Programmable photocurable poly(beta-amino ester) particles for drug delivery

Abstract

Methods and compositions directed to a novel particle system using poly(P-amino ester) (PBAE) resins for applications in drug delivery, where the PBAE particle is fabricated via lithography.

Description

[0001] PROGRAMMABLE PHOTOCURABLE POLY(BETA-AMINO ESTER) PARTICLES FOR DRUG DELIVERY

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 682,666, filed August 13, 2024. The entire specification and figures of the above-referenced application are hereby incorporated, in their entirety by reference.

[0004] STATEMENT OF GOVERNMENT INTEREST

[0005] This invention was made with government support under grant number R35GM147455 awarded by National Institutes of Health (NIH). The government has certain rights in the invention.

[0006] TECHNICAL FIELD

[0007] The technology described herein relates to methods and compositions relating to a novel particle system using poly(β-amino ester) (PBAE) resins for applications in drug delivery.

[0008] BACKGROUND

[0009] Over the past several decades, numerous synthetic polymer systems have been considered to enhance the delivery of drugs for disease treatment. Such polymer systems have been established to control cross linking, encapsulate drugs of various hydrophilicities, and degrade at prescribed rates. However, few of these polymer systems also enable the manufacturing of micro- and nanoparticles with precisely defined, nonspherical shapes. This critical gap impedes the development of next-generation drug-delivery vehicles, as particle shape has been shown to play an important role in nanoparticle delivery and fate.

[0010] SUMMARY OF THE INVENTION

[0011] To address this long-felt need within the field of polymeric particle drug delivery, Applicants have developed a novel and high-throughput method to fabricate programmable drug- loaded micro- and nanoparticles with controllable shape, size, and crosslink density. As described herein, in one preferred embodiment, the PBAE particles of the disclosure are manufactured using via contact photolithography and two-photon lithography. In certain additional embodiments, the PBAE particles are programmable via the parameters of their manufacturing and ratios of components such that their stiffness, shape, size, degradation, and drug release can be precisely tuned. Applicants describe a high-throughput approach for the rapid fabrication of micro- and nanoparticles with engineered shape, elasticity, degradability, and drug release rate for a broad range of drug delivery applications via lithography (i.e., contact, maskless, interferometric, two- photon lithography). Moreover, the photoactivation and / or molecular weight of the PBAE diacrylate monomer can modulate the cross-linking of the PBAE polymer to produce a customized discoidal particles (microscale in dimeter and nanoscale in thickness) with, for example, a tuned drug release profiles among other physical characteristics to enable attachment to cells as drug delivery vehicles generally referred to herein as “backpacks.” In still further embodiments, the PBAE particles of the disclosure can be manufactured to include one or more therapeutic compounds.

[0012] Additional aspects of the present disclosure will be apparent from the claims, specification and figures provided herein.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1. PBAE-dA synthesis and crosslinking, (a) PBAE-dA macromers are formed through the addition of a Michael donor (primary amine) and Michael acceptor (terminal alkene / acrylate). (b) Idealized macromer synthesis based upon molar ratio of diacrylate:amine. (c) Terminal acrylates (yellow) photocrosslink, forming poly acrylate kinetic chains separated by PBAE-dA linkers. Lower molar ratios of diacrylate:amine results in higher molecular weight macromers and thus a larger mesh size.

[0015] Figure 2. Characterization of elastic and shear moduli across PBAE-dA compositions, (a) Naming scheme for two chemical compositions used, (b) Young’s moduli of thin films measured at 37°C (n=3±l S.D.). (c) Representative photorheology data of poly-iso 1.5. This method was used to find the (d) shear moduli and approximate mesh size across each composition (n=3±1 S.D.).

[0016] Figure 3. PBAE-dA particles with microscopic and nanoscopic thicknesses, (a) Visual representation of protocol to print PBAE-dA backpacks on a silicon wafer using contact photolithography. The PBAE-dA resins are dissolved in chloroform (TCM) along with free radical photoinitator, diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide (TPO). (b) Poly-benz 1.5 particles (7x1.7μm) produced through process detailed in part (a), (c) Particles fabricated through maskless photolithography with nanoscopic thickness (690 ± 91nm).

[0017] Figure 4. Degradation is affected by crosslink density and polymer composition, (a) Bulk samples degraded in IM NaOH to exemplify differences in degradation rates between compositions. (n=5±l S.D.). (b) Silicon-wafer bound particles were degraded in lx PBS at 37°C to demonstrate physiologically relevant degradation times.

[0018] Figure 5. Biocompatibility of discoidal PBAE-dA particles, (a) Relative viability of HEK293T cells in the presence of PBAE-dA degradation byproducts (n=5±l S.D.). (b) Primary bone marrow-derived macrophages with poly -benz 1.5 particles bound to their surface (green=cytosol, blue=nuclei, pink=particles).

[0019] Figure 6. Drug release of dexamethasone from PBAE-dA particles, (a) A co-solvent method was used to load hydrophobic dexamethasone into PBAE-dA particles. A biphasic mixture of water, ethanol (EtOH) and chloroform (TCM) was used to dissolve dexamethasone and drive it into PBAE-dA particles as the organic phase evaporates, (b) 7μm-wide hexagonal particles across three compositions (poly-benz 1.5, poly-benz 5, and poly-iso 1.5) possessed no significant differences between thickness (mean ±1 S.D., n=10). (c) The fraction of loaded dexamethasone released from poly-benz 1.5 (n=3), poly-benz 5 (n=2) and poly-iso 1.5 (n=3) particles and (d) cumulative mass released into 1XPBS containing 1 vol.% DMSO.

[0020] Figure 7. Nanoscale 3D printing using two-photon lithography, (a) Two-photon lithography uses rapid pulses of half-energy light which converge at a voxel, providing just enough energy to induce free radical photopolymerization. For example, two 810nm photons combine to activate a photoinitator with a 405nm threshold, (b) Resolution prints of poly-benz 1.5 resin at laser power of 45% and 7500 μm / s (c) and 50% and 7000 μm / s. (d) Dodecahedron printed using poly-benz 1.5 using a scan speed of 7500 μm / s and a laser power of 50% in top-down and (e) side view.

[0021] Figure 8. Biodegradable magnetic microrobot (a) SEM image of Fe-coated micRotini (b) the step-out frequency of micRotinis is around 150 Hz (n=3±l S.D.) (c) External magnet fields endow spatiotemporal control of micRotinis, spelling out “CU”. (d) In 0.1M NaOH, micRotinis delaminate and degrade in ~22 days.

[0022] Figure 9. H-NMR spectra of (a) poly-benz and (b) poly-iso reactants and macromers at molar ratios of 1.2, 1.5, 5, and 10: 1 (diacrylate: amine), with labeled peaks indicating successful aza-Michael addition.

[0023] Figure 10. Biocompatibility of a relatively high concentration of degraded discoidal PBAE-dA particles (a) Relative viability of HEK293T cells in the presence of PBAE-dA degradation byproducts (n=5±l S.D.). Figure 11. The distribution of poly-benz 1.5 particles bound to live primary bone marrow- derived macrophages. The horizontal purple line indicates 1 parti cl e / cell. Particles were associated with macrophages at ratios of 1 : 1, 3: 1, and 5: 1 particles:cell (n=300, solid black line=mean, dashed lines= ±S.D. and 1.96*S.D.

[0024] Figure 12. Primary bone marrow-derived macrophages with surface bound poly-benz 1.5 particles. Cells that were associated with many (> 10) appeared to be less viable, as indicated by decreased green fluorescence (green= live cell cytosol, blue= nuclei, pink= particles).

[0025] Figure 13. Ultraviolet-visible (UV-Vis) spectral calibration curves of dexamethasone in its release media, (a) The UV-Vis spectra for dexamethasone in 1XPBS with 1% DMSO and (b) in pure ethanol, (c) The absorbance at 242nm was measured at various concentrations of dexamethasone and plotted to create calibration curves for dexamethasone in IX PBS with 1% DMSO and (d) in pure ethanol.

[0026] Figure 14. Linearized models of drug release fit to the release of dexamethasone from PBAE-dA backpacks. Across 5 different models, (a) the Korsmeyer-Peppas model fit best to poly- benz 1.5, (b) the Hixson-Crowell fit poly-benz 5, and (c) the Higuchi model best fit poly-iso 1.5.

[0027] Figure 15. Two-photon photolithography printing accuracy at (a) 7500 μm / s scan speed and 45% laser power, and (b) 7000 μm / s at 50% laser power, (c) The error at the lowest printed feature was calculated by comparing the absolute value of the difference between experimentally measured thicknesses and the encoded feature size.

[0028] Figure 16. MicRotini fabrication via two-photon photolithography, (a) The micRotini particle design featured a diameter (D) of 20 μm, length (L) of 30 μm, helix thickness (t) of 3 μm, and number of full helix rotations (n) of 3. (b) Scanning electron microscopy image of several iron-coated micRotinis.

[0029] DETAILED DESCRIPTION OF THE INVENTION

[0030] The following detailed description is provided to aid those skilled in the art in practicing the various embodiments of the present disclosure, including all the methods, uses, compositions, etc., described herein. Even so, the following detailed description should not be construed to unduly limit the present disclosure, as modifications and variations in the embodiments herein discussed may be made by those of ordinary skill in the art without departing from the spirit or scope of the present discoveries. The present disclosure is explained in greater detail below. This disclosure is not intended to be a detailed catalog of all the different ways in which embodiments of this disclosure can be implemented, or all the features that can be added to the instant embodiments. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which variations and additions do not depart from the scope of the instant disclosure. Hence, the following specification is intended to illustrate some particular embodiments of the disclosure, and not to exhaustively specify all permutations, combinations, and variations thereof.

