Article

PeptoSomes for Vaccination: Combining Antigen and Adjuvant in Polypept(o)ide-Based Polymersomes

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Abstract

In this work, the first vaccine is reported based on a PeptoSome, which contains a model antigen (SIINFEKL) and adjuvant (CpG). PeptoSomes are polypept(o)ide-based polymersomes built of a block-copolymer with polysarcosine (PSar) as the hydrophilic block (X n = 111) and poly(benzyl-glutamic acid) (PGlu(OBn)) as the hydrophobic one (X n = 46). The polypept(o)ide is obtained with low dispersity index of 1.32 by controlled ring-opening polymerization. Vesicle formation by dual centrifugation technique allows for loading of vesicles up to 40 mol%. PeptoSomes are characterized by multiangle dynamic light scattering, static light scattering, and cryogenic transmission electron microscopy (cryoTEM). The PeptoSomes have a hydrodynamic radius of 39.2 nm with a low dispersity (µ 2 = 0.1). The ρ-ratio R g/R h of 0.95 already indicates that vesicles are formed, which can be confirmed by cryoTEM. Loaded PeptoSomes deliver the antigen (SIINFEKL) and an adjuvant (CpG) simultaneously into dendritic cells (DCs). Upon cellular uptake, dendritic cells are stimulated and activated, which leads to expression of cluster of differentiation CD80, CD86, and MHCII, but induces excretion of proinflammatory cytokines (e.g., TNFα). Furthermore, DC-mediated antigen-specific T-cell proliferation is achieved, thus underlining the enormous potential of PeptoSomes as a versatile platform for vaccination.

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... 26,100,101 Many studies have reported the synergic effects of polymer-based vaccines delivering both nucleic acid adjuvants and antigens targeting APCs in a single delivery system allowing optimal protective immune response against specific pathogen-like components: for example, recently, Weber et al. employed loaded polypept(o)ide-based polymersomes for simultaneous delivery of CpG motif-containing adjuvant and ovalbumin as antigen model. 102 Upon particle engulfment, they achieved bone marrow derived dendritic cell (BMDC) stimulation and activation with increased expression of CD80, CD86, and MHC class II surface markers and enhanced inflammatory cytokine levels (TNF-a). The co-encapsulation of CpG and antigen greatly promoted antigen-specific T cell proliferation in contrast to free antigen treatment. ...
... In this setting, the Barz group produced the first so-called peptosome synthetic vaccine through combination of the SIINFEKL model peptide antigen and TLR9 danger signal CpG 1826 in a peptide-based polymersome. 102 The B40 nm large peptosomes consisted of a block-copolymer with polysarcosine (PSar) as the hydrophilic block (X n = 111) and poly(benzylglutamic acid) (PGlu(OBn)) as the hydrophobic one (X n = 46). The loaded peptosomes delivered the antigen and danger signal simultaneously into bone marrow derived dendritic cells. ...
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Light scattering is a very powerful method to characterize the structure of polymers and nanoparticles in solution. Recent technical developments have strongly enhanced the possible applications of this technique, overcoming previous limitations like sample turbidity or insufficient experimental time scales. However, despite their importance, these new developments have not yet been presented in a comprehensive form. In addition, and maybe even more important to the broad audience, there lacks a simple-to-read textbook for students and non-experts interested in the basic principles and fundamental techniques of light scattering. As part of the Springer Laboratory series, this book tries not only to provide such a simple-to-read and illustrative textbook about the seemingly very complicated topic of light scattering from polymers and nanoparticles in dilute solution, but also intends to cover some of the newest technical developments in experimental light scattering.
