Author Correction: Self-assembled poly-catenanes from supramolecular toroidal building blocks.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Self-assembled poly-catenanes from supramolecular toroidal building blocks.

Mechanical interlocking of molecules (catenation) is a nontrivial challenge in modern synthetic chemistry and materials science1,2. One strategy to achieve catenation is the design of pre-annular molecules that are capable of both efficient cyclization and of pre-organizing another precursor to engage in subsequent interlocking3-9. This task is particularly difficult when the annular target is composed of a large ensemble of molecules, that is, when it is a supramolecular assembly. However, the construction of such unprecedented assemblies would enable the visualization of nontrivial nanotopologies through microscopy techniques, which would not only satisfy academic curiosity but also pave the way to the development of materials with nanotopology-derived properties. Here we report the synthesis of such a nanotopology using fibrous supramolecular assemblies with intrinsic curvature. Using a solvent-mixing strategy, we kinetically organized a molecule that can elongate into toroids with a radius of about 13 nanometres. Atomic force microscopy on the resulting nanoscale toroids revealed a high percentage of catenation, which is sufficient to yield 'nanolympiadane'10, a nanoscale catenane composed of five interlocked toroids. Spectroscopic and theoretical studies suggested that this unusually high degree of catenation stems from the secondary nucleation of the precursor molecules around the toroids. By modifying the self-assembly protocol to promote ring closure and secondary nucleation, a maximum catenation number of 22 was confirmed by atomic force microscopy.

Enhanced antitumor effect of anti-tissue factor antibody-conjugated epirubicin-incorporating micelles in xenograft models.

For the creation of a successful antibody-drug conjugate (ADC), both scientific and clinical evidence has indicated that highly toxic anticancer agents (ACA) should be conjugated to a monoclonal antibody (mAb) to administer a reasonable amount of ADC to patients without compromising the affinity of the mAb. For ordinary ACA, the conjugation of a mAb to ACA-loaded micellar nanoparticles is clinically applicable. Tissue factor (TF) is often overexpressed in various cancer cells and tumor vascular endothelium. Accordingly, anti-TF-NC-6300, consisting of epirubicin-incorporating micelles (NC-6300) conjugated with the F(ab')2 of anti-TF mAb was developed. The in vitro and in vivo efficacy and pharmacokinetics of anti-TF-NC-6300 were compared to NC-6300 using two human pancreatic cancer cell lines, BxPC3 (high TF expression) and SUIT2 (low TF expression), and a gastric cancer cell line, 44As3 (high TF expression). The intracellular uptake of epirubicin was faster and greater in BxPC3 cells treated with anti-TF-NC-6300, compared with NC-6300. Anti-TF-NC-6300 showed a superior antitumor activity in BxPC3 and 44As3 xenografts, compared with NC-6300, while the activities of both micelles were similar in the SUIT2 xenograft. A higher tumor accumulation of anti-TF-NC-6300 compared to NC-6300 was seen, regardless of the TF expression levels. However, anti-TF-NC-6300 appeared to be localized to the tumor cells with high TF expression. These results indicated that the enhanced antitumor effect of anti-TF-NC6300 may be independent of the tumor accumulation but may depend on the selective intratumor localization and the preferential internalization of anti-TF-NC-6300 into high TF tumor cells.

Effect of combined treatment with the epirubicin-incorporating micelles (NC-6300) and 1,2-diaminocyclohexane platinum (II)-incorporating micelles (NC-4016) on a human gastric cancer model.

Anticancer agent-incorporating polymeric micelles accumulate effectively in tumors via the enhanced permeability and retention effect to exert potent antitumor effects. However, combined use of such micelles has not been elucidated. We compared the effect of combining the epirubicin-incorporating micelle NC-6300 and 1,2-diaminocyclohexane platinum (II) (oxaliplatin parent complex)-incorporating micelle NC-4016 (NCs) with that of epirubicin and oxaliplatin (E/O) in 44As3Luc cells using the combination index method. The in vivo antitumor activities of NCs and E/O were evaluated in mice bearing 44As3Luc xenografts. Pharmacokinetic analysis was performed by high-performance liquid chromatography and mass spectrometry. Cardiotoxicity of NC-6300 and epirubicin was assessed by echocardiography. Neurotoxicity of NC-4016 and oxaliplatin was evaluated by examining the paw withdrawal response to noxious mechanical stimuli. NCs showed a highly synergistic activity equivalent to E/O. In vivo, NCs exhibited higher antitumor activity in the subcutaneous tumor model and longer overall survival in the orthotopic tumor model than E/O (p < 0.001, p = 0.015, respectively). The intratumor concentrations of epirubicin and platinum were significantly higher following NCs than following E/O administration. Moreover, the micelles showed lower cardiotoxicity and neurotoxicity than the corresponding conventional drugs. The combined use of the micelles was associated with remarkable efficacy and favorable toxicities in the human gastric cancer model, and warrants the conduct of clinical trials.

