A Facile Approach for Synthesis and Intracellular Delivery of Size Tunable Cationic Peptide Functionalized Gold Nanohybrids in Cancer Cells

Article abstract of DOI:10.1021/acs.bioconjchem.7b00772

Peptide-based drug delivery systems have become a mainstay in the contemporary medicinal field, resulting in the design and development of better pharmaceutical formulations. However, most of the available reports employ tedious multiple reaction steps fo

Full text of DOI:10.1021/acs.bioconjchem.7b00772

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Article
A facile approach for synthesis and intracellular delivery of size tunable
cationic peptide functionalized gold nanohybrids in cancer cells
Kavita Bansal, Mohammad Aqdas, Munish Kumar, Rajni Bala, Sanpreet Singh,
Javed Naim Agrewala, O. P. Katare, Rohit Kumar Sharma, and Nishima Wangoo
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00772 • Publication Date (Web): 28 Feb 2018
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A Facile Approach for Synthesis and Intracellular Delivery of Size Tunable Cationic
Peptide Functionalized Gold Nanohybrids in Cancer Cells
Kavita Bansal1, Mohammad Aqdas2, Munish Kumar3, Rajni Bala3, Sanpreet Singh2, Javed N.
Agrewala2, O.P. Katare4, Rohit K. Sharma3* and Nishima Wangoo5*
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1Centre for Nanoscience & Nanotechnology, Panjab University, Sectorꢀ25, Chandigarhꢀ160014, India
2CSIRꢀInstitute of Microbial Technology, Chandigarhꢀ160036, India
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3Department of Chemistry & Centre for Advanced Studies in Chemistry, Panjab University, Sectorꢀ14, Chandigarhꢀ160014, India
4University Institute of Pharmaceutical Sciences, Panjab University, Sectorꢀ14, Chandigarhꢀ160014, India
5Department of Applied Sciences, University Institute of Engineering & Technology (U.I.E.T.), Panjab University, Sectorꢀ25, Chandigarhꢀ
160014, India
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*Corresponding authors:
Email: nishima@pu.ac.in (N. Wangoo), rohitksg@pu.ac.in (R.K. Sharma)
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Abstract: Peptide based drug delivery system has found a mainstay in contemporary
medicinal field resulting in the design and development of better pharmaceutical
formulations. However, most of the available reports employ tedious multiple reaction steps
for the conjugation of bioactive cationic peptides with drug delivery vehicles. To overcome
these limitations, the present work describes a oneꢀstep approach for facile and time efficient
synthesis of highly cationic cell penetrating peptide functionalized gold nanoparticles and
their intracellular delivery. The
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nanoconstruct was synthesized by the
reduction of gold metal ions utilizing
cell penetrating peptide (CPP), which
facilitated the simultaneous synthesis of
metal nanoparticles and the capping of
the peptide over the nanoparticle
surface. The developed nanoconstruct
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was thoroughly characterized and tested for intracellular delivery into HeLa cells.
Intriguingly, a high payload of cationic peptide over gold particles was achieved, in
comparison to conventional conjugation methods. Moreover, this method also provides the
ability to control the size and peptide payload of nanoparticles. Produced nanoconstructs
showed enhanced cancer cell penetration (µM) and significant cytotoxic effect compared to
unlabeled gold nanoparticles. Therefore, this novel approach may also have significant future
potential to kill intracellular hidden dreaded pathogens like human immunodeficiency virus,
Mycobacterium tuberculosis, etc.
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Introduction:
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Over the last few years, medicinal chemistry has seen a tremendous rise in the used of
functional peptides. Peptides are enriched with unique properties such as small size, high
specificity, limited toxicity, ease of synthesis and facile surface modification. Owing to their
high biocompatibility and diverse nature, peptides have gained considerable impetus and
become an integral part of targeted drug delivery system. Among peptides, CPPs in
particular, have gained a lot of attention in the recent years. CPP are peptides used to increase
the cellular internalization of high molecular weight molecules for instance DNA, proteins
and fat insoluble drugs1. It has been reported that the transcriptional activator of transcription
(TAT) protein of Human Immunodeficiency Virusꢀ1 (HIVꢀ1) could be efficiently internalized
into cells when present in the surrounding tissue culture media2ꢀ4. The domain which is
responsible for this translocation is a short basic region comprised of 47ꢀ57 residues
(YGRKKRRQRRR) bearing six arginine and two lysine molecules. These positively charged
residues facilitate the cellular uptake by initial interactions of the peptide with negatively
charged residues of heparin sulfate proteoglycans5ꢀ7. TAT is the most frequently used CPP for
functionalization of nanoparticles to increase their overall efficiency and specificity as
delivery system8, 9. Among various nanoparticles, gold nanoparticles (GNPs) are of significant
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interest as delivery vehicle due to their considerable proven bioꢀcompatibility and unique
optical properties10 induced by surface plasmon resonance (SPR) of GNPs surface11.
