Microscale compartments and methods of production thereof

Abstract

A microscale compartment is disclosed. The microscale compartment includes a shell and an internal space. The shell comprises an aliphatic conjugate between a hydrophilic protein component and a hydrophobic polymer component which is capable of forming spheroids. The disclosure further provides a method for preparing a microscale compartment. The method includes assembling a shell comprising an aliphatic conjugate between a hydrophilic protein component and a hydrophobic polymer component which is capable of forming spheroids in a water-in-oil emulsion process. Then adding a crosslinker and passing the emulsion product through a membrane.

Description

Docket No. 11000-11031-PCTMicroscale Compartments and Methods of Production ThereofTECHNICAL FIELD

[0001] This disclosure relates to synthetic organelles and methods of their production.BACKGROUND

[0002] Synthetic organelles are artificially created sub-compartments within cells. Synthetic organelles mimic the natural function of organelles. Synthetic organelles create separate environments within the cell, which enable specific biochemical reactions. Synthetic organelles enable the regulation of the timing and location of cellular processes. Studying synthetic organelles offers insights into how natural organelles function. New therapies can be developed with the help of synthetic organelles, such therapies include delivering drugs or enzymes to specific locations within cells for targeted treatments. Synthetic organelles can influence cells to have novel capabilities.SUMMARY

[0003] In a first aspect, the disclosure provides a microscale compartment. The microscale compartment includes a shell and an internal space. The shell comprises an aliphatic conjugate between a hydrophilic protein component and a hydrophobic polymer component which is capable of forming spheroids.

[0004] In a second aspect, the disclosure provides a method for preparing a microscale compartment. The method includes assembling a shell an aliphatic conjugate between a hydrophilic protein component and a hydrophobic polymer component which is capable of forming spheroids in a water- in-oil emulsion process. Then adding a crosslinker. Finally, passing the emulsion product through a membrane.

[0005] Further aspects and embodiments are provided in the foregoing drawings, detailed description, and claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The following drawings are provided to illustrate certain embodiments described herein. The drawings are merely illustrative and are not intended to limit the scope of claimed inventions and are not intended to show every potential feature or embodiment of the claimed inventions. The drawings are not necessarily drawn to scale; in some instances, certain elements of the drawing may be enlarged with respect to other elements of the drawing for purposes of illustration.

[0007] Figure 1 is a schematic of a synthetic organelle.Docket No. 11000-11031-PCT

[0008] Figure 2 is a schematic of a synthetic organelle.

[0009] Figure 3 is a plasmid map.

[0010] Figure 4 is a plasmid map.

[0011] Figure 5 is a plasmid map.

[0012] Figures 6A and 6B are Temporal Monitoring of Transcription and Translation Reactions via Fluorescence signal detection. Figure 6A depicts the fluorescence signal of DFHBI, which correlates with the increasing concentration of the Broccoli aptamer sequence. This fluorescence enhancement reflects the successful transcription of the aptamer. Figure 6B illustrates the fluorescence signal from mCherry, which increases proportionally with the quantity of mCherry proteins expressed, indicating active translation. Notably, the yellow line, representing the plasmid for mCherry expression in solution, is interrupted due to reaching the detection limit of the instrument.

[0013] Figures 7A-7C show the Visualization of Synthetic Nucleus in a Cell-Free Gene Expression System. This figure illustrates SNs dispersed in a cell-free gene expression solution, viewed under transmitted light. The compartments within the SNs are shown containing different plasmids: Figure 7A displays SNs encapsulating the S plasmid, Figure 7B shows those with the M plasmid, and Figure 7C depicts SNs containing the L plasmid. Scale Bar=20um

[0014] Figures 8A-8C Fluorescence Imaging of DFHBI Signal in Synthetic Nucleus. This figure corresponds to the same samples shown in Figure 7, now imaged using the fluorescence channel to visualize the DFHBI signal. The fluorescence directly correlates with the presence of the translated Broccoli aptamer in the cell free gene expression reaction. Figure 8 A, 8B, and 8C display the DFHBI fluorescence in presence of SNs encapsulating the S (FIG. 8A), M (FIG. 8B), and L (FIG. 8C) plasmids, respectively, illustrating the successful transcription of the Broccoli aptamer across different plasmid sizes. Scale Bar=20 pm

[0015] Figures 9A-9C shoe Fluorescence Imaging of mCherry Signal in SNs Supplemented Cell -Free Gene Expression Reaction. These figures correspond to the same samples shown in Figure 2, now imaged using the fluorescence channel to visualize the mCherry signal. The fluorescence signal is derived from the translation of the Broccoli-mCherry RNA into the mCherry protein. Figures 9 A, 9B, and 9C display the mCherry fluorescence in the cell-free gene expression solution in the presence of SNs encapsulating the S (FIG. 9A), M (FIG. 9B), and L (FIG. 9C) plasmids, respectively, illustrating the mCherry protein expression. Scale Bar=20um

[0016] Figure 10 shows the average DFHBI Fluorescence Signal Analysis inside the SNs. This figure presents the average DFHBI fluorescence signal calculated within the SNs containing the S, M or L plasmid from Figure 8.Docket No. 11000-11031-PCT

[0017] Figure 11 shows the average DFHBI Fluorescence Signal Analysis outside the SNs. This figure presents the average DFHBI fluorescence signal calculated outside the SNs containing the S, M or L plasmid in Figure 8.

[0018] Figure 12 shows a graph depicting the Differential Fluorescence Analysis of SNs. This figure displays the delta fluorescence, which measures the difference in fluorescence intensity between the SNs and the surrounding areas, based on the images shown in Figure 3. The delta fluorescence quantifies the relative concentration of fluorescent markers within the SNs, indicating the distribution and accumulation of these markers as a response to specific reactions involving the SNs.

[0019] Figure 13 shows a graph depicting the average mCherry Fluorescence Signal Analysis. This figure presents the average mCherry fluorescence signal calculated across the entire frame of the images shown in Figure 4. The analysis highlights the overall fluorescence distribution signal, demonstrating that when the mCherry is expressed, it displays a homogeneous distribution throughout the solution, proving its capability to diffuse outside the SN.

[0020] Figures 14A-14B show the temporal Monitoring of Transcription and Translation Reactions via Fluorescence signal detection. Figure 14a depicts the fluorescence signal of DFHBI, which correlates with the increasing concentration of the Broccoli aptamer sequence. This fluorescence enhancement reflects the successful transcription of the aptamer. Figure 14b illustrates the fluorescence signal from mCherry, which increases proportionally with the quantity of mCherry proteins expressed, indicating active translation. Notably, most of the samples shown for Broccoli or mCherry signal an interruption due to reaching the detection limit of the instrument.

[0021] Figure 15 shows the Visualization of Synthetic Nucleus in a Cell-Free Gene Expression System. This figure illustrates SNs dispersed in a cell-free gene expression solution, viewed under transmitted light. Each image is labeled with different letters, where each letter corresponds to a different SN composition. Scale Bar=20um.

[0022] Figure 16 shows the Fluorescence Imaging of DFHBI Signal in Synthetic Nucleus. This figure corresponds to the same samples shown in Figure 9, now imaged using the fluorescence channel to visualize the DFHBI signal. The fluorescence directly correlates with the presence of the translated Broccoli aptamer-mCherry RNA in the cell free gene expression reaction. Each image is labeled with different letters, where each letter corresponds to a different SN composition. Scale Bar=20 pm.

[0023] Figure 17 shows the Fluorescence Imaging of mCherry Signal in Synthetic Nucleus (SNs) Supplemented Cell-Free Gene Expression Reaction. This figure corresponds to the same samples shown in Figure 9, now imaged using the fluorescence channel to visualize the mCherry signal. The fluorescence signal is derived from the translation of the Broccoli-mCherry RNA into the mCherry protein. Each image is labeled with different letters, where each letter corresponds to a different SN composition. Scale Bar=20um.Docket No. 11000-11031-PCT

[0024] Figure 18 shows the Average DFHBI Fluorescence Signal Analysis inside the SNs. This figure presents the average DFHBI fluorescence signal calculated within the SNs containing the M plasmid from Figure 16.

[0025] Figure 19 shows the average DFHBI Fluorescence Signal Analysis outside the SNs. This figure presents the average DFHBI fluorescence signal calculated outside the SNs containing the M plasmid from Figure 16.

[0026] Figure 20 shows the Differential Fluorescence Analysis of SNs. This figure displays the delta fluorescence, which measures the difference in fluorescence intensity between the SNs and the surrounding areas, based on the images shown in Figure 16. The delta fluorescence quantifies the relative concentration of fluorescent markers within the SNs, indicating the distribution and accumulation of these markers as a response to specific reactions involving the SNs.

[0027] Figure 21 shows Average of mCherry Fluorescence Signal Analysis. This figure presents the average mCherry fluorescence signal calculated across the entire frame of the images shown in Figure 17. The analysis highlights the overall fluorescence distribution, with a focus on the solution containing the Synthetic Nucleus compartmentalizing M plasmid, as most of these displays homogeneously distributed fluorescence.

[0028] Figure 22 shows the Temporal Monitoring of Transcription and Translation Reactions via Fluorescence signal detection. Figure 22 depicts the fluorescence signal of DFHBI, which correlates with the increasing concentration of the Broccoli aptamer sequence. This fluorescence enhancement reflects the successful transcription of the aptamer. Figure 22 illustrates the fluorescence signal from mCherry; to show as a negative control the contrast respect the plasmids expressing mCherry. Notably, some of the samples shown for Broccoli signal an interruption due to reaching the detection limit of the instrument.

[0029] Figure 23 shows the Visualization of the SN in a Cell-Free Gene Expression System. This figure illustrates SNs dispersed in a cell-free gene expression solution, viewed under transmitted light. Each image is labeled with different letters, where each letter corresponds to a different SN composition. Scale Bar=20um.

[0030] Figure 24 shows the Fluorescence Imaging of DFHBI Signal in SN. This figure corresponds to the same samples shown in Figure 23, now imaged using the fluorescence channel to visualize the DFHBI signal. The fluorescence directly correlates with the presence of the translated Broccoli aptamer in the cell free gene expression reaction. Each image is labeled with different letters, where each letter corresponds to a different SN composition. Scale Bar=20 pm.Docket No. 11000-11031-PCT

[0031] Figure 25 shows the Average DFHBI Fluorescence Signal Analysis inside the SNs. This figure presents the average DFHBI fluorescence signal calculated within the SNs containing the M plasmid from Figure 24.

[0032] Figure 26 shows the Average DFHBI Fluorescence Signal Analysis outside the SNs. This figure presents the average DFHBI fluorescence signal calculated outside the SNs containing the M plasmid from Figure 24.

[0033] Figure 27 shows the Differential Fluorescence Analysis of SNs. This figure displays the delta fluorescence, which measures the difference in fluorescence intensity between the SNs and the surrounding areas, based on the images shown in Figure 19. The delta fluorescence quantifies the relative concentration of fluorescent markers within the SNs, indicating the distribution and accumulation of these markers as a response to specific reactions involving the SNs.

[0034] Figure 28 shows the Temporal Monitoring of Transcription and Translation Reactions via Fluorescence signal detection. This depicts the fluorescence signal of DFHBI, which correlates with the increasing concentration of the Broccoli aptamer sequence. This fluorescence enhancement reflects the successful transcription of the aptamer. Figure 28 further illustrates the fluorescence signal from mCherry, which increases proportionally with the quantity of mCherry proteins expressed, indicating active translation. Notably, the yellow line, representing the plasmid for mCherry expression in solution, is interrupted due to reaching the detection limit of the instrument.

[0035] Figure 29 shows the Visualization of SN in a Cell-Free Gene Expression System. This figure illustrates SNs dispersed in a cell-free gene expression solution, viewed under transmitted light. Each image is labeled with different letters, where each letter corresponds to a different SN composition. Scale Bar=20um.

[0036] Figure 30 shows the Fluorescence Imaging of DFHBI Signal in SN. This figure corresponds to the same samples shown in Figure 25, now imaged using the fluorescence channel to visualize the DFHBI signal. The fluorescence directly correlates with the presence of the translated Broccoli aptamer in the cell free gene expression reaction. Each image is labeled with different letters, where each letter corresponds to a different SN composition. Scale Bar=20um.

[0037] Figure 31 shows the Fluorescence Imaging of mCherry Signal in Synthetic Nucleus (SNs) Supplemented Cell-Free Gene Expression Reaction. This figure corresponds to the same samples shown in Figure 31 , now imaged using the fluorescence channel to visualize the mCherry signal. The fluorescence signal is derived from the translation of the Broccoli-mCherry RNA into the mCherry protein. Each image is labeled with different letters, where each letter corresponds to a different SN composition. Scale Bar=20um.Docket No. 11000-11031-PCT

[0038] Figure 32 shows the Fluorescence Imaging of DAPI Signal in SNs Supplemented Cell- Free Gene Expression Reaction. This figure corresponds to the same samples shown in Figure 31 , now imaged using the fluorescence channel to visualize the DAPI signal. The fluorescence signal is derived from the specific DNA binding specificity of DAPI dye. Each image is labeled with different letters, where each letter corresponds to a different SN composition. Scale Bar=20um.

