The field of the invention

The present invention relates to a method for eliciting in a subject an immune response against a pathogenic organism and to a method for the treatment or prevention of an infection of a subject by a pathogenic organism. The present invention further relates to self-assembled nanostructures like polymersomes, which can be used in the methods as described herein.

Background of the invention

Infectious diseases - causing about 25% of total annual deaths worldwide - are a major threat to public health, which is enhanced by the emergence of drug resistance and vaccine failures. An emerging strategy for fighting infectious diseases is to inhibit the initial host-pathogen interaction, thus preventing cell invasion as for example in the case of malaria. A large number of human pathogens use the cell surface heparan sulfate proteoglycan for recognition and primary interaction between host and pathogen (1). Plasmodium falciparum, which causes malaria and is responsible for > 600,000 deaths annually uses heparan sulfate as the receptor for initial binding of sporozoites and merozoites to host cells. Highly sulfated polysaccharides such as heparin (closely related to heparan sulfate) or K5 polysaccharide are potent inhibitors of merozoite invasion of red blood cells (RBCs) in vitro; all six tested genetic variants of parasites, which use different invasion pathways for RBC invasion, were inhibited by these sugars (2). However, the use of these polysaccharides for malaria treatment is hindered by short in vivo circulation half-lives (about 30 min to 2 h), limited efficacy, and anticoagulation properties, which led to intracranial bleedings (in case of heparin). Naturally acquired immunity to malaria, and the protection of semi-immune individuals from developing severe forms of the disease, is to a large degree directed against extracellular merozoites, but as yet there is no drug which targets the process of invasion of erythrocytes, although some candidates are known (3). The recent advent of whole attenuated parasite vaccines is accompanied with concerns about production, distribution and safety (4), whilst the limited protection obtained after vaccination with subunit vaccines (5) emphasizes the need for alternative treatment and vaccination strategies.

Nanotechnology provides promising tools for designing innovative structures which could be used to combat complex infections, but as yet has been applied only sparsely to malaria, and was focused on systems for drug or vaccine delivery (6). With other pathogens, mainly bacteria and viruses, few lipid-based nanostructures have been evaluated for inhibition of host-pathogen interactions (7). However, liposomes, which are the most simple membranous nanostructures that ensure lateral mobility of receptors for multivalent ligand interaction, possess poor stability and structural integrity in vivo (7). Polymer-based vesicles (polymersomes) composed of amphiphilic block copolymers have been designed to present viral receptors on their surface for virus-assisted loading of polymersomes (8) or to study viral protein binding (9). In addition, heparin has been used at the surface of solid nanoparticles to achieve long circulation times in blood stream for drug delivery in cancer therapy (10).

Summary of the invention

The present invention provides nanomimics based on self-assembled nanostructures like polymersomes that present attachment receptors to pathogenic organisms and thus mimic host's own cellular structures. It has now surprisingly been found that a pathogenic organism bound to self-assembled nanostructures via an agent, which binds the pathogenic organism and which is covalently bound to the self-assembled nanostructures builds a self-assembled nanostructure/pathogen complex capable to elicit an immune response in a subject similar to an attenuated whole pathogen vaccine. It is assumed that the complex greatly boosts the immunogenicity of the pathogen. Thus, the nanomimics of e.g. host cell membranes are able to produce a drug and vaccine-like dual action against a pathogen. In one embodiment Plasmodium merozoites were used as model to exemplify the concept of efficient blockage of pathogen reinvasion after egress from their host cells in vitro. As an additional benefit, this strategy keeps merozoites artificially extracellular after egress, and thus enhances macrophage up-take. These released merozoites bound to nanomimics will be incapable of entering the host cell and thus provide a strong immunogen, which can elicit an immune response against all merozoite antigens. This would happen during a natural or controlled infection, thus avoiding problems associated with subunit or attenuated merozoite vaccines.

In a first aspect, the present invention relates to a method for eliciting in a subject an immune response against a pathogenic organism, comprising administering to said subject a self- assembled nanostructure comprising a synthetic copolymer and/or a synthetic polymer wherein the synthetic copolymer and/or the synthetic polymer is covalently bound to an agent, which binds to the pathogenic organism, and the pathogenic organism bound to the self-assembled nanostructure elicits an immune response against the pathogenic organism in the subject.

In a further aspect, the present invention relates to a method for the treatment or prevention of an infection of a subject by a pathogenic organism, comprising administering to said subject a self-assembled nanostructure comprising a mixture of a synthetic copolymer and a synthetic polymer comprising:

i) a multiblock copolymer comprising two or more blocks wherein the blocks comprise hydrophilic and hydrophobic blocks; and

ii) a polymer covalently bound to an agent, which binds to the pathogenic organism.

In a further aspect, the present invention relates to a polymersome

comprising a mixture of:

i) a multiblock copolymer comprising two or more blocks wherein the blocks comprise hydrophilic and hydrophobic blocks; and

ii) a hydrophobic polymer covalently bound to an agent,

wherein at least one of the hydrophobic blocks of the multiblock copolymer of i) and the hydrophobic polymer of ii) are the same; or

comprising a mixture of:

i) a multiblock copolymer comprising three or more blocks wherein the blocks comprise hydrophilic and hydrophobic blocks; and

ii) a hydrophobic polymer covalently bound to an agent.

In a further aspect, the present invention relates to a composition for eliciting in a subject an immune response against a pathogenic organism comprising

i) a self-assembled nanostructure comprising a synthetic copolymer and/or a synthetic polymer wherein the synthetic copolymer and/or the synthetic polymer is covalently bound to an agent which binds to the pathogenic organism; and

ii) a pharmaceutically acceptable carrier wherein the pharmaceutically acceptable carrier is not water or a chloroform/water emulsion.

Brief description of the figures

Figure 1) shows the chemical structure of PMOXA-6-PDMS-6-PMOXA (1) and PDMS- heparin (2). Figure 2A) shows a transmission electron microscopy (TEM) image of nanomimics (vesicles) consisting of 25% (w/w) PDMS-heparin. B) Cryogenic-TEM of the same nanomimics (vesicles). Scale bars, 200 nm.

Figure 3A) shows normalized autocorrelation curves from fluorescence correlation

spectroscopy (FCS) data in PBS: free OG488 (circles), free MSP1 ₄₂ -0G488 (triangles), control polymersomes with MSP1 ₂ -0G488 (crosses), and nanomimics (vesicles) with

MSP1 ₂ -0G488 (squares). B) Hydrodynamic diameters (D _H ) obtained from mixtures of MSP1 ₄₂ -0G488 with controls (free heparin or control polymersomes) and nanomimics (vesicles) (striped bars) and the corresponding number of dyes per diffusing species (white bars). Data from dynamic light scattering (DLS) are included for the nanostructures for comparison (black bars). Only when heparin was present on the surface of nanostructures (micelles, vesicles) and mixed with MSP1 ₂ -0G488, the diffusion time shifted to the corresponding nanostructure-size, indicating that MSP1 ₂ -0G488 bound to the

nanostructures. Mixtures with free heparin or control polymersomes yielded no difference to free MSP1 ₄₂ -0G488 diffusion. (Errors are ± standard deviation)

Figure 4A) shows a DIC image of a fixed sample of a merozoite bound with nanomimics (vesicles) on the surface of a RBC, B) fluorescence image of merozoite nucleus stained with DAPI, C) fluorescence image of nanomimics (vesicles) filled with hydrophilic dye

sulforhodamine B (SRB), D) merge of all three images.

Figure 5A) shows a TEM micrograph of an ultrathin section of a control merozoite (scale bar, 500 nm). Rhoptries (rh), nucleus (nu) and dense granules (dg) of merozoites can been seen clearly. B) TEM of ultrathin sections of merozoites with nanomimics (vesicles) on the surface (scale bars, 500 nm) and higher magnification of a surface-bound nanomimic (scale bar 50 nm). Lipid membranes (light) can be distinguished from the polymer membrane of nanomimics (vesicles, dark) and the size of the surface bound nanomimics (vesicles) is in agreement with the diameters of nanomimics (vesicles) (Figure 2A,B). Some nanomimics (vesicles) are indicated with a black arrow.

Figure 6A) shows growth inhibition curves for free heparin and nanomimics (vesicles) as determined using suspension cultures. Free heparin inhibits growth of P. falciparum (3D7). Micelles built from PDMS-heparin and nanomimics (vesicles) inhibit merozoite invasion more efficiently than free heparin (data from 5 independent invasion inhibition experiments, all data points are mean growth ± standard error, from at least 3 duplicate assays for each), presented as percentage of control (PBS)). B) IC5 ₀ values for free heparin (n = 7) compared to heparin loaded nanomimics (vesicles) (nl = 9, n2 = 12) or micelles (nl = 4, n2 = 3) indicating the importance of nanostructured heparin for increased activity. Nanomimics (vesicles) 1 and micelles 1 were built from the same batch of PDMS-heparin, nanomimics (vesicles) 2 were made from two other batches of PDMS-heparin (mean values ± standard error for 3 independent experiments for each sample). C) Comparison of nanomimics (vesicles) and micelles expressed as factor of x difference in IC5 ₀ values for nanostructures compared to free heparin (mean values ± standard error for 3 independent experiments for each sample).

Statistics were analyzed using Student's t-test: *P< 0.05, **P< 0.01, ***P< 0.001.

Figure 7) shows the normalized fractional occupancy of the multiple binding sites (surface- exposed heparin) on nanomimics for the interaction with MSPI ₄₂ -OG488 as determined by a titration experiment using fluorescence cross-correlation spectroscopy (FCCS). Fractional occupancy was calculated and normalized versus total MSPI ₄₂ -OG488 ligand concentration. The experimental curve was fitted with the corresponding formula (Example 1), which yielded a Ka of 12.1 ± 1.6 nM for the interaction of heparin on nanomimics and MSPI ₄₂ -OG488. Figure 8A) dose-response curves from antimalarial suspension assays using soluble short heparin (5 kDa), nanomimics-5% containing less PDMS-heparin and nanomimics-ShortHep, which were built using 25% (w/w) PDMS-ShortHep. All data points are mean growth ± standard error; from at least two duplicate assays for each sample, presented as percentage of control (PBS).

Figure 8B) comparison of the IC5 ₀ , concentration of free heparin, short heparin or

nanostructured-heparin (nanomimics-25%, nanomimics-5%, nanomimics-ShortHep, and micelles-ShortHep) to inhibit parasite growth based on total heparin amount ^g/ml). Statistics were analyzed using Student's t-test: ***p< 0.001, not significant (n.s.).

Detailed description of the invention

The present invention provides self-assembled nanostructures like polymersomes and compositions thereof which can be used in methods for eliciting in a subject an immune response against a pathogenic organism and in methods for the treatment or prevention of an infection of a subject by a pathogenic organism. The term "self-assembled nanostructure" refers to artificial nanostructures that are comprised of a synthetic copolymer and/or a synthetic polymer and includes e.g. polymer vesicles

(polymersomes), micelles, elongated micelles, or nanoparticles. The self-assembled

nanostructures of the present invention are preferably polymersomes.

