Arg-Gly-Asp Research Articles

The importance of tyrosines in multimers of cyclic RGD nonapeptides: towards αvβ6-integrin targeted radiotherapeutics.

In a recent paper in this journal (RSC Med. Chem., 2023, 14, 2429), we described an unusually strong impact of regiospecific exchange of phenylalanines by tyrosines in 10 gallium-68-labeled trimers of certain cyclic RGD peptides, c[XRGDLAXp(NMe)K] (X = F or Y), on non-specific organ uptakes. We found that there was, in part, no correlation of liver uptake with established polarity proxies, such as the octanol-water distribution coefficient (log D). Since this observation could not be explained straightforwardly, we suggested that the symmetry of the compounds had resulted in a synergistic interaction of certain components of the macromolecules. In the present work, we investigated whether a comparable effect also occurred for a series of 5 tetramers labeled with lutetium-177. We found that in contrast to the trimers, liver uptake of the tetramers was well correlated to their polarity, indicating that the unusual observations along the trimer series indeed was a unique feature, probably related to their particular symmetry. Since the Lu-177 labeled tetramers are also potential agents for treatment of a variety of αvβ6-integrin expressing cancers, these were evaluated in mice bearing human lung adenocarcinoma xenografts. Due to their tumor-specific uptake and retention in biodistribution and SPECT imaging experiments, these compounds are considered a step forward on the way to αvβ6-integrin-targeted anticancer agents. Furthermore, we noticed that the presence of tyrosines in general had a positive impact on the in vivo performance of our peptide multimers. In view of the fact that a corresponding rule was already proposed in the context of protein engineering, we argue in favor of considering peptide multimers as a special class of small or medium-sized proteins. In summary, we contend that the performance of peptide multimers is less determined by the in vitro characteristics (particularly, affinity and selectivity) of monomers, but rather by the peptides' suitability for the overall macromolecular design concept, and peptides containing tyrosines are preferred.

Photosensitizer-singlet oxygen sensor conjugated silica nanoparticles for photodynamic therapy and bioimaging.

Intracellular singlet oxygen (1O2) generation and detection help optimize the outcome of photodynamic therapy (PDT). Theranostics programmed for on-demand phototriggered 1O2 release and bioimaging have great potential to transform PDT. We demonstrate an ultrasensitive fluorescence turn-on sensor-sensitizer-RGD peptide-silica nanoarchitecture and its 1O2 generation-releasing-storing-sensing properties at the single-particle level or in living cells. The sensor and sensitizer in the nanoarchitecture are an aminomethyl anthracene (AMA)-coumarin dyad and a porphyrin or CdSe/ZnS quantum dots (QDs), respectively. The AMA in the dyad quantitatively quenches the fluorescence of coumarin by intramolecular electron transfer, the porphyrin or QD moiety generates 1O2, and the RGD peptide facilitates intracellular delivery. The small size, below 200 nm, as verified by scanning electron microscopy and differential light scattering measurements, of the architecture within the 1O2 diffusion length enables fast and efficient intracellular fluorescence switching by the tandem ultraviolet (UV)-visible or visible-near-infrared (NIR) photo-triggering. While the red emission and 1O2 generation by the porphyrin are continually turned on, the blue emission of coumarin is uncaged into 230-fold intensity enhancement by on-demand photo-triggering. The 1O2 production and release by the nanoarchitecture enable spectro-temporally controlled cell imaging and apoptotic cell death; the latter is verified from cytotoxic data under dark and phototriggering conditions. Furthermore, the bioimaging potential of the TCPP-based nanoarchitecture is examined in vivo in B6 mice.