[0039] Figure 33 shows the Average DFHBI Fluorescence Signal Analysis. This figure presents the average DFHBI fluorescence signal calculated within the SNs containing the M plasmid in Figure 30, as these display a homogeneously distributed fluorescence.

[0040] Figure 34 shows the Average DAPI Fluorescence Signal Analysis. This figure presents the average DAPI fluorescence signal calculated within the SNs containing the M plasmid in Figure 30, as these display a homogeneously distributed fluorescence.

[0041] Figure 35 shows the Average DFHBI Fluorescence in the background. This figure presents the average DFHBI fluorescence signal outside the SNs the background or when in the sample do not contain SNs is the average of the full frame. The analysis highlights the overall fluorescence distribution, with a focus on the solution containing the SN compartmentalizing M plasmid, respect the positive (M in solution) or negative (no DNA) control.

[0042] Figure 36 shows the average DAPI fluorescence signal outside the SNs the background or when in the sample do not contain SNs is the average of the full frame. The analysis highlights the overall fluorescence distribution, with a focus on the solution containing the SN compartmentalizing M plasmid, respect the positive (M in solution) or negative (no DNA) control.

[0043] Figure 37 displays the delta fluorescence, which measures the difference in fluorescence intensity between the SNs and the surrounding areas, based on the images shown in Figure 30. The delta fluorescence quantifies the relative concentration of fluorescent markers within the SNs, indicating the distribution and accumulation of these markers as a response to specific reactions involving the SNs.

[0044] Figure 38 displays the delta fluorescence, which measures the difference in fluorescence intensity between the SNs and the surrounding areas, based on the images shown in Figure 32. The delta fluorescence quantifies the relative concentration of fluorescent markers within the SNs, indicating the distribution and accumulation of these markers as a response to specific reactions involving the SNs.

[0045] Figure 39 displays SNs labeled with DY530 dye, displaying their compartmentalization. The SNs are resuspended in DMEM media buffer, highlighting their stability and distribution within the medium. The SNs shown here were produced using the extruder methodDocket No. 11000-11031-PCT under various experimental conditions. Each image is labeled with letters from A to F, corresponding to specific conditions detailed in a table at the beginning of the document. Scale bar= 20 pm.

[0046] Figure 40 presents the size distribution analysis of the SNs represented in Figure 34. Each letter on the histogram plot corresponds to a specific SNs condition depicted in Figure 39. The y- axis illustrates the density of the SNs, representing the probability of finding SNs within a particular diameter range. The x-axis denotes the diameter in micrometers (pm), indicating the size of the SNs. The histogram is constructed with a fixed number of bins set at 10. Additionally, the fitted Gaussian distribution is depicted by the red line, providing a visual representation of the distribution pattern of the SNs sizes.

[0047] Figure 41 is a Visualization of proteinosome-compartmentalized 62 kDa FITC-labeled enzyme, (i) Image acquired using transmitted light, (ii) The same image acquired using the fluorescence FITC filter channel, showing the localization of the labeled enzyme within the proteinosomes. Scale bar= 20 pm.

[0048] Figure 42 is a Visualization of empty proteinosome in a solution mixed with 62 kDa FITC-labeled enzyme, (i) Image acquired using transmitted light, (ii) The same image acquired using the fluorescence FITC filter channel, showing the homogeneous distribution of the labeled enzyme in the solution with the proteinosomes. Scale bar= 20 pm.

[0049] Figure 43 is a Visualization of SNs with Different Contents. The SNs containing plasmid S and T7 polymerase (a, d), SNs containing plasmid S, T7 polymerase, and FITC-labelled luciferase (b, e), or empty SNs (c, f) were photographed using a microscope. The SNs were diluted in the DMEM buffer described earlier. The top images (a-c) were taken in transmitted light, while the bottom images (d-f) were taken under fluorescence with a FITC filter (around 520 nm). Scale bar 50 nm.

[0050] Figure 44 is a Visualization of HEK 293T Cells Transfected with Different Types of SNs. The HEK 293T cells transfected with SNs containing plasmid S, T7 polymerase, and FITC- labelled luciferase (a), SNs containing plasmid S and T7 polymerase (b), or empty SNs (c) are shown in Figure 37. The figure displays overlapped images acquired in transmitted light and fluorescence with a filter for FITC emission around 520 nm. Scale bar 300 pm.

[0051] Figure 45 is a Histogram of Cell Viability Post-Treatment. The histogram shows the average cell viability after treatment with different types of SNs. The negative control (untreated cells) is also shown. Specifically, the viability for cells treated with SNs containing plasmid S, T7 polymerase, and FITC-labelled luciferase is 92% ± 1%, for SNs without luciferase is 90.7% + 1.5%, and for empty SNs is 91.3% ± 1.2%.Docket No. 11000-11031-PCT

[0052] Figure 46 Transmitted Light and Fluorescence Microscopy Images of HEK293T Cells. The transmitted light and fluorescence microscopy images of HEK293T cells are presented to illustrate the effects of synthetic nuclei (SNs) treatment. Panel (a) shows a transmitted light image of HEK293T cells without SNs treatment, displaying typical cell morphology. Panel (b) presents a transmitted light image of HEK293T cells with SNs treatment, with unmodified cellular morphology after a 6-hour incubation with SNs. Panel (c) depicts a fluorescence image of HEK293T cells without SNs treatment. In contrast, panel (d) shows a fluorescence image of HEK293T cells with SNs treatment, demonstrating increased fluorescence due to the transcriptional activity of SNs, which results in the production of broccoli RNA aptamer that binds to DFHBI. The scale bar in each image represents 20 pm.

[0053] Figure 47 shows the Relative DFHBI fluorescence in HEK293T cells. The left bar shows minimal fluorescence in HEK293T cells treated with SNs without DFHBI. The right bar indicates increased fluorescence in HEK293T cells treated with SNs and DFHBI, reflecting the transcriptional activity of SNs producing broccoli RNA aptamer that binds to DFHBI. Error bars represent the standard deviation.

[0054] Figure 48A shows the transcription kinetics of the mRNA containing the aptamer Broccoli, which can be monitored through the fluorescence emitted by the Broccoli aptamer / DFHBI- Molecule complex.

[0055] Figure 48B displays the expression kinetics of mCHerry, which can be monitored through the fluorescence of the protein itself.

[0056] Figure 49A shows SNs after the reacion with PURE express completed in solution with ribosome. In grey the SNs in brightfield, in green the broccoli aptamer-DFHBI complex fluorescence within the SNs and in red the mCHerry protein expressed and diffused in solution.

[0057] Figure 49B shows SNs after the reacion with PURE express without ribosome. In grey the SNs in brightfield, in green the broccoli aptamer-DFHBI complex fluorescence within the SNs and in red channel is missing due to the absence of translation reacrion.DETAILED DESCRIPTION

[0058] The following description recites various aspects and embodiments of the inventions disclosed herein. No particular embodiment is intended to define the scope of the invention. Rather, the embodiments provide non-limiting examples of various compositions, and methods that are included within the scope of the claimed inventions. The description is to be read from the perspective of one of ordinary skill in the art. Therefore, information that is well known to the ordinarily skilled artisan is not necessarily included.Docket No. 11000-11031-PCTDefinitions

[0059] The following terms and phrases have the meanings indicated below, unless otherwise provided herein. This disclosure may employ other terms and phrases not expressly defined herein. Such other terms and phrases shall have the meanings that they would possess within the context of this disclosure to those of ordinary skill in the art. In some instances, a term or phrase may be defined in the singular or plural. In such instances, it is understood that any term in the singular may include its plural counterpart and vice versa, unless expressly indicated to the contrary.

[0060] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to “a substituent” encompasses a single substituent as well as two or more substituents, and the like.

[0061] As used herein, “for example,” “for instance,” “such as,” or “including” are meant to introduce examples that further clarify more general subject matter. Unless otherwise expressly indicated, such examples are provided only as an aid for understanding embodiments illustrated in the present disclosure and are not meant to be limiting in any fashion. Nor do these phrases indicate any kind of preference for the disclosed embodiment.

[0062] As used herein, “microscale compartment” refers to a compartment which is very small or microscopic typically ranging from 0.25 to 999 micrometers (pm) in size.

[0063] As used herein, “synthetic organelle” refers to an artificially created compartment that can be used within a cell.

[0064] As used herein, “synthetic nucleus” or “SN” refers to an artificially created compartment that mimics the functions of a nucleus. The synthetic nucleus includes a template of genetic material, the mechanisms for transcription of that genetic material from the template. For example, the template may be DNA and the mechanism for transcription may be a polymerase. In some embodiments, the mechanism for translation is located within the synthetic nucleus. Such translation mechanisms include ribosomes.

[0065] Microscale compartments, such as synthetic organelles, can be created in a number of ways including lipid-based membranes, protein-based membranes, or polymer-based membranes. The membrane creates a shell surrounding an internal space. One organelle that is useful to create a synthetic analog for is a nucleus. Various processes are initiated and carried out in the nucleus. The internal space is particularly well adapted to hold and store the structures typically used by a nucleus. An information template such as a chromosome, plasmid, or other replicable structure may be housed in the internal space, along with the mechanisms to accomplish that replication, such as polymerases. This setup is intended to facilitate the replication of genetic information within the synthetic nucleus, rather thanDocket No. 11000-11031-PCT replicating the synthetic nucleus itself. This is particularly useful for applications where DNA amplification or replication is required.

[0066] This disclosure describes a microscale compartment comprising a shell and an internal space. The shell comprises an aliphatic conjugate between a protein component and a polymer component which is capable of forming spheroids. In embodiments, the protein component comprises a hydrophilic protein and the polymer component comprises a hydrophobic polymer. In embodiments, the hydrophobic polymer component may be a thermo-responsive polymer. In some embodiments, the thermo-responsive polymer comprises one or more of poly(N-isopropylacrylamide), Poly(N,N- dimethylacrylamide)> (PDMAAm), Poly(N-vinylcaprolactam) (PVCL), or Poly(N,N- diethylacrylamide) (PDEAAm).

[0067] In embodiments, the shell comprises poly(N-isopropylacrylamide) functionalized with N-hydroxy succinimide ester and linked to a polypeptide.

[0068] N-Hydroxy succinimide (NHS) esters are a family of conjugation reagents that selectively react with primary aliphatic amine groups. In the reaction, the carbonyl of the ester group is attacked by a primary aliphatic amine, resulting in a tetrahedral intermediate and the elimination of the NHS as a leaving group. The coupling reaction releases NHS (a weak acid) and forms an amide (a very stable covalent linkage).

[0069] Poly(N-isopropylacrylamide) is widely available, and due to the availability of this modified PNIPAAM containing an NHS group at its end, nano-conjugation of PNIPAAM-NHS with BSA to reconstruct BSA-PNIPAAM as a building block membrane of Synthetic Nucleus (SNs) is used. In some embodiments comprising poly(N-isopropylacrylamide) (PNIPAAm), the poly(N- isopropylacrylamide) (PNIPAAm) comprises one or more of Aminooxy End-Functionalized PNIPAAm, Thiol-Modified PNIPAAm, Vinyl-Modified PNIPAAm, Amine-Modified PNIPAAm, Azido- and Acetylene-Functionalized PNIPAAm, Carboxylated PNIPAAm, or Adamantane - Terminated PNIPAAm (Ad-PNIPAAm). In some embodiments, the poly(N-isopropylacrylamide) (PNIPAAm) is functionalized with one or more of N-hydroxy succinimide ester, adamantane, or mercaptothiazoline.

[0070] The polypeptide may fulfill many diverse options for improved functionality of the synthetic organelle or may be designed to assist in studying the effects of a synthetic organelle.

[0071] In some embodiments, the hydrophilic protein component comprises one or more of bovine serum albumin (BSA), glucose oxidase, [3-cyclodextrin, myoglobin, hemoglobin, poly (dopamine), ferritin, Human serum albumin (HSA), or protamine.

[0072] Further stabilization of the shell is achieved through the addition of a crosslinker to the shell. In some embodiments, the cross-linker O,O’-Bis-[2-(N-succunimyl-succinylamino)-ethyl]-Docket No. 11000-11031-PCT polyethylene glycol (BS(PEG)) is conjugated with the BSA-Poly(N-isopropylacrylamide) (PNIPAAM) to stabilize the SN structure. The shell includes pores, through which molecules pass from the internal space of the synthetic organelle to the cellular space. The size of the pores dictates the size of the molecules which can pass through the pores.

[0073] The pores in the shell of the synthetic organelle allow molecules to pass from the internal space of the synthetic organelle to the cellular space, and to allow molecules to pass from the cellular space into the internal space of the synthetic organelle. Changing the pore size changes the size of the molecules allowed into and out of the internal space of the synthetic organelle. In some embodiments, the pores are sized to allow nucleic acids smaller than between about 40 base pairs and about 80 base pairs to pass through the pores in the shell of the synthetic organelle. In some embodiments, the pores are sized to allow nucleic acids smaller than between about 750 base pairs and about 1,250 base pairs to pass through the pores in the shell of the synthetic organelle. In some embodiments, the pores are sized to allow RNA or proteins smaller than about 2,500 base pairs and about 3,500 base pairs to pass through the pores in the shell of the synthetic organelle.

