Gold nanoparticles (GNPs) stabilized with amino acid L-Cysteine were studied for their cytotoxicity, cell internalization and their ability
as drug delivery cargos for increasing the efficacy of Azidothymidine (AZT) against HIV-1Ba-L virus in vitro. GNPs exhibited low toxicity and
increased cell internalization in peripheral blood mononuclear cells after conjugating with L-cysteine. CC50 of GNPs was improved from
36.49ppm to 57.71ppm after functionalization with L-cysteine. GNP-Lcys demonstrated 70% drug carrying capacity and displayed a sustained
drug release profile under physiological pH. GNPs were capable of halting HIV replication in both pre and post infection assays with an IC50
value of 41.13ppm and 30.93ppm respectively. A statistically significant increase in anti-viral activity of AZT was seen (26.5% increment)
(P<0.01) when it was conjugated to GNP-Lcys. Thus gold nanoparticles can be considered as an alternative anti-viral candidate as well as a
potent drug delivery vehicle for improvising HIV therapeutics (Figure 1).
Keywords: Gold nanoparticles; CC50; Endocytosis; Drug release kinetics; IC50; HIV-1Ba-L; AZT; T cells
Abbreviations: GNPs: Gold Nanoparticles; AZT: Azidothymidine; AIDS: Acquired Immune Deficiency Syndrome; CC50: 50% Cytotoxic
Concentration; HAART: Highly Active Anti-Retroviral Therapy; HIV: Human Immunodeficiency Virus; IC50: 50% Inhibitory Concentration; IL-2:
Interleukin-2; Lcys: L-cysteine; PBMCs: Peripheral Blood Mononuclear Cells; PHA-P: Phytohaemagglutinin-P; SPR: Surface Plasmon Resonance;
TCID50: 50% Tissue Culture Infectivity Dose.
Introduction
Since its inception in 1981, HIV has killed more than a quarter
of a billion people in the past 25 years. An estimate 39 million
people are infected with human immunodeficiency type-1 virus
(HIV-1) worldwide [1,2]. There are more than 125,000 research
articles related to HIV/AIDS that are catalogued in Pubmed
database of the National library of medicine, but the knowledge
about this almost intelligent virus remains incomplete, mosaic of
understanding. Absence of an effective cure or a vaccine clearly
defines HIV/AIDS as one of the most formidable public health
issue of our generation. Antiretroviral therapy continues to be
the mainstay for HIV treatment. Latest minimum three-drugcombination
initiative of Highly Active Anti-Retroviral Therapy
(HAART) though has managed to reduce HIV-1 disease morbidity
and improved life expectancy, but is proving to no longer remain
an obstacle for the HIV. HAART has been currently found to be
associated with disadvantages such as adverse effects, reduced
bioavailability, rapid clearance, emergence of drug resistance viral strains, inconvenient dosage regimen and inability to
provide a functional cure i.e. complete eradication of HIV from
system [3-10].
Recent years have shown tremendous growth of research
and applications in the field of nanoscience and nanotechnology
[11]. Nanoparticles, particularly metallic nanoparticles like
gold and silver nanoparticles are increasingly finding space in
various diagnostic and therapeutic applications [12,13]. Gold
nanoparticles (GNPs) have recently emerged as a promising
drug delivery system (DDS) due to their facile synthesis, ease
of functionalization, biocompatibility and inherent non-toxicity
[14]. Apart from being widely used as a carrier system for
various drug delivery systems, GNPs have also been found to
have potent anti-microbial and anti-viral activities [15-18]. Some
of the noteworthy properties of GNPs used as drug carriers are
high stability, high carrier capacity, feasibility of incorporation of
both hydrophilic and hydrophobic substances and likelihood of
various routes of administration, including oral application and
inhalation [19].
In this study, we try to explore GNPs as potent anti-viral
and drug delivery systems for ferrying ART drugs for better
management of HIV therapy. AZT was attached onto L-cysteine
tagged GNPs and studied for change in its anti-HIV efficacy in vitro.
Efforts were also taken to understand the cell internalization and
drug release pattern of the nano-conjugate.
