Surface Modification of Liposomes Using IR700 Enables Efficient Controlled Contents Release Triggered by Near-IR Light.

Stimuli-responsive liposomes are promising drug carriers for cancer treatment because they enable controlled drug release and the maintenance of desired drug concentrations in tumor tissue. In particular, near-IR (NIR) light is a useful stimulus for triggering drug release from liposomes based on its advantages such as deep tissue penetration and safety. Previously, we found that a silicon phthalocyanine derivative, IR700, conjugated to antibodies, can induce the rupture of the cell membrane following irradiation by NIR light. Based on this finding, we constructed IR700-modified liposomes (IR700 liposomes) and evaluated their drug release properties triggered by NIR light. IR700 liposomes released substantial amounts of encapsulated calcein following irradiation by NIR light. Drug release was substantially suppressed by the addition of sodium azide, suggesting that liposomal membrane permeabilization was mediated by singlet oxygen generated from IR700. Moreover, calcein release from IR700 liposomes triggered by NIR light was promoted under conditions of deoxygenation and the presence of electron donors. Thus, membrane disruption should be induced by the physical change of IR700 from highly hydrophilic to hydrophobic as we previously described, although singlet oxygen can cause a certain level of membrane disruption under normoxia. We also observed that doxorubicin-encapsulated IR700 liposomes exhibited significant cytotoxic effects against CT-26 murine colon carcinoma cells following NIR light exposure. These results indicate that IR700 liposomes can efficiently release anti-cancer drugs following NIR light irradiation even under hypoxic conditions and, therefore, they would be useful for cancer treatment.

Physicochemical Analysis of DPPC and Photopolymerizable Liposomal Binary Mixture for Spatiotemporal Drug Release.

The development of a spatiotemporal drug delivery system with a long release profile, high loading efficiency, and robust therapeutic effects is still a challenge. Liposomal nanocarriers have secured a fortified position in the biomedical field over decades. Herein, liposomal binary mixtures of 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) and photopolymerizable 1,2-bis(10,12-tricosadiynoyl)- sn-glycero-3-phosphocholine (DC8,9PC) phospholipids were prepared for drug delivery applications. The diacetylenic groups of DC8,9PC produce intermolecular cross-linking following UV irradiation. Exposure of the liposomal mixture to 254 nm radiation induces a pore within the lipid bilayer, expediting the release of its entrapped 5,6-carboxyfluorescein dye. The dosage and rate of the released content are highly dependent on the number and size of the induced pore. Photochemical cross-linking studies at different exposure times were reported through the analysis of UV-visible spectrophotometry, nano differential scanning calorimetry, Fourier transform infrared spectroscopy, and Raman spectroscopy. The optimal irradiation time was established after 8 min of exposure, inducing lipid cross-linking with minimal oxidative degradation, which plays an essential role in the pathogenesis of numerous diseases due to the formation of primary and secondary oxidation products, accordingly reducing the encapsulated drug therapeutic level.

Carboxylated phytosterol derivative-introduced liposomes for skin environment-responsive transdermal drug delivery system.

Transdermal drug delivery systems are a key technology for skin-related diseases and for cosmetics development. The delivery of active ingredients to an appropriate site or target cells can greatly improve the efficacy of medical and cosmetic agents. For this study, liposome-based transdermal delivery systems were developed using pH-responsive phytosterol derivatives as liposome components. Succinylated phytosterol (Suc-PS) and 2-carboxy-cyclohexane-1-carboxylated phytosterol (CHex-PS) were synthesized by esterification of hydroxy groups of phytosterol. Modification of phytosterol derivatives on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes was confirmed by negatively zeta potentials at alkaline pH and the change of zeta potentials with decreasing pH. In response to acidic pH and temperatures higher than body temperature, Suc-PS-containing and CHex-PS-containing liposomes exhibited content release at intracellular acidic compartments of the melanocytes at the basement membrane of the skin. Phytosterol-derivative-containing liposomes were taken up by murine melanoma-derived B16-F10 cells. These liposomes delivered their contents into endosomes and cytosol of B16-F10 cells. Furthermore, phytosterol-derivative-containing liposomes penetrated the 3 D skin models and reached the basement membrane. Results show that pH-responsive phytosterol-derivative-containing DMPC liposomes are promising for use in transdermal medical or cosmetic agent delivery to melanocytes.

