Compositions and methods of making polymeric nanoparticles for delivery of therapeutic agents

Abstract

Nanoparticle compositions and methods for preparation thereof are provided. The compositions include nanoparticles comprising anionic polymers, therapeutic agents, and cations. Nanoparticles predominantly fall within a diameter range of 10 nm to 80 nm. Method of preparation involves mixing anionic polymers with cations and therapeutic agents in a microfluidic device to produce nanoparticles.

Description

COMPOSITIONS AND METHODS OF MAKING POLYMERIC NANOPARTICLES FOR DELIVERY OF THERAPEUTIC AGENTSRELATED APPLICATION

[0001] This application claims the benefit to U.S. Provisional Application No. 63 / 632,695, filed on April 11, 2024. The entire teachings of the above applications are incorporated herein by reference.BACKGROUND

[0002] Nucleic acid materials hold immense promise for therapeutically modulating genetic diseases by rectifying defective or absent genes through the expression of exogenously introduced genes. However, their therapeutic efficacy is hindered by challenges such as short half-lives in the bloodstream and difficulties in traversing cell membranes, limiting systemic application critical for addressing disseminated conditions like metastasized tumors and inflamed tissues. To realize the potential of nucleic acid-based therapies, efficient delivery systems suitable for systemic application are imperative. Various strategies have been explored to facilitate nucleic acid delivery, ranging from cationic chemical agents such as cationic lipids, liposomes, polymers, or peptides to physical methods such as electrotransfer. While these approaches offer protection against enzymatic degradation and enhance cellular uptake, their cationic charge may induce severe cytotoxicity and serum inactivation, underscoring the need for improved formulations to enable effective delivery to target cells.

[0003] Thus, there is a growing demand for effective in vivo nucleic acid delivery systems.SUMMARY

[0004] The present disclosure provides compositions comprising nanoparticles for the delivery of nucleic acids and methods of synthesizing said nanoparticles.

[0005] In one aspect, the disclosure provides a composition comprising nanoparticles formed from a plurality of anionic polymers, a therapeutic agent, and a cation, wherein at least 75% of the nanoparticles have a diameter of about 10 nm to about 80 nm as measured by transmission electron microscopy.

[0006] In some embodiments, at least 95% of nanoparticles have a diameter of about 10 nm to about 80 nm as measured by transmission electron microscopy. In some embodiments, at least 98% of the nanoparticles have a diameter of about 10 nm to about 80 nm as measured by transmission electron microscopy.

[0007] In some embodiments, at least 75% of the nanoparticles have a diameter of about 50 nm to about 70 nm as measured by transmission electron microscopy. In some embodiments, at least 95% of the nanoparticles have a diameter of about 50 nm to about 70 nm as measured by transmission electron microscopy. In some embodiments, at least 98% of the nanoparticles have a diameter of about 50 nm to about 70 nm as measured by transmission electron microscopy.

[0008] In some embodiments, at least 75% of the nanoparticles have a diameter of about 50 nm to about 60 nm as measured by transmission electron microscopy. In some embodiments, at least 95% of the nanoparticles have a diameter of about 50 nm to about 60 nm as measured by transmission electron microscopy. In some embodiments, at least 98% of the nanoparticles have a diameter of about 50 nm to about 60 nm as measured by transmission electron microscopy.

[0009] In some embodiments, the nanoparticles have an average diameter of about 48 nm to about 98 nm as measured by dynamic light scattering.

[0010] In some embodiments, the therapeutic agent comprises a nucleic acid. In some embodiments, the therapeutic agent comprises a nucleic acid at least about 100 bases in length. In some embodiments, the therapeutic agent comprises plasmid DNA (pDNA). In some embodiments, the pDNA comprises two or more plasmids. In some embodiments, a first plasmid encodes a CRISPR Cas enzyme and a guide RNA, and a second plasmid encodes a donor oligonucleotide.

[0011] In some embodiments, at least 75% of the nanoparticles have a diameter of about 20 nm to about 30 nm as measured by transmission electron microscopy. In some embodiments, at least 95% of the nanoparticles have a diameter of about 20 nm to about 30 nm as measured by transmission electron microscopy. In some embodiments, at least 98% of the nanoparticles have a diameter of about 20 nm to about 30 nm as measured by transmission electron microscopy.

[0012] In some embodiments, the nanoparticles have an average diameter of about 10 nm to about 30 nm as measured by dynamic light scattering.

[0013] In some embodiments, the therapeutic agent comprises a nucleic acid. In some embodiments, the therapeutic agent comprises a nucleic acid about 20-40 nucleotides in length. In some embodiments, the therapeutic agent comprises small interfering RNA (siRNA).

[0014] In some embodiments, the plurality of anionic polymers comprises one or more natural polymers. In some embodiments, one or more of the plurality of anionic polymers is selected from the group consisting of alginate and hyaluronan (z.e., hyaluronic acid, HA, HyA). In some embodiments, the plurality of anionic polymers comprises alginate. In some embodiments, the plurality of anionic polymers further comprises hyaluronic acid. In some embodiments, the plurality of anionic polymers comprises alginate, hyaluronic acid, or both.

[0015] In some embodiments, the cation comprises a divalent cation. In some embodiments, the cation is a divalent cation. In some embodiments, divalent cation comprises calcium (Ca2+). In some embodiments, the divalent cation is Ca2+.

[0016] In some embodiments, the nucleic acid comprises short interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), guide RNA, DNA, plasmid DNA (pDNA), or any combination of the foregoing. In some embodiments, the nucleic acid comprises or encodes a sequence complementary to a portion of a gene or a transcript associated with a disease, disorder, or condition. In some embodiments, the disease, disorder, or condition comprises a cancer or a metabolic, neurodegenerative, cardiovascular, infectious, or inflammatory disease or disorder.

[0017] In some embodiments, the therapeutic agent comprises a nucleic acid, and wherein the nucleic acid is selected from the group consisting of short interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), guide RNA, DNA and plasmid DNA (pDNA). In some embodiments, the nucleic acid is capable of silencing of a gene associated with a disease, disorder, or condition selected from the group consisting of cancer, a metabolic, a neurodegenerative, a cardiovascular, an infectious, or an inflammatory disease or disorder.

[0018] In some embodiments, the therapeutic agent is RNA, and the RNA and plurality of anionic polymers are in a molar ratio from about 1 : 1 to about 30: 1.

[0019] In some embodiments, the RNA and plurality of anionic polymers are in a molar ratio of about 7: 1.

[0020] In some embodiments, the therapeutic agent is RNA, and the RNA and plurality of anionic polymers comprising alginate are in a molar ratio from about 7: 1 to about 30: 1.

[0021] In some embodiments, the plurality of anionic polymers and the therapeutic agent are in a concentration ratio of about 1 :6.

[0022] In some embodiments, the nucleic acid encodes a pharmaceutically active peptide or protein. In some embodiments, the pharmaceutically active peptide or protein comprises a CRISPR Cas enzyme. In some embodiments, the nucleic acid further encodes a guide RNA. In some embodiments, the nucleic acid further encodes a donor oligonucleotide.

[0023] In some embodiments, the nanoparticles have a zeta potential of about -7.8 mV to about -9.1 mV.

[0024] In some embodiments, the plurality of anionic polymers comprises hyaluronic acid, the cation comprises polyethyleneimine (PEI) or Ca2+, and the therapeutic comprises a nucleic acid; and the nanoparticles comprise about 25 pg / mL to about 115 pg / mL hyaluronic acid, about 79 pg / mL PEI or about 27.75 mg / mL (0.25 M) Ca2+, and about 50 pg / mL nucleic acid. In some embodiments, the nanoparticles comprise about 25 pg / mL hyaluronic acid and about 27.75 mg / mL (0.25 M) Ca2+.

[0025] In some embodiments, the plurality of anionic polymers comprises alginate, the cation comprises Ca2+, and the therapeutic comprises a nucleic acid; and the nanoparticles comprise about 25 pg / mL alginate, about 27.75 mg / mL (0.25 M) Ca2+, and about 3 pg / mL to about 150 pg / mL nucleic acid.

[0026] In some embodiments, the nucleic acid comprises pDNA, and the pDNA is about 5 kilobases (kb) to about 8 kb in length. In some embodiments, the nanoparticles comprise about 3 pg / mL pDNA. In some embodiments, the nanoparticles comprise about 6 pg / mL pDNA. In some embodiments, the nanoparticles comprise about 150 pg / mL pDNA.

[0027] In some embodiments, the nucleic acid comprises siRNA, and the siRNA is about 21 bases in length. In some embodiments, the nanoparticles comprise about 67 pg / mL siRNA.

[0028] In another aspect, the disclosure provides a method for making a nanoparticle composition, the method comprising: (a) in a microfluidic device, mixing: i) a first liquid composition comprising a plurality of anionic polymers; and ii) a second liquid composition comprising a therapeutic agent and a cation, thereby producing a composition of nanoparticles, wherein at least 75% of the nanoparticles have a diameter of about 10 nm to about 80 nm as measured by transmission electron microscopy. In some embodiments, the first and second compositions are mixed in a ratio from about 1 : 1 to about 1 :30. In some embodiments, the first and second compositions are mixed in a ratio of about 1 :7.

[0029] In yet another aspect, the disclosure provides a method for synthesizing nanoparticles, the method comprising: (a) injecting a first solution comprising a plurality of anionic polymers and a buffer into a first inlet of a microfluidic mixer; and (b) injecting a second solution comprising a therapeutic agent, a cation, and the buffer into a second inlet of the microfluidic mixer; thereby producing nanoparticles encapsulating the therapeutic agent, wherein at least 75% of the nanoparticles have a diameter of about 10 nm to about 80 nm as measured by transmission electron microscopy.

[0030] In some embodiments, the microfluidic mixer comprises one or more nanoscale microfluidic channels. In some embodiments, one or more of the nanoscale microfluidic channels is about 1 pm in diameter.

[0031] In some embodiments, the first solution comprises the plurality of anionic polymers at a concentration of about 50 pg / mL to about 230 pg / mL; the buffer at a concentration of about 500 mM; or both.

[0032] In some embodiments, the second solution comprises: the therapeutic agent at a concentration of about 6 pg / mL to about 300 pg / mL; the cation at a concentration of about 158 pg / mL to about 55.5 mg / mL; the buffer at a concentration of about 500 mM; or any combination of the foregoing.

[0033] In some embodiments, the nanoparticles comprise: the plurality of anionic polymers at a concentration of about 25 pg / mL to about 115 pg / mL; the therapeutic agent at a concentration of about 3 pg / mL to about 150 pg / mL; the cation at a concentration of about 79 pg / mL to about 27.75 mg / mL; the buffer at a concentration of about 500 mM; or any combination of the foregoing.

[0034] In some embodiments, the plurality of anionic polymers comprises alginate or hyaluronic acid; the therapeutic agent comprises a nucleic acid; the cation comprises polyethyleneimine (PEI) or Ca2+; the buffer comprises 2-[4-(2-hydroxyethyl)piperazin-l- yl]ethanesulfonic acid (HEPES); or any combination of the foregoing.

[0035] In some embodiments, injecting the first solution and injecting the second solution are performed simultaneously. In some embodiments, the first and second solutions are injected at a flow rate ratio of about 5: 1 to about 1 :5. In some embodiments, the first and second solutions are injected at a flow rate ratio of about 2:1 to 1 :2. In some embodiments, the first and second solutions are injected at a flow rate ratio of about 1 : 1. In some embodiments, the first and second solutions are each injected at a total flow rate of about 12 mL / minute.

[0036] In some embodiments, injecting the first solution and injecting the second solution produces a mixture. In some embodiments, the method further comprises dispensing the mixture from the microfluidic device, thereby producing a dispensed mixture comprising the nanoparticles. In some embodiments, the method further comprises discarding a volume of the dispensed mixture at a start and at an end of dispensing the mixture from the microfluidic device. In some embodiments, the method further comprises incubating the dispensed mixture at room temperature. In some embodiments, the method further comprises incubating the dispensed mixture for a period of about 30 minutes.

[0037] In some embodiments, the method further comprises preparing the first solution by: mixing a polymer solution and a cation solution; gently vortexing the mixed polymer and cation solutions; and allowing the mixed polymer and cation solutions to incubate at room temperature; thereby producing the first solution.

[0038] In some embodiments, the method further comprises storing the nanoparticles at a temperature of about -80°C to about 25°C for a period of up to about 3.5 months.

[0039] In yet another aspect, the disclosure provides a method for delivering a therapeutic agent to a cell, comprising contacting the cell with a composition comprising one or more nanoparticles, each nanoparticle comprising a plurality of anionic polymers, a therapeutic agent, and a cation, wherein at least 75% of the nanoparticles have a diameter of about 10 nm to about 80 nm as measured by transmission electron microscopy.

[0040] In some embodiments, the plurality of anionic polymers comprises alginate, hyaluronic acid, or both.

[0041] In some embodiments, the cation comprises a divalent cation. In some embodiments, the divalent cation comprises calcium (Ca2+).

[0042] In some embodiments, the therapeutic agent comprises a nucleic acid. In some embodiments, the nucleic acid comprises short interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), guide RNA, DNA, plasmid DNA (pDNA), or any combination of the foregoing.

[0043] In some embodiments, the nanoparticles comprise: the plurality of anionic polymers at a concentration of about 25 pg / mL to about 115 pg / mL; the therapeutic agent at a concentration of about 3 pg / mL to about 150 pg / mL; the cation at a concentration of about 79 pg / mL to about 27.75 mg / mL; the buffer at a concentration of about 500 mM; or any combination of the foregoing.

[0044] In some embodiments, the composition comprises the one or more nanoparticles at a concentration of 7.5 pg / mL.BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

[0046] FIGs. 1A-C. Characterization of ENVOYER nanoparticle size. (FIG. 1A)Transmission electron microscopy (TEM) images show ENVOYER nanoparticles were 50- 70 nm in size when freshly prepared, and (FIG. IB) after storage for 24 hours at 4°C. (FIG. 1C) Size distribution of the Envoyer nanoparticles was measured via dynamic light scattering (DLS).

[0047] FIGs. 2A-B. ENVOYER nanoparticles and LIPOFECTAMINE® transfection. (FIG. 2A) Representative fluorescence images show transfection visualized by the intensity of GFP from pDNA (magnification lOx) and (FIG. 2B) western blot.Abbreviations: GFP, green fluorescent protein; pDNA, plasmid DNA.

[0048] FIGs. 3A-B. ENVOYER nanoparticle transfection efficiency. (FIG. 3A)ENVOYER had 16% transfection efficiency using GFP pDNA in PANCI cells confirmed via fluorescence microscopy (magnification lOx), flow cytometry and (FIG. 3B) western blotting techniques. (FIG. 3C) Approximately 83% transfection efficiency was achieved with ENVOYER nanoparticles for pDNA, compared to approximately 63% with LIPOFECTAMINE® in HEK293T cells confirmed with fluorescence microscopy (magnification lOx) and flow cytometry. Abbreviations: FITC, fluorescein; GFP, green fluorescent protein; pDNA, plasmid DNA.

[0049] FIGs. 4A-B. ENVOYER nanoparticles and lipid nanoparticles with encapsulated GFP pDNA. (FIG. 4A) Cellular transduction was quantified by western blot, and (FIG. 4B) yield was visualized by the intensity of GFP pDNA with fluorescence microscopy (magnification lOx). Abbreviations: CaCh, calcium chloride; GFP, green fluorescent protein; LNP, lipid nanoparticle; pDNA, plasmid DNA.

[0050] FIGs. 5A-C. Short-term storage and stability of ENVOYER nanoparticles.(FIG. 5A) Representative microscopic images show GFP expression after 48 hour treatment with 5 |ig GFP pDNA encapsulated ENVOYER nanoparticles stored at 4°C, -20°C, or -80°C(magnification lOx) and corresponding (FIG. 5B) western blot and (FIG. 5C) densitometry. Abbreviations: GFP, green fluorescent protein; hr, hour; RT, room temperature; pDNA, plasmid DNA.

[0051] FIGs. 6A-C. Long-term storage and stability of ENVOYER nanoparticles.(FIG. 6A) Representative microscopic images show GFP expression after 48 hour treatment with 15 pg GFP pDNA ENVOYER nanoparticles stored at 4°C or -80°C for 2 months (magnification lOx) and corresponding (FIG. 6B) western blot and (FIG. 6C) densitometry. Abbreviations: GFP, green fluorescent protein; hr, hour; RT, room temperature; pDNA, plasmid DNA.

[0052] FIGs. 7A-B. Cellular uptake of ENVOYER nanoparticles exposed to amiloride or genistein. (FIG. 7A) Uptake was visualized by the intensity of GFP for pDNA with fluorescence microscopy (magnification lOx) and (FIG. 7B) quantified by western blot. In FIG. 7B, “ +” indicates the addition of a treatment, while indicates its absence. Abbreviations: GFP, green fluorescent protein; pDNA, plasmid DNA.

[0053] FIG. 8. Transmission electron microscopy (TEM) image showing consistent ENVOYER particle size range between 20 and 30 nm. Image was taken with a digital camera system on a JEM1010 TEM. Scale: 50 nm. Magnification: 60000X.

[0054] FIG. 9. TLR4 activity in HEK Blue TLR4 cells. Working concentrations of ENVOYER-encapsulated scrambled siRNA did not significantly activate TLR4 in HEK Blue TLR4 cells. LPS showed dose-dependent activation of TLR4 in HEK Blue TLR4 cells. At the working concentration of the siRNA (about 50 nM to 140 nM), ENVOYER-encapsulated scrambled siRNA did not significantly activate TLR4 compared to LPS. Each experiment was done in duplicates. Abbreviations: LPS, lipopolysaccharide; TLR4, toll-like receptor 4.

[0055] FIGs. 10A-C. Time course of (FIG. 10A) interleukin 6, (FIG. 10B) tumor necrosis factor alpha, and (FIG. 10C) interferon gamma levels in serum measured after injection of 0.1 mg / kg scrambled siRNA encapsulated in either ENVOYER or lipid nanoparticles. Lipopolysaccharide equivalent to 100 pg per mouse (approximately 5 mg / kg) was used as a positive control and saline injection was used as a negative control. Each experimental group included 5 mice. Each colored dot corresponds to an individual mouse. Abbreviations: fFNy, interferon gamma; IL-6, interleukin; LNP, lipid nanoparticles; LPS, lipopolysaccharide; TNFa, tumor necrosis factor alpha.

[0056] FIGs. 11A-D. ENVOYER-encapsulated survivin siRNA reduces survivin mRNA and protein levels in a dose- and time-dependent manner. (FIGs. 11 A, 11B)PANC1 cells were treated with ENVOYER-encapsulated survivin siRNA or scrambled siRNA at concentrations of 15.6, 31.25, 62.5, 125, and 250 nM for 24 hours. Survivin mRNA (FIG. 11 A) and protein (FIG. 1 IB) decreased in a dose-dependent manner. Transfection of 100 nM siRNA using LIPOFECT AMINE® for 24-hours served as a positive control. (FIGs. 11C, 11D) PANCI cells were treated with ENVOYER-encapsulated survivin siRNA 62.5 nM for 3, 6, 24 and 48 hours. Survivin mRNA (FIG. 11C) and protein (FIG. 1 ID) decreased in a time-dependent manner. Transfection of 100 nM siRNA using LIPOFECTAMINE® for 24 and 48 hours were used as positive controls. Each experiment was done independently twice to confirm the findings. Abbreviations: hr, hours; mRNA, messenger RNA; siRNA, small interfering RNA.

