BSA nanoparticles as controlled release carriers for isophethalaldoxime palladacycle complex; Synthesis, characterization,: In vitro evaluation, cytotoxicity and release kinetics analysis

Article abstract of DOI:10.1039/c9nj05847h

A new oxime ligand named isophthalaldoxime (1) was synthesized by the addition of hydroxylamine hydrochloride to isophthalaldehyde. Then, a four nuclear palladacycle complex {Pd₄[(N,C)(NOHCHC₆H₂CHNOH)(μ-Cl)₂]

Full text of DOI:10.1039/c9nj05847h

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BSA nanoparticles as controlled release carriers
for isophethalaldoxime palladacycle complex;
synthesis, characterization, in vitro evaluation,
cytotoxicity and release kinetics analysis†
a
Cite this:DOI: 10.1039/c9nj05847h
Kazem Karami, *a Nasrin Jamshidian,
Afsaneh Hajiaghasia and
b
Zahra Amirghofran
A new oxime ligand named isophthalaldoxime (1) was synthesized by the addition of hydroxylamine
hydrochloride to isophthalaldehyde. Then, a four nuclear palladacycle complex {Pd4[(N,C)(NOHCHC6H2-
CHNOH)(m-Cl)2]2} (2) was prepared and fully characterized using elemental analysis, FT-IR, and NMR
spectroscopy. A desolvation method was employed for the synthesis of protein nanoparticles using
bovine serum albumin (BSA). Then, complex (2) was loaded on the prepared nanoparticles with an
optimized entrapment efficiency (EE) of 97.78% and loading capacity (LC) of 22.68%. FT-IR spectroscopy
and circular dichroism (CD) were used for spectral studies. Field emission scanning electron microscopy
(FESEM) and dynamic light scattering (DLS) measurement was applied for structural studies on the
morphology, size, and zeta potential of BSA nanoparticles (BSA-NPs) and complex-loaded nanoparticles
((2)@BSA-NPs). The results showed the stability and acceptable size of nanoparticles prepared for the
in vitro drug release experiment. Therefore, the in vitro release of (2) from BSA-NPs was studied using
the dialysis bag method. In addition, the release mechanism was investigated by mathematical methods
and the results showed that phase I and II of the release process followed Korsmeyer–Peppas and
Higuchi models, respectively. Finally, the in vitro cytotoxicities of the synthesized nanoparticles, palladium
complex, and complex-loaded nanoparticles were carried out against A549 human lung carcinoma and
K562 human leukemia cell lines using MTT colorimetric assays.
Received 23rd November 2019,
Accepted 30th January 2020
DOI: 10.1039/c9nj05847h
rsc.li/njc
action.4 Indeed, the efficacy of most drugs is limited due to
several reasons such as poor absorption, poor solubility, non-
1. Introduction
Cancer is a class of disease characterized by out-of-control cell specific delivery, short circulating half-life, high toxicity, and high
growth. Approximately 12.7 million people were diagnosed with dosage.5–7 An efficient approach to increase the efficacy and
cancer worldwide in 2008 and this number is expected to reduce the side-effects of anticancer drugs is to incorporate drugs
increase to 21 million by 2030.1,2 There are over 100 different with delivery systems such as nanoparticles.8–10
types of cancer and each is classified by the type of cell that is
Precise diagnosis and effective drug delivery have been
initially affected. Although the clinical treatment for cancer has attracting considerable interest in cancer therapy over the past
made considerable progress with the improvement of surgical decades.11 Researchers have recognized that nanotechnology is the
techniques, the wide application of new technologies, and best solution to overcome the aforesaid problem in developing an
clinical development of new drugs, cancer remains a complicated effective drug. The use of nanomaterials as pharmaceutical drug
medical problem that has not yet been overcome and threatens carriers to increase antitumor efficacy has been studied for more
human health and lives.3
than 30 years. The important goals of nanotechnology are to target
Drug delivery is a promising approach for the biotechnologists the drug at the site of action, to enhance bioavailability without
and pharmacologists to develop a delivery system that can aid the side effects,12 greater safety,13 biocompatibility,14 protecting a drug
drug to its target and maintain its availability for therapeutic from degradation,15 enhancing drug absorption by facilitating
diffusion through epithelium and drug tissue distribution profile,
a Department of Chemistry, Isfahan University of Technology, Isfahan 84156/83111,
and/or improving intracellular penetration and distribution.16
One of the most recent developments in designing drug-
delivery systems has focused on protein-based nanoparticles as
Iran. E-mail: karami@cc.iut.ac.ir
b Immunology Department and Autoimmune Diseases Research Center,
Shiraz University of Medical Sciences, Shiraz, Iran
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9nj05847h drug carriers due to their exceptional characteristics, such as
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extraordinary adsorption capacity, low toxicity, biodegradability,
The major goal of this study is using an albumin nano-
non-immunogenicity, stability for long duration, shelf life, amphi- particle drug delivery system to control the release of the new
philicity, and easy scale up.17,18 In addition, they can be easily palladium complex in a planned manner in order to have the
prepared under soft conditions and a large variety of protocols desired therapeutic effect, protect it from degradation, and improve
based on desolvation, emulsification, thermal gelation,19 nano its function. In this way, a new complex of palladium(II) with a
spray drying,20 the NAB technology (nanoparticle albumin- dioxime ligand derived from isophthalaldehyde was synthesized
bound) or self-assembly without using surfactants or toxic and characterized with spectroscopic methods. Then, BSA-NPs were
organic solvents in their fabrication.21,22
prepared by the desolvation method and were used as a carrier for
Albumin is the most abundant plasma protein and an ideal the synthesized complex. After that, the release mechanism was
candidate for nanoparticle preparation due to its excellent investigated by various mathematical models, including Zero-order,
properties, including extraordinary binding capacity thanks to First-order, Higuchi, Korsmeyer–Peppas, and Hixson–Crowell.
