Search Results (243)

Liposomes modified with slightly acidic pH-sensitive peptides (SAPSp-lipo) are effectively delivered to tumor tissues, followed by cellular uptake in the tumor microenvironment. Although SAPSp-lipo can penetrate tumor tissues via the interspace route between cancer cells and the extracellular matrix (ECM), penetration needs to be enhanced to deliver liposomes into tumor cores comprising malignant cancer cells. To enhance the intratumoral penetration of SAPSp-lipo, we focused on the internalizing RGD peptide (iRGD), which can penetrate tumor tissue, differing from the penetration mechanism of SAPSp. In this study, we developed liposomes modified with iRGD-conjugated SAPSp (SAPSp-iRGD-lipo). Compared with SAPSp-lipo, SAPSp-iRGD-lipo was delivered to deeper regions within both spheroids and tumor tissues. The enhanced penetration was suppressed by a co-treatment with a Neuropilin-1 inhibitor, and the fluorescence signals from intratumorally injected SAPSp-iRGD-lipo were localized in Neuropilin-1-expressing regions, indicating a Neuropilin-1-mediated tumor penetration. Moreover, SAPSp-iRGD-lipo reduced F-actin formation in monolayered cells and was not localized in F-actin-rich regions in tumors, suggesting that SAPSp-iRGD-lipo facilitates tumor penetration through actin depolymerization. In addition, anticancer siRNA delivered by SAPSp-iRGD-lipid nanoparticles effectively induced apoptosis in cells under slightly acidic conditions. Taken together, SAPSp-iRGD-modified nanoparticles represent a novel class of tumor-penetrable and microenvironment-responsive drug carriers capable of efficient intratumoral delivery and therapeutic activity. Full article

Objectives: This paper reports the preclinical evaluation of stable tumor-specific gold nanoparticles (AuNPs) activated by neutron irradiation as a therapeutic option for the treatment of cancers characterized by high tumor angiogenesis. Methods: A selection of promising AuNPs with high avidity to α_vβ₃-expressing glioma (U-87 MG) cells (IC₅₀ = 82–104 nM) were chosen with different surface loading of Arg-Gly-Asp (RGD) peptides as tumor targeting vectors for integrin α_vβ₃, a target which is overexpressed in tissues displaying high tumor angiogenesis. Three different [¹⁹⁸Au]AuNPs were evaluated applying three injection methods, intravenous (i.v.), intraperitoneal (i.p.), and intratumoral (i.t.), each in a group of six U-87 MG xenograft–bearing mice (54 female athymic nude mice in total). Their biodistribution and tumor accumulation was assessed by in vivo imaging within 1–7 days after injection and 7 days after injection by ex vivo measurement. Results: The developed [¹⁹⁸Au]AuNPs exhibited suboptimal biodistribution by i.v. application (accumulation pattern tail > liver > spleen, no significant tumor accumulation) and by i.p. application (accumulation pattern spleen >> liver > pancreas, slight tumor accumulation of <0.3 %ID/g). However, an acceptable biodistribution by i.t. application was observed (5.5 %ID/g in liver, 4.9 %ID/g in spleen, and 3.0 %ID/g in tumor). Conclusions: Despite the very promising in vitro results, the in vivo evaluation suggests that the [¹⁹⁸Au]AuNPs represent a platform for the development of restricted therapeutic strategies. Full article

