Characterization of iRGD-Ligand Modified Arginine-Histidine-Rich Peptides for Nucleic Acid Therapeutics Delivery to αvβ3 Integrin-Expressing Cancer Cells

Efficient and specific delivery of nucleic acid (NA) therapeutics to tumor cells is extremely important for cancer gene therapy. Various therapeutic strategies include delivery of DNA-therapeutics such as immunostimulatory or suicide genes and delivery of siRNA-therapeutics able to silence expression of cancer-related genes. Peptides are a promising class of non-viral vehicles which are biodegradable and can efficiently condense, protect and specifically deliver NA to the cells. Here we designed arginine-histidine-rich peptide carriers consisting of an iRGD ligand to target αvβ3 integrins and studied them as vehicles for DNA and siRNA delivery to cancer cells. Combination of iRGD-modified and unmodified arginine–histidine-rich peptides during NA complexation resulted in carriers with different ligand contents. The NA-binding and protecting properties in vitro transfection efficiency and cytotoxicity of the DNA- and siRNA-polyplexes were studied and the most efficient carrier RGD1 was determined. The ability of the peptides to mediate specific intracellular uptake was confirmed inhuman cervical carcinoma (HeLa), human kidney (293T) and human pancreatic (PANC-1) cell lines with different αvβ3 integrins surface expression. By means of RGD1 carrier, efficient delivery of the herpes simplex virus (HSV-1) thymidine kinase gene to PANC-1 cells was demonstrated. Subsequent ganciclovir treatment led to a reduction of PANC-1 cells’ viability by up to 54%. Efficient RNAi-mediated down-regulation of GFP and VEGFA gene expression was achieved in MDA-MB-231-GFP+ breast cancer and EA.hy926 endothelial cells, respectively, by means of RGD1/siRNA polyplexes. Here we demonstrated that the peptide carrier RGD1 can be considered as promising candidate for development of NA therapeutics delivery systems useful in cancer gene therapy.

Tumor penetrating peptide-functionalized Tenascin-C antibody for glioblastoma targeting

Background: Conjugation to clinical-grade tumor penetrating iRGD peptide is a widely used strategy to improve tumor homing, extravasation, and penetration of cancer drugs and tumor imaging agents. The C domain of extracellular matrix molecule Tenascin-C (TNC-C) is upregulated in solid tumors and represents an attractive target for clinical-grade single-chain antibody-based vehicles for tumor delivery drugs and imaging agents. Objective: To study the effect of C-terminal genetic fusion of the iRGD peptide to recombinant anti-TNC-C single-chain antibody clone G11 on systemic tumor homing and extravasation. Methods: Enzyme-linked immunosorbent assay was used to study the interaction of parental and iRGD-fused anti-TNC-C single-chain antibodies with C domain of tenascin-C and αVβ3 integrins. For systemic homing studies, fluorescein-labeled ScFV G11-iRGD and ScFV G11 antibodies were administered in U87-MG glioblastoma xenograft mice, and their biodistribution was studied by confocal imaging of tissue sections stained with markers of blood vessels and Tenascin C immunoreactivity. Results: In a cell-free system, iRGD fusion to ScFV G11 conferred the antibody a robust ability to bind αVβ3 integrins. The fluorescein labeling of ScFV G11-iRGD did not affect its target binding activity. In U87-MG mice, iRGD fusion to ScFV G11 antibodies improved their homing to tumor blood vessels, extravasation, and penetration of tumor parenchyma. Conclusion: The genetic fusion of iRGD tumor penetrating peptide to non-internalizing affinity targeting ligands may improve their tumor tropism and parenchymal penetration for more efficient delivery of imaging and therapeutic agents into solid tumor lesions.

