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Introduction/Justification: Breast cancer stands as the neoplasm group with the highest incidence rate among women worldwide. Laminin-111 a constituent of the tissue basement membrane, is implicated in the development of breast tumors. Biologically active peptides of laminin-111 such as YIGSR and IKVAV, play crucial roles in tumor growth, metastasis, protease secretion, and angiogenesis. These peptides exert significant influence on various aspects of cancer progression, emphasizing their potential as key targets for therapeutic intervention. Objectives: The aim of the study was to synthesize the bioactive peptides of laminin-111 (YIKVAV and YIGSR) and assess their interactions in triple-negative breast cancer cells. Materials and Methods: The YIKVAV and YIGSR peptides were synthesized through solid-phase peptide synthesis using the Fmoc/tBut strategy. Subsequently, the peptides underwent a characterization and purification process employing high performance liquid chromatography (HPLC) and mass spectrometry. The MDA-MB-231 breast tumor cell line was cultured in supplemented RPMI-1640 medium at 37°C and 5% CO2 until reaching 90% confluence. The growth curve of the MDA-MB-231 cell line was conducted in sextuplicate over a 7-day period, with cell counts performed on days 1, 3, 5, and 7. To assess the effect of peptides on cell proliferation, cells were seeded at a concentration of 2×104 in 6-well plates, with the inclusion of both YIKVAV and YIGSR peptides at 50 µM (n = 6). Cell viability in the presence of YIKVAV and YIGSR was determined using the MTT assay. For this analysis, cells were plated at a concentration of 2×104, with peptide concentration of 50 µM. Spectrophotometric analyses were realized after 24 h and 7 days of incubation at 595 nm. Results: The YIKVAV and YIGSR peptides were efficiently synthesized, yielding approximately 80% for both. Chromatographic analyzes conducted by HPLC and mass spectrometry confirmed the efficiency of the entire synthesis, cleavage, and characterization process of the peptides by the presence of only a single peak corresponding to the synthesized peptides. The growth curve profile determination of MDA-MB-231 cell line revealed exponential growth between days 5 and 7 of cell culture. The results indicate that the YIGSR peptide significantly inhibited cell growth by approximately 45%, whereas the YIKVAV fragment promoted cell growth by approximately 38% and (p < 0.0001) on the seventh day of cell culture. Regarding the MTT analysis after 24 h, no significant differences were observed between the control and treated groups for both fragments, suggesting that the peptides exhibited no toxicity at concentration of 50 µM. Additionally, on the 6th day, a reduction in tumor cell viability was observed for the YIGSR fragment, while an increase in viability was noted for YIKVAV (p < 0.0001). Conclusion: The YIKVAV and YIGSR peptides were effectively synthesized, characterized, and purified. The YIKVAV peptide, at a concentration of 50µM, promoted cell growth, while the YIGSR peptide significantly hindered the growth of the MDA-MB-231 cell line. This observation is consistent with the findings of cell viability assays conducted using MTT. This study holds significance for enhancing our comprehension of peptide actions in their interaction with triple-negative breast cancer cells, with the ultimate aim of proposing more effective targets for treatment.

Introduction/Justification: Peptides are biomolecules that have been associated with various physiological responses and have great potential for the diagnosis or treatment of diseases, including various types of tumors. Several studies have shown that biologically active peptides containing the RGD domain have a high affinity with the αvβ3 integrin adhesion molecule overexpressed in tumor cells, assisting in molecular imaging or targeted radionuclide therapy (TRT) as an anti-tumor agent. Objectives: To obtain an anti-integrin peptide fragment modified by incorporating two spacers — hexa-aminocaproic acid (C6) or dodeca-aminocaproic acid (C12) — and by adding the chelating agent DOTA for subsequent radiolabeling with yttrium-86 (86Y). Materials and Methods: The modified peptides were synthesized by the solid-phase peptide synthesis method using the Fmoc/tBut strategy. In this strategy, the Fmoc group removal step was carried out with 20% 4-methylpiperidine / 80% DMF, and the amino acid coupling step is usually performed with the diisopropylcarbodiimide/1-hydroxybenzotriazole (DIC/HOBt) acylation mixture. In this step, an excess of Fmoc-amino acids and acylating agents of 2.5 times were used relative to the synthesis scale utilized in mmol/g. Peptide cleavage from the resin and removal of side chain protecting groups were carried out using a mixture containing a high concentration of TFA (reagent K). After synthesis and cleavage, the peptides underwent characterization and purification process through high-performance liquid chromatography (HPLC) and mass spectrometry. The radiolabeling process was conducted utilizing cyclotron-produced 86Y, employing a NaOAc buffer (pH = 5.5). The radiochemical reaction was performed at 95°C for 30 min, followed by filtration through a Sep-Pak C18 cartridge for purification and determination of the radiolabeling yield. Results: The DOTA-C6-anti-integrin and DOTA-C12-anti-integrin peptides were efficiently synthesized, and the yields obtained were approximately 12.7% and 26.4%, respectively. Chromatographic analyses obtained by HPLC, as well as mass spectrometry, showed that the entire synthesis, cleavage, and characterization process were carried out properly with visualization of profiles and molecular masses of 1165.3 g/mol and 1249.1 g/mol for the DOTA-C6-anti-integrin and DOTA-C12-anti-integrin peptides, respectively. After the purification process, 14.6 mg and 66.2 mg of pure peptides were obtained. The preliminary 86Y-labeling data indicated a radiochemical yield of approximately 97% for both peptides. Conclusion: The proposed modified anti-integrin peptides were efficiently synthesized, characterized, and purified. Preliminary radiolabeling studies with 86Y demonstrated a high radiochemical yield, paving the way for further exploration in radiochemical studies and assays of affinity to tumor cells.

