Solid-Phase Synthesis of Caged Luminescent Peptides via Side Chain Anchoring.

The synthesis of caged luminescent peptide substrates remains challenging, especially when libraries of the substrates are required. Most currently available synthetic methods rely on a solution-phase approach, which is less suited for parallel synthesis purposes. We herein present a solid-phase peptide synthesis (SPPS) method for the synthesis of caged aminoluciferin peptides via side chain anchoring of the P1 residue. After the synthesis of a preliminary test library consisting of 40 compounds, the synthetic method was validated and optimized for up to >100 g of resin. Subsequently, two separate larger peptide libraries were synthesized either having a P1 = lysine or arginine residue containing in total 719 novel peptide substrates. The use of a more stable caged nitrile precursor instead of caged aminoluciferin rendered our parallel synthetic approach completely suitable for SPPS and serine protease profiling was demonstrated using late-stage aminoluciferin generation.

Imaging and photodynamic therapy of prostate cancer using a theranostic PSMA-targeting ligand.

PURPOSE: Incomplete resection of prostate cancer (PCa) results in increased risk of disease recurrence. Combined fluorescence-guided surgery with tumor-targeted photodynamic therapy (tPDT) may help to achieve complete tumor eradication. We developed a prostate-specific membrane antigen (PSMA) ligand consisting of a DOTA chelator for 111In labeling and a fluorophore/photosensitizer IRDye700DX (PSMA-N064). We evaluated the efficacy of PSMA-tPDT using PSMA-N064 in cell viability assays, a mouse xenograft model and in an ex vivo incubation study on fresh human PCa tissue. METHODS: In vitro, therapeutic efficacy of PSMA-N064 was evaluated using PSMA-positive LS174T cells and LS174T wild-type cells. In vivo, PSMA-N064-mediated tPDT was tested in immunodeficient BALB/c mice-bearing PSMA-positive LS174T xenografts. Tumor growth and survival were compared to control mice that received either NIR light or ligand injection only. Ex vivo tPDT efficacy was evaluated in excised fresh human PCa tissue incubated with PSMA-N064. RESULTS: In vitro, tPDT led to a PSMA-specific light- and ligand dose-dependent loss in cell viability. In vivo, tPDT-induced tumor cell apoptosis, delayed tumor growth, and significantly improved survival (p = 0.004) of the treated PSMA-positive tumor-bearing mice compared with the controls. In fresh ex vivo human PCa tissue, apoptosis was significantly increased in PSMA-tPDT-treated samples compared to non-treated control samples (p = 0.037). CONCLUSION: This study showed the feasibility of PSMA-N064-mediated tPDT in cell assays, a xenograft model and excised fresh human PCa tissue. This paves the way to investigate the impact of in vivo PSMA-tPDT on surgical outcome in PCa patients.

Activity Sensing of Coagulation and Fibrinolytic Proteases.

The blood coagulation cascade is a complex physiological process involving the action of multiple coupled enzymes, cofactors, and substrates, ultimately leading to clot formation. Serine proteases have a crucial role, and aberrations in their activity can lead to life-threatening bleeding disorders and thrombosis. This review summarizes the essential proteases involved in blood coagulation and fibrinolysis, the endogenous peptide sequences they recognize and hydrolyze, and synthetic peptide probes based on these sequences to measure their activity. The information in this review can contribute to developing novel anticoagulant therapies and specific substrates for point-of-care diagnosis of coagulation pathologies.

Readily Accessible Strained Difunctionalized trans-Cyclooctenes with Fast Click and Release Capabilities.

The click reaction between a functionalized trans-cyclooctene (TCO) and a tetrazine (Tz) is a compelling method for bioorthogonal conjugation in combination with payload releasing capabilities. However, the synthesis of difunctionalized TCOs remains challenging. As a result, these compounds are poorly accessible, which impedes the development of novel applications. In this work, the scalable and accessible synthesis of a new bioorthogonal difunctionalized TCO is reported in only four single selective high yielding steps starting from commercially available compounds. The TCO-Tz click reaction was assessed and revealed excellent kinetic rates and subsequently payload release was shown with various functionalized derivatives. Tetrazine triggered release of carbonate and carbamate payloads was demonstrated up to 100 % release efficiency and local drug release was shown in a cellular toxicity study which revealed a >20-fold increase in cytotoxicity.

Luminescent Assay for the Screening of SARS-CoV-2 MPro Inhibitors.

