Enhanced Cytotoxicity to Cancer Cells by Codelivery and Controlled Release of Paclitaxel-loaded Sirolimus-conjugated Albumin Nanoparticles

Article abstract of DOI:10.1111/cbdd.12750

Recently, it is suggested that mTOR signaling pathway is an important mediator in many cancers especially breast cancer. Therefore, effects of sirolimus as a mTOR inhibitor in breast cancer have been studied in combination with paclitaxel with or without controlled release effect. In this work, we prepared a water-soluble formulation of sirolimus-conjugated albumin nanoparticles loaded with paclitaxel, to study the effects of sirolimus concentration when it releases more later than paclitaxel in comparison with sirolimus–paclitaxel-loaded albumin nanoparticles. Also effects of paclitaxel loading on cytotoxic properties of nanoparticles were studied. Sirolimus was succinylated at 42-OH with enzymatic reaction of Candida antarctica lipase B, and then its carboxylic group was activated with EDC/NHS and conjugated to the lysine residues of albumin. Paclitaxel was loaded on albumin surface by nab technique in concentration range of 0–10 μg/mL. Sirolimus-conjugated nanoparticles with 0.01 μg/mL paclitaxel showed lowest cell viability of 44% while it was 53% for non-conjugated nanoparticles in MDA-MB-468 cell lines after 48 h (p-value = 0.003). In MCF-7 cell lines, sirolimus-conjugated nanoparticles with 0.1 μg/mL paclitaxel showed lowest cell viability of 35.69% while it was 48% for non-conjugated nanoparticles after 48 h (p-value = 0.03). We guess that when cancer cell lines arrest in G2-M by anticancer drugs like paclitaxel, Akt activates mTOR to make cells continue living, then inhibiting mTOR can enhance anticancer effects.

Full text of DOI:10.1111/cbdd.12750

Chem Biol Drug Des 2016
Research Article
Enhanced Cytotoxicity to Cancer Cells by Codelivery
and Controlled Release of Paclitaxel-loaded
Sirolimus-conjugated Albumin Nanoparticles
1
2
Hossein Behrouz , Mehdi Esfandyari-Manesh ,
Key words: codelivery, enhanced cytotoxicity, nanotechnology,
paclitaxel, sirolimus
1
3
Mohammad Kazem Khoeeniha , Mohsen Amini ,
Behrang Shiri Varnamkhasti , Fatemeh Atyabi
and Rassoul Dinarvand
2
1,2
Abbreviations: DMAP, 4-dimethylaminopyridine; EDC, 1-
ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl; mTOR,
mammalian target of rapamycin; NHS, N-hydroxysuccinimide;
RF, relative to front.
1
,2,
*
1
Department of Pharmaceutics, Faculty of Pharmacy,
Tehran University of Medical Sciences, P.O. Box 14155-
451, Tehran, Iran
Nanotechnology Research Center, Faculty of Pharmacy,
Tehran University of Medical Sciences, P.O. Box 14155-
6
Received 30 August 2015, revised 19 December 2015 and
accepted for publication 8 February 2016
2
6
451, Tehran, Iran
3
Department of Medicinal Chemistry, Faculty of Pharmacy,
Tehran University of Medical Sciences, P.O. Box 14155-
Sirolimus, a hydrophobic macrolide derived from Strepto-
myces hygroscopicus, is an immunosuppressor which
was originally developed as an antifungal drug, but
recently it has been proved to have potent anticancer
effects in variety of cancers (1). Sirolimus inhibits mam-
malian target of sirolimus (mTOR) by binding to FK506
binding protein 12 (FKBP-12) (2). Many of researches sug-
gest that mTOR is a critical mediator of tumor progression
and may be a novel target in a significant proportion of
patients with breast cancer (3). mTOR is a serine/threonine
protein kinase that regulates cell growth, cell proliferation,
cell motility, cell survival, protein synthesis, and transcrip-
tion and is a member of the phosphatidylinositol 3-kinase-
related kinase protein family (PIKKs) (4).
