Hyperthermia mediated liposomal drug delivery

Abstract

Drug delivery systems have been developed for cancer therapy in an attempt to increase the tumour drug concentration while limiting systemic exposure.

Liposomes have achieved passive targeting of solid tumours through enhanced vascular permeability, which is greatly augmented by hyperthermia. However, anti-tumour efficacy has often been limited by slow release of bioavailable drug within the tumour. Local hyperthermia has become the most widely used stimulus for triggered release of liposomal drugs, through the use of specific lipids, polymers or other modifiers.

A temperature-sensitive liposome containing doxorubicin has been shown to release 100% of contents through stabilized membrane pores within 10–20 s at 41°C. This formulation has exhibited dramatic improvements in pre-clinical drug delivery and tumour regression and is now in clinical trials.

Significantly, recent studies show that this liposome, in combination with local hyperthermia, exhibits vascular shutdown as a mechanism of anti-tumour effect that is not observed with free doxorubicin.

• Liposome
• hyperthermia
• release
• drug delivery
• trigger
• temperature-sensitive
• doxorubicin

Introduction

Normal tissue and organ toxicities pose major limitations to achieving therapeutic levels of drug in the treatment of solid tumours. One solution for optimizing the therapeutic index in systemic chemotherapy is tumour-specific drug delivery [1]. Historically, liposomes have been under development with the objective of meeting four basic requirements for tumour targeted drug delivery. These requirements are ‘Retain (drug), Evade (the body's defenses), Target (tumour tissue and vasculature) and Release (drug specifically in tumours)’ [2]. Temperature-sensitive liposomes, in combination with local hyperthermia, provide targeted control of drug release that may augment chemotherapeutic efficacy in many clinical settings [3]. For induction chemotherapy, a higher local drug concentration is more likely to cause a significant decrease in primary tumour volume. This could downsize a tumour from unresectable to resectable status or permit a less ablative surgery. Dramatic regression could even make primary surgery unnecessary in some cases. In the setting of local recurrence at the primary site, drug resistance is often an important obstacle. Hyperthermia has been shown to reverse resistance to some drugs, and temperature-sensitive release would additionally expose cells to a higher drug concentration [4], [5]. Furthermore, hyperthermia shows synergism with many chemotherapeutic and radiochemotherapeutic regimens [6–8]. It is important to note that temperature-sensitive liposomes are unlikely to be applicable to adjuvant chemotherapy, since disseminated micro-metastases cannot currently be heat-targeted. However, for the local control of solid tumours, hyperthermia mediated liposomal drug delivery has the potential to increase therapeutic ratio by improving efficacy and decreasing side effects.

Liposomes and tumour drug delivery

Liposome technology has expanded over more than 40 years to creatively meet many of the requirements for tumour drug delivery. Initial efforts at passively entrapping the chemotherapeutic agent doxorubicin (Dox) showed significant drug leakage. However, loading and retention were improved by the maintenance of a transmembrane pH gradient (interior acidic, Figure 1(a)) [9]. In vivo, an early obstacle was the rapid entrapment of injected liposomes in the reticulo-endothelial system (RES), including liver, spleen and bone marrow. Papahadjopoulos and Allen discovered that circulation time could be extended from 30 min to 24–48 h in humans by adding polyethylene glycol-derivatized lipids (PEG lipids) to the lipid bilayer (Figure 1(a)) [2], [10], [11]. The PEG coating creates a steric barrier to opsonization and liposome aggregation, which are presumed mechanisms of RES uptake [12]. Sterically stabilized and conventional liposomes have been targeted to the tumour passively, based on the increased vascular permeability associated with aberrant and immature microvessels [13], [14]. Recently, specific targeting has also been achieved by antibody or receptor-mediated targeting to tumour cells [1]. Thus, lipid based drug carrier systems have been developed that can load and retain drug by passive encapsulation and remote pH loading, evade the body's defenses by PEG coating and target the interstitial tissue of tumours through passive or specific means.

Figure 1. (a) Schematic of temperature-sensitive liposome structure. Liposomes are spherical lipid vesicles enclosing an aqueous compartment that contains drug. Amphiphilic drugs can be actively loaded and retained through a pH gradient. The phospholipid bilayer is characterized by a main melting phase transition. Lysolipids can form stable pores at the phase transition temperature. Polymers may be grafted onto lipids to evade immune recognition. Note that only unitary examples of each molecular strategy are shown. (b) Schematic of possible mechanisms involved in combination HT and liposomal therapy for solid tumours [19]. (1) Liposomes (blue circles) preferentially extravasate from pores in tumour vessel walls. (2) HT increases tumour vessel pore size and thus increases tumour liposome extravasation. (3) HT can trigger drug (yellow) release from liposomes in the tumour vessel. (4) HT can trigger liposomal drug release in the tumour interstitium. (5) HT can be directly cytotoxic to tumour cells.