Simultaneous ischemic regions targeting and BBB crossing strategy to harness extracellular vesicles for therapeutic delivery in ischemic stroke

Extracellular vesicles (EVs) derived from adipose-derived stem cells (ADSC-EVs) hold great promise for ischemic stroke treatment, but their therapeutic efficacy is greatly limited due to insufficient targeting ability. Previous reports focused on single ischemic targeting or blood-brain barrier (BBB) penetration, precise delivery to the brain parenchyma has not been fully considered. This study leveraged the targeting ability of RGD peptide and the cell penetrating ability of Angiopep-2 peptide to deliver ADSC-EVs precisely to the impaired brain parenchyma. We found that dual-modified EVs (RA-EVs) significantly enhanced the transcellular permeability across BBB in vitro, and not only targeted ischemic blood vessels but also achieved rapid accumulation in the ischemic lesion area after intravenous administration in vivo. RA-EVs further decreased the infarct volume, apoptosis, BBB disruption, and neurobehavioral deficits. RNA sequencing revealed the molecular regulation mechanism after administration. These findings demonstrate that dual-modification optimizes brain parenchymal targeting and highlights the significance of recruitment and penetration as a previously unidentified strategy for harnessing EVs for therapeutic delivery in ischemic stroke.

Label-free biosensing with singular-phase-enhanced lateral position shift based on atomically thin plasmonic nanomaterials

Rapid plasmonic biosensing has attracted wide attention in early disease diagnosis and molecular biology research. However, it was still challenging for conventional angle-interrogating plasmonic sensors to obtain higher sensitivity without secondary amplifying labels such as plasmonic nanoparticles. To address this issue, we developed a plasmonic biosensor based on the enhanced lateral position shift by phase singularity. Such singularity presents as a sudden phase retardation at the dark point of reflection from resonating plasmonic substrate, leading to a giant position shift on reflected beam. Herein, for the first time, the atomically thin layer of Ge2Sb2Te5 (GST) on silver nanofilm was demonstrated as a novel phase-response-enhancing plasmonic material. The GST layer was not only precisely engineered to singularize phase change but also served as a protective layer for active silver nanofilm. This new configuration has achieved a record-breaking largest position shift of 439.3 μm measured in calibration experiments with an ultra-high sensitivity of 1.72 × 108 nm RIU−1 (refractive index unit). The detection limit was determined to be 6.97 × 10−7 RIU with a 0.12 μm position resolution. Besides, a large figure of merit (FOM) of 4.54 × 1011 μm (RIU∙°)−1 was evaluated for such position shift interrogation, enabling the labelfree detection of trace amounts of biomolecules. In targeted biosensing experiments, the optimized sensor has successfully detected small cytokine biomarkers (TNF-α and IL-6) with the lowest concentration of 1 × 10−16 M. These two molecules are the key proinflammatory cancer markers in clinical diagnosis, which cannot be directly screened by current clinical techniques. To further validate the selectivity of our sensing systems, we also measured the affinity of integrin binding to arginylglycylaspartic acid (RGD) peptide (a key protein interaction in cell adhesion) with different Mn2+ ion concentrations, ranging from 1 nM to 1 mM.

Interaction Regularity of Biomolecules on Mg and Mg-Based Alloy Surfaces: A First-Principles Study

Adsorbed molecules can modulate the behavior of magnesium (Mg) and Mg alloy in biomedical applications. The interaction regularity and mechanism of biomolecules (such as amino acids, dipeptides, and tripeptide) on a Mg(0001) surface, the influence of dipole correction, and the effects of alloying elements and electronic structure were investigated in this study using first-principles calculations. Specifically, the adsorption energy (Eads) of functional groups (-NH2, -COOH and -CN3H4), amino acids (arginine (Arg), glycine (Gly), and aspartic acid (Asp)), dipeptides (arginine–glycine (Arg-Gly), glycine–aspartic acid (Gly-Asp), and arginine–aspartic acid (Arg-Asp)), and arginine–glycine–aspartic acid (RGD) tripeptide were systematically calculated. Dipole correction slightly enhanced the interaction between molecules and Mg surfaces, but the Eads trend remained unchanged. The addition of alloying elements improved the interaction of molecules and Mg-based alloy surfaces. This study will be of fundamental importance in understanding the interaction regularity of molecules on Mg and Mg-based alloy surfaces and provide possibilities for surface modification design of biomedical materials.

