Targeting Peptide Research Articles

Targeted delivery of type I TGF-β receptor-mimicking peptide to fibrotic kidney for improving kidney fibrosis therapy via enhancing the inhibition of TGF-β1/Smad and p38 MAPK pathways

Renal fibrosis is a representative pathological feature of various chronic kidney diseases, and efficient treatment is needed. Interstitial myofibroblasts are a key driver of kidney fibrosis, which is dependent on the binding of TGF-β1 to type I TGF-β receptor (TβRI) and TGF-β1-related signaling pathways. Therefore, attenuating TGF-β1 activity by competing with TGF-β1 in myofibroblasts is an ideal strategy for treating kidney fibrosis. Recently, a novel TβRI-mimicking peptide RIPΔ demonstrated a high affinity for TGF-β1. Thus, it could be speculated that RIPΔ may be used for anti-fibrosis therapy. Platelet-derived growth factor β receptor (PDGFβR) is highly expressed in fibrotic kidney. In this study, we found that target peptide Z-RIPΔ, which is RIPΔ modified with PDGFβR-specific affibody ZPDGFβR, was specifically and highly taken up by TGF-β1-activated NIH3T3 fibroblasts. Moreover, Z-RIPΔ effectively inhibited the myofibroblast proliferation, migration and fibrosis response in vitro. In vivo and ex vivo experiments showed that Z-RIPΔ specifically targeted fibrotic kidney, improved the damaged renal function, and ameliorated kidney histopathology and renal fibrosis in UUO mice. Mechanistic studies showed that Z-RIPΔ hold the stronger inhibition of the TGF-β1/Smad and TGF-β1/p38 pathways than unmodified RIPΔ in vitro and in vivo. Furthermore, systemic administration of Z-RIPΔ to UUO mice led to minimal toxicity to major organs. Taken together, RIPΔ modified with ZPDGFβR increased its therapeutic efficacy and reduced its systemic toxicity, making it a potential candidate for targeted therapy for kidney fibrosis.

Carbon nanotubes functionalized with α-aminoisobutyric acid-containing peptide increase gene delivery efficiency in plant mitochondria

Functionalized carbon nanotubes have shown tremendous promise in the field of plant biotechnology for genetic engineering and cargo delivery; recent findings have shown that they can be delivered within specific organelles, such as mitochondria and chloroplasts, in intact plants. 2-Aminoisobutyric acid is an unnatural amino acid that promotes helical conformation and has been demonstrated to increase membrane permeability. Rational substitution of this amino acid into a mitochondrial targeting peptide induced a helical conformation that, when functionalized onto polymer-coated carbon nanotubes, conferred increased membrane permeability compared with that of the native peptide. The secondary structure was maintained on the surface and, when used to deliver pDNA, led to an increase in gene expression, suggesting that this method may be used to enhance the delivery efficiency of existing functional peptides.

Wideband PRM: Highly Accurate and Sensitive Method for High-Throughput Targeted Proteomics.

Targeted mass spectrometry (MS) approaches, which are powerful methods for uniquely and confidently quantifying a specific panel of proteins in complex biological samples, play a crucial role in validating and clinically translating protein biomarkers discovered through global proteomic profiling. Common targeted MS methods, such as multiple reaction monitoring (MRM) and parallel-reaction monitoring (PRM), employ specific mass spectrometric technologies to quantify protein levels by comparing the transitions of surrogate endogenous (ENDO) peptides with those of stable isotope-labeled (SIL) peptide counterparts. These methods utilizing amino acid analyzed (AAA) SIL peptides warrant sensitive and precise measurements required for targeted MS assays. Compared with MRM, PRM provides higher experimental throughput by simultaneously acquiring all transitions of the target peptides and thereby compensates for different ion suppressions among transitions of a target peptide. However, PRM still suffers different ion suppressions between ENDO and SIL peptides due to spray instability, as the ENDO and SIL peptides were monitored at different liquid chromatography (LC) retention times. Here we introduce a new targeted MS method, termed wideband PRM (WBPRM), that is designed for high-throughput targeted MS analysis. WBPRM employs a wide isolation window for simultaneous fragmentation of both ENDO and SIL peptides along with multiplexed single ion monitoring (SIM) scans for enhanced MS sensitivity of the target peptides. Compared with PRM, WBPRM was demonstrated to provide increased sensitivity, precision, and reproducibility of quantitative measurements of target peptides with increased throughput, allowing more target peptide measurements in a shortened experiment time. WBPRM is a straightforward adaptation to a manufacturer-provided MS method, making it an easily implementable technique, particularly in complex biological samples where the demand for higher precision, sensitivity, and efficiency is paramount.