[0031] Described herein is a novel a system for the fabrication and use of programmable poly-(β- amino ester) (PBAE) particles, referred to interchangeably herein as “backpacks” or “polymeric particles.” In a preferred embodiment, the backpacks particles of the disclosures can be specifically applied for use in cell-specific drug delivery. In this preferred embodiment the backpack particles attached to a cell surface allowing cell-directed delivery. In a preferred embodiment, backpack particles can be attached to the surface of an immune cell thereby permitting effective adoptive immune cell therapy.

[0032] In a preferred embodiment, the polymeric particles comprise poly-(β-amino ester) (PBAE) particles, which generally include a broad class of a class of biodegradable polymer compositions. PBAEs can generally prepared via step-growth polymerization, i.e., Michael addition (MA) reaction. In a preferred embodiment, the particles of the disclosure are fabricated from photocurable PB AE-diacrylate resins (PB AE-dA), which describe an expansive class of polymers, many of which are biocompatible and readily biodegradable. In this preferred aspect, PB AE-dA macromers are synthesized via the aza-Michael addition of acrylates with amines, forming hydrolytically degradable ester bonds.

[0033] As shown in Table 2, Michael addition chemistry can be used to synthesize various PBAEs (B). Diacrylate monomers (lettered) and amine-containing monomers (numbered) can be used to generate a variety PBAEs with varying chemical structure via combinatorial chemistry using Michael addition as shown in C.

[0034] In one embodiment, a preferred PBAE-diacrylate of the disclosure is formed via the Michael addition reaction between 1,4-butanediol diacrylate reacted with isobutylamine, forming a PBAE-diacrylate referred to a “poly-iso.” In another embodiment, a preferred PBAE-diacrylate of the disclosure is formed via the Michael addition reaction between 1,4-butanediol diacrylate reacted with benzhydrazide, forming a PBAE referred to a “poly-benz.” As shown in Table 1 below, different molar concentrations of the acrylate and amine, in this case 1,4-butanediol diacrylate and isobutylamine or benzhydrazide can be used which, as described herein can tune the physical properties, such as density and stiffness of the backpack particle.

[0035] In another preferred embodiment, after PBAE macromer formation, a photoinitiator can be incorporated into the composition, allowing for the free radical polymerization of pendant acrylates in the presence of light. More specifically, in a preferred embodiment, the PBAE- diacrylate macromer can undergo light-initiated crosslinking in the presented of a photoinitiator, such as diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide (TPO).

[0036] As described herein, in one preferred embodiment, the PBAE backpack particles of the disclosure are manufactured using via contact photolithography and two-photon lithography. For example, in one embodiment a PBAE polymer containing a photoinitiator is spin-coated directly on a photolithographic substrate and activated via exposure to UV light, and washed to reveal the formed backpacks which can be further isolated from the same. In the embodiment shown in Figure 4, a silicon wafer can be plasma treated to promote initial adherence of the polymeric resin. Next, a quantity of polyvinyl alcohol (PVA) can be spin coated to the surface of the wafer, after which a quantity of PBAE polymer can be spin coated to the wafer in the presence of chloroform (CHCI3) to a thickness of 100-1600 nm. The wafer can then be heated and cleaned, followed by UV irradiation in a mask aligner, which in this example includes a wavelength of light of 405nm. In one embodiment, the photomask is pressed directly onto the wafer containing spin-coated PBAE, thus slowing the diffusion of oxygen enough for the UV-mediated cross-linking reaction to occur with the photoinitiator present. Post-exposure, the wafer can be heated and developed, for example in a solution of SU-8 developer, followed by one or more wash-steps which reveal the formed backpacks that can be manually isolated.

[0037] In another embodiment, as shown generally in Figure 7 the backpack particles of the disclosure can be fabricated from PBAE-diacrylate monomer containing a photoinitiator via two- photon lithography. In this embodiment, UV light having wavelengths 2x the photoinitiation threshold of the photoinitiator are directed at the focal point of the polymer to initiate crosslinking and formation of the backpack shaped particle, which can further be isolated. As described in the examples herein, the backpack particles displayed favorable characteristics for being retained on the cell surface without altering the cell’s behavior. Tn some embodiments, the polymeric particle is substantially discoidal in shape. In some embodiments, the polymeric particle is discoidal in shape. As used herein, “discoidal” refers to a particle having a disk-like shape, with substantially flat, concave or convex faces. In some embodiments, the PBAE particles have a shape which is a rod, a cylinder, a cube, cuboid, hexahedron, or pyramid.

[0038] In one embodiment, the PBAE particles of the disclosure include at least one secondary compound. In one embodiment, this secondary compound can be selected from secondary compound can be selected from a polarizing agent, a contrast agent, a magnetically responsive material, a cell adhesive molecule, a targeting ligand, a therapeutic compound, or a combination of the aforementioned.

[0039] In certain embodiment, the secondary compound can be loaded into the particle prior to, or after polymerization as generally described herein. For example, in one embodiment the secondary compound can be added directly into the PBAE polymer prior to polymerization. In another embodiment, the secondary compound is coupled to the surface of the PBAE particle after polymerization as generally described herein. In another embodiment, after polymerization and isolation of the PBAE particles of the disclosure, they can be incubated with one or more secondary compounds in a solution containing at least 2 different solvents. In this example, the PBAE particles swell during incubation while at least one of the solvents is evaporated causing the secondary compound to be incorporated into the particle. Exemplary co-solvents can include ethanol, methanol, isopropanol, acetone, DMSO, acetic acid, and acetonitrile

[0040] In another embodiment, the PBAE particles of the disclosure can be fabricated to include one or more a polymerizing agent, also referred to in this embodiment as a co-polymerizing agent which can modulate the polymerization of the PBAE polymer so as to affect the drug delivery characteristics of the particle. In a preferred embodiment, a co- polymerizing agent pentaerythritol tetrakis(3-mercaptopropionate) (PETMP) is added to the PBAE polymer prior to polymerization via lithography fabrication. In this embodiment, the PETMP increases the extent of crosslinking of the polymer which slows the rate of drug release, and further increase the rate of degradation of the polymer.

[0041] In another embodiment, the PBAE particles of the disclosure can be fabricated to include one or more oxygen scavenger. As noted elsewhere in the disclosure, PBAE polymers are sensitive to oxygen. In this embodiment, the addition of an oxygen scavenger to a PBAE resin can enable more consistent particle prints and yields via lithographic processes. In one preferred embodiment, the oxygen scavenger can include triphenylphosphine among others.

[0042] In one embodiment, the PBAE particles of the disclosure include at least one polarizing agent. When the PBAE particles described herein are adhered to a cell surface, they are resistant to phagocytosis, but able to regulate the phenotype of the cell via the polarizing agent(s). Thus, the polymeric particles permit persistence of the desired cell phenotype, which can be beneficial for therapeutic purposes.

[0043] In a referred embodiment, the PBAE particles of the disclosure are configured to adhere to a cell, which preferably is a macrophage cell, i.e., an MO, Ml, M2, Ml -polarized, or M2 -polarized macrophage. The presence of the PBAE particle on the surface of the cell can, by contacting the cell with the polarizing agent, direct or regulate the phenotype of the cell, for example by increasing the likelihood, duration, magnitude, or rate of development Ml or M2 phenotypic characteristics. In some embodiments, the macrophage is substantially driven to an Ml or M2 phenotype by adherence of the PBAE particle. In some embodiments, the phenotype of the macrophage is regulated by the release of the one or more polarizing agent from the PBAE particle, which can include the induced or non-induced release of the cytokine and / or induced or noninduced degradation of the polymeric particle.

[0044] An Ml or Ml -polarized macrophage, also referred to as “killer” macrophages secrete high levels of IL-12 and low levels of IL-10. Ml macrophages can be characterized by the expression of, i.e., CCL3, CCL5, CD80, CCR7, iNOS and INF-g. An M2 or M2 -polarized macrophage, also referred to as a “repair” macrophage, contributes to wound healing and tissue repair. M2 macrophages produce high levels of IL-10. An M2 -polarized macrophage can be characterized by the expression of, for example CCL22, CD206, CD163, YM1, Fizzl, and arginase 1.

[0045] As described herein, a “polarizing agent” is an agent, that when contacted with a macrophage and / or monocyte, alters the likelihood, persistence, magnitude, or rate of development of a particular macrophage phenotype as compared to the absence of the polarizing agent. A polarizing agent can be an Ml-polarizing agent, i.e., it increases the likelihood, persistence, or rate of development of an Ml phenotype, or an M2 -polarizing agent, i.e., it increases the likelihood, persistence, or rate of development of an M2 phenotype. Exemplary Ml and M2 phenotypes are described herein and are well known in the art. Polarizing agents for the Ml and M2 macrophage phenotypes are known in the art, and can include, by way of non-limiting example, the Ml -polarizing Toll-like receptor (TLR) agonists (i.e., LPS, muramyl dipeptide, or lipoteichoic acid); the Ml-polarizing cytokines IFN-g (i.e., NCBI Gene ID: 3458); TNF (i.e., NCBI Gene ID: 7124); IL-12 (i.e., NCBI Gene ID: 3592 and 3593); GM- CSF (i.e., NCBI Gene ID: 1438); IL-1 (i.e., NCBI Gene ID: 3553); IL-6 (i.e., NCBI Gene ID: 3569); CD1 lb (i.e., NCBI Gene ID: 3684) and IL-23 (i.e., NCBI Gene ID: 51561) and the M2- polarizing cytokines IL-4 (i.e., NCBI Gene ID: 3565); IL-10 (i.e., NCBI Gene ID: 3586); glucocortoids (i.e., cortisol, cortisone, prednisone, prednisolone, methylprednisonolone, dexamethasone, betamethasone, triamcinolone, fludrocortisone acetate, and deoxycorticosterone acetate); M-CSF (i.e., NCBI Gene ID: 1435), TGF-beta (i.e. NCBI Gene ID: 7040); IL-6 (i.e., NCBI Gene ID: 3569); and IL-13 (i.e., NCBI Gene ID: 3596). TLR agonists are known in the art and can include, by way of non-limiting example LPS, dsRNA; flagella; bacterial lipoprotein; ssRNA; cpG DNA; bacterial peptidoglycans; profillin; rRNA; imiquimod; resiquimod; IMO- 2055; picibanil; monophsophoryl lipid A (MPL); polyribocytidylic acid (polyPC); CpG-28; MGN1703; glucopyranosyl lipid A; entolimod; and ODN2006.