Biodegradable copper peroxide decorated hollow polydopamine nanocomposite as effective chloroperoxidase delivery carrier for enhancing enzyme dynamic therapy via self-activated cascade reactions

Recently, enzyme dynamic therapy (EDT) has gained enormous attraction in cancer therapy. However, the efficiency of EDT is still largely restricted by the inefficient delivery efficiency of chloroperoxidase (CPO) and the insufficient hydrogen peroxide (H2O2) content in the tumor microenvironment (TME). In the present work, a biodegradable cupper peroxide decorated hollow polydopamine nanocomposite (HPDCP) with surface functionalization of PEGlated tumor targeting peptide RGD (PEG-RGD) was designed for effective CPO delivery. The resultant nanosystem of HPDCP@CPO@PEG-RGD displayed high CPO delivery efficiency to breast tumors without comprising the enzyme activity. Moreover, the nanosystem can dissociate rapidly in acidic TME by supplying substantial H2O2 and simultaneously releasing Cu2+ and CPO, followed by initiating self-activated cascade reactions. At the one hand, the self-supplied H2O2 greatly enhanced the singlet oxygen (1O2) generation via the CPO-mediated enzyme dynamic reaction. At the other hand, substantial hydroxyl radical (•OH) was produced via the Cu2+-driven Fenton reaction for chemodynamic therapy (CDT). Therefore, the anticancer efficiency of EDT was significantly enhanced due to the synergistic EDT/CDT effect. Mechanism study revealed that 1O2/•OH generated by HPDCP@CPO@PEG-RGD synergistically inhibited breast cancer cells by firstly causing severe lipid peroxidation, followed by triggering mitochondria-mediated apoptosis and causing irreversible DNA damage by inhibiting the expression of DNA repair enzyme poly (ADP-ribose) polymerase 1 (PARP-1).

Tumor–homing peptide iRGD-conjugate enhances tumor accumulation of camptothecin for colon cancer therapy

Poor intracellular uptake of therapeutics in the tumor parenchyma is a key issue in cancer therapy. We describe a novel approach to enhance tumor targeting and achieve targeted delivery of camptothecin (CPT) based on a tumor–homing internalizing RGD peptide (iRGD). We synthesized an iRGD–camptothecin conjugate (iRGD–CPT) covalently coupled by a heterobifunctional linker and evaluated its in vitro and in vivo activity in human colon cancer cells. In vitro studies revealed that iRGD–CPT penetrated cells efficiently and reduced colon cancer cell viability to a significantly greater extent at micromolar concentrations than did the parent drug. Furthermore, iRGD–CPT showed high distribution toward tumor tissue, effectively suppressed tumor progression, and showed enhanced antitumor effects relative to the parent drug in a mouse model, demonstrating that iRGD–CPT is effective in vivo cancer treatment. These results suggest that intracellular delivery of CPT via the iRGD peptide is a promising drug delivery strategy that will facilitate the development of CPT derivatives and prodrugs with improved efficacy.

Dual peptides-modified cationic liposomes for enhanced Lung cancer gene therapy by a gap junction regulating strategy

BackgroundGene therapy for lung cancer has emerged as a novel tumor-combating strategy for its superior tumor specificity, low systematical toxicity and huge clinical translation potential. Especially, the applications of microRNA shed led on effective tumor ablation by directly interfering with the crucial gene expression, making it one of the most promising gene therapy agents. However, for lung cancer therapy, the microRNA treatment confronted three bottlenecks, the poor tumor tissue penetration effect, the insufficient lung drug accumulation and unsatisfied gene transfection efficiency. To address these issues, an inhalable RGD-TAT dual peptides-modified cationic liposomes loaded with microRNA miR-34a and gap junction (GJ) regulation agent all-trans retinoic acid (ATRA) was proposed, which was further engineered into dry powder inhalers (DPIs).ResultsEquipped with a rough particle surface and appropriate aerodynamic size, the proposed RGD-TAT-CLPs/ARTA@miR-34a DPIs were expected to deposit into the deep lung and reach lung tumor lesions guided by targeting peptide RGD. Assisted by cellular transmembrane peptides TAT, the RGD-TAT-CLPs/ARTA@miR-34a was proven to be effectively internalized by cancer cells, enhancing gene transfection efficiency. Then, the GJ between tumor cells was upregulated by ARTA, facilitating the intercellular transport of miR-34a and boosting the gene expression in the deep tumor.ConclusionOverall, the proposed RGD-TAT-CLPs/ARTA@miR-34a DPIs could enhance tumor tissue penetration, elevate lung drug accumulation and boost gene transfection efficiency, breaking the three bottlenecks to enhancing tumor elimination in vitro and in vivo. We believe that the proposed RGD-TAT-CLPs/ARTA@miR-34a DPIs could serve as a promising pulmonary gene delivery platform for multiple lung local disease treatments.