Introduction/Justification: Peptides are implicated to various physiological responses and exhibit considerable potential for disease treatment, encompassing diverse types of tumors. The significant therapeutic promise of peptides is related from their characteristics, including the ability to inhibit angiogenesis, induce tumor apoptosis and block of epidermal growth factor receptor (EGFr) signaling. Their relevance is underscored by overexpression in a range of human cancers, notably glioblastoma, which represents the most prevalent and aggressive form of malignant brain tumors. Objectives: This study aims to assess the growth inhibition of rat (C6 cells) and human (U-87 MG cells) glioblastoma tumor cells using peptides that interact with the EGFr. Materials and Methods: The anti-EGFr peptides were synthesized through the solid-phase peptide synthesis using the Fmoc/tBut strategy. Peptide cleavage from the resin was performed using a mixture containing a high concentration of trifluoroacetic acid (reagent K). Subsequently, the peptides underwent a characterization and purification process employing high performance liquid chromatography (HPLC) and mass spectrometry. C6 and U-87 MG cell lines were cultured in supplemented DMEM F-12 medium at 37°C and 5% CO2 until reaching 90% confluence. To assess the effect of peptides on cell proliferation, cells were seeded at a concentration of 5 × 103 in 6-well plates, with the presence of 80 µM of each proposed peptide. Growth curves were performed in sextuplicate over a 7-day period, with cell counts conducted on days 1, 3, 5, and 7. Cell viability in the presence of peptides was determined using the MTT test. For this analysis, cells were plated at a concentration of 5 × 103 in 96-well plates, with peptide concentrations of 80, 120, and 160 µM. Spectrophotometric analyses were performed after 24 h and 7 days of incubation at 595 nm. Results: Anti-EGFr-LP and anti-EGFr-LG peptides were synthesized efficiently with yields of approximately 45 and 98%, respectively. Chromatographic analyzes obtained by HPLC confirmed that the entire synthesis, cleavage, and characterization process of peptides were performed efficiently, as evidenced by the presence of only a single peak corresponding to the synthesized peptides. Following the determination of growth curve profiles of C6 and U-87 MG cell lines, without the presence of peptides, the interaction of the peptides with both tumor cell types was assessed. The results demonstrated that both anti-EGFr-LP and anti-EGFr-LG peptides significantly interacted with and inhibited the growth of C6 and U-87 MG strains (p < 0.0001). Studies conducted with C6 cells showed inhibition percentages of approximately 55.3% and 99.1% for the Anti-EGFr-LP and anti-EGFr-LG peptides, respectively. On the other hand, an inhibition percentage of growth of U-87 MG cells was 44.4% for the Anti-EGFr-LP and 46.4% for the anti-EGFr-LG. Finally, based on the MTT test, the peptides exhibited no toxicity at any of the three concentrations tested. Conclusion: The findings indicate that both proposed peptides, at a minimum concentration (80 µM) effectively reduced the proliferation of tumor cells without inducing toxicity. While further experiments are warranted, the peptides have demonstrated the capability to inhibit tumor cell growth associated with glioblastoma, suggesting a potential therapeutic alternative.