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Poly(2,3-dihydroxypropyl methacrylamide) (P(DHPMA))-based amphiphilic block copolymers have recently proven to form polymer vesicles (polymersomes). In this work, we further expand their potential by incorporating (i) units for pH-dependent disintegration into the hydrophobic membrane and (ii) mannose as targeting unit into the hydrophilic block. This last step relies on the use of an active ester prepolymer. We confirm the stability of the polymersomes against detergents like Triton X-100 and their low cytotoxicity. The incorporation of 2-(2,2-dimethyl-1,3-dioxolane-4-yl)ethyl methacrylate into the hydrophobic block (lauryl methacrylate) allows a pH-responsive disintegration for cargo release. Efficient decomposition of the polymersome structure is monitored by dynamic light scattering. It is thus possible to include an active enzyme (glucose oxidase), which gets only active (is set free) after vesicle disintegration. In addition, the introduction of mannose as targeting structure allows enhanced and selective targeting of dendritic cells.
Article
Surface PEGylation on nanoparticles has greatly helped prolong their blood circulation half-lives. However, The injection of PEGylated nanoparticles into mice induced poly(ethylene glycol) (PEG)-specific IgM antibodies (anti-PEG IgMs), significantly changing PEG-liposomes' pharmacokinetics. In this study, we used various PEG-conjugates to conduct a mechanistic study of anti-PEG IgMs' binding behavior. The conventional belief has been that anti-PEG IgMs bind to PEG main chains; however, our findings reveal that anti-PEG IgMs did not bind to PEG main chains, whereas anti-PEG IgMs did bind to PEG-hydrophobic polymer blocks. The insertion of a hydrophilic polymer between each PEG chain and each hydrophobic polymer block suppressed anti-PEG IgMs' binding. We prove here that hydrophobic blocks are essential to anti-PEG IgMs' binding, and also that anti-PEG IgMs do not bind to intact PEGs without hydrophobic moiety. These results support our conclusion that anti-PEG IgMs exhibit specificity to PEG; however, the presence of a hydrophobic block at a proximity position from each PEG chain is essential for the binding. Also in the present study, we elucidate relations between anti-PEG IgMs and PEGylated nanoparticles. In one of our previous studies, anti-PEG IgMs scarcely affected the pharmacokinetics of PEG-b-poly(β-benzyl l-aspartate) block copolymer (PEG-PBLA) micelles, whereas anti-PEG IgMs significantly decreased PEG-liposomes' blood circulation half-life. Finally, we found that the ratio of anti-PEG IgM molecules to PEG-liposome particles is critical to these pharmacokinetic changes, and that a 10-fold increase in the number of anti-PEG IgM molecules permitted them to capture the PEG-liposome particles, thus leading to the aforementioned changes.
Article
Biological membranes play an essential role in living organisms by providing stable and functional compartments, preserving cell architecture, whilst supporting signalling and selective transport that are mediated by a variety of proteins embedded in the membrane. However, mimicking cell membranes – to be applied in artificial systems – is very challenging because of the vast complexity of biological structures. In this respect a highly promising strategy to designing multifunctional hybrid materials/systems is to combine biological molecules with polymer membranes or to design membranes with intrinsic stimuli-responsive properties. Here we present supramolecular polymer assemblies resulting from self-assembly of mostly amphiphilic copolymers either as 3D compartments (polymersomes, PICsomes, peptosomes), or as planar membranes (free-standing films, solid-supported membranes, membrane-mimetic brushes). In a bioinspired strategy, such synthetic assemblies decorated with biomolecules by insertion/encapsulation/attachment, serve for development of multifunctional systems. In addition, when the assemblies are stimuli-responsive, their architecture and properties change in the presence of stimuli, and release a cargo or allow “on demand” a specific in situ reaction. Relevant examples are included for an overview of bioinspired polymer compartments with nanometre sizes and membranes as candidates in applications ranging from drug delivery systems, up to artificial organelles, or active surfaces. Both the advantages of using polymer supramolecular assemblies and their present limitations are included to serve as a basis for future improvements.
