In recent
years, different types of inorganic nanoparticles (iNPs) with unique
physicochemical properties have emerged. Among these, quantum dots (QDs) have
proved to be very versatile, finding applications in electroluminescent
displays, quantum computing, photovoltaics, solar cells, transistors and
biological imaging. For biological imaging applications, QDs are now excellent
alternatives to organic chromophores, given that they can have similar sizes,
shapes and surface functional groups. A potentially prolific new direction in
inorganic chemistry and nano chemistry could be to combine NPs with small metal
complexes to seek synergistic and/or cooperative effects. In this context,
combining QDs with coordination complexes is being explored as a new strategy
to obtain cooperative systems with improved properties for applications in
sensing, biological imaging and molecular therapy. A prominent area of research
in coordination chemistry is the development of metal complexes that can act as
artificial nucleases. Overall, these synthetic DNA-cleaving reagents operate
using one of two distinct mechanisms.
Keywords: Quantum dots, Nano medicine, Coordination compounds,
Transition metal complexes
INTRODUCTION
Quantum dots are tiny semiconductor particles
a few nanometres in size having optical and electronic particles that differ
from large particles due to quantum mechanics. Their broad excitation bands are
mainly helpful in their applications. Quantum dots are excellent alternatives
to organic chromophores,
given that they have similar sizes, shapes and surface functional groups.
DISCUSSION
In
recent years, different types of inorganic nanoparticles (iNPs) with unique
physicochemical properties have emerged. Among these, quantum dots (QDs) have
proved to be very versatile, finding applications in electroluminescent
displays, quantum computing, photovoltaics, solar cells, transistors and
biological imaging.
Useful physicochemical properties
of QDs
Their broad excitation bands with very high
extinction coefficients and narrow emission bands that can be tuned across a
region of the visible or near-infrared spectrum by varying the size and
composition of the QD with high photo stability. For biological imaging
applications, QDs are now excellent alternatives to organic chromophores. Given
that they can have similar sizes, shapes and surface functional groups. In this
context, some studies have shown that nanosized QDs can be considered generic
curved surfaces that DNA can wrap around. This is important because bending may
open and close certain sites along the double helix, making certain regions of
the DNA more or less accessible. Potentially, this could have widespread
implications and applications because it could lead to artificial regulation of
a wide array of cellular processes for therapeutic and biotechnological
applications, much like protein-DNA interactions do naturally.
In addition to enabling different applications, the effects of DNA-QD interactions need to be considered also from a toxicological point of view. However, there are contradictory reports concerning the ability of QDs to damage DNA in the absence and presence of light as well as their toxicity to cells. A potentially prolific new direction in inorganic chemistry and nanochemistry could be to combine NPs with small metal complexes to seek synergistic and/or cooperative effects. In this context, combining QDs with coordination complexes is being explored as a new strategy to obtain cooperative systems with improved properties for applications in sensing, biological imaging and molecular therapy.
A prominent area of research in coordination
chemistry is the development of metal complexes that can act as artificial
nucleases. Overall, these synthetic DNA-cleaving reagents operate using one of
two distinct mechanisms:
(i) Oxidative scission of deoxyribose
residues through redox chemistry and
(ii) Hydrolysis of the phosphodiester sugar
backbone.
The most classical example of oxidative
DNA-cleavage activity is exemplified by the Cu(II)-1,10-phenanthroline
(1,10-phenantroline = phen) system, which has been utilized as a foot printing
reagent for the evaluation of protein-DNA interactions as well as a probe for
DNA and RNA secondary structure. In this intensively investigated system,
[Cu(phen)2]+ generated in the presence of a reducing
agent and molecular oxygen afford activated oxygen species for DNA cleavage,
whereas the intercalation of phen into the DNA minor groove allows for DNA
targeting.
Recently,
we discovered that QDs cooperate and synergize with the
Cu(II)-1,10-phenanthroline system for DNA cleavage, providing both the first
example of cooperative DNA cleavage between NPs and a small-molecule-based
synthetic metallonuclease and a potentially new approach to develop more
efficient DNA-cleaving systems. Many ligand systems and approaches have been tested
with varying degrees of success to increase both the DNA scission capability
and the affinity of copper metallonucleases for DNA. A popular strategy is to
use bimetallic agents because of the potential cooperative effects that can
arise between the two metal centres. However, an emerging way to design more
powerful synthetic catalysts for a wide range of transformations, including DNA
cleavage, utilizes ligands with hydrogen bonding features resembling those
found in the active sites of metalloenzymes. We have combined the advantages of
dinuclear copper catalysts with those of hydrogen-bonding ligands, and we
exploit QDs as a redox-active protein-like nanostructure to activate strongly
the copper catalysts for DNA cleavage. Two novel (μ-guanazole)-bridged
binuclear copper(II) complexes with 1,10-phenanthroline (phen) or 2,2′-bipyridine (bipy), [Cu2(μ-N2,N4-Hdatrz)(phen)2(H2O)(NO3)4]
(1) and [Cu2(μ-N1,N2-datrz)2(μ-OH2)(bipy)2]-(ClO4)2
(2) (Hdatrz = 3,5-diamino-1,2,4-triazole = guanazole), have been prepared and
characterized by X-ray diffraction, spectroscopy and susceptibility
measurements.