Introduction/Justification: Glioblastomas (GBM) constitute the most prevalent malignant primary tumor in adults and rank as the third most frequent tumors in the central nervous system. The predominant alteration observed in GBM is associated with the tyrosine-kinase pathway, facilitating the connection of growth factors to receptors. Notably, in GBM the overexpression of the epidermal growth factor receptor (EGFr) has opened new treatment perspectives, including molecular targeted therapies, with peptides taking center stage. Recently, radiolabeled peptides with high affinity for EGFr have been employed as potential agents for molecular imaging or targeted radionuclide therapy as anti-tumor agents. Objectives: The aim of the study was to assess the interaction between [131I]I-DEDEYFELV peptide and human glioblastoma cells (U-87 MG), as well as with GBM tissue. Materiais e Métodos: The DEDEYFELV peptide was synthesized through solid-phase peptide synthesis using the Fmoc/tBut strategy. Peptide cleavage from the resin was performed using a mixture containing a high concentration of trifluoroacetic acid (reagent K). Subsequently, the peptides underwent a characterization and purification process employing high performance liquid chromatography (HPLC) and mass spectrometry. The U-87 MG cell line was cultured in supplemented DMEM F-12 medium at 37°C and 5% CO2 until reaching 90% confluence. The neoplastic tissue was surgically removed, histopathologically analyzed, and preserved at -80°C, with its homogenate prepared using PBS buffer (pH 7.4) at 10 mg of tissue/mL. The DEDEYFELV (20 nmol) was radiolabeled with the [131I]NaI radionuclide (18.5 MBq), using the chloramine-T method. The radiochemical yield of the [131I]I-DEDEYFELV was carried out by on Whatmann 3MM strips using 95% MeOH / 5% H2O as eluent. Binding and internalization studies of the [131I]I-DEDEYFELV with tumorigenic cells (U-87 MG) and neoplastic tissue homogenate were evaluated at 1 and 3 h of incubation and measured in an automatic gamma counter. Results: The DEDEYFELV peptide was efficiently synthesized and characterized, with yield of approximately 92%. Chromatographic analyzes obtained by HPLC confirmed that the entire synthesis, cleavage, and characterization process of peptides were performed efficiently, as evidenced by the presence of only a single peak corresponding to the synthesized peptide with molecular mass of 1158.18 g/mol. The radiolabeling process was successful obtained with radiochemical yield > 95%. Binding and internalization studies of the [131I]I-DEDEYFELV conducted with U-87 MG cells showed values of 15.90% ± 1.67 and 54.57% ± 0.90 (n = 5), respectively. On the other hand, the binding percentage of 12.45% ± 0.90 and internalization of 28.41% ± 3.15 (n = 5) were achieved with neoplastic tissue homogenate within a 3-hour period. Conclusion: The proposed peptide was efficiently synthesized, and radiolabeling studies with [131I]NaI exhibited a high radiochemical yield. Binding and internalization studies revealed that the [131I]I-DEDEYFELV peptide has a good affinity for both U-87 MG and neoplastic tissue homogenate, demonstrating higher internalization of [131I]I-DEDEYFELV in human glioblastoma cells. Nevertheless, it is crucial to emphasize that additional in vivo investigations are necessary.