Article
We show the formation of vesicles from amphiphilic block-oligopeptoids, named here peptoidosomes. The peptoids, synthetic analogues to peptides, were synthesised from solid phase synthesis. Their hydrophilic to hydrophobic aspect ratio was designed to favour the formation of vesicles. MALDI-TOF analysis confirmed their formation. Using a range of hydrophilic parts, we analysed their influence on the quality of the actual peptoids generated but also how different hydrophilic part influence vesicle formation. We found peptoids to be quite resistant to swelling but were able to form peptoidosomes for all peptoids investigated.
Article
In this work, activated ester chemistry is employed to synthesize biocompatible and readily functionalizable polymersomes. Via aminolysis of pentafluorophenyl methacrylate-based precursor polymers, an N-(2-hydroxypropyl) methacrylamide (HPMA)-analog hydrophilic block is obtained. The precursor polymers can be versatile functionalized by simple addition of suitable primary amines during aminolysis as demonstrated using a fluorescent dye. Vesicle formation is proven by cryoTEM and light scattering. High encapsulation efficiencies for hydrophilic cargo like siRNA are achieved using dual centrifugation and safe encapsulation is demonstrated by gel electrophoresis. In vitro studies reveal low cytotoxicity and no protein adsorption-induced aggregation in human blood serum occurs, making the vesicles interesting candidates as nanosized drug carriers.
Article
Polypept(o)ides combine the multifunctionality and intrinsic stimuli-responsiveness of synthetic polypeptides with the "stealth"-like properties of the polypeptoid polysarcosine (poly(N-methyl glycine)). This class of block copolymers can be synthesized by sequential ring opening polymerization of α-amino acid N-carboxy-anhydrides (NCAs) and correspondingly of the N-substituted glycine N-carboxyanhydride (NNCA). The resulting block copolymers are characterized by Poisson-like molecular weight distributions, full end group integrity, and dispersities below 1.2. While polysarcosine may be able to tackle the currently arising issues regarding the gold standard PEG, including storage diseases in vivo and immune responses, the polypeptidic block provides the functionalities for a specific task. Additionally, polypeptides are able to form secondary structure motives, e.g., α-helix or β-sheets, which can be used to direct self-assembly in solution. In this feature article, we review the relatively new field of polypept(o)ides with respect to synthesis, characterization, and first data on the application of block copolypept(o)ides in nanomedicine. The summarized data already indicates the great potential of polypept(o)ides.
Article
Nowadays, amphiphilic molecules play an important role in our life. In medical applications, amphiphilic block copolymers have attracted much attention as excipients in drug delivery systems. Here, the polymers are used as emulsifiers, micelles, or polymersomes with a hydrophilic corona block and a hydrophobic core or membrane. The aggregation behavior in aqueous solutions of a series of different amphiphilic block copolypeptoids comprising polysarcosine as a hydrophilic part is here reported. The formation of aggregates is investigated with H-1 NMR spectroscopy and dynamic light scattering, and the determination of the critical micelle concentration (cmc) is performed using pyrene fluorescence spectroscopy. For the different block copolypeptoids cmc values ranging from 0.6 x 10(-6) NI to 0.1 x 10(-3) M are found. The tendency to form micelles increases with increasing hydrophobicity at the nitrogen side chain in the hydrophobic moiety. Furthermore, in the case of the same hydrophobic side chain, a decreasing hydrophilic/lipophilic balance leads to the formation of larger aggregates. The aggregates formed in the buffer are able to solubilize the hydrophobic model compounds Reichardt's dye and pyrene, and exhibit versatile microenvironments. Final investigations about the cytotoxicity reveal that the block copolypeptoids are well tolerated by mammalian cells up to high concentrations.
