WO2020163511A1 - Regio-specific biodegradable nanogels for cargo delivery platform - Google Patents

Abstract

The disclosed technology is directed to regio-specific biodegradable nanogel compositions, and methods of manufacturing and using the same. Specifically, the disclosed technology includes customizing a nanogel network structure with degradable linkages to create a nanogel that is suitable for dmg delivery or tissue scaffold applications. An exemplary nanogel includes a mono-vinyl monomer, a di-vinyl monomer, a chain transfer agent, and an initiator, wherein at least one of the mono-vinyl monomer and the di-vinyl monomer includes a degradable linkage, and the degradable linkage is positioned at a location within the nanogel and/or between covalently interconnected nanogels such that there is selected control of the rate of degradation of the nanogel and/or release of nanogel-contained cargo.

Description

REGIO-SPECIFIC BIODEGRADABLE NANOGELS

FOR CARGO DELIVERY PLATFORM

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority to U.S. Provisional Patent Application No. 63/835,394, entitled “REGIO-SPECIFIC BIODEGRADABLE NANOGELS AS CARGO DELIVERY PLATFORM” and filed on April 17, 2019, and U.S. Provisional Patent Application No. 62/801,292, entitled “REGIO-SPECIFIC BIODEGRADABLE NANOGELS FOR CARGO DELIVERY PLATFORM” and filed on February 5, 2019, both of which are incorporated by reference herein in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant number

R01DE023197 awarded by National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0003] The fields of engineered tissue scaffold removal (Anseth, 2013) and controlled drug delivery (Uhrich, 1999) have benefited from advances in designing new degradable hydrogel systems (Fu, 2010). Extensive work in fabrication of biodegradable hydrogels include crosslinked network construction via radical polymerization from different combinations of amphiphilic block copolymers of polyethylene glycol (PEG) as the hydrophilic segment and polylactide (PLA) as the hydrophobic hydrolytically degradable segment with (meth) acrylate end functionalities (Metters, Polym 2000; Clapper, 2007; Papadopoulos, 2011). Both of the PEG and PLA polymer chains are biocompatible and FDA approved (Lee, 2006). Adding PEG of sufficient length to the copolymer chain enables a high degree of swelling (Metters, J Phys Chem B 2000; Tessmar, 2007) and hydrophobic PLA on the other hand, not only improves the mechanical properties but also adds the biodegradability character to the system. Degradation of PLA and PEG yield lactic acid and glycolic acid, which are both native compounds (Foster, 1880) that can be metabolized and excreted completely from the body (Middleton, 2000; Lee, 2006; Yu, 2012).

[0004] Despite advances, some hydrogel systems suffer from one or more shortcomings including a pronounced degree of nano-structural heterogeneity in the form of spatial distribution of crosslinks on 10-100 nm length scale; defects such as dangling chain ends, loops (primary cyclization), sol fraction, and super-crosslinks (close-spaced crosslinks) (Duesk, 2000; Di Lorenzo, Polym Chem 2015); limited control over degradation variables induced by heterogeneous swelling in hydrolytically degradable gels; degradation by-products comprising non-degradable high molecular weight (long) poly (me th) acrylate or other polymeric chains with highly branched dangling side chains (Metters, J Phys Chem B, Polym 2000); pronounced onset of reverse gelation (burst effect) at certain points during degradation when an un-eroded mass becomes instantly soluble in a solvent; instability in the blood stream; lack of functionality for bio-conjugation; diameter greater than 100 nm, which reduces cellular uptake; poor mechanical properties attributable to lack of crosslinking in the formation of the nano-structure; limitations on choice of compatible monomers due to some polymerization methods; and limitations on type of compatible reducing agent due to some polymerization methods.

SUMMARY

[0005] The disclosed technology is directed to biocompatible reactive compositions, which may be degradable or non-degradable, and methods of manufacturing and using the same. Specifically, regio-specific degradable nanogels disclosed herein may include one or more degradable linkages in select locations in the nanogel structure. The location and/or concentration of the one or more degradable linkages may be selected based on desired nanogel properties and degradation pathways. Different nanogel properties, such as, for example, rate and route of degradation, may be controlled by adjusting the location (e.g., internal, external, internal-external) and/or concentration of the degradable linkage in the nanogel structure. Such improved control over nanogel properties is desirable for various applications of the nanogel. For example, disclosed nanogels offer tremendous control and versatility in hydrolytic degradation for a variety of biomaterial applications, such as, for example, controlled drug delivery and tissue scaffold engineering.

[0006] In several embodiments, regio-specific degradable nanogels disclosed herein may be prepared by free-radical solution polymerization. For example, chain-transfer regulated free-radical polymerization of a dimethacrylate of polyethylene glycol-co- polylactide (PEG-roPLA) with 2-hydroxyethyl methacrylate (HEMA)-coPLA and 2- methoxyethyl methacrylate (MEMA) may produce a nanogel of the present disclosure. The weight-averaged molecular weights of disclosed nanogels may be less than 65 kg/mol with a hydrodynamic radius of less than 6.0 nm and 1.0 < polydispersity index (PDI) < 7.2. The glass transition temperature of bulk nanogels may be at or below room temperature. It is also contemplated that the glass transition temperature may be above room temperature, e.g., for a solid nanogel. The photopolymerized macroscopic networks of disclosed nanogels may exhibit dramatic high dry-state moduli depending on chain flexibility and crosslink density. The swollen-state moduli of disclosed nanogels may be lower than that of dry-state depending on the hydrophilicity of the network. The molecular weight between crosslinks (M_x) of disclosed nanogels evaluated based on mbber elasticity theory may have lower values of M_x by applying non-Gaussian conformation distribution assumption, yet equilibrium swelling theory may predict the same values for M_x regardless of the type of implemented distribution. The incorporation of hydrolytically labile linkages to the backbone and side-chain of disclosed nanogels may result in bulk and surface type erosions (degradations), respectively. The mass loss trends in novel networks of disclosed nanogels may show a sustained release of degraded species (e.g., even under auto-accelerated acid-catalyzed hydrolysis) by delaying or completely bypassing reverse gelation.

[0007] In one implementation, the disclosed technology includes a nanogel for drug delivery or tissue engineering comprising at least one mono vinyl monomer side-chain, at least one divinyl monomer crosslinker, a chain transfer agent, and an initiator, wherein the nanogel has a weight- averaged molecular weight less than 65 kg/mol, a hydrodynamic radius of less than 6.0 nm, and 1.0 < PDI < 7.2. In one implementation, the crosslinker is present at a concentration of from about 10.0 mol to about 90.0 mol%. In one implementation, the side- chain is present at a concentration of from about 10.0 mol to about 90.0 mol%. In one implementation, a dispersed nanogel concentration in a solvent is approximately 30 wt% to 90 wt%. In one implementation, the nanogel has glass transition temperature of about -80 °C to about 25 °C or about 60 °C. In one implementation, a nanogel network has equilibrium swelling of about 5.0 wt% to about 90.0 wt%. In one implementation, the nanogel composition has hydrophilic, amphiphilic, or hydrophobic character. In one implementation, a dry nanogel network has a compressive modulus of about 10 kPa to about 10 GPa. In one implementation, a water-swollen nanogel network has a compressive modulus of about 1 kPa to about 500 MPa.

[0008] In one implementation, the disclosed technology includes a method for synthesizing a nanogel composition for drug delivery or tissue engineering comprising combining at least a di- vinyl monomer and a mono-vinyl monomer, an initiator, and a chain transfer agent to form a mixture, and initiating a free-radical polymerization reaction of the mixture to form the nanogel, wherein the nanogel has a weight- averaged molecular weight less than about 65 kg/mol, a hydrodynamic radius of less than about 6.0 nm, and 1.0 < PDI < 7.2. In one implementation, the mixture is dissolved in a solvent. In one implementation, the free- radical polymerization reaction is one of redox-initiated, thermally-initiated and photo- initiated. In one implementation, the method further comprises the step of performing a degradation process, wherein the degradation process is an acid-catalyzed hydrolysis.

[0009] In one implementation, the disclosed technology includes a method for synthesizing a nanogel composition comprising combining at least a di-vinyl monomer and a mono-vinyl monomer, an initiator, and a chain transfer agent to form a mixture, initiating a free-radical polymerization reaction of the mixture to form the nanogel composition, and forming a degradable linkage in the nanogel composition, wherein the nanogel composition has a weight- averaged molecular weight less than about 65 kg/mol and a hydrodynamic radius of less than about 6.0 nm. In one implementation, the degradable linkage is a hydrolytically labile ester bond. In one implementation, the degradable linkage is embedded in a crosslinker. In one implementation, the degradable linkage is embedded in a side-chain. In one implementation, the degradable linkage is embedded in both a crosslinker and a side-chain. In one implementation, the method includes releasing a covalently bound cargo with the degradable linkage. In one implementation, the method includes releasing a physically encapsulated cargo based on partial or complete degradation of the nanogel-based network density. In one implementation, the method includes altering the hydrophilic/hydrophobic character of the nanogel with the degradable linkage. In one implementation, the method includes altering the polymer network mesh size with the degradable linkage. In one implementation, the method includes reduction of the polymer network mesh size in coordination with cargo release. In one implementation, a degradable nanogel network in the nanogel composition is at least one of bulk and surface eroding material. In one implementation, a degradable nanogel network in the nanogel composition shows burst and sustained increase of molecular weight between crosslinks.

[0010] In one implementation, a non-degradable nanogel network in the nanogel composition has a pH of 4.5 in the surrounding aqueous medium. One example of an aqueous medium is water. While“water” is regularly used in reference to a medium for the disclosed nanogels, it is to be understood that the medium could be any aqueous media.. In one implementation, a non-degradable nanogel network in the nanogel composition has no acidic by-products in water with initial and final pH of about 4.5. In one implementation, a pH of surrounding water changes from about 4.5 to about 3.5. In one implementation, a pH of surrounding water changes from about 4.5 to about 4.0. In one implementation, a pH of surrounding water changes from about 4.5 to about 3.0. In one implementation, a non- degradable nanogel network in the nanogel composition has a linear mass loss profile starting at 0.0% and ending at 0.0% over a broad range of time (e.g., 0.0- 0.0 ± 1.7 (%) during 0-90 days). In one implementation, the bulk eroding nanogel network has an exponential growth rate in mass loss (e.g., 0.0- 92.0 ± 4.5 (%) during 0-90 days). In one implementation, a surface eroding nanogel network has a slow or negligible initial interval that proceeds a transition to a linear or exponential degradation process (e.g., 0.0 - 11.0 ± 3.8 (%) during 0-90 days). In one implementation, a bulk and surface eroding nanogel network has an exponential growth rate (e.g., 0.0- 84.0 ± 15.4 (%) during 0-90 days). In one implementation, degradation by-products comprise a non-degradable crosslinker core; lactic acid, oligomeric poly(methacrylate) chains, and polymeric poly(methacrylate) chains with dangling oligomeric poly(methacrylate) chains. In one implementation, degradation by-products comprise a non-degradable nanogel, lactic acid, and polymeric poly (methacrylate) chains. In one implementation, degradation by products comprise a non-degradable crosslinker core, lactic acid, oligomeric poly(methacrylate) chains, and polymeric poly (methacrylate) chains. In one implementation, localized degradation of linkages can be targeted by at least one of pH, enzymes, and light.

