Thermosensitive Liposome Formulation Research Articles

Triggered release from thermosensitive liposomes improves tumor targeting of vinorelbine.

Triggered drug delivery strategies have been shown to enhance drug accumulation at target diseased sites in comparison to administration of free drug. In particular, many studies have demonstrated improved targetability of chemotherapeutics when delivered via thermosensitive liposomes. However, most studies continue to focus on encapsulating doxorubicin while many other drugs would benefit from this targeted and localized delivery approach. The proposed study explores the therapeutic potential of a thermosensitive liposome formulation of the commonly used chemotherapy drug vinorelbine in combination with mild hyperthermia (39-43°C) in a murine model of rhabdomyosarcoma. Rhabdomyosarcoma, the most common soft tissue sarcoma in children, is largely treated using conventional chemotherapy which is associated with significant adverse long-term sequelae. In this study, mild hyperthermia was pursued as a non-invasive, non-toxic means to improve the efficacy and safety profiles of vinorelbine. Thorough assessment of the pharmacokinetics, biodistribution, efficacy and toxicity of vinorelbine administered in the thermosensitive liposome formulation was compared to administration in a traditional, non-thermosensitive liposome formulation. This study shows the potential of an advanced formulation technology in combination with mild hyperthermia as a means to target an untargeted therapeutic agent and result in a significant improvement in its therapeutic index.

Review of the Delivery Kinetics of Thermosensitive Liposomes.

Thermosensitive liposomes (TSL) are triggered nanoparticles that release the encapsulated drug in response to hyperthermia. Combined with localized hyperthermia, TSL enabled loco-regional drug delivery to tumors with reduced systemic toxicities. More recent TSL formulations are based on intravascular triggered release, where drug release occurs within the microvasculature. Thus, this delivery strategy does not require enhanced permeability and retention (EPR). Compared to traditional nanoparticle drug delivery systems based on EPR with passive or active tumor targeting (typically <5%ID/g tumor), TSL can achieve superior tumor drug uptake (>10%ID/g tumor). Numerous TSL formulations have been combined with various drugs and hyperthermia devices in preclinical and clinical studies over the last four decades. Here, we review how the properties of TSL dictate delivery and discuss the advantages of rapid drug release from TSL. We show the benefits of selecting a drug with rapid extraction by tissue, and with quick cellular uptake. Furthermore, the optimal characteristics of hyperthermia devices are reviewed, and impact of tumor biology and cancer cell characteristics are discussed. Thus, this review provides guidelines on how to improve drug delivery with TSL by optimizing the combination of TSL, drug, and hyperthermia method. Many of the concepts discussed are applicable to a variety of other triggered drug delivery systems.

Harnessing immunotherapy to enhance the systemic anti-tumor effects of thermosensitive liposomes.

Chemotherapy plays an important role in debulking tumors in advance of surgery and/or radiotherapy, tackling residual disease, and treating metastatic disease. In recent years many promising advanced drug delivery strategies have emerged that offer more targeted delivery approaches to chemotherapy treatment. For example, thermosensitive liposome-mediated drug delivery in combination with localized mild hyperthermia can increase local drug concentrations resulting in a reduction in systemic toxicity and an improvement in local disease control. However, the majority of solid tumor-associated deaths are due to metastatic spread. A therapeutic approach focused on a localized target area harbors the risk of overlooking and undertreating potential metastatic spread. Previous studies reported systemic, albeit limited, anti-tumor effects following treatment with thermosensitive liposomal chemotherapy and localized mild hyperthermia. This work explores the systemic treatment capabilities of a thermosensitive liposome formulation of the vinca alkaloid vinorelbine in combination with mild hyperthermia in an immunocompetent murine model of rhabdomyosarcoma. This treatment approach was found to be highly effective at heated, primary tumor sites. However, it demonstrated limited anti-tumor effects in secondary, distant tumors. As a result, the addition of immune checkpoint inhibition therapy was pursued to further enhance the systemic anti-tumor effect of this treatment approach. Once combined with immune checkpoint inhibition therapy, a significant improvement in systemic treatment capability was achieved. We believe this is one of the first studies to demonstrate that a triple combination of thermosensitive liposomes, localized mild hyperthermia, and immune checkpoint inhibition therapy can enhance the systemic treatment capabilities of thermosensitive liposomes.

