Development and Preclinical Evaluation of [211At]PSAt-3-Ga: An Inhibitor for Targeted α-Therapy of Prostate Cancer.

The application of prostate-specific membrane antigen (PSMA)-targeted α-therapy is a promising alternative to ß--particle-based treatments. 211At is among the potential α-emitters that are favorable for this concept. Herein, 211At-based PSMA radiopharmaceuticals were designed, developed, and evaluated. Methods: To identify a 211At-labeled lead, a surrogate strategy was applied. Because astatine does not exist as a stable nuclide, it is commonly replaced with iodine to mimic the pharmacokinetic behavior of the corresponding 211At-labeled compounds. To facilitate the process of structural design, iodine-based candidates were radiolabeled with the PET radionuclide 68Ga to study their preliminary in vitro and in vivo properties before the desired 211At-labeled lead compound was formed. The most promising candidate from this evaluation was chosen to be 211At-labeled and tested in biodistribution studies. Results: All 68Ga-labeled surrogates displayed affinities in the nanomolar range and specific internalization in PSMA-positive LNCaP cells. PET imaging of these compounds identified [68Ga]PSGa-3 as the lead compound. Subsequently, [211At]PSAt-3-Ga was synthesized in a radiochemical yield of 35% and showed tumor uptake of 19 ± 8 percentage injected dose per gram of tissue (%ID/g) at 1 h after injection and 7.6 ± 2.9 %ID/g after 24 h. Uptake in off-target tissues such as the thyroid (2.0 ± 1.1 %ID/g), spleen (3.0 ± 0.6 %ID/g), or stomach (2.0 ± 0.4 %ID/g) was low, indicating low in vivo deastatination of [211At]PSAt-3-Ga. Conclusion: The reported findings support the use of iodine-based and 68Ga-labeled variants as a convenient strategy for developing astatinated compounds and confirm [211At]PSAt-3 as a promising radiopharmaceutical for targeted α-therapy.

Glymphatic-assisted perivascular brain delivery of intrathecal small gold nanoparticles.

Nanoparticles are ultrafine particulate matter having considerable potential for treatment of central nervous system (CNS) disorders. Despite their tiny size, the blood-brain barrier (BBB) restricts their access to the CNS. Their direct cerebrospinal fluid (CSF) administration bypasses the BBB endothelium, but still fails to give adequate brain uptake. We present a novel approach for efficient CNS delivery of 111In-radiolabelled gold nanoparticles (AuNPs; 10-15 nm) via intra-cisterna magna administration, with tracking by SPECT imaging. To accelerate CSF brain influx, we administered AuNPs intracisternally in conjunction with systemic hypertonic saline, which dramatically increased the parenchymal AuNP uptake, especially in deep brain regions. AuNPs entered the CNS along periarterial spaces as visualized by MRI of gadolinium-labelled AuNPs and were cleared from brain within 24 h and excreted through the kidneys. Thus, the glymphatic-assisted perivascular network augment by systemic hypertonic saline is a pathway for highly efficient brain-wide distribution of small AuNPs.

Synthesis and radiolabeling of a polar [125 I]I-1,2,4,5-tetrazine.

Pretargeting imaging has gained a lot of prominence, due to its excellent bioorthogonality and improved imaging contrast compared to conventional imaging. A new iodo tetrazine (Tz) derivative has been synthesized and further developed into the corresponding iodine-125 (125 I) analog (12), via the trimethylstannane precursor. Radiolabeling with either N-chlorosuccinimide or chloramine-T, in either MeCN or MeOH proceeded with a radiochemical conversion (RCC) of >80%. Subsequent deprotection only proved successful, among the tested conditions, when the radiolabeled Tz was stirred in 6-M HCl(aq.) at 60°C for 2.5 h. To the best of our knowledge, this is the first H-tetrazine labeled with iodine. In vivo investigations on the pretargeting ability of 12 are currently under way.

Improved procedures for production and purification of 135La from enriched [135Ba]BaCO3 on a 16.5 MeV cyclotron.

