Search

Doxorubicin (Dox) loaded thermosensitive liposomes (TSLs) have shown promising results for hyperthermia-induced local drug delivery to solid tumors. Typically, the tumor is heated to hyperthermic temperatures (41-42 °C), which induced intravascular drug release from TSLs within the tumor tissue leading to high local drug concentrations (1-step delivery protocol). Next to providing a trigger for drug release, hyperthermia (HT) has been shown to be cytotoxic to tumor tissue, to enhance chemosensitivity and to increase particle extravasation from the vasculature into the tumor interstitial space. The latter can be exploited for a 2-step delivery protocol, where HT is applied prior to i.v. TSL injection to enhance tumor uptake, and after 4 hours waiting time for a second time to induce drug release. In this study, we compare the 1- and 2-step delivery protocols and investigate which factors are of importance for a therapeutic response. In murine B16 melanoma and BFS-1 sarcoma cell lines, HT induced an enhanced Dox uptake in 2D and 3D models, resulting in enhanced chemosensitivity. In vivo, therapeutic efficacy studies were performed for both tumor models, showing a therapeutic response for only the 1-step delivery protocol. SPECT/CT imaging allowed quantification of the liposomal accumulation in both tumor models at physiological temperatures and after a HT treatment. A simple two compartment model was used to derive respective rates for liposomal uptake, washout and retention, showing that the B16 model has a twofold higher liposomal uptake compared to the BFS-1 tumor. HT increases uptake and retention of liposomes in both tumors models by the same factor of 1.66 maintaining the absolute differences between the two models. Histology showed that HT induced apoptosis, blood vessel integrity and interstitial structures are important factors for TSL accumulation in the investigated tumor types. However, modeling data indicated that the intraliposomal Dox fraction did not r

Although melanoma progression and staging is clinically well characterized, a large variation is observed in pathogenesis, progression, and therapeutic responses. Clearly, intrinsic characteristics of melanoma cells contribute to this variety. An important factor, in both progression of the disease and response to therapy, is the tumor-associated vasculature. We postulate that melanoma cells communicate with endothelial cells (ECs) in order to establish a functional and supportive blood supply. We investigated the angiogenic potential of human melanoma cell lines by monitoring the survival of ECs upon exposure to melanoma conditioned medium (CM), under restrictive conditions. We observed long-term (up to 72 h) EC survival under hypoxic conditions upon treatment with all melanoma CMs. No such survival effect was observed with the CM of melanocytes. The CM of pancreatic and breast tumor cell lines did not show a long-term survival effect, suggesting that the survival factor is specific to melanoma cells. Furthermore, all size fractions (up to < 1 kDa) of the melanoma CM induced long-term survival of ECs. The survival effect observed by the < 1 kDa fraction excludes known pro-angiogenic factors. Heat inactivation and enzymatic digestion of the CM did not inactivate the survival factor. Global gene expression and pathway analysis suggest that this effect is mediated in part via the AKT and p38 MAPK/ ERK-1/2 signaling axis. Taken together, these data indicate the production of (a) survival factor/s (< 1 kDa) by melanoma cell lines, which enables long-term survival of ECs and promotes melanoma-induce

Purpose: To evaluate pharmacokinetic profile, biodistribution and therapeutic effect of cationic thermosensitive liposomes (CTSL) encapsulating doxorubicin (Dox) upon mild hyperthermia (HT). Methods: Non-targeted thermosensitive liposomes (TSL) and CTSL were developed, loaded with Dox and characterized. Blood kinetics and biodistribution of Dox-TSL and Dox-CTSL were followed in B16BL6 tumor bearing mice upon normothermia (NT) or initial hyperthermia conditions. Efficacy study in B16BL6 tumor bearing mice was followed with Dox-TSL or Dox-CTSL upon NT or HT. Efficacy study in LLC tumor bearing mice was performed upon two HT conditions. Intravital microscopy was performed on B16BL6 tumors implanted in dorsal-skin fold window-bearing mice. Results: Targeting did not cause faster blood clearance of CTSL compared to TSL. Highest uptake of liposomes was observed in spleen, kidneys and liver. Applying HT prior to CTSL administration increased drug delivery to the tumor and CTSL delivered ∼1.7 fold higher Dox concentration compared to TSL. Efficacy in B16BL6 murine melanoma showed that HT had a significant effect on CTSL in tumor suppression and prolonged survival. Efficacy in LLC Lewis lung carcinoma tumor model demonstrates that two HT treatments hold promises for a successful treatment option. Conclusion: CTSL have potency to increase drug efficacy in tumors due to their targeted and drug release functions.