Transcutaneous drug delivery by liposomes using fractional laser technology.

OBJECTIVE: Transdermal delivery of hydrophilic peptides remains a challenge due to their poor cellular uptake and transdermal penetration. We hypothesize that combination of a CO2 fractional laser to enhance percutaneous absorption and liposomes as transdermal carriers would improve skin penetration of hydrophilic drugs. STUDY DESIGN: NA. METHODS: Liposomes were prepared using membrane fusion lipid dioleoylphosphatidylethanolamine, and used to deliver 5-carboxyfluorescein (CF) and fluorescein isothiocyanate-conjugated ovalbumin (OVA-FITC) as model hydrophilic peptide drugs. Liposome size was estimated by dynamic light scattering. Liposome uptake into murine macrophage cells and penetration or permeation into Yucatan micropig skin after irradiation by CO2 fractional laser at varying energy levels (laser power and exposure duration) were investigated using Franz cell and fluorescence microscopy. Oxidative damage to the irradiated mouse skin was assessed by electron spin resonance. RESULTS: Size of CF and OVA-FITC encapsulated liposomes was 324 ± 75 nm. Cellular uptake of OVA-FITC delivered by liposomes was 10-fold higher (1,370 relative fluorescence units, RFU) than delivered in solution form (130 RFU). Fractional laser irradiation increased skin permeation rate of CF liposomes (0-10%) and OVA-FITC liposomes (4-40%) in a dose-dependent manner. Although peeling off the stratum corneum facilitated CF liposome penetration at low energy levels (2.69-3.29 J/cm2 ; 10-20 W for 500 µs), drug permeation was similar (7-8%) in peeled or untreated skin at higher laser energy levels (6.06 J/cm2 ; 20 W for 1,500 µs). FITC penetrated deeper in the skin after laser irradiation. However, OH, O2-, and VC reactive oxygen species were generated upon irradiation of the skin with a fractional CO2 laser. CONCLUSIONS: Increasing laser power and irradiation, time increased liposome uptake by cells and penetration of peptide drugs across the skin in a dose-dependent manner. High-energy CO2 fractional laser overcomes the rate-limiting barrier function of the stratum corneum. Further investigations are required to establish the safety and efficacy of fractional laser-irradiation assisted delivery of liposome-encapsulated drugs as a transcutaneous drug delivery system. Lasers Surg. Med. 49:525-532, 2017. © 2016 Wiley Periodicals, Inc.

Topical delivery of Avastin to the posterior segment of the eye in vivo using annexin A5-associated liposomes.

Effective delivery to the retina is presently one of the most challenging areas in drug development in ophthalmology, due to anatomical barriers preventing entry of therapeutic substances. Intraocular injection is presently the only route of administration for large protein therapeutics, including the anti-Vascular Endothelial Growth Factors Lucentis (ranibizumab) and Avastin (bevacizumab). Anti-VEGFs have revolutionised the management of age-related macular degeneration and have increasing indications for use as sight-saving therapies in diabetes and retinal vascular disease. Considerable resources have been allocated to develop non-invasive ocular drug delivery systems. It has been suggested that the anionic phospholipid binding protein annexin A5, may have a role in drug delivery. In the present study we demonstrate, using a combination of in vitro and in vivo assays, that the presence of annexin A5 can significantly enhance uptake and transcytosis of liposomal drug carrier systems across corneal epithelial barriers. This system is employed to deliver physiologically significant concentrations of Avastin to the posterior of the rat eye (127 ng/g) and rabbit retina (18 ng/g) after topical application. Our observations provide evidence to suggest annexin A5 mediated endocytosis can enhance the delivery of associated lipidic drug delivery vehicles across biological barriers, which may have therapeutic implications.

Liposome surface functionalization based on different anchoring lipids via Staudinger ligation.