[0057] FIGs. 12A-D. Stability and efficacy of ENVOYER-encapsulated siRNA after freeze-thaw cycles. Western blot analysis of PANCI cells transfected for 48 hours with (FIG. 12A) freshly prepared ENVOYER-encapsulated survivin siRNA (70 nM), (FIG. 12B) ENVOYER-encapsulated survivin siRNA (70 nM) stored at -80°C for 1 month, then thawed, (FIG. 12C) ENVOYER-encapsulated survivin siRNA (70 nM) after 2 freeze-thaw cycles over 3.5 months. ENVOYER-encapsulated scrambled siRNA served as the control. (FIG. 12D) Quantification of survivin protein levels compared to scrambled siRNA. Each experiment was done independently twice to confirm the findings.

[0058] FIGs. 13A-D. Validation of NUF2 knockdown in PANCI cells using transfection with LIPOFECTAMINE® and ENVOYER particles. (FIG 13 A) Western blot analysis with antibodies against NUF2 component of NDC80 kinetochore complex (NUF2), cleaved caspase 3, and beta-actin showing that NUF2 knockdown in PANCI cells using LIPOFECTAMINE® RNAIMAX® transfection with NUF2 siRNA (62.5 nM or 125 nM) reduced NUF2 protein levels and increased cleavage of caspase 3 in a dose-dependent manner compared to transfection with scrambled siRNA (control) using LIPOFECTAMINE® RNAIMAX®. (FIG. 13B) Densitometry analysis of NUF2 and cleaved caspase 3 expression normalized to beta-actin. (FIG. 13C) Western blot analysis with antibodies against NUF2, cleaved caspase 3, phospho-histone H3 (SerlO) and beta-actin showing that NUF2 knockdown in PANCI cells using ENVOYER-encapsulated NUF2 siRNA (125 nM) reduced expression of NUF2 in PANCI cells and increased expression of cleaved caspase 3 and phospho-histone H3 (SerlO) compared to transfection with ENVOYER-encapsulated scrambled siRNA. Addition of genistein to transfection with ENVOYER-encapsulated NUF2 siRNA abrogated the effectiveness of transfection withENVOYER particles. (FIG. 13D) Densitometry analysis of NUF2, cleaved caspase 3, and phospho-histone H3 (SerlO) expression normalized to beta-actin.

[0059] FIGs. 14A-D. Intratumoral injection of ENVOYER-encapsulated NUF2 siRNA inhibits tumor growth in NSG mice. (FIG. 14A) NOD-scid gamma mice were injected intratumorally with siRNA concentration of about 0.2 mg / kg three times per week for 2 weeks. Fifteen days after treatment initiation, mice treated with ENVOYER- encapsulated NUF2 siRNA demonstrated a 60% inhibition in tumor growth compared to mice that received ENVOYER-encapsulated scrambled siRNA. (FIG. 14B) Mean body weight of mice injected with ENVOYER-encapsulated NUF2 siRNA did not change. (FIG. 14C) Analysis of the tumor samples treated with ENVOYER-encapsuled NUF2 siRNA showed a reduction in NUF2 protein expression compared to scrambled siRNA. (FIG. 14D) Immunohistochemistry analysis of tumor sections from mice treated with ENVOYER- encapsulated NUF2 siRNA showed reduced expression of NUF2 and Ki67 and increased expression of cleaved caspase 3 compared with tumor sections from mice treated with ENVOYER-encapsulated scrambled siRNA. Abbreviations: CL, cleaved; siRNA, small interfering RNA.

[0060] FIGs. 15A-B. (FIG. 15 A) Western blot analysis showing expression of full-length cellular tumor antigen p53 (p53) and housekeeping protein actin, cytoplasmic 1 (P-Actin, z.e., ACTB) in HEK cells transfected with CRISPR Cas protein, guide RNA (Gl), and a donor oligonucleotide (Donor) for homology-directed insertion of GFP into the p53 sequence. Cells were treated with ENVOYER nanoparticles (NPs) encapsulating Cas, Gl, and the Donor in the same NPs (1; Gl+Donor NPs), Cas and Gl or the Donor separately encapsulated in NPs (2; Gl NPs + Donor NPs), or Cas, Gl, and the Donor delivered by LIPOFECT AMINE® (Gl + Donor LIPOFECTAMINE®). Ctrl, control. (FIG. 15B) Quantification of expression shown in FIG. 15 A.

[0061] FIG. 16. Western blot analysis of single colonies picked from treatment groups 2 (Gl NPs + Donor NPs) and 3 (Gl + Donor LIPOFECTAMINE®) shown in FIG. 15. The blot shows expression of full-length p53 (indicated by box), GFP-inserted truncated p53, and P- Actin.

[0062] FIG 17. Western blot analysis of full-length p53 expression following reselection of colonies from treatment group 2 (Gl NPs + Donor NPs) colonies with the most p53 knockdown.

[0063] FIGs. 18A-B. (FIG. 18A) Western blot analysis of full-length p53 expression following selection of colonies from treatment group 1 (Gl+Donor NPs). (FIG. 18B) Quantification of FIG. 18 A.

[0064] FIG. 19. Western blot analysis of full-length p53 and GFP-inserted truncated p53 (GFP+p53) expression following re-selection of colonies from treatment group 1 (Gl+Donor NPs).

[0065] FIGs. 20A-B. (FIG. 20 A) Brightfield (top) and fluorescent (bottom) microscopy of cells from treatment group 1 (Gl+Donor NPs) at pre-selection passages 2 and 5 and after re-selection (clone #5-9). (FIG. 20B) Brightfield (top) and fluorescent (bottom) microscopy of control cells. Control at Passage 5 cells underwent the same plating and passaging process at the same time as the Pre-Selection (Gl+Donor) NP cells shown in FIG. 20A. Control HEK293T cells were grown under similar conditions as Clone #5-9 (FIG. 20A) at the time selection for Clone #5-9 was completed.DEFINITIONS

[0066] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related.

[0067] The terms “a” or “an” as used herein in the specification may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more.

[0068] The term “comprise,” or variations such as “comprises” or “comprising,” as used herein may be used to imply the inclusion of a stated element or integer or group of elements or integers, but not the exclusion of any other element or integer or group of elements or integers.

[0069] Reference throughout this specification to “one embodiment”, “an embodiment” or “some embodiments” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases “in one embodiment”, “in an embodiment” or “some embodiments” in various places throughout this specification are not necessarily all referring to the same embodiment(s). Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0070] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “7” is disclosed, then “about 7” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 7 and 10 are disclosed, then 8 and 9 are also disclosed.

[0071] “Nanoparticle”, as used herein, refers to any entity having a diameter of less than 1 micron (pm). Typically, particles have a greatest dimension (e.g., diameter) of 1000 nm or less. In some embodiments, nanoparticles have a diameter of 100 nm or less. In some embodiments, nanoparticles have a diameter of between 20 nm and 100 nm. In some embodiments, nanoparticles have a diameter of between 20 nm and 80 nm. In some embodiments, nanoparticles have a diameter of between 20 nm and 60 nm. In some embodiments, nanoparticles have a diameter of between 20 nm and 40 nm. In some embodiments, the particles are spheres, spheroids, flat, plate-shaped, cubes, cuboids, ovals, ellipses, cylinders, cones, or pyramids. In some embodiments, a population of particles may be relatively uniform in terms of size, shape, charge, and / or composition.

[0072] The terms "incorporated" and "encapsulated" refer to incorporating, formulating, or otherwise including an active agent into and / or onto a composition that allows for release, such as sustained release, of such agent in the desired application. The terms contemplate any manner by which a therapeutic agent or other material is incorporated into a polymer matrix, including chemically or physically couple, in physical admixture, or enveloping the agent in a coating layer.

[0073] As used herein, “mean particle size,” generally refers to the statistical mean particle size (diameter) of the particles in a population of particles. The diameter of an essentially spherical particle may be referred to as the physical or hydrodynamic diameter. The diameter of a non-spherical particle may refer preferentially to the hydrodynamic diameter. As used herein, the diameter of a non-spherical particle may refer to the largest linear distance between two points on the surface of the particle. Mean particle size can bemeasured using methods known in the art, such as transmission electron microscopy and dynamic light scattering.

[0074] As used herein, the term "nucleic acid" refers to the genetic material that undergoes amplification through described amplification techniques, ultimately producing an amplified product comprising a nucleic acid sequence corresponding to the biomarker of interest. In some embodiments, nucleic acid may include DNA (deoxyribonucleic acid) or RNA (ribonucleic acid). In some embodiments, DNA is plasmid DNA (pDNA). In some embodiments, RNA is small interfering RNA (siRNA). In some embodiments, RNA is microRNA (miRNA). In some embodiments, RNA is messenger RNA (mRNA). In some embodiments, RNA is transfer RNA (tRNA). In some embodiments, RNA is guide RNA (gRNA).

[0075] The terms “polypeptide”, “peptide”, and “protein”, as used herein, refer to a molecule composed of amino acids linked by a peptide bond. A “pharmaceutically active peptide or protein” as used herein, has a positive or advantageous effect on the condition or disease state of a subject when administered to the subject in a therapeutically effective amount. Preferably, a pharmaceutically active peptide or protein has curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease or disorder. A pharmaceutically active peptide or protein may have prophylactic properties and may be used to delay the onset of a disease or to lessen the severity of such disease or pathological condition. The term “pharmaceutically active peptide or protein” includes entire proteins or polypeptides, and can also refer to pharmaceutically active fragments thereof. It can also include pharmaceutically active analogs of a peptide or protein. The term “pharmaceutically active peptide or protein” includes peptides and proteins that are antigens, z.e., administration of the peptide or protein to a subject elicits an immune response in a subject which may be therapeutic or partially or fully protective. Examples of pharmaceutically active proteins include, but are not limited to, a clustered regularly interspaced short palindromic repeats-associated nuclease (CRISPR Cas enzyme), a meganuclease, a transcription activator-like effector nuclease (TALEN), a reverse transcriptase, or other protein capable of modifying genomic DNA.

[0076] As used herein, the term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, andalso includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In various aspects, the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the disease from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the disease, e.g., arresting its development; or (iii) relieving the disease, e.g., causing regression of the disease.

[0077] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0078] As used herein, the term “diagnosed” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein.

[0079] As used herein, the terms “administering” and “administration” refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intra-aural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.

[0080] As used herein, the term “subject,” refers to an animal which may, in some embodiments, be a mammal. In many embodiments, a subject is a human. A patient refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects. In one aspect, the subject is a mammal such as a primate, and, in a further aspect, the subject is a human. The term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.). In some embodiments, a subject may display, or have been determined to be at risk of displaying, one or more symptoms or side effects of a particular disease, disorder, condition, or state. In some embodiments, a subject may have been diagnosed with a particular disease, disorder, condition, or state. In some embodiments, a subject may have been determined to be at risk of (e.g., via genetic assessment, observation of certain symptoms or side effects, administration of one or more standard tests or assessments, etc.) a particular disease, disorder, condition, or state. In some embodiments, a subject may be or have been diagnosed, or determined to be at risk of, a relevant disease, disorder, or condition, for example, by virtue of having been subjected to one or more standard tests, or may have been identified, without examination, as one at high risk due to the presence of one or more risk factors. In certain embodiments, a subject is a human.

[0081] As used herein, the term “pharmaceutical composition,” refers to a composition that comprises and / or delivers a compound described herein (and / or, in some embodiments, a therapeutically active metabolite thereof) formulated with a pharmaceutically acceptable excipient, and manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a mammal; often a pharmaceutical composition described herein comprises its active agent (e.g., the compound) in a particular salt form (e.g., a pharmaceutically acceptable salt form). Those skilled in the art will appreciate that, in some embodiments, a pharmaceutical composition may be formulated, e.g., may have a form and / or may include particular excipient(s), for administration by a particular route - for example, for intravenous administration or otherwise as described herein and / or understood by those skilled in the art.

[0082] As used herein, the terms “therapeutic agent” includes any synthetic or naturally occurring biologically active compound or composition of matter which, when administered to an organism (human or nonhuman animal), induces a desired pharmacologic, immunogenic, and / or physiologic effect by local and / or systemic action. The term thereforeencompasses those compounds or chemicals traditionally regarded as drugs, vaccines, and biopharmaceuticals including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs and the like.

[0083] As used herein, the terms “pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier,” refer to an ingredient in a pharmaceutical composition that is understood or considered not to be pharmaceutically “active”. For example, a pharmaceutically acceptable excipient or carrier is or comprises one or more ingredients (e.g., a vehicle capable of suspending or dissolving the active agent) other than a compound described herein and having the properties of being nontoxic and non-inflammatory in a patient. In some embodiments, excipients may include one or more of, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, or waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene, calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (com), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0084] The term “stable” as used herein with reference to the nanoparticles of the present invention, means that these nanoparticles maintain their size, surface charge and composition in physiological conditions and during storage.

[0085] The term “lipid” as used herein refers to a group of organic compounds that are insoluble in water but soluble in nonpolar solvents such as oils and fats. Lipids are essential components of cell membranes and play crucial roles in biological processes such as energy storage, cell signaling, and structural support. Examples of different kinds of lipids include, but are not limited to phospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin), steroids (e.g., estrogen, testosterone, and cortisol), triglycerides, waxese.g., beeswax and carnauba wax), glycolipids (e.g., cerebrosides and gangliosides) and fatsoluble vitamins (e.g., vitamins A, D, E, and K).

[0086] The term "sustained release" as used herein with reference to the nanoparticles of the present invention refers to release of a substance, e.g. therapeutic agent, over an extended period of time in contrast to a bolus type administration in which the entire amount of the substance is made biologically available at one time.

[0087] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related.ABBREVIATIONS

[0088] ANOVA, one way analysis of variance

[0089] Cas, CRISPR-associated protein

[0090] CRISPR, clustered regularly interspaced short palindromic repeats

[0091] DI, deionized

[0092] DLS, dynamic light scattering

[0093] EE, encapsulation efficiency

[0094] FBS, fetal bovine serum

[0095] FDA, United States Food and Drug Administration

[0096] GAPDH, glyceraldehyde 3 -phosphate dehydrogenase

[0097] GFP, green fluorescent protein

[0098] HEPES, 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid

[0099] IACUC; Institutional Animal Care and Use Committee

[0100] IFNy, interferon gamma

[0101] IL-6, interleukin 6

[0102] Ki67, proliferation marker protein Ki-67

[0103] LNPs, lipid nanoparticles

[0104] LPS, Lipopolysaccharide

[0105] mRNA, messenger RNA

[0106] NSG, NOD-scid gamma

[0107] NUF2, kinetochore protein Nuf2

[0108] OD, optical density

[0109] pDNA, plasmid DNA

[0110] PEI, polyethyleneimine

[0111] PKC, protein kinase C

[0112] PLL, poly(Z-lysine)

[0113] PNPs, polymer nanoparticles

[0114] siRNA, small interfering RNA

[0115] TALEN, transcription activator-like effector nuclease

[0116] TBS, Tris-buffered saline

[0117] TBST, TBS 0.1% Tween 20

[0118] TEM, transmission electron microscopy

[0119] TLR4, toll-like receptor 4

[0120] TNFa, tumor necrosis factor alphaDETAILED DESCRIPTION

[0121] A description of example embodiments follows.

[0122] Delivery of pharmaceutical agents to specific targets faces numerous challenges, including systemic toxicity, limited bioavailability, and non-specific distribution within the body. Nanoparticles offer a promising solution to these challenges by providing a versatile platform for targeted drug delivery. Their small size, tunable surface properties, and ability to encapsulate a wide range of pharmaceuticals make nanoparticles ideal candidates for improving drug delivery efficacy. Nanoparticles can protect encapsulated pharmaceuticals from degradation and clearance, prolonging circulation time and enhancing drug bioavailability. Moreover, the ability to modulate nanoparticle properties, such as size, shape, and surface charge, enables tailored approaches for overcoming biological barriers, including the blood-brain barrier and tumor microenvironment.

[0123] Described herein are compositions comprising nanoparticles formed from a plurality of anionic polymers, a therapeutic agent, and a cation. Typically, at least 75% of the nanoparticles have a diameter of 20 nm to 40 nm as measured by transmission electron microscopy. One or more of the plurality of anionic polymers can be selected from the group consisting of alginate and hyaluronic acid (hyaluronan). In some embodiments, the cation is a divalent cation, such as Ca2+.

[0124] In some embodiments, the therapeutic agent is a nucleic acid, such as RNA or DNA. The nucleic acid can be short interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), guide RNA, or plasmid DNA (pDNA). Thenucleic acid can be capable of silencing of a gene associated with a disease, disorder, or condition selected from the group consisting of cancer, a metabolic, a neurodegenerative, a cardiovascular, an infectious, or an inflammatory disease or disorder.

[0125] According to yet another embodiment, the compositions described herein include RNA as the therapeutic agent. The RNA and plurality of anionic polymers can be in a molar ratio from 1 : 1 to 30: 1. In some embodiments, the molar ratio is from 7: 1 to 30: 1. In some embodiments, the molar ratio is 7: 1. In some embodiment, a composition comprises a plurality of anionic polymers consisting of alginate, and the RNA and plurality of anionic polymers are in a molar ratio from 7: 1 to 30: 1.

[0126] Described herein is a method for making a nanoparticle composition. The method involves mixing i) a first liquid composition comprising a plurality of anionic polymers with ii) a second liquid composition comprising a therapeutic agent and a cation, in a microfluidic device, thereby producing a composition of nanoparticles. Typically, at least 75% of the nanoparticles have a diameter of 50 nm to 60 nm as measured by transmission electron microscopy.

[0127] In some embodiments, the first and second compositions are mixed in a ratio of 1 : 1 to 1 :30. In some embodiments, the ratio is about 7:1.

[0128] In embodiments described herein, at least 75% of the nanoparticles have a diameter of 20 nm to 40 nm. Precise control over nanoparticle size is crucial for developing drug delivery systems to achieve desired therapeutic outcomes while minimizing adverse effects. Nanoparticle size has significant impact on the efficacy and safety of nanoparticles. The size of nanoparticles influences various crucial factors including their circulation time, biodistribution, cellular uptake, and release kinetics. Nanoparticles within a specific size range exhibit enhanced permeation and retention effects, facilitating their accumulation at target sites such as tumors with compromised vasculature, thereby improving drug delivery efficiency. Furthermore, smaller nanoparticles possess larger surface area-to-volume ratios, enabling higher drug loading capacity and potentially more efficient drug release kinetics. Conversely, excessively large nanoparticles may face challenges in penetrating biological barriers and suffer from rapid clearance by the reticuloendothelial system, leading to reduced therapeutic efficacy. Moreover, nanoparticle size can influence their interaction with biological components, affecting biocompatibility and potential immunogenicity.

