3D small-scale dosimetry and tumor control of 225Ac radiopharmaceuticals for prostate cancer.

Radiopharmaceutical therapy using α -emitting 225 Ac is an emerging treatment for patients with advanced metastatic cancers. Measurement of the spatial dose distribution in organs and tumors is needed to inform treatment dose prescription and reduce off-target toxicity, at not only organ but also sub-organ scales. Digital autoradiography with α -sensitive detection devices can measure radioactivity distributions at 20-40 µ m resolution, but anatomical characterization is typically limited to 2D. We collected digital autoradiographs across whole tissues to generate 3D dose volumes and used them to evaluate the simultaneous tumor control and regional kidney dosimetry of a novel therapeutic radiopharmaceutical for prostate cancer, [225Ac]Ac-Macropa-PEG4-YS5, in mice. 22Rv1 xenograft-bearing mice treated with 18.5 kBq of [225Ac]Ac-Macropa-PEG4-YS5 were sacrificed at 24 h and 168 h post-injection for quantitative α -particle digital autoradiography and hematoxylin and eosin staining. Gamma-ray spectroscopy of biodistribution data was used to determine temporal dynamics and 213 Bi redistribution. Tumor control probability and sub-kidney dosimetry were assessed. Heterogeneous 225 Ac spatial distribution was observed in both tumors and kidneys. Tumor control was maintained despite heterogeneity if cold spots coincided with necrotic regions. 225 Ac dose-rate was highest in the cortex and renal vasculature. Extrapolation of tumor control suggested that kidney absorbed dose could be reduced by 41% while maintaining 90% TCP. The 3D dosimetry methods described allow for whole tumor and organ dose measurements following 225 Ac radiopharmaceutical therapy, which correlate to tumor control and toxicity outcomes.

Actinium-225 targeted alpha particle therapy for prostate cancer.

Targeted alpha particle therapy (TAT) has emerged as a promising strategy for the treatment of prostate cancer (PCa). Actinium-225 (225Ac), a potent alpha-emitting radionuclide, may be incorporated into targeting vectors, causing robust and in some cases sustained antitumor responses. The development of radiolabeling techniques involving EDTA, DOTA, DOTPA, and Macropa chelators has laid the groundwork for advancements in this field. At the forefront of clinical trials with 225Ac in PCa are PSMA-targeted TAT agents, notably [225Ac]Ac-PSMA-617, [225Ac]Ac-PSMA-I&T and [225Ac]Ac-J591. Ongoing investigations spotlight [225Ac]Ac-hu11B6, [225Ac]Ac-YS5, and [225Ac]Ac-SibuDAB, targeting hK2, CD46, and PSMA, respectively. Despite these efforts, hurdles in 225Ac production, daughter redistribution, and a lack of suitable imaging techniques hinder the development of TAT. To address these challenges and additional advantages, researchers are exploring alpha-emitting isotopes including 227Th, 223Ra, 211At, 213Bi, 212Pb or 149Tb, providing viable alternatives for TAT.

Prostate-Specific Membrane Antigen Targeted StarPEG Nanocarrier for Imaging and Therapy of Prostate Cancer.

The tumor uptake of large non-targeted nanocarriers primarily occurs through passive extravasation, known as the enhanced permeability and retention (EPR) effect. Prior studies demonstrated improved tumor uptake and retention of 4-arm 40 kDa star polyethylene glycol (StarPEG) polymers for cancer imaging by adding prostate-specific membrane antigen (PSMA) targeting small molecule ligands. To test PSMA-targeted delivery and therapeutic efficacy, StarPEG nanodrugs with/without three copies of PSMA-targeting ligands, ACUPA, are designed and synthesized. For single-photon emission computed tomography (SPECT) imaging and therapy, each nanocarrier is labeled with 177Lu using DOTA radiometal chelator. The radiolabeled nanodrugs, [177Lu]PEG-(DOTA)1 and [177Lu]PEG-(DOTA)1(ACUPA)3, are evaluated in vitro and in vivo using PSMA+ PC3-Pip and/or PSMA- PC3-Flu cell lines, subcutaneous xenografts and disseminated metastatic models. The nanocarriers are efficiently radiolabeled with 177Lu with molar activities 10.8-15.8 MBq/nmol. Besides excellent in vitro PSMA binding affinity (kD = 51.7 nM), the targeted nanocarrier, [177Lu]PEG-(DOTA)1(ACUPA)3, demonstrated excellent in vivo SPECT imaging contrast with 21.3% ID/g PC3-Pip tumors uptake at 192 h. Single doses of 18.5 MBq [177Lu]PEG-(DOTA)1(ACUPA)3 showed complete resolution of the PC3-Pip xenografts observed up to 138 days. Along with PSMA-targeted excellent imaging contrast, these results demonstrated high treatment efficacy of [177Lu]PEG-(DOTA)1(ACUPA)3 for prostate cancer, with potential for clinical translation.

