Hidenori Ando

Anti-PEG IgM and complement system are required for the association of second doses of PEGylated liposomes with splenic marginal zone B cells.

The accelerated blood clearance (ABC) phenomenon makes it crucial to use PEGylated liposomes and micelles to deliver drugs. The ABC phenomenon is an immune response against an initial dose of PEGylated liposome, which causes subsequent doses to be rapidly cleared by macrophages in the liver. We recently found that in the early phase of the ABC phenomenon, subsequent doses of PEGylated liposomes were associated with splenic marginal zone (MZ)-B cells and were transported from the MZ to the follicle (FO). In this study, we investigated the underlying mechanisms behind the association of subsequent doses of PEGylated liposomes with MZ-B cells in the spleen. Serum factors, anti-PEG IgM and complement system, were crucial to the association of PEGylated liposomes with MZ-B cells, while the sensitization of MZ-B cells by the first dose of PEGylated liposomes was not significant. It was the complement receptors (CRs) on the MZ-B cells, rather than either the PEG-specific B-cell receptors or the IgM Fc receptors, that were the main contributors to the association between PEGylated liposomes and MZ-B cells. It appeared that anti-PEG IgM would bind to PEGylated liposomes and causes subsequent complement activation, resulting in the formation of immune complexes of PEGylated liposome-anti-PEG IgM-complement. The MZ-B cells then recognized these immune complexes via their CRs. Such an association via CRs might have triggered the transport of the immune complex by MZ-B cells to the FO in the spleen. The information obtained in this study might be useful in the development of an efficient antigen delivery system to usher PEGylated nanoparticles into FO dendritic cells.

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.

Ganglioside inserted into PEGylated liposome attenuates anti-PEG immunity

ABSTRACT 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.

Hepatic Tumor Metastases Cause Enhanced PEGylated Liposome Uptake by Kupffer Cells

Kupffer cells in livers bearing tumor metastases were found to have promoted tumor invasion and exacerbated the metastasis. This implies that the function of Kupffer cells might differ between animals bearing hepatic metastases and those that are healthy. Kupffer cells are considered responsible for the accumulation of liposomes in the liver. In this study, we hypothesized that the alteration in the function of Kupffer cells by hepatic metastasis would also affect the biodistribution of liposomes following intravenous administration. The hepatic accumulation and the blood concentration of PEGylated liposomes were compared between healthy mice and tumor-bearing mice. We noted that hepatic accumulation and elimination from the blood were significantly accelerated in tumor-bearing mice, indicating that our hypothesis was correct. In the tumor-bearing mice, the proportion of Kupffer cells taking up liposomes was significantly increased. Intravenous injection of oxaliplatin (l-OHP) containing PEGylated liposomes decreased the fraction of Kupffer cells, but this administration caused no injury to the hepatocytes. These results suggest that PEGylated liposomes containing l-OHP may have the potential to treat metastatic hepatic cancer-not only via the direct killing of the cancer cells but also via a reduction in tumor-supportive Kupffer cells.

A Novel Strategy to Increase the Yield of Exosomes (Extracellular Vesicles) for an Expansion of Basic Research

Exosomes are tiny extracellular vesicles that are usually harvested in small quantities. Such small yield has been an obstacle for the expansion of the basic research regarding exosome analysis and applications in drug delivery. To increase exosome yield, we attempted to stimulate tumor cells via the addition of liposomes in vitro. Neutral, cationic-bare or PEGylated liposomes were incubated with four different tumor cell lines. The stimulatory effect of liposomal formulations on exosome secretion and cellular uptake propensity of the collected exosome by mother cells or different cells was evaluated. Both neutral and cationic-bare liposomes enhanced exosome secretion in a dose-dependent manner. Fluid cationic liposomes provided the strongest stimulation. Surprisingly, the PEGylation of bare liposomes diminished exosome secretion. Exosomes harvested in the presence of fluid cationic liposomes showed increased cellular uptake, but solid cationic liposomes did not. Our findings indicate that the physicochemical properties of liposomes determine whether they will act as a stimulant or as a depressant on exosome secretion from tumor cells. Liposomal stimulation may be a useful strategy to increase exosome yield, although further preparation to increase the purity of exosomes may be needed. In addition, fine-tuning of the biological properties of induced exosomes could be achieved via controlling the physicochemical properties of the stimulant liposomes.

