Method for introducing a single-chain polymer nanoparticle into a prokaryotic cell.

Abstract

The invention provides a method for introducing a nanoparticle (100) into a prokaryotic cell (10), wherein the method comprises exposing the prokaryotic cell (10) to the nanoparticle (100), wherein: the nanoparticle (100) has an equivalent spherical diameter selected from the range of -20 nm; the nanoparticle (100) comprises a single-chain polymer nanoparticle (101); the nanoparticle (100) comprises a backbone (110), a plurality of intramolecular crosslinks (120), and a plurality of side groups (130) bound to the backbone (110); wherein the backbone (110) comprises n backbone atoms, wherein the n backbone atoms comprise ≥ 0.9*n carbon atoms; each intramolecular crosslink (120) covalently interconnects two backbone locations (112) of the backbone (110); k of the plurality of side groups (130) comprise ligand moieties (140) selected from the group comprising sugar moieties, a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, amino acid moieties, and fatty acid moieties, wherein k / n is selected from the range of 0.05-0.49; the prokaryotic cell (10) comprises a receptor (14) configured to interact with one or more of the ligand moieties (140).

Description

[0001] Method for introducing a single-chain polymer nanoparticle into a prokaryotic cell.

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a method for introducing a nanoparticle into a prokaryotic cell. The invention further relates to a nanoparticle for introduction into a prokaryotic cell. The invention further relates to a method for providing a nanoparticle.

[0004] BACKGROUND OF THE INVENTION

[0005] Nanoparticles for delivering cargo, such as antibiotics, to prokaryotic cells are known in the art. For instance, WELDRICK, PJ et al., Enhanced Clearing of Wound-Related Pathogenic Bacterial Biofilms Using Protease-Functionalized Antibiotic Nanocarriers, CS Appl. Mater. Interfaces 2019, 11, 47, 43902-43919, describes protease-functionalized nanogel carriers of antibiotics. It describes that these antibiotic nanocarriers, surface coated with the protease Alcalase 2.4 L FG, “digest” their way through a biofilm EPS matrix, reach the buried bacteria, and deliver a high dose of antibiotic directly on their cell walls.

[0006] HAMELMANN, NM et al, “Cytosolic Delivery of Single-Chain Polymer Nanoparticles”, ACS Macro Letters, 2021, 10, 1443-1449, describes the synthesis of active ester-functional Single-Chain Polymer Nanoparticles (SCNPs) of approximately 10 nm via intramolecular thiol-Michael addition cross-linking. Cellular uptake of the SCNPs was analyzed, presenting different uptake behavior depending on the degree of functionalization.

[0007] Kroger, APP et al., “Biocompatible Single-Chain Polymer Nanoparticles for Drug Delivery — A Dual Approach”, ACS Applied Materials & Interfaces, 2018, 10, 20946- 30951, describes SCNPs as uniquely sized nanocarriers that are capable of drug encapsulation independent of the polarity of the employed medium.

[0008] Hamelmann NM et al., “Single-Chain Polymer Nanoparticles Targeting the Ookinete Stage of Malaria Parasites”, ACS Infectious Diseases, 2023, 9, 56-64, describes the use of single-chain polymer nanoparticles (SCNPs) to target the Malaria parasite at the ookinete stage, which is one of the stages in the mosquito. It describes that the nanocarrier system provides monodispersed particles of 5-20 nm, via thiol-Michael addition, and that the conjugation of succinic anhydride to the SCNP surface provides negative surface charges that have been shown to increase the targeting ability of SCNPs to Plasmodium berghei ookinetes.

[0009] Hamelmann NM and Paulusse JMJ, “Single-chain polymer nanoparticles in biomedical applications”, Journal of Controlled Release, 2023, 356, 26-42, reviews different crosslinking systems, as well as the preparation of functional SCNPs and the variety of biomedical applications that have been explored.

[0010] SUMMARY OF THE INVENTION

[0011] Antimicrobial resistance has been recognized by the World Health Organization (WHO) as one of the biggest threats to human health. Currently, an estimated 700.000 people die each year due to antimicrobial resistance (AMR), which has been projected to increase to ten million people in 2050. Of particular concern is the emergence of multi-resistant pathogens (“superbugs”), for which hardly any antibiotics may be available. Antibiotic efficiency may be impeded by the formation of a biofilm by the bacteria, a dense matrix of mostly polysaccharides, proteins, lipids and extracellular DNA. This biofilm helps to protect the bacteria from degradation and to impede entry of antimicrobial agents.

[0012] The lack of antimicrobials for multi-resistant pathogens may be particularly pressing in healthcare and agriculture environments. For instance, these pathogens may outcompete other microbes in hospital environments due to their resistances and may consequentially cause hard-to-treat and potentially life-threatening infections in already weakened hospital patients. In the agricultural context, antibiotics may serve to maintain animal health, to limit the spread of diseases in relatively dense animal populations, and to contribute to keeping animal products, such as milk and meat, safe for human consumption, all of which may be detrimentally affected due to a lack of antibiotic options for multi-resistant pathogens.

[0013] Polymeric nanoparticles (NPs) may have emerged as promising candidates for a wide variety of targeted biomedical applications. Polymeric NPs are modular nanomaterials with a high surface to volume ratio and may be suitable for multivalent binding through functional ligands. Such multivalent interactions may allow for tight and selective binding between ligands and targets. Recently, antimicrobial polymeric NPs may have gained widespread attention to overcome challenges associated with conventional antimicrobial therapies. Polymeric NPs may contribute to biofilm penetration and to cargo stability. Antimicrobial NPs may have functional groups with intrinsic antimicrobial activity or may act as a carrier system for antimicrobials.

[0014] For instance, the prior art may describe nanoparticles mimicking the structure of anti -microbial peptides (AMP) of innate immune systems. AMPs may typically have a positive net charge and hydrophobic amino acids, resulting in the formation of amphiphilic structures. This amphiphilicity allows the AMPs to interact with negatively charged bacterial cell membranes, invoking local disruption and subsequent cell lysis. However, since electrostatic interactions between NPs and cell membranes are non-specific, membrane disruption in eukaryotic cells is also observed, which may lead to cytotoxic side-effects. These side effects may prevent such nanoparticles from being applied in applications which would (potentially) involve human or animal exposure to the nanoparticles.

[0015] The prior art may further describe polymeric NPs as carrier-materials for antibiotics. Such NPs may facilitate pharmacokinetic enhancement of existing antibiotics, for instance by increasing the solubility and / or retention at target sites. However, delivering the cargo at the appropriate location may remain challenging. For instance, the prior art may describe NPs configured to deliver a cargo, such as an antibiotic, near or to microbial cells. The microbes may, however, yet minimize or even avoid the antimicrobial effect by (i) degrading the cargo extracellularly, (ii) preventing uptake of the cargo, and / or (iii) if any cargo is taken up, actively exporting the cargo from the cell.

[0016] Further, the application of prior art nanoparticles may be limited due to one or more of (i) nanoparticle instability, such as due to (bio-)degradability, (ii) challenging synthesis processes resulting in low quantities (i.e., lack of scalability), (iii) insolubility, (iv) safety risks, and (v) lack of control of synthesis processes, for instance resulting in substantial nanoparticle size variations within or between batches, which may in turn affect the (predictability) of the performance of the nanoparticles.

[0017] Hence, it is an aspect of the invention to provide an alternative nanoparticle, which preferably further at least partly obviates one or more of above-described drawbacks. The present invention may have as object to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. In particular, the present invention may provide an improved method for introducing a nanoparticle into a prokaryotic cell.

[0018] Hence, in a first aspect, the invention may provide a method for introducing a nanoparticle into a prokaryotic cell. The method may comprise exposing the prokaryotic cell to the nanoparticle, which may lead to uptake of the nanoparticle by the prokaryotic cell depending on the properties of the nanoparticle. The nanoparticle may especially be a singlechain polymer nanoparticle having an equivalent spherical diameter selected from the range of 3-20 nm. The nanoparticle may further comprise a backbone, a plurality of intramolecular crosslinks, and a plurality of side groups (covalently) bound to the backbone, especially a carbon-based backbone. In particular, the backbone may comprise n backbone atoms, wherein the n backbone atoms may comprise > 0.9*n carbon atoms, i.e., wherein at least 90% of the backbone atoms are carbon atoms. Further, each intramolecular crosslink (of the plurality of intramolecular crosslinks) may covalently interconnect two (respective) backbone locations of the backbone, such as two backbone atoms, especially two carbon atoms. The side groups may comprise (functional) moieties that may be recognized by the prokaryotic cell. In particular, k side groups of the plurality of side groups may comprise ligand moieties (individually) selected from the group comprising sugar moieties, a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, amino acid moieties, and fatty acid moieties, especially from the group consisting of sugar moieties, a glycerol moiety, and amino acid moieties. Further, k / n may especially be selected from the range of 0.05-0.49. In other words, for every two backbone atoms, the nanoparticle may comprise (about) 0.1 - 0.98 of the mentioned ligand moieties. As mentioned, the ligand moieties may be recognizable by the prokaryotic cell. Especially, the prokaryotic cell may comprise a receptor configured to interact with (or “recognize”) one or more of the ligand moieties.

[0019] Hence, in specific embodiments, the invention may provide a method for introducing a nanoparticle into a prokaryotic cell, wherein the method comprises exposing the prokaryotic cell to the nanoparticle, wherein the nanoparticle has an equivalent spherical diameter selected from the range of 3-20 nm; the nanoparticle comprises a single-chain polymer nanoparticle; the nanoparticle comprises a backbone, a plurality of intramolecular crosslinks, and a plurality of side groups bound to the backbone; wherein the backbone comprises n backbone atoms, wherein the n backbone atoms comprise > 0.9*n carbon atoms; each intramolecular crosslink covalently interconnects two backbone locations of the backbone; k of the plurality of side groups comprise ligand moieties selected from the group comprising sugar moieties, a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, amino acid moieties, and fatty acid moieties, wherein k / n is selected from the range of 0.05-0.49; and the prokaryotic cell comprises a receptor configured to interact with one or more of the ligand moieties.

[0020] Surprisingly, the inventors have found that such method results in a ready uptake of the nanoparticle by the prokaryotic cell, optionally while carrying a cargo, such as an antibiotic cargo. It appears that ligand moieties may be recognized by receptors of the prokaryotic cell and - without being bound by theory - that the prokaryotic cell may actively take up the nanoparticle, possibly via transporters typically involved in the uptake of the ligand moiety or a related small molecule, despite the nanoparticle being substantially larger than the small molecules such receptors may typically interact with. For instance, a nanoparticle comprising glucose moieties as ligand moieties was observed to be taken up by a prokaryotic cell (see below). In particular, it appears that the nanoparticles may efficiently pass through microbial biofilms and that the nanoparticles may be (essentially) fully taken up by the prokaryotic cell, i.e., (essentially) without extracellular degradation, and may subsequently reside in a soluble fraction surrounded by the outer membrane of the prokaryotic cell, e.g., within the periplasm and / or within the cytoplasm. Since many potential (antimicrobial) targets may be found inside the bacteria, this may open up new strategies to deliver antibiotics and circumvent current and emerging antimicrobial resistance, i.e., existing antibiotics may be rendered more effective (as resistance may be avoided) and additional compounds may be considered as antibiotics (as introduction into the cell is facilitated). The method of the invention may thus facilitate introducing a nanoparticle into a prokaryotic cell, which may facilitate circumventing antimicrobial resistance to known antibiotics, and may further facilitate new antimicrobial strategies.

[0021] The invention may herein primarily be described in the context of antimicrobial activity, such as in the context of the (intracellular) delivery of antibiotics. It will be clear to the person skilled in the art, however, that the invention is not limited to such embodiments. The method of the invention may, for instance, also be employed to delivery RNA molecules, such as sRNA molecules, especially siRNA molecules, into a prokaryotic cell to influence cellular behavior.

[0022] The nanoparticles may be relatively stable (before, during and after introduction into the prokaryotic cell), and may be synthesized in relatively large batches with relatively consistent particle sizes, thereby allowing a single batch to be used for multiple iterations of the method, which may contribute to providing a consistent performance of (the nanoparticles in) the method of the invention.

[0023] The invention may thus provide a method for introducing a (polymeric) nanoparticle, especially a single-chain polymer nanoparticle, into a prokaryotic cell.

[0024] The term “nanoparticle” may herein refer to a particle having a size in the nanometer scale, such as in the range of 1 - 500 nm. A large range of different nanoparticles may be generated, with a large flexibility in, e.g., structural flexibility and (tunable) biocompatibility. Nanoparticle size may play an important role in the performance and behavior of nanoparticles for biomedical applications, such as in the context of cellular uptake, where e.g. smaller particles may be more readily taken up by cells. The term ‘nanoparticle’ may herein also refer to a plurality of (similar) nanoparticles. As the nanoparticles may vary in their arrangement of crosslinks and side groups, the nanoparticles may differ in their exact structure. The term “single-chain polymer nanoparticle” (or “SCNP”) may herein refer to a nanoparticle formed through exclusive intramolecular crosslinking of an individual polymer chain. Such a nanoparticle may thus comprise a (polymer) backbone with crosslinks intramolecularly connecting different backbone locations of the backbone. In particular, an SCNP may be devoid of intermolecular crosslinks with a second polymer backbone. As SCNPs are formed by intramolecular crosslinking (and folding), the size and (size) dispersity of SCNPs may depend on the size and size distribution of the polymers prior to intramolecular crosslinking, and thus on the number of monomers the polymers consist of. The size of an SCNP may thus be controlled by controlling the polymer length prior to intramolecular crosslinking. SCNPs may be soft and semi-flexible nanoparticles, resembling the size and folded structure of proteins. In particular, the structure of an SCNP may be between a flexible polymer and a rigid nanoparticle. These SCNP properties may be beneficial in the context of introduction of nanoparticles into prokaryotic cells via ligand interactions as flexibility may facilitate multivalent target-ligand interactions, while rigidity may generally facilitate cellular uptake.

[0025] The term “prokaryotic cell” may herein generally refer to any archaeal or bacterial cell, especially to a bacterial cell. Prokaryotes may generally be single-celled organisms and may lack a nucleus and other membrane-bound organelles typically found in eukaryotes. The prokaryotic cell may especially comprise a gram-negative bacterial cell.