A Technique to Quantify Very Low Activities in Regions of Interest With a Collimatorless Detector.

We present a new method to measure sub-microcurie activities of photon-emitting radionuclides in organs and lesions of small animals in vivo. Our technique, named the collimator-less likelihood fit, combines a very high sensitivity collimatorless detector with a Monte Carlo-based likelihood fit in order to estimate the activities in previously segmented regions of interest along with their uncertainties. This is done directly from the photon projections in our collimatorless detector and from the region of interest segmentation provided by an x-ray computed tomography scan. We have extensively validated our approach with 225Ac experimentally in spherical phantoms and mouse phantoms, and also numerically with simulations of a realistic mouse anatomy. Our method yields statistically unbiased results with uncertainties smaller than 20% for activities as low as ~111Bq (3nCi) and for exposures under 30 minutes. We demonstrate that our method yields more robust recovery coefficients when compared to SPECT imaging with a commercial pre-clinical scanner, specially at very low activities. Thus, our technique is complementary to traditional SPECT/CT imaging since it provides a more accurate and precise organ and tumor dosimetry, with a more limited spatial information. Finally, our technique is specially significant in extremely low-activity scenarios when SPECT/CT imaging is simply not viable.

Development of CD46 targeted alpha theranostics in prostate cancer using 134Ce/225Ac-Macropa-PEG4-YS5.

Rationale: 225Ac, a long-lived α-emitter with a half-life of 9.92 days, has garnered significant attention as a therapeutic radionuclide when coupled with monoclonal antibodies and other targeting vectors. Nevertheless, its clinical utility has been hampered by potential off-target toxicity, a lack of optimized chelators for 225Ac, and limitations in radiolabeling methods. In a prior study evaluating the effectiveness of CD46-targeted radioimmunotherapy, we found great therapeutic efficacy but also significant toxicity at higher doses. To address these challenges, we have developed a radioimmunoconjugate called 225Ac-Macropa-PEG4-YS5, incorporating a stable PEGylated linker to maximize tumoral uptake and increase tumor-to-background ratios. Our research demonstrates that this conjugate exhibits greater anti-tumor efficacy while minimizing toxicity in prostate cancer 22Rv1 tumors. Methods: We synthesized Macropa.NCS and Macropa-PEG4/8-TFP esters and prepared Macropa-PEG0/4/8-YS5 (with nearly ~1:1 ratio of macropa chelator to antibody YS5) as well as DOTA-YS5 conjugates. These conjugates were then radiolabeled with 225Ac in a 2 M NH4OAc solution at 30 °C, followed by purification using YM30K centrifugal purification. Subsequently, we conducted biodistribution studies and evaluated antitumor activity in nude mice (nu/nu) bearing prostate 22Rv1 xenografts in both single-dose and fractionated dosing studies. Micro-PET imaging studies were performed with 134Ce-Macropa-PEG0/4/8-YS5 in 22Rv1 xenografts for 7 days. Toxicity studies were also performed in healthy athymic nude mice. Results: As expected, we achieved a >95% radiochemical yield when labeling Macropa-PEG0/4/8-YS5 with 225Ac, regardless of the chelator ratios (ranging from 1 to 7.76 per YS5 antibody). The isolated yield exceeded 60% after purification. Such high conversions were not observed with the DOTA-YS5 conjugate, even at a higher ratio of 8.5 chelators per antibody (RCY of 83%, an isolated yield of 40%). Biodistribution analysis at 7 days post-injection revealed higher tumor uptake for the 225Ac-Macropa-PEG4-YS5 (82.82 ± 38.27 %ID/g) compared to other conjugates, namely 225Ac-Macropa-PEG0/8-YS5 (38.2 ± 14.4/36.39 ± 12.4 %ID/g) and 225Ac-DOTA-YS5 (29.35 ± 7.76 %ID/g). The PET Imaging of 134Ce-Macropa-PEG0/4/8-YS5 conjugates resulted in a high tumor uptake, and tumor to background ratios. In terms of antitumor activity, 225Ac-Macropa-PEG4-YS5 exhibited a substantial response, leading to prolonged survival compared to 225Ac-DOTA-YS5, particularly when administered at 4.625 kBq doses, in single or fractionated dose regimens. Chronic toxicity studies observed mild to moderate renal toxicity at 4.625 and 9.25 kBq doses. Conclusions: Our study highlights the promise of 225Ac-Macropa-PEG4-YS5 for targeted alpha particle therapy. The 225Ac-Macropa-PEG4-YS5 conjugate demonstrates improved biodistribution, reduced off-target binding, and enhanced therapeutic efficacy, particularly at lower doses, compared to 225Ac-DOTA-YS5. Incorporating theranostic 134Ce PET imaging further enhances the versatility of macropa-PEG conjugates, offering a more effective and safer approach to cancer treatment. Overall, this methodology has a high potential for broader clinical applications.