the different drug binding sites present in its structure, easy
purification, high solubility in water and pH 7.4 buffer solution
2. Materials and methods
2.1. Materials
(up to 40% w/v), allowing ease of delivery by injection, stability
in the pH range of 4–9, and the capability of heating at 60 1C for
up to 10 h without deleterious effects.23–25
The starting materials and solvents were purchased from Merck
Both bovine serum albumin (BSA) and human serum albumin
and Sigma Aldrich Chemical Companies, and were used
(HSA) are frequently used for preparing nanoparticles of defined
without purification. Reagent grade BSA was obtained from
sizes. Due to the higher availability and lower cost of BSA
Sigma Aldrich Chemical Company and was applied as received.
compared to that of HSA, as well as the high structural and
Tris(hydroxymethyl)-aminomethane (Tris) buffer was analytical
amino acid sequencing similarity (about 75%), we selected BSA
reagent grade, which was obtained from Merck. Doubly distilled
for the synthesis of a nano carrier in this study. BSA is highly
deionized water was used for the preparation of all solutions.
water soluble and binds drugs and inorganic substances non-
covalently, which allows the drug to be transported in the body
2.2. Instruments
and be readily given up at the cell surface.26 Also, the flexibility
FT-IR spectra were recorded on a FT-IR JASCO 680 spectro-
in conformation and presence of reactive groups (thiol, amino,
photometer from 4000–400 cmꢀ1 using KBr pellets. NMR
and carboxylic acid groups) on BSA enable it to bind with
compounds with different structures.27 The BSA-NPs were capable
spectra were recorded in DMSO-d6 at room temperature on a
Bruker spectrometer at 500 MHz (1H) and 125 MHz (13C), and
of not only controlling the drug encapsulation and drug release
but can also extend its circulation in the blood stream.28
the chemical shifts (ppm) are reported according to the internal
TMS standard. Elemental analysis of carbon, hydrogen, and
The mechanism of drug release or pharmacokinetics has
nitrogen was done using a CHNSO Analyzer (Elementar, Vario EL
wide importance in the pharmaceutical industry in the prediction
III). Nanoparticle dispersion was done with an EYELA ultrasonic
of drug release rate and helps researchers in developing effective
cleaner (Japan). A Froilabo SW14 centrifuge (CENSW14000001,
drug formulations and more accurate dosing that saves time and
money.29,30
UK) was used to perform centrifugation. DenaVacuum 5005 freeze
dryer (Iran) was utilized for nanoparticle preparation. Field
Following the side effects observed for platinum-based
Emission Scanning Electron Microscope (FESEM) model Quanta
anticancer drugs, the researchers turned to platinum analogues,
450 FEG, FEI (USA) was applied for nanoparticle morphology
characterization.
In vitro drug release experiments were studied in 0.13 M
phosphate buffer and 100 mM NaCl at pH 7.4 inside a refrigerator
viz., palladium complexes. In this regard, many palladium com-
pounds with trans, chelated, and palladacyclic structures have
been prepared and their pharmacological applications have been
investigated in vitro and in vivo. One of the early studies about the
incubator shaker SHER300. Each experiment was conducted in
use of palladium complexes as anti-cancer agents were done by
Graham.31 Since the hydrolysis of palladium complexes is
triplicate and the UV-Vis spectra were recorded on a Varian Cary
100 UV-Vis spectrophotometer employing a 1 cm path length cell.
very fast and prevents them from reaching biological targets,
CD spectra were recorded on an AvivCircular Dichroism Spectro-
researchers are now inclined to prepare palladacycle complexes
meter, model 215 (USA). A dynamic light scattering (DLS) appara-
tus (NanoQ V2.5.4.0) was applied for the size, polydispersity index
(PDI), and zeta potential characterization of BSA-NPs and complex
(2)@BSA-NPs.