Liposome surface functionalization facilitates numerous potential applications of liposomes, such as enhanced stability, bioactive liposome conjugates, and targeted drug, gene and image agent delivery. Anchoring lipids are needed for grafting ligands of interest and play important roles in ligand grafting density, liposome stability, and liposome chemical and physical characteristics as well. In this report, glyco-functionalized liposome systems based on two kinds of anchoring lipids, phosphatidylethanolamine (PE) and cholesterol (Chol), were prepared by post chemically selective functionalization via Staudinger ligation. The size and stability of the liposomes were confirmed by dynamic light scattering (DLS). Particularly, the impact of anchor lipids on the stability of glyco-functionalized liposomes was investigated by comparing two different anchor lipids, namely Chol-PEG2000-TP and DSPE-PEG2000-TP. In addition, the encapsulation and releasing capacity of the glycosylated liposome based on the two anchoring lipids were investigated by entrapping 5,6-carboxyfluorescein (CF) dye and monitoring the fluorescence leakage, respectively. Furthermore, the density and accessibility of grafted carbohydrate residues on the liposome surface were evaluated for the two anchoring lipid-derived liposomes with lectin binding, respectively.

Effect of PEGylated lipid and Lecinol S-10 on physico-chemical properties and encapsulation efficiency of palmitoleate-palmitoleic acid vesicles.

A vesicle is a microscopic particle composed of a lipid bilayer membrane that separates the inner aqueous compartment from the outer aqueous environment. Palmitoleate-palmitoleic acid vesicles were prepared and their physico-chemical properties were investigated. Moreover, mixed vesicles composed of palmitoleic acid and PEGylated lipid and/or a mixture of phospholipids were also prepared. The stabilizing effects of these double-chain lipids on the formation of palmitoleate-palmitoleic acid vesicles were studied. Stability of the vesicle suspension was examined using particle size and zeta potential at 30 °C. The magnitude of the zeta potential was relatively lower in the vesicle suspension with the presence of phospholipid. Although some of the mixed vesicles that were formed were not very stable, they displayed potential for encapsulating the active ingredient calcein and the encapsulation efficiencies of calcein were encouraging. The palmitoleate-palmitoleic acid-DPPE-PEG2000 vesicle showed the most promising stability and encapsulation efficiency.

Characterization of spherulites as a lipidic carrier for low and high molecular weight agents.

PURPOSE: To develop spherulite formulations to achieve high entrapment efficiency for both small and macromolecules as well as cell-type specific delivery. METHODS: Spherulites of various compositions were prepared, and lipid-PEG was incorporated through post-insertion. Calcein and FITC-labeled albumin were employed as model drugs for small and macromolecules. The spherulites were characterized with respect to entrapment efficiency, size, structure, and release kinetics, and the morphology was examined via cryo-EM. Finally, SV119-decorated spherulites were examined for their selective uptake by cancer cells. RESULTS: The spherulites are 170 ~ 290 nm in size. A loading efficiency of 55 ~ 60% can be consistently achieved for both calcein and albumin under optimized conditions. Cryo-EM shows the onion-like morphology consistent with the structure of multilamellar liposomes. A t(½) of 39.3 h and 69.7 h in cargo release in serum was observed before and after PEG decoration, and incorporation of SV119 led to selective delivery of rhodamine-labeled spherulites to PC-3 tumor cells. CONCLUSIONS: Our optimized formulations may represent a platform with simple preparation approach, relatively small particle size, high drug loading efficiency for both low and high molecular weight agents, and slow release kinetics for selective delivery of various types of therapeutics to target cells.

Towards an understanding of the release behavior of temperature-sensitive liposomes: a possible explanation of the "pseudoequilibrium" release behavior at the phase transition temperature.

It is generally believed that thermal sensitive liposomes (i.e. vesicles) that contain 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000 (DSPE.2000) maintain a constant permeability to ionic molecules when they are heated to the phase transition temperature of the membrane (Tm). However, DPPC:DSPE.2000 liposome systems have been reported to release encapsulated agent in a "pseudoequilibrium" pattern upon temperature rise, whereby there is a rapid release of encapsulated material followed by no further release. Little effort has been made to determine the mechanism of such release behavior. We aim to explore the potential cause of "pseudoequilibrium" release of DPPC:DSPE.2000 liposome systems in response to temperature rise. Using calcein as a hydrophilic marker, the release pattern of DPPC:DSPE.2000 liposome system at Tm has been carefully determined. The potential mechanism of its release behavior has been further explored using two novel assays (i.e. heating-cooling-reheating assay and incubation assay). Our results show that there is a dramatic enhancement of the permeability of DPPC:DSPE.2000 vesicles to ionized molecules (i.e. calcein) during the initial period of heating to 42 °C. This is believed to result from the opening of the hydrophilic pores at the liquid/solid interface. However, after less than 2 min of incubation at this temperature, no further release of calcein is observed, suggesting that the sizes of pores are reduced, restricting any further movement of calcein molecules. On cooling and reheating the DPPC:DSPE.2000 liposomes to 42 °C, no further release of calcein is observed. The incorporation of MSPC (1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine) extends the release period of calcein from the DPPC:MSPC:DSPE.2000 vesicles to more than 30 min, suggesting that the lysolipid stabilizes the pores in the lipid membrane.