The term "polymersome" as used herein refers to a class of artificial vesicles having a membrane structure and an aqueous core. Polymersomes are artificial vesicles with a polymeric membrane, which are typically, but not necessarily always, formed from the self-assembly of dilute solutions of amphiphilic multiblock copolymers i.e multiblock copolymers comprising hydrophilic and hydrophobic blocks, which can be of different types such as diblock and triblock (A-B, A-B-A or A-B-C) or from the self-assembly of dilute solutions of amphiphilic multiblock copolymers and polymers. Polymersomes may also be formed of tetrablock or pentablock copolymers. For triblock copolymers, the central block is often shielded from the environment by its flanking blocks, while diblock copolymers self-assemble into bilayers, placing two hydrophobic blocks tail-to-tail, much to the same effect. Usually, the vesicular membrane has a water-insoluble middle layer and a water-soluble outer layer. The driving force for polymersome formation by self-assembly is considered to be the microphase separation of the insoluble blocks, which tend to associate in order to minimize their contact area with water. Polymersomes possess remarkable properties due to the large molecular weight of the constitutent copolymers. Vesicle formation is favored upon an increase in total molecular weight of the block copolymers. As a consequence, diffusion of the (polymeric) amphiphiles in these vesicles is low compared to vesicles formed by lipids and surfactants. Owing to this less mobility of polymer chains aggregated in vesicle structure, it is possible to obtain stable polymersome morphologies. Unless expressly stated otherwise, the term "polymersome" and "vesicle", are used interchangeably herein, and have the same meaning.

The term "polymer", "synthetic polymer" or "artificial polymer" as used interchangeably herein refers to a man-made polymer that is not a biopolymer, i.e. is not a naturally occurring polymer and includes chemically modified biopolymers, and man-made synthetic or artificial

biopolymers. A polymer refers to a large molecule that contains a plurality of repeating units or monomers and has a polydispersity index, PDI > 1 thus may have a PDI of e.g. 1,00000001, 1,0000001, 1 ,000001, 1,00001 , 1,0001, 1,001, 1,01, 1,1 or higher.

The term "hydrophobic polymer", "synthetic hydrophobic polymer" or "artificial hydrophobic polymer" as used interchangeably herein refers to a polymer comprising hydrophobic repeating units and which has a polydispersity index, PDI > 1.

The term "hydrophilic polymer", "synthetic hydrophilic polymer" or "artificial hydrophilic polymer" as used interchangeably herein refers to a polymer comprising hydrophilic repeating units and which has a polydispersity index, PDI > 1.

The term "copolymer", "synthetic copolymer" or "artificial copolymer" as used interchangeably herein refers to a man-made copolymer of at least two different polymers that are not a biopolymer, i.e. are not a naturally occurring copolymer and includes chemically modified biopolymers, and man-made synthetic or artificial biopolymers.

Preferably the copolymer is a multiblock copolymer.

The term "multiblock copolymer" or "block copolymers" as used interchangeably herein are normally prepared by controlled polymerization of one monomer, followed by chain extension with a different monomer to form e.g. A-B, A-B-A or A-B-C block copolymers. Preferably the multiblock copolymer is an amphiphilic multiblock copolymer i.e. a multiblock copolymer comprising hydrophilic and hydrophobic moieties, e.g. comprising hydrophilic and hydrophobic blocks.

The term "agent" as used herein includes any substance that may be specifically bound by a pathogen. The agent may be any substance, which is identical or similar to a substance of a host cell to which a pathogen binds or may be a substance which mimics a substance of a host cell or its properties so that a pathogen may bind to it or a substance that is secreted by host cells and that binds to a pathogen. Usually, the agent is a substance, which is identical to a substance of a host cell to which a pathogen binds, a substance of a host cell to which a pathogen binds which is chemically modified e.g. chemically modified by polymerization, depolymerization, partial depolymerization, fragmentation, oxidation, reduction, modification with polymer (e.g. PEGylation), O/N-sulfation, O/N-oversulfation, de-O/N-sulfation, N- acetylation, N-deacetylation, carboxyl/amino/alco ho 1/aldehy de-conjugations, disulfide reduction, sulfo-conjuations, etherifications, or esterifications, and which retains the binding properties with respect to the pathogen or a substance which mimics a substance of a host cell or its properties i.e. has a different structure compared to the substance of the host cell but identical or similar properties so that a pathogen bind to it. Examples of agents that may be used in the present invention are host cell proteins or glycoproteins, in particular membrane proteins or fragments thereof, like e.g. band 3 membrane protein or glycophorins of RBCs, CD4 receptor, or peptide fragments thereof; polysaccharides like polysaccharides which are present at the surface of host cells or similar polysaccharides, like e.g. heparan sulfate, heparin, K5 polysaccharide, polymerized glucosamine and uronic acid (glucuronic acid or iduronic acid) or polymerized sialic acid; or sugar molecules like e.g. glucosamine, uronic acid, glucuronic acid, iduronic acid, or sialic acid and chemically modified versions thereof; antibodies, antibody fragments, or aptamers with specific pathogen binding properties. Preferably, an agent is a polysaccharide, more preferably a polysaccharide selected from the group consisting of heparin, K5 polysaccharide or a derivative thereof, most preferably heparin or a derivative thereof. A derivative of e.g. heparin is a purified heparin such as e.g. a heparin purified on an antithrombin III column or a chemically modified heparin such as e.g. partially de-sulfated heparin or heparin bound to a polymer via carboxylic groups, which retains the binding properties of heparin to the pathogen but reduces or eliminates the anticoagulation property of heparin.

The term "eliciting in a subject an immune response" as used herein is equivalent to the terms "evoking in a subject an immune response" or "inducing in a subject an immune response". The immune response is preferably elicited in a subject who has been infected with a pathogen. Thus the present invention also comprises methods and compositions for eliciting an immune response in a subject, which has been infected with a pathogen, thereby treating the infection, preferably treating the infection and/or protecting form future infections by the same pathogen. Usually cells of the immune system mediate an immune response elicited in a subject against a pathogen.

The terms "copolymer and/or polymer covalently bound to an agent" and "polymer covalently bound to an agent" refers to a copolymer and a polymer, respectively, that is covalently attached to an agent, i.e. wherein a functional group of the copolymer and the polymer, respectively, and a functional group of the agent have been covalently bound to each other. The term "pathogenic organism" or "pathogen" as used interchangeably is an organism capable of causing disease in its host. A human pathogen, which is preferred, is capable of causing illness in humans. Pathogenic organisms include viruses, bacteria, fungi, protozoan, worms, prions, or other organisms that are pathogenic to a subject. The term "pathogen" as used herein can be natural or synthetically generated pathogen.

A pathogen may be a virus selected from the group consisting of adenoviruses, herpesviruses, poxviruses, parvoviruses, papovaviruses, hepadnaviruses, orthomyxoviruses, paramyxoviruses, coronaviruses, picornaviruses, reoviruses, togaviruses, flaviviruses, arenaviruses,

rhabdoviruses, filoviruses and retroviruses or selected from the group consisting of known heparin-binding viruses such as Adeno-associated virus type 2, Adenovirus, Coronavirus Coxsackievirus, Cytomegalovirus, Dengue virus, FMDV, HSV-1 and -2, Hepatitis B virus, Hepatitis C virus, HHV-8 (KSHV), HIV-1, HPV, HTLV1, Japanese encephalitis virus, Pseudorabies virus, Respiratory syncytial virus, Rhinovirus, Sindbis virus, Vaccinia virus, West Nile virus, Yellow fever virus (1), preferably selected from the group consisting of HIV-1, Dengue virus, Hepatitis B virus and Hepatitis C virus;

a bacteria selected from the group consisting of Staphylococcus aureus, Streptococcus

(agalactiae, pyogenes, pneumoniae), Neisseria (gonorrhoeae, meningitides), Corynebacteria diphtheriae, Bacillus (anthracis, cereus), Listeria monocytogenes, Escherichia coli,

Salmonella (typhimurium, typhae), Shiella-Calobacter, Yersinia, Pseudomonas aeruginosa, Brucella Haemophilus, influenzae, Legionella, Bordetella, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Treponema pallidum, Borrelia burgdorferi, Leptospira interrogans, Mycobacterium (tuberculosis, leprae, avium), Rickettsia prowazekii, Chlamydia (pneumonia, trachomatis), Helicobacter pylori, Orientia tsutsugamushi, Porphyromonas gingivalis and Mycoplasma pneumoniae or a bacteria selected from the group consisting of known heparin-binding bacteria such as Bacillus anthracis, Bacillus cereus, Borrelia burgdorferi ,Bordetella pertussis, Chlamydia pneumonia, Chlamydia trachomatis,

Haemophilus influenzae, nontypable, Helicobacter pylori, Listeria monocytogenes,

Mycobacterium tuberculosis, Neisseria gonorrhoaea, Neisseria meningitides, Neisseria meningitides, Orientia tsutsugamushi, Porphyromonas gingivalis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus pneumoniae, Yersinia enterocolitica (1);

a fungus selected from the group consisting of Candida albicans, Cryptococcus neoformans, Aspergillus, Histoplasma capsulatum, Coccidioides immitis and Pneumocystis carinii;

a protozoan selected from the group consisting of Entamoeba histolytica, Giardia,

Leishmania, Plasmodium (falciparum, vivax), Trypanosoma (brucei, cruzi), Toxoplasma gondii, Encephalitozoon spp., Neospora caninum, Mxoplasma gondii, and Cryptosporidium or selected from the group consisting of known heparin-binding protozoa such as Giardia lamblia, Leishmania spp., Encephalitozoon spp., Neospora caninum, Plasmodium spp., Toxoplasma gondii, Trypanosoma cruzi (\), preferably Plasmodium spp., more preferably Plasmodium falciparum; or

a worm selected from the group consisting of Trichuris trichiura, Trichinella spiralis, Enterobius vermicularis, Ascaris lumbricoides, Ancylostoma, Stron loides Filaria Onchocerca volvulus Loa loa, Dracuncula medinensis, Schistosoma (mansoni), and Clonorchis sinensis. The pathogen is preferably a virus, a bacteria, a fungus, a protozoan or a worm, more preferably a bacteria, a fungus, a protozoan or a worm, even more preferably a protozoan, most preferably Plasmodium falciparum. Equally preferred are viruses, in particular HIV-1, Dengue virus, Hepatitis B virus and Hepatitis C virus and protozoan, in particular Plasmodium spp., more particular Plasmodium falciparum.

By "comprising" it is meant including, but not limited to, whatever follows the word

"comprising". Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.

By "consisting of it is meant including, and limited to, whatever follows the phrase

"consisting of. Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. By "about" in relation to a given numerical value, such as for % (w/w), temperature and period of time, it is meant to include numerical values within 10% of the specified value. The present invention provides a method for eliciting in a subject an immune response against a pathogenic organism, comprising administering to said subject a self-assembled nanostructure comprising a synthetic copolymer and/or a synthetic polymer wherein the synthetic copolymer and/or the synthetic polymer is covalently bound to an agent, which binds to the pathogenic organism, and the pathogenic organism bound to the self-assembled nanostructure elicits an immune response against the pathogenic organism in the subject. Likewise the invention relates to a self-assembled nanostructure comprising a synthetic copolymer and/or a synthetic polymer wherein the synthetic copolymer and/or the synthetic polymer is covalently bound to an agent, which binds to the pathogenic organism, for use in a method for eliciting in a subject an immune response against a pathogenic organism.

Likewise the invention relates to the use of a self-assembled nanostructure comprising a synthetic copolymer and/or a synthetic polymer wherein the synthetic copolymer and/or the synthetic polymer is covalently bound to an agent, which binds to the pathogenic organism, for the manufacture of a medicament for eliciting in a subject an immune response against a pathogenic organism.

The self-assembled nanostructure used in the method for eliciting in a subject an immune response against a pathogenic organism preferably comprises a synthetic copolymer and a synthetic polymer, wherein the synthetic copolymer or the synthetic polymer is covalently bound to an agent which binds to the pathogenic organism. More preferably the self-assembled nanostructure used in the method for eliciting in a subject an immune response against a pathogenic organism comprises a mixture of a synthetic copolymer and a synthetic polymer comprising:

i) a multiblock copolymer comprising two or more blocks wherein the blocks comprise hydrophilic and hydrophobic blocks; and

ii) a polymer covalently bound to the agent, which binds to the pathogenic organism or a multiblock copolymer covalently bound to the agent, which binds to the pathogenic organism wherein the multiblock copolymer comprises two or more blocks wherein the blocks comprise hydrophilic and hydrophobic blocks.