[0011] In some embodiments, a nanogel is disclosed. The nanogel includes at least one mono vinyl monomer; at least one di vinyl monomer; a chain transfer agent; and an initiator. The at least one mono vinyl monomer includes a degradable linkage. The degradable linkage is positioned at a location within the nanogel selected to control at least one of a rate and a route of degradation of the nanogel.

[0012] In some implementations, a method of synthesizing a nanogel is disclosed. The method includes combining at least a di-vinyl monomer, a mono-vinyl monomer, an initiator, and a chain transfer agent to form a mixture, wherein at least one of the di-vinyl monomer and mono- vinyl monomer comprises a degradable linkage; and initiating a free- radical polymerization reaction of the mixture to form the nanogel, wherein the degradable linkage is selectively positioned within the nanogel to achieve a desired degradation rate of the nanogel.

[0013] These and various other features and advantages will be apparent from a reading of the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1A is a graph of water uptake vs. time for non-degradable nanogel networks, NG₁-NG₄, PEG molecular weight = 600, 2000 g/mol.

[0015] FIG. IB is a graph of water uptake vs. time for non-degradable nanogel networks, NGs-NGs; PEG molecular weight = 600, 2000 g/mol.

[0016] FIG. 2 is a graph of water uptake vs. time for internally degradable nanogel networks, NG9-NG12; PEG molecular weight = 600, 2000 g/mol.

[0017] FIG. 3 is a graph of water uptake vs. time of externally degradable nanogel networks, NGia-NGir,; PEG molecular weight = 600, 2000 g/mol.

[0018] FIG. 4 is a graph of water uptake vs. time of internally-extemally degradable nanogel networks, NG17-NG20; PEG molecular weight = 600, 2000 g/mol.

[0019] FIGS. 5A-D are graphs of compressive modulus (K) vs. time for non- degradable nanogel networks (NGi-NGs).

[0020] FIGS. 6A-B are graphs of compressive modulus (K) vs. time for internally degradable nanogel networks (NG9-NG12).

[0021] FIGS. 7A-B are graphs of compressive modulus (K) vs. time for externally degradable nanogel networks (NG13-NG16).

[0022] FIGS. 8A-B are graphs of compressive modulus (K) vs. time for internally- extemally degradable nanogel networks (NG17-NG20).

[0023] FIGS. 9A-B are graphs of molecular weight between crosslinks (M_x) vs. time for non-degradable nanogel networks (NGi-NGs); M_x calculated based on Rubber Elasticity Theory (Eq 4 and Eq 5) and Gaussian distribution assumption for polymer chain conformations.

[0024] FIGS. 10A-B are graphs of molecular weight between crosslinks (M_x) vs. time for non-degradable nanogel networks (NGi-NGs); M_x calculated based on Equilibrium Swelling Theory (Eq 10) and Gaussian distribution assumption for polymer chain conformations.

[0025] FIGS. 11A-B are graphs of molecular weight between crosslinks (M_x) vs. time for internally degradable nanogel networks (NG9-NG12) based on: (A) Rubber Elasticity Theory, and (B) Equilibrium Swelling. All chains are assumed to possess Gaussian Distribution of Conformations.

[0026] FIGS. 12A-B are graphs of molecular weight between crosslinks (M_x) vs. time for externally degradable nanogel networks (NG13-NG16) based on: (A) Rubber Elasticity Theory, and (B) Equilibrium Swelling. All chains are assumed to possess Gaussian Distribution of Conformations.

[0027] FIGS. 13A-C are graphs of molecular weight between crosslinks (M_x) vs. time for internally-externally nanogel networks (NG17-NG20) based on: (A-B) Rubber Elasticity Theory, and (C) Equilibrium Swelling. All chains are assumed to possess Gaussian Distribution of Conformations.

[0028] FIGS. 14A-B are graphs of molecular weight between crosslinks M_x vs. time for non-degradable nanogel networks (NG1-NG4); M_x calculated based on Rubber Elasticity Theory (Eq 13) and Non-Gaussian Distribution of Conformations.

[0029] FIGS. 15A-B are graphs of molecular weight between crosslinks ( M_x ) vs. time for non-degradable nanogel networks (NGi-NGs); M_x calculated based on Equilibrium Swelling Theory (Eq 14) and Non-Gaussian Distribution of Conformations.

[0030] FIGS. 16A-B are graphs of molecular weight between crosslinks (M_x) vs. time for internally degradable nanogel networks (NG9-NG12) based on: (A) Rubber Elasticity Theory, and (B) Equilibrium Swelling. All chains are assumed to possess Non-Gaussian Distribution of Conformations.

[0031] FIGS. 17A-B are graphs of molecular weight between crosslinks (M_x) vs. time for externally degradable nanogel networks (NG13-NG16) based on: (A) Rubber Elasticity Theory, and (B) Equilibrium Swelling. All chains are assumed to possess Non-Gaussian Distribution of Conformations.

[0032] FIGS. 18A-C are graphs of molecular weight between crosslinks (M_x) vs. time for internally-externally nanogel networks (NG17-NG20) based on: (A-B) Rubber Elasticity Theory, and (C) Equilibrium Swelling. All chains are assumed to possess Non- Gaussian Distribution of Conformations.

[0033] FIGS. 19A-C illustrate (A) Non-degradable active nanogel structure; (B) Polymerized overlapped nanogels create non-degradable network; (C) Sub units: PEGDMA (solid line represents non-degradability), non-degradable active side chain (methacrylate functionalized HEMA), non-degradable side chains (non-functionalized remaining HEMA and MEM A), oligomeric poly (methacrylate) chains connecting PEGDMA primary crosslinkers together to create primary network, polymeric poly(methacrylate) chains connecting nanogels (secondary crosslinker) together creating final macroscopic network.

[0034] FIGS. 20A-C illustrate (A) Internally degradable active nanogel (dashed lines represent degradable linkages (PLA) on both sides of PEG_x units); (B) Degradable macroscopic network formed via polymerizing overlapped internally degradable nanogels; (C) By-products of hydrolysis degradation of internally degradable nanogel network.

[0035] FIGS. 21A-C illustrate (A) Externally degradable active nanogel; (B) Macroscopic network formed by polymerizing overlapped externally degradable nanogels; (C) Schematic structures of degradation by-products.

[0036] FIGS. 22A-C illustrate (A) Intemally-externally degradable active nanogel; (B) Macroscopic network of polymerized overlapped nanogel; (C) Schematic stmctures of hydrolysis degradation by-products.

[0037] FIG. 23 is a graph of mass loss (%) vs. time in DI water and ambient conditions for non-degradable, internally degradable, externally degradable, and intemally- externally degradable nanogel networks of PEG4600 series.

[0038] FIG. 24A illustrates images of swelling internally degradable nanogel network (NG22) of PEG4000 series in DI water from t=0 (dry state) to t=l, 7, and 20 days (swollen state).

[0039] FIG. 24B illustrates images of swelling externally degradable nanogel network (NG23) of PEG4600 series in DI water from t=0 (dry state) to t=l, 7, and 20 days (swollen state).

[0040] FIG. 24C illustrates images of swelling intemally-externally degradable nanogel network (NG24) of PEG4600 series in DI water from t=0 (dry state) to t=l, 7, and 20 days (swollen state).

[0041] FIG. 24D illustrates images of swelling non-degradable nanogel network (NG21) of PEG4600 series in DI water from t=0 (dry state) to t=l, 7, and 20 days (swollen state).

[0042] FIG. 25 is a graph of glass transition temperature (T_g) of non-degradable active nanogels with PEG600DMA as crosslinker and HEMA as side-chain monomer.

[0043] FIG. 26 is a graph of glass transition temperature (T_g) of non-degradable active nanogels with PEG2000DMA as crosslinker and HEMA as side-chain monomer.

[0044] FIG. 27 is a graph of glass transition temperature (T_g) of non-degradable active nanogels with PEG600DMA as crosslinker, HEMA, and MEMA as side-chain monomers.

[0045] FIG. 28 is a graph of glass transition temperature (T_g) of non-degradable active nanogels with PEG2000DMA as crosslinker, HEMA, and MEMA as side-chain monomers. [0046] FIG. 29 is a graph of glass transition temperature (T_g) of internally degradable active nanogels with PEG₆₀₀PLADMA as crosslinker and HEMA as mono functional monomer.

[0047] FIG. 30 is a graph of glass transition temperature (T_g) of internally degradable active nanogels with PEG₂₀₀₀PLADMA as crosslinker and HEMA as side-chain monomer.

[0048] FIG. 31 is a graph of glass transition temperature (T_g) of externally degradable active nanogels with PEG₆₀₀DMA as crosslinker, HEMAPLA, and MEMA as side- chain monomers.

[0049] FIG. 32 is a graph of glass transition temperature (T_g) of externally degradable active nanogels with PEG₂₀₀₀DMA as crosslinker, HEMAPLA, and MEMA as side-chain monomers.

[0050] FIG. 33 is a graph of glass transition temperature (T_g) of internally- extemally degradable active nanogels with PEG₆₀₀PLADMA as crosslinker, HEMAPLA, and MEMA as side-chain monomers.

[0051] FIG. 34 is a graph of glass transition temperature (T_g) of internally- extemally degradable active nanogels with PEG₂₀₀₀PLADMA as crosslinker, HEMAPLA, and MEMA as side-chain monomers.

DETAILED DESCRIPTION

[0052] The disclosed technology includes regio- specific biodegradable nanogels. The regio- specific biodegradable nanogels may include one or more degradable linkages positioned at select locations within the nanogel structure (e.g., internal, external, internal- external). The location and/or concentration of the one or more degradable linkages may be selected to control one or more properties of the nanogel based on the desired nanogel application, such as, for example, tissue repair or cargo release (e.g., drug delivery). As one example, the rate, timing, and/or route/pathway of macroscopic degradation may be controlled by the intemal/external location and/or concentration of the degradable linkages. In this manner, macroscopic degradation of a polymer network can be manipulated at a molecular level.

[0053] Traditionally, the term“nanogel” means a polymer gel particle having any shape with an equivalent diameter of approximately a few to 100 nm.“Nanogel” describes the interconnected localized network structures as well as the physical dimensions of the polymer gel particle. Nanogels are typically soluble/uniformly dispersible in the solvent in which they are made and nanogels may be further made to be soluble in various liquids as necessary depending on the monomers used in their manufacture. However, nanogels can also be prepared in the absence of solvent (in bulk) and subsequently dispersed in an appropriate solvent or monomer composition.

[0054] As used herein, the term“nanogel” is defined as a soluble, porous polymer gel particle or particulate having any shape with an equivalent diameter of about 1 nm to about 200 nm, or greater, so long as the particle remains soluble in a target solvent or a monomer composition with which the nanogel is intended to be used. A nanogel is soluble in that it is uniformly dispersible in the target solvent or monomer composition. In one embodiment, the nanogel of the present disclosure has an equivalent diameter of about 10 nm to about 60 nm. In another embodiment, the diameter of the nanogel is such that it can be visualized by atomic force microscopy, transmission electron microscopy (TEM), or conventional light scattering techniques used for nanogel size characterization.

[0055] A “polymer” is a substance composed of macromolecules. A polymer macromolecule is a molecule of high relative molecular mass, the structure of which comprises the multiple repetition of units derived from molecules of low relative molecular mass.