Experimental
MaterialsHIV-1Ba-L virus and reagents required for carrying out anti-
HIV assay were provided as a generous gift from Dr. Suzanne
Gartner, Dr. Mikulas Popovic and Dr. Robert Gallo through NIH
AIDS Research and Reference Reagent Program, USA. Chloroauric
acid, AZT, L-cysteine, MTT and all other chemical were purchased
from Sigma Aldrich, USA. Glasswares for nanoparticles synthesis
were washed with aqua regia and distilled water to remove
traces of metal contaminants.
Methods
Synthesis of L-cysteine capped gold nanoparticles (GNPs-
Lcys):Procedure to orchestrate GNPs with L-cysteine was adopted
from one of our previous studies [20] where L-cysteine was
incorporated during the synthesis of GNPs. Briefly, 20μl of 0.5%
L-cysteine was added to 10ml of 1% sodium citrate and kept
stirring at 170°C on a magnetic hot plate stirrer. Upon boiling,
Chloroauric acid (HAuCl4) at a final concentration of 100ppm
was added and kept stirring till initiation of color change. For
comparative significance, a set of GNPs were synthesized by
same procedure without the addition of L-cysteine.
Coating GNP-Lcys with AZT:
Here we report drug attachment at room temperature.
100μg/ml of AZT was added to 10ml of GNP-Lcys under closed
environment and kept under continuous stirring for 4 hours. The
concentration of AZT for coating onto GNP-Lcys was determined
based upon optimization studies (data not shown) done in
our laboratory to determine maximum concentration of drug
attachment without affecting the stability of the nanoparticles.
Characteristic determination of GNP-Lcys and GNP-Lcys-
AZT:
Preliminary characterization of nanoparticles and their
conjugates was done by UV-visible spectroscopy using a dual beam
Varian Cary UV-Vis spectrophotometer. Morphological features of
GNPs were studied by Transmission electron microscope (TEM)
(Zeiss Microimaging Gm bH, Germany). Attachment of drug onto
GNPs was analyzed by fourier transformed infrared spectroscopy
(Vertex 80, Bruker, Germany) and zeta potentials were calculated
using Malvern Zetasizer (Brookhaven).
Toxicity and internalization of GNPs-Lcys:
For therapeutic applicability, toxicity of GNPs was inspected
by tissue culture based MTT assay. Briefly, PBMCs were isolated
by density gradient centrifugation using ficol as a density
gradient [21]. PBMCs maintained in RPMI-1640 medium and
supplemented with 10% (v/v) Fetal bovine serum were seeded into 96 flat bottom well plates at concentration of 4x105 cells/
ml at 37°C and 5% CO2. After 24 hours of incubation, medium
was changed and different dilutions of blank GNP and GNP-L-Cys
were added into wells respectively. After overnight incubation
the cells were washed once with PBS and incubated with MTT
dye (5mg/ml) for 4hrs at 37°C. After 4hrs of incubation, Dimethyl
sulfoxide (DMSO) was added to each well and read at 570nm
using a microplate reader (Biotek India Ltd.). Transition of colour
change from yellow to purple is directly proportional to cellular
mitochondrial metabolism and cell viability. Percentage cell
toxicity was determined by following equation:
Where, S = Sample reading and P = Positive control (only
cells) reading. 50% cytotoxic concentration of nanoparticles
synthesized (CC50) was calculated by analyzing the above data
using GRAPH PAD PRISM software, version 3.0.
Endocytosis is known to happen best at 37°C. Thus
internalization of GNPs in PBMCs was studied with respect to
temperature as parameter. Different concentrations of GNPs and
GNP-Lcys below the CC50 value were incubated at different time
points (30min, 60min and 120min) with PBMCs in different 96
well flasks. One set of flask was incubated at 37°C while other flask
was incubated at 4°C - an inhibitory temperature for endocytosis.
After each time point, the supernatant was discarded and cells
were washed twice with PBS to remove excess of GNPs that had
not internalized. Cells were then digested with aqua regia (1
part HNO3 and 3 parts of HCl) for 15 to 20 minutes. The samples
were further diluted to a final volume of 5 ml in distilled water
and 5% of HNO3. The Au content in the samples was measured
by inductively coupled plasma-atomic absorption spectroscopy
(ICP-AES). Results obtained were converted into percentage
concentration internalized. The results obtained were an average
of three replicates from two independent experiments.
