In-depth evaluation of the cycloaddition–retro-Diels–Alder reaction for in vivo targeting with [In]-DTPA-RGD conjugates

IntroductionThe spontaneous copper-free tandem 1,3-dipolar cycloaddition–retro-Diels–Alder (tandem crDA) reaction between cyclic Arg-Gly-Asp-d-Phe-Orn(N₃) [c(RGDfX)] and oxanorbornadiene-DTPA (o-DTPA) or methyloxanorbornadiene-DTPA (mo-DTPA) into two DTPA-c(RGDfX) regioisomers is characterized. Since there is no information on the stability and reaction rate of the tandem crDA reaction in biological media, we set out to characterize these reaction parameters.MethodsThe effects of concentration of the reactants, temperature, pH and reaction environment (serum, blood) on the kinetics of the reaction were determined using ¹¹¹In-labeled oxanorbornadiene-DTPA analogs. The affinity of the radiolabeled conjugate was determined in a solid-phase α_vβ₃ integrin binding assay. Furthermore, the octanol–water partition coefficient was determined and, finally, the biodistribution of the labeled compounds in mice with subcutaneous α_vβ₃-expressing tumors was determined.ResultsFifty percent conversion was reached after 26 h. Kinetic experiments furthermore established that the reaction rate of the tandem crDA reaction follows temperature- and concentration-dependent second-order kinetics, but is independent of the pH of the medium. Affinity of the two [¹¹¹In]DTPA-cRGDfX conjugates for α_vβ₃ integrin is 191 nM. Biodistribution studies showed specific (α_vβ₃-mediated) uptake of [¹¹¹In]DTPA-c(RGDfX) in the tumor and in α_vβ₃-expressing tissues.ConclusionThe tandem crDA reaction using methyl-substituted oxanorbornadiene is a versatile method for a single-step ligation that proceeds independently of pH and also proceeds in serum and blood. Currently, we are further looking into enhancement of reaction kinetics and exploitation of tandem crDA in vivo.     

Strategies for drug targeting in pancreatic cancer

BackgroundPancreatic cancer is expected to replace lung cancer as the second greatest cause of cancer mortality by 2025. It has been a particularly the most lethal kind of cancer.ObjectiveDespite the new innovations, research, and improvements in drug design; there are many hurdles limiting their therapeutic applications such as intrinsic resistance to chemotherapeutics, inability to deliver a sufficient concentration of drug to the target site, lack of effectiveness of drug delivery systems. These are the major contributing factors to limit the treatment. So, the main objective is to overcome these types of problems by nanotechnology and ligand conjugation approach to achieve targeted drug delivery.MethodNanotechnology has emerged as a major approach to develop cancer treatment. Regardless of the severity, there are several issues that restrict the therapeutic impact, including inadequate transport across biological barriers, limited cellular absorption, degradation, and faster clearance.ResultTargeted drug delivery may overcome these obstacles by binding a natural ligand to the surface of nanocarriers, which enhances the drug's capacity to release at the desired site and minimizes adverse effects.ConclusionThis study will investigate the possible outcomes of targeted therapeutic agent delivery in the treatment of pancreatic cancer, as well as the limitations and future prospects.     

Synthesis, fluorescence and biodistribution of a bone-targeted near-infrared conjugate

Enhanced imaging of early-stage bone abnormalities, such as primary tumors or metastases is highly required as the widely-used bone scan frequently lacks the desired sensitivity. Near IR (NIR) fluorescence imaging affords high contrast and enhanced sensitivity, as body tissue expresses minimal autofluorescence at NIR range (600-1200 nm). Indocyanine green (ICG), a biocompatible NIR dye, is widely used in the imaging of various organs, such as liver, heart and blood circulation. We report the preparation and in-vivo testing of a bone-targeting ICG derivative, in comparison to the parent molecule(s). Since ICG itself is chemically unreactive, and could not form conjugates, we prepared two novel ICG conjugatable derivatives. The overall ICG structure was maintained while only a replacement of one or two sulfonate groups with carboxylic acids resulted in new linkers for covalent binding to biomolecules. These derivatives were evaluated for their fluorescence and biodistribution in comparison to ICG and were found to be comparable. One of the novel ICG-derivatives was conjugated to a bone-targeting moiety and this new compound was found to bind to growing regions of the skeleton, and emit fluorescence for as long as two weeks in young mice.     

