Immobilization of liposomes on hydrophobically modified polymer gel particles in batch mode interaction

Immobilization of liposomal phospholipids onto Sephacryl S-1000 gels that were chemically conjugated with hydrophobic alkyl moieties, octyl, dodecyl and hexadecyl, was examined in batch mode interaction. Compared with the octyl gel, the dodecyl and the hexadecyl gels were found to immobilize the three to four times more phospholipids with the less hydrophobic moieties. The encapsulation of a water-soluble marker, with other evidences, suggests that the majority of the immobilized phospholipids maintained liposomal morphology. As the lipid of the interacting liposomes, egg yolk phosphatidylcholine (eggPC), 1,2-dimyristoylphosphatidylcholine (DMPC) and a mixture of DMPC and 1,2-dimyristamido-1,2-deoxyphosphatidylcholine were examined. At 22 °C, DMPC liposomes showed higher extent of immobilization than at 37 °C but not eggPC liposomes, suggesting that the phase of liposomal membrane could have influence on the immobilization. Exchange between the immobilized liposomes and free ones was found to be small, less than 3%. The gel that had been first interacted with liposomes to apparent saturation could further immobilize the newly added liposomes. The rate of this second immobilization was similar to that of the slow adsorption process; the both could be based on the same mechanism, possibly involving rearrangement of the immobilized liposomes on the gel as proposed by Lundahl. As had been observed in the flow mode, the immobilization had preference for smaller liposomes. In application of the system in batch mode, the size distributions of the immobilized liposomes and of those left in the supernatant may differ from that of the originally added liposomes.     

A reduction-triggered delivery by a liposomal carrier possessing membrane-permeable ligands and a detachable coating

To control the cellular uptake of drugs and genes, we synthesized a liposomal carrier possessing membrane-permeable ligands and a detachable poly(ethylene glycol) (PEG) coating. For the detachable coating, a lipid having a thiolytic cleavable spacer (PEG-S-S-DOPE) was synthesized by the reaction of dioleoylphosphatidylethanolamine (DOPE) with a PEG chain via a disulfide linkage. The liposomes were prepared from a mixture of dipalmitoylphosphatidylcholine (DPPC), DOPE, PEG-S-S-DOPE, and cholesteryl hemisuccinate (CHEMS). The octamer (R8 peptide) of arginine was chosen as the membrane-permeable ligand and covalently immobilized onto the CHEMS portion of the liposome surface (PEG-S-S-R8-liposome). The disulfide bond of the PEG chain was cleaved to display the R8 peptides on the liposome surface by adding a reducing agent such as L-cysteine, and thereby internalization of the liposomes was significantly facilitated. When L-cysteine was added to the mixture of cells and the liposome that incorporated plasmids encoding the enhanced green fluorescence protein (pEGFP), the expression of EGFP was low but could be observed in almost 100% of the cells.     

Immobilized liposome chromatography to study drug-membrane interactions. Correlation with drug absorption in humans

For rapid screening of drug-membrane interactions and predicting drug absorption in vivo, unilamellar liposomes were stably immobilized in the pores of gel beads by avidin-biotin binding. Interactions of a diverse set of well-described drugs with the immobilized liposomal membranes were reflected by their elution profiles. The membrane partitioning coefficients (KLM) of the drugs were determined from the retention volumes. The drug retentions on egg phosphatidylcholine (EPC)-phosphatidylserine (PS)-cholesterol (chol) and EPC-PS-phosphatidylethanolamine (PE)-chol columns intended to mimic small intestine membranes were similar, although the positively-charged drugs were more strongly retarded on the negatively-charged liposomes than the negatively-charged drugs. The relationship between log KLM with the drug fraction absorbed in humans showed that the log KLM values obtained with unilamellar liposomes can be used to predict drug passive transcellular absorption, similarly to that previously shown for entrapped multilamellar liposomes. The immobilized liposome chromatography method should be useful for screening compounds at an early stage of the drug discovery process. The avidin-biotin immobilization of the liposomes prolongs the lifetime of the columns.     

