The porphyrin-fullerene nanoparticles to promote the ATP overproduction in myocardium: 25Mg2+-magnetic isotope effect

Article abstract of DOI:10.1016/j.ejmech.2008.07.030

This is a first case ever reported on the fullerene-based low toxic nanocationite particles (porphyrin adducts of cyclohexyl fullerene-C₆₀) designed for targeted delivery of the paramagnetic magnesium stable isotope to the heart muscle providin

Full text of DOI:10.1016/j.ejmech.2008.07.030

European Journal of Medicinal Chemistry 44 (2009) 1554–1569
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
The porphyrin–fullerene nanoparticles to promote the ATP overproduction in
25
myocardium: Mg2þ-magnetic isotope effect
S.M. Rezayat a, S.V.S. Boushehri a, B. Salmanian a, A.H. Omidvari a, S. Tarighat a, S. Esmaeili a, S. Sarkar b,
,
c
d
d
e
e
N. Amirshahi c , R.N. Alyautdin , M.A. Orlova , I.V. Trushkov , A.L. Buchachenko , K.C. Liu ,
*
D.A. Kuznetsov e
a School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
b Research Center for Science and Technology in Medicine, TUMS, Tehran, Iran
c School of Pharmacy, I.M. Sechenov Moscow Medical Academy, Moscow, Russia
d Department of Chemistry, M.V. Lomonosov Moscow State University, Moscow, Russia
e N.N. Semenov Institute for Chemical Physics, Russian Academy of Sciences, Moscow, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
This is a first case ever reported on the fullerene-based low toxic nanocationite particles (porphyrin
adducts of cyclohexyl fullerene-C60) designed for targeted delivery of the paramagnetic magnesium
stable isotope to the heart muscle providing a sharp clinical effect close to about 80% recovery of the
tissue hypoxia symptoms in less than 24 h after a single injection (0.03–0.1 LD50). A whole principle of
this therapy is novel: 25Mg2þ-magnetic isotope effect selectively stimulates the ATP overproduction in
the oxygen-depleted cells due to 25Mg2þ released by the nanoparticles. Being membranotropic cationites,
these ‘‘smart nanoparticles’’ release the overactivating paramagnetic cations only in response to the
metabolic acidic shift. The resulting positive changes in the heart cell energy metabolism may help to
prevent and/or treat the local myocardial hypoxic disorders and, hence, protect the heart muscle from
a serious damage in a vast variety of the hypoxia-caused clinical situations including both doxorubicin
and 1-methylnicotineamide cardiotoxic side effects. Both pharmacokinetics and pharmacodynamics of
the drug proposed make it suitable for safe and efficient administration in either single or multi-injection
(acute or chronic) therapeutic schemes.
Received 25 February 2008
Received in revised form 17 July 2008
Accepted 17 July 2008
Available online 31 July 2008
Keywords:
Porphyrin–fullerenes
Myocardium hypoxia
25Mg2þ-magnetic isotope effect
ATP overproduction
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
ATP formation in myocardium tissue suffering from hypoxia of any
sort.
A development of pharmacologically appropriate, i.e. safe and
efficient, nanocarriers for Mg2þ ions has been initiated by the
recent discovery of a so-called magnesium-25 magnetic isotope
effect in biophysical control over the energy supplying processes in
mitochondria. Thus, the only magnetic isotope of magnesium,
To meet these expectations, a novel pharmaceutical nano-tool
based on the porphyrin-attached fullerene-C60 ‘‘ball’’ (Por-
phylleren-MC16 or PMC16) is now proposed [1,2].
Medicinal hypoxia syndromes, i.e. the drug side effects leading
to sharp oxygen consumption decrease in mammalian tissues,
complicate some anticancer chemotherapeutic procedures. Thus,
a doxorubicin (DXR) induced cardiotoxicity relates predominantly
to the suppression of the ADP oxidative phosphorylation in
myocardial mitochondria [3–5]. That means, the reserve ATP
regeneration pathway, namely the creatine kinase (CK) directed
ADP phosphorylation [6,7], could be chosen as a target for phar-
macological impact having an aim to minimize, i.e. to reduce if not
even exclude, the DXR side effect in a course of (or prior to) the
long-term cytostatic medication.
25Mg, is proven to be a specific over-activator for most Mg2þ
-
dependent reactions of ATP production taking place in a cell.
Noteworthy, this 25Mg2þ-related superactivation of energy
metabolism requires a minute amount of these ions and works
perfectly even in the absence of oxygen (deep tissue hypoxia).
So a desirable application of the above-specified physical/
biophysical phenomenon would be the correction for the decline of
Being Mg2þ-dependent processes, both substrate and oxidative
phosphorylation pathways might be activated up to 2.5-fold more
efficiently by ultramicro-amounts of 25Mg2þ, the only magnetic
magnesium isotope, as compared to non-magnetic 24Mg2þ and
26Mg2þ isotopes [8]. A simple ‘‘endoosmic pressure’’ is to replace
Abbreviations: DXR, doxorubicin; MNA, 1-methylnicotineamide; CK, creatine
kinase; SANS, small angle neutron scattering.
* Corresponding author.
E-mail addresses: rezayat@sina.tums.ac.ir (S.M. Rezayat), sarkar@sina.tums.ac.ir
(S. Sarkar), amirshahi@mail.ru, amupwaxu@gmail.com (R.N. Amirshahi), orlova.r-
adiochem@mail.ru (M.A. Orlova), kuznetsov.icp@mail.ru (D.A. Kuznetsov).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.07.030

