Myrna Luna-Gutiérrez

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.

Design, preparation, in vitro and in vivo evaluation of 99mTc-N2S2-Tat(49-57)-bombesin: A target-specific hybrid radiopharmaceutical

The gastrin-releasing peptide receptor (GRP-r) is over-expressed in various human tumors. Recently, (99m)Tc-EDDA/HYNIC-Lys(3)-bombesin ((99m)Tc-BN) was reported as a radiopharmaceutical with specific cell GRP-r binding and images in breast cancer patients demonstrated distinct radioactivity accumulation in malignant tissue. The HIV Tat-derived peptide has been used to deliver a large variety of cargoes into cells. Therefore, a new hybrid radiopharmaceutical of type (99m)Tc-N(2)S(2)-Tat(49-57)-Lys(3)-bombesin ((99m)Tc-Tat-BN) would increase cell uptake. The aim of this research was to prepare and assess in vitro and in vivo uptake kinetics in cancer cells of (99m)Tc-Tat-BN and to compare its cellular internalization with that of (99m)Tc-BN. Structures of N(2)S(2)-Tat-BN and Tc(O)N(2)S(2)-Tat-BN were calculated by an MM procedure. (99m)Tc-Tat-BN was synthesized and stability studies carried out by HPLC and ITLC-SG analyses in serum and cysteine solutions. In vitro internalization was tested using human prostate cancer PC-3 cells and breast carcinoma cell lines MDA-MB231 and MCF7. Biodistribution was determined in PC-3 tumor-bearing nude mice. Results showed a minimum energy of 271 kcal/mol for N(2)S(2)-Tat-BN and 300 kcal/mol for Tc(O)N(2)S(2)-Tat-BN. (99m)Tc-Tat-BN radiochemical purity was >90%. In vitro studies demonstrated stability in serum and cysteine solutions, specific cell receptor binding and internalization in three cell lines was significantly higher than that of (99m)Tc-BN (p<0.05). The tumor-to-muscle radioactivity ratio was 8.5 for (99m)Tc-Tat-BN and 7 for (99m)Tc-BN. Therefore, this hybrid is potentially useful in breast and prostate cancer imaging.

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.

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.

99m: biokinetics and radiation dosimetry in healthy individualstc-exendin(9-39)/octreotide: biokinetics and radiation dosimetry in healthy individuals

Aim About 90% of insulinomas are benign and 5–15% are malignant. Benign insulinomas express the glucagon-like peptide-1 receptor (GLP-1R, which recognizes exendin-4 and low levels of the somatostatin receptor (SSTR, which recognizes octreotide), whereas malignant insulinomas overexpress SSTR and low levels of GLP-1R. Recently, Lys27(99mTc-EDDA/HYNIC)-exendin(9-39)/99mTc-EDDA/HYNIC-Tyr3-octreotide was formulated to detect 100% of insulinomas. The aim of this study was to estimate the biokinetics and dosimetry of 99mTc-exendin(9-39)/octreotide in four healthy individuals. Participants and methods 99mTc-exendin(9-39)/octreotide was obtained from a lyophilized formulation with radiochemical purities of more than 97%, determined by reversed-phase high-performance liquid chromatography. Whole-body images from four healthy individuals were acquired at 20 min, 2, 6, and 24 h after 99mTc-exendin(9-39)/octreotide administration. Regions of interest were drawn around the source organs on each time frame. Each region of interest was corrected by background, attenuation, scattered radiation, and physical decay. The image sequence was used to extrapolate the 99mTc-exendin(9-39)/octreotide time–activity curves of each organ to adjust the biokinetic model and calculate the total number of disintegrations (N) that occurred in the source regions. N data were the input for the OLINDA/EXM code to calculate internal radiation doses. Furthermore, in a patient suspicious of harboring an insulinoma, whole-body single-photon emission computed tomography/computed tomography images were obtained at 3 h. Results For four healthy individuals, the blood activity showed a half-life value of 1.20 min for the fast component (T1/2 &agr;=ln 2/34.71), 8.7 min for the first slow component (T1/2 &bgr;=ln 2/4.76), and 1.7 h for the second slow component (T1/2 &ggr;=ln 2/0.401). The average equivalent doses calculated for a study using 555 MBq were 15.10, 4.13, 3.08, 2.61, and 1.90 mSv for the kidneys, upper large intestinal wall, lower large intestinal wall, small intestine, and liver, respectively, with an effective dose of 2.33±0.51 mSv. In addition, images from a patient showed an average tumor/heart (blood) ratio of 2.7 at 3 h. Conclusion All the absorbed doses were comparable to those known for most of the 99mTc studies. 99mTc-exendin(9-39)/octreotide obtained from kit formulations showed high tumor uptake in a patient with a malignant lesion, making it a promising imaging radiopharmaceutical to target GLP-1R and SSTR.

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.

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.