Palmitic Acid-Conjugated Radiopharmaceutical for Integrin αvß3-Targeted Radionuclide Therapy.

Peptide receptor radionuclide therapy (PRRT) is an emerging approach for patients with unresectable or metastatic tumors. Our previously optimized RGD peptide (3PRGD2) has excellent targeting specificity for a variety of integrin αvß3/αvß5-positive tumors and has been labeled with the therapeutic radionuclide [177Lu]LuCl3 for targeted radiotherapy of tumors. However, the rapid clearance of [177Lu]Lu-DOTA-3PRGD2 (177Lu-3PRGD2) in vivo requires two doses of 111 MBq/3 mCi to achieve effective tumor suppression, limiting its further clinical application. Albumin binders have been attached to drugs to facilitate binding to albumin in vivo to prolong the drug half-life in plasma and obtain long-term effects. In this study, we modified 3PRGD2 with albumin-binding palmitic acid (Palm-3PRGD2) and then radiolabeled Palm-3PRGD2 with 177Lu. [177Lu]Lu-DOTA-Palm-3PRGD2 (177Lu-Palm-3PRGD2) retained a specific binding affinity for integrin αvß3/αvß5, with an IC50 value of 5.13 ± 1.16 nM. Compared with 177Lu-3PRGD2, the 177Lu-Palm-3PRGD2 circulation time in blood was more than 6 times longer (slow half-life: 73.42 min versus 11.81 min), and the tumor uptake increased more than fivefold (21.34 ± 4.65 %IA/g and 4.11 ± 0.70 %IA/g at 12 h post-injection). Thus, the significant increase in tumor uptake and tumor retention resulted in enhanced efficacy of targeted radiotherapy, and tumor growth was completely inhibited by a single and relatively lowdose of 18.5 MBq/0.5 mCi. Thus, 177Lu-Palm-3PRGD2 shows great potential for clinical application.

An Integrin-αvß6/α5ß1-Bitargeted Probe for the SPECT Imaging of Pancreatic Adenocarcinoma in Preclinical and Primary Clinical Studies.

Pancreatic adenocarcinoma (PA) is one of the deadliest human malignancies. However, early detection, prediction of surgical resectability, and prognosis of PA are challenging with current conventional imaging technologies in the clinic. Molecular imaging technologies combined with novel imaging probes could be useful for early detection and accurate staging of PA. Integrin αvß6 and α5ß1 are found to be overexpressed in PA. In this study, integrin αvß6/α5ß1-bitargeted probes 99mTc-HYNIC-isoDGR (99mTc-isoDGR) and 99mTc-HYNIC-PEG4-PisoDGR2 (99mTc-3PisoDGR2) were prepared and evaluated in the BxPC-3 human pancreatic tumor model. Both subcutaneous and in situ BxPC-3 tumors could be clearly visualized by 99mTc-isoDGR nanoScan SPECT/CT imaging with a high ratio of tumor to background. The blocking study with excess nonradioactive peptide showed a significantly reduced tumor uptake, which confirmed the specificity of 99mTc-isoDGR. Biodistribution results confirmed the imaging results. The dimer tracer 99mTc-3PisoDGR2 significantly enhanced tumor uptake compared with 99mTc-isoDGR, and the spontaneous PA lesion in the mouse model could be clearly visualized by 99mTc-3PisoDGR2. The primary clinical study also verified the ability of 99mTc-3PisoDGR2 for detection of PA. Therefore, SPECT/CT imaging using the integrin αvß6/α5ß1-bitargeted 99mTc-3PisoDGR2 provided a potential approach for the noninvasive detection of PA.

A Dual pH-Responsive DOX-Encapsulated Liposome Combined with Glucose Administration Enhanced Therapeutic Efficacy of Chemotherapy for Cancer.