6451, Tehran, Iran
*Corresponding author: Rassoul Dinarvand,
dinarvand@tums.ac.ir
Recently, it is suggested that mTOR signaling path-
way is an important mediator in many cancers espe-
cially breast cancer. Therefore, effects of sirolimus
as a mTOR inhibitor in breast cancer have been
studied in combination with paclitaxel with or without
controlled release effect. In this work, we prepared a
water-soluble formulation of sirolimus-conjugated
albumin nanoparticles loaded with paclitaxel, to
study the effects of sirolimus concentration when it
releases more later than paclitaxel in comparison
with sirolimus–paclitaxel-loaded albumin nanoparti-
cles. Also effects of paclitaxel loading on cytotoxic
properties of nanoparticles were studied. Sirolimus
was succinylated at 42-OH with enzymatic reaction
of Candida antarctica lipase B, and then its car-
boxylic group was activated with EDC/NHS and con-
jugated to the lysine residues of albumin. Paclitaxel
was loaded on albumin surface by nab technique in
concentration range of 0–10 lg/mL. Sirolimus-conju-
gated nanoparticles with 0.01 lg/mL paclitaxel
showed lowest cell viability of 44% while it was 53%
for non-conjugated nanoparticles in MDA-MB-468 cell
lines after 48 h (p-value = 0.003). In MCF-7 cell lines,
sirolimus-conjugated nanoparticles with 0.1 lg/mL
paclitaxel showed lowest cell viability of 35.69%
while it was 48% for non-conjugated nanoparticles
after 48 h (p-value = 0.03). We guess that when can-
cer cell lines arrest in G2-M by anticancer drugs like
paclitaxel, Akt activates mTOR to make cells con-
tinue living, then inhibiting mTOR can enhance anti-
cancer effects.
An important pathway that is highly active in many cancers
especially in breast cancer and causes cancer pathogene-
sis, progression, and resistance to drug treatment is PI3K/
AKT/mTOR pathway. Dysregulation of PI3K/AKT/mTOR
signaling occurs in various tumor types, including breast
cancer (5–7). In this pathway, there are two mTOR com-
plexes: mTOR complex 1 (mTORC1) and mTOR complex
2
(mTORC2) in which mTOR is the catalytic subunit of this
two structurally distinct complexes (8).
The best recognized positive effector of the mTORC1 is
the growth factor/PI3K/Akt pathway. Akt activates
mTORC1 in the presence of growth factors by inhibiting
tuberous sclerosis complex 2 (TSC2). TSC2 is a protein
that has GTPase-activating protein (GAP) activity toward
Rheb (Ras homolog enriched in brain) and converts Rheb
to inactive GDP-bound state. Rheb in GTP form activates
mTORC1, and thus, Akt activates mTORC1 by inhibiting
activation of TSC2 (9–11).
ª 2016 John Wiley & Sons A/S. doi: 10.1111/cbdd.12750
1

Behrouz et al.
Phosphatase and tensin homolog (PTEN) protein which is
encoded by PTEN gene is a tumor suppressor gene that
regulates the cell cycle and prevents cells from growing
and dividing too rapidly. PTEN is a phosphatase that pref-
erentially dephosphorylates phosphoinositide substrates
Experimental
Materials
Sirolimus as a powder was obtained from Haorui Pharma-
Chem Inc (Shenzhen, China) and paclitaxel USP was pur-
chased from Cipla Pharmaceutical Co. (Mumbai, India). 1-
ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDC),
like phosphatidylinositol (3,4,5)-trisphosphate (PIP ) and
3
therefore inhibits Akt/PI3k pathway activation (12).
4
-Dimethylaminopyridine (DMAP), Candida antarctica
As a negative point, treatment with sirolimus alone, which
is known to selectively inhibit mTORC1, can lead to upreg-
ulation of the Akt and ERK–MAPK pathways through a
PI3K-dependent feedback loop, causing cell survival and
drug resistance (13, 14).
lipase B (Novozym 435 Immobilized), MTT, and human
serum albumin (HSA) were purchased from Sigma (Stein-
heim, Germany).
MDA-MB-468 and MCF-7 cell lines were obtained from
National Cell Bank of Iran (NCBI) at Pasteur Institute of
Iran.
Paclitaxel is one of the cytoskeletal drugs that targets
tubulin and disrupts formation of normal spindles;
therefore, it causes G2/M cell cycle arrest. Paclitaxel is a
potential anticancer agent for several malignancies includ-
ing ovary, breast, head, and neck cancers (15, 16), but it
has many side-effects and also many resistance
responses such as overexpression of multidrug resistance
Methanol and acetonitrile (HPLC grade), N-hydroxysuccini-
mide (NHS), and N,N-dimethylformamide (DMF) were
purchased from Merck (Darmstadt, Germany). Double dis-
tilled water (DDW) was used for all experiments, and all
other solvents and reagents unless otherwise stated were
from Merck (Darmstadt, Germany).
(MDR-1) gene, overexpression of IL-6, changes in beta-
tubulin (target molecule), changes in regulatory apoptotic
proteins and mitosis checkpoint proteins (17).
Sirolimus succinylation at 42-OH
In a report published recently, it was seen that when
sirolimus administered 12 and 24 h after paclitaxel, MDA-
MB-468 breast cancer cell lines demonstrated significantly
more annexin positive and the percentage of cells in sub-
G1 and G2/M increases even further, as compared with
both agents given simultaneously (18).