[0074] The length of the cross-linker is one determinant of the size of the pores. In some embodiments, the crosslinker is O,O’-Bis-[2-(N-succunimyl-succinylamino)-ethyl]-polyethylene glycol (BS(PEG)). In some embodiments, the BS(PEG) is BS(PEG)9. In some embodiments, the BS(PEG) is BS(PEG)2000. In some embodiments, the BS(PEG) is BS(PEG)10000. The longer the BS(PEG) polypeptide, the larger the pores will be. BS(PEG)9 will have the smallest pore size, while BS(PEG)10000 will have the largest pore size. The incorporation of the shorter length crosslinker leads to smaller pore sizes, thus the BS(PEG)9 results in the smallest pore size. The incorporation of the BS(PEG)2000 results in a larger pore size. The incorporation of the BS(PEG)10000 results in the largest pore size.

[0075] Now referring to Figure 1, which is a schematic of a synthetic organelle, in this figure the synthetic organelle is a synthetic nucleus. The synthetic nucleus 101 is constructed as a shell 103 with pores in it such as pore 105. The shell encompasses an internal space and within the space are structures for initiating cellular processes. Such structures include a genetic template and transcription apparatus. The template contains a nucleic acid. In some embodiments, the nucleic acid is a plasmid such as plasmid 107. The plasmid may be any plasmid such as those depicted in Figures 3, 4, and 5. Figure 3 is a plasmid map of a plasmid designated S which has a size of -2,200 base pairs. Figure 4 is a plasmid map of a plasmid designated M which has a size of -3,000 base pairs. Figure 5 is a plasmid map of a plasmid designated E which has a size of -5,000 base pairs. In some embodiments, the genetic template is one of these plasmids. In some embodiments, the genetic template is a chromosome, a gene, a plasmid, an artificial chromosome, any double stranded DNA, or partially double stranded DNA. The transcription apparatus 109 transcribes the information of the genetic template into mRNA. In some embodiments, the transcription apparatus is a polymerase. The plasmid 107 is too large to pass throughDocket No. 11000-11031-PCT the pores in the shell and is therefore retained within the microscale compartment. The polymerase 109 is tethered to the shell and is thus retained within the microscale compartment. The mRNA 111 transcribed from the plasmid 107 is able to freely pass through the pores in the shell. In some embodiments, the size of the mRNA able to pass through the pores is ~60 nucleotides in length. In some embodiments, the size of the mRNA able to pass through the pores is -1,000 nucleotides in length. In some embodiments, the size of the mRNA able to pass through the pores is -3,000 nucleotides in length. In some embodiments, such as that depicted in Figure 1, the mRNA passes through the pores and into a cell where the cellular mechanisms, such as ribosomes 113 take over and produce proteins 115 from the mRNA which came out of the synthetic nucleus.

[0076] In some alternative embodiments, the synthetic nucleus includes the apparatus to enable translation of the mRNA into proteins. In these embodiments, such as that depicted in Figure 2, the ribosomes are located in the internal space created by the shell. The synthetic nucleus 201 is constructed as a shell 203 with pores in it such as pore 205. The shell encompasses an internal space and within the space are structures for initiating cellular processes. Such structures include a genetic template and transcription apparatus. The template contains a nucleic acid. In some embodiments, the nucleic acid is a plasmid such as plasmid 207. The plasmid may be any plasmid such as those depicted in Figures 3, 4, and 5. Figure 3 is a plasmid map of a plasmid designated S which has a size of -2,200 base pairs. Figure 4 is a plasmid map of a plasmid designated M which has a size of -3,000 base pairs. Figure 5 is a plasmid map of a plasmid designated L which has a size of -5,000 base pairs. In some embodiments, the genetic template is one of these plasmids. In some embodiments, the genetic template is a chromosome, a gene, a plasmid, an artificial chromosome, single-stranded DNA or any double stranded DNA. The transcription apparatus 209 transcribes the information of the genetic template into mRNA. In some embodiments, the transcription apparatus is a polymerase. The plasmid 207 is too large to pass through the pores in the shell and is therefore retained within the microscale compartment. The polymerase 209 is tethered within the microscale compartment and is thus retained within the microscale compartment. The mRNA transcribed from the plasmid 207 is then translated by the ribosome 213 in the internal space of the synthetic nucleus, into proteins 215. The proteins are then able to freely pass through the pores in the shell. The size of the proteins able to pass through the pores, such as pore 205 in the shell 203 are similar to the size of the mRNA which is able to pass through the pores in the shell. In some embodiments, the size of the protein able to pass through the pores is -60 nucleotides in length. In some embodiments, the size of the protein able to pass through the pores is -1,000 nucleotides in length. In some embodiments, the size of the protein able to pass through the pores is -3,000 nucleotides in length.

[0077] The microscale compartments are assembled via an oil in water emulsion. Once the shell is formed the oil is removed and the synthetic organelles are placed in a buffer solution to store until use. In some embodiments the addition of the crosslinker occurs during the emulsion process. TheDocket No. 11000-11031-PCT cross-linker is added to the oil phase, and then the supplemented oil phase is used to generate the emulsion. In another method, the oil phase is split in half. One half is supplemented with the crosslinker, while the other half, without the cross-linker, is used to generate the emulsion. Once the emulsion is formed, the second half, supplemented with the cross-linker, is added to the emulsion. This second method allows the emulsion to be generated using the extruder without initiating a cross-linking reaction between the proteins on the surface of the extruder membrane. Moreover, in some cases, the crosslinker can be added to the buffer solution where the SN building block proteins, the polymerase, and the DNA are dissolved.

[0078] It is often desirable to know the size of microscale compartments, and to be able to produce microscale compartments of specific sizes. Knowing the specific size of a microscale compartment enables designs to be created as to where the microscale compartment will be placed. Knowing the size of the microscale compartment can assist in determining the behavior of the microscale compartment. In some embodiments, the microscale compartments are between about 0.25 pm and about 10 pm. In some embodiments, the microscale compartments are between about 2.5 pm and about 7.5 pm. In some embodiments, the microscale compartments are about 5 pm. In some embodiments, the microscale compartments are about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 pm. A novel method for producing microscale compartments of specific sizes utilizes a mechanical system based on the extrusion of microscale compartments, synthetic nuclei (SNs) or proteinosomes through a membrane with a specific pore size exclusion. This extrusion method utilizes a commercially available tool produced by Avanti Polar Lipids (https: / / avantilipids.com / products / equipment / category / mini- extruder). Producing microscale compartments through this method has several benefits including producing micron-sized microscale compartments without using invasive methods that could affect the stability of any DNA encapsulated within the microscale compartment. Additionally, this method is scalable for high production volumes. In some embodiments, the size of the pores in the membrane through which the microscale compartments are extruded are between about 1 pm and about 10 pm. In some embodiments, the size of the pores in the membrane through which the microscale compartments are extruded are between about 2.5 pm and about 7.5 pm. In some embodiments, the size of the pores in the membrane through which the microscale compartments are extruded are about 5 pm.

[0079] Extruding the microscale compartment through the pores in the membrane multiple times increases the consistency of the size of the microscale compartment. The microscale compartments are malleable and a single pass through the pores in the membrane does not give a consistent size of the microscale compartment. By increasing the number of times and microscale compartment is passed through the pores in the membrane, the size of the microscale compartments the consistency of the size of the microscale compartment is increased. In some embodiments, the microscale compartment is passed through the membrane between about 1 time and about 1,000 times.Docket No. 11000-11031-PCTIn some embodiments, the microscale compartment is passed through the membrane between about 3 times and about 12 times.

[0080] The addition of the crosslinker adds stability to the shell of the organelle and assists in determining the size of the pores in the microscale compartment. This additional stability inhibits the malleability of the shell of the microscale compartment. In some embodiments, the crosslinker is added to the shell of the microscale compartment before the microscale compartment is passed through the pores of the membrane. In some embodiments, the crosslinker is added to the shell of the microscale compartment after the microscale compartment is passed through the pores in the membrane.

[0081] A first embodiment of a microscale compartment is a shell and an internal space. The shell comprises an aliphatic conjugate between a protein component and a polymer component which is capable of forming spheroids. In some of these embodiments, the protein is hydrophilic and the polymer is hydrophobic.

[0082] In a second embodiment the hydrophobic component of the microscale compartment of embodiment 1 comprises a thermo-responsive polymer.

[0083] In a third embodiment the thermo-responsive polymer of the microscale compartment of embodiment 2 comprises one or more of poly(N-isopropylacrylamide) (PNIPAAm), Poly(N,N- dimethylacrylamide) (PDMAAm), Poly(N-vinylcaprolactam) (PVCL), or Poly(N,N- diethylacrylamide) (PDEAAm).

[0084] In a fourth embodiment the thermo-responsive polymer of the microscale compartment of embodiment 3 comprises poly(N-isopropylacrylamide) (PNIPAAm).

[0085] In a fifth embodiment the poly(N-isopropylacrylamide) (PNIPAAm), of the microscale compartment of embodiment 4 the poly(N-isopropylacrylamide) comprises one or more of Aminooxy End-Functionalized PNIPAAm, Thiol-Modified PNIPAAm, Vinyl-Modified PNIPAAm, Amine- Modified PNIPAAm, Azido- and Acetylene-Functionalized PNIPAAm, Carboxylated PNIPAAm, or Adamantane-Terminated PNIPAAm (Ad-PNIPAAm).

[0086] In a sixth embodiment the poly(N-isopropylacrylamide) (PNIPAAm) of the microscale compartment of embodiment 4 is functionalized with one of N-hydroxy succinimide ester, adamantane, or mercaptothiazoline.

[0087] In a seventh embodiment the hydrophilic protein compartment of the microscale compartment of embodiment 1 the hydrophilic protein compartment comprises one or more of bovine serum albumin (BSA), glucose oxidase, [3-cyclodextrin, myoglobin, hemoglobin, poly (dopamine), ferritin, Human serum albumin (HSA), or protamine.

[0088] In an eighth embodiment, the shell of the microscale compartment of embodiment 1 comprises pores.Docket No. 11000-11031-PCT

[0089] In a ninth embodiment, the polypeptide linked to the shell of embodiment 2 is Bovine Serum Albumin.

[0090] In a tenth embodiment, the shell of embodiment 2, further comprises a crosslinker.

[0091] In an eleventh embodiment, the crosslinker of embodiment 4 is O,O’-Bis-[2-(N- succunimyl-succinylamino)-ethyl]-polyethylene glycol (BS(PEG)).

[0092] In a twelfth embodiment, the BS(PEG) of embodiment 5 is BS(PEG)9, BS(PEG)2000, or BS(PEG)10000.

[0093] In a thirteenth embodiment, the pores of the microscale compartment of any of embodiments 1-6 are sized to allow Nucleic acids smaller than about 60 base pairs to pass through the pores, and to prevent nucleic acids larger than about 60 base pairs from passing through the pores.

[0094] In a fourteenth embodiment, the pores of the microscale compartment of any of embodiments 1-6 are sized to allow nucleic acids smaller than about 1000 base pairs to pass through the pores, and to prevent nucleic acids larger than about 1000 base pairs from passing through the pores.

[0095] In a fifteenth embodiment, the pores of the microscale compartment of any of embodiments 1-6 are sized to allow RNA or proteins smaller than about 3000 base pairs to pass through the pores.

[0096] In a sixteenth embodiment, the internal space of the microscale compartment of embodiment 1 contains a nucleic acid.

[0097] In a seventeenth embodiment, the nucleic acid of embodiment 10 is a gene.

[0098] In an eighteenth embodiment, the nucleic acid of embodiment 10 is an artificial chromosome.

