pH-dependent stability and fusion of liposomes combining protonatable double-chain amphiphiles with phosphatidylethanolamine

We have prepared a series of novel double-chain amphiphiles with protonatable head groups, including acylated derivatives of various 2-substituted palmitic acids, amino acid conjugates of these species, and 1,2-dioleoyl-3-succinylglycerol. These species can be combined with phosphatidylethanolamine (PE) to prepare reverse-phase evaporation vesicles that are stable and trap hydrophilic solutes at pH 7. At weakly acidic pH values (as high as 6.5, depending on the titratable amphiphilic component), these pH-sensitive vesicles exhibit fusion, with a limited extent of contents mixing and extensive mixing of lipids, accompanied by leakage of aqueous contents. Protons and divalent cations show strong synergistic effects in promoting mixing of both lipids and aqueous contents between pH-sensitive vesicles prepared with any of a variety of double-chain titratable amphiphiles. Calorimetric results indicate that the relative stabilities of different types of pH-sensitive liposomes at low pH cannot be simply correlated with the propensity of the lipids to form a hexagonal II phase under these conditions. Fluorescence measurements demonstrate that single-chain fatty acids, but not double-chain titratable amphiphiles such as N-acyl-2-aminopalmitic acids, are rapidly removed from pH-sensitive vesicles in the presence of other lipid vesicles, serum albumin, or serum. Additionally, pH-sensitive liposomes containing double-chain titratable amphiphiles retain their aqueous contents better than do those containing single-chain amphiphiles in the presence of lipid membranes or albumin. Surprisingly, however, pH-sensitive vesicles of either type show retention of contents in the presence of serum that is comparable to that observed with vesicles composed purely of phospholipids. A model is proposed to explain these latter findings.     

Structural and functional comparisons of pH-sensitive liposomes composed of phosphatidylethanolamine and three different diacylsuccinylglycerols

The titratable, double-chain amphiphiles 1,2-dipalmitoyl-sn-3-succinylglycerol (1,2-DPSG), 1,2-dioleoyl-sn-3-succinylglycerol (1,2-DOSG) and 1,3-dipalmitoylsuccinylglycerol (1,3-DPSG) have been used in combination with phosphatidylethanolamine (PE) to form pH-sensitive liposomes. The effect of the compounds on dielaidoyl PE bilayer stabilization was examined by differential scanning calorimetry. Only 1,2-DPSG showed bilayer stabilization activity; whereas the other two are destabilizers at pH 7.4. All three amphiphiles became strong destabilizers at pH 5.0. The ability of the amphiphiles to stabilize DOPE liposomes was examined by light scattering and calcein entrapment. In general, 1,2-DPSG is the most potent stabilizer of PE bilayers while 1,3-DPSG is the weakest liposome stabilizer. All three compounds can be combined with DOPE to generate liposomes which are stable at neutral and basic pH. At weakly acidic pH, the liposomes are leaky and exhibit extensive lipid mixing, with protons and calcium showing synergistic effects on lipid mixing. DOPE/1,2-DPSG liposomes are stable in human plasma and remain acid-sensitive even after prolonged plasma incubation. Immunoliposomes prepared from either DOPE/1,2-DPSG or DOPE/1,2-DOSG can deliver diphtheria toxin A fragment to the cytoplasm of cultured cells in a process which involves endocytosis of the liposomes. Immunoliposomes prepared with 1,2-DPSG are more effective drug carriers than those prepared with 1,2-DOSG. These results indicate that the bilayer- and, hence the liposome-stabilization activity of the diacylsuccinylglycerol depends on the structure of the compounds. The potential drug delivery activity of the pH-sensitive liposomes composed of these lipids is discussed.     