Briefly, sirolimus (0.1 g, 0.10 mmol) and succinic anhy-
dride (0.054 g, 0.54 mmol) were added to 5 mL of
toluene:dichloromethane (4:1), and then sonicated for
5 min at 40 °C. A total of 250 mg of Candida antarctica
lipase B (NOVOZYM 435 Immobilized) was added to this
solution and shaked for 48 h at 45 °C under argon with
2
00 r.p.m. to obtain sirolimus 42-hemisuccinate (Figure 1).
There are fewer reports of breast cancer treatment that
delivers these two drugs simultaneously or with delay
release of one agent using nanotechnology delivery system
with comparing effects. Thus, we decided to design and
synthesis a water-soluble albumin-based nanocarrier sys-
tem containing paclitaxel and conjugated sirolimus. This
system delivers sirolimus more later than paclitaxel to
breast cancer MCF-7 (PTEN wild type) and MDA-MB-468
The progress of reaction was monitored every 12 h with
TLC; mobile phase dichloromethane:methanol (13:1).
After completion of reaction, the enzyme was filtered off
and washed three times with 5 mL of dichloromethane,
and then the solvent evaporated under reduced pressure
with rotary evaporator (Buchi, Switzerland) to obtain a
concentrated solution and the residue was purified by sil-
ica gel column with dichloromethane: methanol (13:1) as
eluent.
(PTEN mutant) cell lines. Also, the paclitaxel-loaded albu-
min and sirolimus nanoparticles were prepared to deliver
paclitaxel and sirolimus at the same time in comparison
with conjugated formulation.
For non-enzymatic synthesis of succinylated sirolimus,
2
00 mg sirolimus (0.2 mmol), 14 mg DMAP (0.1 mmol),
In this work, albumin was used as carrier, because it has
many benefits including high stability, low toxicity, and
immunogenicity, enabling surface modification and the
presence of receptors in tumor endothelial cells including
gp60, gp30, and g18 that bind albumin and effectively
endocytosis albumin and drug to cancer cells (19, 20).
Also using albumin, we could implement both delay and
burst forms of drug delivery. In other words, we conju-
gated sirolimus to lysine residues using chemical reaction
to achieve delay release and loaded paclitaxel on albumin
surface and inner parts by hydrophobic interactions to
achieve burst release.
and 40 mg succinic anhydride were dissolved in 5-mL
dichloromethane. To this solution, 32 lL dried pyridine
was added and stirred for 2 days at 30 °C under argon.
Other purification steps were as above.
Nuclear magnetic resonance (NMR) spectrum of sirolimus
and succinylated sirolimus after purification was recorded
by Bruker 300 MHz NMR system (Bruker, Germany) to
confirm addition of hemisuccinate group to sirolimus.
Succinylated sirolimus obtained by enzymatic reaction was
used for all other further analyses and reactions.
2
Chem Biol Drug Des 2016

Enhanced Cytotoxicity by Codelivery Anticancers
Figure 1: Succinylation of sirolimus with enzymatic reaction of Candida antarctica lipase B (NOVOZYM 435 Immobilized) for 48 h at
4
5 °C in the presence of succinic anhydride.
Succinylation yield was calculated by this formula (molecu-
lar weight of sirolimus and succinylated sirolimus are
Unreacted sirolimus and DMF were removed by Amicon
Ultra-4 30K centrifugal filter device at 5000 9 g for 20 min
several times using phosphate buffer (10 mM pH = 7.4):
914.172 g/mol and 1000 g/mol, respectively):
weight of dried succinylated sirolimus fraction ꢂ 1000 g/mol
Succinylation yield:
ꢁ 100
weight of sirolimus ꢂ 914:172g=mol
Activation of sirolimus 42-hemisuccinate and
purification of activated ester
acetonitrile:DMSO (15:4:1) and finally with phosphate
buffer (10 mM pH = 7.4). The retained sirolimus-conju-
gated albumin was lyophilized at ꢀ40 °C for 48 h (Christ,
Alpha 2–4 LD, Germany), and filtrate was used for indirect
determination of drug loading.
Carboxylic group of sirolimus hemisuccinate was activated,
and then conjugated to albumin lysine using of EDC and
NHS. For this purpose, 10 mg of sirolimus 42-hemisucci-
nate (0.01 mmol) was added to a solution of 1.5 mg of
EDC (0.01 mmol) and 1.2 mg NHS (0.01 mmol) in 1 mL
of dimethylformamide and the reaction was stirred for 1 h
at room temperature to activate carboxylic group.