[0129] In embodiments described here, the nanoparticles are formed of two or more different types of anionic polymers, such as alginate and hyaluronic acid. This is particularlyimportant due to enhancing the stability, efficacy, and versatility of the nanoparticles for drug delivery applications. Each type of anionic polymer possesses unique physicochemical properties, such as molecular weight, charge density, and hydrophilicity, which can influence nanoparticle formation, stability in biological environments, and interactions with biological targets. By combining different anionic polymers, it is possible to tailor the characteristics of the nanoparticles to meet specific requirements, such as prolonged circulation time, targeted delivery to specific tissues or cells, and controlled release of therapeutic agents.Compositions

[0130] Embodiments described herein are compositions of nanoparticles formed from a plurality of anionic polymers, a therapeutic agent, and a cation. Typically, at least 75% of the nanoparticles have a diameter of 10 nm to 70 nm. In some embodiments, at least 75% of the nanoparticles have a diameter of 20 nm to 40 nm. Particle size can be measured using methods known in the art, such as transmission electron microscopy.

[0131] The terms “ENVOYER” and “ENVOYER nanoparticles” refer to the nanoparticles disclosed herein. When used in reference to a therapeutic agent, e.g., a nucleic acid, the term “ENVOYER-encapsulated” (e.g., ENVOYER-encapsulated siRNA, ENVOYER-encapsulated pDNA) means an ENVOYER nanoparticle comprising the specified therapeutic agent. The term “empty ENVOYER nanoparticles” refers to a composition comprising a plurality of anionic polymers and a cation, but which does not comprise a therapeutic agent (e.g., pDNA or siRNA).

[0132] The anionic polymers described herein can be natural polymers characterized by a negative charge under physiological conditions, typically within the pH range of 7.2 to 7.5, for example, from 7.3 and 7.4. An anionic polymer in this context may be derived from natural polysaccharides. Examples of anionic natural polysaccharides are hyaluronic acid (HA) and alginate (Alg)

[0133] Certain anionic polymers exhibit inherent biological activity or specificity within the human body, thereby augmenting their targeting and uptake by specific cells. For instance, hyaluronic acidleverages hyaluronic acid receptors involved in pivotal biological functions like endocytosis and signal transduction. Notably, receptors such as Cluster of Differentiation 44 (CD44), receptor for hyaluronic acid-mediated motility (RHAMM), and lymphatic vessel endothelial hyaluronan receptor- 1 (LYVE-1) have been identified as key players in mediating the biological effects of hyaluronic acid.

[0134] Conversely, alginate is a plant-derived anionic polymer lacking inherent biological specificity within the human body.

[0135] In view of the above, in one aspect, compositions are in the form of a nanoparticles formed from a plurality of anionic polymers, a therapeutic agent (e.g., a nucleic acid), and a cation. The nanoparticles have a diameter of 20 nm to 40 nm. The nanoparticles comprise the plurality of anionic polymers, the therapeutic agent, and the cation.

[0136] In certain embodiments, the anionic polymer includes at least one polymer selected from the group consisting of hyaluronic acid (HA) and alginate (Alg). In some embodiments, the plurality of anionic polymers includes alginate. In some embodiments, the plurality of anionic polymers includes alginate and hyaluronic acid.

[0137] In certain embodiments, the cation may be a divalent cation or a multivalent cation. In some embodiments, the cation may be a divalent cation, such as Ca2+. In some embodiments the cation is a divalent cation, such as Ca2+.

[0138] In some embodiments, the therapeutic agent is negatively charged (e.g., a nucleic acid). The cation forms interactions, with the negatively charged nucleic acid and the negatively charged anionic polymer, forming a complex comprising the nucleic acid, the cation and the anionic polymers. The interaction with the cation may be in the form of a cation bridge. In certain embodiments, the cation is Ca2+, and the interaction between the nucleic acid and the anionic polymer is mediated by electrostatic interactions with calcium ions. In certain embodiments, the calcium cation is not in the form of calcium phosphate. In certain embodiments, the cation is provided as a salt that is a strong electrolyte, i.e., it is substantially dissociated in aqueous solution. For example, the electrolyte may have a degree of dissociation that is close to 1. In certain embodiments, the cation is Mg2+.

[0139] In certain embodiments, the therapeutic agent is a nucleic acid. The nucleic acid can be DNA, such as a plasmid DNA (pDNA) or an oligodeoxynucleotide (ODN). The nucleic acid can be RNA, such as short interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), or guide RNA (gRNA) (sometimes referred to as single guide RNA (sgRNA). In some embodiments, the nucleic acid is siRNA. In some embodiments, the nucleic acid comprises chemical modifications, e.g., 2'-O-methylation (2'- O-Me), one or more locked nucleic acid (LNA) bases, phosphorothioate linkages, and the like. Additional nucleic acid chemical modifications are well known to those of skill in the art.

[0140] The nucleic acid can be capable of silencing of a gene associated with a genetic disease, disorder, or condition. The disease, disorder, or condition can be selected from the group including cancer, a metabolic, a neurodegenerative, a cardiovascular, an infectious, or an inflammatory disease or disorder. In some embodiments, the nucleic acid comprises a sequence complementary to a portion of a gene or a transcript associated with a disease, disorder, or condition, thereby facilitating hybridization of the nucleic acid with the gene or transcript and silencing of the gene or transcript.

[0141] The molar ratio the of plurality of anionic polymers to the nucleic acid can vary depending on the molecular weight of the anionic polymer, and can be from 100: 1 to 0.01 : 1, from 50:1 to 0.01:1, from 20:1 to 0.01:1, from 18:1 to 0.01:1, from 16:1 to 0.01:1, from 14:1 to 0.01:1, from 12:1 to 0.01:1, from 10:1 to 0.01:1, from 8:1 to 0.01:1, from 6:1 to 0.01:1, from 4:1 to 0.01:1, or from 2:1 to 0.01:1, from 10:1 to 0.05:1, from 5:1 to 0.05:1, from 3:1 to 0.05:1, from 1:1 to 0.05:1, from 10:1 to 1:1, from 5:1 to 1:1, or from 3:1 to 1:1, or said ratio of anionic polymer to RNA or of RNA to anionic polymer is 100:1, 50:1, 20:1, 18:1, 16:1, 14:1, 12:1, 10:1, 8:1, 6:1, 4:1, 2.5:1, 2:1, 1:1, 0.8:1, 0.4:1, 0.25:1, 0.1:1 or 0.08:1.

[0142] In some embodiments, the therapeutic agent is RNA. The RNA and the plurality of anionic polymers can be in a molar ratio from 1:1 to 30: 1. In some embodiments, the RNA and the plurality of anionic polymers are in a molar ratio from 7: 1 to 30: 1. In some embodiments, the RNA and the plurality of anionic polymers are in a molar ratio of 7: 1. In some embodiment, the plurality of anionic polymers includes alginate, wherein the RNA and plurality of anionic polymers are in a molar ratio from 7:1 to 30:1. In some embodiment, the plurality of anionic polymers includes alginate, wherein the RNA and plurality of anionic polymers are in a molar ratio from 1:1 to 30: 1. In some embodiment, the plurality of anionic polymers includes alginate and hyaluronic acid, wherein the RNA and plurality of anionic polymers are in a molar ratio from 1:1 to 30: 1. In some embodiment, the plurality of anionic polymers includes hyaluronic acid, wherein the RNA and plurality of anionic polymers are in a molar ratio from 1 : 1 to 30: 1. In some embodiment, the plurality of anionic polymers includes hyaluronic acid, wherein the RNA and plurality of anionic polymers are in a molar ratio from 1 : 1 to 30: 1.

[0143] In some embodiments, the composition according to present disclosure comprises a nucleic acid encoding at least one pharmaceutically active peptide or protein as the therapeutic agent. The therapeutic agent may encode a protein that needs to be delivered to a cell in a subject. The protein may be selected from the group consisting of a clusteredregularly interspaced short palindromic repeats-associated nuclease (CRISPR Cas enzyme), a meganuclease, a transcription activator-like effector nuclease (TALEN), a reverse transcriptase, or other protein capable of modifying genomic DNA or associated structures, such as histones. Examples of proteins capable of modifying genomic DNA or associated structures include histone methyltransferase, histone demethylases, nucleases (e.g., zinc finger nucleases), lysine acetyltransferases, histone deacetylases, DNA methyltransferases, ten-eleven translocation (TET) dioxygenases, and the like.

[0144] Compositions according to present disclosure include nanoparticles, wherein at least 75% of the nanoparticles have a diameter of 20 nm to 40 nm as measured by transmission electron microscopy. In some embodiments, at least 80% (or 85%, or 90%, or 95%, or 98%, or 99%) of the nanoparticles have a diameter of 20 nm to 40 nm. In some embodiments, at least 98% of the nanoparticles have a diameter of 20 nm to 40 nm.

[0145] Zeta potential is a measurement of surface potential of a particle. In some embodiments, the zeta potential of the produced nanoparticles is a positive value. In some embodiments, the zeta potential of the nanoparticles is more positive than 5 mV.Pharmaceutical Compositions

[0146] In another aspect, described herein is a pharmaceutical composition comprising the nanoparticles of the present invention as defined hereinabove and a pharmaceutically acceptable carrier.

[0147] Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0148] The following exemplification of carriers, modes of administration, dosage forms, etc., are listed as known possibilities from which the carriers, modes of administration, dosage forms, etc., may be selected for use with the present invention. Those of ordinary skill in the art will understand, however, that any given formulation and mode of administration selected should first be tested to determine that it achieves the desired results.

[0149] Methods of administration include, but are not limited to, parenteral, e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, mucosal (e.g., oral, intranasal, buccal, vaginal, rectal, intraocular), intrathecal, topical and intradermal routes.Administration can be systemic or local. In some embodiments, method of administration isintravenous. In some embodiments, method of administration is intraperitoneal. In some embodiments, method of administration is direct injection to an organ affected by the underlying disease or disorder.

[0150] The compositions can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multidose containers, with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen free water, before use. Doses of the compositions may be expressed, for example, as a mass of a composition or component of a composition per mass body weight of a subject, e.g., mg / kg.

[0151] The composition can be formulated for immediate release or sustained release.Compositions for Delivery to Cells

[0152] In a further aspect, compositions are for use in the delivery of a therapeutic (e.g., a nucleic acid) to cells. For this purpose, the plurality of anionic polymers can be in a complex with a cation and the nucleic acid to be delivered to the cells. The plurality of anionic polymers and the cation are as described above.

[0153] The cells, to which the nucleic acid is delivered according to any one of the different aspects of the present invention, can be selected from the group consisting of cells in culture, either adherent to a substrate or in suspension, and cells in a living tissue such as solid tissue and blood, e.g., cells that are part of a living organism. These cells may be diseased cells, such as cancer cells, and therefore, the compositions of the present invention may be useful in gene therapy, for example, wherein the gene therapy comprises controlling the expression level of a gene. The cells can further be selected from various types of cells, such as immune cells, skin cells, stem cells, nerve cells, muscle cells or endothelial cells. The gene therapy may control the expression level by decreasing it or increasing it. The gene therapy may be used to treat diseases, disorders or conditions that are amenable to intervention by decreasing or increasing the expression of certain genes, the aberrant expression of which causes, or is associated with, the diseases, disorders or conditions.

[0154] In some embodiments, the present invention provides a method for prevention or treatment of a disease, disorder or condition in a subject in need thereof, comprisingadministering to said subject a pharmaceutical composition as defined herein above. The administered composition can transduce cells and release the therapeutic agent. The disease, disorder or condition may be associated with a genetic origin. The disease or disorder can be selected from the group consisting of cancer, a metabolic, a neurodegenerative, a cardiovascular, and an infectious or inflammatory disease or disorder.Methods of Microfluidic Preparation of Nanoparticles

[0155] Microfluidic devices offer a highly precise and efficient means for the production of nanoparticles. Microfluidic devices enable the controlled manipulation of fluids at the microscale, allowing for precise control over particle size, uniformity, and composition. The microfluidic approach facilitates the formation of nanoparticles through controlled mixing of lipid or biopolymer components and nucleic acids, resulting in the rapid and homogeneous formation of nanoparticles with tailored characteristics. Furthermore, microfluidic platforms offer advantages such as reduced solvent consumption, shortened processing times, and the potential for integration with downstream processes, making them an attractive option for the streamlined production of nanoparticles for therapeutic applications. Importantly, microfluidic devices allow controlling the size of nanoparticles, which is of paramount importance due to its significant impact on the efficacy and safety of the delivered therapeutic agents. The size of nanoparticles influences various crucial factors including their circulation time, biodistribution, cellular uptake, and release kinetics. Nanoparticles within a specific size range exhibit enhanced permeation and retention (EPR) effects, facilitating their accumulation at target sites such as tumors with compromised vasculature, thereby improving drug delivery efficiency. Furthermore, smaller nanoparticles possess larger surface area-to- volume ratios, enabling higher drug loading capacity and potentially more efficient drug release kinetics.

[0156] An embodiment according to the invention provides a method for making a nanoparticle composition, the method comprising: in a microfluidic device, mixing: i) a first liquid composition comprising a plurality of anionic polymers, with ii) a second liquid composition comprising a therapeutic agent and a cation, thereby producing a composition of nanoparticles, wherein at least 75% of the nanoparticles have a diameter of 10 nm to 70 nm as measured by transmission electron microscopy. In some embodiments, at least 75% of the nanoparticles have a diameter of 20 nm to 40 nm as measured by transmission electron microscopy.

[0157] Some variations of the method may include introducing a first liquid composition comprising a plurality of anionic polymers at a first flow rate through a microfluidic instrument in a first flow stream, moving a second liquid composition comprising a therapeutic agent and a cation at a second flow rate through the microfluidic instrument in a second flow stream, wherein the first liquid composition in the first flow stream and the second fluid composition in the second flow stream are mixed in at least one junction point, wherein a nanoparticle composition is produced. The nanoparticle produced from mixing the first liquid composition and the second fluid composition can be further flown into a straight channel. The first flow rate and the second flow rate may be used to define the molar ratio between the plurality of anionic polymers, the therapeutic agent and the cation in nanoparticles. The first flow rate, the second flow rate, or both the first and second flow rates may be used to control the size of the nanoparticles that are produced. The first flow rate, the second flow rate, or both the first and second flow rates may be set at a target flow rate to achieve a target diameter of the nanoparticles produced by the microfluidic instrument. In some embodiments, the first and second liquid compositions are mixed in a ratio of 1 : 1 to 1 :30. In some embodiments, the ratio is 7: 1.

[0158] Methods of producing nanoparticles according to present invention using a microfluidic device are described herein. A microfluidic device with precise control over fluid flow rate is employed. An anionic polymer solution, comprising one or more anionic polymers such as hyaluronic acid (HA), alginate (Alg), , is introduced into one inlet of the microfluidic device. Simultaneously, a second solution comprising a pre-mixed combination of a therapeutic agent and a cation, is introduced into another inlet. Within the microfluidic device, the solutions are mixed and subjected to controlled flow conditions, facilitating the formation of nanoparticles through the complexation of the anionic polymers with the therapeutic agent via interactions with the cation. This microfluidic-based method enables precise control over particle size, composition, and uniformity, leading to reproducible and scalable production of nanoparticles suitable for therapeutic applications.

[0159] In some embodiments, a microfluidic device comprises a microfluidic cartridge. In some embodiments, the microfluidic device and / or cartridge comprises one or more nanoscale microfluidic channels through which solutions flow. In some examples, the microfluidic channels have a width or diameter of about 1 pm.

[0160] In some embodiments, nanoparticles are produced using the principle of external gelation, z.e., controlled diffusion. As used herein, external gelation refers to a method inwhich a solution comprising a crosslinker (e.g., a pre-mixed combination of a therapeutic agent and a cation, wherein the cation is the crosslinker) is added to a solution comprising an anionic polymer. An example of external gelation is described by Goldshtein et al. (2019).

[0161] In one embodiment, nanoparticles comprising green fluorescent protein (GFP) plasmid DNA (pDNA) are synthesized. Briefly, the synthesis involves the natural polymer and a crosslinker (e.g., Ca2+) to encapsulate 150 ng / pL (final concentration) green fluorescent protein plasmid DNA (GFP-pDNA) in 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES) buffer. Ionizable lipid mix (ILM) is used to encapsulate GFP pDNA to a final concentration of 150 ng / pL.Characterization of Nanoparticles by Transmission Electron Microscopy

[0162] Nanoparticle are applied to 200 mesh carbon coated copper grids that have been pretreated withl% alcian blue ([[A,M,A",A'''-[(29J / ,3 U / -Phthalocyanine-C,C,C,C-tetrayl- K? / 29,KA30,KA31,KA32)tetrakis[methylenethio[(dimethylamino)methylidyne]]]tetrakis[A- methylmethanaminiumato]](2-)]copper(4+) chloride (1 :4)) for 1 minute and subsequently rinsed through 4 drops of DI water. Grids are then placed onto a 10 pl droplet of sample for 1 minute. Excess sample is then wicked off onto filter paper before rinsing through 3 drops of DI water. The sample is then negatively stained by placing the grid onto a drop of 2% Uranyl Acetate 3 or 4 times and wicking off excess stain onto filter paper after each stain treatment. The grids are then thoroughly dried and viewed on a JEOL JEM1010 TEM. Representative images are recorded using an AMT digital camera system.Measurement of Transfection Efficiency

[0163] The efficiency of nanoparticle-mediated delivery of nucleic acid therapeutics can be determined by common methodology in the art. The following is an example protocol for determining efficiency of nanoparticle-mediated delivery of nucleic acid therapeutics.