Brain-Derived Neurotrophic Factor-Loaded Low-Temperature-Sensitive liposomes as a drug delivery system for repairing podocyte damage

Podocytes, cells of the glomerular filtration barrier, play a crucial role in kidney diseases and are gaining attention as potential targets for new therapies. Brain-Derived Neurotrophic Factor (BDNF) has shown promising results in repairing podocyte damage, but its efficacy via parenteral administration is limited by a short half-life. Low temperature sensitive liposomes (LTSL) are a promising tool for targeted BDNF delivery, preserving its activity after encapsulation. This study aimed to improve LTSL design for efficient BDNF encapsulation and targeted release to podocytes, while maintaining stability and biological activity, and exploiting the conjugation of targeting peptides. While cyclic RGD (cRGD) was used for targeting endothelial cells in vitro, a homing peptide (HITSLLS) was conjugated for more specific uptake by glomerular endothelial cells in vivo. BDNF-loaded LTSL successfully repaired cytoskeleton damage in podocytes and reduced albumin permeability in a glomerular co-culture model. cRGD conjugation enhanced endothelial cell targeting and uptake, highlighting an improved therapeutic effect when BDNF release was induced by thermoresponsive liposomal degradation. In vivo, targeted LTSL showed evidence of accumulation in the kidneys, and their BDNF delivery decreased proteinuria and ameliorated kidney histology. These findings highlight the potential of BDNF-LTSL formulations in restoring podocyte function and treating glomerular diseases.

Progress in Core-Shell Magnetic Mesoporous Materials for Enriching Post-Translationally Modified Peptides.

Endogenous peptides, particularly those with post-translational modifications, are increasingly being studied as biomarkers for diagnosing various diseases. However, they are weakly ionizable, have a low abundance in biological samples, and may be interfered with by high levels of proteins, peptides, and other macromolecular impurities, resulting in a high limit of detection and insufficient amounts of post-translationally modified peptides in real biological samples to be examined. Therefore, separation and enrichment are necessary before analyzing these biomarkers using mass spectrometry. Mesoporous materials have regular adjustable pores that can eliminate large proteins and impurities, and their large specific surface area can bind more target peptides, but this may result in the partial loss or destruction of target peptides during centrifugal separation. On the other hand, magnetic mesoporous materials can be used to separate the target using an external magnetic field, which improves the separation efficiency and yield. Core-shell magnetic mesoporous materials are widely utilized for peptide separation and enrichment due to their biocompatibility, efficient enrichment capability, and excellent recoverability. This paper provides a review of the latest progress in core-shell magnetic mesoporous materials for enriching glycopeptides and phosphopeptides and compares their enrichment performance with different types of functionalization methods.

Design of Artificial C-Peptides as Potential Anti-HIV-1 Inhibitors Based on 6-HB Formation Mechanism.

The six-helix bundle (6-HB) is a core structure formed during the membrane fusion process of viruses with the Class I envelope proteins. Peptide inhibitors, including the marketed Enfuvirtide, blocking the membrane fusion to exert inhibitory activity were designed based on the heptads repeat interactions in 6-HB. However, the drawbacks of Enfuvirtide, such as drug resistance and short half-life in vivo, have been confirmed in clinical applications. Therefore, novel design strategies are pivotal in the development of next-generation peptide-based fusion inhibitors. The de novo design of α-helical peptides against MERS-CoV and IAVs has successfully expedited the development of fusion inhibitors. The reported sequences were completely nonhomologous with natural peptides, which can provide some inspirations for the antiviral design against other pathogenic viruses with class I fusion proteins. Here, we design a series of artificial C-peptides based on the similar mechanism of 6-HB formation and general rules of heptads repeat interaction. The inhibitory activity of peptides against HIV-1 was assessed by HIV-1 Env-mediated cell-cell fusion assays. Interaction between artificial C-peptides and target peptides was evaluated by circular dichroism, polyacrylamide gel electrophoresis, size-exclusion chromatography, and sedimentation velocity analysis. Molecular docking studies were performed by using Schrödinger molecular modelling software. The best-performing artificial C-peptide, 1SR, was highly active against HIV-1 env-mediated cell-cell fusion. 1SR binds to the gp41 NHR region, assembling polymer to prevent endogenous 6-HB formation. We have found an artificial C-lipopeptide lead compound with inhibitory activity against HIV-1. Also, this paper enriched both N- and C-teminal heptads repeat interaction rules in 6-HB and provided an effective idea for next-generation peptide-based fusion inhibitors against HIV-1.