Turning down the heat: The case for mild hyperthermia and thermosensitive liposomes

“A single disappointing study does not mean an end to the future of ThermoDox®”, writes Michael Tardugno (CEO of Celsion Corporation), after announcing the termination of Celsion's second Phase III clinical trial. The OPTIMA trial, as it was known, evaluated their thermosensitive liposome (TSL) formulation of doxorubicin (ThermoDox®) in combination with radiofrequency ablation for the treatment of hepatocellular carcinoma (HCC). The purpose of this perspective is to review the case of ThermoDox and to address questions related to its clinical translation. Specifically, what has prevented the clinical translation of this once highly regarded breakthrough technology? Is this the end of TSLs? What can we learn from the challenges faced in the clinical development of this multi-modal therapy? As formulation scientists working in the field, we continue to believe that heat-triggered drug delivery platforms have tremendous potential as chemotherapy. Herein, we highlight potential limitations in the design of many of the Thermodox clinical trials, and we propose that despite these setbacks, TSLs have the potential to become an effective component of cancer therapy.

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 (5min) 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.

Nanoprecipitation preparation of low temperature-sensitive magnetoliposomes

Lysolipid-containing thermosensitive liposomes (LTSL) have gained attention for triggered release of chemotherapeutics. Superparamagnetic iron oxide nanoparticles (SPION) offers multimodal imaging and hyperthermia therapy opportunities as a promising theranostic agent. Combining LTSL with SPION may further enhance their performance and functionality of LTSL. However, a major challenge in clinical translation of nanomedicine is the poor scalability and complexity of their preparation process. Exploiting the nature of self-assembly, nanoprecipitation is a simple and scalable technique for preparing liposomes. Herein, we developed a novel SPION-incorporated lysolipid-containing thermosensitive liposome (mLTSL10) formulation using nanoprecipitation. The formulation and processing parameters were carefully designed to ensure high reproducibility and stability of mLTSL10. The effect of solvent, aqueous-to-organic volume ratio, SPION concentration on the mLTSL10 size and dispersity was investigated. mLTSL10 were successfully prepared with a small size (∼100 nm), phase transition temperature at around 42 °C, and high doxorubicin encapsulation efficiency. Indifferent from blank LTSL, we demonstrated that mLTSL10 combining the functionality of both LTSL and SPION can be successfully prepared using a scalable nanoprecipitation approach.

Determining critical parameters that influence in vitro performance characteristics of a thermosensitive liposome formulation of vinorelbine

Studies have demonstrated the advantages associated with heat-triggered drug delivery via thermosensitive liposomes for the treatment of localized cancer. Challenges that traditional liposomal systems face such as limited drug release and homogeneous distribution throughout the region of interest can potentially be overcome when triggering intravascular drug release. The most prominent example is a thermosensitive liposome formulation of doxorubicin known as ThermoDox®. Many other drugs may benefit from the same targeted and localized delivery approach using thermosensitive liposomes as it can result in a significant improvement in the therapeutic index. Vinorelbine is a semi-synthetic vinca alkaloid which has shown to be active in a broad range of cancers. Several liposome formulations encapsulating vinorelbine have been developed as a means to reduce systemic drug exposure. The present study takes a systematic approach in exploring formulation and drug loading parameters and their influence on performance characteristics of a rapidly releasing thermosensitive liposome formulation of vinorelbine. More broadly, this study shows that trends observed for non-thermosensitive liposome formulations of specific drugs (i.e. vinorelbine) can not be easily translated to their thermosensitive counterparts. The profound impact of the presence of albumin on stability and in vitro release is also highlighted. This is of significance given that a number of recent reports examine drug release in the absence of biologically relevant components. As a result, a strong recommendation emanating from this is a thorough challenge of the liposome formulation in vitro in order to gain a better understanding of its likely behaviour in vivo as well as potential for future clinical translation.

Heat-activated drug delivery increases tumor accumulation of synergistic chemotherapies

