Pharmacokinetics, Tissue Distribution and Therapeutic Effect of Cationic Thermosensitive Liposomal Doxorubicin Upon Mild Hyperthermia.

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.

Enhanced Specificity and Drug Delivery in Tumors by cRGD-Anchoring Thermosensitive Liposomes.

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.

Targeted and heat-triggered doxorubicin delivery to tumors by dual targeted cationic thermosensitive liposomes.

Liposomal nanoparticles can circumvent toxicity of encapsulated chemotherapeutic drugs, but fall short in tumor-specific and efficient intracellular drug delivery. To overcome these shortcomings, we designed a multifunctional dual targeted, heat-responsive nanocarrier encapsulating doxorubicin (Dox) as a chemotherapeutic content. Dox-loaded cationic thermosensitive liposomes (Dox-CTSL) carry targeting functions addressing tumor cells and tumor vasculature and have a heat-responsive lipid bilayer. Targeted Dox-CTSL demonstrated superior uptake by and toxicity to different tumor cell lines and endothelial cells compared to non-targeted TSL. Heat triggered intracellular Dox release in acidic cell compartments was visualized as fluorescent Dox nanobursts by live cell confocal microscopy. In vivo, using high resolution intravital microscopy, we demonstrated that Dox-CTSL upon an external heat-trigger delivered 3-fold higher Dox quantity to tumors than TSL. Dox-CTSL bound specifically to tumor vasculature, which in combination with the heat-triggered drug release caused significant tumor vessel damage, which was not observed when non-targeted TSL were administered. Therefore, Dox-CTSL have strong potency to increase drug efficacy due to targeted delivery and heat-triggered drug release in tumors.

Targeted thermosensitive liposomes: an attractive novel approach for increased drug delivery to solid tumors.

INTRODUCTION: Currently available chemotherapy is hampered by a lack in tumor specificity and resulting toxicity. Small and long-circulating liposomes can preferentially deliver chemotherapeutic drugs to tumors upon extravasation from tumor vasculature. Although clinically used liposomal formulations demonstrated significant reduction in toxicity, enhancement of therapeutic activity has not fully met expectations. AREAS COVERED: Low drug bioavailability from liposomal formulations and limited tumor accumulation remain major challenges to further improve therapeutic activity of liposomal chemotherapy. The aim of this review is to highlight strategies addressing these challenges. A first strategy uses hyperthermia and thermosensitive liposomes to improve tumor accumulation and trigger liposomal drug bioavailability. Image-guidance can aid online monitoring of heat and drug delivery and further personalize the treatment. A second strategy involves tumor-specific targeting to enhance drug delivery specificity and drug internalization. In addition, we review the potential of combinations of the two in one targeted thermosensitive-triggered drug delivery system. EXPERT OPINION: Heat-triggered drug delivery using thermosensitive liposomes as well as the use of tumor vasculature or tumor cell-targeted liposomes are both promising strategies to improve liposomal chemotherapy. Preclinical evidence has been encouraging and both strategies are currently undergoing clinical evaluation. A combination of both strategies rendering targeted thermosensitive liposomes (TTSL) may appear as a new and attractive approach promoting tumor drug delivery.

Intact Doxil is taken up intracellularly and released doxorubicin sequesters in the lysosome: evaluated by in vitro/in vivo live cell imaging.

Doxil, also known as Caelyx, is an established liposomal formulation of doxorubicin used for the treatment of ovarian cancer, sarcoma and multiple myeloma. While showing reduced doxorubicin related toxicity, Doxil does not greatly improve clinical outcome. To become biologically active, doxorubicin needs to be released from its carrier. Uptake and breakdown of the liposomal carrier and subsequent doxorubicin release is not fully understood and in this study we explored the hypothesis that Doxil is taken up by tumor cells and slowly degraded intracellularly. We investigated the kinetics of liposomal doxorubicin (Doxil) in vitro as well as in vivo by measuring cytotoxic effect, intracellular bioavailability and fate of the carrier and its content. To prevent fixation artifacts we applied live cell imaging in vitro and intravital microscopy in vivo. Within 8h after administration of free doxorubicin, 26% of the drug translocated to the nucleus and when reaching a specific concentration killed the cell. Unlike free doxorubicin, only 0.4% of the doxorubicin added as liposomal formulation entered the nucleus. Looking at the kinetics, we observed a build-up of nuclear doxorubicin within minutes of adding free doxorubicin. This was in contrast to Doxil showing slow translocation of doxorubicin to the nucleus and apparent accumulation in the cytoplasm. Observations made with time-lapse live cell imaging as well as in vivo intravital microscopy revealed the liposomal carrier colocalizing with doxorubicin in the cytoplasm. We also demonstrated the sequestering of liposomal doxorubicin in the lysosomal compartment resulting in limited delivery to the nucleus. This entrapment makes the bioavailable concentration of Doxil-delivered doxorubicin significantly lower and therefore ineffective as compared to free doxorubicin in killing tumor cells.

Bcl-2 associated anthanogen-1 (Bag-1) expression and prognostic value in pancreatic head and periampullary cancer.

The expression of anti-apoptosis gene Bcl-2 associated anthanogen-1 (Bag-1), has been associated with outcome in several cancer types, however its prognostic role in pancreatic cancer is unknown. Aim was therefore to evaluate expression of Bag-1 in two anatomically closely related however prognostically different tumours, pancreatic head- and periampullary cancer and correlate expression with outcome. Bag-1 protein expression was studied by immunohistochemistry on original paraffin embedded tissue from 217 patients with microscopic radical resection (R0) of adenocarcinoma of the pancreatic head or periampullary region. Expression was assessed for associations with recurrence free- (RFS), cancer specific- (CSS), overall survival (OS) and conventional prognostic factors. Nuclear Bag-1 was present in 80% of tumours. In 40% Bag-1 resided in the cytosol, which was almost exclusively associated with nuclear expression. Nuclear Bag-1 protein was identified as an independent factor predicting a favourable outcome following radical resection of pancreatic head cancer. Eighteen percent of patients with nuclear Bag-1 were recurrence free and alive 5 years following surgery compared to none of the patients lacking expression. In periampullary cancer Bag-1 was not associated with outcome. In conclusion, Bag-1 was present in the majority of both pancreatic head- and periampullary cancers. However it was only identified as a discriminator of outcome in pancreatic head cancer.

