7/28/2019 (28) the Synthesis and Characterisation of Folic Acid Conjugated Silica Nanocages for Biomedical Applications

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The synthesis and characterisation of folic acid conjugated sil-ica nanocages for cancer cell targeting

K. Patel1, X. Lou1*1School of Chemical Engineering, Curtin University, Bentley, WA, Australia

*Corresponding author: Emailx.lou@curtin.edu.au

Abstract: Zinc oxide (ZnO) template parti-cles of various sizes were prepared. Tem-plates were coated by a silica layer andlater functionalised with active aminogroups. A folic acid (FA) targeting ligandwas then conjugated to the surface of theparticles by virtue of the active surfacefunctional groups. ZnO cores wereleached, leaving a cavity for incorporationof therapeutic and or diagnostic agents.

1 Introduction: Silica hybrids, amongmany other evaluated nanoparticles haveshown great potential for applications inintracellular delivery and optical imaging[1]. The incorporation of cancer cell target-ing ligands to such particles would allow fora reduction in negative side effects seenwith the use of present day chemothera-peutics [2] through increased site specific-ity. The folate receptor, known to be overexpressed by many malignant cell lines, ishighly restricted in most normal tissues [2].It is also known to partake in receptor me-diated endocytosis; whereby folate, the FAsalt pteroylglutamate is transported to thecytosol of the cell [3]. The very high affinity(Kd ~1 nM) FA has for its cell surface re-ceptor makes it an ideal ligand for cancercell targeting [2]. We report herein the syn-thesis and characterisation of FA conju-gated silica nanocages for the site specific

delivery of chemotherapeutic and diagnos-tic agents to cancer cells.

2 Materials & Methods: MonodisperseZnO nanoparticles were synthesised viathe hydrolysis of zinc acetate dihydrate us-ing a reported method [4] with slight modi-fication. The template ZnO particles werecoated by a silica layer using tetraethylor-thosilicate (TEOS) via simple sol-gel proc-ess [5]. The silica layer was functionalisedby an active amino group using the silane

coupling reagent 3-aminopropyl triethoxysi-lane (APTES). Conjugation of FA wasachieved by first preparing the

Nhydroxysuccinimide (NHS) ester of thetargeting ligand, followed by attachment tothe active surface amino groups (Fig. 1).

The particles were then treated with dilutehydrochloric acidsolutionsto remove ZnO,leaving hollow cages for incorporation ofchemotherapeutic and diagnostic agents.

Size and morphology of nanoparticleswere scrutinized at each stage of the syn-thetic process using a Zeiss Neon EsB fo-

cussed ion beam scanning electron micro-scope (SEM) and a J OEL J em2011 trans-mission electron microscope (TEM). Dy-namic light scattering(DLS) was also usedto corroborate size and distribution resultsusing a Zetasizer NanoZS from MalvernInstruments. ATRFTIR and UV-Visible ab-sorbance techniques were used for thecharacterisation of chemical compositions.

Si

O

O

.

.NH2

Amino functionalised nanoparticle

O

OH

R

O

O N

O

O

+

NHS-Folate

Si

O

O

.

.

NH

O

OH

R

O

Folic acid conjugated nanoparticle

ZnO

ZnO

Figure 1 Synthetic mechanism for the conju-gation of NHS-folate (preparation scheme forNHS-Folate not shown) to the active amino

groups on to the nanoparticle surface.3 Results & Discussion: ZnO templateparticles of three average sizes were fabri-cated. DLS results suggested average hy-drodynamic diameters (HDs) of 47020nm(PDI 0.187), 35526nm (PDI 0.143) and22311nm (PDI 0.099) respectively. Theaverage geometric diameter (GD) via SEMshowed a diminution in average size asexpected [6] of 18nm, 22nm, and 8nm re-spectively, all of which agree within ex-perimental error to DLS results. Via DLS,FA conjugation saw an increase in the HDof the largest batch by 837nm, implying a

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7/28/2019 (28) the Synthesis and Characterisation of Folic Acid Conjugated Silica Nanocages for Biomedical Applications

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Figure 2 A) SEM micrograph of FA conjugatedZnO nanoparticle. B) TEM micrograph of hollowFA conjugated silica cages (100nm scale bars).

shell thickness of 41.5nm. Using TEM im-aging (Fig. 3D), the average geometricshell thickness was measured to beroughly 40nm, in agreement within 4% andagain smaller than the HD, as expectedaccording to theory [6]. Shell thickness wasevaluated analogously for smaller particles.