Two
mono (phen)-CuII fragments have been attached in the same compound by means of
a single μ-triazole bridge using the ligand guanazole (guanazole =
3,5-diamino-1,2,4-triazole = Hdatrz), thus yielding two copper centres with
labile coordination sites of facile substitution and a structure suitable for
DNA intercalation.
The
analogous bipy (2, 2′-bipyridine = bipy) ternary compound has been prepared,
resulting in a dinuclear compound that contains a bis (guanazolate) bridge.
In addition to providing a bridge between the
two copper centers, the X-ray crystal structures reveal that the
guanazole/guanazolato provides N−H groups for hydrogen-bonding interactions
with the DNA. To effect DNA cleavage efficiently, these new copper complexes
are combined with water-soluble micelles filled with CdSe-ZnS core-shell QDs.
From a biosafety point of view, phages should
not possess genes associated with bacterial toxins, pathogenicity, antibiotic
resistance or other types of virulence factors. Additionally, phages should
also display a low potential to horizontal gene transfer (transduction).
Therefore, the complete genome needs to be sequenced to determine whether
bacteriophages are suitable to control pathogenic bacteria.
Synthesis of [Cu2(μ-Hdatrz)(phen)2(H2O)2(NO3)4]
An
aqueous solution of Cu (NO3)2·2.5H2O (1.148 g,
5 mmol, 20 mL) was mixed with an aqueous solution of guanazole (0.248 g, 2.5
mmol, 5 mL). A green solution was formed to which an aqueous suspension of
phenantroline·H2O (0.993 g, 5 mmol, 10 mL) was added dropwise. A
dark turbidity was almost immediately observed.
After 2 h of stirring, a black-green
precipitate was filtered off, and the resulting dark green solution was allowed
to stand at room temperature covered with Parafilm. Within 1 month, a few large
black-green crystals, not suitable for X-ray, appeared; they were separated by
filtration.
Synthesis of [Cu2(μ-datrz)2(μ-OH2)(bipy)2](ClO4)2
An
aqueous suspension of bipy (0.156 g, 1 mmol, 20 mL) was slowly added (drop by
drop) to an equimolar aqueous solution of Cu (ClO4)2·6H2O
(0.37 g, 1 mmol, 20 mL). To this mixture, an aqueous solution of guanazole
(0.02 g, 0.25 mmol, 5 mL) was slowly added. The reaction mixture was stirred
for 2 h. A light blue precipitate was formed and filtered off. The remaining
dark green solution was allowed to stand at room temperature. After 1 day, dark
green single crystals of 2 appeared.
Synthesis of micelles filled with QDs and
SPIONs
Micelles
core-shell CdSe-ZnS QD were synthesized, characterized and purified as
described previously. The CdSe-ZnS QD have an average diameter of 5.2 nm (4.0
nm CdSe core diameter and 0.6 nm ZnS shell thickness). The MQDs were prepared
by self-assembly process of PEGylated phospholipids around hydrophobic CdSe-ZnS
core-shell QDs. The water-soluble micelles with encapsulated superparamagnetic
iron oxide nanoparticles of 6 nm as core material were prepared in the same
way.
DNA-Copper complex interaction studies
For QD-CT-DNA
interaction studies, a working solution containing 600 nM QD in cacodylate
buffer (0.1 M, pH 6.0) was prepared. The experiment entailed the addition of
serial aliquots of a CT-DNA stock solution. After each addition, the samples
were excited at 400 nm and emission was recorded between 580 and 700 nm.
Samples
were treated as described above in the presence of MPA. To test for the
presence of reactive oxygen species (ROS) generated during strand scission in
the presence of QD, various reactive oxygen intermediate scavengers were added
to the reaction mixtures.
In
addition, a chelating agent of copper (I), neocuproine (100 μM) was also
assayed. Samples were treated as described above in the presence of MQD. All of
the results are the average of experiments performed at least in triplicate.