Moreover, the ability to functionalize the GNPs by multiple ligands (chemical and
biomolecules) with high loading capacity makes them an excellent candidate for applications
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such as in drug delivery, labeling, sensing and imaging12ꢀ14
.
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For the conjugation of peptides with nanomaterials different approaches are being reported,
for instance, electrostatic interaction between peptide and nanoparticles having opposite
charge, use of thiol or biotin motif peptides for direct conjugation with gold and stratavidin
modified gold nanoparticles respectively15ꢀ17. However, these conjugation techniques suffer
from various shortcomings such as weak peptideꢀnanoparticle interaction and prerequisite of
specific molecules. Moreover, highly stable covalent attachment among peptide and GNPs
requires the use of various coupling reagents and involves tedious multiꢀstep reactions.
Earlier report by Fuente et al.18 encompassed the introduction of TAT peptide onto GNPs via
the carbodiimide coupling reaction. The process involved the formation of tiotropin capped
GNPs in methanol/acetic acid media using sodium borohydride as reducing agent. Here,
tiopronin was used to generate the carboxylic functionalities over GNPs surface, so that
EDC/NHS activated carboxyl groups can form an amide bond with the amine moiety of the
peptide. Moreover, the phase transfer methodology has also been reported for the synthesis of
cationic/ anionic peptides functionalization GNPs using ligand exchange process by
employing 11ꢀaminoꢀ1ꢀundecanethiol19. However, this approach also comprises the multiple
step reaction process. In contrast to the earlier mentioned peptideꢀnanoparticle binding
methodologies, Slocik et al. synthesized GNPꢀpeptide complex by reducing the gold salt
directly with dodecyl A3 peptide (AYSSGAPPMPPF)20 . In another reports, the Rev and
RGD peptides retain their anticancer and αvβ3 integrin targeting efficiency respectively even
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after reducing the gold salt21ꢀ23. Additionally, the antimicrobial peptide conjugated GNPs
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synthesized by one step methodology showed high antimicrobial activity and serum stability
compared to alone peptide24. Indeed, bonding between peptide and GNPs primarily depends
upon the amino acid sequence and studies reveal that it is comparatively difficult to conjugate
the cationic peptides with metallic nanoparticles than anionic peptide25. Peptides having
terminal cationic residue (cysteine, arginine, serine and tryptophan) may lead to aggregation
of negatively charged GNPs in aqueous solution26. DLVO theory implies that the strong
attractive Vander waals forces between charged nanoparticles are responsible for their
aggregation, which can be balanced by generating electrostatic repulsive forces in the
colloidal solution by introduction of opposite charged molecules27. Ojeaꢀjimenez et al.
confirmed this theory by evaluating the effect of various charged molecules on the stability of
citrate capped GNPs. Results confers that the negatively charged molecules ending with Rꢀ
COOꢀ retains the stability of solution by overcoming the Vander waals attraction, whereas
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+
species ended with RꢀCOOHꢀ or RꢀNH3 terminals increase the attractive interaction forces
and resulting the aggregation of nanoparticles28. Hence, a lot of optimizations and multiple
chemical reagents are prerequisite to acquire a stable cationic peptide capped GNPs complex
in aqueous media.
Keeping in view the above mentioned points, we propose a facile, ecoꢀfriendly, oneꢀstep
approach for bioꢀconjugation of highly cationic TAT peptide with GNPs in aqueous medium.
In this method, peptide serves the dual role of reducing as well as stabilizing agent to
synthesize the peptide capped gold nanohybrids. The synthesized conjugates showed
excellent stability in aqueous medium even at room temperature without any further addition
of stabilizing agent. Also, Inꢀvitro results showed enhanced cell penetration and antiꢀ
proliferation effect of TATꢀGNPs hybrids in HeLa cells compared to GNPs alone, thereby
proving their significant potential in drug delivery applications.
