The Potential of the Fibronectin Inhibitor Arg-Gly-Asp-Ser in the Development of Therapies for Glioblastoma.

Glioblastoma (GBM) is the most lethal and common malignant primary brain tumor in adults. An important feature that supports GBM aggressiveness is the unique composition of its extracellular matrix (ECM). Particularly, fibronectin plays an important role in cancer cell adhesion, differentiation, proliferation, and chemoresistance. Thus, herein, a hydrogel with mechanical properties compatible with the brain and the ability to disrupt the dynamic and reciprocal interaction between fibronectin and tumor cells was produced. High-molecular-weight hyaluronic acid (HMW-HA) functionalized with the inhibitory fibronectin peptide Arg-Gly-Asp-Ser (RGDS) was used to produce the polymeric matrix. Liposomes encapsulating doxorubicin (DOX) were also included in the hydrogel to kill GBM cells. The resulting hydrogel containing liposomes with therapeutic DOX concentrations presented rheological properties like a healthy brain. In vitro assays demonstrated that unmodified HMW-HA hydrogels only caused GBM cell killing after DOX incorporation. Conversely, RGDS-functionalized hydrogels displayed per se cytotoxicity. As GBM cells produce several proteolytic enzymes capable of disrupting the peptide-HA bond, we selected MMP-2 to illustrate this phenomenon. Therefore, RGDS internalization can induce GBM cell apoptosis. Importantly, RGDS-functionalized hydrogel incorporating DOX efficiently damaged GBM cells without affecting astrocyte viability, proving its safety. Overall, the results demonstrate the potential of the RGDS-functionalized hydrogel to develop safe and effective GBM treatments.

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System.

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. The Fast Protein Liquid Chromatography (FPLC) system allows high-resolution separation of complex protein mixtures. By adjusting various parameters in FPLC, such as selecting the appropriate purification matrix, regulating the protein sample's temperature, and managing the sample's flow rate onto the matrix and the elution rate, it is possible to ensure the protein's stability and functionality. In this protocol, we will demonstrate the versatility of the FPLC system to purify 6X-His-tagged flap endonuclease 1 (FEN1) protein, produced in bacterial cultures. To improve protein purification efficiency, we will focus on multiple considerations, including proper column packing and preparation, sample injection using a sample loop, flow rate of sample application to the column, and sample elution parameters. Finally, the chromatogram will be analyzed to identify fractions containing high yields of protein and considerations for proper recombinant protein long-term storage. Optimizing protein purification methods is crucial for improving the precision and reliability of protein analysis.

Electrospun fibers modified with polydopamine for enhancing human mesenchymal stem cell culture.

In this study, we coated electrospun polycaprolactone (PCL) fibers with polydopamine (PDA) to modify their hydrophobicity and fabricated a matrix for culturing mesenchymal stem cells (MSCs). Additionally, we incorporated Arg-Gly-Asp (RGD) peptides into PDA to enhance MSCs culture performance on PCL fibers. PDA and RGD were successfully coated in one step by immersing the electrospun fibers in a coating solution, without requiring an additional surface activation process. The characteristics of functionalized PCL fibers were analyzed by scanning electron microscopy with energy-dispersive x-ray analysis, Fourier transform infrared spectroscopy, water contact angle measurement, and fluorescence measurements using a carboxylic-modified fluorescent microsphere. MSCs cultured on the modified PCL fibers demonstrated enhanced cell adhesion, proliferation, and osteogenic- and chondrogenic differentiation. This study provides insight into potential applications for scaffold fabrication in MSCs-based tissue engineering, wound dressing, implantation, and a deeper understanding of MSCs behaviorin vitro.

Multimodal Imaging-Guided Synergistic Photodynamic Therapy Using Carbonized Zn/Co Metal-Organic Framework Loaded with Cytotoxin Against Liver Cancer.