S.M. Rezayat et al. / European Journal of Medicinal Chemistry 44 (2009) 1554–1569
1555
one Mg isotope by another (all of them are stable ones) inside the
CK active site [9]. This is about the magnetic isotope effect of 25Mg2þ
which is now found to be an essential, overactivating element in the
magnesium-dependent ATP production control [8–11]. It makes
a targeted delivery of the above-mentioned magnetic isotope (þ5/2
nuclear spin, 0.85 Bohr magnetron magnetic moment, 11% natural
abundance) towards a hypoxia damaged cells/tissues a truly
important pharmacological task. So the Mg2þ-exchanging nano-
particles would be an appropriate tool for such a targeting.
For this purpose, a low toxic (LD50 ¼ 896 mg/kg, i.v., rats),
amphiphilic (430 mg/ml water, pH 7.40), membranotropic and
cluster forming 1.8 nm fullerene-C60 based particles were now
designed [1,2]. This novel medicinal product, which possesses
marked cationite properties (Fig. 1), is the iron containing
porphyrin monoadduct of a classical buckminster fullerene, buck-
towardstheheartmuscleusingaporphyrinic-fullerenenanoparticles
as the membranotropic Mg2þ-releasing carriers.
2. Materials and methods
2.1. Reagents
DXR, MNA, ATP, ADP, phosphocreatine, creatine, MgCl2, sucrose,
all of analytical grade, were purchased from Merck Corp. USA. All
organic solvents used were kindly supplied by Bio-Rad, Russia. Pure
magnesium isotopes were received in a chloride form (96.8%
isotopic purity, A grade) from Obninsk Radiochemical Center,
Russia. The very same supplier was also responsible for the
following radioactive materials provided: 59FeCl2 (A grade,
910–960 Ci/mmol specific activity), sodium [32P]orthophosphate
(A grade, 600–700 Ci/mmol) and [32P]phosphocreatine (A grade,
280–320 Ci/mmol) were used. Creatine kinase (60–80 U/mg
protein), nuclease S (100–120 U/mg protein) and RNase A (100–
140 U/mg protein) were purchased from Worthington Corp., USA.
Fullerene-C60 was purchased from Lachema Corp. (Czech Republic).
minsterfullerene(C60)-2-(butadiene-1-yl)-tetra(o-g-aminobutyryl-o-
phthalyl)ferroporphyrin. Due to a protocol reason, it has been called
a ‘‘Porphylleren-MC16’’ or, in brief, PMC16.
One would have at least two hopes for this agent to meet. First,
its unique structure may allow the drug to serve as a nanocationite
both in vitro and in vivo which is potentially beneficial in terms of
‘‘smart release’’ of magnesium in hypoxia-caused acidosis.
Secondly, a porphyrin domain of PMC16 is expected to provide
a tissue selective interaction with the heart muscle specific for
porphyrin receptors located in mitochondrial membrane of myo-
cardiocytes [12–14]. This would navigate the drug towards the
heart in a manner of real targeted delivery. In the latter case, the
drug mitochondria intake seems realistic in view of the PMC16
particle size (1.8 nm) [1,2]. The PMC16 pharmacokinetics and the
drug–receptor recognition pattern might prove or disprove that.
According to a conventional up-to-date biopharmacy/pharma-
cology glossary, a medicinal nanoparticle or nanomedicine means
a slowly metabolizing xenobiotic of size of a molecule within
a nano-scale range (rigid structure, 1.0–50.0 nm) manifested by a
marked tropism in biological structures in vivo and in vitro and
capable of penetrating the biological barriers (endothelial, blood/
brain, cell wall, cell compartment membranes) with a following
pharmacological effect to promote [14].
2.2. Animals
Wistar Albino Glaxo (WAG/Sto2J strain) male healthy adult rats
(180–220 g) used were kept under a standard vitaminized diet,
starving for 24 h prior to each one of the test conducted. Three
animals per one experimental point with following 5–6 repetitions
of every measurement were taken as a rule.
2.3. Hypoxia experimental models
The following in vivo experimental hypoxia models were
employed:
(1) Medicinal hypoxia syndrome
A
(1-methylnicotineamide
(MNA), 10–20 mg/kg i.v. once per 24 h);
(2) Medicinal hypoxia syndrome B (DXR 60–80 mg/kg i.v. once per
24 h);
A present work is devoted to biochemical/pharmacological study
on[25Mg]PMC16effectsinratmyocardiumaffectedbyeitherthedrug
(DXR, MNA) induced hypoxia or air oxygen deficit caused hypoxia
with respect to cationite properties of the new nanomedicine tested.
This is a part of the broader research project pronouncing an aim to
develop a novel nanopharmacological approach to prevent and treat
the local tissue hypoxia syndromes (including the DXR promoted
cardiotoxicity side effect) by a targeted delivery of 25Mg2þ cations
(3) The oxygen-depleted inhalation model (10–20 day long expo-
sition of rats to the artificial gas mixture to breath with 12–16%
oxygen/84–88% nitrogen, v/v).
2.4. Isolation of cell compartments
To isolate cell organelle (nuclei, cell wall membranes, mito-
chondria, cytosol), the heart tissue homogenate was conventionally
fractionated by the differential ultracentrifugation in 800 g/
12,000 g/35,000 g/125,000 g regime using the Beckman Spinco
L5-65B Ultracentrifuge, SW27.1 rotor [15,16]. Scalar 0.8–2.0 M
sucrose gradients were applied to isolate and purify the mito-
chondrial membrane fragments (SW 27.5, 110,000 rpm, 2 h) after
the osmotic shock promoted decomposition of organelles and their
nuclease S/RNase A treatment [17]. Protein measurements were
performed by a routine Bradford colorimetric method [18]. When
needed, protein preparations were concentrated on Amicon Diaflo
Y5.0 membranes at 800 psi.
2.5. Isotope analysis
59Fe ( -emitter) and 32P (
g b-emitter) quantitative radioactivity
measurements were conducted in the dioxane LKB scintillation
counting fluids using the LKB SK260 and the Wallac 410B liquid
scintillation counters, respectively.
For the 59Fe-related autoradiography of the organelles isolated,
Fuji RX40 films were employed along with the Farrand XL30
Fig. 1. The PMC16 nanoparticle structure.

1556
S.M. Rezayat et al. / European Journal of Medicinal Chemistry 44 (2009) 1554–1569
transmission electron microscopy unit with a consequent negative
track analysis [19].
First, a diethyl phthalate is to be silicomethylated by trime-
thylsilicochloride in the presence of butyllithium catalyst with
a formation of diethyl-3-(trimethylsilyl)phthalate (Product II, step
1). The latter one is then transformed into diethyl-3-formyl-6-
(trimethylsilyl)phthalate (Product III) by the oxidation of Product II
in the presence of the same mentioned catalyst and dime-
thylformamide (step 2). Product III is then monobromated by
N-bromosuccinimide in the presence of tetrabutylammonium
fluoride so diethyl-6-bromo-3-formylphthalate (Product IV)
formed as a result (step 3). Next, Product IV is to be condensated
with pyrrol in the presence of 2,3-dichloro-5,6-dicyano-1,4-ben-
zoquinone (DDQ) which leads to the formation of Product V, the
Estimations of magnesium stable isotopes (25Mg, 24Mg, 26Mg)
were carried out in the Olivetti Prism DL600 isotope mass spec-
trometer suitable for studies on heterogenous samples of biological
origin [8,10]. For the PMC16 saturation with 25Mg2þ cations, our
original electro-osmotic technique has been applied [1,2].
2.6. Preparation of PMC16 [25Mg2þ
] nanoparticles
4
The aiming compound (Porphylleren-MC16 or PMC16) prepared
by the following method is exemplified in Scheme 1, steps 1–10.
first obtained porphyrin structure (step 4). Then Product
V
Step I
Step II
Step III
CO2Et
CO2Et
CHO
CO2Et
CO2Et
1
CO2Et
CO2Et
1) BuLi, -78°C
2) Me3SiCl
1) BuLi, -78°C
2) DMF
SiMe3
2
SiMe3
3
EtO
O
EtO
EtO
O
OEt
O
Step IV
Br
Step V
Br
O
CHO
CO2Et
CO2Et
1)
2)
NBS
Bu4NF
N
H
N
H
DMF
N
N
[O]
POCl3
H
Br
O
N
4
O
EtO
OEt
OEt
Br
Br
O
O
OEt
O
EtO
5
EtO
EtO
O
EtO
O
EtO
O
OEt
O
Br
OEt EtO
Br
Br
Br
O
O
O
Step VI
N
N
+
-
H
Ph3PCH2CH=CH2 Br
H
N
O
N
N
N
LDA, LiClO4
H
H
N
N
O
O
EtO
EtO
Br
Br
Br
OEt
OEt
OEt
O
O
Br
OEt
OEt
O
O
OEt
O
O
6
7
Step VII
Mg2+
H2N(CH2)3COOH
Mg2+
O
O
O
O
HOOC
(CH2)3
H
O
O
H
N
COOH
EtO
O
EtO
EtO
O
N
H
HOOC
(CH2)3
OEt
H
O
O
N
COOH
(CH2)3
N
(CH2)3
O
O
Step VIII
) aq. NaOH
N
H
N
1
N
H
N
H
N
MgCl2
2)
N
N
H
COOH
(CH2)3
N
(CH2)3
HOOC
O
O
COOH
(CH2)3
(CH2)3
N
O
N
O
O
H
EtO
H
N
O
O
O
HOOC
N
Mg2+
O
OEt
OEt
O
O
H
Mg2+
H
OEt
O
9
8
Scheme 1. The PMC16 synthesis route’s key steps.