Specific inhibition on PAI-1 reduces the dose of Alteplase for ischemic stroke treatment

Administration of recombinant tPA (rtPA, or trade name Alteplase®) is an FDA-approved therapy for acute ischemic stroke (AIS), but poses the risk of hemorrhagic complications. Recombinant tPA can be rapidly inactivated by the endogenous inhibitor, plasminogen activator inhibitor 1 (PAI-1). In this work, we study a novel treatment approach that combines a PAI-1 inhibitor, PAItrap4, with a reduced dose of rtPA to address the hemorrhagic concern of rtPA. PAItrap4 is a highly specific and very potent protein-based inhibitor of PAI-1, comprising of a variant of uPA serine protease domain, human serum albumin, and a cyclic RGD peptide. PAItrap4 efficiently targets and inhibits PAI-1 on activated platelets, and also possesses a long half-life in vivo. Our results demonstrate that PAItrap4 effectively counteracts the inhibitory effects of PAI-1 on rtPA, preserving rtPA activity based on amidolytic and clot lysis assays. In an in vivo murine stroke model, PAItrap4, together with low-dose rtPA, enhances the blood perfusion in the stroke-affected areas, reduces infarct size, and promotes neurological recovery in mice. Importantly, such treatment does not increase the amount of cerebral hemorrhage, thus reducing the risk of cerebral hemorrhage. In addition, PAItrap4 does not compromise the normal blood coagulation function in mice, demonstrating its safety as a therapeutic agent. These findings highlight this combination therapy as a promising alternative for the treatment of ischemic stroke, offering improved safety and efficacy.

Using dynamic biomaterials to study the temporal role of bioactive peptides during osteogenesis

The extracellular matrix is a highly dynamic environment, and the precise temporal presentation of biochemical signals is critical for regulating cell behavior during development, healing, and disease progression. To mimic this behavior, we developed a modular DNA-based hydrogel platform to enable independent and reversible control over the immobilization of multiple biomolecules during in vitro cell culture. We combined reversible DNA handles with a norbornene-modified hyaluronic acid hydrogel to orthogonally add and remove multiple biomolecule-DNA conjugates at user-defined timepoints. We demonstrated that the persistent presentation of the cell adhesion peptide RGD was required to maintain cell spreading on hyaluronic acid hydrogels. Further, we discovered the delayed presentation of osteogenic growth peptide (OGP) increased alkaline phosphatase activity compared to other temporal variations. This finding is critically important when considering the design of OGP delivery approaches for bone repair. More broadly, this platform provides a unique approach to tease apart the temporal role of multiple biomolecules during development, regeneration, and disease progression.

Inhibition effect of Gly-Arg-Gly-Asp-Ser (GRGDS) and Arg-Gly-Asp (RGD) Bioactive Peptides on Angiotensin-Converting Enzyme Activity Purified from Human Serum

Angiotensin-converting enzyme (ACE, EC 3.4.15.1) is a physiological target for researching new antihypertensive drugs, as it is a substantial enzyme in the regulation of blood pressure. Herein, ACE was purified from human serum with affinity chromatography. Vmax and KM values were found as 60.98 (µmol/min)/mL and 0.34 mM, respectively. The effects of Gly-Arg-Gly-Asp-Ser (GRGDS) and Arg-Gly-Asp (RGD) bioactive peptides on purified ACE were researched. Also, captopril, a specific ACE inhibitory, was used as a reference inhibitor. Bioactive peptides, GRGDS and RGD, demonstrated the inhibitory effect on purified ACE with IC50 values of 46.39 µM and 456.46 µM, respectively. Ki values and kind of inhibition for GRGDS and RGD by the Lineweaver-Burk chart were found. The kind of inhibitory for these bioactive peptides was calculated as reversible-competitive inhibitory. Ki values for GRGDS and RGD were obtained as 93.28 µM and 435.67 µM, respectively. The IC50 value of captopril was designated as 1.57 nM. The inhibitory kind of captopril was designated as reversible non-competitive inhibitory and the Ki value was 0.99 nM. In this study, it was concluded that RGD and GRGDS bioactive peptides have the potential to be utilized as ACE inhibitors.