DNA-CLEAVAGE
EXPERIMENTS
DNA-binding and DNA-cleavage
properties
The study of the DNA-binding properties was
carried out for 1 and 2 through a series of techniques: Thermal denaturation,
viscosimetry and fluorescence-based assays. Both complexes were prepared and
isolated as solid products and then dissolved in water for the biological
experiments. The existence of the dinuclear unit of 1 and 2 in solution was
tested by mass spectrometry.
DNA binding properties
The shift
in melting temperatures (ΔTm) resulting from the association of 1
and 2 with CT-DNA. The ΔTm produced by 1 is high and notably larger
than by 2 (20 vs. 5°C), which implies that the stabilization of the DNA double
strand produced by 1 is more pronounced.
DNA cleavage activity
Mechanism of DNA cleavage and role of the
QDs: To clarify
the mechanism (i.e., oxidative vs hydrolytic) of the nuclease activity of
complexes 1 and 2, electrophoresis assays with ROS scavengers. These findings
signal the minor groove of the DNA as the nuclease binding site, but in
compound 1, this specificity is lower than, for instance, in [Cu(phen)2]2+,
presumably because 1 can interact through electrostatic interactions and
hydrogen bonding when the minor groove is inaccessible (vide supra). In the
presence of MQDs, a clear inhibitory effect was found for the superoxide
scavenger Tiron, which indicates that the DNA damage produced under these
conditions occurs by an oxidative mechanism. To identify further the chemical
processes involved in the QD-mediated DNA-cleavage process, we carried out
X-ray photoelectron spectroscopy (XPS) studies.
FUTURE
SCOPE AND AIMS
DNA
binding and cleavage studies reveal that Transition Metal Complexes/compounds
can be used as efficient nucleases because of the cooperative effect of the two
Cu(II) centers and the guanazole ligand, which in addition to providing a
bridge between the two metals can participate in hydrogen-bonding interactions.
Of the two complexes, 1 shows the highest affinity for DNA and binds via
intercalation of the phen ligands. In the presence of oxygen and micromolar
concentrations of MPA or H2O2 as activators, only 1 is
capable of causing DNA cleavage. However, in the presence of nanomolar
concentrations of water-soluble QD-filled micelles, both systems are highly
efficient at cleaving DNA.
Photoinduced electron transfer from PbS
quantum dots to Cobalt (III) Schiff Base Complexes
Recent
advances in the development of therapeutic antitumor and antiviral agents have
focused on compounds that bind to the biological active site of an enzyme.
Although these reversibly bound drugs are susceptible to
nonspecific and potentially undesirable reactions, the success of transition
metal therapeutics, such as cisplatin, has refocused efforts aimed at
investigating new complexes in this broad class. This research has facilitated
an improved understanding of the interactions between complex biological
systems and inorganic coordination complexes. Strategies for designing
prodrugs, drugs that are administered as inactive compounds but are triggered
by some controllable stimulus, have exploited differences in biological
environments, such as pH, redox status and protein expression in achieving a
higher level of specificity and efficacy. Photoreduction of Platinum (IV) is
started by colloidal quantum dots. The electron transfer (PET) following
photoexcitation at 615 nm. The wavelength of light that initiates PET and
subsequent Pt (IV) reduction is within a favorable range (600-1300 nm) for maximal tissue depth
penetration for in vivo applications. QDs possess highly tunable
electrochemical and spectroscopic properties with excitonic transitions in the
low energy visible and near-infrared (NIR) regions.
QDs
have high two-photon cross-sectional efficiencies that surpass those of
traditional organic dyes. Properties such as water solubility, cellular uptake
and selective accumulation in malignant tumors have been tuned to achieve
superior biocompatibility.
These
attributes make QDs favourable candidates as photosensitizers for accessing
multiple redox states of metal-based therapeutics in prodrug designs. Cobalt (III)
Schiff base [Co(III)-SB] complexes of the equatorial tetradentate ligand
bis(acetylacetone)-ethylenediimine (acacen) are known to be potent inhibitors
of a wide array of zinc-dependent proteins, including thermolysin, α-thrombin. Modification
of the acacen backbone to incorporate biomolecular targeting moieties (such as
oligonucleotides) has been shown to selectively target zinc finger
transcription factors. Evidence suggests the inhibition activity is due to
disruption of the protein structure by coordination of Co (III) to active-site
histidine residues. This coordination event occurs via a dissociative ligand
exchange and is strongly dependent on the nature of the axial ligands present
on the Co (III)-SB complex.
Selective enzyme inhibition is observed when
the axial positions are occupied by either sterically hindered
2-methylimidazole or labile amine ligands. In general, the coordination
behavior of cobalt Schiff base complexes is dependent on the oxidation state of
the metal ion. Because of the redox properties of cobalt, axial ligand
coordination of Co (II)-SB complexes has an increased propensity for
dissociation.