Tumor-targeted chemotherapy with the nanopolymer-based drug NC-6004 for oral squamous cell carcinoma.

Cisplatin (CDDP) has been a key drug for chemotherapy in patients with head and neck squamous cell carcinoma. Nephrotoxicity is one of its adverse reactions that are dose limiting. To increase its antitumor effects and reduce such toxicity problems, polymeric micelles carrying CDDP (NC-6004) have been developed. The present study was designed to evaluate the efficacy and safety of NC-6004 for oral squamous cell carcinoma. In vitro antitumor activity was assayed in four oral squamous cell carcinoma cell lines. To investigate the antitumor and nephrotoxic effects of NC-6004, nude mice bearing OSC-19 were administered NC-6004 or CDDP. The in vitro growth-inhibitory effect of NC-6004 was significantly less than that of CDDP. However, both NC-6004 and CDDP showed equivalent antitumor effects in vivo. Mice with CDDP developed renal cell apoptosis; however, those injected with NC-6004 were almost free of renal cell injury. Moreover, in an orthotopic tongue cancer model using OSC-19, NC-6004 reduced the rate of sentinel lymph node metastasis to lower than that with CDDP. In conclusion, considering the potential advantages in terms of noticeable antitumor activity, lymphatic drug delivery and reduced nephrotoxicity, NC-6004 represents a significant structural improvement in the development of a platinum complex.

NC-6300, an epirubicin-incorporating micelle, extends the antitumor effect and reduces the cardiotoxicity of epirubicin.

Epirubicin is widely used to treat various human tumors. However, it is difficult to achieve a sufficient antitumor effect because of dosage limitation to prevent cardiotoxicity. We hypothesized that epirubicin-incorporating micelle would reduce cardiotoxicity and improve the antitumor effect. NC-6300 comprises epirubicin covalently bound to PEG polyaspartate block copolymer through an acid-labile hydrazone bond. The conjugate forms a micellar structure of 40-80 nm in diameter in an aqueous milieu. NC-6300 (10, 15 mg/kg) and epirubicin (10 mg/kg) were given i.v. three times to mice bearing s.c. or liver xenograft of human hepatocellular carcinoma Hep3B cells. Cardiotoxicity was evaluated by echocardiography in C57BL/6 mice that were given NC-6300 (10 mg/kg) or epirubicin (10 mg/kg) in nine doses over 12 weeks. NC-6300 showed a significantly potent antitumor effect against Hep3B s.c. tumors compared with epirubicin. Moreover, NC-6300 also produced a significantly longer survival rate than epirubicin against the liver orthotopic tumor of Hep3B. With respect to cardiotoxicity, epirubicin-treated mice showed significant deteriorations in fractional shortening and ejection fraction. In contrast, cardiac functions of NC-6300 treated mice were no less well maintained than in control mice. This study warrants a clinical evaluation of NC-6300 in patients with hepatocellular carcinoma or other cancers.

Improved anti-tumor activity of stabilized anthracycline polymeric micelle formulation, NC-6300.