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Results and Discussion
Synthesis of size tuned GNPs at basic pH:
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The GNPs were synthesized using chemical methodology employing TAT peptide as
reducing as well as capping agent. The effect of pH and peptide concentration on the
formation of nanoparticles was carefully studied. For this, 20 µl of different concentration of
peptide was used for the synthesis of GNPs at varying pH (9, 10 or 11). It was observed that
the reactions carried at pHꢀ10 having final peptide concentration of 0.16 µM (GP0.16), 0.8 µM
(GP0.8), 1.6 µM (GP1.6) or 3.2 µM (GP3.2) displayed dark red colored solutions, while at 0.08
µM (GP0.08) and 6.4µM (GP6.4) peptide concentrations, violet and light pink color solutions
were obtained respectively. However, pink or violet colored final colloidal solutions were
obtained at pH 9 and 11 [fig.1 (aꢀc)]. It has been reported that the reduction of gold salt
significantly dependent on the pH of the working solution. In the reaction process, phenolic
groups present in side chain of tyrosine (TAT peptide) plays an substantial role in reduction
of the Au(III) ions. Tyrosine shows enhanced reductive capability in aqueous solution at pH
near the pKa value of tyrosine (∼10), where all phenolic moieties are get oxidized into
phenoxide29. Whereas, at pH 9 partial oxidation of phenolic groups leads to incomplete
reduction of gold salt30. Moreover, at high pH (∼11) low yield of GNPs can be due to the
conversion of highly reactive gold ion [AuCl3(OH)]ꢀ to less reactive state [AuCl2(OH)2]ꢀ and
[AuCl(OH)3]ꢀ31.
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In UVꢀvisible spectroscopy, the absorption maxima of the synthesized colloidal solutions
were centered around 515 nm to 530 nm SPR band of GNPs32 which is a clear indicative of
the synthesis of stable GNPs [Fig.2 (dꢀf)]. Furthermore, it was evident from the TEM images
that the synthesized nanoconstructs (GP0.08, GP1.6, GP3.2 at pHꢀ10) were well dispersed, nearly
spherical in shape and had a core particle size of about 30 nm, 15 nm and 10 nm respectively
[Fig. 1 (gꢀi)]. In addition, DLS analysis also confirms the high monoꢀdispersity (PDI<0.5) of
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these nanoconstructs with hydrodynamic size of 42nm, 30.8nm and 18.2nm respectively. Size
analysis data revels that with increase in peptide concentration the size of synthesized
nanoconstructs decreased, which confirms the role of peptide acts as reducing agent.
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TA T pep after heating at 80o
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Fig. 1. Effect of pH and peptide concentration on morphology and kinetics of peptide
syntheized gold nanoparticles. Color (aꢀc) and absorbance spectrum (dꢀf) of gold
nanoparticles synthesized at pH 9,10 and 11 respectively. TEM images (gꢀi) of GP0.08, GP1.6,
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GP3.2 (at pHꢀ10), synthesized nanoparticles are nearly spherical in shape and having mean
particle size of about 30nm, 15 nm and 10 nm respectively at magnification 40000 X. Particle
size distribution (jꢀl) of GP0.08, GP1.6, GP3.2 nanoparticles obtained from DLS analysis (42nm,
30.8nm and 18.2nm respectively). Mean hydrodynamic sizes of the conjugates are usually
larger than actual size of particles. Circular Dichroism spectra (m) of TAT peptide (64µM)
without heating (black) and after heated at 80˚C for 1 hr. in water (red).
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In order to evaluate the reaction kinetics of GNPꢀpep complex, various aliquots were taken at
different time intervals. The reaction in aliquots was ceased by inserting the vessel in ice cold
water. Absorbance of collected aliquots was monitored at 520 λmax (GP1.6) and it was
observed that reduction of gold salt was almost saturated after 60 minutes [Fig. 2 (a, b)].
Furthermore, the rate for synthesis of GNPꢀpep complex decreased with increase in peptide
concentration, which eventually delayed the nucleation and growth process of GNPs.
Consequently, the above studies inferred that a small change in pH and peptide concentration
during synthesis process imparts a significant effect over the structural morphology and
reaction kinetics of synthesized nanoconstructs.
