Three-dimensional cell-culture systems using nanofibrillated bacterial cellulose restores drug metabolism activity in HepG2 hepatocellular carcinoma cells

Three-dimensional (3D) culture more faithfully reproducesin vivo-like cell interactions and functions than conventional two-dimensional (2D) monolayers. Although HepG2 cells are widely used in drug discovery, their 2D cultures exhibit low expression of drug-metabolizing enzymes such as cytochrome P450s (CYPs), limiting utility for toxicity and pharmacokinetic studies. Nano-fibrillated bacterial cellulose (NFBC) is a unique biomaterial that features exceptional homogeneity, high purity, and excellent biocompatibility. We recently employed two NFBC-based 3D culture systems: the Suspension and the OnGel methods. However, the capacity of NFBC to improve hepatocyte-specific functions has yet to be systematically explored. In this study, we aimed to establish a 3D culture platform for HepG2 cells using NFBC to restore hepatic functionality, and drug-metabolizing activity in particular, via comparison with conventional 2D monolayers. Both of these 3D culture systems produced viable HepG2 spheroids with good proliferation. Exploratory microarray profiling suggested broad upregulation of multiple absorption, distribution, metabolism and excretion (ADME) genes in the HepG2 spheroids. Among these, the enzyme Cytochrome P450 3A4 (CYP3A4) showed a significant increase in protein expression and in enzymatic activity in the HepG2 spheroids, which was functionally confirmed by acetaminophen (APAP) toxicity via its bioactivation into toxic metabolite. In addition, the HepG2 spheroids displayed reduced sensitivity to the anticancer drug doxorubicin compared to 2D monolayer. Collectively, these findings demonstrate that NFBC-based 3D culture systems effectively restore liver-specific functions in HepG2 cells, which results in HepG2 spheroids with metabolic competence and physiologically relevant drug responses, and could offer a promising tool for high-throughputin vitrodrug screening.


Spleen-targeting immunization with antigen-loaded PEGylated liposomes enhances the diversity of antigen-specific antibodies


Inflammation-triggering engineered macrophages (MacTriggers) are promising cell-based therapeutic avenues for chemoresistant solid tumors

Chimeric antigen receptor T-cell therapy has shown efficacy against chemoresistant B-cell leukemia and lymphoma but is limited in solid tumors. This study proposes using inflammation-triggering engineered macrophages (MacTriggers) to target chemoresistant tumors. Intravenous MacTriggers infiltrate tumors, inducing inflammation via tumor necrosis factor-alpha (TNF-α), converting the immunosuppressive microenvironment into an immuno-active state, and enhancing anti-tumor immune responses. DOX-resistant murine colon cancer cells (DOX-Resi) were established by repeated in vivo exposure to DOX. IC50 values and mRNA expression of Abcb1a (encoding P-gp) in WT or DOX-Resi cells were evaluated by qPCR. MacTriggers were engineered to release TNF-α upon sensing tumor-associated arginase 1 (Arg1) activity. BALB/c mice with subcutaneous DOX-Resi tumors received intravenous MacTriggers or DOX. Tumor growth, histological changes, and side effects, including cardiotoxicity, were assessed via tumor volume monitoring, immunohistochemistry, and serum cardiac troponin-I measurement. DOX-Resi cells had an IC50 value approximately 2.5 times higher than WT cells, with significantly higher Abcb1a expression. MacTriggers significantly suppressed DOX-Resi tumor growth, while DOX showed limited efficacy. MacTrigger administration did not cause severe side effects, unlike DOX, which induced cardiotoxicity. MacTriggers offer a novel, effective, and safer therapeutic approach for chemoresistant solid tumors, addressing chemotherapy limitations and improving outcomes in drug-resistant cancers.


A spleen-targeted vaccine for SARS-CoV-2 inducting neutralizing antibodies in mice

The development of vaccines against infectious diseases is of the utmost importance to prevent global pandemics such as COVID-19. The application of antigens and adjuvants to efficiently target antigen presenting cells (APCs) is paramount for the development of efficient vaccines. In our previous study, we showed that splenic marginal zone-B (MZ-B) cells are promising APCs in addition to dendritic cells (DCs). In this study we achieved the targeted delivery of sufficient antigen to MZ-B cells by utilizing an intravenous (IV) immunization system we originally developed. This system involves the sequential injection of empty PEGylated liposomes (PEG-Lip) and antigen-containing PEG-Lip within a prescribed interval. Herein, we describe the application of this IV immunization system as a COVID-19 vaccine to induce specific antibodies against SARS-CoV-2. To establish efficacy, SARS-CoV-2 spike proteins were used as an antigen, and α-galactosylceramide (GC) was used as an adjuvant in this study. Three days after priming with empty PEG-Lip, we injected PEG-Lip containing spike protein and α-GC. Our IV immunization system successfully induced higher levels of anti-spike antibodies when spike protein derived from HEK-293, but not E. coli., was injected into mice. The levels were less produced using conventional immunization via subcutaneous (s.c.) injections of complete Freund's adjuvant without priming. Interestingly, a lower dose (0.2 µg) of spike protein antigen encapsulated into PEG-Lip induced a higher level of anti-spike antibodies than that produced using a significantly higher dose (5 µg). The induced anti-spike antibodies inhibited the interaction between the receptor binding domain of the spike protein and the angiotensin-converting enzyme 2. This indicates that the induced antibodies tend to neutralize SARS-CoV-2. Collectively, the specific delivery of spike proteins to spleen, probably MZ-B cells, via nano-carriers could be a promising approach for the development of global pandemic vaccines that require only minimum dosages of antigen.


Application of bacterial-derived long cellulose nanofiber to suspension culture of mammalian cells as a shear protectant

International Journal of Biological Macromolecules


A Stealthiness Evaluation of Main Chain Carboxybetaine Polymer Modified into Liposome


Humoral immune response against SARS-CoV-2 and polyethylene glycol elicited by anti-SARS-CoV-2 mRNA vaccine, and effect of pre-existing anti-polyethylene glycol antibody in patients with hematological and autoimmune diseases.