[0164] PANCI and HEK293T cells are transfected with approximately 5 - 8 pg of either nanoparticles comprising a plurality of anionic polymers, GFP-pDNA and a cation, or using LIPOFECTAMINE® for 24 and 48 hours. Transfection efficiency via GFP expression in cells is quantified by fluorescence microscopy (ECHO), western blotting, and flow cytometry (Attune). 7- Aminoactinomycin D (7AAD) staining is used for detection of apoptotic cells.Measurement of Encapsulation Efficiency

[0165] The yield and encapsulation efficiency (EE) of pDNA can be calculated using PICOGREEN® assay (Themo Fisher). In one exemplary embodiment, PANCI cells are transfected with approximately 4pg of either nanoparticles comprising a plurality of anionic polymers, a cation and GFP-pDNA or lipid nanoparticles comprising GFP-pDNA for 48 hours. Nanoparticle compositions comprising the plurality of anionic polymers and the cation are used as a negative control. The GFP expression is evaluated using fluorescence microscopy and western blotting technique.Nuclease Protection Assay

[0166] The stability of RNA in anionic polymer-based RNA nanoparticles against RNase A digestion, with or without serum presence, is investigated. Aliquots of RNA, with or without serum (10% final, v / v), are incubated with aliquots of enzyme, for 24 hours at 37° C. Afterwards, ethidium bromide (EtBr) exclusion assay can be used to quantify the results (see, for example, US 10,415,035 B2).Assessment of Nucleic Acid Uptake

[0167] Cellular uptake of nanoparticles comprising fluorescent anionic polymers, fluorescent nucleic acids and cations, and internalization of fluorescent nucleic acids and anionic polymers is visualized by fluorescence microscopy (ECHO), western blotting, and flow cytometry (Attune). 7AAD staining was used for detection of apoptotic cells.Cell Viability

[0168] Cytotoxicity of nanoparticles can be assessed by common methodology in the art. Model cells are treated with nanoparticles loaded with therapeutic agents dispersed in a suitable vehicle (test group). A control group of cells is treated with the vehicle alone. Following treatment, cells are incubated for a predetermined period to allow for interaction with nanoparticles and therapeutic cargo. Subsequently, cell viability is assessed using a standard viability assay such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction or AlamarBlue (resazurin, i.e., 7-hydroxy-10-oxidophenoxazin-10- ium-3-one). Absorbance or fluorescence measurements are taken to quantify metabolic activity and cell proliferation. Cytotoxicity of nanoparticles is determined by comparing viability measurements between the test group and the control group.Gene Silencing Studies

[0169] Efficiency of nanoparticle compositions for nucleic acid delivery and inducing effective gene silencing, may be assessed for siRNA against enhanced GFP (eGFP) for investigating the silencing efficacy in eGFP-expressing cells, as a model system. Analysis of silencing at the mRNA level may be performed by qPCR analysis.Stability Studies

[0170] Serum stability of nanoparticles can be assessed by common methodology in the art. To assess whether nucleic acid therapeutic agent in the nanoparticle is stable, nanoparticles are stored, for a specific amount of time in serum. Changes in the molecular weight or degradation products of the encapsulated nucleic acids are monitored, e.g. by microscopy and gel-based assays, to determine stability.

[0171] In one embodiment, freshly prepared nanoparticles comprising GFP-pDNA is equally divided and stored at room temperature, 4°C, -20°C and -80°C. After 24 hours of storage, a 6-well plate of PANCI cells are treated with the nanoparticle samples stored at different conditions. PANCI cells for all the conditions are incubated for 48 hours. The GFP expression is evaluated using fluorescence microscopy and western blotting technique.Statistical Analysis

[0172] Statistical analysis of data can be done by common methodology in the art. In some embodiments, GraphPad Prism 8.0 software is used for constructing graphs and analyzing statistical significance. In some embodiments, FlowJo vlO software is used for flow cytometry analyses. In some embodiments, Student 's t-test and log-rank (Mantel-Cox) test is used to calculate statistical significance.EXEMPLIFICATION

[0173] Gene therapy is a promising clinical approach for treating or preventing genetic diseases by directly targeting disease-causing mutations. Despite the potential for gene therapy in addressing a broad range of genetic diseases, the development of these techniques remains in its early stages. Efficient delivery systems for gene therapy must overcome hurdles such as immune responses, off-target effects, and limited cellular uptake. Biomedical polymeric nanoparticles (PNPs) can enhance gene therapy delivery by encapsulating,protecting, and releasing therapeutic compounds within target cells. However, their clinical translation is hindered by poor efficacy and storage instability.

[0174] To address these challenges, ENVOYER nanoparticles were formulated. ENVOYER nanoparticles are a natural polymer-based delivery system, e.g., non-lipid biopolymeric nanoparticles formulated via external gelation microfluidics. As disclosed herein, ENVOYER’ s characteristics are described and the stability and transfection efficiency of ENVOYER nanoparticles in multiple human cell lines was evaluated.

[0175] The physicochemical characteristics of ENVOYER nanoparticles were determined by transmission electron microscopy, dynamic light scattering and zeta potential measurement. Encapsulation efficiency and ENVOYER stability were assessed. ENVOYER immunogenicity was tested by activation of toll-like receptor 4 (TLR4) in human embryonic kidney (HEK) Blue TLR4 cells transfected with ENVOYER-encapsulated scrambled small interfering RNA (siRNA) and by cytokine activation in mice injected with ENVOYER- encapsulated scrambled siRNA. Time course and dose studies were conducted in PANCI cells to evaluate the knockdown efficiency of ENVOYER-encapsulated survivin siRNA. The effect of ENVOYER-encapsulated NUF2 siRNA on tumor growth was evaluated in an animal tumor model. ENVOYER particles consistently measured 20-30 nm with a surface charge of -8.4 mV and encapsulation efficiency of 95%. At the siRNA working concentration (about 50-140 nM), Storage at -80°C and freeze-thaw cycles did not affect transfection efficiency. ENVOYER-encapsulated scrambled siRNA did not activate TLR4 compared to lipopolysaccharides, or increase inflammation-related cytokines in mice, confirming low immunogenicity. ENVOYER-encapsulated survivin siRNA demonstrated cellular uptake and dose- and time-dependent efficacy. In PANCI cells, 62.5 nM siRNA reduced survivin mRNA and protein levels significantly, with greater reductions at 125 and 250 nM. Effects were observed within 3 hours and persisted over 48 hours. Intratumoral injection of ENVOYER-encapsulated NUF2 siRNA in NOD-scid gamma mice reduced tumor growth by 60% without affecting body weight. Tumor analysis confirmed successful delivery and siRNA target engagement, indicating that ENVOYER nanoparticles are both safe and effective in rodent models of tumorigenesis.

[0176] In HEK293T cells, ENVOYER nanoparticles (50-70 nm) encapsulating green fluorescent protein (GFP) plasmid DNA (pDNA) achieved a transfection efficiency of >80%, outperforming LfPOFECTAMINE® controls (about 60%). In PANCI pancreatic cancer cells, known for being difficult to transfect, ENVOYER nanoparticles also demonstrated superiorefficacy compared to LIPOFECT AMINE® controls. Notably, commonly used commercially available nanoparticles failed to produce GFP expression under similar conditions. Preliminary mechanistic studies suggest that ENVOYER nanoparticle internalization occurs via clathrin-independent endocytosis. Furthermore, ENVOYER nanoparticles were shown to maintain stability and transfection efficiency after storage for 2 months at 4°C and -80°C. Collectively, these findings highlight the potential of ENVOYER nanoparticles as a stable and efficient gene delivery platform for clinical applications in gene therapy.

[0177] ENVOYER encapsulation of DNA or siRNA is a stable and efficient delivery system for gene therapy.Example 1: In vitro characterization of polymeric nanoparticles encapsulating plasmid DNA for gene therapy

[0178] Gene therapy is a promising clinical approach for treating or preventing genetic conditions by directly targeting disease-causing genetic mutations. This innovative strategy has potential applications in managing a broad range of indications such as cancer, infectious diseases, autoimmune diseases, and genetic disorders (Goncalves and Paiva, 2017; Sayed et al., 2022). Despite its promise, gene therapy is still in the early stages of development. A persistent challenge in the field is achieving efficient and targeted delivery of modified therapeutic genetic material, such as messenger ribonucleic acid (mRNA) or deoxyribonucleic acid (DNA), to the intended biological target while minimizing degradation, toxicity, or off-target effects (Goncalves and Paiva, 2017; Uddin etal., 2020; Bulcha et al., 2021). The success of gene delivery vehicles depends on several critical factors, including entrapment efficiency, nanoparticle size, and surface chemistry (Mitchell et al., 2020). Additionally, physiological and biological barriers such as immune responses, circulation dynamics, stability, and clearance pose significant challenges that must be addressed in designing efficient delivery systems (Mitchell et al., 2020; Bulaklak et al., 2020). Overcoming these challenges is key to realizing the full potential of gene therapy.

[0179] Current gene delivery systems include both viral vectors (e.g., retroviruses and adenovirus-associated viruses) and non-viral vectors (e.g., lipoplexes, polymer nanoparticles (PNPs), and inorganic nanoparticles) (Sung and Kim, 2019; Pan et al., 2021). Viral vectors are predominately used in current gene therapy clinical studies due to their natural ability to infect cells and efficiently deliver genetic material (Sung and Kim, 2019; Zhao et al., 2022). However, their use is limited by high immunogenicity, gene cargo size limitations,cytotoxicity, risks of insertional mutagenesis, translational challenges from preclinical to clinical research, and high manufacturing costs (Sung and Kim, 2019; Zhao et al., 2022). Non-viral vectors, in contrast, allow for larger gene load, easier production, and lower risks of immune reactions, cytotoxicity, and mutagenesis (Sung and Kim, 2019; Zhao etal., 2022; Ramamoorth and Narvekar, 2015; Zu and Gao, 2021).

[0180] Due to their simple synthesis and functionality, low immunogenicity, and low toxicity (Roma-Rodrigues et al., 2020), there is now a drive towards the use of these non- viral nanoparticles in clinical gene therapy (Chen et al., 2020). These options offer the potential of a higher transfection efficiency and slower blood clearance with less adverse effects and lower production costs when compared with viral approaches (Chen et al., 2016; Miron-Barroso et al., 2021). A variety of materials can be used to create non-viral nanoparticles including organic materials (e.g., phospholipids), polymers (e.g., chitosan and PLGA), and inorganic materials (e.g., gold and iron) (Mitchell et al., 2020). Thus far, lipid nanoparticles (LNPs) have been particularly promising for use in gene therapy due to low immunogenicity, favorable safety profile compared to viral vectors, biodegradability, stability, superior delivery outcome with mRNA and small interfering ribonucleic acid (siRNA), and surface chemistry (Menon et al., 2022; Mehta et al., 2023). For these reasons, there are currently a number of proposed clinical trials and several underway utilizing LNPs (Roma-Rodrigues et al., 2020). However, formulation for clinical use is difficult since LNPs must withstand physiological environments long enough to reach the target and must remain stable in storage before use (Mendonga er al., 2023). Currently, the shelf life of the majority of LNPs is short and temperature-dependent, necessitating the development of more stable LNPs for long-term storage (Hou et al., 2021). Thus, while LNPs offer therapeutic potential, use in the clinic still requires multi-component formulation development, further reduction of toxicity and immunogenicity, improvement of DNA delivery, enhanced nanoparticle stability in biological environments, and cost-effective production to maximize therapeutic advantages (Mehta et al., 2023; Hald Albertsen et al., 2022).

[0181] Of the non-viral delivery vectors available for gene therapy, PNPs derived from both natural sources offer the same benefits of LNPs without the challenges of suboptimal stability in both storage containers and biological environments. Specifically, PNPs demonstrate the ability to condense genetic material and protect it from degradation, while controlling the release of payloads and facilitating cellular uptake (Niculescu and Grumezescu, 2021). Examples of natural polymers used in nanoparticles for biomedicalapplications include chitosan, hyaluronic acid, dextran, and P-cyclodextrin, all of which exhibit desirable levels of biocompatibility, biodegradability, and toxicity (Chen etal., 2020; Eroglu et cd.. 2017; Galisova et al.^ 2020; Bhardwaj et cd.. 2023).

[0182] To overcome the challenges of current gene delivery systems, ENVOYER nanoparticles, natural polymer-based nanoparticles formulated using a methodology adapted on the principles of external gelation and microfluidics, were developed. Importantly, the polymer used in the scope of this study has already been approved by the United States Food and Drug Administration (FDA) for biomedical application. As described herein, the plasmid DNA (pDNA) encapsulation and subsequent cellular delivery potential of these nanoparticles was examined and reported. In addition to the analysis on the analytical properties of these nanoparticles, their cellular transfection efficiency and stability under various storage conditions was evaluated. Together, the disclosed findings highlight the potential of ENVOYER nanoparticles for clinical applications in gene therapy.MethodsPreparation and characterization of ENVOYER nanoparticles

[0183] The ENVOYER nanoparticles were synthesized at Envoya Inc. (Natick, MA, USA) via a method based on the principle of external gelation. Nanoparticles were synthesized using sodium alginate (Catalog No. #42000501-5G, Merck KGaA, Darmstadt, Germany) and calcium chloride (CaCh) as the crosslinker to encapsulate green fluorescent protein (GFP) pDNA 5757 base pairs (bp) in length (gWiz™, Aldevron, Fargo, ND, USA) in 500 mM 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES; Catalog No.15630-080, Thermo Fisher Scientific Inc., Waltham, MA, USA) buffer at a final concentration of 150 ng / pL. The external gelation methodology of formulation was adapted from a previously described method (Goldshtein et al. 2019) and adjusted for microfluidics technology.

[0184] Alginate nanoparticles encapsulating pDNA were formulated by combining a 600 pg / mL pDNA solution, 1 M CaCh solution, and 50 pg / mL alginate solution in a 1 : 1 :2 volume ratio.

[0185] The 1 M CaCh solution was formed by weighing out solid CaCh and dissolving it EhO. Afterwards, the solution was filtered through a 0.2 pm biological filter to ensure that the solution did not have any biological contaminants or aggregates of salt.

[0186] The pDNA solution was created by diluting a stock solution of the pDNA of interest (e.g., GFP pDNA) in H2O to a concentration of 600 pg / mL. After both solutions were made, they were then mixed in equal volume via pipetting the 1 M CaCh into the 600 pg / mL pDNA solution. Then, the mixture was gently vortexed (e.g., vortexed at a low speed) for 15 seconds, and left to incubate at room temperature for 30 minutes.

[0187] The alginate solution was formed by weighing out solid alginate polymer and dissolving it in HEPES at a concentration of 500 pg / mL of alginate via a stir bar on a stir plate for between 30 minutes to 1 hour. After the alginate was fully dissolved in the HEPES, the solution was filtered through a 0.2 pm biological filter to ensure that the solution did not have any biological contaminants or aggregates of alginate. After filtration, the alginate solution was diluted from 500 pg / mL to 50 pg / mL in HEPES.

[0188] Once the pDNA mixed with CaCh had finished incubating, and the alginate solution was prepared to a 50 pg / mL concentration, the two solutions were taken into syringes at equal volumes, and run through the Ignite NANO ASSEMBLE® nanoparticle formulation instrument (Precision Nanosystems, Vancouver, BC, Canada), which mixed the two solutions via microfluidics. The Ignite was set to dispense the syringes at an equal flow rate ratio, with a flow rate of 12 mL / minute. The starting waste was set to 0.3 or 0.5 mL, depending on whether the syringes were 1 mL or 3 mL syringes, respectively, and the end waste was set to 0.05 mL. Once the Ignite finished dispensing the desired volume, the sample solution was left to sit for 30 minutes at room temperature, after which the particles were considered mature and ready for use. By using the Ignite, the particles were able to maintain a consistent size between batches. The cartridge used for the Ignite mixing was the NxGen NANOASSEMBLR® Ignite Cartridge (Catalog No. #NIN0062, Precision Nanosystems). The tubes used for collection were 15 mL polypropylene tubes.

[0189] A commercially available ionizable lipid mix (ILM), GenVoy-ILM (Catalog No. NWW0041, Precision Nanosystems), was used to encapsulate GFP pDNA to a final concentration of 150 ng / pL according to manufacturer’s instructions to produce lipid nanoparticles (LNPs). The sizes of ENVOYER nanoparticles were assessed by transmission electron microscopy (TEM) via JEM1010 transmission electron microscope (JEOL USA, Inc., Peabody, MA, USA) and dynamic light scattering (DLS) via Zetasizer Nano ZS90 (Malvern Panalytical, Malvern, UK). The size of the ENVOYER nanoparticles depends on the size of the payload. With the payload described (e.g., 5757 bp GFP pDNA), the size of the ENVOYER nanoparticles was around 70 nm in diameter, which is smaller compared toLNPs that have a consistent size (e.g., regardless of how small a payload may be) with an average of over 100 nm in diameter.Cell culture

[0190] The pancreatic tumor cell line, PANCI (CRL-1469-LUC2, American Type Culture Collection (ATCC), Manassas, VA, USA), was cultured in Dulbecco’s Modified Eagle Medium (DMEM; Catalog No. 11965-092, Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Catalog No. A5256801, Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) and 1% penicillinstreptomycin (Catalog No. 15140122, Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA). Human embryonic kidney (HEK) 293T cells (CRL-3216, ATCC, Manassas, VA, USA) were cultured in Minimum Essential Medium (MEM; Catalog No. 32561037, Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% FBS (Catalog No. A5256801, Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) and 1% penicillin-streptomycin (Catalog No. 15140122, Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA). The cells were used to assess cellular uptake and investigate nanoparticle stability. All cells were maintained at 37°C in a humidified incubator with 5% CO2.Transmission electron microscopy

[0191] ENVOYER nanoparticles were applied to 200 mesh carbon coated copper grids that had been pre-treated with 1% alcian blue for one minute and subsequently rinsed with four drops of deionized (DI) water. Grids were placed onto a 10 pL droplet of sample for one minute. Excess sample was wicked onto filter paper before rinsing with three drops of DI water. The sample was negatively stained by placing the grid onto a drop of 2% uranyl acetate three or four times and wicking excess stain onto filter paper after each stain treatment. The grids were thoroughly dried and viewed by TEM on a JEM1010 (JEOL USA, Inc., Peabody, MA, USA) instrument. Representative images were recorded using a digital camera system (AMT Imaging, Woburn, MA, USA).Dynamic light scatering

[0192] ENVOYER nanoparticle size and size distribution were measured by DLS using a ZS90 system (Malvern Panalytical, Malvern, UK). The instrument was set for an equilibration time of 120 seconds and measurement angle of 90° at room temperature (25°C). The ENVOYER nanoparticles were loaded to DTS0012 cuvettes (Malvern Panalytical, Malvern, UK), measured three times back-to-back, and average measurements for each triplicate were reported.Measurement of transfection and encapsulation efficiency

[0193] PANCI and HEK293T cells were transfected overnight with approximately 5 to 8 pg of GFP pDNA encapsulated in either ENVOYER nanoparticles, GenVoy-ILM-derived LNPs, or using LIPOFECT AMINE®. Afterwards, the media was replaced with fresh culture media and cells were collected after 24 and 48 hours. To confirm whether transfection efficiency of ENVOYER nanoparticles was due to the combined effect of the polymer and calcium chloride, rather than solely an artifact of calcium chloride-driven gene transfer, cells were also treated with GFP pDNA (at concentrations similar to ENVOYER nanoparticles) and calcium chloride but without polymers. These cells were subsequently processed in the same manner as those treated with ENVOYER nanoparticles. Transfection efficiency was calculated by quantifying GFP expression in cells using fluorescence microscopy (Revolve, ECHO, San Diego, CA), western blotting, and flow cytometry (Attune Cytpix, Thermo Scientific Inc., Waltham, MA, USA). Encapsulation efficiency (EE) and pDNA yield were calculated using the Quant-iT™ PICOGREEN® (PicoGreen) dsDNA assay (Catalog No. P7589, Thermo Fisher Scientific Inc., Waltham, MA, USA) following manufacturer instructions. Briefly, nanoparticles were diluted in Tris / ethylenediaminetetraacetic acid (EDTA) buffer (TE buffer; Catalog No. P7589B, Thermo Fisher Scientific Inc., Waltham, MA, USA), either with or without Triton-X 100 (X100-100ML, Merck KGaA, Darmstadt, Germany), and then measured for fluorescence in a CLARIOstar plate reader (BMG LABTECH GmbH, Ortenberg, Germany) after adding the diluted PicoGreen reagent (Catalog No. P7589A, Thermo Fisher Scientific Inc., Waltham, MA, USA).Cellular uptake

[0194] HEK293T cells were treated with inhibitors of endocytosis, amiloride (Catalog No. S1811, Selleck Chemicals, Houston, TX, USA) (25 pM) and genistein (Catalog No. S1342, Selleck Chemicals) (150 pM), for 30 minutes and were subsequently transfected with ENVOYER nanoparticles encapsulating 15 pg of GFP pDNA for three hours. The final concentration in the transfection media was 7.5 pg / mL of GFP pDNA. The media in the wells was then replaced with fresh media free from inhibitors and ENVOYER nanoparticles, and the cells were incubated for an additional 24 hours. Fluorescence microscopy performed on a Revolve microscope (ECHO, San Diego, CA, USA) and western blotting were used to quantify transfection efficiency via analysis of GFP expression levels.