Drug kinetics of the nano-conjugate:
The GNP-Lcys-AZT conjugate was purified by extensive
dialysis in nano-pure water for 1 hours using a dialysis membrane
tube (MW cut-off of 3000 Da) to remove and quantitate the excess
amount of unbound AZT. Drug loading efficiency onto GNPs was
calculated using following equation:
To comprehend the drug release pattern, 5 ml of conjugate
was sealed in a two different dialysis tubes and the entire system
was kept at 37ºC in 50 ml phosphate buffer solution at pH 7.2
under mild stirring. To measure the drug release content, 3
ml of sample was periodically removed and replaced with an
equivalent volume of the phosphate buffer solution. The amount
of released AZT was analyzed with spectrophotometer at
266nm. The experiments were performed in triplicate for each
of the samples. The in vitro release data was finally related to
various mathematical models like zero order, first order, higuchi
model etc. to predict the drug release mechanism and kinetics.
Correlation coefficient (R) was calculated for each release model and the best fit release model was selected with highest ‘R’ value
[17].
Anti-HIV activity of GNPs and nano-conjugate:
Isolation and activation of peripheral blood mononuclear
cells (PHA-PBMCs) and Drug susceptibility assay to determine
the anti-viral activity of the conjugate was carried out as per
standard protocols of division of AIDS National Institute of Allergy
and Infectious Diseases, National Institutes of Health [18,22,23].
HIV is known to particularly infect T cells via CD4 receptor based
assistance. For selective growth of T cells from the population
of mononuclear cells and over-expression of surface markers
like CD4, PBMCs were activated with phytohemagglutinin
(PHA-P) and interleukin-2 (IL-2) for 24-72 hours prior to
infection. To determine if GNPs possess anti-HIV activity, the
assay was executed in a pre and post infection format. For preinfection
assay different dilutions of GNPs were incubated with
3X104 TCID50 of HIV-1Ba-L cell free virus for 1hour at 37°C and
then allowed to infect activated PBMCs with 10-1 multiplicity
of infection. Whereas, for post infection assay, same amount of
viral particles were first allowed to infect PBMCs followed by
addition of different dilutions of GNPs. The concentrations of
GNPs selected were less than the CC50 values obtained from MTT
assay. Anti-HIV activity of nano-drug conjugate was studied only
by post infection protocol. HIV infection was assessed after 4
days of incubation by quantifying the viral p24 antigen by ELISA.
Statistical analysis:
All data were analyzed using the GraphPad Prism software
(Version 3.0). Anti-viral experiment was repeated three times
and the data were analyzed by Bonferroni’s multiple comparison
test. A P-value less than 0.05 considered statistically significant.
Results and Discussion
Synthesis of GNP-Lcys
Conversion of chloroauric acid into gold nanoparticles was
seen as a transition in the solution from transparent to wine red
color. Spectral measurements of GNPs demonstrated an intense
peak between 500 to 550 nm (Figure 2) which is characteristic of
GNPs and results due to surface Plasmon resonance (SPR) [24].
As compared to blank GNPs; Lcys functionalized GNPs displayed
a decrease in peak intensity with a prominent blue shift from
528nm to 521nm (Figure 2). Blue shift in SPR is an indication
of change in the surface chemistry of GNPs due to adsorption
of L-cysteine. TEM images (Figure 3) confirmed synthesis of
40-50nm multifaceted cuboidal shaped nanoparticles. It can be
postulated that incorporation of amino acid L-cysteine during
synthesis might have resulted in stable aggregation of small
spherical shaped GNPs due to Ostwald ripening, resulting into
synthesis of large multifaceted cuboidal nano-structures at the
expense of smaller ones. This method of incorporation of linker
concurrent to synthesis produced highly stable nano-complex and
can prove to be an easy and efficient method for functionalization
of nanoparticles.
The infra-red spectrum of GNP-L-Cys was collected in the
course of the reaction in the spectral range of 4000cm-1 to 400cm-1. FTIR spectra of GNPs synthesized with L-cysteine (Figure 4a)
showed presence of many peaks typical of L-cysteine. The most
prominent peak was at 3434.69 cm-1, which represents N-H
stretch of primary and secondary amines. Mild peaks from 1243
– 1122 cm-1can are speculated to be of C-N stretch of aliphatic
amines. Whereas, a narrow peak at 1629.04 cm-1 correspond to
the C=O stretch of carboxylic acid. Peaks at 2924 and 2853 cm-1
suggests C-H stretch of alkanes while, a small peak cluster at
670cm-1 represents the (=C-H) bend of alkanes. All these functional
groups reveal presence of amine group (-NH2) and carboxylic
group (-COOH) of L-cysteine and confirm its incorporation onto
the GNPs during the synthesis. The absence of thiol group at
2500cm-1 suggests that the –SH group of L-cysteine has reacted
with the gold during the formation of GNPs, rendering –NH2 and
–COOH groups free for further attachment of the drug onto GNPs.
