Method and system

Abstract

A method of generating a plurality of different surface-decorated nanoparticles, decorated with different surface decoration, comprising: forming a plurality of microdroplets in a microfluidics device, each microdroplet comprising a nanoparticle and a respectively different macromolecule encoding a different surface decoration molecule; synthesising the surface decoration molecule, within each microdroplet, based on the macromolecule encoding the surface decoration molecule; conjugating the nanoparticle and the surface decoration molecule, within each microdroplet, to form surface decorated nanoparticles.

Description

[0001] METHOD AND SYSTEM

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to a method of generating a plurality of different surfacedecorated nanoparticles, decorated with different surface-decorations; and a microfluidics system for generating a plurality of different surface-decorated nanoparticles, decorated with different surface-decorations. In one specific example, the nanoparticles are protein nanoparticles decorated with proteins or peptides covalently bonded to the nanoparticles.

[0004] BACKGROUND ART

[0005] Nanoparticles as a drug carrier have attracted much interest. For example, nanoparticles can compartmentalize and stabilize therapeutic cargo, which may in turn prolong the circulation lifetimes of drugs and increase the effectiveness of treatment. In particular, protein nanoparticles have attracted interest, for example, due to intrinsic biocompatibility. Surface-decorated human nanoparticles have also attracted interest due to their target specificity. For example, Curcumin-human serum albumin nanoparticles decorated with PDL1 binding peptide for targeting PDL1 -expressing breast cancer cells have been proposed (Hasan et al, 2020).

[0006] Previous attempts at screening therapeutic nanoparticles with diverse compositions for targeting have mostly achieved their diversity through varying lipid and PEG compositions (Dilliard et al., 2021; Sago et al., 2018), or via directed design of targeting ligands against cell-surface markers (Kou et al., 2018). Therefore, there is a need for a high-throughput screening platform to assess the ability of large numbers of protein and peptide -based ligands, to facilitate nanoparticle targeting in a hypothesis-free manner.

[0007] It is an object of the present invention to at least partially solve the above problems.

[0008] SUMMARY OF THE INVENTION

[0009] According to an aspect of the disclosure there is provided a method of generating a plurality of different surface-decorated nanoparticles, decorated with different surface decoration, comprising: forming a plurality of microdroplets in a microfluidics device, each microdroplet comprising a nanoparticle and a respectively different macromolecule encoding a different surface decoration molecule; synthesising the surface decoration molecule, within each microdroplet, based on the macromolecule encoding the surface decoration molecule; conjugating the nanoparticle and the surface decoration molecule, within each microdroplet, to form surface decorated nanoparticles.

[0010] Optionally, each microdroplet comprises a plurality of nanoparticles.

[0011] Optionally, each microdroplet is formed comprising a single macromolecule encoding a surface decoration molecule.

[0012] Optionally, the macromolecule is a polynucleotide.

[0013] Optionally, the macromolecule is a DNA or an RNA molecule.

[0014] Optionally, the polynucleotide is a plasmid.

[0015] Optionally, each microdroplet further comprises an amplification mixture configured to amplify the macromolecules encoding surface decoration molecules.

[0016] Optionally, the method further comprises a step of amplifying the macromolecule encoding a surface decoration molecule, within each microdroplet, by incubating each microdroplet with the amplification mixture for a period of time. Optionally, amplification is ended by denaturing or destroying constituents of the amplification mixture by heating after said period of time. Optionally, the step of amplifying increases the concentration of the macromolecule in each microdroplet by at least 100 times, suitably at least 1000 times.

[0017] Optionally, the step of synthesizing the surface decoration molecule comprises introducing a cell-free synthesis mixture to each microdroplet, the cell-free synthesis mixture being configured to synthesise the surface decoration molecule based on the macromolecule.

[0018] Optionally, the method further comprises incubating each droplet for a period of time with the cell-free synthesis mixture. Optionally, the step of conjugating the nanoparticle and the surface decoration molecule comprises introducing a conjugation mixture configured to conjugate the nanoparticle and the surface decoration molecule. Optionally, the method further comprises incubating the microdroplets with the conjugation mixture for a period of time.

[0019] Optionally, the step of forming the plurality of microdroplets comprises: providing a fist stream of a first fluid in the microfluidics device, the first fluid comprising the nanoparticles; providing a second stream of a second fluid in the microfluidics device, the second fluid comprising the respectively different macromolecules; combining the first and second streams in the microfluidics device to form a third stream comprising the plurality of microdroplets suspended in a third fluid.

[0020] Optionally, the first and second fluids are immiscible with the third fluid. Optionally, the first stream and the second stream are configured such that each microdroplet has a relatively high probability of being formed comprising no more than one macromolecule. Optionally, relative flow rates of the first and second streams and / or relative concentrations of nanoparticles and macromolecules in the first and second streams are configured such that each microdroplet has a relatively high probability of being formed comprising no more than one macromolecule.