[0046] In another embodiment, the PBAE particles of the disclosure are configured to adhere to monocytes, which are the progenitor cells of macrophages. The PBAE particle are modified in the referred embodiment to contain molecules, which facilitate adhesion to the surfaces of the monocytes. The PBAE particles on the surface of the cell can, by contacting the cell with the polarizing agent, direct or regulate the phenotype of the cell, for example by increasing the likelihood, duration, magnitude, or rate of development classical, non-classical, or intermediate phenotypic characteristics. In some embodiments, the phenotype of the monocyte is regulated by the release of the one or more polarizing agent from the PBAE particle, which can include the induced or non-induced release of the cytokine and / or induced or non-induced degradation of the polymeric particle.

[0047] In some embodiments, the PBAE backpack particles described herein can further incorporate a binding ligand, also referred to as cell adhesive molecules. Cell adhesive molecules can be any molecule which binds or adheres to the surface of a cell. Non-limiting examples of suitable cell adhesive molecules include polyelectrolytes, immunoglobulins, ligands for receptors on a cell surface, and / or monocyte targeting and / or macrophage -targeting ligands. Characteristics that can enhance cell adhesion can include high surface free energy, hydrophilic protein content, low surface hydration, and low surface charge density. Exemplary, non-limiting cell adhesive molecules can include poly(glycidyl methacrylate) (PGMA); polycaprolactone (PCL); polydimethylsiloxane (PDMS); poly(hexamethyldisiloxane) (PHMDSO); superhydrophobic perfluoro-substituted PEDOT (PEDOT- F); superhydrophobic polystyrene (PS); plasma-treated poly(methyl methacrylate) (PMMA); plasma- treated poly-3 -hydroxybuty rate (P3HB); phosphatidylethanolamine (PE); and carboxymethyl chitin (CMCH). Cell adhesive molecules can also include, or comprise RGD peptides, collagen, fibronectin, gelatin, and collagen. Cell adhesive molecules can include ligands specific to cell surface proteins exemplified by anti-CDl lb, anti- CD45, anti-CD80, anti-HLA-DR, and anti-CX3CRl. In some embodiments, cell adhesive polyelectrolytes comprise hyaluronic acid, poly(allylamine) hydrochloride, and / or hyaluronic acid modified to comprise aldehyde groups.

[0048] In some embodiments, the PBAE backpack particles described herein can include one or more one or more targeting ligands. Ligands for the receptors on a given cell surface and / or which target a monocyte or macrophage are known in the art and can include natural or synthetic ligands. Exemplary ligands for macrophages and / or monocytes can include, by way of non-limiting example, IL-4; CX3CL1; IL- 17A; IL-17F; M-CSF; GM-CSF; LDL; ApoE; IL-2; IFN-g; Hsp60; Hsp70; complement C5A; leukotriene B4; CCL2; CCL4; CCL3; CCL5; CCL7; CCL8; CXCL8; CXCL9; CXCL10; and / or CXCL11.

[0049] As noted above, particles described herein can include payload molecules, such as therapeutic compositions which can include, but not be limited to: a polypeptide, a nucleic acid, a biologic, small-molecules drugs, imaging molecules, a dye, a diagnostic compositions, a magnetic particle embedded or coating the particle, and the like. The therapeutic molecule can act on the monocyte or macrophage, or on a second cell / cell type. In some embodiments, the therapeutic molecule is present in admixture with the structural PBAE polymer.

[0050] As described herein, certain embodiments of the backpack particles described herein can be disrupted or degraded in a controllable and / or inducible manner. The particles can also be localizable. One approach to providing such functionality is to incorporate into the particle a liposome or nanoparticle that can be disrupted or removed by a controllable external stimulus. For example, echogenic liposomes are known in the art and can be disrupted by ultrasound waves. Magnetic and gold nanoparticles are responsive to magnetic and electromagnetic fields respectively, and this functionality can be used to localize the particles, localize the cells they are adhered to, and / or to disrupt the particles.

[0051] Embodiments of the PBAE backpack particles described herein can further be rationally designed to be controllably degraded, either to control delivery of a therapeutic payload or to regulate the effect of the particle on the carrier cell, such as in a preferred embodiment a cell, such as a stem cell, a macrophage, a monocyte, a T cells, a CAR-T cells, a neutrophil, a B cells, a mast cells, or a combination of the same. In a preferred embodiment, the PBAE backpack particles are programmable, such that the molar ratio of the acrylate and amine components can be varied thereby imparting tunable stiffness and crosslinking density. Moreover, the light-mediated polymerization can be controlled to tune the level of cross-linking of the components of the particles thereby tuning its degradation profde, in particular in vivo degradation. In this manner, the stiffness, shape, size, and degradation of the PBAE backpack particles of the disclosures can the rationally designed, each affecting the delivery of an embedded therapeutic compounds, such as a small molecule drug composition.

[0052] In some embodiments, the PBAE backpack particles can be administered to a subject in need thereof to treat a disease or condition.

[0053] The backpack particles described herein can be administered to a subject having or diagnosed as having one of the conditions described herein. In some embodiments, the methods described herein comprise administering an effective amount of backpack particles described herein. In some embodiments, a therapeutically effective dose of the composition is administered. As used herein, “alleviating a symptom” is ameliorating any condition or symptom associated with the disease. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, injection, or intratumoral administration. Administration can be local or systemic.

[0054] The term “effective amount” as used herein refers to the amount of backpack particles and their associated payload or therapeutic compositions or other therapeutic or diagnostic molecules needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term “therapeutically effective amount” therefore refers to an amount of backpack particles that is sufficient to provide a particular therapeutic effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount”. However, for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.

[0055] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, i.e., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the active ingredient which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[0056] In some embodiments, the backpack particles described herein can be a pharmaceutical composition. In some embodiments, the technology described herein relates to a pharmaceutical composition comprising an PBAE backpack particle as described herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the active ingredients of the pharmaceutical composition comprise a PBAE backpack particle as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist essentially of an PBAE backpack particle as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist of an PBAE backpack particle as described herein. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and / or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent, as described herein.

[0057] In some embodiments, the pharmaceutical composition comprising a PBAE backpack particle as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry or lyophilized products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled- release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS®- type dosage forms and dose-dumping.

[0058] Suitable vehicles that can be used to provide parenteral dosage forms of an PBAE backpack particle composition as disclosed within are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0059] In some embodiments, the PBAE backpack particle compositions described herein is administered as a monotherapy. In some embodiments, the methods described herein can further comprise administering a second agent and / or treatment to the subject as part of a combinatorial therapy. Non-limiting examples of a second agent and / or treatment can include radiation therapy, and surgery. Examples of therapeutic compositions that can be embedded as a payload in the PBAE backpack particles of the disclosure, or in the alternative can be separately administered as part of a combination therapy can be selected from: gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479, FK506, vorinostat, acriflavine, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (i.e., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, i.e., Agnew. Chem. Inti. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholinodoxorubicin, 2- pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5- fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg ); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, i.e., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J ), ABRAXANE® Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; NAVELBINE.RTM. vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-1 1) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb.RTM ); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (i.e., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0060] In certain embodiments, an effective dose of a composition comprising a PBAE backpack particle composition as described herein can be administered to a patient once. In certain embodiments, an effective dose of a composition can be administered to a patient repeatedly. In some embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, i.e., by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.

[0061] The dosage of a PBAE backpack particle composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the composition. The desired dose or amount of activation can be administered at one time or divided into subdoses, i.e., 2-4 subdoses and administered over a period of time, i.e., at appropriate intervals through the day or other appropriate schedule. In some embodiments, administration can be chronic, i.e., one or more doses and / or treatments daily over a period of weeks or months. Examples of dosing and / or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. A composition comprising a PBAE backpack particle as described herein can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.

[0062] The dosage ranges for the administration of the compositions described herein, according to the methods described herein depend upon, for example, the potency of the cells, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for tumor growth or the extent to which, for example, wound healing are desired to be induced. The dosage should not be so large as to cause adverse side effects, such as excessive inflammation or immunosuppression. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.

[0063] The efficacy of a PBAE backpack particle in the treatment of a condition described herein, or to induce a response as described herein can be determined by the skilled clinician. However, a treatment is considered “effective treatment,” as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced i.e., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and / or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and / or are described herein.

[0064] Treatment includes any treatment of a disease in an individual or an animal (some nonlimiting examples include a human or an animal) and includes: (1) inhibiting the disease, i.e., preventing a worsening of symptoms; or (2) relieving the severity of the disease, i.e., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response. It is well within the ability of one skilled in the art to monitor efficacy of administration and / or treatment by measuring any one of such parameters, or any combination of parameters.

[0065] Unless otherwise defined, 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 belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0066] As used herein, “photocurable” refers to a material that forms cross-links when exposed to light energy, and preferably UV light radiation.

[0067] Examples of lithography can include photolithography, soft lithography, proximity lithography, projection lithography, electron-beam lithography, X-ray lithography, interferometric lithography, imprint lithography, deep UV lithography, immersion lithography, extreme UV lithography, and focused ion beam lithography

[0068] As used herein, “photolithography” refers to methods of microfabrication that are used to pattern parts onto a thin film or bulk substrate (or wafer). Photolithography (or photopatterning) can refer to methods that utilize light to transfer a geometric pattern from a photomask to a photosensitive (light-sensitive) chemical photoresist in the desired pattern upon the material under the photoresist.

[0069] As used herein, “maskless lithography” describes a high-resolution of 2D patterning where structure structuring is accomplished by illuminating negative-tone or positive-tone photoresists via light of a well-defined wavelength without the need for a photomask.