BACKGROUND: The acidic microenvironment of cancer can promote tumor metastasis and drug resistance. Acidic tumor microenvironment-targeted therapy is currently an important means for treating tumors, inhibiting metastasis, and overcoming drug resistance. In this study, a dual pH-responsive DOX-encapsulated liposome (DOPE-DVar7-lip@DOX) was designed and fabricated for targeting the acidic tumor microenvironment. On the one hand, the response of acid-sensitive peptide (DVar7) to the acidic tumor microenvironment increased the uptake of liposomes in tumors and prolonged the retention time; on the other hand, the response of acid-sensitive phospholipid (DOPE) to the acidic tumor microenvironment improved the controlled release of DOX in tumors. METHODS: The acid-sensitive peptide DVar7 modified liposomes can be obtained by simple incubation of DSPE-DVar7 with DOX-loaded DOPE liposomes (DOPE-lip@DOX). The tumor targeting of the dual pH-responsive liposome was investigated in vitro and in vivo by near-infrared fluorescence imaging. The tumor therapeutic efficacy of DOPE-DVar7-lip@DOX was evaluated in breast cancer mouse model using the traditional liposome as a control. Moreover, we regulated the tumor microenvironment acidity by injecting glucose to further enhance the therapeutic efficacy of cancer. RESULTS: DVar7 can allosterically insert into the tumor cell membrane in the acidic tumor microenvironment to enhance the tumor uptake of liposomes and prolong the retention time of liposomes in tumor. In addition, the therapeutic efficacy of pH-responsive liposomes can be further enhanced by glucose injection regulating the acidity of tumor microenvironment. DISCUSSION: DVar7 modified acid-sensitive nanocarriers combined with acidity regulation have great potential to improve drug resistance in clinical practice, thus improving the response rate and therapeutic effect of chemotherapy.

Developing PEGylated Reversed D-Peptide as a Novel HER2-Targeted SPECT Imaging Probe for Breast Cancer Detection.

Human epidermal growth factor receptor-2 (HER2)-enriched breast cancer is characterized by strong invasiveness, high recurrence rate, and poor prognosis. HER2-specific imaging can help screening right patients for appropriate HER2-targeted therapies. Previously, we have developed a 99mTc-labeled HER2-targeted H6 peptide for SPECT imaging of breast cancer. However, the poor metabolic stability and high gallbladder uptake hamper its clinical application. In this study, a retro-inverso D-peptide of H6 (RDH6) was designed to increase the metabolic stability. PEGylation was used to improve its water solubility and in vivo pharmacokinetics. The results showed that the D-amino acids in 99mTc-PEG4-RDH6 brought better metabolic stability than 99mTc-PEG4-H6, thus achieving higher tumor uptake. As the length of the PEG chain increases, the hydrophilicity of the probes gradually increased, which may also be the main cause for the decreased liver uptake. Compared with radiotracers modified by PEG4 and PEG12, 99mTc-PEG24-RDH6 had a comparable tumor uptake and the lowest liver radioactivity. The SPECT imaging demonstrated that 99mTc-PEG24-RDH6 could specifically distinguish HER2-positive tumors from HER2-negative tumors with better imaging contrast, which thus has the potential for clinical screening of HER2-positive breast patients.

An Integrin Alpha 6-Targeted Radiotracer with Improved Receptor Binding Affinity and Tumor Uptake.

In this study, we reported a 99mTc-labeled integrin α6-targeted peptide as the molecular imaging probe for tumor imaging by single-photon emission computed tomography (SPECT). We found that replacing Cys-Cys cyclized RWY peptide (sequence: cCRWYDENAC) with lactam-bridged cyclic cKiE peptide (sequence: cKRWYDENAisoE) did not sacrifice the integrin α6-binding affinity and specificity of cKiE radiotracer. To further improve the radiotracer's tumor targeting capability, the dimerized cKiE peptide (termed cKiE2) was designed, and the corresponding radiotracer 99mTc-cKiE2 was evaluated for tumor uptake and in vivo pharmacokinetics properties in tumor models. We found that cKiE2 showed higher binding affinity to integrin α6 than did monomeric RWY or cKiE peptide. The biodistribution results showed that the tumor uptake of 99mTc-cKiE2 was twice higher than that of 99mTc-RWY (3.20 ± 0.12 vs 1.26 ± 0.06 %ID/g, P < 0.001) at 0.5 h postinjection. The tumor to nontargeting tissue ratios were also enhanced in most normal organs. Specificity of 99mTc-cKiE2 for integrin α6 was demonstrated by competitive blocking of tumor uptake with excess cold peptide (3.20 ± 0.24 to 1.38 ± 0.23 %ID/g, P < 0.001). The integrin α6-positive tumors were clearly visualized by 99mTc-cKiE2/SPECT with low background except with a relatively high kidney uptake. The tumor uptake of 99mTc-cKiE2 correlates well with the tumor integrin α6 expression levels in a linear fashion (R2 = 0.9623). We also compared 99mTc-cKiE2 with an integrin αvß3-targeted radiotracer 99mTc-3PRGD2 in the orthotopic hepatocellular carcinoma tumor models. We found that the orthotopic tumor was clearly visualized with 99mTc-cKiE2. 99mTc-3PRGD2 imaging did not show tumor contours in situ as clearly as 99mTc-cKiE2. The tumor-to-liver ratios of 99mTc-cKiE2 and 99mTc-3PRGD2 were 2.20 ± 0.17 and 0.85 ± 0.20. In conclusion, 99mTc-cKiE2 is an improved SPECT radiotracer for imaging integrin α6-positive tumors and has great potential for further clinical application.