A novel pH-sensitive liposome formulation containing oleyl alcohol

pH-sensitive liposomes are designed to undergo acid-triggered destabilization. First generation pH-sensitive liposomes, based on the cone-shaped lipid dioleoylphosphatidylethanolamine (DOPE), have been shown to lose fusogenicity in the presence of serum. Here, we report the design and evaluation of novel serum-resistant pH-sensitive liposome formulations that are based on the composition of egg phosphatidylcholine (PC), cholesteryl hemisuccinate (CHEMS), oleyl alcohol (OAlc), and Tween-80 (T-80). When loaded with the fluorescent probe calcein, these liposomes exhibited excellent stability at pH 7.4 and underwent rapid destabilization upon acidification as shown by calcein dequenching and particle size increase. Adjusting the mole percentages of T-80 and OAlc in the formulation could regulate the stability and pH-sensitive properties of these liposomes. Liposomes with a higher T-80 content exhibited greater stability but were less sensitive to acid-induced destabilization. Meanwhile, formulations with a higher OAlc content exhibited greater content release in response to low pH. The pH-triggered liposomal destabilization did not produce membrane fusion according to an octadecylrhodamine B chloride (R(18)) lipid-mixing assay. Compared to DOPE-based pH-sensitive liposomes, the above formulations showed much better retention of their pH-sensitive properties in the presence of 10% serum. These liposomes were then evaluated for intracellular delivery of entrapped cytosine-beta-D-arabinofuranoside (araC) in KB human oral cancer cells, which have elevated folate receptor (FR) expression. The FR, which is amplified in many types of human tumors, has been shown to mediate the internalization of folate-derivatized liposomes into an acidic intracellular compartment. FR-targeted OAlc-based pH-sensitive liposomes, entrapping 200 mM araC, showed approximately 17-times greater FR-dependent cytotoxicity in KB cells compared to araC delivered via FR-targeted non-pH-sensitive liposomes. These data indicated that pH-sensitive liposomes based on OAlc, combined with FR-mediated targeting, are promising delivery vehicles for membrane impermeable therapeutic agents.     

A novel pH-sensitive liposome formulation containing oleyl alcohol

pH-sensitive liposomes are designed to undergo acid-triggered destabilization. First generation pH-sensitive liposomes, based on the cone-shaped lipid dioleoylphosphatidylethanolamine (DOPE), have been shown to lose fusogenicity in the presence of serum. Here, we report the design and evaluation of novel serum-resistant pH-sensitive liposome formulations that are based on the composition of egg phosphatidylcholine (PC), cholesteryl hemisuccinate (CHEMS), oleyl alcohol (OAlc), and Tween-80 (T-80). When loaded with the fluorescent probe calcein, these liposomes exhibited excellent stability at pH 7.4 and underwent rapid destabilization upon acidification as shown by calcein dequenching and particle size increase. Adjusting the mole percentages of T-80 and OAlc in the formulation could regulate the stability and pH-sensitive properties of these liposomes. Liposomes with a higher T-80 content exhibited greater stability but were less sensitive to acid-induced destabilization. Meanwhile, formulations with a higher OAlc content exhibited greater content release in response to low pH. The pH-triggered liposomal destabilization did not produce membrane fusion according to an octadecylrhodamine B chloride (R₁₈) lipid-mixing assay. Compared to DOPE-based pH-sensitive liposomes, the above formulations showed much better retention of their pH-sensitive properties in the presence of 10% serum. These liposomes were then evaluated for intracellular delivery of entrapped cytosine-β-d-arabinofuranoside (araC) in KB human oral cancer cells, which have elevated folate receptor (FR) expression. The FR, which is amplified in many types of human tumors, has been shown to mediate the internalization of folate-derivatized liposomes into an acidic intracellular compartment. FR-targeted OAlc-based pH-sensitive liposomes, entrapping 200 mM araC, showed ∼17-times greater FR-dependent cytotoxicity in KB cells compared to araC delivered via FR-targeted non-pH-sensitive liposomes. These data indicated that pH-sensitive liposomes based on OAlc, combined with FR-mediated targeting, are promising delivery vehicles for membrane impermeable therapeutic agents.     

Interferon production of L929 and HeLa cells enhanced by polyriboinosinic acid-polyribocytidylic acid pH-sensitive liposomes.

The double-stranded RNA polyinosinic acid-polycytidylic acid (PolyIC) is an inducer of interferons alpha and beta (IFN) genes. With L929 and HeLa cells IFN pretreatment (priming) improves the IFN induction by PolyIC by several orders of magnitude. In the absence of the priming we demonstrate that PolyIC encapsulated into pH-sensitive liposomes (and not into pH-insensitive liposomes) enables L929 cells to secrete IFN efficiently and a low toxicity is observed; on primed cells pH-sensitive liposomes containing PolyIC trigger a high toxicity. With HeLa cells, the absence of the priming PolyIC encapsulated into pH-sensitive liposomes induces weak doses of IFN whereas free PolyIC was ineffective. Our experiments established that a pH drop (from 8 to 5.5) provoked a lipid mixing between pH-sensitive liposomes and cell membranes, likely by a fusion mechanism. Entrapment into pH-sensitive liposomes enhances the effect of PolyIC by several orders of magnitude, which might improve its therapeutic ability as an antitumor or anti-HIV agent.     