Even more preferably the self-assembled nanostructure used in the method for eliciting in a subject an immune response against a pathogenic organism comprises a mixture of a synthetic copolymer and a synthetic polymer comprising: i) a multiblock copolymer comprising two or more blocks wherein the blocks comprise hydrophilic and hydrophobic blocks; and

ii) a polymer covalently bound to the agent, which binds to the pathogenic organism. The present invention also provides a method for the treatment or prevention of an infection of a subject by a pathogenic organism, comprising administering to said subject a self-assembled nanostructure comprising a mixture of a synthetic copolymer and a synthetic polymer comprising:

i) a multiblock copolymer comprising two or more blocks wherein the blocks comprise hydrophilic and hydrophobic blocks; and

ii) a polymer covalently bound to an agent, which binds to the pathogenic organism.

Likewise the invention relates to a self-assembled nanostructure comprising a mixture of a synthetic copolymer and a synthetic polymer comprising:

i) a multiblock copolymer comprising two or more blocks wherein the blocks comprise hydrophilic and hydrophobic blocks; and

ii) a polymer covalently bound to an agent, which binds to the pathogenic organism, for use in the treatment or prevention of an infection of a subject by a pathogenic organism.

Likewise the invention relates to the use of a self-assembled nanostructure comprising a mixture of a synthetic copolymer and a synthetic polymer comprising:

i) a multiblock copolymer comprising two or more blocks wherein the blocks comprise hydrophilic and hydrophobic blocks; and

ii) a polymer covalently bound to an agent, which binds to the pathogenic organism, for the manufacture of a medicament for the treatment or prevention of an infection of a subject by a pathogenic organism.

A particular self-assembled nanostructure, which can be used in the methods of the present invention, comprises a mixture of a synthetic copolymer and a synthetic polymer comprising: i) a multiblock copolymer comprising two or more blocks wherein the blocks comprise hydrophilic and hydrophobic blocks; and

ii) a hydrophobic polymer covalently bound to an agent, which binds to the pathogenic organism. A more particular self-assembled nanostructure which can be used in the methods of the present invention is a polymersome which

comprises a mixture of:

i) a multiblock copolymer comprising two or more blocks wherein the blocks comprise hydrophilic and hydrophobic blocks; and

ii) a hydrophobic polymer covalently bound to an agent,

wherein at least one of the hydrophobic blocks of the multiblock copolymer of i) and the hydrophobic polymer of ii) are the same; or

comprises a mixture of:

i) a multiblock copolymer comprising three or more blocks wherein the blocks comprise hydrophilic and hydrophobic blocks; and

ii) a hydrophobic polymer covalently bound to an agent.

The hydrophobic blocks of the multiblock copolymer of i) and the hydrophobic polymer of ii) are the same when they have the identical hydrophobic polymer structure i.e. if both comprise e.g. poly(dimethylsiloxane) as hydrophobic polymer structure, whereas functional groups of both the hydrophobic block of the multiblock copolymer of i) and the hydrophobic polymer of ii) may be different.

Thus the present invention further provides a polymersome

comprising a mixture of:

i) a multiblock copolymer comprising two or more blocks wherein the blocks comprise hydrophilic and hydrophobic blocks; and

ii) a hydrophobic polymer covalently bound to an agent,

wherein at least one of the hydrophobic blocks of the multiblock copolymer of i) and the hydrophobic polymer of ii) are the same; or

comprising a mixture of:

i) a multiblock copolymer comprising three or more blocks wherein the blocks comprise hydrophilic and hydrophobic blocks; and

ii) a hydrophobic polymer covalently bound to an agent.

In a preferred embodiment the self-assembled nanostructure e.g. the polymersome of the present invention has not a lipid nanostructure in particular not a phospholipid nanostructure, thus does not contain lipids which are polymers with PDI = 1 i.e. has a membrane without lipids, in particular does not contain phospholipids which are polymers with PDI = 1 i.e. has a membrane without phospholipids. Thus the self-assembled nanostructure e.g. the polymersome of the present invention does not comprise one or more lipid or phospholipid mono- or bilayers.

Examples of suitable multiblock copolymers include, but are not limited to, poly(ethyl ethylene)-b-poly(ethylene oxide) (PEE-b-PEO), poly(butadiene)-b-poly(ethylene oxide) (PBD- b-PEO), poly(styrene)-b-poly(acrylic acid) (PS-PAA), poly(2-methyloxazoline)-b-poly(di- methylsiloxane)-b-poly(2-methyloxazoline) (PMOXA-b-PDMS-b-PMOXA), poly(2- methyloxa- zoline)-b-poly(dimethylsiloxane)-b-poly(ethylene oxide) (PMOXA-b-PDMS-b- PEO), poly(ethylene oxide)-b-poly(propylene sulfide)-b-poly(ethylene oxide) (PEO-b-PPS-b- PEO) and a poly(ethylene oxide)-poly(buylene oxide) block copolymer. A block copolymer can be further specified by the average block length of the respective blocks included in a copolymer. Thus PB _M PEO _N indicates the presence of polybutadiene blocks (PB) with a length of M and polyethyleneoxide (PEO) blocks with a length of N. M and N are independently selected integers, which may for example be selected in the range from about 6 to about 60. Thus PB ₃ 5PEO1 ₈ indicates the presence of polybutadiene blocks with an average length of 35 and of polyethyleneoxide blocks with an average length of 18. Likewise, PB1 ₆ PEO24 indicates the presence of polybutadiene blocks with an average length of 16 and of

polyethyleneoxide blocks with an average length of 24. As a further example E ₀ B _P indicates the presence of ethylene blocks (E) with a length of O and butylene blocks (B) with a length of P. O and P are independently selected integers, e.g. in the range from about 10 to about 120. Thus Ei ₆ B ₂ 2 indicates the presence of ethylene blocks with an average length of 16 and of butylene blocks with an average length of 22.

A hydrophobic polymer is preferably selected from the group consisting of polysiloxanes such as poly(dimethylsiloxane) (PDMS), poly(caprolactone) (PCL), poly(methylcaprolactone) (PMCL), poly(menthide), poly(lactide) (PLA), poly(glycolide), poly(lactide-co-glycolide) (PLGA), poly(methylglycolide), poly(isobutylene), poly(styrene), poly(butadiene) (PBD), poly(ethylene), poly(ethyl ethylene) (PEE), poly(isoprene), poly(propylene oxide), polystyrene (PS), poly(methylphenylsilane) (PMPS), poly(2-vinylpyridine) (P2VP), poly(N- isopropylacrylamide) (PNIPAm), poly(propylene sulfide) (PPS), poly(2-(diethylamino) ethyl methacrylate) (PDEA), poly(trimethylene carbonate) (PTMC), poly(methyl methacrylate) (PMMA), perfluoropolyalkyl ethers such as perfluoroalkyl polyether, unsaturated hydrophobic polymers such as a polymer of a conjugated aliphatic or alicyclic diene or partially hydrated derivatives thereof, a polymer of an alkyne or a diaalkyne substituted with lower alkyl or partially hydrated derivatives thereof, a polymer of a conjugated diene or partially hydrated derivatives thereof, poly- 1, 2 -butadiene, poly- 1, 4 -butadiene, polyisoprene, polychloroprene, polypiperylene, or poly-l-trimethylsilyl-propyne.

A hydrophilic polymer is preferably selected from the group consisting of poly(ethylene oxide) (PEO), poly(acrylic acid) (PAA), poly(ethylene glycol) (PEG), poly(2-methyloxazoline) (PMOXA), poly(L- isocyanoalanine(2-thiophen-3-yl-ethyl)amide) (PIAT), poly(4- vinylpyridine) (P4VP), poly(N-vinylpyrrolidone) (PNVP).

Copolymers may comprise combinations of different hydrophobic polymers as hydrophobic block and/or may comprise combinations of different hydrophilic polymers as hydrophilic block. Preferably the hydrophobic polymer has one hydrophobic chain per molecule.

If the multiblock copolymer of the self-assembled nanostructure e.g. of the polymersome comprises a multiblock copolymer comprising two blocks, the two blocks are preferably selected from the group consisting of

poly[styrene-i>-poly(L- isocyanoalanine(2-thiophen-3-yl-ethyl)amide)] (PS-PIAT),

poly(butadiene)-poly(ethylene oxide) (PBD-PEO), poly(ethylene oxide)-poly(caprolactone) (PEO-PCL), poly(ethyl ethylene)-poly( ethylene oxide) (PEE-PEO), poly(ethylene oxide)- poly(lactic acid) (PEO-PLA), poly(isoprene)-poly(ethylene oxide) (PI-PEO), poly(2- vinylpyridine)-poly( ethylene oxide) (P2VP-PEO), poly(ethylene oxide)-poly(N- isopropylacrylamide) (PEO-PNIPAm), poly(styrene)-poly( acrylic acid) (PS-PAA), poly(ethylene glycol)-poly(propylene sulfide) (PEG-PPS), poly(2-methyloxazoline)- poly(dimethylsiloxane) (PMOXA-PDMS), poly(ethylene glycol)-poly(dimethylsiloxane) (PEG- PDMS), or poly(ethylene glycol)-poly(lactide-co-glycolide) (PEG-PLGA).

If the multiblock copolymer of the self-assembled nanostructure e.g. of the polymersome comprises a multiblock copolymer comprising three blocks, the three blocks are preferably selected from the group consisting of poly(2-methyl-2-oxazoline)-poly(dimethylsiloxane)- poly(2-methyl-2-oxazoline) (PMOXA-PDMS-PMOXA), polyethylene glycol)- poly(dimethylsiloxane)-poly(ethylene glycol) (PEG-PDMS-PEG), poly(ethylene oxide)- poly(dimethylsiloxane)-poly(2-methyloxazoline) (PEO-PDMS-PMOXA), poly(ethylene glycol)-poly(lactide-co-glycolide)-poly(ethylene glycol) (PEG-PLGA-PEG), poly(ethylene glycol)-poly(lactic acid)-poly(ethylene glycol) (PEG-PLA-PEG), poly(ethylene glycol)- poly(caprolactone)-poly(ethylene glycol) (PEG-PCL-PEG), poly(2-methyloxazoline)- poly(lactide-co-glycolide)-poly(2-methyloxazoline) (PMOXA-PLGA-PMOXA), poly(2- methyloxazoline)-poly(lactic acid)-poly(2-methyloxazoline) (PMOXA-PLA-PMOXA), poly(2- methyloxazoline)-poly(caprolactone)-poly(2-methyloxazoline) (PMOXA-PCL-PMOXA), and, poly(4-vinylpyridine)-polystyrene-poly(4-vinylpyridine) (P4VP-PS-P4VP) and poly(N- vinylpyrrolidone)-poly(dimethylsiloxane)-poly(N-vinylpyrroli done) (PNVP-PDMS-PNVP). If the multiblock copolymer of the self-assembled nanostructure e.g. of the polymersome is a multiblock copolymer comprising four or more blocks, the four or more blocks are preferably selected from the group consisting of poly(ethylene multiblock oxide)-poly(styrene)- poly(butadiene)-poly(ethylene oxide) (PEO-PS-PB-PEO) and poly(methylphenylsilane)- poly(ethylene oxide) (PMPS-PEO-PMPS-PEO-PMPS).