[0070] As used herein, a “two-photon lithography,” describes a high-resolution method of 3D printing where structuring is accomplished by illuminating negative-tone or positive-tone photoresists via light of a well-defined wavelength without the need for a photomask.

[0071] As used herein, an “acrylate” means a monomeric compound in which the carboxylic acid group is present in the form of an ester, and can preferably include polymerizable diacrylates.

[0072] As used herein, the term “amine” means a primary amine group. For example, an “amine” can include the group — NRR ', where R and R ' are independently selected from hydrogen or alkyl.

[0073] As used herein, a “photoinitiator” refers to a molecule that initiates free radical crosslinking / polymerizing reaction by the use of light. Suitable photoinitiators include, without limitation, benzoin methyl ether, diethoxyacetophenone, a benzoylphosphine oxide, 1- hydroxycyclohexyl phenyl ketone, Darocure® types of photoinitiators, and Irgacure® types of photoinitiators, preferably Darocure® 1173, and Irgacure® 2959. Examples of benzoylphosphine oxide initiators include 2,4,6-trimethylbenzoyldiphenylophosphine oxide (TPO); bis-(2,6- dichlorobenzoyl)-4-N-propylphenylphosphine oxide; and bis-(2,6-dichlorobenzoyl)-4-N- butylphenylphosphine oxide. Reactive photoinitiators which can be incorporated, for example, into a macromer or can be used as a special monomer are also suitable. Examples of reactive photoinitiators are those disclosed in EP 632 329, herein incorporated by reference in its entirety. The polymerization can then be triggered off by actinic radiation, for example light, in particular UV light of a suitable wavelength. The spectral requirements can be controlled accordingly, if appropriate, by addition of suitable photosensitizers.

[0074] As used herein, “Young's modulus (E)” describes a property of a non-spherical elastomeric particle that tells us how easily it can stretch and deform and is defined as the ratio of tensile stress (o) to tensile strain (s). Where stress is the amount of force applied per unit area (o = F / A) and strain is extension per unit length (a = dl / 1). Young's modulus can be expressed as the slope of the linear part of the stress-strain curve for a material under tension or compression.

[0075] As used herein, the term “polymer” refers to oligomers, co-oligomers, polymers and copolymers, such as random block, multiblock, star, grafted, gradient copolymers and combination thereof. The average molecular weight of the polymer, as determined by gel permeation chromatography, can range from 500 to about 500,000, i.e., from 20,000 to about 500,000. In a prefer embodiment, the polymer of the disclosure includes a PBAE polymer.

[0076] As used herein, the term “oxygen scavenger” means a chemical that will remove or inactivate free oxygen or oxygen radicals. In one example, an “oxygen scavenger” can include triphenylphosphine.

[0077] As used herein, the term “polymerizing agent” or “co-polymerizing agent” means a molecule or compound that i) increase the extent of crosslinking of the polymer which slows the rate of drug release and (ii) increase the rate of degradation of the polymer. In one embodiment, a co-polymerizing agent can include pentaerythritol tetrakis(3-mercaptopropionate) (PETMP).

[0078] The terms, “individual,” “patient” and “subject” are used interchangeably herein. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of a disease. A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0079] The terms “compound” and “agent” refer to any entity which is normally not present or not present at the levels being administered and / or provided to a cell, tissue or subject. An agent can be selected from a group comprising: chemicals; small organic or inorganic molecules; signaling molecules; nucleic acid sequences; nucleic acid analogues; proteins; peptides; enzymes; aptamers; peptidomimetic, peptide derivative, peptide analogs, intrabodies; biological macromolecules, extracts made from biological materials such as bacteria, plants, fungi, or animal cells or tissues; naturally occurring or synthetic compositions or functional fragments thereof. In some embodiments, the agent is any chemical, entity or moiety, including without limitation synthetic and naturally occurring non-proteinaceous entities. In certain embodiments the agent is a small molecule having a chemical moiety. For example, chemical moieties include unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties including macrolides, leptomycins and related natural products or analogues thereof. Agents can be known to have a desired activity and / or property or can be selected from a library of diverse compounds.

[0080] As used herein, the term “small molecule” refers to a chemical agent which can include, but is not limited to, a peptide, a peptidomimetic, an amino acid, an amino acid analog, a polynucleotide, a polynucleotide analog, an aptamer, a nucleotide, a nucleotide analog, an organic or inorganic compound (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[0081] As used herein, the terms “protein” and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues. The terms “protein”, and “polypeptide” refer to a polymer of amino acids, including modified amino acids (i.e., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein” and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing. As used herein, the terms “treat,” “treatment,” or “treating,” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, i.e., cancer. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a condition. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term “treatment” of a disease also includes providing relief from the symptoms or side effects of the disease (including palliative treatment).

[0082] As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier i.e., a carrier commonly used in the pharmaceutical industry. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some embodiments, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, i.e., a carrier that the active ingredient would not be found to occur in in nature.

[0083] As used herein, the term “administering,” refers to the placement of a compound as disclosed herein into a subject by a method or route, which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route, which results in an effective treatment in the subject. As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation. The term “consisting of’ refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment. As used herein the term “consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, “i.e.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “i.e.” is synonymous with the term “for example”.

[0084] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0085] The disclosure now being generally described will be more readily understood by reference to the following examples, which are included merely for the purposes of illustration of certain aspects of the embodiments of the present disclosure. The examples are not intended to limit the disclosure, as one of skill in the art would recognize from the above teachings and the following examples that other techniques and methods can satisfy the claims and can be employed without departing from the scope of the claimed disclosure.

[0086] EXAMPLES

[0087] Example 1 : Background, Experimental Overview and Summary Results.

[0088] The ability to control the size, shape, and crosslink density of drug-loadable particles offers an opportunity to improve drug delivery. Applicants have developed a method to fabricate hydrophobic PBAE, also referred to interchangeable in some embodiments as PBAE-dA particles, using two different amine chemistries across a range of stiffnesses. The stiffnesses range 5.8-703.1 MPa and 0.5-442.0 MPa, for poly-benz and poly-iso, respectively. Consequently, a higher stiffness or crosslink density results in a smaller mesh size. Applicants found that the approximate mesh size of the PBAE-dAs ranges 5.4-0.5nm and 6.0-0.6nm for poly-benz and poly-iso, respectively. This relatively small mesh size suggests the PBAE-dAs studied are good candidates for drug loading and release of small molecule drugs and other therapeutic and diagnostic agents.

[0089] Applicants demonstrate in the present disclosures how PBAE-dAs can be used to manufacture drug-loadable particles of well-defined shape with controlled dimensions, stiffnesses, and degradation rates. Applicants assessed the properties of two different hydrophobic PBAE-dA amine chemistries across four crosslink densities. Applicants used these eight PBAE-dA compositions to demonstrate that the mechanical stiffness, degradation and drug release profiles can be programmed to fit a wide range of criteria. The PBAE-dAs of the disclosure represent improvements in rationally designed drug delivery vehicles, wherein the material properties can be programmed with the end goal in mind. Moreover, Applicants have established photolithographic protocols to scalably fabricate microparticles with control of shape and size in 3-dimensions. Altogether, this application provides a novel perspective into PBAE-dAs as a candidate for the drug delivery of small molecule drugs, in which stiffness, shape, size, degradation, and drug release are all parameters that can be programmed.

[0090] In this work, Applicants developed methods to employ contact and maskless photolithography for the fabrication of PBAE-dA micro- and nanoparticles, enabling rapid fabrication of a myriad of particle shapes and sizes. These methods suggest PBAE as a clinically viable option for drug delivery in terms of scalability. The particles studied are relatively nontoxic at reasonable concentrations. Applicants found that approximately 20% of dexamethasone was released from PBAE-dA backpacks in 1% DMSO through a diffusive manner. This suggests that the majority of drug is released upon particle swelling and degradation, which can occur over the course of months. Literature has found that cellular backpacks that release nearly all of its cargo in vitro within 48 hours can polarize macrophages for up to 5 days. Ostensibly, PBAE-dA backpacks that exhibit gradual, degradation-mediated release could potentiate long-lasting, durable results. As described herein, Applicants report a method to leverage these polymers as fully biodegradable resins with nanoscale precision using two-photon lithography. Applicants demonstrate this capability by precisely 3D-printing magnetic microrobots, which can be spatiotemporally controlled via an external rotating magnetic field. PBAE-dAs possess strong potential to serve as a bridge between commercial, nonbiodegradable, hydrophobic resins, and recent advances in in vivo microrobotic drug delivery. Example 2: Fabrication and Loading of PBAE particles.

[0091] The shape of micro- and nanoparticles plays a critical role in particle fate and function in vivo. Particle shape influences cell-particle interactions, biodistribution, immune responses, and particle behavior in fluid flow. Additionally, particle shape is emerging as an essential criterion for medical microrobots, or microscale particle systems that can propel or change shape in response to stimuli for the purposes of biological sensing or drug delivery. Within the past 20 years, interest within the scientific community surrounding particle shape has grown exponentially, reflecting increased appreciation for the fundamental and translational value of nonspherical particles in biological applications. Previous work in the field has explored film stretching, microcontact printing, and plasma etching as approaches to fabricate nonspherical particles with distinct shapes. However, these approaches limit particle throughput or control of material properties, such as degradability. Few methods exist that enable the manufacturing of nonspherical, biodegradable, polymeric micro- and nanoparticles for drug delivery systems at clinically scalable levels.

[0092] To address these needs, Applicants have developed a high-throughput method to fabricate drug-loadable micro- and nanoparticles with controllable shape, size, degradation, drug release, and crosslink density. The particles are fabricated from photocurable poly (β-amino ester) (PBAE), which describes a broad class of polymers that has emerged as a promising vehicle for drug delivery due to its inherent biocompatibility and tunable degradability. PBAE macromolecular monomers (macromers) are synthesized through an aza-Michael step-growth reaction between acrylates with amines or hydrazides, forming hydrolytically degradable ester bonds. The azaMichael synthesis of PBAE macromers is modular, high yielding, performed under mild reaction conditions, capable of being solventless, and lacks side-products or catalysts, satisfying several principles of green chemistry, as defined by Anastas and Warner in 1998, and the requirements of “click chemistry”, as defined by Sharpless in 2001. These principles underline the simplicity and scalability of PBAE macromers for clinical use.

