Method and system

Abstract

A method of generating a plurality of differently surface-modified nanoparticles, the nanoparticles being modified with different surface modifications, comprising: forming a plurality of micro-droplets in a microfluidic device, each micro-droplet comprising a nanoparticle and a respective different macromolecule encoding a different surface modification molecule; synthesizing, within each microdroplet, a surface modification molecule based on a macromolecule encoding the surface modification molecule; the nanoparticles are conjugated with the surface modification molecules within each microdroplet to form surface modified nanoparticles.

Description

Technical Field

[0001] This disclosure relates to a method for generating multiple surface-decorated nanoparticles modified with different surface modifications; and a microfluidic system for generating multiple surface-decorated nanoparticles. In a specific example, the nanoparticles are protein nanoparticles modified with proteins or peptides, which are covalently bound to the nanoparticles. Background Technology

[0002] Nanoparticles have attracted widespread interest as drug carriers. For example, nanoparticles can segment and stabilize therapeutic payloads, which can prolong drug cycle life and improve therapeutic efficacy. In particular, protein nanoparticles have garnered interest, for example, due to their inherent biocompatibility. Surface-modified human nanoparticles have also attracted attention due to their targeting specificity. For instance, it has been proposed to modify curcumin-human serum albumin nanoparticles with PDL1-binding peptides to target PDL1-expressing breast cancer cells (Hasan et al., 2020).

[0003] Previous attempts to target and screen therapeutic nanoparticles with different compositions have mostly achieved diversity by altering lipid and PEG compositions (Dilliard et al., 2021; Sago et al., 2018) or by designing targeted ligands to target cell surface markers (Kou et al., 2018). Therefore, a high-throughput screening platform is needed to evaluate the performance of a large number of protein- and peptide-based ligands in order to facilitate the targeting of nanoparticles in an assumption-free manner.

[0004] The purpose of this invention is to at least partially solve the above-mentioned problems. Summary of the Invention

[0005] According to one aspect of this disclosure, a method is provided for generating a plurality of nanoparticles with different surface modifications, the nanoparticles being modified with different surface modifications, the method comprising: forming a plurality of microdroplets in a microfluidic device, each microdroplet comprising nanoparticles and distinct macromolecules encoding different surface modification molecules; within each microdroplet, synthesizing surface modification molecules based on the macromolecules encoding the surface modification molecules; and conjugating the nanoparticles with the surface modification molecules within each microdroplet to form surface-modified nanoparticles.

[0006] Optionally, each microdroplet comprises multiple nanoparticles.

[0007] Optionally, each microdroplet is formed comprising a single macromolecule encoding surface-modifying molecules.

[0008] Optionally, the macromolecule is a polynucleotide.

[0009] Optionally, the macromolecule is a DNA or RNA molecule.

[0010] Optionally, the polynucleotide is a plasmid.

[0011] Optionally, each microdroplet also includes an amplification mixture of macromolecules configured to amplify surface-modifying molecules.

[0012] Optionally, the method further includes a step of amplifying a macromolecule encoding surface-modified molecules within each microdroplet by incubating each microdroplet with the amplification mixture for a period of time. Optionally, after said period of time, the amplification is terminated by heating to denature or destroy the components of the amplification mixture. Optionally, the amplification step increases the concentration of macromolecules in each microdroplet by at least 100-fold, suitably at least 1000-fold.

[0013] Optionally, the step of synthesizing the surface-modifying molecules includes introducing a cell-free synthetic mixture into each microdroplet, the cell-free synthetic mixture being configured to be based on macromolecular synthetic surface-modifying molecules. Optionally, the method further includes incubating each droplet with the cell-free synthetic mixture for a period of time.

[0014] Optionally, the step of conjugating nanoparticles with surface-modifying molecules includes introducing a conjugated mixture configured to conjugate the nanoparticles with the surface-modifying molecules. Optionally, the method further includes incubating the microdroplets with the conjugated mixture for a period of time.

[0015] Optionally, the step of forming multiple microdroplets includes: providing a first flow of a first fluid in a microfluidic device, the first fluid comprising nanoparticles; providing a second flow of a second fluid in the microfluidic device, the second fluid comprising different macromolecules; and merging the first flow and the second flow in the microfluidic device to form a third flow, the third flow comprising multiple microdroplets suspended in the third fluid.