[0026] The prokaryotic cell may especially be of a pathogenic species, such as (multi-)drug resistant pathogenic species. For instance, in embodiments, the prokaryotic cell may belong to one of the genera Enterococcus, Staphylococcus, Klebsiella, Enterobacter, Escherichia, Acinetobacter, Pseudomonas, and Mycobacterium. In further embodiments, the prokaryotic cell may belong to one of the species Enterococcus faecium, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. In further embodiments, the prokaryotic cell may belong to the class K pneumoniae carbapenemase.

[0027] As mentioned above, the method may comprise exposing the prokaryotic cell to the nanoparticle. The phrase “exposing the prokaryotic cell to the nanoparticle” may herein refer to bringing the nanoparticle and the prokaryotic cell into proximity and allowing interaction between the nanoparticle and the prokaryotic cell. For instance, the method may comprise adding a (drop of a) solution comprising the nanoparticle to a culture comprising the prokaryotic cell. Alternatively or additionally, the method may comprise providing (a solution comprising) the nanoparticle to a surface, for instance as part of a disinfectant. The nanoparticle, especially the SCNP, may have a diameter, especially an equivalent spherical diameter (ESD) selected from the range of 2-40 nm, especially from the range of 2-30 nm, such as from the range of 3-20 nm, especially from the range of 5-10 nm. In further embodiments, the nanoparticle, especially the SCNP, may have a diameter, especially an equivalent spherical diameter (ESD) of at least 3 nm, such as of at least 5 nm, especially of at least 8 nm, such as of at least 10 nm. In further embodiments, the nanoparticle, especially the SCNP, may have a diameter, especially an equivalent spherical diameter (ESD) of at most 50 nm, such as of at most 40 nm, especially of at most 30 nm, such as of at most 20 nm. For instance, in embodiments, the nanoparticle, especially the SCNP, may have a diameter, especially an equivalent spherical diameter (ESD) selected from the range of 5-50 nm, such as from the range of 8-40 nm. The diameter, especially the ESD, of the nanoparticle may be determined via light scattering, such as via dynamic light scattering (DLS). The diameter may thus especially be hydrodynamic diameter. For instance, the diameter may be determined in a dispersion at a nanoparticle concentration of 0.5 mg / mL in a 5 wt% NaCl solution in deionized water (or in a 10 mM HEPES buffer at pH=7.0) using a Malvern Instruments Zetasizer ZS, such as with a 532 nm laser.

[0028] It will be clear to the person skilled in the art that a determination of a nanoparticle dimension may typically be done based on a plurality of nanoparticles. In particular, a plurality of the nanoparticles (in a solution or dispersion) may have a number averaged equivalent spherical diameter dso, wherein dso is selected from the range of 2-40 nm, especially from the range of 2-30 nm, such as from the range of 3-20 nm, especially from the range of 5-10 nm, or especially from the range of 8-15 nm. In further embodiments, dso may be at least 3 nm, such as at least 5 nm, especially at least 8 nm, such as at least 10 nm. In further embodiments, dso may be at most 50 nm, such as at most 40 nm, especially at most 30 nm, such as at most 20 nm. For instance, dso may selected from the range of 5-50 nm, such as from the range of 8-40 nm.

[0029] The equivalent spherical diameter (or ESD) (or “spherical equivalent diameter”) of an (irregularly shaped) three-dimensional shape is the diameter of a sphere of equivalent volume. For a sphere, the diameter is the same as the equivalent spherical diameter. Would a sphere in an xyz-plane with a diameter D be distorted to any other shape (in the xyz-plane), without changing the volume, then the equivalent spherical diameter of that shape would be D.

[0030] As described above, the nanoparticle may comprise a (single) backbone (or “polymer backbone”). The term “backbone” may herein refer to the main chain of the polymer, which main chain may have essentially been in place prior to intramolecular crosslinking. In embodiments, the backbone may comprise a carbon-based backbone, such as wherein at least 90% of backbone atoms (of the backbone) comprise carbon atoms, especially at least 95%, such as at least 99%, including 100%. In particular, in embodiments, the backbone may comprise n backbone atoms, wherein the n backbone atoms comprise > 0.9*n carbon atoms, such as > 0.95*n carbon atoms, especially > 0.99*n carbon atoms, including n carbon atoms. A backbone consisting essentially of carbon atoms, e.g., comprising > 99% carbon atoms, may be relatively stable against (microbial) degradation, which may be beneficial for (i) introducing the full (non-degraded) nanoparticle into a prokaryotic cell and (ii) maintaining the nanoparticle in the prokaryotic cell.

[0031] In specific embodiments, the backbone may comprise a vinyl polymer, i.e., the backbone may be formed through vinyl polymerization. In such embodiments, (essentially) all backbone atoms may comprise carbon atoms.

[0032] However, degradation of (parts of) the nanoparticle may also be desired. For instance, if part of the nanoparticle is to be left extracellularly, or if an (antibiotic) cargo is activated upon (intracellular) release from a remainder of the nanoparticle. Hence, in further embodiments, the backbone may comprise one or more (microbially) degradable groups, especially degradable by the prokaryotic cell. The degradable group may especially be selected from the group comprising an ester, a thioester, an amide, an anhydride, a silyl ether, a disulfide, an orthoester, an imine, an imide, a carbonate, a carbamate, and a thiocarbamate. Hence, in embodiments, the backbone atoms may further comprise one or more of O, S, N, and Si. Especially, in embodiments > 0.001 *n of the backbone atoms may comprise O, S, N, or Si, such as > 0.005*n, especially > 0.01*n. In further embodiments < 0.1*n of the backbone atoms may comprise O, S, N, or Si, such as < 0.05*n, especially < 0.01 *n.

[0033] A method for including biologically and / or chemically degradable groups in a (vinyl) polymer are described in PAULUSSE, JMJ et al., Free Radical Polymers with Tunable and Selective Bio- and Chemical Degradability, J. AM. CHEM. SOC. 2009, 131, 9805-9812, which is hereby herein incorporated by reference.

[0034] The nanoparticle may, in embodiments, further comprise a plurality of intramolecular crosslinks. The term “intramolecular crosslink” herein refers to a crosslink formed within a single molecule, such as between two backbone locations of a polymer backbone. In particular, each intramolecular crosslink may covalently interconnect two (respective) backbone locations of the backbone, i.e., a first intramolecular crosslink may covalently interconnect a first set of two backbone locations and a second intramolecular crosslink may covalently interconnect a second set of two backbone locations. The crosslinks may be formed using side group precursors. For instance, after vinyl polymerization a vinyl polymer may typically have (about) 1 side group precursor for every one to three carbon atoms, most typically (about) 1 side group per two carbon atoms. Part of these side group precursors may be converted to crosslink precursors and subsequently to intramolecular crosslinks, and a (separate) part of these side group precursors may be converted to side groups. Alternatively, part of the side groups may be further converted to crosslink precursors (or vice versa). Regardless of the (order of) operations, the number of possible intramolecular crosslinks may depend on n (the number of backbone atoms) and on the number of side groups.

[0035] The number of intramolecular crosslinks may further contribute to the flexibility / rigidity of the nanoparticle, especially of the SCNP. A nanoparticle having few crosslinks may lack rigidity, which may diminish cellular uptake (see above). A nanoparticle having many crosslinks may lack flexibility, which may diminish (multi -)val ent interactions between ligands and receptors, which may in turn also reduce cellular uptake. Hence, in embodiments, the nanoparticle may comprise m intramolecular crosslinks, especially wherein m is selected from the range comprising 0.01 *n - 0.2*n (or “the range of 0.0 l*n - 0.2*n), such as from the range of 0.02*n - 0.1 *n, especially from the range of 0.03 *n - 0.08*n. It will be clear to the person skilled in the art that the suitable fraction of crosslinks may further depend on the length of the backbone. In particular, long backbones may generally be suitably crosslinked with a relatively lower number of crosslinks than may be selected for short backbones.

[0036] Similarly, the length of the intramolecular crosslinks may (also) contribute to the flexibility / rigidity of the nanoparticle, especially of the SCNP. A nanoparticle having few crosslinks may lack rigidity, which may diminish cellular uptake (see above). A nanoparticle having short crosslinks may lack flexibility, whereas a nanoparticle having long crosslinks may lack rigidity. Hence, in embodiments, the intramolecular crosslinks may have lengths (between the two backbone locations) (individually) selected from the range of 3 - 200 atoms, such as from the range of 4-120 atoms, especially from the range of 8-60 atoms.

[0037] Methods for providing intramolecular crosslinks in a nanoparticle, such as to provide an SCNP, are known in the art. For instance, methods to provide intramolecular crosslinks are described in KROGER, APA and PAULUS SE, JMJ, Single-chain polymer nanoparticles in controlled drug delivery and targeted imaging, Journal of Controlled Release, 2018, 286, pages 326-347, which is hereby herein incorporated by reference. In embodiments, the nanoparticle may further comprise side groups (covalently) bound to the backbone. A subset, especially a proper subset, of these side groups may comprise ligand moieties, especially wherein the ligand moieties are (individually) selected from the group comprising sugar moieties, a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, amino acid moieties, and fatty acid moieties. In particular, in embodiments, (a subset of) k side groups may comprise ligand moieties, wherein the ligand moieties are (individually) selected from the group comprising sugar moieties, a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, amino acid moieties, and fatty acid moieties.

[0038] The side groups may comprise the ligand molecule, a linker (see below) and a connecting part originating from the chemical reaction used for connecting the side group to the SCNP. For example, an amine-functionalized precursor, such as an amine-functionalized sugar moiety, or such as 1 -aminoglycerol, may be used for attaching the side group to the SCNP (see below). In such embodiments, the ligand moiety may be connected to the backbone via (a linker and) an amide group.

[0039] The ligand moieties may be selected for their suitability to interact with a receptor of the prokaryotic cell. A low presence of ligand moieties may reduce the likelihood of interactions with the receptor(s), and may thus slow down or even prevent cellular uptake. However, also a high presence of (specific) ligand moieties may be detrimental to cellular uptake (see below). The number of ligand moieties may especially be selected in dependence of the size of the nanoparticle, such as based on the length of the backbone. Especially, in embodiments, k / n may be selected from the range of 0.03 - 0.7, such as from the range of 0.05- 0.49, especially from the range of 0.08 - 0.45.

[0040] The term “proper subset” may herein refer to a subset comprising at least one member of the (larger) set but not all members of the larger set. For instance, a proper subset of the plurality of side groups may comprise at least one side group but not all side groups of the plurality of side groups.

[0041] Without being bound by theory, the inventors hypothesize that the cellular uptake of the nanoparticle by the prokaryotic cell may result from the receptor recognizing the ligand moiety as a nutrient, e.g., as a carbon source, and initiating uptake of the ligand moiety, and, thereby, the nanoparticle, despite the nanoparticle potentially being substantially larger than the nutrient. It will be clear to the person skilled in the art that prokaryotes, such as bacteria, may vary in the nutrients they can metabolize and in the nutrients they will (actively) import. The ligand moieties may thus be selected based on compounds imported by the prokaryotic cell, especially based on compound actively imported by the prokaryotic cell, such as following recognition by a (cell surface) receptor.

[0042] The ligand moiety may thus (at least partially) resemble a nutrient typically recognized by the receptor. For instance, a glucose moiety may be recognized by a receptor configured to interact with (free-floating) glucose.

[0043] The prokaryotic cell may (thus) comprise a receptor suitable for recognizing the (at least part of) the ligand moieties. In particular, the prokaryotic cell may comprise a receptor configured to interact with (or “recognize”) one or more of the ligand moieties. The receptor may especially be configured suitable for interacting with (or “recognizing”) the ligand moiety outside of the prokaryotic cell. The receptor may thus be a cell surface receptor, such as a receptor arranged in the outer wall or the outer membrane of a prokaryotic cell.

[0044] The prokaryotic cell may further comprise a transporter configured to import a particle interacting with the receptor. Especially, the transporter may be suitable for importing the nanoparticle following interaction between one or more of the ligand moieties and the receptor.

[0045] The ligand moieties may thus be selected based on the receptors of the prokaryotic cell, especially based on the receptors and transporters of the prokaryotic cell. As prokaryotes may substantially vary in their (preferred) nutrients, a wide range of ligand moieties may be considered. In embodiments, the ligand moieties may thus (individually) be selected from the group comprising sugar moieties, a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, amino acid moieties, and fatty acid moieties, especially from the group consisting of a sugar moiety, a glycerol moiety, and an amino acid moiety.

[0046] In further embodiments, the ligand moieties may (at least) comprise sugar moieties, especially (at least) pentose moieties, or especially (at least) hexose moieties. As relatively energy dense carbon sources which may require little to no energy investment to degrade, sugars may be preferred nutrients for many prokaryotes. In particular, the process of carbon catabolite repression favoring the uptake and processing of a sugar, especially of glucose, over other carbon sources may be widespread amongst prokaryotes. As different prokaryotes may be exposed to different sugars in their natural environment, their receptors (and intracellular machinery) may also be attuned to different sugars. The sugar moieties may, for instance, be selected in view of the sugars the (species corresponding to the) prokaryotic cell would be naturally exposed to. For instance, in embodiments, the sugar moieties may be selected from the group comprising a glucose moiety, a xylose moiety, a fructose moiety, a moiety, a galactose moiety, and an arabinose moiety. Further, the sugar moieties may also be selected from disaccharides and / or trisaccharides, such as from the group comprise a sucrose moiety, a raffinose moiety, a trehalose moiety, a lactose moiety, a maltose moiety, an isomaltose moiety, and a panose moiety. In further embodiments, the sugar moieties may comprise a sialic acid moiety, such as a N-acetylneuraminic acid moiety. Sialic acid moieties may comprise a (mono)saccharide at the end of a carbon backbone.