Carboxymethyl chitosan (CMC)-based hydrogels have emerged as promising candidates for wound dressing applications due to their excellent biocompatibility and tunable physicochemical properties. In this study, a novel hydrogel functionalized with hyaluronic acid (HA) and RGD peptides (RGD) was fabricated and evaluated for its structural characteristics and wound-healing potential. Using CMC as the base matrix and EDC/NHS as crosslinking agents, four hydrogel variants were fabricated: CMC gel, CMC-HA gel, CMC-RGD gel, and CMC-HA-RGD gel. The preliminary cell compatibility experiment identified the optimal formulation as 1% CMC, 0.9% HA, and 0.02 mg/mL RGD, crosslinked with 1 vol% EDC and 0.05 wt% NHS. Scanning electron microscopy showed a porous architecture (100–400 μm), conducive to fibroblast viability and proliferation. Zeta potential measurements (|ζ| > 30 mV) indicated colloidal stability of the hydrogel system. Fourier-transform infrared spectroscopy confirmed successful crosslinking and integration of HA and RGD via hydrogen bonding and electrostatic interactions, forming a stable three-dimensional network. Thermogravimetric analysis revealed enhanced thermal stability upon HA/RGD incorporation. CCK-8 assays demonstrated significantly improved cell viability with HA/RGD loading (p < 0.05), while Ki-67 immunofluorescence confirmed enhanced fibroblast proliferation, with the CMC-HA-RGD gel showing the most pronounced effect. In vitro scratch assay results demonstrated that the CMC-HA-RGD hydrogel dressing significantly enhanced cellular migration compared to other carboxymethyl chitosan-based hydrogel groups (p < 0.05). The observed statistically significant improvement in cell migration rate versus controls underscores the distinctive enhancement of synergistic HA and RGD modification in accelerating cellular migration and facilitating wound repair. Collectively, these findings suggest that the CMC-HA-RGD hydrogel possesses favorable physicochemical and biological properties and holds strong potential as an advanced wound dressing for the treatment of chronic and refractory wounds. Full article

Background/Objectives: Conventional therapeutic strategies exhibit limited efficacy against pancreatic cancer, primarily due to its profoundly hypoxic tumor microenvironment and dense fibrotic stroma. Chemodynamic therapy (CDT) holds promise; however, its application in pancreatic cancer is restricted by insufficient endogenous hydrogen peroxide (H₂O₂) levels and the activation of protective autophagy in response to oxidative stress. Methods: To overcome these obstacles, we developed a tumor microenvironment-responsive, pancreatic cancer-targeted CDT nanoamplifier—H-MnO₂/GOX&CQ-iRGD—comprising a hollow mesoporous MnO₂ shell co-loaded with glucose oxidase (GOX) and chloroquine (CQ), and surface-functionalized with the tumor-penetrating peptide iRGD. GOX catalyzes glucose oxidation to generate H₂O₂, enhancing Fenton-like reactions. CQ suppresses autophagy induced by oxidative stress, thereby alleviating therapy resistance. The iRGD peptide targets integrin α_vβ₃, which is overexpressed on pancreatic cancer cells and tumor vasculature, promoting deep tumor penetration and enhanced delivery efficiency. Results: We comprehensively characterized the nanoplatform’s physicochemical properties, tumor microenvironment triggered degradation, controlled drug release, glucose-driven H₂O₂ generation, and hydroxyl radical production in vitro. Cellular studies assessed nanoparticle uptake, intracellular H₂O₂ production, autophagy inhibition, and cytotoxicity. In vivo experiments further demonstrated effective tumor targeting and significant therapeutic outcomes in pancreatic cancer models. Conclusions: This nanoplatform addresses major barriers of CDT—namely, insufficient H₂O₂ levels, autophagy-mediated resistance, and limited intratumoral penetration—offering a promising strategy for pancreatic cancer treatment. Full article

Spinal muscular atrophy (SMA) is a severe neurodegenerative disorder that has an approved treatment that can still be improved. Antisense oligonucleotides (AONs) are currently delivered intrathecally for SMA therapy based on SMN2 gene splicing correction, and high concentrations are required to achieve an improvement of the disease symptoms. In this study, AONs were introduced into SMA fibroblast cell cultures by means of an arginine–histidine-rich peptide carrier that had been decorated with iRGD ligands. Due to the protected and receptor-mediated nature of AON delivery within these complexes, low concentrations can be used. We assessed the RNA-binding characteristics, cytotoxicity, size, and zeta potential of AON/carrier complexes as well as the efficiency of SMN2 gene splicing correction following transfections. After testing a variety of AON/carrier formulations, we selected those that produced the best outcomes. The AON/carrier complexes that were found to be the most effective significantly increased the proportion of full-length SMN transcripts and the quantity of nuclear gems. Thus, we demonstrated the potential of delivering therapeutic AONs into SMA cells using a ligand-modified peptide carrier. Full article

