Heat-Triggered Release of Dexamethasone from Thermosensitive Liposomes Using Prodrugs or Excipients.

Dexamethasone (DXM) is a potent glucocorticoid with an anti-inflammatory and anti-angiogenic activity which is widely clinically used. Systemic side effects limit the long-term use of DXM in patients requiring formulations which deliver and selectively release the drug to the diseased tissues. This in vitro study compares the suitability of DXM and commonly used prodrugs dexamethasone-21-phosphate (DXMP) and dexamethasone-21-palmitate (DP) as well as DXM complexed by 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD) for the use in thermosensitive liposomes (TSL). DXM showed a poor retention and a low final drug:lipid ratio in a 1,2-dipalmitoyl-sn­glycero-3-phosphodiglycerol-based TSL (DPPG2-TSL) and a low-temperature sensitive liposome (LTSL). In contrast to DXM, DXMP and DP were stably retained at 37 °C in TSL in serum and could be encapsulated with high drug:lipid ratios in DPPG2-TSL and LTSL. DXMP showed a rapid release at mild hyperthermia (HT) from both TSL in serum, whereas DP remained incorporated in the TSL bilayer. According to release experiments with carboxyfluorescein (CF), HP-γ-CD and 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) are suitable vehicles for the loading of DXM into DPPG2-TSL and LTSL. Complexation of DXM with HP-γ-CD increased the aqueous solubility of the drug leading to approx. ten times higher DXM:lipid ratio in DPPG2-TSL and LTSL in comparison to un-complexed DXM. Both DXM and HP-γ-CD showed increased release at HT in comparison to 37 °C in serum. In conclusion, DXMP and DXM complexed by HP-γ-CD represent promising candidates for TSL delivery.

Evaluation of release and pharmacokinetics of hexadecylphosphocholine (miltefosine) in phosphatidyldiglycerol-based thermosensitive liposomes.

Hexadecylphosphocholine (HePC, Miltefosine) is a drug from the class of alkylphosphocholines with an antineoplastic and antiprotozoal activity. We previously reported that HePC uptake from thermosensitive liposomes (TSL) containing 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) into cancer cells is accelerated at mild hyperthermia (HT) resulting in increased cytotoxicity. In this study, we compared HePC release of different TSL formulations in serum. HePC showed rapid but incomplete release below the transition temperature (Tm) of investigated TSL formulations in serum. Short heating (5 min) to 42 °C increased HePC release from DPPG2-TSL (Tm = 41 °C) by a factor of two in comparison to body temperature (37 °C). Bovine serum albumin (BSA) induced HePC release from DPPG2-TSL comparable to serum. Furthermore, multilamellar vesicles (MLV) were capable to extract HePC from DPPG2-TSL in a concentration- and temperature-dependent manner. Repetitive exposure of DPPG2-TSL to MLV at 37 °C led to a fast initial release of HePC which slowed down after subsequent extraction cycles finally reaching approx. 50% HePC release. A pharmacokinetic study in rats revealed a biphasic pattern with an immediate clearance of approx. 50% HePC whereas the remaining 50% HePC showed a prolonged circulation time. We speculate that HePC located in the external leaflet of DPPG2-TSL is rapidly released upon contact with suitable biological acceptors. As demonstrated by MLV transfer experiments, asymmetric incorporation of HePC into the internal leaflet of DPPG2-TSL might improve HePC retention in presence of complex biological media and still give rise to HT-induced HePC release.

Effects of Surface Charge, PEGylation and Functionalization with Dipalmitoylphosphatidyldiglycerol on Liposome-Cell Interactions and Local Drug Delivery to Solid Tumors via Thermosensitive Liposomes.