Phase 1/2 of EO2463 immunotherapy as monotherapy and in combination with lenalidomide and/or rituximab in indolent NHL (EONHL1-20/SIDNEY).

7058 Background: Follicular and marginal zone B cell lymphoma (FL; MZL) demonstrate an indolent course with heterogeneous outcomes anda potential for spontaneous remissions indicating immune system intervention. Therapeutic immunization is therefore an attractive approach but new antigens that evoke a strong immune response are needed. EO2463 expands pre-existing memory CD8+ T cells recognizing non-self protein sequences from gut bacteria which cross-react with B cell antigens and can kill HLA-A2 restricted cells (T2) loaded with target peptides. EO2463 includes 4 HLA-A2 synthetically produced epitopes which exhibit molecular mimicry with the B cell markers CD20, CD22, CD37, and CD268 (BAFF-receptor), as well as a the CD4 helper-epitope UCP2 derived from hTERT. EO2463 is being used to drive anti-tumor activity against B cell malignancies. Methods: Patients (pts) with FL and MZL, stage 1-3A, and HLA-A2, are eligible. In the safety lead-in pts with relapsed/refractory (R/R) disease were given EO2463 SC q2 weeks (w) x 4, then q4w, for a max of 12 months; at w7 lenalidomide (20 mg/day for 21/28 days up to 12 cycles) is added (EL), and if no complete remission (CR) at w19, rituximab (375 mg/m2 IV, q1w x 4, then q4w x 4) is also added (ER2). Doses evaluated: 150 μg and 300μg/peptide. Results: 3 pts received 150 μg/peptide and 6 pts 300 μg/peptide (EO2463 1 pat, EL 2 pts, ER2 6 pts): 2 MZL, 7 FL pts with median 2 lines (range 1-4) of prior systemic therapy. No related grade ≥3 adverse events were seen with EO2463 monotherapy. Most common related events were grade 1 to 2 local administration site reactions in 5/9 pts. Adverse events during combination treatment were as expected for R2. PET-CT on w6 after EO2463 monotherapy suggested clinical activity in 4/9 pts (1 PR, 1 pt size reduction 15%, 1 pt 20% reduced tracer uptake, 1 pt 5/6 target lesions reduced metabolic activity). Overall objective responses (OR) were seen in 6/9 pts (67%; 1st OR on EO2463 1 pat, EL 4 pts, ER2 1 pat), with CR in 5/9 (56%) pts; median time to response was 18w (range 8-43w), response ongoing in 5 pts, range 24-76w. Expansion of specific CD8+ T cells against mimic peptides and targeted B cell antigens were detected in all responding pts, incl. 2 pts with no measurable B cells at baseline (prior anti-CD20). Expansion of specific T cells was detected at w5 in 5/6 pts and was maintained as long as currently tested (up to w94, 43w after last EO2463 dose). No decline in expansions were seen after start rituximab. Conclusions: EO2463 (300 μg/peptide) monotherapy, EL, and ER2 are well tolerated, with encouraging clinical activity with EO2463 monotherapy and with subsequent CR in 5/9 pts on combo. Rapid and durable expansion of specific CD8+ T cells was seen in all responding pts, consistent with the preclinical hypothesis. Additional cohorts investigate EO2463 alone or in combination in newly diagnosed or R/R pts. Results will be reported at the meeting. Clinical trial information: NCT04669171 .