[0056] A“crosslinker” or“crosslink” is an intra- and/or inter-chain connection within the nanogel structure. For example, a crosslinker may be a covalent bond or chemical bond joining two polymer chains. A crosslinker has at least two polymerization sites and can connect two or more polymer chains depending on the number of polymerization sites. Cleavage of crosslinks through degradation may release the primary polymeric/oligomeric chains of the nanogel. In some implementations, residual pendent reactive groups (e.g., methacrylate groups) from one nanogel particle may react with a complementary functional group on another nanogel to create a linkage (crosslink) between nanogels in a secondary polymerization process that may form a network of nanogels.

[0057] A“side-chain” is an extension from a backbone of a polymer. For example, a side-chain may be a functional or chemical group extending from the backbone. A side-chain or pendant functionality or chain may be located off of a primary chain or crosslink. A side- chain has a single polymerization site. A side-chain may have an influence on the polymer’s properties and/or reactivity.

[0058] A“copolymer” is a material created by polymerizing a mixture of two, or more, starting compounds. The resultant polymer molecules include the monomers in a proportion which is related both to the mole fraction of the monomers in the starting mixture and to the reaction mechanism.

[0059] A“degradable linkage” is a covalent bond that can be broken reversibly or irreversibly by a stimuli (e.g., pH, light, heat, and other sources of energy).

[0060] Synthetic polymers have a distribution of molecular weights (MW, grams/mole). Polydispersity describes a polymer composed of molecules with a variety of chain lengths and molecular weights. The width of a polymer's molecular weight distribution is estimated by calculating its polydispersity, Mw/Mn, wherein Mw is the weight- average molecular weight and Mn is the number- average molecular weight. The closer this ratio approaches a value of 1, the narrower is the polymer's molecular weight distribution.

[0061] The weight-average molecular weight (Mw) is the average molecular weight of a polydisperse polymer sample, averaged to give higher statistical weight to larger molecules; calculated as Mw=SUM(Mi² Ni)/SUM(Mi Ni), where Mi is the weight of chains with length i and Ni is the number of chains with length i. One technique used to measure molecular weights of polymers is light scattering. A light shining through a very dilute solution of a polymer is scattered by the polymer molecules. The scattering intensity at any given angle is a function of the second power of the molecular weight. Consequently, because of this “square” function, large molecules will contribute much more to the molecular weight calculated than small molecules.

[0062] The number- average molecular weight (Mn) is the average molecular weight of a polydispersed polymer sample, averaged to give equal statistical weight to each molecule; calculated as Mn=SUM(Mi Ni)/SUM(Ni).

[0063] The hydrodynamic radius is the radius of a particle or polymer molecule in solution that is determined from a measurement of mobility or diffusion, for example in viscosity or dynamic light scattering experiments.

[0064] As used herein, the singular forms “a”, “an”, and “the” encompass implementations having plural referents, unless the content clearly dictates otherwise. As used in this specification and the appended claims, the term“or” is generally employed in its sense including“and/or” unless the content clearly dictates otherwise.

[0065] Spatially related terms, including but not limited to, “lower”, “upper”, “beneath”,“below”,“above”,“on top”, etc., if used herein, are utilized for ease of description to describe spatial relationships of an element(s) to another. Such spatially related terms encompass different orientations of the nanogels in addition to the particular orientations depicted in the figures and described herein. For example, if a structure depicted in the figures is turned over or flipped over, portions previously described as below or beneath other elements would then be above or over those other elements.

[0066] In the following description, reference is made to the accompanying drawings that forms a part hereof and in which are shown by way of illustration at least one specific implementation. The following description provides additional specific implementations. It is to be understood that other implementations are contemplated and may be made without departing from the scope or spirit of the present invention. The following detailed description, therefore, is not to be taken in a limiting sense. While the present invention is not so limited, an appreciation of various aspects of the invention will be gained through a discussion of the examples provided below.

[0067] A nanogel disclosed herein may be a polymeric globular nanoparticle with tunable size below 100 nm. A disclosed nanogel may have internal covalent crosslinks, greater free volume than conventional nanogels, and/or controlled reactive and various functional groups. A disclosed nanogel may be prepared with one or more of the following: a mono vinyl monomer, a divinyl monomer, a chain transfer agent, and an initiator. In some examples, FDA approved monomers are used. One or more of the monomers may include a degradable linkage or degradable linking group. A disclosed nanogel may have at least some degree of affinity for water. For example, a disclosed nanogel may be a water dispersible nanogel. A hydrogel having some degree of affinity for water may be degradable by a hydrolytic route. The molecular environment of the nanogel may be hydrophilic, hydrophobic, or amphiphilic. For example, an amphiphilic nanogel may have both hydrophilic and hydrophobic character.

[0068] The components that make up the nanogel and their attachments define a “network” of the nanogel. For example, a nanogel may have an internal network of short polymeric chains, which may be covalently attached, forming the nanogel network. For example, a crosslinked polymer may have two or more connections, on average, between chains such that the sample is, or could be, a single molecule. Limited crosslink connections per chain may be considered lightly crosslinked while numerous crosslinks may be considered highly (or heavily) crosslinked.

[0069] As one example, a disclosed nanogel may include at least one mono vinyl monomer in the nanogel structure. For example, a mono vinyl monomer may be included as a side-chain or a crosslinker (e.g., with more than two polymerization sites) in the nanogel structure. In the example with the mono vinyl monomer included as a crosslinker, the mono vinyl monomer (with its vinyl group still intact) may be added to a crosslinker with (n) number of polymerization sites (e.g., vinyl groups), producing a crosslinker with (n+1) number of vinyl groups. As yet another example, the mono vinyl monomer may be included as an external degradable moiety on the nanogel surface. The mono vinyl monomer may be a hydrophilic monomer. For example, the mono vinyl monomer may contain a hydroxyl functional group (OH) for further functionalization. As one example, the mono vinyl monomer may be 2- hydroxyethyl methacrylate (HEM A). As another example, the mono vinyl monomer may be a degradable copolymer of HEMA and polylactide (HEMAPLA). As another example, the mono vinyl monomer may not contain a hydroxyl group and instead may be used to control the nanogel surface functional groups. For example, the mono vinyl monomer may be 2- methoxyethyl methacrylate (MEMA). In some examples, the mono vinyl monomer may be hydrophobic. In some examples, the mono vinyl monomer may be amphiphilic (e.g., by combining hydrophobic and hydrophilic moieties on the monomer). In some examples, the mono vinyl monomer may be functionalized and used as a means to introduce functionality into the nanogel structure. As one example, a functional group from the mono vinyl monomer may be used as a tether or as a site for secondary polymerizable group placement (or other functionality).

[0070] As another example, a disclosed nanogel may include at least one divinyl monomer in the nanogel structure. For example, a divinyl monomer may be included as a crosslinker or a side-chain in the nanogel structure. In one example, the divinyl monomer may be a non-degradable crosslinker, such as, for example, polyethylene dimethacrylate (PEGDMA). In another example, the divinyl monomer may be a degradable crosslinker, such as, for example, polyethylene glycol-co-polylactide dimethacrylate (PEGPLADMA). In several embodiments, the molecular weight of polyethylene glycol (PEG) and/or poly(lactic acid) or polylactide (PLA) within, for example, PEGDMA or PEGPLADMA, may be varied to adjust amphiphilicity effects on water uptake. In some examples, a pendant reactive or otherwise functionally modified crosslinker may be included and used as a means to introduce functionality into the nanogel structure. For example, a suitably functionalized crosslink may serve as a site for side-chains. As one example, a functional group may be included as a pendant functionality off the divinyl crosslinker and used as a tether or as a site for secondary polymerizable group placement (or other functionality).

[0071] The side-chain and crosslinker concentrations may be varied to achieve different nanogel properties. For example, side-chain concentration may be greater than about 10 mol%, about 20 mol%, about 30 mol%, about 40 mol%, about 50 mol , about 60 mol%, about 70 mol%, or about 80 mol%, and less than about 90 mol%, about 80 mol%, about 70 mol%, about 60 mol%, about 50 mol%, about 40 mol%, about 30 mol%, or about 20 mol%. As another example, crosslinker concentration may be greater than about 10 mol%, about 20 mol%, about 30 mol%, about 40 mol%, about 50 mol%, about 60 mol%, about 70 mol%, or about 80 mol%, and less than about 90 mol%, about 80 mol%, about 70 mol%, about 60 mol%, about 50 mol%, about 40 mol%, about 30 mol%, or about 20 mol%.

[0072] A portion or all of a mono vinyl monomer and/or di vinyl monomer described herein may include a degradable linkage or degradable linking group. For example, a mono vinyl monomer and/or divinyl monomer may be used to introduce a degradable linkage in a select location (e.g., a spatially controlled location) within and/or between nanogel particles. For example, one or more types of degradable linkages may be used with a monovinyl monomer to append reactive functional groups and/or to tether cargo. In some implementations, a nanogel may include a mixture of di vinyl monomers and/or a mixture of mono vinyl monomers (e.g., co-monomers) with at least one of the divinyl monomers and/or at least one of the monovinyl monomers including the degradable linkage, which helps enable complete or partial degradation of the internal nanogel crosslinks. As one example, a mixture of divinyl monomers and/or monovinyl monomers may include two or more monomers (e.g., at least two monovinyl, at least two divinyl, or at least one of both) that include degradable linkages that degrade on different kinetic timeframes.

[0073] A degradable linkage or degradable linking group may be a labile linkage built into a disclosed nanogel that can undergo a stimulus-responsive decomposition activated by pH, temperature, light, etc., or by a pre-configured hydrolytic degradation mechanism. For example, a hydrolytically degradable linkage may be an ester, acid anhydride, orthoester, or an amide. For example, a degradable linkage may include PLA, poly(glycolic acid) (PGA), polycaprolactone (PCL), acid anhydride, and the like.

[0074] In some implementations, a degradable linkage may be added via reaction after the nanogel has been formed. For example, a mono-vinyl monomer may be included in the nanogel structure having suitable functionality to allow the degradable linkage to be added as a post-nanogel modification. As another example, a di-vinyl monomer may be included in the nanogel structure having suitable functionality to allow the degradable linkage to be added as a post-nanogel modification. [0075] Disclosed nanogels may be prepared with a chain transfer agent. The molecular weight of the polymeric/oligomeric chains of the disclosed nanogels may be controlled by the chain transfer agent The chain transfer agent may be, for example, a mercaptan (R-SH). In other examples, the chain transfer agent may be a conventionally used species, such as alkyl thiols, aryl thiols, alkyl halides, and the like. In one example, the chain transfer agent is 1-dodecanethiol.

[0076] Disclosed nanogels may be prepared with an initiator. As an example, a radical initiator may be selected that promotes well-controlled radical production based on thermal, photo, or redox initiation processes. For example, the initiator may be a thermally activated initiator. As another example, the initiator is selected from initiators suitable for controlled radical polymerization processes, which include atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain-transfer polymerization (RAFT). As yet another example, an initiator may be selected that has a dissociation constant suitable for producing a disclosed nanogel. For example, a dissociation constant of an initiator depends on various factors, such as temperature, solvent, stability of the cleavable bond, and the like. As such, the initiator chemical structure and environment directly affect the dissociation constant. As one example, an initiator with a suitable dissociation constant may be an azo compound. For example, the initiator may be 2,2'-azobis(isobutyronitrile) (AIBN). In another example, the initiator may be benzoyl peroxide.