Article
Core-shell structures based on polypept(o)ides combine stealth-like properties of the corona material polysarcosine with adjustable functionalities of the polypeptidic core. Mannose-bearing block copolypept(o)ides (PSar-block-PGlu(OBn)) have been synthesized using 11-amino-3,6,9-trioxa-undecyl-2,3,4,6-tetra-O-acetyl-O-α-D-mannopyranoside as initiator in the sequential ring-opening polymerization of α-amino acid N-carboxyanhydrides. These amphiphilic block copolypept(o)ides self-assemble into multivalent PeptoMicelles and bind to mannose-binding receptors as expressed by dendritic cells. Mannosylated micelles showed enhanced cell uptake in DC 2.4 cells and in bone marrow-derived dendritic cells (BMDCs) and therefore appear to be a suitable platform for immune modulation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Article
Polymersomes are nanoscopic (e.g. nanometer-sized) vesicles formed by amphiphilic block copolymers. They represent the more robust and versatile macromolecular counterparts to the well-established lipid vesicles or liposomes. Recently, considerable efforts have been made to produce them in a uniform and functional manner. New techniques such as artificial endocytosis and electroporation have also been developed to achieve payload encapsulation. In this mini-review, we discuss these and other recent developments in making polymersomes an actual alternative for biomedical applications.
Article
We report the synthesis of polysarcosine-block-polyglutamic acid benzylester (PSar-block-PGlu(OBn)) and polysarcosine-block-polylysine-ε-N-benzyloxycarbonyl (PSar-block-PLys(Z)) copolymers. The novel polypeptoid-block-polypeptide copolymers have been synthesized by ring opening polymerization (ROP) of N-carboxyanhydrides (NCAs). Polymerization conditions were optimized regarding protecting groups, block sequence and length. While the degree of polymerization of the PSar block length was set to be around 200 or 400, PGlu(OBn) and PLys(Z) block lengths were varied between 20 to 75. The obtained block copolypeptides had a total degree of polymerization of 220-475 and dispersity indices between 1.1 and 1.2. Having ensured a non-toxic behavior up to a concentration of 3 mg/mL in HEK293 cells the novel block copolymers have been applied to the synthesis of organic colloids (by miniemulsion polymerization and miniemulsion solvent evaporation process). Colloids of around 100 nm (miniemulsion polymerization) to 200 nm (miniemulsion process) have been prepared. Additionally, PSar-block-PGlu(OBn) copolymers have been used in a drug formulation of an adenylate cyclase inhibitor increasing solubility of the drug, which enhances its bioavailability reducing of intracellular cAMP levels.
Article
Using poly(propylene sulfide) (PPS) and poly(ethylene glycol) (PEG) as components of a nanocarrier platform, we sought to compare immune responses induced by PPS-bl-PEG polymersomes (PSs; watery-core structures, with antigen incorporated within the PSs) and PEG-stabilized PPS nanoparticles (NPs; solid-core structures, with antigen conjugated upon the NP surface). We have previously shown strong CD8+ T cell responses to antigen conjugated to NPs via a disulfide link, and here we investigated the extent to which antigen incorporated within oxidatively-sensitive PSs could induce CD4+ or CD8+ T cell responses. C57BL/6 mice were subcutaneously immunized with free ovalbumin (OVA) as a model antigen, or equivalent doses of OVA-loaded into PSs, conjugated onto NPs, or given as a mixture of the two. Free CpG was used as an adjuvant. Antigen-loaded PSs induced enhanced frequencies of antigen-specific CD4+ T cells in the spleen, lymph nodes and lungs as compared to the NP formulation, whereas antigen-conjugated NPs induced stronger CD8+ T cell responses. Co-administration of both PSs and NPs elicited T cell immunity characteristic of the two nanocarriers at the same time, i.e. both strong CD4+ and CD8+ T cell responses. These results have important implications for particulate-based vaccine design and highlight the potential of using different antigen-delivery systems for the induction of both T helper and cytotoxic T lymphocyte immune responses.