[0047] Especially, the side groups, especially the k side groups, may comprise a first (proper) subset of kl first side groups, wherein each first side group comprises a (single) first ligand moiety. The first ligand moieties may be (individually) selected from the group comprising sugar moieties, especially from the group comprising pentose moieties and hexose moieties. In further embodiments, the first ligand moieties may be selected from the group comprising a glucose moiety, a xylose moiety, a fructose moiety, a moiety, a galactose moiety, and an arabinose moiety. Although sugar moieties may facilitate uptake of the nanoparticle, an excess of sugar moieties may also detrimentally affect the uptake relative to fewer sugar moieties (see below). Hence, in embodiments, ki / n may be selected from the range of 0.03 - 0.4, such as from the range of 0.05-0.3, especially from the range of 0.05-0.25, such as from the range of 0.1-0.2, such as from the range of 0.12-0.18. In further embodiments ki / n may be at least 0.03, such as at least 0.05, especially at least 0.1. In further embodiments ki / n may be at least 0.12, such as at least 0.14. In further embodiments, ki / n may be at most 0.4, such as at most 0.3, especially at most 0.25. In further embodiments, ki / n may be at most 0.2, such as at most 0.18, especially at most 0.16.

[0048] In further embodiments, the ligand moieties may (at least) comprise glycerol moieties. In particular, a nanoparticle comprising solely glycerol moieties as side groups was successfully introduced into a prokaryotic cell using the method of the invention (see below).

[0049] In further embodiments, the ligand moieties may (at least) comprise pyruvate moieties. Pyruvate may be recognized and taken up by many prokaryotes as pyruvate may be a typical entry point into the citric acid cycle via its oxidation to acetyl-CoA.

[0050] In further embodiments, the ligand moieties may (at least) comprise one or more of acetate moieties, lactate moieties and succinate moieties, especially (at least) acetate moieties, or especially (at least) lactate moieties, or especially (at least) succinate moieties). Acetate, lactate and succinate may typically be produced by prokaryotes during growth on sugars, but may also be taken up by prokaryotes, e.g., to be used as carbon sources and / or for redox balancing. In further embodiments, the ligand moieties may (at least) comprise amino acid moieties. Prokaryotes may use amino acids, depending on the amino acid, as one or more of carbon sources, energy sources, nitrogen sources, and sulfur sources, as well as directly as building blocks for proteins. In embodiments, the amino acid moieties may especially be selected from the group comprising an alanine moiety, an arginine moiety, an asparagine moiety, an aspartic acid moiety, a cysteine moiety, a glutamine moiety, a glutamic acid moiety, a glycine moiety, a histidine moiety, an isoleucine moiety, a leucine moiety, a lysine moiety, a methionine moiety, a phenylalanine moiety, a proline moiety, a serine moiety, a threonine moiety, a tryptophan moiety, a tyrosine moiety, and a valine moiety.

[0051] In further embodiments, the ligand moieties may (at least) comprise fatty acid moieties. Prokaryotes may use fatty acids as carbon and / or energy sources, but may also incorporate fatty acids into their cell wall and / or cell membrane. For instance, in embodiments, the fatty acid moieties may be selected from the group comprising a palmitic acid moiety, a palmitoleic acid moiety, and an oleic acid moiety.

[0052] As mentioned above, the side groups may comprise a first subset of kl first side groups comprising sugar moieties. Additionally or alternatively, the side groups may comprise a second subset of k2 second side groups, wherein the k2 second side groups especially comprise non-sugar moieties. Hence, in embodiments, the side groups may comprise a second subset of k2 second side groups, wherein each second side group comprises a (single) second ligand moiety selected from the group comprising a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, amino acid moieties, and fatty acid moieties. In further embodiments, the second ligand moieties may comprise (at least) glycerol moieties. In further embodiments, the second ligand moieties may comprise (at least) acetate moieties. In further embodiments, the second ligand moieties may comprise (at least) pyruvate moieties. In further embodiments, the second ligand moieties may comprise (at least) lactate moieties. In further embodiments, the second ligand moieties may comprise (at least) succinate moieties. In further embodiments, the second ligand moieties may comprise (at least) amino acid moieties. In further embodiments, the second ligand moieties may comprise (at least) fatty acid moieties. In further embodiments, k2 / n may be selected from the range of 0.03-0.49, such as from the range of 0.05-0.44, especially from the range of 0.1-0.4, such as from the range of 0.15-0.35. In further embodiments k2 / n may be at least 0.03, such as at least 0.05, especially at least 0.1. In further embodiments k2 / n may be at least 0.15, such as at least 0.2, especially at least 0.25. In further embodiments, k2 / n may be at most 0.49, such as at most 0.47, especially at most 0.45. In further embodiments, ki / n may be at most 0.4, such as at most 0.35, especially at most 0.3.

[0053] Hence, in embodiments, each ligand moiety may be (individually selected) from the group comprising a sugar moiety, a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, an amino acid moiety, and a fatty acid moiety. In further embodiments, each first ligand moiety may comprise a sugar moiety, especially a hexose moiety, such as a glucose moiety, or especially a pentose moiety. In further embodiments, each second ligand moiety may be (individually selected) from the group comprising a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, an amino acid moiety, and a fatty acid moiety, especially from the group consisting of a sugar moiety, a glycerol moiety, and an amino acid moiety.

[0054] The nanoparticle may, in embodiments, comprise (both) sugar moieties and nonsugar moieties. In further embodiments, kl+k2 < k, especially < 0.9*k. In further embodiments, 0.9*k < kl+k2 < k, such as 0.95*k < kl+k2 < k. Especially, in embodiments, kl+k2=k.

[0055] In further embodiments, the first ligand moieties may be glucose moieties and the second ligand moieties may be glycerol moieties, especially wherein kl+k2 > 0.95*k, such as wherein kl+k2=k. Embodiments involving such nanoparticles may have been found particularly suitable for introduction into the prokaryotic cell.

[0056] As the ligand moieties may serve to interact with (cell surface) receptors, it may be beneficial for the ligand moieties to be attached to the backbone via (flexible) linkers allowing some freedom in movement of the ligand moieties relative to the backbone.

[0057] Hence, in embodiments, the side groups may (further) comprise linkers, wherein the linkers separate the backbone and the ligand moieties. In further embodiments, each linker may comprise a side backbone (or “linker backbone”) comprising 2-30 (side backbone) atoms, such as 3-20 side backbone atoms, especially 4-12 side backbone atoms, especially wherein the side backbone atoms are selected from the group consisting of C, N, S, P, and O.

[0058] For instance, in further embodiments, the linkers may consist of propyl moieties, diethylene glycol moieties or triethylene glycol moieties.

[0059] The length (and flexibility) of the linker may influence the availability of the ligand moiety to the (cell surface) receptor. Effectively, a ligand moiety connected to the backbone via a (relatively) long linker may be more readily available to a receptor than a (same) ligand moiety connected to the backbone via a (relatively) short linker. As described above, an increased availability (or ‘presence’) of ligand moieties may, after a point, detrimentally affect the cellular uptake of the nanoparticle. As this availability may be affected both by the number of ligand moieties and by the linkers via which they are connected to the backbone, these parameters may (synergistically) be selected codependently.

[0060] For instance, in embodiments, the nanoparticle may have relatively long linkers and relatively few ligand moieties, especially relatively few sugar moieties. In particular, in such embodiments: (i) the linkers may comprise at least 8 backbone atoms, such as at least 10 backbone atoms, especially at least 12 backbone atoms, such as at least 16 backbone atoms, and (ii) k / n may be selected from the range of 0.03-0.3, such as from the range of 0.05-0.25, especially from the range of 0.05-0.2. In further embodiments, (i) the linkers may comprise at least 8 backbone atoms, such as at least 10 backbone atoms, especially at least 12 backbone atoms, such as at least 16 backbone atoms, and (ii) ki / n may be selected from the range of 0.03- 0.25, such as from the range of 0.05-0.2, especially from the range of 0.05-0.13.

[0061] For example, in specific embodiments, the first ligand moieties may be glucose moieties, ki / n may be selected from the range of 0.05-0.13, and the linkers may consist of triethylene glycol moieties. Especially, in further embodiments, the side groups may further comprise k2 second side groups comprising glycerol moieties, wherein k2 / n is selected from the range of 0.05-0.44, and wherein ki+k2 < k, especially wherein ki+k2=k.

[0062] Alternatively, in embodiments, the nanoparticle may have relatively short linkers and - in comparison to the nanoparticle with relatively long linkers - relatively many ligand moieties, especially relatively many sugar moieties. In particular, in such embodiments: (i) the linkers may comprise at most 6 backbone atoms, such as at most 5 backbone atoms, especially at most 4 backbone atoms, such as at most 3 backbone atoms, and (ii) k / n may be selected from the range of 0.03-0.3, such as from the range of 0.05-0.25, especially from the range of 0.05-0.2. In further embodiments, (i) the linkers may comprise at least 8 backbone atoms, such as at least 10 backbone atoms, especially at least 12 backbone atoms, such as at least 16 backbone atoms, and (ii) ki / n may be selected from the range of 0.08-0.49, such as from the range of 0.1-0.45, especially from the range of 0.2-0.45.

[0063] For example, in specific embodiments, the first ligand moieties may be glucose moieties, ki / n may be selected from the range of 0.12-0.18, and the linkers may consist of propyl moieties. Especially, in further embodiments, the side groups may further comprise k2 second side groups comprising glycerol moieties, wherein k2 / n is selected from the range of 0.05-0.37, and wherein ki+k2 < k, especially wherein ki+k2=k.

[0064] In further embodiments, the linkers may comprise polymer ethylene glycol linkers. In such embodiments, the polymer ethylene glycol linkers may separate the backbone and the ligand moieties. Especially, each polymer ethylene glycol linker may have a weight selected from the range of 300-4000 Da, such as from the range of 500-3000 Da, especially from the range of 1000-2500 Da.

[0065] Besides the first side groups and the second side groups, the nanoparticle may yet comprise further side groups. For instance, in embodiments, the side groups may comprise a third subset of ks third side groups, wherein each third side group comprises a (single) third ligand moiety selected from the group comprising an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, amino acid moieties, and fatty acid moieties, especially wherein the second ligand moieties differ from the third ligand moieties. In further embodiments, the third ligand moieties may comprise (at least) glycerol moieties. In further embodiments, the third ligand moieties may comprise (at least) acetate moieties. In further embodiments, the third ligand moieties may comprise (at least) pyruvate moieties. In further embodiments, the third ligand moieties may comprise (at least) lactate moieties. In further embodiments, the third ligand moieties may comprise (at least) succinate moieties. In further embodiments, the third ligand moieties may comprise (at least) amino acid moieties. In further embodiments, the third ligand moieties may comprise (at least) fatty acid moieties. In further embodiments, k n may be selected from the range of 0.05-0.4, such as from the range of 0.1- 0.35. In further embodiments, ki+k2+ks < k. In further embodiments, ki+k2+k3=k.

[0066] In further embodiments, the nanoparticle may comprise multiple ligand moieties but may be (essentially) devoid of sugar moieties. For instance, in such embodiments, k2 and ks may be (individually) selected from the range of 0.05-0.44. Especially, in such embodiments, k2+k3 < k. Further, in such embodiments, k2+k3 > 0.8*k, such as > 0.9*k, especially wherein k2+k3=k.

[0067] As described above, the ligand moieties may be selected based on the prokaryotic cell, especially based on the (cell surface) receptors of the prokaryotic cell, or especially based on the transporters of the prokaryotic cell. In practice, a ligand may interact with a (dedicated) receptor and subsequently be imported by a (dedicated) transporter or a single protein (complex) may be responsible for both ligand recognition and transport. The term “receptor” and “transporter” may herein both thus also refer to a protein performing both ligand recognition and ligand transport. Table 1 indicates several known transporters by their abbreviated name and Uniprot identifiers (UniProtKB database, www.uniprot.org) for three different gram-negative bacteria (Escherichia coH, Staphylococcus aureus, and Pseudomonas aeruginosa) as well as the molecules the transporters are configured to transport:

[0068] For instance, the three mentioned bacteria may all have transporters suitable for, potentially dedicated to, glucose transport. Hence, for each of these bacteria a glucose moiety may facilitate uptake of the nanoparticle. Similarly, given that Escherichia coli has multiple transporters suitable for, potentially dedicated to, pyruvate transport, a pyruvate moiety may (also) facilitate uptake of the nanoparticle by Escherichia coli. Similar assessments may be made for other prokaryotes, potentially based on computational identification of receptors and / or transporters, e.g., by identifying the presence of homologs to the transporters indicated in table 1 (see above). It will be clear to the person skilled in the art that the above overview is not exhaustive with respect to known relevant prokaryotes, nor is it necessarily complete regarding the mentioned prokaryotes. The skilled person will be able to (pre-)screen for suitable ligand moieties based on the identified, optionally de novo annotated, receptors and / or transporters of a prokaryote and to subsequently select suitable ligand moieties. Besides ligand moieties, the side groups may further comprise targeting groups and / or neutral groups. The term ‘targeting group’ may herein refer to a group having affinity for the prokaryotic cell and may facilitate bringing the nanoparticle into the vicinity of the prokaryotic cell and / or keeping it there. Such a targeting group may, for example, facilitate selectively targeting a specific (pathogenic) prokaryote over other (non-pathogenic) prokaryotes. For instance, in embodiments, at least part of the side groups may comprise a targeting peptide, wherein the targeting peptide is configured to (specifically) interact with the prokaryotic cell, such as wherein the targeting peptide specifically is configured to target a genus corresponding to the prokaryotic cell, especially a species corresponding to the prokaryotic cell, such as a strain corresponding to the prokaryotic cell. In further embodiments, at least part of the side groups may comprise an aptamer, wherein the aptamer is configured to (specifically) interact with the prokaryotic cell, such as wherein the targeting peptide specifically is configured to target a genus corresponding to the prokaryotic cell, especially a species corresponding to the prokaryotic cell, such as a strain corresponding to the prokaryotic cell.

[0069] The term ‘neutral group’ (or ‘neutral moiety’) may herein refer to a group that may be (essentially) inert with respect to receptors of the prokaryotic cell. A neutral group may, for instance, be selected to influence the hydrophilicity / hydrophobicity (and solubility) of the nanoparticle. In particular, the neutral group(s) may be selected to provide solubility (in water) and to be biocompatible, particularly biocompatible with eukaryotes, such as with humans, or such as with (farm) animals.