Degree of conjugation and sirolimus weight ratio to
albumin were calculated both in direct and indirect method
using below formulae:
Activated NHS-ester of sirolimus hemisuccinate was puri-
fied using INOPAK C-18 SPE columns (bed weight
weight of drug conjugated
Degree of conjugation =
ꢁ 100
weight of drug input
500 mg, 3 mL, RStech Corporation, Republic of Korea).
First cartridge was washed with 10 mL methanol/water
Sirolimus weight ratio to albumin =
weight of sirolimus conjugatedðlgÞ
weight of albumin (mg)
9
2
0/10 and then 10 mL water as preconditioning, and
0 mg sample in 2 mL acetonitrile/water was loaded on
column. Extraction was carried out by acetonitrile/water
70:30) as eluent. Several fractions (each 3 mL) were col-
(
lected, and UV absorbance was checked at 280 nm. The
fraction containing sirolimus activated ester was dried with
rotary evaporator and used for further steps.
Indirect conjugation yield with high-performance
liquid chromatography (HPLC)
For determination of conjugation yield, chloroform was
added to the filtrate (resulted in previous level) in a volume
4
:1 (chloroform:aqueous filtrate) to extract the unreacted
Conjugation of activated sirolimus to albumin
Thirty-three mg of HSA (0.0005 mol) was dissolved in
sirolimus from aqueous filtrate. Chloroform was evaporated,
and then the residual was reconstituted in methanol: water,
and finally it was analyzed by C8 column (4.6 9 150 mm,
pore size 5 lm; NUCLEODUR, Machery-Nagel GmbH Co.
KG) at 57 °C. The mobile phase (methanol:water, 80:20)
was delivered at a flow rate of 0.5 mL/min with a pump
(WellChrom, K-1001, Knauer, Berlin, Germany) and
detected at 277 nm by UV detector.
6
mL of sodium phosphate buffer (10 mM pH = 8.4); after
complete dissolution, 10 mg of activated and purified suc-
cinylated sirolimus (0.01 mol, about 20 times the mole of
albumin) in 1 mL DMF was added dropwise to HSA solu-
tion, and the reaction was remained stirring for 5 h at
room temperature.
Chem Biol Drug Des 2016
3

Behrouz et al.
Direct determination of conjugation yield with
HPLC
bromophenol blue, 30% glycerol) and then mixed thor-
oughly. Also, 2 lL of intact albumin (1 mg/mL) and spike of
albumin:albumin–sirolimus conjugates (1:1) was prepared
similarly. This 20 lL sample solution was loaded on gradient
gel (4-20%, Tris-glycine, non-reducing) and then ran at
120 V for about 1 h. Resulting gel was fixed with solution of
methanol:water:acetic acid (50:40:10) for 5 min and stained
with 0.25% Coomassie Blue for 5 h and destained with
Sirolimus 42-hemisuccinate and the conjugation ratio was
estimated by hydrolysis of conjugate and extracting siroli-
mus (21). For this purpose, 1 mg of the lyophilized siroli-
mus-conjugated albumin was dissolved in 10 mL of lysis
buffer (100 mM acetate buffer pH = 4.5) and stirred for
2
4 h in room temperature. After finishing hydrolysis, siroli-
mus was extracted with 30 mL dichloromethane, and then
the solvent was evaporated, and it was dispersed in ace-
tonitrile. The conjugated sirolimus was quantified by HPLC
with previous conditions.
2
methanol:glacial acetic acid: H O (45:10:45) for overnight.
FT-IR analysis
The lyophilized powder was placed in KBr disks, and its
FT-IR spectra was recorded by a Nicolet (Magna IR-550,
Waltham, MA, USA) spectrophotometer to confirm forma-
tion of amide bonds between carboxylic groups of siroli-
mus hemisuccinate and amine groups of albumin after
conjugation.
Size exclusion chromatography of sirolimus–
albumin conjugate
Albumin conjugates were separated by molecular weight
with TOSOH column (21.5 9 600 mm, 13 lm, TSKgel
2
000SW, TOSOH biosciences) and detected by UV detector
at 280 nm. Mobile phase consisted of 100 mM phosphate
buffer pH = 6.8 and delivered isocratically with at a flow rate
of 1 mL/min with a pump (WellChrom, K-1001, Knauer).
Also, HSA, sirolimus hemisuccinate, and physical mixture
of HSA and sirolimus hemisuccinate were analyzed for
comparing differences.
SDS–PAGE analysis of conjugates
Preparation of paclitaxel-loaded sirolimus-
conjugated albumin nanoparticles
Paclitaxel was loaded in sirolimus-conjugated albumin
nanoparticles using nab technology (21) (Figure 2). For this
purpose, 10 mg of lyophilized albumin–sirolimus conjugate
SDS–PAGE analysis of albumin conjugates was performed
under non-denaturing condition. Briefly, 2 lL of albumin–
sirolimus solution (1 mg/mL) was added to 18 lL loading
buffer (1.5 M Tris–HCL pH = 6.8, 10% SDS, 0.1%
Figure 2: Schematic diagram of paclitaxel loaded in albumin–sirolimus conjugates.