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The CD spectrum of TAT peptide displays a strong negative band at 196 nm (ꢀ13.31
M−1cm−1) and a positive band at 222 nm (+1.62 M−1cm−1), representing the leftꢀhanded 31ꢀ
helix conformation33, 34 [Fig.1 (m)]. Additionally, to evaluate the effect of heating on the
conformation of peptide, the peptide was heated in aqueous solution at 80˚C for 60 min.,
followed by cooling at room temperature (replicating conditions used in the synthesis of the
nanoconstructs). The results illustrate that the peptide retains its conformation with minimal
change in its ellipticity values. Hamley et al.35 also confirmed (using CD spectra and SAXS)
that palmitoylꢀKKFFVLK amphiphile peptide selfꢀassemblies regain their structural
conformation after heating at 55 ˚C followed by cooling. Consequently, this study proves that
the reported method employs a safe and facile approach for the synthesis of peptide gold
nanoconstructs.
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Fig. 2. Image (a) and absorption spectra (b) of various aliquots of proceeding pepꢀGNP
complex reaction mixture, Inset shows the timeꢀdependence absorbance of aliquots at 520
nm. The data are the representative of three independent experiments.
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TAT functionalized gold nanoconstructs bearing negative charge:
In order to confirm the appropriate layering of the peptides over synthesized nanoparticles,
TGA, FTꢀIR and ζꢀpotential studies were carried out. TGA was performed on the lyophilized
sample of pepꢀGNP, GNP and pure peptide, the thermogram of pepꢀGNP conjugate
[Fig.2a(a1)] shows a first weight loss upto 100ºC due to the evaporation of residual and
physisorbed water that can be neglected. Subsequent results show a primary mass loss of
17.13% (I) between 140 to 200 °C followed by a second mass loss peak of 5.45% (II) at
higher temperatures 300ꢀ350 °C. The first primary loss corresponding the capping of
carbonate ions from the potassium carbonate, showing similar weight percent loss (17%) and
temperature range compare to gold salt is reduced only in the presence of K2CO3 [Fig.2a
(a2)]. Secondary loss is due to the capped peptide molecules over the gold surface, also
supported by the DSC data [Fig.S2]. Whole pure peptide gets degraded in the temperature
range of 200ꢀ250°C [Fig.2a (a3)], which is shifted to250ꢀ300 °C for pepꢀGNP, confirms that
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the peptide is strongly bound with the gold surface36, 37
.
In FTꢀIR spectra [Fig. 2a (i, ii)] the characteristic IR absorption peaks of peptides at 1642 cmꢀ
1 (amide I, carbonyl stretch vibration), 1625 (C=C stretch), 1543 cmꢀ1 (amide II) and 1389
cmꢀ1 (amide III, C–N stretch vibration) were found to be present in the corresponding spectra
of pepꢀGNPs, signifying the capping of peptide molecules on the nanoparticles surface.
Intense broad bands in the region between 3300 and 3400 cmꢀ1 were representative of NꢀH
and OꢀH stretching vibrations. The primary aliphatic amine peaks at 1183 cmꢀ1 and 1126 cmꢀ1
completely disappeared in peptideꢀGNPs spectra, depicts the reduction of gold salt by
phenolic group of Tyrosine and free electrons of the amine group respectively29, 38ꢀ40
.
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The high zeta potential values [Fig. 2 b] of the pepꢀGNPs complexes indicated the higher
stability and therefore, their robustness as efficient candidates for drug delivery.
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Fig. 2 Negatively charged GNPꢀpeptide constructs capped with TAT peptide (a) TGA
spectra of (a1) pepꢀGNPs, (a2) GNPs, (a3) pure peptide. (b) FTꢀIR spectra of (i) pure peptide
(ii) pepꢀGNPs, (c) Zeta potential of pepꢀGNPs synthesized at pH 10.
In the present work, reduction of gold salt occurs and electron rich phenolic residues of
tyrosine30 and amine groups of peptide to yield the corresponding nanoconstructs in the
presence of potassium carbonate [Fig. 3]. Besides, carboxylic moieties of the peptide render
the synthesized GNPs stable. Further, physical adsorption of the excess peptide and carbonate
ions in the solution keeps the nano particles well dispersed. In the absence of peptide, only
potassium carbonate is not able to synthesize the stable GNPs [SIꢀ Fig.2 (i)] However, the
GNPs synthesized by peptide showed no aggregation even after three months [SIꢀ Fig.2 (ii)].