Pan-cancer proteogenomics expands the landscape of therapeutic targets

Fewer than 200 proteins are targeted by cancer drugs approved by the Food and Drug Administration (FDA). We integrate Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteogenomics data from 1,043 patients across 10 cancer types with additional public datasets to identify potential therapeutic targets. Pan-cancer analysis of 2,863 druggable proteins reveals a wide abundance range and identifies biological factors that affect mRNA-protein correlation. Integration of proteomic data from tumors and genetic screen data from cell lines identifies protein overexpression- or hyperactivation-driven druggable dependencies, enabling accurate predictions of effective drug targets. Proteogenomic identification of synthetic lethality provides a strategy to target tumor suppressor gene loss. Combining proteogenomic analysis and MHC binding prediction prioritizes mutant KRAS peptides as promising public neoantigens. Computational identification of shared tumor-associated antigens followed by experimental confirmation nominates peptides as immunotherapy targets. These analyses, summarized at https://targets.linkedomics.org, form a comprehensive landscape of protein and peptide targets for companion diagnostics, drug repurposing, and therapy development.

Enhancing the treatment of Staphylococcus aureus infections: A nanosystem with including dual antimicrobial peptide

The unique properties of nanoparticles (NPs) and their modification to selectively target infected tissues or specific pathogens could be an effective approach to revolutionize the treatment of infectious diseases. The objective of this study was to develop poly (lactic-co-glycolic acid) (PLGA) nanoparticles loaded with an antimicrobial peptide (HF-18) and decorated with an antimicrobial peptide (P6.2) for selective targeting against Staphylococcus aureus (S. aureus). Applying the Box-Behnken experimental design, HF-18 loaded PLGA nanoparticles were produced and optimized to have minimum particle size and polydispersity index (PDI) value by selecting primary water phase volume, organic phase volume and secondary water phase volume as independent parameters. Secondary water phase volume had the most significant effect on particle size and PDI. OPT-NP was more effective than HF-18 against most bacterial strains. The non-targeted (OPT-NP) and targeted (P6.2-OPT-NP) systems were shown to be biocompatible at levels that exhibited antibacterial activity. The P6.2-OPT-NP was predominantly taken up by S. aureus, while Escherichia coli (E. coli) used as a negative control showed minimal uptake. Furthermore, co-culture studies indicated that P6.2-OPT-NP was drastically uptake by S. aureus, while no internalized nanoparticles were visualized in fibroblast cells indicating active targeting of bacterial cells by P6.2-OPT-NP. Thus, these findings demonstrate the development of a formulation that selectively targets S. aureus, presenting promising prospects for future therapeutic applications.

Glutathione-depleting Liposome Adjuvant for Augmenting the Efficacy of a Glutathione Covalent Inhibitor Oridonin for Acute Myeloid Leukemia Therapy

BackgroundDiscrepancies in the utilization of reactive oxygen species (ROS) between cancer cells and their normal counterparts constitute a pivotal juncture for the precise treatment of cancer, delineating a noteworthy trajectory in the field of targeted therapies. This phenomenon is particularly conspicuous in the domain of nano-drug precision treatment. Despite substantial strides in employing nanoparticles to disrupt ROS for cancer therapy, current strategies continue to grapple with challenges pertaining to efficacy and specificity. One of the primary hurdles lies in the elevated levels of intracellular glutathione (GSH). Presently, predominant methods to mitigate intracellular GSH involve inhibiting its synthesis or promoting GSH efflux. However, a conspicuous gap remains in the absence of a strategy capable of directly and efficiently clearing GSH.MethodsWe initially elucidated the chemical mechanism underpinning oridonin, a diminutive pharmacological agent demonstrated to perturb reactive oxygen species, through its covalent interaction with glutathione. Subsequently, we employed the incorporation of maleimide-liposomes, renowned for their capacity to disrupt the ROS delivery system, to ameliorate the drug’s water solubility and pharmacokinetics, thereby enhancing its ROS-disruptive efficacy. In a pursuit to further refine the targeting for acute myeloid leukemia (AML), we harnessed the maleic imide and thiol reaction mechanism, facilitating the coupling of Toll-like receptor 2 (TLR2) peptides to the liposomes’ surface via maleic imide. This strategic approach offers a novel method for the precise removal of GSH, and its enhancement endeavors are directed towards fortifying the precision and efficacy of the drug’s impact on AML targets.ResultsWe demonstrated that this peptide-liposome-small molecule machinery targets AML and consequently induces cell apoptosis both in vitro and in vivo through three disparate mechanisms: (I) Oridonin, as a Michael acceptor molecule, inhibits GSH function through covalent bonding, triggering an initial imbalance of oxidative stress. (II) Maleimide further induces GSH exhaustion, aggravating redox imbalance as a complementary augment with oridonin. (III) Peptide targets TLR2, enhances the directivity and enrichment of oridonin within AML cells.ConclusionThe rationally designed nanocomplex provides a ROS drug enhancement and targeted delivery platform, representing a potential solution by disrupting redox balance for AML therapy.