[0016] Optionally, the first fluid, the second fluid, and the third fluid are immiscible. Optionally, the first and second fluids are configured such that each microdroplet has a high probability of forming a mass containing no more than one macromolecule. Optionally, the relative flow rates of the first and second fluids and / or the relative concentrations of nanoparticles and macromolecules in the first and second fluids are configured such that each microdroplet has a high probability of forming a mass containing no more than one macromolecule.

[0017] Optionally, the step of forming multiple microdroplets further includes: providing a fourth flow in a microfluidic device, the fourth fluid comprising an amplification mixture; and merging the fourth flow with the first and second flows in the microfluidic device to form a third flow comprising multiple microdroplets.

[0018] Optionally, additional components (optionally including cell-free synthetic mixtures and / or conjugated mixtures) are introduced into the microdroplets by picoliter injection of the flow of each corresponding component in a microfluidic device.

[0019] Alternatively, the nanoparticles may be formed from one or more proteins.

[0020] Optionally, the modified molecule is a peptide or protein.

[0021] Optionally, the modifying molecules are configured to covalently bind to the nanoparticles.

[0022] Optionally, the modifying molecule is configured to attach to the nanoparticle via non-covalent interactions. Alternatively, the modifying molecule and the nanoparticle are configured to attach to each other via non-covalent interactions between protein-protein affinity tag pairs.

[0023] Optionally, the modifying molecule is configured to bind to the therapeutic target.

[0024] Optionally, the nanoparticles contain, are incorporated into, or are configured to bind therapeutic agents in a covalent or non-covalent manner.

[0025] Optionally, the method forms a stream of microdroplets comprising at least 1,000 distinct macromolecules encoding different surface-modifying molecules.

[0026] According to a second aspect of this disclosure, a method for screening candidate nanoparticles for delivering therapeutic agents to a target is provided, comprising the steps of the method of any of the above aspects.

[0027] Optionally, the screening method may also include determining the binding characteristics of the surface-modified nanoparticles to the target.

[0028] According to a third aspect of this disclosure, a method for generating nanoparticles for delivering therapeutic agents to a target is provided, comprising the steps of the method of any of the foregoing aspects.

[0029] According to a fourth aspect of this disclosure, a microfluidic system is provided for generating a plurality of nanoparticles with different surface modifications, the nanoparticles being modified with different surface modifications, comprising: a microdroplet forming unit configured to form a plurality of microdroplets, each microdroplet comprising nanoparticles and distinct macromolecules encoding surface modification molecules; a synthesis unit configured to introduce a cell-free synthetic mixture into the microdroplets, the cell-free synthetic mixture being configured to synthesize surface modification molecules within each microdroplet based on the macromolecules encoding surface modification molecules; and a conjugation unit configured to introduce a conjugated mixture into the microdroplets, the conjugated mixture being configured to conjugate the nanoparticles with the surface modification molecules within each microdroplet. Attached Figure Description

[0030] Further features of this disclosure will be described below by way of non-limiting examples and in conjunction with the accompanying drawings, wherein:

[0031] Figure 1 The microfluidic system of this disclosure is illustrated schematically;

[0032] Figure 2 Experimental data related to the amplification steps of this disclosure are shown;

[0033] Figure 3 Experimental data related to the synthesis steps of this disclosure are shown;

[0034] Figure 4 Experimental data related to the conjugate steps of this disclosure are shown;

[0035] Figure 5 Experimental data related to the size distribution of nanoparticles are shown;

[0036] Figure 6 Experimental data related to the synthesis steps of this disclosure are shown;

[0037] Figure 7 Experimental data related to the conjugate steps of this disclosure are shown; and

[0038] Figure 8 Experimental data related to the conjugate steps of this disclosure are shown. Detailed Implementation

[0039] Figure 1 The illustration schematically depicts a microfluidic system according to the present disclosure for generating a plurality of nanoparticles with different surface modifications, the nanoparticles being modified with different surface modifications.

[0040] Figure 1 The upper left portion shows the microdroplet generation unit of the microfluidic system, used to generate multiple microdroplets in a microfluidic device. Each microdroplet may include nanoparticles and distinct macromolecules encoding different surface modification molecules.

[0041] As shown in the figure, each microdroplet may comprise multiple nanoparticles. The nanoparticles can be of the same type, for example, formed from the same molecules. For example, nanoparticles can be formed from biomolecules. For example, nanoparticles can be formed from one or more proteins (simple or conjugated), peptides, and / or nucleic acids. Multiple such molecules can combine to form nanoparticles. These molecules can be formed through non-covalent or covalent bonding. In a specific example, the nanoparticles are formed from clusters of human serum albumin.