Purpose: The study aimed to address the non-specific toxicity of cytotoxins (CTX) in liver cancer treatment and explore their combined application with the photosensitizer Ce6, co-loaded into carbonized Zn/Co bimetallic organic frameworks. The goal was to achieve controlled CTX release and synergistic photodynamic therapy, with a focus on evaluating anti-tumor activity against human liver cancer cell lines (Hep G2). Methods: Purified cobra cytotoxin (CTX) and photosensitizer Ce6 were co-loaded into carbonized Zn/Co bimetallic organic frameworks, resulting in RGD-PDA@C-ZIF@(CTX+Ce6). The formulation was designed with surface-functionalization using polydopamine and tumor-penetrating peptide RGD. This approach aimed to facilitate controlled CTX release and enhance the synergistic effect of photodynamic therapy. The accumulation of RGD-PDA@C-ZIF@(CTX+Ce6) at tumor sites was achieved through RGD's active targeting and the enhanced permeability and retention (EPR) effect. In the acidic tumor microenvironment, the porous structure of the metal-organic framework disintegrated, releasing CTX and Ce6 into tumor cells. Results: Experiments demonstrated that RGD-PDA@C-ZIF@(CTX+Ce6) nanoparticles, combined with near-infrared laser irradiation, exhibited optimal anti-tumor effects against human liver cancer cells. The formulation showcased heightened anti-tumor activity without discernible systemic toxicity. Conclusion: The study underscores the potential of utilizing metal-organic frameworks as an efficient nanoplatform for co-loading cytotoxins and photodynamic therapy in liver cancer treatment. The developed formulation, RGD-PDA@C-ZIF@(CTX+Ce6), offers a promising avenue for advancing the clinical application of cytotoxins in oncology, providing a solid theoretical foundation for future research and development.

Revealing the ACE receptor binding properties and interaction mechanisms of salty oligopeptides from Stropharia rugosoannulata mushroom by molecular simulation and antihypertensive evaluation.

The salty oligopeptides from Stropharia rugosoannulata have been proven to be potential ACE inhibitors. To investigate the ACE receptor binding properties and interaction mechanisms of salty oligopeptides, the molecular interaction, dynamics simulation, and antihypertensive evaluation cross-validation strategy were employed to reveal the oligopeptides' binding reactions and modes with the ACE receptor. Single oligopeptide (ESPERPFL, KSWDDFFTR) had exothermic and specific binding reactions with the ACE receptor, driven by hydrogen bonds and van der Waals forces. The coexistence of the multiple oligopeptide molecules did not produce the apparent ACE receptor competition binding reactions. The molecular dynamics simulation verified that the two oligopeptides disturbed the ACE receptor's different residue regions. Both oligopeptides could form stable complexes with the ACE receptor. Based on the classification of 50 oligopeptides' binding modes, ESPERPFL and KSWDDFFTR belonged to different classes, and their receptor binding modes and sites complemented, resulting in a potential synergistic effect on ACE inhibition. The antihypertensive effect of KSWDDFFTR and its distribution in the body were evaluated using SHR rats orally and ICR mice by tail vein injection, and KSWDDFFTR had antihypertensive effects within 8 h. The study provides a theoretical basis for understanding salty oligopeptides' ACE receptor binding mechanism and their antihypertensive effects.

Identification of a novel oligopeptide from defatted walnut meal hydrolysate as a potential neuroprotective agent.