Since the outbreak of SARS-CoV-2 in December 2019 millions of infections have been reported globally. The viral chymotrypsin-like main protease (MPro ) exhibits a crucial role in viral replication and represents a relevant target for antiviral drug development. In order to screen potential MPro inhibitors we developed a luminescent assay using a peptide based probe containing a cleavage site specific for MPro . This assay was validated showing IC50 values similar to those reported in the literature for known MPro inhibitors and can be used to screen new inhibitors.

Theranostic PSMA ligands with optimized backbones for intraoperative multimodal imaging and photodynamic therapy of prostate cancer.

INTRODUCTION: The first generation ligands for prostate-specific membrane antigen (PSMA)-targeted radio- and fluorescence-guided surgery followed by adjuvant photodynamic therapy (PDT) have already shown the potential of this approach. Here, we developed three new photosensitizer-based dual-labeled PSMA ligands by crucial modification of existing PSMA ligand backbone structures (PSMA-1007/PSMA-617) for multimodal imaging and targeted PDT of PCa. METHODS: Various new PSMA ligands were synthesized using solid-phase chemistry and provided with a DOTA chelator for 111In labeling and the fluorophore/photosensitizer IRDye700DX. The performance of three new dual-labeled ligands was compared with a previously published first-generation ligand (PSMA-N064) and a control ligand with an incomplete PSMA-binding motif. PSMA specificity, affinity, and PDT efficacy of these ligands were determined in LS174T-PSMA cells and control LS174T wildtype cells. Tumor targeting properties were evaluated in BALB/c nude mice with subcutaneous LS174T-PSMA and LS174T wildtype tumors using µSPECT/CT imaging, fluorescence imaging, and biodistribution studies after dissection. RESULTS: In order to synthesize the new dual-labeled ligands, we modified the PSMA peptide linker by substitution of a glutamic acid into a lysine residue, providing a handle for conjugation of multiple functional moieties. Ligand optimization showed that the new backbone structure leads to high-affinity PSMA ligands (all IC50 < 50 nM). Moreover, ligand-mediated PDT led to a PSMA-specific decrease in cell viability in vitro (P < 0.001). Linker modification significantly improved tumor targeting compared to the previously developed PSMA-N064 ligand (≥ 20 ± 3%ID/g vs 14 ± 2%ID/g, P < 0.01) and enabled specific visualization of PMSA-positive tumors using both radionuclide and fluorescence imaging in mice. CONCLUSION: The new high-affinity dual-labeled PSMA-targeting ligands with optimized backbone compositions showed increased tumor targeting and enabled multimodal image-guided PCa surgery combined with targeted photodynamic therapy.

Strain-Promoted Azide-Alkyne Cycloaddition-Based PSMA-Targeting Ligands for Multimodal Intraoperative Tumor Detection of Prostate Cancer.

Strain-promoted azide-alkyne cycloaddition (SPAAC) is a straightforward and multipurpose conjugation strategy. The use of SPAAC to link different functional elements to prostate-specific membrane antigen (PSMA) ligands would facilitate the development of a modular platform for PSMA-targeted imaging and therapy of prostate cancer (PCa). As a first proof of concept for the SPAAC chemistry platform, we synthesized and characterized four dual-labeled PSMA ligands for intraoperative radiodetection and fluorescence imaging of PCa. Ligands were synthesized using solid-phase chemistry and contained a chelator for 111In or 99mTc labeling. The fluorophore IRDye800CW was conjugated using SPAAC chemistry or conventional N-hydroxysuccinimide (NHS)-ester coupling. Log D values were measured and PSMA specificity of these ligands was determined in LS174T-PSMA cells. Tumor targeting was evaluated in BALB/c nude mice with subcutaneous LS174T-PSMA and LS174T wild-type tumors using µSPECT/CT imaging, fluorescence imaging, and biodistribution studies. SPAAC chemistry increased the lipophilicity of the ligands (log D range: -2.4 to -4.4). In vivo, SPAAC chemistry ligands showed high and specific accumulation in s.c. LS174T-PSMA tumors up to 24 h after injection, enabling clear visualization using µSPECT/CT and fluorescence imaging. Overall, no significant differences between the SPAAC chemistry ligands and their NHS-based counterparts were found (2 h p.i., p > 0.05), while 111In-labeled ligands outperformed the 99mTc ligands. Here, we demonstrate that our newly developed SPAAC-based PSMA ligands show high PSMA-specific tumor targeting. The use of click chemistry in PSMA ligand development opens up the opportunity for fast, efficient, and versatile conjugations of multiple imaging moieties and/or drugs.

Delivery of Various Cargos into Cancer Cells and Tissues via Cell-Penetrating Peptides: A Review of the Last Decade.