[0042] The nanoparticles are submicron in size, such that the longest dimension of the nanoparticle is less than 1 micrometer. Preferably, the longest dimension does not exceed 200 nm. For example, the longest dimension can be at least 20 nm. In other words, the size of the nanoparticles can be 20-200 nm. For example, this can improve their suitability for intracellular delivery of therapeutic agents.

[0043] Nanoparticles may contain or be configured to contain therapeutic agents. Alternatively or additionally, nanoparticles may be bound to or configured to bind therapeutic agents, such as covalently or non-covalently. In some examples, for use as a therapeutic carrier, nanoparticles may carry therapeutic agents prior to surface modification. In other examples, nanoparticles may not carry therapeutic agents prior to surface modification, and the therapeutic agents may be introduced after surface modification.

[0044] like Figure 1 As shown in the diagram, macromolecules of different colors can be formed from microdroplets, each of which can contribute to the formation of a macromolecule comprising a single encoded surface-modifying molecule. The macromolecules can be polynucleotides, such as DNA or RNA. As also shown, the macromolecules can be plasmids (e.g., formed from DNA or RNA).

[0045] like Figure 1 As shown, the microdroplet generation unit includes a first microfluidic channel providing a first flow of a first fluid comprising nanoparticles. The microdroplet generation unit also includes a second microfluidic channel providing a second flow of a second fluid comprising distinct macromolecules. The first and second flows merge to form a third flow comprising multiple microdroplets suspended in the third fluid. The first, second, and third fluids are immiscible. Preferably, the third fluid is an oil, which may include an emulsion stabilizer.

[0046] The first and second streams can be configured such that each microdroplet has a high probability of generating a product containing no more than one macromolecule. For example, the relative flow rates of the first and second streams and / or the relative concentrations of nanoparticles and macromolecules in the first and second streams can be configured such that each microdroplet has a high probability of generating a product containing no more than one macromolecule. For example, more than 90%, or preferably more than 95%, of the microdroplets generated by this system may contain one macromolecule or may not contain one macromolecule.

[0047] In other examples, the system can be configured to generate microdroplets containing a small number (e.g., less than 10, preferably less than 5) of different macromolecules encoding different modified molecules, such as 2 or 3 different macromolecules. For example, more than 90%, or preferably more than 95%, of the microdroplets generated by the system may contain N or fewer macromolecules, where N is a small number.

[0048] like Figure 1As shown, each microdroplet may also include an amplification mixture of macromolecules configured to amplify surface-modified molecules. As shown, the microdroplet forming unit may include a fourth stream of a fourth fluid comprising the amplification mixture. As shown, the fourth stream may be merged with the first and second streams to form a third stream comprising multiple microdroplets.

[0049] Figure 1 The upper right portion shows the amplification unit of the microfluidic system, which is configured to incubate each microdroplet with the amplification mixture for a period of time. After said period of time, amplification can be terminated by heating to denature or destroy the components of the amplification mixture, for example at 65°C.

[0050] The amplification step is configured to provide higher levels of expression of the modified molecules within the droplets in subsequent synthesis steps. For example, the amplification step may increase the concentration of macromolecules in each microdroplet by at least 100-fold, at least 1000-fold, or at least 5000-fold, depending on the incubation time and the amount of amplification mixture provided.

[0051] Figure 2 The experimental results demonstrate the ability to amplify plasmids encoding GFP proteins from a concentration of 0.1 plasmid / pL, which corresponds to approximately one plasmid per droplet in a typical 14 pL microfluidic droplet. In this experiment, fluorescence intensity (FI) tells us the amount of GFP produced; the data show that even 4 plasmid / pL is still insufficient to produce visible protein without pDNA amplification; however, by amplifying pDNA for 2 hours, a sufficient concentration of DNA was amplified to produce protein levels equivalent to those expected in reactions using only approximately 5000 times more DNA. Amplification was performed using a Genomiphi from Cytiva. TM The V2 DNA amplification kit was used. Following amplification, a mixture of NEBExpress® Cell-free E. coli Protein Synthesis (CFPS) systems from New England Biolabs was introduced to transcribe and translate the gene from the plasmid into a functional and fluorescent GFP. This supports the feasibility of the in vitro method disclosed herein for amplifying a single plasmid within a microfluidic droplet to generate a large number of individual mutants.

