Biocompatible Polymers Modified with d-Octaarginine as an Absorption Enhancer for Nasal Peptide Delivery.

Peptide and protein drugs, which are categorized as biologics, exhibit poor membrane permeability. This pharmacokinetic disadvantage has largely restricted the development of noninvasive dosage forms of biologics that deliver into systemic circulation. We have been investigating the potential use of cell-penetrating peptide-linked polymers as a novel absorption enhancer to overcome this challenge. Since our previous study revealed that biocompatible poly( N-vinylacetamide- co-acrylic acid) modified with d-octaarginine, a typical cell-penetrating peptide, enhanced in vitro permeation of biomolecules such as plasmid DNA and bovine serum albumin through cell membranes, the present study evaluated whether the polymers enhanced in vivo absorption of biologics applied on the mucosa. Mouse experiments demonstrated that d-octaarginine-linked polymers drastically enhanced nasal absorption of exendin-4, whose injection is clinically used. The mean bioavailability was 20% relative to subcutaneous administration, even though it fell short of 1% when exendin-4 alone was administered nasally. The absorption-enhancing function of the polymers was superior to that of sodium caprate and sodium N-(8-(2-hydroxybenzoyl)amino) caprylate, which have been used for humans as an absorption enhancer. In vitro experiments using several biologics with different characteristics revealed that biologics interacted with d-octaarginine-linked polymers and were taken up into cells when incubated with the polymers. The interaction and cellular uptake were enhanced as molecular weights of the biologics increased; however, their charge-dependent in vitro performance was not clearly observed. The current data suggested that biologics formulated with our polymers became an alternative to their conventional invasive parenteral formulations.

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.

Effects of the Chemical Structures of Oligoarginines Conjugated to Biocompatible Polymers as a Mucosal Adjuvant on Antibody Induction in Nasal Cavities.

We have been investigating the potential of oligoarginine-linked polymers as an adjuvant for mucosal vaccination that induces immunoglobulin G (IgG) in systemic circulation and immunoglobulin A (IgA) secreted on the mucosa. Our latest infection experiments demonstrated that mice immunized nasally with a mixture of inactivated influenza viruses and poly(N-vinylacetamide-co-acrylic acid) (PNVA-co-AA) modified with D-octaarginine were perfectly protected from homologous virus infection. On the contrary, virus infection was observed in mice immunized with the antigen alone. This difference was presumably due to insignificant induction of secreted IgA on the nasal mucosa in the latter mice. Since it was unclear whether the current induction level was sufficient for heterologous virus infection, we evaluated the effects of the chemical structures of oligoarginines conjugated to PNVA-co-AA on induction of intranasal IgA. The number and optical activity of the arginine residues and the degree of modification with oligoarginines in the polymer backbone were listed as a factor that would influence IgA induction. Mouse experiments revealed that maximization of the modification resulted in an increase in adjuvant activities of oligoarginine-linked polymers most effectively. Glycine segments inserted between oligoarginines and the polymer backbone were a prerequisite for the maximization. The highest IgA level was observed when antigens were coadministered with diglycine-D-octaarginine-linked PNVA-co-AA.

Modulation of antitumor immunity contributes to the enhanced therapeutic efficacy of liposomal oxaliplatin in mouse model.

Immune modulation of the tumor microenvironment has been reported to participate in the therapeutic efficacy of many chemotherapeutic agents. Recently, we reported that liposomal encapsulation of oxaliplatin (l-OHP) within PEGylated liposomes conferred a superior antitumor efficacy to free l-OHP in murine colorectal carcinoma-bearing mice through permitting preferential accumulation of the encapsulated drug within tumor tissue. However, the contribution of the immune-modulatory properties of liposomal l-OHP and/or free l-OHP to the overall antitumor efficacy was not elucidated. In the present study, therefore, we investigated the effect of liposomal encapsulation of l-OHP within PEGylated liposomes on the antitumor immunity in both immunocompetent and immunodeficient mice. Liposomal l-OHP significantly suppressed the growth of tumors implanted in immunocompetent mice, but not in immunodeficient mice. In immunocompetent mice, liposomal l-OHP increased the tumor MHC-1 level and preserved antitumor immunity through decreasing the number of immune suppressor cells, including regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages, which collectively suppress CD8+ T cell-mediated tumor cells killing. In contrast, free l-OHP ruined antitumor immunity. These results suggest that the antitumor efficacy of liposomal l-OHP is attributed, on the one hand, to its immunomodulatory effect on tumor immune microenvironment that is superior to that of free l-OHP, and on the other hand, to its direct cytotoxic effect on tumor cells.