[0070] For instance, in embodiments, the side groups may comprise a fourth subset of k4 fourth side groups, wherein each fourth side group comprises a (single) neutral moiety selected from the group comprising a glycerol moiety, a 2-hydroxyethyl moiety, an oligoethyleneoxide moiety, a poly(ethyleneoxide) moiety, a methoxyethyl moiety, a 2- hydroxypropyl amide moiety, an N-isopropyl amide moiety, an N,N-dimethyl amide moiety, a carboxybetaine moiety, and a sulfobetaine moiety. In further embodiments, Wn may be selected from the range of 0.05-0.44, such as from the range of 0.1-0.44, especially from the range of 0.2-0.4. In particular, in embodiments, (essentially) all side groups (thus excluding intramolecular crosslinkers) not comprising a ligand moiety may comprise a neutral moiety.

[0071] As the method of the invention may conveniently introduce a nanoparticle into a prokaryotic cell, the method may further be used to introduce a cargo associated with the nanoparticle into the prokaryotic cell. Hence, in embodiments, the nanoparticle may comprise a cargo. In particular, in embodiments, the nanoparticle may be covalently linked to a cargo. In further embodiments, the nanoparticle may encapsulate a cargo, i.e., the cargo may be essentially ‘captured’ in the nanoparticle. For instance, in embodiments, the cargo may comprise an antibiotic, especially an antimicrobial. In particular, the antibiotic may be configured to inhibit growth of the prokaryotic cell and / or to destroy the prokaryotic cell, especially from inside of the prokaryotic cell. For example, the antibiotic may comprise one or more of (a prodrug of) atovaquone, doxorubicin, rifampicin, and Nile red. The mentioned antibiotics may have previously been successfully encapsulated by or conjugated to SCNPs. For instance: KROGER, APA et al., Biocompatible Single-Chain Polymer Nanoparticles for Drug Delivery - A Dual Approach, ACS Appl. Mater. Interfaces, 2018, 10, pages 30946-30951 describes physical encapsulation of Nile red and rifampicin by an SCNP; HAMELMANN, NM et al., Enhancing Cellular Internalization of Single-Chain Polymer Nanoparticles via Polyplex Formation, Biomacromolecules, 2022, 23, pages 5036-5042 describes conjugation of doxorubicin to an SCNP; and HAMELMANN, NM et al., Single-Chain Polymer Nanoparticles Targeting the Ookinete Stage of Malaria Parasites, ACS Infect. Dis. 2023, 9, 1, pages 56-64, describes conjugation of an atovaquone prodrug to an SCNP.

[0072] It will be clear to the person skilled in the art that the mentioned antibiotics are not an exhaustive overview of the available options for antibiotics. Rather, many additional options may be available as the activity of conventional antibiotics against drug-resistant microbes may be improved / restored if these antibiotics can effectively be introduced (and kept) in a prokaryotic cell. Further, compounds that may not have an antibiotic effect when provided to the extracellular environment of a prokaryotic cell may prove to have a growth inhibiting or lethal effect when introduced inside of the prokaryotic cell.

[0073] In embodiments, the antibiotic may comprise a P-lactam based antibiotic, such as a penicillin, or such as a cephalosporin. In further embodiments, the antibiotic may comprise a penicillin selected from the group comprising amoxicillin, ampicillin, aspoxicillin, benzylpenicillin, oxacillin, phenoxymethylpenicillin and piperacillin. In further embodiments, the antibiotic may comprise a cephalosporin selected from the group comprising cefaclor, cefadroxil, cefalexin, cefaloridine, cefalothin, cefapirine, cefazolin, cefdinir, cefepime, cefiderocol, cefotaxime, cefpodoxime, ceftaroline, ceftazidime, ceftibuten and ceftriaxone.

[0074] In further embodiments, the antibiotic may comprise a carbapenem, especially a carapenem selected from the group comprising ertapenem, imipenem and meropenem.

[0075] In further embodiments, the antibiotic may comprise a monobactam, especially aztreonam.

[0076] In further embodiments, the antibiotic may comprise a P-lactamase inhibitor. In further embodiments, the antibiotic may comprise a macrolide, especially a macrolide selected from the group comprising azithromycin, clarithromycin, erythromycin and avilamycin.

[0077] In further embodiments, the antibiotic may comprise a polyene, especially a polyene selected from the group comprising amphotericin B and nystatin.

[0078] In further embodiments, the antibiotic may comprise a quinoline, such as a fluoroquinoline. Especially, the antibiotic may be selected from the group comprising ciprofloxacin, bedaquiline, levofloxacin, moxifloxacin, delafloxacin, anidulafungin, chloramphenicol, clindamycine, rifampin, linezolid, and trimethoprim.

[0079] In further embodiments, the antibiotic may comprise a sulfonamide.

[0080] In further embodiments, the antibiotic may comprise an aminoglycoside, especially an aminoglycoside selected from the group comprising amikacin, apramycin, gentamicin, streptomycin and tobramycin.

[0081] In further embodiments, the antibiotic may comprise a peptide-based antibiotic, such as a peptide-based antibiotic selected from the group comprising capreomycin, daptomycin, bacitracin, colistin and vancomycin.

[0082] In further embodiments, the antibiotic may comprise a tetracycline, especially a tetracycline selected from the group comprising doxycycline, eravacycline, minocycline, tetracycline, and tigecycline.

[0083] In further embodiments, the cargo may comprise an RNA molecule, especially an sRNA molecule, such as a regulatory sRNA molecule. In particular, the introduction of an RNA molecule, especially an sRNA molecule, into a prokaryotic cell may influence cellular behavior, such as by affecting gene expression (e.g., by binding to a complementary mRNA strand).

[0084] The exposure of the prokaryotic cell to the nanoparticle may, for example, occur in vitro or on a surface, such as on a surface in a hospital. In embodiments, the method may be a non-medical method. In particular, the method may be devoid of administration of the nanoparticle to a human or to a (farm) animal.

[0085] In embodiments, the method may especially be a non-medical method.

[0086] In a second aspect, the invention may further provide the nanoparticle as such. Hence, the invention may provide a nanoparticle for introduction into a prokaryotic cell. The nanoparticle may have an equivalent spherical diameter selected from the range of 2-40 nm, such as from the range of 3-20 nm, and may especially comprise a single-chain polymer nanoparticle. The nanoparticle may comprise a backbone, a plurality of intramolecular crosslinks, and a plurality of side groups (covalently) bound to the backbone. The backbone may comprise n backbone atoms, wherein the n backbone atoms comprise > 0.9*n carbon atoms. In embodiments, each intramolecular crosslink may covalently interconnect two (respective) backbone locations of the backbone. In further embodiments, k of the plurality of side groups comprise may comprise ligand moieties (individually) selected from the group comprising sugar moieties, a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, amino acid moieties, and fatty acid moieties, especially wherein k / n is selected from the range of 0.03-0.7, such as from the range of 0.05-0.49, especially from the range of 0.1-0.45.

[0087] Hence, in specific embodiments, the invention may provide a nanoparticle for introduction into a prokaryotic cell, wherein the nanoparticle has an equivalent spherical diameter selected from the range of 3-20 nm, wherein the nanoparticle comprises a single-chain polymer nanoparticle, wherein the nanoparticle comprises a backbone, a plurality of intramolecular crosslinks, and a plurality of side groups bound to the backbone, wherein the backbone comprises n backbone atoms, wherein the n backbone atoms comprise > 0.9*n carbon atoms, wherein each intramolecular crosslink covalently interconnects two backbone locations of the backbone, wherein k of the plurality of side groups comprise ligand moieties selected from the group comprising sugar moieties, a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, amino acid moieties, and fatty acid moieties, wherein k / n is selected from the range of 0.05-0.49.

[0088] The nanoparticle of the invention may be particularly suitable for introduction into a prokaryotic cell, such as into a bacterial cell. Further, the nanoparticle may be efficiently synthesized (see below) and may be relatively stable, both preceding and following the introduction into a prokaryotic cell.

[0089] In embodiments, the side groups may comprise a first subset of ki first side groups. Especially, each first side group may comprise a first ligand moiety selected from the group comprising sugar moieties, especially from the group comprising pentose moieties, or especially from the group comprising hexose moieties, such as glucose moieties. Further, in embodiments, ki / n may be selected from the range of 0.03 - 0.4, such as from the range of 0.05-0.3, especially from the range of 0.05-0.25, such as from the range of 0.1-0.2, such as from the range of 0.12-0.18.

[0090] In further embodiments, the side groups may comprise a second subset of k2 second side groups. Especially, each second side group may comprises a second ligand moiety selected from the group comprising a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, amino acid moieties, and fatty acid moieties. In further embodiments, the second ligand moieties may comprise (at least) glycerol moieties. In further embodiments, the second ligand moieties may comprise (at least) acetate moieties. In further embodiments, the second ligand moieties may comprise (at least) pyruvate moieties. In further embodiments, the second ligand moieties may comprise (at least) lactate moieties. In further embodiments, the second ligand moieties may comprise (at least) succinate moieties. In further embodiments, the second ligand moieties may comprise (at least) amino acid moieties. In further embodiments, the second ligand moieties may comprise (at least) fatty acid moieties. In further embodiments, ki / n may be selected from the range of 0.03-0.49, such as from the range of 0.05-0.44, especially from the range of 0.1-0.4, such as from the range of 0.15-0.35. In further embodiments ki / n may be at least 0.03, such as at least 0.05, especially at least 0.1.

[0091] For instance, in embodiments, the side groups may comprise a first subset of ki first side groups and a second subset of k2 second side groups, wherein each first side group comprises a glucose moiety, and wherein each second side group comprises a glycerol moiety. In further embodiments, ki / n may be selected from the range of 0.05-0.25 and k2 / n may be selected from the range of 0.2-0.44.

[0092] In further embodiments, the side groups may comprise linkers, wherein the linkers separate the backbone and the ligand moieties.

[0093] In specific embodiments, the linkers may consist of propyl moieties and ki / n may be selected from the range of 0.12-0.18.

[0094] In further specific embodiments, the linkers may consist of triethylene glycol moieties and ki / n may be selected from the range of 0.05-0.13.

[0095] In further embodiments, the nanoparticle may comprise a cargo, especially wherein the cargo is covalently bound to the nanoparticle, and / or especially wherein the nanoparticle encapsulates the cargo. The cargo may, in embodiments, especially comprise an antibiotic.

[0096] In embodiments, the nanoparticle may be for use as a medicament, especially for us as an antibiotic. In particular, in embodiments, the nanoparticle may be for use in the treatment of an infection, especially of a bacterial infection. In such embodiments, the ligand moiety may be selected based on the (species of the) prokaryotic cell causing the infection, especially such that the ligand moiety is suitable for interacting with a receptor of the prokaryotic cell. In embodiments, the use may comprise oral administration of the nanoparticle. In further embodiments, the use may comprise nasal administration of the nanoparticle. In further embodiments, the use may comprise topical administration of the nanoparticle, such as on an open wound. In further embodiments, the use may comprise intravenous administration of the nanoparticle.

[0097] Features of the nanoparticle are described in further detail above in the context of the method of the invention.

[0098] In a further aspect, the invention may provide a composition comprising the nanoparticle. The composition may comprise a (suitable) carrier and the nanoparticle, especially in a concentration selected from the range of 50 pg / mL to 20 mg / mL, such as from the range of 100 pg / mL to 10 mg / mL, especially from the range of 200 pg / mL to 5 mg / mL. The carrier may be selected in view of the intended application. For instance, for a composition intended for the cleaning of a surface, e.g., a hospital surface or a desk surface, the carrier may be selected to dry quickly. Alternatively, for a composition intended for administration to a human or animal, the carrier may be selected to be a pharmaceutically acceptable carrier.

[0099] For instance, in embodiments, the composition may be formulated for intravenous injection. In such embodiments, the carrier may be a pharmaceutically acceptable carrier (for intravenous injection). For example, the carrier may comprise water, normal saline (0.9% sodium chloride), a dextrose solution, Ringer’s solution for infusion (isotonic solution of electrolytes), a buffer solution, and / or a suitable medium.

[0100] In further embodiments, the composition may be formulated for topical administration, such as for topical administration onto an (open) wound. In such embodiments, the carrier may be a pharmaceutically acceptable carrier (for topical administration). For example, the carrier may comprise an emulsion (W / O or O / W) with emulsion stabilizers, such as polysorbates, glyceryl stearate or cetearyl alcohol.

[0101] In further embodiments, the composition may be formulated for topical spray administration, such as for topical spray administration onto an (open) wound. In such embodiments, the carrier may be a pharmaceutically acceptable carrier (for topical spray administration). For example, the carrier may comprise solvents such as water and / or alcohols, and a propellant (e.g. hydrocarbons, nitrogen gas).

[0102] In further embodiments, the composition may be formulated for oral or nasal administration. In such embodiments, the carrier may be a pharmaceutically acceptable carrier (for oral and / or nasal administration). For example, the carrier may comprise solvents such as water and / or alcohols.

[0103] In embodiments, the composition may be for use as a medicament, especially for use as an antibiotic. In such embodiments, the nanoparticle may especially comprise an antibiotic. In particular, in embodiments, the composition may be for use in the treatment of an infection, especially of a bacterial infection. In such embodiments, the ligand moiety (of the nanoparticle) may be selected based on the (species of the) prokaryotic cell causing the infection, especially such that the ligand moiety is suitable for interacting with a receptor of the prokaryotic cell. In further embodiments, the composition may comprise a pharmaceutically acceptable carrier (for the type of administration; see above). In embodiments, the use may comprise oral administration of the nanoparticle. In further embodiments, the use may comprise nasal administration of the nanoparticle. In further embodiments, the use may comprise topical administration of the nanoparticle, such as on an open wound. In further embodiments, the use may comprise intravenous administration of the nanoparticle.

[0104] In a further aspect, the invention may provide a use of the nanoparticle or the composition comprising the nanoparticle as a disinfectant.

