Search Results (77)

Monitoring peptide and protein self-association is essential for understanding biological function, formulation stability, and aggregation mechanisms. While size-exclusion chromatography (SEC) is routinely used to quantify protein-size variants under native conditions, its hyphenation to high-resolution mass spectrometry (HRMS) for simultaneous structural characterization remains limited. Here, we report the development and validation of a robust SEC-UV/HRMS method optimized for native-like analysis of bovine serum albumin (BSA) monomers and higher-order oligomers using standard-flow electrospray ionization. Systematic evaluation of source parameters, mobile-phase composition, and chromatographic conditions enabled retention of native BSA structure, minimized in-source unfolding, and enhanced MS sensitivity, allowing detection of oligomers up to the heptamer. A short, narrow-bore 200 Å UHPLC SEC separation column was used. Low-flow separations (~0.05 mL/min) enabled efficient ionization and 10 min run times. An accelerated 60 °C stress-testing protocol demonstrated that SEC-MS can semi-quantitatively monitor oligomerization dynamics, complementing UV-based quantification and revealing transient species not resolved by UV alone. The method showed acceptable linearity, precision, and sample stability, and comparison with SEC-RALS/LALS confirmed molecular-weight trends across aggregation states. Overall, the developed SEC-UV/HRMS workflow provides a rapid, sensitive, and widely accessible approach for UV-based quantification of monomer- and HRMS-based characterizing protein aggregation in research and quality control in pharmaceutical laboratories. Full article

Background: Emerging evidence links the gut microbiome to chronic pain processing. Inulin, a prebiotic fibre, modulates the gut microbiome, while physiotherapy-supported exercise (PSE) improves pain and function. We evaluated the effects of inulin supplementation with and without PSE on knee osteoarthritis (OA) pain. Methods: In a 2 × 2 factorial RCT, 117 community-dwelling adults with knee OA received 6 weeks of: (A) 20 g/day inulin, (B) digital PSE (Joint Academy™), (C) inulin +PSE, or (D) 10 g/day maltodextrin. Primary outcome: pain (Numerical Rating Scale). Secondary: 30 s sit-to-stand (30-CST), timed up and go (TUG), grip strength, and quantitative sensory testing. Serum short-chain fatty acids (SCFAs) and glucagon-like peptide-1 (GLP-1) were measured. The study was not powered to detect synergistic interaction. Results: A total of 117 participants (58.1% female; mean ± SD age = 67.5 ± 9.4 years; BMI = 29.5 ± 5.3 kg/m²; NRS = 3.96 ± 2.67) completed the trial. Pain improved with inulin (baseline-adjusted between-group mean difference (Δ) = −1.11 [95%CI −2.18, −0.04], p = 0.045) and PSE (Δ = −1.55 [95%CI −2.52, −0.58], p = 0.002) compared to placebo, with no synergistic effect. PSE improved TUG (p = 0.02) and 30-CST (p = 0.0004), while inulin improved grip strength (p = 0.002), pressure pain thresholds (p = 0.009) and temporal summation (p = 0.025) compared to placebo and had significantly lower dropout rates (3.6%) compared with PSE (21% p < 0.01). Only inulin increased SCFA butyrate (p = 0.0248) and GLP-1 (p = 0.0109), and higher GLP-1 was associated with improved grip strength, suggesting a gut–muscle link. Conclusions: Inulin and PSE each produced meaningful pain reductions. Only inulin improved pain sensitivity and grip strength, the latter paralleled by increased GLP-1, and had much higher rates of retention compared to PSE. Full article

