Current status of pH-sensitive liposomes in drug delivery

Liposomes are being increasingly utilized to deliver drugs, enzymes, antisense oligonucleotides, and genes to various therapeutic targets. Liposome-mediated delivery of therapeutic molecules has continued to make considerable progress in the transition from the laboratory to the clinic [1], [2], [3], [4]. Circulation lifetimes have been greatly increased by incorporating polymer-coated lipids in the formulation to form sterically stabilized liposomes (SSL) and reduce uptake by macrophages of the reticuloendothelial system (RES) [5], [6], [7]. Advances in drug loading technologies have allowed for loading efficiencies approximating 100% of some amphipathic drugs using pH or ammonium sulfate gradients [8], [9], [10], [11], [12]. In addition, a more detailed understanding of the interactions of different drugs with liposomal membranes has resulted in more stable liposomal drug preparations [13], [14], [15], [16], [17]. Finally, specific targeting to internalizing cell surface receptors has increased both in vitro cytotoxicity [18], [19], [20], [21] and in vivo efficacy of liposomal drugs [4], [22]. These advances have contributed greatly to the development of current liposomal drugs either approved or presently in clinical trials; Doxil® (Alza Corp.; Palo Alto, CA), Evacet™ (Liposome Co.; New Brunswick, NJ), Daunoxome® (Nexstar Pharm.; Boulder, CO), and conventional liposomal vincristine [23], [24], and brightened the prospects for future liposome-based therapies.

However, while significant advances have been made in overcoming many of the barriers associated with liposomal drug delivery, an elusive problem has been the ability to selectively increase the bioavailability of the drug at the target tissue, while maintaining stability in the circulation. For pegylated liposomal doxorubicin (SSL-DOX), the drug is thought to leak very slowly from the carrier and thus be similar to a slow infusion of the drug specifically near the tumor [25], [26]. The exact mechanisms responsible for liposome breakdown remain speculative but may include lysosomal processing by tumor-residing macrophages [27], a destabilizing environment provided by the low pH, lipases, enzymes, and oxidizing agents found in the tumor interstitium [28], or catalysis by free ammonium sulfate [29]. Regardless of the mechanism, the release is slow and may not apply to other classes of drugs, more specifically highly charged water-soluble drugs. In addition, following release from the liposomal carrier, the drug or gene often needs to be delivered to cytosolic or nuclear targets. While amphipathic drugs are able to freely cross biological membranes, DNA, peptides, and water-soluble drugs are likely to be excluded completely from the cell or trapped in internal organelles.

To overcome these problems a variety of approaches have been employed; including complexation of DNA with cationic lipids [30], [31], [32], the design of thermosensitive liposomes capable of releasing their contents in response to small changes in temperature [33], [34], [35], and the development of pH-sensitive liposomes [36], [37], [38]. With regards to vectors, complexation and condensation of nucleic acids with cationic lipids still holds the greatest promise for gene delivery in vivo. The near complete entrapment or complexation of DNA to cationic lipids is considerably greater than the low encapsulation efficiencies observed with neutral or anionic liposomes. However, problems with in vivo stability of the complexes, rapid clearance from the circulation (t_1/2 on the order of minutes), and primary distribution to the lung are just beginning to be addressed and much more work needs to be completed to increase the efficiency of delivery in vivo and make this a viable approach with systemic administration [32], [39], [40], [41]. Thermosensitive liposomes rely on a relatively sharp phase transition in the lipid phase at temperatures only a few degrees above 37° [33], [34], [35]. Packing defects observed at the gel-to-liquid crystalline phase transition result in increased permeability to entrapped solutes or drugs [33], [42]. Thermosensitive liposomes require the addition of an external stimulus (heat) in a spatially well defined region, surrounding and including the tumor. This creates a problem for distant metastasis and thus, may be limited in effectiveness to the primary tumor or substantial and detectable metastasis. Studies are in progress using whole body hyperthermia in an attempt to overcome this problem (Kirpotin, personal communication). The final approach and the focus of this review is the use of pH-sensitive liposomes to release their contents into the cytoplasm following endocytosis. This approach relies on selective destabilization of liposomes following acidification of the surrounding medium. The initial rationale for the design of pH-sensitive liposomes was to exploit the acidic environment of tumors to trigger destabilization of liposomal membranes [43]. However, the sites of greatest acidity in tumors are often the most distant from the tumor microvasculature, where liposomes often fail to reach [44], [45], [46]. In addition, the pH of the tumor interstitium rarely declines below pH 6.5 and therefore, makes it technically difficult to engineer liposomes that become disrupted in such a narrow window of pH. Endosomes and lysosomes, on the other hand, can reach values below 5.0 [47], [48], [49] and liposomes can be internalized by cells on the tumor periphery.

One fate of liposomes upon reaching the target tissues is they can be recognized and taken up by endocytosis [50]. However, upon uptake by the cell, they are eventually delivered to lysosomes (Fig. 1) [47], where the liposome and its contents may be degraded by various hydrolases and peptidases [51], [52]. The result can be diminished or lost biological activity of the delivered drug or macromolecule. pH-Sensitive liposomes have been designed to circumvent this problem by releasing their contents prior to reaching the lysosomes and at least partly, into the cytosol, where they can diffuse to their cytosolic or nuclear targets. Endosomes and lysosomes are acidified by proton-translocating ATPases [48], [53] to an average pH of approximately 5.0 [48], but which can be as low as 4.6 in macrophages [47], [49]. pH-Sensitive liposomes release their contents into the cytosol by one, or a combination, of several potential mechanisms (Fig. 1). These liposomes can be induced to undergo a pH-induced fusion of liposomal membranes with endosomal membranes, directly releasing liposomal contents into the cytosol. Alternatively, liposomes can become destabilized and cause the destabilization of endosomal membranes, resulting in leakage of the drug or liposomal “cargo” into the cytosol. In the case of certain encapsulated molecules, pH-sensitive liposomes may also become destabilized and release their contents solely inside the endosome, where amphipathic molecules may diffuse across endosomal membranes and other molecules (antifolates and nucleoside analogues) may be translocated by membrane transporters located in the endosomal membrane [54], [55], [56], [57].

Four basic classes of pH-sensitive liposomes have been previously described. The first class combines polymorphic lipids, such as unsaturated phosphatidylethanolamines, with mildly acidic amphiphiles that act as stabilizers at neutral pH. Although this class of pH-sensitive liposomes has been the most intensively studied, it may prove to be the least useful for in vivo drug delivery due to the effects of serum components on the formulation stability or pH-sensitivity, and their rapid clearance from the circulation. The second class includes liposomes composed of “caged” lipid derivatives [58], [59], [60], [61], [62]. Acid-induced hydrolysis of specifically engineered chemical bonds results in an increased presence of membrane destabilizing lipid components in the liposome membrane, and thus increased permeability to encapsulated solutes. A third class of pH-sensitive liposomes utilizes pH-sensitive peptides or reconstituted fusion proteins to destabilize membranes at low pH [63], [64], [65], [66], [67]. The final and most current class of pH-sensitive liposomes uses pH-titratable polymers to destabilize membranes following change of the polymer conformation at low pH [38], [68], [69], [70]. All four classes offer unique advantages and disadvantages that may vary in importance depending on the desired application. A careful comparison of these differences, limitations, and potential applications will be the focus of this review.