The effects of vaccination are modified by hematological and autoimmune diseases and/or treatment. Anti-SARS-CoV-2 mRNA vaccine contains polyethylene glycol (PEG), it is largely unknown whether PEG influences the effects of vaccination or induces a humoral response. This study examined whether anti-PEG antibodies before vaccination (pre-existing) influenced the acquisition of SARS-CoV-2 antibodies and evaluated the relationship between the development of anti-SARS-CoV-2 antibodies and anti-PEG antibodies after SARS-CoV-2 vaccination in hematological and autoimmune diseases. Anti-SARS-CoV-2 antibody IgG, anti-PEG IgG, and IgM titers were evaluated in patients with hematological and autoimmune diseases after the second dose of BNT162B2. Anti-PEG IgG and IgM titers were also measured before vaccination to examine changes after vaccination and the relationship with vaccine efficacy. In patients with hematological (n = 182) and autoimmune diseases (n = 96), anti-SARS-CoV-2 and anti-PEG antibody titers were evaluated after a median of 33 days from 2nd vaccination. The median anti-SARS-CoV-2 antibody titers were 1901 AU/mL and 3832 AU/mL in patients with hematological and autoimmune disease, respectively. Multiple regression analysis showed that age and days from 2nd vaccination were negatively associated with anti-SARS-CoV-2 antibody titers. Anti-CD20 antibody treatment was negatively correlated with anti-SARS-CoV-2 antibody titers in hematological disease, and C-reactive protein (CRP) was positively correlated with anti-SARS-CoV-2 antibody titers in autoimmune disease. Baseline anti-PEG antibody titers were significantly higher in patients with autoimmune disease but were not correlated with anti-SARS-CoV-2 antibody titers. Patients with increased anti-PEG IgG acquired higher anti-SARS-CoV-2 antibody titers in patients with autoimmune disease. Anti-SARS-CoV-2 antibody acquisition was suboptimal in patients with hematological disease, but both anti-SARS-CoV-2 antibody and anti-PEG IgG were acquired in patients with autoimmune disease, reflecting robust humoral immune response. Pre-existing anti-PEG antibody titers did not affect anti-SARS-CoV-2 antibody acquisition.


Peritoneal B Cells Play a Role in The Production of Anti-Polyethylene Glycol (PEG) IgM Against Intravenously Injected siRNA-PEGylated Liposome Complexes


Acute kidney injury caused by rhabdomyolysis is ameliorated by serum albumin-based supersulfides donors through antioxidative pathways

Oxidative stress is responsible for the onset and progression of various kinds of diseases including rhabdomyolysis-induced acute kidney injury (AKI). Antioxidants are, therefore, thought to aid in the recovery of illnesses linked to oxidative stress. Supersulfide species have been shown to have substantial antioxidative activity; however, due to their limited bioavailability, few supersulfide donors have had their actions evaluated in vivo. In this study, human serum albumin (HSA) and N-acetyl-L-cysteine polysulfides (NACSn), which have polysulfides in an oxidized form, were conjugated to create a supersulfide donor. HSA is chosen to be a carrier of NACSn because of its extended blood circulation and high level of biocompatibility. In contrast to a supersulfide donor containing reduced polysulfide in HSA, the NACSn-conjugated HSAs exhibited stronger antioxidant activity than HSA and free NACSn without being uptaken by the cells in vitro. The supersulfide donor reduced the levels of blood urea nitrogen and serum creatinine significantly in a mouse model of rhabdomyolysis-induced AKI. Supersulfide donors significantly reduced the expression of oxidative stress markers in the kidney. These results indicate that the developed supersulfide donor has the therapeutic effect on rhabdomyolysis-induced AKI.


Encapsulation of an Antioxidant in Redox-Sensitive Self-Assembled Albumin Nanoparticle for the Treatment of Hepatitis

Hepatitis is an inflammation of the liver caused by the inadequate elimination of reactive oxygen species (ROS) derived from Kupffer cells. Edaravone is clinically used as an antioxidant but shows poor liver distribution. Herein, we report on the design of a Kupffer cell-oriented nanoantioxidant based on a disulfide cross-linked albumin nanoparticle containing encapsulated edaravone (EeNA) as a therapeutic for the treatment of hepatitis. Since the edaravone is bound to albumin, this results in a soluble and stable form of edaravone in water. Exchanging the intramolecular disulfide bonds to intermolecular disulfide bridges of albumin molecules allowed the preparation of a redox responsive albumin nanoparticle that is stable in the blood circulation but can release drugs into cells. Consequently, EeNA was fabricated by the nanoscale self-assembly of edaravone and albumin nanoparticles without the additives that are contained in commercially available edaravone preparations. EeNA retained its nanostructure under serum conditions, but the encapsulated edaravone was released efficiently under intracellular reducing conditions in macrophages. The EeNA was largely distributed in the liver and subsequently internalized into Kupffer cells within 60 min after injection in a concanavalin-A-induced hepatitis mouse. The survival rate of the hepatitis mice was significantly improved by EeNA due to the suppression of liver necrosis and oxidative stress by scavenging excessive ROS. Moreover, even through the postadministration, EeNA showed an excellent hepatoprotective action as well. In conclusion, EeNA has the potential for use as a nanotherapeutic against various types of hepatitis because of its Kupffer cell targeting ability and redox characteristics.


Anti-PEG IgM production induced by PEGylated liposomes as a function of administration route

Modifying the surface of nanoparticles with polyethylene glycol (PEG) is a commonly used approach for improving the in vitro stability of nanoparticles such as liposomes and increasing their circulation half-lives. We have demonstrated that, in certain conditions, an intravenous (i.v.) injection of PEGylated liposomes (PEG-Lip) induced anti-PEG IgM antibodies, which led to rapid clearance of second doses in mice. SARS-CoV-2 vaccines, composed of mRNA-containing PEGylated lipid nanoparticles, have been widely administered as intramuscular (i.m.) injections, so it is important to determine if PEGylated formulations can induce anti-PEG antibodies. If the favorable properties that PEGylation imparts to therapeutic nanoparticles are to be widely applicable this should apply to various routes of administration. However, there are few reports on the effect of different administration routes on the in vivo production of anti-PEG IgM. In this study, we investigated anti-PEG IgM production in mice following i.m., intraperitoneal (i.p.) and subcutaneous (s.c.) administration of PEG-Lip. PEG-Lip appeared to induce anti-PEG IgM by all the tested routes of administration, although the lipid dose causing maximum responses varied. Splenectomy attenuated the anti-PEG IgM production for all routes of administration, suggesting that splenic immune cells may have contributed to anti-PEG IgM production. Interestingly, in vitro experiments indicated that not only splenic cells but also cells in the peritoneal cavity induced anti-PEG IgM following incubation with PEG-Lip. These observations confirm previous experiments that have shown that measurable amounts of PEG-Lip administered i.p., i.m. or s.c. are absorbed to some extent into the blood circulation, where they can be distributed to the spleen and/or peritoneal cavity, and are recognized by B cells, triggering anti-PEG IgM production. The results obtained in this study have important implications for developing efficient PEGylated nanoparticular delivery system.


