Glucagon-like peptide-1 receptor (GLP-1R) agonists are increasingly explored for the treatment of obesity and metabolic disorders, yet the biological consequences of presenting these ligands on the surface of lipid-based nanocarriers remain insufficiently understood. In this study, exendin-4-functionalized nanoemulsions (ex-4 NE) were developed and evaluated in 3T3-L1 pre-adipocytes and adipocytes with respect to cellular uptake and metabolic activity. Both nanoemulsion types were efficiently internalized, however, ex-4 NE displayed higher uptake rates and larger AUC values than non-targeted PEG NE. Uptake was strongly influenced by differentiation stage but only minimally affected by GLP-1R inhibition. Chlorpromazine reduced uptake only in adipocytes differentiated in presence of exendin-4, indicating a shift toward clathrin-mediated routes upon chronic ex-4 treatment, but overall supporting that nanoemulsion uptake is not directly receptor-dependent. Colocalization analysis showed only modestly higher overlap of ex-4 NE with GLP-1R in comparison to the PEG NE. Despite the absence of pronounced receptor-mediated internalization, ex-4 NE exerted substantially stronger GLP-1R-dependent metabolic effects than free exendin-4. When administered early in differentiation, ex-4 enhanced adipogenesis, whereas addition from day 2 markedly reduced lipid accumulation. Notably, 10 nM ex-4 NE produced effects comparable to 1000 nM free ex-4, suggesting enhanced receptor activation via high local ligand density at the nanoemulsion surface, enabling multivalent interactions. At higher NE concentrations, lipid accumulation increased due to lipid substrate supply, independent of GLP-1R signaling. Taken together, these findings indicate that exendin-4 functionalization enhances nanoemulsion activity primarily through amplification of biological responses rather than through pronounced receptor-mediated uptake.

Aim: Nanotechnology showed great potential in selective delivery of therapeutic agents and reducing their side effects.The incorporation of radionuclide in superparamagnetic iron oxide nanoparticles (SPIONs) could lead to the development of multifunctional agents with greater potential in cancer therapy due to the combined effect of therapeutic radiation and hyperthermia.This study aimed to investigate the possibility of 131 I-radiolabeled SPIONs with excellent heating efficiency for nano-brachytherapy of tumors.Materials and methods: SPIONs were synthesized using the polyol method.The biocompatibility of SPIONs was improved by coating with 3-aminopropyltriethoxysilane (APTES). 131I radionuclide was linked to CC49 antibody specific for tumor-associated glycoprotein (TAG-72) before being covalently attached to coated SPIONs.Following intravenous and intratumor injection of the SPIONs complex, biodistribution, retention, and therapeutic effect were investigated on LS174T human colon adenocarcinoma xenografts on NOD-SCID mice.Results: SPIONs were successfully coated with APTES and characterized, by strong heating ability under an oscillating magnetic field.A significant level of radiation and hyperthermia over 45 °C was achieved locally for up to 14 days, demonstrating the stability of the radiolabeling and efficient nanoparticles retention only after intratumor application.Combined radionuclide-hyperthermia therapy showed significant (P < 0.01) suppression of the tumor growth when compared to the control groups and was better than radionuclide or hyperthermia treatments alone.Histopathology analysis proved the necrosis and apoptosis present in treated tumors but neither in nontreated ones nor normal organs.By judging the animals' body mass no general toxicity was observed. Conclusion:Combined therapy by applying coated SPIONs conjugated with 131 I-radiolabeled antibody was shown as a promising modality for tumor therapy.Obtained results justify further investigation in this encouraging nano-brachytherapy approach.