P1216Evaluation of alphavbeta3 integrin-targeted positron emission tomography and photoacoustic tracer for imaging of carotid plaque in apoE–/– mice

Background Cardiovascular disease is the leading cause of death in the world. The majority of cardiovascular events result from the rupture of vulnerable atherosclerotic plaques, which are characterized by high and active macrophage content. The integrin αVβ3 is expressed by activated macrophages and endothelial cells in atherosclerotic lesions and thus is a marker of high-risk plaques. Therefore, 89Zr-RGD-melanin nanoparticle (MNP) positron emission tomography (PET)/photoacoustic imaging (PAI) imaging of αVβ3 expression in plaques might provide a novel noninvasive biomarker of plaque vulnerability. Purpose In this study, the intrinsic photoacoustic signals and the native strong chelating properties with metal ions of MNP, positron-emitting metal ions 89Zr and αVβ3 integrins targeting ability of cyclic c (RGDfC) peptide was employed to construct an efficient nanoplatform. And we evaluated the feasibility of 89Zr-RGD-MNP PET/PAI of αVβ3 expression in vivo and in vitro. Methods We conjugated αVβ3 integrins, cyclic c (RGDfC) peptide, to MNP and chelated the long-lived positron-emitting nuclide 89Zr. The bio-stability and targeting action was detected in macrophages. And the PET/PAI imaging was performed in apoE−/− mice with partial carotid ligation leading to atherosclerosis. In PET imaging, tracer uptake was measured in the stenotic areas of the carotid arteries, as well as on the contralateral side at different time points in vivo. In PAI, photoacoustic signal was measured in the atherosclerosis of the carotid arteries in vivo. Melanin staining and immunohistochemistry of αVβ3 expression were detected in atherosclerosis of the carotid arteries. Results 89Zr-RGD-MNP showed excellent bio-stability and targeting action. PET imaging showed specific tracer accumulation at plaques in the left carotid artery, confirmed by competitive receptor blocking studies and the contrast in the right carotid artery. In the biodistribution studies, the left carotid (5.29%±0.78%) showed higher uptake than the right carotid (2.11%±1.55%). PAI showed the PA signal in the surgery group (452±85 a.u.) were stronger than the control (156±45 a.u.) and blocking group (254±66 a.u). The result was consistent with PET imaging and the presence of nanoparticles, as indicated by pathological examinations. These results presented good in vivo multimodality imaging (PET/PAI) properties. Conclusions We have developed 89Zr-labeled atherosclerotic plaques imaging agents based on the natural melanin nanoparticle. 89Zr-RGD-MNP demonstrates specific tracer accumulation in mice atherosclerotic carotid plaques. In this model, its uptake was associated with αVβ3 expression. 89Zr-RGD-MNP is a potential tracer for noninvasive imaging in atherosclerosis. Acknowledgement/Funding National Natural Science Foundation of China 81770452, 81470401

Synthesis and Characterization of ROSA Dye - A Rhodamine B-type Fluorophore, Suitable for Bioconjugation and Fluorescence Studies in Live Cells

Background: Herein we report the multigram-scale synthesis, characterization and application of a rhodamine B-based fluorophore (ROSA) suitable for fluorescent studies in biological applications. This fluorophore is devoid of rhodamine spirolactone formation and furthermore characterized by a high molar extinction coefficient (ϵ=87250 ± 1630 M-1cm-1) and quantum yield (φ) of 0.589 ± 0.070 in water. Reported here is also the application of ROSA towards synthesis of a ROSA-PEG-GRGDS-NH2 fluorescent probe suitable for live cell imaging of αvβ3 integrins for in vitro assays. Objective: The main objective of this study is to efficiently prepare rhodamine B derivative, devoid of spirolactone formation that would be suitable for bioconjugation and subsequent bioimaging. Methods: Rhodamine B was transformed into rhodamine B succinimide ester (RhoB-OSu) using N-hydroxysuccinimide. RhoB-OSu was further coupled to sarcosine to obtain rhodamine Bsarcosine dye (ROSA) in good yield. The ROSA dye was then coupled to a αvβ3 integrin binding sequence using standard solid-phase conditions. Resulting ROSA-PEG-GRGDS-NH2 probe was used to image integrins on cancer cells. Results: The rhodamine B-sarcosine dye (ROSA) was obtained in multigram scale in good total yield of 47%. Unlike rhodamine B, the ROSA dye does not undergo pH-dependent spirolactone/spirolactam formation as compared with rhodamine B-glycine. It is also characterized by excellent quantum yield (φ) of 0.589 ± 0.070 in water and high molar extinction coefficient of 87250 ± 1630 M-1cm-1. ROSA coupling to the RGD-like peptide was proved to be efficient and straightforward. Imaging using standard filters on multimode plate reader and confocal microscope was performed. The αvβ3 integrins present on the surface of live WM-266-4 (melanoma) and MCF- 7 (breast cancer) cells were successfully imaged. Conclusion: We successfully derivatized rhodamine B to create an inexpensive, stable and convenient to use fluorescent probe. The obtained derivative has excellent photochemical properties and it is suitable for bioconjugation and many imaging applications.