PURPOSE: Previous studies demonstrated the possibility of targeting tumor-angiogenic endothelial cells with positively charged nanocarriers, such as cationic liposomes. We investigated the active targeting potential of positively charged nanoparticles in combination with the heat-induced drug release function of thermosensitive liposomes (TSL). This novel dual-targeted approach via cationic TSL (CTSL) was thoroughly explored using either a novel synthetic phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) or a conventional polyethylene glycol (PEG) surface modification. Anionic particles containing either DPPG2 or PEG were also included in the study to highlight difference in tumor enrichment driven by surface charge. With this study, we aim to provide a deep insight into the main differences between DPPG2- and PEG-functionalized liposomes, focusing on the delivery of a well-known cytotoxic drug (doxorubicin; DOX) in combination with local hyperthermia (HT, 41-43°C). MATERIALS AND METHODS: DPPG2- and PEG-based cationic TSLs (PG2-CTSL/PEG-CTSL) were thoroughly analyzed for size, surface charge, and heat-triggered DOX release. Cancer cell targeting and DOX delivery was evaluated by FACS, fluorescence imaging, and HPLC. In vivo particle behavior was analyzed by assessing DOX biodistribution with local HT application in tumor-bearing animals. RESULTS: The absence of PEG in PG2-CTSL promoted more efficient liposome-cell interactions, resulting in a higher DOX delivery and cancer cell toxicity compared with PEG-CTSL. By exploiting the dual-targeting function of CTSLs, we were able to selectively trigger DOX release in the intracellular compartment by HT. When tested in vivo, local HT promoted an increase in intratumoral DOX levels for all (C)TSLs tested, with DOX enrichment factors ranging from 3 to 14-fold depending on the type of formulation. CONCLUSION: Cationic particles showed lower hemocompatibility than their anionic counterparts, which was partially mitigated when PEG was grafted on the liposome surface. DPPG2-based anionic TSL showed optimal local drug delivery compared to all other formulations tested, demonstrating the potential advantages of using DPPG2 lipid in designing liposomes for tumor-targeted applications.

Mechanistic investigation of thermosensitive liposome immunogenicity and understanding the drivers for circulation half-life: A polyethylene glycol versus 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol study.

Various thermosensitive liposome (TSL) formulations have been described to date and it is currently unclear which are optimal for solid tumor treatment. Sufficient circulation half-life is important and most liposomes obtain this by polyethylene glycol (PEG) surface modification. 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) has been described as a promising alternative which increases TSL circulation half-life and facilitates rapid drug release under mild hyperthermia at 20-30 mol%. The present work describes an investigation of the DPPG2-TSL protein corona, blood cell interactions, complement activation in human plasma/blood and hypersensitivity reactions in rats. Furthermore, accelerated blood clearance (ABC) was investigated to obtain a complete assessment of DPPG2-TSL interactions with components of the blood and identify drivers for circulation half-life. A higher mol% DPPG2 increased Apolipoprotein E (ApoE) adsorption and decreased complement activation and granulocyte interaction in vitro. In contrast to PEG-TSL, DPPG2-TSL showed no ABC effect. In vivo hypersensitivity assessment by eicosanoid measurements, platelet and lymphocyte counting resembled the results of in vitro complement activation assays although here all DPPG2-TSL formulations induced hypersensitive responses upon i.v. administration. Prolonged circulation half-life of DPPG2-TSL may be ApoE-induced and the absent ABC effect demonstrates an advantage over PEG-TSL. Low complement activation in human plasma and blood for 20-30 mol% DPPG2-TSL presents a unique formulation attribute with the potential to strengthen clinical evaluation.

Effective control of tumor growth through spatial and temporal control of theranostic sodium iodide symporter (NIS) gene expression using a heat-inducible gene promoter in engineered mesenchymal stem cells.