Figure 3 ATR-FTIR spectra for amino function-

alised ZnO nanoparticles (ZnO-SiO2-APTES) and

FA conjugated nanoparticles (ZnO-SiO2-FAB4).

ATRFTIR was used to identify suc-cessful coating of silica, amino functionali-zation and conjugation of FA. Strong bandsbetween 1100cm-1 and 1200cm-1 corre-spond to silanol groups (not shown) withinthe silica layer [7]. CH stretching modes at

2929cm

-1

and 2850cm

-1

and the NH bend-ing mode at 1624cm-1 confirm successfulfunctionalization of the silane coupling re-agent and the presence of active aminogroups [8]. FA conjugation was confirmedby the appearance of peaks at 1573cm-1

and 1537cm-1, corresponding to newlyformed amide bonds [8] (NH amide bandII). The NH stretching mode at 3323cm-1intensified, also indicating successful con-

jugation of FA to the nanoparticles.

UV-visible spectroscopy was used toconfirm findings from FTIR. Absorbancepeaks at ~275nm in the spectra of both

Figure 4 UV-Vis absorbance spectra for freeFA (folic acid), FA conjugated ZnO particles(ZnO-SiO2-FA) and hollow FA conjugated silicacages.

hollow FA and non-hollow particles wereslightly blue shifted from the ~280nm ab-

sorbance peak of free FA. This confirmedthe presence of conjugated FA on the par-ticle surfaces. Any absorbance at ~370nmwould correspond to the energy band gapof ZnO. Absence of this peak in the spectraof hollow particles indicated the completeremoval of ZnO.

4 Conclusion: A new class of silica nano-cage structure was produced using a sim-ple approach. The size, and shell thicknesswas easily controllable by varying the size

of template particles and chemical compo-sitions used in reactions. Incorporation ofFA, a cancer cell targeting ligand, shouldallow for the site specific delivery of che-motherapeutic and or diagnostic payloadsthat can easily be held within the hollowcages.

References

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pp 390-396.[2] J . Byrne, T. Betancourt & L. Brannon-Peppas,

Ad. Drug Delivery Rev., 2008, 60, pp 1615-1626.

[3] A. Garcia-Bennett, M. Nees & B. Fadeel,Biochem. Pharmacol., 2011, 81, pp 976-984.

[4] K. Han, Z. Zhao, Z. Xiang, C. Wang, J . Zhang &B. Yang, Mater. Lett., 2007, 61, pp 363-368

[5] X. Lou, T. Schumacher, H. Yang & A.Ding, J .Controlled Release, 2011, 152, pp e65-e67

[6] D. Xuan, X. Sheng-Hua, S. Zhi-Wei & A. Yan,Acta Phys. Chim. Sin., 2010, 26, pp2807-2812.

[7] P. Huang, L. Bao, C. Zhang, J .lin, et al.

Biomaterials, 2011, 32, pp9796-9809.[8] S. Sahu, S. Mallick, S. Santra, T. Maitl, S. Gosh

& P. Pramanik, J . Mater. Sci.: Mater. Med.,2010, 21, pp 1587-1597.

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125022503250

Transmittance(%)

Wavenumber (cm-1)

ATR FT-IR Spect ra for ZnO-SiO2-FA &ZnO-SiO2-APTES Nanopartic les

ZnO-SiO2-FAB4

ZnO-SiO2-APTES

200 300 400 500 600NormalisedAbsorbance

Wavelength (nm)

UV-Vis Spectra of FA ConjugatedNanoparticles

ZnO-SiO2-FA

SiO2-FAB3-Hollow

Folic Acid