[0195] HEK293T cells were transfected with LNPs using LIPOFECTAMINE" as follows. The day before transfection, cells were trypsinized and counted. About 0.5 to 1.25 x 105cells were plated per well in 0.5 mL of complete growth medium. Cell density was about 50-80% confluent (e.g., 70% confluent) on the day of transfection. On the day of transfection, growth medium was removed from cells and replaced with 0.5 mL of transfection medium. Cells were incubated at 37°C in a CO2 incubator for 18-24 hours posttransfection before assaying for transgene expression. Cells typically reached a confluence of about 90% before being assayed.

[0196] Transfection medium was prepared as follows. For each well of cells to be transfected, 0.5 pg of DNA was diluted in 100 pL of Gibco OPTLMEM® I Reduced Serum Media (Catalog No. 31985062, Thermo Fisher Scientific) without serum. For each well of cells, 0.75 to 1.75 pL of LIPOFECTAMINE® LTX Reagent was added into the above diluted OPTLMEM® UNA solution, mixed gently, and incubated 30 minutes at room temperature to form DNA- LIPOFECTAMINE® LTX Reagent complexes. After the 30 minute incubation, 100 pl of the DNA-LIPOFECT AMINE® LTX Reagent complexes was directly added to each well containing cells and mixed gently by rocking the plate back and forth. Complexes did not need to be removed following transfection.Statistical analysis

[0197] GraphPad Prism 8.0 software (GraphPad Software, Boston, MA, USA) was used for constructing graphs and analyzing statistical significance. Flow cytometry analyses wereperformed by FlowJo vlO software (FlowJo, Ashland, OR, USA). Student’s T-test and ANOVA were used to calculate statistical significance.ResultsCharacterization of nanoparticles and size distribution

[0198] TEM images demonstrated that freshly prepared ENVOYER nanoparticles were sized at 50-70 nm (FIG. 1 A). The thread-like products captured by TEM were identified as non-crosslinked biopolymers. The ENVOYER nanoparticle sizes were smaller than those reported for LNPs encapsulating pDNA, which were 120-130 nm in size (Cui et al.. 2022). ENVOYER nanoparticle size was minimally altered after storage at 4°C for 24 hours, measured to be 50-60 nm in size (FIG. IB). The average diameter of the ENVOYER nanoparticles was 72.80 ± 24.76 nm as determined by DLS measurement of size distribution by number (FIG. 1C). The poly dispersity index (PDI) was 0.540, which can be attributed to nanoparticle aggregation and is confirmed by a second peak observed at a much higher diameter when performing size distribution by intensity. The slightly larger size observed with DLS could be due to the assumption that DLS, but not TEM, considered the nanoparticles to be spherical. It is also possibly due to dehydration of samples that occurs when running TEM but not DLS.Transfection efficiencies of ENVOYER nanoparticles in human cell lines

[0199] In PANCI cells, the transfection efficiency of the ENVOYER nanoparticles was greater than that of LIPOFECT AMINE® transfection at both 24 hours and 48 hours posttransfection as confirmed by GFP expression visualized by microscopy (FIG. 2A) and western blotting (FIG. 2B). In PANCI cells, 48 hours post treatment, ENVOYER nanoparticles showed a transfection efficiency of 16% compared to approximately 9% with LIPOFECTAMINE® (FIGs. 3A-3B). Given that PANCI cells are difficult to transfect and are therefore not the recommended cell line to evaluate optimal transfection efficiency, transfection efficiency of ENVOYER nanoparticles was also analyzed in HEK293T cells which are easily transfected and commonly used for this purpose. In HEK293T cells, 48 hours post treatment ENVOYER nanoparticles showed a transfection efficiency of 83% compared to approximately 63% when LIPOFECTAMINE® was used for transfection (FIG. 3C). Interestingly, it was noted that ENVOYER nanoparticles not only showed highertransfection efficiency in the HEK293T cells, but the GFP expression in the transfected cells was also much higher compared to cells transfected using LIPOFECT AMINE® (FIGs. 2A- 2B). Notably, a previous study using transfection media containing FBS reported a GFP expression efficiency of about 30% in the MDA-MB-231 breast adenocarcinoma and HepG2 hepatocellular carcinoma cell lines and a <20% expression efficiency in the primary cardiac fibroblasts with their polymer-based nanoparticles after 72 hours (Goldshtein et al. , 2019). These findings suggest that ENVOYER nanoparticles offer enhanced transfection efficiency and GFP expression compared to previously studied polymer-based nanoparticles.Post-formulation yields and encapsulation efficiencies

[0200] The composition and formulation process for LNPs and ENVOYER nanoparticles differs significantly. For the scope of this study, LNPs were generated according to manufacturer-recommended instructions. Under the given conditions, ENVOYER nanoparticles showed a higher pDNA yield (53.3%) compared to LNPs (13.3%) by PicoGreen assay. The LNPs showed an EE of 96%. However, the EE of ENVOYER nanoparticles could not be determined accurately using the available system and requires further evaluation. One possible explanation is the supposed porous nature of ENVOYER nanoparticles.

[0201] Based on pDNA yield (in the absence of EE for ENVOYER nanoparticles), PANCI cells were treated with LNPs and ENVOYER nanoparticles equivalent to 4 pg of GFP pDNA for 48 hours. ENVOYER nanoparticles showed a robust transfection efficiency compared to LNPs, which showed little to no GFP expression and had a transfection efficiency comparable to untreated cells or cells treated with empty ENVOYER nanoparticles, as confirmed by microscopy and western blotting (FIGs. 4A and 4B). While calcium chloride has been shown under certain conditions to facilitate the introduction of genetic material into cells (Lindell et al., 2004). the present findings demonstrate that treatment with calcium chloride alone (without polymer) did not result in significant transfection of the pDNA (FIGs. 4A and 4B).Impact of storage conditions on ENVOYER nanoparticle stability

[0202] The transfection efficiency of 5 pg GFP pDNA encapsulated ENVOYER nanoparticles was consistent between freshly prepared samples, and samples stored for 24hours at either room temperature, 4°C, -20°C, or -80°C as confirmed by GFP expression analysis using microscopy and western blotting. The GFP signal remained consistent after 24 hours of storage regardless of temperature as visualized by fluorescence microscopy (FIG.5 A) and western blotting (FIG. 5B). Additionally, measurement of GFP by densitometry (FIG. 5C) showed similar levels of GFP between samples treated with fresh ENVOYER nanoparticles and those treated with nanoparticles that had been stored for 24 hours at room temperature, 4°C, -20°C, and -80°C. Furthermore, efficacy of 15 pg GFP pDNA encapsulated ENVOYER nanoparticles after long-term storage (up to 2 months) at 4°C and - 80°C was compared to that of freshly prepared nanoparticles. Long-term storage did not compromise the efficacy of the ENVOYER nanoparticles as confirmed by the GFP expression (FIG. 6A) and corresponding western blot (FIGs. 6B-C) of treated cells after 48 hours. Collectively, these results demonstrate that ENVOYER nanoparticles can be stored at different temperatures without impacting transfection efficiency. In addition, the difference in the GFP intensity observed by treatment with 5 pg or 15 pg of pDNA suggests a dosedependent effect of the ENVOYER nanoparticles.Mechanism of cellular uptake of ENVOYER nanoparticles

[0203] To evaluate whether inhibitors of clathrin-independent endocytosis affected cellular uptake for ENVOYER nanoparticles, cells were next treated with the inhibitors geni stein (a tyrosine kinase inhibitor that inhibits caveolin-dependent endocytosis) and amiloride (a micropinocytosis inhibitor) and analyzed post-treatment GFP expression. Preincubation of HEK293T cells with the inhibitors for 3 hours was followed by 3 hours of exposure to ENVOYER nanoparticles encapsulating GFP pDNA. Cells were incubated for 24 hours after ENVOYER nanoparticle treatment and a significant reduction in GFP expression was observed in inhibitor-treated samples (FIGs. 7A and 7B) compared to those not treated with inhibitors. This suggests that actin polymerization might be critical for the cellular uptake of ENVOYER nanoparticles. The impact of clathrin-dependent endocytosis on the cellular uptake of these nanoparticles is currently under investigation.Discussion

[0204] The results disclosed herein demonstrate that ENVOYER nanoparticles exhibit a higher transfection efficiency for pDNA and maintain a consistent nanoparticle size andmorphology compared to standard LIPOFECTAMINE® and LNPs. Moreover, the transfection efficacy of ENVOYER nanoparticles was preserved even after short- and longterm storage of the nanoparticles at a range of different temperatures. Early observations suggest that the entry of ENVOYER nanoparticles into cells may be supported by clathrin- independent endocytosis.

[0205] Although gene therapy holds immense potential, the lack of durable delivery vectors remains an obstacle. Presently, there is an interest in developing nanoparticle vehicles for gene therapy, especially for use with DNA. Compared to siRNA or mRNA, pDNA needs to cross both the plasma membrane and the nuclear membrane in order to be functionally viable. Therapeutic use of nanoparticle vehicles for DNA delivery will necessitate improvements in key parameters, such as transfection efficiency, compared to other non-viral vectors as well as long-term storage stability for increased application in the clinic (Roma- Rodrigues et al., 2020). Here, ENVOYER nanoparticles were shown to be on track to meet these key parameters, and these nanoparticles continue to be evaluated and developed.

[0206] Nanoparticle material versatility allows for different characteristics that are important in various clinical applications and manufacturing (Roma-Rodrigues etal., 2020). In the development of nanoparticles, the use of different materials and fabrication methods can establish nanoparticle application and utility (Chenthamara et al., 2019). Manipulating nanoparticle size can determine biological fate and transport properties on the molecular scale, which can consequently improve therapeutic drug delivery (Hickey et al. , 2015). PNPs can be synthesized using a combination of natural and synthetic polymers using different methodologies to manipulate parameters such as payload encapsulation, administration route, surface functionalization, and biodegradability (Chenthamara et al., 2019). Currently, there are several FDA-approved polymer-based pharmaceutical products and nanoparticles such as ATRIDOX® (doxycycline hy elate) (see, e.g., FDA, ‘Drug Approval Package: Atridox (Doxycycline Hyclate, 10%) ND A# 50751’ (1998)), LUPRON DEPOT® (see, e.g., FDA, LUPRON DEPOT (leuprolide acetate for depot suspension), 2023), ZOLADEX® Depot, SANDOSTATIN® LAR, and Suprecur MP (Alsaab et al., 2022). ENVOYER nanoparticles are an innovative gene therapy vehicle that have potential for success in biomedical application and clinical purposes.Conclusions

[0207] In the current study, the ENVOYER nanoparticles encapsulating pDNA maintained transfection efficiency and stability across various storage conditions. Furthermore, ENVOYER nanoparticles were more successful in delivering pDNA to targets compared to the commonly used LNPs. Thus, polymer-derived ENVOYER nanoparticles provide an approach to gene therapy using pDNA as the therapeutic modality.Example 2: Effectiveness of non-lipid biopolymeric nanoparticle-encapsulated siRNA in vitro and in vivo

[0208] The field of gene therapy has emerged as a groundbreaking frontier in biomedical research, holding immense promise for the treatment and cure of various genetic disorders and chronic diseases at the molecular level (Sayed et al., 2022). The success of gene therapy is measured by the ability to deliver genetic material safely and precisely to target cells within the body; however, biological and physiological barriers present formidable challenges (Bulaklak etal., 2020). Therefore, efficient delivery systems must overcome hurdles such as immune responses, off-target effects, and limited cellular uptake.

[0209] One area of gene therapy showing particular promise in treating complex diseases, such as cancer, autoimmune diseases, dominant genetic disorders, and infections caused by viruses (Kalita et al., 2022), is the use of small interfering RNAs (siRNA) (Kalita et al.,Parashar et al., 2022). The specificity of siRNA therapeutics lies in their ability to recognize and bind to target sequences in messenger RNA (mRNA), mediating their cleavage and preventing their translation. To date, the United States Food and Drug Administration (FDA) has approved five siRNA agents: patisiran (e.g., ONPATTRO®) and vutrisiran (e.g., AMVUTTRA®) for the treatment of polyneuropathy of hereditary transthyretin-mediated amyloidosis, as well as givosiran (e.g., GIVLAARI®), lumasiran (e.g., OXLUMO®), and inclisiran (e.g., LEQVIOA®), for acute hepatic porphyria, primary hyperoxaluria type 1, and hyperlipidemia, respectively. The therapeutic use of siRNA has been limited by its immunogenicity and inherent instability in serum (Parashar et al., 2022). Thus, there is a growing need to develop an efficient siRNA delivery system targeting cells of diseased tissues (Parashar et al., 2022).

[0210] Researchers have explored various delivery vehicles to address these challenges, primarily focusing on viral vectors and lipid nanoparticles (LNPs) (Shahryari et al., 2021).Although the use of viral vectors as delivery systems has grown considerably in recent years, their use is limited due to poor efficiency and cellular selectivity that generally requires the use of high titers and selective genetic elements, such as promoters, to achieve expression of the therapeutic gene in a given target cell type. Moreover, high titers can induce severe immune responses and lead to cytotoxicity. In addition, if the genetic cargo contains even minimal viral elements it could lead to insertional mutagenesis (Butt et al., 2022). Due to the high cost of viral manufacturing in the clinical setting and the need for high injection titers, viral vectors may be cost prohibitive for many applications (Sung and Kim, 2019; Zhao et al., 2022). LNPs, such as liposomes, solid lipid nanoparticles and nanostructured lipid carriers, are biocompatible carriers primarily formulated from physiologic lipids commonly used as gene delivery vectors. Encapsulating genetic material, such as DNA, mRNA and siRNA, in LNPs provides increased stability for genetic material and offers improved safety profiles over many commonly used viral vectors (Xu et al., 2022). Although LNPs are easier and more cost effective to produce than viral vectors at the scales required for clinical trials, production costs still remain high (Kalita et al., 2022). Additional challenges associated with the use of LNPs include lipid formulation, limited cargo capacity, stimulation of innate and subsequent adaptive immunity, as well as variable stability in biological environments (Hald Albertsen et al.,Mehta et al., 2023; Parhiz et al., 2022). Collectively, while the theoretical benefit of viral vector- or LNP -based delivery systems are well-recognized, translating these benefits into clinical success is an ongoing challenge. Both viral vectors and LNPs suffer from a lack of specificity, the potential for toxicity, immunogenicity, and in many cases low efficacy, highlighting the need to seek next-generation solutions.

[0211] Polymer-based delivery systems may offer solutions to the challenges presented with LNPs and viral vectors. Polymer nanoparticles (PNPs) have been used in biomedicine to deliver pharmaceutical and genetic materials since the late 1980s (Bhardwaj and Jangde, 2023; Rai et al. , 2019). The use of synthetic polymers such as polyethyleneimine (PEI), poly(Z-lysine) (PLL), poly(lactic-co-glycolic acid) (PLGA), and poly(P-amino esters) have been highlighted for their diversity in formulation (Kamaly et al., 2016; Park et al., I ' ). In addition to synthetic polymer-based delivery systems, naturally occurring polysaccharide- based (e.g., chitosan, hyaluronic acid, and agarose) and protein-based (e.g., gelatin, albumin, and collagen) polymers are in development with early results showing increased biocompatibility, easier preparation, and stability in biological fluids (Idrees et al., 2020).However, the challenge of using PNPs is in consistently producing a product of preserved size, shape, zeta potential, encapsulation efficiency, and stability.Disclosed herein is the development of a modality for gene therapy delivery that leverages the benefits and versatility of natural polymers. The disclosed strategy is differentiated by incorporating FDA approved, nontoxic, non-immunogenic, polymers in the PNPs to protect nucleic acid payloads. The biopolymer-based nanoparticles, ENVOYER, are formulated with an external gelation-based microfluidics methodology that offers greater control over particle formation size. As disclosed herein, the consistency of the analytical properties of ENVOYER were examined and its immunogenicity and efficacy in delivering encapsulated siRNA was determined, both in vitro and in vivo.Materials and methodsCell lines and antibodies

[0212] The pancreatic tumor cell line, PANCI (CRL-1469-LUC2, American Type Culture Collection (ATCC), Manassas, VA, USA), was cultured in Dulbecco’s Modified Eagle Medium (DMEM; Catalog No. 11965-092, Gibco, Thermo Fisher Scientific Inc, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Catalog No. A5256801, Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) and 1% penicillinstreptomycin (Catalog No. 15140122, Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA), at 37°C in a humidified atmosphere containing 5% (v / v) CO2.

[0213] HEK Blue toll-like receptor 4 (TLR4) cells (InvivoGen, San Diego, CA, USA) were cultured in Minimum Essential Medium (MEM; Catalog No. 32561037, Gibco, Thermo Fisher Scientific Inc. Waltham, MA, USA) at 37°C in a humidified atmosphere containing 5% (v / v) CO2.

[0214] The antibodies against survivin (Catalog No. 2808S), caspase 3 (Catalog No. 9662S), cleaved caspase 3 (Catalog No. 9664), phospho-histone H3 (SerlO) (Catalog No. 9701 S) and beta-actin (Catalog No. 8457S) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The antibodies against NUF2 component ofNDC80 kinetochore complex (NUF2; Catalog No. 15731-1-AP and Catalog No. HPA076604) were purchased from Proteintech Group, Inc. (Rosemont, IL, USA) and Atlas Antibodies (Soina, Sweden), respectively. The antibody against proliferation marker protein Ki-67 (Ki67; Catalog No. ab 16667) was purchased from Abeam, (Cambridge, UK). IMMPRESS® Horse Anti -Rabbit IgG Polymer Kit, Peroxidase (MP-7401) was from Vector Laboratories, Newark, CA, USA.Formulation of nanoparticles

[0215] ENVOYER particles were formulated at Envoya, Inc. (Natick, MA, USA) from natural polymers sodium alginate (Catalog No. #42000501-5G, Merck KGaA, Darmstadt, Germany) and a crosslinker using a method based on the principle of external gelation adapted for the Ignite NANOASSEMBLR® nanoparticle formulation instrument (Precision Nanosystems, Vancouver, BC, Canada), which is established on the principle of microfluidics technology using NxGen NANOASSEMBLR® Ignite Cartridge (Catalog No. #NIN0062, Precision Nanosystems).

