Keelung Hong

Tumor targeting using anti-her2 immunoliposomes.

We have generated anti-HER2 (ErbB2) immunoliposomes (ILs), consisting of long circulating liposomes linked to anti-HER2 monoclonal antibody (MAb) fragments, to provide targeted drug delivery to HER2-overexpressing cells. Immunoliposomes were constructed using a modular strategy in which components were optimized for internalization and intracellular drug delivery. Parameters included choice of antibody construct, antibody density, antibody conjugation procedure, and choice of liposome construct. Anti-HER2 immunoliposomes bound efficiently to and internalized in HER2-overexpressing cells in vitro as determined by fluorescence microscopy, electron microscopy, and quantitative analysis of fluorescent probe delivery. Delivery via ILs in HER2-overexpressing cells yielded drug uptake that was up to 700-fold greater than with non-targeted sterically stabilized liposomes. In vivo, anti-HER2 ILs showed extremely long circulation as stable constructs in normal adult rats after a single i.v. dose, with pharmacokinetics that were indistinguishable from sterically stabilized liposomes. Repeat administrations revealed no increase in clearance, further confirming that ILs retain the long circulation and non-immunogenicity of sterically stabilized liposomes. In five different HER2-overexpressing xenograft models, anti-HER2 ILs loaded with doxorubicin (dox) showed potent anticancer activity, including tumor inhibition, regressions, and cures (pathologic complete responses). ILs were significantly superior vs. all other treatment conditions tested: free dox, liposomal dox, free MAb (trastuzumab), and combinations of dox+MAb or liposomal dox+MAb. For example, ILs produced significantly superior antitumor effects vs. non-targeted liposomes (P values from <0.0001 to 0.04 in eight separate experiments). In a non-HER2-overexpressing xenograft model (MCF7), ILs and non-targeted liposomal dox produced equivalent antitumor effects. Detailed studies of tumor localization indicated a novel mechanism of drug delivery for anti-HER2 ILs. Immunotargeting did not increase tumor tissue levels of ILs vs. liposomes, as both achieved very high tumor localization (7.0-8.5% of injected dose/g tissue) in xenograft tumors. However, histologic studies using colloidal-gold labeled ILs demonstrated efficient intracellular delivery in tumor cells, while non-targeted liposomes accumulated within stroma, either extracellularly or within macrophages. In the MCF7 xenograft model lacking HER2-overexpression, no difference in tumor cell uptake was seen, with both ILs and non-targeted liposomes accumulating within stroma. Thus, anti-HER2 ILs, but not non-targeted liposomes, achieve intracellular drug delivery via receptor-mediated endocytosis, and this mechanism is associated with superior antitumor activity. Based on these results, anti-HER2 immunoliposomes have been developed toward clinical trials. Reengineering of construct design for clinical use has been achieved, including: new anti-HER2 scFv F5 generated by screening of a phage antibody library for internalizing anti-HER2 phage antibodies; modifications of the scFv expression construct to support large scale production and clinical use; and development of methods for large-scale conjugation of antibody fragments with liposomes. We developed a scalable two-step protocol for linkage of scFv to preformed and drug-loaded liposomes. Our final, optimized anti-HER2 ILs-dox construct consists of F5 conjugated to derivatized PEG-PE linker and incorporated into commercially available liposomal doxorubicin (Doxil). Finally, further studies of the mechanism of action of anti-HER2 ILs-dox suggest that this strategy may provide optimal delivery of anthracycline-based chemotherapy to HER2-overexpressing cancer cells in the clinic, while circumventing the cardiotoxicity associated with trastuzumab+anthracycline. We conclude that anti-HER2 immunoliposomes represent a promising technology for tumor-targeted drug delivery, and that this strategy may also be applicable to other receptor targets and/or using other delivered agents.

Endocytosis of liposomes and intracellular fate of encapsulated molecules: Encounter with a low pH compartment after internalization in coated vesicles

We have compared the intracellular fate of several fluorescent probes and colloidal gold entrapped in negatively charged liposomes. Weakly acidic molecules (carboxyfluorescein) appear in the cytoplasm of CV-1 cells in 30 min; agents that raise lysosomal pH block this process. Highly charged molecules (calcein) and large molecules (FITC-dextran: 18 kd) remain confined to extra-or intracellular vesicles. Thin section electron micrographs show gold-containing liposomes bound to coated pits, in intracellular coated and uncoated vesicles, and in secondary lysosomes, including dense bodies. Free gold was not observed in the cytoplasm. We conclude that negatively charged liposomes are endocytosed and processed intracellularly by the coated vesicle pathway, and acidification of the endocytic vesicle, rather than liposome fusion, permits escape of certain molecules to the cytoplasm.

