in predominance of the protonated species (and therefore of its
cytotoxicity) during the endocytotic pathway, as evidenced by the graph of Figure 10.
It should also be noted that the intrinsic pKa value (9.6) of ALC-0315 represents the absolute
highest ever pKa value for a functional ionizable lipid used in cationic LNPs for immunotherapy.
Being its pKa even higher than that of the ammonium ion (9.25), ALC-0315 expresses a base
strenght about 2 times higher than that of ammonia itself, that is, it possesses ionising powers
twofold stronger than those of an equimolar aqueous solution of ammonia.

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Figure 10. The main fundamental issue that emerges from these calculations is that, before the endocytotic process, the
presence of the protonated form R3NH+ is relatively low and therefore less cytotoxic (4.67%), but then, as soon as the lipid
nanoparticles pass through the phases of lysosomal digestion, LNP disassembling and endosomal escape into the cytosol,
the predominance of the cytotoxic protonated forms jumps up to nearly its maximum value (99.37%). The equilibrium of
equation (1) shifts practically all to the right, that is, towards the production of the cationic and more cytotoxic species
R3NH+.
Therefore, the molecules of ALC-0315, once penetrated and released into the cytosol, after the
disassembly of the LNP envelope, reach maximum predominance of their cationic form, and thus
express the maximum of their cytotoxicity, stimulating the secretion of pro-inflammatory cytokines
and reactive oxygen species (Hou et al., 2021). These ROS, in turn, may have devastating
toxicological consequences including genotoxicity (Yun et al., 2016; Yu et al., 2020), leading to very
serious problems, in the medium and long term, particularly for parenteral applications, as
previously known also to the manufacturer of the medicinal product Comirnaty (BioNTech patent
US 10,485,884 B2, 2019 ). Furthermore, the exceptional penetrability, mobility, chemical reactivity
and systemic accumulation of uncontrollable cationic nanoparticles, with high cytotoxicity levels, in
unpredictable biological locations, even far from the site of inoculation, have predictably resulted in
an unprecedented medical disaster. It should be noted that, with any agent that causes genetic
damage, including cytotoxic anticancer drugs, there is a risk of cancer (including leukemia), and
moreover there is a lifetime limit on the overall dose that can be tolerated. Thus, the prospect of
frequently repeated COVID “booster shots,” and also that of extending mRNA technology to
vaccines against other pathogens or non-infectious diseases, conjures up a very grave public health
risk (Palmer et al., 2022).
CONCLUSIONS AND OUTLOOK
The body of work presented in this review includes several physico-chemical, biochemical and
toxicological evidences which clearly show and irrefutably demonstrate how the medicinal product
named Comirnaty COVID-19 mRNA BNT162b2 vaccine, is not only unsuitable for intramuscular
inoculation, but also endowed with a high degree of toxicity whose devastating consequences can

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manifest themselves both in the short term and in the medium and long term, due to the shedding
effect of LNPs biodistribution and bioaccumulation.
The main reasons for such inadequacy and criticality are represented by the following factors:
- The apparent pKa value (6.09) of the ionizable lipid ALC-0315 is far from having been
optimized for intramuscular delivery. Its value is actually too low to enable a proper
transfection of host cells, despite what EMA incautiously states on page 42 of its
Assessment report dated 19 February 2021, in flagrant contradiction with the same
bibliographic source therein cited (Hassett et al, 2019).
- The intrinsic pKa value (9.6) of the ionizable lipid ALC-0315 is too high, which makes it a
stronger base than ammonia itself (pKa 9.25) in aqueous solution, and makes it thus
completely protonated once released into the cytosol of the host cell, at physiological pH.
- Such elevated cationic charge, acquired by ALC-0315 after its endosomal escape, stimulates
the formation of pro-inflammatory cytokines and reactive oxygen species, that can disrupt
the mitochondrial membrane and release its content, cause RNA mistranslation,
polymerization of proteins and DNA, DNA mutations, destruction of the nuclear
membrane and consequent release of its content (Yu et al., 2020).
- This kind of “troyan horse” mechanism determines the apparently low toxicity of the LNP
before internalization, facilitating its penetration into the host cell. However, once penetrated
and released into the cytosol, the LNP gets disassembled and its cationic lipid fragments are
free to re-express their intrinsically elevated pKa and therefore their maximum intrinsic
toxicity.
- An elevated chemical toxicity of the ionizable cationic LNPs, maximized in the Comirnaty
vaccine by the extreme ionizing power of ALC-0315, nevertheless must be expected also in
other and future vaccines that use the same delivery technology, namely the mRNA cationic
LNP-based platform, regardless of whether they be directed against the spike protein,
another SARS-CoV-2 antigen, or a different antigen or disease altogether.
- There is overwhelming scientific evidence that the lipid-nanoparticles used in the COVID-19
vaccines have been found to induce significant inflammatory cytokine secretion and
macrophage inflammatory proteins with cell death. This pro-inflammatory effect of the
cationic lipid-nanoparticles would increase the vaccine adjuvant immunogenicity of the
COVID-19 mRNA vaccines and add to the adverse events caused by the toxicity of the
spike protein itself (Ndeupen et al., 2021; Parry et al., 2023).
A Final Word
It is to be considered a matter of priority that thorough and long-term studies should be carried
out in the appropriate institutional, clinical and forensic locations, especially in relation to any
causal or con-causal links between what is presented here and the wide pathological heterogeneity
of serious or lethal adverse events that have occurred, and are still occurring, after vaccinations, in
order to adopt and expedite all appropriate corrective and preventive actions to protect public
health, including discontinuing vaccinations with the COVID-19 mRNA-LNP-based vaccines in
accordance with the precautionary principle, and in the light of

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Article 10 of the Nuremberg Code
:
During the course of the experiment the scientist in charge must be prepared to terminate the
experiment at any stage, if he has probable cause to believe, in the exercise of the good faith,
superior skill and careful judgment required of him, that a continuation of the experiment is
likely to result in injury, disability, or death to the experimental subject.
Funding and conflicts of interest
The author declares that he has not received any funding to influence what he says here and that the
research was conducted in the absence of any commercial or financial relationship that could be
construed as a potential conflict of interest.
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