[0105] In a further aspect, the invention may provide a synthesis method for providing the nanoparticle of the invention. The synthesis method may comprise a polymerization step, a crosslinking step and optionally a functionalization step. The polymerization step may comprise co-polymerizing (via radical polymerization) a first monomer and a second monomer to provide a precursor polymer, especially wherein the first monomer comprises a first polymerization group, and wherein the second monomer comprises a second polymerization group. In further embodiments, the first polymerization group and the second polymerization group may be (independently) selected from the group comprising an acrylate moiety, an acrylamide moiety, a styrene moiety, a methacrylate moiety and a methacrylamide moiety. The second monomer may further comprise a side precursor group. The method, especially the polymerization stage, may comprise providing the first monomer and the second monomer in a ratio (first monomer: second monomer) selected from the range of 0.05:0.95 - 0.2:0.8, wherein amounts of the first monomer and second monomer may be selected such that the precursor polymer has a (number average) molecular weight selected from the range of 20-300 kDa, such as from the range of 30-150 kDa, especially from the range of 45-100 kDa. The polymerization stage may result in a precursor polymer comprising a (single) backbone and side group precursors. The crosslinking step may comprise exposing the precursor polymer to a crosslinking composition, wherein the crosslinking composition is configured to (i) provide a plurality of crosslink precursor groups, and (ii) to crosslink (sets of two of) the crosslink precursor groups to provide intramolecular crosslinks. In particular, the crosslinking step may comprise one of: (i) converting a part of the side group precursors to crosslink precursor groups (e.g., by attaching crosslink precursor groups to the precursor polymer), and (ii) deprotecting crosslink precursor groups present in the precursor polymer. Depending on the second monomer, the precursor polymer may comprise ligand moieties or may comprise placeholder groups configured to react with a (first) reactant to provide a side group comprising a ligand moiety. Hence, in embodiments, each side precursor group comprises a ligand moiety selected from the group comprising a sugar moiety, a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, an amino acid moiety, and a fatty acid moiety, especially from the group consisting of a sugar moiety, a glycerol moiety, and an amino acid moiety. Alternatively, in embodiments, the method may further comprise the functionalization step, wherein the functionalization step comprises exposing the precursor polymer to first reactants, wherein each first reactant comprises a reactant group and a ligand moiety. Especially, in such embodiments, the reactant group may be configured to react with (at least part of) the side precursor groups, and wherein the ligand moiety is selected from the group comprising a sugar moiety, a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, an amino acid moiety, and a fatty acid moiety, especially from the group consisting of a sugar moiety, a glycerol moiety, and an amino acid moiety.

[0106] Hence, in specific embodiments, the invention may provide a synthesis method for providing a nanoparticle of the invention, wherein the method comprises: a polymerization step comprising co-polymerizing a first monomer and a second monomer to provide a precursor polymer, wherein the first monomer comprises a first polymerization group, and wherein the second monomer comprises a side precursor group and a second polymerization group, wherein the first polymerization group and the second polymerization group are independently selected from the group comprising an acrylate moiety, an acrylamide moiety, a styrene moiety, a methacrylate moiety and a methacrylamide moiety, wherein a ratio of the first monomer to the second monomer is selected from the range of 0.05:0.95 - 0.2:0.8, wherein amounts of the first monomer and second monomer are selected such that the precursor polymer has a (number average) molecular weight selected from the range of 30-150 kDa; a crosslinking step comprising exposing the precursor polymer to a crosslinking composition, wherein the crosslinking composition is configured to (i) provide a plurality of crosslink precursor groups, and (ii) crosslink the (deprotected) crosslink precursor groups to provide intramolecular crosslinks; and wherein: (a) each side precursor group comprises a ligand moiety selected from the group comprising a sugar moiety, a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, an amino acid moiety, and a fatty acid moiety, especially from the group consisting of a sugar moiety, a glycerol moiety, and an amino acid moiety; or (b) the method further comprises a functionalization step comprising exposing the precursor polymer to first reactants, wherein each first reactant comprises a reactant group and a ligand moiety, wherein the reactant group is configured to react with at least part of the side precursor groups, and wherein the ligand moiety is selected from the group comprising a sugar moiety, a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, an amino acid moiety, and a fatty acid moiety, especially from the group consisting of a sugar moiety, a glycerol moiety, and an amino acid moiety.

[0107] The method of the invention may be particularly suitable for synthesizing the nanoparticle of the invention.

[0108] In embodiments, the method may comprise a polymerization step, a crosslinking step and, optionally, a functionalization step. The polymerization step may especially be executed before the crosslinking step and, if applicable, before the optional functionalization step. In further embodiments, the crosslinking step may be arranged before the functionalization step. In further embodiments, the functionalization step may be arranged before the crosslinking step. In embodiments, the method may further comprise a plurality of functionalization steps (see below). In such embodiments, (all of) the functionalization steps may be arranged before the crosslinking step, (all of) the functionalization steps may be arranged after the crosslinking step, or a first part of the functionalization steps may be arranged before the crosslinking step and a second part of the functionalization steps may be arranged after the crosslinking step.

[0109] The polymerization step may comprise co-polymerizing a first monomer and a second monomer (via radical polymerization) to provide a precursor polymer. The polymerization step may especially comprise co-polymerizing the first monomer and the second monomer via vinyl polymerization. In particular, the first monomer may comprise a first polymerization group and the second monomer may comprise a second polymerization group, wherein the first polymerization group and the second polymerization group are suitable for co-polymerization. In embodiments, the first polymerization group and the second polymerization group are (independently) selected from the group comprising an acrylate moiety, an acrylamide moiety, a styrene moiety, a methacrylate moiety and a methacrylamide moiety. The polymerization step may comprise providing the first monomer and the second monomer in a ratio (first monomer: second monomer) selected from the range of 1 :20 to 1 :4, such as selected from the range of 1 :15 to 1 :5, especially from the range of 1 : 12 to 1 :8. The polymerization stage may especially comprise providing amounts of the first monomer and the second monomer such that the (resulting) precursor polymer has a molecular weight selected from the range of 20-300 kDa, such as from the range of 30-150 kDa, especially from the range of 45-100 kDa. In embodiments, the polymerization step may comprise co-polymerizing a first monomer, a second monomer and a third monomer (via radical polymerization) to provide a precursor polymer. Especially, the third monomer may comprise a third polymerization group selected from the group comprising an acrylate moiety, an acrylamide moiety, a styrene moiety, a methacrylate moiety and a methacrylamide moiety. In particular, the polymerization step may comprise co-polymerizing two or more monomers (via radical polymerization) to provide a precursor polymer. Especially, each of the two or more monomers may comprise a (respective) polymerization group (independently) selected from the group comprising an acrylate moiety, an acrylamide moiety, a styrene moiety, a methacrylate moiety and a methacrylamide moiety.

[0110] After the polymerization stage, the precursor polymer may comprise a (single) backbone, side group precursors, and, optionally, (protected) crosslink precursor groups (see below).

[0111] The polymerization stage may conveniently be performed at room temperature under mild conditions, which may facilitate the introduction of relatively sensitive cargo (e.g., an RNA molecule or a peptide).

[0112] In embodiments, the second monomer may comprise a side precursor group. The second precursor group may comprise a ligand moiety or may comprise a reactive group suitable to react with a first reactant (see below) to provide a side group comprising the ligand moiety.

[0113] Hence, in embodiments, (at least part of) the side precursor groups may comprise a ligand moiety selected from the group comprising a sugar moiety, a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, an amino acid moiety, and a fatty acid moiety. Especially, each side precursor group may comprise a ligand moiety selected from the group comprising a sugar moiety, a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, an amino acid moiety, and a fatty acid moiety. In such embodiments, the method may generally not comprise the functionalization step.

[0114] In embodiments, the crosslinking step may comprise exposing the precursor polymer to a crosslinking composition. The crosslinking composition may be configured to provide a plurality of (available) crosslink precursor groups. For instance, in embodiments, the crosslinking composition may be configured to provide a plurality of (available) crosslink precursor groups by reacting second reactants to side group precursors, wherein the second reactants comprise crosslink precursor groups. Alternatively, in embodiments, the crosslinking composition may be configured to provide a plurality of (available) crosslink precursor groups by deprotecting (protected) crosslink precursor groups present in the precursor polymer. The crosslinking composition may further be configured to crosslink (sets of two of) the crosslink precursor groups to provide intramolecular crosslinks. Hence, the crosslinking step may comprise (i) providing (available) crosslink precursor groups, especially by reacting second reactants to side group precursors, or especially by deprotecting (protected) crosslink precursor groups, and (ii) crosslinking (sets of two) crosslink precursor groups to provide intramolecular crosslinks.

[0115] During the crosslinking step a concentration of precursor polymer may be selected such that (essentially) only intramolecular crosslinks are formed. For instance, in embodiments, the crosslinking step comprise crosslinking the precursor groups in a reaction mixture, wherein the reaction mixture comprises the precursor polymer in a concentration selected from the range of 1-20 g / L, such as from the range of 2-10 g / L, especially from the range of 3-7 g / L.

[0116] For instance, in embodiments the first monomer may further comprise a (protected) crosslink precursor group, such as a (protected) thiol group. In such embodiments, the crosslinking step may comprise deprotecting the (protected) crosslink precursor group, such as by exposing the precursor polymer to a deprotection agent. For instance, in embodiments, the first monomer may comprise xanthate methacrylate, and the crosslinking step may comprise (a) exposing the precursor polymer to ethanolamine to provide (or “deprotect”) a thiol group, and (b) exposing the precursor polymer to a crosslinking composition, such as to phosphine and methacrylate, to provide an intramolecular crosslink using two (deprotected) thiol groups.

[0117] The crosslinking step may (also) be carried out at room temperature under mild conditions, which may be beneficial in view of compatibility with sensitive cargo molecules.

[0118] The method may, in embodiments, comprise a functionalization step comprising exposing the precursor polymer to first reactants. In embodiments, each first reactant may comprise a reactant group and a ligand moiety, wherein the reactant group is configured to react with at least part of the side precursor groups, and wherein the ligand moiety is selected from the group comprising a sugar moiety, a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, an amino acid moiety, and a fatty acid moiety. In further embodiments, the method may comprise a plurality of functionalization steps, wherein the functionalization steps comprise providing different first reactants, especially wherein the first reactants of different functionalization steps differ in ligand moieties.

[0119] In further embodiments, the side precursor groups may comprise pentafluorophenyl moieties and the reactant groups may comprise amine groups. In such embodiments, the pentafluorophenyl may function as ‘leaving group’, and the ligand moiety may become connected to the backbone via an amide group.

[0120] In further embodiments, the polymerization step may comprise (radical) (copolymerization of vinyl monomers, such as functional (meth)acrylate, (meth)acrylamide or styrene monomers, providing handles for intramolecular crosslinking, and providing the selected ligand moieties directly or indirectly via subsequent post-polymerization functionalization.

[0121] In specific embodiments, the first monomer comprises xanthate methacrylate, the side precursor groups comprise pentafluorophenyl moieties, the crosslinking step comprises exposing the precursor polymer to ethanolamine, and the method comprises the functionalization step, wherein the reactant group comprises an amine group.

[0122] BRIEF DESCRIPTION OF THE DRAWINGS

[0123] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which: Fig. 1A-B schematically depict embodiments of the nanoparticle. Fig. 2 schematically depicts an embodiment of the method of the invention. Fig. 3-4 depict experimental results obtained with an embodiment of the method of the invention. The schematic drawings are not necessarily on scale.

[0124] DETAILED DESCRIPTION OF THE EMBODIMENTS

[0125] Fig. 1A-B schematically depict embodiments of the nanoparticle 100 for introduction into a prokaryotic cell 10 (see Fig. 2). The nanoparticle 100 may have a diameter, e.g., an equivalent spherical diameter, selected from the range of 2-40 nm, especially from the range of 3-20 nm, such as from the range of 8-15 nm. In the depicted embodiments, the nanoparticle 100 comprises a single-chain polymer nanoparticle 101, wherein the nanoparticle 100 comprises a backbone 110, a plurality of intramolecular crosslinks 120, and a plurality of side groups 130 (covalently) bound to the backbone 110. The backbone 110 may comprise n backbone atoms, wherein the n backbone atoms comprise > 0.9*n carbon atoms, such as > 0.99*n carbon atoms, especially (essentially) only carbon atoms. As depicted in Fig. 1A-B, each intramolecular crosslinks 120 may covalently interconnect two (respective) backbone locations 112 of the backbone 110. For instance, in Fig. 1 A, a first intramolecular crosslink 110 covalently interconnects two first backbone locations 112,112a and a second intramolecular crosslink 110 covalently interconnects two second backbone locations 112,112b. In embodiments, k of the plurality of side groups 130 may comprise ligand moieties 140, such as ligand moieties (individually) selected from the group comprising sugar moieties, a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, amino acid moieties, and fatty acid moieties. In further embodiments, k / n may be selected from the range of 0.05-0.49. The ligand moieties 130 in the nanoparticle 100 may all be the same, such as schematically depicted in Fig. 1 A, but may also differ.

[0126] In further embodiments, the nanoparticle 100 may comprise m intramolecular crosslinks 120, wherein m is selected from the range comprising 0.02*n - 0.1 *n (or “the range of 0.01 *n - 0.2*n), and wherein the intramolecular crosslinks 120 have lengths between the two (respective) backbone locations 112 selected from the range of 4-120 atoms.

[0127] In the depicted embodiments, the ligand moieties 140 are depicted arranged at the end of (linear) linkers 135. In particular, in the depicted embodiments, the side groups 130 comprise linkers 135, wherein the linkers 135 separate the backbone 110 and the ligand moieties 140.

[0128] In embodiments, each linker 135 may comprise a side backbone comprising (individually selected) 3-20 (side backbone) atoms, especially wherein the (side backbone) atoms are selected from the group consisting of C, N, S, P, and O. For instance, in embodiments, (at least part of) the linkers 135 may consist of propyl moieties, di ethylene glycol moieties or triethylene glycol moieties.

[0129] In further embodiments, the side groups 130, especially the linkers 135, may comprise polymer ethylene glycol linkers, wherein the polymer ethylene glycol linkers separate the backbone 110 and the ligand moieties, wherein each polymer ethylene glycol linker has a weight selected from the range of 500-3000 Da.

[0130] In the depicted embodiments, the nanoparticle 100 further comprises a cargo 150. In embodiments, the nanoparticle 100 may encapsulate the cargo 150, such as to (non- covalently) physically encapsulate the cargo. Additionally or alternatively, in embodiments, the nanoparticle 100 may be covalently bound to the cargo 150. The cargo may comprise essentially any (small) molecule that is desired to be introduced into the prokaryotic cell. For instance, in embodiments, the cargo may comprise an antibiotic. In further embodiments, the cargo may comprise an RNA molecule, especially an sRNA molecule, or especially an siRNA molecule.