Article
Hydrophobic helical peptides having alternating hydrophobic amino acid and Aib in the sequence were synthesized to construct supramolecular systems. Three types of supramolecular systems were constructed by the peptides and the derivatives in different environments. First, the dispersion of TFA · H-(Ala-Aib)8-OBzl in water was studied by dynamic light scattering, which suggests the formation of a vesicular structure with an average diameter of 76 nm. We call the peptide assembly in water “peptosome”. Second, Boc-Ser(Ant)-(Ala-Aib)8-OMe spanned the phospholipid bilayer membrane and formed a helix-bundle structure. The bundle structure was supported by ion-channel formation in the membrane. Third, Boc-(Ala-Aib)8-OMe and Boc-(Leu-Aib)8-OBzl formed a two-dimensional crystal at the air-water interface. Boc-(Ala-Aib)12-OBzl also formed a monolayer in a solid state at the air-water interface, but the helix orientation was perpendicular to the interface, which presents a contrast to the parallel orientation of the former hexadecapeptides.
Article
The accelerated blood clearance phenomenon involving anti-PEG IgM production has been recognized as an important issue for the design and development of PEGylated liposomes. Here, we show that empty PEGylated liposomes and Doxil, PEGylated liposomes containing doxorubicin, both caused anti-PEG IgM production and thereby a rapid clearance of the second and/or third dose of Doxil in Beagle dogs in a lipid-dose, inverse-dependent manner. It appears that the pharmacokinetic profile of the second and third administration of Doxil reflected the presence of anti-PEG IgM circulating in the blood. Doxil plus an excess amount of empty PEGylated liposomes rather enhanced the production of anti-PEG IgM compared to Doxil of the same doxorubicin dose. During sequential administration, increasing the lipid dose of Doxil in each dose by the addition of empty PEGylated liposomes strongly attenuated the magnitude of the ABC phenomenon during the effectuation phase of a second and third dose of Doxil. Our results suggest that the pre-clinical study of anti-cancer drug-containing PEGylated liposomes with dogs must be carefully designed and performed with monitoring of the anti-PEG IgM and liposomal drugs circulating in the blood.
Article
Rising from the shortcomings of modern day therapeutics there is a need for a controlled approach in carrier-mediated drug delivery. Polymeric vesicles, also called polymersomes, are powerful tools to address issues of efficacy, specificity, and controlled release of drugs to diseased tissues. These recent, biomimetic structures are able to overcome the body's natural defences, remaining stable for extended time in circulation, have tuneable membrane properties, allowing the control of membrane permeability and therefore of drug release, and have the potential to be functionalized for active targeting of specific tissues, reducing undesirable side effects. Extensive work has been carried out in order to attain multifunctional polymeric vesicles that respond to precise triggers (e.g., temperature, pH, redox, magnetic field, etc) with a spatial and temporal monitoring what may enable unprecedented control of drug release in the body. These versatile structures can be loaded with different type of (bio)molecules and nanoparticles, from drugs to contrast agents for medical imaging, and are able to accommodate them in different subcompartments of the vesicle (i.e., hydrophobic membrane and hydrophilic core). Multimodal targeted delivery system could be obtained from this unique platform, with abilities in both drug delivery and medical imaging contrast enhancement, widening the perspectives toward theranostics. Polymersomes offer a promising route toward more effective treatments with fewer side effects and superior outcomes. WIREs Nanomed Nanobiotechnol 2012, 4:525–546. doi: 10.1002/wnan.1183 For further resources related to this article, please visit the WIREs website.