[0077] Disclosed nanogels may be prepared via free-radical solution (homogeneous) polymerization. For example, free-radical polymerization of PEG-co-PLA (backbone) and HEMA-co-PLA/MEMA (side-chains) may produce a disclosed nanogel. The large surface to volume ratio of regio-specific biodegradable nanogels enables the nanogels to be candidates for multivalent bioconjugation either on their surface or their interior, or both, which is beneficial for drag delivery applications or precursors for tissue scaffold.

[0078] The weight-averaged molecular weights of disclosed nanogels may be less than 65 kg/mol and/or with a hydrodynamic radius of less than 6.0 nm and/or with a glass transition temperature at or below room temperature. In one example, the weight- averaged molecular weight of a disclosed nanogel is less than 65 kg/mol. In one example, the weight- averaged molecular weight of a disclosed nanogel is greater than about 0.5 kg/mol. In some examples, the weight- averaged molecular weight of a disclosed nanogel may be greater than about 0.3 kg/mol, about 0.5 kg/mol, about 1 kg/mol, about 10 kg/mol, about 20 kg/mol, or about 30 kg/mol and less than about 65 kg/mol, about 55 kg/mol, about 45 kg/mol, about 35 kg/mol, about 25 kg/mol, about 15 kg/mol, about 5 kg/mol, or about 1 kg/mol.

[0079] In another example, a disclosed nanogel may have a hydrodynamic radius of less than 13.0 nm. In some examples, the hydrodynamic radius of a disclosed nanogel may be greater than about 1.0 nm, about 1.5 nm, about 2.0 nm, about 2.5 nm, about 3.0 nm, about

3.5 nm, about 4.0 nm, about 4.5 nm, or about 5.0 nm, and less than about 12.5 nm, about 12.0 nm, about 11.5 nm, about 11.0 nm, about 10.5 nm, about 10.0 nm, about 9.5 nm, about 9.0 nm, about 8.5 nm, about 8.0 nm, about 7.5 nm, about 7.0 nm, about 6.5 nm, about 6.0 nm, about

5.5 nm, about 5.0 nm, or about 4.5 nm. In some examples, the hydrodynamic radius of a disclosed nanogel may be from about 1.0 nm to about 5.0 nm.

[0080] In yet another example, a disclosed nanogel may have a glass transition temperature at or below room temperature. As one example, the glass transition temperature of a disclosed nanogel may be less than about 25 °C, less than about 15°C, less than about 5°C, less than about -5°C, less than about -15°C, less than about -25°C, or less than about -35°C, and more than about -80 °C, more than about -70 °C, more than about -60 °C, more than about -50 °C, more than about -40 °C, more than about -30 °C, more than about -20 °C, more than about -10 °C, more than about 0 °C, or more than about 10 °C. For example, the glass transition temperature may be between about -80°C to about 25°C. As another example, the glass transition temperature may be between about -45 °C to about 25 °C. As another example, the glass transition temperature may be between about -45 °C to about 5°C. As yet another example, the glass transition temperature may be at or above room temperature. For example, the glass transition temperature may be greater than about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, or about 55°C and less than about 60°C, about 55°C, about 50°C, about 45°C, about 40°C, about 35°C, or about 30°C.

[0081] In another example, a disclosed nanogel may have equilibrium swelling greater than about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt , about 30 wt%, about 35 wt%, about 40 wt , about 45 wt%, about 50 wt%, about 55 wt%, about 60 wt%, about 65 wt%, about 70 wt%, about 75 wt%, about 80 wt%, or about 85 wt%, and less than about 90 wt%, about 85 wt%, about 80 wt%, about 75 wt%, about 70 wt%, about 65 wt%, about 60 wt%, about 55 wt%, about 50 wt , about 45 wt%, about 40 wt%, about 35 wt%, about 30 wt%, about 25 wt%, about 20 wt%, about 15 wt%, or about 10 wt%.

[0082] Nanogels described herein may include non-degradable, internally degradable, externally degradable, and internally-extemally degradable nanogels. The type of nanogel may depend on the location of the degradable linkage (e.g., whether the crosslinker and/or side-chains are degradable). For example, non-degradable nanogels may include both a non-degradable crosslinker and non-degradable side-chains, internally degradable nanogels may include a degradable crosslinker and non-degradable side-chains, externally degradable nanogels may include a non-degradable crosslinker with at least one degradable side-chain monomer, and internally-externally degradable nanogels may include a degradable crosslinker and at least one degradable side-chain monomer.

[0083] The incorporation of hydrolytically degradable linkages (e.g., PLA) to different regions of the molecular structure (backbone, side-chain, and both) of disclosed nanogels may result in bulk, surface, and bulk/surface tandem erosions, respectively. In some embodiments, the rate of erosion may be tuned according to the degree of hydrophilicity and labile linkage concentration. The mass loss trends of disclosed nanogels show sustained release of degraded species even under auto-accelerated acid catalyzed hydrolysis by delaying or completely bypassing reverse gelation.

[0084] The disclosed technology encompasses versatility in the nanogel design in terms of chemical (hydrophilic, hydrophobic, and amphiphilic), mechanical (glass transition temperature), structural, and degradation properties. A disclosed nanogel with one order of magnitude higher structural homogeneity, which allows for more realistic prediction of its behavior, is superior to the current state of the art.

[0085] The structural versatility of nanogels disclosed herein allows improved control over nanogel properties to achieve desired properties for various nanogel applications. For example, a nanogel may be used as a tissue scaffold, for quick-release drug delivery, slow- release drag delivery, and the like. In each of these exemplary applications, different rates and routes of degradation may be desirable. As described herein, the rates and routes (e.g., spatial pathway) of degradation (e.g., internal to external, external to internal, bulk, surface, etc.) of a disclosed nanogel may be controlled by the location and/or concentration of one or more degradable linkages within the nanogel structure. As such, the location and/or concentration of one or more degradable linkages may be selected based on the particular desired application. As one example, by controlling the route of macroscopic degradation, drug dose released may be quantitatively measured. Further, controlling the route of macroscopic degradation enables control over the nature (e.g., molecular weight, bio-compatibility, concentration, etc.) and clearance of degradation byproducts. [0086] In several embodiments, the structure or architecture and/or environment of the nanogel may be selected to limit or control water access, which influences the rate of degradation. For example, the location of the degradable linkage and its molecular environment (e.g., hydrophilic, hydrophobic, amphiphilic) affects the orientation of the surrounding water molecules and their access to the degradable linkage. As one example, the range of amphiphilicity for a nanogel in an amphiphilic environment is determined by the polarity/molecular heterogeneity of the mono vinyl and/or di vinyl monomers. In this manner, altering one or both of the location of the degradable linkage and the molecular environment or nature of the nanogel allows improved control over the rate and or timing of degradation of the nanogel.

[0087] As one example, for drug or other cargo delivery, nanogel degradation can occur to release drugs or other cargo via direct cleavage of covalent attachments to the cargo and/or to chemically degrade the nanogel network density (e.g., based on internal nanogel crosslink decomposition) or release effectively intact basic nanogel structural components (e.g., based on degradation of connections formed between nanogels) or through both internal and external degradation of nanogels to create primary oligomeric/polymeric chains of well- controlled molecular weight along with any released cargo. As one example, nanogel degradation may release a covalently bound cargo via a degradable linkage. As another example, partial or complete degradation of the nanogel-based network density may release a physically encapsulated cargo. In some instances, cargo may be released by both physical and chemical means. For example, physical release of one cargo may be used in conjunction with hydrolytic (chemical) release of a covalently tethered separate cargo (e.g., an independent release (staged or otherwise) of two separate cargos from within a single nanogel). As another example, independent release of two separate cargos may result from mixing two separate nanogels each carrying different cargos with their own release profiles. The degradation rate, location, and behavior (e.g., cargo release) may be determined by the selection of the labile linkage (PLA, PGA, PCL, acid anhydride, etc.) and the local structure that alters the environment and kinetics of the degradation, as does the overall bulk nanogel structure. By controlling the degradation rate and location, rapid, extended or staged degradation can occur with resulting control in the physical/mechanical property de-evolution of the nanogel-based polymer networks as well as controlled cargo release behavior. In one example, reduction of the polymer network mesh size helps control with cargo release. As such, nanogels of different structure and/or carrying different cargo can be mixed and polymerized in various ways to selectively and controllably release cargo in coincident, staged, or other complex manners.

[0088] The disclosed technology is directed toward polymer characteristics and degradation kinetics of networks constructed from reactive nanogels with regio-specifically (e.g., selectively located within the nanogel network) degradable linkages. As one example, design and characterization of nanogel-based hydrogels that include the copolymerization of polyethylene glycol dimethacrylate) with 2-hydroxyethyl methacrylate (PEG-HEMA) and these same co-monomers with 2-methoxyethyl methacrylate (PEG-HEMA-MEMA) were investigated based on structural location of hydrolytically degradable polyQactic acid) (PLA) linkages. The results of equilibrium swelling (e.g., Examples 10-13), mass loss (e.g., Examples 14-18), and compressive modulus (dry/swollen) (e.g., Examples 28-32) demonstrate an interplay between hydrophilic/hydrophobic effects associated with PEG-PLA linkages, PLA location, and the crosslinking density that appear to dominate many of predicted property trends. Consequently, nanogel-based networks with shorter PEG and lower crosslinker concentration showed lower mass swelling rate, higher glass transition temperature (Tg), and lower compressive modulus reduction (see., e.g., Examples 9 and 14-22).

[0089] The disclosed technology includes polymeric globular nanogels with tunable size below 100 nm with large surface to volume ratio that are candidates for multivalent bioconjugation either on their surface or their interior, or both, which make them suitable for drug delivery applications or precursors for tissue scaffolds. In several embodiments, the disclosed technology utilizes free radical solution (homogeneous) polymerization as a simple route to synthesize a wide range of polymeric nanoparticles for a variety of applications such as shrinkage stress reduction and enhanced mechanical properties, surface morphology modification for polymer gradient materials, and precursors for macroscopic network formation. In some embodiments, nanogels are formed by free-radical polymerization of at least a di-vinyl and a mono-vinyl monomer in a relatively concentrated solution (e.g., good solvent) in the presence of an initiator and chain transfer agent. As used herein,“good solvent” may refer to a solvent with either the same or nearly the same solubility parameter as the polymer used at a specific temperature. A good solvent may be determined by the nanogel’s degree of water compatibility. For example, an amphiphilic nanogel may be compatible with numerous solvents because it has both hydrophilic and hydrophobic character. As another example, a good solvent may range from highly to moderately hydrophilic. In several implementations, a good solvent may be determined visually when the mixture becomes clear before and/or during polymerization. As examples, methyl ethyl ketone (MEK), hexane, acetone, acetonitrile, dimethylformamide (DMF), and/or dimethyl sulfoxide (DMSO) may be used as solvents. In some implementations, water is not used as a solvent due to the hydrolytically labile nature of the disclosed nanogels’ components. One or more of the following benefits over known methods of making nanogels are achieved by these approaches: i) a larger selection of monomers are usable, ii) highly crosslinked nanogels (« 100 nm) are easily attainable, (increasing the crosslinker concentration helps create more compact particles), iii) stable one-phase reaction reduces or eliminates the need for surfactant and monomer droplet formation, therefore polymerization site may shrink to the size of a swollen monomer, iv) instead of coagulation that happens at early stage of miniemulsion polymerization, microgel formation as a result of macroradicals crosslinking, happens at high conversions in homogeneous solution polymerization (late stage of reaction), and v) the kinetic chain length is controlled and reduced by the concentration of chain transfer agent to maintain the nanoscale dimension of the growing particle, additional branching may also be introduced to the nanogel structure.