Varying the RGD concentration on a hyaluronic acid hydrogel influences dormancy versus proliferation in brain metastatic breast cancer cells.

A majority of breast cancer deaths occur due to metastasis of cancer cells to distant organs. In particular, brain metastasis is very aggressive with an extremely low survival rate. Breast cancer cells that metastasize to the brain can enter a state of dormancy, which allows them to evade death. The brain microenvironment provides biophysical, biochemical, and cellular cues, and plays an important role in determining the fate of dormant cancer cells. However, how these cues influence dormancy remains poorly understood. Herein, we employed hyaluronic acid (HA) hydrogels with a stiffness of ~0.4 kPa as an in vitro biomimetic platform to investigate the impact of biochemical cues, specifically alterations in RGD concentration, on dormancy versus proliferation in MDA-MB-231Br brain metastatic breast cancer cells. We applied varying concentrations of RGD peptide (0, 1, 2, or 4 mg/mL) to HA hydrogel surfaces and confirmed varying degrees of surface functionalization using a fluorescently labeled RGD peptide. Post functionalization, ~10,000 MDA-MB-231Br cells were seeded on top of the hydrogels and cultured for 5 days. We found that an increase in RGD concentration led to changes in cell morphology, with cells transitioning from a rounded to spindle-like morphology as well as an increase in cell spreading area. Also, an increase in RGD concentration resulted in an increase in cell proliferation. Cellular dormancy was assessed using the ratio of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK) to phosphorylated p38 (p-p38) positivity, which was significantly lower in hydrogels without RGD and in hydrogels with lowest RGD concentration compared to hydrogels functionalized with higher RGD concentration. We also demonstrated that the HA hydrogel-induced cellular dormancy was reversible. Finally, we demonstrated the involvement of β1 integrin in mediating cell phenotype in our hydrogel platform. Overall, our results provide insight into the role of biochemical cues in regulating dormancy versus proliferation in brain metastatic breast cancer cells.

Multifunctional Injectable Hydrogel Microparticles Loaded with miR-29a Abundant BMSCs Derived Exosomes Enhanced Bone Regeneration by Regulating Osteogenesis and Angiogenesis.

The study investigated whether both the osteogenic and angiogenic potential of Exos (Exosomes) can be enhanced by overexpression of exosomal miRNA (microRNA) and to confirm whether Exos loaded in HMPs (Hydrogel microparticles) exert long-term effects during new bone formation. BMSCs and Exos are successfully obtained. In vitro and in vivo experiments confirmed that HDAC4 (Histone deacetylase 4) is inhibited by miR-29a overexpression accompanied by the upregulation of RUNX2 (Runt-related transcription factor 2) and VEGF (Vascular Endothelial Growth Factor), thereby enhancing osteogenic and angiogenic capabilities. The HMP@Exo system is synthesized from HB-PEGDA (Hyperbranched Poly Ethylene Glycol Diacrylate)- and SH-HA (Sulfhydryl-Modified Hyaluronic Acid)-containing Exos using a microfluidic technique. The HMP surface is modified with RGD (Arg-Gly-Asp) peptides to enhance cell adhesion. The system demonstrated good injectability, remarkable compatibility, outstanding cell adhesion properties, and slow degradation capacity, and the sustained release of Agomir-29a-Exos (Exosomes derived from Agomir-29a transfected BMSCs) from HMPs enhanced the proliferation and migration of BMSCs and HUVECs (Human Umbilical Vein Endothelial Cells) while promoting osteogenesis and angiogenesis. Overall, the findings demonstrate that the HMP@Exo system can effectively maintain the activity and half-life of Exos, accompanied by overexpression of miR-29a (microRNA-29a). The injectable system provides an innovative approach for accelerating fracture healing by coupling osteogenesis and angiogenesis.