Investigation of anti-PEG antibody response to PEG-containing cosmetic products in mice


A mouse model for studying the effect of blood anti-PEG IgMs levels on the in vivo fate of PEGylated liposomes

The presence of anti-polyethylene glycol (PEG) antibodies in the systemic circulation might have potential implications for the therapeutic activity of PEGylated products in vivo in the clinic. In order to study the effect of pre-existing anti-PEG antibodies on the in vivo fate and the therapeutic efficiency of PEGylated therapeutics, we developed a BALB/c mouse model by virtue of the intraperitoneal (i.p.) inoculation of hybridoma cells (HIK-M09 and HIK-M11), secreting monoclonal anti-PEG IgM, mimicking the presence of pre-existing anti-PEG antibodies in the blood. In the model, the titers of anti-PEG IgM in the blood increased as a function of hybridoma cells numbers and time after i.p. inoculation. The in vivo levels of anti-PEG IgM decreased in a dose-dependent manner, following i.v. administration of empty PEGylated liposomes. C26 tumor-bearing mice with measurable levels of anti-PEG IgM, receiving i.v. injection of DiR-labeled empty PEGylated liposomes, showed lower levels of liposomal tumor accumulation and higher levels of liver and spleen accumulation, compared to C26 tumor-bearing mice without measurable anti-PEG IgM. This specifies that the presence of anti-PEG IgM in the murine circulation induced accelerated blood clearance of PEGylated liposomes and reduced their tumor accumulation. The biodistribution and antitumor efficacy of commercially available doxorubicin (DXR)-containing PEGylated liposomes, Doxil®, were scrutinized in the anti-PEG IgM mouse model. In C26 tumor-bearing mice having circulating anti-PEG IgM, at 24 h after injection almost no DXR was observed in blood and tumor, and increased DXR accumulation was observed in spleen and liver, compared to tumor-bearing mice with no circulating anti-PEG IgM. The antitumor efficacy of Doxil® was significantly compromised in the C26 tumor-bearing mice in the presence of anti-PEG IgM. These results demonstrate that the anti-PEG IgM mouse model could be a useful prognostic indicator for the therapeutic effectiveness of different formulations of PEGylated therapeutics in pre-clinical studies.


I.p.-injected cationic liposomes are retained and accumulate in peritoneally disseminated tumors


The therapeutic effect of HSA dimer-doxorubicin complex against human pancreatic tumour


Doxorubicin embedded into nanofibrillated bacterial cellulose (NFBC) produces a promising therapeutic outcome for peritoneally metastatic gastric cancer in mice models via intraperitoneal direct injection


Incorporating gangliosides into PEGylated cationic liposomes that complexed DNA attenuates anti-PEG antibody production, but not anti-DNA antibody production in mice

Gangliosides (glycosphingolipids) reduce antibody production by inhibiting B-cell receptor (BCR) signaling. We have shown that a copresentation of gangliosides and polyethylene glycol (PEG) on the same liposomes suppresses anti-PEG IgM production in mice. In addition, we recently observed that pDNA incorporated in PEGylated cationic liposomes (PCLs) induces anti-DNA IgM, which could be a hurdle to the development of efficient gene delivery systems. Therefore, the focus of this study was to determine if the copresentation of gangliosides and DNA on the same PCL would suppress antibody production against DNA. PCLs including DNA induced both anti-PEG IgM production and anti-DNA IgM production. The extent of anti-PEG and anti-DNA IgM production was likely dependent on the immunogenicity of the complexed DNA. Treatment of clodronate-containing liposomes, which causes a depletion of phagocytic cells, suppressed anti-PEG IgM production from PCLs that did not include DNA but failed to suppress anti-PEG IgM production from PCLs that complexed DNA (PCLD). Both anti-PEG IgM and anti-DNA IgM was induced in T-cell-deficient nude mice as well as in normal mice following treatment with PCLs and PCLD, respectively. These results indicate that phagocytic cells contribute to anti-PEG IgM production but not to anti-DNA IgM production, while T-cells do not contribute to any form of antibody production. The copresentation of gangliosides and DNA significantly reduced anti-PEG IgM production but unfortunately did not reduce anti-DNA IgM production. It appears that the immunosuppressive effect of gangliosides, presumably via the CD22 signaling pathway, is limited only to anti-PEG immunity.


Nucleic acids delivered by PEGylated cationic liposomes in systemic lupus erythematosus-prone mice: a possible exacerbation of lupus nephritis in the presence of pre-existing anti-nucleic acid antibodies


Neutralization of acidic tumor microenvironment (TME) with daily oral dosing of sodium potassium citrate (K/Na Citrate) increases therapeutic effect of anti-cancer agent in pancreatic cancer xenograft mice model


Blood retention and antigenicity of polycarboxybetaine-modified liposomes

Zwitterionic polycarboxybetaines (PCBs) have gained attention as alternative stealth polymers whose liposomal formulation and protein conjugates were reported not to elicit anti-polymer antibodies. Here, we studied the blood retention and antigenicity of liposomes modified with PCBs focusing on their chemical structures and doses. We compared PCBs with either 1 or 3 (PCB1 or PCB3) spacer carbons between the carboxylate and ammonium groups. PCB3-modified liposomes had a short blood retention, whereas PCB1-modified liposomes demonstrated extended blood retention that was somewhat superior to PEGylated liposome. This confirmed the excellent non-fouling nature of PCB1 reported previously. Interestingly, PCB1-liposome as well as PCB3-liposome elicited specific IgMs toward each PCB. The dose-dependent production of specific IgMs to PCB-liposomes was similar to that of PEGylated liposome, i.e., high doses of PCB-liposomes reduced the production of specific IgMs, termed immunological tolerance. These results indicate the importance of investigating the effect of dose to clarify the existence of antigenicity of stealth polymers.


