Dibenzocyclooctyne Group Research Articles

AIEgens Cross‐linked Iron Oxide Nanoparticles Synchronously Amplify Bimodal Imaging Signals in Situ by Tumor Acidity‐Mediated Click Reaction

AbstractActivatable dual‐modal molecular imaging probes present a promising tool for the diagnosis of malignant tumors. However, synchronously enhancing dual‐modal imaging signals under a single stimulus is challenging. Herein, we propose an activatable bimodal probe that integrates aggregation‐induced emission luminogens (AIEgens) and iron oxide nanoparticles (IOs) to synergistically enhance near‐infrared fluorescence (NIRF) intensity and magnetic resonance (MR) contrast through a tumor acidity‐mediated click reaction. Tumor acidity‐responsive IOs containing dibenzocyclooctyne groups (termed cDIOs) and AIEgens containing azide groups (termed AATs) can be covalently cross‐linked in response to tumor acidity, which leads to a simultaneous enhancement in NIRF intensity (≈12.4‐fold) and r2 relaxivity (≈2.8‐fold). cDIOs and AATs were effectively activated in mice orthotropic breast tumor, and the cross‐linking prolonged their retention in tumor, further augmenting the bimodal signals and expanding imaging time frame. This facile strategy leverages the inherent properties of probes themselves and demonstrates promise in future translational studies.

AIEgens Cross-linked Iron Oxide Nanoparticles Synchronously Amplify Bimodal Imaging Signals in Situ by Tumor Acidity-Mediated Click Reaction.

Activatable dual-modal molecular imaging probes present a promising tool for the diagnosis of malignant tumors. However, synchronously enhancing dual-modal imaging signals under a single stimulus is challenging. Herein, we propose an activatable bimodal probe that integrates aggregation-induced emission luminogens (AIEgens) and iron oxide nanoparticles (IOs) to synergistically enhance near-infrared fluorescence (NIRF) intensity and magnetic resonance (MR) contrast through a tumor acidity-mediated click reaction. Tumor acidity-responsive IOs containing dibenzocyclooctyne groups (termed cDIOs) and AIEgens containing azide groups (termed AATs) can be covalently cross-linked in response to tumor acidity, which leads to a simultaneous enhancement in NIRF intensity (≈12.4-fold) and r2 relaxivity (≈2.8-fold). cDIOs and AATs were effectively activated in mice orthotropic breast tumor, and the cross-linking prolonged their retention in tumor, further augmenting the bimodal signals and expanding imaging time frame. This facile strategy leverages the inherent properties of probes themselves and demonstrates promise in future translational studies.

Sensitive NIR Fluorescence Identification of Bacteria in Whole Blood with Bioorthogonal Nanoprobes for Early Sepsis Diagnosis.

Sepsis is one of the leading causes of death worldwide. The disease progression of sepsis is very fast, and there is a 7-9% increase in mortality every hour. Therefore, rapid and sensitive detection of pathogenic bacteria is crucial for the timely treatment of sepsis as well as the reduction of mortality. Herein, we present a sensitive near-infrared (NIR) fluorescence identification and a rapid magnetic capture based on bioorthogonal nanoprobes for the detection of multiple bacteria in whole blood. The nanoprobes with NIR fluorescence/magnetic properties were modified with dibenzocyclooctyne groups and used to capture and recognize the bacteria via bioorthogonal reaction. The magnetic nanoprobes showed superparamagnetic properties with a saturation magnetization as high as 63 emu/g. Through clicking with the azide groups inserted on the bacteria walls by metabolic engineering, the bioorthogonal magnetic nanoprobes allow fast and broad-spectrum capture of both Gram-positive and Gram-negative bacteria. The bioorthogonal NIR fluorescent nanoprobes with a maximum emission at 900 nm can effectively avoid background interference, further enabling sensitive identification of the bacteria in whole blood. The detection limit was as low as 4 CFU/mL in less than 2.5 h and the nanoprobes were successfully applied to the detection of bacteria in blood samples from the patients with sepsis, showing the potential application in early sepsis diagnosis and clinical studies.