Biodistribution of pH-sensitive immunoliposomes

Liposomes composed of either dioleoylphosphatidylethanolamine and oleic acid (pH-sensitive) or dioleoylphosphatidylcholine and oleic acid (pH-insensitive) were injected into C3H and Balb/c mice in order to determine the tissue distribution of both the lipid and the aqueous content. The lipid component was monitored by use of [3H]cholestanyl ether and the aqueous content was monitored by use of encapsulated 125I-tyraminyl-inulin. The pH-insensitive liposomes injected into both types of mice were rapidly cleared from the blood stream followed by accumulation primarily in the liver, followed by the spleen. The presence of a monoclonal antibody on the liposome surface caused a slight acceleration in liver accumulation, though generally gave the same profile as the antibody-free liposomes. pH-sensitive liposomes were leaky upon exposure to the mouse plasma following injection. The lipid component, though, displayed a large amount (e.g., 50-70% in C3H mice) of accumulation in the lung for up to 6 h, followed by a subsequent appearance in the liver and spleen. The presence of monoclonal antibody had no effect on the tissue distribution profile. These results indicate that the pH-sensitive liposomes, although ineffective as an aqueous drug delivery agent, may be effective as a means of delivering lipophilic drugs to the lung.     

Interactions of mammalian cells with lipid dispersions containing novel metabolizable cationic amphiphiles

Several novel cationic amphiphiles, based on a hydrophobic cholesteryl or dioleoylglyceryl moiety, have been prepared whose hydrophobic and cationic portions are linked by ester bonds to facilitate efficient degradation in animal cells. Dispersions combining such cationic species with phosphatidylethanolamine (PE), certain structural analogues of PE or diacylglycerol can mediate efficient transfer of both nonexchangeable lipid probes and the DNA plasmid pSV2cat into cultured mammalian (CV-1 and 3T3) cells. The abilities of different types of cationic lipid dispersions to mediate transfection of mammalian cells with pSV2cat could not be directly correlated with their abilities to coalesce with other membranes, as assessed by their ability to intermix lipids efficiently with large unilamellar phosphatidylcholine/phosphatidylserine vesicles in the presence or absence of DNA. The cytotoxicities toward CV-1 cells of dispersions combining PE with most of the degradable cationic amphiphiles studied here compare favorably with those reported previously for similar dispersions containing other types of cationic amphiphiles. Fluorescent analogues of two of the diacylglycerol-based cationic amphiphiles examined in this study are shown to be readily degraded after incorporation into CV-1 cells from PE/cationic lipid dispersions.     

Interaction of pH-Sensitive Liposomes With Blood Components

Abstract

Avoidance of lysosomal degradation of drugs entrapped in liposomes has been one of the major efforts in liposome research. The achievement of high drug deliver}' efficiency using pH-sensitive liposomes over the pH-insensitive liposomes has greatly influenced our strategies in liposome drug delivery. The success of pH-sensitive liposomes in delivering compounds such as fluorescence dye, anti-cancer reagents, toxins and DNA to target cells with high efficiency in vitro shows a great potential to apply the same strategy to in vivo systems. Using human plasma as a simplified model for blood, we have systematically examined the interaction of pH-sensitive liposomes composed of dioleoylphosphatidyl-ethanolamine (DOPE) and oleic acid (OA) with plasma components. Our results show that the bilayer structure of liposomes in plasma depends on their sizes. Small liposomes (d<200nm) were stabilized by plasma components while the larger ones (d>600nm) were rapidly lysed upon the exposure to plasma. Such differences in their stability in plasma may derive from their differences in lipid packing which determines the surface pressure of the membrane. Using purified serum proteins, we found that albumin such as bovine serum albumin (BSA) lyse liposomes by extracting OA from the bilayer. However, BSA induced lysis could be blocked by lipoproteins including HDL, LDL and VLDL, but not by immunoglobulins. Further studies with purified components of HDL demonstrated that apoAl, not the lipids of the HDL, contains the stabilization activity. The extraction of OA from liposomes and the insertion of plasma components into the bilayer modified the bilayer properties such that plasma stabilized liposomes were no longer pH sensitive. Using dipalmitoylsuccinylglycerol (DPSG), a double-chain pH senser for DOPE liposomes, we could preserve 50% pH sensitivity after plasma treatment. The potential application of such liposomes and other essential properties of pH-sensitive liposomes for drug delivery in vivo are also discussed.     