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.

Evaluation of a D-Octaarginine-linked polymer as a transfection tool for transient and stable transgene expression in human and murine cell lines.

Poly(N-vinylacetamide-co-acrylic acid) coupled with d-octaarginine (VP-R8) promotes the cellular uptake of peptides/proteins in vitro; however, details of the transfection efficacy of VP-R8, such as the cell types possessing high gene transfer, are not known. Herein, we compared the ability of VP-R8 to induce the cellular uptake of plasmid DNA in mouse and human cell lines from different tissues and organs. A green fluorescent protein (GFP)-expression plasmid was used as model genetic material, and fluorescence as an indicator of uptake and plasmid-derived protein expression. Three mouse and three human cell lines were incubated with a mixture of plasmid and VP-R8, and fluorescence analysis were performed two days after transfection. To confirm stable transgene expression, we performed drug selection three days after transfection. A commercially available polymer-based DNA transfection reagent (PTR) was used as the transfection control and standard for comparing transgene expression efficiency. In the case of transient transgene expression, slight-to-moderate GFP expression was observed in all cell lines transfected with plasmid via VP-R8; however, transfection efficiency was lower than using the PTR for gene delivery. In the case of stable transgene expression, VP-R8 promoted drug-resistance acquisition more efficiently than the PTR did. Cells that developed drug resistance after VP-R8-mediated gene transfection expressed GFP more efficiently than cells that developed drug resistance after transfection with the PTR. Thus, VP-R8 shows potential as an in vitro or ex vivo nonviral transfection tool for generating cell lines with stable transgene expression.

Mechanism on antigen delivery under mucosal vaccination using cell-penetrating peptides immobilized at multiple points on polymeric platforms.

We have developed an aggregate of D-octaarginine immobilized at multiple points on a co-polymer of N-vinylacetamide and acrylic acid. Previous studies revealed that immunoglobulin G and A were induced when mice were inoculated with influenza virus antigens under coadministration with the D-octaarginine-immobilized polymers as a mucosal vaccine adjuvant. Infection experiments demonstrated that mice vaccinated with a mixture of inactivated influenza viruses and the polymers were protected from infection with mouse-adapted infectious viruses. In the present study, we investigated the mechanism on antigen delivery under mucosal vaccination using the polymers. Two-hour retention of fluorescein-labeled ovalbumin (F-OVA) on the nasal mucosa was observed when applied with the polymers; nevertheless F-OVA was eliminated less than 10 min under polymer-free conditions. F-OVA mixed with the polymers was vigorously taken up into murine dendritic cells. Electrophoresis and dynamic light scattering analysis indicated that OVA interacted with the polymers. The uptake of F-OVA was hardly ever inhibited by the addition of an excess amount of intact OVA. The results suggested that viral antigens were accumulated on the mucosa and delivered into dendritic cells under basolateral membranes via dendrites extending to the mucosal surface and/or subsequent to their permeation through epithelial cells, when they were coadministered with D-octaarginine-immobilized polymers.

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.

Quantitative determination of polysulfide in albumins, plasma proteins and biological fluid samples using a novel combined assays approach.

Hydrogen sulfide (H2S) signaling involves polysulfide (RSSnSR') formation on various proteins. However, the current lack of sensitive polysulfide detection assays poses methodological challenges for understanding sulfane sulfur homeostasis and signaling. We developed a novel combined assay by modifying Sulfide Antioxidant Buffer (SAOB) to produce an "Elimination Method of Sulfide from Polysulfide" (EMSP) treatment solution that liberates sulfide, followed with methylene blue (MB) sulfide detection assay. The combined EMSP-MB sulfide detection assay performed on low molecular weight sulfur species showed that sulfide was produced from trisulfide compounds such as glutathione trisulfide and diallyl trisulfide, but not from the thiol compounds such as cysteine, cystine and glutathione. In the case of plasma proteins, this novel combined detection assay revealed that approximately 14.7, 1.7, 3.9, 3.7 sulfide mol/mol released from human serum albumin, α1-anti-trypsin, α1-acid glycoprotein and ovalbumin, respectively, suggesting that serum albumin is a major pool of polysulfide in human blood circulation. Taken together with the results of albumins of different species, the liberated sulfide has a good correlation with cysteine instead of methionine, indicating the site of incorporation of polysulfide is cysteine. With this novel sulfide detention assay, approximately 8,000, 120 and 1100 µM of polysulfide concentrations was quantitated in human healthy plasma, saliva and tear, respectively. Our promising polysulfide specific detection assay can be a very important tool because quantitative determination of polysulfide sheds light on the functional consequence of protein-bound cysteine polysulfide and expands the research area of reactive oxygen to reactive polysulfide species.