Cationic thermosensitive liposomes: a novel dual targeted heat-triggered drug delivery approach for endothelial and tumor cells.

Developing selectively targeted and heat-responsive nanocarriers holds paramount promises in chemotherapy. We show that this can be achieved by designing liposomes combining cationic charged and thermosensitive lipids in the bilayer. We demonstrated, using flow cytometry, live cell imaging, and intravital optical imaging, that cationic thermosensitive liposomes specifically target angiogenic endothelial and tumor cells. Application of mild hyperthermia led to a rapid content release extra- and intracellularly in two crucial cell types in a solid tumor.

Angiogenesis: a prognostic determinant in pancreatic cancer?

Angiogenesis has been associated with disease progression in many solid tumours, however the statement that tumours need angiogenesis to grow, invade and metastasise seems no longer applicable to all tumours or to all tumour subtypes. Prognostic studies in pancreatic cancer are conflicting. In fact, pancreatic cancer has been suggested an example of a tumour in which angiogenesis is less essential for tumour progression. The aim of the present study was therefore to measure angiogenesis in two anatomically closely related however prognostically different types of pancreatic cancer, pancreatic head and periampullary cancer, and investigate its relation with outcome. Vessels were stained by CD31 on original paraffin embedded tissue from 206 patients with microscopic radical resection (R0) of pancreatic head (n=98) or periampullary cancer (n=108). Angiogenesis was quantified by microvessel density (MVD) and measured by computerised image analysis of three randomly selected fields and investigated for associations with recurrence free survival (RFS), cancer specific survival (CSS), overall survival (OS) and conventional prognostic factors. MVD was heterogeneous both between and within tumours. A higher MVD was observed in periampullary cancers compared with pancreatic head cancers (p<.01). Furthermore, MVD was associated with lymph node involvement in pancreatic head (p=.014), but not in periampullary cancer (p=.55). Interestingly, MVD was not associated with RFS, CSS or with OS. In conclusion, angiogenesis is higher in periampullary cancer and although associated with nodal involvement in pancreatic head cancer, pancreatic cancer prognosis seems indeed angiogenesis independent.

Enriching lipid nanovesicles with short-chain glucosylceramide improves doxorubicin delivery and efficacy in solid tumors.

For amphiphilic anticancer drugs, such as the anthracyclin doxorubicin (Dox), uptake by tumor cells involves slow diffusion across the plasma membrane, a limiting factor in clinical oncology. Previously, we discovered that preinsertion of short-chain sphingolipids such as N-octanoyl-glucosylceramide (GC) in the tumor cell membrane enhances cellular Dox uptake. In the present study, we apply this strategy in vitro and in vivo by coadministering GC and Dox in a lipid nanovesicle (LNV). GC enrichment of Dox-LNVs strongly enhanced in vitro cytotoxicity toward B16 melanoma and A431 carcinoma, as evidenced by 6-fold decreased IC(50) values compared with Dox-LNVs. This correlated with enhanced cellular Dox uptake observed by confocal microscopy. Intravital optical imaging in window chamber-bearing mice with orthotopically implanted B16 melanoma demonstrated enhanced GC-mediated Dox delivery to tumor cells. Treatment of nude mice bearing human A431 xenografts with 6 mg/kg GC-Dox-LNVs almost doubled the tumor growth delay compared with Dox-LNVs. A second administration of 5 mg/kg after 3 d induced even 3-fold delay in tumor growth, while no systemic toxicity was found. GC-enriched Dox-LNVs displayed superior in vitro and in vivo antitumor activity, without systemic toxicity. This new drug delivery concept, aiming at increased membrane permeability for amphiphilic drugs, provides an opportunity to improve cancer chemotherapy.

Differential expression and prognostic value of HMGA1 in pancreatic head and periampullary cancer.

The high mortality rate and minimal progress made in the treatment of pancreatic cancer over the last few decades, warrant an alternative approach. Treatment protocols should be individualised to the patient guided by prognostic markers. A particularly interesting target would be the architectural transcription factor high mobility group A1 (HMGA1), that is low or undetectable in normal tissue, induced during neoplastic transformation and consequently often exceptionally high in cancer. The aim of the current study was therefore to determine the differential expression of HMGA1 in pancreatic head and periampullary cancer and investigate its relation with outcome. HMGA1 expression was determined by immunohistochemistry on original paraffin embedded tissue from 99 pancreatic head- and 112 periampullary cancers (with R0). Expression was investigated for associations with recurrence free (RFS), cancer specific (CSS) and overall survival (OS) and conventional prognostic factors. HMGA1 was expressed in 47% and 26% of pancreatic head- and periampullary cancer, respectively and associated with poor RFS, CSS and OS in periampullary cancer. CSS 5years following surgery was 25% and 44% for patients with tumours which were positive or negative for HMGA1 protein, respectively. HMGA1 expression was not associated with survival in pancreatic head cancer. In conclusion HMGA1 was identified as an independent prognostic marker predicting poor outcome in periampullary cancer. Although expressed to a higher extent as compared to periampullary cancer, HMGA1 was not associated with survival in pancreatic head cancer.