PET imaging of the glucagon-like peptide-1 receptor (GLP-1R) using radiolabeled exendin is a promising imaging method to detect insulinomas. However, high renal accumulation of radiolabeled exendin could hamper the detection of small insulinomas in proximity to the kidneys and limit its use as a radiotherapeutic agent. Here, we report two new exendin analogues for GLP-1R imaging and therapy, designed to reduce renal retention by incorporating a cleavable methionine-isoleucine (Met-Ile) linker. We examined the renal retention and insulinoma targeting properties of these new exendin analogues in a nude mouse model bearing subcutaneous GLP-1R-expressing insulinomas. NOTA or DOTA was conjugated via a methionine-isoleucine linker to the C-terminus of exendin-4 (NOTA-MI-exendin-4 or DOTA-MI-exendin-4). NOTA- and DOTA-exendin-4 without the linker were used as references. The affinity for GLP-1R was determined in a competitive binding assay using GLP-1R transfected cells. Biodistribution of [68Ga]Ga-NOTA-exendin-4, [68Ga]Ga-NOTA-MI-exendin-4, [177Lu]Lu-DOTA-exendin-4, and [177Lu]Lu-DOTA-MI-exendin-4 was determined in INS-1 tumor-bearing BALB/c nude mice, and PET/CT was acquired to visualize renal retention and tumor targeting. For all tracers, dosimetric calculations were performed to determine the kidney self-dose. The affinity for GLP-1R was in the low nanomolar range (<11 nM) for all peptides. In vivo biodistribution revealed a significantly lower kidney uptake of [68Ga]Ga-NOTA-MI-exendin-4 at 4 h post-injection (p.i.) (34.2 ± 4.2 %IA/g), compared with [68Ga]Ga-NOTA-exendin-4 (128 ± 10 %IA/g). Accumulation of [68Ga]Ga-NOTA-MI-exendin-4 in the tumor was 25.0 ± 8.0 %IA/g 4 h p.i., which was similar to that of [68Ga]Ga-NOTA-exendin-4 (24.9 ± 9.3 %IA/g). This resulted in an improved tumor-to-kidney ratio from 0.2 ± 0.0 to 0.8 ± 0.3. PET/CT confirmed the findings in the biodistribution studies. The kidney uptake of [177Lu]Lu-DOTA-MI-exendin-4 was 39.4 ± 6.3 %IA/g at 24 h p.i. and 13.0 ± 2.5 %IA/g at 72 h p.i., which were significantly lower than those for [177Lu]Lu-DOTA-exendin-4 (99.3 ± 9.2 %IA/g 24 h p.i. and 45.8 ± 3.9 %IA/g 72 h p.i.). The uptake in the tumor was 7.8 ± 1.5 and 11.3 ± 2.0 %IA/g 24 h p.i. for [177Lu]Lu-DOTA-MI-exendin-4 and [177Lu]Lu-DOTA-exendin-4, respectively, resulting in improved tumor-to-kidney ratios for [177Lu]Lu-DOTA-MI-exendin-4. The new exendin analogues with a Met-Ile linker showed 2-3-fold reduced renal retention and improved tumor-to-kidney ratios compared with their reference without the Met-Ile linker. Future studies should demonstrate whether [68Ga]Ga-NOTA-MI-exendin-4 results in improved detection of small insulinomas in close proximity to the kidneys with PET/CT. [177Lu]Lu-DOTA-MI-exendin-4 might open a window of opportunity for exendin-based radionuclide therapy.

A correction to this paper has been published: https://doi.org/10.1007/s11060-021-03769-9

Radiolabeled gastrin analogues have been proposed for theranostics of cholecystokinin subtype 2 receptor (CCK2R)-positive cancer. Peptide radioligands based on other receptor antagonists have displayed superior pharmacokinetics and higher biosafety than agonists. Here, we present DGA1, a derivative of the nonpeptidic CCK2R antagonist Z-360 carrying an acyclic tetraamine, for [99mTc]Tc labeling. Preclinical comparison of [99mTc]Tc-DGA1 with [99mTc]Tc-DG2 (CCK2R-agonist reference) was conducted in HEK293-CCK2R/CCK2i4svR cells and mice models, qualifying [99mTc]Tc-DGA1 for further study in patients with CCK2R-positive tumors and single-photon emission computed tomography/CT.

