Nallely Jiménez-Mancilla

Multifunctional targeted therapy system based on 99mTc/177Lu-labeled gold nanoparticles-Tat(49–57)-Lys3-bombesin internalized in nuclei of prostate cancer cells

Radiolabeled gold nanoparticles may function simultaneously as radiotherapy and thermal ablation systems. The gastrin-releasing peptide receptor (GRP-r) is overexpressed in prostate cancer, and Lys(3) -bombesin is a peptide that binds with high affinity to the GRP-r. HIV Tat(49-57) is a cell-penetrating peptide that reaches the DNA. In cancer cells, (177) Lu shows efficient crossfire effect, whereas (99m) Tc that is internalized in the cancer cell nuclei acts as an effective system of targeted radiotherapy because of the biological Auger effect. The aim of this research was to evaluate the in vitro potential of (99m) Tc-labeled and (177) Lu-labeled gold nanoparticles conjugated to Tat(49-57)-Lys(3) -bombesin peptides ((99m) Tc/(177) Lu-AuNP-Tat-BN) as a plasmonic photothermal therapy and targeted radiotherapy system in PC3 prostate cancer cells. Peptides were conjugated to AuNPs (5 nm) by spontaneous reaction with the thiol group of cysteine (Cys). The effect on PC3 cell viability after laser heating of the AuNP-Tat-BN incubated with the cancer cells was conducted using an Nd:YAG laser pulsed for 5 ns at 532 nm (0.65 W/cm(2) ). For the (99m) Tc/(177) Lu-AuNP-Tat-BN to be obtained, the (177) Lu-DOTA-Gly-Gly-Cys and (99m) Tc-HYNIC-octreotide radiopeptides were first prepared and added simultaneously to a solution of AuNP-Tat-BN. (99m) Tc/(177) Lu-AuNP-Tat-BN (20 Bq/cell) was incubated with PC3 cells, and the effect on the cell proliferation was evaluated after 3 days. Fluorescence images of (99m) Tc/(177) Lu-AuNP-Tat-BN internalized in nuclei of PC3 were also obtained. After laser irradiation, the presence of AuNP-Tat-BN caused a significant increase in the temperature of the medium (46.4 vs 39.5 °C of that without AuNP) resulting in a significant decrease in PC3 cell viability down to 1.3%. After treatment with (99m) Tc/(177) Lu-AuNP-Tat-BN, the PC3 cell proliferation was inhibited. The nanosystem exhibited properties suitable for plasmonic photothermal therapy and targeted radiotherapy in the treatment of prostate cancer.

Laser Heating of Gold Nanospheres Functionalized with Octreotide: In Vitro Effect on HeLa Cell Viability

OBJECTIVE The aim of this study was to assess the effect of laser heating a well-characterized gold nanoparticle (AuNP)-octreotide system on HeLa cell viability, to evaluate its potential as a suitable agent for plasmonic photothermal therapy. BACKGROUND DATA Octreotide is a synthetic peptide derivative of somatostatin with an effect on the survival of HeLa cells. Peptides bound to AuNPs are biocompatible and stable multimeric systems with target-specific molecular recognition. METHODS Octreotide was conjugated to AuNPs (∼20 nm) by spontaneous reaction with the thiol groups. The nanoconjugate was characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), ultraviolet visible spectroscopy (UV-Vis), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy. Irradiation experiments were conducted using an Nd:YAG laser pulsed for 5 ns at 532 nm with a repetition rate of 10 Hz for up to 6 min while delivering an average irradiance of 0.65 W/cm(2). HeLa cells were incubated at 37°C (1) with AuNP-citrate, (2) with AuNP-octreotide, or (3) without nanoparticles. RESULTS After laser irradiation, the presence of AuNP caused a significant increase in the temperature of the medium (48°C vs. 38.3°C of that without AuNP). The AuNP-octreotide system resulted in a significant decrease in cell viability of up to 6 % compared with the AuNP-citrate system (15.8±2.1%). Two possible mechanisms could be at play: (1) octreotide alone exerts an effect on survival HeLa cells, or (2) the release of heat (∼727°C per nanoparticle) in the membranes or cytoplasm of the cells caused by the interaction between AuNP-octreotide and somatostatin receptors reduced viability. CONCLUSIONS The AuNP-octreotide system exhibited properties suitable for plasmonic photothermal therapy in the treatment of cervical cancer.