Anthracyclines have long been considered to be among the most active agents clinically available for the treatment of breast cancer despite their toxicity. To improve their pharmacological profiles, a new macromolecular prodrug, denoted NC-6300, was synthesized. NC-6300 comprises epirubicin covalently bound to polyethylene-glycol polyaspartate block copolymer through an acid-labile hydrazone bond. The conjugate forms a micellar structure spontaneously in aqueous media with a diameter of 60-70 nm. The block copolymers are partially substituted with hydrophobic benzyl groups to stabilize the micellar structure. The present study was designed to confirm that polymeric micelles incorporating epirubicin through an acid-labile linker improve the therapeutic index and achieve a broad range of therapeutic doses. Pharmacokinetic studies in rats showed highly enhanced plasma retention of NC-6300 compared with native epirubicin. The maximal tolerated doses in mice of NC-6300 and native epirubicin were 25 and 9 mg/kg, respectively, when administered three times with a 4-day interval between each dose. NC-6300 at 15 and 20 mg/kg with the same administration schedule regressed a Hep3B human hepatic tumor with slight and transient bodyweight loss. Remarkably, NC-6300 also inhibited growth of an MDA-MB-231 human breast tumor at the same dosage. In contrast, native epirubicin at 7 mg/kg administered three times with a 4-day interval was only able to slow tumor growth. Tissue distribution studies of NC-6300 showed efficient free epirubicin released in the tumor at 74% by area under the concentration-time curve (AUC) evaluation, supporting the effectiveness of NC-6300. In conclusion, NC-6300 improved the potency of epirubicin, demonstrating the advantage of NC-6300 attributable to the efficient drug release in the tumor.

Controlled release of a protein kinase inhibitor UCN-01 from liposomes influenced by the particle size.

A protein kinase inhibitor UCN-01 binds with high affinity to human alpha 1-acid glycoprotein (hAGP) which may compromise the drugs therapeutic effectiveness. Liposomal formulations of UCN-01 have been evaluated as a means of reducing the impact of binding to hAGP. However, in an initial study, UCN-01 was released rapidly from liposomes added to rat plasma containing hAGP. The purpose of this study was to develop a liposomal formulation of UCN-01 that only slowly released drug. Liposomes composed of lipids with a high phase transition temperature and having an average particle size of 120 nm and above reduced leaking of UCN-01 when the formulations were evaluated by adding to rat plasma containing hAGP. Furthermore, formulations composed of larger liposomes were also more effective in vivo; in tests in which liposomal preparations were injected together with hAGP into rats, more UCN-01 was retained in liposomes for 24h after administration of 155 nm liposomes as compared to 112 nm liposomes.

Development of wrapped liposomes: novel liposomes comprised of polyanion drug and cationic lipid complexes wrapped with neutral lipids.

Novel wrapped liposomes comprised of polyanion drug and cationic lipid complexes wrapped with neutral lipids were prepared using an efficient, innovative procedure. In this study, dextran fluorescein anionic (DFA) was used as an example of a polyanionic compound. During the process, neutral lipids accumulated around the complexes and eventually covered the complexes. The resulting liposomes were 120-140 nm in diameter and the encapsulation efficiency was up to 90%. In fetal bovine serum, DFA/cationic lipid complexes degraded rapidly but the wrapped liposomes were considerably more stable. Following intravenous administration to rats, DFA/cationic lipid complexes were rapidly eliminated whereas the wrapped liposomes exhibited a much longer blood half-life. These data suggest that DFA is located on the surface of the complexes, but DFA is present inside the wrapped liposomes. The drug-delivery properties of the wrapped liposomes established in the present study suggests that formulations based on this technology could offer important advantages for the administration of many types of drug including antisense oligonucleotides, plasmids and siRNAs which may therefore lead to improved therapeutic effectiveness of this range of drugs. The method of preparation of the wrapped liposomes is so simple that it should be straightforward to adapt to a manufacturing scale.

Improved formulations of antisense oligodeoxynucleotides using wrapped liposomes.

Previously, we demonstrated that wrapping dextran fluorescein anionic/cationic lipid complexes with neutral lipids produced a stable formulation that markedly increased the duration of the compound in plasma after intravenous administration to rats. The improved drug-delivery properties of the wrapped liposomes (WL) relative to other formulations suggested that this technology could offer important advantages for the administration of other polyanionic drugs, including antisense oligodeoxynucleotides (ODN). In the present study, we investigated the value of WL for formulating fluorescence-labeled phosphorothioated ODN (F-ODN). WL encapsulating F-ODN/cationic lipid complexes were prepared efficiently using similar methodology to that used in our earlier study. Studies confirmed that these WL were stable in vitro. Following intravenous administration to mice, free F-ODN and naked F-ODN/cationic lipid complexes were rapidly eliminated whereas administration of the WL resulted in high blood concentrations of drug that were maintained for several hours. Additional studies were conducted in mice that were inoculated with tumor cells (Caki-1 xenograft model, human kidney); in these experiments, intravenous administration of WL delivered 13 times more F-ODN to the tumor site than achieved after injection of free F-ODN.