Article
While current subunit vaccines successfully induce humoral immune responses, a need exists for vaccine strategies to elicit strong cell-mediated immunity to address diseases such as cancer and chronic viral infection. Polymersomes are stable vesicles composed of self-assembling block copolymers with tunable degradation properties allowing delivery of both hydrophilic (within vesicle interior) or hydrophobic (within vesicle membrane) payload molecules. Here we apply oxidation-sensitive nanoscale polymersomes for both antigen and adjuvant delivery to dendritic cell (DC) endosomes. Calcein-loaded polymersomes were observed to release their payload initially in multiple DC endosomal compartments and subsequently within the cytosol. With either the Toll-like receptor agonists gardiquimod or R848 as payloads within the polymersomes, release resulted in DC activation, as indicated by induction of inflammatory cytokine expression and upregulation of DC maturation surface markers: for example, the ability of gardiquimod to induce IL-6 and IL-12 cytokine expression by DCs was enhanced 10-fold when loaded within polymersomes. With the model antigen ovalbumin as a payload, release resulted in CD8(+) T cell cross-priming by promoting protein antigen cross-presentation through MHC I, as indicated by activation of OT-I CD8(+) T cells. Our results demonstrate that oxidation-sensitive polymersomes can function as a vaccine delivery platform for inducing cell-mediated antigen-specific immune responses.
Article
Polypeptide block copolymers with different block length ratios were obtained by sequential ring-opening polymerization of benzyl-L-glutamate and propargylglycine (PG) N-carboxyanhydrides. Glycosylation of the poly(PG) block was obtained by Huisgens cycloaddition "click" reaction using azide-functionalized galactose. All copolymers were self-assembled using the nanoprecipitation method to obtain spherical and wormlike micelles as well as polymersomes depending on the block length ratio and the nanoprecipitation conditions. These structures display bioactive galactose units in the polymersome shell, as proven by selective lectin binding experiments.
Article
Unlabelled: The in vivo efficacy of doxorubicin (DOX)-loaded poly(γ-benzyl l-glutamate)-block-hyaluronan (PBLG(23)-b-HYA(10))-based polymersomes (PolyDOX) was evaluated. Samples were efficiently labeled with technetium-99m radionuclide with good stability for in vivo studies. PolyDOX enhanced circulation time compared to free DOX. Biodistribution studies revealed selective accumulation of PolyDOX in the Ehrlich ascites tumor (EAT) as a result of passive accumulation and active targeting (CD44-mediated endocytosis) in EAT-bearing mice. Toxicity studies demonstrated PolyDOX is a safe drug carrier, and no hemolysis was observed with PolyDOX equivalent to 200 μg/mL of free DOX. PolyDOX dominantly controlled tumor growth by delaying doubling time of EATs compared to free DOX over 30 days after treatment. PolyDOX also increased life span six times more than free DOX. Hence, it is reasonable to expect that higher DOX levels attributable to PolyDOX improve the therapeutic index and reduce side effects due to site-specific drug accumulation. From the clinical editor: In this preclinical project, doxorubicin loaded polymersomes enhanced intracellular uptake of doxorubicin in a murine model of Ehrlich Ascites Tumor (EAT) through CD44 receptor mediated endocytosis, resulting in prolonged Tumor Doubling Time and increase in life span of mice.
Article
Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs act as immune adjuvants, accelerating and boosting antigen-specific immune responses. CpG motifs promote the induction of Th1 and pro-inflammatory cytokines and support the maturation/activation of professional antigen presenting cells (particularly plasmacytoid dendritic cells). These effects are optimized by maintaining close physical contact between the CpG ODN and the immunogen. Co-administering CpG ODN with a variety of vaccines has improved the resultant humoral and/or cellular immune responses, culminating in enhanced protective immunity in rodent and primate challenge models. Ongoing clinical studies indicate that CpG ODN are safe and well-tolerated when administered as adjuvants to humans, and that they can support increased vaccine-specific immune responses.
