Thermosensitive liposomes (TSL) belong to the category of triggered nanoparticle drug delivery systems (DDS) where a drug associated with the DDS is released in response to an external trigger ^[1][2][3][4][1,2,3,4]. TSL are triggered by heat and release the encapsulated drug when exposed to mild hyperthermia (HT), typically ~40–43 °C. TSL were first described more than four decades ago ^[5][6][7][8][5,6,7,8]. Since then, numerous TSL formulations combined with various drugs have been described, as summarized in prior reviews ^{[9][10][11][12][13][14][15]}[9,10,11,12,13,14,15]. TSL are most often administered systemically, e.g., by intravenous infusion, and then circulate in the blood stream for an extended duration. Combined with localized hyperthermia, TSL enable loco-regional drug delivery (Figure 1). This enables the delivery of a large drug dose to a targeted tissue region (e.g., tumor) while reducing systemic toxicities. Therefore, TSL are attractive as a therapeutic strategy in cancer patients where loco-regional drug delivery is beneficial, but less useful in metastatic cancer patients that require systemic therapy. While TSL have been most widely investigated for drug delivery in cancer therapy, additional potential clinical applications include the delivery of antibiotics ^[16][17][16,17], the treatment of inflammatory diseases [18], and the treatment of blood clots [19].

TSL enable two different delivery approaches: extravascular triggered release, and intravascular triggered release (Figure 2) ^{[14][20][21][22][23]}[14,20,21,22,23]. Extravascular triggered release requires the extravasation of the TSL, followed by HT-triggered release of the encapsulated agent ^[14][24][14,24]. This extravasation is based on TSL accumulation within the tumor interstitium facilitated by enhanced permeability and retention (EPR) ^{[14][25][26][27][28]}[14,25,26,27,28]. Several recent papers described the limitations of the EPR effect, such as high intra- and inter-tumor variability, and an apparent upper delivery limit ^[14][27][29][14,27,29]. SomRecent reviews highlight the need for delivery strategies that do not rely on EPR ^[14][27][29][14,27,29].

Intravascular triggered release is a strategy where drug release occurs in the microvasculature while the TSL pass through the heated tumor, and does not require the EPR effect (Figure 2a) ^{[6][20][21][22][23]}[6,20,21,22,23]. Many of the more recent TSL formulations are based on intravascular triggered release, and such TSL have demonstrated superior delivery efficacy, with up to 25× higher drug delivery compared to unencapsulated drugs [31]. Compared to non-triggered nanoparticle drug delivery systems, TSL based on intravascular triggered delivery demonstrate superior tumor drug uptake (Figure 3). In addition, the direct comparison of TSL with extra- versus intra-vascular triggered delivery strongly suggests that the latter is superior ^{[22][24][32][33]}[22,24,32,33] (Figure 2b).

Tissue Transit Time

For TSL based on intravascular triggered release, the dynamics of blood flow through the tumor vasculature is of primary relevance. Blood/plasma with TSL enter a tumor segment through a supplying artery, pass through tumor capillaries, and exit the tumor segment through a draining vein. The average time that plasma spends within a tumor segment is termed the ‘tissue transit time’ (TT) (compared to plasma, red blood cells move significantly slower through capillaries, and thus remain for longer within the tumor segment [115]). The drug release from TSL, and drug extraction by tumor tissue, can only occur during this tissue transit time. Figure 4 visualizes the transit time between supplying artery and draining vein of a small mouse tumor segment. In human tumors, the mean transit time through a tumor has been measured for various tumor types. This mean tumor transit time varies widely, and is ~2 s for primary hepatocellular carcinoma [116], ~3 s for head and neck and prostate tumors ^[117][118][117,118], ~11 s for renal cell carcinoma [119], ~25 s for metastases to the liver [116], and ~30 s for breast cancer [120]. Furthermore, transit time and perfusion vary spatially within tumors such that transit time can be locally within a tumor considerably higher or lower than these mean values that were averaged over the whole tumor. As plasma with TSL enters capillary vessels within a heated tumor region, the TSL start releasing the drug and the drug is then extracted by the tumor (Figure 5b,c). Therefore, the plasma drug concentration varies along the vasculature as plasma flows between the supplying artery and draining vein of a tumor segment—in other words, a concentration gradient develops along the tumor microvasculature between the supplying artery and draining vein. Figure 5 illustrates schematically this microvascular concentration gradient along a representative capillary connecting the supplying artery and the draining vein of a tumor segment. For free (unencapsulated) drug, plasma drug concentration decreases as drug is extracted (Figure 5a). For TSL, drug is first released by hyperthermia, and then the released (free) drug is extracted (Figure 5b,c). Ideally, TSL completely release the encapsulated drug during the transit time to maximize tumor drug uptake—i.e., TSL that release their drug within seconds are preferable (Figure 5b).