[0090] With free-radical polymerization, the distribution of crosslinks in the nanogel is not homogeneous because primary cyclization and intramolecular crosslinking are favored at early stages of reaction (the local concentration of pendant vinyl groups inside a macroradical coil is much higher than their overall concentration in the mixture), but then cyclizations may be replaced by intermolecular crosslinking due to steric and excluded volume effects, as a result the degree of crosslinking may decrease outward. The nanogel formation disclosed herein results in a reduced level of heterogeneity to sub-nano scale, even though the nanoscopic network may still have defects such as loops, dangling chains, and super-crosslinks. The sol fraction is soluble in a reaction solvent and is separated from nanogel particles after a precipitation step. Furthermore, when a high concentration of purified active nanogels (e.g., > 50 wt%) is dispersed in a good solvent, a continuous phase of overlapped swollen nanogels (confluent or densely packed) may be created, where nanogel-nanogel interactions may be immobilized through steric stabilization in the overlapped volume. The continuous phase may then fixed in place by radical crosslinking (photo-polymerization) and a uniform macroscopic network with reduced level of heterogeneity (e.g., <10 nm) may be created having mechanical properties in quantitative agreement with rubber elasticity theory. Due to nanogels overlapping, unreacted nanogels, dangling nanogels, and loops have low probabilities, although multiple crosslinking can occur between adjacent nanogels. The pre-defined distance between vinyl groups in active nanogels may increase the number of effective crosslinks in the final macroscopic network.

[0091] The disclosed technology includes the following with respect to degradable hydrogels: 1) hydrolytically degradable nanogel precursors (« 100 nm), overlapped beyond percolation threshold, are able to create a macroscopic degradable network with a decreased level of heterogeneity compared to conventional crosslinked networks, and 2) the type of macroscopic erosion can be pre-modulated by the location of hydrolytically labile linkages in the nanogel structure. As one example, amphiphilic degradable active nanogels are disclosed, synthesized with secondary methacrylate functionalization, enabling them to create a secondary crosslinked network. The degradation of such hydrogels was investigated based on structural location of PLA linkages. As another example, a series of nanogels are disclosed, synthesized according to several experimental variables: 1) addition of degradable lactide linkage to i) crosslinker structure within the nanogel (bulk degradation), and/or ii) side chain structure (surface degradation); 2) two different molar percentages of the crosslinker (10 mol%, 50 mol%); 3) three different molecular weight PEGs (600, 2000, and 4600 g/mol); and 4) mono-functional biocompatible monomers, 2-hydroxyethyl methacrylate (HEMA) and 2- methoxyethyl methacrylate (MEMA) (e.g., Examples 1-3). In several examples described herein (e.g., Examples 6-9), the analysis of disclosed nanogel structures is performed in terms of molecular weight (M_w), degree of branching (MH-a), polydispersity index (PDI), and glass transition temperature (T_g). In examples described herein (e.g., Examples 5, 10-18, and 28-32), the macroscopic hydrogels underwent hydrolytic degradation and experimental/observational results are presented in terms of equilibrium water uptake (%W), mass loss (%), mechanical property (compressive modulus K), and pH value. The nanogels disclosed herein provide numerous possibilities in tissue engineering and controlled drug delivery.

[0092] In some embodiments, nanogels disclosed herein may be linked to form a network of nanogels. For example, nanogels with dangling polymerization sites may be covalently attached to form a micro- or macroscopic network of covalently attached nanogels. Nanogels may be coupled by one or more crosslinkers reacting with the nanogels’ dangling polymerization sites (e.g., methacrylate groups); however, polymerizable nanogels disclosed herein may connect to each other without the use of crosslinkers. The level of connections between the nanogels by incorporation of one or more crosslinkers may depend on the crosslinker properties, such as, for example, number of polymerization sites, flexibility, stability/affinity of the polymerization sites in the crosslinker and nanogels, concentration, and the like.

EXAMPLES

[0093] In order to illustrate the disclosure, the following examples are included. However, it is to be understood that these examples do not limit the disclosure and are only meant to suggest exemplary methods of practicing the disclosure.

[0094] The following materials were used in the examples. Polyethylene glycol (M_w = 600 g/mol) (REϋ_doo) (Sigma Aldrich), polyethylene glycol (M_w = 2000 g/mol) (PEG2000) (Sigma Aldrich), polyethylene glycol (M_w = 4600 g/mol) (PEG4600) (Sigma Aldrich), hydroxyethyl methacrylate (HEMA) (ESSTECH), ethylene glycol methyl ether methacrylate (MEMA) (Sigma Aldrich), 3,6-dimethyl-l,4-dioxane-2,5-dione (D,L Lactide) (Sigma Aldrich), and 2-isocyanatoethyl methacrylate (IEM) (TCI) were used as monomers. The thermal initiator was 2,2’-azobisisobutyronitrile (AIBN) (Sigma Aldrich). 1-Dodecanethiol was the chain transfer agent and 2,2-dimethoxy-2-phenylacetophenone (DMPA) (Sigma Aldrich) was used as photo initiator. Dibutyltin dilaurate (Sigma Aldrich) was the catalyst; however, other organotin compounds or catalysts may be used. Butylated hydroxytoluene (BHT) was added as an inhibitor. Methyl ethyl ketone (MEK) (Fisher Scientific), hexane (Fisher Chemical), and acetone were used as solvents. All the materials are used as received. For cell biocompatibility study, Minimum Essential Medium (MEM), 10000 international unit (IU)/mL penicillin, and 10000 pg/mL streptomycin antibiotic solutions were purchased from Gibco Life Technologies. Fetal bovine serum (FBS) was purchased from Sigma- Aldrich.

EXAMPLE 1

Non-degradable Monomer Synthesis

[0095] In one example, a non-degradable crosslinker was synthesized for use with a nanogel disclosed herein. In this example, for non-degradable crosslinker PEGDMA synthesis, methacrylate functionality was added to both sides of PEG (600, 2000, 4600 g/mol). PEG and IEM with 1 to 2 molar ratio were added to 6-fold excess of methylene chloride relative to monomer mass. The catalyst (dibutyltin dilaurate) (0.2 wt% relative to monomer mass) was added to the mixture, and reaction was carried out under ambient condition for 48 hours. The reaction reached 100% conversion and methacrylate functionalized PEG was precipitated out by drop- wise addition of the mixture to 10-fold excess of hexane (relative to mixture volume). The monomer was re-dispersed in acetone and inhibitor amount of BHT was added to the solution before removing the solvent (acetone) under high vacuum. The other non-degradable monomers (HEMA and MEMA) were used as received.

EXAMPLE 2

Degradable Monomer Synthesis

[0096] In one example, a degradable crosslinker was synthesized for use with a nanogel disclosed herein. In this example, degradable crosslinkers were synthesized by adding one PLA block on each side of the PEG via ring-opening polymerization reaction of lactide with PEG (Sawhney, 1993), and further functionalization of PLA-PEG-PLA copolymer on both sides with methacrylate groups PEGPLADMA (MA-PLA-PEG-PLA-MA). PEG to lactide molar ratio was 1 to 4.5. To avoid oxidation, mixture was under N2 purge 30 minutes prior to reaction till the end. Reaction temperature was 140° C. After almost 30 minutes the lactide was melted, then catalyst (dibutyltin dilaurate) (0.2 wt% relative to monomer mass) was added to the reaction mixture. N2 was continuously purging throughout the reaction time. After 4 h of reaction the mixture was cooled down to room temperature before exposing it to oxygen, then the unreacted lactide was removed via Kugelrohr Distillation Apparatus ( Bl)CHI) under high vacuum at 180 °C. IEM and 2-fold (relative to mass) of DCM was added to the product (PLA-PEG-PLA), then the catalyst (dibutyltin dilaurate) (0.2 wt% relative to monomer mass) was added to the mixture. The molar ratio of PLA-PEG-PLA to IEM was 1 to 2. After the reaction reached 100% conversion at room temperature, drop-wise precipitation step in 10-fold excess of hexane (relative to volume) was performed to purify the product from unreacted species. Degradable side-chain monomer HEMA-PLA was also synthesized following the same procedure. HEMA to lactide molar ratio was 1 to 2.

EXAMPLE 3 Nanogel Synthesis

[0097] In one example, nanogels were synthesized via free radical solution polymerization. The non-degradable nanogel batch included PEGDMA (10, 50 mol%) as primary crosslinker, HEMA (45, 25 mol%) and MEMA (45, 25 mol%) as mono-vinyl monomers. For internally degradable nanogels, degradable crosslinker PEGPLADMA (10, 50 mol%) and non-degradable mono-functional monomers were used. For externally degradable nanogels, crosslinker (PEGDMA) was non-degradable but one of the side-chain monomers was degradable (HEMAPLA). For internally-extemally degradable nanogels, both crosslinker (PEGPLADMA) and one of the side-chain monomers (HEMAPLA) were degradable. For the non-degradable, internally degradable, externally degradable, and internally-extemally degradable nanogels, the following were used: AIBN (1 wt% relative to monomer mass) as thermal initiator, 1-dodecanethiol (15-30 mol%) as chain transfer agent, and 8-10 fold excess of MEK (relative to monomer mass) as the solvent. Higher volume of solvent, 10-12 fold excess was used for internally-extemally degradable nanogels to avoid macrogellation due to copolymerization of higher molecular weight monomers. Also, for PEG4600 nanogel series, 30 mol% of chain transfer agent was used instead of 15 mol%, to control nanogel size and avoid macrogellation. Reaction temperature was 80° C, and reflux condenser was utilized to maintain the initial solvent amount during the reaction. Stirring rate was set to 200 rpm. The reaction was carried out isothermally for 3 hours and reached almost complete conversion (> 90%) for all nanogels.

[0098] For post-functionalizing the nanogel with methacrylate groups, IEM (equimolar to half of HEMA or HEMAPLA content) was added to the reaction mixture after it was cooled down to room temperature, then 3 drops of catalyst (dibutyltin dilaurate) (0.2 wt% relative to monomer mass) was added and reaction was carried out at ambient condition for two days. After disappearance of isocyanate peak, drop-wise precipitation step in 10-fold excess of hexane (relative to mixture volume) was performed. The precipitate (active nanogel) was re-dispersed in acetone and inhibitor amount of BHT was added to the acetone-nanogel solution before removing acetone under high vacuum. An opaque viscous fluid was obtained after complete removal of acetone. Active nanogel is considered as a multi-functional crosslinker for creating a final macroscopic network.