CD46-Targeted Theranostics for PET and 225Ac-Radiopharmaceutical Therapy of Multiple Myeloma.

PURPOSE: Multiple myeloma is a plasma cell malignancy with an unmet clinical need for improved imaging methods and therapeutics. Recently, we identified CD46 as an overexpressed therapeutic target in multiple myeloma and developed the antibody YS5, which targets a cancer-specific epitope on this protein. We further developed the CD46-targeting PET probe [89Zr]Zr-DFO-YS5 for imaging and [225Ac]Ac-DOTA-YS5 for radiopharmaceutical therapy of prostate cancer. These prior studies suggested the feasibility of the CD46 antigen as a theranostic target in multiple myeloma. Herein, we validate [89Zr]Zr-DFO-YS5 for immunoPET imaging and [225Ac]Ac-DOTA-YS5 for radiopharmaceutical therapy of multiple myeloma in murine models. EXPERIMENTAL DESIGN: In vitro saturation binding was performed using the CD46 expressing MM.1S multiple myeloma cell line. ImmunoPET imaging using [89Zr]Zr-DFO-YS5 was performed in immunodeficient (NSG) mice bearing subcutaneous and systemic multiple myeloma xenografts. For radioligand therapy, [225Ac]Ac-DOTA-YS5 was prepared, and both dose escalation and fractionated dose treatment studies were performed in mice bearing MM1.S-Luc systemic xenografts. Tumor burden was analyzed using BLI, and body weight and overall survival were recorded to assess antitumor effect and toxicity. RESULTS: [89Zr]Zr-DFO-YS5 demonstrated high affinity for CD46 expressing MM.1S multiple myeloma cells (Kd = 16.3 nmol/L). In vitro assays in multiple myeloma cell lines demonstrated high binding, and bioinformatics analysis of human multiple myeloma samples revealed high CD46 expression. [89Zr]Zr-DFO-YS5 PET/CT specifically detected multiple myeloma lesions in a variety of models, with low uptake in controls, including CD46 knockout (KO) mice or multiple myeloma mice using a nontargeted antibody. In the MM.1S systemic model, localization of uptake on PET imaging correlated well with the luciferase expression from tumor cells. A treatment study using [225Ac]Ac-DOTA-YS5 in the MM.1S systemic model demonstrated a clear tumor volume and survival benefit in the treated groups. CONCLUSIONS: Our study showed that the CD46-targeted probe [89Zr]Zr-DFO-YS5 can successfully image CD46-expressing multiple myeloma xenografts in murine models, and [225Ac]Ac-DOTA-YS5 can effectively inhibit the growth of multiple myeloma. These results demonstrate that CD46 is a promising theranostic target for multiple myeloma, with the potential for clinical translation.

Highly efficient color-tunable organic co-crystals unveiling polymorphism, isomerism, delayed fluorescence for optical waveguides and cell-imaging.