[0021] Optionally the step of forming the plurality of microdroplets further comprises: providing a fourth stream of a fourth in the microfluidics device, the fourth fluid comprising the amplification mixture; and combining the fourth stream with the first and second streams in the microfluidics device to form the third stream comprising the plurality of microdroplets.

[0022] Optionally, additional constituents, optionally including the cell-free synthesis mixture of and / or the conjugation mixture, are introduced to the microdroplets by picoinjection of a stream of each respective constituent in the microfluidics device.

[0023] Optionally, the nanoparticle is formed from one or more proteins.

[0024] Optionally, the decorating molecule is a peptide or a protein. Optionally, the decorating molecule is configured to covalently bond to the nanoparticle.

[0025] Optionally, the decorating molecule is configured to attach to the nanoparticle by non- covalent interactions. Optionally, the decorating molecule and nanoparticle are configured to attach to each other by non-covalent interactions between protein-protein affinity tag pairs.

[0026] Optionally, the decorating molecule is configured to bind to a therapeutic target.

[0027] Optionally, the nanoparticle either contains internally, is bound to, or configured to bind to a therapeutic agent - either covalently or non-covalently.

[0028] Optionally, the method forms a stream of microdroplets comprising at least 1 ,000 respective different macromolecules encoding different surface decoration molecules.

[0029] According to a second aspect of the disclosure there is provided a method of screening candidate nanoparticles for delivering a therapeutic agent to a target comprising the steps of the method of any preceding aspect.

[0030] Optionally, the method of screening further comprises determining a binding characteristic of the surface-decorated nanoparticles with the target.

[0031] According to a third aspect of the disclosure there is provided method of generating nanoparticles for delivering a therapeutic agent to a target comprising the steps of the method of any preceding aspect.

[0032] According to a fourth aspect of the disclosure there is provided a microfluidics system for generating a plurality of different surface decorated nanoparticles, decorated with different surface decoration, comprising: microdroplet forming unit configured to form a plurality of microdroplets, each microdroplet comprising a nanoparticle and a respectively different macromolecule encoding a surface decoration molecule; a synthesising unit configured to introduce a cell-free synthesis mixture to the microdroplets, the cell-free synthesis mixture configured to synthesise the surface decoration molecule based on the macromolecule encoding the surface decoration molecule, within each microdroplet; a conjugating unit configured to introduce a conjugating mixture to the microdroplets, the conjugating mixture configured to conjugate the nanoparticle and the surface decoration molecule, within each microdroplet.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Further features of the disclosure will be described below, by way of non-limiting examples and with reference to the accompanying drawings, in which:

[0035] Fig. 1 schematically shows a micro fluidics system of the disclosure;

[0036] Fig. 2 shows experimental data relating to an amplification step of the disclosure;

[0037] Fig. 3 shows experimental data relating to a synthesis step of the disclosure;

[0038] Fig. 4 shows experimental data relating to a conjugation step of the disclosure;

[0039] Fig. 5 shows experimental data relating to the size distribution of nanoparticles;

[0040] Fig. 6 shows experimental data relating to a synthesis step of the disclosure;

[0041] Fig. 7 shows experimental data relating to a conjugation step of the disclosure; and Fig. 8 shows experimental data relating to a conjugation step of the disclosure.

[0042] DETAILED DESCRIPTION

[0043] Fig. 1 schematically shows a micro fluidics system for generating a plurality of different surface-decorated nanoparticles, decorated with different surface decorations, according to the disclosure.

[0044] The top left portion of Fig. 1 shows a microdroplet generating unit of the microfluidics system, for generating a plurality of microdroplets in a microfluidics device. Each microdroplet may comprise a nanoparticle and a respectively different macromolecule encoding a different surface decoration molecule.

[0045] As shown each microdroplet may comprise a plurality of nanoparticles. The nanoparticles may be of the same type, for example, formed from the same molecules. The nanoparticles may be formed from biomolecules, for example. The nanoparticles may be formed from one or more proteins (simple or conjugated), polypeptides and / or nucleic acids, for example. A plurality of such molecules may combine to form the nanoparticles. These may be bonded non-covalently or covalently. In a specific example, the nanoparticles are formed from clusters of human serum albumin.

[0046] The nanoparticles are sub-micron in size, such that the longest dimension of the nanoparticles is smaller than 1 micron. Preferably, the longest dimension does not exceed 200 nm. The longest dimension may be at least 20 nm, for example. In other words, the nanoparticles may have a size of 20-200 nm. This may improve their suitability for intracellular delivery of a therapeutic agent for example.

[0047] The nanoparticles may contain internally or be configured to contain internally a therapeutic agent. Alternatively, or additionally, the nanoparticles may be bound to, or configured to bind to a therapeutic agent - e.g. either covalently or non-covalently. In some examples to be used as a therapeutic carrier, the nanoparticles may carry a therapeutic agent prior to surface decoration. In other examples, the nanoparticles may not carry a therapeutic agent prior to surface decoration, and the therapeutic agent may be introduced after surface decoration.

