The synthesis and characterisation of folic acid conjugated silica nanocages for cancer cell targeting

K. Patel1, X. Lou1*

School of Chemical Engineering, Curtin University, Bentley, WA, Australia *Corresponding author: Email x.lou@curtin.edu.au

Abstract: Zinc oxide (ZnO) template particles of various sizes were prepared. Templates were coated by a silica layer and later functionalised with active amino groups. A folic acid (FA) targeting ligand was then conjugated to the surface of the particles by virtue of the active surface functional groups. ZnO cores were leached, leaving a cavity for incorporation of therapeutic and or diagnostic agents. 1 Introduction: Silica hybrids, among many other evaluated nanoparticles have shown great potential for applications in intracellular delivery and optical imaging [1]. The incorporation of cancer cell targeting ligands to such particles would allow for a reduction in negative side effects seen with the use of present day chemotherapeutics [2] through increased site specificity. The folate receptor, known to be over expressed by many malignant cell lines, is highly restricted in most normal tissues [2]. It is also known to partake in receptor mediated endocytosis; whereby folate, the FA salt pteroylglutamate is transported to the cytosol of the cell [3]. The very high affinity (K d ~1 nM) FA has for its cell surface receptor makes it an ideal ligand for cancer cell targeting [2]. We report herein the synthesis and characterisation of FA conjugated silica nanocages for the site specific delivery of chemotherapeutic and diagnostic agents to cancer cells. 2 Materials & Methods: Monodisperse ZnO nanoparticles were synthesised via the hydrolysis of zinc acetate dihydrate using a reported method [4] with slight modification. The template ZnO particles were coated by a silica layer using tetraethylorthosilicate (TEOS) via simple sol-gel process [5]. The silica layer was functionalised by an active amino group using the silane coupling reagent 3-aminopropyl triethoxysilane (APTES). Conjugation of FA was achieved by first preparing the

Nhydroxysuccinimide (NHS) ester of the targeting ligand, followed by attachment to the active surface amino groups (Fig. 1). The particles were then treated with dilute hydrochloric acid solutions to remove ZnO, leaving hollow cages for incorporation of chemotherapeutic and diagnostic agents. Size and morphology of nanoparticles were scrutinized at each stage of the synthetic process using a Zeiss Neon EsB focussed ion beam scanning electron microscope (SEM) and a JOEL Jem2011 transmission electron microscope (TEM). Dynamic light scattering (DLS) was also used to corroborate size and distribution results using a Zetasizer NanoZS from Malvern Instruments. ATRFTIR and UV-Visible absorbance techniques were used for the characterisation of chemical compositions.
O . O Si . O NH2 O O N O

ZnO

+
O

OH

Amino functionalised nanoparticle

O OH O HN O Si O . .

NHS-Folate

ZnO

Folic acid conjugated nanoparticle

Figure 1 Synthetic mechanism for the conjugation of NHS-folate (preparation scheme for NHS-Folate not shown) to the active amino groups on to the nanoparticle surface.

3 Results & Discussion: ZnO template particles of three average sizes were fabricated. DLS results suggested average hydrodynamic diameters (HDs) of 47020nm (PDI 0.187), 35526nm (PDI 0.143) and 22311nm (PDI 0.099) respectively. The average geometric diameter (GD) via SEM showed a diminution in average size as expected [6] of 18nm, 22nm, and 8nm respectively, all of which agree within experimental error to DLS results. Via DLS, FA conjugation saw an increase in the HD of the largest batch by 837nm, implying a

Normalised Absorbance

UV-Vis Spectra of FA Conjugated Nanoparticles ZnO-SiO2-FA SiO2-FAB3-Hollow Folic Acid

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

200

shell thickness of 41.5nm. Using TEM imaging (Fig. 3D), the average geometric shell thickness was measured to be roughly 40nm, in agreement within 4% and again smaller than the HD, as expected according to theory [6]. Shell thickness was evaluated analogously for smaller particles.
100 Transmittance (%) 99 98 97 96 95 94 3250 2250 Wavenumber (cm-1) 1250 ZnO-SiO2-FAB4 ZnO-SiO2-APTES ATR FT-IR Spectra for ZnO-SiO2-FA & ZnO-SiO2-APTES Nanoparticles

300 400 Wavelength (nm)

500

600

Figure 4 UV-Vis absorbance spectra for free FA (folic acid), FA conjugated ZnO particles (ZnO-SiO 2 -FA) and hollow FA conjugated silica cages.

hollow FA and non-hollow particles were slightly blue shifted from the ~280nm absorbance peak of free FA. This confirmed the presence of conjugated FA on the particle surfaces. Any absorbance at ~370nm would correspond to the energy band gap of ZnO. Absence of this peak in the spectra of hollow particles indicated the complete removal of ZnO. 4 Conclusion: A new class of silica nanocage structure was produced using a simple approach. The size, and shell thickness was easily controllable by varying the size of template particles and chemical compositions used in reactions. Incorporation of FA, a cancer cell targeting ligand, should allow for the site specific delivery of chemotherapeutic and or diagnostic payloads that can easily be held within the hollow cages. References
[1] M. Mulner, A. Schallon, A. Walther, R. Freitag & A. H. E. Muller, Biomarcomolecules, 2010, 11, pp 390-396. [2] J. Byrne, T. Betancourt & L. Brannon-Peppas, Ad. Drug Delivery Rev., 2008, 60, pp 16151626. [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.

Figure 3 ATR-FTIR spectra for amino functionalised ZnO nanoparticles (ZnO-SiO 2 -APTES) and FA conjugated nanoparticles (ZnO-SiO 2 -FAB4).

ATRFTIR was used to identify successful coating of silica, amino functionalization and conjugation of FA. Strong bands between 1100cm-1 and 1200cm-1 correspond to silanol groups (not shown) within the silica layer [7]. CH stretching modes at 2929cm-1 and 2850cm-1 and the NH bending mode at 1624cm-1 confirm successful functionalization of the silane coupling reagent and the presence of active amino groups [8]. FA conjugation was confirmed by the appearance of peaks at 1573cm-1 and 1537cm-1, corresponding to newly formed amide bonds [8] (NH amide band II). The NH stretching mode at 3323cm-1 intensified, also indicating successful conjugation of FA to the nanoparticles. UV-visible spectroscopy was used to confirm findings from FTIR. Absorbance peaks at ~275nm in the spectra of both