Targeted delivery of methotrexate to skeletal muscular tissue by thermosensitive magnetoliposomes

Thermosensitive magnetoliposomes (TMs) encapsulated with methotrexate (MTX) were prepared with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol by reverse-phase evaporation. Encapsulation efficiency of MTX and hydrophilic magnetite Fe₂O₃-glu, liposome particle size, ζ-potential, and in vitro and in vivo drug release were studied. More than 80% of loaded MTX was released from TMs within 30 min when the environmental temperature increased from 37 °C to 41 °C, while 60% of the drug was remained inside TMs for up to 24 h at 37 °C. Furthermore, the pharmacokinetics and tissue distribution study showed that TMs significantly increased the accumulation of MTX in the skeletal muscular tissue when exposed to an external constant magnetic field and heated to 41 °C compared to the absence of the magnetic field and heating. Therefore, the results in this study suggested that TMs prepared by reverse-phase evaporation can archive a good magnetic targeting effect and fast drug release in response to hyperthermia, which implies their great potential of application in cancer therapy.     

Near-Infrared Image-Guided Delivery and Controlled Release Using Optimized Thermosensitive Liposomes

Purpose

To engineer optimized near-infrared (NIR) active thermosensitive liposomes to potentially achieve image-guided delivery of chemotherapeutic agents.

Methods

Thermosensitive liposomes were surface-coated with either polyethylene glycol or dextran. Differential scanning calorimetry and calcein release studies were conducted to optimize liposomal release, and flow cytometry was employed to determine the in vitro macrophage uptake of liposomes. Indocyanine green (ICG) was encapsulated as the NIR dye to evaluate the in vivo biodistribution in tumor-bearing mice.

Results

The optimized thermosensitive liposome formulation consists of DPPC, SoyPC, and cholesterol in the 100:50:30 molar ratio. Liposomes with dextran and polyethylene glycol demonstrated similar thermal release properties; however in vitro macrophage uptake was greater with dextran. Non-invasive in vivo NIR imaging showed tumor accumulation of liposomes with both coatings, and ex vivo NIR imaging correlated well with actual ICG concentrations in various organs of healthy mice.

Conclusions

The optimized thermosensitive liposome formulation demonstrated stability at 37?°C and efficient burst release at 40 and 42?°C. Dextran exhibited potential for application as a surface coating in thermosensitive liposome formulations. In vivo studies suggest that liposomal encapsulation of ICG permits reliable, real-time monitoring of liposome biodistribution through non-invasive NIR imaging.     

Rates of Systemic Degradation and Reticuloendothelial System Uptake of Calcein in the Dipalmitoylphosphatidylcholine Liposomes with Soybean-Derived Sterols in Mice

The systemic degradation and the reticuloendothelial system (RES) uptake of calcein entrapped in dipalmitoylphosphatidylcholine (DPPC) liposomes with soybean-derived sterols (SS) were examined after intravenous administration to mice by measuring the free and liposomal calcein levels in the blood. The results indicate that the rates of systemic degradation and the RES uptake of liposomes decrease with the addition of SS in DPPC liposomes since the SS has the ability to stabilize the liposomes. The rate of uptake by RES is larger than the rate of systemic degradation. The rate of leakage of calcein from liposomes by incubation in plasma in vitro is almost the same as that of systemic degradation in vivo.     

Synergic fabrication of multifunctional liposomes nanocomposites for improved radiofrequency ablation combination for liver metastasis cancer therapy

The field of biomedical research has recently been interested in nanoplatforms with various functionalities, such as cancer drug carriers and MRI and optical imaging, as well as thermal treatment, among other things. As a result of the present investigation, a unique multifunctional liposome (MFL) was established in this investigation. Using radiofrequency-induced imaging and drug release based on magnetic field impact, a dual drug delivery targeted with tumor multi-mechanism treatment was made more effective. The C60 (fullerene) surface was coated with iron nanocomposites to establish the proposed nanosystems, and PEGylation was used (Fe₃O₄-C60-PEG₂₀₀₀). For fullerene radiofrequency-triggered drug release, thermosensitive DPPC liposomes with folate-DSPE-PEG₂₀₀₀ enveloped the binary nanosystems and doxorubicin (DOX). The in vitro cytotoxicity of the nanocomposites was confirmed by the liver metastasis in HT-29 colon cancer cells using radiofrequency. The flow cytometry analysis confirmed the apoptosis cell death mechanism. The thermal treatment combined chemotherapeutic MFL nano framework transformed radiofrequency radiation from thermoresponsive liposomes, which was noticed both in vivo and in vitro. Due to their superior active tumor targeting and magnetic targeting characteristics, the MFL could also selectively destroy cancerous liver cells in highly co-localized targets.     