containing a strong C–M s-bond to overcome the problem of
fast hydrolysis kinetics.32
Many palladium compounds with oxime ligands have been
synthesized and their anticancer activity has been studied. In
our previous studies, palladium complexes with various ligands
including amines, phosphorus ylides, and oximes were provided
2.3. Synthesis and methodology
that showed acceptable results against different cancer cell
2.3.1. Synthesis of oxime ligand: NOHCHC6H4CHNOH (1).
lines.33–35 Also, given the importance of protein nanocarriers, There are several methods for the preparation of oximes, including
the in vitro release of palladium trimer complexes with oxime the reduction of nitro compounds,37 oxidation of amines,38 addition
ligands encapsulated in either albumin or algal cellulose nano- of NOCl to alkenes,39 and addition of hydroxylamine to aldehydes or
particles has been reported in one of our previous works.36
ketones.40 According to the last method (Scheme 1), 0.16 g (4 mmol)
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Scheme 1 Synthesis of oxime ligand (1) and the palladium complex (2).
sodium hydroxide and 0.34 g (4 mmol) hydroxylamine hydro-
2.3.3. Preparation of nanoparticles by the desolvation
chloride were dissolved in a solution of distilled water and method. BSA-NPs were prepared using the well-known desolvation
ethanol (50 : 10 mL) on an ice bath and were stirred for 10 min. technique as reported before.41 This method is a thermo-
Then, 0.13 g (1 mmol) isophthalaldehyde was dissolved in 5 mL dynamically driven self-assembly process used for polymeric
ethanol and was later added dropwise to the stirred solution to materials. Both hydrophilic and hydrophobic drugs can be
achieve an opaline solution. The resulting solution was stirred encapsulated into nanoparticles employing this technique.42,43
for 4 h at 0 1C and was then stirred at room temperature for 2 h. As a general working (Scheme 2), bovine serum albumin powder
Eventually, the reaction mixture was refluxed for 2 h at 60 1C. (200 mg) was dissolved in 2 mL deionized water. The pH was
The final transparent solution was left to slowly concentrate by made to 7.2 with 0.01 M NaOH and the solution was left to stir at
solvent evaporation at room temperature overnight. Very fine 500 rpm at room temperature (25 1C) for 10 min to equilibrate.
needle crystals of (1) were obtained and were dissolved in Subsequently, by continuous dropwise addition of 8.0 mL ethanol
acetone. The acetone solution was filtered, evaporated to a small by a syringe pump at the rate of 1.0 mL minꢀ1 as a desolvating
volume, and precipitated with hexane, which was collected and agent, an opalescent suspension was achieved, which indicates the
dried in air to give (1) a white shining solid. Yield: 85%, FT-IR (KBr formation of the nanoparticles (step i). Ethanol changes the
pellet, cmꢀ1): n(O–H) = 3430, n(C–Haromatic) = 2998, n(CQN) = 1638, tertiary structure of the protein and during the addition of ethanol
n(CQC) = 1489, d(O–H) = 1321, n(N–O) = 945, d (C–H) = 680. to the solution, the albumin is phase separated due to its
1H-NMR (DMSO-d6, ppm): 11.31 (s, 2Ha), 8.14 (s, 2Hb), 7.79 diminished water solubility.44 Since the formed nanoparticles
(s, 1Hc), 7.57 (dd, 2Hd, 3J = 7.7 Hz, 4J = 1.5 Hz), 7.40 (t, 1He, were not sufficiently stabilized and could consequently redissolve
3J = 7.7 Hz). 13C{H} NMR (DMSO-d6, ppm): 148.15 (CQN carbon), again after dispersion with water, cross-linking was implemented,
133.98, 129.56, 127.56, 124.62 (aromatic carbons). Anal. calc. for which is the major step in the desolvation method. In this step,
C8H8N2O2: C, 58.53; H, 4.91; N, 17.06%. Found: C, 57.84; H, 4.05; N, 37.5 mL 50% aqueous solution of glutaraldehyde (0.2 mL
17.26%.
50% Gta per mg of BSA)45 was added gradually for the stabilization
2.3.2. Synthesis of the palladium complex: {Pd4[(N,C)- and cross linking of the amino moieties in lysine residues and the
(NOHCHC6H2CHNOH)(l-Cl)2]2} (2). LiCl (0.020 g, 0.48 mmol) guanidine side chains in arginine of BSA via a condensation
was added to a stirred orange solution of PdCl2 (0.042 g, reaction with the aldehyde group (step ii). The mixture was
0.24 mmol) in MeOH (20 mL) and refluxed at 50 1C for 40 min, maintained under stirring condition for 12 h.
during which the color of the solution turned to a transparent
2.3.4. Purification of the nanoparticles. In order to eliminate
dark red, which indicates the formation of Li2[PdCl4]. This the non-desolvated albumin, the excess cross-linking agent, and
solution was allowed to cool to 25 1C. Then, the oxime ligand organic solvent, the resulting nanoparticles were purified by three
(1) (0.020 g, 0.12 mmol) and NaOAc (0.020 g, 0.24 mmol) were successive centrifugations (16 000 rpm, 20 min). The first
added to cool down the solution, which results in a dark brown centrifugation supernatant was used for the determination of
suspension immediately. This suspension was refluxed at 50 1C non-desolvated albumin. After that, between each centrifugation,
for 7 h. After 1 h, the color changed to light yellow and at the end the supernatant solution was thrown away and the pellets were
of this period, a green slurry was obtained that was left to cool washed with the original volume of deionized water (step iii).