Partially polymerized liposomes: stable against leakage yet capable of instantaneous release for remote controlled drug delivery.

A critical issue for current liposomal carriers in clinical applications is their leakage of the encapsulated drugs that are cytotoxic to non-target tissues. We have developed partially polymerized liposomes composed of polydiacetylene lipids and saturated lipids. Cross-linking of the diacetylene lipids prevents the drug leakage even at 40 °C for days. These inactivated drug carriers are non-cytotoxic. Significantly, more than 70% of the encapsulated drug can be instantaneously released by a laser that matches the plasmon resonance of the tethered gold nanoparticles on the liposomes, and the therapeutic effect was observed in cancer cells. The remote activation feature of this novel drug delivery system allows for precise temporal and spatial control of drug release.

Fluorescence investigation of the retinal delivery of hydrophilic compounds via liposomal eyedrops.

We examined the feasibility of using submicron-sized liposomes (ssLips) for retinal delivery of hydrophilic compounds, which would also have a wide range of applications. To evaluate the uptake into conjunctival cell line and the intraocular behavior of hydrophilic compound-containing ssLips after eyedrop application, fluorometric investigation was carried out by using a hydrophilic fluorescence probe, 5(6)-carboxyfluorescein (CF). A relatively high amount of CF (>50%) could be incorporated into an internal phase of ssLips by a calcium acetate gradient method. CF being entrapped within the liposomes markedly enhanced both the uptake of CF into conjunctival cells and CF-oriented emission in the retina in mice after eyedrop application, while the free CF did not clear delivery efficiency in both in vitro and in vivo study. In addition, the cellular uptake and luminescence intensity in the retina were higher when a ssLip formulation composed of L-α-distearoyl phosphatidylcholine was applied than when a ssLip formulation composed of egg phosphatidylcholine was applied. Consequently, ssLips of appropriate composition were considered to have good potential to carry hydrophilic compounds into the retina.

Ultrasound-triggered herceptin liposomes for breast cancer therapy.

The functionalization of liposomes with monoclonal antibodies is a potential strategy to increase the specificity of liposomes and reduce the side-effects associated with chemotherapeutic agents. The active targeting of the Human Epidermal growth factor Receptor 2 (HER2), which is overexpressed in HER2 positive breast cancer cells, can be achieved by coating liposomes with an anti-HER2 monoclonal antibody. In this study, we synthesized calcein and Doxorubicin-loaded immunoliposomes functionalized with the monoclonal antibody Trastuzumab (TRA). Both liposomes were characterized for their size, phospholipid content and antibody conjugation. Exposing the liposomes to low-frequency ultrasound (LFUS) triggered drug release which increased with the increase in power density. Trastuzumab conjugation resulted in enhancing the sensitivity of the liposomes to LFUS. Compared to the control liposomes, TRA-liposomes showed higher cellular toxicity and higher drug uptake by the HER2 + cell line (SKBR3) which was further improved following sonication with LFUS. Combining immunoliposomes with LFUS is a promising technique in the field of targeted drug delivery that can enhance efficiency and reduce the cytotoxicity of antineoplastic drugs.

Novel Liposome Aggregate Platform (LAP) system for sustained retention of drugs in the posterior ocular segment following intravitreal injection.