Surface bioengineering of diverse orthopaedic implants with optional functions via bioinspired molecular adhesion and bioorthogonal conjugations

In this work, we reported an upgraded mussel-inspired strategy for surface bioengineering of osteoimplants by combination of mussel adhesion and bioorthogonal click chemistry. The main idea of this strategy is a mussel-inspired synthetic peptide containing multiple 3,4-dihydroxy-L-phenylalanine (DOPA) units and a dibenzocyclooctyne (DBCO) terminal (DOPA-DBCO). According to the mussel adhesion mechanism, the DOPA-DBCO peptide could stably adhere onto a variety of material surface, leaving the residual DBCO groups on the surface. Then, the DBCO residues could be employed for a second-step bioorthogonal conjugation with azide-capping biomolecules through bioorthogonal click chemistry, finally leading to the biomodified surfaces. To demonstrate the generality of our strategy for surface biomodification of diversified orthopaedic materials including metallic and polymeric substrates, we here conceptually conjugated some typical azide-capping biomolecules on both metal and polymeric surfaces. The results definitely verified the feasibility for engineering of functional surfaces with some essential requirements of osteoimplants, for example, the ability to facilitate cell adhesion, suppress bacterial infection, and promote osteogenesis. In a word, this study indicated that our novel surface strategy would show broad applicability for diverse osteoimplants and in different biological scenarios. We can also image that the molecular specificity of bioorthogonal conjugation and the universality of mussel adhesion mechanism may jointly provide a versatile surface bioengineering method for a wider range of biomedical implants.

Enhancement of Probe Density in DNA Sensing by Tuning the Exponential Growth Regime of Polyelectrolyte Multilayers.

Surface-based biosensing devices benefit from a dedicated design of the probe layer present at the transducing interface. The layer architecture, its physicochemical properties, and the embedding of the receptor sites affect the probability of binding the analyte. Here, the enhancement of the probe density at the sensing interface by tuning the exponential growth regime of polyelectrolyte multilayers (PEMs) is presented. PEMs were made of poly-l-lysine (PLL), with appended clickable dibenzocyclooctyne (DBCO) groups and oligo(ethylene glycol) chains, and poly(styrene sulfonate) (PSS). The DNA probe loading and target hybridization efficiencies of the PEMs were evaluated as a function of the PLL layer number and the growth regime by a quartz crystal microbalance (QCM). An amplification factor of 25 in the target DNA detection was found for a 33-layer exponentially grown PEM compared to a monolayer. A Voigt-based model showed that DNA probe binding to the DBCO groups is more efficient in the open, exponentially grown films, while the hybridization efficiencies appeared to be high for all layer architectures. These results show the potential of such engineered gel-like structures to increase the detection of bio-relevant analytes in biosensing systems.

Proximity-constructed bifunctional DNA probes for identification of stem-like biomarker in breast cancer

CD44, a stem-like biomarker, plays a critical role in maintenance of stem-like phenotype in breast cancer and is associated with migration, invasion and apoptosis resistance of breast tumors. Herein, we designed an effective sensing method using proximity-constructed bifunctional DNA probes for electrochemical identification of CD44. In our design, hybridization of capture probes was stabilized by target CD44 binding-induced proximity, thereby initiating subsequent cyclic strand displacement reactions. As a result, signaling probes were produced and dual-labeled with dibenzocyclooctyne group and CdTe quantum dots (QDs). These signaling probes performed two functions, specifically enrichment on azide-functionalized magnetic beads via click chemistry and generation of amplified electrochemical signaling relied on QDs. With aid of these bifunctional probes, the sensing method allowed not only highly specific identification of target CD44 even in serum samples and breast cancer cells but also monitoring of CD44 expression changes upon siRNA treatment. The linear range for CD44 identification was 0.1–1000 ng/mL and the detection limit was 0.0792 ng/mL, which displayed an improved sensitivity compared to previous reports. Therefore, the method may provide a powerful tool for CD44 identification and have a great potential in revealing stem-like properties in breast cancer in the future.