Data Independent Acquisition to Inform the Development of Targeted Proteomics Assays Using a Triple Quadrupole Mass Spectrometer.

Mass spectrometry based targeted proteomics methods provide sensitive and high-throughput analysis of selected proteins. To develop a targeted bottom-up proteomics assay, peptides must be evaluated as proxies for the measurement of a protein or proteoform in a biological matrix. Candidate peptide selection typically relies on predetermined biochemical properties, data from semi-stochastic sampling, or by empirical measurements. These strategies require extensive testing and method refinement due to the difficulties associated with prediction of peptide response in the biological matrix of interest. Gas-phase fractionated (GPF) narrow window data-independent acquisition (DIA) aids in the development of reproducible selected reaction monitoring (SRM) assays by providing matrix-specific information on peptide detectability and quantification by mass spectrometry. To demonstrate the suitability of DIA data for selecting peptide targets, we reimplement a portion of an existing assay to measure 98 Alzheimer's disease proteins in cerebrospinal fluid (CSF). Peptides were selected from GPF-DIA based on signal intensity and reproducibility. The resulting SRM assay exhibits similar quantitative precision to published data, despite the inclusion of different peptides between the assays. This workflow enables development of new assays without additional up-front data acquisition, demonstrated here through generation of a separate assay for an unrelated set of proteins in CSF from the same dataset.

In vivo phage display identifies novel peptides for cardiac targeting

Heart failure remains a leading cause of mortality. Therapeutic intervention for heart failure would benefit from targeted delivery to the damaged heart tissue. Here, we applied in vivo peptide phage display coupled with high-throughput Next-Generation Sequencing (NGS) and identified peptides specifically targeting damaged cardiac tissue. We established a bioinformatics pipeline for the identification of cardiac targeting peptides. Hit peptides demonstrated preferential uptake by human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and immortalized mouse HL1 cardiomyocytes, without substantial uptake in human liver HepG2 cells. These novel peptides hold promise for use in targeted drug delivery and regenerative strategies and open new avenues in cardiovascular research and clinical practice.

Ru(II)-Catalyzed C(sp2)-H Activation Annulation: Synthesis of Fluorescent Benzoisoquinolonyl Acetate/Peptides from N-Arylamides and Ethyne at Room Temperature.

Benzoisoquinolones are aryl ring extended isoquinolinone derivatives, which are constituents of alkaloid natural products. This report describes the synthesis of novel benzoisoquinolone amino acid/peptide derivatives from the respective N-aryl amino esters/peptides through Ru-catalyzed C(sp2)-H annulation at room temperature. The N-terminal amide acts as an intrinsic directing group and coordinates with the active Ru(II) catalyst for the C-H bond activation and annulation of the aryl ring to produce benzoisoqunolone derivatives. Importantly, these benzoisoquinolinones exhibit fluorescence (QY ∼35%) in protic polar solvents, possibly due to charge transfer, and exhibit cell internalization to the cell nucleus without any significant cytotoxicity to human cell lines (HEK293T). Hence, our results are exceptional to transform standard amino acids/peptides into fluorescent peptides at room temperature in the late stage, which could be applicable for tracking specific target peptides by fluorescence microscopy.

Neurogranin modulates the rate of association between calmodulin and target peptides

The best-known mode of action of calmodulin (CaM) is binding of Ca2+ to its N- and C-domains, followed by binding to target proteins. An underappreciated facet of this process is that CaM is typically bound to proteins at basal levels of free Ca2+, including the small, intrinsically disordered, neuronal IQ-motif proteins called PEP-19 and neurogranin (Ng). PEP-19 and Ng would not be effective competitive inhibitors of high-affinity Ca2+-dependent CaM targets at equilibrium because they bind to CaM with relatively low affinity, but they could influence the time course of CaM signaling by affecting the rate of association of CaM with high-affinity Ca2+-dependent targets. This mode of regulation may be domain specific because PEP-19 binds to the C-domain of CaM, whereas Ng binds to both N- and C-domains. In this report, we used a model CaM binding peptide (CKIIp) to characterize the preferred pathway of complex formation with Ca2+-CaM at low levels of free Ca2+ (0.25–1.5 μM), and how PEP-19 and Ng affect this process. We show that the dominant encounter complex involves association of CKIIp with the N-domain of CaM, even though the C-domain has a greater affinity for Ca2+. We also show that Ng greatly decreases the rate of association of Ca2+-CaM with CKIIp due to the relatively slow dissociation of Ng from CaM, and to interactions between the Gly-rich C-terminal region of Ng with the N-domain of CaM, which inhibits formation of the preferred encounter complex with CKIIp. These results provide the general mechanistic paradigms that binding CaM to targets can be driven by its N-domain, and that low-affinity regulators of CaM signaling have the potential to influence the rate of activation of high-affinity CaM targets and potentially affect the distribution of limited CaM among multiple targets during Ca2+ oscillations.

