Dual-stimuli responsive and reversibly activatable theranostic nanoprobe for precision tumor-targeting and fluorescence-guided photothermal therapy

The integrated functions of diagnostics and therapeutics make theranostics great potential for personalized medicine. Stimulus-responsive therapy allows spatial control of therapeutic effect only in the site of interest, and offers promising opportunities for imaging-guided precision therapy. However, the imaging strategies in previous stimulus-responsive therapies are ‘always on' or irreversible ‘turn on' modality, resulting in poor signal-to-noise ratios or even ‘false positive' results. Here we show the design of dual-stimuli-responsive and reversibly activatable nanoprobe for precision tumour-targeting and fluorescence-guided photothermal therapy. We fabricate the nanoprobe from asymmetric cyanine and glycosyl-functionalized gold nanorods (AuNRs) with matrix metalloproteinases (MMPs)-specific peptide as a linker to achieve MMPs/pH synergistic and pH reversible activation. The unique activation and glycosyl targetibility makes the nanoprobe bright only in tumour sites with negligible background, while AuNRs and asymmetric cyanine give synergistic photothermal effect. This work paves the way to designing efficient nanoprobes for precision theranostics.


Preparation of CTAB@AuNRs
CTAB@AuNRs was prepared according to Wang

Synthesis of H 2 N-GPLGVRGC-SH peptide modified asymmetric cyanine (Pep-Acy)
To a DMSO solution of the asymmetric cyanine (Acy) prepared according to our previous work 2  Supplementary Fig. 1b).  Fig. 19b).

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The mixture of NHS (1.27 g, 11.03 mmol) and EDC•HCl (2.10 g, 10.95 mmol) was added to the DMSO solution of 11-mercaptoundecanoic acid (1.06 g, 9.99 mmol) at room temperature under N 2 atmosphere. After stirring for 12 h, D-glucosamine hydrochloride (2.16 g, 10.02 mmol) and DIPEA (5.0 mL, 30.25 mmol) were added. The reaction mixture was continually stirred for an additional 8 h, and then poured into water (200 mL). The precipitation was collected by filtration and re-dissolved in 1.0 mol/L NaOH solution (100 mL). After that, the resulting solution was filtered to remove the residue and then acidified to pH 5.0 with 1 mol/L HCl solution. The  Fig. 2c).

Preparation of Pep-Acy@AuNRs
10 mL of CTAB@AuNRs solution was first centrifuged twice at 8000 rpm for 5 min to remove the excess CTAB and then re-suspended in the same volume of high-purity water. To the purified CTAB@AuNRs solution, 2 mL anhydrous DMSO solution of Pep-Acy (2 mg, 1.56 μmol) was added dropwise under vigorous stirring. After stirring for 24 at room temperature in the dark, the solution was centrifuged at 8000 rpm for 5 min at room temperature to remove the excess Pep-Acy. The resulting residue was washed three times with DMSO-water (1:9, v/v) to further remove the unbound Pep-Acy, and then re-dispersed in high-purity water to obtain Pep-Acy@AuNRs. The prepared Pep-Acy@AuNRs was kept in dark for further use.

Quantitative analysis of Pep-Acy on the surface of AuNRs and calculation of the fluorescence quenching efficiency
To determine the concentration of Pep-Acy bound to the surface of AuNRs, UV-vis titration of asymmetric cyanine (Acy) was carried out at pH 7.4 ( Supplementary Fig. 7). Then, the solution of Pep-Acy/Glu@AuNRs at 60 μg/mL (as Au, determined by AAS) was treated with DTT (20 mM) and NaCl (4 M) for 30 min (the strong binding ability of DTT to AuNRs makes Pep-Acy depart from AuNRs 3-5 ), and then the resulting mixture was centrifuged. The supernatant was collected and its absorbance at ca. 525 nm was measured by UV-vis-NIR spectroscopy. The concentration of the Acy in the supernatant was determined with a standard calibration method and then the amount of Pep-Acy in the above Pep-Acy/Glu@AuNRs was further calculated.
Besides, each AuNR can be assumed to a cylindrical middle and two spherical segments 6 .
The radius (r) and height (h) of cylindrical middle were 5.56 nm and 40.03 nm, respectively, while S29 the base radius (r) and height (a) of spherical segment were 5.56 nm and 5.04 nm, respectively (determined by TEM images, Supplementary Fig. 3a). Hence, the volume of the AuNR was calculated as follow: V = πa (3r 2 +a 2 )/3 +πr 2 h = 4508.89 nm 3 . The amount of Au of each AuNR was determined by the formula: amount of Au/AuNR = ρ Au VM Au /NA = 5.24 × 10 7 / NA, Where ρ Au = 59 atoms/nm 3 , is the density of gold atoms in the bulk; M Au = 197 g/mol, is the molar mass of Au. As the amount of Au in the AuNRs solution is 60 μg/mL, the number of the gold rods in this solution was calculated to be 1.45 × 10 -9 NA/L. Finally, the grafting density of peptide-probe on each AuNR surface was calculated to be 6234.
To calculation of the fluorescence quenching efficiency, the solution of Pep-Acy/Glu@AuNRs (60 μg/mL, as Au) was treated with DTT (20 mM) and NaCl (4 M) for 30 min and adjusted to pH 6.0 to recover the de-quenched state. Then, the fluorescence spectra of Pep-Acy/Glu@AuNRs in its de-quenched state and quenched state (untreated with DTT, pH 7.4) were recorded (Supplementary Fig. 8a and 8b). The fluorescence quenching efficiency (QE) was calculated via the formula: QE = (1-β) × 100%, where β is the ratio of fluorescence intensity of the quenched to completely de-quenched state 5 .

Evaluation of cytotoxicity and in vitro photothermal therapy
Standard MTT assay was carried out to evaluate the cytotoxicity and in vitro photothermal therapy. Briefly, both SCC-7 and 293T cells were separately seeded into a 96-well plate at a density of 1 × 10 4 cells per well and incubated overnight, then the medium was replaced with fresh medium containing a series of concentration of CTAB@AuNRs, Pep-Acy@AuNRs or Pep-Acy/Glu@AuNRs (7.5, 15, 30, 60 and 120 μg mL -1 ). Each concentration was done in quintuplicate. The cells incubated with no AuNRs were used as the blank control. After incubation S30 for 24 h, the medium was removed and fresh medium containing MTT (0.5 mg mL -1 ) was added to each well followed by incubation for an additional 4 h. Afterwards, the medium was removed and rinsed with cold PBS, then 100 μL DMSO was added to each well to dissolve the formazan crystals generated by living cells. Finally, the absorbance at 570 nm of each well was recorded on a microplate reader before the plate was vibrated for 20 min in the dark. The relative cell viability (%) was then calculated by the following formula: viability (%) = (the average absorbance of test group / the average absorbance of the blank control) × 100.
For photothermal therapy, SCC-7 cells were seeded in 96-well plate at 1 × 10 4 cells per well and incubated for 24 h until adherence, then incubated with Pep-Acy/Glu@AuNRs (60 μg mL -1 ) for an additional 12 h. After that, the cells in each well were subjected to irradiation with an 808 nm laser at various power densities (0.2, 0.4 and 0.6 w cm -2 ) for different times (3, 6 and 10 min).
The cells irradiated under the same conditions but without AuNRs were employed as blank controls. Each experiment group was done in quintuplicate as well. After irradiation, the cells were further incubated for 12 h and then the cell viability was evaluated following the similar procedure mentioned above.