Cell-free protein synthesis andin situimmobilization of deGFP-MatB in polymer microgels for malonate-to-malonyl CoA conversion

In the present work, microgels were utilized as a cell-free reaction environment to produce a functional malonyl-CoA synthetase (deGFP-MatB) under geometry-controlled transcription and translation. Our approach combines the straight-forward optimization of overall protein yield of an E. coli-based cell-free protein synthesis (CFPS) system based on concentration screening of magnesium and potassium glutamate, DNA as well as polyethylene glycol (PEG), and its innovative usage in microgel-based production of a key enzyme of the polyketide synthesis pathway. After partial modification of the carboxyl groups of hyaluronic acid (HA) with 5′-methylfuran groups via 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM)-activation, these were further functionalized with dibenzocyclooctyne (DBCO) and nitrilotriacetic acid (NTA) groups by bio-orthogonal [4+2] Diels–Alder cycloaddition to yield a bifunctional macromer. After coupling the DBCO groups with azide-functionalized DNA, containing the genetic information for deGFP-MatB, via strain-promoted azide–alkyne cycloaddition (SPAAC), the DNA-/NTA-functionalized HA macromer was utilized as base material together with maleimide-functionalized PEG (PEG-mal2) as the crosslinker to form bifunctional microgels utilizing water-in-oil (W/O) microemulsions. As-formed microgels were incubated with nickel sulfate to activate the NTA groups and provide binding sites for deGFP-MatB, which contained six histidine residues (His-tag) for that purpose. The optimized CFPS mixture was loaded into the microgels to initiate the formation of deGFP-MatB, which was detected by a clear increase in fluorescence exclusively inside the microgel volume. Functionality of both, the bound and the decoupled enzyme was proven by reaction with malonate to yield malonyl CoA, as confirmed by a colorimetric assay.

Strategies for conjugating iridium(III) anticancer complexes to targeting peptides via copper-free click chemistry

We report the synthesis and characterization of novel pentamethylcyclopentadienyl (Cp*) iridium(III) complexes [(Cp*)Ir(4-methyl-4′-carboxy-2,2′-bipyridine)Cl]PF6 (Ir-I), the product (Ir-II) from amide coupling of Ir-I to dibenzocyclooctyne-amine, and its conjugate (Ir-CP) with the cyclic nona-peptide c(CRWYDENAC). The familiar three-legged ‘piano-stool’ configuration for complex Ir-I was confirmed by its single crystal X-ray structure. Significantly, copper-free click strategy has been developed for site-specific conjugation of the parent complex Ir-I to the tumour targeting nona-cyclic peptide. The approach consisted of two steps: (i) the carboxylic acid group of the bipyridine ligand in complex Ir-I was first attached to an amine functionalized dibenzocyclooctyne group via amide formation to generate complex Ir-II; and (ii) the alkyne bond of dibenzocyclooctyne in complex Ir-II underwent a subsequent strain-promoted copper-free cycloaddition with the azide group of the modified peptide. Interestingly, while complex Ir-I was inactive towards A2780 human ovarian cancer cells, complex Ir-II exhibited moderate cytotoxic activity. Targeted complexes such as Ir-CP offer scope for enhanced activity and selectivity of this class of anticancer complexes.

PEGylation and surface functionalization of liposomes containing drug nanocrystals for cell-targeted delivery

Liposomal formulations have important therapeutic applications in anti-cancer treatments but current formulations suffer from serious side effects, high dosage requirements and prolonged treatment. In this study, PEGylated azide-functionalized liposomes containing drug nanocrystals were investigated with the aim of increasing the drug payload and achieving functionalization for targeted delivery. Liposomes were characterized using cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), small and ultra-small angle neutron scattering (SANS/USANS) and small and wide angle X-ray scattering (SAXS/WAXS). Cryo-TEM experiments revealed the dimensions of the nanocrystal-loaded liposomes and the change of shape from spherical to elongated after the formation of nanocrystals. Results from SANS/USANS experiments confirmed the asymmetric particle shape. SAXS/WAXS experiments confirmed that the crystalline drug only occurred in freeze-thawed samples and correlated with a new unidentified polymorphic form of ciprofloxacin. Using a small molecule dye, dibenzocyclooctyne (DBCO)-cy5, specific conjugation between DBCO groups and surface azide groups on the liposomes was confirmed; this indicates the promise of this system for tumour-targeted delivery.

Unique Fluorescent Imaging Probe for Bacterial Surface Localization and Resistant Enzyme Imaging.