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Fig. 3 Schematic representation of peptide functionalized gold nanoparticles synthesis
GNPs synthesis follows twoꢀstep reaction mechanism, firstly reduction of ionic gold (Au3+)
into metallic gold nuclei (Au0), subsequently the growth of nuclei in to bigger particles by
coalescence41
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High and regulated payload of peptide on peptide-GNP constructs:
Owing to the presence of arginine in the TAT peptide, Bradford assay was employed to
calculate the amount of peptide capped on the GNPs surface. Arginine produces blue color on
reacting with Bradford reagent, which provides a characteristic absorption band at 590 nm.
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Firstly, a calibration curve [Fig. 4(a)] of TAT was obtained by plotting the absorbance ratio at
42, 43
590 nm and 450 nm (A590/A450
)
followed by calculation of unreacted amount of peptide
remaining in the supernatant of reaction mixture [Table 1]. Interestingly, graph [Fig. 4(b)]
reveals the amount of peptide capped to nanoparticles gives a linear regression with the
concentration of the peptide added during the nanoparticle synthesis. Thereby, it is possible
to control the loading amount of peptide over GNPs.
Additionally, the number of peptide molecules capped over a single GNP was also calculated
by considering the core size of nanoparticle obtained by TEM analysis. The average number
of gold atoms (N) in one gold nanoparticle were calculated by Eq.1 assuming that all the
synthesized particles are spherical in shape and have uniform fcc structure, here
density for fcc gold (19.3 g/cm3) and
ρ is the
M
stands for atomic weight of gold (197 g/mol). Total
number of gold nanoparticles (Nt) and the molar concentration (C) of the nanoparticles in
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solution was calculated using Eq. 2,3, here W is weight of gold solution taken (Mol.
wt.HAuCl4.3H2O=393.83), NA is the Avogadro's constant and V is the volume of the reaction
6
solution44, 45. Remarkably, a high payload of TAT peptide i.e. 144, 427 and 24 molecules was
found to be conjugated on one nanoparticle of the synthesized GP0.08, GP1.6 and GP3.2
nanoconstructs respectively. TGA studies also confirmed the high loading of peptide
molecules over the gold nanoconstruct (SI). Sanz et al. reported that the addition of more
than 1 µg/ml (0.7 µM) of TAT peptide leads to aggregation of GNPs during covalent
coupling process and number of TAT chains bound to GNPs is directly proportional to the
initial concentration of peptide in reaction mixture46. Hence, our proposed methodology gives
the benefits of functionalization the GNPs with approximately 4.5 times higher payload of
TAT peptide (GP3.2) in a facile manner. Furthermore, approximately 83% of added peptide
was utilized in reduction and capping of GNPs, which reflects its high utility to be used as
novel procedure for synthesis of cationic peptide functionalized gold conjugates.
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ꢄ
14
15
ꢀ = ꢁꢂꢃ = 30.89602ꢇꢈ…………………. (Eq.1)
ꢅꢆ
ꢊ ×ꢋ
ꢌ
ꢀꢉ = ꢈꢍꢈ.ꢎꢈ×ꢋ ……………………………… (Eq.2)
ꢋ
ꢐ
ꢊ (ꢒꢓ)
16 ꢏ = ꢑ×ꢋ
=
…………… (Eq.3)
ꢈꢍꢈ.ꢎꢈ ×ꢑ(ꢔ)×ꢋ
ꢌ
17
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5
(a)
(b)
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1
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0
0
0
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6
0.08 0.16
0.8
1.6
3.2
11
Concentration of peptide (µM)
Concentration of peptide (µM)
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Fig. 4 Quantification of the amount of peptide capped on GNPs. (a) Standard calibration
plot and the amount of peptide capped to nanoparticles (b) of TAT peptide.
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Table 1 Calculated amount of peptide capped over GNPs
7
Quantity of peptide
capped on gold
particles (µg)
Quantity of peptide
added (µg)
Quantity of peptide in
supernatant (µg)
8
9
GP0.08
GP0.16
GP0.8
GP1.6
GP3.2
0.624
1.284
0.140
0.266
0.399
1.138
3.572
0.484
1.017
10
11
12
6.240
5.841
12.480
24.960
11.342
21.388
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High colloidal stability of GNP-peptide constructs at physiological conditions:
Colloidal GNPs are highly susceptible to aggregation in the presence of electrolytes.