Enhanced EphB2-Specific Peptide Inhibitors through Stabilization of Polyproline II Helical Structure.

Ephrin (Eph) receptors are the largest family of receptor tyrosine kinases. Interactions between Eph receptors and their membrane-bound ephrin protein ligands are associated with many developmental processes as well as various cancers and neurodegenerative diseases. With significant crosstalk between different Eph receptors and ephrin ligands, there is an urgent need for high-affinity ligands that bind specifically to individual Eph receptors to interrogate and modulate their functions. Here, we describe the rational development of potent EphB2 receptor inhibitors derived from the EphB2 receptor-specific SNEW peptide. To improve inhibitory potency, we evaluated 20+ cross-linkers with the goal of spanning and stabilizing a single polyproline II helical turn observed when SNEW binds to the EphB2 receptor. Of the cross-linkers evaluated, an 11-atom cross-linker, composed of a rigid 2,7-dimethylnaphthyl moiety between two cysteine residues, was found to yield the most potent inhibitor. Analysis of the cyclized region of this peptide by NMR and molecular dynamics simulations suggests that cross-linking stabilizes the receptor-bound polyproline II helix structure observed in the receptor-peptide complex. Cross-linked SNEW variants retained binding specificity for EphB2 and showed cross-linker-dependent resistance to trypsin proteolysis. Beyond the discovery of more potent EphB2 receptor inhibitors, these studies illustrate a novel cyclization approach with potential to stabilize polyproline II helical structure in various peptides for specific targeting of the myriad protein-protein interactions (PPIs) mediated by polyproline II helices.

Reactive oxygen species-responsive nano-platform with dual-targeting and fluorescent lipid-specific imaging capabilities for the management of atherosclerotic plaques

Atherosclerosis (AS), a pathological cause of cardiovascular disease, results from endothelial injury, local progressive inflammation, and excessive lipid accumulation. AS plaques rich in foam cells are prone to rupture and form thrombus, which can cause life-threatening complications. Therefore, the assessment of atherosclerotic plaque vulnerability and early intervention are crucial in reducing the mortality rates associated with cardiovascular disease. In this work, A fluorescent probe FC-TPA was synthesized, which switches the fluorescence state between protonated and non-protonated, reducing background fluorescence and enhancing imaging signal-to-noise ratio. On this basis, FC-TPA is loaded into cyclodextrin (CD) modified with phosphatidylserine targeting peptide (PTP) and coated with hyaluronic acid (HA) to construct the intelligent responsive diagnostic nanoplatform (HA@PCFT). HA@PCFT effectively targets atherosclerotic plaques, utilizing dual targeting mechanisms. HA binds strongly to CD44, while PTP binds to phosphatidylserine, enabling nanoparticle aggregation at the lesion site. ROS acts as a smart release switch for probes. Both in vitro and in vivo evaluations confirm impressive lipid-specific fluorescence imaging capabilities of HA@PCFT nanoparticles (NPs). The detection of lipid load in atherosclerotic plaque by fluorescence imaging will aid in assessing the vulnerability of atherosclerotic plaque. Statement of significanceCurrently, numerous fluorescent probes have been developed for lipid imaging. However, some challenges including inadequate water solubility, nonspecific distribution patterns, and fluorescence background interference, have greatly limited their further applications in vivo. To overcome these limitations, a fluorescent molecule has been designed and synthesized, thoroughly investigating its photophysical properties through both theoretical and experimental approaches. Interestingly, this fluorescent molecule exhibits the reversible fluorescence switching capabilities, mediated by hydrogen bonds, which effectively mitigate background fluorescence interference. Additionally, the fluorescent molecules has been successfully loaded into nanocarriers functionalized with the active targeting abilities, which has significantly improved the solubility of the fluorescent molecules and reduced their nonspecific distribution in vivo for an efficient target imaging in atherosclerosis. This study provides a valuable reference for evaluating the performance of such fluorescent dyes, and offers a promising perspective on the design of the target delivery systems for atherosclerosis.