In certain embodiments, the self-assembled nanostructure e.g. the polymersome may contain one or more compartments (or otherwise termed "multicompartments"). In a particular embodiment the self-assembled nanostructure e.g. the polymersome contains only one compartment i.e. does not contain multicompartments. In another particular embodiment the self-assembled nanostructure e.g. the polymersome comprise two or more, preferably two compartments wherein the two or more compartments are identical i.e. comprise the same multiblock copolymer and polymer, preferably hydrophobic polymer covalently bound to an agent which binds to the pathogenic organism. In another particular embodiment the self- assembled nanostructure e.g. the polymersome contains two or more compartments formed spontaneously and simultaneously e.g. the inner compartment is not formed independently and then encapsulated in another compartment. In another particular embodiment the self- assembled nanostructure e.g. the polymersome is a monolayer. Compartmentalization of the vesicular structure of polymersome allows for the co-existence of complex reaction pathways in living cell and helps to provide a spatial and temporal separation of many activities inside a cell. Accordingly, more than one type of agent may be incorporated in the polymersome carrier. The different agents may have the same or different isoforms. Each compartment may also be formed of a same or a different amphiphilic polymer. In various embodiments, two or more different agents are integrated into the circumferential membrane of the amphiphilic polymer. Each compartment may encapsulate at least one agent.

In the case where the polymersome carrier contains more than one compartment, the compartments may comprise an outer block copolymer vesicle and at least one inner block copolymer vesicle, wherein the at least one inner block copolymer vesicle is encapsulated inside the outer block copolymer vesicle. By "encapsulated" it is meant that the inner vesicle is completely contained inside the outer vesicle and is surrounded by the vesicular membrane of the outer vesicle. The confined space surrounded by the vesicular membrane of the outer vesicle forms one compartment. The confined space surrounded by the vesicular membrane of the inner vesicle forms another compartment.

Preferably the multiblock copolymer of the self-assembled nanostructure e.g. of the polymersome comprises three or more blocks wherein the blocks may comprise hydrophilic and hydrophobic blocks as outlined above. More preferably the multiblock copolymer of the self-assembled nanostructure e.g. of the polymersome comprises three or more blocks selected form the group consisting of PMOXA-PDMS-PMOXA, PEG-PDMS-PEG, PEG-PCL-PEG, PEG-PLGA-PEG, PMOXA-PLGA-PMOXA, or PMOXA-PCL-PMOXA. More preferably the multiblock copolymer of the self-assembled nanostructure e.g. of the polymersome comprises the PMOXA-PDMS-PMOXA copolymer.

Preferably the polymer of the self-assembled nanostructure e.g. the hydrophobic polymer of the polymersome provided by the present invention is a polymer selected form the group consisting of PDMS, PCL, PMCL, PLGA, or PLA, or is more preferably PDMS. The diameter of the self-assembled nanostructure e.g. of the polymersomes ranges from about 1 nm up to about 20 μιη, preferably from about 10 nm up to about 2 μιη, preferably from about 20 nm up to about 0,2 μιη.

The mixture of the multiblock copolymer and the polymer covalently bound to an agent of the self-assembled nanostructure may vary according to the desired structure of the self-assembled nanostructure. Usually mixtures of about 99.999 to about 65% (w/w) of the multiblock copolymer and of about 0.001 to about 35% (w/w) of the polymer covalently bound to an agent mainly form vesicles. From about 65 to about 45% (w/w) of the multiblock copolymer and from about 35 to about 55% (w/w) of the polymer covalently bound to an agent a mixture of vesicles/micelles/elongated micelles is formed. About 45 to about 0.001% (w/w) of the multiblock copolymer and about 55 to about 99.999%) (w/w) of the polymer covalently bound to an agent forms mainly aggregated micelles. The mixture of the multiblock copolymer and the polymer covalently bound to an agent of the polymersome provided by the present invention comprises at least about 45 % (w/w), preferably at least about 65 % (w/w), more preferably at least about 75 % (w/w) of the multiblock copolymer. Particular useful are mixtures of the multiblock copolymer and the polymer covalently bound to an agent of the polymersome comprising about 65 to about 95 % (w/w) of the multiblock copolymer and about 35 to about 5 % (w/w) of the polymer covalently bound to an agent, preferably about 65 to about 75 % (w/w) of the multiblock copolymer and about 35 to about 25 % (w/w) of the hydrophobic polymer covalently bound to an agent. The present invention provides a composition for eliciting in a subject an immune response against a pathogenic organism comprising

i) a self-assembled nanostructure comprising a synthetic copolymer and/or a synthetic polymer wherein the synthetic copolymer and/or the synthetic polymer is covalently bound to an agent which binds to the pathogenic organism; and

ii) a pharmaceutically acceptable carrier wherein the pharmaceutically acceptable carrier is not water or a chloroform/water emulsion.

As self-assembled nanostructure, the self-assembled nanostructure or the polymersome as described supra are usually comprised by the composition. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible except water or a chloroform/water emulsion. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Pharmaceutically acceptable carriers include phosphate buffered saline (PBS), saline, mannitol, lactose, fructose, sucrose, sorbitol, xylitol, maltodextrin, dextrates, dextrins, lactitol, inositol, trehalose, maltose, raffinose, [alpha]-, [beta]- and [gamma]-cyclodextrins, gum arabic, sodium alginate, propylene glycol alginate, agar, gelatin, tragacanth, xanthan gum, starch, lectins, urea, chitosan, chitosan glutamate, cellulose and cellulose derivatives, or organic amines. Preferred pharmaceutically acceptable carriers are PBS or saline, more preferably PBS. The use of mixtures of more than one of the pharmaceutically acceptable carriers to provide desired release profiles or for the enhancement of stability is within the scope of this invention.

As administration route of the self-assembled nanostructure e.g. of the polymersome in the methods described supra and as administration route for the composition one or more routes of administration using one or more of a variety of methods known in the art can be used. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Preferred routes of administration include oral,

intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous routes of administration, for example by injection or infusion or by non-invasive administration. More preferred routes of administration are intravenous or subcutaneous, whereas the self-assembled nanostructure e.g. the polymersome is injected. In alternative embodiments, the injection of the composition may include intradermal injection.

The immune response level may be further heightened or boosted by including an adjuvant in the composition including the polymersome carrier carrying the pathogen-binding agent. In such embodiments, the self-assembled nanostructure e.g. the polymersome and the adjuvant are administered simultaneously to the subject. In alternative embodiments, the adjuvant may be administered separately from the administration of the self-assembled nanostructure e.g. of the polymersome. The adjuvant may be administered before, simultaneously, or after the administration of the self-assembled nanostructure e.g. of the polymersome. The polymers and copolymers such as multiblock copolymers of the self-assembled

nanostructure or the polymersome can be synthetized according to methods known in the art (see, e.g. 11).

The assembling of the nanostructure or the polymersome can be obtained by techniques known in the art such as the bulk rehydration technique (see, e.g. 11). A particular useful technique of the present invention for the assembling of a polymersome comprises the following steps: a) dissolving a multiblock copolymer and a hydrophobic polymer covalently bound to an agent in a solvent;

b) mixing the dissolved multiblock copolymer and the hydrophobic polymer covalently bound to an agent in the desired ratio;

c) removing the solvent; and

d) hydrating the polymer mixture obtained in c).

The solution for hydration (d) used is usually selected from the group consisting of PBS or saline. A particular useful technique of the present invention for obtaining a hydrophobic polymer covalently bound to a polysaccharide comprises the following steps:

a) ion exchange to obtain tetrabutylammonium salt of a polysaccharide;

b) mixing tetrabutylammonium salt of the polysaccharide containing e.g. a reducing end (aldehyde) with hydrophobic polymer, containing at least one primary amino group, in a suitable solvent;

c) adding reducing agent;

d) purify hydrophobic polymer bound to the polysaccharide.

This technique of the present invention for obtaining a hydrophobic polymer covalently bound to a polysaccharide is particularly useful for obtaining a hydrophobic polymer covalently bound to a heparin or a derivative thereof. The solvent used is usually selected form the group consisting of methanol, ethanol, dichloromethane, chloroform, tetrahydrofuran, or

dimethylsulfoxide. The reducing agent is usually selected form the group consisting of 2- picoline borane, sodium cyanoborohydride, sodium triacetoxyborohydride, pyridine-borane, Ti(Oi-Pr) ₄ / NaBH ₄ , Zn(BH ₄ ) ₂ /Si0 ₂ , Bu ₃ SnH/Si0 ₂ , or PhSiH ₄ /Bu ₂ SnCl ₂ .

Ion exchange can be performed according to methods known in the art (see, e.g. 12). Examples

Example 1: Materials and Methods

PMOXA-6-PDMS-6-PMOXA (1) synthesis. PMOXA-6-PDMS-6-PMOXA (1, Fig. 1) was synthesized according to previously published protocols (13). Briefly, bifunctional

poly(dimethylsiloxane) (PDMS from ABCR GmbH, Karlsruhe, Germany) was vacuum stripped in a Schlenkflask overnight. Anhydrous hexane was subsequently added and the stirred solution was dried by bubbling argon through it for 1 h. After bubbling, freshly distilled triethylamine (TEA) was added and the mixture was cooled to -20 °C. PDMS was then reacted with trifluoromethanesulfonic acid (Tfsa) for 3 h at -20 °C resulting in a bifunctional triflic PDMS macroinitiator. The reaction mixture was filtered through a cooled G4 filter under argon. From the filtrate hexane was removed under vacuum and replaced by dry ethylacetate. Adding distilled 2-methyl-2-oxazoline resulted in a cationic ring opening polymerisation of poly(2- methyl-2-oxazoline) (PMOXA) on the PDMS macroinitiator. Termination was performed after 60 h at 40 °C by adding a 2:8 mixture of TEA:water resulting in bifunctional OH-terminated PMOXA-6-PDMS-6-PMOXA. Finally, the solvent was removed by vacuum distillation.

Purification was performed by re-solubilising the polymer in ethanol/water 1 : 1 mixture and ultrafiltration through a 5 kDa membrane. The final product was dried under vacuum.

PDMS-heparin (2) synthesis. Heparin sodium salt from porcine intestinal mucosa (15 kDa, 197 U/mg, 375095) was purchased from Merck KGaA (Darmstadt, Germany) and

aminopropyl-terminated poly(dimethylsiloxane) (PDMS(NH ₂ ) ₂ ) (5 kDa, AB109371) from ABCR GmbH (Karlsruhe, Germany).

The tetrabutylammonium salt of heparin was obtained using a published protocol (12). Briefly, 500 mg heparin sodium salt were dissolved in a minimum amount of water (approximately 3 ml) and passed through a freshly packed Dowex® Marathon™ MSC column (H ⁺ form, 6 ml, Sigma- Aldrich, 428787). The pH was adjusted to pH 7 using a tetrabutylammonium hydroxide solution (54.0-56.0% in H ₂ 0, Sigma-Aldrich, 86863). After reducing the volume on a rotary vacuum evaporator, the product was dialyzed against water for at least 48 hours (Spectrum Laboratories Inc., Rancho Dominguez, CA, USA, MWCO 3.5 - 5 kDa). The product was subsequently dried under vacuum.

For PDMS-heparin (2, Fig. 1) synthesis, 100 mg of tetrabutylammonium salt of heparin (~ 22 kDa) was dissolved in a 50 ml round-bottom flask in 25 ml dichloromethane (DCM) and a hundred- fold excess of PDMS(NH ₂ )2 was added under stirring. Furthermore, a 10-fold excess of 2-picoline borane (Sigma- Aldrich, 654213) was dissolved in a small amount of DCM, added to the reaction mixture and stirred for 7 days at room temperature (25 °C). After the 3rd and 5th day of the reaction another 10-fold excess of 2-picoline borane were added, respectively. After 7 days, DCM was evaporated on a rotary evaporator and the product was washed in diethyl ether. The washed product was dried under vacuum and redissolved in a minimum amount of ethanol. Adding the solution dropwise into 4 x 15 ml diethyl ether in glass centrifugal tubes precipitated the product; unreacted PDMS(NH ₂ )2 and reducing agent were soluble in diethyl ether. The precipitate was collected by centrifugation for 5 min (2000 RCF) and discarding the supernatant. This precipitation process was repeated two times. The final product was dissolved in ethanol, filtered and dried under vacuum. For 'H-NMR about 20 mg of the product was dissolved in 10% ethanol, passed through a freshly packed Dowex® Marathon™ MSC column (Na ⁺ form, 2ml, Sigma- Aldrich, 428787), dried on a rotary evaporator and redissolved in D ₂ 0/acetone-D ₆ (v/v 3: 1).