[0052] Figure 2 A shows that the plasmid can be amplified in vitro to increase the DNA concentration by more than 5000 times. Figure 2B shows the detection of GFP production in diluted plasmid DNA mixtures based on a microplate reader. The blue, red, and purple curves represent CFPS reactions with 960, 4, and 0.4 units of pDNA (plastid) per pL, respectively. The orange and green curves represent solutions with 1 and 0.1 units of pDNA per pL, respectively, both amplified for 2 hours using an in vitro plasmid amplification kit before initiating the CFPS reaction.

[0053] Figure 1 The lower right section shows the synthesis unit of the microfluidic system, which is used to synthesize macromolecules based on encoded surface-modifying molecules within each microdroplet.

[0054] As shown in the figure, the synthesis unit is configured to introduce a cell-free synthesis mixture into 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 be based on macromolecular synthetic surface-modifying molecules. The cell-free synthesis mixture may be a cell-free protein synthesis (CFPS) mixture, which is configured to express proteins or peptides encoded by macromolecules.

[0055] Cell-free synthetic mixtures can be injected into microdroplets, for example, via picoliter injection. Figure 4 The upper part illustrates the picoliter injection process (although not specifically for cell-free synthetic mixtures). As microdroplets pass through the inlet, the aqueous stream of the cell-free synthetic mixture is fused with the aqueous microdroplets. This fusion can be facilitated by an electric field configured to agitate the surface of the water-oil interface between the microdroplets and the flow of the cell-free synthetic mixture.

[0056] In one experimental example, a co-flow microfluidic device was used to encapsulate a single plasmid in each microfluidic droplet (Agresti et al., 2010; Holstein et al., 2021). For this purpose, the plasmid DNA containing the gene for GFP was diluted to a concentration such that, on average, each droplet would contain less than one plasmid when forming the microfluidic droplets. Since the plasmid cannot be present in partial form, most droplets contained zero plasmid copies, and approximately 10% of the droplets should contain one plasmid. Droplets were formed from a plasmid solution, with the plasmid and the same mixture previously used for plasmid amplification co-encapsulated and stored in an incubation chamber for amplification. The microfluidic droplets were formed as a water-in-oil emulsion, stabilized by a PEG-PTFE triblock surfactant (EP2077912A1) dispersed in the oil phase of HFE-7500. After 6 hours of incubation, the droplets were exposed to a 65°C temperature shock for 10 minutes to inactivate the DNA amplification system.

[0057] Next, the CFPS reagent was introduced into the droplet using the picoliter injection technique described below. Figure 4The droplets were incubated again for 6 hours to induce GFP expression, and then imaged using a wide-field microscope. The reagents used were the same as those used in [previous study]. Figure 2 The batch experiments were identical, and the concentrations were adjusted to be the same. For example... Figure 3 As shown, approximately 5% of the droplets contain GFP, while the remaining droplets do not contain any fluorescent material, indicating that they do not contain any plasmids. Figure 3 This diagram illustrates the generation of GFP in a microfluidic droplet by a single capture plasmid. The green panel in the middle shows a droplet containing the GFP protein observed via 488 fluorescence. The left panel shows the droplet visualized using bright-field microscopy. The right panel shows an overlay of fluorescence and bright-field signals.

[0058] This demonstrates the ability to capture individual plasmid variants in microfluidic droplets and amplify them prior to addition to a CFPS mixture, ultimately resulting in a large number of functional proteins.

[0059] Figure 1 The lower left portion shows the system's conjugated unit, which is used to conjugate nanoparticles with surface-modifying molecules within each microdroplet to form surface-modified nanoparticles.

[0060] As shown in the figure, the conjugated unit is configured to introduce a conjugated mixture, configured to conjugate the nanoparticles with the surface-modifying molecules, into the microdroplet, and the microdroplet is incubated with the conjugated mixture for a period of time. The conjugated mixture can be injected into the microdroplet, for example, by picoliter injection as described above. Figure 4 The upper part shows the picoliter injection process for the conjugated mixture.

[0061] Conjugated mixtures can be configured to form covalent bonds between nanoparticles and modifying molecules. When modified nanoparticles are used in therapeutic applications, covalent bonding between the nanoparticles and modifying molecules can be advantageous because, for example, changes in in vivo pH are unlikely to disrupt the covalent bond. Covalent bonds between nanoparticles and modifying molecules can be achieved using carbodiimide-amine coupling, non-natural amino acid click chemistry coupling, succinimidyl thioether conjugation, glutaraldehyde-amino coupling, and / or hydroxysuccinimidyl-amine coupling. For example, the conjugated mixture may include a crosslinking agent. The crosslinking agent can be configured to form covalent bonds between protein nanoparticles and protein or peptide modifying molecules. Suitable crosslinking agents are known to those skilled in the art and include carbodiimides, succinimidides, hydroxysuccinimidides, and glutaraldehyde. In one embodiment, the crosslinking agent is (1-ethyl-3-(3-dimethylaminopropyl))carbodiimide.