Efficiency of succinylated gelatin and amino acid infusions for kidney uptake reduction of radiolabeled αvβ6-integrin targeting peptides: considerations on clinical safety profiles

Purpose68Ga-Trivehexin is an investigational PET radiopharmaceutical (NCT05799274) targeting αvβ6-integrin for PET imaging of carcinomas. 177Lu-D0301 is a structurally related therapeutic peptide tetramer. However, it showed considerable kidney uptake in rodents, impeding clinical applicability. We therefore evaluated the impact of different kidney protection strategies on the biodistribution of both agents in normal and tumor-bearing mice.MethodsEx-vivo biodistribution of 68Ga-Trivehexin (90 min p.i.) and 177Lu-D0301 (90 min and 24 h p.i.) was determined in healthy C57BL/6N and H2009 (human lung adenocarcinoma) xenografted CB17-SCID mice without and with co-infusion of 100 µL of solutions containing 2.5% arginine + 2.5% lysine (Arg/Lys), 4% succinylated gelatin (gelofusine, gelo), or combinations thereof. Arg/Lys was injected either i.p. 30 min before and after the radiopharmaceutical, or i.v. 2 min before the radiopharmaceutical. Gelo was administered either i.v. 2 min prior activity, or pre-mixed and injected together with the radiopharmaceutical (n = 5 per group). C57BL/6N mice were furthermore imaged by PET (90 min p.i.) and SPECT (24 h p.i.).ResultsKidney uptake of 68Ga-Trivehexin in C57BL/6N mice was reduced by 15% (Arg/Lys i.p.), 25% (Arg/Lys i.v.), and 70% (gelo i.v.), 90 min p.i., relative to control. 177Lu-D0301 kidney uptake was reduced by 2% (Arg/Lys i.p.), 41% (Arg/Lys i.v.), 61% (gelo i.v.) and 66% (gelo + Arg/Lys i.v.) 24 h p.i., compared to control. Combination of Arg/Lys and gelo provided no substantial benefit. Gelo furthermore reduced kidney uptake of 177Lu-D0301 by 76% (90 min p.i.) and 85% (24 h p.i.) in H2009 bearing SCID mice. Since tumor uptake was not (90 min p.i.) or only slightly reduced (15%, 24 h p.i.), the tumor/kidney ratio was improved by factors of 3.3 (90 min p.i.) and 2.6 (24 h p.i.). Reduction of kidney uptake was demonstrated by SPECT, which also showed that the remaining activity was located in the cortex.ConclusionsThe kidney uptake of both investigated radiopharmaceuticals was more efficiently reduced by gelofusine (61–85%) than Arg/Lys (25–41%). Gelofusine appears particularly suitable for reducing renal uptake of αvβ6-integrin targeted 177Lu-labeled peptide multimers because its application led to approximately three times higher tumor-to-kidney ratios. Since the incidence of severe adverse events (anaphylaxis) with succinylated gelatin products (reportedly 0.0062–0.038%) is comparable to that of gadolinium-based MRI or iodinated CT contrast agents (0.008% and 0.04%, respectively), clinical use of gelofusine during radioligand therapy appears feasible if similar risk management strategies as for contrast agents are applied.

Zwitterionic targeting nanodrugs prepared from poly(propyleneimine) dendrimers for enhanced anti-tumor therapy