Doxorubicin is a clinically important anthracycline chemotherapeutic agent that is used to treat many cancers. Nanomedicine formulations including Doxil® and ThermoDox® have been developed to mitigate doxorubicin cardiotoxicity. Doxil is used clinically to treat ovarian cancer, AIDS-related Kaposi's sarcoma, and multiple myeloma, but there is evidence that therapeutic efficacy is hampered by lack of drug release. ThermoDox is a lipid-based heat-activated formulation of doxorubicin that relies on externally applied energy to increase tissue temperatures and efficiently trigger drug release, thereby affording therapeutic advantages compared to Doxil. However, elevating tissue temperatures is a complex treatment process requiring significant time, cost, and expertise compared to standard intravenous chemotherapy. This work endeavors to develop a companion therapeutic to ThermoDox that also relies on heat-triggered release in order to increase the therapeutic index of doxorubicin. To this end, a thermosensitive liposome formulation of the heat shock protein 90 inhibitor alvespimycin has been developed and characterized. This research demonstrates that both doxorubicin and alvespimycin are potent anti-cancer agents and that heat amplifies their cytotoxic effects. Furthermore, the two drugs are proven to act synergistically when cancer cells are treated with the drugs in combination. The formulation of alvespimycin was rationally designed to exhibit similar pharmacokinetics and drug release kinetics compared to ThermoDox, enabling the two drugs to be delivered to heated tumors at similar efficiencies resulting in control of a particular synergistic ratio of drugs. In vivo measurements demonstrated effective heat-mediated triggering of doxorubicin and alvespimycin release from thermosensitive liposomes within tumor vasculature. This treatment strategy resulted in a ~10-fold increase in drug concentration within tumors compared to free drug administered without tumor heating.

Photothermally Amplified Therapeutic Liposomes for Effective Combination Treatment of Cancer.

Near-infrared photothermal therapy has been investigated extensively with regard to selective tumor eradication, yet its clinical translation has been limited because of the absence of FDA-approvable agents with effective phototherapeutic function and minimal systemic toxicity. In this work, we developed photothermally amplified therapeutic liposomes in an attempt to synergize chemotherapy and hyperthermia for effective cancer phototherapy. The anticancer drug cisplatin and the photothermal agent indocyanine green (ICG) were encapsulated in a thermosensitive liposomal formulation at the lipid/ICG ratio maximizing the ICG loading efficiency. These liposomes released cytotoxic cisplatin molecules selectively via ICG-mediated photothermal stimulation. In phototherapeutic studies, these liposomes amplified therapeutic effects both in vitro in cancer cells and in vivo in mouse tumor models significantly over chemotherapy or photothermal therapy alone. We believe that these photothermally amplified therapeutic liposomes composed solely of already FDA-approved components (cisplatin, ICG, and phospholipids) have enormous potential for clinical translation in treating various tumors compatible with laser irradiation.

Thermosensitive Liposomes for Image-Guided Drug Delivery.

Liposomes have been employed as cancer therapy clinically since the 1990s, with the primary benefit of reduced toxicity but no appreciable efficacy improvement. Thermosensitive liposomes (TSLs) are specifically formulated such that they release the encapsulated drug when exposed to hyperthermic temperatures in the fever range (~40-42°C) and have been investigated as cancer therapy for several decades, with first clinical trials initiated in the last decade. Combined with localized hyperthermia, TSLs allow precise drug delivery to a targeted region. Typically, the targeted tissue is exposed to localized hyperthermia facilitated by an image-guided hyperthermia device. Thus, TSLs enable image-guided drug delivery where drug is delivered to a tissue region identified by medical imaging. Recent TSL formulations are based on the more recent paradigm of intravascular triggered release, where drug is released rapidly (within seconds) while TSLs pass through the vasculature of the heated tissue region. The drug released within the blood then extravasates and is taken up by cancer cells. These TSLs enable up to 20-30 times higher tumor drug uptake compared to infusion of unencapsulated drug, and the dose locally delivered to the heated region can be modulated based on heating duration. This chapter reviews various TSL formulations, the different anticancer agents that have been encapsulated, as well as targeted cancer types. Further, the various hyperthermia devices that have been used for image-guided hyperthermia are reviewed, focusing on those that have been employed in human patients.

Tumor microenvironment determines response to a heat-activated thermosensitive liposome formulation of cisplatin in cervical carcinoma

Significant heterogeneity in the tumor microenvironment of human cervical cancer patients is known to challenge treatment outcomes in this population. The current standard of care for cervical cancer patients is radiation therapy and concurrent cisplatin (CDDP) chemotherapy. Yet this treatment strategy fails to control loco-regional disease in 10–30% of patients. In order to improve the loco-regional control rate, a thermosensitive liposome formulation of CDDP (HTLC) was developed to increase local concentrations of drug in response to mild hyperthermia (HT). The HTLC formulation in combination with local HT demonstrated a significant therapeutic advantage in comparison to free drug and Lipoplatin™ in ME-180 and SiHa xenograft models of human cervical cancer, as well as in four distinct cervical patient-derived xenograft models. Differential response to HTLC+HT treatment was observed between the ME-180 and SiHa tumor models. Tumor doubling time, in vitro cell sensitivity, and tumor drug accumulation were found to be non-predictive of treatment efficacy. Rather, tumor microenvironment parameters, in particular elevated levels of both tumor hypoxia and associated stromal content, were found to serve as the overriding factors that limit drug efficacy. The prognostic value of these markers may enable stratification of cervical cancer patients for implementation of personalized medicine in the clinical setting.