[0062] Alternatively, the modifying molecule can be configured to attach to the nanoparticle via non-covalent interactions, such as non-covalent interactions between protein-protein affinity tag pairs. Typically, the interaction between affinity tag pairs is achieved by the surface-modifying molecule containing a fusion domain (such as albumin-binding protein or streptavidin-binding peptide), and the nanoparticle surface being modified with a corresponding affinity ligand targeting that fusion affinity domain. Alternatively, the nanoparticle surface may contain a fusion domain, and the modifying molecule may contain a corresponding affinity ligand. The interaction between the fusion domain and the affinity ligand allows the modifying molecule to attach to the nanoparticle surface non-covalently.

[0063] As previously mentioned, the sequential addition of reagents to microfluidic droplets can be most efficiently achieved through a technique known as picoliter injection (US11358105B2, (Abate et al., 2010)). Picoliter injection relies on the fusion of the aqueous stream and the aqueous droplets as they flow through the inlet. This fusion is mediated by an electric field that perturbs the surface of the water-oil interface between the droplets and the injected stream, enabling the controlled fusion of the two solutions.

[0064] Figure 4 (Top row) Shows wide-field time-lapse images of the picoliter injection technique. Injections were run at 0.77 kHz under typical operating conditions, meaning that 462,000 additional droplets could be injected during a 10-minute run. To demonstrate the compatibility of picoliter injection with the methods disclosed herein, the ability to inject a crosslinking agent (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)) into droplets containing BSA nanoparticles and GFP protein was tested. The injection was performed, and the droplets were collected in an imaging chamber for fluorescence detection.

[0065] Figure 4 A picoliter injection method is shown, illustrating the injection of a mixture containing EDC (red) into a droplet containing BSA nanoparticles and GFP protein (green). The top row shows a bright-field time-lapse image demonstrating the injection of the EDC solution (bottom inlet) into a droplet containing BSA and GFP. The scale bar in the fluorescence and bright-field images is 40 µm.

[0066] like Figure 4(Bottom row) As can be seen, almost all droplets simultaneously contain BSA nanoparticles and GFP (green, left) as well as an EDC crosslinking agent (red, right). A fluorescent dye (Alexa Fluor-647) that does not overlap with the GFP spectrum was added to the EDC mixture to allow visualization. Droplets injected via the picoliter injection device were almost 100% successfully injected with the crosslinking agent. To achieve the unique surface modifications of the nanoparticles in this library, the crosslinking agent must be added sequentially after protein generation. This picoliter injection capability supports multiple steps in the methods of this disclosure that rely on the sequential addition of various reagents within the confined space of a microfluidic droplet, which collectively promotes the combinatorial nature of the methods of this disclosure for generating different modified nanoparticles.

[0067] As mentioned, the nanoparticles in this disclosure can be composed of protein building blocks to allow for rapid functionalization and biocompatibility. One type of particle is made from bovine serum albumin (BSA). BSA nanoparticles can be prepared via a multi-step batch procedure in which a solution of BSA (80 mg / mL) in H₂O is titrated with ethanol at a flow rate of 1 mL / min using a syringe pump. By adding 8 mL of ethanol to 3 mL of the BSA solution, the BSA is desolvated in a controlled manner due to its insolubility in ethanol. The desolvated BSA forms particles with a diameter of 20-50 nm, which can then be covalently stabilized by adding various crosslinking agents. The compatibility of two different crosslinking methods with this disclosure was tested. The first method uses glutaraldehyde (50% aqueous solution), which is added to the BSA particle mixture at a rate of 0.2 µL per mg BSA. The crosslinking reaction proceeds for 18 hours, followed by quenching with an excess of 1 M TRIS-HCl (pH 7.4). The nanoparticles were then centrifuged (13,400 rpm, 40 min) to the bottom of a Falcon tube, the supernatant was discarded, the nanoparticle precipitate was resuspended in fresh H2O and centrifuged again, and the nanoparticles were washed with H2O. This washing step was repeated three times to remove excess glutaraldehyde. The particles were characterized immediately and again by dynamic light scattering (DLS) at 24 hours and 7 days to determine size and stability. At time point 0, the particle diameter was determined to be 30 ± 12 nm; after 24 hours, the particle diameter remained at 30 ± 12 nm, indicating particle stability. Measurements after 7 days showed that the BSA particle size increased to 52 ± 20 nm, which may be attributed to the swelling property of the particles during storage in aqueous solution. The DLS curves showing the particle size distribution are shown in the figure. Figure 5 As shown in the image.