Purpose: The tumor homing characteristics of mesenchymal stem cells (MSCs) make them attractive vehicles for the tumor-specific delivery of therapeutic agents, such as the sodium iodide symporter (NIS). NIS is a theranostic protein that allows non-invasive monitoring of the in vivo biodistribution of functional NIS expression by radioiodine imaging as well as the therapeutic application of 131I. To gain local and temporal control of transgene expression, and thereby improve tumor selectivity, we engineered MSCs to express the NIS gene under control of a heat-inducible HSP70B promoter (HSP70B-NIS-MSCs). Experimental Design: NIS induction in heat-treated HSP70B-NIS-MSCs was verified by 125I uptake assay, RT-PCR, Western blot and immunofluorescence staining. HSP70B-NIS-MSCs were then injected i.v. into mice carrying subcutaneous hepatocellular carcinoma HuH7 xenografts, and hyperthermia (1 h at 41°C) was locally applied to the tumor. 0 - 72 h later radioiodine uptake was assessed by 123I-scintigraphy. The most effective uptake regime was then selected for 131I therapy. Results: The HSP70B promoter showed low basal activity in vitro and was significantly induced in response to heat. In vivo, the highest tumoral iodine accumulation was seen 12 h after application of hyperthermia. HSP70B-NIS-MSC-mediated 131I therapy combined with hyperthermia resulted in a significantly reduced tumor growth with prolonged survival as compared to control groups. Conclusions: The heat-inducible HSP70B promoter allows hyperthermia-induced spatial and temporal control of MSC-mediated theranostic NIS gene radiotherapy with efficient tumor-selective and temperature-dependent accumulation of radioiodine in heat-treated tumors.

Development and evaluation of an isolated limb infusion model for investigation of drug delivery kinetics to solid tumors by thermosensitive liposomes and hyperthermia.

The combined administration of thermosensitive liposomes (TSLs) and hyperthermia (HT) has been increasingly shown to be a powerful tool for the treatment of solid tumors. At present, it is hypothesized that the circulation of TSLs through the vasculature of a heated tumor results in the rapid release of the entrapped drug, followed by its uptake and distribution within the tumor microenvironment. However, simple questions on the transport kinetics of TSLs through the heated tumor and how much drug is retained upon passage of TSLs through the tumor microcirculation have not been investigated in an experimental setting to-date. The present work describes a novel methodology for investigating these parameters by isolated limb infusion (ILI), developed in a rat model of sarcoma. This approach was used to assess the efficacy of Doxorubicin (Dox) delivery by TSL in a heated (42°C) tumor following a single passage of TSL through the tumor vasculature. Analysis of the effluent post-ILI, whole-tumor histological sections, and tissue homogenates revealed that upon a single passage, Dox delivery by TSL at 42°C did not exceed delivery under conventional (i.e. free Dox) or physiological (i.e. TSL at 37°C, or normothermia; NT) conditions. In fact, mathematical modeling demonstrated that at least thirteen passages are required to obtain the intratumoral Dox levels typically achieved using TSL (i.e. ~5%ID/g). Overall, this work investigates TSL-based determinants for achieving efficacious drug delivery using a model of ILI in tumor-bearing rats and the results bear important implications for TSL disposition in vivo.

Comparing the therapeutic potential of thermosensitive liposomes and hyperthermia in two distinct subtypes of breast cancer.

Local drug delivery of Doxorubicin (Dox) with thermosensitive liposomes (TSL) and hyperthermia (HT) has shown preclinically to achieve high local drug concentrations with good therapeutic efficacy. Currently, this is clinically studied for treatment of chest wall recurrence of breast cancer, however with various outcomes. This study examines the potency of neoadjuvant TSL HT combination therapy in two orthotopic mouse models of human breast cancer, MDA-MB-231 and T-47D, which morphologically correlate to mesenchymal and epithelial phenotypes, respectively. Both cell lines showed improved in vitro chemosensitivity and Dox uptake at HT. Dox-loaded TSL (TSLDox) was stable in vitro in FBS, BALB/c-nu plasma and human plasma, although release of the drug at HT was incomplete for the latter two. Combination treatment with TSLDox and HT in vivo was significantly more effective against MDA-MB-231 tumors, whereas T-47D tumors showed no significant therapeutic response. Ex vivo investigation revealed a higher mean vessel density and poorly differentiated extracellular matrix (ECM) in MDA-MB-231 tumors relative to T-47D tumors. Although in vitro results of the TSLDox and HT treatment were favorable for both cell types, the therapeutic efficacy in vivo was remarkably different. The well-differentiated and slowly-growing T-47D tumors may provide a microenvironment that limits drug delivery to the target cell and therefore renders the therapy ineffective. Mesenchymal and invasive MDA-MB-231 tumors display higher vascularization and less mature ECM, significantly enhancing tumor response to TSLDox and HT treatment. These results yield insight into the efficacy of TSL treatment within different tumor microenvironments, and further advance our understanding of factors that contribute to heterogeneous therapeutic outcomes in clinical trials.