Background: Host cell proteins (HCPs) are process-related impurities that must be monitored in biopharmaceutical products due to their potential impact on product quality and patient safety. Targeted LC–MS/MS approaches such as multiple reaction monitoring (MRM) enable protein-specific HCP quantification but are difficult to apply in highly multiplexed assays because of retention time (RT) variability across complex bioprocess matrices. Methods: Here, we show that conventional RT-scheduled MRM workflows lack transferability when applied to heterogeneous drug substances and process intermediates. Using a targeted assay comprising 240 peptides corresponding to 97 CHO-derived HCPs, RT shifts of several minutes resulted in truncated chromatographic peaks and peptide signal loss, even when wide scheduling windows were used. To overcome this limitation, a scout-triggered MRM (st-MRM) acquisition strategy based on event-driven monitoring was implemented. Results: This approach enabled robust peptide detection across diverse matrices within a single injection, without method re-optimization. Absolute quantification using stable isotope-labeled peptides spanned six orders of magnitude, with HCPs quantified down to 2.9 ppm in purified drug substances. Conclusion: Overall, st-MRM improves the robustness and transferability of highly multiplexed targeted proteomics workflows for HCP analysis. Full article

Background/Objectives: Chronic wounds represent a significant clinical burden and require multimodal treatment strategies targeting inflammation, infection, moisture balance, and tissue remodeling, as defined by the TIME framework. This study aimed to evaluate the therapeutic potential of innovative hydrogel dressings enriched with fungal biomass, designed to exploit natural bioactive compounds—such as antimicrobial peptides and proteolytic enzymes—to enhance wound healing while maintaining high biocompatibility. Methods: Hydrogel dressings incorporating selected fungal biomasses were fabricated and characterized for physicochemical and biological performance. Key material properties relevant to wound care, including hydrophilicity and porosity, were analyzed to assess exudate management capacity and maintenance of a moist wound environment. Antimicrobial activity was tested against common wound pathogens, and species–pathogen interactions were evaluated using generalized linear modeling. In vitro biocompatibility was assessed using human keratinocytes and compared with conventional silver nanoparticle–based dressings. Results: The developed hydrogels demonstrated properties suitable for clinical application, including superhydrophilicity and high porosity, supporting effective exudate control and moisture retention. Significant broad-spectrum antimicrobial activity was observed, particularly against Staphylococcus aureus and Pseudomonas aeruginosa, with effects dependent on fungal species. Statistical modeling revealed highly significant interactions between fungal species and pathogens in inhibition zones (p < 0.001). Hydrogels containing Pleurotus ostreatus and Agaricus bisporus showed broad activity against Escherichia coli, P. aeruginosa, and S. aureus, whereas Enterococcus faecalis exhibited resistance. Fungal biomass–based dressings displayed superior keratinocyte biocompatibility compared to silver nanoparticle controls. Conclusions: Fungal biomass–reinforced hydrogels offer a promising, safer, multifunctional alternative for infected chronic wound management, supporting both antimicrobial action and tissue regeneration. Full article

Therapeutic peptides have gained significant attention due to their high specificity, potency, and safety profiles in treating various diseases. However, their clinical application via the oral route remains challenging. Peptides are inherently unstable in the gastrointestinal environment, where they are rapidly degraded by proteolytic enzymes and acidic pH, leading to poor bioavailability. Additionally, their large molecular size and hydrophilicity restrict passive diffusion across the epithelial barriers of the gastrointestinal tract. These limitations have traditionally necessitated parenteral administration, which reduces patient compliance and convenience. The oral cavity, comprising the buccal and sublingual mucosa, offers a promising alternative for peptide delivery. Its rich vascularization allows for rapid systemic absorption while bypassing hepatic first-pass metabolism. Furthermore, the mucosal surface provides a relatively permeable and accessible site for drug administration. However, the oral cavities also present significant barriers: the mucosal epithelium limits permeability, the presence of saliva causes rapid clearance, and enzymes in saliva contribute to peptide degradation. Therefore, innovative strategies are essential to enhance peptide stability, retention, and permeation in this environment. Nanoparticle-based delivery systems, including lipid-based carriers such as liposomes and niosomes, as well as polymeric nanoparticles like chitosan and PLGA, offer promising solutions. These nanocarriers protect peptides from enzymatic degradation, enhance mucoadhesion to prolong residence time, and facilitate controlled release. Their size and surface properties can be engineered to improve mucosal penetration, including through receptor-mediated endocytosis or by transiently opening tight junctions. Among these, niosomes have shown high encapsulation efficiency and sustained release potential, making them particularly suitable for oral peptide delivery. Despite advances, challenges remain in translating these technologies clinically, including ensuring biocompatibility, scalable manufacturing, and patient acceptance. Nevertheless, the oral cavity’s accessibility, combined with nanotechnological innovations, offers a compelling platform for personalized, non-invasive peptide therapies that could significantly improve treatment outcomes and patient quality of life. Full article