Stabilization of cationic liposome-plasmid DNA complexes by polyamines and poly(ethylene glycol)-phospholipid conjugates for efficient in vivo gene delivery

Stable complexes of cationic liposomes with plasmid DNA were prepared by (1) including a small amount of poly(ethylene glycol)‐phospholipid conjugate or (2) condensing the DNA with polyamines prior to the formation of liposome‐plasmid complexes. These preparations were stable for months at 4°C and gave reproducible high transfection activity for in vivo gene delivery after intravenous injection in mice. Under these conditions, the expression of marker gene (luciferase) was primarily in the lungs (reaching values up to 3 ng expression per mg tissue protein), but also in other tissues to a lesser extent. Non‐stabilized formulations lost all their transfection activity in 4 days. In these formulations cholesterol, not dioleoylphosphatidylethanolamine, was the helper lipid effective for sustaining high transfection activity in vivo. These new developments in formulation technology should enhance the potential for liposome‐mediated gene therapy.

Thermosensitive liposomes: Extravasation and release of contents in tumor microvascular networks

PURPOSE The purpose of this study was to determine whether hyperthermic exposure would accelerate drug release from thermosensitive sterically stabilized liposomes and enhance their extravasation in tumor tissues. MATERIALS AND METHODS In vivo fluorescence video microscopy was used to measure the extravasation of liposomes, as well as release of their contents, in a rat skin flap window chamber containing a vascularized mammary adenocarcinoma under defined thermal conditions (34 degrees, 42 degrees, and 45 degrees C). Images of tissue areas containing multiple blood vessels were recorded via a SIT camera immediately before, and for up to 2 h after i.v. injection of two liposome populations with identical lipid composition: one liposome preparation was surface labeled with Rhodamine-PE (Rh-PE) and the other contained either Doxorubicin (Dox) or calcein at self-quenching concentrations. The light intensity of the entire tissue area was measured at 34 degrees C (the physiological temperature of the skin) for 1 h, and at 42 degrees or 45 degrees C for a second hour. These measurements were then used to calculate the fluorescent light intensity arising from each tracer (liposome surface label and the released contents) inside the vessel and in the interstitial region. RESULTS The calculated intensity of Rh-PE for the thermosensitive liposomes in the interstitial space (which represents the amount of extravasated liposomes) was low during the first hour, while temperature was maintained at 34 degrees C and increased to 47 times its level before heating, when the tumor was heated at 42 degrees or 45 degrees C for 1 h. The calculated intensity of the liposome contents (Dox) in the interstitial space was negligible at 34 degrees C, and increased by 38- and 76-fold, when the tumor was heated at 42 degrees and 45 degrees C for 1 h, respectively. Similar values were obtained when calcein was encapsulated in liposomes instead of Dox. A similar increase in liposome extravasation was seen with nonthermosensitive liposomes, but negligible release of Dox occurred when the window chamber was heated to 45 degrees C for 1 h. Extravasation of liposomes continued after heating was stopped, but content release stopped after removal of heat. Release of Dox from extravasated liposomes was also seen if heating was applied 24 h after liposome administration, but no further enhancement of liposome extravasation occurred in this case. CONCLUSIONS Our data suggest that hyperthermia can be used to selectively enhance both the delivery and the rate of release of drugs from thermosensitive liposomes to targeted tissues.

Recognition of liposomes by cells: In vitro binding and endocytosis mediated by specific lipid headgroups and surface charge density

