Nanotheranostics through Mitochondria-targeted Delivery with Fluorescent Peptidomimetic Nanohybrids for Apoptosis Induction of Brain Cancer Cells

Overview: Malignant brain tumors remain one of the greatest challenges faced by health professionals and scientists among the utmost lethal forms of cancer. Nanotheranostics can play a pivotal role in developing revolutionary nanoarchitectures with multifunctional and multimodal capabilities to fight cancer. Mitochondria are vital organelles to eukaryotic cells, which have been recognized as a significant target in cancer therapy where, by damaging the mitochondria, it will cause irreparable cell death or apoptosis. Methods: We designed and produced novel hybrid nanostructures comprising a fluorescent semiconductor core (AgInS2, AIS) and cysteine-modified carboxymethylcellulose (termed thiomer, CMC_Cys) conjugated with mitochondria-targeting peptides (KLA) forming a macromolecular shell for combining bioimaging and for inducing brain cancer cell (U-87 MG) death. Results: The optical and physicochemical properties of the nanoconjugates demonstrated suitability as photoluminescent nanostructures for cell bioimaging and intracellular tracking. Additionally, the results proved a remarkable killing activity towards glioblastoma cells of cysteine-bearing CMC conjugates coupled with KLA peptides through the half-maximal effective concentration values, approximately 70-fold higher compared to the conjugate analogs without Cys residues. Moreover, these thiomer-based pro-apoptotic drug nanoconjugates displayed higher lethality against U-87 MG cancer cells than doxorubicin, a model drug in chemotherapy, although extremely toxic. Remarkably, these peptidomimetic nanohybrids demonstrated a relative “protective effect” regarding healthy cells while maintaining high killing activity towards malignant brain cells. Conclusion: These findings pave the way for developing hybrid nanoarchitectures applied as targeted multifunctional platforms for simultaneous imaging and therapy against cancer while minimizing the high systemic toxicity and side-effects of conventional drugs in anticancer chemotherapy.


C o n t r o l A I S @ C M C A I S @ C M C _ K L A A I S @ C M C _ K L A R 7 A I S @ C M C _ C y s A I S @ C M C _ C y s _ K L A A I S @ C M C _ C y s _ K L
MitoTracker® Deep Red FM was supplied by Thermo Fisher Scientific (USA). Unless otherwise specified, all chemicals, reagents, and precursors were used as supplied without any additional purification procedure. All solutions used deionized water with the minimum resistivity of 18 MΩ cm (DI water, Millipore Simplicity™), and the processes were performed at room temperature (RT, 23 ± 2 ºC).

Synthesis of AIS@CMC colloidal solution
The synthesis of ternary quantum dots of Ag-In-S (AIS QDs) using carboxymethylcellulose (CMC) as both stabilizing ligand and functionalization agent was performed as follows. CMC polymer solution (0.04 wt. %) was prepared in DI water and homogenized by magnetic stirring.
Then, 0.33 mL of AgNO 3 (10 mM) was added to 48 mL of the polymer solution and vigorously stirred for 1 min. Following, 1.33 mL of In(NO 3 ) 3 ⋅xH 2 O (10 mM) were pipetted into the reaction flask and stirred for 1 min ([Ag:In] molar ratio = 1:4). Finally, 2.16 mL of Na 2 S⋅9H 2 O (10 mM) was rapidly poured into the reaction flask and vigorously stirred for 10 min. The colloidal solution was submitted to thermal treatment for annealing/growth of AgInS 2 QDs by heating at S12 100 ± 5 ˚C for 10 min. The resulting AgInS 2 colloidal dispersion was referred to as AIS@CMC, and it was dialyzed using a cellulose membrane with cut-off (MWCO) of 12 kDa (Sigma-Aldrich) for 24 h against distilled water with two changes of the dialysate for eliminating unreacted precursors. The nanoconjugate solution was stored in plastic flasks at RT.

Functionalization of AIS@CMC QD with L-cysteine
EDC was used as a zero-length crosslinker for CMC functionalization with Cys residues by amide bond formation. In brief, 50 mL of AIS@CMC suspension previously prepared was dried in an oven using hot air at 40 ± 1 º C to achieve a volume of 23.5 mL by solvent evaporation.