EXAMPLE 4

Macroscopic Network Formation

[0099] Mixtures of active-nanogel: solvent with 50:50 wt% ratio were prepared (acetone and DCM were used to disperse PEG600-PEG2000 and PEG4600 nanogel series, respectively). DMPA (0.2 wt% relative to total mass) was added to each mixture. A thin disc mold was placed between two glass slides (Diameter = 5 mm; Thickness = 1.5 mm) and nanogel mixtures were injected into the mold. The disc-shaped macroscopic network was formed after photo polymerization with UV light (365 nm; irradicance = 15.5 mW/cm²) (all disc samples displayed conversions greater than 90%). EXAMPLE 5

Macroscopic Swelling and Degradation

[00100] The discs (n = 3) were weighed subsequent to drying under house vacuum in desiccator for 7-14 days at ambient temperature. Their weights were then measured on analytical balance to insure complete solvent removal. Samples lost half of their weights due to solvent evaporation. The weights were also monitored during hydrolytical degradation at room temperature in buffer (pH = 3.0), by carefully blotting the samples to remove the excess water on the surface. The percent water uptake (%W) was measured for each sample by using (Eq 1) (Lester 2003; Wu 2010):

[00101] W = (Ms - Mi /Ms) x 100 (1)

[00102] Ms : Mass after Swelling

[00103] Mi : Initial Dry Mass

[00104] The average density of each network was calculated by using sample dry mass and volume of the mold (p x (diameter/2)² x thickness), assuming no significant shrinkage or equal shrinkage after drying for all networks. For mass loss study, PEG4600 nanogels were chosen due to shorter degradation time, and their mass loss was monitored at room temperature in DI water. The % Mass Loss was calculated gravimetrically by measuring the initial and final dry mass of the polymer specimen (polymer samples were dried at room temperature under house vacuum for 2-3 weeks) and using (Eq 2):

[00105] % Mass Loss = ((Mi - M_f)/Mi) x 100 (2)

[00106] Mi : Initial Dry Mass

[00107] M_f : Final Dry Mass

EXAMPLE 6

Measurements

[00108] Mid-IR spectroscopy (Nexus 670, Nicolet, Madison, WI) was used to calculate the conversion of the methacrylate carbon-carbon double bond (815 cm ¹) during nanogel synthesis, and isocyanate group (2270 cm ¹) conversion during nanogel post functionalization. Triple-detection gel permeation chromatography (GPC; Viscotek) with differential refractive index, viscosity, and light scattering detectors was employed for the analysis of nanogel weight and number averaged molecular weights, M_w (g/mol) and M_n (g/mol), polydispersity index (PDI = M_w/M_n), and average hydrodynamic radius R_h (nm). Tetrahydrofuran (THF) was used as diluent with a flow rate of 1 mL/min at 35° C in a series of three columns spanning molecular weight of 10⁴ - 10⁷ calibrated with a 65 kg/mol poly(methyl methacrylate) standard.

[00109] Proton nuclear magnetic resonance ( Ή NMR; Varian 500 MHz) and MestReNova 5.2.4 software were employed to determine and analyze the structural composition of the nanogel. DMA (Dynamic Mechanical Analysis) tests were performed using a TA instruments (DMA 8000, Perkin Elmer) to determine the glass transition temperature (T_g) of the nanogel. Nanogel (10-15 mg) (n = 2) was placed inside a steel pocket and was tested between the clamps. The glass transition temperature was determined as the position of the maximum on the tan 5 vs. temperature plot. The temperature range was from -150 °C to 100 °C with a ramping rate of 3 °C/min at a frequency of 1 Hz and tan d were recorded as a function of temperature. A preheating cycle was applied with a ramping rate of 10 °C/min.

[00110] The compressive modulus of the photopolymerized nanogel network before (dry) and after swelling in buffer solution pH = 3.0 were measured at room temperature using a mechanical testing machine (MTS; MiniBionix II). Disc samples (n = 3) were compressed with 10 N load at a constant rate of 1 mm/min. The modulus (K) was calculated from the slope of the linear region of the stress vs. strain curve. StatPlus v5.9.91 (AnalystSoft Inc) was used for statistical analysis. After passing normality (Shapiro-Wink W), one-way Analysis of Variance (ANOVA) and post hoc test (Tukey B) was performed, and null hypothesis was rejected if the - value was less than 0.05.

EXAMPLE 7

Nanogel GPC Analysis

[00111] The M_w, M_n, R_h, PDI, and the degree of branching/crosslinking portrayed by Mark Houwink exponent measured by GPC, are important determinants of physical and mechanical properties of nanogel particles (Tables 1-5). In the examples, when PEG length was constant, increasing the concentration of crosslinker from 10 to 50 mol% increased the molecular weight and hydrodynamic radius of the nanogels. This effect was expected due to increased number of pendant double bonds on the backbone and overall addition of higher number of monomers to the nanogel structure. Also, by keeping the crosslinker concentration constant while increasing the PEG molecular weight from 600 to 2000 g/mol, the molecular weight of the nanogel increased. At a constant PEG length, polydispersity index decreased when the concentration of the crosslinker increased from 10 to 50 mol%, due to larger M_n values (Table 1). When crosslinker concentration increases, M_n increases accordingly, due to higher molecular weight species (Mi) and their higher mole fractions (xi). 10-fold excess of solvent was used to synthesize the internally-externally degradable nanogels, increasing the solvent increases the extent of intra-molecular crosslinking and cyclization rather than intermolecular crosslinking, and consequently creates more compact particles with relatively lower molecular weights (Table 4). By increasing the crosslinker concentration, it was expected to see lower MH-a values due to increased branching/crosslinking and more hard-sphere like behavior, yet GPC results showed the opposite trend mainly in non-degradable and internally degradable nanogels (Tables 1 and 2), which, without meaning to be limited by theory, might be due to the change in solvent-particle interaction, arising from increased hydrophilicity character with higher concentration of hydrophilic crosslinker. The MH-a can take up different values depending on the quality of the solvent, 0 < MH-a < 0.5, MH-a =0.5 (theta solvent), and MH-a > 0.8 represent semi-rigid sphere (highly branched), an unperturbed Gaussian chain (flexible random coil), and flexible chain (less branched), respectively (Hiemenz, 2007). The NG11 (Table 2) had the lowest molecular weight among all the nanogels synthesized in this study, which might be related to water/THF ensemble, causing hydrolytic degradation during GPC sample preparation and overnight runs (Lyu, 2009).

[00112] Table 1 discloses GPC Results for Non-Degradable Nanogels: copolymers of PEGDMA (PEG molecular weight = 600, 2000 g/mol) and HEMA (NG₁-NG₄); copolymers of PEGDMA, HEMA, and MEMA (NG₅-NG₈).

TABLE 1

Nanogel Name M_n M_w M_w/M_n R_h MH - a

NGi PEG600DM A/HEM A( 10:90) 5,700 12,000 2.1 2.4 0.35

NG₂ PEG_6QODM A/HEM A(50:50) 32,000 32,000 1.0 3.7 0.81

NG₃ PEG2000DM A/HEMA( 10 : 90) 5,900 20,000 3.4 3.5 0.37

NG4 PEG_2OOODMA/HEMA(50:50) 37,000 41,000 1.1 5.7 0.49

NGs PEG_6QODM A/HEM A/MEM( 10:45:45) 4,200 15,000 3.6 2.8 0.42 NG₆ PEG₆OODMA/HEMA/MEMA(50:25:25) 25,000 27,000 1.1 3.8 0.82

NG₇ PEG_2OOODMA/HEMA/MEMA(10:45:45) 11,000 25,000 2.2 4.3 0.54

NGs PEG2000DM A/HEMA/MEM A(50:25:25) 42,000 46,000 l.l 5.7 0.50

[00113] Table 2 discloses GPC Results for Internally Degradable Nanogels: crosslinker stmcture contains degradable PLA linkages; copolymers of PEGPLADMA (PEG molecular weight = 600, 2000 g/mol) and HEMA (NG₉-NG₁₂).

TABLE 2

Nanogel Name M_n M_w M_w/M_n R_h MH-a

NG₉ PEG600PLADM A/HEM A( 10:90) 8,600 36,000 4.2 3.3 0.41

NG10 PEG600PLADM A/HEM A(50 : 50) 39,000 62,000 1.6 6.0 0.61

NG11 PEG2000PLADM A/HEMA( 10 :90) 800 2,000 2.4 1.5 0.21

NG12 PEG2000PLADM A/HEM A(50 : 50) 41,000 41,000 1.0 5.5 0.83

[00114] Table 3 discloses GPC Results for Externally Degradable Nanogels: the side-chain monomers contain degradable PLA linkages; copolymers of PEGDMA (PEG molecular weight = 600, 2000 g/mol), HEMAPLA, and MEMA (NG₁₃-NG₁₆).

TABLE 3

Nanogel Name M_n M_w M_w/M_n R_h MH-a

NGi₃ PEG™ DM A/H EM A PL A/M EM A

3,000 24,000 7.2 2.2 0.51 (10:45:45) Nanogel Name M_n M_w M_w/M_n R_h MH-a

NGi₄ PEGeooDMA/HEMAPLA/MEMA

5.900 9,400 1.6 1.9 0.33

(50:25:25)

NGis PEG₂₀₀₀ D M A/ HEM A PL A/M EM A

4.900 19,000 3.9 3.6 0.44

(10:45:45)

NG₁₆ PEG₂₀₀₀DMA/HEMAPLA/MEMA

42,000 46,000 1.1 5.9 0.52

(50:25:25)

[00115] Table 4 discloses GPC Results for Internally-Extemally Degradable Nanogels: crosslinker and one of the side-chain monomers contain degradable PLA linkages; copolymers of PEGPLADMA (PEG molecular weight = 600, 2000 g/mol), HEMAPLA, and MEMA (NG17-NG20).

TABLE 4

Nanogel Name M_n M_w M_w/M_n R_h MH-a

NGi₇ PEGeooPLADMA/HEMAPLA/MEM

1,800 6,000 3.4 2.1 0.38

A (10:45:45)

NGis PEGeooPLADMA/HEMAPLA/MEM

3,000 6,000 1.8 2.3 0.23 A (50:25:25)

NG19 PEG2000PLADMA/HEMAPLA/MEM

1,400 3,000 2.2 1.8 0.28 A (10:45:45)

NG20 PEG2000PLADMA/HEMAPLA/MEM

5,700 8,000 1.4 3.0 0.13

A (50:25:25) [00116] Table 5 discloses GPC Results for PEGeoo Nanogel Series: non-degradable (NG₂I), internally degradable (NG₂₂), externally degradable (NG₂₃), and internally-extemally degradable (NG₂₄).

TABLE 5

Nanogel Name M_n M_w M_w/M_n R_h MH-a

NG₂I PEG₄₆OODMA/HEMA/MEMA

13,000 15,000 1.2 3.3 0.71

50:25:25)

NG₂₂ PEG_46OOPLADMA/HEMA/MEMA

19,000 24,000 1.3 3.4

(50:25:25)

NG₂₃ PEG₄₆₀₀DMA/HEMAPLA/MEMA

13,000 16,000 1.2 3.2 0.74 (50:25:25)

NG₂₄ PEG₄₆₀₀PLADMA/HEMAPLA/ME

MA 6,700 18,000 1.1 3.2 -

(50:25:25)

EXAMPLE 8

Nanogel ¾ NMR Analysis

[00117] ¹ H NMR spectra of nanogels help to verify the structures particularly for low M_w particles. The ratio of di -vinyl group C=CH₂ (peak at 5.7 or 6.2) to PEG length PEG- 0CH₂CH₂0- (peak at 3.64) was calculated for a PEGeoo and PEG₂ooo series (Table 6-9). This ratio is correlated with functional group density of the active nanogel. Increasing the PEG concentration from 10 to 50 mol reduces this ratio. This trend was observed for all nanogel groups except for NG₂ and NG16, due to unreacted residual IEM trapped in the precipitated nanogel. There was a decreasing effect on (peak 6.2/peak 3.64) ratio, when PEG molecular weight increased from 600 to 2000 g/mol, while concentration of the crosslinker remained constant. In all four nanogel categories, the nanogel with highest concentration of crosslinker and highest molecular weight of PEG had the lowest functional group density (NG4, NGx, NG12, and NG20), except for NG16 (Table 6-9).