Liposome-encapsulated midazolam for oral administration

The oral administration of midazolam has often been used for sedation in pediatric patients. However, oral administration of an intravenous formulation of midazolam is difficult for younger pediatric patients because of its bitter taste. Liposomes have been developed as vesicles encapsulating various kinds of drugs to serve as a medical drug-delivery system. Thus, the aim of the present study was to produce pH-sensitive liposomes encapsulating midazolam and to evaluate its pharmacokinetics on rabbits. Liposome-encapsulated midazolam was produced from hydrogenated L-α-phosphatidylcholine, cholesterol, dipalmitoylphosphatidic acid, and midazolam. The capacity of liposomes to encapsulate midazolam (encapsulation efficiency), stability of encapsulation, and release efficiency were evaluated in vitro. Further, the produced liposome-encapsulated midazolam solution was orally administered to rabbits in vivo. As a result, midazolam was encapsulated by liposomes with a high encapsulation efficiency and was stably encapsulated in a physiological medium. Further, the produced liposomes rapidly and effectively released midazolam in an acidic medium in vitro. When the liposome-encapsulated midazolam solution was orally administered to rabbits, the time to achieve the maximum plasma concentration of midazolam after administration was slightly longer, but both the maximum plasma concentration and area under the concentration-time curve were higher than those receiving midazolam solution. In conclusion, we produced pH-sensitive liposome-encapsulated midazolam, which remained stable in a physiological medium and showed efficient release in an acidic environment. The results suggest that it is possible to clinically use liposome-encapsulated midazolam for oral administration as a useful drug-delivery vehicle.     

In vitro cytotoxicity of liposome-encapsulated doxorubicin: dependence on liposome composition and drug release.

We have investigated the in vitro cytotoxicity of free doxorubicin (DOX) and liposome-entrapped DOX (L-DOX) against a human ovarian carcinoma cell line (OV-1063) using a colorimetric assay. DOX was encapsulated in the inner water phase of liposomes by an ammonium sulfate-generated proton gradient. Liposomes varied in phospholipid composition but were of a similar size. It was found that the cytotoxic activity of L-DOX is substantially decreased when liposomes containing phospholipids of high phase-transition temperature (Tm) are used. The type of negatively charged headgroup did not have any significant influence on the cytotoxicity observed. Experiments using resin beads that bind free and protein-bound DOX, but do not interact with L-DOX, indicated that the cytotoxic effect is mediated by the release of drug from the liposomes into the extracellular medium; no evidence was found for direct cellular uptake of liposome-encapsulated drug. The use of the ionophore nigericin to induce the release of DOX from high-Tm liposomes increased cytotoxicity to a level comparable to free DOX, suggesting that 'remote release' techniques may substantially improve the efficiency of liposome-mediated drug delivery and allow for the full exploitation of the favorable pharmacokinetic properties of specific high-Tm formulations.     

Study of phospholipase A2 specificity in substrate-containing structures having different supramolecular organization

The specificity of snake venom phospholipase A2(PLA2) towards a number of phospholipid (PL) substrates, e. g., phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) organized in Triton X-100 mixed micelles, liposomes and proteoliposomes was studied. PC was shown to be more rapidly hydrolyzed in micelles. For other PLs, the rate of hydrolysis decreased in the following sequence: PC greater than PI greater than PE greater than PG. The incorporation into micelles of a non-hydrolyzable by PLA2 sphinogomyelin which, similar to PC, has a choline group, resulted in an increase of PLA2 specificity towards PL that are known to be devoid of this group: PE greater than PI greater than PG greater than PC. Quite a different picture was observed in bilayer liposomal structures: PI congruent to PE greater than PC greater than PG. The incorporation of cytochrome P-450 into liposomes caused the acceleration of PE and PG hydrolysis. The course of the PLA2-catalyzed hydrolysis in model membrane structures seems to be governed primarily by the supramolecular organization and localization of the substrate in the bilayer, but not by its chemical structure.     