Synthesis
Several
batches of NIR light absorbing PbS were synthesized. We selected PbS QDs. The
Co (III)-SB complexes were synthesized and characterized according to
literature. To prepare the PbS QD/Co (III)-SB complex samples, we transferred a
methanolic solution of the Co(III)-SB complex into a scintillation vial and
dried it under nitrogen before adding 1.4 × 10−5 M PbS QDs in CHCl3. The vial was
shaken until the Co (III)-SB complex was dissolved and the solution was allowed
to equilibrate for 24 h before measurements
were taken. In summary, It has been shown that that selective photoexcitation
of PbS QDs within mixtures of the QDs and Co (acacen)(Im)2 increases the axial
ligand reactivity of the Co (III)-SB complex. It is proposed that the mechanism
for this observation is electron transfer from the PbS QDs to Co (III), given
that
(i)
The
dissociation of axial ligands is a documented and well-understood consequence
of reduction of Co (III) to Co (II) in this Co (III)-SB complex.
(ii)
Electron
transfer from the bottom of the conduction band of PbS QDs of this size to Co (III)
is energetically favorable by ∼100
meV while energy transfer is not thermodynamically possible.
(iii)
Addition of
the Co (III)-SB complex to the QDs quenches their PL while exposure of the QDs
to the acacen molecule without the redox-active Co(III) center does not quench
their PL, and
(iv)
Increasing
the reduction potential of the Co (III) center within the Co (III)-SB complex
by changing the axial ligand makes the Co (III)-SB complex a less efficient
quencher of the PL of the QDs.
We can improve the electronic coupling
between the QD and the Co (III)-SB complex by using a QD coating that minimizes
steric repulsion at the surface of the QD or by functionalizing the Co (III)-SB
complex such that it can more closely approach the QD. Results of such studies
offer a unique route for light activation of a Co (III)-SB protein inhibitor
via NIR excitation and suggest that the
development of inorganic therapeutic agents may be specifically coupled to a
biologically active site by cooperative redox binding ligation. A new binding
strategy of linking quantum dots (QDs) to magnetic nanoparticles (MNPs) using
DNA interaction with metal coordination bonding was developed. Platinum was
selected for binding QDs to DNA. This nanoconjugate acts as a new probe for
diagnosis with its double modalities, fluorescence and magnetic property. Owing
to the high quantum yield and excellent photostability, QDs have been
intensively investigated as a new probe for bioimaging and biosensor
applications. The high potential medical utility of QDs in the areas of imaging
and diagnosis has led to extensive exploration of methods to functionalize
these nanoparticles. This effort has generated improved methods for surface
modification and bioconjugation of QDs with various bioactive molecules,
including small molecules, DNA and antibodies and the employment
of the functionalized QDs for visualizing biological events in vitro and
in vivo. In addition, because of the excellent optical properties of
QDs, they have been utilized in approaches to create novel biologically
interesting nanoconjugate probes. Various probes of this type, exemplified by
gold nanoparticle (AuNP)-QD conjugates, have been devised to detect specific
DNA, RNA and peptides, as a part of new techniques for cancer diagnosis. Nanomaterials
comprised of conjugates between MNPs and QDs, which have both fluorescent and
magnetic properties, have been investigated in the context of tools for the
diagnosis of multiple cancers. A new strategy has been explored for binding QDs
and MNPs taking advantage of metal coordination of DNA molecules as the key bonding
component. This choice was inspired by platinum-based cisplatin, one of the
most widely used anticancer drugs for the treatment of ovarian, testicular and
head and neck cancers. The studies aimed at understanding the mode of action of
this drug have shown that a cisplatin 1,2-intrastand d(GpG) cross link occurs
on DNA to cause apoptosis of cancer cells. The central platinum (Pt)(II) of
cisplatin, which possesses two ammonia and two chloride ligands coordinated
with square planar geometry, forms stable coordination bonds with guanine N7
sites of DNA. Studies have shown that the nanoconjugates are more stable in
vivo than are simple Pt complexes. Only a few approaches using strong
coordination bonding character of the Pt (II) complex to generate nanoconjugates
or nano assemblies have been reported.