Free radical damage and oxidative stress are thought to play a crucial role in the development of neurodegenerative diseases. Walnut peptides, especially walnut oligopeptides, have been shown to protect nerve cells from oxidative stress and inflammatory damage, as well as improve memory function. In this study, walnut peptides were obtained from walnut meal through enzymatic hydrolysis, ultrafiltration, and gel filtration chromatography. A novel oligopeptide called AQ was successfully isolated and its chemical structure was identified as AASCDQ using ESI-MS/MS. AQ demonstrated remarkable scavenging activity against O2- free radicals (81.00%), DPPH free radicals (79.40%), and ABTS free radicals (67.09%) at a concentration of 1 mg mL-1. Furthermore, AQ exhibited strong neuroprotective effects against hydrogen peroxide-induced damage in SH-SY5Y cells, reducing cell injury and apoptosis. AQ also effectively inhibited the secretion of pro-inflammatory factors NO (IC50 = 46.03 ± 0.32 µM) and suppressed the expression of IL-6 and TNF-α in RAW264.7 cells stimulated by LPS. In vivo experiments demonstrated that AQ promoted angiogenesis in the quail chick chorioallantoic membrane assay and reduced ROS accumulation in Caenorhabditis elegans, thereby extending its lifespan. The anti-inflammatory mechanism of AQ was further confirmed by western blotting. In summary, the novel oligopeptide AQ possesses potential neuroprotective effects, including antioxidant, anti-inflammatory, angiogenic, and anti-aging properties, making it a promising candidate for the development of functional foods and pharmaceutical products.

The tick Ixodes scapularis has five different GPCRs specifically activated by ACP (adipokinetic hormone/corazonin-related peptide).

Insects have about 50 neuropeptide genes and about 70 genes, coding for neuropeptide G protein-coupled receptors (GPCRs). An important, but small family of evolutionarily related insect neuropeptides consists of adipokinetic hormone (AKH), corazonin, and AKH/corazonin-related peptide (ACP). Normally, insects have one specific GPCR for each of these neuropeptides. The tick Ixodes scapularis is not an insect, but belongs to the subphylum Chelicerata, which comprises ticks, scorpions, mites, spiders, and horseshoe crabs. Many of the neuropeptides and neuropeptide GPCRs occurring in insects, also occur in chelicerates, illustrating that insects and chelicerates are evolutionarily closely related. The tick I. scapularis is an ectoparasite and health risk for humans, because it infects its human host with dangerous pathogens during a blood meal. Understanding the biology of ticks will help researchers to prevent tick-borne diseases. By annotating the I. scapularis genome sequence, we previously found that ticks contain as many as five genes, coding for presumed ACP receptors. In the current paper, we cloned these receptors and expressed each of them in Chinese Hamster Ovary (CHO) cells. Each expressed receptor was activated by nanomolar concentrations of ACP, demonstrating that all five receptors were functional ACP receptors. Phylogenetic tree analyses showed that the cloned tick ACP receptors were mostly related to insect ACP receptors and, next, to insect AKH receptors, suggesting that ACP receptor genes and AKH receptor genes originated by gene duplications from a common ancestor. Similar duplications have probably occurred for the ligand genes, during a process of ligand/receptor co-evolution. Interestingly, chelicerates, in contrast to all other arthropods, do not have AKH or AKH receptor genes. Therefore, the ancestor of chelicerates might have lost AKH and AKH receptor genes and functionally replaced them by ACP and ACP receptor genes. For the small family of AKH, ACP, and corazonin receptors and their ligands, gene losses and gene gains occur frequently between the various ecdysozoan clades. Tardigrades, for example, which are well known for their survival in extreme environments, have as many as ten corazonin receptor genes and six corazonin peptide genes, while insects only have one of each, or none.

Photodynamically Tumor Vessel Destruction Amplified Tumor Targeting of Nanoparticles for Efficient Chemotherapy.