Preparation and Characterization of Polyethylene-Glycol-Modified Salmon Calcitonins

The conjugation of salmon calcitonin (sCT) by covalent linkage of polyethylene glycol (PEG) was attempted to overcome several disadvantages of sCT as a therapeutic drug, namely its rapid clearance from blood circulation and enzymatic degradation. The polymer employed was succinimidyl carbonate monomethoxypolyethylene glycol (12 kDa). Superose HR size-exclusion chromatography was applied to separate the PEGylated sCTs (mono-PEG-sCT and di-PEG-sCT) from the unmodified sCT. The PEGylation of sCT was verified by an electrophoresis gel stained with iodine and by MALDI-TOF mass spectrometry. The molecular weights of mono-PEG-sCT and di-PEG-sCT were determined to be 16,094 and 29,077 Da, respectively. PEGylated sCTs showed a substantially improved stability in rat liver homogenates as compared to the intact sCT, indicating that PEG molecules protected sCT from various degrading enzymes. These PEGylated sCTs exhibited similar biological activity to the intact sCT by adenosine cyclic 3′,5′-phosphate (cAMP) assay. In clearance studies in the rat, PEGylated sCTs had significantly longer circulating half-lives than the intact sCT (11.2 min for mono-PEG-sCT and 54.0 min for di-PEG-sCT versus 4.7 min for intact sCT).     

Genome-free Viral Capsids as Carriers for Positron Emission Tomography Radiolabels

PURPOSE: We have developed a modular synthetic strategy to append imaging agents to a viral capsid. PROCEDURES: The hollow protein shell of bacteriophage MS2 (mtMS2) was labeled on its inside surface with [18F]fluorobenzaldehyde through a multistep bioconjugation strategy. An aldehyde functional group was first attached to interior tyrosine residues through a diazonium coupling reaction. The aldehyde was further elaborated to an alkoxyamine functional group, which was then condensed with n.c.a. [18F]fluorobenzaldehyde. Biodistribution of the radioactive MS2 conjugates was subsequently evaluated in Sprague-Dawley rats. RESULTS: Relative to fluorobenzaldehyde, fluorine-18-labeled MS2 exhibited prolonged blood circulation time and a significantly altered excretion profile. It was also observed that additional small molecule cargo installed inside the capsids did not alter the biodistribution. CONCLUSIONS: These studies provide further insight into the pharmacokinetic behavior of nanomaterials and serve as a platform for the future development of targeted imaging and therapeutic agents based on mtMS2.     

Engineering Luminescent Quantum Dots for In Vivo Molecular and Cellular Imaging

Semiconductor quantum dots are luminescent nanoparticles that are under intensive development for use as a new class of optical imaging contrast agents. Their novel properties such as optical tunability, improved photostability, and multicolor light emission have opened new opportunities for imaging living cells and in vivo animal models at unprecedented sensitivity and spatial resolution. Combined with biomolecular engineering strategies for tailoring the particle surfaces at the molecular level, bioconjugated quantum dot probes are well suited for imaging single-molecule dynamics in living cells, for monitoring protein–protein interactions within specific intracellular locations, and for detecting diseased sites and organs in deep tissue. In this article, we describe the engineering principles for preparing high-quality quantum dots and for conjugating the dots to biomolecular ligands. We also discuss recent advances in using quantum dots for in vivo molecular and cellular imaging.     

量子点与多肽LyP-1的连接、表征及对肿瘤细胞的识别

利用水相合成的量子点(Quantum dots,QDs)纳米粒子,通过交联剂[N-succinimidyl3-(2-pyridyldithio)propionate,SPDP]将其与多肽LyP-1(CGNKRTRGC)进行连接以形成一种纳米荧光探针。毛细管电泳、吸收光谱以及荧光光谱测试结果表明,LyP-1已成功地连接到了QDs表面,所得荧光探针能较好地识别SPCA-1肺腺癌细胞,但不识别HL-60原髓细胞白血病成淋巴细胞。