Phthalocyanines (Pcs) are well-established photosensitizers in photodynamic therapy, valued for their strong light absorption, high singlet oxygen generation, and photostability. Recent advances have focused on covalently conjugating Pcs, particularly zinc phthalocyanines (ZnPcs), with a wide range of small bioactive molecules to improve selectivity, efficacy, and multifunctionality. These conjugates combine light-activated reactive oxygen species (ROS) production with targeted delivery and controlled release, offering enhanced treatment precision and reduced off-target toxicity. Chemotherapeutic agent conjugates, including those with erlotinib, doxorubicin, tamoxifen, and camptothecin, demonstrate receptor-mediated uptake, pH-responsive release, and synergistic anticancer effects, even overcoming multidrug resistance. Beyond oncology, ZnPc conjugates with antibiotics, anti-inflammatory drugs, antiparasitics, and antidepressants extend photodynamic therapy’s scope to antimicrobial and site-specific therapies. Targeting moieties such as folic acid, biotin, arginylglycylaspartic acid (RGD) and epidermal growth factor (EGF) peptides, carbohydrates, and amino acids have been employed to exploit overexpressed receptors in tumors, enhancing cellular uptake and tumor accumulation. Fluorescent dye and porphyrinoid conjugates further enrich these systems by enabling imaging-guided therapy, efficient energy transfer, and dual-mode activation through pH or enzyme-sensitive linkers. Despite these promising strategies, key challenges remain, including aggregation-induced quenching, poor aqueous solubility, synthetic complexity, and interference with ROS generation. In this review, the examples of Pc-based conjugates were described with particular interest on the synthetic procedures and optical properties of targeted compounds. Full article

Background and aim: ANGPTL3 is a hepatokine acting as a negative regulator of lipoprotein lipase (LPL) through its N-terminal domain. Besides this activity, the C-terminal domain of ANGPTL3 interacts with integrin αVβ3. Since integrins are involved in inflammation and in the initiation of atherosclerotic plaque, the aim of our study was to evaluate the potential direct pro-inflammatory action of ANGPTL3 through the interaction of the fibrinogen-like domain and integrin αVβ3. Methods: We utilized cultured THP-1 human-derived macrophages and evaluated their pro-inflammatory phenotype in response to treatment with human recombinant ANGPTL3 (hANGPTL3). By Western blot, RT-qPCR, biochemical analysis, and ELISA assays, we determined the expression of genes and proteins involved in lipid metabolism and inflammatory response as well as intracellular cholesterol and triglyceride levels. In addition, we evaluated the effect of hANGPTL3 on the cellular cholesterol efflux process. Results: Incubation of THP-1-derived macrophages with 100 ng/mL of hANGPTL3 increased the mRNA expression of the pro-inflammatory cytokines IL-1β, IL-6, and TNFα (respectively, 1.87 ± 0.08-fold, 1.35 ± 0.11-fold, and 2.49 ± 0.43-fold vs. control). The secretion of TNFα, determined by an ELISA assay, was also induced by hANGPTL3 (1.98 ± 0.4-fold vs. control). The pro-inflammatory effect of hANGPTL3 was partially counteracted by co-treatment with the integrin αVβ3 inhibitor RGD peptide, reducing the mRNA levels of IL-1β (3.35 ± 0.35-fold vs. 2.54 ± 0.25-fold for hANGPTL3 vs. hANGPTL3 + RGD, respectively). Moreover, hANGPTL3 reduced cholesterol efflux to apoA-I, with a parallel increase in the intracellular triglyceride and cholesterol contents by 31.2 ± 2.8% and 20.0 ± 4.1%, respectively, compared to the control. Conclusions: ANGPTL3 is an important liver-derived regulator of plasma lipoprotein metabolism, and overall, our results add a new important pro-inflammatory activity of this circulating protein. This new function of ANGPTL3 could also be related to triglyceride and cholesterol accumulation into macrophages. Full article