A Novel Folate-Targeted Nanoliposomal System of Doxorubicin for Cancer Targeting

Targeted drug delivery systems for cancer improves anti-tumor efficacy and reduces systemic toxicity by restricting availability of cytotoxic drugs within tumors. Targeting moieties, such as natural ligands (folic acid, transferrin, and biotin) which are overexpressed on tumors, have been used to enhance liposome-encapsulated drug accumulation within tumors and resulted in better control. In this report, we explored the scope of targeting ligand folic acid, which is incorporated in liposome systems using folic acid-modified cholesterol (CPF), enabled highly selective tumor-targeted delivery of liposome-encapsulated doxorubicin and resulted in increased cytotoxicity within tumors. Folate-tagged poloxamer-coated liposomes (FDL) were found to have significantly higher cellular uptake than conventional poloxamer-coated liposomes (DL), as confirmed by fluorometric analysis in B16F10 melanoma cells. Biodistribution study of the radiolabeled liposomal system indicated the significantly higher tumor uptake of FDL as compared to DL. Anti-tumor activity of FDL against murine B16F10 melanoma tumor-bearing mice revealed that FDL inhibited tumor growth more efficiently than the DL. Taken together, the results demonstrated the significant potential of the folate-conjugated nanoliposomal system for drug delivery to tumors.     

Targeted delivery and triggered release of liposomal doxorubicin enhances cytotoxicity against human B lymphoma cells.

Dioleoylphosphatidylethanolamine (DOPE)-containing liposomes that demonstrated pH-dependent release of their contents were stabilized in the bilayer form through the addition of a cleavable lipid derivative of polyethylene glycol (PEG) in which the PEG was attached to a lipid anchor via a disulfide linkage (mPEG-S-S-DSPE). Liposomes stabilized with either a non-cleavable PEG (mPEG-DSPE) or mPEG-S-S-DSPE retained an encapsulated dye at pH 5.5, but treatment at pH 5.5 of liposomes stabilized with mPEG-S-S-DSPE with either dithiothreitol or cell-free extracts caused contents release due to cleavage of the PEG chains and concomitant destabilization of the DOPE liposomes. While formulations loaded with doxorubicin (DXR) were stable in culture media, DXR was rapidly released in human plasma. pH-Sensitive liposomes, targeted to the CD19 epitope on B-lymphoma cells, showed enhanced DXR delivery into the nuclei of the target cells and increased cytotoxicity compared to non-pH-sensitive liposomes. Pharmacokinetic studies suggested that mPEG-S-S-DSPE was rapidly cleaved in circulation. In a murine model of B-cell lymphoma, the therapeutic efficacy of an anti-CD19-targeted pH-sensitive formulation was superior to that of a stable long-circulating formulation of targeted liposomes despite the more rapid drug release and clearance of the pH-sensitive formulation. These results suggest that targeted pH-sensitive formulations of drugs may be able to increase the therapeutic efficacy of entrapped drugs.     

pH-sensitive liposomes mediate cytoplasmic delivery of encapsulated macromolecules

Negatively charged liposomes are endocytosed by the coated vesicle system and accumulate in acidic intracellular vesicles. Liposomes that become unstable at acidic pH improve cytoplasmic delivery of membrane-impermeant macromolecules such as calcein (CAL) and FITC dextran (18 or 40 kDa). Oleic acid (OA): phosphatidylethanolamine (PE) (3:7 mole ratio) liposomes become permeable to CAL at pH less than 7.0. Control liposomes of phosphatidylserine:PE or OA:phosphatidylcholine are stable at pH 4-8. OA:PE liposomes promote cytoplasmic delivery of encapsulated CAL to CV-1 cells, as evidenced by the emergence of diffuse, cytoplasmic CAL fluorescence. Delivery requires metabolic energy and is partially inhibited by chloroquine or monensin, which raise the pH of intracellular vesicles.     

Preclinical Studies with Doxorubicin Encapsulated in Polyethyleneglycol-Coated Liposomes

Abstract

Doxorubicin (DOX) has been encapsulated with high efficiency in the water phase of small-sized lipid vesicles. Plasma-induced drug leakage from these vesicles is minimal when hydrogenated phosphatidylcholine is present as the main component. A prolonged circulation time of liposome-encapsulated DOX is observed in animal models when a small fraction of polyethyleneglycol-derivatized phospholipid (PEG) is present in the liposome bilayer. Using these PEG-coated liposomes, we found that the concentration of DOX in tumor implants of the mouse M-109 carcinoma is significantly enhanced by liposome delivery. The antitumor activity of liposome-encapsulated DOX in a lung metastases model of the M-109 carcinoma is superior to that of free DOX. The minimal lethal dose of DOX to tumor-free mice was substantially increased by encapsulation in PEG-coated liposomes, indicating that toxicity is reduced. We also found that the vesicant of DOX after intradermal injection is prevented by liposome encapsulation. These preclinical observations, suggesting that encapsulation of DOX in PEG-coated liposomes may lead to a significant improvement of the therapeutic index of DOX, have led to the initiation of clinical trials in cancer patients.     