Cell-penetrating peptides (CPPs), also known as protein transduction domains, are a class of diverse amino acid sequences with the ability to cross cellular membranes. CPPs can deliver several bioactive cargos, including proteins, peptides, nucleic acids and chemotherapeutics, into cells. Ever since their discovery, synthetic and natural CPPs have been utilized in therapeutics delivery, gene editing and cell imaging in fundamental research and clinical experiments. Over the years, CPPs have gained significant attention due to their low cytotoxicity and high transduction efficacy. In the last decade, multiple investigations demonstrated the potential of CPPs as carriers for the delivery of therapeutics to treat various types of cancer. Besides their remarkable efficacy owing to fast and efficient delivery, a crucial benefit of CPP-based cancer treatments is delivering anticancer agents selectively, rather than mediating toxicities toward normal tissues. To obtain a higher therapeutic index and to improve cell and tissue selectivity, CPP-cargo constructions can also be complexed with other agents such as nanocarriers and liposomes to obtain encouraging outcomes. This review summarizes various types of CPPs conjugated to anticancer cargos. Furthermore, we present a brief history of CPP utilization as delivery systems for anticancer agents in the last decade and evaluate several reports on the applications of CPPs in basic research and preclinical studies.

Photosensitizer-based multimodal PSMA-targeting ligands for intraoperative detection of prostate cancer.

Incomplete resection of prostate cancer (PCa) occurs in 15%-50% of PCa patients. Disease recurrence negatively impacts oncological outcome. The use of radio-, fluorescent-, or photosensitizer-labeled ligands to target the prostate-specific membrane antigen (PSMA) has become a well-established method for the detection and treatment of PCa. Methods: Here, we developed and characterized multimodal [111In]In-DOTA(GA)-IRDye700DX-PSMA ligands, varying in their molecular composition, for use in intraoperative radiodetection, fluorescence imaging and targeted photodynamic therapy of PCa lesions. PSMA-specificity of these ligands was determined in xenograft tumor models and on fresh human PCa biopsies. Results: Ligand structure optimization showed that addition of the photosensitizer (IRDye700DX) and additional negative charges significantly increased ligand uptake in PSMA-expressing tumors. Moreover, an ex vivo incubation study on human tumor biopsies confirmed the PSMA-specificity of these ligands on human samples, bridging the gap to the clinical situation. Conclusion: We developed a novel PSMA-targeting ligand, optimized for multimodal image-guided PCa surgery combined with targeted photodynamic therapy.

Activation of cell-penetrating peptide fragments by disulfide formation.

Three cell-penetrating peptides (CPPs), Tat, Pep-3 and penetratin, were split into two parts and each fragment was terminated with a cysteine residue, to allow disulfide bridge formation, as well as a fluorescent label, for visualization and quantitative analysis. After disulfide formation between two complementary CPP fragments, cellular uptake of the resulting conjugates was observed. As confirmed by in vitro experiments, the conjugated peptides showed uptake activity comparable to the native CPP sequences, while the truncated peptides were hardly active. Until now, this split CPP strategy has only been demonstrated for oligo-arginine CPPs, but here we demonstrate that it is also applicable to other cell-penetrating peptides. This wider applicability may help in the design of new activatable cell-penetrating peptides for, e.g., targeted drug delivery.

Self-recovering dual cross-linked hydrogels based on bioorthogonal click chemistry and ionic interactions.

The biocompatible, injectable and high water-swollen nature of hydrogels makes them a popular candidate to imitate the extracellular matrix (ECM) for tissue engineering both in vitro and in vivo. However, commonly used covalently cross-linked hydrogels, despite their stability and tunability, are elastic and deteriorate as bulk material degrades which would impair proper cell function. To improve these deficiencies, here, we present a self-recovering cross-linked hydrogel formed instantaneously with functionalized poly(ethylene glycol) as a basis. We combine covalent cross-links introduced via a strain-promoted azide-alkyne cycloaddition (SPAAC) click reaction and non-covalent links between phosphonate groups and calcium ions. By adjusting the ratios of non-covalent and covalent cross-links, we synthesized these dual cross-linked (DC) hydrogels that displayed storage moduli below ∼2000 Pa and relaxation times from seconds to minutes. The gels recovered to 41-96% of their initial mechanical properties after two subsequent strain failures. Cryo-scanning electron microscopy revealed that DC hydrogels containing approximately equal amounts of covalent and non-covalent cross-links displayed phase separation. Finally, we functionalized the DC hydrogels by incorporating an integrin binding motif, RGDS, to provide a biocompatible environment for human mesenchymal stem cells (HMSCs) by facilitating adhesion inside the gel network. Inside these DC gels HSMCs displayed a viability up to 73% after five days of cell culture.