Photofunctional co-crystal engineering strategies based on donor-acceptor π-conjugated system facilitates expedient molecular packing, consistent morphology, and switchable optical properties, conferring synergic 'structure-property relationship' for optoelectronic and biological functions. In this work, a series of organic co-crystals were formulated using a twisted aromatic hydrocarbon (TAH) donor and three diverse planar acceptors, resulting in color-tunable solid and aggregated state emission via variable packing and through-space charge-transfer interactions. While, adjusting the strength of acceptors, a structural transformation into hybrid stacking modes ultimately results in color-specific polymorphs, a configurational cis-isomer with very high photoluminescence quantum yield. The cis-isomeric co-crystal exhibits triplet-harvesting thermally activated delayed fluorescence (TADF) characteristics, presenting a key discovery in hydrocarbon-based multicomponent systems. Further, 1D-microrod-shaped co-crystal acts as an efficient photon-transducing optical waveguides, and their excellent dispersibility in water endows efficient cellular internalization with bright cell imaging performances. These salient approaches may open more avenues for the design and applications of TAH based co-crystals.

Coded aperture and Compton imaging for the development of 225 Ac-based radiopharmaceuticals.

BACKGROUND: Targeted alpha-particle therapy (TAT) has great promise as a cancer treatment. Arguably the most promising TAT radionuclide that has been proposed is 225 Ac. The development of 225 Ac-based radiopharmaceuticals has been hampered due to the lack of effective means to study the daughter redistribution of these agents in small animals at the preclinical stage. PURPOSE: The ability to directly image the daughters, namely 221 Fr and 213 Bi, via their gamma-ray emissions would be a boon for preclinical studies. That said, conventional medical imaging modalities, including single photon emission computed tomography (SPECT) based on nonmultiplexed collimation, cannot be employed due to sensitivity limitations. METHODS: As an alternative, we propose the use of both coded aperture and Compton imaging with the former modality suited to the 218-keV gamma-ray emission of 221 Fr and the latter suited to the 440-keV gamma-ray emission of 213 Bi. RESULTS: This work includes coded aperture images of 221 Fr and Compton images of 213 Bi in tumor-bearing mice injected with 225 Ac-based radiopharmaceuticals. CONCLUSIONS: These results are the first demonstration of visualizing and quantifying the 225 Ac daughters in small animals through the application of coded aperture and Compton imaging.

Evaluation of 134Ce/134La as a PET Imaging Theranostic Pair for 225Ac α-Radiotherapeutics.

225Ac-targeted α-radiotherapy is a promising approach to treating malignancies, including prostate cancer. However, α-emitting isotopes are difficult to image because of low administered activities and a low fraction of suitable γ-emissions. The in vivo generator 134Ce/134La has been proposed as a potential PET imaging surrogate for the therapeutic nuclides 225Ac and 227Th. In this report, we detail efficient radiolabeling methods using the 225Ac-chelators DOTA and MACROPA. These methods were applied to radiolabeling of prostate cancer imaging agents, including PSMA-617 and MACROPA-PEG4-YS5, for evaluation of their in vivo pharmacokinetic characteristics and comparison to the corresponding 225Ac analogs. Methods: Radiolabeling was performed by mixing DOTA/MACROPA chelates with 134Ce/134La in NH4OAc, pH 8.0, at room temperature, and radiochemical yields were monitored by radio-thin-layer chromatography. In vivo biodistributions of 134Ce-DOTA/MACROPA.NH2 complexes were assayed through dynamic small-animal PET/CT imaging and ex vivo biodistribution studies over 1 h in healthy C57BL/6 mice, compared with free 134CeCl3 In vivo, preclinical imaging of 134Ce-PSMA-617 and 134Ce-MACROPA-PEG4-YS5 was performed on 22Rv1 tumor-bearing male nu/nu-mice. Ex vivo biodistribution was performed for 134Ce/225Ac-MACROPA-PEG4-YS5 conjugates. Results: 134Ce-MACROPA.NH2 demonstrated near-quantitative labeling with 1:1 ligand-to-metal ratios at room temperature, whereas a 10:1 ligand-to-metal ratio and elevated temperatures were required for DOTA. Rapid urinary excretion and low liver and bone uptake were seen for 134Ce/225Ac-DOTA/MACROPA. NH2 conjugates in comparison to free 134CeCl3 confirmed high in vivo stability. An interesting observation during the radiolabeling of tumor-targeting vectors PSMA-617 and MACROPA-PEG4-YS5-that the daughter 134La was expelled from the chelate after the decay of parent 134Ce-was confirmed through radio-thin-layer chromatography and reverse-phase high-performance liquid chromatography. Both conjugates, 134Ce-PSMA-617 and 134Ce-MACROPA-PEG4-YS5, displayed tumor uptake in 22Rv1 tumor-bearing mice. The ex vivo biodistribution of 134Ce-MACROPA.NH2, 134Ce-DOTA and 134Ce-MACROPA-PEG4-YS5 corroborated well with the respective 225Ac-conjugates. Conclusion: These results demonstrate the PET imaging potential for 134Ce/134La-labeled small-molecule and antibody agents. The similar 225Ac and 134Ce/134La-chemical and pharmacokinetic characteristics suggest that the 134Ce/134La pair may act as a PET imaging surrogate for 225Ac-based radioligand therapies.