PDMS-ShortHep (3) synthesis. PDMS-6/oc£-short-heparin (PDMS-ShortHep) was synthesized similarly to PDMS-heparin (2) but using shorter heparin starting material (LMWH, 4 - 6 kDa, Fluka, 51550) for the synthesis instead of full-length heparin (15 kDa). Polymersome and nanomimic formation. Control polymersomes (PMOXA-£-PDMS-£- PMOXA only) were formed by the film rehydration technique (11). 1 ml of ΡΜΟΧΑ-δ- PDMS-δ-ΡΜΟΧΑ (6 mg/ml in ethanol) was put in a 5 ml round bottom flask and ethanol was removed on a rotary evaporator (100 mbar, 40 °C, 120 RPM). Subsequently, the thin polymer film was rehydrated using PBS or 1 mM sulforhodamine b (SRB, Sigma- Aldrich, SI 402) in PBS and stirred at least overnight.

Nanomimics-25% ( PMOXA-6-PDMS-6-PMOXA with 25 wt% PDMS-heparin ),

nanomimics-5% (PMOXA-6-PDMS-6-PMOXA with 5 wt% PDMS-heparin) and micelles were prepared using the bulk rehydration technique (11). PMOXA-6-PDMS-6-PMOXA (6 mg/ml) and PDMS-heparin (4 mg/ml) were dissolved in ethanol (both yielded clear solutions) and mixed in a 5 ml round-bottom flask in the desired ratio. The solvent was evaporated on a rotary evaporator (100 mbar, 40 °C, 120 RPM) and the film was further dried at high vacuum overnight. The polymer film was subsequently destroyed using a spatula. The bulk polymer was hydrated in PBS and stirred for at least 12 hours. To yield fluorescent nanomimics, the polymer mixture was hydrated using a 1 mM SRB (Sigma- Aldrich, SI 402) solution in PBS. Nanomimics-ShortHep and PDMS-ShortHep micelles were prepared using the same method but a mixture of PMOXA-6-PDMS-6-PMOXA and 25 wt% (w/w) of PDMS-ShortHep or pure PDMS-ShortHep, respectively.

The polymersome-, nanomimic- and micelle solutions were extruded 15 times through a 0.1 μιη nucleopore track-etch membrane (Whatman, GE Healthcare, UK) using a LIPEX™ extruder (Northern Lipids Inc., Canada). The final solution of nanostructures was passed through a size-exclusion column (SEC) containing Sepharose ^® 2B (Sigma-Aldrich, 2B300) equilibrated with PBS . Control polymersomes (PMOXA-6-PDMS-6-PMOXA) or nanomimics were incubated with 200 nM Bodipy630 (hydrophobic dye) in PBS after the vesicle formation procedure for FCS/FCCS.

Protein labeling. Water-soluble proteins were fluorescently labeled via amine-groups using Oregon Green ^® 488 Carboxylic Acid, Succinimidyl Ester, 5-isomer (OG488-NHS, Invitrogen, Carlsbad, CA, USA, 0-6147) using established protocols (Invitrogen). The buffer of

Plasmodium falciparum major surface protein 1-42 ( MSPI ₄₂ (3D7)) was exchanged to 0.1 M carbonate buffer (pH 8.3) at a concentration of 2 mg/ml by using Amicon Ultra-0.5ml (10K) centrifugal filters. OG488-NHS was dissolved in water-free DMSO (19.6 mM) and a 12-fold excess of OG488-NHS was added to the protein solution. After shaking for at least 2 hours at room temperature, free OG488 was separated from labeled proteins on HiTrap Desalting columns (Sephadex G25) using PBS as running buffer. The labeled proteins were stored in aliquots at -20 °C. Farndale microassay. The Farndale microassay was performed based on Farndale et. al. (14) with slight modifications. The dimethylmethylene blue (DMMB)-solution was prepared as suggested (15). 250 μΐ of the DMMB-solution was pipetted into 96-well plates. 50 μΐ of PBS, heparin standards (20 μg/ml, 10 μg/ml, 7.5 μg/ml, 5 μg/ml, 2.5 μg/ml), and diluted nanomimic- samples were mixed with the DMMB-solution in duplicates. The UV-Vis absorbance was measured from 395 nm to 595 nm immediately after mixing. The heparin standard curve was generated using the absorbance at 525 nm and this curve was used as a calibration curve. In the nanomimic samples a baseline correction (exponential decay) was performed and the corrected absorbance value at 525 nm was used to calculate the amount of surface accessible heparin. Farndale microassay was also performed using tetrabutylammonium salt of heparin (short or long heparin), PDMS-heparin, and PDMS-ShorHep in ethanol to estimate the number of repeating units for heparin in the heparin-containing block copolymers.

UV-Vis Spectrometry. UV-Vis absorbance measurements were performed on a SpectraMax Plus M5e (Molecular Devices, Sunnyvale, California) using 0.1 ml cuvettes or 96-flat-bottom- well plates. Nuclear magnetic resonance. Ή-NMR spectra were recorded on a Bruker DPX-400 NMR spectrometer in D ₂ 0, D ₂ 0/acetone-D ₆ -mixture or CDCI ₃ at room temperature. The

spectrometer was operated at 400 MHz and 16 or 128 NMR cycles were recorded for each sample. Gel permeation chromatography. Gel permeation chromatography (GPC) was performed on a Viscotek GPCmax system equipped with two PLgel Mixed-c 5 μιη columns (300 x 7.5 mm), THF as eluent (flow rate = lmL min-1) at 40 °C and recorded by refractive-index (RI).

Polymer molecular weights and polydispersity indices (PDI) were determined using polystyrene standards for calibration.

Transmission electron microscopy. Nanostructure samples were negatively stained with 2% uranyl acetate for transmission electron microscopy (TEM) imaging. Ultrathin sections of parasites were stained with a mixture of 4% uranyl acetate/methylcellulose (ratio 1 :9). Imaging was carried out on a transmission electron microscope (Philips CM 100) at an acceleration voltage of 80 kV. Size measurements were performed using ImageJ software.

Cryogenic transmission electron microcopy. 4 μΐ of nanomimic sample (3mg/ml) was adsorbed on a holey carbon-coated grid (Quantifoil, Germany), blotted with a Whatman 1 filter paper and vitrified in liquid nitrogen-cooled liquid ethane using an FEI Vitrobot MK4 (FEI company, Netherlands). Frozen grids were transferred to a Philips CM200-FEG electron microscope, which was operated at an acceleration voltage of 200 kV. Digital electron micrographs were recorded with a 4k x 4k TemCam-F416 CMOS camera (TVIPS company, Germany).

Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy. Fluorescence correlation spectroscopy (FCS) and fluorescence correlation spectroscopy (FCCS) measurements were performed on a commercial Confocor2 (Carl Zeiss, Jena, Germany) using an Ar+ laser for the 488 nm wavelength, a HeNe laser for 543 nm and a HeNe laser for 633 nm. The laser beam was focused onto the sample through a 40x C-Apochromat water immersion objective with a numeric aperture of 1.2 and the appropriate filter sets.

Finally, the fluorescence intensity was recorded with an avalanche photodiode (APD). All measurements were performed at 20 °C.

Typically, 5 μΐ samples were used. Measurement series of 30 x 5 s (fast diffusing species) or 30 x 10 s (slow diffusing species) were taken for each sample. Raw data were processed on the ConfoCor3 software. R Statistics was used for normalization and graphs. The following fit function was used to fit samples with one component and including triplet state:

TD is the diffusion time. T represents the fraction of fluorophores in triplet state with the corresponding triplet time T _tr i _p . N is the number of particles and R the structural parameter. R was set to 5 if fit results yielded R smaller than 3 or bigger than 8. The relation between x-y dimension of the confocal volume (co _xy ) and x _D was used to calculate diffusion coefficients (D).

6Jxy 2

^" CD =

4D

Einstein- Stokes equation was utilized to calculate hydrodynamic radii (RH). k _B is the

Boltzmann's constant, T the absolute temperature, and η the viscosity of the surrounding medium. ksT

D =

R _H was calculated for each of the 30 curves, and data are presented as mean ± standard deviation. To obtain nanomimic concentrations, calibration of the confocal volume was needed. This was obtained by measuring a series of dye solutions with known concentrations from 1 nM to 100 nM. A linear fit of dye concentration versus N - obtained from amplitudes of FCS curves - yielded the size of the confocal volume (approximately 0.5 fl). This calibrated value was subsequently used to determine nanomimic concentrations.

For binding studies, MSP1 ₄₂ -0G488 was mixed with controls or nanomimics, incubated under shaking at 37 °C for 2 hours, cooled to RT (20 °C), put on the cover slide, incubated 5 min and FCS curves were recorded. Autocorrelation curves that could not be fitted due to big diffusing aggregates were excluded from the mean (max. 3 of 30 curves, 10%).

For FCCS measurments, the lasers were simultaneously focused onto the sample through a 40x C-Apochromat water immersion objective with a numeric aperture of 1.2 and the appropriate filter sets (Green: BP 505-550 nm, red: LP 650 nm) to separate the two colors (green/red). All measurements were performed at 20 °C. FCCS calibration data to test the relative minimal cross-correlation amplitude, which is given by the cross-talk from one channel to the other, was estimated using a mixture of the two used dyes (OG488 and Bodipy630) and the relative maximum cross-correlation amplitude using a standard sample (IBA standard, double labeled DNA). Different concentrations of MSP1 ₄₂ -0G488 were mixed with diluted control polymersomes (PMOXA-6-PDMS-6-PMOXA) or nanomimics-25% that were both stained with Bodipy630, immediately added onto the sample plate, and incubated at 20 °C on the glass plate for 5 min before recording fluorescence intensity fluctuations in both detection channels (green/red) 3 Ox for 10 seconds each. This procedure was repeated in three independent experiments for each concentration. Intensity fluctuations recorded in both channels were auto- (FCS) and cross-correlated (FCCS) on the ConfoCor3 software to yield auto-correlation and cross-correlation curves. R Statistics was used for preparing graphs.

FCCS data was analyzed using the following procedure. All auto- and cross-correlation curves were fitted using a one-component model without triplet state:

1 1

G(x)fit = l + ½ Where TD is the diffusion time. N is the number of particles, and R the structural parameter (aspect ratio of detection volume), which was fixed to 5.

Relative cross-correlation amplitudes (RCA) were calculated to obtain the fractional occupancy of the binding sites on nanomimics (32):

G _c (0)-1

RCA =

G _r<5 (0)-1

Where G _c (0) is the cross-correlation amplitude and G _{r g} (0) the auto-correlation amplitudes of the respective red or green auto-correlation curves. The average data of normalized RCA (n = 3 for each concentration) and corresponding standard errors were plotted against the total ligand concentration ( MSP1 ₄₂ -OG488) and fit using the following formula in QtiPlot (http://soft.proindependent.com/qtiplot.html) to obtain IQ for the interaction (26, 33).

[AB] _ \Ao ] + \ Bo } +K _d -^( lA ₀ ] + \ B ₀ } +Ka) ² →{A ₀ } lB ₀ }

RCA

2 \A ₀ )

Where [AB] is the complex concentration, [Ao] the accessible heparin concentration (2.8 nM fixed), [B ₀ ] the MSPI ₄₂ -OG488 concentration, and IQ the dissociation constant.

Quantitative fluorescence correlation spectroscopy (FCS) measurements were used to calculate concentrations of nanomimics in solution by first measuring a series of known dye

concentrations to calibrate the confocal volume (26).