Lanthanum-135 (135La) is a favorable Auger electron emitter with a high Auger electron yield and low gamma emission, making it promising for Auger electron radiotherapy. However, successful application requires reliable and scalable 135La production. Up to now, metallic natural barium (natBa) is a commonly used target material, but this material is sensitive to moisture and oxidation. BaCO3 has also been tested, due to its higher chemical stability. However, BaCO3 has poor thermal conductivity, limiting the applicable current and making high yield production challenging. In this study, we pressed a mixture of enriched [135Ba]BaCO3 and fine aluminum (Al) powder to provide a stable target with improved thermal conductivity compared to pure BaCO3. After 4 h of irradiation with a 16.5 MeV proton beam at 20 µA current, 1.62 ± 0.18 GBq was produced from a 200 mg [135Ba]BaCO3:Al (1:2, w/w) target. This corresponded to a saturation yield of 11.91 ± 1.31 GBq (or 596 ± 66 MBq/µA). A purification procedure involving initial precipitation, followed by a single composite column containing a layer of TK200 resin and a second layer of branched DGA resin was developed, with 97.1 ± 3.6 % decay corrected 135La recovery. [135La]LaCl3 was obtained in an effective molar activity of 79.6 ± 25.3 MBq/nmol (DOTA titration), 104.0 ± 40.4 MBq/nmol (DTPA titration) and 186.5 ± 83.8 MBq/nmol (CHX-A″-DTPA titration), and a radionuclidic purity (RNP) of >99.9 % at end of purification, hereby demonstrating a purity suitable for radiopharmaceutical use.

Covalent core-radiolabeling of polymeric micelles with 125I/211At for theranostic radiotherapy.

Astatine-211 (211At) is one of the most promising α-emitters for targeted alpha therapy, especially of cancer metastases. However, the lack of a stable isotope, frequent in vivo deastatination, and limited radiochemical knowledge makes it challenging to apply. Here, we report a new strategy for radiolabeling the lipophilic core of polymeric micelles (PMs) with covalently bound 211At. The PMs were radiolabeled via either an indirect synthon-based method or directly on the amphipathic block copolymer. The radiochemistry was optimized with iodine-125 (125I) and then adapted for 211At, enabling the use of both elements as a potential theranostic pair. PMs that were core-radiolabeled with both 125I or 211At were prepared and characterized, based on a PEG(5k)-PLGA(10k) co-polymer. The stability of the radiolabeled PMs was evaluated in mouse serum for 21 h, showing radiochemical stability above 85%. After in vivo evaluation of the 211At- labeled PMs, 4-5 % ID/g of the 211At could still be detected in the blood, showing a promising in vivo stability of the PMs. Further, 211At-labeled PMs accumulated in the spleen (20-30 %ID/g) and the liver (2.5- 5.5 %ID/g), along with some detection of 211At in the thyroid (3.5-9 %ID/g). This led to the hypothesis that deastatination takes place in the liver, whereas good stability of the 211At core-radiolabel was observed in the blood.

Bioorthogonal Click of Colloidal Gold Nanoparticles to Antibodies In vivo.

Combining nanotechnology and bioorthogonal chemistry for theranostic strategies offers the possibility to develop next generation nanomedicines. These materials are thought to increase therapeutic outcome and improve current cancer management. Due to their size, nanomedicines target tumors passively. Thus, they can be used for drug delivery purposes. Bioorthogonal chemistry allows for a pretargeting approach. Higher target-to-background drug accumulation ratios can be achieved. Pretargeting can also be used to induce internalization processes or trigger controlled drug release. Colloidal gold nanoparticles (AuNPs) have attracted widespread interest as drug delivery vectors within the last decades. Here, we demonstrate for the first time the possibility to successfully ligate AuNPs in vivo to pretargeted monoclonal antibodies. We believe that this possibility will facilitate the development of AuNPs for clinical use and ultimately, improve state-of-the-art patient care.

Novel Bifunctional [16]aneS4-Derived Chelators for Soft Radiometals.

The field of targeted radionuclide therapy is rapidly growing, highlighting the need for wider radionuclide availability. Soft Lewis acid ions, such as radioisotopes of platinum, rhodium and palladium, are particularly underdeveloped. This is due in part to a lack of compatible bifunctional chelators. These allow for the practical bioconjugation to targeting vectors, in turn enabling radiolabeling. The [16]andS4 macrocycle has been reported to chelate a number of relevant soft metal ions. In this work, we present a procedure for synthesizing [16]andS4 in 45% yield (five steps, 12% overall yield), together with a selection of strategies for preparing bifunctional derivatives. An ester-linked N-hydroxysuccimide ester (NHS, seven steps, 4% overall yield), an ether-linked isothiocyanate (NCS, eight steps, 5% overall yield) and an azide derivative were prepared. In addition, a new route to a carbon-carbon linked carboxylic acid functionalized derivative is presented. Finally, a general method for conjugating the NHS and NCS derivatives to a polar peptide (octreotide) is presented, by dissolution in water:acetonitrile (1:1), buffered to pH 9.4 using borate. The reported compounds will be readily applicable in radiopharmaceutical chemistry, by facilitating the labeling of a range of molecules, including peptides, with relevant soft radiometal ions.