4
Chem Biol Drug Des 2016

Enhanced Cytotoxicity by Codelivery Anticancers
Figure 3: FT-IR analysis of succinylated
ꢀ1
sirolimus, peaks of 1694 cm (terminal
ꢀ1
carboxylic acid) and 1738 cm ester
linkage appear as a result of ester bond
formation after succinylation and peaks of
ꢀ1
1
725 and 1642 cm in native sirolimus are
due to ketone and amide groups in
sirolimus.
containing about 0.96 mg sirolimus was dissolved in 98 mL
of 10 mM phosphate buffer pH = 7.4 (final concentration of
sirolimus = 100 nM) and then 2 mL of paclitaxel in chloro-
form with different concentrations of 500, 50, 5, and
Morphology of nanoparticles
Morphology of nanoparticles was characterized by
transmission electron microscopy (Zeiss EM 900, Jena,
Germany) and scanning electron microscopy (FE-SEM,
S4160, Hitachi, Japan).
0.5 lg/mL was added dropwise under probe sonicator
(Misonix, Newtown, CT, USA) in 2 min and then homoge-
nized with high pressure homogenizer (GEA, Parma, Italy)
for five cycles. Chloroform was evaporated with rotary evap-
orator, and this solution was lyophilized for further use.
Briefly, for TEM, 100 lL of sample was prepared for imag-
ing by drying nanoparticles on a copper grid that is coated
with a thin layer of carbon.
For obtaining nanoparticles containing loaded paclitaxel
and sirolimus, 10 mg albumin was dissolved in 98 mL of
For SEM, 100 lL of nanoparticles in water was spread on
aluminum stub and was allowed to be dry. The samples
were then coated with a thin layer of gold using the sput-
ter coater and examined by SEM.
10 mM phosphate buffer pH = 7.4 and 2 mL of sirolimus
(0.5 mg/mL) and 2 mL of different concentrations of pacli-
taxel (as said previous) was added dropwise in 2 min.
Other steps were same as above.
Sample C2 was used for both SEM and TEM.
Measurement of size, size distribution, and zeta
potential of nanoparticles
Lyophilized nanoparticles were added to 50 mM phosphate
buffer PH = 7.4, and then the size, polydispersity index,
and zeta potential nanoparticles were determined by
Zetasizer ZS (Malvern Instruments, Malvern, UK).
In vitro cytotoxicity of nanoparticles
Cytotoxicity of five samples of the conjugated and the
non-conjugated nanoparticles with 100 nM conjugated sir-
olimus and 0–10 lg/mL loaded paclitaxel was evaluated
against MDA-MB-468 and MCF-7 breast cancer cell lines
for 48 h, and the cell viability was assessed by MTT assay.
Cytotoxicity of free sirolimus (100 nM) and free paclitaxel
(0-–10 lg/mL) was also evaluated to compare effects.
Table 1: Comparison of direct and indirect methods in evaluation
of conjugation
Cells were cultured in suitable flasks and were counted
3
2
4 h before performing tests. About 5 9 10 of cells
Number of sirolimus
molecules conjugated weight ratio
Method to albumin
Direct About 6
Sirolimus/albumin
(100 lL) were transferred to each well of 96-well plate and
incubated in 37 °C with 5% CO2 for 24 h. After reaching
to a constant and steady level of culture, samples and free
drug were added to each plate in four replicates for each
sample. After 24 and 48 h, MTT was added and absor-
bance of each well was read at 570 nm. Cell viability was
Conjugation
percent
(lg/mg)
90
120
30
39.8
Indirect About 8
Chem Biol Drug Des 2016
5

Behrouz et al.
calculated by dividing mean OD of sample wells to mean
OD of negative control wells.
Results and Discussion
Synthesis of sirolimus 42-hemisuccinate
The succinylation process of sirolimus with conventional
chemical reactions using reagents like DMAP and pyridine
had some disadvantages such as degradation of sirolimus,
poor yield, and lack of regioselectivity. These problems
were mostly due to using strong bases like DMAP and
pyridine causing ring opening of sirolimus, especially in the
presence of water (22).
Using one-step enzymatic reaction with lipase, the siroli-
mus 42-hemisuccinate was synthesized with good yield.
NMR results and TLC plate confirmed this claim. In TLC
analyzing, two spots were seen with RF = 0.8 and 0.7 and
comparing with standard sirolimus spot (RF = 0.8), it
assumed that succinic group attached to sirolimus caused
it to become more hydrophilic.