The development of so-called Proticles opens attractive possibilities for new drug delivery systems. Proticles are nanoparticles (NPs), which are formed by self-assembly of negatively charged oligonucleotides in combination with the positively charged peptide protamine. Polyethylene glycol (PEG) is a widely known pharmaceutical agent to stop particle growth and prolong circulation half-life of drug delivery systems. Therefore, two different NP formulations - one PEGylated and one non-PEGylated - were used in this work to gain information about the biological stability and half-life in circulation of Proticles. Thus, this study presents data of in vitro stability and in vivo pharmacokinetics of both, non-PEGylated and PEGylated Proticles radiolabeled with 111InCl3. The study demonstrated that successful radiolabeling of both Proticle-formulations was performed resulting in high radiochemical yields (> 85 %). Furthermore, the influence of PEGylation on the in vitro stability of 111In-radiolabeled NPs was investigated. No significant difference due to PEGylation was found. Unlike in vitro results, non-PEGylated 111In-Proticles seemed to degrade faster in vivo than PEGylated 111In-proticles, resulting in significantly higher blood values (111In-PEG-proticles: 0.23 ± 0.01 % ID/g 1 h p.i.; 111In-proticles: 0.06 ± 0.01 % ID/g 1 h p.i.; p < 0.05). Visualized by SPECT imaging urinary excretion represented the major pathway of elimination for both NP-formulations. In conclusion, this study provides data indicating a positive influence of PEG-derivatization on the biodistribution and pharmacokinetics of Proticles. These results form the basis for further developments as drug delivery and active drug targeting devices.

Due to large homology of human and canine EGFR, dogs suffering from spontaneous EGFR+ cancer can be considered as ideal translational models. Thereby, novel immunotherapeutic compounds can be developed for both human and veterinary patients. This study describes the radiolabeling of a canine anti-EGFR IgG antibody (can225IgG) with potential diagnostic and therapeutic value in comparative clinical settings. Can225IgG was functionalized with DTPA for subsequent chelation with the radionuclide 99mTc. Successful coupling of 10 DTPA molecules per antibody on average was proven by significant mass increase in MALDI-TOF spectroscopy, gel electrophoresis and immunoblots. Following functionalization and radiolabeling, 99mTc-DTPA-can225IgG fully retained its binding capacity towards human and canine EGFR in flow cytometry, immuno- and radioblots, and autoradiography. The affinity of radiolabeled can225IgG was determined to KD 0.8 ±0.0031 nM in a real-time kinetics assay on canine carcinoma cells by a competition binding technique. Stability tests of the radiolabeled compound identified TRIS buffered saline as the ideal formulation for short-term storage with 87.11 ±6.04% intact compound being still detected 60 minutes post radiolabeling. High stability, specificity and EGFR binding affinity pinpoint towards 99mTc-radiolabeled can225IgG antibody as an ideal lead compound for the first proof-of-concept diagnostic and therapeutic applications in canine cancer patients.

Since therapeutic peptides and oligonucleotides are gathering interests as active pharmaceutical ingredients (APIs), nanoparticulate drug delivery systems are becoming of great importance. Thereby, the possibility to design drug delivery systems according to the therapeutic needs of APIs enhances clinical implementation. Over the last years, the focus of our group was laid on protamine-oligonucleotide-nanoparticles (so called proticles), however, the possibility to modify the size, zeta potential or loading efficiencies was limited. Therefore, at the present study we integrated a stepwise addition of protamine (titration) into the formation process of proticles loaded with the angiogenic neuropeptide secretoneurin (SN). A particle size around 130 nm was determined when proticles were assembled by the commonly used protamine addition at once. Through application of the protamine titration process it was possible to modify and adjust the particle size between approx. 120 and 1200 nm (dependent on mass ratio) without influencing the SN loading capacity. Dynamic light scattering pointed out that the difference in particle size was most probably the result of a secondary aggregation. Initially-formed particles of early stages in the titration process aggregated towards bigger assemblies. Atomic-force-microscopy images also revealed differences in morphology along with different particle size. In contrast, the SN loading was only influenced by the applied mass ratio, where a slight saturation effect was observable. Up to 65% of deployed SN could be imbedded into the proticle matrix. An in-vivo biodistribution study (i.m.) showed a retarded distribution of SN from the site of injection after the application of a SN-proticle formulation. Further, it was demonstrated that SN loaded proticles can be successfully freeze-dried and resuspended afterwards. To conclude, the integration of the protamine titration process offers new possibilities for the formulation of proticles in order to address key parameters of drug delivery systems as size, API loading or modified drug release.