Vaccination with OVA-bound nanoparticles encapsulating IL-7 inhibits the growth of OVA-expressing E.G7 tumor cells in vivo.

Immunotherapy has gained special attention due to its specific effects on tumor cells and systemic action to block metastasis. We recently demonstrated that ovalbumin (OVA) conjugated to the surface of nanoparticles (NPs) (OVA­NPs) can manipulate humoral immune responses. In the present study, we aimed to ascertain whether vaccination with OVA-NPs entrapping IL-7 (OVA-NPs-IL-7) are able to induce antitumor immune responses in vivo. Pretreatment with a subcutaneous inoculation of OVA-NPs delayed the growth of thymic lymphoma cells expressing a model tumor antigen OVA (E.G7-OVA), and OVA-NPs-IL-7 substantially blocked the growth of E.G7-OVA tumor cells, although NPs-IL-7 alone had a meager effect, as assessed by the mean tumor size and the percentage of tumor-free mice. However, pretreatment with OVA-NPs-IL-7 failed to reduce the growth of parental thymic tumor cells, suggesting that the antitumor effect was antigen-specific. A tetramer assay revealed that vaccination with OVA-NPs-IL-7 tended to enhance the proportion of cytotoxic T cells (CTLs) specific for OVA. When the tumor-free mice inoculated with OVA-NPs-IL-7 plus EG.7 cells were rechallenged with E.G7-OVA cells, they demonstrated reduced growth compared with that in the control mice. Thus, a single subcutaneous injection of OVA-NPs-IL-7 into mice induced tumor-specific and also memory-like immune responses, resulting in regression of tumor cells. Antigens on NPs entrapping IL-7 would be a promising carrier to develop and enhance immune responses, including humoral and cellular immunity as well as a method of drug delivery to a specific target of interest.

OVA-bound nanoparticles induce OVA-specific IgG1, IgG2a, and IgG2b responses with low IgE synthesis.

There is an urgent requirement for a novel vaccine that can stimulate immune responses without unwanted toxicity, including IgE elevation. We examined whether antigen ovalbumin (OVA) conjugated to the surface of nanoparticles (NPs) (OVA-NPs) with average diameter of 110nm would serve as an immune adjuvant. When BALB/c mice were immunized with OVA-NPs, they developed sufficient levels of OVA-specific IgG1 antibody responses with low levels of IgE synthesis, representing helper T (Th)2-mediated humoral immunity. OVA-specific IgG2a and IgG2b responses (i.e., Th1-mediated immunity) were also induced by secondary immunization with OVA-NPs. As expected, immunization with OVA in alum (OVA-alum) stimulated humoral immune responses, including IgG1 and IgE antibodies, with only low levels of IgG2a/IgG2b antibodies. CD4-positive T cells from mice primed with OVA-NPs produced substantial levels of IL-21 and IL-4, comparable to those from OVA-alum group. The irradiated mice receiving OVA-NPs-primed B cells together with OVA-alum-primed T cells exhibited enhanced anti-OVA IgG2b responses relative to OVA-alum-primed B cells and T cells following stimulation with OVA-NPs. Moreover, when OVA-NPs-primed, but not OVA-alum-primed, B cells were cultured in the presence of anti-CD40 monoclonal antibody, IL-4, and IL-21, or LPS plus TGF-ß in vitro, OVA-specific IgG1 or IgG2b antibody responses were elicited, suggesting that immunization with OVA-NPs modulates B cells to generate IgG1 and IgG2b responses. Thus, OVA-NPs might exert their adjuvant action on B cells, and they represent a promising potential vaccine for generating both IgG1 and IgG2a/IgG2b antibody responses with low IgE synthesis.

Combination therapy with dendritic cell vaccine and IL-2 encapsulating polymeric micelles enhances intra-tumoral accumulation of antigen-specific CTLs.