Article
We investigated supramolecular assemblies of various hydrophobic helical peptides. The assemblies were formed at the air/water interface or in aqueous medium. The hexadecapeptide, Boc-(Ala-Aib)s-OMe (BA16M), was reported to take alpha-helical structure by X-ray analysis. Several derivatives were prepared, which have the repeating sequence of Ala-Aib, Lys(Z)-Aib or Leu-Aib, or have the terminal chemically modified. CD spectra of the peptides indicated helical conformation in ethanol solution. The surface pressure-area isotherms of the peptide monolayers showed an inflection at the surface area corresponding to the cross section along the helix axis, and the monolayers were collapsed by further compression. All the helical peptides oriented their helix axis parallel to the air/water interface on the basis of the results of transmission IR spectra and RAS of the monolayers transferred onto substrates. A small mound was observed in the isotherm of BA16M and other derivatives, which was ascribed to the phase transition from the liquid state to the solid state. One mol% of FITC-labeled peptide was mixed into the monolayers to visualize the phase separation of the solid and liquid states at the surface pressure of the coexisting region. Various shapes of the dark domain were observed at the top of the mound in the isotherms by fluorescence microscopy. The helical peptides formed two-dimensional crystals at the air/water interface when they were compressed to the solid state. An amino-terminated helical peptide, HA16B, was suspended in an aqueous medium by a sonication method and transparent dispersion was obtained. The dynamic light scattering measurement of the dispersion revealed the particle size of 75 nm with a narrow size distribution. The molecular assembly of the helical peptide in water was called "Peptosome", because it takes a vesicular structure.
Article
PEG-modified lipid vesicles have already shown considerable utility in delaying vesicle clearance from the circulation. They are, however, limited in their ability to stably integrate high molar ratios of PEG-lipid due to the high curvature and micellar preference of the very large hydrophilic PEG chain. Polymersomes, by contrast, are vesicles composed entirely of PEG-based block copolymer amphiphiles that are not only more proportionately designed, but also have already been shown to considerably broaden the range of vesicle properties (e.g. stability). Here, polymersomes composed of varying length copolymer chains were injected into rats and found to have in vivo circulation times, tau(1/2), up to about two-fold longer than PEGylated, or Stealth, liposomes. The dependence of tau(1/2) on PEG molecular weight is nonetheless limited by uptake into the liver and spleen-as with liposomes. In vitro incubations of polymersomes in plasma indicate gradual opsonization through plasma protein adsorption, such that, when vesicles are held in an optical trap and presented to a phagocyte, rapid engulfment occurs only after incubation times of similar magnitude to tau(1/2). The stealthiness introduced to liposomes through PEGylation is thus extended here with completely synthetic polymersomes.
Article
We recently reported that a second dose of polyethylene glycol (PEG) (M.W. 2000)-modified liposomes (mPEG2000-liposomes) is rapidly cleared from the blood and accumulates in the liver when injected twice in the same rat or mouse at several-day intervals (referred to as the "accelerated blood clearance (ABC) phenomenon"). In the present study we observed that a high dose (5 micromol/kg) of conventional liposomes (CL: without a PEG-coating) can induce the same phenomenon, while a low lipid dose (0.001 micromol/kg) did not. The induction of the phenomenon by mPEG2000-liposomes decreased with increasing first dose (0.001-5 micromol/kg). We observed a strong inverse relationship between the dose of initially injected PEG2000-liposomes and the extent to which the ABC phenomenon was induced: the higher the dose the smaller the phenomenon. Increasing the PEG density at the liposome surface beyond 5 mol% attenuated rather than induced the induction of the phenomenon, but elongation of the PEG chain length up to M.W. 5000, had no effect. In a series of hematological, serum-biochemical and histopathological safety evaluations we observed neither acute toxicity nor any signs of hepatic damage during the induction of the ABC phenomenon. Morphological examination of the liver by transmission electron microscopy (TEM) showed extensive accumulation of the second dose of mPEG2000-liposomes in the Kupffer cells, even already after 15 min, suggesting that the PEG liposomes had somehow lost the protective effect of the surface-grafted PEG against rapid clearance. The observations reported in this paper may have a considerable impact on the design and engineering of PEGylated liposomal formulations for use in multiple drug therapy.