Listeriolysin O enhances cytoplasmic delivery by Her-2 targeting liposomes

To enhance cytoplasmic delivery of liposomal contents to breast cancer cells, the authors have attached the pore-forming protein, listeriolysin O (LLO), to thermosensitive liposomes. The antibody trastuzumab (Herceptin^®) was also conjugated with the outer surface of the liposomes, resulting in highly specific binding and internalization into mammary epithelial cells that overexpress the human epidermal growth factor receptor 2 (Her-2). The liposomes were preloaded with a marker fluorescent dye, and the effect of LLO on the distribution of dye within the cells was monitored using fluorescence microscopy. Owing to the thermosensitive nature of the liposomes, hyperthermia at 42°C triggered the release of the encapsulated fluorescent calcein from the endocytosed liposomes into the interior of the endosomes. LLO, when conjugated to these liposomes, subsequently formed pores in the endosomal membrane, allowing calcein to flow out of the endosomal compartment into the cytoplasm. Her-2–targeted liposomes bearing LLO delivered a 22-fold greater concentration of calcein to mammary epithelial cells that overexpress Her-2 compared to cells with normal Her-2 expression. Thus, the addition of LLO to preformed liposomes offers a method for significantly enhancing delivery of liposomal contents to the cytoplasm of targeted cells.     

Bilayer Composition,Temperature, Speciation Effects and the Role of Bilayer Chain Ordering on Partitioning of Dexamethasone and its 21-Phosphate

Purpose

Models to predict membrane-water partition coefficients (K_p) as a function of drug structure, membrane composition, and solution properties would be useful. This study explores the partitioning of dexamethasone (Dex) and its ionizable 21-phosphate (Dex-P) in liposomes varying in acyl chain length, physical state, and pH.

Methods

DMPC:mPEG DMPE, DPPC:mPEG DPPE, and DSPC:mPEG DSPE (95:5 mol%) liposomes were prepared by thin film hydration. K_p values for Dex and Dex-P were determined from pH 1.5–8 by equilibrium dialysis and equilibrium solubility (Dex).

Results

Dex K_p values at 25°C were 705?±?24, 106?±?11, and 58?±?9 in DMPC, DPPC, and DSPC, increasing to 478?±?20 in DPPC liposomes at 45°C. Both neutral and anionic species contributed to the K_p of Dex-P versus solution pH (1.5–8). A linear correlation was found between the natural logarithm of K_p and the inverse of bilayer free surface area (1/a_free) where a_free is a parameter reflecting chain ordering that depends on bilayer composition and temperature.

Conclusions

Models of the pH dependence of partitioning of ionizable compounds must include contributions of both neutral and ionized species. Bilayer free surface area may be an important variable to predict K_p of drug molecules versus lipid composition and temperature.

     

Cholate-Induced Disruption of Calcitonin-Loaded Liposomes: Formation of Trypsin-Resistant Lipid-Calcitonin-Cholate Complexes

Purpose. The work was performed to obtain a better understanding why the oral administration of calcitonin (CT)-loaded liposomes to rats results in a hypocalcemia, while liposomes are normally disrupted in the gastro-intestinal tract and cannot protect the hormone from enzymatic digestion. Methods. In vitro comparisons between the stability of calcein and CT-loaded liposomes in the presence of cholate solutions led to an interpretation of the results observed. By means of gel filtration, turbidimetry, and fluorescence measurements, the interactions between CT and lipids were studied after sonicated liposomes had been broken down by cholate. Results. Experiments showed that CT in the external medium of a liposome suspension had no effect on the vesicles. Gel filtration of cholate-treated liposomes loaded with calcein and CT resulted in a total separation of calcein from the lipid fraction for detergent concentrations higher than 4 mM. However, 50% of the CT was reencapsulated even when the cholate-to-phospholipid molar ratio was increased up to 100. Incubation of cholate-solubilized liposomes with 1% trypsin resulted in a partial CT-breakdown. Conclusions. These results strongly suggest that during membrane solubilization by cholate, lipid-CT complexes are formed which retain most of the CT initially embedded in the liposomal membrane, and which offer some protection to CT under the action of trypsin. The existence of these complexes could be one of the reasons for the reported hypocalcemia in rats after oral administration of CT-loaded liposomes.     