Background The significant progress in nanotechnology provides a wide spectrum of nanosized material for various applications, including tumor targeting and molecular imaging. The aim of this study was to evaluate multifunctional liposomal nanoparticles for targeting approaches and detection of tumors using different imaging modalities. The concept of dual-targeting was tested in vitro and in vivo using liposomes derivatized with an arginine-glycine-aspartic acid (RGD) peptide binding to αvβ3 integrin receptors and a substance P peptide binding to neurokinin-1 receptors. Methods For liposome preparation, lipids, polyethylene glycol building blocks, DTPA-derivatized lipids for radiolabeling, lipid-based RGD and substance P building blocks and imaging labels were combined in defined molar ratios. Liposomes were characterized by photon correlation spectroscopy and zeta potential measurements, and in vitro binding properties were tested using fluorescence microscopy. Standardized protocols for radiolabeling were developed to perform biodistribution and micro-single photon emission computed tomography/computed tomography (SPECT/CT) studies in nude mice bearing glioblastoma and/or melanoma tumor xenografts. Additionally, an initial magnetic resonance imaging study was performed. Results Liposomes were radiolabeled with high radiochemical yields. Fluorescence microscopy showed specific cellular interactions with RGD-liposomes and substance P-liposomes. Biodistribution and micro-SPECT/CT imaging of 111In-labeled liposomal nanoparticles revealed low tumor uptake, but in a preliminary magnetic resonance imaging study with a single-targeted RGD-liposome, uptake in the tumor xenografts could be visualized. Conclusion The present study shows the potential of liposomes as multifunctional targeted vehicles for imaging of tumors combining radioactive, fluorescent, and magnetic resonance signaling. Specific in vitro tumor targeting by fluorescence microscopy and radioactivity was achieved. However, biodistribution studies in an animal tumor model revealed only moderate tumor uptake and no additive effect using a dual-targeting approach.

BACKGROUNDVasoactive intestinal peptide (VIP) receptors are overexpressed in a broad variety of tumours. For the detection of these tumours, novel chemically modified and shortened VIP derivatives were designed.MATERIALS AND METHODS1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-derivatised VIP analogues were radiolabelled with (111)In and in vitro and in vivo behaviour was evaluated using stability and internalisation assays, as well as an initial biodistribution study.RESULTSRadiolabelling of the VIP analogues resulted in high radiochemical yields, without need for further purification steps. Stability of the VIP derivatives was variable and cell uptake studies in VIP receptor-positive cell lines revealed that only a limited number of derivatives were internalised. In the tumour mouse model, no specific tumour targeting was shown.CONCLUSIONSince the tested VIP derivatives exhibited impaired in vitro and in vivo characteristics alternative modifications to increase their stability while retaining receptor affinity should be considered to enable the use of synthetic VIP analogues for tumour targeting.