slowly and collected by filtration, washed with acetone and Then, the redispersion step in deionized water (20 mL) was
hexane, and dried in air to give a green precipitate. Yield: 79%, performed in an ultrasonic bath for 30 minutes (step iv). The
FT-IR (KBr pellet, cmꢀ1): n(O–H) = 3470, n(C–Haromatic) = 2967, product was dried in a freeze dryer with a cycle of 24 h at the shelf
n(CQN) = 1630, n(CQC) = 1575, d(O–H) = 1330, n(N–O) = 1022, temperature of ꢀ55 1C and then incubated at 4 1C in the dark
d(C–H) = 692. 1H-NMR (DMSO-d6, ppm): 10.67 (s, 2Ha), 8.08 (step v). Average particle sizes were measured by scanning
(s, 2Hb), 7.76 (s, 1Hc), 6.95 (s, 1He). 13C{H} NMR (DMSO-d6, electron microscopy (SEM) and dynamic light scattering (DLS).
ppm): 155.30 (Ci), 154.95 (CQN carbon), 140.77, 136.74, 122.53 The samples were dispersed in distilled water for DLS analyses.
(aromatic carbons). Anal. calc. for C16H12N4O4Pd4Cl4: C, 21.55; H,
1.36; N, 6.28%. Found: C, 21.03; H, 1.49; N, 6.78%.
2.3.5. Loading of the palladium complex onto BSA-NPs.
The BSA nanoparticles were loaded with the complex (2) and
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Scheme 2 Preparation and purification of the nanoparticles by the desolvation method.
were checked for the loading efficiency. The loading process 2.4. Cytotoxicity assessment
was done by the preparation of nanoparticle solutions by
The cytotoxic effects of complex (2), BSA-NPs, and complex
(2)@BSA-NPs on A549 human lung carcinoma and K562 human
leukemia cell lines were assessed using MTT [3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay.
A predetermined number of A549 cells (7000 cells per well) and
K562 (15 000 cells per well) in 96-well culture plates was seeded in
triplicate and then, 10 mL of the compounds at final concentra-
tions of 0.01 to 200 mg mLꢀ1 was added. The cells were incubated
at 37 1C in a humidified CO2 incubator for 48 h and then, 10 mL
of the MTT solution (5 mg mLꢀ1, Sigma) was added to each well.
After 4 h incubation at the same conditions, the supernatant was
removed and 150 mL dimethyl sulfoxide (DMSO) was added.
After that, the optical density (OD) was detected at 570 nm with a
reference wavelength of 630 nm on a microplate reader (BioTek,
USA). As the negative control, the solvent (DMSO) at concentrations
equal to that in the test wells was used. To determine the percentage
of cell growth inhibition, the following formula was used: Inhibition
(%) = 100 ꢀ [(OD of compounds/OD of negative control) ꢁ 100]. The
IC50 (half maximal inhibitory concentration) value of different
compounds was calculated using the Curve Expert software.
dissolving 25 mg in 1 mL deionized water. Then, the volumes
of 50–400 mL of complex (2) stock solution (2 ꢁ 10ꢀ3 M) were
added to the nanoparticle solutions, the final volume was
adjusted with deionized water to 2 mL, and was magnetically
stirred for 12 h (600 rpm) at room temperature. After this, there
is an adsorption equilibrium between the complex (2) and the
surface of the nanoparticles. These suspensions were trans-
mitted to polypropylene centrifuge tubes and centrifuged at
12 000 rpm for 20 min. The supernatants were separated and
their absorbance was measured using an ultraviolet spectro-
photometer to determine the loading efficiency.
2.3.6. ‘‘In vitro’’ drug release experiment. Complex (2)@BSA-
NPs was evaluated for in vitro release of the drug from the protein
nanoparticles by using a dialysis membrane. For this purpose,
10 mg complex (2)@BSA-NPs was taken and dispersed in
2 mL phosphate buffer (0.13 M) solution. The dialysis bags
with molecular weight cutoff 12 kDa were plunged in double-
distilled water for 12 h at room temperature before use. Then,
complex (2)@BSA-NPs suspension (10 mg/2 mL) was poured
into it and suspended in thick glass round storage bottles of
borosilicate Pyrex glass with blue GL45 screw cap containing
40 mL of the receiving phase (phosphate buffer pH 7.4). The
dialysis system was placed in an incubator shaker maintained
at 37 1C and 170 rpm for 48 h under constant floating and
stirring conditions during the experiment. At predefined time
3. Results and discussion
3.1. Spectral characterization of the ligand and the palladium
complex
intervals, 2 mL of the release medium was removed and FT-IR spectrum of ligand (1) (Fig. 1) clearly shows the absence
replaced with equal volumes of fresh phosphate buffer solution. of aldehyde CQO band (observed at 1696 cmꢀ1 in the initial
The amount of drug in the release medium was determined by aldehyde spectrum) and the appearance of oxime CQN band at
UV-Vis spectrophotometry at 303 nm and averaged from three 1638 cmꢀ1. Also, the absorption bands due to OH stretching,
independent experiments. The obtained release percentage was OH deformation, and N–O stretching vibrations were observed
plotted against time.