Utilization of Click Chemistry to Study the Effect of Poly(ethylene) Glycol Molecular Weight on the Self-Assembly of PEGylated Gambogic Acid Nanoparticles for the Treatment of Rheumatoid Arthritis

Many small-molecule drugs exhibit poor aqueous solubility, and various approaches have been developed to improve their solubility and delivery. Chemical conjugation of an insoluble drug to a hydrophilic polymer can promote the self-assembly into nanoparticles (NPs) to increase the apparent solubility and improve the pharmacokinetics of the drug. However, majority of the reports in the field disclose only one composition of the conjugate, while accumulating evidence suggests that structure-activity relationship (SAR) studies must be conducted to identify an optimal construct. In this study, we employed a click chemistry platform to robustly conjugate short-chain methoxypolyethylene glycol (mPEG) of three different molecular weights to a small molecule anti-inflammatory drug, gambogic acid (GA), and studied the SAR. NPs formed with mPEG550 and mPEG5000, referred to as NP-550 and NP-5000, respectively, had larger mean diameters (130.0 ± 16.9 nm and 143.0 ± 0.1 nm, respectively) and higher critical micellar concentrations (CMCs, 9.5 μg mL-1 and 10.5 μg mL-1, respectively) compared to NPs formed with mPEG2000 (NP-2000, mean diameter = 97.8 ± 5.0 nm and CMC = 6.6 μg mL-1). NP-2000 and NP-5000 did not cause significant hemolytic toxicity, whereas NP-550 and free GA induced 90% and 60% hemolysis, respectively. NP-2000 was selected for further studies due to its improved safety, small size and low CMC. In cultured inflammatory macrophages, NP-2000 exhibited activity comparable to free GA in suppressing tumor necrosis factor-α. In mice, NP-2000 showed 185-fold improved drug exposure compared to free GA after intraperitoneal delivery. Treatment with free GA showed little anti-inflammatory activity compared to vehicle control in a murine model of rheumatoid arthritis. In contrast, NP-2000 significantly reduced the paw inflammation by 27% from day 15 to day 29. NP-2000 showed no visible signs of toxicity in mice, while free GA elicited significant irritation at the injection site. Our work emphasizes the importance of performing SAR studies for developing an optimal drug-polymer conjugate for self-assembly into NPs. We also demonstrate a unique application of click chemistry to robustly synthesize a small library of conjugates for the SAR study.


Albumin domain mutants with enhanced Aβ binding capacity identified by phage display analysis for application in various peripheral Aβ elimination approaches of Alzheimer's disease treatment

Deposition of amyloid protein, particularly Aβ , is a major contributor to the onset of Alzheimer's disease (AD). However, almost no deposition of Aβ in the peripheral tissues could be found. Human serum albumin (HSA), the most abundant protein in the blood, has been reported to inhibit amyloid formation through binding Aβ, which is believed to play an important role in the peripheral clearance of Aβ. We identified the Aβ binding site on HSA and developed HSA mutants with high binding capacities for Aβ using a phage display method. HSA fragment 187-385 (Domain II) was found to exhibit the highest binding capacity for Aβ compared with the other two HSA fragments. To elucidate the sequence that forms the binding site for Aβ on Domain II, a random screening of Domain II display phage biopanning was constructed. A number of mutants with higher Aβ binding capacities than the wild type were identified. These mutants exhibited stronger scavenging abilities than the wild type, as revealed via in vitro equilibrium dialysis of Aβ experiments. These findings provide useful basic data for developing a safer alternative therapy than Aβ vaccines and for application in plasma exchange as well as extracorporeal dialysis.


Long-term storage of PEGylated liposomal oxaliplatin with improved stability and long circulation times in vivo

Liposomal anticancer drugs have been developed with improved clinical effects and reduced side effects. We have developed a PEGylated liposomal formulation of oxaliplatin that has anticancer effects in animal models of colorectal cancer with a favorable toxicity profile. To move this formulation into clinical development, a formulation that is stable during long term storage is needed, which has similar pharmacokinetics and therapeutic activity against solid tumors to the original formulation. In this study, we found that PEGylated liposomal oxaliplatin showed no changes in particle size after long term storage (12 months at 2-8 °C), but phospholipid degradation had occurred. Hence, the stored formulation had compromised membrane integrity, resulting in decreased in vivo circulation times of the liposomes. To improve the stability during long-term storage, a screening study to obtain an appropriate stabilizer was carried out. The buffer 2-morpholinoethansulfonic acid (MES) attenuated not only phospholipid degradation but also oxaliplatin degradation, unlike most other excipients. After 12 months storage at 2-8 °C in the presence of MES only slight degradation of phospholipids in PEGylated liposomal oxaliplatin occurred, resulting in similar in vivo pharmacokinetic profiles of the encapsulated oxaliplatin to the original formulation. Long term stability of PEGylated liposomal oxaliplatin was achieved by addition of MES, resulting in long circulation half-lives in vivo, a property which would be expected to lead to increased suppression of tumor growth and reduced side effects. Our formulation may now be suitable for clinical development.


発酵ナノセルロース(NFBC)の大量生産とその医療応用

発酵ナノセルロース(NFBC)の大量生産とその医療応用

月刊バイオインダストリー


Co-administration of liposomal l-OHP and PEGylated TS shRNA-lipoplex: A novel approach to enhance anti-tumor efficacy and reduce the immunogenic response to RNAi molecules

Many therapeutic strategies have been applied in efforts to conquer the development and/or progression of cancer. The combination of chemotherapy and an RNAi-based approach has proven to be an efficient anticancer therapy. However, the feasibility of such a therapeutic strategy has been substantially restricted either by the failure to achieve the efficient delivery of RNAi molecules to tumor tissue or by the immunostimulatory response triggered by RNAi molecules. In this study, therefore, we intended to investigate the efficacy of using liposomal oxaliplatin (liposomal l-OHP) to guarantee the efficient delivery of RNAi molecules, namely shRNA against thymidylate synthase (TS shRNA) complexed with cationic liposome (TS shRNA-lipoplex), to solid tumors, and to suppress the immunostimulatory effect of RNAi molecules, TS shRNA, following intravenous administration. Herein, we describe how liposomal l-OHP enhanced the intra-tumor accumulation of TS shRNA-lipoplex and significantly reduced the immunostimulatory response triggered by TS shRNA. Consequently, such enhanced accumulation of TS shRNA-lipoplex along with the cytotoxic effect of liposomal l-OHP led to a remarkable tumor growth suppression (compared to mono-therapy) following systemic administration. Our results, therefore, may have important implications for the provision of a safer and more applicable combination therapy of RNAi molecules and anti-cancer agents that can produce a more reliable anti-tumor effect.