[0093] After synthesis, PBAE macromers can undergo free radical photopolymerization, forming crosslinked bulk networks. Previous work has established libraries of photocrosslinkable PBAE macromers, of which the macromer chemistry can be used to determine polymer charge and hydrophilicity. Further, researchers have investigated how to form complex three-dimensional structures from PBAE via microstereolithography and digital light processing. However, these studies aimed to fabricate macroscopic scaffolds for tissue engineering. To Applicants knowledge, no work has previously described fabrication of nonspherical PBAE micro / nanoparticles, nor with feature resolution below 55 microns, which can be a size threshold for particles used in drug delivery and for medical microrobots.

[0094] Applicants demonstrate a high-throughput approach for the rapid fabrication of micro- and nanoparticles with precisely engineered shape, elasticity, degradability, and drug release rates for a broad range of drug delivery applications. To demonstrate the manufacturability of this polymer and its versatility in drug delivery applications, Applicants fabricated micro- and nanoparticles using three distinct photolithography techniques: contact lithography, maskless lithography, and two-photon lithography. These methods illustrate the inherent trade-off between particle throughput and shape tunability. Contact lithography offers the highest throughput but most limited design flexibility, while maskless lithography enhances flexibility of patterning, and two- photon lithography enables precise three-dimensional patterning at the expense of slower production rates. Applicants harnessed the throughput of contact lithography to fabricate large batches of cellular “backpacks” which are discoidal microparticles that bind to the surface of cells. On the other hand, Applicants used two-photon lithography coupled with metal evaporation to fabricate fully biodegradable magnetic microrobots. These materials and methods open the door to the scalable manufacturing of biocompatible, biodegradable, and mechanically tunable polymers into precisely defined, non-spherical shapes — addressing a longstanding gap in the field of particle-based drug delivery.

[0095] PBAE macromers are formed through the aza-Michael addition of an amine or a hydrazide with the terminal alkene of an acrylate group (Figure la). Applicants use diacrylates in this system, which permit functionalization on both ends of the molecule and thus chain formation. Previous work has demonstrated that tuning the molar ratio of diacrylate:amine can determine the average molecular weight of PBAE macromers. For example, assuming 100% completion of reaction, a 2: 1 ratio of diacrylate:amine would result in macromers with two diacrylates sandwiching one amine (Figure lb). Similarly, a 1.5:1 molar ratio would have chains of three diacrylates and two amines, and a 1 : 1 molar ratio would theoretically form a singular, long chain. Conversely, adding excess diacrylate (such as in a 5:1 molar ratio) would result in a polymer that most closely resembles a diacrylate network with occasional PBAE linkers.

[0096] Here, Applicants explore a wide range of di acrylate: amine molar ratios (1.2: 1, 1.5: 1, 5: 1, and 10: 1). H-NMR was used to confirm that lower molar ratios result in the formation of more ester bonds, indicating aza-Michael completion (Figure 9). Applicants include relatively high ratios of diacrylate: amine (i.e., 5:1 and 10: 1) in this study because i) some drug delivery targets such as the bladder of gastrointestinal tract do not require biodegradable vehicles, and ii) to map out an expansive range of material characteristics, such that researchers can extrapolate between the data presented to select a material that best suits their needs in terms of degradation, drug release, and stiffness. For simplicity, Applicants refer to all compositions that Applicants use as PBAE-diacrylates (PBAE-dAs), because in the 5:1 and 10: 1 conditions, the concentration of free diacrylate likely surpasses that of PBAE-dA macromers.

[0097] After macromer synthesis, a free radical photoinitator, diphenyl(2,4,6-trimethylbenzoyl) phosphine oxide (TPO), is added which facilitates photocrosslinking of PBAE-dA terminal acrylates and thus formation of polyacrylate kinetic chains. In this step, PBAE-dA macromers act as bridges between polyacrylate chains, directly influencing the mesh size of the cured resin (Figure 1c). Simply, incorporating fewer diacrylates relative to amines increases the average molecular weight of PBAE-dA macromers, thus increasing polymer mesh size.

[0098] Applicant, in one embodiment used 1,4-butanediol diacrylate in combination with either isobutylamine or benzhydrazide, to form “poly-iso” or “poly-benz”, respectively (Figure 2a). Applicants selected these diacrylate and primary amine combinations based their hydrophobic nature for microstereolithography. Both poly-iso and poly-benz macromers were synthesized in molar ratios of 1.2: 1, 1.5: 1, 5: 1, and 10: 1 (diacrylate: amine). Henceforth, they will be referred to as either poly-iso or poly-benz followed by the number of moles of diacrylate per one mole of amine. For example, poly-benz 1.5 is a macromer composed of a ratio of 1.5 moles 1,4-butanediol diacrylate in combination with 1 mole of benzhydrazide. The Young’s moduli of thin films (30- 100μm) were measured at 37°C via dynamic mechanical analysis (Figure 2b). Figure 2b demonstrates that increasing the molar ratio of diacrylate to amine increases the stiffness of the material, reaching a plateau as the material approaches a pure diacrylate. This corroborates understanding that a higher diacrylate: amine ratio results in a more densely crosslinked polymer network. Further, poly-benz is consistently more flexible than poly-iso, spanning 5.8-703.1 MPa and 0.5-442.0 MPa, respectively.

[0099] Photorheology was used to determine the shear moduli (G) of each PB AE-dA composition after the storage and loss moduli reach a plateau (Figure 2c). Applicants then approximated the mesh size of each composition (Figure 2d, Supplementary Equation 1). The calculated mesh size decreases with increasing concentration of diacrylates, which was hypothesized. The equation used relies upon the theory of perfect elasticity, which means that the calculated values are likely more accurate for the lower diacrylate: amine ratios that are elastomeric, rather than the stiffer compositions.

[0100] Discoidal particles, often referred to as cellular backpacks, have rapidly gained popularity as drug delivery vehicles due to their ability to attach to immune cell surfaces, exploit chemotactic migration, and evade phagocytosis. Applicants set out to establish a novel, scalable method of fabricating backpacks with high uniformity and precisely defined properties. Contact photolithography was employed to fabricate PBAE-dA backpacks (Figure 3a). In this process, PBAE-dAs act as a negative photoresist, depositing particles where the photomask is transparent (Figure 3b). Applicants used a photomask with 7μm-wide hexagons and spin coated each PBAE- dA resin to achieve a film thickness of ~1.7μm, though the film thickness can be adjusted by changing the concentration of PBAE-dA in solvent or spin speed. Because the viscosity of PBAE- dA macromers increases with their molecular weight, Applicants diluted molar ratios of 1.2: 1 and 1.5:1 (di acrylate: amine) with chloroform, which evaporates throughout spin coating and bake steps. Additionally, each PBAE-dA composition achieved particle formation at different dosages of ultraviolet light due to differences in availability of pendant acrylates (Table 1).

[0101] One complication of free radical photopolymerization is oxygen quenching free radical photoinitiators. Applicants observed that portions of resin that made robust contact with the photomask were more crosslinked than compared to surrounding regions where oxygen was present at the interface, suggesting oxygen was unable to reach these areas. For this reason, it is essential to maintain a protocol with consistent contact, or, as is the case with most mask aligners, to use a setting that facilitates the release of nitrogen gas, which supplies an inert environment and excludes oxygen.

[0102] Cured 1 cm3samples of poly -iso and poly-benz of four different molar ratios (1.2, 1.5, 5, 10) were degraded in accelerated conditions (1 M NaOH at 37°C) to highlight the distinctions between degradation rate based upon chemical composition (Figure 4a). Basic conditions accelerate degradation of polymer due to higher availability of nucleophiles to facilitate hydrolytic degradation. Increasing the molar ratio of diacrylate:amine increases the crosslink density of the polymer and thus restricts the penetration of water, slowing degradation. Further, poly-benz samples degraded at a higher rate than poly-iso samples.

[0103] To illustrate physiologically relevant degradation times for microparticles, Applicants degraded poly-iso 1.2 and poly-benz 1.5 particles in IX phosphate buffered saline (PBS) at 37°C (Figure 4b). The particles possessed widths of 7μm and thicknesses of 1.75 ± 0.29μm and 1.79 ± 0.18μm for poly-iso 1.2 and poly-benz 1.5, respectively. Throughout degradation, the particles were surface bound to silicon wafers leaving one side inaccessible to water penetration. Applicants found that both poly-iso 1.2 and poly-benz 1.5 particles swell considerably around 15 days, indicating penetration of water. Subsequently, the particles can undergo hydrophilic degradation.

[0104] Applicants found that poly-benz particles and poly-iso particles degrade swell considerably around 20 days and degrade within 4 months. Applicants hypothesize that free particles suspended in solution or in vivo would degrade faster because both faces of the backpacks would be available to an aqueous environment. The degradation of PBAEs is highly pH dependent, and buffer or acidic solutions can delay degradation, which could have interesting implications for in vivo drug delivery, particularly in acidic environments such as the tumor microenvironment.

[0105] Literature has documented the biocompatibility of PBAE as intact tissue scaffolds and nanoparticles, however the cytotoxic effects of byproducts from PBAE-dA degradation has been unclear. PBAE-dA degradation releases carboxylic acids and ester-cleaved PBAE-dA segments. Applicants performed an MTT assay, which employs cellular metabolism as an indicator of viability, using cell media infused with degraded PBAE-dA byproducts. Applicants found no significant changes in the viability of HEK293T cells when a seeding density of ~1 fully degraded backpack per 3 cells (2.6xl0‘5ng resin / cell) was used, assuming a backpack size of 7x1.7μm (Figure 5a). However, when an increased concentration of ~3 backpacks per cell (2.0xl0'4ng / cell) was used, statistically significant toxicity was observed in poly-benz 5 composition (padj=O.Ol 3) (Figure 10).

