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Table 2 In vitro effect of citrate–Mn3O4 NPs on haemoglobin and
different blood cells. Data are expressed as mean ꢀ standard deviation
[n ¼ 12]. P values were determined by Student's unpaired t-test
radical pathways, we have performed the BR decomposition
study (as shown in Fig. 2) in presence of a radical initiator
(H2O2, a source of cOH radical) and a radical scavenger (ethanol)
separately. As shown in the inset of Fig. 3b, in both cases, a
slower catalytic rates have been observed, which validates the
role of cOH radicals in the catalytic process. Although, slower
rate in case of H2O2 seems unexpected, however, this result can
be explained by the fact that, H2O2 itself can inuence the
conversion of Mn+3 to Mn+4 states to a great extent and conse-
quently diminish the number of active catalytic sites on the NPs
surface. Therefore, we hypothesized that, the origin of such
unprecedented catalytic activity might be initiated by the
conversion of Mn3+ to Mn+4 states at the NPs surface and
subsequent formation of reactive oxygen species19 (such as cOH
radicals), that ultimately leads to the decomposition of the
analyte, BR.
Having developed the highly active citrate–Mn3O4 NPs for BR
decomposition, we sought to test their in vitro effectiveness by
adding the catalyst to human peripheral blood specimens
collected from hyperbilirubinemia patients. Our primary
objective was to evaluate the BR levels of the blood specimens
with and without treatment by citrate–Mn3O4 NPs. Fig. 4
represents the assay results, performed over blood specimens of
hyperbilirubinemia patients. As shown in Fig. 4a, average BR
level (total, conjugated and unconjugated) in the blood speci-
mens of 12 patients, treated with citrate–Mn3O4 NPs, reduced
down remarkably with respect to the reference (Table 1). It has
also been observed that conjugated portion of the total BR
decreases more than its unconjugated counterpart, this is
expected, as the water soluble glucuronic acid conjugated
portion get higher chance to interact with NPs than the albumin
bound unconjugated part. This observation is further sup-
ported by our initial ndings that catalytic decomposition of
HSA bonded BR is slower compared to free BR (Fig. S7 in ESI†).
In the same blood specimens where we have evaluated BR level,
simultaneously, we have also checked the effects of citrate–
Mn3O4 NPs on other liver function parameters such as total
protein, albumin, globulin, alkaline phosphatase, g-glutamyl
transferase, SGOT and SGPT. As shown in Fig. 4b and c, the
change in the parameters upon interaction with citrate–Mn3O4
NPs is statistically insignicant (Table S1,† P value >0.05),
except in case of SGPT. Exact reason for the increase of SGPT
upon interaction with the NPs is not known in the present time
Reference
specimen
NP treated
specimen
Parameters
P value
Haemoglobin (gm dLꢃ1
)
8.70 ꢀ 1.93
2.97 ꢀ 0.64
8.36 ꢀ 1.85 0.70
2.50 ꢀ 0.67 0.13
RBC count (ꢂ106/mL)
Total leukocyte count (ꢂ109/L) 14.23 ꢀ 9.01 13.86 ꢀ 8.83 0.92
and needs further investigation. In case of different haemato-
logical parameters such as haemoglobin, red blood cell (RBC)
count and total leukocyte count (Fig. 4d and Table 2), it has
been found that there is also insignicant variation (P value
>0.05) upon treatment of the blood specimens with citrate–
Mn3O4 NPs. Nominal variation in the count of RBC and total
leukocyte cells in the NP treated specimens, directly indicates
the blood biocompatibility of citrate–Mn3O4 NPs. Moreover, we
have also evaluated the effectiveness of citrate–Mn3O4 NPs
against the conventional blue light used in phototherapy,
towards BR decomposition. As shown in Fig. S8b in ESI,†
citrate–Mn3O4 NPs exhibit comparable efficiency against blue
light along with an added advantage of dark reactivity.
In summary, we have for the rst time demonstrated that
highly water-soluble citrate–Mn3O4 NPs can catalytically
decompose yellow aqueous solution of bilirubin to its colour-
less oxidative break down products in a very quick time and
most importantly, without any photo-activation. Mechanistic
studies on the catalytic process have resulted in greater
understanding of the catalytic cycle and additional insight into
the active sites of the nanoparticles involved. Furthermore, the
remarkable in vitro reactivity of the catalyst towards the
suppression of bilirubin level in the whole blood specimens of
hyperbilirubinemia patients, without much affecting other
important blood constituents, represents a great promise of
citrate–Mn3O4 NPs in direct therapeutic applications against
hyperbilirubinemia.
The authors would like to thank Prof. Anjan Kr. Dasgupta at
Department of Biochemistry, University of Calcutta and Prof.
Avijit Hazra of IPGME&R, Kolkata, for insightful discussions. A.
G. thanks UGC, India, for fellowship. N. G. thanks CSIR, India,
for fellowship. We thank Department of Science and Tech-
nology (DST), Government of India, for nancial grants DST/
TM/SERI/2k11/103 and SB/S1/PC-011/2013.
Table 1 In vitro effect of citrate–Mn3O4 NPs on the bilirubin level of
blood specimen. Data are expressed as mean ꢀ standard deviation [n
¼ 12]. P values indicating statistical significance, were determined by
Student's unpaired t-test. P values less than 0.05 considered statisti-
cally significant
Notes and references
1 (a) J. Kapitulnik, Mol. Pharmacol., 2004, 66, 773–779; (b)
J. Fevery, Liver Int., 2008, 28, 592–605.
Reference
specimen
NP treated
specimen
2 R. Brodersen, J. Biol. Chem., 1979, 254, 2364–2369.
3 X. Wang, J. R. Chowdhury and N. R. Chowdhury, Curr.
Paediatr., 2006, 16, 70–74.
Parameters (mg dLꢃ1
)
P value
Total bilirubin
Conjugated bilirubin
Unconjugated
bilirubin
7.01 ꢀ 6.80
4.40 ꢀ 4.81
2.62 ꢀ 2.36
0.42 ꢀ 0.29
0.10 ꢀ 0
<0.0001
<0.0001
0.0001
4 C. N. Van Der Veere, M. Sinaasappel, A. F. McDonagh,
´
P. Rosenthal, P. Labrune, M. Odievre, J. Fevery, J. Otte,
0.32 ꢀ 0.29
¨
P. McClean, G. Burk, V. Masakowski, W. Sperl,
5078 | RSC Adv., 2014, 4, 5075–5079
This journal is © The Royal Society of Chemistry 2014