[0131] Fig. IB schematically depicts an embodiment with side groups 130 comprising different ligand moieties 140. In particular, in the depicted embodiment, the side groups 130 comprise a first subset 31 of ki first side groups 131 and a second subset 32 of k2 second side groups 132. In further embodiments, each first side group 131 may comprise a (single) first ligand moiety 141 (individually) selected from the group comprising sugar moieties, especially pentose moieties, or especially hexose moieties. The first ligand moieties 141 are, for explanatory purposes, schematically depicted as hexose moieties, such as glucose moieties. In further embodiments, each second side group 132 may comprise a (single) second ligand moiety 142 selected from the group comprising a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, amino acid moieties, and fatty acid moieties.

[0132] Especially, ki / n may be selected from the range of 0.05-0.3, such as from the range of 0.05-0.25, especially from the range of 0.1-0.2. Further, in embodiments, ki / n may be selected from the range of 0.05-0.44, such as from the range of 0.1-0.4. In the depicted embodiment, the side groups 130 may consist of the first side groups 131 and the second side groups 132, i.e., in the depicted embodiment ki+k2=k. In further embodiments, the nanoparticle may comprise further side groups, i.e., in further embodiments, wherein ki+k2 < k.

[0133] Hence, Fig. IB schematically depicts an embodiment wherein the nanoparticle 100 comprises first side groups 131 and second side groups 132, wherein the first side groups 131 comprise sugar moieties, and wherein the second side groups comprise non-sugar ligand moieties.

[0134] Fig. 2 schematically depicts an embodiment of the method for introducing a nanoparticle 100 into a prokaryotic cell 10, such as into a gram -negative bacterium 11. As schematically depicted, the method may comprise exposing the prokaryotic cell 10 to the nanoparticle 100, resulting in an interaction between the nanoparticle 100 and a (cell surface) receptor 11, following which the prokaryotic cell 10 may import the nanoparticle 100. As a prokaryotic cell 10 may be exposed to a plurality of nanoparticles 100, a plurality of the nanoparticles 100 may end up in the prokaryotic cell 10, as schematically depicted in Fig. 2. The prokaryotic cell 10 may thus comprise a receptor 14 configured to interact with (or “recognize”) one or more of the ligand moieties 140. The prokaryotic cell 10 may further comprise a transporter configured to import (or “suitable for importing”) the ligand moiety 140 upon interaction with the receptor 14, especially to import the nanoparticle 100 when the ligand moiety 140 interacts with the receptor 14. The ligand moieties 140 of the nanoparticle 100 may thus especially be selected based on the (cell surface) receptors 14 and transporters of the prokaryotic cell 10.

[0135] The nanoparticle 100 of Fig. 2 may, for instance, comprise the nanoparticle schematically depicted in Fig. 1A or in Fig. IB (see description above). Hence, in embodiments, the nanoparticle 100 may have an equivalent spherical diameter selected from the range of 5-20 nm, wherein the nanoparticle 100 comprises a single-chain polymer nanoparticle 101, wherein the nanoparticle 100 comprises a backbone 110, a plurality of intramolecular crosslinks 120, and a plurality of side groups 130 (covalently) bound to the backbone 110, wherein the backbone 110 comprises n backbone atoms, wherein the n backbone atoms comprise > 0.9*n carbon atoms, wherein each intramolecular crosslink 120 covalently interconnects two (respective) backbone locations 112 of the backbone 110, wherein k of the plurality of side groups 130 comprise ligand moieties 140 [individually] selected from the group comprising sugar moieties, a glycerol moiety, an acetate moiety, a pyruvate moiety, a lactate moiety, a succinate moiety, amino acid moieties, and fatty acid moieties, wherein k / n is selected from the range of 0.05-0.49.

[0136] Experiments

[0137] Unless specified otherwise, the experiments described hereinafter were performed using the following materials and methods.

[0138] Materials - BFs-Et2O (46%), 3-amino-l,2-propanediol (1 -aminoglycerol, 97%), 3 -bromo- 1 -propanol (97%), 2-[2-(2-chloroethoxy)ethoxy]ethanol (96%), Pd / C 10%, NaOMe in MeOH 25 wt%, NaNs (>99.5%), DMSO (anhydrous, 99.9%), Concanavalin A from Canavalia ensiformis (Type VI) corresponding to Uniprot ID A8WDZ4, methyl a-D- mannopyranoside, Amberlyst 15 (H), chloroform-d (99.8% atom % D) and DMSO-de (99.9 atom % D) were purchased from Sigma-Aldrich. Fluorescent label 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF) and SnakeSkin™ dialysis tubing (10K MWCO) were purchased from ThermoFisher Scientific. P-D-glucose pentaacetate (>99%) was purchased from Fluka BioChemika. Tetrahydrofuran (THF, >99%), di chloromethane (DCM, >99%), ethyl acetate (EtOAc, >99%), heptane (>99%) and methanol (>99%) were purchased from LPS B.V. N,N- dimethylformamide (DMF, >99.8%) was purchased from VWR. Chloroform (>99.8%) was purchased from Merck. Disposable PD-10 desalting columns were purchased from GE healthcare All chemicals were used without further purification unless stated otherwise. When stated as dry, solvents were treated with molecular sieves (3 A) 24 h before usage and stored under nitrogen.

[0139] Analytical techniques - 'H-NMR (400 MHz),13C NMR (101 MHz) and19F- NMR (376 MHz) spectra were recorded on a Bruker 400 spectrometer. Size exclusion chromatography (SEC) analysis was performed on a Waters e2695 Separations Module equipped with an Agilent PLgel 5 pm MIXED-D 300x7.5 mm column and Waters photodiode array detector (PDA 2998), fluorescence detector (FLR 2475) and refractive index detector (RI 2414). DMF with 50 mM LiCl was employed as eluent. Samples for SEC were filtered using a GE Healthcare Whatman SPARTAN 13 / 0.2 RC 0.2 pm syringe filter prior to measurements. UV-measurements were performed on a Shimadzu 1 UV-2401PC UV-VIS Recording Spectrophotometer. Mass spectra were obtained on an Advion Expression-L Compact Mass Spectrometer featured with either electrospray ionization (ESI) or Atmospheric Pressure Chemical Ionization (APCI).

[0140] Synthesis of glucose ligand precursors - Eight different glucose ligand precursors were synthesized; successful synthesis of the compounds was confirmed with NMR,13CNMR and ESLMS. Specifically, the following glucose ligands were synthesized (see detailed procedures below):

[0141] Compound 1 Compound 5

[0142] Compound 3 Compound 7

[0143] Compound 1 : A 250 mL three neck flask was charged with 3 -bromo- 1 -propanol

[0144] (1.43 g, 10.2 mmol, 2.0 eq.) and brought under nitrogen. Dry dichloromethane (“DCM”) (80 mL) was added, followed by the addition of P-D-glucose-pentaacetate (2.0 g, 5.1 mmol, 1.0 eq.). After dissolving, BF3-Et2O (46%, 1.8 mL, 14.6 mmol, 2.9 eq.) was added dropwise by syringe in 5 min. The mixture was stirred overnight, after which the light yellow solution was diluted with 300 mL DCM, transferred to a 2 L beaker and sat. NaHCCh (400 mL) was slowly added over 30 min. The organic layer was separated, dried over MgSCU, filtered and concentrated under reduced pressure. Flash chromatography (SiCL, 20% — 50% EtOAc in heptane) yielded a colorless oil, which slowly solidified to an off-white waxy solid (yield: 1.25g, 52%). Compound 2: A 250 mL three neck flask was charged with 2-[2-(2- chloroethoxy)ethoxy]ethanol (2.6 g, 15.4 mmol, 2 eq.) and brought under nitrogen. Dry DCM was added (120 mL), followed by P-D-glucose-pentaacetate (3.0 g, 7.7 mmol, 1 eq.). After dissolving, BF3-Et2O (3.3 g, 23. 1 mmol, 3 eq.) was added dropwise by dropping funnel in 20 min and the solution was stirred overnight, after which the yellow solution was diluted with 450 mL DCM, transferred to a 2L beaker and sat. NaHCOs (600 mL) was slowly added over 120 min. The organic layer was separated and concentrated under reduced pressure. The residue was redissolved in 100 mL EtOAc and washed consecutively with 5 x 30 mL ELO, 2 x 30 mL 10% NH4CI and 1 x 30 mL brine, dried over MgSCU, filtered and concentrated under reduced pressure. Flash chromatography (SiCL, 25% — 100% EtOAc in heptane) yielded a colorless oil (yield: 2.3g, 61%).

[0145] Compound 3: A 100 mL three neck flask was charged with compound 1 (1.25 g, 2.6 mmol, 1 eq.), brought under nitrogen and dissolved in 13 mL dry DMF. NaNs (0.85g, 13.0 mmol, 5 eq.) was added and the mixture was stirred overnight at 50° C. DMF was removed under reduced pressure and the residue was dissolved in 50 mL ethyl acetate. The mixture was washed consecutively with 2 x 20 mL H2O, 5 x 20 mL 10% NH4CI, 20 mL brine, dried over MgSO4, filtered and concentrated under reduced pressure to yield a colorless oil (yield: 1.23 g, 98%).

[0146] Compound 4: A 250 mL three neck flask was charged with compound 2 (2.35 g, 4.7 mmol, 1 eq.), brought under nitrogen and dissolved in 80 mL dry DMF. NaNs (L53g, 23.5 mmol, 5 eq.) was added and the mixture was stirred overnight at 50° C. DMF was removed under reduced pressure and the residue was dissolved in 150 mL ethyl acetate. The mixture was washed consecutively with 1 x 25 mL H2O, 5 x 50 mL 10% NH4CI, 50 mL brine, dried over MgSO4, filtered and concentrated under reduced pressure to yield a colorless oil (yield: 2.3 g, 96%).

[0147] Compound 5: A 100 mL flask was charged with compound 3 (0.50 g, 1.16 mmol) and dissolved in 20 mL 20 mM NaOMe in MeOH and stirred overnight. The mixture was neutralized with Amberlyst 15 (H), filtered, rinsed with MeOH and concentrated under reduced pressure to a sticky colorless solid (yield: 0.3 g, quant).

[0148] Compound 6: A 250 mL flask was charged with compound 4 (2.3 g, 4.6 mmol) and dissolved in 80 mL 20 mM NaOMe in MeOH and stirred overnight. The mixture was neutralized with Amberlyst 15 (H), filtered, rinsed with MeOH and concentrated under reduced pressure to a sticky brown solid (yield: 1.5 g, 98%). Compound 7: A 100 mL flask was charged with compound 5 (0.20 g, 0.76 mmol) and 15 mL MeOH. The solution was purged with nitrogen for 15 min, followed by addition 15 mg Pd / C 10 wt%. The flask was placed in an autoclave and filled with 8 bar H2 and stirred overnight. Next, the solution was purged with nitrogen for 15 min and filtered over Celite S, rinsed with MeOH and concentrated to yield a yellowish oil, which was suspended in water and lyophilized (yield: 0.18 g, quant).

[0149] Compound 8: A 100 mL flask was charged with compound 6 (0.35 g, 1.0 mmol) and 15 mL MeOH. The solution was purged with nitrogen for 15 min, followed by addition 30 mg Pd / C 10 wt%. The flask was placed in an autoclave and filled with 8 bar H2 and stirred overnight. Next, the solution was purged with nitrogen for 15 min and filtered over Celite S, rinsed with MeOH and concentrated to yield a yellowish oil, which was suspended in water and lyophilized (yield: 0.30 g, 93%).

[0150] Formation of pentafluorophenyl-based SCNPs - pentafluorophenyl SCNPs (PFP-SCNPs) were synthesized as described in KROGER, APA et al., Pentafluorophenyl- based single-chain polymer nanoparticles as a versatile platform towards protein mimicry, Polymer chemistry, 2020, 11, pages 6056-6065, which is hereby herein incorporated by reference. Specifically, a co-polymer (600 mg, DP=230, 0.24 mmol xanthate groups, 1 eq.), containing pentafluorophenyl and xanthate pendant side-groups was used. The xanthate moieties were deprotected by ethanolamine (87 pL, 1.44 mmol, 6 eq.) to obtain free thiols. After precipitation of the polymer in cold MeOH (130 mL), the polymer was redissolved in Tetrahydrofuran (“THF”) (11 mL) and intramolecularly crosslinked via a thiol-Michael addition, by slow addition of the co-polymer to a solution of 1,4-butanediol diacrylate (47 mg, 0.24 mmol, 1 eq.) as crosslinker and tri(w-butyl)phosphine (10 mg, 0.04 mmol, 0.2 eq.) as initiator in DCM (130 mL). After stirring for an additional 2 hours, the remaining thiols were end-capped by methyl acrylate (1 mL, 12 mmol, 50 eq.). The SCNPs were obtained as a white powder after repeated precipitations in MeOH (60 mL) (310 mg, 53% yield). For these SCNPs about 10% of the used monomers comprised xanthate side groups and were crosslinked after formation of the precursor polymer. The PFP-SCNPs may comprise (about) 0.05 intramolecular crosslinks per 2 backbone atoms and (about) 0.9 side groups per 2 backbone atoms.

[0151] Glucose functionalized SCNPs - A range of SCNPs with increasing glucose ligand density was made from the PFP-SCNPs. For instance, for the 19% substituted short linker glyco-SCNPs (SCNP-S19; see below), 40 mg of PFP-SCNPs (0.14 mmol PFP, 1 eq.) was dissolved in 3 mL dry THF under nitrogen atmosphere, 77 pL of triethylamine (0.55 mmol, 4 eq.) was added, followed by 3-aminopropyl P-D-glucopyranoside (compound 7, 11.5 mg, 0.048 mmol, 0.35 eq.) from a 20 mg / mL stock solution in dry DMF and another 0.4 mL dry DMF was added to reach a total of 1 mL. The solution was stirred for 24h at 45° C. Conversion was determined by19F NMR analysis, followed by the addition of 3-amino-l, 2, -propanediol (138 mg, 1.5 mmol, 11 eq., 16 wt% solution in DMF) to endcap the remaining reactive PFP- groups. After stirring overnight at room temperature, the mixture was dialyzed against 1 wt% NaCl for 24 hours, followed by another 48 hours against demineralized water. The clear solutions were lyophilized to yield a white powder (20 mg, 75% yield). The remaining glyco- SCNPs (see below) were made analogously but with different amounts of the respective glucose ligand precursor and optionally with a different glucose ligand precursor i.e., with compound 8 instead of compound 7.

