Theranostic is an approach combining diagnosis and therapy to increase the precision and efficacy of cancer treatment. Different nanoparticle (NP) platforms have been engineered to obtain both functions by uniting them in one agent; however none of these can overcome the limitation in specificity of NP targeting, which is still a major challenge in cancer treatment. Plasmonic nanobubbles (PNB) are a new class of nano-agent that was developed to overcome this limitation. A PNB is a vapor nanobubble transiently generated around superheated gold NPs by a short laser pulse. The rapidly expanding bubble mechanically disrupts the surrounding tissue and achieves micrometer precision in tissue ablation. In general, PNB therapy relies on receptor-mediated endocytosis of gold NPs and formation of gold clusters within the cells [1],[2]. However, variability in the cellular uptake and subsequent aggregation of gold NPs may be detrimental to reproducibility of PNB. In this study, M13 and T4 phage display platforms are used to synthesize well-defined gold nano-aggregates and their efficiency in formation of gold nano-aggregates are evaluated.
Two different T4 phage templates were designed by displaying cysteine and gold binding A3 peptide (AYSSGAPPMPPF) (A: Alanine, Y: Tyrosine, S: Serine, G: Glycine, P: Proline, M: Methionine, F: Phenylalanine, L: Leucine, K: Lysine, H: Histidine, R: Arginine, V: Valine, D: Aspartic acid) on small outer capsid (SOC) protein of phage through the homologous recombination between T4-Z1 mutant phage and pRH recombination plasmid. Ph.D. Peptide Display Cloning System (Biolabs Inc.) was used to design M13 phage templates. Self assembly of gold NP was directed by displaying gold binding peptide sequence (VSGSSPDS) on p8 major coat protein of phage. M13 phage templates were also engineered to display RGD peptides (ACDCRGDCFC, ACRGDGWC, GRGDSP) (C; Cysteine, W; Tryptophan) on p3 minor coat protein to target cancer cells through integrin protein. Recombinant phages were analyzed by PCR and also verified via DNA sequencing. The affinities of RGD displaying M13 phages were tested against human integrin alpha 5 protein fragment by phage-ELISA and M13 phages displaying cyclic RGD peptides (with two and four cysteines) showed significant binding affinity to integrin.
Gold NPs were synthesized according to the protocol of Zahr and Blum and formation of gold nano-aggregates was initially studied by using cysteine bearing T4 phage template [3]. Conjugation of gold NPs to phage capsid was monitored by using UV-Vis spectrometer and the growth of plasmon at 600 nm was recorded. After 3 days of reaction, the samples were visualized by transmission electron microscopy (TEM) and formation of phage-templated gold nano-aggregates was confirmed.
Herein, it was shown that cysteine-displaying T4 phage was successfully used as a template to develop well-defined gold nano-aggregates. The number of functional moieties on phage surface can be controlled at gene level and make us to avoid the use of irreproducible functionalization steps. The future work will focus on optimization of gold nano-aggregate formation for each phage design and their efficiency in PNB generation will be investigated.
We would like to acknowledge that T4Z1 mutant phage and pRH recombination plasmid were kindly supplied by Prof. Lindsay W. Black (University of Maryland, US).
References:
[1] Wagner, D.S., Delk, N.A., Lukianova-Hleb, E.Y., Hafner, J.H., Farach-Carson, M.C., Lapotko, D.O. Biomaterials 2010, 31, 7567-7574.
[2] Lukianova-Hleb, E.Y., Lapotko, D.O. IEEE Journal of Selected Topics in Quantum Electronics 2014, 20, 7300412- 7300412.
[3] Zahr, O.K., Blum, A.S., NanoLetters 2012, 12, 629-633.