S.M. Rezayat et al. / European Journal of Medicinal Chemistry 44 (2009) 1554–1569
1557
COOH
COOH
2Mg2+
O
2Mg2+
O
HOOC
O
O
HOOC
O
O
(CH2)3
HN
(CH2)3
HN
O
O
O
O
(CH2)3
(CH2)3
O
O
NH
NH
O
O
O
O
Step IX
N
N
C60, Pt
H
H
N
N
pyridine,
N
N
sonication
H
H
N
N
O
O
H
H
O
O
N
N
HN
HN
O
O
O
O
(CH2)3
(CH2)3
HOOC
2Mg2+
2Mg2+
(CH2)3
O
O
O
O
O
O
(CH2)3
O
O
HOOC
COOH
COOH
9
10
FeCl2
DMF, o-C6H4Cl2
Δ
Step X
COOH
HOOC
(CH2)3
2Mg2+
O
O
O
O
(CH2)3
NH
O
HN
O
N
O
O
O
N
N
Fe
N
O
H
O
N
HN
(CH2)3
O
(CH2)3
2Mg2+
11
O
O
HOOC
O
O
COOH
Scheme 1. (continued).
transforms into Product VI (step 5) in the presence of dime-
thylformamide and POCl3.
evaporation and a consequent dissolving in 10 mM sodium-phos-
phate (pH 9.60) buffer containing 15 mM EDTA. The very same way
to concentrate the resulted [25Mg]PMC16 was applied to the first
alkaline–water extraction run.
Noteworthy, the diethyl phthalate was initiated by butyllithium
at ꢂ78 ꢀC followed by the quenching of carbanion with trime-
thylsilyl chloride. The resulting diethyl-3-(trimethylsilyl)-phthalate
was subsequently treated with butyllithium and DMF (dime-
thylformamide) leading to the corresponding benzaldehyde. Its
reaction with N-bromosuccinimide in the presence of Bu4NF gave
a diethyl-6-bromo-3-formylphthalate which was a final precursor
for the substituted porphyrin synthesis. Then 5,10,15,20-tetrakis[4-
Consequently, Product VI transforms into Product VII by the
one-step combined treatment with lithium diisopropylamide (LDA)
and lithium perchlorate (step 6). Gamma-aminobutyric acid is to
induce the transformation of Product VII into Product VIII as indi-
cated in Scheme 1 (step 7). Simple addition of NaOH and MgCl2
transforms Product VIII into Product IX which, in turn, then
transforms into the fullerene porphyrin adduct by the treatment
with a pure C60-fullerene, Pt catalyst (powder suspension), here
a simple pyridine sonication is involved. Eventually, the final
PMC16 nanostructure is to be formed from Product X by incubation
(combined simultaneous treatment) with FeCl2, o-dichlorobenzene
and dimethylformamide (step 10).
bromo-2,3-bis(ethoxycarbonyl)phenyl]-21H,23H-porphine
was
synthesized by the routine Lewis acid-catalyzed condensation of
this precursor with pyrrol followed by treatment with DDQ
according to the standard Lindsay procedure (Scheme 1, steps 1–4).
Another peculiarity of this synthetic route to stress out (Scheme
1, steps 5–10) is that the Vilsmeier–Haak formylation of the
porphyrin obtained was carried out conventionally in the DMF/
POCl3 mixture with a following promotion of the Witting reaction
conducted with allyltriphenyl-phosphonium bromide yielding
butadienyl-substituted porphyrin F. A classical copper-catalyzed
reaction of the latter with an excess of gamma-aminobutyric acid
then resulted in the formation of 2-(buta-1,3-dienyl)-5,10,15,20-
tetrakis[4-(3-carboxypropylamino)-2,3-bis(ethox-
PMC16 ‘‘loading’’ with 25Mg2þ cations (saturation,
PMC16[Mg2þ]4) is done according to a following procedure. Up to
600–800 mg of PMC16 is dissolved in 1.0 ml mixture of pyr-
idine:CS2:chloroform (1:2:7) at room temperature. Then 4 volumes
of 2.5 M solution of pure 25MgCl2 (98.6% isotopic purity) in 0.2 M
NaOH has been added to the PMC16-containing organic mixture.
After 30 min of extensive shaking (22–25 ꢀC), the water-soluble
phase was separated by centrifugation at 15,000 rpm, 20 min,
22 ꢀC, carefully collected and then lyophilized or evaporated with
a following mass spec and atomic absorption spec control.
The remaining organic phase was repeatedly (2–3ꢁ) subjected
to Mg2þ alkaline–water extraction, the organic phase-containing
25MgCl2 traces were then re-utilized using the fast rotor
ycarbonyl)phenyl]-21H,23H-porphine. An alkaline hydrolysis of
this product, along with a magnesium chloride induced cation