This study presents nanofractionation analytics coupled with in vivo profiling of zebrafish embryo paralysis and lethality in response to toxins in cone snail venoms. The focus of this study is on the development of this approach using venoms of Conus marmoreus, Conus ebraeus, and Conus bandanus. In brief, cone snail venoms were separated using reversed-phase chromatography following high-resolution nanofractionation on microplates with parallel mass spectrometry, enabled via a post-column flow split. All collected fractions were dried overnight, followed by assays on zebrafish embryos. For the paralysis assessment, we monitored swimming behavior and swimming distance and found that exposure to cone snail toxins led to paralysis and decreased movement and swim distance. To correlate the masses of eluted toxins with their paralyzing effects and potency, we compared the fractionation retention time versus normalized swimming distance. This allowed identification of the masses of toxins with paralyzing bioactivity, which were predominantly conopeptides. To assess lethality, zebrafish embryos were exposed to fractionated toxins for 24 h, after which they were inspected. The lethal doses and correlated toxins were identified by comparing retention times of fractionation versus the lethal dose values calculated for each fraction. We found that the most lethal venom was from C. bandanus, displaying the largest number of lethal peptides, followed by C. marmoreus and C. ebraeus. On the other hand, the most paralytic venom was from C. ebraeus, presenting a higher number of peptides with non-lethal paralytic effects, followed by C. bandanus and C. marmoreus. This study provides a pipeline to rapidly identify paralytic and lethal cone snail venom toxins using the zebrafish embryo model. Full article

Nisin, a food preservative lantibiotic produced by Lactococcus lactis, exhibits potent antimicrobial activity against a wide range of Gram-positive pathogens, including antibiotic-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA). This study explores the development of a novel nano drug delivery platform comprising nisin-loaded niosomes, formulated via microfluidic mixing, and integrated into fast-dissolving oral films for targeted buccal administration. Microfluidic synthesis enabled the precise control of critical parameters including the flow rate ratio, surfactant composition, and lipid concentration, resulting in uniform niosomal vesicles with optimal size distribution (100–200 nm), low polydispersity index, and high encapsulation efficiency. Span 40 and Span 60 were employed as non-ionic surfactants, stabilized with cholesterol to improve bilayer rigidity and drug retention. The encapsulated nisin demonstrated improved physicochemical stability over time and protection against proteolytic degradation, thus preserving its antimicrobial potency. The niosomal suspensions were subsequently incorporated into polymer-based oral films as a final dosage form composed of polyvinyl alcohol (PVA) as the primary film-forming polymer, polyethylene glycol 400 (PEG400) as a plasticizer, and sucralose and mint as a sweetener and flavoring agent, respectively. A disintegrant was added to accelerate film dissolution in the oral cavity, facilitating the rapid release of niosomal nisin. The films were cast and evaluated for thickness uniformity, mechanical properties, disintegration time, surface morphology, and drug content uniformity. The dried films exhibited desirable flexibility, rapid disintegration (<30 s), and consistent distribution of nisin-loaded vesicles. In vitro antimicrobial assays confirmed that the bioactivity of nisin was retained post-formulation, showing effective inhibition zones (16 mm) against Bacillus subtilis. This delivery system offers a promising platform for localized antimicrobial therapy in the oral cavity, potentially aiding in the treatment of dental plaque, oral infections, and periodontal diseases. Overall, the integration of microfluidic-synthesized nisin niosomes into oral films presents a novel, non-invasive strategy for enhancing the stability and therapeutic efficacy of peptide-based drugs in mucosal environments. Physicochemical characterization of the niosomes and niosome films was performed using Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) to evaluate thermal stability and scanning electron microscopy (SEM) to assess surface morphology. In vitro peptide release studies demonstrated sustained release from both niosomal suspensions and film matrices, and the resulting data were further fitted to established kinetic models to elucidate the underlying drug release mechanisms. This delivery system offers a promising platform for localized antimicrobial therapy in the oral cavity, potentially aiding in the treatment of dental plaque, oral infections, and periodontal diseases. Overall, the integration of microfluidic-synthesized nisin niosomes into oral films presents a novel, non-invasive strategy for enhancing the stability and therapeutic efficacy of peptide-based drugs in mucosal environments. Full article