Investigation of Particle Accumulation, Chemosensitivity and Thermosensitivity for Effective Solid Tumor Therapy Using Thermosensitive Liposomes and Hyperthermia.

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 reach therapeutic relevant concentrations in the tumor tissue in a 2-step delivery protocol due to the leaking of the drug from its liposomal carrier providing an explanation for the observed lack of efficacy.

In depth study on thermosensitive liposomes: Optimizing formulations for tumor specific therapy and in vitro to in vivo relations.

In numerous studies, thermosensitive liposomes (TSLs) for local heat-triggered delivery of Doxorubicin (Dox) to tumors have been investigated, with TSLs having different lipid formulations, drug loading methodology and testing procedures. To gain more insight in these parameters, we investigated TSLs with four variable DSPC-DPPC lipid ratios (50, 60, 70 or 80% DPPC and 5 mol% of DSPE-PEG2000) using either ammonium sulfate or a citrate buffer for Dox loading. Ammonium sulfate loading of Dox yielded more stable TSLs than citrate loading. At 37 °C, leakage was unnoticeable for all ammonium sulfate TSLs. At 42 °C, complete release occurred within seconds, except for 50% DPPC TSLs, where slow and incomplete release was observed in vitro but also in vivo using a dorsal skinfold window chamber. In contrast to in vitro assays, blood kinetics studies indicated a burst release of Dox upon injection and higher leakage for all TSLs. In therapeutic studies, hyperthermia in combination with TSLs repressed BFS-1 sarcoma growth. Our study shows that prediction of therapeutic efficacy purely based on differences found in vitro is difficult, instead, parameters obtained from pharmacokinetic studies in vivo, and the exact timing of the delivery protocol need to be taken into account.

Formulation and optimization of idarubicin thermosensitive liposomes provides ultrafast triggered release at mild hyperthermia and improves tumor response.

Drug delivery through thermosensitive liposomes (TSL) in combination with hyperthermia (HT) has shown great potential. HT can be applied locally forcing TSL to release their content in the heated tumor resulting in high peak concentrations. To perform optimally the drug is ideally released fast (seconds) and taken up rapidly by tumor cells. The aim of this study was to develop a novel thermosensitive liposome formulation of the anthracycline idarubicin (IDA-TSL). The hydrophobicity of idarubicin may improve its release from liposomes and subsequently rapid cellular uptake when combined mild hyperthermia. Here, we investigated a series of parameters to optimize IDA-TSL formulation. The results show that the optimal formulation for IDA-TSL is DPPC/DSPC/DSPE-PEG (6/3.5/0.5 mol%), with ammonium EDTA of 6.5 pH as loading buffer and a size of ~85 nm. In vitro studies demonstrated minimal leakage of ~20% in FCS at 37 °C for 1h, while an ultrafast and complete triggered release of IDA was observed at 42 °C. On tumor cells IDA-TSL showed comparable cytotoxicity to free IDA at 42 °C, but low cytotoxicity at 37 °C. Intravital microscopy imaging demonstrated an efficient in vivo intravascular triggered drug release of IDA-TSL under mild hyperthermia, and a subsequent massive IDA uptake by tumor cells. In animal efficacy studies, IDA-TSL plus mild HT demonstrated prominent tumor growth inhibition and superior survival rate over free IDA with HT or a clinically used Doxil treatment. These results suggest beneficial potential of IDA-TSL combined with local mild HT.