Static and dynamic light scattering. Dynamic light scattering (DLS) was performed on a Zetasizer Nano ZSP (Malvern Instruments Ltd, UK) at 20 °C. Static and dynamic light scattering (SLS/DLS) was carried out to determine the hydrodynamic (R _h ), the radius of gyration (R _g ) and the value p = R _g /Rh of nanomimics in solution. SLS and DLS experiments were performed on an ALV goniometer (ALV GmbH, Germany), equipped with an ALV He- Ne laser (λ = 632.8 nm). Measurements were performed in 10 mm cylindrical quartz cells at angles of 30 - 140° at 20 °C. Data processing was performed using ALV static & dynamic fit and plot software (version 4.31 10/01). SLS data were processed using the Berry-model.

Viability MTS assay. HeLa cells (2Ό00 cells per well) were seeded in a 96-well plate, and incubated at 37°C, 5 % C0 ₂ for 24 h in DMEM containing 10 % fetal calf serum and 1% Penicillin Streptomycin. After 24 hours, nanomimics were added to triplicate wells at concentrations ranging from about 50-300 μg/mL in a total volume of 100 μΐ^ per well (90 μΐ^ media mixed with 10 μΐ _^ nanomimic solution in PBS). Cells were incubated in the presence of nanomimics for an additional 24 h. Cell viability was determined using the MTS assay. Briefly, 20 μΐ, MTS assay solution was added to each well and incubated at 37 °C for 3 hours. Cell viability was determined by measuring absorbance at 490 nm using a microplate reader SpectraMax Plus M5e (Molecular Devices, Sunnyvale, California) and comparing to a PBS control (100 % cell viability) to obtain the percentage of living cells. All samples were corrected against controls containing only media and PBS or an SRB solution in PBS.

Suspension culture assay. Plasmodium falciparum 3D7 strain was maintained in culture as described elsewhere (16). Malaria culture medium (MCM) was RPMI medium supplemented with 0.5 % Albumax (17). Synchronization was performed using 5 % sorbitol (18).

Invasion inhibition experiments were carried out in standard 24-well flat-bottom culture plates (Falcon 353047, Becton Dickinson, NJ, USA). Starting parasitemia was about 0.1 % ring/trophozoite stages at 5 % hematocrit in MCM and parasitemia reached ~ 2 - 4 % after 96 hours. For the suspension culture assay with nanomimics-5%, nanomimics-ShortHep, and micelles-ShortHep (Figure 8) MCM was supplemented with 0.5 % CellMaxx bovine albumin instead of 0.5 % Albumax and starting parasitemia was about 0.2 %. Total volume added to each well was 500 μΐ of parasite culture plus 55 μΐ PBS or test samples. Plates were placed in a plastic box with wet paper (for humidification) and cultured under continuous and

simultaneous rocking (140 RPM, ProBlot 25 Rocker, Labnet International Inc., NJ, USA) and shaking (105 RPM, Lab-Therm LT-W, Kuhner, Switzerland) at 37 °C for 96 hours by fixing the ProBlot 25 Rocker onto the shaking plate of the Lab-Therm shaking incubator. The maximum tilt angle was increased to 15° by putting one side of the plastic box on a 3 cm thick spacer. This setup ensures continuous suspension of RBCs and iRBCs over the 96-hour incubation period. Each sample was tested in at least 3 independent assays in duplicates. After 96 hours, parasitemia was determined by flow cytometry (FACSCalibur, BD Biosciences) using dihydroethidium to stain parasite DNA. In total, 100Ό00 cells were counted for each well. Data are presented as mean growth ± SEM with respect to PBS controls. Statistical comparison of free heparin versus nanostructured heparin was performed using Student's t-test (two-tailed, type 2) in Microsoft Excel. Graphs were drawn using QtiPlot

(http://soft.proindependent.com/qtiplot.html). To obtain IC ₅ o values, experimental growth inhibition curves were fitted using logistic or exponential curves in QtiPlot. Preparation of parasites for fluorescence microscopy. Mature 3D7 parasites (trophozoites/schizonts) were purified by Percoll density gradient (19). Then, purified late stages were mixed again with RBCs to yield a mixture at 20 % parasitemia. Polymersomes or nanomimics (both filled with fluorescent dye SRB) were then added to this mixture and incubated at 37 °C for 3 hours under static conditions. After incubation, cells were fixed using 2 % paraformaldehyde/0.2 % glutaraldehyde in phosphate buffer 0.1 M (pH 7.4) at 4 °C overnight. Then, fixed samples were centrifuged at high speed (13.5 kRPM) and redissolved in a small amount of PBS and finally mounted on a slide using Vectashield supplemented with D API (Vector Laboratories) .

Preparation of parasites for TEM and 3D-SIM. For TEM and 3D-SIM imaging, merozoites were mechanically released from mature schizonts using a published protocol (20). Briefly, 3D7 mature parasites were purified by Percoll density gradient and incubated with 10 μΜ E-64 inhibitor. After 6 - 8 hours incubation, mature schizonts were filtered through 1.2 μιη filters to release merozoites mechanically, immediately mixed with nanomimics and incubated for 20 min at 37 °C. Then, merozoites were fixed in 2% paraformaldehyde/0.2 % glutaraldehyde in phosphate buffer 0.1 M (pH 7.4) at 4 °C overnight. For TEM, samples were prepared according to Tokuyasu (21). Briefly, merozoite-nanomimic complexes were washed in PBS, embedded in 12 % gelatin and thin blocks were infiltrated with 2.3 M sucrose overnight at 4°C. Each centrifugation step was performed at high speed (13.5 kRPM). Ultrathin sections (80 to 100 nm) were prepared on a FC7/UC7-ultramicrotome (Leica) at -120 °C. For 3D-SIM fixed merozoites were collected by fast centrifugation (13.4 kRPM) and then mounted on a slide using Vectashield with DAPI as mounting medium.

Fluorescence microscopy and super-resolution 3D structured illumination microscopy (3D-SIM). Fluorescence micrographs were taken on a Leica DM 5000B fluorescence microscope. Image processing was performed on GIMP software.

3D-SIM was performed on a microscope system (Delta Vision OMX-Blaze version 4; Applied Precision, Issaquah, WA) equipped with 405, 445, 488, 514, 568 and 642 nm solid-state lasers. Images were acquired using a Plan Apo N 60 ^x 1.42 NA oil immersion objective lens

(Olympus) and 4 liquid-cooled sCMOs cameras (pco Edge, full frame 2560 x 2160; Photometries). Exciting light was directed through a movable optical grating to generate a fine-striped interference pattern on the sample plane. The pattern was shifted laterally through five phases and three angular rotations of 60° for each z section. Optical z-sections were separated by 0.125 μιη. The laser lines 405 and 568 nm were used for 3D-SIM acquisition. Exposure times were typically between 3 and 50 ms, and the power of each laser was adjusted to achieve optimal intensities of between 7,000 and 10,000 counts in a raw image of 15-bit dynamic range at the lowest laser power possible to minimize photobleaching. Multichannel imaging was achieved through sequential acquisition of wavelengths by separate cameras. Raw 3D-SIM images were processed and reconstructed using the Delta Vision OMX

SoftWoRx software package (Applied Precision [22, 23)). The resulting size of the reconstructed images was 128 x 128 px from an initial set of 64 x 64 raw images. The channels were aligned in the image plane and around the optical axis using predetermined shifts as measured using a target lens and the SoftWoRx alignment tool. The channels were then carefully aligned using alignment parameter from control measurements with 0.5 μιη diameter multi- spectral fluorescent beads (Invitrogen, Molecular Probes).

Anticoagulation property of nanomimics. Anticoagulation property of heparin-containing samples was measured using a chromogenic anti-Xa assay (Biophen Heparin (LRT) kit and manual) at the University Hospital Basel (Prof. D. Tsakiris). The detection limit is 0.1 Ul/ml.

Experiment on "vaccine-like" action of nanomimics with surface-bound immunogen. 3x2

BALB/c mice (12 weeks old) were immunized on day 0 with either a) 150 μΐ of 0.1 mg/ml MSP1 ₄₂ (3D7) in PBS intravenously (i.v.), b) 150 μΐ of 0.1 mg/ml MSPI42 (3D7) in PBS containing 50% Sigma- Adjuvant® subcutaneously (s.c), or c) 150 μΐ of nanomimics-25% that were first incubated with 0.1 mg/ml MSPI ₄₂ (3D7) in PBS and then injected i.v. On day 24 a second shot was given via the same routes for a)-c) but with slightly lower protein

concentrations (150 μΐ, 0.065 mg/ml each). All mice were terminally bled on day 31. The collected blood was centrifuged, the sera was separated and stored at -20°C. Induced IgG antibody titers were quantified using ELISA. Briefly, Nuc-Maxisorb ELISA plates were coated with 3 μg/ml MSPI ₄₂ (3D7) in PBS (50 μΐ/well) at 4° for 48 h. Plates were washed and blocked with 3% BSA in PBS (100 μΐ/well) at RT for 1 h. Test-sera were diluted 1 : 10 in 1% BSA TNT and serially diluted on the plate up to 1 :20'048. Serum dilutions were incubated at RT for 2 h. Plates were washed and incubated with anti-mouse IgG alkaline phosphate labeled antibodies (1 :5000) 1% BSA TNT at RT for lh. Plates were developed with lmg/ml pNPP in carbonate buffer pH 8.6 for 20 minutes and absorbance was measured at 405nm. Antibody endpoint titers are given for the last dilution where the absorbance was two times the absorbance of the negative control.

Example 2: Block copolymer synthesis

Heparan sulfate has been shown to play a critical role as a receptor for the initial attachment of merozoites to RBCs (2); therefore we selected the closely related heparin, which is a potent inhibitor of merozoite invasion of RBCs in vitro (2), for exposure on the exterior surface of nanomimics. To produce model nanomimics three different block copolymers were

synthesized. The biocompatible, polymersome-forming ABA block copolymer poly(2-methyl- 2-oxazoline)-¾/oc^-poly(dimethylsiloxane)-¾/oc^-poly(2-met hyl-2-oxazoline) (ΡΜΟΧΑ-δ- PDMS-6-PMOXA, Fig. 1, 1) was synthesized as previously published (13), and the PDMS- heparin block copolymer (Fig. 1, 2) and PDMS-ShortHep were synthesized by coupling a commercial PDMS block (5 kDa) with commercial heparin polysaccharide of mean starting molecular weight of 15 kDa or 5 kDa, respectively. A critical step was the solubilization of heparin in organic solvents, which is not possible with commercial sodium salt of heparin, but was needed for PDMS-heparin synthesis and nanomimic formation. Therefore, it was first hydrophobized by ion exchange from sodium to tetrabutylammonium (12), which exchanged again with sodium during nanomimic purification. ^-NMR after reaction, purification and ion exchange indicated successful coupling of PDMS with heparin. The reductive amination used for PDMS-heparin synthesis is a mild reaction; the reducing agent is specific to imines (24). This specific reaction ensured that heparin was only modified with PDMS at its natural anchor point - leaving the rest of the chain unmodified. Farndale microassay in ethanol was used to estimate the length of heparin chains and the corresponding number of repeating units per PDMS block. PDMS-heparin was found to have an average of 23 ± 10 monosaccharide repeating units per PDMS-chain of 65 silxoane units, whereas PDMS-ShortHep had only 6 ± 3 monosaccharide repeating units per PDMS-chain of 65 silxoane units. Clearly, PDMS-Hep contained a 3- to 4-fold longer heparin chain compared to PDMS-ShortHep, while the hydrophobic blocks were similar. Example 3: Preparation and characterization of nanomimics

Nanomimics were self-assembled by the bulk rehydration technique using a mixture of

PMOXA-6-PDMS-6-PMOXA with PDMS-heparin block copolymers. To expose long or short heparin chains on nanomimics, they were formed using mixtures of 75 wt% ΡΜΟΧΑ-δ- PDMS-6-PMOXA with 25 wt% PDMS-heparin (nanomimics-25%) or 25 wt% PDMS-

ShortHep (nanomimics-ShortHep-25%). To test the effect of the number of heparin chains on nanomimic antimalarial potency, another mixing ratio of the functional copolymer (heparin- containing) and polymersome-forming copolymer was used with 95 wt% PMOXA-¾-PDMS-¾- PMOXA and 5 wt% PDMS-heparin (nanomimics-5%). For comparison, pure PDMS- ShortHep-based micelles (100 wt% PDMS-ShortHep) were formed as well. Since heparin (mean 15 kDa) is a longer polymer than PMOXA (max. 0.8 kDa) in PMOXA-6-PDMS-6- PMOXA and PDMS blocks were similar in both copolymers (mean 5 kDa) only a small part of heparin was shielded by PMOXA whilst the rest was accessible for protein interaction.