The application of high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) in the analysis of peptide therapeutics demonstrates its capacity to achieve high sensitivity and selectivity, which are essential qualities for the expanding peptide therapeutic industry. Given the challenges posed by hydrophilic peptides in reversed-phase chromatography, we investigated the necessity of a derivatization procedure to improve chromatographic separation and quasimolecular ion fragmentation during MS/MS detection. We investigated how eight different derivatizing agents react with a hydrophilic lysine- and arginine-containing human ezrin peptide-1 (HEP-1) to identify the most suitable one. The results showed that the reaction of HEP-1 with propionic anhydride proceeds most rapidly and completely, providing a high and reproducible yield of the product, which has sufficient retention on the RP column. The 4-propionylated derivative of HEP-1, compared to the other derivatives considered, demonstrates the most pronounced MS/MS fragmentation. The retention time of 2.42 min allows the separation of the substance from the interfering components of the blood plasma matrix and provides a limit of quantification of 5.00 ng/mL, which allows the use of this derivatizing agent for subsequent applications in pharmacokinetic studies, and this approach can improve the analytical parameters of similar peptides in other HPLC-MS/MS studies. Full article

Positron emission tomography (PET) imaging targeting glypican-3 (GPC3) holds promise for improving the detection and characterization of hepatocellular carcinoma (HCC). Preclinical and early clinical studies have largely utilized high-molecular-weight antibodies radiolabeled with isotopes such as ⁸⁹Zr and ¹²⁴I, demonstrating high affinity and tumor uptake but suffering from prolonged circulation times and suboptimal signal-to-background ratios. To address these limitations, interest has shifted toward low-molecular-weight vectors—synthetic peptides and small antibody fragments—labeled with shorter-lived radionuclides (e.g., ⁶⁸Ga and ¹⁸F) to enable rapid pharmacokinetics and same-day imaging protocols. Emerging platforms such as affibodies and aptamers offer further advantages in target affinity and reduced immunogenicity. However, clinical translation requires rigorous validation: larger, histologically confirmed cohorts, head-to-head comparison with CT/MRI, and correlation with hard clinical endpoints. Moreover, leveraging GPC3 expression as a biomarker could guarantee a deeper knowledge of tumor biology—differentiation grade and vascular invasion risk—and guide theranostic strategies. While β-emitters (⁹⁰Y, ¹⁷⁷Lu) have been explored for GPC3-directed therapy, their efficacy is influenced by oxygenation and cell-cycle status, whereas α-emitters (²²⁵Ac) may overcome these constraints, albeit with challenges in radionuclide selection and daughter nuclide management. Finally, dual-targeting probes combining GPC3 and prostate-specific membrane antigen (PSMA) have demonstrated superior uptake and retention in murine models, suggesting a versatile approach for future clinical diagnostics and therapy planning. Full article