Clinical translation of a PSMA inhibitor for 99mTc-based SPECT

BACKGROUND Prostate-specific membrane antigen (PSMA) is highly over-expressed in advanced prostate cancers. 68Ga-labeled PSMA inhibitors (iPSMA) are currently used for prostate cancer detection by PET imaging. The availability of simple, efficient and reproducible radiolabeling procedures is essential for developing new SPECT radiopharmaceuticals for clinical translation. The aim of this research was to prepare 99mTc-EDDA/HYNIC-Lys(Nal)-Urea-Glu (99mTc-EDDA/HYNIC-iPSMA) obtained from lyophilized kit formulations and evaluate the in vitro and in vivo radiopharmaceutical binding to prostate cancer cells over-expressing PSMA, as well as the 99mTc-EDDA/HYNIC-iPSMA normal biodistribution in humans and the preliminary uptake in patients with prostate cancer. METHODS 99mTc labeling was performed by adding sodium pertechnetate solution and a 0.2M phosphate buffer (pH 7.0) to a lyophilized formulation containing HYNIC-iPSMA, EDDA, tricine, mannitol and stannous chloride. The radiochemical purity was evaluated by reversed-phase HPLC and ITLC-SG analyses. Stability studies in human serum were performed by size-exclusion HPLC. In vitro cell uptake was tested using prostate cancer cells (LNCaP) with blocked and non-blocked receptors. Biodistribution and tumor uptake were determined in LNCaP tumor-bearing nude mice with blocked and non-blocked receptors, and images were obtained using a micro-SPECT/CT. Whole-body images from three healthy men and two patients with histologically-confirmed prostate cancer (one of them with a previous 68Ga-PSMA-617scan) were acquired at 1h and 3h after 99mTc-EDDA/HYNIC-iPSMA administration with radiochemical purities of >98%. RESULTS In vitro and in vivo studies showed high radiopharmaceutical stability in human serum, specific recognition for PSMA, high tumor uptake (10.22±2.96% ID/g at 1h) with rapid blood clearance and mainly kidney elimination. Preliminary images in patients demonstrated the ability of 99mTc-EDDA/HYNIC-iPSMA to detect tumors and metastases of prostate cancer as well as 68Ga-PSMA-617 does. CONCLUSIONS The results obtained in this study warrant further dosimetry and clinical studies to determine the specificity and sensitivity of 99mTc-EDDA/HYNIC-iPSMA.

Design and biological evaluation of 99mTc-N2S2-Tat(49–57)-c(RGDyK): A hybrid radiopharmaceutical for tumors expressing α(v)β(3) integrins

UNLABELLED The α(ν)β(3) integrin is over-expressed in the tumor neovasculature and the tumor cells of glioblastomas. The HIV Tat-derived peptide has been used to deliver various cargos into cells. The aim of this research was to synthesize and assess the in vitro and in vivo uptake of (99m)Tc-N₂S₂-Tat(49-57)-c(RGDyK) ((99m)Tc-Tat-RGD) in α(ν)β(3) integrin positive cancer cells and compare it to that of a conventional (99m)Tc-RGD peptide ((99m)Tc-EDDA/HYNIC-E-[c(RGDfK)]2). METHODS The c(RGDyK) peptide was conjugated to a maleimidopropionyl (MP) moiety through Lys, and the MP group was used as the branch position to form a thioether with the Cys(12) side chain of the Tat(49-57)-spacer-N₂S₂ peptide. (99m)Tc-Tat-RGD was prepared, and stability studies were carried out by size exclusion HPLC analyses in human serum. The in vitro affinity for α(v)β(3) integrin was determined by a competitive binding assay. In vitro internalization was determined using glioblastoma C6 cells. Biodistribution studies were accomplished in athymic mice with C6 induced tumors that had blocked and unblocked receptors. Images were obtained using a micro-SPECT/CT. RESULTS (99m)Tc-Tat-RGD was obtained with a radiochemical purity higher than 95%, as determined by radio-HPLC and ITLC-SG analyses. Protein binding was 15.7% for (99m)Tc-Tat-RGD and 5.6% for (99m)Tc-RGD. The IC50 values were 6.7 nM ((99m)Tc-Tat-RGD) and 4.6 nM ((99m)Tc-RGD). Internalization in C6 cells was higher in (99m)Tc-Tat-RGD (37.5%) than in (99m)Tc-RGD (10%). Biodistribution studies and in vivo micro-SPECT/CT images in mice showed higher tumor uptake for (99m)Tc-Tat-RGD (6.98% ± 1.34% ID/g at 3h) than that of (99m)Tc-RGD (3.72%±0.52% ID/g at 3h) with specific recognition for α(v)β(3) integrins. CONCLUSIONS Because of the significant cell internalization (Auger and internal conversion electrons) and specific recognition for α(v)β(3) integrins, the hybrid (99m)Tc-N₂S₂-Tat(49-57)-c(RGDyK) radiopharmaceutical is potentially useful for the imaging and possible therapy of tumors expressing α(v)β(3) integrins.

Synthesis and evaluation of Lys¹(α,γ-Folate)Lys³(¹⁷⁷Lu-DOTA)-Bombesin(1-14) as a potential theranostic radiopharmaceutical for breast cancer.

The aim of this work was to synthesize Lys(1)(α,γ-Folate)-Lys(3)((177)Lu-DOTA)-Bombesin (1-14) ((177)Lu-Folate-BN), as well as to assess its potential for molecular imaging and targeted radiotherapy of breast tumors expressing folate receptors (FR) and gastrin-releasing peptide receptors (GRPR). Radiation absorbed doses of (177)Lu-Folate-BN (74 MBq, i.v.) estimated in athymic mice with T47D-induced breast tumors (positive to FR and GRPR), showed tumor doses of 23.9±2.1 Gy. T47D-tumors were clearly visible (Micro-SPECT/CT images). (177)Lu-Folate-BN demonstrated properties suitable as a theranostic radiopharmaceutical.