It is
known that the nature and specificities of reactions of metal-ligand complexes
depend greatly on the properties of both the metal and chelating ligands. As a
result, the strength of the coordination bond and the related ease of ligand
substitution can be easily tuned using specific ligands. Studies would be aimed
at developing a new approach for conjugating QDs and MNPs. Studies have used
cis-dichlorobis-(dimethylsulfoxide)-platinum(II) (cis-PtII(DMSO)2Cl2), which
contains a weakly bonded DMSO ligand that serves as a leaving group in a
substitution reaction with a diamine, forming a (diamine)-QD–PtCl2 complex. The
key proposal is that the N7 nitrogen of guanine bases in DNA linked MNPs would
then substitute for the chloride ligands in the resulting complex to form a
QD–Pt (II)–MNP–DNA nanoconjugate. Cd Se quantum dots are prepared by
phosphorus-free methods using oleic acid as stabilizing surface ligand. Ligand
exchange is monitored quantitatively by 1H NMR spectroscopy gives about 30
monodentate ligands per nanocrystal, with a ligand density of 1.8−2.3 nm−2. The arrangement of nanosized
objects into more complex structures remains a challenging target. One of the
preconditions for such higher-level organization is the introduction of a
limited number of attachment points. This offers the opportunity to link up
spherical nanoparticles using covalent bonds or specific donor−acceptor interactions to generate,
for example, divalent or trivalent nanoparticles that can be the building
blocks for linear or branched arrangements. We are especially interested in the
functionalization of colloidal semiconductor quantum dots (QDs), in particular
CdSe and were looking for ways of restricting the number of surface ligands by
macrocyclic attachments, using coordination chemistry principles for the
construction of stable nanocrystal-organic conjugates. The successful
functionalization of nanoparticles requires an understanding of their surface
chemistry. Semiconductor nanocrystals are stabilized by surfactant-type surface
ligands which determine the growth rate, stability, and solubility of the
nanocrystals.
While CdSe prepared using trioctylphosphine
(TOP) and trioctylphosphine oxide (TOPO) are frequently represented with a
surface coverage of TOPO, it is now known that the surface is mainly covered by
TOP oxidation products, alkyl phosphinates and phosphonates, which coordinate
to metal surface sites as bridging ligands. Nanocrystals prepared under
phosphorus-free conditions using long-chain carboxylic acids such as stearic or
oleic acid are stabilized by a layer of metal carboxylate. The nature of ligand
binding has been explored mainly by NMR Spectroscopy, as well as luminescence
and isotopic labelling methods. Macrocyclic compounds with a rigid aromatic
framework, such as phthalocyanines, subphthalocyanines and porphyrins are
synthetically readily accessible and can easily be derivatized to give
compounds with a predetermined number of anchorpoints (usually 0-4). Hence, we propose to
synthesize such macrocyclic molecules. Such ligands are expected to bind to
nanocrystal surface sites by substitution of the surfactant ligands present
from the synthesis stage. For a given concentration, due to the chelate effect
polydentate ligands should bind to the surface of a nanocrystal significantly
more strongly than monodentate ligands. We would focus on ligands of type subphthalocyanines possess a cup-shaped structure, which
suggested a good geometric match with nanocrystals with diameters in the 2-3 nm range; in addition they
contain a functional group perpendicular to the macrocyclic core which offers
the possibility of connecting to other ligand-decorated nanoparticles by
covalent bonds. We will focus on porphyrins of this type which would be
expected to act as monodentate ligands oriented perpendicular to the
nanocrystal surface.
Although
the central ring in porphyrins of this type is flattened, there is sufficient
flexibility both in the ring and in the functionalized substituents X to allow
these compounds to attach themselves to nanocrystals flat-on, i.e., parallel to
the crystal surface. Alternatively, these ligands may adopt a perpendicular
orientation bridging between two adjacent quantum dots. The structures and
properties of the CdSe-macrocyclic constructs have been evaluated
using a combination of 1HNMR, absorption and fluorescence spectroscopies.
Relative ligand binding strength
Oleic
acid (octadec-9-enoic acid) possesses a cis-vinylene moiety which acts as a convenient
1H NMR marker. Preliminary qualitative experiments would be performed to
establish the relative binding preference of carboxylic acids, phosphonic acids
and thiols.
Number of ligands per nanoparticle
The
number of oleate ligands per nanoparticle and hence the ligand density would be
evaluated using a combination of UV-vis and 1H NMR spectroscopies.
Reactions of subphthalocyanines with CdSe QDs
In a
series of initial reactions, the binding behavior of subphthalocyanine would be
explored. This ligand is decorated with three meta-pyridyl substituents which
give a bite angle that should be geometrically well-matched for binding to
nanocrystals of 2-3 nm diameter, provided there are accessible
Lewis acidic surface sites. Treatment of a solution of CdSe QDs in chloroform
gave a deeply colored solution. Quenching of the QDs fluorescence was observed.
The nanoparticles were precipitated with methanol, isolated by centrifugation
and repeatedly washed with acetone until the washings were free of
subphthalocyanine by UV/vis spectroscopy.