2. Impact of TSL Properties on Drug Delivery

The methods for preparation and loading of various TSL formulations with different agents has been reviewed extensively in prior reviews ^{[9][10][11][12][13][14][15]}[9,10,11,12,13,14,15]. Additionally, the factors and mechanisms that affect drug release from TSL have been summarized in detail in earlier publications ^[9][11][12][9,11,12]. Here, we focus on reviewing how TSL properties such as release kinetics and plasma stability affect drug delivery is focused on. These properties depend both on the TSL formulation and the drug. For example, the same formulation will have varying release kinetics depending on which drug is encapsulated [121]. In addition, the buffer used to measure release affects release kinetics [121], highlighting the importance of selecting an appropriate buffer (e.g., plasma) (Figure 6d).

2.1. TSL Release Kinetics

The early TSL formulations had comparably slow release (within minutes to hours) ^[122][123][122,123]. In addition, heating to >42 °C was required to achieve substantial release. This is disadvantageous since temperatures above 43 °C may result in reduced blood flow [124] that would also reduce the inflow of TSL-encapsulated drug. The first fast-release TSL formulations were published in the early 1990s, demonstrating substantial release within a few seconds after heating to >41 °C ^[125][126][125,126]. Such rapid release is required to take full advantage of the intravascular triggered release paradigm, as discussed above. The first fast-release formulation with substantial release at lower temperatures (40 °C) was presented around the year 2000 ^{[31][127][128]}[31,127,128], and formed the basis for the first commercial TSL formulation (ThermoDox®) that has been employed in several human clinical trials ^{[129][130][131][132][133][134][135]}[129,130,131,132,133,134,135]. Several additional fast-release TSL formulations encapsulating various agents have been presented within the last two decades ^{[136][137][138]}[136,137,138]. Recent studies confirm that fast-release TSL that release within a few seconds can deliver substantially higher drug amounts compared to slower releasing formulations ^[20][21][22][20,21,22]. However, for most of these TSL formulations, release kinetics is not known within the time scale relevant for intravascular triggered release (e.g., within the first few seconds), owing to limitations of conventional methods used for measuring the release kinetics. The most widely used method for measuring TSL release kinetics employs a buffer pre-heated to the desired temperature, where a small volume of TSL is added, typically under stirring ^{[113][123][137][139][140][141][142][143]}[113,123,137,139,140,141,142,143]. Release is quantified usually using spectrophotometry, since optical properties (e.g., fluorescence) change when the drug is released from TSL. Due to the time required for mixing of the TSL with the buffer, the first reported time points are typically between 8–20 s. This time is substantially longer than many typical tumor transit times, making these measurements of limited value. There have been two methods presented to measure TSL release kinetics at short (second) time scales. The first method employed a small-diameter tube within which TSL solution was passed through heated water for a specific time, and the released drug was quantified in the sample exiting the tube [125]. An advantage of this method is that various quantification methods can be employed on these samples. In a second method, a glass capillary tube was heated by a Peltier element, and release was quantified by measuring fluorescence along the tube by either microscopic or macroscopic fluorescence imaging (Figure 6a) ^[121][144][121,144]. This method provides data at high temporal resolution not possible with other methods (Figure 6b–d), but is limited to fluorescent agents. In both methods, it is important to select thin-walled tubes and to validate sufficiently rapid heating of the solution passing though the tube to target temperature [121]. To compare the release kinetics of TSL formulations, a recent study suggested using a characteristic release time based on a linear approximation of the TSL release kinetics [20]. As noted earlier, TSL only spend a few seconds within a tumor (=transit time), and for most TSL, the release kinetics within those first seconds can be adequately represented by a linear approximation (Figure 7). Ideally, this release time would be smaller than the transit time to maximize release and tissue drug uptake (Figure 5). The amount of drug released during tumor transit (Figure 5) can be estimated by the ratio of transit time to release time (can bsee found in original contextSupplementary Material File S1, Equation (S2)). A recent study demonstrated that TSL with rapid release (i.e., short release time) can deliver substantially more drug to tissue than TSL with slow release (Figure 8) [20]. Table 1 summarizes the release times of published fast-release TSL formulations. In most cases, the exact release times could not be determined owing to limitations of methods used to quantify the release kinetics, as described above.