[0099] In a nineteenth embodiment, the nucleic acid of embodiment 10 is a plasmid.[000100] In a twentieth embodiment the plasmid of embodiment 13 is about 2.2 Kb.[000101] In a twenty-first embodiment the plasmid of embodiment 13 is about 3 Kb.[000102] In a twenty-second embodiment the plasmid of embodiment 13 is about 5 Kb.[000103] In a twenty-third embodiment, the internal space of the microscale compartment of any of embodiments 1-16 contains a polymerase.[000104] In a twenty-fourth embodiment, the polymerase of embodiment 17 is retained within the microscale compartment.[000105] In a twenty-fifth embodiment, the microscale compartment of any of embodiments 1- 18 is between about 0.25 pm and about 10 pm in diameter.Docket No. 11000-11031-PCT[000106] In a twenty-sixth embodiment, the microscale compartment of any of embodiments 1- 18 is between about 3 pm and about 7 pm.[000107] In a twenty-seventh embodiment, the microscale compartment of any of embodiments 1-18 is about 5 pm in diameter.[000108] A twenty-eighth embodiment is a method for preparing a microscale compartment. The method includes assembling a shell comprising an aliphatic conjugate between a hydrophilic protein component and a hydrophobic polymer component which is capable of forming spheroids. Then adding a crosslinker. Finally, passing the emulsion product through a membrane.[000109] In a twenty-ninth embodiment, the microscale compartment prepared by the method of embodiment 28 is between about 1 pm and about 10 pm in diameter.[000110] In a thirtieth embodiment, the microscale compartment prepared by the method of embodiment 28 is between about 3 pm and about 7 pm.[000111] In a thirty-first embodiment, the microscale compartment prepared by the method of embodiment 28 is about 5 pm in diameter.[000112] In a thirty-second embodiment, the pores in the membrane through which the microscale compartment in any of embodiments 28-31 are passed, are between about 1 pm and about 10 pm in diameter.[000113] In a thirty-third embodiment, the pores in the membrane through which the microscale compartment in any of embodiments 28-32 are passed, are about 5 pm in diameter.[000114] In a thirty-fourth embodiment, the crosslinker added to the shell of the microscale compartment prepared by the method of any of embodiments 28-33 is O,O’-Bis-[2-(N-succunimyl- succinylamino) -ethyl] -polyethylene glycol BS(PEG).[000115] In a thirty-fifth embodiment, the BS(PEG) added to the shell of the microscale compartment of embodiment 28 is BS(PEG)9, BS(PEG)2000, or BS(PEG)10000.[000116] In a thirty-sixth embodiment, the addition of the crosslinker added to the shell of the microscale compartment prepared by the method of any of embodiments 28-35, is added after the emulsion process.[000117] In a thirty-seventh embodiment, the emulsion product of the method to prepare the microscale compartment of embodiment 36 is passed through the membrane between about 2 times and about 10 times.[000118] In a thirty-eighth embodiment, the emulsion product of the method to prepare the microscale compartment of embodiment 37 is passed through the membrane between about 3 times and about 9 times.Docket No. 11000-11031-PCT[000119] In a thirty-ninth embodiment, the addition of the crosslinker added to the shell of the microscale compartment prepared by the method of any of embodiments 28-38, is added before the emulsion process.[000120] In a fortieth embodiment, the emulsion product of the method to prepare the microscale compartment of embodiment 39 is passed through the membrane between about 2 times and about 10 times.[000121] In a forty-first embodiment, the emulsion product of the method to prepare the microscale compartment of embodiment 40 is passed through the membrane between about 3 times and about 9 times.[000122] In a forty-second embodiment the shell produced by the method of any of embodiments 28-41 is assembled by one or more of an oil in water emulsion process, sonication, pipetting, microfluidics, extrusion, solution agitation, or bead beating.ExamplesStabilization and Functional Assessment of Ester-Modified PNIPAAM-Based synthetic nucleus for Gene Expression[000123] First, Poly(N-isopropylacrylamide) functionalized with N-hydroxysuccinimide ester (PNIPAAM-NHS), that previously never been used for a proteinosome assembly, was employed to fabricate synthetic nuclei (SNs) capable of encapsulating plasmids of three sizes, designated as S: 2,2Kb DNA, ~60bp RNA, M: 3 Kb DNA, 1Kb RNA, L: 5Kb DNA, ~3Kb RNA. S plasmid contained Broccoli aptamer, M - Broccoli aptamer + mCherry, L - Broccoli aptamer + TOL2, plasmid maps can be found at the end of the document.[000124] The SNs were initially assembled via a water-in-oil emulsion technique; following stabilization, the oil phase was removed to transfer the SNs into a buffer solution following standard protocol which consist of 8mg / ml BSA-PNIPAAM dissolved in lx hepes buffer pH8 together with the purified plasmid with a final concentration of ~300ng / ul and 250 / ul final concentration of T7 polymerase. To stabilize the proteinosome, a cross-linker, O,O'-Bis-[2-(N-succinimyl-succinylamino)- ethyl] -polyethylene glycol (BS(PEG)) with molecular weights 708.71 (BS(PEG)9) was used. Subsequently, these SNs were tested in cell-free gene expression systems including H.(RExpjvss and mammalian cell-free expression systems, accordingly to the manufacturer’s protocol. The reaction's progress was monitored over 3 hours using the fluorescence spectrophotometer Tecan spark at 37°C, assessing fluorescence markers indicative of Broccoli aptamer transcription (by addition of DFHBI fluorophore) and mCherry protein expression (Figure 1). Upon confirming active gene expression, the SN-containing solution was transferred to a microscope capillary chamber to localize and verify fluorescence signals.Docket No. 11000-11031-PCT[000125] Distinct fluorescence behaviors were observed and are illustrated in Figures 2, 3, and 4. Phase-contrast images of SNs encapsulating different plasmids were acquired under transmitted light (Figure 2) and used to determine the spatial coordinates of SNs within the image frame. In Figure 3, fluorescence emitted from the DFHBI-Broccoli aptamer complex is depicted, demonstrating successful transcription across all three types of SN, and revealing Broccoli aptamer diffusion characteristics dependent on molecular size. To analyze the source of fluorescence inside versus outside the SNs when molecules are uniformly distributed within the frame, spatial coordinates obtained under transmitted light are utilized to localize SNs in the fluorescence channel. This method proves to be efficient, particularly in the case of the small Broccoli aptamer (produced by plasmid S), due to its small nucleotide count (60bp), resulting in homogeneous fluorescence distribution throughout the solution (Figure 3i). Conversely, larger RNA molecules originating from the aptamer combined with mCherry (Ikb, produced by plasmid M) (Figure 3ii) or the Tol2 sequence (3kb, obtained from the plasmid L) (Figure 3iii) remained confined within the SNs, attributed to the small pore sizes formed by the short(9) BS(PEG) chains in the SN membrane. Furthermore, since RNA transcribed within the SNs can also serve as a template for translation reactions, the mCherry fluorescence signal produced from plasmid M has been analyzed, as shown in Figure 4. Using the images acquired in transmitted light in Figure 2, the localization of the SNs can be mapped and the fluorescence signal in each area of the image. Specifically, our interest is to observe the fluorescence outside the SNs (Figure 5) and inside them (Figure 6). Differences in fluorescence levels are observable when RNA cannot diffuse, due to its size, outside the SNs (for RNA produced by SNs containing the M plasmid or the L plasmid), resulting in a higher signal level inside compared to the outside.[000126] The structure of SNs in RNA diffusibility studies were observed and compared by following the fluorescence behavior. Specifically, the data was normalized to compare results from different batches. For this purpose, each experiment included both the sample being studied and a control sample without DNA.[000127] The normalization is based on the hypothesis that RNA, being transcribed inside the SNs, should be more concentrated there and thus exhibit higher fluorescence signals. The maximum expected fluorescence values inside the SNs were set to one. Consequently, the minimum values are assumed to be outside the SNs and are normalized towards zero. Zero represents the fluorescence values observed in the negative control without DNA.[000128] By normalizing the fluorescence signals this way, the values inside the SNs (set to one) were compared with those outside (ranging between zero and one, with zero representing the background signal from the no-DNA control). The normalized values inside the SNs were related to those outside. If the difference tends towards one, it indicates that most, if not all, of the fluorescence — and thus the RNA — is localized inside the SNs. Conversely, if the difference tends towards zero, itDocket No. 11000-11031-PCT suggests that there are no significant differences between the inside and outside, indicating that the RNA can diffuse freelv.[000129] As expected, Figure 7 shows that RNA transcribed by SNs containing plasmid S is short enough to pass through the pores in the SNs membrane, resulting in values close to zero. In contrast, for RNA produced inside SNs encapsulating plasmids M or L, the signals tend towards one, indicating that the fluorescence signals from the broccoli RNA-DFHBI complex are predominantly concentrated inside the SNs. This is due to the greater length of the RNA produced, which exceeds the pore size of the SNs.[000130] SNs have the ability to transcribe long RNA within them and facilitate the diffusion of the transcribed RNA into the cellular cytosol when the SNs are inside cells. This ability has been optimized by altering the SNs’ structure, as discussed in the following sections. Specifically, a direct but not straightforward connection between the cross-linker length and the pore size of the SNs membrane will be demonstrated.[000131] The ability of RNA of varying lengths to diffuse through optimized SNs membranes with respect to the previously described when BS(PEG)9 has been used. As shown in Figure 7, SNs formed using BS(PEG)9 and compartmentalizing plasmid S are capable of transcribing RNA that can fully diffuse outside the SNs. These SNs and their resulting data derived from the difference of fluorescence signal values between the inside and outside of the SNs will serve as a positive model representing a system that allows perfect RNA diffusion.[000132] Conversely, the SNs containing plasmids M or L and their results, as shown in Figure 6, will represent models of complete inability for RNA to diffuse out of the SNs. These latter models are particularly useful for comparing various structural optimizations in SNs aimed at improving the diffusion of even longer RNAs.[000133] Regarding the data about the mCherry protein, both the images (figure 4) and the total fluorescence distribution plots (figure 8) indicate that mCherry can diffuse freely in and out of the SNs. This observation is attributed to the shorter size of the mCherry protein polymer compared to its respective mRNA. The ease of diffusion highlights the influence of molecular size on the permeability of the SNs membrane and reinforcing the importance of optimizing SNs structure for different molecular targets.[000134] Conclusion of Stabilization and Functional Assessment of Ester-Modified PNIPAAM- Based SN for Gene Expression[000135] PNIPAAM modified with NHS, with a molecular weight of approximately 5 kDa, constructs stable SNs that efficiently sustain transcription reactions and subsequent protein expression using encapsulated T7 polymerase and DNA templates. Short cross-linkers limit the diffusion of largeDocket No. 11000-11031-PCTRNA molecules. Short RNA sequences, such as the 63 base pair core domain of the Broccoli aptamer, can be effectively transcribed within and diffuse out of the SNs. This capability opens new possibilities for RNA-based therapeutic applications, including anti-miRNA, miRNA, and shRNA strategies. Furthermore, the transcription of the Broccoli aptamer-mCherry RNA within these SNs results in the translation of mCherry protein, which then diffuses freely. This underscores the potential of these systems in medical applications, particularly in the delivery and functional expression of therapeutic RNA molecules.[000136] Stabilization and Functional Assessment of SNs Capable of Transcribing and Allowing Diffusion of lOOObp RNA.[000137] PNIPAAM-NHS was used to fabricate SNs encapsulating the M plasmid. The SNs were initially assembled via a water-in-oil emulsion technique; following stabilization, the oil phase was removed to transfer the SNs into a buffer solution. To stabilize the SNs, a cross-linker named (BS(PEG)n where n is the number of polyethylene glycol repetition. The molecular weight of (BS(PEG) varies depending on the specific product. For example, BS(PEG)9 has a molecular weight of 708.71, as specified by the manufacturer. Other variants such as BS(PEG)2000 or BS(PEG) 10000 have higher molecular weights, reflecting longer polyethylene glycol chains.[000138] Various conditions were used to assemble the SNs. The results of different concentrations of BS(PEG)2000 or BS(PEG) 10000, or BSA-PNIPAAM, are presented below:[000139][000140] Subsequently, these SNs were analyzed in a PurExpress cell-free gene expression system. The reaction's progress was monitored over 5 hours at 37°C, assessing fluorescence markers indicative of Broccoli aptamer transcription and mCherry protein expression (Figure 9). Upon confirming active gene expression, the SN-containing solution was transferred to a microscope capillary chamber to localize and verify fluorescence signals.[000141] Distinct fluorescence behaviors were observed and are illustrated in Figures 10, 11, and 12. Figure 10 presents phase-contrast images of SNs encapsulating different plasmids. In Figure 11, fluorescence derived from the DFHBI-Broccoli aptamer-mCherry RNA complex is depicted,Docket No. 11000-11031-PCT demonstrating successful transcription across all assembled SNs. It is possible to observe how the BS(PEG) length affects the size of the pores on the SN membrane, allowing for the diffusion of broccoli-mCherry RNA, leading to a homogenous fluorescence distribution throughout the solution (Figure 12i). Moreover, to verify that the RNA transcribed within the SNs can also work as a template for the translation reaction of its protein, mCherry fluorescence signal produced from plasmid M has been analyzed as shown in Figure 12.[000142] As in the previous analysis, the fluorescence values were analyzed inside (Figure 13) and outside (Figure 14) the SNs, derived from the signal produced by the interaction between the broccoli aptamer and DHFBI. Using the same method described for Figure 6, RNA diffusion in SNs compartmentalizing plasmids M, was analyzed as shown in Figure 15. A high ratio of fluorescence inside versus outside the SNs suggests RNA entrapment, whereas values near zero indicate an even distribution, suggesting free diffusion. Negative values would imply RNA dilution outside the SNs, indicating a tendency for RNA to localize more externally than within the SNs. In this case, the normalized fluorescence signal representing the RNA diffusion behavior from the same plasmid M but compartmentalized in SNs produced with BS(PEG)9 to provide a comparison with SNs that do not allow the diffusion of longer RNA was included.[000143] Additionally, both the images (Figure 12) and the total fluorescence distribution plots (Figure 16) indicate that mCherry can diffuse freely in and out of the SNs. This observation is attributed to the shorter size of the mCherry protein polymer compared to its respective mRNA. The ease of diffusion highlights the influence of molecular size on the permeability of the SNs membrane, indicating the importance of optimizing SNs structure for different molecular targets.[000144] Commercially available PNIPAAM-NHS with an approximately 5 kDa, effectively constructs stable SNs utilizing both the already tested BS(PEG)2000 and the longer BS(PEG) 10000, previously unexplored in this capacity, as cross-linkers to stabilize the SNs structure. These findings demonstrate not only the stability of these SNs but also their compatibility in hosting active transcription reactions. Encapsulating the M plasmid under varying concentrations of nano-conjugate protein (BSA- PNIPAAM) and BS(PEG) cross-linkers enables transcription and subsequent protein expression.