Ganglioside inserted into PEGylated liposome attenuates anti-PEG immunity

Despite the clinical introduction of a vast number of polyethylene glycol (PEG)-conjugated therapeutics, conjugated PEG is also known for an unfortunate inclination toward immunogenicity. Immunogenicity of PEG, manifested by the robust production of anti-PEG IgM, is known to compromise the therapeutic efficacy and/or reduce the tolerance of PEGylated therapeutics. In the present study, we inserted ganglioside into the membrane of PEGylated liposome (PL) to prepare ganglioside-modified PEGylated liposomes (G-PL), and investigated its efficacy in attenuating the anti-PEG IgM response against PL. A single intravenous injection of G-PL significantly attenuated the anti-PEG IgM production, compared with that of naïve PL. In addition, pretreatment with G-PL substantially alleviated the anti-PEG IgM response elicited by a subsequent dose of PL, presumably via inducing B cell tolerance, and as a consequence, this modification abrogated/attenuated the incidence of the rapid clearance of subsequently administrated PL. These results indicate that incorporating gangliosides in PEGylated liposome membrane not only prevents the immunogenicity of PEG but also induces the tolerance of B cells to subsequent doses of the immunogenic PL. Consequently, liposomal membrane modification with ganglioside might represent a promising approach to attenuating the immunogenicity of PEGylated liposomes while preserving their therapeutic efficacy, particularly upon repeated administration.


Improvement of intratumor microdistribution of PEGylated liposome via tumor priming by metronomic S-1 dosing

The efficient delivery of nanocarrier-based cancer therapeutics into tumor tissue is problematic. Structural abnormalities, tumor vasculature heterogeneity, and elevated intratumor pressure impose barriers against the preferential accumulation of nanocarrier-based cancer therapeutics within tumor tissues and, consequently, compromise their therapeutic efficacy. Recently, we have reported that metronomic S-1, orally available tegafur formulation, dosing synergistically augmented the therapeutic efficacy of oxaliplatin (l-OHP)-containing PEGylated liposome without increasing the toxicity in animal model. However, the exact mechanism behind such synergistic effect was not fully elucidated. In this study, therefore, we tried to shed the light on the contributions of metronomic S-1 dosing to the enhanced accumulation and/or spatial distribution of PEGylated liposome within tumor tissue. Tumor priming with metronomic S-1 treatment induced a potent apoptotic response against both angiogenic endothelial cells and tumor cells adjacent to tumor blood vessels, resulting in enhanced tumor blood flow via transient normalization of tumor vasculature, along with alleviation of intratumor pressure. Such a change in the tumor microenvironment imparted by S-1 treatment allows efficient delivery of PEGylated liposome to tumor tissue and permits their deep penetration/distribution into the tumor mass. Such a priming effect of S-1 dosing can be exploited as a promising strategy to enhance the therapeutic efficacy of nanocarrier-based cancer therapeutics suffering from inadequate/heterogeneous delivery to tumor tissues.


Gene silencing using 4'-thioDNA as an artificial template to synthesize short-hairpin RNA without inducing a detectable innate immune response

The development of a versatile technique to induce RNA interference (RNAi) without immune stimulation in vivo is of interest as existing approaches to trigger RNAi, such as small interfering RNA (siRNA) and plasmid DNA (pDNA) expressing short hairpin RNA (shRNA), present drawbacks arising from innate immune stimulation. To overcome them, an intelligent shRNA expression device (iRed) designed to induce RNAi was developed. The minimum sequence of iRed encodes only the U6 promoter and shRNA. A series of iRed comprises a polymerase chain reaction (PCR)-amplified 4'-thioDNA in which any one type of adenine (A), guanine (G), cytosine (C), or thymine (T) nucleotide unit was substituted by each cognate 4'-thio derivatives, i.e., dSA iRed, dSG iRed, dSC iRed, and ST iRed respectively. Each modified iRed acted as a template to transcribe shRNA with RNAi activity. The highest shRNA yield was generated using dSC iRed that exerted gene silencing activity in an orthotopic mouse model of mesothelioma. Reducing the minimal structure required to transcribe shRNA and the presence of the 4'-thiomodification synergistically function to abrogate innate immune response induced by dsDNA. The iRed will introduce a new approach to induce RNAi without inducing a detectable innate immune response.


Comprehensive analysis of PEGylated liposome-asscociated proteins relating to the accelerated blood clearance phenomenon by combination with shotgun analysis and conventional methods

PEGylated liposome, sterically stabilized by polyethylene glycol (PEG), results in reduced recognition of the liposome by the mononuclear phagocyte system. Recently, we reported regarding the accelerated blood clearance (ABC) phenomenon that PEGylated liposome is cleared very rapidly from blood circulation upon repeated injection. Anti-PEG IgM production and subsequent complement activation were crucial in causing the ABC phenomenon. However, there still remains the possibility that unknown plasma factors might affect the fate of PEGylated liposome that is subjected to the ABC phenomenon. A label-free approach to shotgun analysis is a great tool for characterizing proteins in a biological system. In this study, therefore, a shotgun analysis was employed to identify plasma protein bound on PEGylated liposome after the ABC phenomenon was induced in the mouse model. The analysis revealed that immunoglobulin and complement components (C1 and C3) are the major proteins. Subsequent analysis with enzyme-linked immunosorbent assay and Western blotting showed that the immunoglobulin was IgM and that the complement system was mainly activated via an anti-PEG IgM-mediated classical pathway. These results support our earlier assumptions-anti-PEG IgM and complement activation were the major causes of the ABC phenomenon. Our proposed analytical strategy would be expected to provide useful information for the development and design of the nanocarrier drug delivery system.


Novel Submicronized Rebamipide Liquid with Moderate Viscosity: Significant Effects on Oral Mucositis in Animal Models

This study aimed at developing a novel rebamipide liquid for an effective treatment of oral mucositis. The healing effects of a variety of liquids comprising submicronized rebamipide crystals were investigated using a rat cauterization-induced oral ulcer model. Whereas 2% rebamipide liquid comprising micro-crystals did not exhibit significant curative effect, 2% rebamipide liquids comprising submicronized crystals with moderate viscosities exhibited healing effects following intra-oral administration. The 2% and 4% optimized rebamipide liquids showed significant healing effects in the rat oral ulcer model (p<0.01). In addition, in the rat radiation-induced glossitis model, whereby the injury was caused to the tongue by exposing only around the rat's snout to a 15 Gy of X-irradiation, the 2% optimized rebamipide liquid significantly reduced the percent area of ulcerated injury (p<0.05). In conclusion, the submicronized rebamipide liquid with moderate viscosity following intra-oral administration showed better both healing effect in the rat oral ulcer model and preventive effect in the rat irradiation-induced glossitis model.