Treatment of Prostate Cancer with CD46-targeted 225Ac Alpha Particle Radioimmunotherapy.

PURPOSE: Radiopharmaceutical therapy is changing the standard of care in prostate cancer and other malignancies. We previously reported high CD46 expression in prostate cancer and developed an antibody-drug conjugate and immunoPET agent based on the YS5 antibody, which targets a tumor-selective CD46 epitope. Here, we present the preparation, preclinical efficacy, and toxicity evaluation of [225Ac]DOTA-YS5, a radioimmunotherapy agent based on the YS5 antibody. EXPERIMENTAL DESIGN: [225Ac]DOTA-YS5 was developed, and its therapeutic efficiency was tested on cell-derived (22Rv1, DU145), and patient-derived (LTL-545, LTL484) prostate cancer xenograft models. Biodistribution studies were carried out on 22Rv1 tumor xenograft models to confirm the targeting efficacy. Toxicity analysis of the [225Ac]DOTA-YS5 was carried out on nu/nu mice to study short-term (acute) and long-term (chronic) toxicity. RESULTS: Biodistribution study shows that [225Ac]DOTA-YS5 agent delivers high levels of radiation to the tumor tissue (11.64% ± 1.37%ID/g, 28.58% ± 10.88%ID/g, 29.35% ± 7.76%ID/g, and 31.78% ± 5.89%ID/g at 24, 96, 168, and 408 hours, respectively), compared with the healthy organs. [225Ac]DOTA-YS5 suppressed tumor size and prolonged survival in cell line-derived and patient-derived xenograft models. Toxicity analysis revealed that the 0.5 µCi activity levels showed toxicity to the kidneys, likely due to redistribution of daughter isotope 213Bi. CONCLUSIONS: [225Ac]DOTA-YS5 suppressed the growth of cell-derived and patient-derived xenografts, including prostate-specific membrane antigen-positive and prostate-specific membrane antigen-deficient models. Overall, this preclinical study confirms that [225Ac]DOTA-YS5 is a highly effective treatment and suggests feasibility for clinical translation of CD46-targeted radioligand therapy in prostate cancer.

CD46 targeted 212Pb alpha particle radioimmunotherapy for prostate cancer treatment.

We recently identified CD46 as a novel prostate cancer cell surface antigen that shows lineage independent expression in both adenocarcinoma and small cell neuroendocrine subtypes of metastatic castration resistant prostate cancer (mCRPC), discovered an internalizing human monoclonal antibody YS5 that binds to a tumor selective CD46 epitope, and developed a microtubule inhibitor-based antibody drug conjugate that is in a multi-center phase I trial for mCRPC (NCT03575819). Here we report the development of a novel CD46-targeted alpha therapy based on YS5. We conjugated 212Pb, an in vivo generator of alpha-emitting 212Bi and 212Po, to YS5 through the chelator TCMC to create the radioimmunoconjugate, 212Pb-TCMC-YS5. We characterized 212Pb-TCMC-YS5 in vitro and established a safe dose in vivo. We next studied therapeutic efficacy of a single dose of 212Pb-TCMC-YS5 using three prostate cancer small animal models: a subcutaneous mCRPC cell line-derived xenograft (CDX) model (subcu-CDX), an orthotopically grafted mCRPC CDX model (ortho-CDX), and a prostate cancer patient-derived xenograft model (PDX). In all three models, a single dose of 0.74 MBq (20 µCi) 212Pb-TCMC-YS5 was well tolerated and caused potent and sustained inhibition of established tumors, with significant increases of survival in treated animals. A lower dose (0.37 MBq or 10 µCi 212Pb-TCMC-YS5) was also studied on the PDX model, which also showed a significant effect on tumor growth inhibition and prolongation of animal survival. These results demonstrate that 212Pb-TCMC-YS5 has an excellent therapeutic window in preclinical models including PDXs, opening a direct path for clinical translation of this novel CD46-targeted alpha radioimmunotherapy for mCRPC treatment.