at 3430, 1321, and 1083 cmꢀ1, respectively, all of which are in
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Fig. 1 Merged FT-IR spectra of the ligand (red) and the complex (blue) using KBr pellet.
accord with the previously reported oxime derivatives.46,47 The palladacyclic carbon atoms and CQN carbon atom was
CQN oxime stretch decreased from 1638 cmꢀ1 in the free detected at 154.95 ppm. The signals that appeared at 140.77,
ligand to 1630 cmꢀ1 in complex (2), which indicates that the 136.74, and 122.53 ppm (2Cd, Cc and Ce, respectively) were
nitrogen atoms of the CQN groups participate in the complex assigned to other phenyl carbon atoms.
formation. Another important band in the FT-IR spectrum of
complex (2) appeared at 617 cmꢀ1 due to Pd–N stretching
3.2. Nanoparticles’ structural studies
vibration.
3.2.1. FT-IR spectroscopy. FT-IR spectroscopy is a simple
In the 1H-NMR spectrum of ligand (1) (Fig. S1, ESI†), the method that provides further information about the changes in
peaks corresponding protons of N–OH, NQCH, and internal the secondary structure of BSA and its characteristic bands after
aromatic C–H between two oxime groups appeared as singlets cross-linking with glutaraldehyde and conversion to the nano
at 11.31, 8.14, and 7.79 ppm, respectively. In the 1H-NMR structure.26,48 The IR spectra of pure BSA, unloaded BSA-NPs,
spectrum of complex (2) (Fig. S2, ESI†), these peaks shifted to and (2)@BSA-NPs are presented in Fig. 2. In the IR spectrum of
lower chemical shifts (10.67, 8.08, and 7.76 ppm, respectively), proteins, there are a number of amide bands due to vibrations
indicating the shielding of these protons after complex for- of the peptide moiety. As can be seen in Fig. 2, four characteristic
mation. The C–H protons in the ortho position of the oxime peaks were observed for BSA, including amide I (CQO stretching
groups were in resonance as doublet of doublets at 7.57 with vibrations) at 1649 cmꢀ1, amide II (coupling of N–H bending and
3J = 7.7 Hz and 4J = 1.5 Hz. In the 1H-NMR spectrum of complex C–N stretching vibrations) at 1536 cmꢀ1, side chain COOꢀ at
(2), these protons do not appear, which is consistent with Pd(II) 1385 cmꢀ1, and amide III (C–N stretching/N–H bending vibrations)
coordination in the proposed structure. Finally, the C–H proton at 1241 cmꢀ1 49
These bands are sensitive to changes in the
.
in the meta position of the oxime groups was observed as a secondary BSA structure due to hydrogen bonding and since COOꢀ
triplet at 7.40 with 3J = 7.7 Hz. This peak appeared as a singlet at and N–H groups are involved in hydrogen bonding, this sensitivity
6.95 ppm in the spectrum of complex (2). It confirms the is more for the amide I band.
removal of adjacent carbon protons due to Pd(II) coordination.
Compared to BSA, the characteristic bands shifted to lower
The 13C{H} NMR spectrum of ligand (1) (Fig. S3, ESI†) shows wavenumbers (1647, 1529, 1383, and 1232 cmꢀ1) in BSA-NPs
that the carbons of CQN groups resonate at 148.15 ppm. The spectrum. These slight shifts indicated no conformational
signals observed at 133.98, 129.56, 127.56, and 124.62 ppm are changes in the secondary BSA structure due to cross linking with
assigned to the phenyl carbon atoms (2Cd, Cc, 2Cf, and Ce, glutaraldehyde and nanoparticle preparation. This confirmed a
respectively). In the 13C{H} NMR spectrum of complex (2) chemical cross linking between tyrosine residues that could
(Fig. S4, ESI†), the signal at 155.30 ppm belongs to the result in the overall stability of BSA NPs.
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Fig. 2 Merged FT-IR spectra of pure BSA (solid line), BSA-NPs (dotted line), and (2)@BSA-NPs (dashed line) using KBr pellet.