[0152] An overview of the amount (in eq.) of compound 7 (for short linkers) and compound 8 (for long linkers), the percentage of side groups comprising a glucose-ligand, and the zeta potential for 8 different SCNPs are provided in table 2:

[0153] Specifically, “S” indicates that the glucose-containing side groups 130,131 contain ‘short’ propyl moi eties as linkers 135 and “L” indicates that the glucose-containing side groups 130,131 contain Tong’ triethylene glycol moieties as linkers 135. The number following “S” or “L” indicates the percentage of side groups comprising a glucose moiety. For these eight nanoparticles 100, the remaining side groups 130, especially second side groups 132, comprise glycerol moieties. These SCNPs may hereinafter also be referred to as ‘glyco- SCNPs’ or ‘glucose-functionalized SCNPs’. In addition, a nanoparticle 100 comprising (essentially) only glycerol moieties was generated using the process as described above for the glyco-SCNPs but without the step of adding the glucose ligand precursors; this nanoparticle 100 is hereinafter referred to as SCNP-0. UsingXH Diffusion-Ordered Spectroscopy (DOSY) NMR in D2O the following hydrodynamic radii of SCNP-0 and the eight glyco-SCNPs were determined: SCNP-0 - 5.7 nm; SCNP-7 - 6.5 nm; SCNP-S19 - 6.8 nm; SCNP-S30 - 7.0 nm; SCNP-S55 - 7.9 nm; SCNP-L7 - 6.7 nm; SCNP-L19 - 7.8 nm; SCNP-L30 - 8.0 nm; and SCNP-L55 - 9.0 nm.

[0154] Comparative example - An SCNP with 45% of side groups comprising tertiary amines and 55% of side groups comprising glycerolamide moieties was used as a comparative example; this nanoparticle is hereinafter referred to as SCNP -45. SCNP -45 was generated as described in KROGER, APA et al., Pentafluorophenyl-based single-chain polymer nanoparticles as a versatile platform towards protein mimicry, Polymer chemistry, 2020, 11, pages 6056-6065, which is hereby herein incorporated by reference.

[0155] Fluorescent labelling - Glucose-functionalized SCNPs (between 7-8 mg) were dissolved in 2 mL of 0.1 M carbonate-bicarbonate buffer (pH 9.7). 5-DTAF (0.02 eq. to SCNPs, 10 mg / mL stock solution in dry DMSO) was added and the solutions were stirred under ambient conditions overnight, after which they were purified by elution with water from a PD10 column and subsequently lyophilized to yield yellow powders (90% yield).

[0156] Concanavalin A precipitation assay - Concanavalin A (Con A) was dissolved in HEPES Buffer pH 7.4 (140 mM NaCl, 20 mM HEPES, 1.0 mM MgCh, 2.5 mM KC1, 1.8 mM CaCh and 1 mM MnCh) to reach a 60 mM concentration (assuming tetramers of 104 kDa). The Glyco-SCNPs were dissolved in the buffer to reach 17 pM concentrations. Equal volumes (150 pL) of SCNP-solution and Con A were thoroughly mixed and incubated for 20h at r.t at 500 rpm. The samples were centrifuged (15 min x 7000 rpm) and the supernatant was removed. The pellets were washed with 200 pL cold buffer, followed by centrifugation (15 min x 7000 rpm). The supernatant was removed and the washing step was repeated. The pellets were dissolved in 600 pL 100 mM methyl a-D-mannopyranoside to help dissolving any aggregates, incubated for 2 h at r.t at 1400 rpm, diluted with 500 pL buffer and the UV-absorbance was measured at 285 nm ( max). Measurements are the average of 6 experiments for each data point.

[0157] Concanavalin A turbidimetric assay - Con A was dissolved in HEPES Buffer pH 7.4 (140 mM NaCl, 20 mM HEPES, 1.0 mM MgCh, 2.5 mM KC1, 1.8 mM CaCl2and 1 mM MnCh) to reach a 60 mM concentration (assuming tetramers of 104 kDa). Glyco-SCNPs were dissolved in the buffer to reach 2.83 pM concentrations. Con A solution was added to a micro-cuvette (75 pL) and placed in the UV-spectrophotometer. Glyco-SCNPs were added (75 pL), mixed by pipetting up and down and the absorbance was measured at 420 nm for 10 minutes. The initial linear part of the graph was determined by a linear fit up until the timepoint where the coefficient of determination (R2) dropped below 0.99. The final slope was determined between t = 400 and t = 600 s. All measurements were performed in triplicate. Bacteria culture and FACS analysis - E. coli BL21 strain was transformed with a pET-mcherry cloning vector and selected on LB-Agar with 50 pg / mL Ampicillin (Ap). The cells were pre-cultured overnight in LB-Ap 50pg / mL at 37°C at 200rpm, then they were centrifuged at 5000 g for 5 min and resuspended in M63 minimal medium supplemented with Glucose 0.4% (w / v), MgSCU 1 mM, Ap 50 pg / mL and IPTG 100 pM. The cells were inoculated at 0.5 uOD in 2 mL of M63 supplemented medium in a 12 wells plate. The SCNPs were added at t = 0 h at a concentration of 3 pM and incubated for 20 h at 37° C at 100 rpm. The cultures were collected, centrifuged at 5000 g for 5 min and resuspended in phosphate buffered saline (PBS). The uptake of the SCNPs by bacteria was analyzed on a MACSQuant® Analyzer 10 Flow Cytometer using excitation and emission filter of 488-525 / 50 nm for the SCNPs, 561- 6115 / 20 nm for the mCherry and 561-561 / 10 nm for the scattering. Experiments were conducted in triplicate.

[0158] S. aureus cells were pre-cultured overnight in LB-Ap 50pg / mL at 37°C at 200rpm, then they were centrifuged at 5000 g for 5 min and resuspended in M63 minimal medium supplemented with Glucose 0.4% (w / v), MgSCU 1 mM, Ap 50 pg / mL and IPTG 100 pM. The cells were inoculated at 0.5 uOD in 2 mL of M63 supplemented medium in a 12 wells plate. The SCNPs were added at t = 0 h at a concentration of 3 pM and incubated for 20 h at 37° C at 100 rpm. The cultures were collected, centrifuged at 5000 g for 5 min and resuspended in phosphate buffered saline (PBS). The uptake of the SCNPs by bacteria was analyzed on a MACSQuant® Analyzer 10 Flow Cytometer using excitation and emission filter of 488-525 / 50 nm for the SCNPs and 561-561 / 10 nm for the scattering. Experiments were conducted in triplicate.

[0159] Bacteria fractionation - For the fractionation of the bacteria, the three experiments were pooled together and the ODeoonm was measured. 1 uOD was taken for the whole cell fraction analysis and 5 uOD was used for the fractionation steps. The whole cells were centrifuged at 3000 g for 5 min. The supernatant was discarded and the pellet resuspended in Laemmli buffer IX. Two types of fractionation were performed, one to separate the soluble fractions and the membranes and a second one to also separate the cytoplasm and the periplasm. For the fractionation of the soluble membranes, 5 uOD of cells were resuspended in 400pL buffer M (lOmM Tris HC1 pH7.4, 5mM EDTA, 20% sucrose) and sonicated 2x30 sec, amplitude 80% (Fisherbrand 120). The lysates were centrifuged at 1600 g for lOmin to remove debris and unbroken cells. The supernatant containing the membranes and the soluble fractions were ultracentrifuged at 130000 g for 45’ with a Fiberlite F50L rotor. The pellet (membranes) was resuspended in Laemmli buffer IX. The supernatant (soluble fractions) was collected and Laemmli 4X buffer was added in corresponding quantity. For the fractionation of the periplasm, cytoplasm and membranes, 5 uOD of cells were resuspended in 34 pL TES buffer (0.2M Tris / HCl pH 8, 0.5 mM EDTA, 0.5 M saccharose) with 35 pg of Lysozyme. The cells were put on ice for 15 min and then 120 pL of TES (1 / 2 diluted) was added. The cell were then again put on ice for 1 h. The cells were centrifuged at 5000 g for 5 min and the supernatant (periplasm) was collected. Laemmli 4X buffer was added in corresponding quantity. The pellet was resuspended in buffer M and sonicated 2x30 sec, amplitude 80%. The lysates were centrifuged at 1600 g for 10 min. The pellet was resuspended in Laemmli buffer IX. The supernatant (soluble fractions) was collected and Laemmli 4X buffer was added in corresponding quantity.

[0160] Bacteria gel analysis - A Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) with 15% acrylamide was performed to analyze all the fractions. The equivalent of 0.25 uOD was loaded for all the fractions. The gel was imaged using FluorChem M hardware (Proteinsimple) to detect the SCNPs. Afterwards, the proteins were transferred onto nitrocellulose membranes using a wet blotting apparatus (BioRad). Membranes were blocked with 5% milk in PBST (Phosphate Buffered Saline; 0.05% Tween 20) and incubated with polyclonal rabbit anti-OmpF antibody (Sanbio, dilution 1 :2000) or a monoclonal mouse anti-Mbp anti-body (BioRad, dilution 1 : 1000). After incubation, the membranes was washed 2 x 10 min and subsequently incubated for 1 h with peroxidase-labeled anti-mouse or anti-rabbit antibody (1 :2000, Sigma). Membranes were developed by homemade enhanced chemiluminescence and scanned using FluorChem M hardware (Proteinsimple). The western-blots were quantified by Image Studio Lite using the same size region of interest for the different lanes. The Western-blots were quantified by Image Studio Lite using the same size region of interest for the different lanes.

[0161] Experiment 1 - lectin binding assays

[0162] A quantitative precipitation assay was performed using concanavalin A (Con A) to evaluate the glucose accessibility on the SCNP surface. Con A is a tetrameric lectin (27 kDa per monomer) with four glucose binding sites at neutral pH. Upon binding of Con A to the glyco-SCNPs, aggregates can form, which can be separated and quantified by UV absorbance to give an indication of the availability of the ligands for receptor-binding. Experiments were conducted with Con A in 3.5: 1 excess compared to the glyco-SCNPs, i.e., the glucose moieties were in high stochiometric excess compared to Con A. Lowering the SCNP concentration an order of magnitude did not yield observable precipitations, whereas higher concentrations of SCNPs resulted in increased scattering. When low amounts of glucose-ligands (SCNP-S7 and SCNP-L7) were incorporated, no measurable Con A precipitates were formed. Upon increasing the ligand density to 19% (SCNP-S19 and SCNP-L19), both the short and long linker SCNPs were observed to bind around 1 Con A tetramer, ultimately reaching 2.6-2.7 con A tetramers per glyco-SCNP at the maximum ligand density (75% of the total available con A in solution, as summarized in table 3 :

[0163] These results demonstrate that even upon increasing the number of glucose ligands on the SCNPs, the ligands are still accessible for lectin-binding. However, binding efficiency may drop, as observed by the increasing amount of glucose ligands per lectin for the highest substituted SCNPs. No significant difference between the two linkers was observed (for any of the ligand densities).

[0164] A turbidimetry assay with Con A in 20-fold excess to glyco-SCNPs was performed to probe the rate of SCNP-Con A cluster formation. Coagulation was induced and monitored over time by measuring the increased absorbance at 420 nm. The lowest substituted glyco-SCNPs (SCNP-S7 and SCNP-L7) did not yield any increased turbidity. Upon increased first ligand moiety density, rapid complexation and subsequent precipitation was observed. No difference between short and long linker glyco-SCNPs was observed until the highest substitution grade. However, a difference between the short and long linker glyco-SCNPs was observed at later timepoints. Whereas SCNP-S19 to SCNP-S55 reach a similar plateau value within the experiment’s timeframe, SCNP-L19 to SCNP-L55 show a 2-3 fold higher plateau, the difference between short and long linker being more pronounced upon increasing first ligand moiety density, as summarized in table 4: where the “final slope” refers to the slope from t=400s to t=600s, and wherein At=600 refers to the absorbance at 420 nm at t=600.

[0165] Experiment 2 - cellular uptake in E. coli bacteria

[0166] The entire set of glyco-SCNPs, SCNP-0, and SCNP-45 was evaluated for cellular association in E. coli by flow cytometry.

[0167] Fig. 3 schematically depicts fluorescence F (in a.u.) versus the amount of glucose moieties G (as percentage of total side groups) for the glyco-SCNPs, for SCNP-0, and for SCNP-45, with SCNP-0 referring to SCNP-0, S referring to SCNPs with short linkers, L referring to SCNPS with long linkers, and SCNP-45 referring to SCNP-45 (see above).

[0168] For the glyco-SCNPs with a short linker (SCNP-S7 to SCNP-S55), a gradual increase in cellular association was observed upon increasing glucose-content, with an optimum at about 30% (SCNP-S30). However, the highest substituted SCNP-S55 does not display increased cellular association when comparing to SCNP-0. Regarding the long linker glyco-SCNPs, SCNP-L7 and SCNP-L19 show similar results as their short-linker counterparts, but a strong decrease in cellular association is observed for SCNP-L30, with a 5 fold difference with SCNP-S30. For the highest glucose-incorporation, the cellular association is again similar for both. Thus, even though the long linker SCNPs displayed stronger lectin association in the turbidity assays compared to the short linker versions (see above), a different trend is observed for the flow cytometry. It thus appears that the cellular association (and cellular uptake; see below) is determined by a combination of the amount of glucose moieties and the linker length, where shorter linkers may beneficially be combined with (relatively) higher numbers of glucose moieties.