Characterisation of electroosmotic flow in capillary electrochromatography columns

Immobilized liposome chromatography (ILC) has been proven to be a useful method for the study or rapid screening of drug-membrane interactions. To obtain an adequate liposomal membrane phase for ILC, unilamellar liposomes were immobilized in gel beads by avidin-biotin binding. The retardation of 15 basic drugs on the liposome column could be converted to membrane partitioning coefficients, K(LM). The effects of small or large unilamellar liposomes and multilamellar liposomes on the drug-membrane partitioning were compared. The K(LM) values for both small and large liposomes were similar, but higher than those for the multilamellar liposomes. The basic drugs showed stronger partitioning into negatively charged liposomes than into either neutral liposomes or positively charged liposomes. The membrane fluidity of the immobilized liposomes was modulated by incorporating cholesterol into the liposomal membranes, by changing the acyl chain length and degree of unsaturation of the phospholipids, and by changing the temperature for ILC runs. Our data show that K(LM) obtained using ILC correlated well with those reported by batch studies using free liposomes. It is concluded that negatively charged or cholesterol-containing large unilamellar liposomes are suitable models for the ILC analysis of drug-membrane interactions.     

A reusable liposome array and its application to assay of growth-hormone-related peptides

We describe a reusable liposome array based on the formation of cleavable disulfide cross-links between liposomes and the surface of a glass slip. The N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP)-modified liposomes encapsulating a pH-sensitive fluorescence dye were immobilized on a 3-mercaptopropyltrimethoxysilane (MTS)-modified glass slip through the formation of disulfide bonds. The regeneration of a used slip was performed by the lysis of immobilized liposomes with Triton X-100 and the cleavage of disulfide bonds by reduction with TCEP, followed by immobilization of SPDP-modified liposomes. The regeneration steps did not affect the fluorescence intensity of re-immobilized liposomes. The liposome array was applied to simultaneous quantification of growth hormone related peptides, i.e., GHRF and somatostatin, in a mixture. After optimizing the assay condition, the method allowed quantification of GHRF and somatostatin in concentration ranges from 0.5 × 10⁻⁹ to 0.5 × 10⁻⁷ g/mL with detection limits of 2 × 10⁻¹⁰ and 3 × 10⁻¹⁰ g/mL, respectively.     

Covalent immobilization of liposomes on plasma functionalized metallic surfaces

A method was developed to functionalize biomedical metals with liposomes. The novelty of the method includes the plasma-functionalization of the metal surface with proper chemical groups to be used as anchor sites for the covalent immobilization of the liposomes. Stainless steel (SS-316) disks were processed in radiofrequency glow discharges fed with vapors of acrylic acid to coat them with thin adherent films characterized by surface carboxylic groups, where liposomes were covalently bound through the formation of amide bonds. For this, liposomes decorated with polyethylene glycol molecules bearing terminal amine-groups were prepared. After ensuring that the liposomes remain intact, under the conditions applying for immobilization; different attachment conditions were evaluated (incubation time, concentration of liposome dispersion) for optimization of the technique. Immobilization of calcein-entrapping liposomes was evaluated by monitoring the percent of calcein attached on the surfaces. Best results were obtained when liposome dispersions with 5mg/ml (liposomal lipid) concentration were incubated on each disk for 24h at 37°C. The method is proposed for developing drug-eluting biomedical materials or devices by using liposomes that have appropriate membrane compositions and are loaded with drugs or other bioactive agents.     

Development of liposomal immunosensor for the measurement of insulin with femtomole detection

The monitoring of insulin is of great relevance for the management of diabetes, the detection of pancreatic islet-cell malfunction, the definition of hypoglycemia, and the diagnosis of insulinoma. A liposomal immunosensing system for the determination of insulin was developed in this study. The insulin sensor was constructed by the immobilization of anti-insulin antibodies on the inner wall of the microcapillary immunoseparator. Liposomes tagged with anti-insulin and encapsulating a fluorescent dye were used as the detectable label. In the presence of insulin, sandwich immunocomplexes were formed between the immobilized antibodies in the column, the sample of insulin, and the antibody-tagged sulforhodamine B-dye-loaded liposomes. Signals generated by lysing the bound liposomes with 30 mM n-octyl-β-d-glucopyranoside were measured by a fluorescence detector. The detected signal was directly proportional to the amount of insulin in the test sample. The liposomal immunosensing system successfully detected as low as 136 attomole. MeOH (30%) was used for the regeneration of antibody-binding sites in the microcapillary after each measurement, which allowed the immunoseparator to be used for at least 70 repeated assays. The antibody activity in this proposed microcapillary immunoseparator could be well maintained for at least 1 week. The calibration curve for insulin in Tris-buffered saline had a linear dynamic range of 10 pM-10 nM, and the total assay time was less than 30 min. The coefficient of variation for triplicate measurements was <5.00%, which indicated that well-reproducible results can be obtained by this newly developed method.     