The spectrum of (2)@BSA-NPs shows all the characteristic and 222 nm due to n–p* and p–p* transitions of the peptide
peaks of BSA-NPs. Also, a strong band appeared at 3300–3500 cmꢀ1 bond carbonyl group, which are protein a-helical structure
and an obvious change in the O–H stretching was observed in characteristic feature. Compared with BSA, the characteristic
comparison with the unloaded BSA-NPs due to oxime ligand OH CD signal did not change in the BSA-NPs spectrum. The results
group. These observations confirmed the stability of complex (2) in reveal that the a-helical predominant structure of BSA did not
the formulation and its loading onto the synthesized nanoparticles. change after nanoparticle preparation and confirm the structural
3.2.2. Circular dichroism measurement. Circular dichro- stability of the synthesized nanoparticles.50
ism spectroscopy is a powerful, fast, and sensitive photometric
3.2.3. Morphology and size studies. The morphological
technique for monitoring and evaluating protein structural investigation of the nanoparticles was conducted using FE-SEM
changes. We measured pure BSA and unloaded BSA-NPs CD and the obtained images are illustrated in Fig. 4a–c. As can be
spectra to examine the structural correctness and stability of seen, natural protein (Fig. 4a) has a disordered structure, while
this protein on nano structure conversion. As seen in Fig. 3, the for BSA-NPs (Fig. 4b), a spherical shape with a smooth surface
CD spectrum of BSA shows two negative intense peaks near 208 and an average diameter of 91.77 nm was observed. Besides,
complex (2)@BSA-NPs (Fig. 4c) were very orderly sphere-shaped
with a rough surface and mean size of 97.01 nm due to palladium
complex loading. It is obvious that the size of the nanoparticle
support (BSA-NPs) changes, while the general morphology
does not.
The obtained size of the spherical nanoparticles and palladium
complex loaded nanoparticles determined by dynamic light
scattering was 229 and 378 nm, respectively (Fig. 5). These
values were larger than the size of 91.77 and 97.01 nm, respectively,
measured by SEM due to particle constriction during dehydration.
These differences are consistent with the previous literature.8,15 The
low PDI values verify their narrow size distribution.
3.2.4. Zeta potential measurements. Zeta potentiometry
provide surface charge of the synthesized NPs, which can confirm
their stability against aggregation. Electrostatically stabilized
nanosuspensions have minimum zeta potential of ꢂ30 mV while
this value is not less than ꢂ20 mV for sterically stabilized
Fig. 3 Circular dichroism spectra of pure BSA (black) and desolvated BSA-
NPs (red) in acetonitrile at 5 ꢁ 10ꢀ6 M concentration.
nanosuspensions. Based on Fig. S5 (ESI†), the zeta potential for
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Fig. 5 Particle size distxribution of BSA-NPs (a) and (2)@BSA-NPs (b)
determined by dynamic light scattering.
Fig. 4 SEM images and the histogram of the particle size distribution in
Digimizer for pure BSA (a), BSA-NPs (b and b0), and (2)@BSA-NPs (c and c0).
structure have potential to participate in hydrogen bonds, it can be
concluded that this compound binds to nanoparticles through
non-covalent encapsulation.
BSA-NPs was ꢀ21.5 mV, suggesting strong repulsive forces
In order to determine the loading capacity of nanoparticles
and entrapment efficiency of the synthesized palladium
complex, the amount of free Pd(II) complex (2) present in the
clear supernatant after differential centrifugation was deter-
mined by an ultraviolet spectrophotometer. The amount of
the loaded Pd(II) complex on the BSA-NPs was calculated by
subtracting the concentration of the free Pd(II) complex in the
supernatant from its initial concentration ([Pd(II) complex]initial) and
finally, the entrapment efficiency was obtained using eqn (1):
between them and electrostatic stabilization.
3.3. Drug release studies
3.3.1. Calibration curve. For the calibration curve, accurate
volumes of complex (2) (50–400 mL) were added to the beakers
containing 2 mL buffer. Thus, solutions in the concentration
range of 50–250 ppm were obtained and their absorption was
recorded by a UV-Vis spectrophotometer. This way, five point
calibration curves were plotted in the abovementioned concen-
tration range. As can be seen in Fig. S6 (ESI†), there is a linear
relationship between the absorbance and the corresponding
complex (2) concentration, and the absorption coefficient
obtained with the determination coefficient (R2) exceeds 0.99.
3.3.2. Calculation of the loading capacity and entrapment
efficiency. BSA has internal hydrophobic cavity surfaces with
natural affinities for small molecules such as metal complexes
or drugs. Due to the existence of charged groups, BSA nano-
particles can bind to metal complexes non-covalently through
electrostatic interactions for their effective delivery to various
affected areas of the body.51 Also, as these nanoparticles have a
monolithic structure, they may be used as a matrix in which drugs
Entrapment efficiency ð%Þ
(1)
½PdðIIÞ complexꢃ initial ꢀ ½PdðIIÞ complexꢃ free
¼
ꢁ 100
½PdðIIÞ complexꢃ initial
where [Pd(II) complex]initial was varied between 56 and 280 mg mLꢀ1
(50–400 mL). The loading capacity of the nanoparticles was calcu-
lated using eqn (2):
Loading capacity ð%Þ
(2)
½PdðIIÞ complexꢃ initial ꢀ ½PdðIIÞ complexꢃ free
¼
ꢁ 100
½BSA-NPsꢃ total
can be physically entrapped.52 The possible interactions in drug where [BSA-NPs]total is the total amount of nanoparticles recovered.