Coating of AZT onto GNP-Lcys
Spectral observation of the conjugate after AZT attachment;
showed an increase in the peak intensity with a red-shift of 3nm
in wavelength (521nm to 524nm) as shown in the Figure 1.
This could probably be due to the change in surface chemistry
of the GNPs after impregnation with AZT. Zeta potential value
showed a decrease in charge from -11.7mV to -15.21 mV after conjugation with AZT; thus suggesting adsorption of the drug
onto the nanoparticles. Decrease in the zeta value could be due
to the presence of benzene ring in the AZT. This explanation also
undermines the possible interaction of the azide group of AZT
with the nano-linker intermediate and decrease the positive
charge in the system. The size of the gold nanoparticles- linker
intermediate according to zeta measurements was further found
to increase from 42.8nm to 71.52 nm after conjugation with AZT,
which further confirms attachment of AZT onto GNPs.
FTIR spectrum of GNP-Lcys and GNP-Lcys-AZT was further
compared to obtained a better understanding of attachment
between the drug and nano-linker intermediate. IR-spectra of
GNP-Lcys-AZT (Figure 4b) showed a peak shift of N-H stretch
from 3434 to 3447 cm-1. The spectra also showed a prominent
peak shift of (C=O) carboxylic stretch from 1629 to 1638cm-1.
Corresponding peaks from 1383 to 1022 cm-1 were also damped
which represents C-O stretch. Emergence of peaks at 435 cm-1 is typical of C-C stretch and suggests incorporation of aromatic
groups of AZT. These spectral shifts suggest adsorption of
AZT onto GNP-Lcys and involve interaction of amine group of
L-Cysteine with the alcoholic group (CH2-OH) of the sugar present
in AZT. A decrease in peak transmittance corresponding to the
azide group of AZT may be due to a negative induction effect of
carboxylic group of the linker.
Drug release kinetics
In the process of designing an efficient carrier system for
drug delivery, it is imperative that the candidate carries high
amount of drug payload as well as provide control over its
release to maintain the required therapeutic concentration in the
system. From equation 2, percent drug loading efficiency of GNPLcys
was found to be 70%. Such high amount of payload carrying
capacity might be due to high surface to volume ratio of GNPs;
making them favourable candidate for drug delivery system.
Drug release studies at physiological pH demonstrated a constant
concentration of drug released by the nano-conjugate w.r.t. time
(Figure 5). Mathematical models applied to this data suggested
that the GNP-Lcys-AZT conjugate followed zero order release
kinetics. Zero order release kinetics highlights a constant drug
release profile, where the amount of drug release is independent
of the drug concentration loaded or left to be released and ideal
for maintaining constant therapeutic concentration of the drug in
circulation and prolonged pharmacological action.
Toxicity and internalization of GNPs
Percent cellular toxicity with respect to increase in log
concentrations of blank GNPs and GNP-Lcys is shown in Figure
6. Both the nanoparticles displayed high cell survival at lower
concentrations, which progressively worsened at higher
concentrations. However, GNPs after functionalization with
L-cysteine demonstrated higher CC50 values as compared to
blank GNPs, suggesting that GNP-Lcys were more biocompatible
than GNPs alone.
The initial contact and subsequent crossing of the
nanoparticles through the cell membrane is a critical process that
is expected to exert a profound effect on the cells and tissues. To
study the effect of gold nanoparticle size and surface composition
on cellular uptake, ICP-AES was employed to detect and quantify the intracellular Au content on PBMCs. Inductively coupled
plasma atomic emission spectroscopy (ICP-AES), as compared
to other imaging techniques, is a more sensitive and accurate
method to quantify small amounts of gold nanoparticles within
the cells. Nanoparticle uptake was also studied as a function
of temperature. Numerous investigators have shown in many
systems that endocytosis does not occur below 10°C [25]. Thus
nanoparticle uptake was studied at two different temperatures
i.e. 37°C and 4°C. As seen in Figure 7a & 7b, GNPs showed higher
amount of cell internalization at 37°C as compared to 4C. Thus
GNPs preferentially follow receptor mediated endocytosis to
enter cells. However, it was noticed that presence of L-cysteine
onto GNPs increased the cellular uptake by 10%. Thus it can be
concluded that L-cysteine increases the cell uptake profile of
nanoparticle conjugate and might improve drug delivery.