Pharmacokinetic and Pharmacological Profiles of Free and Liposomal Recombinant Human Erythropoietin After Intravenous and Subcutaneous Administrations in Rats

Purpose. Recombinant human erythropoietin (Epo) is used frequently through intravenous (i.v.) and subcutaneous (s.c.) administration for the clinical treatment of the last stage of renal anemia. We encapsulated Epo in liposomes to develop an alternative administration route. The purpose of our study was to evaluate the pharmacokinetics and the pharmacological effects of liposomal Epo in comparison with the Epo after i.v. and s.c. administration to rats. Methods. Epo was encapsulated in liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and soybean-derived sterol mixture (SS) prepared by the reversed-phase evaporation vesicle method. After filtration through a 0.1 m polycarbonate membrane, liposomes were gel filtered (Epo/liposomes). Results. Epo/liposomes showed higher pharmacological activity than Epo/liposomes before gel filtration after i.v. administration to rats. Non-encapsulated Epo lost its activity, whereas encapsulated Epo in liposomes retained it. The pharmacological effects of Epo/liposomes were greater than those of Epo after i.v. administration. Epo/liposomes afforded 3–9 times higher AUC, lower clearance and lower steady-state volume of distribution than Epo after both i.v. and s.c. administrations. Epo/liposomes had an improved pharmacokinetic profile compared with Epo. S.c. administration of Epo/liposomes at 7 h may penetrate primarily (40% of dose) through the blood as a liposome and partly (7% of dose) in lymph. Conclusions. Epo/liposomes may reduce the frequency of injections required for a certain reticulocyte effect in comparison to Epo. The lower clearance of Epo/liposomes may increase the plasma concentrations of Epo, which increases the efficacy.     

Rates of Systemic Degradation and Reticuloendothelial System (RES) Uptake of Thermosensitive Liposome Encapsulating Cisplatin in Rats

The systemic degradation and reticuloendothelial system (RES) uptake of cisplatin (CDDP)-encapsulated thermosensitive liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and di-stearoylphosphatidylcholine (DSPC) (DPPC/DSPC = 9/1, 7/3, and 5/5, w/w) after intravenous administration to rats were examined by measuring the platinum (Pt) levels in the blood and RES (liver and spleen). The blood liposome level profile showed first-order rate elimination for each liposome administration. The elimination rate (K _e1) was faster when the content of DSPC was lower (K _e1: 1.3/hr for 9/1-liposomes, 0.7/hr for 7/3-liposomes, 0.5/hrfor5/5-liposomes). On the other hand, the RES liposome level profile showed distribution of liposomes followed by elimination therefrom. The RES level of the liposomes was lower when the content of DSPC was smaller (maximal level: 25% for 9/1-liposomes at 1 hr, 32% for 7/3-liposomes at 1 hr, 37% for 5/5-liposomes at 2 hr). The kinetic analysis demonstrated that the RES uptake rate (K _res) was almost the same among the liposomes (0.4/hr), while the systemic degradation rate (K _deg; K _e1 – K _res) became larger as the content of DSPC decreased (0.9/hr for 9/1-liposomes, 0.3/hr for 7/3-liposomes, and 0.1/hr for 5/5-liposomes) and that the RES liposome distribution amount was dependent not only on the K _res but also on the K _deg and the rate of RES liposome degradation. The K _deg for each type of liposome corresponded with the systemic CDDP release rate.     