Optochemical sensing by immobilizing fluorophore-encapsulating liposomes in sol–gel thin films

The chemical stability of optochemical sensors depends largely on the physiochemical properties of the supportive matrix of the sensor and on the method used to immobilize sensing reagents to the supportive matrix of the sensor. Leaking of physically immobilized sensing reagents from the matrix support decreases the stability of the sensor and its overall usefulness. Covalent immobilization eliminates leakage of the sensing reagent from the support but may lead to alteration of spectral properties and loss of analyte response. This paper presents a new method for physical immobilization of polar fluorescence dyes in a sensing support. The method is based on the immobilization of fluorescent dye encapsulating liposomes in a sol–gel film of micrometer thickness. The encapsulation of the dye molecules in the liposomes effectively increases the molecular dimensions of the sensing reagent, thus preventing its leakage from the matrix support. This paper describes the analytical properties of a pH sensor fabricated by immobilizing carboxyfluorescein-encapsulating liposomes in a sol–gel thin film. The sensor shows excellent stability with respect to dye leaking which in turn leads to high reproducibility and sensitivity of about 0.01 pH units. The linear dynamic range of the sensor is between pH 6 and 7.5 and its response time is at the sub-seconds time scale.     

Liposomal glyco-microarray for studying glycolipid–protein interactions

A microarray enables high-throughput interaction screening of numerous biomolecules; however, fabrication of a microarray composed of cellular membrane components has proven difficult. We report fabrication of a liposomal glyco-microarray by using an azide-reactive liposome that carries synthetic and natural glycolipids via chemically selective and biocompatible liposome immobilization chemistry. Briefly, liposomes carrying anchor lipid dipalmitoylphosphatidylethanolamine (DPPE)-PEG(2000)-triphenylphosphine and ganglioside (GM1 or GM3) were prepared first and were then printed onto an azide-modified glass slide so as to afford a liposomal glyco-microarray via Staudinger ligation. Fluorescent dye release kinetics and fluorescence imaging confirmed successful liposome immobilization and specific protein binding to the intact arrayed glycoliposomes. The liposomal glyco-microarray with different gangliosides showed their specific lectin and toxin binding with different binding affinity. The azide-reactive liposome provides a facile strategy for fabrication of either a natural or a synthetic glycolipid-based membrane-mimetic glycoarray. This liposomal glyco-microarray is simple and broadly applicable and thus will find important biomedical applications, such as studying glycolipid-protein interactions and toxin screening applications.     

Reduction-sensitive liposomes from a multifunctional lipid conjugate and natural phospholipids: reduction and release kinetics and cellular uptake

The development of targeted and triggerable delivery systems is of high relevance for anticancer therapies. We report here on reduction-sensitive liposomes composed of a novel multifunctional lipidlike conjugate, containing a disulfide bond and a biotin moiety, and natural phospholipids. The incorporation of the disulfide conjugate into vesicles and the kinetics of their reduction were studied using dansyl-labeled conjugate 1 in using the dansyl fluorescence environmental sensitivity and the Fo?rster resonance energy transfer from dansyl to rhodamine-labeled phospholipids. Cleavage of the disulfide bridge (e.g., by tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), l-cysteine, or glutathione (GSH)) removed the hydrophilic headgroup of the conjugate and thus changed the membrane organization leading to the release of entrapped molecules. Upon nonspecific uptake of vesicles by macrophages, calcein release from reduction-sensitive liposomes consisting of the disulfide conjugate and phospholipids was more efficient than from reduction-insensitive liposomes composed only of phospholipids. The binding of streptavidin to the conjugates did not interfere with either the subsequent reduction of the disulfide bond of the conjugate or the release of entrapped molecules. Breast cancer cell line BT-474, overexpressing the HER2 receptor, showed a high uptake of the reduction-sensitive doxorubicin-loaded liposomes functionalized with the biotin-tagged anti-HER2 antibody. The release of the entrapped cargo inside the cells was observed, implying the potential of using our system for active targeting and delivery.     