Use of polyglycerol (PG), instead of polyethylene glycol (PEG), prevents induction of the accelerated blood clearance phenomenon against long-circulating liposomes upon repeated administration

The accelerated blood clearance (ABC) phenomenon accounts for the rapid systemic clearance of PEGylated nanocarriers upon repeated administrations. IgM production against the polyethylene glycol (PEG) coating in PEGylated liposomes is now known to be responsible for such unexpected pharmacokinetical alterations. The ABC phenomenon poses a remarkable clinical challenge by reducing the therapeutic efficacy of encapsulated drugs and causing harmful effects due to the altered tissue distribution pattern of the drugs. In this study, we investigated the in vivo performance of liposomes modified with polyglycerol (PG) upon repeated injection, and the in vivo therapeutic efficacy of such liposomes when they encapsulated a cytotoxic agent, doxorubicin (DXR). Repeated injection of PEG-coated liposomes in rats induced the ABC phenomenon, while repeated injection of PG-coated liposomes did not. In addition, DXR-containing PG-coated liposomes showed antitumor activity that was superior to that of free DXR and similar to that of DXR-containing PEG-coated liposomes upon repeated administration. These results indicate that polyglycerol (PG) might represent a promising alternative to PEG via enhancing the in vivo performance of liposomes by not eliciting the ABC phenomenon upon repeated administration.


The co-delivery of oxaliplatin abrogates the immunogenic response to PEGylated siRNA-lipoplex.

In vivo application of siRNA/PEGylated cationic liposome complex (lipoplex) is impeded by two main obstacles: cytokine responses and anti-PEG IgM responses to PEGylated siRNA-lipoplex. Here, we investigated whether co-administration of oxaliplatin (l-OHP) abrogates the cytokine release and anti-PEG IgM production by PEGylated siRNA-lipoplex. Free l-OHP was administered either simultaneously or 30 min prior to PEGylated siRNA-lipoplex administration, and cytokine response and anti-PEG IgM production were evaluated. In addition, the effect of the liposomal encapsulation of l-OHP on the immunogenic response of PEGylated siRNA-lipoplex was investigated. Simultaneous co-administration of free l-OHP with PEGylated siRNA-lipoplex caused a significant reduction in anti-PEG IgM production, along with an increase in the cytokine response. Free l-OHP injected prior to the lipoplex injection, however, successfully reduced cytokine release and anti-PEG IgM response. Platination of siRNA by simultaneously administered free l-OHP might facilitate the dissociation of double-stranded siRNA to single-stranded siRNA, resulting in the inducement of a potent immuno-stimulation of siRNA via endosomal toll-like receptors (TLRs). On the other hand, encapsulation of l-OHP into the siRNA-lipoplex resulted in a reduction of both anti-PEG IgM production and cytokine responses. Our results suggest that, besides the expected therapeutic efficacy of co-administration, encapsulation of l-OHP into the PEGylated siRNA-lipoplex has great potential for minimizing the immunostimulation of PEGylated siRNA-lipoplex, resulting in a safe, applicable, and compliant treatment regimen for sequential clinical administration.


Sequential administration with oxaliplatin-containing PEG-coated cationic liposomes promotes a significant delivery of subsequent dose into murine solid tumor.

Recently, we designed a PEG-coated cationic liposome to achieve dual targeting delivery of l-OHP to both tumor endothelial cells and tumor cells in a solid tumor. The targeted liposomal l-OHP formulation showed an efficient antitumor activity in a murine tumor model after three sequential liposomal l-OHP injections. This led us to assume that prior dosing with liposomes might enhance the intra-tumoral accumulation of a subsequent dose, and hence improve the therapeutic efficacy of entrapped l-OHP. The present study shows that while a single liposomal l-OHP injection does not enhance tumor accumulation of subsequent test-PEG-coated cationic liposomes, two sequential injections of liposomal l-OHP do. Cumulative cytotoxic effects of l-OHP delivered by PEG-coated cationic liposomes led to deep diffusion of a subsequent dose of liposomal l-OHP in solid tumor presumably as a result of the enlarged intra-tumoral interstitial space. Our study suggests that sequential injections of a targeted liposomal anticancer drug is of significant clinical and practical importance in enhancing the delivery of adequate quantities of anticancer agents into intractable solid tumors, and thereby may achieve a significant anticancer efficacy.


Effect of co-administration of tacrolimus on the pharmacokinetics of multiple subcutaneous administered interferon-alpha in rats.

Repeated administration of exogenous proteinic compounds triggers the production of specific antibodies. This reaction is limits not only pharmacokinetic studies in animal but also development of human or humanized proteins as drugs. We investigated the effect of co-administration of tacrolimus on pharmacokinetic of human interferon-alpha (h-IFN) following multiple subcutaneous administration in rats. h-IFN was administered at a dose of 5 million IU/kg. For some experiments, tacrolimus was also either subcutaneously or intravenously injected in rats at a dose of 0.001 or 0.5 mg/kg as well as with administration of h-IFN. Multiple administration of h-IFN without co-administration of tacrolimus induced IgG response at 2 or 3 weeks following first administration in the short dosing interval (daily, once per 3 days, or once per a week), irrespective of the dosing interval. Both intravenous and subcutaneous administration of tacrolimus (0.5 mg/kg) with multiple h-IFN injections successfully suppressed IgG response against h-IFN. Interestingly, in lower doses (0.001 mg/kg), intravenous co-administration of tacrolimus showed much stronger suppressive effect than subcutaneous co-administration. Intravenous co-administration of tacrolimus (0.001 mg/kg) may be a promising way to assess crossover pharmacokinetic study of human or humanized proteinic formulations with multiple dosing schedules in an experimental animal.


Oxaliplatin targeting to angiogenic vessels by PEGylated cationic liposomes suppresses the angiogenesis in a dorsal air sac mouse model.

Oxaliplatin (trans-l-diaminocyclohexane oxalatoplatinum, l-OHP) is a third-generation platinum analogue with proven anti-tumor activity against many tumor cell lines, however it does not show sufficient anti-tumor activity in vivo when used alone. In order to overcome this problem and to achieve an anti-angiogenic therapy with l-OHP, the drug was encapsulated into PEG-coated cationic liposomes, which were designed to target the newly formed vessels, and its anti-angiogenic activity was evaluated in an in vivo mouse dorsal air sac (DAS) assay. For the DAS assay, chambers filled with tumor cells were implanted underneath the dorsal skin. l-OHP encapsulated in PEG-coated cationic liposomes (5 mg/kg mice) was intravenously injected once on day 1, 2, 3 or 4 after chamber implantation. On the fifth day after chamber implantation, animals were sacrificed and tumor-angiogenesis was evaluated. Liposome-encapsulated l-OHP completely suppressed angiogenesis in the skin when it was administered day 3 after chamber implantation. Under similar experimental conditions, neither l-OHP encapsulated in PEG-coated neutral liposomes, nor free l-OHP, nor "empty" (no drug containing) PEG-coated cationic liposomes showed such strong suppressive effect. The present study suggests that the liposomal formulation of l-OHP, which targeted to angiogenic vessels, has a remarkable in vivo anti-angiogenic activity and the formulation may become a promising novel approach to achieve anti-angiogenic therapy.