Characterization of quantum dots, thiomers, peptidomimetic nanoassemblies, and nanocarriers
Ultraviolet-visible (UV-Vis) and photoluminescence (PL) spectroscopies were applied for accessing the absorption and emission properties of all nanoconjugates, respectively. UV−vis spectra were acquired at 190 nm < λ < 700 nm in transmission mode using the Lambda EZ-210 spectrometer (PerkinElmer Inc., USA). PL emission spectra were collected using FluoroMax-Plus -CP (Horiba Scientific, Japan) with λ excitation = 350 nm and λ emission = 400-800 nm. Quantum yield (QY) parameter was estimated according to the comparative procedure using Rhodamine 6G (Sigma, USA) in ethanol as the standard at  exc = 488 nm [1]. The time-correlated single-S13 photon counting setup from Horiba (DeltaDiode -pulsed laser peak at 375 ± 10 nm) was used for collecting the photoluminescence lifetime decay at λ emission = 625 nm. The fluorescence decay curves were fitted by the multiexponential analysis using Eq. S1, and the average lifetime decay, τ av , was calculated according to the Eq. S2.
where Ai is the relative amplitude of the decay components associated with the PL lifetimes  i .

The morphological characterization of QD nanostructures was based on images obtained using
Tecnai G2-20-FEI (FEI Company, USA) transmission electron microscope (TEM) coupled with energy-dispersive x-ray spectroscopy (EDX, EDAX detector) for elemental chemical analysis at the accelerating voltage of 200 kV. For preparing samples, AIS@CMC QD suspension was centrifuged and washed with DI water using Amicon® Ultra Centrifuge Filter (30 kDa cut-off cellulose membrane, Sigma-Aldrich, 4 cycles × 5 min, 12,000 rpm). The retained material was resuspended in 450 L of DI water, dropped onto carbon-coated copper grids (Electron Microscopy Sciences, USA) and dried at room temperature. The size and size-distribution data of quantum dots were assessed based on the TEM images with at least 100 randomly selected nanoparticles using DigitalMicrograph TM image processing software (Gatan, Inc.), and the PdI was calculated according to Eq. S3 [2].
where σ is the standard deviation, and d is the mean particle diameter.
Also, atomic force microscopy (AFM) images of the AIS@CMC QDs were obtained using XE-70 (Parker Systems Inc., USA). The instrument was operated in non-contact tapping mode (frequency = 325 kHz). The scanning rate was 1.0 Hz, and the images were acquired with a 528 × 528 pixel resolution. The samples were prepared as described for TEM experiments; however, the retained material was dropped onto plastic molds and dried in a hot-air oven at 40 ± 1 º C for S14 X-ray photoelectron spectroscopy (XPS) analysis was performed using Mg-Kα as the excitation source (Amicus spectrometer, Kratos Analytical, Japan). All peak positions were corrected based on C 1s binding energy (285 eV). For sample preparation, a concentrated colloidal medium of QD was dropped onto a plastic mold and dried in an oven (see AFM sample preparation). In the sequence, samples were dehydrated in absolute ethanol (3 immersions of 30 s each) and dried in a conventional vacuum desiccator at room temperature for 2 h.
Fourier Transformed Infrared Spectroscopy (FTIR) spectra were obtained using attenuated total reflectance (ATR, 4000-850 cm −1 using 32 scans and a 4 cm −1 resolution -Nicolet 6700, Thermo-Fischer, USA) with background subtraction. Films of conjugates were prepared as described for AFM analysis. All FTIR experiments were conducted in duplicate (n = 2) unless expressly noted.
Analyses of zeta potential and DLS (dynamic light scattering) were carried at RT using the ZetaPlus instrument with a minimum of ten replicates (Brookhaven Instruments Corporation, UK, 35 mW red diode laser light, wavelength λ = 660 nm).
For estimating the degree of insertion of Cys residues in AIS_CMC QDs, the Ellman's method was applied following a previous publish protocol with slight modifications [3].

S15
where ε is the molar coefficient at 412 nm, A is the absorbance at 412 nm, l is the path length (cm), and c is the molar concentration.   Neubauer Chamber using a microscope (CH30, Olympus Corporation, Japan). The PL steadystate emission spectra of the cell-containing solutions were collected under λ excitation = 360 nm (slit = 5 nm), and results were expressed by the ratio of PL intensity per cell. As a control, the autofluorescence of nanoconjugate-free suspended cells was measured under the same conditions.
Plots of the data of PL intensity per cell versus Time were fitted to pseudo-first-order law (Eq. S5) and pseudo-second-order law and its linear form (Eq. S6 and Eq. S7, respectively) aiming at semi-quantitatively comparing the uptake kinetics of the samples [4].
Where k 1 is assigned to the pseudo-first-order rate constant (min -1 ), k 2 is the pseudo-second-order rate constant (cps -1 .min −1 ), and PL eq and PL (t) are the photoluminescence intensity (cps) at equilibrium and at time t (min), respectively.