Relocation of active acetylcholinesterase to liposome-gel conjugate

Relocation of a glycosylphosphatidylinositol (GPI)-anchored protein acetylcholinesterase (AChE) in its enzymatically active form from proteovesicles containing human erythrocyte ghost membrane proteins onto a liposome-gel conjugate was examined. Liposomes of 1,2-dimyristoylphosphatidylcholine (DMPC) were immobilized on Sephacryl S-1000 gel that was chemically modified to bear hydrophobic octyl moieties. Upon coincubation of the liposome-gel conjugate with freely suspended proteovesicles prepared from erythrocyte ghosts, 50% of the AChE left the proteovesicles and immobilized onto the liposome-gel conjugate in 18 h. When the proteovesicles were immobilized and interacted with freely suspended plain liposomes, approximately 2% of the AChE appeared in the liposome fraction. The relocation of AChE apparently possesses strong preference for the liposome-gel conjugate, suggesting that the hydrophobic moieties on the gel could assist the relocation.     

Azide-reactive liposome for chemoselective and biocompatible liposomal surface functionalization and glyco-liposomal microarray fabrication

Chemically selective liposomal surface functionalization and liposomal microarray fabrication using azide-reactive liposomes are described. First, liposome carrying PEG-triphenylphosphine was prepared for Staudinger ligation with azide-containing biotin, which was conducted in PBS buffer (pH 7.4) at room temperature without a catalyst. Then, immobilization and microarray fabrication of the biotinylated liposome onto a streptavidin-modified glass slide via the specific streptavidin/biotin interaction were investigated by comparing with directly formed biotin-liposome, which was prepared by the conventional liposome formulation of lipid-biotin with all other lipid components. Next, the covalent microarray fabrication of liposome carrying triphenylphosphine onto an azide-modified glass slide and its further glyco-modification with azide-containing carbohydrate were demonstrated for glyco-liposomal microarray fabrication via Staudinger ligation. Fluorescence imaging confirmed the successful immobilization and protein binding of the intact immobilized liposomes and arrayed glyco-liposomes. The azide-reactive liposome provides a facile strategy for membrane-mimetic glyco-array fabrication, which may find important biological and biomedical applications such as studying carbohydrate-protein interactions and toxin and antibody screening.     

Liposome chromatography: liposomes immobilized in gel beads as a stationary phase for aqueous column chromatography

Liposomes have been used as a stationary phase for column chromatography with an aqueous mobile phase. They were immobilized in the pores of carrier gel beads by two methods: (A) hydrophobic ligands were coupled to the matrix of gel beads, which then were packed into a column and liposomes were applied and became associated with the ligands by hydrophobic interaction; and (B) phospholipids and detergent were dialysed in the presence of gel beads; many of the liposomes that formed in the pores of the beads were sterically immobilized by the gel matrix. Proteoliposomes containing red cell glucose transport protein in the lipid bilayers were immobilized in a column by method A. This column retained D-glucose longer than L-glucose. In contrast to L-glucose, D-glucose was transported into and out of the immobilized liposomes, causing an increased retention. Liposomes with (stearylamine)+ or (phosphatidylserine)- in their lipid bilayers were immobilized by method B and the gel beads were packed into a column. A protein of opposite charge was applied in excess. Under suitable conditions, the protein molecules became close-packed on the liposome surfaces. Ion-exchange chromatographic experiments with proteins showed that these sterically immobilized liposomes were also stable enough to be used as a stationary phase. The loss of lipids was 5-23% in the first run at high protein load and with sodium chloride gradient elution but was lower in subsequent runs. It is proposed that water-soluble molecules can be separated and their interactions with liposome surfaces studied by chromatography on immobilized liposomes in detergent-free aqueous solution. Membrane proteins can be inserted and ligands can be anchored in the lipid bilayers for chromatographic purposes.     

Chromatographic purification steps involved in the synthesis of prodrug-liposome-antibody-complexes

Summary The cytostatic effect of the widely used antitumor drug 1-β-D-arabinofuranosyl cytosine (ara C) can be improved by its chemical derivatization to lipophilic prodrugs. We have incorporated these prodrugs together with lipophilic biotin derivatives into membranes of unilamellar liposomes. Monoclonal antibodies were coupled to the biotin residues of the liposomes via avidin-biotin complexation resulting in prodrug-liposome-antibody complexes whichin vitro preferably bind to cells selectively recognized by the immobilized antibodies. The results open a promising way of drug targeting. The components and liposomal derivatives used for the stepwise preparation of the prodrug-liposome antibody complex are purified by means of preparative liquid chromatography. Lipophilic membrane components are chromatographed on silica gel, antibodies on hydroxylapatite and liposomal derivatives on Ultrogel AcA 22 columns. Concentration and desalting are achieved by ultrafiltration. The purification process can be quantitatively pursued by labelling with radioactive components.     