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S.M. Rezayat et al. / European Journal of Medicinal Chemistry 44 (2009) 1554–1569
metathesis and a reaction between the butadienyl moiety and the
C60-fullerene combined with metalation of the porphyrin core with
FeCl2 were then completed in the eventual formation of the goal
product [1].
(Nanofinder Co., Russia) technology as described by the manufac-
turer [21].
2.10. ATP synthesis rate measurements
To identify the CS2-extracted resulting product, the fast atom
bombardment (FAB) mass spectra were registered in Varian GT800
LC–MS machine and then processed in the HP6100-J2A analytical
unit using a Sigma Delta Chem AX2000 data base. In addition,
a combined MALDI (matrix assisted laser desorption/ionization)
and FI/FD (field ionization/field desorption) techniques were also
employed to identify the product structure using the MALDI-
QX90A/Finnigan Matt and the MS400/Olivetti tools, respectively,
with a following automated spectra resolution in the above-
mentioned HP analytical unit but applying another data base
software, LabRun-2002 [1,12,14,20]. For pharmacokinetic and
autoradiographic studies, special samples of [59Fe]PMC16 (280–
320 Ci/mmol) were obtained using the A grade 59FeCl2 (910–960 Ci/
mmol, Obninsk Radiochemical Center, Russia) as a precursor.
Once mitochondria were isolated, they were rapidly submitted
to the Triton X-100 treatment (2.0%, v/v, þ4 ꢀC, 2 h, 10 mM Tris–HCl
pH 7.45) with a following extraction of a total pool of the low
molecular mass compounds by the addition of 10 vols ice-cold
acetone. The resulting acetone-soluble material was then frac-
tionated by our original HPLC procedure (ODS-S5CN stationary
phase/10–60% linear pyridine gradient mobile phase, Altex 1800
15 ꢁ 280 mm column, 22 ꢀC, 2000 psi, Gilson W100 UV-detecting
HPLC system) in order to get both ATP/ADP ratio values and the ATP
production rate estimated as g
-[32P]ATP cpm/mg protein [22]. In all
in vivo [32P]orthophosphate ATP labelling tests, animals were
sacrificed in 20–180 min time interval after a single 80–100 mCi/kg
i.v. injection of the label used. A CK activity was measured as
described earlier [8].
2.7. PMC16 measurement in biological material
2.11. Heart muscle oxygen consumption measurements
A pure CS2 was applied as a universal PMC16 extragent in
studies on lyophilized samples of either isolated cell compartments
or a crude heart tissue homogenates, 8.0–12.0 w/v, 22–25 ꢀC,
extensive shaking. The resulting extracts were repeatedly treated
with 7.5 vols pyridine (3–5ꢁ); all post-extraction pyridine portions
were then collected, combined, and concentrated in rotor evapor-
Both heart tissue homogenates and the isolated mitochondrial
samples were subjects for [O2] and [D
Hþ] parameters’ estimation. A
BioRad OxyAnalyser SJ80 has been employed for the tissue/mito-
chondrial oxygen consumption tests. In these studies, the phos-
phorescence-based oxygen analyzer was superior to the
conventional oxygen electrode system in that it provided an
accurate, sensitive and reproducible quantitation of [O2].
Measurements in this study were performed over periods up to
60 min, thus ensuring cell viability. Measurements on re-aerated
suspensions showed that the probe was stable and did not interfere
with cell function. Stoichiometric titration of oxygen with ascorbate
in the presence of ascorbate oxidase (2 ascorbate þ O2 / 2
dehydroascorbate þ 2 H2O) was done as described [23]. The tissue
free protons’ content was potentiometrically evaluated in homog-
enates using a Pt/Ar microelectrode in an extensively aerated cell
coupled with the [O2] detecting unit [23,24].
izer. A 15–20 ml aliquots of the PMC16-containing concentrated
extracts was fractionated by HPLC using a CosmoSil 5-PBB (the
PentaBromBenzene-modified silica gel suitable for separation of
porphyrinic fullerenes), 20 ꢁ 250 mm column at 22 ꢀC, 1200–
1500 psi, 10 ml/min, with a pure 1,2,4-trichlorobenzene as a mobile
phase [20]. The final PMC16 quantification was performed plani-
graphically with an automatized correction for molar extinction
indexes once the elution profile got recorded by absorption at 180,
210, 320, and 470 nm in Perkin Elmer 266QL HPLC Analytical
System.
2.8. The PMC16 in vitro tests
2.12. Pharmacokinetics and pharmacodynamics
For the drug nano-size measurements as well as for the pH-
dependent PMC16 cluster formation direct monitoring, a two-
dimensional arrays of nanoparticles and their conglomerates were
registered by the atomic force microscopy in the Zelenograd-ERA
M3 (Russia) AFM system [20]. Apart from AFM, the PMC16 cluster
sizes were also estimated by gel filtration on Sephadexes G10, G15
and G50 within an eluent pH range of 5.0–9.5 [1]. The PMC16-
related Mg2þ release kinetics has been simultaneously tested,
within the same pH range, by evaluation of the Mg/Fe ratios using
a routine flame atomic absorption spectrophotometry method
(DX800-B6 Carl Zeiss AAS, Carl Zeiss Jana). SANS spectra of PMC16
monomers and dimers were registered and analysed in SANS 2TD
accelerator at the International Nuclear Research Institute, Dubna,
Russia, with a kind technical assistance provided by Dr. M.N.
Osmolov.
All nine major pharmacokinetics parameters along with 12
additional ones (Table 1) were estimated by the standard
Table 1
The [Mg]PMC16 pharmacokinetics in rat
T1/2 ¼ 9.0 h
C0 ¼ 62
mg/ml
Tmax ¼ 2.5 h
Cmax ¼ 260 ꢃ 83 ng/ml
VP ¼ 16.2 ml/kg
VC ¼ 12.4 ml
Cl ¼ 32 ꢃ 4 ml/min/kg
k ¼ 0.685
V1 ¼0.08 ml
28 ꢃ 4.3%
Renal excretion
Hepatic excretion(metabolization)
Plasma proteins binding
Blood cells uptake
16 ꢃ 4.0%
1.2 ꢃ 0.3%
Lymphocytes
Erythrocytes
Tissue-specific accumulation
Myocardium
Brain
Urine-eliminating PMC16 metabolites (258 ꢃ 4.0
Alanyl-depleted derivatives
Deacetylated derivatives
Cyclohexyl-C60
PMC16 urine content
C60 urine content
28.6 ꢃ 5.5%
8.0 ꢃ 3.2%
2.9. Biomaterial morphology tests
18.4 ꢃ 3.40%
0.6 ꢃ 0.02%
For morphological tracing of PMC16 particles inside the organ-
elle membranes, a metal-chelating iron response AFM toolbox
along with a simple imaging AFM biological version [14,20] was
employed. The in situ morphology of mitochondria in myocardio-
cytes was observed by a routine transmission electron microscopy
using a Pt contrasting coating [16]. A laser contrast confocal
microscopy was additionally employed using a Nanofinder-S16E
mg/ml)
56.4 ꢃ 8.7%
27.0 ꢃ 6.1%
16.2 ꢃ 3.3%
462 ꢃ 11
2.9 ꢃ 0.1
m
g/ml
m
g/ml
Single 20 mg/kg i.v. injection (M ꢃ SEM, n ¼ 6) 24 h monitoring presented.

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procedures specified clearly in Section 3. To identify/quantify the
PMC16 and its metabolites in any biomaterial tested (urine, blood
cells and plasma, tissue homogenates, cell compartments),
a lyophilized CS2/acetone extracts were used for chromato-mass
spectrometry procedure (GC–FAB/MS, LC–MALDI, FI/FD–MS)
[1,12,14].
This technique allows to detect even the water-insoluble PMC16
metabolites including a pure C60 [1].
Blood plasma transferrin, hemoglobin, myoglobin and liver
ferritin isolation shots were carried out using a fast double
immunoprecipitation technique, Bio-Rad 1167JS Analytical Kit, as
recommended by the manufacturer [25].
Ficoll mini-column preparative fractionation of cells was applied
to erythrocytes and lymphocytes isolation [26].
Fig. 2. PMC16 cationite properties and nanocluster formation as a function of pH. Big
arrow shows the normal homeostasis edge point.
A possible sorption of the drug tested on blood plasma proteins
has been studied using the [59Fe]PMC16 species according to
Ref. [27].
All the results obtained were statistically treated with respect to
a classical two-compartment drug distribution model [28,29].
(Fig. 2). At the same time, the PMC16 cluster formation is also found
to be a function of pH (Figs. 2–4). These findings look attractive in
the light of the myocardial hypoxia molecular pathogenesis data,
according to which a tissue acidosis is a direct and natural meta-
bolic consequence of the hypoxia of any sort [2,7,11,16]. So the
acidosis induced release of 25Mg2þ is what we may expect in case of
the [25Mg]PMC16 in vivo administration. The amphiphilic character
of this agent [1,2] is also in favor of such expectations since the
ambivalent solubility of the drug correlates normally with its
membranotropic properties [14,17,20]. And, indeed, the PMC16
biomembrane uptake has been eventually found (Fig. 5). The AFM
technical approach we used to obtain these data [14,20] is a late
modification of the most efficient earlier developments performed
in biophysical studies on living cells and their compartments
[39–42]. Besides, a non-allergic and anti-inflammation properties
of most fullerene derivatives ever tested [37,38] as well as their
generally high level of ‘‘biocompatibility’’ [39] make PMC16 suit-
able for the in vivo safety and pharmacological activity studies.
Both morphological (Fig. 6) and biochemical (Fig. 7) patterns
show a clear and positive effect of [25Mg]PMC16 on the hypoxia
promoted myocardial cell energy metabolism disorders. Addition-
ally, this has been proven by a high level of synergism manifested in
the [25Mg]PMC16 hypoxia treated cases, i.e. by a synergism
between the CK activity, ATP production rate, magnesium isotopes
contents in the heart mitochondria, myocardial O2 consumption
2.13. Statistics
A conventional non-parametric statistical treatment (for ‘‘n’’
equal to 6 or lower) was performed using the Sigma Biostat A6
software package to evaluate the significance of experiment/
control differences. A Penmann–Dalbreaux approximation tech-
nique has then been employed once the standard error of the mean
(SEM) values were found to be not higher than 6.5% of the mean
(0.020–0.065 M limit range) whatever pair compared. To manage
this, the HP LabRun-06 software operating graph-design algorithm
has been processed in the HP6100-J2A Analytical Unit to perfec-
tionize all graphics [19,20,29].
3. Results and discussion
A not-too-long lasting history of the medicinal use of fullerene-
based nanoparticles includes a clear indication of their very low
acute toxicity [20,30] as well as a variety of data that reveal the
fullerene derivatives’ anti-malignant [31], anti-viral [32] and
bacteriostatic [33] properties. It would be safe to say, however, that
the main stream trends in a modern fullerenes’ nanopharmacology
research deal with the targeted drug delivery problem due to
marked capabilities of these membranotropic nanoagents to serve
as specific carriers for organic/bioorganic pharmaceuticals [34,35].
As for a tissue targeting mechanism, magnetic and immuno-
vectoring navigation paths alone were tested so far with respect to
this very peculiar family of nanomedicines [20,36].
Let us remind that the latter term (medicinal nanoparticles,
nanomedicines) stands for the biologically active nano-size
synthetic structures capable of moving across the main types of
biological barriers with further targeting towards a certain recep-
tor(s) inside or outside the cell and without being instantly
destructed by surrounding enzymes [14,37–42].
To the best of our knowledge, no research but ours was in fact
devoted to the C60-fullerene derivatives’ cationite activity [1,2]. On
the other hand, this is exactly the activity needed to provide
a delivery of 25Mg2þ cations towards a hypoxia suffering cells and
tissues. According to our basic standpoint, a targeted delivery
mentioned may help to compensate the hypoxia-caused ATP losses
owing to a known 25Mg-related overactivation of both oxidative
and substrate ADP phosphorylation paths of the ATP synthesis in
a cell [8–11].
and the tissue acidosis ([DH[]) degree (Figs. 8 and 9).
The MNA-induced tissue hypoxia comes with inhibitor’s
complete, selective and irreversible suppression of the ADP
oxidative phosphorylation in mitochondria as long as this agent is
the NAD/NADP precursing antimetabolite [8,9]. Unlike MNA, DXR
provides much slighter inhibitory effect towards ATP synthase and
cytochrome oxidase in mitochondria [4,5] which makes both drugs
comparable to each other in terms of the depth of medicinal
hypoxia induced. Whatever hypoxia model tested, the
[
25Mg]PMC16 prophylactic or corrective use look at an efficient way
to either prevent or correct the ATP-depleting metabolic disorders
(Figs. 6–9) and, hence, to protect the heart muscle in general.
Another remarkable peculiarity of PMC16 nanoparticles is a fact
of their days-long lasting retaining in the heart tissue (Fig. 10) and,
particularly, inside the heart mitochondrial membranes (Figs. 11
and 12E). In our opinion, the most probable explanation for this
phenomenon is an existence of the PMC16 high-affinity protein
receptor in these membranes, i.e. a presence of the signaling
protein responsible for a selective drug–protein recognition. A
mere protein signaling function of that type is known for a number
of porphyrin metabolites [12–14,16].
As seen from our data presented in Figs. 1 and 2, the PMC16
possesses an essential Mg2þ-carrying cationite potential. Note-
worthy, the product may act as a so-called smart nanoparticle being
capable of releasing magnesium in response to the pH acidic shift
A total lack of the PMC16 long-lasting ‘‘trapping’’ observed in
tests conducted on several other tissues (liver, lungs, kidneys,
skeletal muscles) is in a good accordance with an above stated
assumption (Fig. 12). A relatively fast systemic clearance of this

