Radionuclide-functionalized drug delivery vehicles capable of being imaged via positron emission tomography (PET) are of increasing interest in the biomedical field as they can reveal the in vivo behavior of encapsulated therapeutics with high sensitivity. However, the majority of current PET-guided theranostic agents suffer from poor retention of radiometal over time, low drug loading capacities, and time-limited PET imaging capability. To overcome these challenges, we have developed hollow microcapsules with a thin (<100 nm) multilayer shell as advanced theranostic delivery systems for multiday PET tracking in vivo. The 3 μm capsules were fabricated via the aqueous multilayer assembly of a natural antioxidant, tannic acid (TA), and a poly(N-vinylpyrrolidone) (PVPON) copolymer containing monomer units functionalized with deferoxamine (DFO) to chelate the 89 Zr radionuclide, which has a half-life of 3.3 days. We have found using radiochromatography that (TA/PVPON-DFO) 6 capsules retained on average 17% more 89 Zr than their (TA/PVPON) 6 counterparts, which suggests that the covalent attachment of the DFO to PVPON provides stable 89 Zr chelation. In vivo PET imaging studies performed in mice demonstrated that excellent stability and imaging contrast were still present 7 days postinjection. Animal biodistribution analyses showed that capsules primarily accumulated in the spleen, liver, and lungs with negligible accumulation in the femur, with the latter confirming the stable binding of the radiotracer to the capsule walls. The application of therapeutic ultrasound (US) (60 s of 20 kHz US at 120 W cm −2 ) to Zr-functionalized capsules could release the hydrophilic anticancer drug doxorubicin from the capsules in the therapeutic amounts. Polymeric capsules with the capability of extended in vivo PET-based tracking and US-induced drug release provide an advanced platform for development of precisiontargeted therapeutic carriers and could aid in the development of more effective drug delivery systems.

Breast cancer continues to be the most lethal cancer type in women and one of the most diagnosed. Understanding Breast cancer receptor status is one of the most vital processes for determining treatment options. One type of breast cancer, human epidermal growth factor receptor 2 (HER2) positive, has approved receptor-based therapies including trastuzumab and pertuzumab that can significantly increase the likelihood of survival. Current methods to determine HER2 status include biopsies with immunohistochemical staining and/or fluorescence in situ hybridization. However, positron emission tomography (PET) imaging techniques using 89Zr-trastuzumab or 89Zr-pertuzumab are currently in clinical trials for a non-invasive, full body diagnostic approach. Although the antibodies have strong specificity to the HER2 positive lesions, challenges involving long post-injection time for imaging due to the blood circulation of the antibodies and matching of long-live isotopes leading to increased dose to the patient leave opportunities for alternative PET imaging probes. Peptides have been shown to allow for shorter injection-to-imaging time and can be used with shorter lived isotopes. HER2 specific peptides under development will help improve the diagnosis and potentially therapy options for HER2 positive breast cancer. Peptides showing specificity for HER2 could start widespread development of molecular imaging techniques for HER2 positive cancers.

Nitric oxide (NO) mimetics and other agents capable of enhancing NO/cGMP signaling have demonstrated efficacy as potential therapies for Alzheimer's disease. A group of thiol-dependent NO mimetics known as furoxans may be designed to exhibit attenuated reactivity to provide slow onset NO effects. The present study describes the design, synthesis, and evaluation of a furoxan library resulting in the identification of a prototype furoxan, 5a, which was profiled for use in the central nervous system. Furoxan 5a demonstrated negligible reactivity toward generic cellular thiols under physiological conditions. Nonetheless, cGMP-dependent neuroprotection was observed, and 5a (20 mg/kg) reversed cholinergic memory deficits in a mouse model of passive avoidance fear memory. Importantly, 5a can be prepared as a pharmaceutically acceptable salt and is observed in the brain 12 h after oral administration, suggesting potential for daily dosing and excellent metabolic stability. Continued investigation into furoxans as attenuated NO mimetics for the CNS is warranted.