Lymph node metastases are common in advanced ovarian cancer and are associated with poor prognosis. Accurate intraoperative identification of lymph node metastases remains a challenge in ovarian cancer surgery due to the lack of tumor-specific intraoperative imaging tools. Here, we developed a gonadotropin-releasing hormone receptor (GnRHR)-targeted near-infrared (NIR) fluorescent probe, GnRHa-PEG-Rh760, through conjugation of a GnRH analog peptide with the Rh760 fluorophore and polyethylene glycol (PEG). A non-targeted probe (PEG-Rh760) served as control. In mouse models of subcutaneous xenografts, peritoneal and lymph node metastases derived from ovarian cancer cells, GnRHa-PEG-Rh760 showed superior tumor-specific accumulation. NIR fluorescence imaging revealed strong fluorescence signals localized to primary tumors, peritoneal lesions, and metastatic lymph nodes with no off-target signals in normal lymph nodes. The spatial co-localization between the NIR fluorescence of GnRHa-PEG-Rh760 and tumor-derived bioluminescence clearly confirmed the probe’s target specificity. GnRHa-PEG-Rh760 mainly accumulated in the tumor and liver and was gradually cleared at 96 h post-injection. The retention of fluorescence signals in normal ovary tissue further validated GnRHR-mediated binding of the probe. Notably, GnRHa-PEG-Rh760 exhibited excellent biocompatibility with no observed systemic toxicity as evidenced by hematologic and histopathologic analyses. These data demonstrate the potential of GnRHa-PEG-Rh760 as an intraoperative imaging agent, providing real-time fluorescence imaging guidance to optimize surgical precision. This study highlights the value of receptor-targeted molecular imaging probes in precision cancer surgery. Full article

Novel peptides based on common amino acid building blocks may serve as possible drug candidates; however, their flexible structures may require stabilization via the incorporation of conformational constraints. The insertion of unusual amino acids is a feasible option that may provide improved pharmacokinetic and pharmacodynamic properties of such peptide-type drugs. The stereochemical purity of these kinds of building blocks must be verified by an efficient separation technique, such as high-performance liquid chromatography. Here, we present and discuss the results of the stereoselective separation mechanism of ß-methylated phenylalanine (ß-MePhe), tyrosine (ß-MeTyr), 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (ß-MeTic), and cyclohexylalanine (ß-MeCha) together with non-methylated Phe, Tyr, Tic, and Cha applying Cinchona alkaloid-based chiral stationary phases (CSPs). The studied zwitterionic CSPs acting as ion exchangers provided optimal performance in the polar ionic mode when methanol or a mixture of methanol and acetonitrile was utilized as the mobile phase together with organic acid and base additives. It was found that the basicity of small amines applied as mobile phase additives did not directly influence the chromatographic ion exchange concept. However, the size of the amines and their concentration led to a reduced retention time following the principles of ion exchange chromatography. On the basis of a systematic study of the effects of the eluent composition on the chromatographic behavior, important structure–retention and enantioselectivity relationships could be revealed. Through a temperature study, it has become evident that the composition of the eluent and the structure of analytes markedly affect the thermodynamic properties. Full article