loading are hydrogen bonding and p–p stacking interactions.53 As shown in Fig. S7 (ESI†), there is a dependence on the
Since the oxime functional groups in the palladium complex (2) entrapment efficiency and [Pd(II) complex]initial. The percentage
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entrapment efficiency was increased along with the increase in This is because of the constant rate release of complex (2) into the
the amount of initial Pd(II) complex, which ranged between release medium and can be related to the adsorbed Pd(II) complexes
96.63–97.78% for 56–220 mg mLꢀ1 and decreased with the onto the nanoparticle surface, which is controlled by a diffusion
increase in the amount of initial Pd(II) complex to 280 mg mLꢀ1
.
process due to the electrostatic interaction. In the second phase, the
On the other hand, at 220 mg mLꢀ1 concentration of the Pd(II) release rate decreases within 45 h. Essentially, there are two
complex, the maximum amount of the metal complex was loaded possibilities for this observation. The first is increasing the diffusion
onto the surface of the nanoparticle successfully. According to pathways’ length and the second is the entrapment of complex (2)
eqn (2), the loading capacity of the Pd(II) complex was found to be into the BSA-NPs. Thus, the synthesized nanoparticles are capable of
22.68% on BSA-NPs when [Pd(II) complex]initial = 220 mg mLꢀ1
Actually, this parameter helps us to know the nanoparticle drug
.
sustained release of complex (2) without burst release.
Another noticeable point is that the free complex (2) release
content after separation from the medium. It is clear that the amount is higher than that of the loaded one on the nanoparticles’
nanoparticles with concentration lower than 280 mg mLꢀ1 surface, confirming their loading and decrease in the release
have better drug loading content. So, the optimized suspension strength. In other words, the entrapped drug in the inner core of
[Pd(II) complex]initial = 220 mg mLꢀ1 was washed by centrifugation the BSA-NPs diffuses slowly from the polymer matrix to the release
as described above and after freezing, the product was dried in a medium. These observations are consistent with the various studies
freeze dryer with a cycle of 24 h at the shelf temperature of ꢀ40 1C in which BSA-NPs has been used in drug delivery systems.54
and then incubated at 4 1C in the dark for the investigation of its
3.3.4. Release mechanism study. In this section, model
morphology by SEM.
dependent methods were used to get further insight and to
3.3.3. In vitro drug release study. The cumulative release of select a kinetic model for the release mechanism characterization,
the synthesized Pd(II) complex is calculated using eqn (3):
which can be useful in the control of the release process. The
applied method is based on fitting the release data with different
mathematical functions that describe the dissolution profile and
compare the obtained parameters.55–57 The equations of these
functions are given below.58,59
Volumeof samplewithdrawnðmlÞ
Cumulativedrugreleaseð%Þ ¼
BathvolumeðmlÞ
ꢁ Pðt ꢀ 1Þ þ Pt
(3)
Zero-order: f = k0ꢄt
First-order: f = 1 ꢀ exp(ꢀkt)
Higuchi: f = kHꢄt0.5
where Pt is the release percentage at time t and P(t ꢀ 1) is the
release percentage before ‘t’. Fig. 6 shows the cumulative
release profile over a period of 45 h in the pH 7.4 phosphate
buffer solution (0.13 M) at 37 1C. The release profile of complex
(2) from the BSA nanoparticles showed a single phase pattern
including an initial burst release in the first 10 h, followed by a
sustained release to the end of the experimental period. The
initial burst release could be due to the dissolution of the
adsorbed complex at or beneath the surface of the nano-
particles. After this, the cumulative release value is fixed.
For complex (2) loaded BSA-NPs, a biphasic release pattern is
Korsmeyer–Peppas: f = kKPꢄtn
Hixson–Crowell: f = [1 ꢀ (1 ꢀ kHCꢄt)3]
Actually, a statistical calculation was performed and the
observed. In the first phase, 36.70% of release occurs within 20 h. statistical indicators of the data fitting were compared for
Fig. 6 In vitro drug release profile of free palladium complex (blue) and palladium complex loaded on BSA-NPs (red) over 48 h in phosphate buffer (pH =
7.4, 37 1C) analyzed by the dialysis method.
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Fig. 7 Experimental (dashed line) and models predicted (colored lines) the cumulative release profile for the phase I release process.
Fig. 8 Experimental (dashed line) and models predicted (colored lines) the cumulative release profile for the phase II release process.
Table 1 Data of %entrapment efficiency and loading content of (2)@BSA-NPs
selecting the best model. Table S1 (ESI†) shows the experi-
mental and predicted release values for complex (2)@BSA-NPs
that were used for drawing the cumulative drug release (%) vs.
time (h) plots shown in Fig. 7 and 8. The results of data fitting
with the mentioned equations are presented in Tables S2 and
S3 (ESI†).