[0216] Alginate ENVOYER nanoparticles encapsulating siRNA were formulated by combining a 270 pg / mL of siRNA solution, 1 M CaCh solution, and 50 pg / mL alginate solution in a 1 : 1 :2 volume ratio.

[0217] The 1 M CaCh solution was formed by weighing out solid CaCh and dissolving it H2O. Afterwards, the solution was filtered through a 0.2 pm biological filter to ensure that the solution did not have any biological contaminants or aggregates of salt.

[0218] The siRNA solution was created by diluting a stock solution of the siRNA of interest (e.g., NUF2 siRNA) in H2O to a concentration of 270 pg / mL. After both solutions were made, they were then mixed in equal volume via pipetting the 1 M CaCh into the 270 pg / mL pDNA solution. Then, the mixture was gently vortexed (e.g., vortexed at a low speed) for 15 seconds, and left to incubate at room temperature for 30 minutes.

[0219] The alginate solution was formed by weighing out solid alginate polymer and dissolving it in HEPES at a concentration of 500 pg / mL of alginate via a stir bar on a stir plate for between 30 minutes to 1 hour. After the alginate was fully dissolved in the HEPES, the solution was filtered through a 0.2 pm biological filter to ensure that the solution did not have any biological contaminants or aggregates of alginate. After filtration, the alginate solution was diluted from 500 pg / mL to 50 pg / mL in HEPES.

[0220] Once the siRNA mixed with CaCh had finished incubating, and the alginate solution was prepared to a 50 pg / mL concentration, the two solutions were taken into syringes at equal volumes, and run through the Ignite NANOASSEMBLR®, which mixed the two solutions via microfluidics. The Ignite NANOASSEMBLR® was set to dispense the syringes at an equal flow rate ratio, with a flow rate of 12 mL / minute. The starting waste was set to 0.3 or 0.5 mL, depending on whether the syringes were 1 mL or 3 mL syringes, respectively, and the end waste was set to 0.05 mL. Once the Ignite NANOASSEMBLR®finished dispensing the desired volume into a 15 mL polypropylene tube, the sample solution was left to sit for 30 minutes at room temperature, after which the particles were considered mature and ready for use.

[0221] Considerations for selection of polymers included the ease of access to the polymer and its compatibility with the crosslinking agent and encapsulated siRNA regarding the polymer’s charge. Briefly, the nanoparticles were synthesized by injecting the polymer solution in HEPES buffer (Catalog No. 15630-080, Thermo Fisher Scientific Inc., Waltham, MA, USA) through one inlet of the microfluidic mixer, and the crosslinker and siRNA solution through the other inlet. The nanoparticles were collected in the sample collection tube. The initial and final waste volumes were discarded. The waste volume resulted from the mixing of the polymer with the nucleic acid and crosslinker, and was discarded to ensure that all particles were mixed in exactly the same conditions, and that the last bit of mixing from the syringes did not interfere with the production of a consistent mixture. If waste volumes were not discarded, since the syringes were at their ends (e.g., having had already dispensed the majority of their contents), the mixture could be prone to not include equal volumes of each solution (e.g., the nucleic acid solution and the crosslinker solution) or to contain air. Therefore, the last bit of particle volume was discarded as a precaution.

[0222] A starting concentration of 20 mM target siRNA (NUF2 siRNA, Catalog No. AM16706 or survivin siRNA, Catalog No. 4390824, both were purchased from Thermo Fisher Scientific Inc., Waltham, MA, USA) or scrambled siRNA (Catalog No. AM4611, Thermo Fisher Scientific Inc. Waltham, MA, USA) was used for the encapsulation. The siRNAs used were about 21 bases in length.

[0223] The microfluidics method described above results in ENVOYER nanoparticles with a consistent size. Usually, microfluidics methods are used for lipid nanoparticles (LNPs) and not sugar-based polymers, e.g., alginate. The flow conditions (1 : 1 with payload) and the nano-chambers used e.g., NxGen NANOASSEMBLR® Ignite Cartridge) modify the microfluidics to be suitable for sugar polymers. Use of different polymers will necessitate different flow rates, e.g, for longer polymers that are more branched.

[0224] The flow rates used (e.g, 1 : 1 compared to payload) and the nature of the crosslinker that flows with the payload determines the size of the ENVOYER nanoparticles. Unlike LNPs, which are essentially a shell with a payload inside, ENVOYER nanoparticles are more similar to a yarn ball with yam embedded inside (e.g., siRNA) and surrounded by outer layers (e.g., the polymer), resulting in a “tighter” or more compact package.

[0225] The ENVOYER nanoparticles encapsulating siRNA are smaller compared to the ENVOYER nanoparticles encapsulating pDNA because the pDNA over 5000 bp and the siRNA size is smaller than 1000 bp.Physicochemical characterization of ENVOYER nanoparticlesTransmission electron microscopy (TEM)

[0226] For TEM studies, ENVOYER samples were applied to 200 mesh carbon coated copper grids that had been pretreated with 1% alcian blue for 1 minute and subsequently rinsed through 4 drops of deionized water. The grids were then placed onto a 10 pL droplet of sample for 1 minute. The excess sample was then wicked off onto filter paper before rinsing through 3 drops of deionized water. The sample was then negatively stained by placing the grid onto a drop of 2% uranyl acetate 3 or 4 times and wicking off excess stain onto filter paper after each stain treatment. The grids were then thoroughly dried and viewed on a JEM1010 transmission electron microscope (JEOL USA, Inc., Peabody, MA, USA). Images were taken with a digital camera system (Advanced Microscopy Techniques, Woburn, MA, USA). Transmission electron microscopy was performed at Boston University’s Chobanian and Avedisian School of Medicine.Dynamic light scattering

[0227] ENVOYER particle size and size distribution were measured by dynamic light scattering (DLS) using the Zetasizer Pro (Malvern Panalytical, Malvern, UK). ENVOYER particles were loaded into a ZEN0040 cuvette (Malvern Panalytical, Malvern, UK) and then measured at an angle of 173° with the instrument set for an equilibration time of 120 seconds at room temperature (25°C). The reported measurement is the average of three automated measurements performed back-to-back. Dynamic light scattering was performed in part at the Harvard University Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Coordinated Infrastructure Network (NNCI).Measurement of zeta potential

[0228] To measure the zeta potential of the nanoparticle, the sample was placed in a DTS1070 cell (Malvern Panalytical, Malvern, UK) and measured using the Zetasizer Pro (Malvern Panalytical, Malvern, UK) with the instrument set for an equilibration time of 120 seconds at room temperature (25°C). The reported measurement is the average of three automated measurements performed back-to-back. Zeta potential measurements were performed in part at the Harvard University CNS.Determination of encapsulation efficiency

[0229] Quant-iTTM RIBOGREEN® assay kit (Catalog No. R11490, Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the amount of siRNA encapsulated in the nanoparticles by measuring the difference between the concentration of siRNA in a preparation without the polymer / crosslinker and the amount of non-entrapped siRNA remaining in the suspension after the formulation of the nanoparticles using the polymer / crosslinker. The siRNA encapsulation efficiency (EE) was calculated using the following equation.EE% = (siRNA concentration in formulation without polymer - siRNA concentration in formulation with polymer) / (siRNA concentration in formulation without polymer) x 100Assessment of ENVOYER immunogenicity

[0230] Human embryonic kidney (HEK) Blue TLR4 cells (InvivoGen, San Diego, CA, USA), which comprise a reporter for TLR4-dependent NF-KB activation, were seeded at a concentration of 10,000 cells / well in 96-well plate and allowed to adhere overnight. ENVOYER-encapsulated scrambled siRNA was added to the 96-well plate at a final polymer concentration range of 0.7 nM to 175 nM. Lipopolysaccharide (LPS) at an initial concentration of 10 ng / ml (the molarity of LPS could not be accurately determined due to the heterogeneous nature of LPS impacting the molecular weight) and diluted 1 :3 was used as a positive control. Each condition was assessed in duplicate. The cells were incubated for 24 hours at 37°C. Then, TLR4 activity was assessed by evaluating alkaline phosphatase levels using a para-nitrophenylphosphate substrate (Catalog No. 0405- 1KT, VWR, Radnor, PA,USA) in diethanolamine substrate buffer (Catalog No. 34064, Thermo Fisher Scientific Inc., Waltham, MA, USA) and reading the absorbance at optical density (OD) 405 nm. Absorbance was corrected to account for cytotoxicity, measured using CELL TITER 96® AQueous One Solution (Catalog No. G3582, Promega Corporation, Madison, WI, USA) and the final OD for each condition was determined relative to untreated cells (control).

[0231] To assess the immunogenicity of ENVOYER in animals, 65 C56BL / 6 mice (5 mice / group / timepoint; Charles River Laboratories, Wilmington, MA, USA) aged 6-8 weeks were injected intraperitoneally (IP) with 0.1 mg / kg scrambled siRNA encapsulated in either ENVOYER or LNP. LPS equivalent to 100 pg per mouse (approximately 5 mg / kg) was used as a positive control and saline injection was used as a negative control. Serum was collected at 2, 4, 12, and 24 hours post-injection; serum for the negative control was collected at 24 hours only. Pro-inflammatory cytokine levels (tumor necrosis factor alpha (TNFa), interleukin 6 (IL-6), and interferon gamma (IFNy)) levels were analyzed by the Luminex™ 200 System (Merck KGaA, Darmstadt, Germany) using Mouse PROCARTAPLEX® Mix & Match 6-Plex kit (Catalog No. PPX-06-MXU64YH, Thermo Fisher Scientific Inc., Waltham, MA, USA). A standard curve was run for each of the analytes along with the serum samples.Time course and dose studies

[0232] PANCI cells were seeded in a 6-well plate at a density of 4 x 105cells / well and allowed to adhere overnight. On the day of treatment, the growth media were replaced with antibiotics-free media. For the dose response experiments, PANCI cells were transfected with ENVOYER-encapsulated survivin siRNA or scrambled siRNA at concentrations of 15.6, 31.25, 62.5, 125, and 250 nM based on the starting concentration of the siRNA for 24 hours.

[0233] For the time course study, cells were transfected with ENVOYER-encapsulated survivin siRNA (62.5 nM) for 3, 6, 24 and 48 hours. To evaluate the knockdown efficiency of the target gene (positive control), PANCI cells were transfected with survivin siRNA (100 nM) using LIPOFECTAMINE® RNAIMAX® (Catalog No. 13778-075, Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA) for 24 and 48 hours. Cells transfected with ENVOYER-encapsulated scrambled siRNA (62.5 nM) served as a negative control.

[0234] At the end of the incubation period, the cells were harvested and lysed for their protein contents using radioimmunoprecipitation assay (RIP A) buffer (Catalog No. 89900,Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with protease and phosphastase inhibitor (Catalog No. A32959, Thermo Fisher Scientific Inc., Waltham, MA, USA). RNA was extracted using RNEASY® Plus Mini Kit (Catalog No. 74134, Qiagen, Hilden, Germany) following manufacturer instructions.

[0235] Survivin mRNA expression was determined with quantitative polymerase chain reaction (qPCR), using a TAQMAN® assay (Catalog No. 4331182, Thermo Fisher Scientific Inc., Waltham, MA, USA) with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the house-keeping gene (Catalog No. 4326317E, Thermo Fisher Scientific Inc., Waltham, MA, USA).

[0236] Survivin protein levels were quantitated by western blot analysis with beta-actin as a loading control.Assessment of ENVOYER stability

[0237] PANCI cells were seeded in a 6-well plate at a density of 4 x 105cells / well and allowed to adhere overnight. To evaluate the stability of ENVOYER-encapsulated siRNA after freeze-thaw cycles, 70 nM of freshly prepared ENVOYER-encapsulated survivin siRNA was used for transfecting PANCI cells and the rest of the preparation was stored at -80°C for 1 month, then thawed to transfect cells, and refrozen at -80°C for additional 2.5 months before being thawed again for PANCI cell transfection (z.e., a total of 2 freeze-thaw cycles over 3.5 months). After each thawing, PANCI cell cultures were transfected with a volume of thawed formulations equivalent to 70 nM of survivin siRNA and incubated for 48 hours. The expression of survivin protein levels were subsequently evaluated via western blot analysis.Effect ofNVF2 siRNA on PANCI cells delivered via LIPOFECTAMINE® and ENVOYER particles

[0238] NUF2, an Ndc80 kinetochore complex component that can stabilize microtubule attachment and is closely related to cell apoptosis, is upregulated in many human cancers, including pancreatic cancer (Hu et cd.. 2015). PANCI cells were transfected with 62.5 nM and 125 nM (final concentration) of siRNA against NUF2 using LIPOFECTAMINE® RNAIMAX® (Catalog No. 13778-075, Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA) for 48 hours. In an independent experiment, 125 nM of NUF2 siRNA encapsulated in ENVOYER particles (formulated as mentioned above) were added toPANCI cells for 48 hours. The cells were then harvested and lysed. Protein expression was determined by western blot analysis using antibodies against NUF2, cleaved caspase 3, phospho-histone H3, and beta-actin (as the loading control).Cellular internalization of ENVOYER particles

[0239] ENVOYER-encapsulated NUF2 siRNA were prepared using the Ignite NANOASSEMBLR® (Precision Nanosystems, Vancouver, BC, Canada) as described above. PANCI cells were transfected with ENVOYER particles corresponding to 125 nM siRNA in the presence and absence of genistein (Catalog No. SI 342, Selleck Chemicals, Selleck, Houston, TX, USA) which is a tyrosine kinase inhibitor that inhibits caveolae-dependent endocytosis (Hu et al., 2015). The cells were treated for 30 minutes with 150 pM genistein. Then, ENVOYER-encapsulated NUF2 siRNA was added to the cells for 3 hours. After the incubation period with both genistein and ENVOYER, the media was replaced with fresh complete media and the cells were incubated for 48 hours. Then the cells were lysed, and the lysate was analyzed by western blot analysis to evaluate the expression of NUF2, cleaved caspase 3 and phospho-histone H3 (SerlO).Effects of ENVOYER-encapsulated NUF2 siRNA on an animal tumor model

[0240] Six NOD-scid gamma (NSG) immunodeficient female mice aged 6-8 weeks (Jackson Laboratories, Bar Harbor, ME, USA) were housed (3 mice per cage) and allowed to acclimate for 2 days.

[0241] To evaluate the effects of ENVOYER particles in a tumor model, PANCI cells were mixed with MATRIGEL® and directly implanted in the left flank of NSG mice (n=6). The mice were divided into 2 groups (n=3 per group) with an average tumor volume of 500 mm3per group.

[0242] ENVOYER encapsulated- NUF2 siRNA or scrambled siRNA (control) were prepared as described above. Mice were dosed intratumorally at an siRNA concentration of about 0.2 mg / kg 3 times / week (volume equivalent to 100 pL) for 2 weeks. Mouse body weight was measured daily, and tumor volume was calculated three times weekly using the standard formula.Volume = (Length x Width2) / 2

[0243] At the end of the study (after 2 weeks of dosing, or 6 doses), tumors from both groups were isolated. Half of each tumor was frozen, and the other half was fixed with 2% paraformaldehyde and embedded in paraffin. Proteins were extracted from the frozen tumors to evaluate the expression of NUF2 using western blot analysis.Immunohistochemistry

[0244] Immunohistochemistry was performed as per the standard operating procedure at iHisto, Inc. (Salem, MA, USA). Briefly, formalin-fixed paraffin embedded tumors were cut into 4-pm sections. The sections were deparaffinized and hydrated. For heat-induced epitope removal, the slides were submerged in an antigen retrieval buffer (citrate-based, pH 6), and heated in a pressure cooker to 110°C for 15 minutes. The slides were allowed to cool for 20 minutes and then water was slowly added to the retrieval container to bring the slides to room temperature. Then, the slides were washed Tris-buffered saline (TBS), washed with TBS 0.1% Tween 20 (TBST), and incubated in peroxidase blocking solution. The slides were washed again in TBS and TBST, then incubated with 2.5% normal horse serum. Excess serum was removed from the sections and primary antibody (Ki67, NUF2, or cleaved caspase 3) diluted in 2.5% normal horse serum was applied and the sections were incubated overnight at 4°C. IMMPACT® DAB EqV Substrate Kit (SK-4103-400, Vector Laboratories, Newark, CA, USA) was applied to the sections and the stain was allowed to develop. The slides were counterstained with hematoxylin, dehydrated and coverslipped using a xylene-based mounting medium.Ethics statement and statistical analysis

[0245] This study was approved by the Institutional Animal Care and Use Committee (IACUC; Approval number 22-0329-2 and the approval date is March 29, 2022) and all animal procedures were carried out following their recommendations. A veterinarian was accessible during the course of the study to minimize animal suffering.

[0246] Statistical analyses were done using GraphPad Prism 8.0 software (Boston, MA, USA).

[0247] Student’ s t-test was used to calculate statistical significance between groups. For the immunogenicity and time and dose experiments, the differences among groups weredetermined using one way analysis of variance (ANOVA). Statistical significance was determined if p-value was <5%.ResultsCharacterization of ENVOYER-encapsulated siRNA

[0248] TEM images indicated the ENVOYER particles formulated through micro fluidics-adapted external gelation methodology had sizes in the range of 20-30 nm (FIG. 8). Particle sizes were consistent across 2 independent studies. DLS analysis of the ENVOYER particles showed a peak between 10-30 nm. An additional peak was observed at >1000 nm, probably due to particle aggregation. The zeta potential (z) measurements showed a slightly anionic surface charge for the particles (about 8.4 mV, Range: -7.8 to -9.1). The encapsulation efficiency of the particles was 95%.Immunogenicity of ENVOYER particles

[0249] LPS showed dose dependent activation of TLR4 in HEK Blue TLR4 cells. ENVOYER-encapsulated scrambled siRNA showed minimal dose dependent activation of TLR4 but was cytotoxic at the highest polymer concentrations (175 nM and 58.3 nM; FIG.9). At these ENVOYER concentrations, the theoretical concentration of the scrambled siRNA would be between 1.25 pM and 0.42 pM, respectively, which is much higher than the optimal siRNA concentration used for silencing. At the working concentration of the siRNA (about 50 nM to 140 nM), ENVOYER-encapsulated scrambled siRNA did not significantly activate TLR4 compared to LPS.