Efficient tumor-targeted drug delivery is still a challenging and currently unbreakable bottleneck in chemotherapy for tumors. Nanomedicines based on passive or active targeting strategy have not yet achieved convincing chemotherapeutic benefits in the clinic due to the tumor heterogeneity. Inspired by the efficient inflammatory-cell recruitment to acute clots, we constructed a two-component nanosystem, which is composed of an RGD-modified pyropheophorbide-a (Ppa) micelle (PPRM) that mediates the tumor vascular-targeted photodynamic reaction to activate local coagulation and subsequently transmits the coagulation signals to the circulating clot-targeted CREKA peptide-modified camptothecin (CPT)-loaded nanodiscs (CCNDs) for amplifying tumor targeting. PPRM could effectively bind with the tumor vasculature and induce sufficient local thrombus by a photodynamic reaction. Local photodynamic reaction-induced tumor target amplification greatly increased the tumor accumulation of CCND by 4.2 times, thus significantly enhancing the chemotherapeutic efficacy in the 4T1 breast tumor model. In other words, this study provides a powerful platform to amplify tumor-specific drug delivery by taking advantage of the efficient crosstalk between the PPRM-activated coagulation cascade and clot-targeted CCND.

Integrin αVß3 antagonist-c(RGDyk) peptide attenuates the progression of ossification of the posterior longitudinal ligament by inhibiting osteogenesis and angiogenesis.

BACKGROUND: Ossification of the posterior longitudinal ligament (OPLL), an emerging heterotopic ossification disease, causes spinal cord compression, resulting in motor and sensory dysfunction. The etiology of OPLL remains unclear but may involve integrin αVß3 regulating the process of osteogenesis and angiogenesis. In this study, we focused on the role of integrin αVß3 in OPLL and explored the underlying mechanism by which the c(RGDyk) peptide acts as a potent and selective integrin αVß3 inhibitor to inhibit osteogenesis and angiogenesis in OPLL. METHODS: OPLL or control ligament samples were collected in surgery. For OPLL samples, RNA-sequencing results revealed activation of the integrin family, particularly integrin αVß3. Integrin αVß3 expression was detected by qPCR, Western blotting, and immunohistochemical analysis. Fluorescence microscopy was used to observe the targeted inhibition of integrin αVß3 by the c(RGDyk) peptide on ligaments fibroblasts (LFs) derived from patients with OPLL and endothelial cells (ECs). The effect of c(RGDyk) peptide on the ossification of pathogenic LFs was detected using qPCR, Western blotting. Alkaline phosphatase staining or alizarin red staining were used to test the osteogenic capability. The effect of the c(RGDyk) peptide on angiogenesis was determined by EC migration and tube formation assays. The effects of the c(RGDyk) peptide on heterotopic bone formation were evaluated by micro-CT, histological, immunohistochemical, and immunofluorescence analysis in vivo. RESULTS: The results indicated that after being treated with c(RGDyk), the osteogenic differentiation of LFs was significantly decreased. Moreover, the c(RGDyk) peptide inhibited the migration of ECs and thus prevented the nutritional support required for osteogenesis. Furthermore, the c(RGDyk) peptide inhibited ectopic bone formation in mice. Mechanistic analysis revealed that c(RGDyk) peptide could inhibit osteogenesis and angiogenesis in OPLL by targeting integrin αVß3 and regulating the FAK/ERK pathway. CONCLUSIONS: Therefore, the integrin αVß3 appears to be an emerging therapeutic target for OPLL, and the c(RGDyk) peptide has dual inhibitory effects that may be valuable for the new therapeutic strategy of OPLL.

[Syntheses and Structure-Activity Relationship Studies of Antitumor Bicyclic Hexapeptide RA-VII Analogues].

A series of antitumor bicyclic hexapeptide RA-VII analogues modified at residue 2, 3, or 6 were prepared by the chemical transformation of the hydroxy, methoxy, or carboxy groups or the aromatic rings of natural peptides RA-II, III, V, VII, and X. Analogues with modified side chains or peptide backbones, which cannot be prepared by the chemical transformation of their natural peptides, and newly isolated peptides from Rubia cordifolia roots were synthesized by using protected cycloisodityrosines prepared by the degradation of bis(thioamide) obtained from RA-VII or the diphenyl ether formation of boronodipeptide under the modified Chan-Lam coupling reaction conditions. Studies of the conformational features of the analogues and the newly isolated peptides and their relationships with cytotoxic activities against the HCT-116, HL-60, KATO-III, KB, L1210, MCF-7, and P-388 cell lines revealed the following: the methoxy group at residue 3 is essential for the potent cytotoxic activity; the methyl group at Ala-2 and Ala-4 but not at D-Ala-1 is required to establish the bioactive conformation; the N-methyl group at Tyr-5 is necessary for the peptides to adopt the active conformation preferentially; and the orientation of Tyr-5 and/or Tyr-6 phenyl rings has a significant effect on the cytotoxic activity.