Figure 4: Molecular weight analysis of synthesized albumin–
sirolimus in comparison with native albumin by SDS–PAGE
gradient gel (4–20%, Tris-glycine, non-reducing).
Figure 5: Chromatogram of size exclusion
HPLC, sirolimus-albumin peak elutes earlier
(retention time = 10 min) than native albumin
indicating higher molecular weight.
Figure 6: FT-IR spectrum of native albumin
ꢀ1
(
1660 cm indicating C=O stretch vibration)
and albumin–sirolimus which amide peaks
ꢀ1
are intensified and 1717 cm shows ketone
ꢀ1
groups of sirolimus, peak in 1735 cm (not
good resolution) could be due to ester
bonds in sirolimus.
6
Chem Biol Drug Des 2016

Enhanced Cytotoxicity by Codelivery Anticancers
In chemical reaction, there is no selectivity through hydro-
xyl groups, but to some extent 42-OH is more susceptible
to react with succinic anhydride as it is more free and
does not have any steric hindrance compared to other
hydroxyl groups. But in enzymatic reaction, there is high
selectivity to 42-OH (23).
Activation of sirolimus 42-hemisuccinate and
purification of its NHS-ester
One of the strategies for activating of carboxylic group and
make it reactive to amine groups and hydroxyl groups is
using EDC and NHS. EDC is a type of carbodiimide that
reacts with carboxylic acids and makes it reactive to NHS.
NHS causes formation of stable amine reactive linker.
Excess unreacted EDC and NHS causes formation of albu-
min aggregates due to cross-linking of albumin carboxylic
groups with amine groups of other albumin molecules; thus,
separation of EDC and NHS from sirolimus NHS-ester is
important before conjugation step. EDC and NHS were suc-
cessfully isolated from sirolimus 42-hemisuccinate-activated
ester with solid phase extraction (C-18 resin).
In enzymatic synthesis, 80 mg (succinylation yield: 73%)
of sirolimus 42-hemisuccinate was obtained after purify-
ing it with silica column, but in chemical synthesis it
was <40 mg (succinylation yield: 18%). NMR studies
showed:
H NMR (CDCl3, 300 MHz) 4.716 (m, 1H,
H2COC=O), 4212 (d, 1H, H2COH), 2.678 (s, 4H,
OC=OCH2CH2C=O).
In FT-IR spectroscopy of succinylated sirolimus, peaks
ꢀ
1
of 1694 cm
(
indicated terminal carboxylic acid
Sirolimus conjugation to albumin and yield of
reaction
In indirect method of conjugation yield, after extraction of
filtrate with chloroform and redispersion of pellet in 5 mL
methanol:water, concentration of this part was 1.2 mg/
mL meaning that about 6.0 mg of 10 mg was not
ꢀ
1
1
ꢀ1
1690–1760 cm
)
and 1738 cm
ester linkage
ꢀ
(
1735–1750 cm ) as a result of succinylation and peaks
ꢀ
1
ꢀ1
of 1642 cm
and 1725 cm in native sirolimus are
related to amide and ketone groups, respectively (Fig-
ure 3).
Table 2: Sample preparation and related size, PDI, and zeta potentials
Paclitaxel
Chloroform/phosphate
Sample
(lg/mL)
Sirolimus (nM)
Conjugated/non-conjugated
buffer
Size
PDI
Zeta potential
C
C
C
C
C
nC
nC
nC
nC
nC
1
2
3
4
5
10
1
0.1
0.01
0
10
1
0.1
0.01
0
100
100
100
100
100
100
100
100
100
100
Conjugated
Conjugated
Conjugated
Conjugated
2/100
2/100
2/100
2/100
2/100
2/100
2/100
2/100
2/100
2/100
315
204
269
187
199
298
305
201
197
212
0.37
0.42
0.18
0.26
0.43
0.23
0.34
0.21
0.12
0.15
29.9
27.0
16.1
12.0
Conjugated
ꢀ29.9
1
2
3
4
5
Non-conjugated
Non-conjugated
Non-conjugated
Non-conjugated
Non-conjugated
28.1
24.5
22.6
21.2
ꢀ21.2
Figure 7: TEM of nanoparticles in scale of
00, 150, and 80 nm.
2
Chem Biol Drug Des 2016
7

Behrouz et al.
Figure 8: SEM of nanoparticles in scale of
lm, 750 nm, and 500 nm.
1
conjugated and 4.0 mg sirolimus (0.004 mol, 8 mol per
determination of molecular weight of each band, we calcu-
lated distance of each band from the lowest band and the
difference between 63 and 75 KD band in marker was
about 6 mm; thus, each millimeter is about 2 KD
difference in molecular weight. Average of difference
between albumin and albumin–sirolimus band is 3 mm
(about 6 KD). Also, spike of albumin and albumin–sirolimus
conjugates clearly shows molecular weight change after
conjugation of sirolimus.