Neuroprotective Effect of Curcumin-Loaded RGD Peptide-PEGylated Nanoliposomes.

Curcumin is known for its anti-inflammatory, neuroprotective, and antioxidant properties, but its use in biological applications is hindered by its sensitivity to light, oxygen, and temperature. Furthermore, due to its low water solubility, curcumin has a poor pharmacokinetic profile and bioavailability. In this study, we evaluated the potential application of curcumin as a neuroprotective agent encapsulated in RGD peptide-PEGylated nanoliposomes developed from salmon-derived lecithin. Salmon lecithin, rich in polyunsaturated fatty acids, was used to formulate empty or curcumin-loaded nanoliposomes. Transmission electron microscopy, dynamic light scattering, and nanoparticle tracking analysis characterizations indicated that the marine-derived peptide-PEGylated nanoliposomes were spherical in shape, nanometric in size, and with an overall negative charge. Cytotoxicity tests of curcumin-loaded nanoliposomes revealed an improved tolerance of neurons to curcumin as compared to free curcumin. Wild-type SH-SY5Y were treated for 24 h with curcumin-loaded nanoliposomes, followed by 24 h incubation with conditioned media of SH-SY5Y expressing the Swedish mutation of APP containing a high ratio of Aβ40/42 peptides. Our results revealed significantly lower Aβ-induced cell toxicity in cells pre-treated with RGD peptide-PEGylated curcumin-loaded nanoliposomes, as compared to controls. Thus, our data highlight the potential use of salmon lecithin-derived RGD peptide PEGylated nanoliposomes for the efficient drug delivery of curcumin as a neuroprotective agent.

225Ac-iPSMA-RGD for Alpha-Therapy Dual Targeting of Stromal/Tumor Cell PSMA and Integrins

Prostate-specific membrane antigens (PSMAs) are frequently overexpressed in both tumor stromal endothelial cells and malignant cells (stromal/tumor cells) of various cancers. The RGD (Arg-Gly-Asp) peptide sequence can specifically detect integrins involved in tumor angiogenesis. This study aimed to preclinically evaluate the cytotoxicity, biokinetics, dosimetry, and therapeutic efficacy of 225Ac-iPSMA-RGD to determine its potential as an improved radiopharmaceutical for alpha therapy compared with the 225Ac-iPSMA and 225Ac-RGD monomers. HEHA-HYNIC-iPSMA-RGD (iPSMA-RGD) was synthesized and characterized by FT-IR, UV-vis, and UPLC mass spectroscopy. The cytotoxicity of 225Ac-iPSMA-RGD was assessed in HCT116 colorectal cancer cells. Biodistribution, biokinetics, and therapeutic efficacy were evaluated in nude mice with induced HCT116 tumors. In vitro results showed increased DNA double-strand breaks through ROS generation, cell apoptosis, and death in HCT116 cells treated with 225Ac-iPSMA-RGD. The results also demonstrated in vivo cytotoxicity in cancer cells after treatment with 225Ac-iPSMA-RGD and biokinetic and dosimetric properties suitable for alpha therapy, delivering ablative radiation doses up to 237 Gy/3.7 kBq to HCT116 tumors in mice. Given the phenotype of HCT116 cancer cells, the results of this study warrant further dosimetric and clinical studies to determine the potential of 225Ac-iPSMA-RGD in the treatment of colorectal cancer.

An intravenous pancreatic cancer therapeutic: Characterization of CRISPR/Cas9n-modified Clostridium novyi-Non Toxic.