[0106] The likelihood of achieving the concentrations studied in Figure 5a and 10 in particle-based drug delivery systems is low because i) particle degradation happens gradually, ii) interstitial fluid constantly provides clearance and iii) in most drug delivery scenarios, cell populations significantly outnumber particles.

[0107] To explore using PBAE-dA backpacks for macrophage-mediated drug delivery, Applicants associated poly-benz 1.5 particles with C57BL / 6 primary bone marrow-derived macrophages (BMDMs). Applicants confirmed the successful association of PBAE-dA particles with BMDMs via fluorescent confocal microscopy (Figure 5b). When associated with BMDMs at 1: 1, 3: 1, and 5: 1 particle-to-cell ratios, Applicants observed 1.3±1.6, 2.9±2.7, and 3.3±3.2 particles per live cell, respectively (mean±l S.D., n=300 cells) (Figure 11). Applicants found that at a 5: 1 seeding density of particles:cells, cells that associated with numerous particles (i.e., up to 25) appeared to undergo apoptosis (Figure 12). In one embodiment, PBAE-dA backpacks for macrophage-mediated drug delivery, particles should be associated to cells around 1 : 1-3: 1 ratio of particles:cells for optimal cell carrier viability.

[0108] Applicants assessed in vitro release of a model hydrophobic drug, dexamethasone, to assess any differences in release between amine chemistries (i.e., poly-benz 1.5 vs. poly-iso 1.5) and molar ratio (i.e., poly-benz 1.5 vs. poly-benz 5). Applicants performed a co-solvent drug loading method, wherein an aqueous phase and organic phase are combined (Figure 6a). Dexamethasone is soluble in ethanol and chloroform (TCM) but remains insoluble in water. Chloroform was added to swell PBAE-dA particles for drug loading and bring them to the bottom of the tube, thus reducing particles loss on the tube walls. Ethanol, which has a boiling point of 78°C, is less volatile than chloroform and was used to dissolve and transfer dexamethasone. As the tubes are heated, the organics evaporate, driving dexamethasone into the particles.

[0109] Because diffusion is significantly affected by the particle surface area to volume ratio, Applicants identified contact photolithography protocols that yield particles with statistically insignificant differences in height (Figure 6b). Then, the particles were submerged in 50 pL of IX PBS containing 1 vol.% dimethyl sulfoxide (DMSO). The ultraviolet-visible absorption spectra were measured periodically and analyzed with a calibration curve to reveal the concentration of dexamethasone (Figure 13). The release media used, IX PBS 1 vol.% DMSO, was selected to resemble physiological conditions, while providing a controlled environment to measure drug release. It should be emphasized that the kinetics of in vitro drug release rely upon the composition of the release solution; the inclusion of organic solvents such as DMSO accelerates release while allowing reliable measurement concentrations. The fraction (Figure 6c) and mass (Figure 6d) of dexamethasone released were plotted and fitted to a Weibull model, which can encapsulate plateaus in drug release (Figure 13).

[0110] At 120 hours Applicants lyophilized the particles and submerged them in absolute ethanol, which facilitates the release of drug remaining within the particles. Applicants found that despite reaching a plateau after 96 hours, only -26%, 19%, and 14% of dexamethasone that was released from poly-benz 1.5, poly-benz 5, and poly-benz 5, respectively. This suggests that the remaining -74-86% of drug would be released via particle swelling and degradation. Applicants hypothesize that the dexamethasone near the particle-liquid interface was able to permeate into the supernatant, while imbedded drug remained entrapped within the polymer mesh. Applicants previous found nearly complete release of dexamethasone from linear (un-crosslinked), drug-conjugated PBAE chains within a day. Although the exact PBAEs used differ, this suggests that the crosslinked network structure is responsible for drug retention, and not material alone. Still, material can make a difference; Applicants observed a higher fraction of release for poly-benz 1.5 compared to polyiso 1.5, potentially because the more strongly hydrophobic poly-iso composition results in stronger drug retention. Further, Applicants observed a higher fraction of release in poly-benz 1.5 particles compared to poly-benz 5, which could be explained by the smaller mesh size reported in Figure 2d. Assessment of the mass of drug released over time demonstrates that poly-benz 5 : 1 had a lower loading efficiency when compared to poly-iso 1.5 and poly-benz 1.5, which corroborates with the tighter mesh size described in Figure 2d.

[0111] To further characterize the release profiles of each PBAE-dA composition, Applicants fit the fraction of release over time to linearized 0-order, 1storder, Korsmeyer-Peppas, Hixson- Crowell, and Higuchi equations (Figure 14). Applicants found that poly-benz 1.5 best fits a Korsmeyer-Peppas model (R2=0.99) and had an n-value of 0.65, suggesting anomalous diffusion that is mediated by swelling and diffusion. Poly-benz 5 best fit Hixson-Crowell (n=0.95), and yielded a knc=0.047, which suggests that degradation is erosion-mediated, but that erosion is relatively slow. Poly-iso 1.5 fit to the Higuchi model had a R2=0.96 indicating diffusion-mediated release with weak polymer / drug interactions. These fits are specific to the drug and polymer combination and provide hypotheses surrounding the physical phenomena governing drug release in each respective polymer.

[0112] Despite considerable advancements in microrobots designed for in vivo drug delivery, there are limited biodegradable options for high resolution resins. One of the most promising methods to actuate microrobots for drug delivery is with magnetic fields, which are biorthogonal and can reach deep regions of tissue. Researchers have investigated using gelatin methacrylate, chitosan, and even patient-derived blood to print biodegradable magnetic microrobots. However, these systems are either hydrophilic, or have low printing resolutions, which creates a gap between other researchers who have been printing complex shapes using hydrophobic commercial resins.

[0113] To fill this gap, Applicants investigated printing microparticles from poly-benz 1.5, a flexible (E=4.1±1.6 MPa) and biodegradable PBAE-dA composition using two-photon lithography (Figure 7a). Applicants systematically varied laser intensity, scan speed, and substrates to identify conditions that resulted in submicron printing resolutions. Rectangles were designed with widths equal to the radii of overlying circles, increasing from left to right: 0.1, 0.15, 0.2, 0.25, 0.5, 0.75, 1, 1.5, 2, & 2.5 μm. The resolution prints revealed that a 45% laser power and laser scan speed of 7500 μm / s yielded shapes down to 500nm in width (Figure 7b) and 16% error between the theoretical and measured values (Figure 15). 50% laser power and 7000μm / s scan speed provide each voxel with more energy, and as a result, shapes down to 200nm can be printed, but with some bubble formation from overheating and 47% error (Figure 7c, 15). To the best of Applicants knowledge, this is the first time that PB AE is being published as a resin for two-photon lithography. This method results in a printing resolution that is >100-fold smaller than microstereolithography of PBAEs. This is not surprising as two-photon lithography is a slower and more precise process. To test the stability of poly-benz 1.5 prints in 3-dimensions, Applicants printed a series of dodecahedrons. Applicants found that several print parameters resulted in clear structures but selected a laser power of 50% and 7500μm / s scan speed (Figure 7d) and 65% at 7000μm / s (Figure 7e) as examples.

[0114] Applicants fabricated cylindrical shapes with spiral ridges, and sputter coated iron onto their surfaces to develop a fully biodegradable magnetic microrobot, which Applicants called micRotinis, due to their semblance to rotini pasta (Figure 8a, 16). Applicants demonstrated micRotinis moving in water under a 5mT rotating magnetic field, changed the frequency of the magnetic field, and performed particle tracking. In doing this, Applicants were able to identify 150 Hz as the frequency that micRotinis have the highest velocity, also known as the step-out frequency (Figure 8b).

[0115] To illustrate spatiotemporal control over the micRotinis, Applicants placed micRotinis in water and directed them to spell “CU” by changing the orientation of the 5 mT rotating magnetic field (Figure 8c). Finally, Applicants placed micRotinis in a slightly accelerated degradation media, 0.1 M NaOH, at 37°C and imaged them periodically using a widefield microscope to demonstrate that the particles are biodegradable. The micRotinis undergo delamination of the iron shell, making the polymer susceptible to degradation, which occurred within 22 days (Figure 8d). Example 3: Materials and Methods.

[0116] Materials: 1,4-butatediol diacrylate, benzhydrazide, isobutylamine, chloroform, diphenyl(2,4,6-trimethylbenzoyl) phosphine oxide (TPO), polyvinyl alcohol (MW 13,000-28,000 87-89% hydrolyzed), and sodium hydroxide were purchased from Sigma-Aldrich. SU-8 developer was purchased from Kayaku,. Silicon wafers were purchased from University Wafer. Dexamethasone was purchased from MedChem Express. Dulbecco’s Modified Eagle Medium (DMEM) High Glucose, dimethyl sulfoxide (DMSO), Penicillin-streptomycin, fetal bovine serum, macrophage colony-stimulating factor (M-CSF), calcein AM dye, nile red dye, NucBlue Live Ready Probes Reagent (Hoechst 33342) were purchased from Thermo Fisher Scientific. Dulbecco’s phosphate buffered saline (dPBS), phosphate buffered salied (PBS) tablets, and trypsin were acquired from VWR. Immersol immersion oil was purchased from Zeiss.