Therapy of melanoma using T-cells with genetically introduced T-cell receptors (TCRs) directed against a tumor-selective cancer testis antigen (CTA) NY-ESO1 demonstrated clear antitumor responses in patients without side effects. Here, we exploited the concept of TCR-mediated targeting through introduction of single-chain variable fragment (scFv) antibodies that mimic TCRs in binding major histocompatibility complex-restricted CTA. We produced scFv antibodies directed against Melanoma AntiGEn A1 (MAGE A1) presented by human leukocyte antigen A1 (HLA-A1), in short M1/A1, and coupled these TCR-like antibodies to liposomes to achieve specific melanoma targeting. Two anti-M1/A1 antibodies with different ligand-binding affinities were derived from a phage-display library and reformatted into scFvs with an added cysteine at their carboxyl termini. Protein production conditions, ie, bacterial strain, temperature, time, and compartments, were optimized, and following production, scFv proteins were purified by immobilized metal ion affinity chromatography. Batches of pure scFvs were validated for specific binding to M1/A1-positive B-cells by flow cytometry. Coupling of scFvs to liposomes was conducted by employing different conditions, and an optimized procedure was achieved. In vitro experiments with immunoliposomes demonstrated binding of M1/A1-positive B-cells as well as M1/A1-positive melanoma cells and internalization by these cells using flow cyto

Prostate-specific membrane antigen (PSMA) is overexpressed in prostate cancer (PCa) and a promising target for molecular imaging and therapy. Nanobodies (single-domain antibodies, VHH) are the smallest antibody-based fragments possessing ideal molecular imaging properties, such as high target specificity and rapid background clearance. We developed a novel anti-PSMA Nanobody (JVZ-007) for targeted imaging and therapy of PCa. Here, we report on the application of the 111In-radiolabeled Nanobody for SPECT/CT imaging of PCa. Methods: A Nanobody library was generated by immunization of a llama with 4 human PCa cell lines. Anti-PSMA Nanobodies were captured by biopanning on PSMA-overexpressing cells. JVZ-007 was selected for evaluation as an imaging probe. JVZ-007 was initially produced with a c-myc-hexahistidine (his) tag allowing purification and detection. The c-myc-his tag was subsequently replaced by a single cysteine at the C terminus, allowing site-specific conjugation of chelates for radiolabeling. JVZ-007-cmyc- his was conjugated to 2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (p-SCN-DTPA) via the lysines, whereas JVZ-007-cys was conjugated to maleimide-DTPA via the C-terminal cysteine. PSMA targeting was analyzed in vitro by cell-binding experiments using flow cytometry, autoradiography, and internalization assays with various PCa cell lines and patient-derived xenografts (PDXs). The targeting properties of radiolabeled Nanobodies were evaluated in vivo in biodistribution and SPECT/CT imaging experiments, using nude mice bearing PSMA-positive PC-310 and PSMA-negative PC-3 tumors. Results: JVZ-007 was successfully conjugated to DTPA for radiolabeling with 111In at room temperature. 111In-JVZ007-c-myc-his and 111In-JVZ007-cys internalized in LNCaP cells and bound to PSMA-expressing PDXs and, importantly, not to PSMA-negative PDXs and human kidneys. Good tumor targeting and fast blood clearance were obse