Therefore, it is essential to examine the stability of synthesized nanohybrids in physiological
ionic conditions, in order for them to be used as potential drug carriers47. For this, the
stability of different pepꢀGNP complexes were examined in phosphate buffer saline solutions
and the change in SPR band at 520 nm was detected, which is a characteristic feature of
GNPs stability. As the stability of GNPs decreases, it leads to agglomeration and the
absorption maximum is shifted to higher wavelength. The percentage change in absorbance
intensity of GNPsꢀpeptide complexes [Fig. 5] was calculated at increasing concentrations of
PBS (1X=150 mM of NaCl and 10 mM of phosphate). All the studied conjugates showed
high stability in 1X PBS solution which corresponds to the tonicity of physiological fluids.
As expected, GP1.6 exhibited maximum stability in PBS solutions owing to high loading of
peptide molecules over their surface. Consequently, all the synthesized pepꢀGNPs conjugates
of different sizes are applicable for biological based systems.
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120
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20
0
GP 3.2
GP 1.6
GP 0.08
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0X
1X
2X
3X
4X
5X
Concentration of PBS
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Fig. 5 Stability studies of conjugates in phosphate buffer saline (PBS). Percentage change
in intensity of GNPꢀpep1, GNPꢀpep3 and GNPꢀpep5 at different concentrations of phosphate
buffer saline solution (0ꢀ5X)
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Anti-proliferation and enhanced cellular uptake of GNP-peptide complex:
MTT results showed that the viability percentage of Hela cells decreased significantly
(p<0.05 and p<0.001) from 70% to 50% (0.2µM) after treating with GNPꢀPep conjugates for
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6h and 24h respectively, which illustrate that the survival of cells is time dependent [Fig. 6 (a,
b)]. While, cells treated with the bare GNPs and alone TAT peptide exhibit high survival rate.
Additionally, the results suggest that the GP1.6 conjugate showed the maximum damage to the
cell, which can be explained by its highest loading of peptide over their surface amongst the
studied conjugates.
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Fig.6 Cell viability studies for GNPꢀpep nanoconstructs by MTT assay. Dose and time
dependent cytotoxicity of bare GNPs, TAT and peptide capped GNPs (GP0.08, GP1.6,
GP3.2) on the HeLa cells at different time interval of 6 h (a) and 24 h (b)
Furthermore, to visualize the cellular internalization of bare and GP1.6 conjugate, tagging with
FITC was done48. As shown in Fig.7, the uptake of bare GNPs in HeLa cells was negligible
compared to peptide capped gold nanoconstructs. These images reveal that the TAT peptide
functionalized over the GNPs surface in GP1.6 conjugate is actually responsible for its
increased cellular uptake. Moreover, Confocal Zꢀstack images clearly indicate that the GNPꢀ
pep was efficiently internalized with in the cell (Fig. S4). Hence, inꢀvitro results clearly
indicate that the TAT does not lose its penetrating efficiency during the synthesis process49,
50. Therefore, in nutshell, peptide and GNPs hybrids synthesized by the reported facile
approach are highly efficient to be used as carrier vehicles for delivery of antiꢀcancer drug
and other biological molecules with in the cells.
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Lysotracker
FITC
DAPI
Merge
Zoom
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Fig.7. Cellular uptake studies by Fluorescence spectrophotometry cellular internalization
of unꢀstimulated (a1ꢀa5), bare GNPs (b1ꢀb5) and GP1.6 (c1ꢀc5) in HeLa cells after 6h treatment.
In fluorescence images green, blue and red corresponding FITC tagged GNP conjugates, cell
nuclei and lysosomal compartments respectively.