Background/Objectives: Gastrin-releasing peptide receptor is a promising target for cancer diagnosis and therapy. However, the high pancreas uptake of reported GRPR-targeted radioligands limits their clinical applications. Our group previously reported one ⁶⁸Ga-labeled GRPR antagonist, [⁶⁸Ga]Ga-TacsBOMB5 (⁶⁸Ga-DOTA-Pip-[D-Phe⁶,NMe-Gly¹¹,Leu¹³ψThz¹⁴]Bombesin(6–14)), and two agonists, [⁶⁸Ga]Ga-LW01110 (⁶⁸Ga-DOTA-Pip-[D-Phe⁶,Tle¹⁰,NMe-His¹²,Thz¹⁴]Bombesin(6–14)) and [⁶⁸Ga]Ga-LW01142 (⁶⁸Ga-DOTA-Pip-[D-Phe⁶,His⁷,Tle¹⁰,NMe-His¹²,Thz¹⁴]Bombesin(6–14)) showing minimal pancreas uptake. Thus, in this study, we prepared their ¹⁷⁷Lu-labeled analogs, evaluated their therapeutic potentials, and compared them with the clinically evaluated [¹⁷⁷Lu]Lu-AMBA. Methods: GRPR binding affinities were determined by in vitro competition binding assay using PC-3 prostate cancer cells. Longitudinal SPECT/CT imaging and ex vivo biodistribution studies were conducted in PC-3 tumor-bearing mice. Dosimetry data were calculated from the biodistribution results. Results: The K_i(GRPR) values of Lu-TacsBOMB5, Lu-LW01110, Lu-LW01142, and Lu-AMBA were 12.6 ± 1.02, 3.07 ± 0.15, 2.37 ± 0.28, and 0.33 ± 0.16 nM, respectively. SPECT/CT images and biodistribution results demonstrated good tumor accumulation of [¹⁷⁷Lu]Lu-TacsBOMB5, [¹⁷⁷Lu]Lu-LW01110, and [¹⁷⁷Lu]Lu-LW01142 at early time points with rapid clearance over time. The pancreas uptake of all three [Thz¹⁴]Bombesin(6–14)-derived ligands was significantly lower than that of [¹⁷⁷Lu]Lu-AMBA at all time points. The calculated absorbed doses of [¹⁷⁷Lu]Lu-TacsBOMB5, [¹⁷⁷Lu]Lu-LW01110, and [¹⁷⁷Lu]Lu-LW01142 in PC-3 tumor xenografts were 87.1, 312, and 312 mGy/MBq, respectively, higher than that of [¹⁷⁷Lu]Lu-AMBA (79.1 mGy/MBq), but lower than that of the previously reported [¹⁷⁷Lu]Lu-RM2 (429 mGy/MBq). Conclusions: Our data suggest that [¹⁷⁷Lu]Lu-TacsBOMB5 and [¹⁷⁷Lu]Lu-LW01142 reduce radiation exposure to the pancreas. However, further optimizations are needed for both radioligands to prolong their tumor retention and enhance treatment efficacy. Full article

Peptide and protein (PP) therapeutics are highly specific and potent biomolecules that treat chronic and complex diseases. However, their oral delivery is significantly hindered by enzymatic degradation, instability, and poor permeability through the gastrointestinal (GI) epithelium, resulting in low bioavailability. Various strategies have emerged as transformative solutions to address existing challenges, offering enhanced protection, stabilization, and absorption of PPs. These strategies primarily focus on two major challenges: protecting the PP against harsh conditions and enhancing permeation across the intestinal membrane. Innovative approaches such as pH modulation and incorporation of enzyme inhibitors are usually used to mitigate proteolytic degradation of PP during transit across the GI tract. In a similar vein, absorption enhancers and prodrug strategies facilitate epithelial transport, while targeted delivery systems focus on specific areas of the GI tract to enhance absorption. Likewise, mucus-penetrating and mucoadhesive strategies have enhanced retention and interaction with epithelial cells, effectively overcoming barriers like the mucus layer and tight epithelial junctions. Furthermore, structural modifications such as lipidation, peptide cyclization, and polyethylene glycosylation are promising alternatives to render stability, prolong circulation time, and membrane permeability. In particular, functional biomaterials, active targeting, and lymphatic transport strategies have provided new platforms for oral PP delivery. Advancing in materials science, nanotechnology, and the disruption of medical devices holds new frontiers to overcome barriers. Despite substantial advancements, the limited success in clinical translation underscores the urgency of innovative strategies. This review presents oral PPs as a promising platform, highlighting the key barriers and strategies to transform their therapeutic landscapes. Full article