Fibronectin (FN), a primary component of the extracellular matrix (ECM), features multiple structural domains closely linked to various cellular behaviors, including migration, spreading, adhesion, and proliferation. The FN3 domain, which contains the RGD sequence, is critical in tissue repair because it enables interaction with integrin receptors on the cell surface. However, the large molecular weight of wild-type FN presents challenges for its large-scale production through heterologous expression. Therefore, this study focused on cloning the FN3 functional domain of full-length FN for expression and validation. This study selected Bacillus subtilis as the expression host due to its prominent advantages, including efficient protein secretion, absence of endotoxins, and minimal codon bias. The recombinant vector pHT43-FN3 was successfully constructed through homologous recombination technology and transformed into Bacillus subtilis WB800N. The FN3 protein was successfully expressed after induction with IPTG. Following purification of the recombinant FN protein using a His-tag nickel column, SDS-PAGE analysis showed that the molecular weight of FN3 was approximately 27.3 kDa. Western blot analysis confirmed the correct expression of FN3, and the BCA protein assay kit determined a protein yield of 5.4 mg/L. CCK8 testing demonstrated the good biocompatibility of FN3. In vitro cell experiments showed that FN3 significantly promoted cell migration at a 20 μg/mL concentration and enhanced cell adhesion at 10 μg/mL. In summary, this study successfully utilized Bacillus subtilis to express the FN3 functional domain peptide from FN protein and has validated its ability to promote cell migration and adhesion. These findings not only provide a strategy for the expression of FN protein in B. subtilis, but also establish an experimental foundation for the potential application of FN3 protein in tissue repair fields such as cutaneous wound healing and cartilage regeneration. Full article

Background/Objectives: Tetrastatin, the globular non collagenous (NC1) domain of the α4 chain of collagen IV, was previously demonstrated to inhibit melanoma progression. We identified the minimal active sequence (QKISRCQVCVKYS: QS-13) that reproduced the anti-tumor effects of whole Tetrastatin and demonstrated its anti-angiogenic activity mediated through αvβ3 and α5β1 binding. As QS-13 peptide was not fully soluble in aqueous solution, we designed new peptides with better water solubility. The present work aimed to investigate the interactions of ten QS-13-derived peptides, exhibiting improved hydro-solubility, with αvβ3 and α5β1 integrins. Methods: Using bioinformatics tools such as GROMACS, VMD, and the Autodock4 suite, we investigated the ability of the substituted peptides to bind αvβ3 and α5β1 integrins in silico. Results: We demonstrated in silico that all substituted peptides were able to bind both integrins at the RGD-binding site and determined their theoretical binding energy. Conclusions: The new soluble peptides should be able to compete with natural integrin ligands such as fibronectin, but also FGF1, FGF2, IGF1, and IGF2. Taken together, these findings suggest that the QS-13-derived peptides are reliable anti-angiogenic and anti-tumor agents. Full article

Background: In tissue engineering, developing injectable hydrogels with tailored mechanical and bioactive properties remains a challenge. This study introduces an injectable hydrogel composite for soft tissue regeneration, composed of oxidized alginate (OA) and N-succinyl chitosan (NSC) cross-linked via Schiff base reaction, reinforced with graphene oxide (GOx) and cyclic arginylglycylaspartic acid (c-RGD). The objective was to create a multifunctional platform combining injectability, bioactivity, and structural stability. Methods: The OA/NSC/GOx-cRGD hydrogel was synthesized through Schiff base cross-linking (aldehyde-amine reaction). Characterization included FTIR (C=N bond at 1650 cm⁻¹), Raman spectroscopy (D/G bands at 1338/1567 cm⁻¹), SEM (porous microstructure), and rheological analysis (shear-thinning behavior). In vitro assays assessed fibroblast viability (MTT) and macrophage TNF-α secretion (ELISA), while ex-vivo injectability and retention were evaluated using chicken cardiac tissue. Results: The hydrogel exhibited shear-thinning behavior (viscosity: 10 to <1 Pa·s) and elastic-dominated mechanics (G′ > G″), ensuring injectability. SEM revealed an interconnected porous structure mimicking native extracellular matrix. Fibroblast viability remained ≥95%, and TNF-α secretion in macrophages decreased by 80% (30 vs. 150 pg/μL in controls), demonstrating biocompatibility and anti-inflammatory effects. The hydrogel adhered stably to cardiac tissue without leakage. Conclusions: The OA/NSC/GOx-cRGD composite integrates injectability, bioactivity, and structural stability, offering a promising scaffold for tissue regeneration. Its modular design allows further functionalization with peptides or growth factors. Future work will focus on translational applications, including scalability and optimization for dynamic biological environments. Full article