[0048] As shown by the different coloured macromolecule in Fig. 1, each microdroplet may be formed comprising a single macromolecule encoding a surface decoration molecule. The macromolecule, may be a polynucleotide such as DNA or RNA. As shown, the macromolecule may be a plasmid (formed from DNA or RNA, for example).

[0049] As shown in Fig. 1, the microdroplet generating unit comprises a first micro fluidic channel comprising a first stream of a first fluid, the first fluid comprising the nanoparticles. The microdroplet generating unit further comprises a second microfluidic channel providing a second stream of a second fluid, the second fluid comprising the respectively different macromolecules. The first and second streams are combined to form a third stream comprising the plurality of microdroplets suspended in a third fluid. The first and second fluids are immiscible with the third fluid. Preferably, the third fluid is oil, which may comprise an emulsion stabilizing agent.

[0050] The first stream and the second stream may be configured such that each microdroplet has a relatively high probability of being generated comprising no more than one macromolecule. For example, relative flow rates of the first and second streams and / or relative concentrations of nanoparticles and macromolecules in the first and second streams may be configured such that each microdroplet has a relatively high probability of being generated comprising no more than one macromolecule. For example, more than 90%, or preferably more than 95%, of microdroplets generated by the system may comprise one or no macromolecules.

[0051] In other examples, the system may be configured to generate microdroplets having a small number (e.g. less than 10, preferably less than 5) of different macromolecules encoding different decorating molecules, e.g. 2 or 3 different macromolecules. For example, more than 90%, or preferably more than 95%, of microdroplets generated by the system may comprise N or fewer macromolecules, where N is the small number.

[0052] As shown in Fig. 1, each microdroplet may further comprise an amplification mixture configured to amplify the macromolecules encoding surface decoration molecules. As shown, the microdroplet forming unit may comprise a fourth stream of a fourth fluid, the fourth fluid comprising the amplification mixture. As shown, the fourth stream may be combined with the first and second streams to form the third stream comprising the plurality of microdroplets.

[0053] The top right portion of Fig. 1 shows an amplification unit of the micro fluidics system configured to incubate each microdroplet with the amplification mixture for a period of time. Amplification may be ended by denaturing or destroying constituents of the amplification mixture by heating after said period of time, e.g. at 65 degrees C.

[0054] The amplification is configured to provide higher levels of expression of the decorating molecule within the droplet in a subsequent synthesis step. For example, the step of amplifying may increase the concentration of the macromolecule in each microdroplet by at least 100 times, at least 1,000 times, or at least 5,000 times, depending on incubation time and the amount of provided amplification mixture.

[0055] Fig. 2 shows experimental results demonstrating the ability to amplify a plasmid encoding the GFP protein from a concentration of 0.1 plasmid / pL, which corresponds to ~1 plasmid per droplet in typical 14pL micro fluidic droplets. In this experiment, fluorescence intensity (F.I.) tells us the quantity of GFP produced, and the data shows that without pDNA amplification, even 4 plasmids / pL is too little DNA to produce visible protein, but by amplifying the pDNA for 2 hours, a sufficient concentration of DNA is amplified to be able to produce protein at levels expected in a reaction with -5000 times more DNA alone. Amplification was performed using the Genomiphi™ V2 DNA Amplification kit from Cytiva. After amplification, the NEBExpress® Cell-free E. coli Protein Synthesis (CFPS) System mixture from New England Biolabs was introduced to enable the transcription and translation of the genes from the plasmids into functional and fluorescent GFP. This supports the feasibility of our in vitro method of amplifying a single plasmid within a micro fluidic droplet to produce large amounts of individual mutants.

[0056] Fig. 2 A shows that plasmids may be amplified in vitro to raise DNA concentration more than 5000 times. Fig. 2B shows plate reader-based detection of GFP production from a mixture of dilute plasmid DNA. The blue, red, and purple curves represent CFPS reactions in the presence of 960, 4, and 0.4 units of pDNA (plasmids) per pL respectively. The orange and green curves, represent solutions of 1 and 0.1 units of pDNA per pL, each of which was amplified using an in vitro plasmid amplification kit for 2 hours before initiating the CFPS reaction.

[0057] The bottom right of Fig. 1 shows a synthesising unit of the microfluidics system for synthesising the surface decoration molecule, within each microdroplet, based on the macromolecule encoding the surface decoration molecule.

[0058] As shown, the synthesising unit is configured to introduce a cell-free synthesis mixture to each microdroplet and incubate the microdroplets with the cell-free synthesis mixture for a period of time. The cell-free synthesis mixture is configured to synthesise the surface decoration molecule based on the macromolecule. The cell-free synthesis mixture may be a cell-free protein synthesis (CFPS) mixture configured to express a protein or peptide encoded by the macromolecule.