One in eight women will be diagnosed with breast cancer in their lifetime and approximately 25% of those cases will be HER2-positive. Current methods for diagnosing HER2-positive breast cancer involve using IHC and FISH from suspected cancer biopsies to quantify HER2 expression. HER2 PET imaging could potentially increase accuracy and improve the diagnosis of lesions that are not available for biopsies. Using two previously discovered HER2-targeting peptides, we modified each peptide with the chelator DOTA and a PEG2 linker resulting in DOTA-PEG2-GSGKCCYSL (P5) and DOTA-PEG2-DTFPYLGWWNPNEYRY (P6). Each peptide was labeled with 68Ga and was evaluated for HER2 binding using in vitro cell studies and in vivo tumor xenograft models. Both [68Ga]P5 and [68Ga]P6 showed significant binding to HER2-positive BT474 cells versus HER2-negative MDA-MB-231 cells ([68Ga]P5; 0.68 ± 0.20 versus 0.47 ± 0.05 p < 0.05 and [68Ga]P6; 0.55 ± 0.21 versus 0.34 ± 0.12 p < 0.01). [68Ga]P5 showed a higher percent injected dose per gram (%ID/g) binding to HER2-positive tumors two hours post-injection compared to HER2-negative tumors (0.24 ± 0.04 versus 0.12 ± 0.06; p < 0.05), while the [68Ga]P6 peptide showed significant binding (0.98 ± 0.22 versus 0.51 ± 0.08; p < 0.05) one hour post-injection. These results lay the groundwork for the use of peptides to image HER2-positive breast cancer.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer related death with a 5-year survival rate at 11%. New systemic therapies for patients with PDAC are desperately needed. Upregulation or exposure of a new protein on the tumor cell surface can serve as a therapeutic or diagnostic target. Here, we highlight our recently developed strategy to induce localization of the reticular protein calreticulin (CALR) to the cell surface in PDAC cells and its subsequent detection using a novel radiolabeled peptide. Methods: Surface translocation of CALR was detected by flow cytometry, western blot, and total internal reflection fluorescence (TIRF) microscopy in PDAC cells treated with either doxorubicin or gemcitabine. The radionuclide-binding chelator ‘DOTA’ was covalently linked to a CALR-specific peptide ‘KLGFFKR’ and then labeled with 68Ga. Samples were analyzed on HPLC with an average radiolabeling efficiency of 93%. Mice bearing Panc02 allografts were treated with doxorubicin for 24h, injected with ~3 MBq (5 μg) of radiopeptide, and sacrificed after 1 hour to determine biodistribution. Results: Using flow cytometry, we found that treating PDAC cells with doxorubicin or gemcitabine increased both the total number and the median fluorescence intensity of surface staining for live cells expressing CALR. When membrane proteins were isolated from PDAC cells treated with doxorubicin at various time points, a peak in surface CALR protein was detected at 30 minutes with persistent expression lasting for 24 hours. TIRF microscopy showed that Panc02 cells treated with doxorubicin had approximately a 2-fold higher surface CALR expression as detected by enhanced membrane fluorescence compared to controls. In vivo, our novel [68Ga]-CALR peptide showed rapid clearance through the kidneys with no significant uptake in vital organs (n=4). In Panc02 allograft-bearing mice treated intratumorally with either vehicle or doxorubicin, biodistribution analysis after radiopeptide injection showed a significant increase in radiopeptide uptake in the treated tumors (n=6, p &lt; 0.05). Conclusions: CALR is translocated to the cell surface in PDAC cells, where it can subsequently be targeted by a novel radiopeptide agent. Future studies are needed to determine if induced CALR can be targeted for therapeutic effect. Citation Format: Rachael Guenter, Maxwell Ducharme, Brendon Herring, Tejeshwar C. Rao, Odalyz Montes, Tyler McCaw, Herbert Chen, Suzanne E. Lapi, Benjamin Larimer, J. Bart Rose. Using a novel [68Ga]-radiolabeled peptide to detect cell surface expression of calreticulin in pancreatic adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3578.