Activatable cell-penetrating peptides: 15 years of research.

An important hurdle for the intracellular delivery of large cargo is the cellular membrane, which protects the cell from exogenous substances. Cell-penetrating peptides (CPPs) can cross this barrier but their use as drug delivery vehicles is hampered by their lack of cell type specificity. Over the past years, several approaches have been explored to control the activity of CPPs that can be primed for cellular uptake. Since the first report on such activatable CPPs (ACPPs) in 2004, various methods of activation have been developed. Here, we provide an overview of the different ACPPs strategies known to date and summarize the benefits, drawbacks, and future directions.

PSMA-targeting agents for radio- and fluorescence-guided prostate cancer surgery.

Despite recent improvements in imaging and therapy, prostate cancer (PCa) still causes substantial morbidity and mortality. In surgical treatment, incomplete resection of PCa and understaging of possible undetected metastases may lead to disease recurrence and consequently poor patient outcome. To increase the chance of accurate staging and subsequently complete removal of all cancerous tissue, prostate specific membrane antigen (PSMA) targeting agents may provide the surgeon an aid for the intraoperative detection and resection of PCa lesions. Two modalities suitable for this purpose are radionuclide detection, which allows sensitive intraoperative localization of tumor lesions with a gamma probe, and fluorescence imaging, allowing tumor visualization and delineation. Next to fluorescence, use of photosensitizers may enable intraoperative targeted photodynamic therapy to eradicate remaining tumor lesions. Since radiodetection and optical imaging techniques each have their own strengths and weaknesses, a combination of both modalities could be of additional value. Here, we provide an overview of recent preclinical and clinical advances in PSMA-targeted radio- and fluorescence-guided surgery of PCa.

Click to enter: activation of oligo-arginine cell-penetrating peptides by bioorthogonal tetrazine ligations.

Cell-penetrating peptides are able to transport a wide variety of cargo across cell membranes. Although promising, they are not often considered for therapeutic purposes as they lack controllable activity and cell selectivity. We have developed an activation strategy based on a split octa-arginine cell-penetrating peptide (CPP) that can be activated by means of bioorthogonal ligation. To this end we prepared two non-penetrating tetra-arginine halves, functionalized either with a tetrazine or with a complementary bicyclo[6.1.0]nonyne (BCN) group. We demonstrate that an active octa-arginine can be reconstituted in situ upon mixing the complementary split peptides. The resulting activated peptide is taken up as efficiently as the well-established cell-penetrating peptide octa-arginine. The activation of the oligo-arginines can also be achieved using trans-cyclooctene (TCO) as a ligation partner, while norbornene appears too kinetically slow for use in situ. We further show that this strategy can be applied successfully to transport a large protein into living cells. Our results validate a promising first step in achieving control over cell penetration and to use CPPs for therapeutic approaches.

Comparison of Bioorthogonally Cross-Linked Hydrogels for in Situ Cell Encapsulation.

Hydrogels are water-saturated polymer networks and extensively used in drug delivery, tissue repair engineering, and cell cultures. For encapsulation of drugs or cells, the possibility to form hydrogels in situ is very much desired. This can be achieved in numerous ways, including use of bioorthogonal chemistry to create polymer networks. Here we report a set of bioorthogonally clickable polymers that was designed with the aim to find a combination that could rapidly encapsulate cells in a three-dimensional manner to improve the preparation of hydrogels as tissue mimics. To this end, tetrazine (Tet), trans-cyclooctene (TCO), azide (N3), dibenzocyclooctyne (DBCO), bicyclo[6.1.0]nonyne (BCN), 3,4-dihydroxyphenylacetic acid (DHPA), and norbornene (Norb) were grafted to four-armed poly(ethylene)glycol (star-PEG) polymers of 10 kDa. Inverted vial tests and rheology demonstrated that hydrogels formed within seconds from combinations of TCO-Tet, BCN-DHPA, and BCN-Tet. Hydrogels from DBCO-N3, DBCO-DHPA, and BCN-N3 formed in the range of minutes, whereas the Norb-Tet ligation required multiple hours to form a gel. After this comparison, we chose to prepare hydrogels via DBCO-N3 and BCN-N3 and employed them for human mesenchymal stem cell (HMSC) cultures for a period of 5 days. We additionally incorporated RGDS and MMP cleavable peptide (MMPcp) motifs in these gels to stimulate cell adhesion and add degradability. Both DBCO and BCN gel systems including the functional peptide motifs allowed HMSCs to be viable and spread in 5 days. The DBCO-based hydrogel could trap cells at different depths due to its fast gelation process, while the slower gelation of the BCN-based hydrogel led to cell sedimentation.