Carbohydrate based biomarkers enable hybrid near infrared fluorescence and 64Cu based radio-guidance for improved surgical precision.

Increasing numbers of lung tumors are identified at early disease stages by diagnostic imaging in screening programs, but difficulties in locating these during surgical intervention has prevented an improved treatment outcome. Surgical biomarkers that are visible on diagnostic images, and that provide the surgeon with real-time image guidance during the intervention are thus highly warranted to bridge diagnostic precision into enhanced therapeutic outcome. In this paper, a liquid soft tissue marker for near infrared fluorescence and radio-guidance is presented. The biocompatible marker is based on the carbohydrate ester, sucrose acetate isobutyrate, ethanol, and a multifunctional naphthalocyanine dye, which enable near infrared fluorescence image-guided resection at short, medium and long tissue depths. Naphthalocyanine dyes have high quantum yields and may further act as chelators of radionuclides. Upon injection of the liquid marker, a gel-like depot is formed in situ at the site of injection, wherein the fluorescent dye and radionuclide is retained. The radiolabeled markers were optimized for minimal fluorescence quenching and high retention of the positron emission tomography radionuclide 64Cu. The performance of the radiolabeled marker was tested in vivo in mice, where it displayed high photostability over a period of 4 weeks, and high retention of 64Cu for 48 hours. The retention and biodistribution of 64Cu was quantified via PET/CT, and the fluorescence emission by an in vivo imaging system. The presented data demonstrate proof-of-concept for naphthalocyanine markers as multimodal imaging agents that can bridge the precision of diagnostic imaging into surgical interventions.

Effective Intratumoral Retention of [103 Pd]AuPd Alloy Nanoparticles Embedded in Gel-Forming Liquids Paves the Way for New Nanobrachytherapy.

Local application of radioactive sources as brachytherapy is well established in oncology. This treatment is highly invasive however, due to the insertion of millimeter sized metal seeds. The authors report the development of a new concept for brachytherapy, based on gold-palladium (AuPd) alloy nanoparticles, intrinsically radiolabeled with 103 Pd. These are formulated in a carbohydrate-ester based liquid, capable of forming biodegradable gel-like implants upon injection. This allows for less invasive administration through small-gauge needles. [103 Pd]AuPd nanoparticles with sizes around 20 nm are prepared with radiolabeling efficiencies ranging from 79% to >99%. Coating with the hydrophobic polymer poly(N-isopropylacrylamide) leads to nanoparticle diameters below 40 nm. Dispersing the nanoparticles in ethanol with water insoluble carbohydrate esters gives "nanogels", a low viscosity liquid capable of solidifying upon injection into aqueous environments. Both nanoparticles and radioactivity are stably retained in the nanogel over 25 days (>99%) after formation in aqueous buffers. Animals bearing CT26 murine tumors are injected intratumorally with 25 MBq of the 103 Pd-nanogel, and display tumor growth delay and significantly increase median survival times compared with control groups. Excellent retention in the tumor of both the 103 Pd and the nanoparticle matrix itself is observed, demonstrating a potential for replacing currently used brachytherapy seeds.

Corrigendum to 'quantitative determination of 64Cu-liposome accumulation at inflammatory and infectious sites: Potential for future theranostic system': [journal of controlled release 327 (2020) 737-746].

BACKGROUND: Therapeutic interventions for infectious and inflammatory diseases are becoming increasingly challenging in terms of therapeutic resistance and side-effects. Theranostic systems to ameliorate diagnosis and therapy are therefore highly warranted. The pathophysiological changes in inflammatory lesions provide an attractive basis for extravasation and accumulation of PEGylated liposomes. The objective of this study was to provide direct quantitative information on the theranostic potential of radiolabeled liposome for accumulation in inflammatory models using position emission tomography (PET). METHOD: Preclinical murine models of inflammation (turpentine and LPS), infection (Staphylococcus aureus) and collagen-induced arthritis (CIA) was established and monitored using bioluminescence imaging (BLI). Across all models PET imaging using radiolabeled PEGylated liposomes (64Cu-liposomes) were performed and evaluated in terms of accumulation properties in inflammatory and infectious lesions. RESULTS: BLI demonstrated that the inflammatory and infectious models were successfully established and provided information on lesion pathology. Activity of 64Cu-liposomes were increased in inflammatory and infectious lesions between early (10-min or 3-h) and late (24-h) PET scans, which validates that a continuous extravasation and accumulation of long circulation PEGylated liposomes occurs. CONCLUSION: The theranostic potential of long circulating PEGylated radiolabeled liposomes was shown in multiple preclinical models. Impressive accumulation was seen in both inflammatory and infectious lesions. These results are encouraging towards advancing PEGylated liposomes as imaging and drug delivery systems in inflammatory and infectious diseases.