[000145] A significant breakthrough of this research is the proven capability of longer RNA sequences, around 1000 base pairs, to be not only transcribed but also to diffuse freely outside the SNs. This was monitored through the fluorescence of the Broccoli RNA aptamer linked to the mCherry RNA sequence, which diffused effectively outside the SN membranes with longer cross-linkers.[000146] It's essential to note the differences in our experimental approaches and results compared to similar previous studies. For instance, the analysis used BS(PEG)2000 in combination with a 5 kDa NHS-modified PNIPAAm to explore the diffusion capabilities of RNA from synthetic nucleus. This specific setup allowed us to observe RNA diffusion outside the SNs.Docket No. 11000-11031-PCT[000147] In contrast, a related study, such as the one by M. Gao et al., employed BS(PEG)2000 with a mercaptothiazoline-activated PNIPAAm with a molecular weight around 14 kDa. Their results indicated that the RNA, while successfully transcribed, remained entrapped within the proteinosome, or SNs structures and did not diffuse.[000148] The combination of shorter PNIPAAM with longer cross-linkers such as BS(PEG) is the key to achieving both successful transcription and effective diffusion of longer RNA molecules. This strategy highlights the importance of optimizing polymer and cross-linker dimensions to enhance the functional capabilities of synthetic nuclei in gene therapy applications.[000149] Furthermore, the diffusion of the mCherry protein was confirmed, as it was distributed throughout the solution, underscoring the functional permeability of the SN membrane facilitated by the use of shorter PNIPAAM modified with NHS. This adaptability suggests a broad potential for therapeutic applications, particularly in gene therapy, where controlled diffusion and robust expression of therapeutic molecules are crucial.[000150] Stabilization and Functional Assessment of the SN Capable of Transcribing and Allowing Diffusion of 3000bp RNA.[000151] Similarly, for the M plasmid, PNIPAAM-NHS was also used to create SNs encapsulating the L plasmid. As with previous assemblies, the SNs were formed via a water-in-oil emulsion technique and transferred into a buffer solution after stabilization. Various conditions were tested to optimize the assembly process, focusing on different concentrations and combinations of BS(PEG)2000 or BS(PEG)10000 with BSA-PNIPAAM.[000152][000153] Also in this case, these SNs were tested in a cell-free gene expression system, monitoring Broccoli aptamer transcription over 5 hours at 37°C, as shown in Figure 17. Following confirmation of gene expression, the SNs were analyzed under a microscope in a capillary chamber to verify fluorescence signals.[000154] Distinct fluorescence behaviors were observed and are illustrated in Figures 18 and 19. Figure 18 presents phase-contrast images of SNs encapsulating different plasmids. In Figure 19,Docket No. 11000-11031-PCT fluorescence derived from the DFHBI-Broccoli aptamer complex is depicted, demonstrating successful transcription across all assembled SNs and the followed RNA diffusion outside the SNs membrane. As in the previous analysis, we examined the fluorescence values inside (Figure 20) and outside (Figure 21) the SNs, derived from the signal produced by the interaction between the broccoli aptamer and DHFBI. Using the same method described for Figure 6, RNA diffusion in SNs compartmentalizing plasmids L, was analyzed as shown in Figure 19. A high ratio of fluorescence inside versus outside the SNs suggests RNA entrapment, whereas values near zero indicate an even distribution, suggesting free diffusion. Negative values would imply RNA dilution outside the SNs, indicating a tendency for RNA to localize more externally than within the SNs.[000155] Also in this case, we included the normalized fluorescence signal representing the RNA diffusion behavior from the same plasmid L but compartmentalized in SNs produced with BS(PEG)9 to provide a comparison with SNs that do not allow the diffusion of longer RNA.[000156] Additionally, both the images (Figure 12) and the total fluorescence distribution plots (Figure 16) indicate that mCherry can diffuse freely in and out of the SNs. This observation is attributed to the shorter size of the mCherry protein polymer compared to its respective mRNA. The ease of diffusion highlights the influence of molecular size on the permeability of the SNs membrane, supporting our findings on RNA diffusion and reinforcing the importance of optimizing SNs structure for different molecular targets.[000157] The capability of PNIPAAM-NHS modified SNs to not only stabilize and encapsulate the L plasmid but also to support the transcription and diffusion of RNA sequences exceeding 3000 base pairs.[000158] Utilizing various concentrations of BS(PEG)2000 and BS(PEG)10000, the study successfully identified optimal conditions that promote the effective transcription of long RNA sequences and their subsequent diffusion through the SN membrane.[000159] The observed diffusion of such long RNA strands, as evidenced by significant fluorescence compared to the SNs assembled with the BS(PEG)9, highlights the potential of these SNs as a promising platform for nucleic acid therapies. This ability to manage and facilitate the transcription and diffusion of long RNA molecules sets a foundational precedent for using SN in therapeutic applications where longer RNA sequences are integral.[000160] Analysis of Fluorescence Signal Distribution and DNA Localization in Synthetic MicroCompartments Using BS(PEG)10000[000161] Notable differences in fluorescence signaling for both M and L plasmids were observed, particularly under conditions utilizing BS(PEG)10000. The presence of a diminished fluorescence signal from both broccoli aptamer-DFHBI and mCherry was noted within the SNsDocket No. 11000-11031-PCT compared to the background. These findings were further supported by histogram analyses, which demonstrated a negative delta fluorescence. This suggests a higher concentration of fluorescence molecules outside the SNs compared to the inside.[000162] The underlying cause of this phenomenon remains unclear. However, one possible explanation could be the presence of DNA outside the SNs, combined with a lack of reaction within the SNs. To further investigate, SNs prepared with BS(PEG)10000 were utilized in a series with the following legend:[000163][000164] These SNs have been used to complete cell-free gene expression reactions. The signals from broccoli aptamer and mCherry were monitored using a spectrophotometer, as shown in Figure 23.[000165] At the conclusion of these reactions, 30 pM DAPI was introduced to the solution to label the DNA and determine its location — whether still compartmentalized within the SNs or dispersed in the solution. The experiments were conducted in a microscope capillary, facilitating direct observation under a microscope. First, SNs in the solution were localized and mapped using transmitted light (Figure 24). Fluorescence from inside and outside the SNs was then measured, as shown for the broccoli-aptamer in Figures 28 and 30, and DAPI in Figures 29 and 31. This data enabled the calculation of delta fluorescence for the aptamer (Figure 32)and DAPI signals (Figure 33), providing insights into the distribution and localization of these molecules relative to the SNs.[000166] These observations reaffirmed the higher concentration of the broccoli aptamer signal outside the SNs, but interestingly, DAPI staining indicated the presence of DNA within the SNs.[000167] As previously noted in results such as those depicted in Figure 10, under specific conditions — particularly when BS(PEG) 10000 at a concentration of 0.55mM is used — the fluorescence of the Broccoli aptamer-DFHBI complex shows the location of the SNs, suggesting a reduced concentration of RNA within the SNs.[000168] To ensure that this phenomenon was not due to DNA diffusion out of the SNs, which could potentially lead to transcription occurring externally, DAPI staining, a DNA fluorescent intercalator, was utilized to confirm the presence of DNA within the SNs post-cell-free gene expression reactions. This crucial step demonstrated that the DNA remains within the SNs, affirming that the center of the transcription activity is indeed still located within the confines of the SNs. This finding cruciallyDocket No. 11000-11031-PCT supports the integrity and functionality of the SNs in maintaining the essential components for transcription within their structure, reinforcing their potential in gene expression studies and therapeutic applications.[000169] Innovative Extrusion Method for Size Control of SNs in Emulsion Systems[000170] To control the size of the SNs, a novel method based on the extrusion of the previously generated oil-in-water emulsion of SNs through a commercially available extruder was used. The commercially available extruder was an AV ANTIPOLAR that forces the emulsion to pass through a membrane with pores of a defined diameter. The pore size used was 5 pm.[000171] Different numbers of passes of the emulsion through the membrane were performed specifically 1, 3, 5, 6, 9, or 12 passes. Additionally, the addition of the cross-linker during the emulsion formation or after passing through the extruder was tested to determine whether the timing of the addition of the cross-linker would have different effects. In the latter case, the water-in-oil emulsion was generated with half of the oil volume. Once the emulsion was extruded, it was resuspended by adding the second half of the oil in which the cross-linker had been dissolved. In all cases, the crosslinker used was BS(PEG)9, while the SNs contained ssDNA labeled with DY530 to observe its presence inside. The various types of emulsions generated have been classified according to the following legend:[000172][000173] After the production process, the SNs were further processed by removing the oil and replacing it with DMEM media, supplemented with 10% FBS and lx penicillin-streptomycin. The prepared samples were then loaded onto a capillary glass and examined under a fluorescence microscope. This examination focused on tracking the DY530 fluorescence dye, as illustrated in Figure 34.[000174] Subsequently, the captured images were analyzed to determine the size distribution of the SNs. The size data were plotted in a histogram and subjected to Gaussian fitting to calculate the mean size and standard deviation of the SNs (fig 35), confirming that size was reduced to 6 pm+-2um.[000175] A novel method for the production of SNs was developed that provides precise control over the size of the SNs. This method was developed by adapting a technique typically used for lipid extrusion. By employing a commercial extruder, which forces an oil-in-water emulsion of SNs to passDocket No. 11000-11031-PCT through a membrane with defined pore sizes (5 pm in this case), the size of the SNs were effectively standardized.[000176] The number of extrusion passes — specifically 1, 3, 5, 6, 9, or 12 — allowed for further refinement of size distribution from 10.5 pm to 2.5 pm, demonstrating that the frequency of extrusion directly influences the uniformity and precision of the final SN sizes.[000177] Additionally, the timing of cross-linker addition, either before or after extrusion, was explored to assess its impact on the final structure of the SNs. The resulting data, highlighted by the DY530 fluorescence dye tracking within the SNs and analyzed through Gaussian fitting of size distributions, confirmed the effectiveness of this approach. This method not only ensures a consistent and controlled size of SNs but also represents a scalable technique suitable for large-scale production.[000178] By leveraging membrane extrusion — a process traditionally utilized for lipids — a robust framework for the systematic production of DNA-containing SNs was adapted and validated. This technology is thus positioned as a formidable tool for therapeutic and research applications where precise size control is paramount.[000179] Enzyme Encapsulation and Retention in SNs[000180] SNs are capable of hosting an active transcription reaction, demonstrating the presence of DNA within the SNs themselves. SNs are also capable of retaining enzymes within their compartments. FITC was used to label a 62 kDa enzyme.[000181] After labeling, the enzyme was encapsulated within the SNs during their assembly. The enzymes are localized and entrapped inside the SNs only when introduced during the assembly process. As shown in Figure 36, the labeled enzymes were successfully confined within the SNs after the SNs were transferred into DMEM complete media buffer.[000182] Conversely, when empty proteinosomes or SNs were created and then diluted in DMEM complete media buffer containing the FITC-labeled 62 kDa enzyme, the enzymes diffused throughout the solution, including into the SNs. This demonstrated that when assembled in a specific way, SNs can effectively keep the enzymes stably confined within their membrane. Otherwise, a distribution of fluorescence similar to that in the control with empty SNs would have been observed.[000183] Characterization and Treatment of SNs[000184] SNs were characterized, and some of those SNs were used these to treat cells to monitor two key aspects: 1) the capability of cellular uptake, and 2) the cytotoxicity of the SNs on the treated HEK 293T cells.[000185] Production of SNs[000186] The SNs were produced in three different batches:Docket No. 11000-11031-PCT[000187] Batch 1: Containing plasmids S and the T7 polymerase.[000188] Batch 2: Containing plasmids S, the T7 polymerase, and FITC-labelled luciferase.[000189] Batch 3: Empty SNs (known as proteinosomes).[000190] The incorporation of FITC-labelled luciferase in Batch 2 was intended to facilitate the monitoring and verification of the presence of SNs using fluorescence microscopy.[000191] Preparation and V erification[000192] Three batches of SNs were prepared using the novel extrusion method. After removing the oil, the SNs are produced as water-in-water compartments. To verify the presence of SNs, the water- in-water compartments were aliquoted into DMEM media buffer, complete with 10% FBS (fetal bovine serum), lx Penicillin-Streptomycin (Pen Strep) resistance, and lx pyruvic acid. This media was chosen as it is the cell culture media that will be used later for treating the HEK 293T cells with SNs.[000193] As shown in Figure 38, all proteinosome batches were prepared. Fluorescence microscopy confirmed the presence of SNs only in the batch prepared with FITC-labelled luciferase, demonstrating the effectiveness of FITC incorporation for visualization purposes.[000194] Treatment and Evaluation of SNs in HEK 293T Cells[000195] Once the SNs were assembled and their presence verified under the microscope, four batches of each type of SNs (approximately 200 pl in total for each type) were diluted in 5 ml of the DMEM media previously described and used to treat the HEK 293T cells. The treatment and cell washing processes are described in the treatment protocol.[000196] During the washing steps, the cells treated with the empty SNs were lost. Each sample was prepared in triplicate to ensure reproducibility.[000197] After washing away, the SNs that were not taken up by the cells and adding fresh media, the cells were observed under the microscope to evaluate the presence of SNs. Although only the SNs containing the FITC-labelled luciferase were observable, Figure 38 shows images of the cells treated with each type of SNs. These same cells were then analyzed to assess cell viability and compared to control cells, which underwent the same operational steps (e.g., media removal, PBS washes, etc.) but were not treated with SNs.[000198] Cell Viability Assessment Post-Treatment[000199] After treatment, the cells were detached, and cell viability was measured for each of the three samples for each type of SNs used for treatment. As observed, there are no significant differences between the treated and untreated samples. Figure 40 shows the plotted values in the histogram.