[0059] The cell-free synthesis mixture may be injected into the microdroplets, e.g. by picoinjection. The top part of Fig. 4 shows a picoinjection process (though not for a cell- free synthesis mixture). An aqueous stream of the cell-free synthesis mixture is fused with aqueous microdroplets as the microdroplets pass an inlet. The fusion may be facilitated by an electric field configured to disrupt the surface of the aqueous-oil interface of the microdroplets and the cell-free synthesis mixture stream.

[0060] In an experimental example, a co-flow microfluidic device was used for the entrapment of a single plasmid per microfluidic droplet (Agresti et al., 2010; Holstein et al., 2021). To do this, plasmid DNA containing the gene for GFP was diluted to a concentration such that upon microfluidic droplet formation, less than 1 plasmid on average would be contained per droplet. Since a fraction of a plasmid cannot be present, most droplets contain zero copies of plasmid, and roughly 10% of droplets should contain one plasmid. The droplets were formed from solutions of plasmids that were co-encapsulated with the same mixture used for plasmid amplification as before and stored in an incubation chamber for amplification. The microfluidic droplets were formed as water-in-oil emulsions, stabilized by a PEG-PTFE triblock surfactant (EP2077912A1) dispersed in an oil phase of HFE- 7500. After 6 hours of incubation, the droplets were exposed to a 65°C temperature shock for 10 min to kill the DNA amplification machinery.

[0061] Next, reagents for CFPS were introduced to the droplets using the picoinjection technique described later (Fig. 4). The droplets were again incubated for 6 hours to allow for GFP expression before being imaged via widefield microscopy. The reagents used were the same as those used in Fig. 2 for the bulk experiments, and concentrations were adjusted to be the same as well. As can be observed in Fig. 3, roughly 5% of droplets contained GFP and the rest were void of any fluorescent material - indicating they did not contain any plasmid. Fig. 3 shows the production of GFP in micro fluidic droplets from a single trapped plasmid. The centre panel, in green, shows droplets seen via 488 fluorescence containing GFP protein. The left panel shows droplets visualized via bright field microscopy. The right panel shows an overlay of the fluorescent and bright field signals.

[0062] This demonstrates the capability to trap single plasmid variants in micro fluidic droplets and the ability to amplify them before adding CFPS mixture to finally yield substantial amounts of functional protein.

[0063] The bottom left portion of Fig. 1 shows a conjugation unit of the system for conjugating the nanoparticle and the surface decoration molecule, within each microdroplet, to form surface-decorated nanoparticles. As shown, the conjugation unit is configured to introduce a conjugation mixture configured to conjugate the nanoparticle and the surface decoration molecule, to the microdroplet and incubate the microdroplets with the conjugation mixture for a period of time. The conjugation mixture may be injected into the microdroplets, e.g. by picoinjection, as described above. The top part of Fig. 4 shows a picoinjection process for a conjugation mixture.

[0064] The conjugation mixture may be configured to form covalent bonds between the nanoparticles and the decorating molecules. Attachment of the nanoparticles and the decorating molecules using covalent bonds may be advantageous when the decorated nanoparticles are to be used in a therapeutic setting, because the covalent attachment is unlikely to be broken by, for example, pH changes in the body. Covalent bonds between the nanoparticles and the decorating molecules may be achieved using carbodiimide-amine coupling, unnatural amino acid click-chemistry coupling, succinimidyl thioether conjugation, glutaraldehyde-amino coupling and / or hydroxysuccinimide-amine coupling. The conjugation mixture may comprise a crosslinking agent for example. The crosslinking agent may be configured to form covalent bonds between a protein nanoparticle and protein or peptide decorating molecule, for example. Suitable crosslinking agents are known to the person skilled in the art and include carbodiimides, succinimides, hydroxysuccinimides, and glutaraldehyde. In one embodiment, the crosslinking agent is ( 1 -ethyl-3 -(3 -dimethylaminopropyl) carbodiimide.

[0065] Alternatively, the decorating molecules may be configured to attach to the nanoparticles by non-covalent interactions, for example by non-covalent interactions between proteinprotein affinity tag pairs. Typically, the interaction between affinity tag pairs is achieved where the surface decorating molecule contains a fusion domain (such as albumin binding protein or a streptavidin binding peptide), and the nanoparticle surface is decorated with the respective affinity ligand for the fusion affinity domain. Alternatively, the nanoparticle surface may contain the fusion domain and the decorating molecule may contain the respective affinity ligand. The interaction between the fusion domain and the affinity ligand attaches the decorating molecule non-covalently to the nanoparticle surface. As mentioned previously, the sequential addition of reagents into microfluidic droplets may most effectively be achieved by a technique called picoinjection (US11358105B2, (Abate et al., 2010)). Picoinjection relies on the fusion of an aqueous stream with aqueous droplets as they pass by an inlet. This fusion is mediated by an electric field which disrupts the surface of the aqueous-oil interfaces of the droplet and the injection streams and allows for the controlled fusion of the two solutions.