Dynamic light scattering (DLS) revealed hydrodynamic nanomimic diameters (D _H ) of about 132 ± 34 nm (average of 8 independent samples ± SD). DLS and static light scattering (SLS) performed on four representative nanomimic samples provided R _g /R _h values οΐρ = 0.90 - 0.99, as expected close to the value of 1.0 for ideal hollow spheres (vesicles) (25), and thus indicating the vesicular structure of nanomimics. Transmission electron microscopy (TEM) and cryogenic-TEM (cryo-TEM) confirmed these vesicle sizes and illustrated the membranous structure of these nanomimics (Fig. 2). TEM micrographs showed collapsed nanomimics (D = 1 11 ± 38 nm), which clearly distinguished them from filled nanoparticles (Fig. 2A).

Nanomimics retained their spherical structure (D = 101 ± 33 nm) during cryo-TEM imaging (Fig. 2B), and revealed a vesicle-in- vesicle structure and a membrane thickness of 10.9 ± 1.4 nm. Self-assembly of PDMS-heparin copolymer alone yielded aggregated micelles with D _H of about 83 ± 26 nm (DLS).

Hydrophilic sulforhodamine B (SRB) was encapsulated in the aqueous core of nanomimics for visualization and both D _H and the concentration of the nanomimics were obtained with fluorescence correlation spectroscopy (FCS) by comparing diffusion time of the free dye with that of the encapsulated dye. Farndale microassays, in which positively charged dye molecules align on heparin chains resulting in a metachromatic shift in the absorption spectrum, were performed to quantify the amount of surface-accessible heparin after purification. Calibration curves were produced with the same heparin solution that was subsequently used in in vitro assays (Example 6) to ensure comparability of assays. Due to the random character of the self- assembly process underlying nanomimic formation, not all PDMS-heparin added led to surface- exposed heparin in the final nanomimics; part of the heparin chains will face towards the vesicle core, few will be hidden in the membrane or in the nanomimic core (vesicle-in- vesicle structures), and few free PDMS-heparin or free heparin chains not incorporated in the membrane were removed during purification. Importantly, nanomimic formation yielded detectable amounts of heparin being incorporated in the membrane after purification, whereas formation with PMOXA-¾-PDMS-¾-PMOXA and the tetrabutylammonium salt of heparin did not. In total, three independent PDMS-heparin and three PMOXA-6-PDMS-6-PMOXA batches were used. Typical nanomimic samples, theoretically containing 2.5 mg/ml ΡΜΟΧΑ-δ- PDMS-6-PMOXA and 0.833 mg/ml PDMS-heparin copolymer, yielded 57 ± 13 μ^πιΐ (mean ± SD) surface-exposed heparin after purification.

To validate the toxicity of nanomimics on cultured cells, a cell viability test was performed using HeLa cells and a representative nanomimic sample (2.9 mg/ml PMOXA-¾-PDMS-¾- PMOXA). This test demonstrated the absence of any cell toxicity of nanomimics up to the maximum concentration of 290 μg/ml PMOXA-6-PDMS-6-PMOXA and 4.9 μg/ml surface- exposed heparin, which was an even higher concentration than the highest concentration used in the antimalarial assays (Example 6). Example 4: Nanomimics bind Plasmodium merozoite proteins involved in initial attachment

FCS also allows the analysis of protein binding to nanoobjects by comparing diffusion times of freely diffusing and bound proteins labeled with fluorescent molecules (26). For the FCS measurements, OregonGreen® was used to label the P. falciparum (clone 3D7) major surface protein 1-42 ( MSP1 ₄₂ -0G488), which has been identified as the ligand for heparin-like receptors (2). The difference in diffusion times between MSP1 ₄₂ -0G488 and MSPI ₄₂ - OG488 bound to nanomimics was used to verify that the surface of nanomimics contained heparin molecules, and that they were able to bind merozoite proteins (Fig. 3A, B). No binding of MSPl ₄₂ -OG488occurred with polymersomes without heparin. In contrast, nanomimics and PDMS-heparin micelles bound significant amounts of MSP1 ₄₂ -0G488.

The interaction of heparin exposed on nanomimics with MSP1 , in particular the processed 42 kDa fragment MSPI ₄₂ , which is one of the most abundant merozoite surface protein that binds HS and heparin, was analyzed in detail. Fluorescence cross-correlation spectroscopy (FCCS) was selected as a suitable single-molecule detection method to evaluate receptor- ligand interactions (26, 32, 33). First, FCCS calibration data were recorded. Both, pure PMOXA-6-PDMS-6-PMOXA polymersomes-Bodipy630 mixed with MSP1 ₄₂ -0G488, and nanomimics-25%-Bodipy630 incubated with free Oregon Green 488 (OG488) yielded no significant cross-correlation amplitudes. Therefore, no unspecific binding of MSP1 ₄₂ -0G488 to the surface of non-functionalized polymersomes (without heparin) was observed, nor did the dye OG488 bind nanomimics. To study the interaction of MSP1 ₄ 2 with heparin on nanomimics, red fluorescent nanomimics-25%-Bodipy630 were mixed with different concentrations of green fluorescent MSP1 ₄₂ -0G488, and the diffusion of fluorescent species were recorded in two detection channels simultaneously. Titration of MSP1 ₄ 2-0G488 into a solution of nanomimics, and analysis of the corresponding relative cross-correlation amplitudes yielded a typical ligand-receptor saturation curve (Figure 7). At the highest MSP1 ₄₂ -0G488 concentration tested, four MSP1 ₄ 2-0G488 were bound to each nanomimic, which was calculated by comparing the signal per molecule (CPM = 6.8 kHz) of MSP l ₄₂ -OG488 to CPM of the nanomimic- MSPl ₄₂ -OG488 complex (CPM = 29 kHz). A constant

concentration of 2.8 nM MSPl ₄ 2-OG488-binding heparins on nanomimics in the titration experiment was calculated. This value was fixed for the fitting of the normalized ligand- receptor saturation curve (Figure 7). Fitting the saturation curve of MSP1 ₄ 2-0G488 with heparin on nanomimics-Bodipy630 (Figure 7) yielded a IQ of 12.1 ± 1.6 nM for the interaction, confirming the hypothesis that the interaction of MSP1 ₄ 2 with nanomimics is based on a high affinity interaction. This helps to explain the potent invasion inhibitory effect of nanomimics against malaria parasites (Figure 6, 8). Example 5: Nanomimics block invasion of and expose Plasmodium merozoites. To test whether our nanomimics could competitively bind freshly egressed merozoites in vitro, we incubated a mixture of RBCs and late stages of infected RBCs (iRBCs) with these nanomimics for three hours. During this time, merozoites start to be released and invade new RBCs. After incubation with fluorescent nanomimics, nanomimics-5%, and nanomimics-ShortHep fluorescence imaging showed both binding of nanomimics to merozoites and lack of invasion (Fig. 4). In addition, agglutination of merozoites and nanomimics, and nanomimic binding to iRBC just during merozoite egress were observed, whereas intact iRBCs and RBCs did not bind nanomimics. Polymersomes without heparin did not bind any of the cells present (RBCs, iRBCs, or merozoites). The merozoite-nanomimic interaction was further examined by super- resolution 3D structured illumination microscopy (3D-SIM), and electron microscopy (EM) of ultrathin sections of merozoite-nanomimic complexes (Fig. 5B). 3D reconstruction of merozoites with surface-bound nanomimics showed that several dozens of nanomimics bound to a single merozoite. The EM images of ultrathin sections revealed nanomimics attached to the outer membrane of merozoites (Fig. 5B), which was not found in a preparation without nanomimics (Fig. 5 A). Example 6: Efficacy of invasion inhibition by nanomimics. The inhibitory effect of nanomimics on the parasite life cycle was determined by a growth inhibition assay using a P. falciparum (clone 3D7) suspension culture in 24-well plates, which better mimics the in vivo situation by using suspension cultures with a higher hematocrit than usually used for measurements of drug effects (typically 1 % (27)). Invasion-inhibition curves with free heparin, nanomimics, and PDMS-heparin micelles (Fig. 6) show a significant difference in IC5 ₀ values between free heparin and nanomimics or PDMS-heparin micelles (Fig. 6A). IC5 ₀ values dramatically decreased from 37.4 ± 4.7 μg/ml (2.5 μΜ for free heparin) to 0.197 ± 0.047 μg/ml for the best preparation of nanomimics (13 nM of heparin on nanomimics2) (Fig. 6B). This corresponded to a decrease in IC5 ₀ value of more than two orders of magnitude (Fig. 6C), which indicates a very highly efficient inhibitory effect of the nanomimics. The highest concentration of nanomimics tested contained about 60 μg/ml of the polymersome-forming PMOXA-¾-PDMS-¾-PMOXA and 1.3 μg/ml surface-accessible heparin. Polymersomes consisting of PMOXA-6-PDMS-6-PMOXA without PDMS-heparin had no effect on the parasite life cycle at similar concentrations (60 μg/ml). The IC5 ₀ value for free heparin was two fold higher than previously published (2), most probably due to the higher hematocrit (5%) and suspension culture. Nanomimics with different ratios of PDMS-heparin to ΡΜΟΧΑ-δ-PDMS- δ-ΡΜΟΧΑ were also tested in order to find the optimum mixture. A mixture of 1 :3 provided the best balance in terms of efficacy and control over self-assembly (spherical vesicles (Fig. 2). Nanomimics were also significantly more effective than micelles self-assembled from PDMS- heparin only, most likely because the membranous structure of the nanomimics allowed lateral diffusion of receptors for multivalent interactions (7). Considering that the active inhibitors are nanomimics themselves, then IC5 ₀ value can also be presented in 'nanomimic-concentrations' as determined by FCS, and results in an IC ₅ o value of 0.27 ± 0.09 nM (5 independent samples, 3 invasion inhibition assays), which corresponds to an IC ₅ o shift of ~ 10,000 fold compared to free heparin.

When less heparin chains were exposed on a single nanomimic, which was the case for nanomimics-5%, more nanomimics were needed to obtain the same antimalarial effect as with nanomimics-25% (same amount of heparin was necessary in the final assay, Fig. 8), which becomes visible when these samples are compared based on the nanomimic concentration (nanomimics-25% IC5 ₀ ~ 0.3 nM and nanomimics-5% IC5 ₀ ~ 4.4 nM) rather than the total heparin content (Fig. 8). Therefore, nanomimics with less heparin chains, but with identical lengths, are less potent invasion inhibitors in comparison to nanomimics with more heparin chains on the surface.