Enteral Absorption of Insulin in Rats from Mucoadhesive Chitosan-Coated Liposomes

Purpose. The mucoadhesiveness of polymer-coated liposomes was evaluated to develop a novel drug carrier system for oral administration of poorly absorbed drugs such as peptide drugs. Methods. Multilamellar liposomes consisting of dipalmitoylphosphatidylcholine (DPPC) and dicetyl phosphate (DCP) (DPPC:DCP = 8:2 in molar ratio) were coated with chitosan (CS), polyvinyl alcohol having a long alkyl chain (PVA-R) and poly (acrylic acid) bearing a cholesteryl group. The adhesiveness of the resultant polymer-coated liposomes to the rat intestine was measured in vitro by a particle counting method with a Coulter counter. The CS-coated liposomes containing insulin were administered to normal rats and the blood glucose level was monitored. Results. The existence of polymer layers on the surface of liposomes was confirmed by measuring the zeta potential of liposomes. The CS-coated liposomes showed the highest mucoadhesiveness and the degree of adhesion was dependent on the amount of CS on the surface of the liposomes. The blood glucose level of rats was found to be significantly decreased after administration of the CS-coated liposomes containing insulin. The lowered glucose level was maintained for more than 12h after administration of the liposomal insulin, which suggested mucoadhesion of the CS-coated liposomes in the intestinal tract of the rats.     

A two-component drug delivery system using Her-2-targeting thermosensitive liposomes

We report on a new method for enhancing the specificity of drug delivery for tumor cells, using thermosensitive immunoliposomes. The liposomes are conjugated to the antibody trastuzumab (Herceptin^®), which targets the human epidermal growth factor receptor 2 (Her-2), a cell membrane receptor overexpressed in many human cancers. Being thermosensitive, the liposomes only release their contents when heated slightly above body temperature, allowing for the possibility of tissue targeting through localized hyperthermia. Using self-quenching calcein, we demonstrate the release of liposome contents into cell endosomes after brief heating to 42°C. To further increase targeting specificity, we incorporate the concept of a two-component delivery system that requires the interaction of two different liposomes within the same endosome for cytoplasmic delivery. Experimental evaluation of the technique using fluorescently labeled liposomes shows that a two-component delivery system, combined with intracellular disruption of liposomes by hyperthermia, significantly increases specificity for Her-2-overexpressing tumor cells.     

Thermosensitive magnetic liposomes with doxorubicin cell-penetrating peptides conjugate for enhanced and targeted cancer therapy

To specifically deliver cytotoxic drug to tumor cells and enhance cellular uptake is the key for effective cancer therapy. In this paper, we described a novel drug targeting system, which is designed to combine features of biological (cell-penetrating peptides, CPPs) and physical (magnetic) drug targeting for use in the magnetic hyperthermia-triggered release. A doxorubicin–CPPs conjugate (DOX-CPPs) was loaded into thermosensitive magnetic liposomes (TSMLs) (DOX-CPPs/TSMLs), and in vitro DOX-CPPs thermosensitive release activity, anti-proliferation effect, in vivo targeted delivery as well as in vivo antitumor activity were determined. The results demonstrated that the DOX-CPPs/TSMLs showed good physicochemical properties, effective anti-proliferation effect in MCF-7 cells in vitro. Additionally, in vivo study, DOX-CPPs/TSMLs under AC magnetic field displayed superior in vivo targeted delivery efficacy, antitumor efficacy in an MCF-7 xenograft murine model. In conclusion, the application of DOX-CPPs/TSMLs under AC magnetic field may provide a strategy for the selective and efficient delivery of drug.     

Effect of soybean-derived sterols on the in vitro stability and the blood circulation of liposomes in mice

The stability and long lifetime in blood circulation of liposomes are important when using them as a reservoir for drugs. A soybean-derived sterol mixture (SS) was used to stabilize and extend the blood circulation lifetime of liposomes. In the present study, the effects of SS on the stability and lifetime in blood of liposomes entrapping calcein were investigated by measuring the leakage of calcein and the concentration of calcein in blood. The results indicate that SS has a greater ability to stabilize the DPPC liposomes than cholesterol which is usually used as a stabilizer. The stabilizing effect was greatest at a molar ratio of DPPC and SS of 7: 4.     

Targeted thermosensitive liposomes: an attractive novel approach for increased drug delivery to solid tumors

Introduction: Currently available chemotherapy is hampered by a lack in tumor specificity and resulting toxicity. Small and long-circulating liposomes can preferentially deliver chemotherapeutic drugs to tumors upon extravasation from tumor vasculature. Although clinically used liposomal formulations demonstrated significant reduction in toxicity, enhancement of therapeutic activity has not fully met expectations.