Potency of a small molecule that targets the molluscum contagiosum virus processivity factor increases when conjugated to a tripeptide.

We recently developed compound FC-7269 for targeting the Molluscum contagiosum virus processivity factor (mD4) and demonstrated its ability to inhibit viral processive DNA synthesis in vitro and cellular infection of an mD4-dependent virus (Antiviral Res 211, 2023,105520). However, despite a thorough medicinal chemistry campaign we were unable to generate a potent second analog as a requisite for drug development. We overcame this impasse, by conjugating a short hydrophobic trivaline peptide to FC-7269 to produce FC-TriVal-7269 which significantly increased antiviral potency and reduced cellular toxicity.

A humanized trivalent Nectin-4-targeting nanobody drug conjugate displays potent antitumor activity in gastric cancer.

BACKGROUND: Gastric cancer represents a highly lethal malignancy with an elevated mortality rate among cancer patients, coupled with a suboptimal postoperative survival prognosis. Nectin-4, an overexpressed oncological target for various cancers, has been exploited to create antibody-drug conjugates (ADCs) to treat solid tumors. However, there is limited research on Nectin-4 ADCs specifically for gastric cancer, and conventional immunoglobulin G (IgG)-based ADCs frequently encounter binding site barriers. Based on the excellent tumor penetration capabilities inherent in nanobodies (Nbs), we developed Nectin-4-targeting Nb drug conjugates (NDCs) for the treatment of gastric cancer. RESULTS: An immunized phage display library was established and employed for the selection of Nectin-4-specific Nbs using phage display technology. Subsequently, these Nbs were engineered into homodimers to enhance Nb affinity. To prolong in vivo half-life and reduce immunogenicity, we fused an Nb targeting human serum albumin (HSA), resulting in the development of trivalent humanized Nbs. Further, we site-specifically conjugated a monomethyl auristatin E (MMAE) at the C-terminus of the trivalent Nbs, creating Nectin-4 NDC (huNb26/Nb26-Nbh-MMAE) with a drug-to-antibody ratio (DAR) of 1. Nectin-4 NDC demonstrated excellent in vitro cell-binding activities and cytotoxic efficacy against cells with high Nectin-4 expression. Subsequent administration of Nectin-4 NDC to mice bearing NCI-N87 human gastric cancer xenografts demonstrated rapid tissue penetration and high tumor uptake through in vivo imaging. Moreover, Nectin-4 NDC exhibited noteworthy dose-dependent anti-tumor efficacy in in vivo studies. CONCLUSION: We have engineered a Nectin-4 NDC with elevated affinity and effective tumor uptake, further establishing its potential as a therapeutic agent for gastric cancer.

A bicentric retrospective study of the correlation of EAU BCR risk groups with 18F-PSMA-1007 PET/CT detection in prostate cancer biochemical recurrence.

The European Association of Urology (EAU) has proposed a risk stratification for patients harboring biochemical recurrence (BCR) after radical prostatectomy: ISUP < 4 and PSA doubling time (PSAdt) > 12 months for low risk, and ISUP ≥ 4 or PSAdt ≤ 12 months for high risk. This dual-center retrospective study aims to investigate the correlation between the EAU risk stratification for BCR following radical prostatectomy and the detection rate of lesions using 18F-PSMA-1007 PET/CT. Among the 71 included patients (58 high-risk, 13 low-risk), with a median PSA level of 1.43 ng/ml, PET/CT demonstrated a significantly higher positivity in the high-risk group compared to the low-risk group (72.4% vs. 38.0%, p = 0.026). Analysis of recurrence sites revealed a similar proportion of pelvic-confined disease in both groups (24.1% vs. 23.1%, p = 0.935), but a significantly higher incidence of metastatic disease in the high-risk group (51.7% vs. 15.4%, p = 0.017), with detailed findings indicating an increased prevalence of bone metastases in the high-risk BCR group (37.8% vs. 7.7%, p = 0.048). Therefore, PSMA PET/CT offers valuable insights for treatment decisions, aligning with the evolving landscape of prostate cancer management.