Immunotherapeutic Targeting and PET Imaging of DLL3 in Small-Cell Neuroendocrine Prostate Cancer.

Effective treatments for de novo and treatment-emergent small-cell/neuroendocrine (t-SCNC) prostate cancer represent an unmet need for this disease. Using metastatic biopsies from patients with advanced cancer, we demonstrate that delta-like ligand 3 (DLL3) is expressed in de novo and t-SCNC and is associated with reduced survival. We develop a PET agent, [89Zr]-DFO-DLL3-scFv, that detects DLL3 levels in mouse SCNC models. In multiple patient-derived xenograft models, AMG 757 (tarlatamab), a half-life-extended bispecific T-cell engager (BiTE) immunotherapy that redirects CD3-positive T cells to kill DLL3-expressing cells, exhibited potent and durable antitumor activity. Late relapsing tumors after AMG 757 treatment exhibited lower DLL3 levels, suggesting antigen loss as a resistance mechanism, particularly in tumors with heterogeneous DLL3 expression. These findings have been translated into an ongoing clinical trial of AMG 757 in de novo and t-SCNC, with a confirmed objective partial response in a patient with histologically confirmed SCNC. Overall, these results identify DLL3 as a therapeutic target in SCNC and demonstrate that DLL3-targeted BiTE immunotherapy has significant antitumor activity in this aggressive prostate cancer subtype. SIGNIFICANCE: The preclinical and clinical evaluation of DLL3-directed immunotherapy, AMG 757, and development of a PET radiotracer for noninvasive DLL3 detection demonstrate the potential of targeting DLL3 in SCNC prostate cancer.

Prostate-Specific Membrane Antigen Targeted Deep Tumor Penetration of Polymer Nanocarriers.

Tumoral uptake of large-size nanoparticles is mediated by the enhanced permeability and retention (EPR) effect, with variable accumulation and heterogenous tumor tissue penetration depending on the tumor phenotype. The performance of nanocarriers via specific targeting has the potential to improve imaging contrast and therapeutic efficacy in vivo with increased deep tissue penetration. To address this hypothesis, we designed and synthesized prostate cancer-targeting starPEG nanocarriers (40 kDa, 15 nm), [89Zr]PEG-(DFB)3(ACUPA)1 and [89Zr]PEG-(DFB)1(ACUPA)3, with one or three prostate-specific membrane antigen (PSMA)-targeting ACUPA ligands. The in vitro PSMA binding affinity and in vivo pharmacokinetics of the targeted nanocarriers were compared with a nontargeted starPEG, [89Zr]PEG-(DFB)4, in PSMA+ PC3-Pip and PSMA- PC3-Flu cells, and xenografts. Increasing the number of ACUPA ligands improved the in vitro binding affinity of PEG-derived polymers to PC3-Pip cells. While both PSMA-targeted nanocarriers significantly improved tissue penetration in PC3-Pip tumors, the multivalent [89Zr]PEG-(DFB)1(ACUPA)3 showed a remarkably higher PC3-Pip/blood ratio and background clearance. In contrast, the nontargeted [89Zr]PEG-(DFB)4 showed low EPR-mediated accumulation with poor tumor tissue penetration. Overall, ACUPA conjugated targeted starPEGs significantly improve tumor retention with deep tumor tissue penetration in low EPR PC3-Pip xenografts. These data suggest that PSMA targeting with multivalent ACUPA ligands may be a generally applicable strategy to increase nanocarrier delivery to prostate cancer. These targeted multivalent nanocarriers with high tumor binding and low healthy tissue retention could be employed in imaging and therapeutic applications.

Synthesis and Preliminary Biological Assessment of Carborane-Loaded Theranostic Nanoparticles to Target Prostate-Specific Membrane Antigen.