[000200] Evaluation of Transcriptional Activity of SNs in HEK293T CellsDocket No. 11000-11031-PCT[000201] After testing the uptake capacity of HEK293T cells for the SNs and confirming the non-toxicity of SNs to the cells, the transcriptional activity of the SNs when incorporated into the cytoplasm of HEK293T cells was assessed. Specifically, after 6 hours of treatment with the SNs, the cells were first washed with PBS, and then new culture medium was added. This time, however, the culture medium also contained the molecule 3,5-difluoro-4-hydroxybenzylidene imidazolone (DFHBI). DFHBI is a molecule that, when it binds to the broccoli RNA aptamer, emits fluorescence with an absorption wavelength of 482 nm and an emission wavelength of 507 nm. This DFHBI is used to evaluate the transcriptional activity of the SNs for the broccoli aptamer.[000202] Specifically, the SNs assembled contain both T7 polymerase and plasmid S, which transcribes, under the T7 promoter, the RNA "broccoli aptamer". Thus, if the SNs are active inside the cell and can transcribe RNA using their own T7 polymerase, and nucleotides and Mg diffuse passively from the cytosol into the SNs through the pores present on the SNs, an increase in fluorescence within the cells compared to the untreated control was observed. The success of SNs' activity within the HEK293T cells is shown in Figure 41, where fluorescence signals can be observed in some cells treated with SNs and DFHBI compared to untreated cells. Fluorescence signals were then recorded, and the mean and standard deviation were calculated. These values were plotted as shown in Figure 42, where the difference between SN-treated samples and untreated samples can be observed.[000203] The study demonstrated that our size-reduced SNs, ranging between 1 and 5 pm and produced by extrusion, have the capability to be taken up by HEK 293T cells. The cells were treated with SNs containing plasmid S and T7 polymerase, SNs with plasmid S, T7 polymerase, and FITC- labelled luciferase, as well as empty SNs (proteinosomes). Post-treatment observations confirmed that the SNs were successfully internalized by the cells. Importantly, cell viability assays indicated no significant differences in viability between treated and untreated cells, suggesting that the presence of SNs did not adversely affect cell vitality. This conclusion underscores the biocompatibility and potential utility of our SNs for cellular applications.[000204] Additionally, further experiments showed that SNs could effectively transcribe the broccoli RNA aptamer within the cytoplasm of HEK293T cells. Fluorescence microscopy confirmed the presence of active transcription, indicated by increased fluorescence in cells treated with SNs and DFHBI compared to untreated controls. This highlights the functionality of SNs in facilitating intracellular transcription, further supporting their potential for advanced cellular applications.[000205] SNs modified with PNIPAAM-NHS have innovative and functional capabilities. SNs have substantial potential across several dimensions crucial for advancing nucleic acid therapies.[000206] Stabilization and Functional Assessment: PNIPAAM-NHS constructs stable SNs that efficiently facilitate transcription reactions and subsequent protein expression. This includes the unique capability of these SNs to support the transcription and diffusion of short RNA sequences such as theDocket No. 11000-11031-PCT63 base pair core domain of the Broccoli aptamer. These attributes are critical for developing RNA- based therapeutic applications like anti-miRNA, miRNA, and shRNA strategies.[000207] Control of RNA Diffusion: SNs stabilize and effectively release longer RNA sequences, around 1,000 base pairs and up to about 12,000 base pairs. These findings underline the SNs' utility in therapies requiring the delivery and sustained expression of substantial RNA molecules, offering a robust platform for the controlled diffusion and expression of therapeutic agents.[000208] Optimization of Polymer and Cross-Linker Dimensions: Importantly, the precise optimization of polymer length (PNIPAAM) and cross-linker length (BS(PEG)) is essential for enhancing the transcription and diffusion capabilities of SNs. This optimization allows for a broader range of RNA molecules to be effectively processed within these systems, promising greater flexibility, and efficacy in nucleic acid therapies.[000209] Transcription Integrity and DNA Localization: By employing DAPI staining, it was confirmed that DNA remains within the SNs during transcription, ensuring that the transcription center is maintained within the SNs. This is pivotal for the integrity of gene expression processes, particularly in scenarios where external transcription could compromise therapeutic efficacy.[000210] Scalable Production through Novel Methods: A novel production method using membrane extrusion, typically associated with lipid particles, has been adapted for SNs. This method allows precise control over the size of SNs and is scalable, which is essential for clinical and commercial applications. The controlled size distribution enhances the reproducibility and reliability of SNs in therapeutic contexts.[000211] In addition to RNA, the capability of SNs to host an active transcription reaction was verified, demonstrating the presence of DNA within the SNs themselves. An important next step was to verify the capability of SNs to retain enzymes within their compartments. To address this, FITC was used to label a 62 kDa enzyme. After labeling, the enzyme was encapsulated within the SNs during their assembly. This experiment aimed to show that the enzymes could be localized and entrapped inside the SNs only when introduced during the assembly process. As shown in Figure 36, the labeled enzymes were successfully confined within the SNs after the SNs were transferred into DMEM complete media buffer. Conversely, when empty proteinosomes were prepared and then diluted in DMEM complete media buffer containing the FITC-labeled 62 kDa enzyme, the enzymes diffused throughout the solution, including into the SNs. This demonstrated that when assembled in a specific way, SNs can effectively keep the enzymes stably confined within their membrane. Otherwise, a distribution of fluorescence similar to that in the control with empty SNs would have been observed.[000212] Expanding RNA Size Capabilities: By finely tuning the combination of PNIPAAM length and BS(PEG) cross-linker length, the range of RNA sizes that can be efficiently transcribed and diffused from SNs can be accommodated and potentially extend. This finding suggests the excitingDocket No. 11000-11031-PCT possibility of adapting the SN platform to facilitate the expression and release of even larger RNA molecules than currently demonstrated. Expanding the capability to handle an increased range of RNA sizes could further broaden the applicability of SNs in a wider array of therapeutic contexts, particularly those requiring the delivery of very large RNA or DNA sequences that are beyond the reach of existing delivery systems.[000213] Uptake and Cytotoxicity Assessment: Once the SNs were assembled and their presence verified under the microscope, four batches of each type of SNs (approximately 200 pl in total for each type) were diluted in 5 ml of the DMEM media previously described and used to treat the HEK 293T cells. The treatment and cell washing processes are described in the transfection protocol. During the washing steps, the cells treated with the empty SNs were lost, likely due to a too low confluence in this triplicate. Each sample was prepared in triplicate to ensure reproducibility.[000214] After washing away, the SNs that were not taken up by the cells and adding fresh media, the cells were observed under the microscope to evaluate the presence of SNs. Although only the SNs containing the FITC-labelled luciferase were observable, Figure 36 shows images of the cells treated with each type of SNs. These same cells were then analyzed to assess cell viability and compared to control cells, which underwent the same operational steps (e.g., media removal, PBS washes, etc.) but were not treated with SNs. The size -reduced SNs, ranging between 1 and 5 pm, demonstrated their capability to be taken up by the cells. Importantly, cell viability assays indicated no significant differences between treated and untreated cells, suggesting that the presence of SNs did not adversely affect cell vitality. Additionally, experiments demonstrated the transcriptional activity of SNs within the cytoplasm of HEK293T cells. Fluorescence microscopy confirmed active transcription through increased fluorescence in cells treated with SNs and DFHBI, compared to untreated controls. This further supports the functionality of SNs in intracellular transcription, enhancing their potential for advanced cellular applications.[000215] In another experiment, SNs were produced containing the M plasmid and then tested either in the presence or absence of ribosomes. As shown in Figures 48A and 48B, when ribosomes are present in solution, the kinetics of mCherry expression can be observed. In contrast, when ribosomes are absent, this is not detectable. Furthermore, transcription is always observed, regardless of whether ribosomes are present. Figures 48 A and 48B. PURE Express reaction with or without Ribosomes in solution. Figure 48A shows the transcription kinetics of the mRNA containing the aptamer Broccoli, which can be monitored through the fluorescence emitted by the Broccoli aptamer / DFHBI-Molecule complex. Figure 48B displays the expression kinetics of mCHerry, which can be monitored through the fluorescence of the protein itself.[000216] SNs after the PURE Express reaction with ot without Ribosomes in solution Figure 49A shows SNs after the reacion with PURE express completed in solution with ribosome. In grey theDocket No. 11000-11031-PCTSNs in brightfield, in green the broccoli aptamer-DFHBI complex fluorescence within the SNs and in red the mCHerry protein expressed and diffused in solution. Figure IB shows SNs after the reacion with PURE express without ribosome. In grey the SNs in brightfield, in green the broccoli aptamer- DFHBI complex fluorescence within the SNs and in red channel is missing due to the absence of translation reaction.[000217] The synthesis of these results underscores the revolutionary potential of SNs as a powerful tool for nucleic acid therapies. The ability of SNs to encapsulate and control the diffusion of both short and long RNA sequences opens up significant opportunities in gene therapy, particularly in treatments where the expression of long proteins is crucial, a challenge that current technologies fail to meet effectively. Moreover, the stability and controlled release capabilities of SNs make them highly suitable for applications requiring high concentrations of RNA sustained over time, such as in treatments involving miRNA or anti-miRNA therapies.[000218] The development and refinement of SNs represents a significant advancement in the field of gene therapy. The unique properties of these particles provide a versatile, efficient, and cost- effective solution for the challenges currently faced in delivering and expressing therapeutic nucleic acids, particularly those of substantial length and concentration. These findings pave the way for SNs to become a cornerstone technology in advancing the efficacy and accessibility of nucleic acid-based treatments, setting a new standard for therapeutic interventions in the molecular medicine landscape.[000219] Protocol for BSA conjugation with Poly(N-isopropylacrylamide)-NHS (PNIPAAM- NHS)[000220] Objective: To conjugate commercially available Poly(N-isopropylacrylamide)-NHS (PNIPAAM-NHS) with native Bovine Serum Albumin (BSA), forming BSA-PNIPAAM as a building block membrane of Synthetic Nuclei (SNs).[000221] Materials:• Native Bovine Serum Albumin (BSA)• Poly(N-isopropylacrylamide)-NHS (PNIPAAM-NHS, 5 KDa)• lx Phosphate Buffered Saline (PBS) buffer (pH 8)• lx Phosphate Buffered Saline (PBS) buffer (pH 7.4)• 50 KDa cut-off Amicon ultra-centrifugation tubes• Centrifuge capable of 4000 xg• Cold storage (4°C)[000222] Procedure:1. React Bovine Serum Albumin (BSA) with Poly(N-isopropylacrylamide)-NHS (PNIPAAM- NHS): o Weigh 100 mg of native Bovine Serum Albumin (BSA).Docket No. 11000-11031-PCT o Calculate and weigh the amount of Poly(N-isopropylacrylamide)-NHS (PNIPAAM- NHS) required to maintain a molar ratio of 1:3 (BSA).2. Dissolve Poly(N-isopropylacrylamide)-NHS: o Dissolve the Poly(N-isopropylacrylamide)-NHS (PNIPAAM-NHS) in lx Phosphate Buffered Saline (PBS) buffer (pH 8) at 4°C until fully dissolved.3. Conjugation Reaction: o Slowly add native Bovine Serum Albumin (BSA) dropwise to the Poly(N- isopropylacrylamide)-NHS (PNIPAAM-NHS) solution. o Allow the reaction mixture to incubate and react for 12 hours at 4°C.4. Concentration of Reaction Mixture: o Transfer the reaction mixture to a 50 KDa cut-off Amicon ultra-centrifugation tube, o Centrifuge at 4000 xg at 4°C for 15 minutes.5. Resuspension and Washing: o Resuspend the concentrated BSA-PNIPAAM in lx Phosphate Buffered Saline (PBS) buffer (pH 7.4). o Centrifuge again at 4000 xg at 4°C for 15 minutes. o Repeat the resuspension and centrifugation step 7 times to thoroughly wash the conjugate.6. Final Concentration: o For the final centrifugation step, increase the centrifugation time to 30 minutes. o Aim to achieve a high concentration of BSA-PNIPAAM, approximately 200 mg / ml in a final volume of 0.5 ml.[000223] Notes:• N-Hydroxy succinimide (NHS) esters are a family of conjugation reagents that selectively react with primary aliphatic amine groups. In the reaction, the carbonyl of the ester group is attacked by a primary aliphatic amine, resulting in a tetrahedral intermediate and the elimination of the NHS as a leaving group. The coupling reaction releases NHS (a weak acid) and forms an amide (a very stable covalent linkage). This reaction strategy is already used in Synthetic Nucleus (SNs) to conjugate the cross-linker BS(PEG)n with BSA-Poly(N- isopropylacrylamide) (PNIPAAM) to stabilize the SN structure.• Poly(N-isopropylacrylamide) used in the literature for producing proteinosomes typically involves mercaptothiazoline-terminated PNIPAAM. However, due to the availability of modified PNIPAAM containing an NHS group at its end, this protocol utilizes the described chemical mechanism to nano-conjugate PNIPAAM-NHS with BSA to reconstruct BSA- PNIPAAM as a building block membrane of Synthetic Nucleus (SNs). Some examples: o Lopez-Cuevas P, Xu C, Severn CE, Oates TCL, Cross SJ, Toye AM, Mann S, Martin P. Macrophage Reprogramming with Anti-miR223 -Loaded Artificial Protocells Enhances In Vivo Cancer Therapeutic Potential. Adv Sci (Weinh). 2022 Dec;9(35): e2202717. doi: 10.1002 / advs.202202717. Epub 2022 Oct 31. PMID: 36314048; PMCID: PMC9762313. o Gao M, Wang D, Wilsch-Brauninger M, Leng W, Schulte J, Morgner N, Appelhans D, Tang TD. Cell Free Expression in Proteinosomes Prepared from Native Protein- PNIPAAm Conjugates. Macromol Biosci. 2024 Mar;24(3): e2300464. doi: 10.1002 / mabi.202300464. Epub 2023 Nov 23. PMID: 37925629. o Yuting Zhou, Jianmin Song, Lei Wang, Xuting Xue, Xiaoman Liu, Hui Xie, and Xin Huang. In Situ Gelation-Induced Death of Cancer Cells Based on Proteinosomes.Biomacromolecules 2017 18 (8), 2446-2453. DOI: 10.1021 / acs . biomac ,7b00598 o Huang, X., Li, M., Green, D. et al. Interfacial assembly of protein-polymer nanoconjugates into stimulus-responsive biomimetic protocells. Nat Commun 4, 2239 (2013). https: / / doi.org / 10.1038 / ncomms3239Docket No. 11000-11031-PCT o Zambrano, A., Fracasso, G., Gao, M. et al. Programmable synthetic cell networks regulated by tuneable reaction rates. Nat Commun 13, 3885 (2022). https: / / doi.org / 10.1038 / s41467-022-31471-5• Ensure all steps involving Poly(N-isopropylacrylamide)-NHS (PNIPAAM-NHS) and Bovine Serum Albumin (BSA) are performed at 4°C to maintain stability.• Maintain the pH of the Phosphate Buffered Saline (PBS) buffer accurately to ensure optimal reaction conditions.[000224] Protocol for Measuring RNA Expression Activity Using Cell-Free Expression System with SNs Compartmentalizing Plasmids S, M, and L.[000225] The aim of this protocol is to measure the activity of RNA expression using a cell-free expression system (PURE Express System) with synthetic nuclei (SNs) compartmentalizing plasmids S, M, and L. This method allows for the visualization of RNA transcription and protein expression in a controlled, cell-free environment.