A novel Liposome Aggregate Platform (LAP) system for prolonged retention of drugs in the posterior eye segment after intravitreal injection (IVT) was developed and evaluated. Calcein, FITC-dextran-4000 (FD4) and Flurbiprofen (FLB), were encapsulated in negatively charged liposomes, and mixed with protamine to produce the LAP. The lipid/protamine ratio was fixed, in order to have a convenient aggregation rate permitting IVT injection and also a sustained release of liposome-entrapped molecules (in vitro) from LAP. In vitro release studies confirmed the potential of LAP system consisted of HPC/DPPG/Chol liposomes and protamine (at 1:1 w/w to lipid), to delay calcein, FD4 and FLB release, compared to free liposomes. In vivo studies demonstrated increased vitreous retention of liposomes and LAP for all molecules, compared to the corresponding solutions; however the retention of FD4 is similar for non-aggregated liposomes and LAP, and calcein retention is only slightly increased by LAP compared to liposomes. The later result may be connected with the visible ocular inflammation caused by both dyes; interestingly inflammation was moderately reduced when dyes were entrapped in liposomes and even more when in LAP. No visible inflammation was demonstrated for FLB, and the LAP system significantly increased the retention of FLB in the ocular tissues (compared to non-aggregated liposomes). Taking into account the capability of the novel LAP system to decrease inflammatory reactions towards calcein and FD4, and prolong the retention of FLB in ocular tissues, it is concluded that such systems, after further optimization, may be considered as promising effective and safe approaches for treatment of posterior segment ocular pathologies.

Electrostatically mediated liposome fusion and lipid exchange with a nanoparticle-supported bilayer for control of surface charge, drug containment, and delivery.

The loading and containment of cargo within nanoparticles and their efficient delivery to cells represent a primary challenge in nanomedicine. We report lipid exchange between free and mesoporous silica nanoparticle-supported lipid bilayers as an effective means of containing cargo, controlling charge, and directing delivery to mammalian cells. The delivery of a membrane-impermeable dye (calcein) and a chemotherapeutic drug (doxorubicin) are demonstrated. Exchanged lipid bilayers minimized premature drug release, and an overall positive charge on the supported lipid bilayer effected enhanced delivery.

Cytoplasmic delivery of liposomal contents mediated by an acid-labile cholesterol-vinyl ether-PEG conjugate.

An acid-cleavable PEG lipid, 1'-(4'-cholesteryloxy-3'-butenyl)-omega-methoxy-polyethylene[112] glycolate (CVEP), has been developed that produces stable liposomes when dispersed as a minor component (0.5-5 mol %) in 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Cleavage of CVEP at mildly acidic pHs results in dePEGylation of the latently fusogenic DOPE liposomes, thereby triggering the onset of content release. This paper describes the synthesis of CVEP via a six-step sequence starting from the readily available precursors 1,4-butanediol, cholesterol, and mPEG acid. The hydrolysis rates and release kinetics from CVEP/DOPE liposome dispersions as a function of CVEP loading, as well as the cryogenic transmission electron microscopy and pH-dependent monolayer properties of 9:91 CVEP/DOPE mixtures, also are reported. When folate receptor-positive KB cells were exposed to calcein-loaded 5:95 CVEP/DOPE liposomes containing 0.1 mol % folate-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene[76] glycolamide (folate-PEG-DSPE), delivery of the calcein cargo to the cytoplasm of the cells was observed as determined by fluorescence microscopy and flow cytometry. Fluorescence resonance energy transfer analysis of lipid mixing in these cells was consistent with membrane-membrane fusion between the liposome and endosomal membranes.

Liposome-cell interaction: transfer and intracellular release of a trapped fluorescent marker.

When small, unilamellar lipid vesicles containing a high concentration of the fluorescent dye 6-carboxyfluorescein are incubated with either frog retinas or human lymphocytes, fluroescence distributes widely throughout each cell. Since "self-quenching" largely prevents the dye from fluorescing as long as it remains sequestered in vesicles, it is clear that a considerable amount of dye is released from the vesicles and diluted into the much larger volume of the cell.

Nanostraw membrane stamping for direct delivery of molecules into adhesive cells.

Delivering ions and molecules into living cells has become an important challenge in medical and biological fields. Conventional molecular delivery, however, has several issues such as physical and chemical damage to biological cells. Here, we present a method of directly delivering molecules into adhesive cells with an Au-based nanostraw membrane stamp that can physically inject a target molecule into the cytoplasm through a nanostraw duct. We successfully delivered calcein target molecules into adhesive cells with high efficiency (85%) and viability (90%). Furthermore, we modeled the molecular flow through Au nanostraws and then demonstrated the control of calcein flow by changing the concentration and geometry of Au nanostraws. Our Au membrane stamping provides a new way of accessing the cytoplasm to modulate cellular functions via injected molecules.