Liposomalization of oxaliplatin induces skin accumulation of it, but negligible skin toxicity

&NA; Liposomalization causes alteration of the pharmacokinetics of encapsulated drugs, and allows delivery to tumor tissues through passive targeting via an enhanced permeation and retention (EPR) effect. PEGylated liposomal doxorubicin (Doxil®, Lipo‐DXR), a representative liposomal drug, is well‐known to reduce cardiotoxicity and increase the anti‐tumor activity of DXR, but to induce the hand‐foot syndrome (HFS) as a result of skin DXR accumulation, which is one of its severe adverse effects. We have developed a new liposomal preparation of oxaliplatin (l‐OHP), an important anti‐tumor drug for treatment of colorectal cancer, using PEGylated liposomes (Lipo‐l‐OHP), and showed that Lipo‐l‐OHP exhibits increased anti‐tumor activity in tumor‐bearing mice compared to the original preparation of l‐OHP. However, whether Lipo‐l‐OHP causes HFS‐like skin toxicity similar to Lipo‐DXR remains to be determined. Administration of Lipo‐l‐OHP promoted accumulation of platinum in rat hind paws, however, it caused negligible morphological and histological alterations on the plantar surface of the paws. Administration of DiI‐labeled empty PEGylated liposomes gave almost the same distribution profile of dyes into the dermis of hind paws with DXR as in the case of Lipo‐DXR. Treatment with Lipo‐l‐OHP, Lipo‐DXR, DiI‐labeled empty PEGylated liposomes or empty PEGylated liposomes caused migration of CD68+ macrophages into the dermis of hind paws. These findings suggest that the skin toxicity on administration of liposomalized drugs is reflected in the proinflammatory characteristics of encapsulated drugs, and indicate that Lipo‐l‐OHP with a higher anti‐cancer effect and no HFS may be an outstanding l‐OHP preparation leading to an improved quality of life of cancer patients. Graphical abstract Figure. No caption available. HighlightsPEGylated liposomal oxaliplatin induced negligible skin toxicity on rat hind paws.Hand‐foot syndrome‐like skin toxicity was caused by PEGylated liposomal doxorubicin.PEGylated liposomes distributed into rat hind paw skin tissues, especially dermis.Macrophages migrated to dermis to engulf the drug‐ and vehicle‐containing liposomes.The skin toxicity depends on proinflammatory characteristics of encapsulated drugs.

Liposome co-incubation with cancer cells secreted exosomes (extracellular vesicles) with different proteins expressions and different uptake pathways

We recently showed that in vitro incubation of cells with liposomes of varying compositions can increase exosome secretion and increase the yield of harvested exosomes (extracellular vesicles, EVs). This might foster their potential therapeutic implementations. In the current study, we investigated the surface proteins and the uptake of the harvested exosomes (EVs) to see if the incubation of cells with liposomes would change the biological properties of these exosomes (EVs). Interestingly, exosomes (EVs) induced by solid cationic liposomes lacked some major exosome marker proteins such as CD9, flotillin-1, annexin-A2 and EGF, and subsequently had lower levels of cellular uptake upon re-incubation with donor cancer cells. However, exosomes (EVs) induced under normal condition and by fluid cationic liposomes, displayed the entire spectrum of proteins, and exhibited higher uptake by the donor cancer cells. Although endocytosis was the major uptake pathway of exosomes (EVs) by tumor cells, endocytosis could occur via more than one mechanism. Higher exosome uptake was observed in donor B16BL6 cells than in allogeneic C26 cells, indicating that donor cells might interact specifically with their exosomes (EVs) and avidly internalize them. Taken together, these results suggest a technique for controlling the characteristics of secreted exosomes (EVs) by incubating donor cancer cells with liposomes of varying physiochemical properties.

A Cell Assay for Detecting Anti-PEG Immune Response against PEG-Modified Therapeutics