Areas covered: Low drug bioavailability from liposomal formulations and limited tumor accumulation remain major challenges to further improve therapeutic activity of liposomal chemotherapy. The aim of this review is to highlight strategies addressing these challenges. A first strategy uses hyperthermia and thermosensitive liposomes to improve tumor accumulation and trigger liposomal drug bioavailability. Image-guidance can aid online monitoring of heat and drug delivery and further personalize the treatment. A second strategy involves tumor-specific targeting to enhance drug delivery specificity and drug internalization. In addition, we review the potential of combinations of the two in one targeted thermosensitive-triggered drug delivery system.

Expert opinion: Heat-triggered drug delivery using thermosensitive liposomes as well as the use of tumor vasculature or tumor cell-targeted liposomes are both promising strategies to improve liposomal chemotherapy. Preclinical evidence has been encouraging and both strategies are currently undergoing clinical evaluation. A combination of both strategies rendering targeted thermosensitive liposomes (TTSL) may appear as a new and attractive approach promoting tumor drug delivery.     

Effects of soybean-derived sterol and its glucoside mixtures added in dipalmitoylphosphatidylcholine liposomes on the blood circulation and hepatic cellular distribution in mice

The effects of a soybean-derived sterol mixture (SS) and their glucoside mixture (SG) in dipalmitoylphosphatidylcholine (DPPC) liposomes on blood circulation and hepatic distribution were investigated by measuring the leakage of calcein after intravenous administration to mice. Four kinds of liposomes were prepared: liposomes consisting only of DPPC (DPPC liposomes); and DPPC liposomes containing SS, cholesterol (Ch) or SG (molar ratio of DPPC/X = 7:2, X = SS, Ch, SG; SS, Ch and SG liposome), respectively. The area under the blood concentration-time curve (AUC) was greater in the order, SS liposomes > DPPC liposomes > SG liposomes, and the order is the same for lower membrane fluidity. Hepatic cellular distribution of SG liposomes 2 h after intravenous injection was significantly high compared with that of DPPC and SS liposomes. The results indicated that SS liposomes were stable in the blood circulation; however, SG liposomes were not stable and appeared to have an enhancing effect on hepatic uptake. This difference might indicate that hepatic accumulation is primarily governed by the glucose group of SG.     

Heat-Induced Drug Release Rate and Maximal Targeting Index of Thermosensitive Liposome in Tumor-Bearing Mice

To evaluate the rate of drug release at the tumor and maximal drug targeting after administration of thermosensitive liposomes with hy-perthermia, a theoretical and experimental method was derived assessing the fraction of drug released from liposomes in a single pass through the heated tumor, F, and the drug targeting index when drug release occurs completely in response to heat (F = 1), DTI_max. The F and DTImax were evaluated for four types of liposomes (LUV-1 and LUV-2, thermosensitive large unilamellar liposomes; LUV-3, a nonthermosensitive large unilamellar liposome; and SUV-1, a thermosensitive small unilamellar liposome) using reported data on blood liposome levels and tumor drug levels after the liposomes were administered to tumor bearing mice. DTI_max values for LUV-1 and SUV-1 were approximately 6, while the value for LUV-2 with a relatively large systemic clearance was only 2.3. The F values for LUV-1, LUV-2, and SUV-1 with hyperthermia were 0.71, 1.17, and 0.34, respectively, whereas the values for these liposomes without hyperthermia and for LUV-3 with or without hyperthermia were nearly zero. These results confirm earlier findings that LUV-1 and LUV-2 release CDDP almost completely at the heated tumor and that the large DTI value obtained in LUV-1 (DTI = 4.6) was due to its high heat sensitivity and its small systemic clearance.     

Effect of Freezing Rate on the Stability of Liposomes During Freeze-Drying and Rehydration