PEG-detachable polyplex micelles based on disulfide-linked block catiomers as bioresponsive nonviral gene vectors

PEG-based polyplex micelles, which can detach the surrounding PEG chains responsive to the intracellular reducing environment, were developed as nonviral gene vectors. A novel block catiomer, PEG-SS-P[Asp(DET)], was designed as follows: (i) insertion of biocleavable disulfide linkage between PEG and polycation segment to trigger PEG detachment and (ii) a cationic segment based on poly(aspartamide) with a flanking N-(2-aminoethyl)-2-aminoethyl group, P[Asp(DET)], in which the Asp(DET) unit acts as a buffering moiety inducing endosomal escape with minimal cytotoxicity. The polyplex micelles from PEG-SS-P[Asp(DET)] and plasmid DNA (pDNA) stably dispersed in an aqueous medium with a narrowly distributed size range of approximately 80 nm due to the formation of hydrophilic PEG palisades while undergoing aggregation by the addition of 10 mM dithiothreitol (DTT) at the stoichiometric charge ratio, indicating the PEG detachment from the micelles through the disulfide cleavage. The PEG-SS-P[Asp(DET)] micelles showed both a 1-3 orders of magnitude higher gene transfection efficiency and a more rapid onset of gene expression than PEG-P[Asp(DET)] micelles without disulfide linkages, due to much more effective endosomal escape based on the PEG detachment in endosome. These findings suggest that the PEG-SS-P[Asp(DET)] micelle may have promising potential as a nonviral gene vector exerting high transfection with regulated timing and minimal cytotoxicity.     

Interfacial interactions of hydrophobic peptides with lipid bilayers

Four hydrophobic laminin-related peptides and their corresponding parent peptides were synthesized to use them to target liposomes to tumoral cells. The peptide sequence was YIGSR((NH(2))), and hydrophobic residues linked to the alpha-amino terminal end were decanoyl, myristoyl, stearoyl, and cholesteryl-succinoyl. Before use in biological systems, a physicochemical study was carried out in order to determine their interaction with DPPC bilayers that could compromise both the toxicity and the stability of liposomal preparations. The experiments were based on DSC, fluorescence polarization, outer-membrane destabilization, and vesicle leakage. These peptides showed in general a low interaction with the vesicles, promoting in all cases the rigidification of bilayers. This lack of strong disturbances in the ordered state of phospholipid molecules seems more likely due to the similarity of peptide acyl chains with those of lipids than to the absence of interactions. The bulkiness of cholesteryl derivative as well as its tendency toward aggregation resulted in weak interaction levels except in thermograms. The binding of peptides to the surface of liposomes loaded with doxorubicin resulted in preparations with good entrapment yields and small size, required for long circulating vesicles (especially for the myristoyl derivative). The alternative method based on the reaction of parent peptide to the surface of liposomes through an amide linkage was slightly more efficient when the peptide was linked to the carboxy-terminal end of the DSPE-PEG-COOH-containing liposomes. Nevertheless, the final decision must be made with the simplicity of the procedure and reduction in losses during all the steps of the processes taken into consideration.     

Physico-chemical characterization of liposomes with covalently attached hepatitis A VP3(101-121) synthetic peptide.

The covalent conjugation of a 20-mer peptide belonging to the VP3 capsid protein of hepatitis A virus to the surface of preformed liposomes was investigated. Three different bonds (disulfide, thioether and amide) were established between the peptide sequence and liposomes bearing at their surface appropriate reactive groups. The effect of the relative concentration of the N-[4-(p-maleimidophenyl)butyryl]dipalmitoylphosphatidylethanolamine anchor in liposomes on stability during coupling of the peptide sequence was studied. The interaction of the three liposomal preparations with phospholipids in a biomembrane model system, monolayers at the air-water interface, is also reported. The results showed that although the peptides associate with liposomes in similar yields for the three strategies studied, differences can be observed when their interaction with phospholipid monolayers composed of dipalmitoylphosphatidylcholine is analysed.     