Fluorescent, Plasmonic, and Radiotherapeutic Properties of the 177Lu–Dendrimer-AuNP–Folate–Bombesin Nanoprobe Located Inside Cancer Cells:

The integration of fluorescence and plasmonic properties into one molecule is of importance in developing multifunctional imaging and therapy nanoprobes. The aim of this research was to evaluate the fluorescent properties and the plasmonic–photothermal, therapeutic, and radiotherapeutic potential of 177Lu–dendrimer conjugated to folate and bombesin with gold nanoparticles in the dendritic cavity (177Lu–DenAuNP–folate–bombesin) when it is internalized in T47D breast cancer cells. The intense near-Infrared (NIR) fluorescence emitted at 825 nm from the conjugate inside cells corroborated the usefulness of DenAuNP–folate–bombesin for optical imaging. After laser irradiation, the presence of the nanosystem in cells caused a significant increase in the temperature of the medium (46.8°C, compared to 39.1°C without DenAuNP–folate–bombesin, P < 0.05), resulting in a significant decrease in cell viability (down to 16.51% ± 1.52%) due to the 177Lu–DenAuNP–folate–bombesin plasmonic properties. After treatment with 177Lu–DenAuNP–folate–bombesin, the T47D cell viability decreased 90% because of the radiation-absorbed dose (63.16 ± 4.20 Gy) delivered inside the cells. The 177Lu–DenAuNP–folate–bombesin nanoprobe internalized in cancer cells exhibited properties suitable for optical imaging, plasmonic–photothermal therapy, and targeted radiotherapy.

In vitro and in vivo synergistic effect of radiotherapy and plasmonic photothermal therapy on the viability of cancer cells using 177Lu–Au-NLS-RGD-Aptamer nanoparticles under laser irradiation

This research aimed to evaluate the photothermal and radiotherapeutic effect of the 177Lu–Au-RGD-NLS-Aptamer anti-angiogenic nanosystem on the viability of U87MG cancer cells by using in vitro and in vivo models, as well as to assess the synergic effect of both therapies. In vitro results demonstrated a decrease in cell viability to 2.14 ± 0.27% after the treatment with photothermal therapy plus radiotherapy. These results correlated with the observed in vivo therapeutic response in mice with U87MG-induced tumors, in which 177Lu–Au-RGD-NLS-Aptamer under laser irradiation inhibited tumor progression. The combination of radiotherapy and thermotherapy in one nanoradiopharmaceutical could be potentially useful for cancer treatment.

Estimation of the effectiveness ratio (α/β) for resistant cancer cells in U87MG human glioblastoma

Glioblastoma contains self-renewing, tumorigenic cancer stem-like cells that contribute to tumor initiation and therapeutic resistance. The aim of this research was to estimate and compare the effectiveness ratio (α/β) of stem-like cells and differentiated glioma cells derived from the U87MG glioblastoma cell line. Cell survival experiments were obtained in a dose range of 0-20 Gy (13.52 ± 0.09 Gy/h) as a hyperfractionationated accelerated radiotherapy scheme. Biochemical characterization of the post-irradiated cells was performed by flow cytometry analysis and the percentage of stem-like cells that resisted irradiation was determined by the CD133 expression. Results showed that U87MG stem-like cells are highly proliferative and more radioresistant than the U87MG adherent group (with a lesser stem-like character), this in association with the calculated α/β ratio of 17 and 14.1, respectively.

Study of the Optical Properties of Functionalized Gold Nanoparticles in Different Tissues and Their Correlation with the Temperature Increase

Mie theory explains the interaction of light with a gold nanoparticle (AuNP) through the absorption , scattering , and extinction cross sections. These parameters have been calculated in the case of AuNPs dispersed in homogeneous media, but not for specific tissues. The aim of this research was to theoretically obtain the optical cross sections (,, and ) of functionalized AuNPs in liver and colon tissues through Mie theory and correlate them with the temperature increase observed experimentally in tissues containing AuNPs under plasmonic photothermal irradiation using a Nd-YAG laser (λ = 532 nm). Calculations showed that represents % of at 532 nm. The value for a functionalized AuNP in water was 365.66 nm2 (94% of the theoretical maximum value at 522.5 nm), 404.24 nm2 in colon (98% of the theoretical maximum value at 525 nm), and 442.39 nm2 in liver (96% of the theoretical maximum value at 525 nm). Therefore, nanoparticles irradiated at 532 nm are very close to their resonance value. These results correlated with the experimental irradiation of functionalized AuNPs in different tissues, where the average temperature increase showed the pattern liver > colon > water. The temperature increase observed ( up to 13°C) is sufficient to produce cellular death.