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Conclusion:
We hereby report a one pot and single step process for the functionalization of GNPs by a
cell penetrating peptide (TAT) and show its intracellular internalization. In the conventional
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approaches, the citrate reduced negatively charged GNPs aggregate in the presence of highly
cationic TAT peptide. To overcome this problem, we have synthesized the GNPꢀTAT
complex by direct reduction of gold salt with the TAT peptide, which subsequently capped
over the gold particle surface and was confirmed by FTꢀIR. To optimize the reaction
conditions, the synthesis was carried out at different pH and peptide concentrations. The
optimum results were obtained at pH 10 and size tunable goldꢀpeptide constructs were
obtained at different peptide concentrations. The functionalization procedure is highly
efficient as approximately 83% of added peptide was utilized in reduction and
functionalization of GNPs. A high loading of peptide on GNPS was achieved and the
calculated number of TAT peptide molecules conjugated over one nanoparticle of GP 0.08, GP
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and GP nanoconstructs are 144, 427 and 24 respectively. Zeta potential and stability
1.6
studies confirmed their high suitability for biological studies. The viability of cervical cancer
cells (HeLa) in MTT assay was significantly reduced after treating with the GP 0.08, GP 1.6 and
GP3.2 nanoconstructs compared to bare GNPs. The cellular uptake and cell viability assay
results confirmed that the TAT peptide retains its cell penetrating efficiency even after
reducing the gold salt. This clearly confirms that, the peptide here serves the role of reducing
agent, capping agent as well as cell penetrating agent. Hence, our method gives a new
approach to functionalize highly cationic peptides with GNPs in a facile and time efficient
manner. Moreover, the synthesized conjugates hold good potential to be used as a carrier
vehicle in the drug delivery, biosensor and bio imaging systems.
Experimental details
Materials and methods:
Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4.3H2O), trisodium citrate dehydrate
(TSC), FmocꢀArg(Pbf)ꢀOH, FmocꢀLys(Boc)ꢀOH, dimethylformamide, N,N,N’,N’ꢀ
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TetramethylꢀOꢀ(benzotriazolꢀ1ꢀyl)uroniumtetrafluoroborate
(TBTU),
N,N
diisopropylethylamine (DIEA), piperidine, trifluoroacetic acid, diethyl ether
triisopropylsilane, acetonitrile, 3ꢀ(4,5ꢀDimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀDiphenyltetrazolium
Bromide (MTT), 4,6ꢀdiamidinoꢀ2ꢀphenylindole dihydrochloride (DAPI), Sodium dodecyl
sulfate (SDS), Fluorescein isothiocyanate (FITC), Paraformaldehyde (PFA) Bradford reagent
and potassium carbonate (K2CO3) were acquired from Sigma Aldrich (India). All the reagents
used were of analytical grade and used without further purifications. All the experiments
were carried out using MilliꢀQ water having a resistivity of 18 Mꢁ cm. The glassware was
rinsed with aqua regia prior to use.
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Synthesis of TAT peptide (YGRKKRRQRRR):
The peptide was synthesized using the standard fluorenylmethyloxycarbonyl (Fmoc) solidꢀ
phase peptide synthesis based strategy. Initially, Wang resin was employed followed by
subsequent coupling steps using FmocꢀArg(pbf)ꢀOH, FmocꢀGln(Trt)ꢀOH, FmocꢀLys(Boc)ꢀ
OH, FmocꢀGlyꢀOH, FmocꢀTyr(tꢀbut)ꢀOH,TBTU and DIEA. The intermediate
Fmocdeprotection steps were carried out using 20% piperidine. The final deprotection step
was
performed
using
standard
protocol
(95%
trifluoroacetic
acid/2.5%
triisopropylsilane/2.5% ELGA water) for 6h at room temperature. The crude peptide was
analyzed by reverse phase high performance liquid chromatography (RPꢀHPLC) on a C18
column and then characterized by matrix assisted laser desorptionꢀionization/timeꢀofꢀflight
(MALDIꢀTOF) mass spectrometry [Fig.S1].
Synthesis of peptide reduced/functionalized gold nanoparticles (pep-GNPs):
Briefly, an aqueous solution of 0.4 mM HAuCl4 (5 mL) was heated at 80˚C followed by the
addition of 20 µl of peptide solution (varying concentration) and pH was adjusted using 0.5
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M K2CO3. Initially, the color of the solution changed from light yellow to pink and finally to
red, indicating the formation of GNPs. The reaction was further continued for 60 minutes in
order to complete the synthesis of nanoparticles and allowed to cool at room temperature.
The synthesized colloids were centrifuged (10,000 rpm for 15 minutes) and washed with DI
water. The supernatant and pellet were collected separately and stored at 4˚C for analysis.
The optimal reaction conditions were found by varying the pH and concentration ratio of gold
ions/ peptide. As a reference, citrate capped GNPs were also synthesized by following the
Turkevich method 41[SI].
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Characterization techniques:
The UV–visible absorbance spectra of the nanoparticles were recorded using a JascoVꢀ530
UV Vis spectrophotometer using 1 cm path length quartz cuvettes with 0.1 nm resolution.