Anti-HIV activity of GNPs and nano-conjugate
As shown in Figure 8a, GNPs showed increase in the
antiviral activity with increasing concentrations for both pre
and post-infection assay with an IC50 value of 41.13ppm and
30.93ppm respectively. The IC50 values were calculated from
the log-concentration-response curves using GRAPH PAD PRISM
software, version 3.0. Thus gold nanoparticles were found to
exert anti-HIV activity even at early stages of viral replication. It
can be hypothesized that the exposed sulphur bearing residues of
the glycoprotein knobs would be attractive sites for nanoparticle
interaction. In case of GNPs, the anti-HIV activity might mainly
be due to their polyanion surface which possesses the ability to
bind to the positively charged amino acids in the V3 loop/second
binding site of the viral envelope glycoprotein gp120. Post-entry
inhibition studies revealed that gold nanoparticles (GNPs) have
other sites of intervention on the viral life cycle, besides fusion
or entry. Gold nanoparticles are known to complex with electron
donating groups containing sulphur, oxygen or nitrogen that
are generally present in thiols or phosphates in the amino acids
and nucleic acid. Thus they are likely to inhibit post-entry stages
of infection by blocking HIV-1 proteins other than gp120, like
reverse transcriptase enzyme, proteases etc.
To assess if gold nanoparticles can act synergistically
with the available anti-retrovirals, the synthesized nano-drug
conjugate was subjected to drug-susceptibility assay. As the
drug (AZT) used for conjugation act on the viral life cycle post
viral entry, we performed the post-infection drug susceptibility
assay to determine their activity. Comparative analysis revealed
a noteworthy reduction in p24 antigen production in nanoconjugate
challenged culture as compared to p24 levels in
cultures challenged with AZT alone. From Figure 8b, a substantial
26.55% increase in activity was seen when AZT was fired with GNP-Lcys. The results presented here demonstrate that the
therapeutic efficacy of a molecule can increased or improvised by
conjugating them to gold nanoparticles.
Conclusion
An ideal retroviral agent should act directly on the virus along with acting on other replicating stages prior to integration of proviral cDNA. It should be absorbable by uninfected cells in order to provide a barrier to infection by residual active virus and be effective at non-cytotoxic concentration. Our gold nanoparticles were found to comply all these criteria’s. Having such a varied panel of targets and showing both pre and post infection anti-HIV activities, along with improvising the efficacy of the conjugated drugs, gold nanoparticles can be possible alternative to currently available therapeutic management of HIV/AIDS.
Acknowledgements
The authors wish to acknowledge technical support from
SAIF laboratory, IIT, Bombay and Tata Institute of Fundamental
Research, Mumbai. We also thank Indian Council of Medical
Research, Govt. of India for providing fellowship to the
corresponding author.
Contribution of authors
Rohan Kesarkar: Designed, Performed and analyzed study
and wrote paper.
Sailee Shroff and Madhuri Yeole: Performed study;
particularly nanoparticle synthesis and collected data.
Abhay Chowdhary: Designed and analyzed study.
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Figure 1: Graphical abstract.
Figure 1: Graphical abstract.
Figure 2: Comparative spectral shift with respect to change in surface plasmon resonance of GNPs upon interaction with L-cysteine and AZT respectively.
Figure 3: TEM images showing multifaceted cuboidal shaped GNPs.
Figure 4: FTIR spectra of (A) GNP-Lcys (B) GNP-Lcys-AZT.
Figure 5: Drug release pattern of AZT from GNP-Lcys-AZT conjugate at physiological pH.
Figure 6: Percent cell toxicity shown by GNPs and GNP-Lcys on PBMCs
Figure 7: Percent uptake of nanoparticles by cells at (A) 37°C and (B) 4°C.
Figure 8: (A) Pre and post infection anti-HIV activity shown by GNP, (B) Anti-HIV activity shown by GNP-Lcys-AZT conjugate w.r.t. AZT.