[Pd(II) complex]initial
EE (%)
LC (%)
56
96.63
96.66
97.17
97.78
97.65
4.83
8.21
15.54
22.68
28.71
110
170
220
280
Several parameters have been investigated and discussed to
prove the best model for drug release. The first and foremost
parameter is the correlation coefficient (R2) and according the best.64 The values of AIC for the release steps are given in
to statistical calculation rules, the best model has the nearest Table 1; the lowest AIC value was obtained by fitting the data
R2 to 1.60–62 Without a doubt, the important point to note is the with the Korsmeyer–Peppas model for phase I and Higuchi
number of parameters obtained from the models compared so model for phase II.
that models with different numbers of parameters should be
The important issue after determining a better kinetic
compared with the adjusted R2.63 As can be seen in Table 1, the model is to identify the mechanism of drug release from the
obtained R2-adj values are in the range of ꢀ0.1728 to 0.9647 and surface of the nanoparticles’ support. Generally, there are two
ꢀ0.0403 to 0.8659 for phase I and II release steps, respectively. So, release mechanism categories, including Fickian diffusion65
the largest and closest values to 1 belong to the Korsmeyer–Peppas and non-Fickian diffusion or anomalous transport.66 The determi-
and Higuchi models for phases I and II, respectively.
nation of mechanism type is possible by using the parameters
The next parameter is the Akaike information criterion obtained from the Korsmeyer–Peppas function. In this function, n is
(AIC), which is based on the concept of entropy and shows the diffusional coefficient, which is the characteristic of the release
the extent of information loss caused by the use of a statistical mechanism and depends on the shape of the support. For the
model. The lesser the information lost in a model, the higher spheres, values of n less than 0.43 indicate that the release
the quality of the model. Thus, the model with the lowest AIC is process is controlled by Fickian diffusion, while values between
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Fig. 9 Growth inhibitory effects of complex (2), bovine serum albumin nanoparticles (BSA-NPs), and complex (2) loaded NPs (complex (2)@BSA-NPs) on
A549 and K562 tumor cell lines measured by the MTT colorimetric assay. DMSO at concentrations equal to that in the test wells was used as the negative
control. Percentage of cell growth inhibition was determined according to the formula described in Section 2.4. Data represent mean ꢂ standard
deviation (SD) of two independent experiments performed at least in triplicate. The values of SD that were less than 2% are not shown.
0.43–1 are an indication of the non-Fickian diffusion mechanism.67 and purified. The morphology, particle size, and stability
In this study, the n value obtained using the Korsmeyer–Peppas analysis studied by SEM, DLS, and CD spectroscopy indicated
equation for release phase I equals 0.223, which suggests that the that the BSA-NPs were quite stable, spherical in shape, and
release of complex (2) from the nanoparticles is controlled by approximately monodisperse. The synthesized nanoparticles
Fickian diffusion. Besides, the release phase II data have a good were used as the carrier for the palladium complex. The results
fitting to the Higuchi model, which describes the drug release as showed that the size of the nanoparticles did not change
a diffusion process based on the Fickian diffusion equation. significantly after loading and did not exceed the acceptable
According to these results, the release process of complex (2) range for drug delivery applications. Also, the MTT colorimetric
from BSA-NPs is achieved by the diffusion mechanism.
assay was used to evaluate the effects of the prepared nano-
structure support on the growth of two tumor cell lines. Finally,
mathematical equations were used to investigate the mechanism
3.4. In vitro cytotoxicity evaluation
We used the MTT colorimetric assay to evaluate the effects of of palladium complex release from the nanoparticles’ surface.
various concentrations of the free complex (2), BSA-NPs, and The results of these studies showed a two-phase mechanism for
complex (2)@BSA-NPs on the growth of two tumor cell lines this release that followed kinetic models Korsmeyer–Peppas and
including A459 and K562 cells after 48 h. As shown in Fig. 9, Higuchi separately. The data obtained from these equations
complex (2) had strong cytotoxicity against both the cell lines, showed that the preferred mechanism in this system was non-
especially the K562 cells, with IC50 values of 26.4 ꢂ 1.6 mg mLꢀ1 diffusion release mechanism. Given the good results of this
(K562) and 128.7 ꢂ 4.8 mg mLꢀ1 (A549). These effects were dose study, we will use other palladium compounds in future works
dependent and increased from o1% at 1 mg/m + l to 61% for to study their release from the surface of protein nanoparticles.
A549 and 81.3% for K562 at 200 mg mLꢀ1. The BSA-NPs had no
significant cytotoxic effect at the concentrations used. The
Conflicts of interest
effect of complex (2)@BSA-NPs on the cell growth was also
evaluated. As seen in Fig. 9, although this complex inhibited
There are no conflicts to declare.
19.9% (K562) and 11.8% (A549) of the cell growth at the
maximum concentration (200 mg mLꢀ1), in contrast to free
complex (2), it showed a weak inhibitory effect on the cells. This
finding may suggest that for observing the cytotoxic effect of
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for complex (2)@BSA-NPs, more time is needed to overcome the
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