Incorporation of simvastatin in PLLA membranes for guided bone regeneration: effect of thermal treatment on simvastatin release.

Electrospun membranes based on biodegradable polymers are promising materials to be used for guided bone regeneration (GBR) therapy. The incorporation of osteostimulatory compounds can improve the biofunctionality of those membranes, making them active players in bone regeneration. Simvastatin has been shown to promote osteogenic differentiation both in vitro and in vivo. However, in most of these systems, the drug was quickly released, not matching the pace of bone regeneration. The aim of this study was to develop poly(l-lactic acid) (PLLA) membranes containing simvastatin (SV) that have a prolonged drug release rate, compatible with GBR applications. To this end, SV was mixed with PLLA and electrospun. The membranes were subjected to a thermal treatment in order to increase the crystallinity of PLLA. Morphological, structural and chemical properties of the electrospun membranes were characterized. The effect of the thermal treatment on the release profile of SV was evaluated by near physiological release experiments at 37 °C. The osteostimulatory potential was determined by in vitro culture of the membranes with rat bone marrow stromal cells (rBMSCs). The results confirmed that the thermal treatment led to an increase in polymer crystallinity and a more sustained release of SV. In vitro assays demonstrate cellular proliferation over time for all the membranes and a significant increase in osteogenic differentiation for the membranes containing SV subjected to thermal treatment.

Dodging Endosomes: Effective Cytosolic Antibody Delivery.

On the inside: New methodologies for delivering antibodies right into the cytosol of cells either directly across the plasma membrane or by allowing the antibody to escape from endosomes have been proposed recently by the Cardoso/Hackenberger and Futaki groups, respectively.

Nanostructured raspberry-like gelatin microspheres for local delivery of multiple biomolecules.

Multicompartment particles, which are particles composed of smaller building units, have gained considerable interest during the past decade to facilitate simultaneous and differential delivery of several biomolecules in various applications. Supercritical carbon dioxide (CO2) processing is an industrial technology widely used for large-scale synthesis and processing of materials. However, the application of this technology for production of multicompartment particles from colloidal particles has not yet been explored. Here, we report the formation of raspberry-like gelatin (RLG) microparticles composed of gelatin nanoparticles as colloidal building blocks through supercritical CO2 processing. We show that these RLG microparticles exhibit a high stability upon dispersion in aqueous media without requiring chemical cross-linking. We further demonstrate that these microparticles are cytocompatible and facilitate differential release of two different model compounds. The strategy presented here can be utilized as a cost-effective route for production of various types of multicompartment particles using colloidal particles with suitable interparticle interactions. STATEMENT OF SIGNIFICANCE: Multicompartment particles have gained considerable interest during the past decade to facilitate simultaneous and differential delivery of multiple biomolecules in various biomedical applications. Nevertheless, common methods employed for the production of such particles are often complex and only offer small-scale production. Here, we report the formation of raspberry-like gelatin (RLG) microparticles composed of gelatin nanoparticles as colloidal building blocks through supercritical CO2 processing. We show that these microparticles are cytocompatible and facilitate differential release of two model compounds with different molecular sizes, promising successful applications in various biomedical areas. Summarizing, this paper presents a novel strategy that can be utilized as a cost-effective route for production of various types of multicompartment particles using a wide range of colloidal building blocks.

A peptide functionalized nanomotor as an efficient cell penetrating tool.

A nanomotor based strategy for fast cellular entry and cargo delivery is presented. The concept focuses on integrating tat peptide, a basic domain of HIV-1 tat protein, with state of the art nanomotors which possess attractive autonomous properties, facilitating cellular penetration and uptake. The rapid cellular internalization process leads to higher delivery efficiency.

Coiled-Coil-Mediated Activation of Oligoarginine Cell-Penetrating Peptides.

A supramolecular approach was undertaken to create functionally activatable cell-penetrating peptides. Two tetra-arginines were assembled into an active cell-penetrating peptide by heterodimerizing leucine zippers. Three different leucine-zipper pairs were evaluated: activation was found to depend on the association constant of the coiled-coil peptides. The weaker-binding peptides required an additional disulfide linkage to induce cell-penetrating capability, whereas for the most-stable coiled-coil no additional stabilization was needed. The latter zipper pair was used to show that the induced formation of the coiled coils allows control over the uptake of an oligoarginine CPP-conjugated cargo protein.