[0068] Crosslinked nanoparticles were formed using a similar method, with EDC used as the crosslinking agent instead of glutaraldehyde, because glutaraldehyde produced some undesirable autofluorescence effects in downstream characterization (Ma et al., 2016). Similar to the protocol for preparing BSA particles using glutaraldehyde, the crosslinking agent EDC was added to the solution at a ratio of 0.02 mg / mg BSA. The reaction was carried out at room temperature with constant stirring at 1500 rpm for 3 hours. The particles were then collected and washed in the same manner as described above. At this ratio, BSA formed particles with a diameter of 129 ± 26 nm. Therefore, EDC and glutaraldehyde represent two different crosslinking methods for altering the morphology of BSA nanoparticles used in downstream experiments. After establishing the procedure for preparing BSA nanoparticles, the nanoparticles were subsequently introduced into a CFPS mixture containing a plasmid encoding GFP (experimental conditions as described above). Figure 2 The objective is to determine whether BSA nanoparticles (BSANP) will have any negative impact on the in vitro molecular biology methods disclosed herein.

[0069] From Figure 6 The results showed that adding up to 16% of a 12 mg / mL BSA NP solution had no effect on the ability to generate GFP from the plasmid in vitro (as measured by 488 fluorescence intensity over time). This indicates that the nanoparticles are fully compatible with CFPS and that the encapsulation of nanoparticles using the mechanism of in vitro protein production is fully compatible with the workflow of our invention. Furthermore, the biocompatibility of the cross-linked nanoparticles in HEK293 cell culture was tested. Adding particle concentrations of 10 mg / mL and 2 mg / mL, along with soluble BSA (10 mg / mL), to the cell culture medium and exposing HEK293 cells to pH 5, 7, and 9 for 24 hours resulted in complete cell viability. Both EDC and glutaraldehyde-crosslinked BSA nanoparticles maintained cell viability. Overall, the cell viability indicates that the nanoparticles should possess extremely high biocompatibility. As a final step to demonstrate the feasibility of the method disclosed herein, the ability to functionalize the nanoparticles by covalently coupling proteins to the nanoparticle surface was demonstrated. It has been shown that EDC can be injected into microscale droplets containing BSA and GFP, but due to the nanoscale size of BSA particles, it is difficult to easily visualize the crosslinking of GFP with its surface. Instead, the reaction is carried out in batches, and then the crosslinked particles are flowed through a microfluidic device mounted on a single-molecule sensitive confocal microscope (Krainer et al., 2023), which can detect nanoscale to microscale particles in multiple fluorescence channels.

[0070] Fluorescently labeled BSA nanoparticles (15 mg / mL, containing 1% Alexa Fluor-647) were mixed with GFP (0.25 mg / mL) and 17 mM EDC. After the coupling reaction proceeded for 30 minutes, the mixture was washed by centrifugation as previously described. Then, it can be processed as follows... Figure 7 The particles were characterized using confocal detection, as shown. First, control particles without GFP were characterized. Figure 7 A) showed a significant peak in the 647 nm channel (BSA, red), but no significant peak in the 488 nm channel (GFP, cyan). Next, the GFP-functionalized particles were characterized ( Figure 7 (B) shows significant peaks in both fluorescence channels. A peak is defined as significant if the signal is more than 3 standard deviations above the mean. The cutoff is 200 photons for the 647 nm channel and 856 photons for the 488 nm channel. The number of peaks for each channel is N488nm = 1171 and N647nm = 1058. Crucially, 165 peaks overlap in both channels, indicating GFP-modified BSA particles. This means that 15.6% of the BSA particles were successfully modified with GFP. The significant number of peaks at 488 nm stems from the fact that EDC is a non-specific crosslinking agent, thus allowing for the easy formation of GFP oligomers in addition to those attached to the BSA surface. Further optimization of different crosslinking agents and reaction schemes could improve the specificity and efficiency of BSA surface coupling, but it is evident that covalent functionalization of BSA nanoparticles using protein components is readily achievable.