Clostridium novyi has demonstrated selective efficacy against solid tumors largely due to the microenvironment contained within dense tumor cores. The core of a solid tumor is typically hypoxic, acidic, and necrotic-impeding the penetration of current therapeutics. C. novyi is attracted to the tumor microenvironment and once there, can both lyse and proliferate while simultaneously re-activating the suppressed immune system. C. novyi systemic toxicity is easily mitigated by knocking out the phage DNA plasmid encoded alpha toxin resulting in C. novyi-NT; but, after intravenous injection spores are quickly cleared by phagocytosis before accomplishing significant tumor localization. C. novyi-NT could be designed to accomplish intravenous delivery with the potential to target all solid tumors and their metastases in a single dose. This study characterizes CRISPR/Cas9 modified C. novyi-NT to insert the gene for RGD, a tumor targeting peptide, expressed within the promoter region of a spore coat protein. Expression of the RGD peptide on the outer spore coat of C. novyi-NT indicates an increased capacity for tumor localization of C. novyi upon intravenous introduction based on the natural binding of RGD with the αvβ3 integrin commonly overexpressed on the epithelial tissue surrounding a tumor, and lead to immune stimulation.

Dual peptide functionalization of Zn alloys to enhance endothelialization for cardiovascular applications

A new generation of fully bioresorbable metallic Zn-based alloys could be used for stenting applications; however, the initial surface degradation delays stent re-endothelialization. Thus, this work proposes a dual strategy to control the corrosion and accelerate the endothelialization of ZnMg and ZnAg biodegradable alloys. First, a stable polycaprolactone (PCL) coating is obtained and followed by its functionalization with either linear RGD (Arg-Gly-Asp) or REDV (Arg-Glu-Asp-Val) peptides or a dual peptide-based platform combining both sequences (RGD-REDV). Scratching tests showed neither delamination nor detachment of the polymeric coating. Potentiodynamic polarization (PDP) and electrochemical impedance spectroscopy (EIS) measurements confirmed the corrosion resistance after PCL coating by revealing lower current density and higher absolute impedance values. X-ray photoelectron spectroscopy (XPS) and fluorescent microscopy confirmed the correct peptide immobilization onto PCL coated Zn alloys. The functionalized samples exhibited enhanced human umbilical vein endothelial cells (HUVEC) adhesion. The higher number of adhered cells to the functionalized surfaces with the RGD-REDV platform demonstrates the synergistic effect of combining both RGD and REDV sequences. Higher corrosion resistance together with enhanced endothelialization indicates that the dual functionalization of Zn alloys with PCL and peptide-based RGD-REDV platform holds great potential to overcome the clinical limitations of current biodegradable metal stents.

Ultrasound imaging of renal fibrosis using gold nanoparticles

Abstract Introduction Microbubble ultrasound contrast agents have limitations due to their short circulation half-life and poor acoustic attenuation. In our previous study, pH-responsive gold nanoparticles (2∼3 nm) were optimized for ultrasound fluorescence molecular imaging of mouse kidneys. Renal fibrosis is associated with increased expression of the integrin αvβ3 receptor in neovascular endothelial cells. To address this, gold nanoparticles (cRGD-GSH-AuNPs) were synthesized by introducing RGD peptide, which specifically binds to integrin. Purpose The purpose of this study was to evaluate the potential of cRGD-GSH-AuNPs as a contrast agent for targeted ultrasound imaging of renal fibrosis. Methods Cyclic arginine-glycine-aspartic (cRGD) targeting integrin αvβ3 receptors was linked with glutathione (GSH)-modified gold nanoparticles (AuNPs) to create cRGD-GSH-AuNPs. After characterizing their physical properties and verifying their biocompatibility, GSH-AuNPs and cRGD-GSH-AuNPs were injected into mice with the renal fibrosis model. The renal imaging effect was evaluated by ultrasound and fluorescence imaging, and the targeting of the particles was confirmed by pathological tissue staining. Results The average size of cRGD-GSH-AuNPs determined by electron microscopy was 1.7 ± 0.2 nm, and the particles maintained biological stability after modification. The average ultrasound intensity of cRGD-GSH-AuNPs in the renal fibrosis group (22 ± 2 dB) was significantly higher than that of GSH-AuNPs in the fibrosis group (3.5 ± 3 dB), cRGD-GSH-AuNPs in the control group (1.5 ± 1 dB), and GSH-AuNPs in the control group (2 ± 1.5 dB) (all, P < 0.05). Intravenous injection of cRGD-GSH-AuNPs significantly enhanced the ultrasound signal intensity in fibrotic kidneys (see Figure 1). Conclusions Gold nanoparticles targeting integrin αVβ3 receptors achieve specific non-invasive imaging of renal fibrosis, and are a promising ultrasound contrast agent for the evaluation of renal fibrosis.Figure 1