Purpose: To develop RGD-targeted thermosensitive liposomes with increased tumor retention, improving drug release efficiency upon mild hyperthermia (HT) in both tumor and angiogenic endothelial cells. Methods: Standard termosensitive liposomes (TSL) and TSL containing a cyclic Arg-Gly-Asp (cRGD) pentapeptide with the sequence Arg-Cys-D-Phe-Asp-Gly (RGDf[N-Met]C) were synthetized, loaded with Dox and characterized. Temperature- and time-dependent drug release profiles were assessed by fluorometry. Intracellular Dox delivery was studied by flow cytometry and confocal microscopy. Cytotoxic effect of TSL and RGD-TSL was studied on B16Bl6 melanoma, B16F10 melanoma and HUVEC. Intravital microscopy was performed on B16Bl6 tumors implanted in dorsal-skin fold window-bearing mice. Pharmacokinetic and biodistribution of Dox-TSL and Dox-RGD-TSL were followed in B16Bl6 tumor bearing mice upon normothermia or initial hyperthermia conditions. Results: DLS and cryo-TEM revealed particle homogeneity and size of around 85 nm. Doxorubicin loading efficiency was >95%as assessed by spectrofluorometry. Flow cytometry and confocal microscopy showed a specific uptake of RGD-TSL by melanoma and endothelial cells when compared to TSL and an increased doxorubicin delivery. High resolution intravital microscopy demonstrated specific accumulation of RGD-TSL to the tumor vasculature. Moreover, application of hyperthermia resulted in massive drug release from RGD-TSL. Biodistribution studies showed that initial hyperthermia increases Dox uptake in tumors from TSL and RGD-TSL. Conclusion: RGD-TSL have potency to increase drug efficacy due to higher uptake by tumor and angiogenic endothelial cells in combination with heat-triggered drug release.

Liposomal chemotherapy offers several advantages over conventional therapies, including high intratumoral drug delivery, reduced side effects, prolonged circulation time and the possibility to dose higher. The efficient delivery of liposomal chemotherapeutics relies however on the enhanced permeability and retention (EPR) effect, which refers to the ability of macromolecules to extravasate leaky tumor vessels and accumulate in the tumor tissue. Using a panel of human xenograft tumors, we evaluated the influence of the EPR effect on liposomal distribution in vivo by injection of pegylated liposomes radiolabeled with 111In. Liposomal accumulation in tumors and organs was followed over time by SPECT/CT imaging. We observed that fast growing xenografts, which may be less representative of tumor development in patients, showed higher liposomal accumulation as compared to slow growing xenografts. Additionally, several other parameters determining the EPR effect were evaluated, such as blood and lymphatic vessel density, intratumoral hypoxia, and the presence of macrophages. The investigation of various parameters showed a few correlations. Although hypoxia, proliferation and macrophage presence were associated with tumor growth, no hard conclusions or predictions could be made regarding the EPR effect or liposomal uptake. However liposomal uptake was s

Aims: With the aim to improve peptide receptor radionuclide therapy effects in patients with gastroenteropancreatic neuroendocrine tumor (GEPNET) liver metastases we explored the effect of intra-arterial (IA) administration of [111In-DTPA]octreotide (111In-DTPAOC) on tumor uptake in an animal model and in a patient study. Methods: Preclinical study: After administering 111In-DTPAOC intra-venously (IV) or IA, biodistribution studies were performed in rats with a hepatic somatostatin receptor subtype 2 (sst2)-positive tumor. Clinical study: 3 patients with neuroendocrine liver metastases were injected twice with 111In-DTPAOC. The first injection was given IV, and 2 weeks later, the second was injected IA (hepatic artery). Planar images of the abdomen were made up to 72 hours after injection. Blood samples were taken and urine was collected. Pharmacokinetic modeling was performed on the IV and IA data of the same patient. Based on this model, additional 177Lu dosimetry calculations for IV and IA administrations were performed. Results: The preclinical study showed a two-fold higher 111In-DTPAOC tumor uptake after IA administration than after IV injection. Patient data showed a large variability in radioactivity increment in liver metastases after IA administration compared with IV administration. Renal radioactivity was not significantly lower after IA administration; 177Lu dosimetry simulations in 1 patient using a maximum kidney radiation dose of 23Gy showed IA administration resulted in a mean increase in tumor radiation dose of 2.9-fold. Conclusion: Preclinical and clinical data both indicate that IA administration of radiolabeled somatostatin analogs via the hepatic artery can significantly increase radionuclide uptake in GEPNET, sst2-positive, liver metastases up to 72 hours postinjection, although the effect of IA administration can differ between patients.