[0071] In another replicate of this experiment, it was demonstrated that the covalent linking method between nanoparticles and surface modifiers works well when click chemistry is employed, as shown in the results. Figure 8As shown. In this experimental version, BSA was modified with the click chemistry reagent dibenzocyclooctyne (DBCO) using the following protocol: BSA was dissolved at 6.6 mg / mL (100 µM) in 100 mM Tris buffer (pH 7.5) with 1 mM TCEP and incubated at room temperature for 10 min. DMSO (2 or 4 µL) containing 50 mM maleimide-DBCO was added to the 100 µM BSA solution (100 µL). The solution was incubated at room temperature for 2 h. The labeled BSA was purified using a 0.5 mL Zeba spin desalting column at 40 K MWCO. This BSA was then used to form nanoparticles via ethanol desolvation, as described above. Independently, the fusion protein SNAP-GFP was prepared via cell-free protein synthesis in the same manner as described above. Then, SNAP-GFP was made click-responsive by incubation with 1.5 molar equivalents of µM benzylguanine-PEG(4)-Azide, thus obtaining azide-SNAP-GFP. Subsequently, according to Figure 4 The method described involves introducing azide-SNAP-GFP into microdroplets containing DBCO-modified BSA particles via picoliter injection. The nanoparticle-SNAP-GFP mixture was then incubated in water-in-oil droplets for 12 hours, after which the emulsion was ruptured and the aqueous phase was extracted. The fluorescence intensity of the resulting aqueous phase was then analyzed by flow cytometry. Figure 8 (A, B, C) shows histograms of 488 fluorescence signals, where block A shows unreacted nanoparticles, block B shows nanoparticles incubated with non-click reactive SNAP-GFP, and block C shows nanoparticles that have undergone click chemical modification. Figure 8 The relative integrated intensity above the noise cutoff value (350 intensity units, represented by the black dashed line in A, B, and C) in the quantization histogram of patch D is shown. Compared to unreacted particles, the integrated 488 nm fluorescence signal on the nanoparticles increased by 1737% (or 646% relative to non-click reactive GFP), demonstrating the effectiveness of click chemistry. The average particle sizes in patches A, B, and C were confirmed by DLS to be 282 nm, 303 nm, and 299 nm, respectively. This experiment supports the use of click chemistry as a method for attaching modified macromolecules to the surface of nanoparticles, utilizing the previously outlined approach.

[0072] This disclosure provides a unique method for creating libraries of diverse, covalently modified, protein-based nanoparticles. This workflow relies on a combination of microfluidic and molecular biology techniques, integrating both for a novel application.

[0073] The project upon which this application is based has been funded by the European Union's Horizon 2020 research and innovation program (Marie Sklodowska-Curie grant agreement number 101023060).

[0074] The project upon which this application is based has been funded by the European Research Council (ERC) under the EU Horizon 2020 research and innovation program (funding agreement number 101001615).

[0075] This work was supported by the Harding Distinguished Postgraduate Scholars Programme at the University of Cambridge.

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[0086] Sago, CD, Lokugamage, MP, Paunovska, K, Vanover, DA, Monaco, CM, Shah, NN, Castro, MG, Anderson, SE, Rudoltz, TG, Lando, GN, Tiwari, PM, Kirschman, JL, Willett, N, Jang, YC, Santangelo, PJ, Bryksin, AV, & Dahlman, JE (2018). High-throughputin in vivo screen of functional mRNA delivery identifies forendothelial cell gene editing nanoparticles. Proceedings of the National Academy of Sciences of the United States of America, 115(42). https: / / doi.org / 10.1073 / pnas.1811276115

Claims

1. A method for generating multiple nanoparticles with different surface modifications, wherein the nanoparticles are modified using different surface modifications, comprising: Multiple microdroplets are formed in a microfluidic device, each microdroplet comprising nanoparticles and distinct macromolecules encoding different surface modification molecules; Within each microdroplet, the surface-modifying molecules are synthesized based on macromolecules encoding the surface-modifying molecules; The nanoparticles are conjugated with the surface-modifying molecules within each microdroplet to form the surface-modified nanoparticles.

2. The method according to claim 1, wherein, Each microdroplet contains multiple nanoparticles.

3. The method according to any one of the preceding claims, wherein, Each microdroplet is formed, which includes a single macromolecule encoding surface-modifying molecules.