[0066] Widefield time-lapse images of the picoinjection technique are shown in Fig. 4 (top row). Under typical operating conditions, the injection operates at 0.77kHz, meaning 462,000 droplets could be injected with an additional solution over the course of 10 minutes of operation. To demonstrate the compatibility of picoinjection to the method of present disclosure, the ability to inject a crosslinking agent (l-Ethyl-3 -(3 -dimethylaminopropyl) carbodiimide, (EDC)) into droplets containing BSA nanoparticles and GFP protein was tested. This injection was carried out and droplets were collected in an imaging chamber for fluorescent detection.

[0067] Fig. 4 shows a picoinjection method showing the injection of an EDC-containing mixture (red) into droplets containing BSA nanoparticles and GFP protein (green). The top row shows bright field time-lapse images showing the injection of the EDC solution (bottom inlet) into droplets containing BSA and GFP. The scale bar in fluorescent and brightfield images is 40pm.

[0068] As may be seen in Fig. 4 (bottom row) nearly all droplets contain both BSA nanoparticles with GFP (green, left) and EDC crosslinking agent (red, right). A fluorescent dye (AlexaFluor-647), which does not spectrally overlap with GFP, was added to the EDC mixture to allow for visualization. Nearly 100% of droplets that passed through the picoinjection device were successfully injected with the crosslinking agent. To achieve unique surface decorations for nanoparticles in our library, crosslinking agents must be added sequentially after protein production. This picoinjection capability supports multiple steps in the method of the disclosure which rely on the sequential addition of various reagents within the confinement of microfluidic droplets, which overall facilitates the combinatorial nature the disclosed approach to generating diversely decorated nanoparticles. As mentioned, the nanoparticles in the disclosed method may be made up of protein building blocks, to allow for rapid functionality and biological compatibility. One such type of particle is those made from bovine serum albumin (BSA). BSA nanoparticles can be produced through a multistep bulk procedure in which a solution of BSA (80mg / mL) in H2O is titrated with ethanol at a flow rate of Iml / min using a syringe pump. By adding 8mL of ethanol to 3mL of BSA solution, the BSA is desolvated in a controlled manner due to its insolubility in ethanol. The desolvated BSA forms particles of 20-50nm in diameter, which may then be stabilized covalently through the addition of various crosslinking agents. Two different crosslinking methods were tested for compatibility with the disclosed method. The first was glutaraldehyde (50% aqueous solution), which was added to the BSA particle mixture as 0.2pL per mg of BSA. The crosslinking reaction was carried out for 18hrs, before being quenched with the addition of excess IM TRIS- HC1 (pH 7.4). The nanoparticles were subsequently washed with H2O by centrifuging the nanoparticles (13,400rpm for 40 minutes) to the bottom of a Falcon tube, removing the supernatant, resuspending the nanoparticle pellet in fresh H2O, and centrifuging again. The washing step was repeated 3 times to remove excess glutaraldehyde. The particles were characterized immediately and again after 24 hours and 7 days via dynamic light scattering (DLS) to determine their size and stability. At time point zero, the particles were determined to be 30±12nm in diameter, and after 24 hours they were still 30±12nm in diameter, indicating that the particles are stable. The measurement after 7 days showed an increase in BSA particle size to 52±20nm which may be attributed to the nature of the particles swelling during storage in aqueous solution. The DLS curves showing the size distribution of the particles are shown in Fig. 5.

[0069] A similar method was used to form crosslinked nanoparticles with EDC as a cross-linking agent rather than glutaraldehyde since glutaraldehyde yielded some undesirable autofluorescence effects during downstream characterization (Ma et al., 2016). Similar to the protocol of making BSA particles with glutaraldehyde, the crosslinking agent EDC was added to the solution in a ratio of 0.02mg per mg of BSA. The reaction was carried out at room temperature for 3 hours with constant stirring at 1500rpm. Then, the particles were harvested and washed in the same way above. In this ratio, the BSA formed particles that were 129±26nm in diameter. Thus, EDC and glutaraldehyde represent two different crosslinking methods for changing the morphology of BSA nanoparticles used in downstream experiments. With the workflow to produce BSA nanoparticles established, the nanoparticles were then introduced into a CFPS mixture containing GFP coding plasmids (same experimental conditions as Fig. 2). The aim was to determine whether the BSA nanoparticles particles (BSA NPs) would have any negative effect on the disclosed in vitro molecular biology method.