[0117] PBAE-dA Macromer Synthesis: Macromers were synthesized in 1.2: 1, 1.5: 1, 5:1, or 10: 1 molar ratios of 1,4-butanediol diacrylate:isobutylamine or benzhydrazide. The 1,4-butanediol diacrylate was preheated to 60°C stirring at 300 rpm for 15 min in a 20 mL scintillation vial prior to the addition of primary amine. Once combined, the monomers reacted at 90°C for 48 hrs, stirring at 300 rpm in an oil bath. The resulting macromers were tested using H NMR (Bruker 400 MHz NMR spectrometer) in deuterated chloroform. The 1.2: 1 and 1.5: 1 macromers were dissolved to achieve a 10 wt.% solution of resin in chloroform. Free radical photoinitiator, diphenyl (2,4,6- trimethylbenzoyl) phosphine oxide (TPO) was sonicated into the resins at either 0.85 wt.% if a 5: 1 or 10: 1 molar ratio or 3 wt.% if at 1.2: 1 or 1.5: 1.

[0118] Dynamic Mechanical Analysis: Thin films tensile testing was conducted using a DMA 850 (TA Instruments). Thin, rectangular films (5 mm x 10 mm x 30-60 μm) were prepared via contact photolithography using a Karl Suss MJB3 mask aligner equipped with a mercury arc lamp. All macromers contained 0.85 wt.% of TPO and any chloroform used to dissolve the resin was evaporated on a hot plate at 65°C before performing photolithography. The thickness of each sample specimens was measured three times using a Bruker Dektak stylus profilometer (vertical measurement range: 524 μm, horizontal length of measurement: 500 μm, duration of measurement: 10 s, stylus force: 3 mg). Samples with standard deviation < 5.0 μm were used and the average thickness value was reported into TRIOS software upon measurement. Each rectangular strip was conditioned at 37°C for 60 s, and then elongated to break at a strain rate of 5% / min and a sampling rate of 1 data point / s. The stress-strain curves were plotted, and the Young’s moduli were reported using the slope of the linear elastic regime.

[0119] Photorheology: Photorheology was conducted using an Ares G2 rheometer (TA instruments). Samples were measured at room temperature to mimic photolithography conditions. The PBAE-dA macromers were placed between two 8 mm plates, with a 150 μm gap. The samples were irradiated with 405 nm light at an intensity of 3 mW / cm2. The sample was subject to 0.05% strain at 1 Hz for 250 s.

[0120] Contact Photolithography of PBAE-dA Particles: PBAE-dA micro / nanoparticles were fabricated on 2” silicon wafer substrates. For experiments where adhesion to Si wafer was preferred (i.e., imaging and backpack degradation) the wafers were plasma treated (AXIC Inc., PlasmaSTAR) for 5 min at 100 W and 350 mTorr < 15min before being spin coated with PBAE- dA. For experiments where separation from the Si wafer was preferred (i.e., MTT or drug release) the substrates were first coated with a sacrificial layer of 2 wt.% poly (vinyl alcohol) dissolved in deionized water (DIW). All spin coat parameters include an acceleration step of 500 rpm for 5 s. The PVA was spin coated at 3000 rpm for 1 min. 700pL-lmL of the PBAE-dA macromers were spin coated onto the substrates for 30 s, at 2000 rpm to reveal final film thicknesses ~1.7 μm. Wafers were then placed on a hot plate at 60°C for 1 min, and the perimeter of the wafer was gently wiped with acetone to remove the raised edge bead. The resin-coated silicon substrates were exposed in a mask aligner (Karl Suss MJB3) in contact mode with 365 and 405 nm light to achieve final dosages listed in Table 1. PBAE-dA-coated wafers were then placed again on a hot plate (60°C for 1 min) and subsequently developed in SU-8 developer for 2 min. Finally, the substrates were gently rinsed with isopropyl alcohol (IP A) and dried with nitrogen to reveal the cured micro / nanoparticles. After photolithography, the wafers were submerged in absolute ethanol and were agitated on a shake plate at lOOrpm for 1 hour to remove residues from the photolithography process (i.e. SU-8 developer, photoinitator, chloroform). Particles were removed from the wafers by pipetting DIW with 0.1% tween 20 solution onto the surface of the wafers, thereby dissolving PVA and allowing the particles to be collected through gentle aspiration.

[0121] Accelerated Bulk Degradation: PBAE-dA macromer with 0.85 wt.% TPO were pipetted into 1 cm3cubic molds and cured using an Original Prusa CW 1 S under 405nm light for 15 minutes to achieve a final dosage of 7.29 J / cm2. The polymer cubes were removed from the mold (n=5) and placed in 10 mL of IM sodium hydroxide at 37°C. Periodically, the samples were removed, rinsed gently in deionized water, dried in vacuo, massed, and returned to their respective conical tubes. After the average mass loss for a PBAE-dA composition (i.e., poly-benz 1.5: 1) passed an increment of 10%, the NaOH was replaced to ensure a constant dilute solution.

[0122] Physiological Degradation of Microparticle PBAE-dA: PBAE-dA particles were fabricated on plasma-treated silicon substrates and placed in lx PBS at 37°C. Three locations per wafer were identified and marked. The particles were measured with stylus profilometry 10 times (vertical measurement range: 84 μm, horizontal length of measurement: 1000 μm, duration of measurement: 10 s, stylus force: 3 mg) and possessed statistically insignificant differences in height. The wafers were periodically removed and imaged on a Keyence VHX 7000 at 500x magnification with full coaxial light. The images were converted to 8-bit and a threshold was applied in ImageJ. The “fill holes” function was used, and the particles were analyzed with edge particles excluded.

[0123] Co-solvent Drug Loading and Release of Doxorubicin: Dexamethasone was dissolved at 25mg / mL in absolute ethanol. In nonstick Eppendorf tubes, 1 ,5xl06particles in 673 pL ultrapure water, lOOpL chloroform, and 27pL dexamethasone solution were combined. The tubes were left uncapped in a VorTemp (Invitrogen) shaking at 120rpm and 65°C overnight. In the morning, the tubes were centrifuged at 250xg for 10 minutes and the supernatant was aspirated. Then each tube was briefly washed with 700pL of DMSO, which was sufficient to dissolve any drug not encapsulated within the particles. Each tube was then washed twice with ultrapure water, replaced with 50pL of IX PBS lvol% DMSO, and placed in an oven at 37°C. At each time point, 5-20pL of supernatant was removed, transferred to a new Eppendorf tube, measured using a NanoDrop 2000 (Thermo Scientific), and the same volume of fresh media was replaced. At the end of the study, lOOpL of ethanol was added to each tube and the UV-Vis spectra was measured at 1, 4, and 24 hrs to confirm a plateau of drug release. The UV-vis absorbance peak at 242 nm indicating the presence of dexamethasone was compared to a calibration curve to identify the concentration of dexamethasone in the media. Particles not containing dexamethasone were also fabricated and were measured at the same time as the drug-loaded particles. The blank particles were compared with the drug-loaded particles to confirm that polymer degradation was not contributing to the absorbance intensity at 242 nm.

[0124] Cytotoxicity Assay: Thin films of known mass were degraded in DMEM High Glucose cell media at 37C. After 6-10 months, the films were rinsed with DIW, dried in vacuo, and remeasured to calculate the mass that had degraded. Pure cell media (no PBAE-dA) was also left to degrade in a sterile environment for the same amount of time. HEK239T cells were seeded in a 96 well plate at a concentration of 2.5xl04cells / well in lOOuL DMEM media and incubated overnight. Experimental conditions were applied, including equal volumes of degraded and fresh cell media within each well. 24 hours later the cells were labeled, solubilized, and their absorbance at 570 nm was measured in a Tecan plate reader.

[0125] Macrophage-particle Association: C57BL / 6 bone marrow-derived macrophage progenitor cells were isolated using previously described methods. 2.0xl05C57BL / 6 bone marrow-derived macrophages were seeded into a glass-bottom 24-well plate in DMEM / F-12 bone marrow media (1 vol.% penicillin-streptomycin, 10 vol.% fetal bovine serum, and 20 ng / mL macrophage colony-stimulating factor) and allowed to incubate overnight. The media was aspirated, and the wells were washed with dPBS and replaced with ImL serum and macrophage colony-stimulating factor free DMEM / F-12. Cells were stained with 2 pL per well of Calcein AM and incubated for 1.5 hrs. The supernatant was aspirated and replaced with serum free BMM-. A hemocytometer was used to count the particles, and 0: 1, 1 : 1, 3 : 1, and 5: 1 particles:cell seeded were added to each well (n=3). The particles were then pelleted at 300xg for 8 min. The well plate was incubated for 4 hrs to allow for particle association. Then, 2 drops per well of Hoechst 33342 stain was added to each well and was left to incubate at room temperature for 30 min. Each well was washed twice with dPBS and replaced with cell media.

[0126] Imaging and Image Processing: Images were taken on a Nikon SDC Spinning Disc Confocal Microscope under environmental conditions; green channel = cytosol (Calcein AM), blue channel = nuclei (Hoechst 33342), red channel = particles (Nile red). To limit bias in cell counts, MATLAB was used to randomly crop 1 image per well to a 2000 μm x 2000 μm image. The number of particles per cell was counted in each image and plotted. Two-Photon Lithography: 2 inch silicon wafer substrates were first plasma-treated in a March Jupiter III for 5 min at 100 W and 350 mTorr. Then 500 pL of poly-benz 1.5: 1 with 3 wt.% TPO as a 10 wt.% solution in chloroform was deposited onto the wafer and placed on a hot plate at 60°C for 30 min to allow the chloroform to evaporate. Immersion oil was placed directly on the resin and into a NanoScribe GT2 for lithography. After lithography, the substrates were placed back on the hot plate for 1 min at 60°C. and developed in SU-8 developer at 60°C for 15 minutes, gently rinsed with isopropyl alcohol, and dried with nitrogen.

[0127] SEM Images: 3 nm of platinum was deposited onto the two-photon printed substrates with a Leica EM ACE600. The images were taken on a Hitachi TM-4000PlusE-2 at 5-10 kV. Statistical Methods: Statistics were performed using Origin and Excel.

[0128] TABLES

[0129] Table 1. Approximate dosage and conditions required for different PBAE compositions to fabricate nano / microparticles. Ideal dosages are subject to slight changes due to batch-to-batch variability.