Particle size distribution and Zeta potential were analyzed using Malvern Zetasizer. FTꢀIR
spectra of lyophilized powder of pepꢀGNPs complex and of pure peptide were measured. on
Thermo Scientific Nicolet iS50 FTꢀIR spectrophotometer in the range from 500 cmꢀ1 to 4500
cmꢀ1. Thermogravimetric analysis (TGA) was performed on the lyophilized samples using
PerkinElmer TGA instrument. Transmission electron microscopy (TEM) studies were carried
out using Hitachi (Model Hꢀ7500) transmission electron microscope operating at an
accelerating voltage of 100 kV. Samples for TEM studies were prepared by placing a drop of
the nanoparticles on carbonꢀcoated TEM grids and allowed to dry for 5 min at room
temperature before analysis. For cyclic dichroism (CD) analysis purified proteins were
dialysed into distilled water and secondary structure analysis were performed using JASCO Jꢀ
815 CD spectrometer at 64 µm concentration in 1mm pathꢀlength quartz cuvette. CD data
were recorded at 20 °C in far UV range 190ꢀ250 nm at data pitch 0.5 nm, scanning speed 50
nm/min and bandwidth 1 nm.
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Cell viability assay:
The HeLa cells were seeded into 96ꢀwell plates at 1x104 cells/well, and incubated overnight
at 37°C in a humidification incubator with 5% CO2. Later, the cells were treated with
different concentrations (0.1, 0.2, 0.4 µM) of GP0.08, GP1.6, GP3.2, bare GNPs (TSC reduced)
and TAT peptide, followed by incubation for 6h and 24h. Subsequently, the cells were
incubated for 3ꢀ4h with MTT solution followed by the stop solution (50% dimethyl
formamide + 20% SDS in water). The absorbance of the purple colored product was
measured at 572 nm. Percentage viability of cells was calculated according to the following
equation.
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ꢞꢛꢟꢠꢡꢢ (ꢜꢣꢖꢚꢜꢖꢤ)
% ꢕꢖꢗꢗ ꢘꢙꢚꢛꢙꢗꢙꢜꢝ =
× 100
ꢞꢛꢟꢠꢡꢢ(ꢕꢥꢦꢜꢣꢥꢗ)
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Cellular uptake studies:
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For evaluating the cellular internalization of bare and peptide fictionalized GNPs were firstly
tagged with fluorophore FITC (SI). For this, HeLa cells (1x105/ml) were plated overnight on
coverslips, FITC labeled pepꢀGNPs and bare GNPs conjugates (200ꢂl of 0.4 µM) were added
into each well and incubated at 37°C for 6h. The cells were then treated with 300 nM
Lysotraker Red (Invitrogen, Carlsbad, CA) for 30 mins to stain the acidic organelles. After
staining, the cells were washed twice with 1XPBS followed by fixing with 4% PFA for 10
mins. The cells were washed thrice with PBS and the coverslip was mounted on the slide
using mounting reagent (Slow fade with DAPI, Invitrogen, Carlsbad, CA). The coverslips
were sealed and observed under the Nikon A1 confocal microscope (Nikon, Tokyo, Japan)
using 488 nm (FITCꢀtagged gold particle) and 561 nm. The extent of nanoparticles
internalization was observed by analyzing FITC signal in the cells.
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Statistical analysis:
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Statistical testing was performed by one way ANOVA for group analysis using GraphPad
Prism (GraphPad Software, San Diego, CA). Di
level of p < 0.05.
fferences were considered significant at a
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Acknowledgements: Authors thank financial support from UGCꢀBSR, India. KB and MA
thank Panjab University, Chandigarh and INSPIRE Program, Department of Science and
Technology (DST) respectively, for research fellowships. We are also grateful to Mr. Deepak
Bhatt for confocal imaging and Mr. Randeep Sharma for TEM facility at CSIRꢀInstitute of
Microbial Technology, Chandigarh, India.
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Supporting information available at DOI: Synthesis of TSC reduced GNPs, Tagging with
fluorophore, TGAꢀDSC spectra and Confocal Zꢀstack cellular uptake images of pepꢀGNPs,
MALDIꢀTOF spectrum of TAT peptide.
Conflict of interest: Authors declare no conflict of interest.
ORCID ID: Dr. Nishima Wangoo: 0000-0001-8691-1072
References
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(4)
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Vives, E., Brodin, P., and Lebleu, B. (1997) A truncated HIVꢀ1 Tat protein basic
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