Dendritic cell (DC) vaccine is a promising immunotherapy for cancer due to its ability to induce antigen-specific CTLs efficiently. However, a number of clinical studies have implied insufficient therapeutic benefits with the use of MHC class 1 restricted peptide-pulsed DC vaccine. To enhance the clinical efficacy, we examined combination therapy of DC vaccine pulsed with OVA peptide and intravenous low dose unmodified IL-2 (IL-2 solution) administration against EG7 tumor-bearing mice. Unexpectedly, no additional effects of IL-2 solution were observed on CTL induction and the therapeutic effects of DC vaccine, possibly because of the short half-life of IL-2 in plasma. Therefore, we generated IL-2-encapsulating polymeric micelles (IL-2 micelle), which showed prolonged IL-2 retention in the circulation after intravenous administration compared with IL-2 solution. When mice were treated with OVA peptide-pulsed DCs in combination with IL-2 micelle, OVA-specific CTLs were efficiently induced in the spleen in comparison with DC vaccine combined with IL-2 solution or DC vaccine alone. In addition, combination therapy of DC vaccine and IL-2 micelle against EG7 tumor-bearing mice induced the efficient accumulation of antigen-specific CTLs into the tumor and marked anti-tumor effects. Thus, the administration of IL-2 micelle can significantly enhance DC vaccine efficacy against tumors.

Assessment of antitumor activity and acute peripheral neuropathy of 1,2-diaminocyclohexane platinum (II)-incorporating micelles (NC-4016).

Oxaliplatin, a third-generation platinum compound incorporating oxalate and 1,2-diaminocyclohexane platinum, has been widely used in chemotherapy regimens for the treatment of metastatic colorectal cancer. Because of its wide spectrum of antitumor activity, oxaliplatin has been applied for the treatment of other carcinomas. However, the antitumor activity of single-agent oxaliplatin is insufficient. To increase its antitumor effects, polymeric micellar nanoparticles incorporating 1,2-diaminocyclohexane platinum (NC-4016) have been developed. The present study was designed to evaluate the efficacy of NC-4016 and its association with peripheral neuropathy, which is a primary dose-limiting factor in oxaliplatin therapy. The in vitro antitumor activity of NC-4016 was investigated using human carcinoma cell lines. To investigate the antitumor effects of NC-4016 in vivo, nude mice bearing the human carcinoma cell line KB were administered NC-4016 or oxaliplatin. The in vitro growth-inhibiting effect of NC-4016 was significantly weaker than that of oxaliplatin. However, the antitumor efficacy of NC-4016 was superior to that of oxaliplatin in vivo. Moreover, we compared the severity of peripheral neuropathy induced by oxaliplatin and NC-4016 in a rat model. Oxaliplatin, NC-4016, or 5% glucose (control) were administered by a single tail vein injection. In the oxaliplatin-treated rats, neither mechanical nor heat allodynia was observed during the experimental period, whereas cold hyperalgesia/allodynia was observed from day 1 to 7. Conversely, cold hyperalgesia/allodynia was not observed in the NC-4016-treated rats. The present study demonstrated that the antitumor efficacy of NC-4016 was superior to that of oxaliplatin in a mouse model of human carcinoma cell line KB. In addition, NC-4016-treated rats did not develop acute cold hypersensitivity, which is frequently experienced by patients after oxaliplatin administration.

NC-6301, a polymeric micelle rationally optimized for effective release of docetaxel, is potent but is less toxic than native docetaxel in vivo.

Drug release rate is an important factor in determining efficacy and toxicity of nanoscale drug delivery systems. However, optimization of the release rate in polymeric micellar nanoscale drug delivery systems has not been fully investigated. In this study NC-6301, a poly(ethylene glycol)-poly(aspartate) block copolymer with docetaxel (DTX) covalently bound via ester link, was synthesized with various numbers of DTX molecules bound to the polymer backbone. The number of DTX molecules was determined up to 14 to achieve an optimal release rate, based upon the authors' own pharmacokinetic model using known patient data. Efficacy and toxicity of the formulation was then tested in animals. When administered three times at 4-day intervals, the maximum tolerated doses of NC-6301 and native DTX were 50 and 10 mg/kg, respectively, in nude mice. Tissue distribution studies of NC-6301 in mice at 50 mg/kg revealed prolonged release of free DTX in the tumor for at least 120 hours, thus supporting its effectiveness. Furthermore, in cynomolgus monkeys, NC-6301 at 6 mg/kg three times at 2-week intervals showed marginal toxicity, whereas native DTX, at 3 mg/kg with the same schedule, induced significant decrease of food consumption and neutrophil count. NC-6301 at 50 mg/kg in mice also regressed a xenografted tumor of MDA-MB-231 human breast cancer. Native DTX, on the other hand, produced only transient and slight regression of the same tumor xenograft. NC-6301 also significantly inhibited growth of OCUM-2MLN human scirrhous gastric carcinoma in an orthotopic mouse model. Total weight of metastatic lymph nodes was also reduced. In conclusion, NC-6301 with an optimized release rate improved the potency of DTX while reducing its toxicity.