The effect of thermosensitive liposomal formulations on loading and release of high molecular weight biomolecules

Thermosensitive liposomes are clinically-relevant nanocarriers which have been used to deliver chemotherapeutic agents to tumors in combination with local hyperthermia. However, the encapsulation and release of macromolecular therapeutic agents (proteins, nucleic acids, bioactive polymers) is often hindered by their instability during the liposome formation as well as by the low encapsulation efficiency.The objective of this study was to investigate the influence of the thermosensitive liposomal formulation on the encapsulation and release of low and high molecular weight hydrophilic drugs, in order to identify the key parameters to control during nanocarrier design, depending on the specific drug delivery application.Thermosensitive liposomes with different formulations were prepared through the combinations of different lipids, including dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), cholesterol (Chol), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (P-Lyso-PC), and the PEGylated lipid distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(PEG)-2000 (DSPE-PEG2000). The thin film hydration method was used for liposome preparation and loading of different water soluble molecules. The encapsulation efficiency and release profiles were investigated for a low molecular weight compound such as carboxyfluorescein (CF), proteins (albumin), and hydrophilic polymers which do not interact with the lipid bilayer, such as a linear dextran and a poly(ethylene glycol)-based star polymer. An optimised liposomal formulation [DPPC/P-lyso-PC/DSPE-PEG2000 90/10/4 (mol/mol) (LTSL)] was chosen for further application in encapsulating therapeutic proteins, such as lysozyme and the brain-derived neurotrophic factor (BDNF), which are recognized as drug carriers and potential therapeutic agents for kidney diseases and neurological disorders.

Surrogate MRI markers for hyperthermia-induced release of doxorubicin from thermosensitive liposomes in tumors

The efficacy of systemically applied, classical anti-cancer drugs is limited by insufficient selectivity to the tumor and the applicable dose is limited by side effects. Efficacy could be further improved by targeting of the drug to the tumor. Using thermosensitive liposomes (TSL) as a drug carrier, targeting is achieved by control of temperature in the target volume. In such an approach, effective local hyperthermia (40–43°C) (HT) of the tumor is considered essential but technically challenging. Thus, visualization of local heating and drug release using TSL is considered an important tool for further improvement. Visualization and feasibility of chemodosimetry by magnetic resonance imaging (MRI) has previously been demonstrated using TSL encapsulating both, contrast agent (CA) and doxorubicin (DOX) simultaneously in the same TSL. Dosimetry has been facilitated using T1-relaxation time change as a surrogate marker for DOX deposition in the tumor. To allow higher loading of the TSL and to simplify clinical development of new TSL formulations a new approach using a mixture of TSL either loaded with DOX or MRI-CA is suggested. This was successfully tested using phosphatidyldiglycerol-based TSL (DPPG2-TSL) in Brown Norway rats with syngeneic soft tissue sarcomas (BN175) implanted at both hind legs. After intravenous application of DOX-TSL and CA-TSL, heating of one tumor above 40°C for 1h using laser light resulted in highly selective DOX uptake. The DOX-concentration in the heated tumor tissue compared to the non-heated tumor showed an almost 10-fold increase. T1 and additional MRI surrogate parameters such as signal phase change were correlated to intratumoral DOX concentration. Visualization of DOX delivery in the sense of a chemodosimetry was demonstrated. Although phase-based MR-thermometry was affected by CA-TSL, phase information was found suitable for DOX concentration assessment. Local differences of DOX concentration in the tumors indicated the need for visualization of drug release for further improvement of targeting.

Thermosensitive liposomal cisplatin in combination with local hyperthermia results in tumor growth delay and changes in tumor microenvironment in xenograft models of lung carcinoma*