A 64 Cu-porphyrin-based dual-modal molecular probe with integrin αv ß3 targeting function for tumour imaging.

Pyropheophorbide-a (Pyro) is a promising multifunctional molecule for multimodal tumour imaging and photodynamic therapy, but its clinical applications are seriously restricted by the limited tumour accumulation capability. Here, we designed and synthesized a small-molecule probe that achieved specific dual-modal tumour imaging based on Pyro. Briefly, a novel molecule combining Pyro, an RGD dimer peptide (3PRGD2 ) and 64 Cu, was designed and synthesized, and the obtained molecule, 64 Cu-Pyro-3PRGD2 , exhibited high tumour specificity in both positron emission tomography and optical imaging in vivo. c (RGDfk) peptide blocking significantly reduced the efficacy of the probe, which confirmed the integrin αV ß3 targeting of this molecular probe. 64 Cu-Pyro-3PRGD2 had very low accumulation in normal organs and could be rapidly cleared through kidney metabolism, which prevented the potential damage to adjacent normal tissues. Overall, combining tumour targeting, dual-modal imaging, and biosafety, 64 Cu-Pyro-3PRGD2 has the potential for clinical use as a molecular imaging probe for tumour diagnosis.

Improved in Vivo Targeting Capability and Pharmacokinetics of 99mTc-Labeled isoDGR by Dimerization and Albumin-Binding for Glioma Imaging.

Previously, we successfully developed the c(phg-isoDGRk) peptide as a novel integrin α5ß1-targeted SPECT imaging probe 99mTc-HisoDGR for Glioma imaging. However, the fast clearance of 99mTc-HisoDGR in blood reduced its tumor accumulation and retention, which would be the obstacles for further clinical application. Dimerization and albumin-binding strategies have been proven as effective approaches to improve tumor targeting capability and blood circulation time of radiotracers. In this study, the novel PEGylated dimeric isoDGR peptides (termed 3PisoDGR2) and its analogue with an albumin binder (termed AB-3PisoDGR2) were designed, and the corresponding radiotracers 99mTc-3PisoDGR2 and 99mTc-AB-3PisoDGR2 were fabricated and assessed for tumor-targeting and in vivo pharmacokinetics properties in subcutaneous and orthotopic tumor models. The dimerization of isoDGR peptide provided higher binding affinity to tumor cells and longer blood circulation time than the original monomeric isoDGR peptide, resulting in twice increased tumor uptake (99mTc-3PisoDGR2 2.51 ± 0.17 %ID/g vs 99mTc-PisoDGR 1.17 ± 0.21 %ID/g, P < 0.01) at 0.5 h post-injection (p.i.) and enhanced tumor to nontargeting tissue ratios (T/NT) in most normal organs. The blocking study indicated that the tumor uptake was receptor-mediated specifically. NanoScanSPECT/CT imaging of 99mTc-3PisoDGR2 in glioma tumor-bearing model showed clear visions of tumors with low background, except high uptake in excretion system including kidneys and bladder at all detected time points (0.5, 1, and 2 h p.i.). The orthotopic glioma tumor could also be clearly visualized by nanoScanSPECT/CT imaging with 99mTc-3PisoDGR2. The addition of albumin-binding entity further prolonged blood circulation time and reached higher tumor uptake for 99mTc-AB-3PisoDGR2. However, since 99mTc-AB-3PisoDGR2 is less capable of passing BBB than 99mTc-3PisoDGR2, 99mTc-3PisoDGR2 is preferable for the in situ glioma imaging. In conclusion, 99mTc-3PisoDGR2 represents an improved molecular probe for integrin α5ß1-targeted tumor imaging, showing more potential for further clinical application.