[000226] Materials:• PURE Express System (New England Biolabs, NEB)• SNs compartmentalizing plasmid S (for aptamer transcription)• SNs compartmentalizing plasmid M (for mCherry expression)• SNs compartmentalizing plasmid L (for mCherry expression)• 3,5-Difluoro-4-hydroxybenzylidene imidazolinone (DFHBI)• mq Water[000227] Protocol for Measuring RNA Expression Activity Using Cell-Free Expression System with SNs Compartmentalizing Plasmids S, M, and L[000228] Procedure:1. Preparation of Reaction Mixture: o Follow the standard PURE Express System protocol provided by NEB for setting up the reaction mixture.2. Substitution of Plasmid with SNs: o Instead of adding plasmid DNA directly to the reaction mixture, add the SNs compartmentalizing plasmid S, M or L (plasmids are used as positive controls around 2ul of DNA solution concentrate at 3.5ug / ul) o The final volume of SNs is normally used at 5.75ul3. Reaction Setup: o Mix the following components in a microcentrifuge tube:■ PURE Express Solution A■ PURE Express Solution B■ SNs containing plasmid S, M, or L■ Add 10 |1M DFHBI to the final reaction mixture. o The total volume should be adjusted to 25 pl or as per the NEB protocol guidelines.4. Incubation: o Incubate the reaction mixture at 37°C for 2 hours to allow transcription and translation to occur.5. Fluorescence Measurement for Aptamer Transcription: o After the incubation period, measure the fluorescence to assess the expression of the aptamer (for plasmid S, M and L).Docket No. 11000-11031-PCT6. Fluorescence Measurement for mCherry Expression: o Similarly, measure the fluorescence for mCherry expression (in the case of plasmid M).[000229] Protocol for Measuring RNA Expression Activity Using Cell-Free Expression System with SNs Compartmentalizing Plasmids S, M, and L[000230] To achieve a DNA concentration in the final reaction that is at least 10 times higher than the concentration in the SNs dispersion, and to ensure comparable fluorescence signals between the encapsulated and control reactions, follow these steps:[000231] Encapsulated DNA Calculation1. Initial DNA Solution: o Start with 6 pl of DNA solution at 3.5 pg / pl. o Total DNA = 6 pl * 3.5 pg / pl = 21 pg.2. Encapsulation Efficiency: o Encapsulation efficiency is 40%. o Encapsulated DNA = 21 pg * 0.4 = 8.4 pg.3. Encapsulated DNA in 60 pl: o The encapsulated DNA (8.4 pg) is in 60 pl of solution.4. Volume Used for Reaction: o Use 5.75 pl of the SNs solution. o Encapsulated DNA in 5.75 pl = 8.4 pg * (5.75 pl / 60 pl) = 0.805 pg.5. Final Reaction Volume: o The final reaction volume is 12.5 pl. o DNA concentration in the final reaction = 0.805 pg / 12.5 pl = 0.0644 pg / pl.[000232] Control DNA Calculation1. Desired DNA Concentration: o To have a concentration at least 10 times that in the SNs dispersion: 0.0644 pg / pl * 10 = 0.644 pg / pl.2. Preparing Control Reaction: o To achieve a concentration of 0.644 pg / pl in the final 12.5 pl reaction:■ Required DNA amount = 0.644 pg / pl * 12.5 pl = 8.05 pg. o Volume of 3.5 pg / pl DNA solution required = 8.05 pg / 3.5 pg / pl = 2.3 pl.[000233] Summary of the Protocol1. For Encapsulated DNA Reaction: o Prepare SNs using 6 pl of 3.5 pg / pl DNA solution, resulting in 8.4 pg of encapsulated DNA in 60 pl. o Use 5.75 pl of the SNs solution in a 12.5 pl final reaction volume. o The concentration of DNA in this reaction will be 0.0644 pg / pl.2. For Control DNA Reaction: o To match the reaction conditions and ensure a comparable fluorescence signal, prepare a control reaction with a DNA concentration 10 times higher. o Use 2.3 pl of 3.5 pg / pl DNA solution in a 12.5 pl final reaction volume to achieve a concentration of 0.644 pg / pl.Docket No. 11000-11031-PCT[000234] Protocol for Assembly of Proteinosomes and Synthetic Nuclei (SNs) Using Mini¬Extruder Avanti[000235] This protocol is to produce SNs and proteinosomes using a mechanical system based on the extrusion of SNs or proteinosomes through a membrane with a specific pore size exclusion. This extrusion method utilizes a commercially available tool produced by Avanti Polar Lipids (https: / / avantilipids.com / products / equipment / category / mini-extruder). This method was developed to address two key challenges: the need to produce micron-sized SNs (>5 pm) without using invasive methods that could affect the stability of the DNA encapsulated within the SNs (e.g., sonication), and to ensure that the method is scalable for high production volumes.Inner Solution Components (final concentrations):• HEPES Buffer (50mM, pH 8)• DNA Plasmid (-150 ng / pl)• Biotinilated linear dsDNA (lOpM)• Streptavidin- Biotinilated linear dsDNA (lOpM)• Streptavidin (lOpM)• T7 Polymerase (5000 units)• BSA-PNIPAAM (16 mg / ml or desired concentration)• Milli-Q WaterOil Solution Components:• 2-Ethyl-l -hexanol• BS(PEG)n Cross-Linker (0.5mM)Extrusion Equipment:• Mini-Extruder (as per manufacturer’ s instructions)• Polycarbonate Membrane (5 pm pore size, 19 mm diameter)Other Materials:• Ethanol (75%, 50%, 25%)• 5 ml Vials• Pipettes and Tips• Centrifuge• Syringes• Vortex MixerCalculations for Inner Solution1. DNA Concentration: o To obtain 150 ng / pl final concentration in 60 pl solution, add 3 pl of 3000 ng / pl DNA plasmid.2. T7 Polymerase Units:Docket No. 11000-11031-PCT o To achieve 5000 units in 60 pl solution, add 6 pl of 50,000 units / ml T7 polymerase.3. HEPES Buffer: o To achieve a final concentration of 50mM in 60 pl solution using 500mM stock, add 6 pl of 500mM HEPES buffer.4. BSA-PNIPAAM: o For a final concentration of 16 mg / ml in 60 pl solution, calculate based on the stock concentration of BSA-PNIPAAM.Preparation of Inner Solution*1. Calculate the volume of Milli-Q water required to achieve a final volume of 60 pl after adding all components.2. Add the required volume of Milli-Q water to a 5 ml vial.3. Add 6 pl of 500mM HEPES buffer to the vial.4. Add BSA-PNIPAAM to achieve the desired final concentration.5. Add 6 pl of 50,000 units / ml T7 polymerase.6. Add 3 pl of 3000 ng / pl DNA plasmid.7. Vortex the 60 pl completed solution for at least 30 seconds.8. Place the vial at 4°C to rest while preparing the oil solution.Preparation of Oil Solution1. Aliquot 898 pl of 2-ethyl-l -hexanol into a vial.2. Add 2 pl of 250mM BS(PEG)n cross-linker.3. Vortex the solution for 1 minute to achieve a final concentration of 0.5mM cross-linker.Assembly of the Mini-Extruder1. Assemble the mini extruder according to the manufacturer’ s instructions (https: / / avantilipids.com / divisions / equipment-products / mini-extruder-assembly-instructions).2. Cut polycarbonate membranes to a diameter of 19 mm using a pincher.Formation of Pre-Emulsion and Extrusion1. Transfer the oil solution into the 5 ml vial containing the 60 pl inner solution.2. Gently mix with a pipette to form a pre-emulsion.3. Transfer the pre-emulsion to the syringe component of the extruder kit.4. Assemble the extruder by attaching the second syringe and membrane component.5. Extrude the mixture below 20°C to ensure PNIPAAM remains in its hydrophilic state.6. Perform 5 extrusions to form size-controlled SNs (1-5 pm). Only 5 extrusions are performed to prevent excessive resistance and potential membrane clogging due to the cross-linker reaction.Post-Extrusion Processing1. Transfer the white emulsion into a new 5 ml vial and leave at 4°C for 12 hours.2. Add 3 ml of 75% ethanol to the emulsion and mix until the emulsion turns transparent.3. Centrifuge at 4000xg for 4 minutes at 4°C.4. Carefully remove the supernatant, leaving around 100 pl to avoid disturbing the pellet, which may now be transparent.5. Resuspend the pellet in 2 ml of 50% ethanol.6. Repeat the process with 25% ethanol and twice with Milli-Q water.7. Resuspend the final pellet in the desired volume of water.Docket No. 11000-11031-PCT8. Check the density of SNs under the microscope.Depending on the starting DNA we can have different approaches to encapsulate the DNA within SNs:* incorporating Biotin-Linear dsDNA Instead of the PlasmidPreparation of Inner Solution with Streptavidin-Biotinylated Linear dsDNA1. Calculate the volume of Milli-Q water required to achieve a final volume of 60 pl after adding all components.2. Add the required volume of Milli-Q water to a 5 ml vial.3. Add 6 pl of 500mM HEPES buffer to the vial.4. Add BSA-PNIPAAM to achieve the desired final concentration.5. Add 6 pl of 50,000 units / ml T7 polymerase.6. Add 1 pM of streptavidin-biotinylated linear dsDNA.7. Vortex the 60 pl completed solution for at least 30 seconds.8. Place the vial at 4°C to rest while preparing the oil solution.** incorporating Biotin-Linear dsDNA in a Second StepPreparation of Inner Solution with Streptavidin1. Calculate the volume of Milli-Q water required to achieve a final volume of 60 pl after adding all components.2. Add the required volume of Milli-Q water to a 5 ml vial.3. Add 6 pl of 500mM HEPES buffer to the vial.4. Add BSA-PNIPAAM to achieve the desired final concentration.5. Add 6 pl of 50,000 units / ml T7 polymerase.6. Add 0.5 pM of streptavidin.7. Vortex the 60 pl completed solution for at least 30 seconds.8. Place the vial at 4°C to rest while preparing the oil solution.Post- Assembly Incorporation of Biotin-Linear dsDNA1. After completing the washing steps, resuspend the SNs in water to form a water-in-water compartment.2. Add an excess of biotin-linear dsDNA to the resuspended SNs.3. Allow the mixture to incubate for 1 hour to ensure diffusion and binding of the biotin-linear dsDNA within the SNs.4. Repeat the SNs washing steps to eliminate any unreacted biotin-linear dsDNA.[000236] The shell of the synthetic organelle or synthetic nucleus includes surface attachment molecules. Many different molecules are capable of being attached to the surface, each of these molecules will impart different functionalities.[000237] The specific molecules or chemical complexes intended for attachment on the surface of synthetic nuclei (SNs) using NHS (N-hydroxy succinimide) esters and NH2 (amino) groups. This reaction, commonly referred to as NHS ester-amino coupling, is extensively used for attaching molecules to the surface of proteins. The information will be included in the intellectual property (IP)Docket No. 11000-11031-PCT documentation. We have already employed this reaction method with promising preliminary results using PNIPAAm-NHS. We demonstrated the ability to generate stable and active SNs, where "active" refers to SNs capable of compartmentalizing functional RNA transcription within a cell-free gene expression system or HEK293T cells. Building on these results, we aim to explore the feasibility of attaching various molecules to the surface of SNs using the NHS ester and NH2 groups’ reaction mechanism. Further validation steps are necessary to verify the stability and activity of SNs modified with other molecules or chemical complexes.[000238] The following molecules or chemical complexes are considered for surface attachment on SNs using the NHS-NH2 reaction mechanism:1. Fluorescent Markers: For visualization and tracking purposes.2. Small molecules for targeting Ligands: To enhance specificity and binding affinity to target cells or tissues.3. Therapeutic Agents: Including small molecules or drugs for direct delivery to target sites.4. Enzymes: For catalytic activities on the surface of SNs.5. Antibodies: To cellular targeting or detection capabilities.6. Polymers: Such the already described PNIPAAm, to modify surface properties or functionality.[000239] The following molecules or chemical complexes are considered but not limited to for surface attachment on SNs using the NHS-NH2 reaction mechanism:[000240] Immune Response Activators1. Imiquimod: o Activates immune cells through TLR7 and TLR8, inducing proinflammatory cytokine production and showing antiviral and antitumor efficacy (Schon et al., 2006).2. a-Galactosylceramide (a-GalCer): o Triggers strong innate and adaptive immune responses by binding to CD Id molecules on dendritic cells, activating NKT cells and promoting cytokine production (Fujii et al., 2006).3. a-C-Galactosylceramide (a-C-GalCer): o An analogue of a-GalCer that binds more stably to dendritic cells, inducing stronger and more prolonged immune responses (Fujii et al., 2006).4. Toll-like receptor 7 / 8 (TLR7 / 8) Agonists: o Activate innate immune cells and enhance lymphatic delivery while minimizing systemic inflammation when conjugated to block copolymers (Van Herck et al., 2018).5. Neuropilin-1 (NRP1) Antagonists: o Reduce TGF[3 production in regulatory T cells, enhancing antitumor activity and modulating immune responses (Powell et al., 2018).6. Monolaurin: o Exhibits antimicrobial and immunomodulatory properties, affecting cytokine production and immune cell activity (Silva et al., 2018).[000241] Immune Response Repressors1. Glycerol Monolaurate (GML):Docket No. 11000-11031-PCT o Suppresses T cell signaling by altering lipid dynamics, leading to reduced production of cytokines such as IL-2, IFN-y, TNF-a, and IL-10 (Zhang et al., 2016).2. Glycogen Synthase Kinase 3P (GSK3P) Inhibitors: o Reduce monocyte adhesion and migration across endothelial cells, thereby diminishing inflammatory leukocyte responses (Rom et al., 2012).3. Flurbiprofen: o A biphenyl scaffold that binds to PD-L1, potentially modulating immune checkpoints and reducing immune responses (Bailly & Vergoten, 2020).[000242] Cell targeting:1 . DARPins (Designed Ankyrin Repeat Proteins) are a class of synthetic protein-binding molecules that are engineered for high specificity and affinity.2. Single-Chain Variable Fragments (scFvs)3. Nanobodies are single-domain antibody fragments derived from the heavy-chain antibodies.4. Small molecules for eg: a. Targeting Beta Cells i. Phogrin-Green Fluorescent Protein Chimeras: Targeted to pancreatic beta cells using the rat insulin II promoter, specifically expressed in beta cells for visualization and analysis (Bright et al., 2002). ii. OncoFAP Derivatives: Used for targeting beta cells with high specificity, utilized in diagnostic and therapeutic applications (Millul et al., 2021). b. Targeting Fibroblasts c. Targeting areas within tumors. i. Carbonic anhydrase IX (CAIX) (Zhang et al., 2016). d. Increasing the cellular uptake: i. Cationic Branched Polypeptides: These polypeptides, particularly those with cationic side chains, increase cellular uptake through mechanisms such as macropinocytosis and caveolae / lipid raft-mediated endocytosis. They are effective at internalizing large molecular complexes and particles (Szabo et al., 2017). ii. Cell-Penetrating Peptides (CPPs): CPPs like TAT, Antennapedia (Antp), and Transportan significantly enhance the uptake of various cargos, including relatively large nanoparticles and liposomes. They promote cellular uptake by different pathways, including macropinocytosis, which can accommodate large particles (Vives et al., 2003). iii. Arginine-Rich Peptides: These peptides, when used in conjunction with other carriers, have been shown to facilitate the uptake of relatively large molecular complexes by enhancing endocytosis and macropinocytosis (Maiolo et al., 2005). iv. Penetratin: A well-studied CPP, Penetratin enhances the uptake of large molecular complexes and nanoparticles by reducing membrane dipole potential and promoting escape from endo-lysosomal compartments (Batta et al., 2020). v. Taurine-Modified D-Peptides:Taurine-conjugated D-peptides significantly boost the cellular uptake of these peptides by promoting macropinocytosis and dynamin-dependent endocytosis, which can internalize large particles (Zhou et al., 2018). vi. Multivalent Cell-Penetrating Peptides: Multivalency increases the cellular uptake efficiency of CPPs like Tat and Transportan, which can facilitate the internalization of larger particles and aggregates (Eggimann et al., 2013). vii. MK2 Inhibitory Peptide Nano-Polyplexes: These nano-polyplexes utilize a cationic peptide and an anionic polymer to enhance cellular uptake throughDocket No. 11000-11031-PCT macropinocytosis and clathrin-mediated endocytosis, demonstrating effectiveness in internalizing larger particles (Kilchrist et al., 2016). viii. gH625 Peptide: This membranotropic peptide enhances the uptake of nanoparticles by escaping the endo-lysosomal pathway and delivering the particles into the cytosol, effectively internalizing particles of various sizes(Guarnieri et al., 2017).[000243] The invention has been described with reference to various specific and preferred embodiments and techniques. Nevertheless, it is understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