Purpose: Liposomes have been proposed to be a means of selectively targeting cancer sites for diagnostic and therapeutic applications. The focus of this work was the evaluation of radiolabeled PEGylated liposomes derivatized with varying amounts of a cyclic arginyl-glycyl-aspartic acid (RGD) peptide. RGD peptides are known to bind to alpha(v)beta(3) integrin receptors overexpressed during tumor-induced angiogenesis.Methods: Several liposomal nanoparticles carrying the RGD peptide targeting sequence (RLPs) were synthesized using a combination of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, cholesterol, diethylenetriaminepentaacetic acid-derivatized lipids for radiolabeling, a polyethylene glycol (PEG) building block, and a lipid-based RGD building block. Relative amounts of RGD and PEG building blocks were varied. In vitro binding affinities were determined using isolated alpha(v)beta(3) integrin receptors incubated with different concentrations of RLPs in competition with iodine-125-labeled cyclo-(-RGDyV-). Binding of the indium-111-labeled RLPs was also evaluated. Biodistribution and micro single photon emission computed tomography/computed tomography imaging studies were performed in nude mice using different tumor xenograft models.Results: RLPs were labeled with indium-111 with high radiochemical yields. In vitro binding studies of RLPs with different RGD/PEG loading revealed good binding to isolated receptors, which was dependent on the extent of RGD and PEG loading. Binding increased with higher RGD loading, whereas reduced binding was found with higher PEG loading. Biodistribution showed increased circulating time for PEGylated RLPs, but no dependence on RGD loading. Both biodistribution and micro single photon emission computed tomography/computed tomography imaging studies revealed low, nonspecific tumor uptake values.Conclusion: In this study, RLPs for targeting angiogenesis were described. Even though good binding to alpha(v)beta(3) integrin receptors was found in vitro, the balance between PEGylation and RGD loading clearly requires optimization to achieve targeting in vivo. These data form the basis for future development and provide a platform for the investigation of multimodal approaches.

Radiolabeled PEGylated liposomal nanoparticles (NPs) open new possibilities for a variety of applications including diagnosis, drug delivery, targeted therapy, and monitoring treatment effects. Here we describe the characterization of liposomal NPs (liposomes and micelles) derivatized with the somatostatin analogue tyrosine-3-octreotide as a proof of concept for tumor targeting. NPs were radiolabeled with indium-111, and targeting properties were evaluated in vitro on rat pancreatic tumor cells (AR42J), demonstrating specific binding and IC50 values in the low nanomolar range. Biodistribution studies were performed in Lewis rats and compared to single-photon emission computed tomography images. Moderate tumor uptake was found in xenografted nude mice (<2.5% ID/g tissue) as compared to control. Micelles and liposomes revealed comparable pharmacokinetics and targeting properties. This study provides insight into tumor-targeting characteristics of peptide-derivatized liposomal NPs and can serve as a basis for further improvement of these constructs.From the Clinical Editor: The authors investigated tumor-targeting characteristics of peptide-derivatized liposomal NPs. Similar radiolabeled PEGylated liposomal NPs open new possibilities for a variety of applications including diagnosis, drug delivery, targeted therapy, and treatment monitoring. (C) 2012 Elsevier Inc. All rights reserved.

Inhalative administration of vasoactive intestinal peptide (VIP) is a promising approach for the treatment of severe lung diseases. However, the clinical use of VIP is limited by the fact that the peptide is prone to rapid degradation mechanisms and proteolytic digestion. Accordingly, VIP exhibits a very short period of activity in the lung. To overcome this problem, we have designed a liposomal drug delivery system for VIP and characterized it in terms of its potential to protect VIP from enzymatic cleavage. The proteolytic conditions of the lung, the target site of aerosolic administered VIP, were mimicked by bronchoalveolar lavage fluid (BALF), a lung surfactant solution, obtained by fiberoptic bronchoscopy. Thus, the stability of VIP was assessed by its resistance to enzymatic degradation in BALF, using a combination of high pressure liquid chromatography with mass spectrometry. We found that free VIP was rapidly digested, whereas liposomal-associated VIP remained intact. By fluorescence spectroscopic techniques using fluorescent-labelled VIP we got strong indications that the tight association of VIP with the lipid membrane is only minimally affected upon incubation with BALF. Loading capacity and stability of EtCy3-VIP loaded liposomes were measured by fluorescence fluctuation spectroscopy. Finally, the protective properties of the liposomes were also expressed in the maintained biological activity of the peptide incubated with BALF.