Development of targeting and high drug loading drug delivery system has been gaining great interest. Zwitterionic modification of the terminal amine groups on G2 poly(propyleneimine) (G2 PPI) dendrimers was achieved via the thiol-ene click reaction, followed by the introduction of the anti-cancer drug paclitaxel and targeting peptides c(RGDfC), then the micelles were formed through self-assembly behavior, subsequently doxorubicin was encapsulated internally, finally the targeting drug-loaded micelles PPIMPRC-DOX were synthesized. The PPIMPRC-DOX micelles were irregular spherical with good monodispersity. The average particle size from TEM image was 63.05 nm and the hydrodynamic size was 93.1 nm, both of which were relatively uniform. The critical micelle concentration was 48.52 μg/mL, the total drug loading efficiency was 32.45 %. In vitro release experiments revealed that within the mildly acidic environment of tumors, PPIMPRC-DOX micelles exhibited enhanced release efficiency and increased accumulation of DOX. The anti-nonspecific protein adsorption experiment proved that the interaction between the PPIMPRC-DOX micelles and fibrinogen was very weak, and they had good anti-nonspecific protein adsorption ability. The cytotoxicity assessments using the MTT assay on HeLa cells and A549 cells revealed obvious cytotoxic effects of PPIMPRC-DOX micelles on both cells. Hence, the utilization of zwitterionic G2 PPI dendrimers in constructing PPIMPRC-DOX micelles holds promise as a prospective nanodrug delivery system.

Purification of angiotensin converting enzyme inhibitory peptides and antihypertensive effect generated from Indonesian traditional fermented beef (Cangkuk).

Traditional fermented meat products can be considered a source of bioactive peptides. Cangkuk, a traditional Indonesian fermented beef product is one source of angiotensin converting enzyme (ACE) inhibitory peptides. This study aimed to identify ACE-inhibitory peptides from Cangkuk and analyze their antihypertensive effects. The water-soluble fraction of Cangkuk was fractionated to obtain ACE-inhibitory peptides using an ethanol solvent at several concentrations and solid-phase extraction with an OASIS HLB cartridge followed by purification with reversed-phase high-performance liquid chromatography (RP-HPLC). HPLC-MS was used to identify target peptides, followed by automatic protein sequencer analysis to detect peptide sequences. Antihypertensive effects were analyzed on the water-soluble fraction and synthesized peptides. The animal model comprised 14-16-week-old male spontaneously hypertensive rats (SHRs) (~320 g average body weight) with mean systolic blood pressures (SBPs) higher than 190 mmHg. All oral doses of peptides were 1 mL in volume. Distilled water was used as a control. The antihypertensive activities of the sample and control were observed by measuring the SBP at 0, 2, 4, 6, 8 and 24 h after oral administration. Two sequences of ACE inhibitory peptides were found, EAPLNPKANR (IC50 value of 44.6 μmol/L) and IVG (IC50 value of 97.3 μmol/L). The water-soluble fraction demonstrated an antihypertensive effect on SHRs after oral administration at 100 mg/kg body weight, maximally lowering the SBP by 14.9 mmHg 8 h after administration. The tripeptide IVG showed the highest reduction of SBP, 24.76±2.1 mmHg 8 h after administration. The decapeptide EAPLNPKANR showed the highest reduction of SBP, 21.0±1.9 mmHg, 8 h after administration. All the samples differed significantly from the control (p<0.01). Cangkuk has potential as a functional food ingredient acting as an antihypertensive agent.

Tumor Microenvironment-Responsive Nanocapsule Delivery CRISPR/Cas9 to Reprogram the Immunosuppressive Microenvironment in Hepatoma Carcinoma.

Cancer immunotherapy has demonstrated significant efficacy in various tumors, but its effectiveness in treating Hepatocellular Carcinoma (HCC) remains limited. Therefore, there is an urgent need to identify a new immunotherapy target and develop corresponding intervention strategies. Bioinformatics analysis has revealed that growth differentiation factor 15 (GDF15) is highly expressed in HCC and is closely related to poor prognosis of HCC patients. The previous study revealed that GDF15 can promote immunosuppression in the tumor microenvironment. Therefore, knocking out GDF15 through gene editing could potentially reverse the suppressive tumor immune microenvironment permanently. To deliver the CRISPR/Cas9 system specifically to HCC, nanocapsules (SNC) coated with HCC targeting peptides (SP94) on their surface is utilized. These nanocapsules incorporate disulfide bonds (SNCSS) that release their contents in the tumor microenvironment characterized by high levels of glutathione (GSH). In vivo, the SNCSS target HCC cells, exert a marked inhibitory effect on HCC progression, and promote HCC immunotherapy. Mechanistically, CyTOF analysis showed favorable changes in the immune microenvironment of HCC, immunocytes with killer function increased and immunocytes with inhibitive function decreased. These findings highlight the potential of the CRISPR-Cas9 gene editing system in modulating the immune microenvironment and improving the effectiveness of existing immunotherapy approaches for HCC.