A metronomic schedule of cyclophosphamide combined with PEGylated liposomal doxorubicin has a highly antitumor effect in an experimental pulmonary metastatic mouse model.

Metronomic chemotherapy is a novel approach to the control of advanced cancer, as it appears to preferentially inhibit endothelial cell activity in the growing vasculature of tumors. Doxorubicin-containing sterically stabilized liposomes (DXR-SL) accumulate in large amounts in tumor tissue, resulting in enhanced antitumor effects of the encapsulated DXR. In the present study, it was hypothesized that metronomic chemotherapy may further augment the accumulation of DXR-SL, improving its therapeutic efficacy. This study tests the antitumor efficacy for the combination of a metronomic cyclophosphamide (CPA)-dosing schedule with sequential intravenous injections of DXR-SL in the treatment of lung metastatic B16BL6 melanoma-bearing mice. Three dosing schedules for the combination of metronomic CPA injections (s.c. 170 mg/kg every 6 days) plus either a low or a high dose of DXR-SL (i.v. 1 or 5 mg/kg every 6 days) were set: Schedule I, DXR-SL was given 3 days before the first CPA treatment; Schedule II, DXR-SL and CPA were given simultaneously; and, Schedule III, DXR-SL was given 3 days after the first CPA treatment. Lung weight and median survival time (MST) were evaluated. As expected, both the dosing schedule as well as the dose of DXR-SL improved therapeutic efficacy. Schedule I with the low DXR dose and Schedule II with the low or high DXR dose significantly increased MST, compared with regular metronomic CPA therapy. Under the dosing schedules (Schedule I with the low DXR dose and Schedule II with the high DXR), there was a strong relationship between increased MST and decreased lung weight. However, Schedule I with high DXR dose resulted in significantly lower lung weights, but did not increase MST, suggesting that chemotherapy may result in increased toxicity in some conditions. Although treatment regimens require optimization, the results of the present study may prove useful in further explorations of combining metronomic chemotherapy with liposomal anticancer drugs in the treatment of solid tumors.


Combined effect of liposomalization and addition of glycerol on the transdermal delivery of isosorbide 5-nitrate in rat skin.

In this report, we investigated the combined effect of drug liposomalization and addition of glycerol on the transdermal delivery of isosorbide 5-nitrate (ISN) in rat abdominal skin in vitro. Occlusive application of both liposomal and aqueous ISN solution, with and without addition of 5% glycerol, showed that drug liposomalization and addition of glycerol has far-reaching implications for ISN permeation and accumulation in 4 and 8 weeks old rat abdominal skin. Using 8 weeks old rat abdominal skin, the optimal concentration of glycerol to be added to liposomal ISN was found to be 5%. The ISN mean values permeated through and accumulated in stripped 8 weeks old rat abdominal skin from those formulations described above were not significant different, which might indicate the combined effect of glycerol and liposomal ISN resides solely in the stratum corneum (SC). Based on previous reports, the enhancement effect of glycerol might be due to an increase in the SC hydration, and perhaps due to subtle changes in the lipid organization caused by penetration of liposomal lipids within the SC intercellular spaces. These data might provide evidence that glycerol action on SC is useful to facilitate skin permeation and accumulation of drugs formulated in liposome.


Disappearance of the angiogenic potential of endothelial cells caused by Argonaute2 knockdown.

Argonaute2 (Ago2), a component protein of RNA-induced silencing complex, plays a central role in RNA interference. We focused on the involvement of Ago2 in angiogenesis. Human umbilical vein endothelial cells (HUVECs) stimulated with several growth factors such as vascular endothelial growth factor were used for angiogenesis assays. We applied polycation liposomes for transfection of small interfering RNA (siRNA) to determine the biological effects of siRNA for Ago2 (siAgo2) on HUVECs. The proliferation study indicated that siAgo2 significantly suppressed the growth of HUVECs compared with control siRNA. TUNEL staining showed a certain population of HUVECs treated with siAgo2 underwent apoptosis. Furthermore, the treatment with siAgo2 suppressed the tube formation of HUVECs and significantly reduced the length of the tubes. These present data demonstrate that siAgo2 inhibited indispensable events of angiogenesis in vitro. This is the first report suggesting that Ago2 is required for angiogenesis.

Biochemical and Biophysical Research Communications


Injection of PEGylated liposomes in rats elicits PEG-specific IgM, which is responsible for rapid elimination of a second dose of PEGylated liposomes.

Steric stabilization of the surface of liposomes by a PEG conjugated lipid results in reduced recognition of the liposomes by the cells of the mononuclear phagocyte system and consequently extended their circulation times (t(1/2) approximately 20h in rat). Recently, we reported on the "accelerated blood clearance phenomenon", causing "invisible" PEGylated liposomes to be cleared very rapidly from the circulation upon repeated injection. In addition, we reported that certain serum factor(s) secreted into the blood after the first dose of PEGylated liposomes play an essential role in the phenomenon. The aim of the present study was to identify the major serum protein(s) responsible for the phenomenon and to unravel their mode of action. The amount of protein binding to PEGylated liposomes during incubation with serum hardly correlated with the extent of the phenomenon. PEGylated liposomes incubated with serum obtained from rats pre-injected 5 days before with the same liposomes showed a much more complex pattern of bound proteins than when incubated with naïve serum, as revealed by 2D-PAGE and SDS-PAGE. Subsequent analysis with LC-MS/MS and Western blot showed that the major pre-treated serum protein binding to PEGylated liposomes was IgM. Semi-quantitative analysis showed that larger amount of IgM bound to PEGylated liposomes compared to conventional liposomes. It was further demonstrated that PEGylated liposomes could activate the complement system due to IgM binding when incubated in serum from rats pre-injected with PEGylated liposomes, while conventional liposomes were not. These findings suggest that the selective binding of IgM to the second injected PEGylated liposomes and the subsequent complement activation by IgM resulted in the accelerated clearance and enhanced hepatic uptake of the second injected PEGylated liposomes. Based on the results described here, we are drawing attention to the potential occurrence of unexpected immune reactions upon intravenous administration of PEGylated liposomes or other particles and, by extension, PEGylated proteins and DNAs.