[00118] Table 6 discloses ¹ H NMR Results for Non-Degradable Nanogels (NGi-

NGs).

TABLE 6

Nanogel Name Peak 6.2 ppm/Peak 3.64 ppm

NGi PEG₆ooDM A/HEMA( 10:90) 0.0359

NG₂ PEG₆OODMA/HEMA(50 : 50) 0.0370

NG PEG2000DM A/HEM A( 10:90) 0.0255

NG4 PEG2000DM A/HEM A(50: 50) 0.0036

NG₅ PEG₆OODM A/HEMA/MEM A( 10:45 :45) 0.0368

NGe PEG_6OODMA/HEMA/MEMA(50:25 :25) 0.0108

NGv PEG2000DM A/HEM A/MEM A( 10:45:45) 0.0201

NGs PEG2000DM A/HEM A/MEM A(50 : 25:25) 0.0063

[00119] Table 7 discloses Ή NMR Results for Internally Degradable Nanogels (NG9-NG12).

TABLE 7

Nanogel Name Peak 6.2 ppm/Peak 3.64 ppm

NG₉ PEG600PLADM A/HEM A( 10:90) 0.1862

NG10 PEG₆OOPLADMA/HEMA(50:50) 0.0587 Nanogel Name Peak 6.2 ppm/Peak 3.64 ppm

NGn PEG2000PLADM A/HEMA( 10 :90) 0.0089

NG12 PEG _OOOPLADMA/HEMA(50:50) 0.0064

[00120] Table 8 discloses ¹ H NMR Results for Externally Degradable Nanogels (NG13-NG16).

TABLE 8

Nanogel Name Peak 6.2 ppm/Peak 3.64 ppm

NGn PEG₆OODMA/HEMAPLA/MEMA(10:45:45) 0.0594

NGi₄ PEG600DMA/HEM APLA/MEM A(50:25:25) 0.0094

NGi₅ PEG_2OOODMA/HEMAPLA/MEMA( 10:45:45) 0.0129

NG16 PEG OOODMA/HEMAPLA/MEMA(50:25:25) 0.0169

[00121] Table 9 discloses ¹ H NMR Results for Intemally-Externally Degradable Nanogels (NG17-NG20).

TABLE 9

Nanogel Name Peak 6.2 ppm/Peak 3.64 ppm

NGn PEG600PLADM A/HEM APLA/MEM A( 10:45:45) 0.0293

NG18 PEG600PLADM A/HEM APLA/MEM A(50:25:25) 0.0141

NG19 PEG_2OOOPLADMA/HEMAPLA/MEMA(10:45:45) 0.0210

NG20 PEG_2OOOPLADMA/HEMAPLA MEMA(50:25:25) 0.0029 EXAMPLE 9

Nanogel DMA Characterization

[00122] In the examples, the nanogels had T_g at or below room temperature (Figs. 25-34; where Fig. 32 -level = 0.02258) and Tan d less than unity, which is an indication of elastic behavior (Mahlin, 2009). The highest (25.2 ± 0.71 °C) and lowest (-77.7 ± 0.07 °C) average glass transition temperatures belonged to externally degradable nanogel PEG_6OODMA/HEMAPLA/MEMA(10:45:45) (NGI₃) (Fig. 31; -level = 0.00154) and non- degradable nanogel PEG₂oooDMA/HEMA(50:50) (NG3) (Fig. 26; p-level between groups = 0.00056.), respectively. The flexibility of the backbone and the T_g of constituent monomers were the dominant factors in nanogel T_g values. Increasing the crosslinker concentration from 10 to 50 mol% decreased the T_g 21.2 ± 0.41 °C between NGi and NG2 (Fig 25; /2-level between groups = 0.00054), as opposed to increasing it, and 41.3 ± 0.85 °C between NG3 and NG4 (Fig 26), which is due to the increased fraction of the lower T_g monomer (PEG) compared to higher T_g monomer (HEMA) in the co-polymer composition (Verhoeven, 1989; Fernandez- Garcia, 2000). This trend was also observed between NG5 and NG₆ with decreasing T_g of 14.9 + 0.55 °C (Fig 27; -level = 0.00098), and NG7 and NGs difference was 21.8 ± 19.1 °C (Fig 28; p- level = 0.24812). Furthermore, in non-degradable nanogel category of PEG/HEMA, increasing PEG molecular weight from 600 to 2000 g/mol at constant crosslinker concentration, resulted in a T_g reduction of about 47.3 ± 0.92 °C and -67.3 ± 0.22 °C at 10 and 50 mol% crosslinker concentration, respectively. The same trend was observed for PEG/HEMA/MEMA non- degradable nanogel category. Glass transition temperature of PEGs decreases with increasing their molecular weight, as a result of increased flexibility of the longer PEG length. Consequently, nanogels with longer PEGs in their backbone had lower T_gs. Replacing half of HEMA with MEMA in the nanogel structure, while keeping the crosslinker concentration constant at 10 mol% (45 mol% MEMA content), had a mixed effect on T_g. The T_g difference between NGi and NG5 was 5.35 + 0.55 °C, and between NG3 and NG7 was 6.1 ± 1.1 °C. On the other hand, the T_g difference when crosslinker concentration was 50 mol% (25 mol% MEMA content) (Figs. 25-28) increased 0.9 ± 0.41 °C from NG2 to NG7, and 13.4 ± 19.1 °C between NG4 and NGs, which is an indication of dominant effect of higher PEG concentration on reducing T_g. Addition of PLA to the backbone and side chain of the nanogel structure had an overall increasing effect on the T_g compared to controls. In internally degradable nanogel category, there was statistically significant difference between the T_g of NG9 and NG10, but when PEG molecular weight increased from 600 to 2000 g/mol in NGn and NG12, there was a -12.2 °C reduction in T_g (Fig. 29, p-level = 0.0559; and Fig. 30, p- level = 0.01909). Furthermore, the dependency of T_g on nanogel molecular weight did not exhibit a conventional trend, due to the dominant effect of nanogel composition. The effect of PLA addition to both crosslinker and side-chain monomer, had more dominant effect on raising the T_g compared to control, when PEG was longer (PEG molecular weight = 2000 g/mol) (Fig. 33, / level = 0.00942; and Fig. 34, /?-level = 0.00119).

EXAMPLE 10

Equilibrium Swelling Analysis

[00123] The amount of water uptake of a polymeric material is often correlated with its free volume and chain polarity. The amount of unbound water, which is correlated with free volume in the network only contributes to mass gain as opposed to swelling, but what causes swelling is the amount of bound water, which is directly correlated with the polarity of the polymer chains. Water uptake measurement is based on mass gain during swelling, therefore volume change data is needed to decouple the effect of free volume and chains polarity. In common crosslinked systems, where a crosslinker is directly mixed with a monofunctional monomer and polymerized to form a macroscopic network, increasing the crosslinker concentration decreases free volume available in the bulk and decreases water uptake. Equilibrium degree of swelling is considered where water uptake vs. time curve plateaus for a long period of time, which its value is solvent and temperature dependent, and is obtained from balancing the osmotic drive to dilute the polymer and the entropic resistance to chain extension. Equilibrium degree of swelling of a polymer provides valuable information regarding biomedical and pharmaceutical applications including: the solute diffusion coefficient, surface properties and surface molecule mobility, mechanical properties, and optical properties (contact lens). Two types of free volumes were considered: first, the intra-particle free volume associated with nanogel particle itself and is governed by concentration of the primary crosslinker and its functional group density; second, the inter-particle free volume affected by the concentration of the nanogel (secondary crosslinker) and its functional group density.

[00124] Concentration of nanogel in the medium determines the distance between dispersed particles and the level of network confluency (nanogel loading > 20 wt%). Functional group density of nanogel particle is determined with the ratio of number of attached methacrylate groups on the particle surface to nanogel molecular weight. The post functionalization of all nanogels in this study was performed by addition of IEM to more accessible hydroxyl groups on side-chains (equimolar to half of HEMA or HEMAPLA). By increasing the concentration of HEMA or HEMAPLA, the number of functional groups surrounding the nanogel particle increase.

EXAMPLE 11 Non-Degradable Networks

[00125] Referring to FIGS. 1A and IB, in non-degradable networks, increasing the concentration of hydrophilic primary crosslinker (PEGDMA) from 10 to 50 mol had an overall increasing effect on water uptake, although the primary mesh size had significantly decreased. In FIGS. 1A and IB, graphs 100 show water uptake vs. time for non-degradable nanogel networks, NG1-NG4, in FIG. 1A and NGs-NGs in FIG. IB. In both graphs, the PEG molecular weight = 600, 2000 g/mol.

[00126] The simultaneous increase in hydrophilic character of the backbone (more PEG content) counter-balances the tighter mesh size yet draws more water molecules into polymer network. It is important to note, when the concentration of the primary crosslinker increases, concentration of side-chain monomer(s), HEMA and MEMA decreases, leading to lower secondary functional group density, and consequently lower secondary mesh size. The lower concentration of polar side-chains (HEMA and MEMA) was entirely shielded by higher concentration of PEG. At the same time, nanogel molecular weight increased due to higher concentration of crosslinker, as a result, secondary functional group density decreases furthermore, which creates much larger secondary mesh size.

[00127] The larger secondary void volume and higher hydrophilicity of the nanogel have dominant collective impact on raising the equilibrium water uptake than reverse effect of smaller primary mesh size and lower polar side chain content. The amount of water uptake also increased when PEG molecular weight increased from 600 to 2000 g/mol at constant crosslinker concentration. The longer PEG crosslinkers possess amplified hydrophilicity due to greater oxygen content, more primary free volume due to larger distance of active chains, and less resistance to extension relative to shorter PEG (a polymer chain can be stretched by nearly a factor of N^1/2; N is the number of repeat units). Comparing two non-degradable control groups, PEGDMA HEMA and PEGDMA/HEMA/MEMA indicated that, replacing half of HEMA with MEMA slightly decreased equilibrium water uptake when PEG length was shorter (PEG600), yet did not have a significant effect in cases of longer PEGs (PEG2000). [00128] Referring to FIG. 2, adding hydrophobic PLA linkages to both sides of PEG in internally degradable networks decreased the degree of swelling (day 56) compared to non- degradable counterparts, except for PEG2oooPLADMA/HEMA(50:50) (NG12), which water uptake did not change compared to the control (p- level = 0.52). Adding PLA on both sides of PEG increases primary void volume yet decreases the polarity of polymer backbone. In FIG. 2, a graph 200 shows water uptake vs. time for internally degradable nanogel networks, NG9- NG12; PEG molecular weight = 600, 2000 g/mol. In addition, increasing the degradable crosslinker concentration lowered swelling in a greater extent compared to control when core PEG segment was shorter (600 g/mol). The value of equilibrium water uptake for PEG600PLADM A/HEM A( 10 : 90) (NG9) and PEG₆ooPLADMA/HEMA(50:50) (NG10) on day 2 was statistically the same (p- level = 0.25), which shows that these two systems are equivalent in terms of total void volume and chain polarity.