Basement membrane regulates fibronectin organization using sliding focal adhesions driven by a contractile winch

We have discovered that basement membrane and its major components can induce rapid, strikingly robust fibronectin organization. In this new matrix assembly mechanism, α5β1 integrin-based focal adhesions slide actively on the underlying matrix towards the ventral cell center through the dynamic shortening of myosin IIA-associated actin stress fibers to drive rapid fibronectin fibrillogenesis distal to the adhesion. This mechanism contrasts with classical fibronectin assembly based on stable/fixed-position focal adhesions containing αVβ3 integrins plus α5β1 integrin translocation into proximal fibrillar adhesions. On basement membrane components, these sliding focal adhesions contain standard focal adhesion constituents but completely lack classical αVβ3 integrins. Instead, peripheral α3β1 or α2β1 adhesions mediate initial cell attachment, but over time are switched to α5β1 integrin-based sliding focal adhesions to assemble fibronectin matrix. This basement membrane-triggered mechanism produces rapid fibronectin fibrillogenesis, providing a mechanistic explanation for the well-known widespread accumulation of fibronectin at many organ basement membranes.

Design of an Amphiphilic Poly(aspartamide)-mediated Self-assembled Nanoconstruct for Long-Term Tumor Targeting and Bioimaging

Biodegradable polymers have been developed for the targeted delivery of therapeutics to tumors. However, tumor targeting and imaging are usually limited by systemic clearance and non-specific adsorption. In this study, we used poly(amino acid) derivatives, such as poly(succinimide), to synthesize a nanomicelle-forming poly(hydroxyethylaspartamide) (PHEA, P) modified sequentially with octadecylamine, polyethylene glycol (PEG, P), and glycine (G) to design PHEA-PEG-glycine (PPG) nanoparticles (NPs). These PPG NPs were further tethered to cyclic Arg-Gly-Asp (cRGD) sequences for formulating tumor-targeting PPG-cRGD NPs, and then loaded with IR-780 dye (PPG-cRGD-IR-780) for visualizing tumor homing. cRGD cloaked in PPG NPs could bind specifically to both tumor endothelium and cancer cells overexpressing αvβ3 integrins. PPG-cRGD NPs exhibited enhanced physiological stability, cellular viability, and targeted intracellular uptake in cancer cells. In addition, PPG-cRGD NPs offered enhanced systemic circulation, leading to preferential tumor targeting and prolonged fluorescence tumor imaging for nearly 30 days. Nevertheless, non-targeted formulations demonstrated premature systemic clearance with short-term tumor imaging. Histochemical analysis showed no damage to normal organs, reaffirming the biocompatibility of PHEA polymers. Overall, our results indicated that PPG-cRGD NPs, which were manipulated to obtain optimal particle size and surface charge, and were complemented with tumor targeting, could improve the targeted and theranostic potential of therapeutic delivery.