Emergence of antibiotic bacterial resistance has caused serious clinical issues worldwide due to increasingly difficult treatment. Development of a specific approach for selective visualization of resistant bacteria will be highly significant for clinical investigations to promote timely diagnosis and treatment of bacterial infections. In this article, we present an effective method that not only is able to selectively recognize drug resistant AmpC β-lactamases enzyme but, more importantly, is able to interact with bacterial cell wall components, resulting in a desired localization effect on the bacterial surface. A unique and specific enzyme-responsive cephalosporin probe (DFD-1) has been developed for the selective recognition of resistance bacteria AmpC β-lactamase, by employing fluorescence resonance energy transfer with an "off-on" bioimaging. To achieve the desired localization, a lipid-azide conjugate (LA-12) was utilized to facilitate its penetration into the bacterial surface, followed by copper-free click chemistry. This enables the probe DFD-1 to be anchored onto the cell surface. In the presence of AmpC enzymes, the cephalosporin β-lactam ring on DFD-1 will be hydrolyzed, leading to the quencher release, thus generating fluorescence for real-time resistant bacterial screening. More importantly, the bulky dibenzocyclooctyne group in DFD-1 allowed selective recognition toward the AmpC bacterial enzyme instead of its counterpart ( e.g., TEM-1 β-lactamase). Both live cell imaging and cell cytometry assays showed the great selectivity of DFD-1 to drug resistant bacterial pathogens containing the AmpC enzyme with significant fluorescence enhancement (∼67-fold). This probe presented promising capability to selectively localize and screen for AmpC resistance bacteria, providing great promise for clinical microbiological applications.

Clickable Cubosomes for Antibody-Free Drug Targeting and Imaging Applications.

The combination of copper-free click chemistry with metabolic labeling offers new opportunities in drug delivery. The objective of this study was to determine whether cubosomes functionalized with azide or dibenzocyclooctyne (DBCO) groups are able to undergo copper-free click chemistry with a strained cyclooctyne or azide, respectively. Phytantriol-based cubosomes were functionalized using phospholipids bearing an azide or DBCO group. The modified cubosome dispersions were characterized using dynamic light scattering, cryo-TEM, and small-angle X-ray scattering. The efficiency of "clickability" was assessed by reacting the cubosomes with a complementary dye and determining bound and unbound dye via size exclusion chromatography. The clickable cubosomes reacted specifically and efficiently with a click-Cy5 dye with minor changes to the size, shape, and structure of the cubosomes. This indicates that cubosomes can retain their unique internal structure while participating in copper-free click chemistry. This proof of concept study paves the way for the use of copper-free click chemistry and metabolic labeling with cubosomes for targeted drug delivery and imaging.

Synthesis and Application of Injectable Bioorthogonal Dendrimer Hydrogels for Local Drug Delivery.

We developed novel dendrimer hydrogels (DH)s on the basis of bioorthogonal chemistry, in which polyamidoamine (PAMAM) dendrimer generation 4.0 (G4) functionalized with strained alkyne dibenzocyclooctyne (DBCO) via PEG spacer (Mn = 2,000 g/mol) underwent strain-promoted azide-alkyne cycloaddition (SPAAC) with polyethylene glycol bisazide (PEG-BA) (Mn= 20,000 g/mol) to generate a dendrimer-PEG cross-linked network. This platform offers a high degree of functionality and modularity. A wide range of structural parameters including dendrimer generation, degree of PEGylation, loading density of clickable DBCO groups, PEG-BA chain length as well as the ratio of clickable dendrimer to PEG-BA and their concentrations can be readily manipulated to tune chemical and physical properties of DHs. We used this platform to prepare an injectable liquid DH. This bioorthogonal DH exhibited high cytocompatibility and enabled sustained release of the anticancer drug 5-fluorouracil (5-FU). Following intratumoral injection, the DH/5-FU formulation significantly suppressed tumor growth and improved survival of HN12 tumor-bearing mice by promoting tumor cell death as well as by reducing tumor cell proliferation and angiogenesis.