Integrin αvß3 and LHRH Receptor Double Directed Nano-Analogue Effective Against Ovarian Cancer in Mice Model.

Introduction: The high mortality rate of malignant ovarian cancer is attributed to the absence of effective early diagnosis methods. The LHRH receptor is specifically overexpressed in most ovarian cancers, and the integrin αvß3 receptor is also overexpressed on the surface of ovarian cancer cells. In this study, we designed LHRH analogues (LHRHa)/RGD co-modified paclitaxel liposomes (LHRHa-RGD-LP-PTX) to target LHRH receptor-positive ovarian cancers more effectively and enhance the anti-ovarian cancer effects. Methods: LHRHa-RGD-LP-PTX liposomes were prepared using the thin film hydration method. The morphology, physicochemical properties, cellular uptake, and cell viability were assessed. Additionally, the cellular uptake mechanism of the modified liposomes was investigated using various endocytic inhibitors. The inhibitory effect of the formulations on tumor spheroids was observed under a microscope. The co-localization with lysosomes was visualized using confocal laser scanning microscopy (CLSM), and the in vivo tumor-targeting ability of the formulations was assessed using the IVIS fluorescent imaging system. Finally, the in vivo anti-tumor efficacy of the formulations was evaluated in the armpits of BALB/c nude mice. Results: The results indicated that LHRHa-RGD-LP-PTX significantly enhanced cellular uptake in A2780 cells, increased cytotoxicity, and hand a more potent inhibitory effect on tumor spheroids of A2780 cells. It also showed enhanced co-localization with endosomes or lysosome in A2780 cells, improved tumor-targeting capability, and demonstrated an enhanced anti-tumor effect in LHRHR-positive ovarian cancers. Conclusion: The designed LHRHa-RGD-LP-PTX liposomes significantly enhanced the tumor-targeting ability and therapeutic efficacy for LHRH receptor-positive ovarian cancers.

Enzymatic functionalization of decellularized tilapia skin scaffolds with enhanced skin regeneration.

The decellularized tilapia skin (dTS) has gained significant attention as a promising material for tissue regeneration due to its ability to provide unique structural and functional components that support cell growth, adhesion, and proliferation. However, the clinical application of dTS is limited by its low mechanical strength and rapid biodegradability. Herein, we prepare a novel RGD (arginine-glycine-aspartic acid) functionalized dTS scaffold (dTS/RGD) by using transglutaminase (TGase) crosslinking. The developed dTS/RGD scaffold possesses excellent properties, including a medium porosity of ∼59.2%, a suitable degradation rate of approximately 80% over a period of two weeks, and appropriate mechanical strength with a maximum tensile stress of ∼46.36 MPa which is much higher than that of dTS (∼32.23 MPa). These properties make the dTS/RGD scaffold ideal for promoting cell adhesion and proliferation, thereby accelerating skin wound healing in a full-thickness skin defect model. Such an enzymatic cross-linking strategy provides a favorable microenvironment for wound healing and holds great potential for application in skin regeneration engineering.

AS1411 aptamer/RGD dual functionalized theranostic chitosan-PLGA nanoparticles for brain cancer treatment and imaging.