[0070] The results from Fig. 6 show that an addition of up to 16% of 12mg / mL BSA NP solution has no effect on the ability to produce GFP from plasmids in vitro (as measured by 488 fluorescence intensity over time). This indicates that the nanoparticles are fully compatible with CFPS and that the entrapment of nanoparticles with machinery for in vitro protein production is completely compatible with our proposed invention workflow. Additionally, the crosslinked nanoparticles were tested for biocompatibility in HEK293 cell culture. Particles concentrations of lOmg / mL and 2mg / mL as well as soluble BSA at lOmg / mL were introduced into cell culture media, and upon 24 hours of exposure to HEK293 cells in pH 5, 7, and 9 the cells were found to be fully viable. The viability of cells was maintained with both EDC and Glutaraldehyde crosslinked BSA nanoparticles. Overall, the cellular viability shows that the nanoparticles should be highly biocompatible. As a final step to indicate the feasibility of the disclosed method, the ability to functionalize our nanoparticles by covalently coupling a protein to their surface is demonstrated. It has already been shown that EDC can be injected into droplets containing BSA and GFP on the microscale, but due to the nanoscale size of the BSA particles, it is not possible to easily visualize the crosslinking of GFP to their surface. Instead, the reaction was carried out in bulk and then the crosslinked particles flowed through a micro fluidic device mounted on top of a single-molecule sensitive confocal microscope (Krainer et al., 2023) which enables the detection of nanometer to micrometer scale particles in multiple fluorescent channels.

[0071] Fluorescently labelled BSA nanoparticles (15mg / mL, 1% AlexaFluor-647 containing) were mixed with GFP (0.25mg / mL) and 17mM EDC. The coupling was carried out for 30 minutes before centrifugation washing steps were used as previously described. The particles could then be characterized via confocal detection as shown in Figure 7. First, control particles without GFP were characterized (Fig. 7 A), showing significant peaks in the 647nm channel (BSA, red) but none in the 488nm channel (GFP, cyan). Next, the particles functionalized with GFP were characterized (Figure 7B), showing significant peaks in both fluorescent channels. Peaks were defined as significant if the signal was greater than 3 standard deviations above the mean. For the 647nm channel, this cutoff was 200 photons, for the 488nm channel it was 856 photons. The number of peaks for each channel was N488nm = 1171, N647nm =1058. Crucially 165 peaks overlapped in both channels, indicating BSA particles that were decorated with GFP. This translated to 15.6% of BSA particles successfully being decorated with GFP. The significant number of 488nm peaks comes from the fact that EDC is a non-specific crosslinking agent and thus oligomers of GFP could easily form in addition to GFP attaching to the surface of BSA particles. Further optimization of different crosslinking agents and reaction schemes could improve the specificity and the efficiency of coupling to the BSA surface, but it can be seen that covalent functionalization of the BSA nanoparticles with protein components can readily be achieved.

[0072] In another iteration of the experiment, it was demonstrated that that the covalent method of linkage between the nanoparticles and surface modification agents works very well when click-chemistry is used - the results of which are presented in Fig. 8. In this version of the experiment, BSA was modified with the click-chemistry reagent dibenzocyclooctyne (DBCO) via the following protocol: BSA was dissolved at 6.6 mg / mL (100 pM) in 100 mM Tris buffer pH 7.5 with 1 mM TCEP and incubated at room temperature for 10 minutes. 50 mM Maleimide-DBCO in DMSO (2 or 4 pL) was added to 100 pM BSA solution (100 pL). The solution was incubated at room temperature for 2 hours. The labelled BSA was purified by 0.5 mL Zeba spin desalting column 40K MWCO. The BSA was then used to form nanoparticles via ethanol desolvation as described above. Independently, the fusion protein SNAP-GFP was produced via cell-free protein synthesis in the same manner as described above. The SNAP-GFP was then made click reactive via incubation with 1.5 molar equivalent of pM Benzyl guanine-PEG(4)-Azide resulting in Azide-SNAP-GFP. The Azide-SNAP-GFP was then introduced via picoinjection into microdroplets containing DBCO-modified BSA particles via the method outlined in Figure 4. Nanoparticles and the Azide-SNAP-GFP mixture were then incubated in water-in-oil droplets for 12 hours before the emulsions were broken and the aqueous phase was extracted. The resulting aqueous phases were then analysed for fluorescence intensity using flow cytometry. Histograms of the 488 fluorescent signals are shown in Fig. 8 (A, B, C), where panel A shows non-reacted nanoparticles, panel B shows nanoparticles that have been incubated with non-click reactive SNAP-GFP and panel C shows nanoparticles where click chemistry decoration has taken place. Fig. 8, panel D quantifies the relative integrated intensity of the histograms above the noise cut-off (350 intensity units, indicated by the dashed black line in A, B, C). The click-chemistry is demonstrated to work by the 1737% increase in integrated 488nm fluorescent signal on the nanoparticles vs non-reacted particles (or 646% vs non-click reactive GFP). The particles in panels A, B, and C we confirmed to be 282nm, 303nm, and 299nm respectively on average via DLS. This experiment supports the use of click-chemistry as a means of attaching decoration macromolecules to nanoparticle surfaces using the method previously outlined.