The Fmoc/t-Bu solid-phase synthesis of three difficult peptide sequences (a 9-mer, 15-mer, and 24-mer) was performed using N,N'-diisopropylcarbodiimide/1-hydroxybenzotriazole as coupling reagent on polystyrene, Tentagel, and ChemMatrix resins. In order to obtain an insight into the specific role of the elevated temperature and/or the electromagnetic field for peptide syntheses carried out using microwave irradiation, peptide couplings and Fmoc-deprotection steps were studied under microwave and conventionally heated conditions at the same temperature. While room temperature couplings/deprotections generally produced the difficult peptides in rather poor quality, excellent peptide purities were obtained using microwave heating at a temperature of 86 degrees C for both the coupling and deprotection steps in only 10 and 2.5 min reaction time, respectively. While for most amino acids no significant racemization was observed, the high coupling temperatures led to considerable levels of racemization for the sensitive amino acids His and Cys. It was demonstrated for all three peptide sequences that when performing the coupling/deprotection steps at the same reaction temperature using conventional heating, nearly identical results in terms of both peptide purity and racemization levels were obtained. It therefore appears that the main effect of microwave irradiation applied to solid-phase peptide synthesis is a purely thermal effect not related to the electromagnetic field.

Drug delivery of protein and peptide-based drugs, which represent a growing and important therapeutic class, is hampered by these drugs' very short half-lives. High susceptibility towards enzymatic degradation necessitates frequent drug administration followed by poor adherence to therapy. Among these drugs is vasoactive intestinal peptide (VIP), a potent systemic and pulmonary vasodilator, which is a promising drug for the treatment of idiopathic pulmonary arterial hypertension (IPAH). Encapsulation of VIP into the nanoparticle matrix of biodegradable protamine-oligonucleotide nanoparticles (proticles) protects the peptide against rapid enzymatic degradation. Additionally, the nanoparticle matrix will be able to sustain drug release. Proticles consist of 18mer non-sense oligonucleotides and protamine, a polycationic arginine-rich peptide. VIP encapsulation occurs during self-assembly of the components. Within the present study, we evaluate nanoparticle size (hydrodynamic diameter) and zeta potential of VIP-loaded proticles as well as encapsulation efficiency and VIP release. Further, the pharmacological VIP response of "encapsulated VIP" is investigated using an ex vivo lung arterial model system. We found satisfying encapsulation efficiency (up to 80%), VIP release (77-87%), and an appropriate nanoparticle size (177-251 nm). Investigations on rat pulmonary arteries showed a modified VIP response of proticle-associated VIP. We noted differences in the profile of artery relaxation where VIP proticles lead to a 20-30% lower relaxation maximum than aqueous VIP solutions followed by prolonged vasodilatation. Our data indicate that proticles could be a feasible drug delivery system for a pulmonary VIP depot formulation. (C) 2008 Elsevier B.V. All Fights reserved.

A polymer-grafted liposomal formulation that has the potential to be developed for aerosolic pulmonary delivery of vasoactive intestinal peptide (VIP), a potent vasodilatory neuropeptide, is described. As VIP is prone to rapid proteolytic degradation in the microenvironment of the lung a proper delivery system is required to increase the half-life and bioavailability of the peptide. Here we investigate structural parameters of unilamellar liposomes composed of palmitoyl-oleoyl-phosphatidylcholine, lyso-stearyl-phosphatidylglycerol and distearyl-phosphatidyl-ethanolamine covalently linked to polyethylene glycol 2000, and report on VIP–lipid interaction mechanisms. We found that the cationic VIP is efficiently entrapped by the negatively charged spherical liposomes and becomes converted to an amphipathic α-helix. By fluorescence spectroscopy using single Trp-modified VIP we could show that VIP is closely associated to the membrane. Our data suggest that the N-terminal random-coiled domain is embedded in the interfacial headgroup region of the phospholipid bilayer. By doing so, neither the bilayer thickness of the lipid membrane nor the mobility of the phospholipid acyl chains are affected as shown by small angle X-ray scattering and electron spin resonance spectroscopy. Finally, in an ex vivo lung arterial model system we found that liposomal-associated VIP is recognized by its receptors to induce vasodilatory effects with comparable high relaxation efficiency as free VIP but with a significantly retarded dilatation kinetics. In conclusion, we have designed and characterized a liposomal formulation that is qualified to entrap biologically active VIP and displays structural features to be considered for delivery of VIP to the lung.