The opportunistic bacteria Serratia proteamaculans are able to penetrate human cells. It was previously shown that the bacterial surface protein OmpX promotes bacterial adhesion. In addition, infection with bacteria that synthesize the OmpX protein enhances the expression of EGFR and β1 integrin involved in the invasion of S. proteamaculans. Therefore, this work was aimed at determining the mechanism of interaction of S. proteamaculans with receptors of eukaryotic cells. Both integrin-linked kinase (ILK) and EGFR tyrosine kinase have been shown to be involved in the invasion of these bacteria. During infection, EGFR is first phosphorylated at Tyr845, which is carried out by c-Src kinase transmitting a signal from nearby receptors. The S. proteamaculans invasion depends on c-Src and focal adhesion kinase (FAK), which can both transmit a signal between β1 integrin and EGFR and participate in cytoskeletal rearrangements. These bacteria have been shown to interact with integrin not through the RGD binding site, and integrin binding to the RGD peptide enhances adhesion, invasion, and expression of α5 and β1 integrin subunits in response to infection. On the other hand, bacterial adhesion and increased expression of integrins during infection are caused by OmpX. Thus, OmpX interacts with integrins, and the participation of the α5 and β1 integrin subunits in the S. proteamaculans invasion allows us to assume that the receptor of OmpX is α5β1 integrin. Full article

Background/Objectives: Integrin α_Vβ₃ plays a crucial role in tumor angiogenesis and cancer progression, making it a key target for radiolabeled probes used in imaging and therapy. A previously developed probe, [¹²⁵I]bcRGD, exhibited high selectivity for α_Vβ₃ but limited tumor accumulation due to rapid blood clearance. This study aimed to address this issue through two strategies: (1) conjugating albumin-binding molecules to enhance systemic circulation and (2) dimerizing RGD peptides to improve binding affinity via multivalency effects. Methods: Three [¹²⁵I]bcRGD derivatives were synthesized: [¹²⁵I]bcRGD_pal (with palmitic acid), [¹²⁵I]bcRGD_iba (with 4-(p-iodophenyl)butyric acid), and [¹²⁵I]bcRGD_dimer (a dimeric bicyclic RGD peptide). Their physicochemical properties, α_Vβ₃-selectivity, albumin-binding capacity, and biodistribution were assessed in vitro and in vivo using tumor-bearing mice. Tumor models included α_Vβ₃-high U-87 MG and α_Vβ₃-low A549 xenografts. Results: [¹²⁵I]bcRGD_pal and [¹²⁵I]bcRGD_iba exhibited prolonged blood retention (30-fold and 55-fold vs. [¹²⁵I]bcRGD, respectively) and increased tumor accumulation (3.9% ID/g and 3.6% ID/g at 2 h, respectively). Despite improved systemic circulation, tumor-to-blood ratios remained low (<1), indicating limited tumor retention. [¹²⁵I]bcRGD_dimer achieved significantly greater tumor accumulation (4.2% ID/g at 2 h) and favorable tumor-to-blood (22) and tumor-to-muscle (14) ratios, with a 5.4-fold higher uptake in U-87 MG tumors compared to A549 tumors. Conclusions: Dimerization was more effective than albumin binding in enhancing bcRGD’s tumor-targeting potential. The dimeric probe demonstrated improved tumor accumulation, favorable pharmacokinetics, and preserved integrin selectivity. These findings provide a foundation for further structural optimization of bicyclic RGD peptides for integrin α_Vβ₃-targeted imaging and therapy applications. Full article