[0073] The present disclosure provides a unique method to create a diverse, covalently decorated, library of protein-based nanoparticles. The workflow relies on a combination of micro fluidic and molecular biology techniques which are put together for a novel application.

[0074] The project leading to this application has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 101023060.

[0075] The project leading to this application has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 101001615).

[0076] This work was supported by the University of Cambridge Harding Distinguished Postgraduate Scholars Programme.

[0077] References:

[0078] Zahra Hasanpoor, Ah Mostafaie, Iraj Nikokar, Zuhair Mohammed Hassan. (2020) Curcumin-human serum albumin nanoparticles decorated with PDL1 binding peptide for targeting PDL1 -expressing breast cancer cells. International Journal of Biological Macromolecules, 159, 137-153 http s : / ' / do i . or g / 10 ,1016 / j . tj bi o ac .2020.04.130

[0079] Abate, A. R., Hung, T., Marya, P., Agresti, J. J., & Weitz, D. A. (2010). High-throughput injection with micro fluidics using picoinjectors using picoinjectors. Proceedings of the National Academy of Sciences of the United States of America, 107(45).

[0080] Agresti, J. J., Antipov, E., Abate, A. R., Ahn, K., Rowat, A. C., Baret, J.-C., Marquez, M., Klibanov, A. M., Griffiths, A. D., & Weitz, D. A. (2010). Ultrahigh-throughput screening in drop-based microfluidics for directed evolution. Proceedings of the National Academy of Sciences of the United States of America, 107(9), 4004-4009.

[0081] Dilliard, S. A., Cheng, Q., & Siegwart, D. J. (2021). On the mechanism of tissue-specific mRNA delivery by selective organ targeting nanoparticles. Proceedings of the National Academy of Sciences of the United States of America, 118(52).

[0082] Holstein, J. M., Gylstorff, C., & Hollfelder, F. (2021). Cell-free Directed Evolution of a

[0083] Protease in Microdroplets at Ultrahigh Throughput. ACS Synthetic Biology, 10(2).

[0084] Kou, L., Bhutia, Y. D., Yao, Q., He, Z., Sun, J., & Ganapathy, V. (2018). Transporter- guided delivery of nanoparticles to improve drug permeation across cellular barriers and drug exposure to selective cell types. In Frontiers in Pharmacology (Vol. 9, Issue JAN).

[0085] Krainer, G., Saar, K. L., Arter, W. E., Welsh, T. J., Czekalska, M. A., Jacquat, R. P. B., Peter, Q., Traberg, W. C., Pujari, A., Jayaram, A. K., Challa, P., Taylor, C. G., van der Linden, L. M., Franzmann, T., Owens, R. M., Alberti, S., Klenerman, D., & Knowles, T. P. J. (2023). Direct digital sensing of protein biomarkers in solution. Nature Communications, 14(1). https:Z / doi,org / 10,1038 / s41467-023-35792-x

[0086] Ma, X., Sun, X., Hargrove, D., Chen, J., Song, D., Dong, Q., Lu, X., Fan, T. H., Fu, Y., & Lei, Y. (2016). A Biocompatible and Biodegradable Protein Hydrogel with Green and Red Autofluorescence: Preparation, Characterization and in Vivo Biodegradation Tracking and Modeling. Scientific Reports, 6. Sago, C. D., Lokugamage, M. P., Paunovska, K., Vanover, D. A., Monaco, C. M., Shah, N.

[0087] N., Castro, M. G., Anderson, S. E., Rudoltz, T. G., Lando, G. N., Tiwari, P. M.,

[0088] Kirschman, J. L., Willett, N., Jang, Y. C., Santangelo, P. J., Bryksin, A. V., & Dahlman, J. E. (2018). High-throughput in vivo screen of functional mRNA delivery identifies nanoparticles for endothelial cell gene editing. Proceedings of the National Academy of Sciences of the United States of America, 115(42). https: / / doi.org / 10.1073 / pnas.18112 / 6115

Claims

CLAIMS1. A method of generating a plurality of different surface-decorated nanoparticles, decorated with different surface decoration, comprising: forming a plurality of microdroplets in a micro fluidics device, each microdroplet comprising a nanoparticle and a respectively different macromolecule encoding a different surface decoration molecule; synthesising the surface decoration molecule, within each microdroplet, based on the macromolecule encoding the surface decoration molecule; conjugating the nanoparticle and the surface decoration molecule, within each microdroplet, to form surface decorated nanoparticles.

2. The method of claim 1, wherein each microdroplet comprises a plurality of nanoparticles.

3. The method of any preceding claims, wherein each microdroplet is formed comprising a single macromolecule encoding a surface decoration molecule.