Conventional chemotherapy and poor targeted delivery in brain cancer resulting to poor treatment and develop resistance to anticancer drugs. Meanwhile, it is quite challenging to diagnose/detection of brain tumor at early stage of cancer which resulting in severity of the disease. Despite extensive research, effective treatment with real-time imaging still remains completely unavailable, yet. In this study, two brain cancer cell specific moieties i.e., AS1411 aptamer and RGD are decorated on the surface of chitosan-PLGA nanoparticles to improve targeted co-delivery of docetaxel (DTX) and upconversion nanoparticles (UCNP) for effective brain tumor therapy and real-time imaging. The nanoparticles were developed by a slightly modified emulsion/solvent evaporation method. This investigation also translates the successful synthesis of TPGS-chitosan, TPGS-RGD and TPGS-AS1411 aptamer conjugates for making PLGA nanoparticle as a potential tool of the targeted co-delivery of DTX and UCNP to the brain cancer cells. The developed nanoparticles have shown an average particle size <200 nm, spherical in shape, high encapsulation of DTX and UCNP in the core of nanoparticles, and sustained release of DTX up to 72 h in phosphate buffer saline (pH 7.4). AS1411 aptamer and RGD functionalized theranostic chitosan-PLGA nanoparticles containing DTX and UCNP (DUCPN-RGD-AS1411) have achieved greater cellular uptake, 89-fold improved cytotoxicity, enhanced cancer cell arrest even at lower drug conc., improved bioavailability with higher mean residence time of DTX in systemic circulation and brain tissues. Moreover, DUCPN-RGD-AS1411 have greatly facilitated cellular internalization and higher accumulation of UCNP in brain tissues. Additionally, DUCPN-RGD-AS1411 demonstrated a significant suppression in tumor growth in brain-tumor bearing xenograft BALB/c nude mice with no impressive sign of toxicities. DUCPN-RGD-AS1411 has great potential to be utilized as an effective and safe theranostic tool for brain cancer and other life-threatening cancer therapies.

Dynamic RGD ligands derived from highly mobile cyclodextrins regulate spreading and proliferation of endothelial cells to promote vasculogenesis.

A thiolated RGD was incorporated into the threaded allyl-ß-cyclodextrins (Allyl-ß-CDs) of the polyrotaxane (PR) through a thiol-ene click reaction, resulting in the formation of dynamic RGD ligands on the PR surface (dRGD-PR). When maintaining consistent RGD density and other physical properties, endothelial cells (ECs) cultured on dRGD-PR exhibited significantly increased cell proliferation and a larger cell spreading area compared to those on the non-dynamic RGD (nRGD-PCL). Furthermore, ECs on dRGD-PR demonstrated elevated expression levels of FAK, p-FAK, and p-AKT, along with a larger population of cells in the G2/M stage during cell cycle analysis, in contrast to cells on nRGD-PCL. These findings suggest that the movement of the RGD ligands may exert additional beneficial effects in promoting EC spreading and proliferation, beyond their essential adhesion and proliferation-promoting capabilities, possibly mediated by the RGD-integrin-FAK-AKT pathway. Moreover, in vitro vasculogenesis tests were conducted using two methods, revealing that ECs cultured on dRGD-PR exhibited much better vasculogenesis than nRGD-PCL in vitro. In vivo testing further demonstrated an increased presence of CD31-positive tissues on dRGD-PR. In conclusion, the enhanced EC spreading and proliferation resulting from the dynamic RGD ligands may contribute to improved in vitro vasculogenesis and in vivo vascularization.

The design of an RGD in situ sustained delivery system utilizing scallop byssal protein through genetic engineering.