Prolonged circulation and in vivo efficacy of recombinant human granulocyte colony-stimulating factor encapsulated in polymeric micelles.

To improve the pharmacokinetics of granulocyte colony-stimulating factor (G-CSF) and decrease dosing frequency, polyethylene glycol polyglutamate block copolymers were used as delivery carriers. The block copolymers are partially substituted with hydrophobic octyl or benzyl groups to form a micellar structure in aqueous media and encapsulate the protein. G-CSF is encapsulated in the polymeric micelles with a diameter of 60-70nm. The present study was designed to evaluate the plasma pharmacokinetics, G-CSF release and in vivo efficacy of G-CSF-encapsulating micelles. Pharmacokinetic studies in rats showed highly enhanced plasma retention of the micelles compared with native G-CSF. The AUC (area under the curve) of the octyl-based polymer formulation showed a 5-fold increase, compared with native G-CSF. Size-exclusion chromatography of the blood from rats injected with the micelles demonstrated the release of G-CSF from the micelles in the blood circulation. The pharmacokinetic behavior supports the in vivo studies showing that the micelles display a comparable efficacy to PEGylated G-CSF. Simultaneous pharmacokinetic analysis of released and encapsulated G-CSF plasma levels showed that the G-CSF release occurs with the first-order kinetics and the half-life is 4.8h. In conclusion, G-CSF is endowed by the polymeric micelles with prolonged half-life and increased efficacy without any chemical modification.

A nanoparticle system specifically designed to deliver short interfering RNA inhibits tumor growth in vivo.

Use of short interfering RNA (siRNA) is a promising new approach thought to have a strong potential to lead to rapid development of gene-oriented therapies. Here, we describe a newly developed, systemically injectable siRNA vehicle, the "wrapsome" (WS), which contains siRNA and a cationic lipofection complex in a core that is fully enveloped by a neutral lipid bilayer and hydrophilic polymers. WS protected siRNA from enzymatic digestion, providing a long half-life in the systemic circulation. Moreover, siRNA/WS leaked from blood vessels within tumors into the tumor tissue, where it accumulated and was subsequently transfected into the tumor cells. Because the transcription factor KLF5 is known to play a role in tumor angiogenesis, we designed KLF5-siRNA to test the antitumor activity of siRNA/WS. KLF5-siRNA/WS exhibited significant antitumor activity, although neither WS containing control scrambled-siRNA nor saline containing KLF5-siRNA affected tumor growth. KLF5-siRNA/WS inhibited Klf5 expression within tumors at both mRNA and protein levels, significantly reducing angiogenesis, and we detected no significant acute or long-term toxicity. Our findings support the idea that siRNA/WS can be used to knock down specific genes within tumors and thereby exert therapeutic effects against cancers.

Reducing the impact of binding of UCN-01 to human alpha1-acid glycoprotein by encapsulation in liposomes.

A liposomal formulation of UCN-01 was studied to prevent binding of drug to human alpha1-acid glycoprotein (hAGP). The release of drug from liposomes added to various media was investigated by monitoring the concentration of UCN-01 in different fractions. Protein bound UCN-01 was separated from liposomal UCN-01 and free UCN-01 by gel chromatography and the drug content in the fractions was measured by high-performance liquid chromatography. Also, the blood levels of hAGP bound drug and drug retained in liposomes were assessed after intravenous administration to rats of UCN-01 liposomes together with hAGP. In media containing hAGP, but not rat AGP, UCN-01 was released from liposomes. When UCN-01 liposomes were mixed with rat plasma plus hAGP, the UCN-01 in the liposomes was only gradually released so that some drug remained in the liposomes, and therefore not bound to hAGP, for up to 24 h. After the mixture of liposomal UCN-01 and hAGP was injected into rats, some UCN-01 was retained in liposomes for several hours. Encapsulation of UCN-01 into liposomes is an effective method of preventing binding of UCN-01 to hAGP.