Article
Polymersomes are self-assembled polymer shells composed of block copolymer amphiphiles. These synthetic amphiphiles have amphiphilicity similar to lipids, but they have much larger molecular weights, so for this reason--along with others reviewed here--comparisons of polymersomes with viral capsids composed of large polypeptide chains are highly appropriate. We summarize the wide range of polymers used to make polymersomes along with descriptions of physical properties such as stability and permeability. We also elaborate on emerging studies of in vivo stealthiness, programmed disassembly for controlled release, targeting in vitro, and tumor-shrinkage in vivo. Comparisons of polymersomes with viral capsids are shown to encompass and inspire many aspects of current designs.
Article
DNA vaccination with mammalian-expressible plasmid DNA encoding protein antigens is known to be an effective means to elicit cell-mediated immunity, sometimes in the absence of a significant antibody response. This may be contrasted with protein vaccination, which gives rise to antibody responses with little evidence of cell-mediated immunity. This has led to considerable interest in DNA vaccination as a means to elicit cell-mediated immune responses against conserved viral antigens or intracellular cancer antigens, for the purpose of therapeutic vaccination. However, almost all current vaccines are used prophylactically and work by producing antibodies rather than cell mediated immune responses. In the present study we have therefore explored the combination of DNA and protein forms of an antigen using two exemplary prophylactic vaccine antigens, namely inactivated influenza virion and hepatitis-B surface antigen. We studied the effects of various combinations of DNA and protein on the antibody response. Co-administration of soluble forms of DNA and protein representations of the same antigen gave rise to the same level of antibody response as if protein were administered alone. In contrast, we found that when these antigens are entrapped in the same liposomal compartment, that there was a strong synergistic effect on the immune response, which was much greater than when either antigen was administered alone, or in various other modes of combination (e.g. co-administration as free entities, also pooled liposomal formulations where the two materials were contained in separate liposomal vehicles in the same suspension). The synergistic effect of liposomally co-entrapped DNA and protein exceeded, markedly, the well known adjuvant effects of plasmid DNA and liposomes. We have termed this new approach to vaccination 'co-delivery' and suggest that it may derive from the simultaneous presentation of antigen via MHC class-I (DNA) and MHC class-II (protein) pathways to CD8+ and CD4+ cells at the same antigen presenting cell--a mode of presentation that would commonly occur with live viral pathogens. We conclude that co-delivery is a very effective means to generate protective antibody responses against viral pathogens.
Article
No substantial progress has really been observed during the past 50 years in fighting cancer, and the way we currently detect and treat cancer is similar to 30 years ago. Cancer nanotechnology will change the situation radically. Progress in developing nanoparticles of biodegradable polymers for new-concept chemotherapy is reviewed here by using the in vitro and in vivo experimental results obtained in my laboratory as a proof-of-concept demonstration. The prospects of using multifunctional nanoparticles for targeting, diagnosing, therapy delivery and result-reporting as a possible solution for cancer detection and treatment are also described. I believe that cancer will become curable using targeted and sustained chemotherapy by such nanoparticles at the earliest stage of disease. High efficacy and low side effects can be achieved, since high drug concentrations can be delivered selectively to the cancer cells, leaving healthy cells untouched. Thus, the required amount of the drug can be minimized.
Article
Nonionic amphiphilic copolypeptides, which were composed of hydrophilic poly(sarcosine) and hydrophobic poly(gamma-methyl L-glutamate) blocks, were synthesized with varying chain lengths of the blocks. The polypeptides having a suitable hydrophilic and hydrophobic balance were found to form vesicular assemblies of 100 nm size in buffer, which was evidenced by the TEM observation, the DLS analysis, and the encapsulation experiment. The genuine peptide vesicles, peptosomes, were labeled with a near-infrared fluorescence (NIRF) probe. In vivo retention in blood experiment showed long circulation of the peptosome in rat blood as stable as the PEGylated liposome. NIRF imaging of a small cancer on mouse by using the peptosome as a nanocarrier was successful due to the EPR effect of the peptosome. Peptosome is shown here as a novel excellent nanocarrier for molecular imaging.
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