Reaction of metal-free porphyrin derivatives
with CdSe nanoparticles
The interaction of pyridine-substituted free-base
porphyrins as well as their metal complexes with CdSe and Cd/ZnS core-shell particles made by the TOPO
route would be intensively studied, since we are interested in the interactions
of carboxylate-type CdSe nanocrystals with carboxylate-substitutedporphyrins. Preliminary
studies were carried out with 5,10,15,20-tetra(4-decyloxyphenyl) porphyrin
which carries only alkyl substituents but no surface-binding functional groups.
It quickly becomes evident that metal-free porphyrins become metalated in the
presence of CdSe nanoparticles over time at room temperature. The insertion of
Cd2+ into the ring system is evidenced by a visible color change;
this was confirmed by the UV-vis spectra. The conversion of the metal-free
into 5,10,15,20-tetra(4-decyloxyphenyl)-porphyrinato cadmium as a typical
example.
Monodentate porphyrin ligands and CdSe
nanoparticles
The
binding of monodentate porphyrin ligands would be first examined
Tetradentate porphyrin ligands and CdSe
nanoparticles
While
monodentate porphyrin ligands were readily able to substitute oleate ligands,
their steric requirements are moderate since they bind to the nanoparticle
surface in “upright” position. Polydentate ligands, on the other hand, have the
potential of covering large sections of the nanocrystal surface by lying flat and
since we are interested in the question whether or not a nanoparticle could be
essentially encapsulated by porphyrins and what binding mode would be adopted.
The tetrasubstituted porphyrins are therefore synthesized.
Why
Porphorins were chosen
Porphyrins were chosen due to the flexibility
of the molecule. It is well-known that, in the case of tetraphenylporphyrins,
the phenyl groups in the meso positions lie perpendicular to the tetrapyrrolic
ring. Such a ligand is therefore able to bind parallel to the nanoparticle
surface. Of course, the phenyl substituents are free to rotate, and suitable
geometries will also be present.
PREPARATION
OF CDSE NANOCRYSTALS
CdSe
nanocrystals were produced by a modification of a literature procedure. To
cadmium oxide (300 mg, 2.34 mmol) in octadecene (20 mL) was added oleic acid (2
mL). The mixture was stirred under vacuum for 10 min, and then N2
was introduced. The mixture was heated to 250°C and stirred at this temperature
until a clear solution was obtained, which was then left to cool to ca. 120°C.
Selenium powder (100 mg, 1.27 mmol) was added and the mixture heated to 240°C,
causing the color to change from yellow to orange. Heating was stopped when the
color of the solution was deemed the right shade of orange for the desired
nanocrystal size (after various experimental trials). The solution was
immediately cooled on an ice bath, and toluene (10 mL) was added. The solution
was then transferred to two large centrifuge tubes with filtration (syringe
filter: 0.22 μL). The volume of both tubes was adjusted to 30 mL with toluene.
The addition of acetone (20 mL) caused the formation of a white precipitate of
unreacted starting material. The tubes were centrifuged (1400 rpm), the orange
solutions were collected, and the white precipitate was discarded. The solution
(25 mL) was again placed in a large centrifuge tube. Methanol (25 mL) was
added, followed by centrifugation. An orange oil separated at the bottom, which
was collected; the clear top solvent layer was discarded. This process was
repeated until all the solution was processed in this way. The separated orange
oils were combined and transferred to a smaller centrifuge tube with toluene
(total volume 7 mL). Methanol (7 mL) was added and the tube centrifuged. Again,
the orange oil was decanted. The volume was made up to 7 mL in dichloromethane,
and the same volume of ethanol was added, followed by centrifugation. This
process was repeated until a thick orange oil or powder was obtained. Finally,
acetone (14 mL) was added, and the tube was sonicated for several minutes and
then centrifuged. This was repeated five times. A free-flowing orange powder was
finally obtained after drying under vacuum and stored under N2. Encapsulation
of zinc-rifampicin complex into transferrin conjugated silver quantum dots improves
its antimycobacterial activity and stability and facilitates drug delivery into
macrophages.
In
order to improve the chemotherapy of tuberculosis, there is an urgent requirement
and hence synthesis of a novel anti TB drug complex consisting of zinc and
rifampicin (ZnRIF) and encapsulated it into transferrin conjugated silver
quantum dots (ZnRIFTf QD) to improve delivery in macrophages. Tuberculosis
(TB), caused by Mycobacterium tuberculosis (M. tb), is one of the
world’s major health problems. In combination chemotherapy, the “first line”
therapy is of three or four drugs, i.e., isoniazid, rifampicin, pyrazinamide
and ethambutol, followed by the less efficacious and more expensive “second
line” drugs, which include capreomycin, kanamycin, amikacin,
paraaminosalicylicacid, ciprofloxacin.
