Plasmonic nanobubbles rapidly detect and destroy drug-resistant tumors

Abstract

The resistance of residual cancer cells after oncological resection to adjuvant chemoradiotherapies results in both high recurrence rates and high non-specific tissue toxicity, thus preventing the successful treatment of such cancers as head and neck squamous cell carcinoma (HNSCC). The patients' survival rate and quality of life therefore depend upon the efficacy, selectivity and low non-specific toxicity of the adjuvant treatment. We report a novel, theranostic in vivo technology that unites both the acoustic diagnostics and guided intracellular delivery of anti-tumor drug (liposome-encapsulated doxorubicin, Doxil) in one rapid process, namely a pulsed laser-activated plasmonic nanobubble (PNB). HNSCC-bearing mice were treated with gold nanoparticle conjugates, Doxil, and single near-infrared laser pulses of low energy. Tumor-specific clusters of gold nanoparticles (solid gold spheres) converted the optical pulses into localized PNBs. The acoustic signals of the PNB detected the tumor with high specificity and sensitivity. The mechanical impact of the PNB, co-localized with Doxil liposomes, selectively ejected the drug into the cytoplasm of cancer cells. Cancer cell-specific generation of PNBs and their intracellular co-localization with Doxil improved the in vivo therapeutic efficacy from 5-7% for administration of only Doxil or PNBs alone to 90% thus demonstrating the synergistic therapeutic effect of the PNB-based intracellular drug release. This mechanism also reduced the non-specific toxicity of Doxil below a detectable level and the treatment time to less than one minute. Thus PNBs combine highly sensitive diagnosis, overcome drug resistance and minimize non-specific toxicity in a single rapid theranostic procedure for intra-operative treatment.

Keywords: cancer cell; plasmonic nanobubble.

Figure 1

(A): Separate administration of gold NP conjugates and encapsulated drug; (B): cancer cell self-assembles mixed clusters of the drug carriers and gold NPs during receptor-mediated endocytosis; (C): diagnostic function is provided by the selective generation of PNB around the cluster of gold NPs with a single laser pulse and remote real-time detection of the acoustic response of the PNB; single gold NPs in normal (green) cells have a higher threshold of PNB generation and thus do not produce a PNB under a low level of laser pulse fluence; (D): selective therapeutic effect is provided by explosive localized disruption of drug carrier and endosome by PNB and the ejection of the drug (blue dots) into cytoplasm.

Figure 2

PNB-enhanced endosomal escape in vitro. (A): Confocal microscopy images of cancer (green boarder) and normal (adjacent) cells show NPs (blue) and Doxil (red) co-localized in the large mixed clusters (inset) only in cancer cell while adjacent normal cells show non-specific uptake of NPs and Doxil (scale bar is 5 μm); (B): time-resolved optical scattering image of the same sample shows PNBs selectively generated with a broad single laser pulse (780 nm, 70 ps, 40 mJ/cm²) only in cancer cell and their co-localization with NP-Doxil clusters; (C): PNB lifetime (blue bars) and cell death level (red bars) measured in 72 h after laser treatment for cancer (solid bars) and normal (hollow bars) cells after the application of several treatment modes (PNB+D: single laser pulse, NPs and Doxil, PNB: single laser pulse and NPs; NP+D: NPs and Doxil (* p < 0.001, ** p<0.05, *** p > 0.05); D: Doxil, intact: non-treated cells); (D): DL50 dose for cancer cells as function of drug dose and PNB lifetime.

Figure 3

NP-C225, solid gold 60 nm spheres conjugates in HNSCC-bearing mouse: transmission electron microscopy image of a tumor (A) and adjacent muscle tissue (B) in 24 h after systemic injection of NP-C225 to mouse. (C): Histogram of the PNB lifetime obtained for individual cells in slices of tumor for cancer (green) and normal (grey) cells after being extracted from mice in 24 hours after systemic administering of NP-C225.

Figure 4

PNBs in HNSCC-bearing mouse. (A): Functional diagram of the experimental set up, optical fiber delivers short laser pulse through the surface of the tissue, PNBs are detected with ultrasound detector, which signals, the scan of the fiber and laser pulse generation are controlled by computer program; (B): acoustic responses to single laser pulses (70ps, 780 nm, 40 mJ/cm²) obtained from a tumor (green) and adjacent muscle (black) in mouse systemically treated with 60 nm solid gold sphere conjugates, NP-C225; (C): Spectrum of PNB amplitudes of acoustic responses obtained from a squamous cell carcinoma tumor (solid green) and normal tissue (hollow black circles) of a mouse systemically treated with C225-conjugated 60 nm gold spheres, tumor in intact animal (solid black squares) and (40 mJ/cm² ); (D): Ddependence of the amplitudes of acoustic responses upon laser pulse fluence (energy per square unit) for the same animals obtained from the tumor (green) and adjacent muscle (black) (* p < 0.001); (E): acoustic amplitudes obtained during the scans of the tumor, muscle and blood of NP-treated animals and of a tumor of an intact animal (* p < 0.001); (F): dependence of PNB lifetime (blue, optical response obtained from individual tumor cells in vitro) and the acoustic amplitude of PNB (green, acoustic response obtained from a tumor in animal) as function of NP size (20 nm and 60 nm) and in vivo administration route (local intra-tumor injection and intra-venous injection) under identical laser excitation (single pulse,70 ps,780 nm, 40 mJ/cm²).

Figure 5

PNB therapeutics of HNSCC tumor in mouse. Tumor depth profiles in (A-C) show tumor (green) and necrotic tumor (yellow) for the treatment modes: (A, E, solid red): PNB+D: small PNBs (40 mJ/cm² @780 nm, 70 ps, single pulse, NP: gold 60 nm spheres conjugated to C225) and Doxil (1 mg/kg); (B, E, hollow red): PNBs alone; (C, E, hollow black): Doxil+NP; (D): Level of necrosis (red bars) at 0.3 mm depth and acoustic signals of PNBs (green bars) for the listed treatment modes (p < 0.001, ** p > 0.05). (E): Dependence of the level of necrosis in tumor upon tissue depth (PNB+D: solid red, PNB: hollow red, NP+D: hollow black, D: magenta, intact tumor: blue).