Release of liposome-encapsulated calcein from liposome entrapping gelatin-carboxymethylcellulose films: a presentation of different possibilities.

Liposome entrapment in films consisting of gelatin (GEL) or GEL/sodium carboxymethylcellulose (NaCMC) mixtures, as a method to alter drug release kinetics from polymeric films and/or incorporate sensitive bioactive molecules in solid films, was investigated. Bulk or thin complex (liposome trapping) films were formed by crosslinking (with glutaraldehyde) solutions of GEL or GEL/NaCMC in presence of calcein-encapsulating or rhodamine-labeled liposomes (Rho-Lip). Rho-Lip were observed by confocal microscopy to be homogenously distributed in the films. Calcein release from films was evaluated for periods up to 25 d, and it was found that several possibilities, concerning the release of the liposome-encapsulated molecule from the films, are offered; (i) Release can be sustained, if large liposomes are entrapped in the films. In this case the liposome-encapsulated molecules are released from the films only after they have been released from the vesicles, and the release can be controlled by modifying the film composition, the network density and/or the film geometry. (ii) Intact small unilamellar liposomes (SUV) can be released from the polymeric films depending on their swelling degree. The later can be controlled by modulating the film composition and amount of crosslinker. Film composition also affects the integrity of the film-entrapped liposomes during the crosslinking process, possibly due its effect on the density of the polymeric network of the film.

Prolongation of residence time of liposome by surface-modification with mixture of hydrophilic polymers.

The objective of this study is to evaluate the biodistribution characteristics of liposomes surface-modified with the mixture of polyethylene glycol (PEG) and polyvinyl alcohol (PVA) as a drug carrier for passive targeting of drugs. The liposomes (egg phosphatidylcholine:cholesterol=55:40, molar ratio) modified with both PEG and PVA (4:1 molar ratio) (PEG4%/PVA1% liposome) provided the largest AUC, which could be attributed to the smallest hepatic clearance of the liposomes. The liver perfusion studies clearly indicated that lower hepatic disposition of PEG4%/PVA1% liposome was ascribed to the decrease in its hepatic uptake via receptor-mediated endocytosis. Furthermore, the amounts of whole serum proteins and of opsonins such as complement C3 and immunoglobulin G adsorbed on PEG4%/PVA1% liposome were significantly smaller than those on the liposome solely modified with PEG (PEG5% liposome). On the other hand, several proteins were adsorbed at larger amount on PEG4%/PVA1% liposome than PEG5% liposome, and the protein identification by LC-MS/MS suggested that some of those proteins including albumin might function as dysopsonins. The decrease in the adsorbed amount of several opsonins and the increase in the adsorbed dysopsonins would be responsible for its lower affinity to the liver and long residence in the systemic circulation of PEG4%/PVA1% liposome.

Glucose-sensitive liposomes incorporating hydrophobically modified glucose oxidase.

Glucose-sensitive liposomes were prepared by incorporating hydrophobically modified glucose oxidase (EC 1.1.3.4.) into the liposomal bilayer of dioleoylphosphatidylethanolamine and cholesteryl hemisuccinate. For the release test, calcein, a fluorescence marker, was entrapped in the liposomes. The liposomes were stable under neutral conditions in terms of calcein release but an extensive release was observed under acidic conditions. In the experiment of glucose concentration-dependent calcein release, no release was observed for 180 min when the suspension of liposome was free of glucose. With a glucose concentration of 50 mg/dL, no appreciable amount of calcein was released for the first 20 min, and then the release rate was accelerated. At 200 mg/dL glucose concentration which is diagnostic and indicative for insulin-dependent diabetes, the lag time of calcein release became shorter and a faster response was obtained. When glucose concentration further increased to 400 mg/dL, the calcein release rate and the degree of release in 180 min were almost the same as the values when the glucose concentration was 200 mg/dL. The glucose concentration-dependent release is due to pH change, since the suspension of liposomes became acidic during the release experiments.