Multimodal soft tissue markers for bridging high-resolution diagnostic imaging with therapeutic intervention.

Diagnostic imaging often outperforms the surgeon's ability to identify small structures during therapeutic procedures. Smart soft tissue markers that translate the sensitivity of diagnostic imaging into optimal therapeutic intervention are therefore highly warranted. This paper presents a unique adaptable liquid soft tissue marker system based on functionalized carbohydrates (Carbo-gel). The liquid state of these markers allows for high-precision placement under image guidance using thin needles. Based on step-by-step modifications, the image features and mechanical properties of markers can be optimized to bridge diagnostic imaging and specific therapeutic interventions. The performance of Carbo-gel is demonstrated for markers that (i) have radiographic, magnetic resonance, and ultrasound visibility; (ii) are palpable and visible; and (iii) are localizable by near-infrared fluorescence and radio guidance. The study demonstrates encouraging proof of concept for the liquid marker system as a well-tolerated multimodal imaging marker that can improve image-guided radiotherapy and surgical interventions, including robotic surgery.

Preclinical evaluation of cationic DOTA-triarginine-lipid conjugates for theranostic liquid brachytherapy.

Liquid brachytherapy is an emerging technology for internal radiation therapy where liquids containing radionuclides are administered directly into solid tumors. These technologies are less invasive than conventional brachytherapy, and can potentially improve the dose coverage and homogeneity of the radioactivity distribution within the tumor. For this purpose, we have developed a novel cationic micelle system for delivery of a range of radionuclides. The system is applicable for emitters of alpha, beta or photon radiation, and enables dose-mapping via theranostic nuclear imaging. Methods: The cationic micelles were developed as linear surfactants comprising the chelator DOTA, a triarginine sequence and a palmitoyl or stearoyl fatty acid chain. The critical micelle concentration of the surfactants was determined, and the micelles were radiolabelled with 64Cu or 177Lu in high radiochemical purity (>95%). The tumor retention and biodistribution of the 64Cu-radiolabeled surfactants, administered as micelles or formulated in liposomes, were investigated in vivo by PET/CT in a tumor bearing mouse model. Results: The interaction of the micelles with anionic lipid membranes was demonstrated to be favourable, using a liposome partition assay. In vivo, the surfactants formulated both as cationic micelles and liposomes displayed the best intratumoral retention, with micelles providing more homogeneous activity distribution. Conclusion: A cationic, surfactant-based drug delivery system was developed and demonstrated promise as a vehicle for liquid brachytherapy when formulated as micelles or in liposomes. The system enables accurate dosimetry due to the flexible radiochemistry of DOTA.

A solid support generator of the Auger electron emitter rhodium-103m from [103Pd]palladium.

Auger electron therapy is an attractive modality for targeting microscopic tumors. Rhodium-103 m (103mRh, T½â€¯= 56.1 min) is a promising Auger electron emitter that can be obtained as the decay product of palladium-103 (103Pd, T½â€¯= 16.99 days). 103Pd was chelated in a lipophilic derivative of the 16aneS4 macrocycle and the complex was trapped on a C18 cartridge. Elution with dilute hydrochloric acid gave radiochemically pure 103mRh. We hypothesize this to be through a combination of the Szilard-Chalmers effect and transient ionization.

Remote loading of liposomes with a 124I-radioiodinated compound and their in vivo evaluation by PET/CT in a murine tumor model.