[0169] SCNP-0 as well as the entire set of glyco-SCNPs may have backbones 110 each comprising (about) 484 backbone atoms (based on molecular weight prior to functionalization), i.e., n was (about) 484 for these SCNPs. In total, these SCNPs may comprise (about) 12 intramolecular crosslinks and (about) 218 side groups. For each of these SCNPs, the number of glucose moieties were estimated based on ligand substitution as determined by19F NMR spectroscopy. The estimated number of glucose moieties, glycerol moieties and corresponding ki / n and k2 / n values are summarized in table 5:

[0170] As depicted in Fig. 3, these SCNPs may associate with the prokaryotic cell 10. Further, these SCNPs may be taken up by the prokaryotic cell 10 (see Fig. 4 and discussion below). Hence, in specific embodiments, each first side group 131 may comprise a glucose moiety, and each second side group 132 may comprise a glycerol moiety, wherein ki / n is selected from the range of 0.05-0.25, and wherein k2 / n is selected from the range of 0.2-0.44. In further embodiments, ki / n may be selected from the range of 0.1-0.18, such as from the range of 0.12-0.18, and the linkers 135 may consist of propyl moieties. Alternatively, in further embodiments, ki / n is selected from the range of 0.05-0.13, and the linkers 135 consist of triethylene glycol moieties.

[0171] Subsequently, the cellular location of the SCNPs was determined by fractionation experiments. Specifically, fractionation experiments were performed with SCNP- 0, SCNP-S30, and SCNP-45. SCNP-45 had previously revealed high cellular uptake and entry into the cytosol of eukaryotic endothelial cells, as described in KROGER, APA et al., Pentafluorophenyl-based single-chain polymer nanoparticles as a versatile platform towards protein mimicry, Polymer chemistry, 2020, 11, pages 6056-6065. SCNP-45 was observed by flow cytometry to have a comparable cellular association as SCNP-S30.

[0172] E. coll cells were incubated with SCNP-0, SCNP-S30 and SCNP-45, after which bacteria were lysed and the different bacterial compartments were separated by centrifugation. The different fractions were put onto an SDS-page gel as described above.

[0173] Fig. 4 schematically depicts the gel electrophoresis results of the fractionation experiment, where ‘Cell’ indicates a total fraction, ‘Mb’ indicates an outer membrane fraction, ‘sol’ indicates a soluble fraction, ‘Cyto’ indicates a cytoplasmic fraction, and ‘Peri’ indicates a periplasmic fraction. Anti-MbP is an antibody against periplasmic maltose binding protein and Anti-OmpF is an antibody against the porin of the external membrane, which antibodies were used for loading and quality control of the fractionation. Periplasmic Maltose-binding Protein (MbP) is only found in the soluble fractions, not in the membrane fraction, indicating that the membrane fraction is not contaminated by the soluble fraction (see Anti-MbP line). The Outermembrane protein F (OmpF) is found in the soluble fraction for all four samples, although in only minute amounts compared to the membrane fraction (see anti-OmpF line). This means small leakages from the membrane into the soluble fraction occurred during separation. However, as the SCNPs are only found in either the membrane or the soluble fraction for all the samples, this leakage does not appear to have affected the fractioning of the SCNPs.

[0174] As depicted in Fig. 5, despite the high cellular association SCNP-45 was found to fully resides with the (outer) membrane fraction, with no uptake into the soluble fractions.

[0175] By contrast, both the nanoparticles 100 SCNP-0 and glyco-SCNP-S30 were observed to have been into a soluble fraction of E. coli, with no SCNPs found residing with the membranes. By normalizing the signal for the intrinsic fluorescence of the SCNPs, the uptake of SCNP-S30 is determined to be 2-fold higher than that observed for SCNP-0, which is in line with the above-described association differences observed with flow cytometry. Hence, the glucose moieties may substantially contribute to the cellular uptake of the SCNPs in E. coli.

[0176] Experiment 3 - cellular uptake in S. aureus bacteria

[0177] The same experiments conducted on E. coli, a gram-negative bacterium, were also realized on Staphylococcus aureus, a gram-positive bacterium. The tested SCNPs included the best performing SCNP for E. coli, which was SCNP-S30. SCNPs with only glycerol (SCNP- 0) and with the charge (SCNP-45) were also tested as the wall of gram-positive bacteria is different from that of gram-negative bacteria and differences could be observed.

[0178] FACS and microscopy results showed a clear association of the SCNP-45 and SCNP-S30 with the bacteria, and a minor association of SCNP-0 with the bacteria. S. aureus may be known to present relatively few glycerol receptors, e.g. compared to glucose receptors, while E. coli may have an abundance of both receptors. Accordingly, the nanoparticle may be functionalized with specific ligand moieties to target specific bacteria.

[0179] Fractionation of the bacteria into an outer membrane fraction and a soluble fraction showed that SCNP-45 remained at the membrane, which was also observed earlier for E. coli. Only a minor association between the bacteria and SCNP-0 was observed, and the fractionation revealed that the particle remained at the membrane as well. Without being bound by theory, this is hypothesized to be due to the relatively low number of glycerol receptors available to internalize SCNP-0 in S. aureus. By contrast, SCNP-S30 was found to be predominantly present in the cytoplasm of S. aureus, while also some presence at the membrane was observed. Regardless, the experiments demonstrate the successful internalization of SCNP-S30 in S. aureus.

[0180] These results demonstrate that nanoparticles 100 equipped with the appropriate ligand moieties 140 may be internalized by a prokaryotic cell, such as by gram -negative Escherichia coli bacteria or gram-positive S. aureus bacteria. The term “plurality” refers to two or more. Furthermore, the terms “a plurality of’ and “a number of’ may be used interchangeably.

[0181] The terms “substantially” or “essentially” herein, and similar terms, will be understood by the person skilled in the art. The terms “substantially” or “essentially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially or essentially may also be removed. Where applicable, the term “substantially” or the term “essentially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. Moreover, the terms ’’about” and “approximately” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. For numerical values it is to be understood that the terms “substantially”, “essentially”, “about”, and “approximately” may also relate to the range of 90% - 110%, such as 95%-105%, especially 99%-101% of the values(s) it refers to.

[0182] The term “comprise” also includes embodiments wherein the term “comprises” means “consists of’.

[0183] The term “and / or” especially relates to one or more of the items mentioned before and after “and / or”. For instance, a phrase “item 1 and / or item 2” and similar phrases may relate to one or more of item 1 and item 2. The term “comprising” may in an embodiment refer to “consisting of’ but may in another embodiment also refer to “containing at least the defined species and optionally one or more other species”.

[0184] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0185] The devices, apparatus, or systems may herein amongst others be described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation, or devices, apparatus, or systems in operation.

[0186] The term “further embodiment” and similar terms may refer to an embodiment comprising the features of the previously discussed embodiment, but may also refer to an alternative embodiment.

[0187] It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

[0188] Use of the verb “to comprise” and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, “include”, “including”, “contain”, “containing” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0189] The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

[0190] The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In a device claim, or an apparatus claim, or a system claim, enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

[0191] The invention also provides a control system that may control the device, apparatus, or system, or that may execute the herein described method or process. Yet further, the invention also provides a computer program product, when running on a computer which is functionally coupled to or comprised by the device, apparatus, or system, controls one or more controllable elements of such device, apparatus, or system.

[0192] The invention further applies to a device, apparatus, or system comprising one or more of the characterizing features described in the description and / or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterizing features described in the description and / or shown in the attached drawings. Moreover, if a method or an embodiment of the method is described being executed in a device, apparatus, or system, it will be understood that the device, apparatus, or system is suitable for or configured for (executing) the method or the embodiment of the method, respectively.

[0193] The various aspects discussed in this patent can be combined in order to provide additional advantages. Further, the person skilled in the art will understand that embodiments can be combined, and that also more than two embodiments can be combined. Furthermore, some of the features can form the basis for one or more divisional applications.

Claims

CLAIMS:

1. A method for introducing a nanoparticle (100) into a prokaryotic cell (10), wherein the method comprises exposing the prokaryotic cell (10) to the nanoparticle (100), wherein:- the nanoparticle (100) has an equivalent spherical diameter selected from the range of 3-20 nm,- the nanoparticle (100) comprises a single-chain polymer nanoparticle (101),- the nanoparticle (100) comprises a backbone (110), a plurality of intramolecular crosslinks (120), and a plurality of side groups (130) bound to the backbone (110),- the backbone (110) comprises n backbone atoms, wherein the n backbone atoms comprise > 0.9*n carbon atoms, each intramolecular crosslink (120) covalently interconnects two backbone locations (112) of the backbone (110), k of the plurality of side groups (130) comprise ligand moieties (140) selected from the group consisting of sugar moieties, a glycerol moiety, and amino acid moieties, wherein k / n is selected from the range of 0.05-0.49, and- the prokaryotic cell (10) comprises a receptor (14) configured to interact with one or more of the ligand moieties (140).

2. The method according to claim 1, wherein the side groups (130) comprise a first subset (31) of ki first side groups (131), wherein each first side group (131) comprises a first ligand moiety (141) selected from the group comprising sugar moieties, and wherein ki / n is selected from the range of 0.05-0.25.

3. The method according to claim 2, wherein ki / n is selected from the range of 0.1 - 0.2.

4. The method according to any one of the preceding claims 2-3, wherein the side groups (130) comprise a second subset (32) of k2 second side groups (132), wherein each second side group (132) comprises a second ligand moiety (142) selected from the group comprising a glycerol moiety and amino acid moieties, wherein k2 / n is selected from the range of 0.05-0.44, and wherein ki+k2 < k.

5. The method according to claim 4, wherein the first ligand moi eties (141) are glucose moi eties, and wherein the second ligand moi eties (142) are glycerol moi eties.

6. The method according to any one of the preceding claims, wherein the nanoparticle (100) has a size selected from the range of 8 - 15 nm.

7. The method according to any one of the preceding claims, wherein the backbone(110) comprises a vinyl polymer, wherein all backbone atoms comprise carbon atoms.

8. The method according to any one of the preceding claims, wherein the nanoparticle (100) comprises m intramolecular crosslinks (120), wherein m is selected from the range of 0.02*n - 0.1 *n, and wherein the intramolecular crosslinks (120) have lengths selected from the range of 4-120 atoms.

9. The method according to any one of the preceding claims, wherein the side groups (130) comprise linkers (135), wherein the linkers (135) separate the backbone (110) and the ligand moieties (140), wherein each linker (135) comprises a side backbone comprising 3- 20 atoms, wherein the atoms are selected from the group consisting of C, N, S, P, and O.

10. The method according to claim 9, wherein the linkers (135) consist of propyl moieties, diethylene glycol moieties or triethylene glycol moieties.

11. The method according to claims 5 and 10, wherein ki / n is selected from the range of 0.12-0.18, and wherein the linkers (135) consist of propyl moieties.

12. The method according to claims 5 and 10, wherein ki / n is selected from the range of 0.05-0.13, and wherein the linkers (135) consist of triethylene glycol moieties.

13. The method according to any one of the preceding claims 1-8, wherein the side groups (130) comprise polymer ethylene glycol linkers, wherein the polymer ethylene glycol linkers separate the backbone (110) and the ligand moieties (140), wherein each polymer ethylene glycol linker has a weight selected from the range of 500-3000 Da.

14. The method according to any one of the preceding claims, wherein the prokaryotic cell (10) comprises a gram negative bacterium (11).

15. The method according to any one of the preceding claims, wherein the nanoparticle (100) comprises a cargo (150), wherein the cargo (150) comprises an antibiotic.

16. The method according to any one of the preceding claims 1-14, wherein the nanoparticle (100) comprises a cargo (150), wherein the cargo comprises an RNA molecule.

17. A nanoparticle (100) for introduction into a prokaryotic cell (10), wherein the nanoparticle (100) has an equivalent spherical diameter selected from the range of 3-20 nm, wherein the nanoparticle (100) comprises a single-chain polymer nanoparticle (101), wherein the nanoparticle (100) comprises a backbone (110), a plurality of intramolecular crosslinks (120), and a plurality of side groups (130) bound to the backbone (110), wherein the backbone (110) comprises n backbone atoms, wherein the n backbone atoms comprise > 0.9*n carbon atoms, wherein each intramolecular crosslink (120) covalently interconnects two backbone locations (112) of the backbone (110), wherein the side groups (130) comprise a first subset (31) of ki first side groups (131) and a second subset (32) of k2 second side groups (132), wherein each first side group (131) comprises a glucose moiety, and wherein each second side group (132) comprises a glycerol moiety, and wherein the side groups (130) comprise linkers (135), wherein the linkers (135) separate the backbone (110) and the ligand moieties (140), wherein the linkers (135) consist of propyl moieties, wherein ki / n is selected from the range of 0.12-0.18, and wherein k2 / n is selected from the range of 0.2-0.44.

18. A synthesis method for providing a nanoparticle (100) as defined in claim 18, wherein the method comprises: a polymerization step comprising co-polymerizing a first monomer and a second monomer to provide a precursor polymer, wherein the first monomer comprises a first polymerization group, and wherein the second monomer comprises a side precursor group and a second polymerization group , wherein the first polymerization group and the second polymerization group are selected from the group comprising an acrylate moiety, an acrylamide moiety, a styrene moiety, a methacrylate moiety and a methacrylamide moiety, wherein a ratio of the first monomer to the second monomer is selected from the range of 1 :20 to 1 :4, whereinamounts of the first monomer and second monomer are selected such that the precursor polymer has a molecular weight selected from the range of 30-150 kDa; a crosslinking step comprising exposing the precursor polymer to a crosslinking composition, wherein the crosslinking composition is configured to (i) provide a plurality of crosslink precursor groups, and (ii) crosslink the crosslink precursor groups to provide intramolecular crosslinks (135); and wherein: each side precursor group comprises a ligand moiety selected from the group consisting of a sugar moiety, a glycerol moiety, and an amino acid moiety; or the method further comprises a functionalization step comprising exposing the precursor polymer to first reactants, wherein each first reactant comprises a reactant group and a ligand moiety, wherein the reactant group is configured to react with at least part of the side precursor groups, and wherein the ligand moiety is selected from the group consisting of a sugar moiety, a glycerol moiety, and an amino acid moiety.

19. The synthesis method according to claim 18, wherein the first monomer comprises xanthate methacrylate, wherein the side precursor groups comprise pentafluorophenyl moieties, wherein the crosslinking step comprises exposing the precursor polymer to ethanolamine, and wherein the method comprises the functionalization step, wherein the reactant group comprises an amine group.

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