Treatment efficacy of a heat-activated thermosensitive liposome formulation of cisplatin (CDDP), known as HTLC, was determined in xenograft models of non-small-cell lung carcinoma. The short-term impact of local hyperthermia (HT) on tumor morphology, microvessel density and local inflammatory response was also evaluated. The HTLC formulation in combination with local HT resulted in a significant advantage in therapeutic effect in comparison with free drug and a non-thermosensitive liposome formulation of CDDP (i.e. LipoplatinTM) when administered at their maximum tolerated doses. Local HT-induced widespread cell necrosis and a significant reduction in microvessel density in the necrotic regions of tumors. CD11b-expressing innate leukocytes were demonstrated to infiltrate and reside preferentially at the necrotic rim of tumors, likely as a means to phagocytose-damaged tissue. Colocalization of CD11b with a marker of DNA damage (i.e. γH2AX) revealed a small portion of CD11b-expressing leukocytes that were possibly undergoing apoptosis as a result of HT-induced damage and/or the short lifespan of leukocytes. Overall, HT-induced tissue damage (i.e. at 24-h post-treatment) alone did not result in significant improvements in treatment effect, rather, the enhancement in tumor drug availability was correlated with improved therapeutic outcomes.

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance.

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect resulting in significant reductions in systemic toxicity. Nonetheless, insufficient release of encapsulated drug from liposomes has limited their clinical efficacy. Temperature-sensitive liposomes have been engineered to provide site-specific release of drug in order to overcome the problem of limited tumor drug bioavailability. Our lab has designed and developed a heat-activated thermosensitive liposome formulation of cisplatin (CDDP), known as HTLC, to provide triggered release of CDDP at solid tumors. Heat-activated delivery in vivo was achieved in murine models using a custom-built laser-based heating apparatus that provides a conformal heating pattern at the tumor site as confirmed by MR thermometry (MRT). A fiber optic temperature monitoring device was used to measure the temperature in real-time during the entire heating period with online adjustment of heat delivery by alternating the laser power. Drug delivery was optimized under magnetic resonance (MR) image guidance by co-encapsulation of an MR contrast agent (i.e., gadoteridol) along with CDDP into the thermosensitive liposomes as a means to validate the heating protocol and to assess tumor accumulation. The heating protocol consisted of a preheating period of 5 min prior to administration of HTLC and 20 min heating post-injection. This heating protocol resulted in effective release of the encapsulated agents with the highest MR signal change observed in the heated tumor in comparison to the unheated tumor and muscle. This study demonstrated the successful application of the laser-based heating apparatus for preclinical thermosensitive liposome development and the importance of MR-guided validation of the heating protocol for optimization of drug delivery.

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 (6/3.5/0.5mol%), with ammonium EDTA of 6.5 pH as loading buffer and a size of ~85nm. 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.

Aptamer-Modified Temperature-Sensitive Liposomal Contrast Agent for Magnetic Resonance Imaging.

A novel aptamer modified thermosensitive liposome was designed as an efficient magnetic resonance imaging probe. In this paper, Gd-DTPA was encapsulated into an optimized thermosensitive liposome (TSL) formulation, followed by conjugation with AS1411 for specific targeting against tumor cells that overexpress nucleolin receptors. The resulting liposomes were extensively characterized in vitro as a contrast agent. As-prepared TSLs-AS1411 had a diameter about 136.1 nm. No obvious cytotoxicity was observed from MTT assay, which illustrated that the liposomes exhibited excellent biocompatibility. Compared to the control incubation at 37 °C, the liposomes modified with AS1411 exhibited much higher T1 relaxivity in MCF-7 cells incubated at 42 °C. These data indicate that the Gd-encapsulated TSLs-AS1411 may be a promising tool in early cancer diagnosis.

Development of a bone targeted thermosensitive liposomal doxorubicin formulation based on a bisphosphonate modified non-ionic surfactant

Bone is among the most common sites of metastasis in cancer patients, so it is an urgent need to develop drug delivery systems targeting tumor bone metastasis with the feature of controlled release. This study aimed to delivery of thermosensitive liposomal doxorubicin to bone for tumor metastasis treatment. First, Brij78 (polyoxyethylene stearyl ether) was conjugated with Pamidronate (Pa). By incorporating Pa-Brij78 to DPPC/Chol liposomes, we developed Pa surface functionalized liposomes. The Pa-Brij78/DPPC/Chol liposomes (PB-liposomes) exhibited a stronger binding affinity to hydroxyapatite (HA), a major component of bone, than Brij78/DPPC/Chol liposomes (B-liposomes). Doxorubicin (Dox) was then encapsulated in PB-liposomes and the results demonstrated complete release of Dox from PB-liposomes or the complex of HA/PB-liposomes within 10 min at 42 °C. Next, human lung cancer A549 cells were treated with the thermosensitive complex of HA/PB-liposomes/Dox to mimic tumor bone metastasis treatment through bone targeted delivery of therapeutic agents. Pre-incubation of HA/PB-liposomes/Dox with mild heat at 42 °C induced subsequent higher cytotoxicity to A549 cells than incubation of the same complex at 37 °C, suggesting more active drug release triggered by heat. In conclusion, we synthesized a novel surfactant Pa-Brij78 and it has the potential to be used for development of a bone targeted thermosensitive liposome formulation for treatment of tumor bone metastasis.