Currently, gold nanorods can be synthesized in a wide range of sizes. However, for the intended biological applications gold nanorods with approximate dimensions 50 nm × 15 nm are used. We investigate by computer simulation the effect of particle dimensions on the optical and thermal properties in the context of the specific applications of photoacoustic imaging. In addition we discuss the influence of particle size in overcoming the following biophysical barriers when administrated in vivo: extravasation, avoidance of uptake by organs of the reticuloendothelial system, penetration through the interstitium, binding capability and uptake by the target cells. Although more complex biological influences can be introduced in future analysis, the present work illustrates that larger gold nanorods, designated by us as 'nanobig rods', may perform better at meeting the requirements for successful in vivo applications compared to their smaller counterparts, which are conventionally used.

Multimodal bioluminescence (BLI) and single-photon emission computed tomography/computed tomography (SPECT/CT) imaging were investigated as means to monitor somatostatin receptor subtype 2 (SST 2)-positive neuroendocrine tumors as both a subcutaneously implanted and a liver metastasis animal model in mice and rats. Ultimately, such a model will be of use for studying SST 2-targeted peptide receptor radionuclide therapy (PRRT). CA20948 cells were transfected with a green fluorescent protein/luciferase plasmid construct. Cells were inoculated subcutaneously in the shoulder of nude mice: nontransfected cells in the left shoulder and transfected cells in the right shoulder. BLI, SPECT/CT imaging, biodistribution analysis, and ex vivo autoradiography of the tumors were performed. BLI and SPECT/CT imaging were also performed on an intrahepatic tumor model in the rat. Caliper volume measurement of transfected tumors could be correlated with BLI measurements (R 2=.76). SPECT/CT imaging showed high levels of accumulation of 111In-DTPA-octreotide in control and transfected tumors, which was confirmed by biodistribution analysis and autoradiography. Subcapsular inoculation of transfected cells in rat liver resulted in an intrahepatic tumor, which could be visualized by both SPECT/CT and BLI. Transfection of CA20948 tumor cells did not alter the growth properties of the cell line or the expression of SST 2. Transfected tumors could be clearly visualized by BLI and SPECT/CT imaging. The transfected SST 2-positive tumor cell line could represent a novel preclinical model for tumor monitoring in studies that aim at further optimizing PRRT for neuroendocrine tumors.

Traditionally, the goal of nanoparticle-based chemotherapy has been to decrease normal tissue toxicity by improving drug specificity to tumors. The enhanced permeability and retention effect can permit passive accumulation into tumor interstitium. However, suboptimal delivery is achieved with most nanoparticles because of heterogeneities of vascular permeability, which limits nanoparticle penetration. Furthermore, slow drug release limits bioavailability. We developed a fast drug-releasing liposome triggered by local heat that has already shown substantial antitumor efficacy and is in human trials. Here, we show that thermally sensitive liposomes (Dox-TSL) release doxorubicin inside the tumor vasculature. Real-time confocal imaging of doxorubicin delivery to murine tumors in window chambers and histologic analysis of flank tumors illustrates that intravascular drug release increases free drug in the interstitial space. This increases both the time that tumor cells are exposed to maximum drug levels and the drug penetration distance, compared with free drug or traditional pegylated liposomes. These improvements in drug bioavailability establish a new paradigm in drug delivery: rapidly triggered drug release in the tumor bloodstream. Copyright

