Stable and reproducible synthesis of gold nanorods

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Stable and reproducible synthesis of gold nanorods for biomedical applications: A comprehensive study Uzma Azeem Awan1, 2, ShaukatAli3, Mehreen Rehman1, Nashmia Zia1, Syeda Sohaila Naz1, Muhammad Ovais1, and Abida Raza1* 1

Nanotheragnostic Research Labs, National Institute of Laser and Optronics, Islamabad, Pakistan Department of Biotechnology, University of Azad Jammu and Kashmir Muzaffarabad, Pakistan 3 Medical Toxicology Lab. Department of Zoology, University of Azad Jammu and Kashmir Muzaffarabad, Pakistan

2

*Corresponding Author email: [email protected] ORCID ID: 0002-4414-1070 Abstract Gold nanorods (GNRs) are ideal choice in biomedical research due to their amenability of synthesis, tunable plasmonic properties, less toxicity and ease of detection but their diverse biological applications necessitate stable structure. Despite two decades’ efforts made towards reproducible anisotropic structures synthesis, still we have not achieved kinetic control during GNRs growth. Current study is an attempt to apprehend thermodynamic and kinetic parameters for synthesizing mono-disperse, reproducible and highly stable GNRs with desired aspect ratios. Effects of various growth parameters and assay steps on the facile and reproducible synthesis of GNRs are analyzed. GNRs’ environmental and biological colloidal stability is studied through UV-vis spectroscopy based particle instability parameter (PIP60°C) and alkaline pH can trigger colloidal instability. GNRs remain stable at higher salt concentration, physiological and slightly acidic pH. GNRs can be stored in 0.001 M CTAB for 03 months without compromising their stability. PEGylated GNRs are quite stable in cellular media solution (PIP 0.1). There is no shift

absorption spectra of CTAB-GNRs is observed with

in wave length (785 nm) and absorption intensity of

increase in number of washes. Absorption intensity

GNRs is observed after one month (Figure: 7B).

12 IET Review Copy Only

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Optimum concentration of CTAB is 0.001 M for

at physiological pH [22] but in current study GNRs

storage of GNRs.

are stable at acidic and physiological pH, even after

3.3.2

GNRs stability and pH

one month (Figure 8).

We have studied GNRs for their stability in

3.3.3

Effect of temperature on GNRs

acidic and alkaline environment initially for 2 h and

Gold nanorods are stable at 28-60°C with no

then for 1 month. Absorption spectra after 2 h present

significant change in absorbance intensity and

stable GNRs at almost all pH except 14. Under acidic

wavelength at 2 minutes heating (PIP < 0.1) making

conditions GNRs are stable after one month.

them a good candidate for heat assisted external

However, absorption intensity decreases considerably

stimuli

at neutral pH with slight blue shift (decrease in size).

reported

At alkaline conditions GNRs aggregate after 1

However, significant change in absorbance at 90-

month. PIP values greater than 0.2 indicate

100°C is recorded (Figure 9). LSPR blue shift is

aggregation of particles at alkaline pH. At alkaline

observed, probably due to disruption of CTAB

pH, considerable uncertainty in the stability of GNRs

bilayer. Both length and width become broader after

is observed. Therefore, it may be assumed that acidic

thermal treatment. Surface melting phenomenon may

and neutral pH conditions are suitable to keep GNRs

be involved in shortening of LSPR and increase in

stable. Extracellular environment of tumor tissue is

average diameter. Possible explanation may be that

often acidic [34]. Gold nanorods for use in cancer

tips of GNRs are more reactive than the body, hence

theranostics must not aggregate at acidic and

at higher temperature the tips of smaller GNRs would

physiological pH in order to deliver their payload at

like to dissolve first, the resultant cluster deposit on

the site of action. In previous studies GNRs aggregate

the body of the larger GNRs [36].


responsive GNRs

experiments.

instability

above

Earlier 40°C

study [35].


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Figure 6: Overall stability of GNRs in response to different growth parameters. The aspect ratio of GNRs decrease by increasing seed concentration in growth solution and increase by increasing Ag+ ion and salt concentration in growth solution. Sphere formation is observed, when pH of GNRs growth solution is raised. 3.3.4

Effect of ionic concentration on GNRs

electronic double layer along the particle surface

stability

introduce repulsive force among nanoparticles,

GNRs must be stable in buffer solution at

minimize aggregation and

potentially lead to

physiological pH for best use in biological systems.

stabilization of GNRs [17]. GNRs remain resistant to

In current study two plasmon bands of GNRs change

extreme low and high salt concentrations. We may

slightly from 5 to 10 mM NaCl concentration

assume that GNRs show non-monotonic behavior in

indicative of particles stability (PIP < 0.1), while

ionic solution [37].