[0130] Table 2 Michael addition chemistry can be used to synthesize various PBAEs (B). Diacrylate monomers (lettered) and amine-containing monomers (numbered) can be used to generate a variety PBAEs with varying chemical structure via combinatorial chemistry using Michael addition as shown in C. Reproduced from Karlsson J, et al., Poly(beta-amino ester)s as gene delivery vehicles: challenges and opportunities. REFERENCES

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Claims

CLAIMSWhat is claimed is1. A programmable polymeric composition comprising:- a polymeric particle formed from a poly-(β-amino ester) (PBAE) polymer;- a photoinitiator; and- wherein the PBAE particle is formed via lithography, and further optionally functionalized with a secondary compound.

2. The composition of claim 1, wherein the PBAE polymer is formed via aza-Michael addition of an amine or a hydrazide with an alkene on a terminal acrylate.

3. The composition of claim 2, wherein the amine is selected from isobutylamine, or said hydrazide is benzhydrazide.

4. The composition of claim 2, wherein the acrylate is 1,4-butanediol diacrylate.

5. The composition of claim 2, wherein the PBAE is selected from Group B of Table 2.

6. The composition of claim 2, wherein the acrylate is a diacrylate monomer.

7. The composition of claim 2, wherein the amine is selected from the amine-containing monomers 1-94 of Table 2.

8. The composition of claim 2, wherein the acrylate is selected from the diacrylate monomers A- PP of Table 2.

9. The composition of claim 1, wherein the lithography is selected from: photolithography, or two- photon lithography, soft lithography, proximity lithography, projection lithography, electron-beam lithography, X-ray lithography, interferometric lithography, imprint lithography, deep UV lithography, immersion lithography, extreme UV lithography, and focused ion beam lithography.

10. The composition of claim 1, wherein the secondary compound is incorporated directly into the PBAE polymer prior to polymerization.11 . The composition of claim 1 , wherein the secondary compound is coupled to the surface of the PBAE particle after polymerization.

12. The composition of claim 1, wherein the secondary compound is integrated into the PBAE particle after polymerization via incubation of the particle and the secondary compound in a cosolvent, wherein at least one co-solvent is evaporated causing the secondary compound to be incorporated into the particle.

13. The composition of any of claims 1 to 12, wherein the secondary compound can be selected from a polarizing agent, a contrast agent, a magnetic particle, a cell adhesive molecule, a targeting ligand, a therapeutic compound, or a combination of the same.

14. The composition of claim 13, wherein the therapeutic compound is selected from a polypeptide, a nucleic acid, a biologic, a small-molecules drug, an imaging molecule, a dye, a diagnostic composition, , or a combination of the same.

15. The composition of claim 1, further comprising an oxygen scavenger.

16. The composition of claim 15, wherein the oxygen scavenger is triphenylphosphine.

17. The composition of claim 1, further comprising a co-polymerizing agent.

18. The composition of claim 17, wherein the co-polymerizing agent is pentaerythritol tetrakis(3- mercaptopropi onate) (PETMP).

19. The composition of claim 1, wherein the density of crosslinks between the PBAE polymers is modulated by the exposure to radiation during fabrication via photolithography or two-photon lithography.

20. The composition of claim 2, wherein the molar ratio of acrylate and amine or a hydrazide is modulated to fabricate the particle having a desired stiffness and degradation characteristics in vivo.

21. The composition of claim 1, wherein the particle binds to an immune cell.

22. The composition of claim 21, wherein the immune cell is selected from a macrophage, a monocyte, a T cell, a CAR-T cell, a neutrophil, a B cell, a mast cell, a dendritic cell, an NK cell, or a combination of the same.

23. A pharmaceutical composition comprising the PBAE particle of any of claims 1 to 22.

24. A method of treating a disease or condition comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of claim 23, or the PBAE particle of any of claims 1 to 22.

25. A kit comprising the pharmaceutical composition of claim 23, or the PBAE particle of any of claims 1 to 22, a container for holding the same, and instructions for use.

26. A method of fabricating a polymeric particle comprising:- reacting an amine compound and an acrylate compound to form a poly(β-amino ester) (PBAE) diacrylate macromer;- adding a photoinitiator to the PBAE diacrylate macromer;- optionally adding a secondary compound;- spin-coating the polymer to a substrate;- applying patterned radiation to the substrate causing polymerization of the PBAE- diacrylate monomers; and- washing the substrate and removing the shaped PBAE particles.

27. The method of claim 26, wherein the PBAE-diacrylate monomer is formed via aza-Michael addition of an amine or a hydrazide with an alkene on a terminal acrylate.

28. The method of claim 27, wherein the amine is selected from isobutylamine, or said hydrazide is benzhydrazide.

29. The method of claim 27, wherein the acrylate is 1,4-butanediol diacrylate.

30. The method of claim 27, wherein the PBAE is selected from Group B of Table 2.

31. The method of claim 27, wherein the acrylate is a diacrylate monomer.

32. The method of claim 27, wherein the amine is selected from the amine-containing monomers 1-94 of Table 2.

33. The method of claim 27, wherein the acrylate is selected from the diacrylate monomers A-PP of Table 2.

34. The method of claim 26, wherein the secondary compound is added directly into the PBAE polymer prior to polymerization.

35. The method of claim 26, wherein the secondary compound is coupled to the surface of the PBAE particle after polymerization.

36. The method of claim 26, further comprising incubating particle and the secondary compound in a co-solvent, wherein the co-solvent is evaporated causing the secondary compound to be incorporated into the particle.

37. The method of any of claims 26 to 36, wherein the secondary compound can be selected from an antibody, a contrast agent, a magnetic particle, a cell adhesive molecule, a targeting ligand, a therapeutic compound, or a combination of the same.

38. The composition of claim 37, wherein the therapeutic compound is selected from a polypeptide, a nucleic acid, a biologic, small-molecules drugs, an imaging molecule, a dye, a diagnostic composition, , or a combination of the same.

39. The method of claim 26, wherein the density of crosslinks in the PBAE polymer is modulated by the exposure to radiation during fabrication via photolithography or two-photon lithography.

40. The method of claim 27, wherein the molar ratio of acrylate and amine is modulated to fabricate the particle having a desired stiffness and degradation characteristics in vivo.41 . The method of claim 26, wherein PBAE diacrylate monomer is diluted with a solvent.

42. The method of claim 26, wherein the PBAE particle is less than 2 um thick.

43. The method of claim 26, further comprising adding a co-polymerizing agent.

44. The method of claim 43, wherein the co-polymerizing agent is pentaerythritol tetrakis(3- mercaptopropionate) (PETMP).

45. The composition of claim 26, further comprising adding an oxygen scavenger.

46. The composition of claim 45, wherein the oxygen scavenger is triphenylphosphine.

47. A pharmaceutical compositions comprising the PBAE particle fabricated by the method of any of claims 26 to 46.

48. A method of treating a disease or condition comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of claim 47, or the PBAE particle of any of claims 26 to 46.

49. A kit containing the composition of any of claims 1-23, a container for holding the composition and instructions for use.

50. A programmable delivery system comprising:- a polymeric particle formed from a poly-(0-amino ester) (PBAE) polymer;- a photoinitiator;- an cell capable of bonding with the polymeric particle; and- wherein the PBAE particle is formed via lithography, and further optionally functionalized with a secondary compound.

51. The system of claim 50, wherein the PBAE polymer is formed via aza-Michael addition of an amine with an alkene on a terminal acrylate.

52. The system of claim 51, wherein the amine is selected from isobutylamine, or benzhydrazide.

53. The system of claim 51, wherein the acrylate is 1,4-butanediol diacrylate.

54. The system of claim 51, wherein the PBAE is selected from Group B of Table 2.

55. The system of claim 51, wherein the acrylate is a diacrylate monomer.

56. The system of claim 51, wherein the amine is selected from the amine-containing monomers 1-94 of Table 2.

57. The system of claim 51, wherein the acrylate is selected from the diacrylate monomers A-PP of Table 2.

58. The system of claim 50, wherein the lithography is selected from: photolithography or two- photon lithography, soft lithography, proximity lithography, projection lithography, electron-beam lithography, X-ray lithography, interferometric lithography, imprint lithography, deep UV lithography, immersion lithography, extreme UV lithography, and focused ion beam lithography.

59. The system of claim 50, wherein the secondary compound is incorporated directly into the PBAE polymer prior to polymerization.

60. The system of claim 50, wherein the secondary compound is coupled to the surface of the PBAE particle after polymerization.

61. The system of claim 50, wherein the secondary compound is integrated into the PBAE particle after polymerization via incubation of the particle and the secondary compound in a co-solvent, wherein at least one co-solvent is evaporated causing the secondary compound to be incorporated into the particle.

62. The system of any of claims 50 to 61, wherein the secondary compound can be selected from a polarizing agent, a contrast agent, a cell adhesive molecule, a targeting ligand, a therapeutic compound, a magnetic particle, or a combination of the same.

63. The system of claim 62, wherein the therapeutic compound is selected from a polypeptide, a nucleic acid, a biologic, a small-molecules drug, an imaging molecule, a dye, a diagnostic composition, or a combination of the same.

64. The system of claim 50, further comprising an oxygen scavenger.

65. The system of claim 64, wherein the oxygen scavenger is triphenylphosphine.

66. The system of claim 50, further comprising a co-polymerizing agent.

67. The system of claim 66, wherein the co-polymerizing agent is pentaerythritol tetrakis(3- mercaptopropionate) (PETMP).

68. The system of claim 50, wherein the density of crosslinks between the PBAE polymers is modulated by the exposure to radiation during fabrication via photolithography or two-photon lithography.

69. The system of claim 51, wherein the molar ratio of acrylate and amine is modulated to fabricate the particle having a desired stiffness and degradation characteristics in vivo.

70. The system of claim 50, wherein the cell is selected from a macrophage, a monocyte, a T cell, a CAR-T cell, a neutrophil, a B cell, a mast cell, a dendritic cell, an NK cell, or a combination of the same.

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