With these advances, liposomal delivery of doxorucibin has been shown to reduce cardiotoxicity, myelosuppression and alopecia in comparison with free doxorubicin [15]. In addition, long-circulating liposomes exhibit extensive accumulation in tumours [14]. However, the therapeutic effectiveness of many liposomes has been limited by low drug bioavailability within the tumour. Drug release from most liposomes is passive and slow, minimizing the effective drug concentration. This can be an advantage for cell cycle-specific drugs, such as vincristine, which require a longer exposure to relatively low drug concentrations [16], [17]. However, non-cell cycle specific drugs, such as doxorubicin and cisplatin, may not achieve cytotoxic local concentrations with passive release [18], [19]. Therefore, although targeting the liposome to the tumour is obviously an important feature of drug delivery, targeting liposomal drug release is paramount. To obtain controlled release, the carrier/drug relationship must change from the stable state (required for circulatory transport and delivery) to one of quick instability and release at the tumour site [20]. In 1978, Yatvin et al. [21] introduced the concept of temperature-sensitive liposomes that could take advantage of hyperthermia (HT) to trigger this change.

Liposomes and hyperthermia

The selective effects of HT on tumour vasculature have been exploited to augment liposomal drug delivery to tumours in a temperature-dependent manner. Despite the fact that many different drugs and liposomes have been tested in a variety of tumour models, these studies consistently show that HT increases liposomal drug delivery to tumours and enhances anti-tumour effect [22]. The mechanisms of these benefits include improved vascular perfusion and increased extravasation of liposomes (Figure 1(b)). Pre-clinical research conducted by Kong et al. [23], [24] utilised tumour bearing skin-fold window chamber models in nude mice to quantify liposomal extravasation rates as a function of liposome size, temperature, tumour vs. normal micro-vessels and timing between HT and liposome administration. This work demonstrated a ∼2–4-fold increase in uptake of (non-thermally sensitive) liposomes in heated tumours as opposed to non-heated tumours (Figure 2) [23]. In addition, studies in cats with spontaneous vaccine-associated soft tissue sarcomas demonstrated a 2–16-fold increase in liposomal delivery to tumours with the use of hyperthermia [25].

Figure 2. Extravasation of 100-nm liposomes from SKOV-3 (human ovarian carcinoma) tumour vessels at 34°C (a) and 42°C (b) at 60 min after injection, in a mouse window chamber model. Minimal extravasation of liposomes was seen at 34°C (a). At 42°C, focal perivascular fluorescent spots developed (b) [23].

Temperature-sensitive liposome (TSL) technology

To further improve the combination of liposomal drug delivery, many groups have used specific lipids, polymers or other modifiers to achieve temperature-sensitive release of contents. Most temperature-sensitive liposomes take advantage of the inherent increase in permeability associated with the acyl-chain melting phase transition [26–30]. For example, dipalmitoyl phosphatidylcholine (DPPC) converts from solid (crystalline) to liquid phase at 41°C with concurrent leakage of contents. Moreover, the doxorubicin release associated with the DPPC phase transition can be increased 20-fold by incorporating micelle-forming lysolipids like monostearoyl phosphatidylcholine (MSPC) into the bilayer [31]. Evidence from liposome permeability after dialysis and observations of the material properties of giant liposomes have led to the conclusion that the lysolipid release mechanism consists of stabilized pore structures at the interfaces between solid and liquid phase lipids at grain boundaries (Figure 1(a), phases not shown) [32–34]. These liposomes retain contents at temperatures below and above the main phase transition while facilitating rapid release of contents at temperatures near the main phase transition [3], [32]. The new TSL formulation containing doxorubicin (Dox-TSL) has exhibited complete drug release within ∼10–20 s at mild hyperthermic temperatures (40–42°C) that are readily achievable in the clinic [20], [31], [35]. The unique triggered pore release mechanism is ideal for water-soluble drugs, which will be retained in the solid phase liposome (in the blood-borne delivery phase) until they are released in tumour vasculature and tissue as a consequence of hyperthermia (Figure 1(b)).