aggregation is observed from 10-100 mM. Minimal

3.3.5

Stability of GNRs in cellular media

aggregation of GNRs is observed when salt

solutions

concentration is above 100 mM (PIP > 0.1)

Recent studies have demonstrated that NPs

(Figure10). At salt concentration 100 mM), the Cl-anions are

biomedical applications [39]. These biomolecules

sufficient enough to completely bind the positively

mostly

charge surface of GNRs, the phenomena lead to

nanoparticle

neutralization of surface charges. Consequently, an

interactions, Au-N and Au-S bonding [40]. Behavior

interacts


include

with

biological

proteins surface

that

systems

adsorb

through

[38].

to

the

electrostatic

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of GNRs in serum albumin (BSA and HSA), whole

while serum albumins are negatively charged at

serum

neutral pH. Hence, the electrostatic interaction

(FBS)

and

immunoglobulin

protein

at

physiological pH is investigated. Very little change in

between

wavelength and absorption intensity of GNRs is

nanoparticles may cause CTAB bilayer disruption.

observed in presence of FBS, BSA and HSA.

Immunoglobulin are membrane proteins, these may

However, GNRs are more stable in immunoglobulin

bind to GNRs by inserting their Fc region into CTAB

protein (PIP < 0.1). PEG coated GNRs are more

bilayer and interact with GNRs. GNRs surface layer

stable in proteins solution than CTAB coated at PIP

is

value less than 0.1 (Figure11). It is hypothesized that

Interactions

these proteins have ability to form a stable protein

hydrophobic surface are only possible in non-

corona on the GNRs surface which prevent nanorods

pegylated area.

not

aggregation CTAB-GNRs are positively charged


oppositely

disrupted

charged

during

between

proteins

protein

plasma

and

adsorption.

proteins

and


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Figure 7: Particle instability parameter (PIP) value as a stability index. A). PIP versus number of washes. Less aggregation up to 3-time washing is observed; B) PIP versus time (months) plot; GNRs stability study up to 04 months. GNRs in 0.001 M CTAB solution are stable for 3 months at room temperature (22-25°C) without compromising considerable change in the LSPR peak intensity (PIP > 0.1). UV-Vis spectroscopic analysis shows decrease in peak intensity without a notable wavelength shift in LSPR peak (810-817 nm).


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Figure 8: Stability of GNRs with respect to pH. (A) UV–Vis spectra of synthesized GNRs after 2 h at room temperature; (B) after 1 month at room temperature. Loss of Gaussian shape at pH 8-14 is visible; (C) PIP values plot of GNRs stability after 2 h and 1 month shows particle degradation at pH 10-14 (PIP > 0.1) after 2 h. GNRs remain stable in acidic condition for 1 month (PIP < 0.1).



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A

Figure 9: (A). Plot of LSPR absorption peak wavelength in response to temperature. Decrease in LSPR is observed when temperature is raised above 60°C. Statistical analysis shows no significance difference in LSPR when heated from 28 to 60°C for 2 min (p 0.1); (B). UV–Vis absorption spectra of GNRs in response to different salt concentrations at room temperature.


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Figure 11: Stability studies of GNRs in cellular media. Significant difference between CTAB coated and PEG coated GNRs is observed at p< 0.05 in case of three serum proteins.

acidic pH. PEGylated GNRs are comparatively more 4.

Conclusion stable in cellular media solution (PIP < 0.1) than We have developed a facile and efficient CTAB coated GNRs. Results suggest that current

method for GNRs synthesis by optimizing reactant optimized conditions have

great potential for

concentrations, reaction pH and using less amount of controlled production of mono-disperse and tunable CTAB. The diameter of GNRs is 20-50 nm and their GNRs with high reproducibility. Environmental and LSPR peak varies from 682 to 906 nm with change in biological stability make gold nanorods a good amount of seed solution and silver ions. Quantitative candidate for a wide array of biomedical application. analysis of GNRs colloidal stability was studied by 5.

Acknowledgements

recording LSPR spectral response against various We are thankful to Pakistan Science environmental and biological parameters at particle Foundation Islamabad, PK, for financial assistance instability parameter (PIP) > 0.1. The GNRs are (PSF/Res/C-NILOP/Med (330). Part of study is stable for 3 months in 0.001 M CTAB without facilitated through Pak-Norway grant PK3004, compromising peak intensity and wavelength. GNRs Planning Commission of Pakistan. TEM work is remain stable at temperature below 60°C, at carried out on NORTEM JEOL JEM-2100, TEM physiological salt concentration, neutral and slightly Gemini Centre, Norwegian University of Science and



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Technology (NTNU), Norway, Courtesy of Prof. Dr.

Noor Muhammad Butt, PINSAT Islamabad, Pakistan

Catharina de Lange Davies (Norwegian counterpart

for technical support.

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