Background/Objectives: Cm-p5 and its cyclic monomeric and dimeric analogues are known for their antifungal, antibacterial, antiviral, and antibiofilm activities. Previously, our cyclization method produced a mixture of peptides that were difficult to separate, which was then improved by a selective synthesis of the parallel dimer and its differentiation from the antiparallel by comparison of the retention times in RP-HPLC. Methods: Here, we developed a more reliable identification method for the Cm-p5 dimer identification, which included chymotrypsin proteolytic digestion and sequencing of the different fragments by ESI-MSMS. We also improved our cyclization methods to specifically produce higher amounts of the desired cyclic variant, either cyclic monomer or dimer. Results: We show that liquid phase oxidation with 20% DMSO or iodine oxidation yields only the cyclic analogue. However, the on-resin oxidation with iodine showed greater efficacy and efficiency. Additionally, liquid phase cyclization yields the antiparallel dimer in high EtOH or peptide concentration, indicating a kinetic control. On the other hand, the parallel dimer was preferentially produced in 5% of TFE and low peptide concentration without the formation of the cyclic analogue indicating a thermodynamic control. Conclusions: In conclusion, we report that chymotryptic digestion combined with ESI-MS and MS/MS allows an unambiguous differentiation of Cm-p5 dimers. Here, we develop more selective and efficient methods for the synthesis of cyclic and dimeric analogues of Cm-p5. Full article

Background/Objectives: Overexpressed in various solid tumors, the gastrin-releasing peptide receptor (GRPR) is a promising target for cancer diagnosis and therapy. However, the high pancreas uptake of the current clinically evaluated GRPR-targeted radiopharmaceuticals limits their applications. In this study, we replaced the Pro¹⁴ residue in our previously reported GRPR-targeted LW02056 and ProBOMB5 with 4,4-difluoroproline (diF-Pro) to obtain an agonist LW02060 (DOTA-Pip-[D-Phe⁶,Tle¹⁰,NMe-His¹²,diF-Pro¹⁴]Bombesin(6–14)) and an antagonist LW02080 (DOTA-Pip-[D-Phe⁶,NMe-Gly¹¹,Leu¹³(ψ)diF-Pro¹⁴]Bombesin(6–14)), respectively. Methods/Results: The binding affinities (K_i) of Ga-LW02060, Ga-LW02080, Lu-LW02060, and Lu-LW02080 were measured by in vitro competition binding assays using PC-3 cells and were found to be 5.57 ± 2.47, 21.7 ± 6.69, 8.00 ± 2.61, and 32.1 ± 8.14 nM, respectively. The ⁶⁸Ga- and ¹⁷⁷Lu-labeled ligands were obtained in 36–75% decay-corrected radiochemical yields with >95% radiochemical purity. PET imaging, SPECT imaging, and ex vivo biodistribution studies were conducted in PC-3 tumor-bearing mice. Both [⁶⁸Ga]Ga-LW02060 and [⁶⁸Ga]Ga-LW02080 enabled clear tumor visualization in PET images at 1 h post-injection (pi). Tumor uptake values of [⁶⁸Ga]Ga-LW02060 and [⁶⁸Ga]Ga-LW02080 at 1 h pi were 16.8 ± 2.70 and 7.36 ± 1.33 %ID/g, respectively, while their pancreas uptake values were 3.12 ± 0.89 and 0.38 ± 0.04 %ID/g, respectively. Compared to [¹⁷⁷Lu]Lu-LW02080, [¹⁷⁷Lu]Lu-LW02060 showed higher tumor uptake at all time points (1, 4, 24, 72, and 120 h pi). However, fast tumor clearance was observed for both [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080. Conclusions: Our data demonstrate that [⁶⁸Ga]Ga-LW02060 is promising for clinical translation for the detection of GRPR-expressing tumor lesions. However, further optimizations are needed for [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080 to prolong tumor retention for therapeutic applications. Full article