Boron neutron capture therapy (BNCT) is an encouraging therapeutic modality for cancer treatment. Prostate-specific membrane antigen (PSMA) is a cell membrane protein that is abundantly overexpressed in prostate cancer and can be targeted with radioligand therapies to stimulate clinical responses in patients. In principle, a spatially targeted neutron beam together with specifically targeted PSMA ligands could enable prostate cancer-targeted BNCT. Thus, we developed and tested PSMA-targeted poly(lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-b-PEG) nanoparticles (NPs) loaded with carborane and tethered to the radiometal chelator deferoxamine B (DFB) for simultaneous positron emission tomography (PET) imaging and selective delivery of boron to prostate cancer. Monomeric PLGA-b-PEGs were covalently functionalized with either DFB or the PSMA ligand ACUPA. Different nanoparticle formulations were generated by nanoemulsification of the corresponding unmodified and DFB- or ACUPA-modified monomers in varying percent fractions. The nanoparticles were efficiently labeled with 89Zr and were subjected to in vitro and in vivo evaluation. The optimized DFB(25)ACUPA(75) NPs exhibited strong in vitro binding to PSMA in direct binding and competition radioligand binding assays in PSMA(+) PC3-Pip cells. [89Zr]DFB(25) NPs and [89Zr]DFB(25)ACUPA(75) NPs were injected to mice with bilateral PSMA(-) PC3-Flu and PSMA(+) PC3-Pip dual xenografts. The NPs demonstrated twofold superior accumulation in PC3-Pip tumors to that of PC3-Flu tumors with a tumor/blood ratio of 25; however, no substantial effect of the ACUPA ligands was detected. Moreover, fast release of carborane from the NPs was observed, resulting in a low boron delivery to tumors in vivo. In summary, these data demonstrate the synthesis, characterization, and initial biological assessment of PSMA-targeted, carborane-loaded PLGA-b-PEG nanoparticles and establish the foundation for future efforts to enable their best use in vivo.

Developing membrane-derived nanocarriers for ex vivo therapy of homologous breast cancer cells.

Aim: The primary aim of this study was to develop biomimetic nanocarriers for specific homologous targeting of the anticancer drugs ammonium pyrrolidine dithiocarbamate (PDTC) and doxorubicin. Methods: Membranous nanovesicles were synthesized from a breast cancer cell line (MCF7) by syringe extrusion process and were loaded with PDTC and doxorubicin. Besides their abilities for self-homing, the drug-loaded nanovesicles showed anti-cell proliferative effects via the generation of reactive oxygen species. Results: The nanovesicles demonstrated efficient internalization via homologous targeting. Delivery of PDTC showed a higher killing effect for homologous cell targeting than other cell types. Experimental results demonstrated increased antiproliferative potency of PDTC, which induced apoptosis via reactive oxygen species generation. Conclusion: The developed membrane-derived nanocarrier is an attractive biocompatible system for ex vivo targeted drug delivery.

Effect of Secondary Structure and Side Chain Length of Hydrophobic Amino Acid Residues on the Antimicrobial Activity and Toxicity of 14-Residue-Long de novo AMPs.

Herein we report the efficacy and toxicity of three de novo designed cationic antimicrobial peptides (AMPs) LL-14, VV-14 and ßß-14, where side chains of the hydrophobic amino acids were reduced gradually. The AMPs showed broad-spectrum antimicrobial activity against three pathogens from the ESKAPE group and two fungal strains. This study showed that side chains which are either too long or too short increase toxicity and lower antimicrobial activity, respectively. VV-14 was found to be non-cytotoxic and highly potent under physiological salt concentrations against several pathogens, especially Salmonella typhi TY2. These AMPs acted via membrane deformation, depolarization, and lysis. The activity of the AMPs is related to their ability to take on amphipathic helical conformations in the presence of microbial membrane mimics. Among AMPs with the same charge, hydrophobic interactions between the side chains of the residues with cell membrane lipids determine their antimicrobial potency and cytotoxicity. Strikingly, an optimum hydrophobic interaction is the crux of generating highly potent non-cytotoxic AMPs.

A conformational tweak for enhanced cellular internalization, photobleaching resistance and prolonged imaging efficacy.

A strategy of conformational tweaking regulates the condensed state behavior of naphthalimide skeletal isomers (NSIs) and enhances their photophysical properties, cellular uptake and prolonged imaging capability. This salient approach results in a large Stokes shift (>120 nm), rapid cellular internalization, photobleaching resistance, and efficient bioimaging of the ribbon-like nano-assembly superior to that of its electronically similar micro-flower isomer.

Nerve growth factor from Indian Russell's viper venom (RVV-NGFa) shows high affinity binding to TrkA receptor expressed in breast cancer cells: Application of fluorescence labeled RVV-NGFa in the clinical diagnosis of breast cancer.