Long circulating liposomes entrapping iodinated and radioiodinated compounds offer a highly versatile theranostic platform. Here we report a new methodology for efficient and high-yield loading of such compounds into liposomes, enabling CT/SPECT/PET imaging and 131I-radiotherapy. Methods: The CT contrast agent diatrizoate was synthetically functionalized with a primary amine, which enabled its remote loading into PEGylated liposomes by either an ammonium sulfate- or a citrate-based pH transmembrane gradient. Further, the amino-diatrizoate was radiolabeled with either 124I (t1/2 = 4.18 days) for PET or 125I (t1/2 = 59.5 days) for SPECT, through an aromatic Finkelstein reaction. Results: Quantitative loading efficiencies (>99%) were achieved at optimized conditions. The 124I-labeled compound was remote-loaded into liposomes, with an overall radiolabeling efficiency of 77 ± 1%, and imaged in vivo in a CT26 murine colon cancer tumor model by PET/CT. A prolonged blood circulation half-life of 19.5 h was observed for the radiolabeled liposomes, whereas injections of the free compound were rapidly cleared. Lower accumulation was observed in the spleen, liver, kidney and tumor than what is usually seen for long-circulating liposomes. Conclusion: The lower accumulation was interpreted as release of the tracer from the liposomes within these organs after accumulation. These results may guide the design of systems for controlled release of remote loadable drugs from liposomes.

Remote-loading of liposomes with manganese-52 and in vivo evaluation of the stabilities of 52Mn-DOTA and 64Cu-DOTA using radiolabelled liposomes and PET imaging.

Liposomes are nanoparticles used in drug delivery that distribute over several days in humans and larger animals. Radiolabeling with long-lived positron emission tomography (PET) radionuclides, such as manganese-52 (52Mn, T½=5.6days), allow the imaging of this biodistribution. We report optimized protocols for radiolabeling liposomes with 52Mn, through both remote-loading and surface labeling. For comparison, liposomes were also remote-loaded and surface labeled with copper-64 (64Cu, T½=12.7h) through conventional means. The chelator DOTA was used in all cases. The in vivo stability of radiometal chelates is widely debated but studies that mimic a realistic in vivo setting are lacking. Therefore, we employed these four radiolabeled liposome types as platforms to demonstrate a new concept for such in vivo evaluation, here of the chelates 52Mn-DOTA and 64Cu-DOTA. This was done by comparing "shielded" remote-loaded with "exposed" surface labeled variants in a CT26 tumor-bearing mouse model. Remote loading (90min at 55°C) and surface labeling (55°C for 2h) of 52Mn gave excellent radiolabeling efficiencies of 97-100% and 98-100% respectively, and the liposome biodistribution was imaged by PET for up to 8days. Liposomes with surface-conjugated 52Mn-DOTA exhibited a significantly shorter plasma half-life (T½=14.4h) when compared to the remote-loaded counterpart (T½=21.3h), whereas surface-conjugated 64Cu-DOTA cleared only slightly faster and non-significantly, when compared to remote-loaded (17.2±2.9h versus 20.3±1.2h). From our data, we conclude the successful remote-loading of liposomes with 52Mn, and furthermore that 52Mn-DOTA may be unstable in vivo whereas 64Cu-DOTA appears suitable for quantitative imaging.

Neodymium-140 DOTA-LM3: Evaluation of an In Vivo Generator for PET with a Non-Internalizing Vector.

140Nd (t1/2 = 3.4 days), owing to its short-lived positron emitting daughter 140Pr (t1/2 = 3.4 min), has promise as an in vivo generator for positron emission tomography (PET). However, the electron capture decay of 140Nd is chemically disruptive to macrocycle-based radiolabeling, meaning that an in vivo redistribution of the daughter 140Pr is expected before positron emission. The purpose of this study was to determine how the delayed positron from the de-labeled 140Pr affects preclinical imaging with 140Nd. To explore the effect, 140Nd was produced at CERN-ISOLDE, reacted with the somatostatin analogue, DOTA-LM3 (1,4,7,10- tetraazacyclododecane, 1,4,7- tri acetic acid, 10- acetamide N - p-Cl-Phecyclo(d-Cys-Tyr-d-4-amino-Phe(carbamoyl)-Lys-Thr-Cys)d-Tyr-NH2) and injected into H727 xenograft bearing mice. Comparative pre- and post-mortem PET imaging at 16 h postinjection was used to quantify the in vivo redistribution of 140Pr following 140Nd decay. The somatostatin receptor-positive pancreas exhibited the highest tissue accumulation of 140Nd-DOTA-LM3 (13% ID/g at 16 h) coupled with the largest observed redistribution rate, where 56 ± 7% (n = 4, mean ± SD) of the in situ produced 140Pr washed out of the pancreas before decay. Contrastingly, the liver, spleen, and lungs acted as strong sink organs for free 140Pr3+. Based upon these results, we conclude that 140Nd imaging with a non-internalizing vector convolutes the biodistribution of the tracer with the accumulation pattern of free 140Pr. This redistribution phenomenon may show promise as a probe of the cellular interaction with the vector, such as in determining tissue dependent internalization behavior.