Adipose-derived stromal cells (ASCs) possess significant regenerative potential, playing a key role in tissue repair and angiogenesis. During wound healing, ASC interacts with the extracellular matrix by recognizing arginylglycylaspartic acid (RGD) motifs, which are crucial for mediating these functions. This study investigates how RGD exposure influences ASC behavior, with a focus on angiogenesis. To mimic the wound-healing environment, ASC were cultured in a porcine gelatin sponge, an RGD-exposing matrix. Transcriptomics revealed that ASC cultured in gelatin exhibited an upregulated expression of genes associated with inflammation, angiogenesis, and tissue repair compared to ASC in suspension. Pro-inflammatory and pro-angiogenic factors, including IL-1, IL-6, IL-8, and VEGF, were significantly elevated. Functional assays further demonstrated that ASC-conditioned media enhanced endothelial cell migration, tubulogenesis, and reduced endothelial permeability, all critical processes in angiogenesis. Notably, ASC-conditioned media also promoted vasculogenesis in human vascular organoids. The inhibition of ASC-RGD interactions using the cyclic peptide cilengitide reversed these effects, underscoring the essential role of RGD-integrin interactions in ASC-mediated angiogenesis. These findings suggest that gelatin sponges enhance ASC’s regenerative and angiogenic properties via RGD-dependent mechanisms, offering promising therapeutic potential for tissue repair and vascular regeneration. Understanding how RGD modulates ASC behavior provides valuable insights into advancing cell-based regenerative therapies. Full article

Oncolytic adenoviruses derived from human serotype 5 (Ad5) are being developed to treat cancer. Treatment efficacy could be affected by pre-existing or induced neutralizing antibodies (NAbs), in particular in repeat administration strategies. Several oncolytic adenoviruses that are currently in clinical development have modified fiber proteins to increase their infectivity. One example is Ad5-∆24.RGD, which carries a cyclic RGD peptide insert in the fiber protein to allow cell entry via integrins. The effect of anti-Ad5 NAbs on anticancer efficacy could be different for oncolytic adenoviruses with RGD-modified fibers than for unmodified Ad5-based viruses. Here, we determine pre-existing and elicited NAb titers in the serum of patients with glioblastoma who were treated by delivering Ad5-∆24.RGD to the tumor and to the surrounding tumor-infiltrated brain. We show that intracranial infusion of Ad5-∆24.RGD induced mainly neutralization of adenovirus native tropism. Infection of cells with RGD-modified virus was significantly less affected. In cerebrospinal fluid, neutralizing activity against RGD-mediated infection remained very low. Thus, the RGD-mediated alternative cell entry route allowed to bypass pre-existing and induced anti-Ad5 neutralization. Interestingly, in the course of these experiments, we discovered that the serum of most humans promotes the uptake of RGD-modified adenovirus in human cells. The until now unidentified infection-stimulating factor seems distinct from serum proteins known to promote Ad5 infection. Together, our work supports the utility of RGD-modified oncolytic adenoviruses for the treatment of cancer in humans. Since these viruses hardly induced neutralization, they seem particularly suitable for repeat administration treatments. Full article

Under benign conditions, bone tissue can regenerate itself without external intervention. However, this regenerative capacity can be compromised by various factors, most importantly related with the extent of the injury. Critical-sized defects, exceeding the body’s natural healing ability, demand the use of temporary or permanent devices like artificial joints or bone substitutes. While titanium is a widely used material for bone replacement, its integration into the body remains limited. This often leads to the progressive loosening of the implant and the need for revision surgeries, which are technically challenging, are commonly associated with high complication rates, and impose a significant economic burden. To enhance implant osseointegration, numerous studies have focused on the development of surface functionalization techniques to improve the response of the body to the implant. Yet, the challenge of achieving reliable and long-lasting prostheses persists. In this work, we address this challenge by applying a robust and versatile biofunctionalization process followed by the decoration of the material with oligopeptides. We immobilize four different peptides (RGD, CS-1, IKVAV, PHSRN) on R-THAB^® functionalized surfaces and find them to be highly stable in the long term. We also find that RGD is the best-performing peptide in in vitro cell cultures, enhancing adhesion, proliferation, and osteogenic differentiation of mesenchymal stem cells. To assess the in vivo effect of RGD-decorated Ti-6Al-4V implants, we develop a calvarial model in murine hosts. We find that the RGD-decoration remains stable for 1 week after the surgical procedure and reduces post-implantation macrophage-related inflammation. These results highlight the potential of peptide decoration on R-THAB^® functionalized surfaces to expedite the development of novel metallic biomaterials with enhanced biocompatibility properties, thereby advancing the field of regenerative medicine. Full article