Although bioactive peptides enhancing bone healing have demonstrated effectiveness in treating bone defects, in vivo instability poses a challenge to their clinical application. Currently reported peptide delivery systems do not meet the demands of bone tissue repair regarding stability and peptide release efficacy. Herein, the self-assembling recombinant chimeric protein (Sbp5-2RGD) is developed by genetic engineering with cell adhesion peptide RGD as the targeted peptide and a newly discovered scallop byssal-derived protein Sbp5-2 that can assemble into wet stable films as the structural domain. In vitro studies show that the Sbp5-2RGD film exhibits excellent extensibility and biocompatibility. In vitro and in vivo degradation experiments demonstrate that the film remains stable due to the layer-by-layer degradation mode, resulting in sustained delivery of RGD in situ for up to 4 weeks. Consequently, the film can effectively promote osteogenesis, which accelerates bone defect healing and the implants osseointegration. Cell-level studies further show that the film up-regulates the expression of genes and proteins (ALP, OCN, OSX, OPN, RUNX2, VEGF) associated with osteogenesis and angiogenesis. Overall, this novel protein film represents an intelligent platform for peptide immobilization, protection, and release through its self-assembly, dense structure, and degradation mode, providing a therapeutic strategy for bone repair.

Investigation of Basolateral Targeting Micelles for Drug Delivery Applications in Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a complex disorder characterized by uncontrolled renal cyst growth, leading to kidney function decline. The multifaceted nature of ADPKD suggests that single-pathway interventions using individual small molecule drugs may not be optimally effective. As such, a strategy encompassing combination therapy that addresses multiple ADPKD-associated signaling pathways could offer synergistic therapeutic results. However, severe off-targeting side effects of small molecule drugs pose a major hurdle to their clinical transition. To address this, we identified four drug candidates from ADPKD clinical trials, bardoxolone methyl (Bar), octreotide (Oct), salsalate (Sal), and pravastatin (Pra), and incorporated them into peptide amphiphile micelles containing the RGD peptide (GRGDSP), which binds to the basolateral surface of renal tubules via integrin receptors on the extracellular matrix. We hypothesized that encapsulating drug combinations into RGD micelles would enable targeting to the basolateral side of renal tubules, which is the site of disease, via renal secretion, leading to superior therapeutic benefits compared to free drugs. To test this, we first evaluated the synergistic effect of drug combinations using the 20% inhibitory concentration for each drug (IC20) on renal proximal tubule cells derived from Pkd1flox/-:TSLargeT mice. Next, we synthesized and characterized the RGD micelles encapsulated with drug combinations and measured their in vitro therapeutic effects via a 3D PKD growth model. Upon both IV and IP injections in vivo, RGD micelles showed a significantly higher accumulation in the kidneys compared to NT micelles, and the renal access of RGD micelles was significantly reduced after the inhibition of renal secretion. Specifically, both Bar+Oct and Bar+Sal in the RGD micelle treatment showed enhanced therapeutic efficacy in ADPKD mice (Pkd1fl/fl;Pax8-rtTA;Tet-O-Cre) with a significantly lower KW/BW ratio and cyst index as compared to PBS and free drug-treated controls, while other combinations did not show a significant difference. Hence, we demonstrate that renal targeting through basolateral targeting micelles enhances the therapeutic potential of combination therapy in genetic kidney disease.

Light-triggered AND logic tetrapeptide dynamic covalent assembly.

We demonstrate light-triggered dynamic covalent assembly of a linear short tetrapeptide containing two terminal cysteine residues in an AND logic manner. A photobase generator is introduced to accomplish light-mediated pH regulation to increase the reduction potential of thiols in the tetrapeptide, which activates its oxidative polymerization through disulfide bonds. Interestingly, it is elucidated that under light irradiation, mere co-existence of photobase generator and the oxidizing agent permits the polymerization performance of this tetrapeptide. Hence, a light-triggered AND logic dynamic covalent assembly of a tetrapeptide is achieved. Further, upon redox response, the reversible aggregation and disaggregation can be transformed for numerous times due to the dynamic covalent feature of disulfide bond. As a comparison, no assembly occurs for a short peptide containing one terminal cysteine residue under the same stimuli condition. This work offers a new approach to remotely control programmable molecular assembly of short linear peptides based on dynamic covalent bond, holding great potential in wide bioapplications.