PET imaging with copper-64 as a tool for real-time in vivo investigations of the necessity for cross-linking of polymeric micelles in nanomedicine.

Polymeric micelles in nanomedicine are often cross-linked to prevent disintegration in vivo. This typically requires clinically problematic chemicals or laborious procedures. In addition, cross-linking may interfere with advanced release strategies. Despite this, it is often not investigated whether cross-linking is necessary for efficient drug delivery. We used positron emission tomography (PET) imaging with 64 Cu to demonstrate general methodology for real-time in vivo investigations of micelle stability. Triblock copolymers with 4-methylcoumarin cores of ABC-type (PEG-PHEMA-PCMA) were functionalized in the handle region (PHEMA) with CB-TE2A chelators. Polymeric micelles were formed by dialysis and one half was core cross-linked (CL) by UV light and the other half was not (nonCL). Both CL and nonCL were radiolabeled with 64 Cu and compared in vivo in tumor-bearing mice, with free 64 Cu as control. Accumulation in relevant organs was quantified by region of interest analysis on PET images and ex vivo counting. It was observed that CL and nonCL showed limited differences in biodistribution from each other, whereas both differed markedly from control (free 64 Cu). This demonstrated that 4-methylcoumarin core micelles may form micelles that are stable in circulation even without cross-linking. The methodology presented here where individual unimers are radiolabeled is applicable to a wide range of polymeric micelle types.

Optimized procedures for manganese-52: Production, separation and radiolabeling.

Pressed chromium-powder cyclotron targets were irradiated with 16MeV protons, producing 52Mn with average yields of 6.2±0.8MBq/µAh. Separation by solid-phase anion exchange from ethanol-HCl mixtures recovered 94.3±1.7% of 52Mn and reduced the chromium content by a factor of 2.2±0.4×105. An additional AG 1-X8 column was used to remove copper, iron, cobalt and zinc impurities from the prepared 52Mn in 8M HCl. The macrocyclic chelator DOTA was rapidly radiolabeled with 52Mn in aq. ammonium acetate (pH 7.5R.T.) with a radiochemical yield >99% within 1min and was stable for >2 days in bovine serum. The improved separation and purification methodology facilitates the use of 52Mn in basic science and preclinical investigations.

Transferrin receptor expression and role in transendothelial transport of transferrin in cultured brain endothelial monolayers.

Receptor-mediated transcytosis of the transferrin receptor has been suggested as a potential transport system to deliver therapeutic molecules into the brain. Recent studies have however shown that therapeutic antibodies, which have been reported to cross the brain endothelium, reach greater brain exposure when the affinity of the antibodies to the transferrin receptor is lowered. The lower affinity of the antibodies to the transferrin receptor facilitates the dissociation from the receptor within the endosomal compartments, which may indicate that the receptor itself does not necessarily move across the endothelial cells by transcytosis. The aim of the present study was to investigate transferrin receptor expression and role in transendothelial transferrin transport in cultured bovine brain endothelial cell monolayers. Transferrin receptor mRNA and protein levels were investigated in endothelial mono-cultures and co-cultures with astrocytes, as well as in freshly isolated brain capillaries using qPCR, immunocytochemistry and Western blotting. Transendothelial transport and luminal association of holo-transferrin was investigated using [125I]holo-transferrin or [59Fe]-transferrin. Transferrin receptor mRNA expression in all cell culture configurations was lower than in freshly isolated capillaries, but the expression slightly increased during six days of culture. The mRNA expression levels were similar in mono-cultures and co-cultures. Immunostaining demonstrated comparable transferrin receptor localization patterns in mono-cultures and co-cultures. The endothelial cells demonstrated an up-regulation of transferrin receptor mRNA after treatment with the iron chelator deferoxamine. The association of [125I]holo-transferrin and [59Fe]-transferrin to the endothelial cells was inhibited by an excess of unlabeled holo-transferrin, indicating receptor mediated association. However, over time the cell associated [59Fe]-label exceeded that of [125I]holo-transferrin, which could indicate release of iron in the endothelial cells and receptor recycling. Luminal-to-abluminal transport of [125I]holo-transferrin across endothelial cell monolayers was low and not inhibited by unlabeled holo-transferrin. This indicated that transendothelial transferrin transport was independent of transferrin receptor-mediated transcytosis.