[0250] IP injection of LPS to C56BL / 6 mice showed a maximum induction in IL-6 (average about 19,000 pg / mL) and TNFa (average about 500 pg / mL) at 2 hours post injection, with a steady decrease thereafter. A similar trend was observed in the LNP treated group, but the extent of cytokine induction was lower than that observed with LPS. IFNy levels increased at 4 and 12 hours post-injection of LPS and LNP, respectively. In the mice injected with ENVOYER-encapsulated scrambled siRNA, the levels of all three cytokines were similar to the levels observed in mice injected with saline (FIG. 10).Dose and time course studies with ENVOYER particles

[0251] PANCI cells transfected with ENVOYER-encapsulated survivin-specific siRNA reduced survivin mRNA and protein levels in a dose- and time-dependent manner. As shown in FIG. 11 A, the 15.6 and 31.25 nM doses did not significantly decrease survivin mRNA levels compared to its level in the control sample (1.03±0.04 and 0.93±0.03, respectively vs. 1.0); however, the 62.5, 125, and 250 nM doses significantly reduced survivin mRNA (0.34±0.02; 0.14±0.003; 0.08±0.001; respectively). The decrease in mRNA levels in these doses were reflected in protein expression (FIG. 1 IB): a significant reduction (about 50%) in survivin protein levels were observed for siRNA doses of 62.5, 125, and 250 nM, whereas increasing doses of ENVOYER-encapsulated negative siRNA did not modulate survivin mRNA (FIG. 11 A) or protein levels (FIG. 1 IB).

[0252] The time course studies showed a statistically significant decrease in survivin mRNA levels compared to control levels as early as 3 hours after transfection with ENVOYER-encapsulated survivin-specific siRNA with a continued decrease at all timepoints measured (FIG. 11C, 3 hours, 0.74±0.14; 6 hours, 0.52±0.41; 24 hours, 0.3 ± 0.01; 48 hours, 0.16 ± 0.016). The decrease in mRNA levels at these timepoints were also reflected in protein expression (FIG. 1 ID). ENVOYER-encapsulated scrambled siRNA did not impact mRNA or protein levels at any timepoint tested. Although the siRNA concentration (100 nM) used for transfection with LIPOFECTAMINE® was higher than the siRNA concentration (62.5 nM) in ENVOYER nanoparticles used for transfection in the time course study (based on the initial concentration of siRNA used for lipofection and nanoparticle formulation, respectively), siRNA delivered by ENVOYER particles showed comparable silencing efficiency to that of LIPOFECTAMINE® siRNA delivery in relation to the mRNA levels at 24 hours (0.19±0.09 vs. 0.30±0.01) and 48 hours (0.05±.03 vs 0.16±0.016, FIG. 11C).ENVOYER s ’ stability and transfection efficiency across freeze-thaw cycles and time

[0253] As shown in FIG. 12, the knockdown efficiency of survivin gene via ENVOYER was consistent (about 80 to 90% knockdown) between freshly prepared ENVOYER- encapsulated siRNA and ENVOYER-encapsulated siRNA stored at -80°C for 1 month or 3.5 months as confirmed by western blot analysis.Effect of cellular internalization of ENVOYER-encapsulated NUF2 siRNA

[0254] Transfection of NUF2 siRNA using LIPOFECTAMINE" reduced NUF2 protein levels and increased caspase 3 cleavage after 48 hours in a dose-dependent manner (FIG. 13 A, B). This validated the sensitivity of PANCI cells to NUF2 knockdown. ENVOYER- encapsulated NUF2 siRNA particles (125 nM) also knocked down NUF2 protein levels in PANCI cells. Furthermore, cleaved caspase 3 levels and phospho-histone H3 (SerlO) levels increased in these cells (FIG. 13C, D).

[0255] To understand the probable cellular uptake mechanism, PANCI cells were treated with ENVOYER-encapsulated NUF2 siRNA in the presence of genistein. The reduction in NUF2 protein levels and impact on subsequent proliferation and apoptosis markers were abrogated in the presence of genistein (FIG. 13C, D). These preliminary data suggest that ENVOYER particles are internalized in the cells via active endocytosis.In vivo tumor reduction with ENVOYER-encapsulated NUF2 siRNA

[0256] Intratumoral injection of ENVOYER-encapsulated NUF2 siRNA were tested on NSG mice bearing subcutaneous PANCI tumors. Fifteen days after treatment initiation (6 doses), mice treated with ENVOYER-encapsulated NUF2 siRNA demonstrated a 60% inhibition in tumor growth compared to mice that received ENVOYER-encapsulated scrambled siRNA (FIG. 14A). Importantly, intratumoral injection of ENVOYER- encapsulated NUF2 siRNA was well tolerated by the mice even after repeated dosing. This was demonstrated by a consistency in body weight and lack of any clinical abnormalities (FIG. 14B). Due to size of the tumors in the control group, the study could not be performed for an extended period. Analysis of the tumor samples treated with NUF2 siRNA-encapsuled ENVOYER particles showed a reduction in NUF2 protein expression compared to scrambled siRNA (FIG. 14C). Immunohistostaining of tumors of mice treated with ENVOYER- encapsuled NUF2 siRNA showed reduced NUF2 and Ki67 staining and increased cleaved caspase 3 staining in comparison to tumors of mice treated with ENVOYER-encapsulated scrambled siRNA (FIG. 14D).Discussion

[0257] According to a recent review of FDA-approved nanoparticle-based therapy, 29% utilize polymeric formulations (Namiot et al., 2023). They provide the possibility oftransporting bioactive compounds to specific tissues, cells and cell compartments. PNPs can be based on proteins (e.g., silk, collagen, gelatin, P-Casein, zein, and albumin), polypeptides (e.g., such as elastin-like polypeptide), or polysaccharides (chitosan, alginate, heparin, hyaluronic acid, pullulan, and dextran) (Moazzam et cd., 2024; Nitta and Numata, 2013). Natural PNPs also have several advantages over synthetic polymers, such as biocompatibility, biodegradability and renewable sources (Nitta and Numata, 2013).

[0258] There are several challenges to formulating and upscaling LNPs, including particle size and variability among batches (Liu et al., 2018). LNPs require complicated formulation strategies to balance the types and ratios of lipids to reduce toxicity and maintain stability while forming the appropriately sized particles (Tomeh etal., 2022). The size range of LNP for mRNA delivery is 50-200 nm (Guevara et al., 2020; Hassett et al., 2021). The particle size of LNP-encapsulated plasmid DNA is 120-130 nm (Cui et al., 2022). Compared to LNPs, PNPs are easier to formulate and produce in the larger quantities required for Good Laboratory Practice (United States Code of Federal Regulations, Part 58) and clinical studies. ENVOYER particles are formulated with external gelation-based methodology that offers greater control over particle formation size, which can be more efficiently sorted with the microfluidics for higher and more cost-effective yields. TEM and DLS analysis of the ENVOYER particles revealed a mean hydrodynamic diameter between 10-30 nm, with an additional peak greater than 1000 nm seen with DLS, suggesting particle aggregation. The encapsulation efficiency of the particles was 95% which is very comparable to encapsulation seen with LNPs.

[0259] The delivery of siRNA has to overcome several extracellular and intracellular barriers. Extracellular barriers include intravascular degradation, renal clearance, plasma protein binding mononuclear phagocyte system entrapment and membrane impermeability (Wang et al., 2010). Nanoparticle systems less than 100 nm usually avoid entrapment by monocytes and macrophages (Wang et al., 2010). In the current study, ENVOYER particles were demonstrated to maintain a consistent size, ranging from 20 to 30 nm (Cui et al., 2022), suggesting that they can avoid monocyte and macrophage entrapment.

[0260] Intracellular barriers to siRNA delivery include endosome entrapment; therefore, siRNA vehicles should enhance their payload accumulation in the target tissue and protect it from degradation during delivery (Moazzam et al., 2024). Based on the initial data, the ENVOYER particles are internalized into the cells through endocytosis. Genistein is a tyrosine-kinase inhibitor which causes local disruption of the actin network at the site ofendocytosis and inhibits the recruitment of dynamin II, both known to be indispensable events in the caveolae-mediated cellular uptake mechanism (Vercauteren etal., 2010). The exposure of the cells to genistein prior to treatment with the ENVOYER particles inhibited the efficacy of the ENVOYER-encapsulated siRNA particles.

[0261] siRNA can also trigger TLR4-dependent or -independent responses of the innate immune system and the production of cytokines (Wu etal., 2012). ENVOYER particles encapsulating scrambled siRNA were shown to be non-immunogenic. At the working concentration of the siRNA (about 50 nM to 140 nM), ENVOYER-encapsulated scrambled siRNA did not significantly activate TLR4 compared to LPS. Furthermore, blood levels of pro-inflammatory cytokines did not increase up to 24 hours following IP injection of ENVOYER-encapsulated scrambled siRNA to mice.

[0262] The data shows that ENVOYER-encapsulated survivin siRNA are internalized by the cells and act in a dose- and time-dependent manner. Transfection of cells with ENVOYER-encapsulated survivin siRNA showed that the threshold dose for decreasing survivin mRNA and protein levels was 62.5 nM, with higher concentrations (125 and 250 nM) further decreasing both mRNA and protein levels. A time course analysis showed that ENVOYER-encapsulated survivin-targeted siRNA decreased survivin mRNA and protein levels as early as 3 hours post-transfection and this trend continued throughout the 48-hour incubation period. Two freeze-thaw cycles and storage of ENVOYER-encapsulated survivin siRNA at -80°C for over three months did not impact the transfection efficiency of the particles.

[0263] Transfection of PANC 1 cells with NUF2 siRNA either by LIPOFECTAMINE® or ENVOYER reduced NUF2 protein levels and subsequently increased the levels of cleaved caspase 3 and phospho-histone H3 (SerlO). Histone H3 SerlO phosphorylation has been reported to be mediated by the pro-apoptotic kinase protein kinase C (PKC) 5 during apoptosis (Park and Kim, 2012), suggesting that the knockdown of NUF2 leads to reduction in cellular proliferation and induced apoptosis.

[0264] Intratumoral injection of ENVOYER-encapsulated NUF2 siRNA in NSG mice showed inhibition of tumor growth by 60% but did not induce any changes in body weight or other clinical parameters. Furthermore, evaluation of the tumors from mice that received ENVOYER-encapsulated NUF2 siRNA showed reduced NUF2 levels as well as a reduction in the proliferative marker, Ki67, and an increase cleaved caspase 3 levels. These findings confirm successful delivery of the particles to the target tissue and target engagement by thesiRNA, indicating that ENVOYER nanoparticles are both safe and effective in rodent models of tumorigenesis. Collectively, these data show that ENVOYER encapsulation of siRNA is a stable and efficient delivery system for gene therapy.Conclusions

[0265] This study provides evidence that the ENVOYER delivery platform is stable and effective in modulating targeted genetic material using siRNA both in vitro and in vivo. Moreover, this study provides the supportive framework that biopolymer-based nanoparticles can potentially offer many advantages including ease of formulation, increased stability, lower immunogenicity compared to LNP (as shown in the current study) and the ability to easily customize the delivery of targeted information for treatment-specific diseases.Example 3: CRISPR-Cas9 ENVOYER Nanoparticles MethodsPreparation and characterization of ENVOYER nanoparticles

[0266] The CRISPR-Cas9 ENVOYER nanoparticles were synthesized at Envoya Inc. (Natick, MA, USA) via a method based on the principle of external gelation. Nanoparticles were synthesized using sodium alginate (Catalog No. #42000501-5G, Merck KGaA, Darmstadt, Germany) and calcium chloride (CaCh) as the crosslinker to encapsulate plasmids encoding Cas9 and guide RNA (G1 plasmid) and donor template (Donor plasmid), together or separately, in 500 mM 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES; Catalog No. 15630-080, Thermo Fisher Scientific Inc., Waltham, MA, USA) at the final concentrations shown in Table 1. Plasmids were prepared with the OriGene CRISPR KN2.0 Gene Knockout Kit (OriGene Technologies, Inc., Rockville, MD, USA; Catalog No. KN400003), using the G1 plasmid (p53 gRNA vector 1 in pCas-Guide CRISPR vector, where the pCas-Guide CRISPR vector is approximately 8 kilobases (kb) in length; OriGene Technologies, Inc. Catalog No. KN400003G1) and the Donor plasmid (linear donor DNA containing LoxP-EFlA-tGFP-P2A-Puro-LoxP; cassette sequence only is 2739 bp; OriGene Technologies, Inc. Catalog No. KN400003D).Table 1. CRISPR-Cas9 ENVOYER Nanoparticle Formulations.

[0267] Alginate nanoparticles encapsulating a CRISPR-Cas9 system (e.g., G1 + Donor NPs) were formulated by combining a 12.5 pg / mL guide and Cas9 (Gl) plasmid solution, 12.5 pg / mL donor plasmid solution, 1 M CaCh solution, and 50 pg / mL alginate solution in a 1 : 1 :2:4 volume ratio.

[0268] In addition, alginate nanoparticles separately encapsulating Gl plasmid or Donor plasmid were formulated by combining 12.5 pg / mL plasmid solution, 1 M CaCh solution, and 50 pg / mL alginate solution in a 1 : 1 :2 volume ratio.

[0269] The 1 M CaCh solution was formed by weighing out solid CaCh and dissolving it EhO. Afterwards, the solution was filtered through a 0.2 pm biological filter to ensure that the solution did not have any biological contaminants or aggregates of salt.

[0270] The CRISPR-Cas9 solution was created by diluting a stock solution of the guide and Cas9 (Gl) plasmid and the donor plasmid in EhO to a concentration of 12.5 pg / mL each and then combining them in equal volumes.

[0271] After the CaCh and CRISPR-Cas9 solutions were made, they were then mixed in equal volume (e.g., 1 M CaCh solution mixed with one of the CRISPR-Cas9 solutions shown in Table 1) via pipetting the 1 M CaCh into the CRISPR-Cas9 solution. Then, the mixture was gently vortexed (e.g., vortexed at a low speed) for 15 seconds, and left to incubate at room temperature for 30 minutes.

[0272] The alginate solution was formed by weighing out solid alginate polymer and dissolving it in HEPES at a concentration of 500 pg / mL of alginate via a stir bar on a stir plate for between 30 minutes to 1 hour. After the alginate was fully dissolved in the HEPES, the solution was filtered through a 0.2 pm biological filter to ensure that the solution did not have any biological contaminants or aggregates of alginate. After filtration, the alginate solution was diluted from 500 pg / mL to 50 pg / mL in HEPES.

[0273] Once the CRISPR-Cas9 solution mixed with CaCh had finished incubating, and the alginate solution was prepared to a 50 pg / mL concentration, the two solutions were taken into syringes at equal volumes, and run through the Ignite NANOASSEMBLR® nanoparticleformulation instrument (Precision Nanosystems, Vancouver, BC, Canada), which mixed the two solutions via microfluidics. The Ignite was set to dispense the syringes at an equal flow rate ratio, with a flow rate of 12 mL / minute. The starting waste was set to 0.3 or 0.5 mL, depending on whether the syringes were 1 mL or 3 mL syringes, respectively, and the end waste was set to 0.05 mL. Once the Ignite finished dispensing the desired volume, the sample solution was left to sit for 30 minutes at room temperature, after which the particles were considered mature and ready for use. The cartridge used for the Ignite mixing was the NxGen NanoAssemblr Ignite Cartridge (Catalog No. #NIN0062, Precision Nanosystems). The tubes used for collection were 15 mL polypropylene tubes.Transfection

[0274] HEK293T cells were treated with inhibitors of endocytosis, amiloride (Catalog No. S1811, Selleck Chemicals, Houston, TX, USA) (25 pM) and genistein (Catalog No. S1342, Selleck Chemicals) (150 pM), for 30 minutes and were subsequently transfected with ENVOYER nanoparticles (formulated as described above) for three hours. The final concentration of the nanoparticles in the transfection media was 7.5 pg / mL. The media in the wells was then replaced with fresh media free from inhibitors and ENVOYER nanoparticles, and the cells were incubated for an additional 24 hours.

[0275] HEK293T cells were transfected with G1 plasmid and the Donor plasmid using LIPOFECTAMINE® as follows. The day before transfection, cells were trypsinized and counted. About 0.5 to 1.25 x 105cells were plated per well in 0.5 mL of complete growth medium. Cell density was about 50-80% confluent (e.g., 70% confluent) on the day of transfection. On the day of transfection, growth medium was removed from cells and replaced with 0.5 mL of transfection medium. Cells were incubated at 37°C in a CO2 incubator for 18-24 hours post-transfection before assaying for transgene expression. Cells typically reached a confluence of about 90% before being assayed.

[0276] Transfection medium was prepared as follows. For each well of cells to be transfected, 1.25 pg of G1 guide / Cas9 plasmid and 2.5 pg of Donor plasmid was diluted in 100 pL of Gibco OPTLMEM® I Reduced Serum Media (Catalog No. 31985062, Thermo Fisher Scientific) without serum. For each well of cells, 0.75 to 1.75 pL of LIPOFECTAMINE® LTX Reagent was added into the above diluted OPTI-MEM®:G1 / Donor solution, mixed gently, and incubated 30 minutes at room temperature to form DNA-LIPOFECTAMINE® LTX Reagent complexes. After the 30 minute incubation, 100 pl of the DNA-LIPOFECTAMINE® LTX Reagent complexes was directly added to each well containing cells and mixed gently by rocking the plate back and forth. Complexes did not need to be removed following transfection.Results

[0277] HEK cells were transfected with ENVOYER nanoparticles (NPs) encapsulating G1 and the Donor plasmids (treatment group 1; Gl+Donor NPs), NPs encapsulating G1 plasmid and NPs encapsulating the Donor plasmid (treatment group 2; G1 NPs + Donor NPs); or the G1 plasmid and the Donor plasmid delivered via LIPOFECTAMINE® (treatment group 3; G1 + Donor LIPOFECTAMINE®). The guide RNA was complementary to a region of p53 which, when cleaved by Cas and inserted with the Donor, resulted in reduced expression of full-length p53 and expression of a GFP -tagged, truncated p53 protein (FIGs. 15A-B). The Donor was a long double-stranded DNA (dsDNA) encoding green fluorescent protein (GFP) and facilitated homology-directed repair (HDR) following cleavage by Cas.

[0278] After 5 passages in culture, p53 expression was stably reduced in all transfected cells (FIGs. 15A-B). Cells transfected under each of conditions 1 (Gl+Donor NPs), 2 (G1 NPs + Donor NPs), and 3 (G1 + Donor LIPOFECTAMINE®) were plated in 96-well plates. Single colonies were selected from 1 (Gl+Donor NPs) and 3 (G1 + Donor LIPOFECTAMINE®) and assayed for colony formation and GFP expression (Tables 2-3). Each sample collected from the 96-well plates was plated into its own well in a 24-well plate (one plate per treatment group).Table 2. Single Colony Selection from 96-well Plates - Gl+Donor LIPOFECT AMINE®Table 3. Single Colony Selection from 96-well Plates - G1 NPs + Donor NPs

[0279] Cells in treatment group 2 (G1 NPs + Donor NPs) had partial knockdown of full- length p53, while cells in treatment group 3 (G1 + Donor LIPOFECT AMINE®) had full knockdown (FIG. 16). The partial knockdown in NPs-treated cells was most likely due to thecolonies having multiple cell populations, so colonies with the most p53 knockdown were reisolated and tested.