1
mol albumin or 8 molecules of sirolimus per albumin)
was conjugated to albumin. In direct method, 0.085 mg
ꢀ5
sirolimus (9.2
after lysis of 1 mg albumin–sirolimus conjugates
9 10 mol) was detected by HPLC
ꢀ
5
(
about 1.5 9 10 mol). It means that about six mole-
cules of sirolimus were conjugated to each albumin
molecule. Other parameters such as encapsulation effi-
cacy and sirolimus/albumin weight ratio are shown in the
Table 1.
In SE-HPLC, albumin peak was seen at 13.5 min,
whereas albumin–sirolimus peak was seen at 10 min. As
in this method higher molecular weight molecules elute
earlier, it can be concluded that albumin–sirolimus conju-
gation was successful and it has higher molecular weight
than albumin (Figure 5).
Sirolimus is very unstable in anionic buffers, especially in
phosphate and HEPES buffer, and rapidly undergoes
chemical hydrolysis, but it has the best stability in acetate
buffer among other buffers (22,24); thus, the acetate buffer
was used instead of phosphate buffer in hydrolysis buffer
to decrease these degradation products. In direct method,
the detected sirolimus was lower than indirect method and
it can be concluded that after 24 h, sirolimus was
degraded to some degree.
FT-IR analysis of albumin–sirolimus conjugates
After conjugation of sirolimus to albumin, peaks of amide
ꢀ
1
bonds in 1656 cm
(C=O stretch vibrations), peak of
ꢀ
1
Also, as PI (isoelectric pH) of albumin is 5.2, we chose
higher pH values (pH = 8.5) for conjugation of sirolimus to
albumin to make sure that all lysine moieties have con-
verted from –NH3 + to –NH2. This makes amine groups
more nucleophile. In variety of articles, this pH is used for
conjugation (25).
1717 cm due to ketone groups of sirolimus and its
shoulder, peak of 1735 cm
ꢀ1
related to ester bond in
conjugated sirolimus show that sirolimus successfully has
conjugated to albumin by amide bond, while ester bond
proves that sirolimus has successfully succinylated and
then conjugated (Figure 6).
Conjugation analysis of albumin–sirolimus by gel
permeation chromatography and SDS–PAGE
Gel permeation chromatography and SDS–PAGE were
performed to analyze molecular weight of conjugate.
Preparation of paclitaxel-loaded sirolimus-
conjugated albumin nanoparticles
The water-soluble formulation with rapid release properties
was prepared by physical loading of paclitaxel using nab
technique. Chloroform was chosen as solvent because it
is a good solvent for paclitaxel and also it has low boiling
point and high vapor pressure, so it can be easily removed
by rotary evaporator. As chloroform is not miscible with
water, its volume was adjusted 2/100 in proportion to
aqueous phase to prevent albumin aggregation and also
Each sirolimus is about 1 kD, and about eight molecules
of sirolimus are conjugated to each albumin, and there-
fore, the final conjugate must be 75 kD. In SDS–PAGE
analysis, albumin conjugated sirolimus was placed upper
than native albumin near 75 kD marker (Figure 4). For
8
Chem Biol Drug Des 2016

Enhanced Cytotoxicity by Codelivery Anticancers
Table 3: Comparison of cytotoxicity of conjugated and non-conjugated samples on MDA-MB-468 and MCF-7 cell lines after 24 and 48 h
MDA-MB-468 cell viability
After 24 h
MCF-7 cytotoxicity cell viability
After 24 h
Sample
After 48 h
After 48 h
C
C
C
C
C
nC
nC
nC
nC
nC
1
2
3
4
5
67.11 ꢃ 6.05
62.15 ꢃ 9.47
58.73 ꢃ 5.21
56.47 ꢃ 3.15
55.77 ꢃ 6.02
69.82 ꢃ 7.12
70.98 ꢃ 3.89
59.25 ꢃ 0.90
61.44 ꢃ 6.50
75.24 ꢃ 7.34
48.39 ꢃ 8.18
50.21 ꢃ 3.13
46.96 ꢃ 5.53
44.40 ꢃ 3.43
54.08 ꢃ 9.51
50.25 ꢃ 4.87
49.86 ꢃ 1.30
47.73 ꢃ 5.19
53.65 ꢃ 1.19
73.05 ꢃ 3.47
55.79 ꢃ 4.15
56.51 ꢃ 2.9
38.18 ꢃ 1.84
38.75 ꢃ 5.82
35.69 ꢃ 1.87
38.58 ꢃ 3.77
53.34 ꢃ 6.73
38.98 ꢃ 3.10
40.19 ꢃ 2.27
48.56 ꢃ 7.80
47.22 ꢃ 5.72
45.47 ꢃ 3.90
54.30 ꢃ 1.91
57.78 ꢃ 2.42
57.61 ꢃ 6.04
55.18 ꢃ 5.06
58.22 ꢃ 2.96
65.35 ꢃ 3.60
65.35 ꢃ 12.62
62.75 ꢃ 4.13
1
2
3
4
5
better sonication and homogenization of nanoparticles in
further steps (Table 2).