Metal
ions have many important physiological functions in the body. Transition metal
complexes exhibit unique and interesting properties such as changing oxidation
states and the ability to form specific interactions with other biomolecules.
It was shown that some metal drug complexes are more potent as compared to pure
drug. Toward this end, the interactions of some antibiotics with transition
metals have been studied. Among them, zinc is known to exhibit antibacterial
activity. Zinc oxide (ZnO) nanoparticles are even more efficient along with
reduced toxicity. Rifampicin (RIF), a broad-spectrum antibiotic, is one of the
most effective first line drugs against TB. Semiconductor nanocrystals, also
known as quantum dots (QDs), have become an important tool in biomedical
research, especially for quantitative and long-term fluorescence imaging and
detection. In view of this goal, we synthesized a zincrifampicin (ZnRIF)
complex and checked its activity against non-pathogenic Mycobacterium
smegmatis, and Mycobacterium bovis BCG, which behave like pathogenic
M. tb. RIF is chosen since it coordinates with metal ions through
chemical groups i.e. two phenolic and two aliphatic OH groups and the presence
of additional nitrogen and oxygen donor atoms provide this compound interesting
properties for studying its coordination behavior with transition metal ions.
Synthesis
of ZnRIF complex and its conjugation to transferrin coupled QDs was studied by
UV Visible Spectroscopy, Transmission Electron Microscopy (TEM), Fourier
Transform Infrared Spectroscopy (FTIR), photoluminescence, X-ray diffraction
(XRD), X-ray Fourier Transform Infrared Spectroscopy (FTIR) UVVis Spectra
analysis of the ZnRIF complex Photoluminescence analysis of the ZnRIF complex X-ray
photoelectron spectroscopy X-ray powder diffraction analysis of the ZnRIF complex
go to: Photoelectron Spectroscopy (XPS), and Nuclear Magnetic Resonance (NMR).
We observed that conjugation of ZnRIF complex to transferrin coupled QDs.
RESULTS
Fourier Transformation Infrared Spectroscopy
(FTIR)
The
structure of the ZnRIF complex was confirmed by several physicochemical
methods. The IR spectrum of ZnRIF was compared with that of free RIF. In agreement
with a previous report, the spectrum of free RIF showed characteristic peaks
at3482 (OH), 2880,1726 (C=O), 1646(C=N), 1247(COC) and 808(CH) cm. A
characteristic broad band at 3474 cm was observed with the synthesis and
characterization of Zn-RIF complex.
1.
UV-Vis
spectra analysis of the Zn-RIF Complex UV-V
2.
Photoluminescence
analysis of the Zn-RIF
3.
X-ray photoelectron
spectroscopy
4.
X-ray diffraction
(XRD) analysis of ZnRIF COMPLEX
NMR spectroscopy1H
NMR spectrum analysis of RIF and the ZnRIF complex
was carried out in DMSO-D6using a Bruker AVANCE 400 NMR spectrometer.
Tetramethylsilane (TMS) was used as an internal standard. All peaks arising
from unbound RIF ligand were clearly resolved with the phenolic hydroxyl
protons appearing at 9-10 (2 H) ppm. In the ZnRIF spectrum these peaks were
absent due to replacement with zinc. By comparing the splitting patterns, it
can be concluded that the metal to ligand ratio is 1:1.
Characterization of ZnRIF complex
Transition
metals can exhibit a wide variety of coordination properties and reactivities,
which can be used to form complex with drugs as ligands. Previously, several
studies have reported improved therapeutic properties of several metal
complexes against M. tb. RIF, which is considered the cornerstone in the
short course TB treatment regimen, exhibits detrimental side effects. To
address these issues, we employed a strategy in which Zn was complexed with RIF
to form a ZnRIF complex, which was subsequently encapsulated in transferrin conjugated
silver QDs to yield the ZnRIFTf QD conjugate. Detailed physicochemical analyses
confirmed the formation and encapsulation of ZnRIF complex in transferrin coupled
QDs. We demonstrated that encapsulation of transferrin on the surface of quantum
dots enhanced the binding efficiency of drug molecules. Then we showed that transferrin
conjugated and ZnRIF encapsulated silver QDs successfully targeted to the
macrophages, remaining stable for up to 48 h and significantly reducing the
bacterial burden inside the macrophages.
Quantum
dots (QDs) are nano scale semiconductor crystals with sizes of 1-10 nm. QDs
provide excellent tools for sensing, imaging, drug delivery and therapy due to
their optical properties, broad excitation range, well defined emission wavelengths
and their ability to attain different shapes thus providing an excellent
structure for coating with various biomolecules. Development of nanoscale drug
delivery system that allows a slow release of drug over prolonged periods of
time is important to thus avoid burst effects. Our results show that ZnRIFTf QDs
meet these criteria.