Purpose. In the present study we examined the effect of the freezing protocol on carboxyfluorescein (CF) retention in liposomes after freeze-drying and rehydration. Methods. Liposomes were frozen slowly at 0.5°C/min, or quickly by submerging the samples in boiling nitrogen before freeze-drying. The thermal behaviour of the frozen dispersions was analysed by Modulated Temperature Differential Scanning Calorimetry (MTDSC). The dried cakes were analysed by SEM, MTDSC and FTIR. The % encapsulated CF of the (re)hydrated liposomes was determined by fluorimetry after GPC, their vesicle size was measured by the Dynamic Light scattering Technique and their bilayer transition was studied by DSC. Results. Slow freezing resulted in a markedly higher CF retention after freeze-drying and rehydration as compared to quick freezing. The effect of the freezing rate depended on the lipid composition and was most pronounced for rigid liposomes. The damage caused by quick freezing did not occur after a freezing/thawing cycle. The freezing protocol did not influence the interaction between the phospholipids and the lyoprotectants (sucrose, trehalose or glucose) in the freeze-dried state. However, analysis by DSC of dipalmitoylphosphatidylcholine (DPPC): dipalmitoylphosphatidylglycerol (DPPG) =10:1 and DPPC liposome dispersions showed that the freezing protocol affected the bilayer melting characteristics of these liposomes after freeze-drying and rehydration. Conclusions. A proper design of the freezing protocol is essential to achieve optimal stability of rigid liposomes during a freeze-drying and rehydration cycle.     

Preparation and characterization of Tegafur magnetic thermosensitive liposomes

The purpose was to study the preparation and properties of tegafur magnetic thermosensitive liposomes. The method was to employ an improved chemical coprecipitation method for preparing nano-magnetic particles and a reverse-phase evaporation and ultrasonic method for preparing tegafur magnetic thermosensitive liposomes. The results showed that tegafur magnetic thermosensitive liposomes were prepared successfully. They had comparatively strong magnetism and superparamagnetism, and their temperature showed a linear positive correlation with dosages and the field strength under a current value. The conclusion was that tegafur magnetic thermosensitive liposomes with comparatively small particle size, superparamagnetism and comparatively strong magnetism were prepared successfully.     

Effects of sialic acid derivative on long circulation time and tumor concentration of liposomes

The effects of a sialic acid derivative, Neu5Aβ-PA, on the blood circulation and tissue distribution of liposomes composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol) and Neu5Aβ-PA were investigated compared with liposomes composed of DPPC, Chol and monosialoganglioside GM₁, in mice and rats. When liposomes containing Neu5Aβ-PA were intravenously administered into mice, the plasma concentration of liposomes containing Neu5Ac/3-PA was increased, and the liver and spleen uptakes were decreased; there was no significant difference in tissue distribution between liposomes containing Neu5Aβ-PA (DPPC/Chol/Neu5Aβ-PA= 10: 10:3) and those containing GM₁, (DPPC/Chol/GM₁= 10:10:1). On the other hand, the plasma concentration of liposomes containing Neu5Aβ-PA was significantly greater than that of liposomes containing GM₁, at all times determined in rats, and was about 30.7- and 10.3-fold that of liposomes containing GM₁, at 6 and 24 h, respectively. The liver and spleen uptakes of liposomes containing Neu5Aβ-PA at 6 h were significantly reduced compared with those of liposomes containing GM, in rats. The tumor accumulation of liposomes was also examined. The liver/tumor ratio of liposomes containing Neu5Aβ-PA was similar to that of liposomes containing GM₁ in mice and lower than that of liposomes containing GM₁ in rats.     

Use of the Post-Insertion Technique to Insert Peptide Ligands into Pre-Formed Stealth Liposomes with Retention of Binding Activity and Cytotoxicity

Purpose: Simple methods for the large-scale manufacture of ligand-targeted liposomes will be needed if clinical trials are to proceed. We tested a recently developed technology for inserting peptide ligands into preformed Stealth liposomes. Antagonist G-targeted liposomes (PLG) were prepared and loaded with doxorubicin and their cellular association and cytotoxicity were evaluated using the human small cell lung cancer H69 cell line. Methods: The hexapeptide antagonist G was covalently coupled via a thioether bond to the terminus of polyethylene glycol (PEG) in micelles formed from maleimide-derivatized poly(ethylene glycol) (M_r 2000) distearoylphosphatidylethanolamine followed by transfer into preformed liposomes during a one-step incubation. For cellular association, we used radiolabeled liposomes. Cytotoxicity was evaluated using the MTT in vitro proliferation assay. Results: The postinsertion approach to the formation of peptide-targeted liposomes led to the production of PLG bearing a maximum of approximately 0.3 g antagonist G/mol phospholipid. These liposomes had increased cellular association to H69 cells relative to nontargeted liposomes and, when loaded with doxorubicin, they resulted in similar levels of cytotoxicity to those obtained by conventional coupling techniques. Conclusions: The postinsertion technique is a simple, effective means for the production of biologically active peptide-targeted liposomes.