Development of pH-sensitive liposomes that efficiently retain encapsulated doxorubicin (DXR) in blood.

We have reported that targeted, pH-sensitive sterically stabilized liposomes are able to increase the cytotoxicity of DXR in vitro against B lymphoma cells, but the rate of release of DXR in plasma was too rapid to permit the results to be extended to in vivo applications. The purpose of the study reported here is two-fold. First, to understand the mechanism of the rapid release of DXR from pH-sensitive sterically stabilized liposomes (PSL) in human plasma. Second, to reformulate the above liposomes to improve their drug retention, while retaining their pH sensitivity. The stability of the PSL formulations in human plasma was evaluated by comparing the rate of release of encapsulated DXR with that of HPTS, a water-soluble fluorescent marker. Since DXR, but not HPTS, a water soluble-less membrane permeable fluorescence marker, was rapidly released from liposomes in the presence of plasma, the rapid release of DXR is likely caused by the diffusion of DXR molecules through the lipid bilayer, not by the disruption of the membrane. In order to develop more stable PSL formulations, various molar ratios of the membrane rigidifying lipid, hydrogenated soy HSPC and/or CHOL, were added to the lipid composition and the rate of release of encapsulated solutes and pH-sensitivity were evaluated. The compositions that showed the best drug retention and pH-sensitivity were a mixture of DOPE/HSPC/CHEMS/CHOL/mPEG(2000)-DSPE at a molar ratio of 4:2:2:2:0.3 and DOPE/HSPC/CHEMS/CHOL at a molar ratio of 4:2:2:2. Our formulations, if targeted to internalizing antigens on cancer cells, may increase intracellular drug release rates within acidic compartment, resulting in a further increase in the therapeutic efficacy of targeted anticancer drug-containing liposomes.


Gene Expression in Primary Cultured Mouse Hepatocytes with a Cationic Liposomal Vector, TFL-3: Comparison with Rat Hepatocytes

We recently reported that a cationic liposomal vector, TFL-3, could be used to achieve significant gene expression in primary cultured rat hepatocytes (Nguyen et al., Biol. Pharm. Bull., 26, 880-885 (2003)). A combination of hepatocyte transplantation and hepatocyte-targeted gene transfer represents a potentially important strategy for expanding treatment options for liver disease. A widely applied approach to support cross-species is necessary before human applications can be realized. Therefore, in this study, we examined the utility of TFL-3 in another species of rodent hepatocytes, namely mouse hepatocytes. Gene expression in mouse hepatocytes by TFL-3 was successful and the level was higher than those in rat hepatocytes that we recently reported on. Interestingly, it appears that both the degree and rate of gene expression were dependent on the incubation time prior to lipofection as well as on the density of cells per dish, but these parameters were independent of the amount of pDNA associated with the cells. These significantly suggest that the culture time prior to and following lipofection, which are related to the biological condition of the cells, may be one of major factors that affect gene expression in hepatocytes and non- or less dividing cells.


HEPC-based liposomes trigger cytokine release from peripheral blood cells: effects of liposomal size, dose and lipid composition


Efficacy of Intrathecal Liposomal Fasudil for Experimental Cerebral Vasospasm after Subarachnoid Hemorrhage

To investigate the safety and efficacy of liposomal fasudil in a sustained-release form for the prevention of cerebral vasospasm after subarachnoid hemorrhage (SAH). Eighteen rats were divided into three groups, each of which received 2.5 mg/kg of liposomal fasudil, 5 mg/kg of liposomal fasudil, or drug-free liposomes after SAH. Next, experimental SAH was induced in 15 dogs by injection of autologous arterial blood into the cisterna magna twice after baseline vertebral angiography. In six dogs, 0.94 mg/kg of liposomal fasudil was injected into the cisterna magna (treatment group). In four dogs, drug-free liposomes were similarly injected (placebo group), and the remaining five dogs were not treated with liposomal injection after SAH (control group). Angiography was repeated on Day 7, and cerebrospinal fluid was collected before the dogs were killed. A high dose of liposomal fasudil caused no significant changes in mean arterial blood pressure and did not induce seizures during the observation period. Gross and microscopic examination of the brains revealed no abnormalities, but severe vasospasm was noted in the rat basilar artery, mainly in the group treated with drug-free liposomes. Likewise, in the canine placebo and control groups, significant vasospasm occurred in the basilar artery on Day 7. In the treatment group, vasospasm in the basilar artery was significantly ameliorated (P < 0.01). In vivo, 90% of fasudil was released from liposomes in the cerebrospinal fluid. A single injection of intrathecal liposomal fasudil is safe and effective for the prevention of vasospasm in experimental SAH.


Neuroprotection by intrathecal application of liposome-entrapped Fasudil in a rat model of ischemia

Pharmacological treatment for cerebral ischemia cannot attain sufficiently high concentrations of the drugs in the cerebrospinal fluid (CSF) without precipitating systemic side effects. The objective of this study is the development of a liposomal drug delivery system that maintains effective concentrations of protein kinase inhibitors fasudil in the CSF, resulting in neuroprotection against cerebral ischemia. Focal cerebral ischemia in rats was induced by middle cerebral artery occlusion using an intraluminal suture technique. Treated rats received 0.25 mg liposome-entrapped fasudil via the cisterna magna 2 hours after ischemic insult. Control rats received drug-free liposomes. Neurological condition and the infarct size were assessed at 24 and 72 hours after ischemia. The concentration of liposome-entrapped fasudil in the CSF was measured before sacrifice. Treated animals showed significantly improved neurological outcomes after the 24-hour observation period compared to the control group (p < 0.001). Treatment with 0.25 mg liposomal fasudil resulted in a reduction in the infarct area (24 hours: 29.0 +/- 4.4%, 72 hours: 28.1 +/- 3.9% of total brain slices) compared to controls (49.6 +/- 4.6%, p < 0.001), but there was no statistical difference between 24 and 72 hours. At 24 hours post-administration, CSF concentrations of liposome-entrapped fasudil were 45.4 +/- 31.5 micrograms/ml (20% of the injected dose). A single intrathecal injection of liposomal fasudil can maintain a therapeutic drug concentration in the CSF over a period of time, significantly decreasing infarct size in a rat model of acute ischemia.


A combinatorial approach to producing sterically stabilized(Stealth)immunoliposomal drugs


A novel plasma factor initiating complement activation on cetylmannoside-modified liposomes in human plasma