Nerve growth factor (NGF) is a minor and neglected component of snake venom. Present study describes the purification and characterization of a NGF isoform (RVV-NGFa) from Indian Russell's viper venom (RVV). RVV-NGFa showed a protonated molecular ion [MH+] at m/z 17388.725 Da. The RVV-NGFa induced neuritogenesis in PC-12 cells but did not show cytotoxicity in mammalian cells, hemolytic activity, platelet modulation, and interference in blood coagulation system which are the characteristic pharmacological properties of RVV. By ELISA and immunofluorescence microplate reader assay, the RVV-NGFa showed appreciable binding to TrkA receptor expressed in breast cancer MDA-MB-231 and MCF-7 cells; nevertheless, pre-incubation of cells with anti-TrkA (and not TrkB or TrkC) or anti-p75NTR antibody significantly decreased (p < 0.05) this binding. The RVV-NGFa demonstrated insignificant binding with non-cancerous cells (HEK-293, L6) lacking TrkA receptor. The binding of RVV-NGFa to TrkA receptor of breast cancer cells resulted in internalization of ligand (RVV-NGFa)-receptor (TrkA) complex to cell cytoplasm in a time-dependent manner. The spectrofluorometric study demonstrated an interaction between RVV-NGFa and cytosolic domain of the purified TrkA receptor. The fluorescence (FITC)-tagged RVV-NGFa depicted a strong fluorescence signal that was observed under a fluorescence microscope and determined by fluorescence microplate reader assay post binding to breast cancer cells; but no fluorescence signal was detected after incubating FITC-RVV-NGFa with non-cancerous L6 and HEK-293 cells. The clinical application of FITC/fluorescence nanoparticle tagged RVV-NGFa for the ex vivo and in vivo diagnosis of breast cancer is highly promising.

Rationally designed antimicrobial peptides: Insight into the mechanism of eleven residue peptides against microbial infections.

The widespread abuse of antibiotics has led to the use of antimicrobial peptides (AMPs) as a replacement for the existing conventional therapeutic agents for combating microbial infections. The broad-spectrum activity and the resilient nature of AMPs has mainly aggrandized their utilization. Here, we report the design of non-toxic, non-hemolytic and salt tolerant undecapeptides (AMP21-24), derived by modification of a peptide P5 (NH2-LRWLRRLCONH2) reported earlier by our group. Our results depict that the designed peptides show potency against several bacterial as well as fungal strains. Circular dichroism (CD) spectroscopy in combination with molecular dynamic (MD) simulations confirm that the peptides are unstructured. Intrinsic tryptophan fluorescence quenching as well as interaction studies using isothermal calorimetry (ITC) of these peptides in the presence of biological microbial membrane mimics establish the strong microbial membrane affinity of these AMPs. Membrane permeabilization assay and cytoplasmic membrane depolarization studies of Pseudomonas aeruginosa and Candida albicans in the presence of AMPs also hint towards the AMP-membrane interactions. Leakage of calcein dye from membrane mimic liposomes, live cell NMR and field emission scanning electron microscopy (FESEM) studies suggest that the AMPs may be primarily involved in membrane perturbation leading to release of intracellular substances resulting in subsequent microbial cell death. Confocal laser scanning microscopy (CLSM) shows localization of the peptides throughout the cell, indicating the possibility of secondary mode of actions. Electrostatic interactions seem to govern the preferential binding of the AMPs to the microbial membranes in comparison to the mammalian membranes as seen from the MD simulations.

Peptide Hydrogels as Platforms for Sustained Release of Antimicrobial and Antitumor Drugs and Proteins.

A charged synthetic peptide-based noncytotoxic hydrogelator was employed in encapsulation, storage, and sustainable release of different kinds of drugs, namely, ciprofloxacin (CP), an antibiotic; 5-fluorouracil (5-FU), an anticancer drug and proteins like lysozyme and bovine serum albumin (BSA). Hydrogelation of the peptide and its coassembly with the drug molecules were studied to obtain mechanistic details. All of the different cargos were capable of sustained and efficient release from the delivery platform. The drugs were found to retain their activity post release, while the proteins showed complete retention of their secondary structure. While about 80% CP was released at physiological pH over a period of 3 days, 5-FU was better released (73%) at an acidic pH (5.5) in comparison to the physiological pH (68%). Lysozyme was better released (82%) than BSA (43%) owing to the smaller size of the former and negative charge on the latter. Such biocompatible multicargo-releasing platforms from simple economically viable biomaterials, capable of sustained and tissue-specific release of cargo, are extremely promising in topical delivery of therapeutics.