4. The method according to any one of the preceding claims, wherein, The macromolecule is a polynucleotide.

5. The method according to claim 4, wherein, The macromolecule is a DNA or RNA molecule.

6. The method according to claim 4 or 5, wherein, The polynucleotide is a plasmid.

7. The method according to any one of the preceding claims, wherein, Each microdroplet also includes an amplification mixture of macromolecules configured to amplify the encoded surface-modifying molecules.

8. The method according to claim 7, further comprising the following step: The macromolecule encoding the surface-modified molecule is amplified within each microdroplet by incubating each microdroplet with the amplification mixture for a period of time.

9. The method according to claim 8, wherein, After a certain period of time, the amplification is terminated by heating to denature or destroy the components of the amplification mixture.

10. The method according to claim 8 or 9, wherein, The amplification step increases the concentration of the macromolecule in each microdroplet by at least 100-fold, and appropriately at least 1000-fold.

11. The method according to any one of the preceding claims, wherein, The step of synthesizing the surface-modifying molecule includes introducing a cell-free synthetic mixture into each microdroplet, the cell-free synthetic mixture being configured to synthesize the surface-modifying molecule based on the macromolecule.

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

13. The method according to any one of the preceding claims, wherein, The step of conjugating the nanoparticles with the surface-modifying molecules includes introducing a conjugated mixture configured to conjugate the nanoparticles with the surface-modifying molecules.

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

15. The method according to any one of the preceding claims, wherein, The steps for forming the plurality of microdroplets include: A first flow of a first fluid is provided in the microfluidic device, the first fluid comprising the nanoparticles; A second flow of a second fluid is provided in the microfluidic device, the second fluid comprising the respective macromolecules; In the microfluidic device, the first flow and the second flow are combined to form a third flow, the third flow comprising the plurality of microdroplets suspended in a third fluid.

16. The method according to claim 15, wherein, The first fluid, the second fluid, and the third fluid are immiscible.

17. The method according to claim 15 or 16, wherein, The first and second streams are configured such that each microdroplet has a high probability of forming a substance containing no more than one macromolecule.

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

19. The method according to any one of claims 15-18, when also dependent on claim 7, wherein, The step of forming the plurality of microdroplets further includes: A fourth fluid is provided in the microfluidic device, the fourth fluid comprising the amplification mixture; and In the microfluidic device, the fourth stream is combined with the first and second streams to form the third stream comprising the plurality of microdroplets.

20. The method according to any one of the preceding claims, wherein, In the microfluidic device, additional components are introduced into the microdroplets by picoliter injection of each corresponding component, the additional components optionally including the cell-free synthetic mixture of claim 12 and / or the conjugate mixture of claim 14.

21. The method according to any one of the preceding claims, wherein, The nanoparticles are formed from one or more proteins.

22. The method according to any one of the preceding claims, wherein, The modified molecule is a peptide or protein.

23. The method according to any one of the preceding claims, wherein, The modifying molecule is configured to covalently bind to the nanoparticles.

24. The method according to any one of claims 1-22, wherein, The modified molecules are configured to attach to the nanoparticles via non-covalent interactions.

25. The method according to claim 24, wherein, The modified molecules and the nanoparticles are configured to attach to each other via non-covalent interactions between protein-protein affinity tag pairs.

26. The method according to any one of the preceding claims, wherein, The modified molecule is configured to bind to the therapeutic target.

27. The method according to any one of the preceding claims, wherein, The nanoparticles contain, are bound to, or are configured to be covalently or non-covalently bound to a therapeutic agent.

28. The method according to any one of the preceding claims, wherein, The method forms a stream of microdroplets comprising at least 1,000 distinct macromolecules, each encoding a different surface-modifying molecule.

29. A method for screening candidate nanoparticles for delivering therapeutic agents to a target, comprising the steps of the method of any of the preceding claims.

30. The method of claim 29, further comprising determining the binding characteristics of the surface-modified nanoparticles to the target.

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

32. A microfluidic system for generating multiple nanoparticles with different surface modifications, said nanoparticles being modified with different surface modifications, comprising: A microdroplet forming unit is configured to form multiple microdroplets, each microdroplet comprising nanoparticles and distinct macromolecules encoding surface modification molecules; A synthesis unit configured to introduce a cell-free synthesis mixture into the microdroplets, the cell-free synthesis mixture being configured to synthesize the surface-modifying molecules within each microdroplet based on macromolecules encoding the surface-modifying molecules; as well as A conjugated unit is configured to introduce a conjugated mixture into the microdroplets, the conjugated mixture being configured to conjugate the nanoparticles with the surface-modifying molecules within each microdroplet.

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