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Fig. 3. Laser contrast and atomic force microscopy. PMC16 nanoclusters immobilized on acetyl cellulose membrane [40,44]. (a) LCM, pH ¼ 7.00; (b) LCM, pH ¼ 8.40; (c) AFM,
pH ¼ 8.80.
nanomedicine along with a nearly perfect tissue clearance esti-
mated for numerous PMC16 ‘‘non-trapping’’ tissues listed above
seems an additional argument to prove a high potential of PMC16
for the heart-selective 25Mg2þ targeted delivery (Figs. 13 and 14).
The heart muscle tissue manifests its high capability to retain
the drug non-metabolized molecules for longer than a week. After
a single i.v. injection (20–30 mg/kg), about 18.0% of the whole
amount of the drug injected remains in myocardium for 12 days. At
the same time, there is no sign of tissue-specific accumulation of
this drug (no 24 h or longer retaining) in skeletal muscles, kidneys,
lungs and liver (Figs. 10–12).
Considering the isolation of purified receptor a non-urgent
although important task for further studies, we may gain, never-
theless, a rather obvious advantage of the very fact of the heart
muscle specific PMC16 reception.
Once we do have a reliable information on the drug receptor
presence in the heart (Figs. 10–12), then, the receptor dependent
targeted drug delivery should be expected. That means, extra low
drug doses multiple administration scheme may lead to accumu-
lation of the drug molecules exclusively inside the so-called
drug-specific receptor-rich body compartments whereas the rest of
the whole organism may remain practically drug-free due to
a normal elimination mechanism.
This statement has been proven indeed by the heart muscle
targeted delivery of [25Mg]PMC16 which we have reached by
developing of a special scheme of the multiple long-term admin-
istration of low drug doses. In Fig. 13, a true efficiency of this
scheme (0.4 mg/kg, i.v. [10 days] – 0.2 mg/kg, i.v. [6 days] – 0.1 mg/
kg, i.v. – .up to 24 days monitored) is presented. The scheme
proposed comes from our understanding of pharmacological
meaning of the PMC16 heart muscle specific reception. Particularly,
this scheme provides a saturation of majority if not of all of the
heart-located PMC16-specific receptors in case of the gradual
administration of drug doses minimal but enough to get this satu-
ration reached. Meanwhile, all other receptor-lacking body
compartments are not in a position to retain the drug as long as its
concentration is below an accumulation-required elimination limit.
That’s the extra low doses are for. All together, this creates a unique
condition for the heart drug targeting even without any effort to
manage the artificially designed targeted delivery path like, for
example, the external magnetic field navigation or the antibody
involving liposome drug delivery.
Fig. 4. Small angle neutron scattering image of the PMC16 mono/dimer, pH 6.50 (scale bar: 1.0 nm).

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1561
Fig. 5. The PMC16 nanoparticles/clusters atomic force micrographs: (a) suspension, 400 mg/ml 1.5 mM K2HPO4 (pH 10.0); 10 nm scale bar; (b) diaflo YM 1.0 membrane remained
pellets, 400 mg/ml 1.5 mM K2HPO4 (pH 9.0) suspension used; 100 nm scale bar; (c) rat myocardial mitochondria membrane lipid layer. 0.1 LD50 PMC16, 10 h / 0.8 LD50 DXR, 10 h;
50 nm scale bar.
In a contrast to these known drug delivery paths [42–44], the
porphyrin tissue receptors docking way look far more preferable
due to ability of 1.8 nm PMC16 particles to intervene the cell (Figs.
6, 11, and 12) for further possible coupling with corresponding
affinity proteins located in mitochondrial membranes of myo-
cardiocytes [6,14,16].
Noteworthy, the only reason why these low doses of PMC16 are
enough to promote an essential pharmacological effect in the
hypoxia damaged myocardial cells, i.e. to promote a marked
activation of the ATP synthesis, is the efficient delivery of 25Mg
isotopes known as the universal ATP production overactivating
agents [8–11]. So despite a low tissue drug concentration, the
energy metabolism rehabilitation is to be taken care of by a very
small amount of an extremely active ATP regeneration promoter
which is a magnetic magnesium isotope of 25Mg. As a cationite
carrier for magnesium, the PMC16 is certainly a right nano-tool to
provide a proper pharmacological application of the 25Mg2þ
-
related magnetic isotope effect. Thus, being capable for a tissue-
specific targeting, this tool allows not to waste paramagnetic all
over the whole organism which helps to avoid the unpredictable
side effects dealing with ATP hyperproduction in cells apart from
myocardiocytes and lymphocytes. Other advantages of the PMC16
medicinal use, as seen from everything stated above, are the drug
smart magnesium release, a long-term drug–receptor retaining in
target tissue, and a high biological activity of minute amounts of
25Mg2þ delivered.
These and related statements are, as a matter of fact, supported
by a complex but positive correlation revealed between the heart
tissue 25Mg content values and the tissue respiration parameters
([ATP], [ATP]/[ADP], Y/Yo; [DO2], [D
Hþ]) measured in both DXR- and
MNA-induced hypoxia chronic experiments (Figs. 8 and 9).
The data presented may serve as a background for a new
nanopharmacological approach to prevent and correct a number of
Fig. 6. Electron transmitting microphotograms of the rat myocardiocytic perinuclear
areas. A,C: PMC16-related hypoxia preventing effect (30 mg/kg [25Mg]PMC16, i.v. /
12 h / 20 mg/kg MNA, i.v. / 12 h / test); B: MNA hypoxia model (20 mg/kg MNA /
12 h / test); D: control (healthy intact rat tissue).
Fig. 7. The effect of a PMC16-targeted delivery of Mg2þ on the doxorubicin pre-sup-
pressed ATP production in rat myocardium (0.8 LD50 DXR i.v., 6 h / PMC16, i.v., 6 h).