A number of other strategies have been employed to increase the thermal sensitivity of liposomes. Lindner et al. [36] recently developed an alkylphosphocholine formulation that releases at the melting phase transition in a similar manner to the MSPC-containing formulation [36]. Another group has encapsulated block co-polymers which disrupt membranes from the interior at higher temperatures [37], [38]. Alternatively, temperature-sensitive polymers can be grafted to lipids, causing bilayer disruption at the coil-globule transition temperature of the polymer [39–41]. Most groups have encapsulated drugs that are well characterized and widely used in oncology, such as doxorubicin, cisplatin and methotrexate. However, the mechanism of action must not be ignored in these studies, as the dramatic change in drug delivery can alter the type of response as well the magnitude of response. For example, Chen et al. [42] recently showed that the Dox-TSL formulation exhibits vascular shutdown as a mechanism of anti-tumour effect that is not observed with free doxorubicin.

Temperature-sensitive liposomes in vivo

Pre-clinical studies using Dox-TSL combined with local 42°C HT have shown superior results in comparison with other Dox liposome formulations in a mouse model bearing flank xenografts of human squamous cell carcinoma (FaDu). Using tumour growth delay (time to 5× tumour volume) as an endpoint, the lysolipid-containing TSL (referred to as LTSL or Dox-TSL) was clearly more effective than a traditional TSL (TTSL), a non-thermal sensitive formulation (NTSL) and free Dox (Figure 3) [31]. In fact, 17 out of 20 animals receiving Dox-TSL showed complete regression (up to 6 weeks) [19], [31].

Figure 3. Individual tumour volume growth curves after 5 mg kg⁻¹ doxorubicin plus heating at 42°C for 1 h. Each line represents an individual mouse in the treatment group. (a) free doxorubicin; (b) NTSL (non-thermally sensitive liposome); (c) TTSL (traditional temperature-sensitive liposome); (d) LTSL (lysolipid-containing temperature-sensitive liposome, also referred to as Dox-TSL). The endpoint was time until tumours reached five times initial tumour volume or 60 days. The differences in local control rates between LTSL and TTSL were highly significant (p ≤ 0.0001) [31].

The difference in effectiveness was correlated to a significant increase in drug delivery to the tumour [19]. This can be attributed to the rapid and complete release properties of the Dox-TSL formulation. In general, the local concentration of Dox depends on the difference between the rate of drug release from the liposome and the rate of drug clearance through the micro-circulation. Importantly, drug release may occur intravenously or after extravasation of the liposome, thereby exposing both endothelial and tumour cells to doxorubicin (Figure 1(b)). When Dox is rapidly released from the TSL [35], the peak concentration of free Dox in solid tumours can be ∼30-fold higher than after intravenous injection of free Dox and at least 2-fold higher than after intravenous injection of Dox-TTSL [19]. At such a high local concentration, Dox is able to shut down blood flow in tumours within 24 h [42]. Thus, the mechanisms of tumour regression are likely to be related to the anti-vascular effects of Dox-TSL in addition to the anti-neoplastic effects.

Based on the success of these pre-clinical studies, as well as experience in a clinical trial using conventional liposomal doxorubicin (Evacet™) and HT as neoadjuvant therapy for locally advanced breast cancer, new clinical trials are currently underway [43]. A phase I/II trial evaluating Dox-TSL for the treatment of chest wall recurrence of breast cancer will be followed by a trial testing Dox-TSL in patients with locally advanced breast cancer. At the National Cancer Institute, another phase I trial will evaluate Dox-TSL in combination with radiofrequency thermal ablation for liver metastases. Future directions include pre-clinical and clinical studies using TSLs containing cisplatin in the treatment of solid tumours, as well as MRI monitoring of liposomal drug release and distribution [44].

Conclusion

In conclusion, hyperthermia mediated liposomal drug delivery may be an effective means to achieving increased tumour drug concentration and greater clinical response to chemotherapy. Previous advancements in liposome technology include efficient loading and retention of drug, increased circulation time and passive or specific targeting to tumours. However, the efficacy of many formulations has been limited by the uncontrolled release of bioavailable drug at the tumour site. Triggered drug release through hyperthermia provides targeted bioavailability of encapsulated drug. In addition, hyperthermia has been shown to significantly improve vascular perfusion and increase the extravasation of liposomes in tumours. Temperature-sensitive liposome formulations have utilized this synergism between hyperthermia and drug delivery. In particular, the Dox-TSL displays unique ultrafast drug release through membrane pore formation. Pre-clinical studies conducted using Dox-TSL in combination with local hyperthermia showed a significant increase in drug uptake and tumour regression. As this drug delivery system moves forward into clinical trials, continuing research will broaden the understanding and applicability of hyperthermia mediated liposomal drug delivery.

Acknowledgements

This work was supported by grants from the National Institutes of Health/National Cancer Institute (CA42745 and CA87630) and the Department of Defense (DAMD 17-03-1-0348).