Docket No. 11000-11031-PCTWHAT IS CLAIMED IS:

1. A microscale compartment comprising a shell and an internal space; wherein the shell comprises an aliphatic conjugate between a protein component and a polymer component, wherein the aliphatic compartment is capable of forming spheroids.

2. The microscale compartment of claim 1 , wherein the polymer component comprises a hydrophobic component.

3. The microscale compartment of claim 2, wherein the hydrophobic polymer component comprises one or more of poly(N-isopropylacrylamide) (PNIPAAm), Poly(N,N-dimethylacrylamide) (PDMAAm), Poly(N-vinylcaprolactam) (PVCL), or Poly(N,N-diethylacrylamide) (PDEAAm).

4. The microscale compartment of claim 3, wherein the thermo-responsive polymer comprises poly (N -isopropylacrylamide) (PNIPAAm) .

5. The microscale compartment of claim 4, wherein the poly(N-isopropylacrylamide) comprises one or more of Aminooxy End-Functionalized PNIPAAm, Thiol-Modified PNIPAAm, Vinyl- Modified PNIPAAm, Amine-Modified PNIPAAm, Azido- and Acetylene-Functionalized PNIPAAm, Carboxylated PNIPAAm, or Adamantane-Terminated PNIPAAm (Ad-PNIPAAm).

6. The microscale compartment of claim 4, wherein the poly(N-isopropylacrylamide) is functionalized with one of N-hydroxy succinimide ester, adamantane, mercaptothiazoline, or glucose oxidase.

7. The microscale compartment of claim 1, wherein the hydrophilic protein compartment comprises one or more of bovine serum albumin (BSA), glucose oxidase, P-cyclodextrin, myoglobin, hemoglobin, poly(dopamine), ferritin, Human serum albumin (HSA), or protamine.

8. The microscale compartment of claim 1, wherein the shell comprises pores.

9. The microscale compartment of claim 1, wherein the shell further comprises a crosslinker.Docket No. 11000-11031-PCT10. The microscale compartment of claim 2, wherein the crosslinker is O,O’-Bis-[2-(N-succunimyl- succinylamino) -ethyl] -polyethylene glycol (BS(PEG ).

11. The microscale compartment of claim 3, wherein the BS(PEG) is BS(PEG)9, BS(PEG)2000, or BS(PEG)10000.

12. The microscale compartment of any of claims 1-6, wherein the pores are sized to allow Nucleic acids smaller than about 60 base pairs to pass through the pores, and to prevent nucleic acids larger than about 60 base pairs from passing through the pores.

13. The microscale compartment of any of claims 1-6, wherein the pores are sized to allow nucleic acids smaller than about 1000 base pairs to pass through the pores, and to prevent nucleic acids larger than about 1000 base pairs from passing through the pores.

14. The microscale compartment of any of claims 1-6, wherein the pores are sized to allow RNA or proteins smaller than about 3000 base pairs to pass through the pores.

15. The microscale compartment of claim 1, wherein the internal space contains a nucleic acid.

16. The microscale compartment of claim 11, wherein the nucleic acid comprises a gene.

17. The microscale compartment of claim 11, wherein the nucleic acid is a plasmid.

18. The microscale compartment of claim 12, wherein the chromosome is an artificial chromosome.

19. The microscale compartment of claim 12, wherein the plasmid is about 2.2 Kb.

20. The microscale compartment of claim 12, wherein the plasmid is about 3 Kb.

21. The microscale compartment of claim 12, wherein the plasmid is about 14 Kb.

22. The microscale compartment of any of claims 1-16, wherein the internal space contains a polymerase.Docket No. 11000-11031-PCT23. The microscale compartment of any of claim 17, wherein the polymerase is retained within the microscale compartment.

24. The microscale compartment of any of claims 1-18, wherein the microscale compartment is between about 0.5 pm and about 10 pm in diameter.

25. The microscale compartment of claim 19, wherein the microscale compartment is between about 3 pm and about 7 pm.

26. The microscale compartment of claim 20, wherein the microscale compartment is about 5 pm in diameter.

27. A method for preparing a microscale compartment comprising: assembling a shell comprising an aliphatic conjugate between a hydrophilic protein component and a hydrophobic polymer component which is capable of forming spheroids; adding a crosslinker; and passing the emulsion product through a membrane.

28. The method according to claim 27, wherein the shell is assembled by one or more of an oil in water emulsion process, sonication, pipetting, microfluidics, extrusion, solution agitation, or bead beating.

29. The method according to claim 27, wherein the microscale compartment is between about 0.25 pm and about 10 pm in diameter.

30. The method according to claim 29, wherein the microscale compartment is between about 3 pm and about 7 pm.

31. The method according to claim 30, wherein the microscale compartment is about 5 pm in diameter.

32. The method according to any of claims 27-32, wherein the pores in the membrane through which the microscale compartment is passed, are between about 1 pm and about 10 pm in diameter.Docket No. 11000-11031-PCT33. The method according to claim 32, wherein the pores in the membrane through which the microscale compartment is passed, are about 5 pm in diameter.

34. The method according to any of claims 27-33, wherein the crosslinker is O,O’-Bis-[2-(N- succunimyl-succinylamino)-ethyl] -polyethylene glycol BS(PEG).

35. The method according to claim 34, wherein the BS(PEG) is BS(PEG)9, BS(PEG)2000, or BS(PEG)10000.

36. The method according to any of claims 27-35, wherein the crosslinker is added after the emulsion process.

37. The method according to claim 36, wherein the emulsion product is passed through the membrane between about 1 times and about 12 times.

38. The method according to claim 37, wherein the emulsion product is passed through the membrane between about 3 times and about 9 times.

39. The method according to any of claims 27-38, wherein the crosslinker is added before the emulsion process.

40. The method according to claim 39, wherein the emulsion product is passed through the membrane between about 2 times and about 10 times.

41. The method according to claim 40, wherein the emulsion product is passed through the membrane between about 3 times and about 9 times.

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