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dealing with a classical two-compartment drug distribution model
[27–29].
The routine pharmacokinetics analysis reveals the following key
parametric values, which are all listed in Table 1.
T1=2 ¼ 9:0 h; Tmax ¼ 2:5 h; Cmax ¼ 260 ng=ml:
Cl ¼ D=S ¼ 32 ml=min=kg
where D – dose, S – square under pharamacological curve. Vp
stands for a drug distribution volume which is a ratio of a total
clearance (C1) to the drug elimination constant (k), i.e. to the
constant of the rate of a drug blood plasma concentration decrease
registered at the steady state point.
Vp ¼ 16 ml=kg; k ¼ 0:685 Vp ¼ Cl=k ¼ D=ðk ꢁ SÞ
Besides, Vp ¼ Cb/Cp1 where Cb – a total content of the drug in
Fig. 8. Synergism of the mitochondrial matrix CK activity, PMC16-related magnesium
cations influx and the free protons excess degree. Isolated rat myocardial mitochondria
pre-treated with 0.25 DL50 PMC16.
a whole body, Cp1 – drug plasma concentration.
Co ¼ 62
mg;/ml, this is the drug concentration which could be
reached in the very moment of its injection.
myocardial hypoxic disorders. The way proposed includes the heart
muscle targeted delivery of magnetic 25Mg2þ isotope carrying by
membranotropic cation-exchanging fullerene-based nanoparticles,
PMC16. The heart drug targeting depends on the PMC16 tissue-
specific reception while the magnesium release is just a response to
hypoxia-caused metabolic acidosis. That makes an agent a tested
‘‘smart medicinal nanoparticle’’. All findings listed look promising
and require further extensive studies.
Vc ¼ 12.4 ml, T1/2 ¼ k(Vc/Cl) which is actual for the steady state
moment.
Thus, Vc is a drug distribution volume at the steady state
condition. That is, this is a volume in which the drug is supposed to
be distributed once the steady state status is in fact reached if its
concentration in this volume would become the same as in plasma
or in a whole blood of the animal [29].
V1 ¼ D/Co ¼ 0.08 ml, V1 – the initial distribution volume (a
As per the pharmacokinetics/pharmacodynamics patterns, even
though they may look guite self-sufficient (Table 1; Figs. 10–18),
some of them might be pointed out.
central compartment volume).
So if we would neglect the drug concentration measurement in
an early time step after the injection (10–30 min), the resulting
PMC16 pharmacokinetic model would be mistakenly defined as
a single compartment one. We escaped such an error by direct
detailing of the very first steps of the drug concentration dynamics
(Figs. 14 and 15). The error mentioned would come to about 10–12%
by clearance alone [27].
Thus, the blood plasma drug concentration dynamics (20 mg/kg
[
25Mg]PMC16, i.v.) has been started to investigate, 10 min after
a single injection. In s semi-logarithmic coordinate (Fig. 14),
a concentration time dependence (C ¼ f(T)) is found to be describ-
able by two consequent exponential functions. That means, we are
Fig. 9. Synergism of the ATP yield, oxygen consumption and the PMC16-related Mg2þ release in the rat heart muscle tissue (30 mg/kg PMC16, i.v., 12 h exposition). A: zero spin
magnesium test; B: magnetic magnesium test.

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1563
It should be stressed out that the PMC16 shows a marked tissue-
specific accumulation exclusively in the heart muscle (18.4%) and in
the brain (0.6%). This indicates to the above-discussed drug
reception mechanism and, not less importantly, perhaps to the
blood–brain barrier penetration abilities as well. Looking at the
results listed in Table 1, it is noteworthy to outline that a complete
(z98%) drug elimination out of the heart tissue has been reached
by 20th day after a single intravenous injection of therapeutic dose
(0.1 DL50, 80–85 mg/kg body weight) in rats. A systemic clearance
level, however, is far lower (blood cells 10 days retention, Fig. 17;
blood plasma 1 day retention, Fig. 14). At the same time, all
parenchymatic organs tested (skeletal muscle, kidney, lung, liver,
brain) do not retain the drug and/or its metabolites for longer than
16–18 h (Fig. 12; Table 1).
In myocardium, the separate and targeted PMC16 delivery of
magnetic (25Mg) and non-magnetic (26Mg) isotopes provides an
extremely high level of ATP production once the magnetic isotope
has been applied (Figs. 8 and 9). The difference in 25Mg/26Mg
activation effect values is estimated as high as 2.2–2.5-fold. This
might be elevated even up to 3.5 when the PMC16 in vivo does
increase from 20 to 30–35 mg/kg (i.v.).
The PMC16 high-safety expectations correlate nicely with what
we found investigating the drug related iron metabolic turnover
(Fig. 15). A single [59Fe]PMC16 injection leads to a consequent time-
dependent appearance of the gamma-emitting label in such natural
metabolism participants as heme (hemoglobin, myoglobin),
hepatic tissue ferritin and blood plasma transferrin. A kinetics of
the latter processes fits the general level of the PMC16 hepatic
excretion specified above (Table 1). All together, this is a factor
favorable to the metabolically useful and, therefore, potentially safe
biotransformation of the PMC16 porphyrin domain [45].
Fig. 10. A highly selective targeting of PMC16 nanoparticles towards the rat heart
muscle in a course of the long-term administration of an extra low drug dosage.
The differences between the drug distribution levels within
central and distant compartments are important to be tested in the
saturation doses’ estimation.
As seen from Table 1, renal excretion of PMC16 is equal to 28.5%
and hepatic excretion equals to 16.0%. The nanodrug tested has no
affinity to the plasma proteins (1.2% binding) whereas it provides
a rather high level of the blood cells uptake: 28.6% for lymphocytes
and 8.0% for erythrocytes. The hepatic metabolism leads to the
formation of biologically safe urine-eliminated fullerene deriva-
tives such as alanyl-depleted PMC16 (54.6% of total metabolites
amount), deacetylated PMC16 derivatives (27.0%) and cyclohexyl-
C60-fullerene (16.2%). In a 24 h-collected urine, about 3% of pure
C60-fullerene has also been found (Fig. 16).
Both water-soluble and water-insoluble metabolites (products
of the PMC16 metabolic degradation) were detected and identified
in urine (see Section 2) due to their extraction by CS2/acetone out of
the lyophilized urine dry matter (24 h-collected urine) [1,12,14].
Drug hydrolytic degradation observed is one of the common
xenobiotic metabolism pathways, in addition to alkylation, oxida-
tion and conjugation [26,27,31]. Hepatic hydrolases and trans-
ferases are major enzyme families involved [31,35,36].
The drug distribution volume (Vp) does not necessarily corre-
spond to some real volume. Vp is just a volume needed for an equal,
homogenous, distribution of the drug in the very same concen-
tration as in plasma or in total blood. The PMC16 is found to posses
almost a zero affinity towards the plasma proteins (PP binding
degree ¼ 1.2%). Its renal excretion level is 28% and hepatic excretion
(metabolizing extent) is 16%.
The PMC16 blood cell in vivo uptake is up to 36% (24 h moni-
toring): 28% in lymphocytes and 8% in erythrocytes.
The urine excreting nine metabolites is found to have the
following ones (Table 1; Fig. 16):
Alanyl-depleted PMC16 species – 56%,
Deacylated PMC16 species – 28%,
Cyclohexyl-C60 – 16%.
In a classical Ruggher’s rat blood cells monitoring experiment,
the PMC16 uptake has been tested in lymphocytes and erythrocytes
separately using the [59Fe]PMC16 preparations with not less than
470–520 Ci/mmol specific activity (30 mg/kg or 10–12 Ci/kg, i.v.
single injection with a following 280 h long-term gradual in-cell
label estimations). The lymphocytic drug retention time does not
exceed the 280 h limit getting to a highest drug content level
within 80–120 h time interval. In erythrocytes, the total retention
time is about 120 h with the 1–40 h covered drug cell concentration
peak (Fig. 17).
So unlike blood plasma proteins, blood cells may retain an
unmetabolized drug for not longer than 120 h (erythrocytes) and
nearly 200 h (lymphocytes). A total drug blood cells uptake,
however, is rather low being estimated as 7.5–8.5% of the whole
dose injected (Table 1, Figs. 15 and 17). According to Refs. [2,36–38],
the saturation of lymphocyte membranes with fullerene deriva-
tives of many kinds would hardly cause any immunological
Fig. 11. The heart muscle cell compartment retaining distribution of [59Fe]PMC16
caused by a single i.v. administration in rats (30 mg/kg, 470–520 Ci/kg).