Processing of exogenous liposome-encapsulated antigens in vivo generates class I MHC-restricted T cell responses.

Acid-sensitive liposomes have been developed for cytosolic delivery of encapsulated substances. We now demonstrate delivery of liposome-encapsulated Ag into the class I MHC Ag processing pathway in peritoneal macrophages in vitro using several types of acid-sensitive liposomes, including those composed of dioleoylphosphatidylethanolamine (DOPE)/palmitoylhomocysteine, DOPE/cholesterol hemisuccinate, DOPE/dioleoylsuccinylglycerol, and DOPE/dipalmitoylsuccinylglycerol. Our previous studies showed that acid-resistant liposomes (dioleoylphosphatidylcholine/dioleoylphosphatidylserine) did not engender class I-mediated presentation in vitro. However, in vivo immunization with OVA encapsulated in acid-resistant as well as acid-sensitive liposomes generated class I MHC-restricted T cell responses, as determined by subsequent in vitro cytotoxicity assays using OVA-transfected target cells. Target lysis by these cells was OVA- and class I MHC (Kb)-specific. This response was not generated by immunization with equivalent amounts of soluble OVA. Thus, a pathway for in vivo class I processing of Ag encapsulated in acid-resistant liposomes has been missed in vitro, perhaps because it is dependent on specific populations of APC or interactions between cells that have not been reconstituted in vitro. This pathway may explain the ability of many exogenous particulate Ag (liposomes, bacteria, parasites, and mammalian cells) to generate class I MHC-restricted T cell responses.     

Phagocytosis of liposomes by macrophages: intracellular fate of liposomal malaria antigen

Liposomes containing a synthetic recombinant protein were phagocytosed by macrophages, and the internalized protein was recycled to the cell surfaces where it was detected by enzyme-linked immunosorbent assay. The transit time of the liposome-encapsulated protein from initial phagocytosis of liposomes to appearance of protein on the surfaces of macrophages was determined by pulse-chase experiments. The macrophages were pulsed with liposomes containing protein and chased with empty liposomes, and vice versa. The amount and rate of protein antigen expression at the cell surfaces depended on the quantity of encapsulated protein ingested by the macrophages. Although liposomes were rapidly taken up by macrophages, the liposome-encapsulated protein was antigenically expressed for a prolonged period (at least 24 h) on the cell surface. Liposomes were visualized inside vacuoles in the macrophages by immunogold electron microscopy. The liposomes accumulated along the peripheries of the vacuoles and many of them apparently remained intact for a long time (greater than 6 h). However, nonliposomal free protein was also detected in the cytoplasm surrounding these vacuoles, and it was concluded that the free protein in the cytoplasm was probably en route to the macrophage surface. Exposure of the cells to ammonium chloride did not inhibit the appearance of liposomal antigenic epitopes on the cell surface, and this suggests that expression of the liposomal antigenic epitopes at the surface was not a pH-sensitive phenomenon. There was no significant effect of a liposomal adjuvant, lipid A, on the rate or extent of surface expression of the liposomal protein.     

Characterization of a novel pH-sensitive peptide that enhances drug release from folate-targeted liposomes at endosomal pHs

Although liposomes have proven useful for the delivery of drugs and gene therapy vectors, their potencies are often compromised by poor unloading following uptake into their target cells. We have consequently explored the properties of a novel 29-residue amphipathic peptide that was designed by arrangement of hydrophobic and hydrophilic residues to disrupt liposomes at lower peptide concentrations than previously tested peptides. The peptide was indeed found to promote pH-dependent liposome unloading with improved efficiency. A peptide of the same sequence, but half the length, however, promoted pH-dependent permeabilization only at much higher concentrations. Further characterization of the longer peptide revealed that release of liposome contents (i) occurred at a pH of ∼6, (ii) became less efficient as the size of the encapsulated cargo increased, and (iii) was moderately suppressed in cholesterol-containing liposomes. Use of this peptide to enhance the cytotoxicity of cytosine arabinoside encapsulated in folate-targeted liposomes demonstrated an increase in drug potency of ∼30-fold. Gene expression by a serum-stable folate-targeted liposomal vector was also measurably enhanced by inclusion of the peptide. We conclude that intracellular unloading of liposomal contents can be significantly improved by co-encapsulation of an optimally designed, pH-sensitive peptide.