We investigated the interaction of liposomes of different surface properties with two mammalian cell lines, CV1, an African green monkey kidney cell line, and J774, a murine macrophage-like cell line. Cell surface binding and endocytosis of liposomes were quantified by fluorometry, using the liposome-encapsulated pH-sensitive fluorescent dye, pyranine, and the lipid marker rhodamine-PE. The liposome uptake was dependent both on the surface properties of the liposomes and on the cell line. Negatively charged phospholipids incorporated into egg phosphatidylcholine (PC)/cholesterol (C) (2:1) liposomes were recognized by the two cell lines to different extents depending on the lipid headgroup and its charge density in the liposome bilayer. Inclusion of 9% phosphatidylserine (PS), phosphatidylglycerol (PG), or phosphatidic acid (PA) promoted the uptake by CV1 cells more than 20-fold. Increasing the content of these negatively charged lipids beyond 9% did not further enhance the uptake. In contrast, 9% monosialoganglioside GM1, phosphatidylinositol (PI), or phosphatidylethanolamine conjugated to poly(ethylene glycol) (PEG-PE) did not promote the uptake. Inclusion of 9% PS, PG, PA or PI in PC/C liposomes did not enhance the uptake by J774 cells, but a drastic enhancement was observed when increasing concentrations of these anionic lipids were incorporated in the liposome bilayer. At least 50% PS, PG, or PI was needed to reach the level of uptake seen with CV1 cells. The uptake of liposomes containing 50% PS by J774 cells was inhibited by poly-anions which are the competing ligands for scavenger receptors, but the uptake by CV1 was not inhibited. Different mechanisms of liposome uptake by these two cell lines are suggested from the different patterns of uptake and the competition with various poly-anions. The differences observed in the uptake rate of liposomes with different lipid compositions seemed to be primarily due to the differences in the binding between liposomes and cell membrane components. The in vitro interaction of various liposomes with these cell lines, especially CV1 cells, shows significant similarities to the in vivo clearance rates of the liposomes.

Liposomes with detachable polymer coating: destabilization and fusion of dioleoylphosphatidylethanolamine vesicles triggered by cleavage of surface-grafted poly(ethylene glycol)

Plasma‐stable liposomes (100 nm) were prepared from dioleoylphosphatidylethanolamine (DOPE) and 3–6 mol% of a new disulfide‐linked poly(ethylene glycol)‐phospholipid conjugate (mPEG‐DTP‐DSPE). In contrast to similar preparations containing non‐cleavable PEG‐phospholipid conjugate, thiolytic cleavage of the grafted polymer chains facilitated rapid and complete release of the liposome contents. Furthermore, the detachment of PEG from DOPE liposomes resulted in liposomal fusion. Finally, while formulation of pH‐sensitive DOPE/cholesterol hemisuccinate liposomes with mPEG‐DTP‐DSPE abolished the pH sensitivity, cleavage of the PEG chains completely restored this property. These are the first examples of new useful properties of liposomes grafted with cleavable polymer.

Therapeutic efficacy of anti-ErbB2 immunoliposomes targeted by a phage antibody selected for cellular endocytosis.

Many targeted cancer therapies require endocytosis of the targeting molecule and delivery of the therapeutic agent to the interior of the tumor cell. To generate single chain Fv (scFv) antibodies capable of triggering receptor-mediated endocytosis, we previously developed a method to directly select phage antibodies for internalization by recovering infectious phage from the cytoplasm of the target cell. Using this methodology, we reported the selection of a panel of scFv that were internalized into breast cancer cells from a nonimmune phage library. For this work, an immunotherapeutic was generated from one of these scFv (F5), which bound to ErbB2 (HER2/neu). The F5 scFv was reengineered with a C-terminal cysteine, expressed at high levels in Escherichia coli, and coupled to sterically stabilized liposomes. F5 anti-ErbB2 immunoliposomes were immunoreactive as determined by surface plasmon resonance (SPR) and were avidly internalized by ErbB2-expressing tumor cell lines in proportion to the levels of ErbB2 expression. F5-scFv targeted liposomes containing doxorubicin had antitumor activity and produced significant reduction in tumor size in xenografted mice compared to nontargeted liposomes containing doxorubicin. This strategy should be applicable to generate immunotherapeutics for other malignancies by selecting phage antibodies for internalization into other tumor types and using the scFv to target liposomes or other nanoparticles.

Endocytosis of liposomes by macrophages: binding, acidification and leakage of liposomes monitored by a new fluorescence assay