Background: Although widely fragmented BMs have been associated with adverse outcome in several cancer types, comparatively little is known with respect to its effect on the prognosis of pancreatic cancer. The aim of the current study was therefore to determine the prognostic value of tumour basement membrane (BM) continuity in two anatomically closely related, however, prognostically different tumours, pancreatic head-and periampullary cancer. Methods: Tumour BM continuity was determined by immunohistochemical staining of its two major components, laminin and collagen type IV. Associations were made with recurrence free survival (RFS), cancer-specific survival (CSS), overall survival (OS) and conventional prognostic factors. Results: Fifty-nine and 61% of pancreatic head and periampullary tumours, respectively, showed limited BM laminin expression. Whereas 43% and 41% of pancreatic head and periampullary cancers, respectively, showed limited BM collagen type IV expression. Limited BM laminin was associated with poor outcome following curative resection of pancreatic head cancer (P=0.034, 0.013 and 0.017 for RFS, CSS and OS, respectively). Two and a half times as many patients with 25% BM laminin were recurrence free and alive 5 years following resection compared with those with limited BM laminin. Although staining patterns of both BM components were weakly correlated with each other, BM collagen type IV expression was not significantly associated with outcome in either tumour type.Conclusion: Discontinuous BMs, determined by laminin expression, are associated with poor outcome following curative resection of pancreatic head cancer.

This study sought to compare lumen dimensions as assessed by 3D quantitative coronary angiography (QCA) and by intravascular ultrasound (IVUS) or optical coherence tomography (OCT), and to assess the association of the discrepancy with vessel curvature. Coronary lumen dimensions often show discrepancies when assessed by X-ray angiography and by IVUS or OCT. One source of error concerns a possible mismatch in the selection of corresponding regions for the comparison. Therefore, we developed a novel, real-time co-registration approach to guarantee the point-to-point correspondence between the X-ray, IVUS and OCT images. A total of 74 patients with indication for cardiac catheterization were retrospectively included. Lumen morphometry was performed by 3D QCA and IVUS or OCT. For quantitative analysis, a novel, dedicated approach for co-registration and lumen detection was employed allowing for assessment of lumen size at multiple positions along the vessel. Vessel curvature was automatically calculated from the 3D arterial vessel centerline. Comparison of 3D QCA and IVUS was performed in 519 distinct positions in 40 vessels. Correlations were r = 0.761, r = 0.790, and r = 0.799 for short diameter (SD), long diameter (LD), and area, respectively. Lumen sizes were larger by IVUS (P < 0.001): SD, 2.51 ± 0.58 mm versus 2.34 ± 0.56 mm; LD, 3.02 ± 0.62 mm versus 2.63 ± 0.58 mm; Area, 6.29 ± 2.77 mm2versus 5.08 ± 2.34 mm2. Comparison of 3D QCA and OCT was performed in 541 distinct positions in 40 vessels. Correlations were r = 0.880, r = 0.881, and r = 0.897 for SD, LD, and area, respectively. Lumen sizes were larger by OCT (P < 0.001): SD, 2.70 ± 0.65 mm versus 2.57 ± 0.61 mm; LD, 3.11 ± 0.72 mm versus 2.80 ± 0.62 mm; Area 7.01 ± 3.28 mm2versus 5.93 ± 2.66 mm2. The vessel-based discrepancy between 3D QCA and IVUS or OCT long diameters increased with increasing vessel curvature. In conclusion, our comparison of co-registered 3D QCA and invasive imaging data suggests a bias to