PurposeImmunogenicity of PEGylated proteins and nanomedicines represents a potential impediment against their development and use in clinical settings. The purpose of this study is to develop a method for detecting anti-PEG immunity of PEGylated proteins and/or nanomedicines using flow cytometry.MethodsThe binding of fluorescence-labeled mPEG-modified liposomes to HIK-G11 cells, PEG-specific hybridoma cells, or spleen cells was evaluated by flow cytometry for detecting immunogenicity of PEGylated therapeutics.ResultsThe fluorescence-labeled methoxy PEG (mPEG)-modified liposomes were efficiently bound to HIK-G11 cells. Such staining with fluorescence-labeled mPEG-modified liposomes was significantly inhibited in the presence of either non-labeled mPEG-modified liposomes or mPEG-modified ovalbumin (OVA) but not polyglycerol-modified liposomes. In addition, we found that mPEG-modified liposomes, highly immunogenic, caused proliferation of PEG-specific cells, while hydroxyl PEG-modified liposomes, less immunogenic, scarcely caused. Furthermore, after intravenous injection of mPEG-modified liposomes, the percentage of PEG-specific cells in the splenocytes, as determined by flow cytometry, corresponded well with the production level of anti-PEG antibodies, as determined by ELISA.ConclusionsPEG-specific B cell assay we introduced may become a useful method to detect an anti-PEG immune response against PEGylated therapeutics and clarify the mechanism for anti-PEG immune responses.

Intratumoral Visualization of Oxaliplatin within a Liposomal Formulation Using X-ray Fluorescence Spectrometry

Microsynchrotron radiation X-ray fluorescence spectrometry (μ-SR-XRF) is an X-ray procedure that utilizes synchrotron radiation as an excitation source. μ-SR-XRF is a rapid, nondestructive technique that allows mapping and quantification of metals and biologically important elements in cell or tissue samples. Generally, the intratumor distribution of nanocarrier-based therapeutics is assessed by tracing the distribution of a labeled nanocarrier within tumor tissue, rather than by tracing the encapsulated drug. Instead of targeting the delivery vehicle, we employed μ-SR-XRF to visualize the intratumoral microdistribution of oxaliplatin (l-OHP) encapsulated within PEGylated liposomes. Tumor-bearing mice were intravenously injected with either l-OHP-containing PEGylated liposomes (l-OHP liposomes) or free l-OHP. The intratumor distribution of l-OHP within tumor sections was determined by detecting the fluorescence of platinum atoms, which are the main elemental components of l-OHP. The l-OHP in the liposomal formulation was localized near the tumor vessels and accumulated in tumors at concentrations greater than those seen with the free form, which is consistent with the results of our previous study that focused on fluorescent labeling of PEGylated liposomes. In addition, repeated administration of l-OHP liposomes substantially enhanced the tumor accumulation and/or intratumor distribution of a subsequent dose of l-OHP liposomes, presumably via improvements in tumor vascular permeability, which is also consistent with our previous results. In conclusion, μ-SR-XRF imaging efficiently and directly traced the intratumor distribution of the active pharmaceutical ingredient l-OHP encapsulated in liposomes within tumor tissue. μ-SR-XRF imaging could be a powerful means for estimating tissue distribution and even predicting the pharmacological effect of nanocarrier-based anticancer metal compounds.

Reactivity of IgM antibodies elicited by PEGylated liposomes or PEGylated lipoplexes against auto and foreign antigens

Abstract Polyethylene glycol (PEG) is an attractive tool for the development of nanoparticle‐based cancer therapy since it endows nanoparticles with extended‐circulation properties. Nevertheless, recent reports have revealed that intravenous injection of either PEGylated liposomes (SLs) or PEGylated lipoplex (PLpx) could elicit an anti‐PEG immunoglobulin (IgM) response in a T cell‐independent (TI) manner that would substantially compromise the in vivo fate of PEGylated products upon repeated administration. In the same context, viral or bacterial infections trigger the production of polyreactive IgM that binds both self and foreign antigens. The polyreactivity of IgM elicited by SLs or PLpx, to bacteria and other polymers, however, is yet to be elucidated. In this study, the polyreactivity of IgM elicited by SLs or PLpx was challenged against different bacteria (TI antigens) and against synthetic polymer composed of repetitive structures (PVP‐360 or FITC‐dextran). Results demonstrated that anti‐PEG IgM elicited by either SLs or PLpx showed no reactivity to various bacteria examined, while the IgM showed remarkable reactivity to both PVP‐360 and FITC‐dextran. In addition, interestingly, anti‐PEG IgM elicited by either SLs or PLpx showed no antinuclear antibody‐like immune reactivity, and, therefore, treatment with either SLs or PLpx was not expected to exacerbate autoimmune diseases such as systemic lupus erythematosus. Collectively, our findings could provide information supporting the safety of PEGylated nanoparticle‐based pharmaceutics, particularly in patients with autoimmune diseases. Graphical abstract Figure. No Caption available.