Lectin recognition of liposomes containing neoglycolipids. Influence of their lipidic anchor and spacer length

Eleven neoglycolipids were synthesized with a membrane anchor (cholesterol or Guerbet alcohols) attached, via a hydrophilic spacer, to a potential lectin-recognized sugar moiety (N-acetylglucosamine: GlcNAc). Neoglycolipids G24-0, G28-0 and G32-0, with a C₂₄, C₂₈ and C₃₂ Guerbet alcohol residue, had no spacer, while neoglycolipids G24-3, G28-3, G32-3 contained a triethoxy spacer. Cholesteryl neoglycolipids chol-1 to chol-4 and chol-7 contained one to four and seven ethoxy units. All the cholesteryl neoglycolipids were incorporated into liposomes, while, for the Guerbet derivatives, the presence (or not) of a spacer and the length of their alkyl chains played an important role for obtaining mixed liposomes. The abilities of liposomes to be recognized by wheat germ agglutinin (WGA) were measured as an increase of the absorbance at 450 nm. Significant WGA-induced aggregations were obtained with liposomes containing neoglycolipids chol-3, chol-4, chol-7, G28-3 and G32-3. As neoglycolipid G24-3 was not recognized, the accessibility of its GlcNAc moiety for WGA depended not only on the spacer length but on the nature of the anchor. The WGA-induced aggregations increased with increasing lectin concentrations until a maximum value, which depended on the nature of the neoglycolipid. The highest aggregation obtained with G28-3 and G32-3 occurred for a WGA–neoglycolipid mole ratio superior to the ratio observed with chol-3, chol-4 and chol-7. A preincubation of WGA with free GlcNAc did not inhibit the aggregation between WGA and mixed liposomes, demonstrating a greater affinity of WGA for the neoglycolipid GlcNAc than for free GlcNAc.     

Effect of adsorption of bovine serum albumin on liposomal membrane characteristics

The effect of adsorption of bovine serum albumin (BSA) on the membrane characteristics of liposomes at pH 7.4 was examined in terms of zeta potential, micropolarity, microfluidity and permeability of liposomal bilayer membranes, where negatively charged L-alpha-dipalmitoylphosphatidylglycerol (DPPG)/L-alpha-dipalmitoylphosphatidylcholine (DPPC), negatively charged dicetylphosphate (DCP)/DPPC and positively charged stearylamine (SA)/DPPC mixed liposomes were used. BSA with negative charges adsorbed on negatively charged DPPG/DPPC mixed liposomes but did not adsorb on negatively charged DCP/DPPC and positively charged SA/DPPC mixed liposomes. Furthermore, the adsorption amount of BSA on the mixed DPPG/DPPC liposomes increased with increasing the mole fraction of DPPG in spite of a possible electrostatic repulsion between BSA and DPPG. Thus, the adsorption of BSA on liposomes was likely to be related to the hydrophobic interaction between BSA and liposomes. The microfluidity of liposomal bilayer membranes near the bilayer center decreased by the adsorption of BSA, while the permeability of liposomal bilayer membranes increased by the adsorption of BSA on liposomes. These results are considered to be due to that the adsorption of BSA brings about a phase separation in liposomes and that a temporary gap is consequently formed in the liposomal bilayer membranes, thereby the permeability of liposomal bilayer membranes increases by the adsorption of BSA.     

Enhanced Anticancer Efficacy by ATP‐Mediated Liposomal Drug Delivery

A liposome‐based co‐delivery system composed of a fusogenic liposome encapsulating ATP‐responsive elements with chemotherapeutics and a liposome containing ATP was developed for ATP‐mediated drug release triggered by liposomal fusion. The fusogenic liposome had a protein–DNA complex core containing an ATP‐responsive DNA scaffold with doxorubicin (DOX) and could release DOX through a conformational change from the duplex to the aptamer/ATP complex in the presence of ATP. A cell‐penetrating peptide‐modified fusogenic liposomal membrane was coated on the core, which had an acid‐triggered fusogenic potential with the ATP‐loaded liposomes or endosomes/lysosomes. Directly delivering extrinsic liposomal ATP promoted the drug release from the fusogenic liposome in the acidic intracellular compartments upon a pH‐sensitive membrane fusion and anticancer efficacy was enhanced both in vitro and in vivo.