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Fig. 12. The cell compartment retaining distribution of [59Fe]PMC16 caused by a single i.v. administration in rats (30 mg/kg, 470–520 Ci/kg). A: lungs; B: kidneys; C; liver; D: brain;
E: heart muscle; F: skeletal muscle.
dysfunctions. Under a normal metabolic condition, no 25Mg2þ
release is expected in [25Mg2þ]PMC16 administration. Unless the
hypoxia determined acidosis ‘‘commands’’ so, no paramagnetic
magnesium intervention would affect the ATP synthesis in
lymphocytes. This should be treated as an additional proof in favor
of the drug safety.
So the PMC16 hepatic turnover relates predominantly to the
porphyrin domain decomposition leading to the formation of the
biologically neutral heme precursors (Figs. 15 and 16). Along with
a high rate of the renal drug elimination (Table 1), this allows to
consider the PMC16 particles safe enough for a low dose chronic
administration.
The data presented are in a good accordance with a pre-calcu-
lated pattern [2] estimating the efficiency of our scheme of the
myocardium reception dependent drug targeted delivery (Figs. 10
and 13) as equal to about 75%. In other words, nearly 75% of all
single injection inserted drug molecules are retained inside the
receptor-rich heart muscle whereas the rest of the drug pool is in
fact distributed between the hepatic metabolism reservoir
(16–17%) and the limited blood cells uptake (7–9%).
Since the PMC16 looks to be a promising tool for myocardium
protection in some drug-induced hypoxia cases (Figs. 5–7), we
were unable to escape from being involved into certain pharma-
cokinetic tests on experimental hypoxia models. The most valuable
part of this segment of our work is shown in Fig. 18. No matter what
particular model of hypoxia tested, just very slight, moderate,
changes in the PMC16 pharmacokinetics were observed. Thus,
hypoxia promotes nearly 8–8.5% increase of T1/2 elevating it from

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1565
Fig. 15. The PMC16-related 59Fe turnover in rat (20 mg/kg, 380–420 Ci/kg
59Fe]PMC16, single i.v. injection).
[
metabolism. In contrary, the MNA-promoted hypoxia leads to
reversed phenomenon which is up to 80% deacetylation slowing
down with roughly 60–65% suppression of the porphyrin domain
decay rate.
So the hypoxia conditions, whatever mechanism is behind it,
make the drug more resistant to the hepatic metabolic attack. This
is considered as an essential and positive supplement to ‘‘phar-
macological image’’ of the drug tested [46,47].
The data presented fit our standpoint on essential pharmaco-
logical perspectives of the PMC16 as a nanomedicinal agent even
though extensive further studies are required. For time-being,
however, these results meet some major expectations usual for the
new drug pre-clinical trial anyway.
Thus, the most promising key point about the drug tested is that
it may provide a truly remarkable (approx. 70%) sliding down of the
heart tissue oxygen consumption requirement (Figs. 8 and 9). The
drug clearly promotes an essential fall of the myocardium sensi-
tivity to oxygen hunger whatever cause beyond. What seems
clinically valid about it, the drug allows to avoid or minimize all
pathological consequences of hypoxia in the heart like the
contractile strength weakness or arrhythmic disorders.
As per the toxicity/safety patterns of PMC16, they make it
suitable for further pre-clinical study since its hepatic metabolism
turnover leads to the conversion of the drug porphyrin domain into
a normal heme molecule while the cyclohexyl fullerene residues
are to be totally eliminated by kidneys with no sign of acute toxicity
of these C60-derivatives. The data presented in this paper give
a firm reason to expect that the new nanodrug family proposed
would attract attention of medical and veterinary professionals
engaged in the fields of sports and military medicine, veterinary
pharmacology, extreme physiology studies and all clinical disci-
plines that deal with pre-ischaemic and ischaemic heart function
disorders as long as a pathogenesis of the latter is about to deplete
a myocardium oxygen availability/consumption.
Fig. 13. The rat myocardium tissue respiration affected by DXR and MNA in a course of
25Mg2þ]PMC16 administration (0.4 / 0.2 / 0.1 mg/kg i.v.). DXR, 80 mg/kg/24 h, i.v.;
MNA, 20 mg/kg/24 h, i.v.
[
9.0 h to about 9.7–9.8 h. Besides, both renal and hepatic excretion
processes got slower and changed from 28.5% to 22.0% and from
16.0% to 12.2%, respectively. There is no any serious hypoxia-related
impact on the drug blood cells uptake at all. As for the formation of
particular drug metabolites, they themselves as well as their ratios
are absolutely the same compared to the intact, normal, tissue
metabolism conditions (Table 1, Figs. 15, 16, and 18).
In a separate series of experiments, such specific drug metab-
olism parameters as the PMC16 59Fe loss degree and the PMC16
hepatic deacetylation degree were investigated for correlation with
the hepatic oxygen consumption level (Fig. 18). In the inhalation
oxygen-depleted hypoxia, the deep porphyrin domain decompo-
sition does not practically occur while the drug deacetylation rate
had decreased by approx. 25% compared to the intact hepatic
There are actually the very few precedent studies close enough
to the work we have done [20,48–52]. Thus, some bacteriostatic
agents based on the oxypiperidine-modified fullerenes are found to
be hepatotoxic in rats and rabbits although there is no proof for the
high drug systemic clearance rate found for any tissue-specific
accumulation [20,52]. Amino acid modifications of fullerene-C60
,
on a contrary, are found safe due to their fast and complete hepatic
metabolism followed by a total renal excretion in rats [49,50]. The
latter agents show no trends for the long-term tissue-selective
retaining of any sort in either acute [49,53] or chronic [53] exper-
iments in mice and rats. Purine nucleosides were used to func-
tionalize the fullerene-C60 surface with following tests of the
resulting products for their anticancer activity in hepatoma
possessing rats [50]. No asymmetrical distribution of these agents
Fig. 14. Systemic clearance: a multiexponential two-compartment dynamics of the
blood plasma [Mg]PMC16 concentration after a single 20 mg/kg i.v. injection in rats.