Abstract 18397: Enhanced Ultrasound Imaging of Thrombotic Microvascular Obstruction Using GpIIb/IIIa-Targeting Gold Nanoparticles

Introduction: Thrombotic microvascular obstruction (MVO) poses a diagnostic challenge due to the lack of specific imaging techniques in clinical practice. This study introduces a novel ultrasound imaging method that utilizes gold nanoparticles (AuNPs) modified with the GPIIb/IIIa-targeting peptide Arg-Gly-Asp (RGD) to enhance the contrast of ultrasound imaging for microembolization. Methods: Targeted gold nanoparticles (RGD-AuNPs) and untargeted gold nanoparticles (GSH-AuNPs) were synthesized using the glutathione reduction method from chloroauric acid. A rat model of MVO was established by infusing microthrombi into the left hindlimb via the right femoral artery. The rats were randomly assigned to receive either RGD-AuNPs or GSH-AuNPs injected via an angiocatheter. Ultrasound imaging was performed on the left hindlimb for three hours at five-minute intervals, and the average video intensity of the images was measured. Histological staining and immunohistochemistry were performed to validate the co-localization of microthrombi and RGD-AuNPs. Results: In the RGD-AuNPs+MVO group, enhanced ultrasound signals were visible at 15 minutes and progressively increased over three hours. The average video intensity in this group was approximately three times higher than that in the GSH-AuNPs+MVO group (11.88±3.68 vs. 3.39±2.67, p<0.001). No significant difference in average video intensity was observed between the GSH-AuNPs+MVO group and the GSH-AuNPs control group (3.39±2.67 vs. 3.50±2.09, p>0.05), or between the GSH-AuNPs control group and the RGD-AuNPs control group (3.50±2.09 vs. 2.95±2.18, p>0.05). Histological staining and immunohistochemistry confirmed the co-localization of microthrombi and RGD-AuNPs. Conclusions: Gold nanoparticles modified with the RGD peptide effectively enhance the signal of ultrasound imaging, enabling targeted imaging of thrombotic microvascular obstruction. This approach holds promise for improving the diagnosis and monitoring of MVO in clinical settings.

Chitosan nanomedicine containing RGD peptide and PAD4 inhibitor based on phenyl boronate coupling inhibition of primary tumor growth and lung metastasis

Stimulus-responsive nanodrugs have been extensively studied and their structural changes in the cells are important for controlled intracellular drug release. Histone citrullination of peptidylarginine deiminase 4 (PAD4) regulates the expression of tumor suppressor genes. In our previous study, compounds such as YW3–56 (356) were developed as potent PAD4 inhibitors with excellent anti-tumor activity in vitro and in vivo. To enhance the antitumor activity and improve the bioavailability, we further optimized the structure by modifying the phenylboronic acid moiety to the PAD4 inhibitor (4B). Taking advantage of the oxidative stress responsiveness of the phenylboronic acid moiety, in this study, we covalently attached 4B to RGD sequence peptide modified chitosan (K-CRGDV) to construct this new oxidative stress responsive nanodrug (K-CRGDV-4B). The modification of RGD sequence peptide conferred the nanodrug the ability to actively target tumors. The release mechanism was verified by UV-Vis spectroscopy, NMR. The anti-tumor and anti-metastatic properties of K-CRGDV-4B were demonstrated by in vitro cytotoxicity assay and in vivo mouse Lewis lung cancer metastasis model. In addition, K-CRGDV-4B modulates the ratio of immune cells in LLC tumor-bearing mice. Immunosuppressive proteins such as PD1 were inhibited, while IFN-γ and IFN-β, which are stimulators of tumor immune responses, were upregulated. Overall, K-CRGDV-4B is a stimulus-responsive nanodrug that responds to the tumor microenvironment by inhibiting PAD4 activity, blocking the formation of neutrophil extracellular traps (NETs), and improving the tumor immune microenvironment.