The interaction of liposomes with macrophage cells was monitored by a new fluorescence method (Hong, K., Straubinger, R.M. and Papahadjopoulos, D., J. Cell Biol. 103 (1986) 56a) that allows for the simultaneous monitoring of binding, endocytosis, acidification and leakage. Profound differences in uptake, cell surface-induced leakage and leakage subsequent to endocytosis were measured in liposomes of varying composition. Pyranine (1-hydroxypyrene-3,6,8-trisulfonic acid, HPTS), a highly fluorescent, water-soluble, pH sensitive dye, was encapsulated at high concentration into the lumen of large unilamellar vesicles. HPTS exhibits two major fluorescence excitation maxima (403 and 450 nm) which have a complementary pH dependence in the range 5-9: the peak at 403 nm is maximal at low pH values while the peak at 450 nm is maximal at high pH values. The intra- and extracellular distribution of liposomes and their approximate pH was observed by fluorescence microscopy using appropriate excitation and barrier filters. The uptake of liposomal contents by cells and their subsequent exposure to acidified endosomes or secondary lysosomes was monitored by spectrofluorometry via alterations in the fluorescence excitation maxima. The concentration of dye associated with cells was determined by measuring fluorescence at a pH independent point (413 nm). The average pH of cell-associated dye was determined by normalizing peak fluorescence intensities (403 nm and 450 nm) to fluorescence at 413 nm and comparing these ratios to a standard curve. HPTS-containing liposomes bound to and were acidified by a cultured murine macrophage cell line (J774) with a t1/2 of 15-20 min. The acidification of liposomes exhibited biphasic kinetics and 50-80% of the liposomes reached an average pH lower than 6 within 2 h. A liposomal lipid marker exhibited a rate of uptake similar to HPTS, however the lipid component selectively accumulated in the cell; after an initial rapid release of liposome contents, 2.5-fold more lipid marker than liposomal contents remained associated with the cells after 5 h. Coating haptenated liposomes with antibody protected liposomes from the initial release. The leakage of liposomal contents was monitored by co-encapsulating HPTS and p-xylene-bis-pyridinium bromide, a fluorescence quencher, into liposomes. The time course of dilution of liposome contents, detected as an increase in HPTS fluorescence, was coincident with the acidification of HPTS. The rate and extent of uptake of neutral and negatively charged liposomes was similar; however, liposomes opsonized with antibody were incorporated at a higher rate (2.9-fold) and to a greater extent (3.4-fold).(ABSTRACT TRUNCATED AT 400 WORDS)

N-isopropylacrylamide copolymers for the preparation of pH-sensitive liposomes and polymeric micelles

Hydrophobically-modified copolymers of N-isopropylacrylamide bearing a pH-sensitive moiety were investigated for the preparation of pH-responsive liposomes and polymeric micelles. The copolymers having the hydrophobic anchor randomly distributed within the polymeric chain were found to more efficiently destabilize egg phosphatidylcholine (EPC)/cholesterol liposomes than the alkyl terminated polymers. Release of both a highly-water soluble fluorescent contents marker, pyranine, and an amphipathic cytotoxic anti-cancer drug, doxorubicin, from copolymer-modified liposomes was shown to be dependent on pH, the concentration of copolymer, the presence of other polymers such as polyethylene glycol, and the method of preparation. Both polymers were able to partially stabilize EPC liposomes in human serum. These polymers were found to self-assemble to form micelles. The critical association concentration was low (9--34 mg/l) and influenced by the position of the alkyl chains. In phosphate buffered saline, the micelles had a bimodal size distribution with the predominant population having a mean diameter of 35 nm. The polymeric micelles were studied as a delivery system for the photosensitizer aluminum chloride phthalocyanine, (AlClPc), currently evaluated in photodynamic therapy. pH-Responsive polymeric micelles loaded with AlClPc were found to exhibit increased cytotoxicity against EMT-6 mouse mammary cells in vitro than the control Cremophor EL formulation.

Thermosensitive sterically stabilized liposomes : formulation and in vitro studies on mechanism of doxorubicin release by bovine serum and human plasma

AbstractPurpose. To formulate thermosensitive sterically stabilized liposomes and to study the effects of plasma and serum components in vitro. Methods. The rate of release of encapsulated doxorubicin (Dox) from liposomes of various compositions was followed by fluorometric assay at 37°, 42° and 45°C, in buffer and also in both calf serum and human plasma up to 50% by volume. Results. The optimal composition for the maximal differential release of doxorubicin between 37°C and 42°C in human plasma was a mixture of dipalmitoylphosphatidylcholine/hydrogenated soy phosphatidylcholine/cholesterol and distearoylphosphatidylethanolamine derivatized with polyethylene glycol at a molar ratio of 100:50:30:6. In experiments designed to study the mechanism causing increased permeability of liposomes in bovine serum, we found two different distinct release patterns: a slow linear rise of rate of Dox release for fluid liposomes and fast exponential rise reaching plateau within 5 minutes for solid phase (rigid) liposomes. This release of Dox from rigid but not fluid liposomes was inhibited by pre-heating serum at 55°C for 30 minutes or by addition of EDTA (but not EGTA) or antiserum to the C3 component of complement. Conclusions. A formulation of sterically stabilized liposomes with the proper thermal sensitivity in human plasma has been obtained. In addition, the results suggest that complement may play an important role in the interaction of rigid but not fluid liposomes with bovine serum. Human plasma did not show this effect.