Improved Cellular Specificity of Plasmonic Nanobubbles versus Nanoparticles in Heterogeneous Cell Systems

The limited specificity of nanoparticle (NP) uptake by target cells associated with a disease is one of the principal challenges of nanomedicine. Using the threshold mechanism of plasmonic nanobubble (PNB) generation and enhanced accumulation and clustering of gold nanoparticles in target cells, we increased the specificity of PNB generation and detection in target versus non-target cells by more than one order of magnitude compared to the specificity of NP uptake by the same cells. This improved cellular specificity of PNBs was demonstrated in six different cell models representing diverse molecular targets such as epidermal growth factor receptor, CD3 receptor, prostate specific membrane antigen and mucin molecule MUC1. Thus PNBs may be a universal method and nano-agent that overcome the problem of non-specific uptake of NPs by non-target cells and improve the specificity of NP-based diagnostics, therapeutics and theranostics at the cell level

Tunable Plasmonic Nanoprobes for Theranostics of Prostate Cancer

Theranostic applications require coupling of diagnosis and therapy, a high degree of specificity and adaptability to delivery methods compatible with clinical practice. The tunable physical and biological effects of selective targeting and activation of plasmonic nanobubbles (PNB) were studied in a heterogeneous biological microenvironment of prostate cancer and stromal cells. All cells were targeted with conjugates of gold nanoparticles (NPs) through an antibody-receptor-endocytosis-nanocluster mechanism that produced NP clusters. The simultaneous pulsed optical activation of intracellular NP clusters at several wavelengths resulted in higher optical contrast and therapeutic selectivity of PNBs compared with those of gold NPs alone. The developed mechanism was termed &#8220;rainbow plasmonic nanobubbles.&#8221; The cellular effect of rainbow PNBs was tuned in situ in target cells, thus supporting a theranostic algorithm of prostate cancer cell detection and follow-up guided destruction without damage to collateral cells. The specificity and tunability of PNBs is promising for theranostic applications and we discuss a fiber optic platform that will capitalize on these features to bring theranostic tools to the clinic.</p

Influence of targeting vectors on NP scattering amplitude (red) and PNB lifetime (blue) in individual target (solid bars) and non-target (hollow bars) cells.

<p>A: Target (HN31) and non-target (NOM9) cells identically treated with bare 60 nm gold NSPs and NSP-Panitumumab conjugates (antibody specific to EGFR that is overexpressed in HN31 cells); B: Effects of EGFR-specific antibodies C225 and Panitumumab as targeting vectors in HN31 cell model show 5 different combinations of the two antibodies; C: Effects of single and dual targeting antibodies against PSMA and EGFR (C225) in C4-2B cell model applied in combination with dual simultaneous optical excitation (so called rainbow PNB method) show synergistic enhancement of PNB lifetime in the rainbow mode.</p

Images and signals of gold NPs and PNBs in co-culture of target (HN31, labeled with Green Fluorescent Protein for identification) and non-target (NOM9) cells identically treated with 60 nm gold NSP-C225 conjugates (specific to EGFR that is overexpressed in target cells).

<p>A: overlay of bright field, fluorescent and scattering images shows target cells (green) and gold NPs (red) that can be found in both types of cells (the arrows show NP clusters in non-target cells); B: time-resolved scattering image of the same field shows PNB images (bright white spots) only in target cells; C,D: optical scattering time-responses of individual target (C) and non-target (D) cells show the PNB-specific signal only for target cell and the definition of the PNB lifetime of PNBs; time is measured from the moment of the exposure to the excitation laser pulse.</p

Parameters of PNBs generated around gold NP clusters in water for gold nanoshells.

<p>A: PNB generation threshold fluence of the excitation laser pulse as function of NP cluster size (measured through optical scattering amplitude of NP cluster image for individual clusters); B: PNB lifetime and scattering brightness as function of the NP cluster size (measured through optical scattering amplitude of NP cluster image) at specific fluence of the excitation pulse (778 nm, 22 mJ/cm²).</p

Cell models and conditions of their treatment with NP and laser pulses.

<p>Cell models and conditions of their treatment with NP and laser pulses.</p

Cell population-averaged levels of optical scattering signals obtained for individual target (solid bar) and non-target (hollow bar) cells in six cell models represented by target/non-target cells/molecular targets:

<p>Squamous cell carcinoma, HN31/NOM9/EGFR (treated with 50 nm NS-Panitumumab conjugates); Lung cancer, A549/Fibroblast/EGFR (treated with 60 nm NSP-C225 conjugates); Epithelial cancer, HES/HS5/MUC1 (treated with 60 nm NSP-214D4 conjugates); Prostate cancer, C2-4B/HS5/PSMA (treated with 60 nm NSP-anti-PSMA conjugates); Leukemia, J32/JRT3-T3.5/CD3 and human T-cells, T-cell/BM/CD3 (treated with 60 nm NSP-OKT3 conjugates) for: Row A (red): gold NP amplitude of scattering image of gold NPs (a metric for the uptake of NPs by cells; Row B (purple): time-resolved scattering image amplitudes of PNBs; Row C (blue): PNB lifetimes. The ratio of the signals for target/non-target cell is shown for each parameter and cell model and indicates the cellular specificity of NPs (row A) and PNBs (rows B,C).</p