For the
synthesis of ZnRIF complex, 18 mg (0.095 mM) of hydrated zinc nitrate and 80 mg
(0.097 mM). rifampicin in methanol and stirred for 3-4
h. The precipitate was dried, weighed and dissolved in dH O to do the
experiments. ZnRIF complex was
characterized by Fourier Transform Infrared Spectroscopy (FTIR) (Nicolet iS5,
Thermo Scientific, India), UV Visible (Epoch, BioTek, Germany), X-ray photoelectron
spectroscopy (XPS) (S/N: 10001, Prevac, Poland), Powder X-ray diffraction (XRD)
(Shimatzu6100, Japan) and Nuclear Magnetic Resonance (NMR) (AVANCE 400, Bruker,
Switzerland). Silver QDs were synthesized as described previously. Briefly,
silver QDs were synthesized by reduction of silver nitrate (1 mM) by addition
of excess of ice-cold sodium borohydride (2 mM) solution by vigorous stirring
at room temperature. QDs were synthesized in less than a minute reaction time.
The stoichiometric ratio of silver nitrate to sodium borohydride is very
critical for the synthesis of QDs.
To
synthesize ZnRIF encapsulated transferrin conjugated silver QDs, 1 mg/ml of
transferrin and 500 μg/ml of ZnRIF complex were added together with 1 mM
concentration of silver nitrate. Then the reaction mixture was reduced by
excess ice-cold sodium borohydrate solution.
Characterization of ZnRIF encapsulated
transferrin conjugated QDs (TfQDs) Cytotoxicity assay
RAW 264.7 cells (1 × 10 cells/well) were grown in 24 well plates for 24 h followed by treatment with different concentrations of drug complexes for another 24 h. Cell viability was determined by MTT assay as described previously.
CONCLUSION
Future scope and aims
DNA binding and cleavage studies reveal that
Transition Metal Complexes/compounds can be used as efficient nucleases because
of the cooperative effect of the two Cu (II) centres and the guanazole ligand, which in addition to providing a bridge between
the two metals can participate in hydrogen-bonding interactions. Of the
complexes, some show the highest affinity for DNA and binds via intercalation
of the phen ligands. In the presence of
oxygen and micromolar concentrations of MPA or H2O2 as activators, only some
are capable of causing DNA cleavage. So, a large amount of studies can be done
using this transition metal complexes as quantum dots.
1. Yi DK, Selvan T (2005)
Silica-coated nanocomposites of magnetic nanoparticles and quantum dots. J Am
Chem Soc 127: 4990-4991.
2. Sun YP, Zhou B (2006)
Quantum-sized carbon dots for bright and colorful photoluminescence. J Am Chem
Soc 128: 7756-7757.
3. Sun YP, Fu KP (2002)
Functionalized carbon nanotubes: Properties and applications. Acc Chem Res 35:
1096-1104.
4. Juzhenas P (2008) Quantum dots and
nanoparticles for photodynamic and radiation therapies of cancer. Adv Drug Del
Rev 60: 1600-1614.
5. Tvrdy K, Kamat PV (2011)
Photoinduced electron transfer from semiconductor quantum dots to metal oxide
nanoparticles. Proc Natl Acad Sci 108: 29-34.
6. Bijuand V, Otoh T (2008)
Semiconductor quantum dots and metal nanoparticles: Syntheses, optical
properties and biological applications. Anal Bioanal Chem 391: 2469-2495.
7. Beverly A, Strobl JS (2009)
Cadmium-containing nanoparticles: Perspectives on pharmacology and toxicology
of quantum dots. Toxicol Appl Pharmacology 238: 280-288.
8. Smit AM, Duan H (2008)
Bioconjugated quantum dots for in vivo molecular and cellular imaging. Adv Drug
Del Rev 60: 1226-1240.
QUICK LINKS
- SUBMIT MANUSCRIPT
- RECOMMEND THE JOURNAL
-
SUBSCRIBE FOR ALERTS
RELATED JOURNALS
- Proteomics and Bioinformatics (ISSN:2641-7561)
- Journal of Microbiology and Microbial Infections (ISSN: 2689-7660)
- Advances in Nanomedicine and Nanotechnology Research (2688-5476)
- Food and Nutrition-Current Research (ISSN:2638-1095)
- Journal of Genetics and Cell Biology (ISSN:2639-3360)
- Journal of Veterinary and Marine Sciences (ISSN: 2689-7830)
- Journal of Womens Health and Safety Research (ISSN:2577-1388)