Nanomimics-ShortHep were also very potent in invasion inhibition, although the exposed heparin chains were short. The antimalarial activity of these nanomimics with short, but more heparin chains on the surface (25% is based on wt%) was similar to nanomimics-25%, which were assembled using less, but longer heparin block copolymers. For soluble heparin it has previously been reported that the antimalarial activity drops for very short chains with a number of repeat units (monosaccharides) below six (2). Our findings of very potent

ShortHep-based nanomimics can be explained by the multivalent presentation of heparin chains on a single nanomimic, which allow strong enough interactions to block invasion. In case of both, long and short heparin block copolymers, the known flexibility and fluidity of PDMS- based membranes can further promote recruitment of more heparin chains upon initial binding of one chain to the parasite by diffusion of PDMS-heparin within the nanomimic membrane to yield stronger multivalent binding.

This higher inhibitory activity of nanomimics compared to free heparin (> 2 orders of magnitude) can be explained by multivalent interactions between a multitude of heparin molecules on a single nanomimic with a multitude of MSPI ₄₂ molecules (Fig. 3B, max. 12 MSP1 ₄₂ -0G488 per nanomimic measured) confirming results from binding assays. Indeed, nanomimics represent 'macroscopic' objects when compared to free heparin, and thus the binding of a few nanomimics is sufficient to block merozoites, whilst a very large number of free heparin molecules (-10,000 times more) is required for such an effect.

Example 7: Reduced anticoagulation properties of nanomimics. Anticoagulation property of heparin is a major drawback when heparin is intended for another medical purpose.

Therefore, heparin-containing samples (nanomimics) were tested for anticoagulation property and the obtained values compared to the surface-exposed heparin amount measured by

Farndale microassays. Control vesicles (only PMOXA-¾-PDMS-¾-PMOXA), four independent samples of nanomimics-25% (26 μg/ml, 78 μg/ml, 102 μg/ml, 146 μg/ml) and one sample nanomimics- 12.5% (50 μg/ml) were measured. Only in one sample, nanomimics-25% (102 μg/ml heparin), a slight anticoagulation activity (0.15 Ul/ml) was observed; all other samples did not reach activities above the detection limit. To calculate the maximum expectable percentage of anticoagulation property for nanomimic samples, the heparin concentration was converted from 102 μg/ml to 19.7 Ul/ml (heparin used for polymer synthesis, 193 Ul/mg) and compared to the measured activity (0.15 Ul/ml), which resulted in a value of 0.8 %. Therefore, the whole procedure of synthesis and nanomimic formation yields a final nanostructure with much reduced anticoagulation property (only 0.8 % of the expected activity is left). Example 8: "Vaccine-like" action of nanomimics with surface-bound immunogen. A preliminary experiment was performed to assess the "vaccine-like" action of nanomimics. For simplicity, only one merozoite protein was used for this initial test (not the whole pathogen) and these nanomimic-protein complexes were injected intravenously to mimic the situation when nanomimics with surface bound immunogen (in the real case it would be the whole pathogen) would appear in the bloodstream after successful inhibition of merozoite invasion in vivo. In more detail, nanomimic- MSPl ₄₂ complexes were injected intravenously and production of anti- MSPl ₄ 2 IgG antibodies was assessed. Measured ELISA endpoint titers show clearly that anti- MSPl ₄₂ IgG antibodies were produced after injection of nanomimic- MSPI ₄₂ complexes intravenously (Endpoint titer for both mice 640 ELISA units). Controls with intravenously applied MSPI42 in PBS also induced anti- MSPl ₄ 2 IgG antibodies

(Endpoint titer for both mice 1280 ELISA units). Due to the small sample size (only 2 mice per group) no conclusion about the significance of this difference can be drawn at this stage. As expected a standard immunization protocol with subcutaneous injection of MSPI ₄₂ in adjuvants did induce a much stronger antibody titer (one mouse 10240, and one mouse >20480 ELISA units). Nevertheless, these data demonstrate that nanomimics with surface bound immunogen can elicit an immune response in a subject (here mice), which was demonstrated by the appearance of anti- MSPl ₄₂ IgG antibodies after intravenous injection of nanomimic- MSPI ₄₂ complexes. Similarly, invasion inhibition of parasite invasion in vivo will expose not only one protein (as demonstrated here with MSPI ₄₂ ), but the whole pathogen - with all its proteins - in the bloodstream. Even if antibody production will be similarly low as

demonstrated here with one protein on nanomimics, antibodies to multiple merozoite proteins could still lead to a highly inhibitory antibody mixture against different merozoite surface proteins, when the whole merozoite is exposed after invasion inhibition by nanomimics in vivo. Furthermore, with the whole pathogen exposed on nanomimics, the route of the complex will be determined by the pathogen, which is much bigger than the nanomimics, compared to here, where one merozoite protein is much smaller than the nanomimics. This could change the fate and final immune response against merozoites when blocked by nanomimics in vivo.

Conclusion

Nanomimic-merozoite complexes (Fig. 4,5) are expected to be taken up and processed by antigen-presenting cells, and hence the whole merozoite could serve as a potent immunogen. In a preliminary experiment (Example 8) the appearance of antibodies against one merozoite protein was found after applying nanomimics-protein complexes in mice intravenously.

Additionally, as soluble heparin can reverse the two major pathogenic events in P. falciparum infections - sequestration and resetting - in vitro and in vivo (28, 29) it would be expected that nanomimics might have a similar effect. Since the interaction between sporozoites and hepatocytes also involves heparan sulfate as attachment receptors (30), it is expected that nanomimics could also prevent liver cell invasion by sporozoites.

Thus our strategy of interrupting the parasite life cycle using nanomimics and then

subsequently eliciting an immune response represents a promising alternative to current drug and vaccination strategies (31). Further, nanomimics offer theoretically a unique possibility of encapsulating high concentrations of adjuvants or other immune-modulators, which could be released after phagocytosis to enhance immunogenicity. We have used these nanomimics for malaria as proof of principle, but a variety of other pathogens also use host cell heparan sulfate for initial attachment (1); therefore this technology could be directly applied to inhibit other infections and could potentially provide a strong immunological boost. List of references :

1. Pavao MSG (2011) Glycans in Diseases and Therapeutics (Springer- Verlag, Berlin Heidelberg, Berlin Heidelberg).

2. Boyle MJ, Richards JS, Gilson PR, Chai W, Beeson JG (2010) Interactions with

heparin-like molecules during erythrocyte invasion by Plasmodium falciparum merozoites. Blood 115:4559-4568.

3. Srinivasan P et al. (2013) Disrupting malaria parasite AMA1-RON2 interaction with a small molecule prevents erythrocyte invasion. Nat Commun 4:2261.

4. Richards JS, Beeson JG (2009) The future for blood- stage vaccines against malaria.

Immunol Cell Biol 87:377-390.

5. The RTS,S Clinical Trials Partnership (2012) A Phase 3 Trial of RTS,S/AS01 Malaria Vaccine in African Infants. N Engl J Med 367:2284-2295.

6. Santos-Magalhaes NS, Mosqueira VCF (2010) Nanotechnology applied to the

treatment of malaria. Adv Drug Deliver Rev 62:560-575.

7. Bricarello DA, Patel MA, Parikh AN (2012) Inhibiting host-pathogen interactions using membrane-based nanostructures. Trends Biotechnol 30:323-330.

8. Graff A, Sauer M, Van Gelder P, Meier W (2002) Virus-assisted loading of polymer nanocontainer. Proc Natl Acad Sci USA 99:5064-5068.

9. Nazemi A, Haeryfar SMM, Gillies ER (2013) Multifunctional dendritic

sialopolymersomes as potential antiviral agents: their lectin binding and drug release properties. Langmuir 29:6420-6428.

10. Zhang J et al. (2013) Long-circulating heparin- functionalized magnetic nanoparticles for potential application as a protein drug delivery platform. Mol Pharmaceutics 10:3892- 3902.

11. Kita-Tokarczyk K, Grumelard J, Haefele T, Meier W (2005) Block copolymer

vesicles— using concepts from polymer chemistry to mimic biomembranes. Polymer 46:3540-3563.

12. Liu J et al. (1994) New approaches for the preparation of hydrophobic heparin

derivatives. J Pharm Sci 83: 1034-1039.

13. Kumar M, Grzelakowski M, Zilles J, Clark M, Meier W (2007) Highly permeable

polymeric membranes based on the incorporation of the functional water channel protein Aquaporin Z. Proc Natl Acad Sci USA 104:20719-20724.

14. Farndale RW, Sayers CA, Barrett AJ (1982) A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. Connect Tissue Res 9:247-248.

15. Barbosa I et al. (2003) Improved and simple micro assay for sulfated

glycosaminoglycans quantification in biological extracts and its use in skin and muscle tissue studies. Glycobiology 13:647-653.

16. Trager W, Jensen JB (1976) Human malaria parasites in continuous culture. Science 193:673-675.

17. Dorn A, Stoffel R, Matile H, Bubendorf A, Ridley RG ( 1995) Malarial haemozoin/β- haematin supports haem polymerization in the absence of protein. Nature 374:269-271.

18. Lambros C, Vanderberg JP (1979) Synchronization of Plasmodium falciparum

erythrocytic stages in culture. J Parasitol 65:418-420.

19. Rivadeneira EM, Wasserman M, Espinal CT (1983) Separation and concentration of schizonts of Plasmodium falciparum by Percoll gradients. J Protozool 30:367-370. 20. Boyle MJ et al. (2010) Isolation of viable Plasmodium falciparum merozoites to define erythrocyte invasion events and advance vaccine and drug development. Proc Natl Acad Sci USA 107: 14378-14383.

21. Tokuyasu KT (1973) A technique for ultracryotomy of cell suspensions and tissues. J Cell Biol 57:551-565.

22. Gustafsson MG (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198:82-87.

23. Schermelleh L et al. (2008) Subdiffraction Multicolor Imaging of the Nuclear Periphery with 3D Structured Illumination Microscopy. Science 320: 1332-1336.

24. Sato S, Sakamoto T, Miyazawa E, Kikugawa Y (2004) One-pot reductive amination of aldehydes and ketones with a-picoline-borane in methanol, in water, and in neat conditions. Tetrahedron 60:7899-7906.

25. Stauch O, Schubert R, Savin G, Burchard W (2002) Structure of artificial cytoskeleton containing liposomes in aqueous solution studied by static and dynamic light scattering. Biomacromolecules 3:565-578.

26. Rigler P, Meier W (2006) Encapsulation of fluorescent molecules by functionalized polymeric nanocontainers: investigation by confocal fluorescence imaging and fluorescence correlation spectroscopy. J Am Chem Soc 128:367-373.

27. Persson KEM, Lee CT, Marsh K, Beeson JG (2006) Development and optimization of high-throughput methods to measure Plasmodium falciparum-specific growth inhibitory antibodies. J Clin Microbiol 44: 1665-1673.

28. Vogt AM et al. (2003) Heparan sulfate on endothelial cells mediates the binding of Plasmodium falciparum-mfected erythrocytes via the DBL1 alpha domain of PfEMPl . B lood 101 :2405-2411.

Vogt AM et al. (2006) Release of sequestered malaria parasites upon injection of a glycosaminoglycan. PLoS Pathog 2:853-863.

30. McCormick CJ, Tuckwell DS, Crisanti A, Humphries MJ, Hollingdale MR (1999) Identification of heparin as a ligand for the A-domain of Plasmodium falciparum thrombospondin-related adhesion protein. Mol Biochem Parasitol 100: 111-124. Bijker EM et al. (2013) Protection against malaria after immunization by chloroquine prophylaxis and sporozoites is mediated by preerythrocytic immunity. Proc Natl Acad Sci USA 110:7862-7867.

Bacia K, Kim SA, Schwille P (2006) Fluorescence cross-correlation spectroscopy in living cells. Nat Methods 3:83-89.

Sasaki A, Kinjo M (2010) Monitoring intracellular degradation of exogenous DNA using diffusion properties. J Control Release 143: 104-111.