Lipid-based nanocarriers or liposomes have been proven successful in the delivery of chemotherapeutic agents and are currently applied clinically in the treatment of various types of cancer. Liposomes offer the advantage of a high drug payload, decreased drug toxicity and enhanced drug accumulation at tumor sites. Increased accumulation is due to the relatively leaky tumor vasculature that allows liposome extravasation. Between different types of tumors and even within one tumor, vascular permeability and thus liposome extravasation may differ greatly. Furthermore, upon accumulation of liposomes in the tumor area, drug bioavailability is not guaranteed. At present, these are the major issues for clinically used liposomal drugs. Mild hyperthermia (HT), the heating of tumor tissue to temperatures of up to 43°C, has been developed in the past decades as an established and efficacious treatment modality in combination with chemo- and radiotherapy. HT can be used to further improve liposomal chemotherapy in two ways: HT is known to increase vascular permeability in solid tumors and may therefore increase levels of liposome accumulation, and thermosensitive liposomes have been developed that can be triggered to release their contents upon hyperthermia. By applying these two strategies, drug delivery to tumors can be strongly enhanced.

Lipid-based nanocarriers have proven successful in the delivery of mainly chemotherapeutic agents, and currently they are being applied clinically in the treatment of various types of cancer. These drug delivery systems achieve increased therapeutic efficacy by altering the pharmacokinetics and biodistribution of encapsulated drugs, resulting in decreased drug toxicity and enhanced accumulation in tumor tissue. This increased accumulation is due to the relatively leaky immature vasculature of a tumor. After the clinical relevance of such drug delivery systems was demonstrated, research in this area focused on optimization, both by cell specific targeting and including controlled and triggered release concepts within the carrier. These more advanced targeted nanocarriers in general have clearly shown their potential in various animal tumor models and await clinical application. The development of targeted nanocarriers in which therapeutic and imaging agents are merged into a single carrier will certainly be of importance in the near future. Indeed, scientists active in the field of imaging (e.g. nuclear and magnetic resonance imaging) have already started to exploit nanocarriers for molecular imaging. Image-guided drug delivery using these multifunctional nanocarriers, containing therapeutic and imaging agents, will ultimately allow for online monitoring of tumor location, tumor targeting levels, intratumoral localization and drug release kinetics prior and during radio- and/or chemotherapeutic treatment. This review describes the current status and challenges in the field of nanocarrier-aided drug delivery and drug targeting and discusses the opportunities of combining imaging probes with these drug carriers and the potential of these multifunctional lipid-based nanocarriers within image-guided drug delivery.

Applied Sciences

Many advanced molecular imaging agents are currently being investigated preclinically. Especially, liposomes, have proven to be very promising carrier systems for diagnostic agents for use in single-photon emission computed tomography (SPECT) or magnetic resonance imaging (MRI), as well as for therapeutic agents to treat diseases such as cancer. In this study, nanosized liposomes were designed and labeled with the radionuclides, holmium-166 (both a beta- and gamma-emitter and also highly paramagnetic) or technetium-99m, and coloaded with paramagnetic gadolinium allowing multimodality SPECT and MR imaging and radionuclide therapy with one single agent. Methods: Diethylenetriaminepentaacetic acid bisoctadecylamide (an amphiphilic molecule with a chelating group suitable for labeling with radionuclides) and gadoliniumacetylacetonate (GdAcAc) (a small lipophilic paramagnetic molecule) were incorporated in liposomes. The liposomes were characterized by measuring their mean size and size distribution, gadolinium content, and radiochemical stability after incubation in human serum at 37°C. The MRI properties (in vitro) were determined by use of relaxivity measurements at 1.5 and 3.0 Tesla in order to evaluate their potency as imaging agents. Results: The liposomes were successfully labeled with holmium-166, resulting in a high labeling efficiency (95% ± 1%) and radiochemical stability (> 98% after 48 hours of incubation), and coloaded with GdAcAc. Labeling of liposomes with technetium-99m was somewhat less efficient (85% ± 2%), although their radiochemical stability was sufficient (95% ± 1% after 6 hours of incubation). MRI measurements showed that the incorporation of GdAcAc had a strong effect on the MRI relaxivity. Conclusions: The synthesized liposomes allow for multimodality imaging and therapy, which makes these new agents highly attractive for future applications, Radiation, Radionuclides and Reactors, Applied Sciences