(sample C
while in loading paclitaxel on albumin surface, an increase
in zeta potential was seen from +16.1 mV for sample C
5
) can be related to phosphate buffer pH (26),
1
to 29.9 mV for sample C4. This trend was seen for non-
conjugated samples as well (Table 2).
Measurement of size and size distribution of
nanoparticles
An increase in size of nanoparticles was seen with
increase in paclitaxel loading; thus, size of nanoparticles
Morphology studies
was from 187 nm for sample
taxel = 0.01 lg/mL) to 315 nm for sample C
paclitaxel = 10 lg/mL). Same trend was seen for non-
conjugated samples. Negative quantities of zeta potential
for albumin–sirolimus nanoparticles with no paclitaxel
C
4
(loaded pacli-
(loaded
As it is observed in TEM, paclitaxel-loaded albumin–siro-
limus nanoparticles had 150 nm particle size and mor-
phology of them was almost spherical, spongy surface,
and a thin layer around it with size of about 150 nm
(Figure 7).
1
Figure 9: Comparison of conjugated and
non-conjugated formulations in MDA-MB-
4
68 and MCF-7 cell lines after 24 and 48 h.
Chem Biol Drug Des 2016
9

Behrouz et al.
In SEM, also most of the nanoparticles were globular
shaped with porous surface and narrow size distribution.
This porous surface was mostly because of chloroform
evaporation effect (Figure 8). The rate of solvent extraction
or evaporation has significant effects on the porosity of the
nanoparticles, which, in turn, significantly affects the drug
release from the nanoparticles (27).
be explained by paclitaxel effect on activating PI3K/Akt and
ERK–MAPK pathway and also release of survivin (30).
Conclusion
Sirolimus was successfully succinylated using Candida
antarctica lipase B with good yield, and it was conjugated to
albumin with aid of EDC and NHS. Paclitaxel as second
drug was loaded on albumin using nab technique. The
microscopic images show good morphology, and the parti-
cle size was between 187 and 315 nm. The cytotoxicity
studies showed that after 48 h, nanoparticles with 0.01 and
0.1 lg/mL paclitaxel were more cytotoxic on MDA-MB-468
and MCF-7 cell lines, respectively, also nanoparticles with
higher or zero concentration of paclitaxel did not show any
higher effects. The most important result in work was that it
seems that paclitaxel concentration ratio to sirolimus and
time of sirolimus release after paclitaxel are two important
factors in cytotoxic effects. Higher concentration of cyto-
toxic drugs not always generates higher response; thus,
maybe in future, administration of low-dose paclitaxel with
sirolimus in constant time intervals can have best effects in
cancer patients with less side-effects of paclitaxel. Codeliv-
ery of anticancers using controlled release pattern is an
effective strategy for enhance cytotoxicity and less side-
effects.
In vitro cytotoxicity of paclitaxel-loaded
albumin–sirolimus nanoparticles
Cytotoxicity of five samples of the conjugated and the
non-conjugated nanoparticles with 100 nM conjugated sir-
olimus and different concentrations of loaded paclitaxel (0,
0
4
.01, 0.1, 1, 10 lg/mL) were evaluated against MDA-MB-
68 and MCF-7 breast cancer cell lines. The results
showed highest cell death with 0.01 lg/mL paclitaxel in
MDA-MB-468 cell lines and with 0.1 lg/mL paclitaxel in
MCF-7 cell lines.
Also, samples of constant 100 nM sirolimus (non-conju-
gated) with different concentrations of loaded paclitaxel (0,
0
.01, 0.1, 1, 10 lg/mL) were evaluated to see how delayed
release of sirolimus affects cytotoxicity in comparison with
burst release of both drugs at the same time.
Nanoparticles of 0.01 lg/mL paclitaxel showed lowest cell
viability of 44.40 ꢃ 3.43% while it was 53.65 ꢃ 1.19% for
non-conjugated nanoparticles in MDA-MB-468 cell lines
after 48 h (p-value = 0.0035). Nanoparticles of 0.01 lg/
mL paclitaxel showed significantly lower cell viability after
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