4. The method of any preceding claim, wherein the macromolecule is a polynucleotide.

5. The method of claim 4, wherein the macromolecule is a DNA or an RNA molecule.

6. The method of claim 4 or 5, wherein the polynucleotide is a plasmid.

7. The method of any preceding claim, wherein each microdroplet further comprises an amplification mixture configured to amplify the macromolecules encoding surface decoration molecules.

8. The method of claim 7, further comprising a step of amplifying the macromolecule encoding a surface decoration molecule, within each microdroplet, by incubating each microdroplet with the amplification mixture for a period of time.

9. The method of claim 8, wherein amplification is ended by denaturing or destroying constituents of the amplification mixture by heating after said period of time.

10. The method of any one of claims 8 or 9, wherein the step of amplifying increases the concentration of the macromolecule in each microdroplet by at least 100 times, suitably at least 1000 times.

11. The method of any preceding claim, wherein the step of synthesizing the surface decoration molecule comprises introducing a cell-free synthesis mixture to each microdroplet, the cell-free synthesis mixture being configured to synthesise the surface decoration molecule based on the macromolecule.

12. The method of claim 11, further comprising incubating each droplet for a period of time with the cell-free synthesis mixture.

13. The method of any preceding claim, wherein the step of conjugating the nanoparticle and the surface decoration molecule comprises introducing a conjugation mixture configured to conjugate the nanoparticle and the surface decoration molecule.

14. The method of claim 13, comprising incubating the microdroplets with the conjugation mixture for a period of time.

15. The method of any preceding claim, wherein the step of forming the plurality of microdroplets comprises: providing a fist stream of a first fluid in the microfluidics device, the first fluid comprising the nanoparticles; providing a second stream of a second fluid in the microfluidics device, the second fluid comprising the respectively different macromolecules; combining the first and second streams in the microfluidics device to form a third stream comprising the plurality of microdroplets suspended in a third fluid.

16. The method of claim 15, wherein the first and second fluids are immiscible with the third fluid.

17. The method of claim 15 or 16, wherein the first stream and the second stream are configured such that each microdroplet has a relatively high probability of being formed comprising no more than one macromolecule.

18. The method of claim 17, wherein relative flow rates of the first and second streams and / or relative concentrations of nanoparticles and macromolecules in the first and second streams are configured such that each microdroplet has a relatively high probability of being formed comprising no more than one macromolecule.

19. The method of any one of claims 15 to 18, when also dependent on claim 7, wherein the step of forming the plurality of microdroplets further comprises: providing a fourth stream of a fourth in the microfluidics device, the fourth fluid comprising the amplification mixture; and combining the fourth stream with the first and second streams in the microfluidics device to form the third stream comprising the plurality of microdroplets.

20. The method of any preceding claim, wherein additional constituents, optionally including the cell-free synthesis mixture of claim 12 and / or the conjugation mixture of claim 14, are introduced to the microdroplets by picoinjection of a stream of each respective constituent in the microfluidics device.

21. The method of any preceding claim, wherein the nanoparticle is formed from one or more proteins.

22. The method of any preceding claim, wherein the decorating molecule is a peptide or a protein.

23. The method of any preceding claim, wherein the decorating molecule is configured to covalently bond to the nanoparticle.

24. The method of any one of claims 1 to 22, wherein the decorating molecule is configured to attach to the nanoparticle by non-covalent interactions.

25. The method of claim 24, wherein the decorating molecule and nanoparticle are configured to attach to each other by non-covalent interactions between protein-protein affinity tag pairs.

26. The method of any preceding claim, wherein the decorating molecule is configured to bind to a therapeutic target.

27. The method of any preceding claim, wherein the nanoparticle either contains internally, is bound to, or configured to bind to a therapeutic agent - either covalently or non-covalently.

28. The method of any preceding claim, wherein the method forms a stream of microdroplets comprising at least 1 ,000 respective different macromolecules encoding different surface decoration molecules.

29. A method of screening candidate nanoparticles for delivering a therapeutic agent to a target comprising the steps of the method of any preceding claim.

30. The method of claim 29, further comprising determining a binding characteristic of the surface-decorated nanoparticles with the target.

31. A method of generating nanoparticles for delivering a therapeutic agent to a target comprising the steps of the method of any preceding claim.

32. A micro fluidics system for generating a plurality of different surface decorated nanoparticles, decorated with different surface decoration, comprising: microdroplet forming unit configured to form a plurality of microdroplets, each microdroplet comprising a nanoparticle and a respectively different macromolecule encoding a surface decoration molecule; a synthesising unit configured to introduce a cell-free synthesis mixture to the microdroplets, the cell-free synthesis mixture configured to synthesise the surface decoration molecule based on the macromolecule encoding the surface decoration molecule, within each microdroplet;a conjugating unit configured to introduce a conjugating mixture to the microdroplets, the conjugating mixture configured to conjugate the nanoparticle and the surface decoration molecule, within each microdroplet.

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