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.

Advanced therapeutic approach for the treatment of malignant pleural mesothelioma via the intrapleural administration of liposomal pemetrexed.

Malignant pleural mesothelioma (MPM) is an aggressive cancer that proliferates in the pleural cavity. Pemetrexed (PMX) in combination with cisplatin is currently the approved standard care for MPM, but a dismal response rate persists. Recently, we prepared various liposomal PMX formulations using different lipid compositions and evaluated their in vitro cytotoxicity against human mesothelioma cells (MSTO-211H). In the present study, we investigated the in vivo therapeutic effect of our liposomal PMX formulations using an orthotopic MPM tumor mouse model. PMX encapsulated within either cholesterol-containing (PMX/Chol CL) or cholesterol-free (PMX/Non-Chol CL) cationic liposome was intrapleurally injected into tumor-bearing mice. PMX encapsulated in cholesterol-free liposomes (PMX/Non-Chol CL) drastically inhibited the tumor growth in the pleural cavity, while free PMX and PMX encapsulated in cholesterol-containing liposomes (PMX/Chol CL) barely inhibited the tumor growth. The enhanced in vivo anti-tumor efficacy of PMX/Non-Chol CL was credited, on the one hand, for prolonging the retention of cationic liposomes in the pleural cavity via their electrostatic interaction with the negatively charged membranes of tumor cells, but on the other hand, it was charged with contributing to a higher drug release from the "fluid" liposomal membrane following intrapleural administration. This therapeutic strategy of direct intrapleural administration of liposomal PMX, along with the great advances in CL-guided therapeutics, might be a promising therapeutic approach to conquering the poor prognosis for MPM.

2-Methacryloyloxyethyl phosphorylcholine polymer (MPC)-coating improves the transfection activity of GALA-modified lipid nanoparticles by assisting the cellular uptake and intracellular dissociation of plasmid DNA in primary hepatocytes.

We previously reported that modification of GALA peptide on the surface of liposomes enhanced fusion with endosomal membrane, and cytoplasmic release of encapsulated macromolecules. We report herein that an additional coating of GALA-modified liposomes with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer resulted in a two order of magnitude enhancement in the transfection activity of encapsulating plasmid DNA (pDNA). Quantification of the delivered gene copies in whole cells and isolated nuclei revealed that the increase of transfection activity can be attributed to improved efficiencies in cellular uptake and post-nuclear delivery processes. Imaging studies revealed that the intracellular dissociation of pDNA from the lipid envelope is enhanced by GALA modification and further coating with MPC polymer in a stepwise manner. The MPC polymer-coating decreased the zeta-potential of GALA-modified liposomes, suggesting that it assisted in the functional display of negatively charged GALA on the cationic liposomes by providing shielding from mutual electrostatic interactions. Collectively, these data indicate that MPC polymer-coating induced the fusogenic activity of the GALA-modified envelope with endosomes, leading to a more effective cytoplasmic release pDNA. The extensive fusion of the lipid envelope may also reduce electrostatic interactions between mRNA and cationic lipid components, thereby resulting in an enhancement in the translation process.

Envelope-type lipid nanoparticles incorporating a short PEG-lipid conjugate for improved control of intracellular trafficking and transgene transcription.

Lipid envelope-type nanoparticles are promising carriers for gene delivery. The modification of liposomes with polyethyleneglycol (PEG) can often be useful in liposomal formation and pharmacokinetics. However, there is a dilemma concerning the use of PEG because of its poor intracellular trafficking properties. To overcome this problem, in the present study, we report on a strategy for improving the intracellular trafficking of PEG-modified lipid particles by incorporating a short PEG lipid. The findings presented here show that the incorporation of tetra(ethylene)glycol (TEG)-conjugated cholesterol into a liposome composition is useful in controlling the number of lipid envelopes, resulting in an improvement in particle uniformity with a reduced particle size. The TEG-modified lipid particles were found to enhance transfection activity by more than 100-fold. This increase is attributed to an enhancement of cellular uptake, and nuclear transcription by improving intracellular decoating. Moreover, the use of a various short PEG lipids in lipid particle formation showed a clear threshold polymerization degree (less or equal 25: PEG1100), for achieving stimulated transfection activity. Collectively, the use of short PEG lipid promises to be useful in developing an efficient non-viral gene vector.