Cyclopropenone-masked dibenzocyclooctyne end-functionalized polymers from reversible addition-fragmentation chain transfer polymerization

Functional reversible addition-fragmentation chain transfer polymerization (RAFT) agent was developed to have a cyclopropenone-masked dibenzocyclooctyne end group. RAFT polymerization was then explored on three typical types of vinyl monomers in free radical polymerization such as styrene, methyl methacrylate, and tert-butyl acrylate. The living polymerization behaviors were demonstrated for all three cases, which guaranteed the production of well-defined polystyrene, poly (methyl methacrylate), and poly (tert-butyl acrylate) end-functionalized with cyclopropenone-masked dibenzocyclooctyne groups from the convenient RAFT polymerization technique. Under UV irradiation, the cyclopropenone-masked dibenzocyclooctyne end group could be quantitatively changed to dibenzocyclooctyne clickable group, which was widely used in the metal-free strain-promoted azide-alkyne cycloaddition (SPAAC) click reaction. Subsequently, using the dibenzocyclooctyne end-functionalized polymers as building blocks, a practical and efficient metal-free method was developed for the formation of topological polymers including block and bottlebrush polymers based on the SPAAC click reaction.

Tadpole-shaped polymers based on UV-induced strain promoted azide-alkyne cycloaddition reaction

In the present study, we synthesized well-defined tadpole-shaped polystyrene (PS) via the combination of atom transfer radical polymerization (ATRP) and UV-induced strain promoted azide-alkyne cycloaddtion (SPAAC) reaction. A di-bromo ATRP initiator (Br-ini-Br) containing cyclopropenone-masked dibenzocyclooctyne group was used to prepare the linear PS with a cyclopropenone-masked dibenzocyclooctyne in the middle of the chain and bromo groups at both ends (Br-PS-Br). Then we used the single electron transfer-nitroxide radical coupling (SET-NRC) reaction to transfer the bromo end groups to azide groups (N3-PS-N3). After UV irradiation, the dibenzocyclooctyne group was quantitatively released, and intramolecularly reacted with alternative azide end group to produce the tadpole-shaped PS based on SPAAC reaction.

T-shaped and H-shaped polymers constructed from UV-induced strain promoted azide-alkyne cycloaddition reaction

Topological polymers with T-shaped and H-shaped molecular architecture were built on the combination of atom transfer radical polymerization (ATRP) and UV-induced strain promoted azide-alkyne cycloaddition (SPAAC) reaction. In the presence of a cyclopropenone-masked dibenzocyclooctyne functionalized dibromo ATRP initiator, reactive polystyrene (PS) was synthesized to have a cyclopropenone-masked dibenzocyclooctyne group in the middle of polymer chain. After releasing dibenzocyclooctyne from deprotecting cyclopropenone-masked dibenzocyclooctyne under UV irradiation, T-shaped and H-shaped topological polymers were constructed by reacting PS with mono-end and di-end azide functionalized poly(ethylene oxide) (PEO), respectively, based on the SPAAC reaction.

In Vivo Targeting of Metabolically Labeled Cancers with Ultra-Small Silica Nanoconjugates.

Unnatural sugar-mediated metabolic labeling of cancer cells, coupled with efficient Click chemistry, has shown great potential for in vivo imaging and cancer targeting. Thus far, chemical labeling of cancer cells has been limited to the small-sized azido groups, with the large-sized and highly hydrophobic dibenzocyclooctyne (DBCO) being correspondingly used as the targeting ligand. However, surface modification of nanomedicines with DBCO groups often suffers from low ligand density, difficult functionalization, and impaired physiochemical properties. Here we report the development of DBCO-bearing unnatural sugars that could directly label LS174T colon cancer cells with DBCO groups and subsequently mediate cancer-targeted delivery of azido-modified silica nanoconjugates with easy functionalization and high azido density in vitro and in vivo. This study, for the first time, demonstrates the feasibility of metabolic labeling of cancer cells with large-sized DBCO groups for subsequent, efficient targeting of azido-modified nanomedicines.

A clickable and photocleavable lipid analogue for cell membrane delivery and release.

For drug delivery purposes, the ability to conveniently attach a targeting moiety that will deliver drugs to cells and then enable controlled release of the active molecule after localization is desirable. Toward this end, we designed and synthesized clickable and photocleavable lipid analogue 1 to maximize the efficiency of bioconjugation and triggered release. This compound contains a dibenzocyclooctyne group for bioorthogonal derivatization linked via a photocleavable 2-nitrobenzyl moiety at the headgroup of a synthetic lipid backbone for targeting to cell membranes. To assess delivery and release using this system, we report fluorescence-based assays for liposomal modification and photocleavage in solution as well as through surface immobilization to demonstrate successful liposome functionalization and photoinduced release. In addition, fluorophore delivery to and release from live cells was confirmed and characterized using fluorescence microscopy and flow cytometry analysis in which 1 was delivered to cells, derivatized, and photocleaved. Finally, drug delivery studies were performed using an azide-tagged analogue of camptothecin, a potent anticancer drug that is challenging to deliver due to poor solubility. In this case, the ester attachment of the azide tag acted as a caging group for release by intracellular esterases rather than through photocleavage. This resulted in a dose-dependent response in the presence of liposomes containing delivery agent 1, confirming the ability of this compound to stimulate delivery to the cytoplasm of cells.