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Fig. 16. PMC16 metabolites resulting in the drug hepatic turnover.
was found except for their marked tendency of hepatocyte uptake
[50,53]. Riboflavin–fullerene and pyrrolidine–fullerene adducts are
to be accumulated predominantly in the secretory epithelial cells
while the rest of the mammalian cell/tissue pool remains a non-
hosting compartment for these chemicals [20,48,51,53].
It should be mentioned that some water-soluble C60-fullerene
derivatives are found to be capable of selective accumulation inside
mammalian mitochondria [54] which corresponds roughly to our
above listed data on PMC16 pharmacokinetics (Figs. 5, 11, and 12E).
However, no porphyrin adducts of fullerene-C60 were ever
studied for pharmacological properties prior to our work.
As per the PMC16 zero affinity to the blood plasma proteins,
there are some data on the unique (extra low) affinity of the pol-
yalkylated heterocyclic adducts of fullerene-C60 to acidic proteins.
Even though this might look unusual, there is a firm chemical
background beyond that [20,21,36,38]. In short, a hydrophobic
fullerene and
a heavily carboxylated (negatively charged)
porphyrin domain of the drug are hardly compatible with most

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1567
porphyrin adducts of fullerene-C60 tested, the more functionalized
the product is, the less acute toxicity level it shows, and vice versa
[20,44,46]. An acute toxicity of a pure non-functionalized cyclo-
hexyl-C60, for example, is equal to DL50 ¼ 577–840 mg/kg, i.v., for
different lab mammals (rats, mice, guinea pigs, hamsters), which is
far less toxic than such a common pharmaceutical as benzodiaze-
pine anxiolytics or penicillin group antibiotics [20,30].
Secondly, numerous data specified in available literature show
the polyphenyl-, oligophenyl-, sorbitol-, heterocyclic-, porphyrin-,
and amino acid-modified negatively charged fullerenes as perfectly
non-allergenic products with no tendency to intervene in reactions
of immunological response of any sort [20,30,34–37]. This is what
we consider an additional argument supporting the statement on
a good safety potential of the nanoparticles tested.
Concerning the blood–brain barrier (BBB) permeability of the
drug, the following should be stressed out. According to our rough
estimations, about 0.6% of the [59Fe] label is to appear in the brain
tissue homogenate fractions including some cell compartments
isolated (Table 1; Fig. 12D) when the [59Fe]PMC16 species were i. v.
administered to rat. But unlike in myocardium, the 0.55–0.60% drug
content did not allow to find out a non-destructed (non-metabo-
lized, intact) nanoparticle in biological membranes of the brain
using a direct tracing/detection techniques (AFM, SANS, HPLC(CG)–
FAB/MS, MALDI, FI/FD–MS – see Section 2). So the question about
the BBB permeability to PMC16, as a matter of fact, is still to be
answered in forthcoming studies.
As seen from the numerous data [30,36,42,44], a vast majority of
the fullerene adducts ever tested are unable to penetrate the BBB in
mammals. At the same time, some porphyrin-modified derivatives
of C60 are found to be the BBB-crossing agents although with a very
low permeability degree estimated (0.05–0.08% of the i.v. injected
intact agent [14]).
Fig. 17. The PMC16 blood cells uptake in rats. Single i.v. injection, 30 mg/kg
59Fe]PMC16, 470–520 Ci/kg.
[
abundant functional radicals of the major blood plasma proteins
(albumin and globulin sub-populations first).
So we are treating a lack of the marked drug–protein affinity
(blood plasma proteins) as a quite expected result except the
obvious case of a high-affinity recognition of the drug particle by
the cardiac-specific membrane receptor(s) known for their
porphyrin-binding properties [2,14,16,44–47], Figs. 5 and 10–12.
Replying some other drug toxicology concerns, the following
two statements are to be made.
First, the mass spectrometrically identified PMC16 metabolites
(eliminated through the renal excretion) are found to be the low
toxic urine-eliminating compounds in numerous studies conducted
in the past 3–4 years [20,36,44,46]. Thus, a monograph [20] alone
contains a summarizing analysis of over 1000 individual fullerene-
C60 derivatives’ acute toxicity and biological activity patterns.
Indeed, some of them are not safe for chronic use although a very
few of them have a truly high acute toxicity level. There is a regu-
larity to be mentioned here: once the cyclohexyl-interfaced
In all of these and some other related studies, however, the
direct monitoring of the intact drug molecule in a cell (for example,
the FAB-chromato-mass spec technique) was not employed so the
data quoted are not totally reliable. Therefore, once again, the drug
Fig. 18. The hypoxia-affected PMC16 metabolic decay in rat. A: chemically induced hypoxia (0.005–0.5 DL50 MNA, 12 h); B: oxygen-depleted inhalation hypoxia (15%, O2, 1–10 days).
(1) The drug hepatic deacetylation degree. (2) The drug 59Fe loss degree.

1568
S.M. Rezayat et al. / European Journal of Medicinal Chemistry 44 (2009) 1554–1569
5. The PMC16 metabolic turnover relates predominantly to the
porphyrin domain decomposition leading to the formation of
the biologically neutral heme precursors. Along with a high
enough rate of the renal drug elimination, this allows to
consider the nanodrug safe for a low dose chronic use.
BBB permeability problem is far from being solved due to the
above-specified technological limitations.
The present work is simply focused on the [25Mg2þ]PMC16
biological activity in the heart.
Regardless of its high affinity to myocardium-specific receptors
[55], a pure intact heme is found of being totally non-suitable for
Mg2þ transportation to the heart due to the following
circumstances:
Acknowledgements
(a) it has no cationite properties, and
(b) it is clearly a subject for fast decompositive biotransformation
in vivo (Fig. 15).
This work was supported by the TCO-TUMS NanoBioMedicine
Committee and, partly, by the Russian Fundamental Research
Support Foundation (Moscow, Russia). The PMC16 synthesis and
the nanoparticles 25Mg2þ loading were performed owing to
a technical assistance provided by the Division of Analytical
Chemistry, La Sapienza University, Rome, Italy (Prof. L. Campanella)
and by the Division of Organic Chemistry, M.V. Lomonosov Moscow
State University, Moscow, Russia (Prof. M.A. Yurovskaya). Special
thanks to Dr. M.N. Osmolov, the International Nuclear Research
Institute at Dubna, Russia, for his exceptional help in getting and
analyzing the nanoparticles SANS images. Dr. A. Gupta, Bangalore
Center for Cell Biology, India, is to be thanked for his kind help in
English style correction.
Instead, a C60-fullerene nucleus protects the Mg2þ-exchanging
porphyrin domain from outside enzymatic attack (Figs. 15 and 18)
as long as a heavily carboxylated porphyrin K derivative is engaged
in the drug structure (Fig. 1).
Hence, the specific peculiarities of PMC16 structure as a combi-
nation of porphyrin and fullerene domains provide an advantage of
a simultaneous possession of amphiphilic/membranotropic, cation
exchanging and low toxic properties associated with a relatively
low rate of metabolic decomposition.
As the heart muscle specific PMC16 receptor hypothesized due
to a long-term retention of the drug in mitochondria (Figs. 11 and
12E), this receptor was indeed isolated in our parallel study [55]. It
turns out that this is a hydrophobic tryptophan-rich 17.6 kDa
monomer protein located in an external membrane of the myo-
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