Indocyanine green loaded liposome nanocarriers for photodynamic therapy using human triple negative breast cancer cells

The goal of the current research is to evaluate the potential of photodynamic therapy (PDT) in the treatment of triple negative breast cancer (TNBC) with the development of a theranostic thermosensitive liposome platform to deliver indocyanine green (ICG) as the near-infrared (NIR) photosensitizer excited by an 808 nm diode laser. In the PDT protocol, an optimized thermosensitive liposome formulation is investigated to formulate ICG as the photosensitizer, which is exited by laser light at the wavelength of 808 nm delivered by a fiber-coupled laser system. ICG in both free solution and thermosensitive liposomal formulation were evaluated as the NIR photosensitizer and compared in the PDT treatment on a panel of triple negative breast cancer cell lines along with the nontumorigenic mammary epithelial cell line MCF-10A. In addition to cytotoxicity, and clonogenic survival assessment, the role of DNA double strand break damage was evaluated. Both MTT and clonogenic assays revealed that PDT using ICG inhibited the growth of several TNBC cell lines as well as the non-tumorigenic human breast epithelial cell line MCF-10A; and the liposomal formulation of ICG did not compromise the in vitro treatment potency, though free ICG performed slightly more effective in certain cell lines, but was not statistically significant. Cell viability was dose dependent in regards to ICG concentration and irradiation energy. Interestingly, PDT using the described protocol was more potent to inhibit the growth of MDA-MB-468 and HCC-1806 cells, coinciding with the observation that these cells are more sensitive toward DNA damaging agents. In comparison, cell lines HCC-70, BT-549, and MCF-10A were found to have less of an inhibitory effect. Furthermore, substantial DNA double strand breaks (DSBs) were observed 30 min after the PDT treatment via a γ-H2AX staining assay. PDT induced DNA damage has the potential to lead to mutagenicity, which may have various responses depending on the repair capabilities of the cells. Our results suggest that PDT using indocyanine green loaded liposomes were effective in inhibiting tumor cell growth to varying extents with higher responses observed for MDA-MB-468 and HCC-1806 cells.

Heat-activated thermosensitive liposomal cisplatin (HTLC) results in effective growth delay of cervical carcinoma in mice

Cisplatin (CDDP) has been identified as the primary chemotherapeutic agent for the treatment of cervical cancer, but dose limiting toxicity is a key issue associated with its clinical application. A suite of liposome formulations of CDDP has been developed in efforts to reduce systemic toxicity, but their therapeutic advantage over the free drug has been modest due to insufficient drug release at the tumor site. This report describes the development of a novel heat-activated thermosensitive liposome formulation containing CDDP (HTLC) designed to release approximately 90% of the loaded drug in less than 5min under mild heating conditions (42°C). Physico-chemical characteristics of HTLC were assessed in terms of gel to liquid crystalline phase transition temperature (Tm), drug loading efficiency, particle size, and stability. The pharmacokinetic profile and biodistribution of HTLC in non-tumor-bearing mice were evaluated over a 24h period. A sophisticated spatio-temporal elucidation of HTLC release in tumor-bearing mice was achieved by way of real-time monitoring using a magnetic resonance (MR) imaging protocol, wherein a custom-built laser-based conformal heat source was applied at the tumor volume to trigger the release of HTLC co-encapsulated with the MR contrast agent gadoteridol (Gd-HP-DO3A). MR thermometry (MRT) demonstrated that a relatively uniform temperature distribution was achieved in the tumor volume using the external laser-based heating setup. In mice bearing subcutaneously-implanted ME-180 cervical tumors, the combination of HTLC and heat resulted in a 2-fold increase in tumor drug levels at 1h post-administration compared to HTLC without heating. Furthermore, the overall tumor accumulation levels for the HTLC groups (with and without heat) at 1h post-injection were significantly higher than the corresponding free CDDP group. This translated into a significant improvement in therapeutic efficacy evaluated as tumor growth delay (p<0.05) for the heated HTLC treatment group compared to the unheated HTLC, heated or unheated free CDDP, and saline groups. Overall, findings from this study demonstrate that a heat-activated, triggered release formulation of CDDP results in a significant enhancement in the therapeutic index of this drug.