[0280] Treatment group 2 (G1 NPs + Donor NPs) colonies 3 and 6 were chosen for reselection, and were plated into a 96-well plate. From there, cells from 4 wells that displayed GFP expression, each of which had as close to a single colony as possible, were chosen for passaging and western blotting. The results (FIG. 17) indicate that some of the colonies that survived the puromycin selection had partial implementation of the full CRISPR-Cas9 system. Partial implementation of the full CRISPR-Cas9 system would be avoided by encapsulating both the guide and donor plasmids into a single particle (e.g., as in treatment group 1).

[0281] After puromycin selection and before single colony selection, cells in treatment group 1 (Gl+Donor NPs) showed a major knockdown of full-length p53 (FIGs. 18A-B). Cells were plated and a colony (clone #5) was re-plated. All 10 of the clone #5 colonies had full p53 knockout, and had expression of truncated p53 as in the treatment group 3 (G1 + Donor LIPOFECT AMINE®) colonies. GFP-inserted truncated p53 is still tagged by the p53 antibody; therefore, western blot analysis of GFP (approximately 33 kDa) would not yield any results. GFP expressed under the p53 promoter in re-selected Clone #5-9 yields a modest signal (FIGs. 20A-B).Example 4: Hyaluronic Acid Polymer ENVOYER NanoparticlesPreparation and characterization of hyaluronic acid polymer (HyA) ENVOYER nanoparticles

[0282] Hyaluronic acid (also known as hyaluronan) nanoparticles encapsulating plasmid DNA (pDNA) were formulated by combining a pDNA solution, a crosslinker solution (e.g., polyethylenimine (PEI), a cationic polymer, or calcium chloride (CaCh)), and a hyaluronic acid (HA) polymer solution in a 1 : 1 :2 volume ratio, with all solutions prepared in Hank’s Buffered Solution (HBS) buffer. The concentration of pDNA was dependent on the length of the plasmid sequence (5757 bp), and the concentrations of crosslinker and polymer were adjusted to change the nitrogen to phosphate (N / P) ratio of the final particle formulation.

[0283] Varying HA concentrations and crosslinkers were tested in nanoparticle formulations utilizing a starting concentration of 200 pg / mL pDNA (Table 4). Transfection efficiencies were tested in HEK293T cells 48 hours post-transfection and were determined using flow cytometry.Table 4. HA nanoparticle formulations.

[0284] The 1 M CaCh solution was formed by weighing out solid CaCh and dissolving it in HBS buffer. Afterwards, the solution was filtered through a 0.2 pm biological filter to ensure that the solution did not have any biological contaminants or aggregates of salt.

[0285] The PEI stock solution was formed by weighing out a PEI solution (50% w / w) and diluting it in HBS buffer to the desired concentration. Afterwards, the solution was filtered through a 0.2 pm biological filter to ensure that the solution did not have any biological contaminants or aggregates of salt. This filtered solution was then used as a stock solution, and the required crosslinker concentration solution is created via dilution using HBS buffer.

[0286] The pDNA solution was created by diluting a stock solution of the pDNA of interest in HBS buffer to a concentration of 200 pg / mL.

[0287] After both the crosslinker and the pDNA solutions were made to the required concentrations, they were then mixed in equal volume via dropwise addition of the pDNA solution to the crosslinker solution over 20 seconds. Then, the mixture was gently triturated by pipette and left to incubate at room temperature for 30 minutes.

[0288] The hyaluronic acid solution was formed by weighing out solid hyaluronic acid between 1 to 2 gg and dissolving it in an amount of HBS buffer to form the stock solution. After the hyaluronic acid was fully dissolved in the HBS buffer, the stock solution was filtered through a 0.2 gm biological filter to ensure that the solution did not have any biological contaminants or aggregates of hyaluronic acid. With this stock solution, the respective required polymer concentration solutions for particle formulation were prepared by simple dilutions with HBS buffer.

[0289] Once the pDNA mixed with the crosslinker had finished incubating, and the hyaluronic acid solution at the desired concentration was prepared, the two solutions were taken into syringes at equal volumes, and run through the Ignite NANO ASSEMBLE® nanoparticle formulation instrument (Precision Nanosystems), which mixed the two solutions via microfluidics. The Ignite was set to dispense the syringes at an equal flow rate ratio, with a flow rate of 12 mL / min. The starting waste was set to 0.3 or 0.5 mL, depending on whether the syringes were 1 mL or 3 mL syringes, and the end waste was always set to 0.05 mL. Once the Ignite finished dispensing the desired volume, the sample solution was left to sit for one hour at room temperature, after which the particles were considered mature and ready for use.REFERENCESReferences from Examples

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[0346] Schoenmaker, L., Witzigmann, D., Kulkarni, J. A., Verbeke, R., Kersten, G., Jiskoot, W., et al. (2021). mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability. Int. J. Pharm. 601, 120586. doi: 10.1016 / J.UPHARM.2021.120586

[0347] Zielinska, A., Carreiro, F., Oliveira, A. M., Neves, A., Pires, B., Nagasamy Venkatesh, D., et al. (2020). Polymeric Nanoparticles: Production, Characterization, Toxicology and Ecotoxicology. Molecules 25. doi: 10.3390 / MOLECULES25163731

[0348] Myers Squibb, B. (n.d.). Abraxane®.

[0349] AMVUTTRA Prescribing Information. 2022.

[0350] FDA. ‘Drug Approval Package: Atridox (Doxycycline Hy elate, 10%) ND A# 50751’ (1998).

[0351] GIVLAARI Prescribing Information. 2023.

[0352] LEQVIO Prescribing Information. 2023.

[0353] ONPATTRO prescribing information. 2023.

[0354] FDA. LUPRON DEPOT (leuprolide acetate for depot suspension) 2023.

[0355] OXLUMO Prescribing Information. 2023.INCORPORATION BY REFERENCE; EQUIVALENTS

[0356] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0357] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A composition comprising nanoparticles formed from a plurality of anionic polymers, a therapeutic agent, and a cation, wherein at least 75% of the nanoparticles have a diameter of about 10 nm to about 80 nm as measured by transmission electron microscopy.

2. The composition of claim 1, wherein at least 95% of nanoparticles have a diameter of about 10 nm to about 80 nm as measured by transmission electron microscopy.

3. The composition of claim 1, wherein at least 98% of the nanoparticles have a diameter of about 10 nm to about 80 nm as measured by transmission electron microscopy.

4. The composition of claim 1, wherein at least 75% of the nanoparticles have a diameter of about 50 nm to about 70 nm as measured by transmission electron microscopy.

5. The composition of claim 1, wherein at least 95% of the nanoparticles have a diameter of about 50 nm to about 70 nm as measured by transmission electron microscopy.

6. The composition of claim 1, wherein at least 98% of the nanoparticles have a diameter of about 50 nm to about 70 nm as measured by transmission electron microscopy.

7. The composition of claim 1, wherein at least 75% of the nanoparticles have a diameter of about 50 nm to about 60 nm as measured by transmission electron microscopy.

8. The composition of claim 1, wherein at least 95% of the nanoparticles have a diameter of about 50 nm to about 60 nm as measured by transmission electron microscopy.

9. The composition of claim 1, wherein at least 98% of the nanoparticles have a diameter of about 50 nm to about 60 nm as measured by transmission electron microscopy.

10. The composition of claim 1, wherein the nanoparticles have an average diameter of about 48 nm to about 98 nm as measured by dynamic light scattering.

11. The composition of any one of claims 1-10, wherein the therapeutic agent comprises a nucleic acid.

12. The composition of any one of claims 1-10, wherein the therapeutic agent comprises a nucleic acid at least about 100 bases in length.

13. The composition of any one of claims 1-10, wherein the therapeutic agent comprises plasmid DNA (pDNA).

14. The composition of claim 13, wherein the pDNA comprises two or more plasmids.

15. The composition of claim 13, wherein at least 75% of the nanoparticles have a diameter of about 20 nm to about 30 nm as measured by transmission electron microscopy.

16. The composition of claim 13, wherein at least 95% of the nanoparticles have a diameter of about 20 nm to about 30 nm as measured by transmission electron microscopy.

17. The composition of claim 13, wherein at least 98% of the nanoparticles have a diameter of about 20 nm to about 30 nm as measured by transmission electron microscopy.

18. The composition of claim 13, wherein the nanoparticles have an average diameter of about 10 nm to about 30 nm as measured by dynamic light scattering.

19. The composition of claim 11, wherein the therapeutic agent comprises a nucleic acid about 20-40 nucleotides in length.

20. The composition of claim 19, wherein the therapeutic agent comprises small interfering RNA (siRNA).

21. The composition of any one of claims 1-10, wherein the plurality of anionic polymers comprises one or more natural polymers.

22. The composition of any one of claims 1-10, wherein one or more of the plurality of anionic polymers is selected from the group consisting of alginate and hyaluronic acid.

23. The composition of any one of claims 1-10, wherein the plurality of anionic polymers comprises alginate.

24. The composition of claim 23, wherein the plurality of anionic polymers further comprises hyaluronic acid.

25. The composition of any one of claims 1-10, wherein the plurality of anionic polymers comprises alginate, hyaluronic acid, or both.

26. The composition of any one of claims 1-10, wherein the cation comprises a divalent cation.

27. The composition of claim 26, wherein the divalent cation comprises calcium (Ca2+).

28. The composition of any one of claims 1-10, wherein the cation is a divalent cation.

29. The composition of claim 28, wherein the divalent cation is Ca2+.

30. The composition of claim 11, wherein the nucleic acid comprises short interferingRNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), guide RNA, DNA, plasmid DNA (pDNA), or any combination of the foregoing.

31. The composition of claim 30, wherein the nucleic acid comprises or encodes a sequence complementary to a portion of a gene or a transcript associated with a disease, disorder, or condition.

32. The composition of claim 31, wherein the disease, disorder, or condition comprises a cancer or a metabolic, neurodegenerative, cardiovascular, infectious, or inflammatory disease or disorder.

33. The composition of claim 11, wherein the nucleic acid is selected from the group consisting of short interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), guide RNA, DNA and plasmid DNA (pDNA).

34. The composition of claim 33, wherein the nucleic acid is capable of silencing of a gene associated with a disease, disorder, or condition selected from the group consisting of cancer, a metabolic, a neurodegenerative, a cardiovascular, an infectious, or an inflammatory disease or disorder.

35. The composition of claim 11, wherein the nucleic acid is RNA, and wherein the RNA and the plurality of anionic polymers are in a molar ratio from about 1 : 1 to about 30: 1.

36. The composition of claim 35, wherein the RNA and plurality of anionic polymers are in a molar ratio of about 7: 1.

37. The composition of claim 35, wherein the plurality of anionic polymers comprises alginate, and wherein the RNA and plurality of anionic polymers are in a molar ratio from about 7: 1 to about 30: 1.

38. The composition of any one of claims 1-10, wherein the plurality of anionic polymers and the therapeutic agent are in a concentration ratio of about 1 :6.

39. The composition of claim 11, wherein the nucleic acid encodes a pharmaceutically active peptide or protein.

40. The composition of claim 39, wherein the pharmaceutically active peptide or protein comprises a CRISPR Cas enzyme, and wherein the nucleic acid further encodes a guide RNA.

41. The composition of claim 40, wherein the nucleic acid further encodes a donor oligonucleotide.

42. The composition of claim 14, wherein a first plasmid encodes a CRISPR Cas enzyme and a guide RNA, and wherein a second plasmid encodes a donor oligonucleotide.

43. The composition of any one of claims 1-10, wherein the nanoparticles have a zeta potential of about -7.8 mV to about -9.1 mV.

44. The composition of any one of claims 1-10, wherein: the plurality of anionic polymers comprises hyaluronic acid, the cation comprises polyethyleneimine (PEI) or Ca2+, and the therapeutic comprises a nucleic acid; and wherein the nanoparticles comprise: about 25 pg / mL to about 115 pg / mL hyaluronic acid, about 79 pg / mL PEI or about 27.75 mg / mL (0.25 M) Ca2+, and about 50 pg / mL nucleic acid.

45. The composition claim 44, wherein the nanoparticles comprise about 25 pg / mL hyaluronic acid and about 27.75 mg / mL (0.25 M) Ca2+.

46. The composition of any one of claims 1-10, wherein: the plurality of anionic polymers comprises alginate, the cation comprises Ca2+, and the therapeutic comprises a nucleic acid; and wherein the nanoparticles comprise: about 25 pg / mL alginate, about 27.75 mg / mL (0.25 M) Ca2+, and about 3 pg / mL to about 150 pg / mL nucleic acid.

47. The composition of claim 46, wherein the nucleic acid comprises pDNA, and wherein the pDNA is about 5 kilobases (kb) to about 8 kb in length.

48. The composition of claim 47, wherein the nanoparticles comprise about 3 pg / mL pDNA.

49. The composition of claim 47, wherein the nanoparticles comprise about 6 pg / mL pDNA.

50. The composition of claim 47, wherein the nanoparticles comprise about 150 pg / mL pDNA.

51. The composition of claim 46, wherein the nucleic acid comprises siRNA, and wherein the siRNA is about 21 bases in length.

52. The composition of claim 51, wherein the nanoparticles comprise about 67 pg / mL siRNA.

53. A method for making a nanoparticle composition, the method comprising:(a) in a microfluidic device, mixing: i) a first liquid composition comprising a plurality of anionic polymers; and ii) a second liquid composition comprising a therapeutic agent and a cation, thereby producing a composition of nanoparticles, wherein at least 75% of the nanoparticles have a diameter of about 10 nm to about 80 nm as measured by transmission electron microscopy.

54. The method of claim 53, wherein the first and second compositions are mixed in a ratio from about 1 : 1 to about 1 :30.

55. The method of claim 53, wherein the first and second compositions are mixed in a ratio of about 1 :7.

56. A method for synthesizing nanoparticles, the method comprising:(a) injecting a first solution comprising a plurality of anionic polymers and a buffer into a first inlet of a microfluidic mixer; and(b) injecting a second solution comprising a therapeutic agent, a cation, and the buffer into a second inlet of the microfluidic mixer; thereby producing nanoparticles encapsulating the therapeutic agent, wherein at least 75% of the nanoparticles have a diameter of about 10 nm to about 80 nm as measured by transmission electron microscopy.

57. The method of claim 56, wherein the microfluidic mixer comprises one or more nanoscale microfluidic channels.

58. The method of claim 57, wherein one or more of the nanoscale microfluidic channels is about 1 pm in diameter.

59. The method of claim 56, wherein the first solution comprises: a) the plurality of anionic polymers at a concentration of about 50 pg / mL to about 230 pg / mL; b) the buffer at a concentration of about 500 mM; or c) both a) and b).

60. The method of claim 56, wherein the second solution comprises: a) the therapeutic agent at a concentration of about 6 pg / mL to about 300 pg / mL; b) the cation at a concentration of about 158 pg / mL to about 55.5 mg / mL; c) the buffer at a concentration of about 500 mM; or d) any combination of the foregoing.

61. The method of claim 56, wherein the nanoparticles comprise: a) the plurality of anionic polymers at a concentration of about 25 pg / mL to about 115 pg / mL; b) the therapeutic agent at a concentration of about 3 pg / mL to about 150 pg / mL; c) the cation at a concentration of about 79 pg / mL to about 27.75 mg / mL; d) the buffer at a concentration of about 500 mM; or e) any combination of the foregoing.

62. The method of claim 56, wherein: a) the plurality of anionic polymers comprises alginate or hyaluronic acid; b) the therapeutic agent comprises a nucleic acid; c) the cation comprises polyethyleneimine (PEI) or Ca2+; d) the buffer comprises 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES); or e) any combination of the foregoing.

63. The method of any one of claims 56-62, wherein injecting the first solution and injecting the second solution are performed simultaneously.-n -64. The method of any one of claims 56-62, wherein the first and second solutions are injected at a flow rate ratio of about 5: 1 to about 1 :5.

65. The method of any one of claims 56-62, wherein the first and second solutions are injected at a flow rate ratio of about 2: 1 to 1 :2.

66. The method of any one of claims 56-62, wherein the first and second solutions are injected at a flow rate ratio of about 1 :1.

67. The method of any one of claims 56-62, wherein the first and second solutions are each injected at a total flow rate of about 12 mL / minute.

68. The method of any one of claims 56-62, wherein injecting the first solution and injecting the second solution produces a mixture.

69. The method of claim 58, the method further comprising dispensing the mixture from the microfluidic device, thereby producing a dispensed mixture comprising the nanoparticles.

70. The method of claim 69, the method further comprising discarding a volume of the dispensed mixture at a start and at an end of dispensing the mixture from the microfluidic device.

71. The method of claim 69, the method further comprising incubating the dispensed mixture at room temperature.

72. The method of claim 69, the method further comprising incubating the dispensed mixture for a period of about 30 minutes.

73. The method of any one of claims 56-62, the method further comprising preparing the first solution by: a) mixing a polymer solution and a cation solution; b) gently vortexing the mixed polymer and cation solutions; and c) allowing the mixed polymer and cation solutions to incubate at room temperature; thereby producing the first solution.

74. The method of any one of claims 56-62, the method further comprising storing the nanoparticles at a temperature of about -80°C to about 25°C for a period of up to about 3.5 months.

75. A method for delivering a therapeutic agent to a cell, comprising contacting the cell with a composition comprising one or more nanoparticles, each nanoparticle comprising a plurality of anionic polymers, a therapeutic agent, and a cation, wherein at least 75% of the nanoparticles have a diameter of about 10 nm to about 80 nm as measured by transmission electron microscopy.

76. The method of claim 75, wherein the plurality of anionic polymers comprises alginate, hyaluronic acid, or both.

77. The method of claim 75, wherein the cation comprises a divalent cation.

78. The method of claim 77, wherein the divalent cation comprises calcium (Ca2+).

79. The method of claim 75, wherein the therapeutic agent comprises a nucleic acid.

80. The method of claim 79, wherein the nucleic acid comprises short interfering RNA(siRNA), microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), guide RNA, DNA, plasmid DNA (pDNA), or any combination of the foregoing.

81. The method of claim 75, wherein the nanoparticles comprise: a) the plurality of anionic polymers at a concentration of about 25 pg / mL to about 115 pg / mL; b) the therapeutic agent at a concentration of about 3 pg / mL to about 150 pg / mL; c) the cation at a concentration of about 79 pg / mL to about 27.75 mg / mL; d) the buffer at a concentration of about 500 mM; or e) any combination of the foregoing.

82. The method of claim 75, wherein the cation comprises polyethyleneimine (PEI).

83. The method of any one of claims 75-82, wherein the composition comprises the one or more nanoparticles at a concentration of 7.5 pg / mL.

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