Efficient method for iodine radioisotope labeling of cyclooctyne-containing molecules using strain-promoted copper-free click reaction

Herein we report an efficient method for iodine radioisotope labeling of cyclooctyne-containing molecules using copper-free click reaction. For this study, radioiodination using the tin precursor 2 was carried out at room temperature to give 125I-labeled azide ([125I]1) with high radiochemical yield (85%) and excellent radiochemical purity. Dibenzocyclooctyne (DBCO) containing cRGD peptide and gold nanoparticle were labeled with [125I]1 at 37°C for 30min to give triazoles with good radiochemical yields (67–95%). We next carried out tissue biodistribution study of [125I]1 in normal ICR mice to investigate the level of organ accumulation which needs to be considered for pre-targeted in vivo imaging. Large amount of [125I]1 distributed rapidly in liver and kidney from bloodstream and underwent rapid renal and hepatobiliary clearance. Moreover [125I]1 was found to be highly stable (>92%) in mouse serum for 24h. Therefore [125I]1 could be used as a potentially useful radiotracer for pre-targeted imaging. Those results clearly indicated that the present radiolabeling method using copper free click reaction would be quite useful for both in vitro and in vivo labeling of DBCO group containing molecules with iodine radioisotopes.

Well-defined cyclopropenone-masked dibenzocyclooctyne functionalized polymers from atom transfer radical polymerization

Two functional atom transfer radical polymerization (ATRP) initiators (I-2 and I-3) were developed bearing a cyclopropenone-masked dibenzocyclooctyne group. ATRP was then explored on three main kinds of monomers for radical polymerization including acrylates, styrenics, and methacrylates based on these novel initiators. By a standard ATRP protocol, the polymerization behavior demonstrated the living characteristics for all three cases and the corresponding well-defined cyclopropenone-masked dibenzocyclooctyne end or middle functionalized polymers were produced conveniently. Since UV irradiation of the cyclopropenone-masked dibenzocyclooctynes could quantitatively release the dibenzocyclooctynes widely used in strain promoted azide-alkyne cycloaddition bioorthogonal click reaction, these novel ATRP initiators and the resultant well-defined polymers should play an important role in the preparation of topological polymers and bio-synthetic polymer conjugates and many other related fields.

64Cu-labeled somatostatin analogues conjugated with cross-bridged phosphonate-based chelators via strain-promoted click chemistry for PET imaging: in silico through in vivo studies.

Somatostatin receptor subtype 2 (sstr2) is a G-protein-coupled receptor (GPCR) that is overexpressed in neuroendocrine tumors. The homology model of sstr2 was built and was used to aid the design of new somatostatin analogues modified with phosphonate-containing cross-bridged chelators for evaluation of using them as PET imaging radiopharmaceuticals. The new generation chelators were conjugated to Tyr3-octreotate (Y3-TATE) through bioorthogonal, strain-promoted alkyne azide cycloaddition (SPAAC) to form CB-TE1A1P–DBCO–Y3-TATE (AP) and CB-TE1K1P–PEG4–DBCO–Y3-TATE (KP) in improved yields compared to standard direct conjugation methods of amide bond formation. Consistent with docking studies, the clicked bioconjugates showed high binding affinities to sstr2, with Kd values ranging from 0.6 to 2.3 nM. Selected isomers of the clicked products were used in biodistribution and PET/CT imaging. Introduction of the bulky dibenzocyclooctyne group in AP decreased clearance rates from circulation. However, the additional carboxylate group and PEG linker from the KP conjugate significantly improved labeling conditions and in vivo stability of the copper complex and ameliorated the slower pharmacokinetics of the clicked somatostatin analogues.