Introduction/Justification: Peptides are implicated in various physiological responses and hold significant potential as targeting molecules, especially in cancer diagnosis or treatment. Radiolabeled peptides have been investigated for their potential as theranostic agents, holding considerable promise for precisely targeting tumorigenic cells. Previous studies indicate that biologically active peptides exhibit a high affinity for the Epidermal Growth Factor receptor (EGFr), which is overexpressed in various tumor cells, including glioblastoma, the most prevalent and aggressive malignant brain tumor. Objectives: To evaluate the in vitro interactions involving two radiolabeled peptide inhibitors targeting the EGFr overexpressed in glioblastoma cells. Materials and Methods: Two EGFr-targeting peptide inhibitors, anti-EGFr-LP and anti-EGFr-LG, were radiolabeled with [131I]NaI (11.1–14.8 MBq) using the chloramine T method (room temperature; reaction time = 120 s). The radiochemical yield (RCY) (n = 8) and stability (n = 3) were evaluated using ascending chromatography on TLC-SG strips and acetonitrile/water (95:5) as eluent. C6 and U-87 MG glioblastoma cell lines were cultured in supplemented DMEM medium (5% CO2 atmosphere; 37°C) until reaching ∼85% confluence. Subsequently, aliquots of 2 x 10^6 C6 or U-87 MG cells were incubated with each radiopeptide (37°C) under agitation (500 rpm). In vitro binding and internalization percentages were assessed at 1 and 3 h post-incubation (n = 6). Data were expressed as ‘mean ± standard deviation’ and the statistical analysis was performed using GraphPad Prism software. Results: The RCY of [131I]I-anti-EGFr-LP and [131I]I-anti-EGFr-LG were 92.92 ± 3.42 and 97.80 ± 1.08, respectively. Both 131I-labeled peptides were radiochemically stable over 24 h. The in vitro interaction between C6 cells and [131I]I-anti-EGFr-LP showed binding percentages of 4.80 ± 0.37% (1 h) and 5.87±1.21% (3 h), with no statistically significant difference (p = 0.1519). The internalization percentages, within the bound fractions, increased from 64.45 ± 4.19% (1 h) to 75.15±1.60% (3 h) (p < 0.0001). For the [131I]I-anti-EGFr-LG, the data were of the same order of magnitude. The binding percentages increased from 3.95±0.33% (1 h) to 6.03 ± 0.66 (3 h) (p < 0.0001) and the internalization percentages, among the bound fractions, were 62.57±5.53% (1 h) and 64.04±3.21% (3 h), with no statistically significant difference (p = 0.5959). The in vitro interaction between U-87 MG cells and [131I]I-anti-EGFr-LP showed an increment of the binding percentages from 6.50 ± 0.93% (1 h) to 8.03 ± 0.29% (3 h) (p < 0.0001), but the internalization percentages, within the bound fractions, showed no statistically significant difference (p = 0.2791), 68.98 ± 2.23% (1 h) and 73.02±6.57% (3 h). For the [131I]I-anti-EGFr-LG, the binding percentages were 10.97±1.48 (1 h) and 11.28 ± 0.84 (3 h), with no statistically significant difference (p > 0.6724). The internalization percentages, among the bound fractions, were also statistically similar (p > 0.3596), 68.21 ± 0.16% (1 h) and 65.36 ± 3.56 (3 h). Conclusion: The in vitro interaction data revealed high affinity of [131I]I-anti-EGFr-LP and [131I]I-anti-EGFr-LG for the C6 and U-87 MG glioblastoma cell lines, which are known to overexpress EGFr. These preliminary findings support the potential use of these peptide inhibitors as specific peptide-based targeting molecules for EGFr, with potential applications as theranostic agents.

The expression of prostate-specific membrane antigen (PSMA) is upregulated in prostate cancer (PCa) cells and PSMA-ligands have been radiolabeled and used as radiopharmaceuticals for targeted radionuclide therapy (TRT), single photon emission computed tomography (SPECT) or positron emission tomography (PET) molecular imaging, and radioguided surgery in PCa patients. Herein, we aimed at radiolabeling the PSMA-I&S cold kit with 99mTc, resulting in a radiopharmaceutical with high radiochemical yield (RCY) and stability for SPECT imaging and radioguided surgery in PCa malignancies. Various pre-clinical assays were conducted to evaluate the [99mTc]Tc-PSMA-I&S obtained by the cold kit. These assays included assessments of RCY, radiochemical stability in saline, lipophilicity, serum protein binding (SPB), affinity for LNCaP-PCa cells (binding and internalization studies), and ex vivo biodistribution profile in naive and LNCaP-PCa-bearing mice. The radiopharmaceutical was obtained with good RCY (92.05% ± 2.20%) and remained stable for 6 h. The lipophilicity was determined to be −2.41 ± 0.06, while the SPB was ∼97%. The binding percentages to LNCaP cells were 9.41% ± 0.57% (1 h) and 10.45% ± 0.45% (4 h), with 63.12 ± 0.93 (1 h) and 65.72% ± 1.28% (4 h) of the bound material being internalized. Blocking assays, employing an excess of unlabeled PSMA-I&S, resulted in a reduction in the binding percentage by 2.6 times. The ex vivo biodistribution profile confirmed high accumulation of [99mTc]Tc-PSMA-I&S in the tumor and the tumor-to-contralateral muscle ratio was ∼6.5. In conclusion, [99mTc]Tc-PSMA-I&S was successfully obtained by radiolabeling the cold kit using freshly eluted [99mTc]NaTcO4, exhibiting good RCY and radiochemical stability. The preclinical assays demonstrated that the radiopharmaceutical shows favorable characteristics for SPECT imaging and radioguided surgery in PCa patients.