[00129] On the other hand, the value of water uptake was 20.9% higher in PEG2000PLADM A/HEMA(50 : 50) (NG12) than in PEG₂oooPLADMA/HEMA( 10:90) (NGn). Although unchanged total void volume also seems like a reasonable assumption here, yet polarity increase is the main contributor to invite more water inside the former network NG12 than the latter NGn. Also, for NGn , a 6.6% reduction in W (p-level = 0.006) was observed, and there was a visual indication of mass loss, therefore the degraded mass did not leave the sample as a result of high secondary crosslinking density.

[00130] In NG12, the water uptake increased about 5.0% from day 2 to 56 and never reached a plateau. This observation is an indication of gradually increasing void volume due to degradation and being occupied simultaneously by more penetrated water molecules. For NGn, the opposite trend (deswelling) was observed, since W decreased from 51% (day 2) to 44.5% (day 56). As it was discussed before, the secondary mesh size in this nanogel is much tighter than in NG12 due to higher HEMA content and consequently higher secondary crosslinker, therefore the impenetrable and non-degradable outer shell is able to trap the degraded species inside the network and shift the osmotic drive. NG9 and NG10 remained at equilibrium level of water content until the end of study, which shows a mass balance between water entering the network and degraded mass leaving the network. EXAMPLE 12

Externally Degradable Networks

[00131] Referring to FIG. 3, a graph 300 shows water uptake vs. time of externally degradable nanogel networks, NGia-NGir,; PEG molecular weight = 600, 2000 g/mol. The amount of water uptake of PEG₆ooDMA/HEMAPLA/MEMA(10:45:45) (NG13) decreased about 10% compared to the control (NGs) is shown. On the other hand, in PEG600DM A/HEM APLA/MEMA(50:25:25) (NGI₄), a 13% increase in W compared to its non-degradable counterpart (Nϋb) was observed, also NG14 had 45% more water content than NGi3 (FIG. 3).

[00132] Without being limited to any one theory, the reason for higher W in NG14 than in NG₆ may be related to hard sphere behavior of NG14 particles (MH- = 0.33 < 0.5) (Table 3), which lowers the packing density and increases the void volumes between particles. PEG_2OOODMA/HEMAPLA/MEMA(10:45:45) (NG15) and

PEG2_OOODMA/HEMAPLA/MEMA(50:25:25) (NG16) networks, had 5% decrease and no change in water uptake compared to their controls, respectively. Comparing these nanogels within the group showed that decreasing the concentration of HEMAPLA increased swelling in a greater extent when molecular weight of PEG was lower (600 g/mol). Based on these observations, the more dominant factor in reducing water sorption is, to what extent the hydrophilicity of the backbone is affected by shielding effect of PLA on the side-chains. All the nanogel networks in this group remained at equilibrium state until day 56.

EXAMPLE 13

Internally-Externally Degradable Networks

[00133] In FIG. 4, a graph 400 shows water uptake vs. time of intemally-externally degradable nanogel networks, NG17-NG20; PEG molecular weight = 600, 2000 g/mol. The initial water content (day 2) of PEG₆ooPLADMA/HEMAPLA MEMA(10:45:45) (NGn) was 10%, which was the lowest among all other groups, yet it continued swelling linearly with lower rate than the initial rate without reaching equilibrium plateau until the end of the study (day 56), with final W of 32% (FIG. 4).

[00134] The initial low water uptake is due to significant increase of PLA content both in the backbone and side-chains, and the linear increase of W from day 2, is a result of higher inward diffusion rate of water than outward diffusion rate of degraded species. In PEG₆OOPLADM A/HEM APLA/MEMA(50:25:25) (NGis), initial water uptake reached 31% (day 2) and gradually decreased to 17% (day 56). Higher initial swelling compared to NGn is a result of enhanced polarity (more PECrao content), yet the reduction in W overtime represents the underlying degradation process and the actual reduction in mass. In this type of network, more PLA units may be cleaved for erosion of the network and consequently more water concentration may be needed based on hydrolysis kinetics. The initial water uptake of PEG2000PLADM A/HEMAPLA/MEMA( 10:45 :45) (NG19) was 47%, which immediately reached equilibrium. The NG19 samples sustained their equilibrium state at 47% water content until day 56, this behavior indicates a continuous mass balance between degradation and diffusion, in other words, the residence time of degraded polymers approaches zero due to larger secondary mesh size. As observed from equilibrium swelling of non-degradable samples, larger secondary mesh size had the dominant effect on water diffusion. PEG_2OOOPLADMA/HEMAPLA/MEMA(50:25 :25) (NG20) network had the highest initial water content of 84% among all the networks in this study, water continued diffusing into the network until day 12 when blotting/weighing (mechanical stress) turned the sample into several pieces. All the pieces were transferred into the water but it was clear after this point on the erosion would be enhanced due to smaller sample size. On day 20 very small residual pieces were left from the whole sample, which made gravimetric measurement no longer feasible. The continuation of swelling after day 2 is an indication of uninterrupted and rapid primary and secondary crosslinker bond cleavage, due to higher initial water content and high concentration of PLA. The microscopic reverse gelation time happened after day 20 when the original network was completely dissolved in water. In conclusion, this is a premature reverse gelation time due applying external stress to the network during weight measurement.

EXAMPLE 14

Compressive Modulus Analysis

[00135] Two independent contributing factors may be considered for understanding the mechanical strength of a swollen hydrogel network: i) crosslinking density and ii) polymer volume fraction. The compressive modulus of a polymer network is directly proportional to crosslinking density, polymer volume fraction, and inversely proportional to the molecular weight between crosslinks (M_c) (Eq 3-5). Also the polymer volume fraction (f₂) is inversely proportional to the degree of swelling (Q_v) (Eq 3-5). Based on this knowledge, for hydrogels composed of nanogel particles, the macroscopic compressive modulus should be inversely correlated with the number averaged molecular weight between primary and secondary crosslinks (M_x). For a network composed of crosslinked Gaussian chains this relationship is determined through mbber elasticity theory and correlation between Young’s modulus (E), macroscopic compressive modulus (K), and shear modulus (Eq 3-5). These equations are valid under the following assumptions: i) no defects in the network (no loops, no dangling ends, and no multiple crosslinks), ii) crosslinked Gaussian chains are still Gaussian, iii) each junction point moves in proportion to the macroscopic deformation (affine junction assumption), iv) free energy gradient is purely entropic (ideal elastomer), and v) conservation of volume during deformation. In fact assumption (v) meets the experimental condition in this study, since the modulus was measured in the linear regime of stress-strain curve, where extension ratio approaches 1 (l 1).

[00136] 3 K (1 - 2v) = 2G (1 + v) (3)

(4)

[00139] In Eq 3, v is the Poisson’s ratio and its value is strongly dependent on packing and connectivity of the material (Greaves, 2011). Eq 4 and Eq 5 are used for dry and swollen hydrogels, respectively, Eq 5 indicates that compressive modulus of a swollen network is reduced by a factor of (f2)^1/3 compared to dry network. The other parameters in Eq 4-5 are density of dry macroscopic network (p), gas constant (R), and temperature (T). The same level of packing in dry state of macroscopic nanogel networks in this study is a safe assumption due to confluent nature of these networks, yet they differ in degree of crosslinking (connectivity), therefore the real values of Poisson’s ratio in dry state of each network is different. Additionally V_dry and V_swoiien is different for each sample due to the effect of swelling on packing, therefore assumption of constant v (vary polymer ~ 0.3; Visotropic hydrogel ~ 0.45) for all samples in dry and equilibrium swollen state is a poor assumption, however it is an inevitable alternative due to lack of shear modulus (G) or Young’s modulus (E) data. In addition, an assumption was made that dry and swollen networks are both isotropic due to quasi-homogeneous distribution of crosslinks. Polymer volume fraction (F2) can be obtained gravimetrically by using Eq 6-8.

[00142] f₂ = (8) [00143] Here, Q_m is mass swelling ratio, M_s network swollen mass, M_d network dry mass, p_p polymer density (equal to p in Eq 4 and 5), and p_s water density (1 g/cm³). M_x was evaluated for swollen hydrogels based on equilibrium swelling theory or Flory-Rehner equation (Eq 10) by assuming: i) isotropic swelling, ii) no contribution of network in AG_m (N -> ¥) using Flory-Huggins theory (Eq 9), and iii) free energy gradient of mixing and distortion are purely entropic, or in other words c = 0 and AG_d = -TAS_d, respectively.

[00146] In Eq 7, k is Boltzmann constant, (fi) solvent volume fraction, and c solvent- polymer interaction parameter. In Eq 10,

is molar volume of the solvent (18 cm³/mol for water). The value of M_x for dry network using Eq 10 is mathematically undefined, since (F2 = 1), and also it violates the underlying thermodynamics concepts of this relationship, since neither mixing nor distortion occur in dry sample, thus EqlO is not the best model to evaluate M_x history from dry state all the way to degraded state in hydrogel systems, therefore our analysis is predominantly based on M_x values using Eq 4-5 and compressive modulus measurements.

EXAMPLE 15

Non-Degradable Network

[00147] Referring to FIG. 5A-D, graphs 500 show compressive modulus (K) vs. time for non-degradable nanogel networks (NGi-NGs). In non-degradable hydrogels, the decrease of compressive modulus from dry to swollen state is due to

factor (the larger the value of Q_m the lower the f₂), not increasing M_x. The first observation is that, K values of dry samples in all four categories of nanogel network in this study are two orders of magnitude higher than the compressive modulus of networks obtained by radical polymerization of functionalized PEG-co-PLA macromer only. Samples with lower primary crosslinking (10 mol%) and higher secondary crosslinking had higher dry modulus, although this trend was less pronounced and even reversed when PEG length changed from 600 to 2000 g/mol. For PEG₂₀₀₀DM A/HEM A/MEM A(50:25:25) (NGs) and PEG₂oooDMA/HEMA/MEMA( 10:45:45) (NG₇), the difference in modulus was DK ~ 71.5 MPa with former having higher modulus than the latter. These trends reveal the dominant effect of primary crosslinking, when PEG length is long, and that of secondary crosslinking, when PEG is short. Another contributor to high modulus of nanogel networks with lower primary crosslinking (higher secondary crosslinking) might be due to higher level of chain entanglements in the overlapped regions. In addition, K values for NG7 did not change statistically all throughout the experiment (p-level between groups = 0.31). NG7 networks had average water uptake of 50%, therefore, based on Eq 5, constant K after swelling with no degradation involved, may be related to the microstmcture of these gels, which under compressive stress squeeze the water out. Replacing half of HEMA with MEMA had mixed effects on dry K values.

[00148] The graphs 500 in FIGS. 5 A and 5B show a PEG600 nanogel series in this group. The graphs in FIGS. 5C and 5D show adding MEMA increased the modulus as opposed to PEG2000 series. The increasing modulus effect might be due to more reactivity of MEMA monomers compared to HEMA during nanogel synthesis, since MEMA radicals are relatively more stable than HEMA (due to less electron withdrawing effect of methoxy compared to hydroxyl), as a result MEMA monomers are consumed at early stages of particle formation due to less stability, and HEMAs at later stages. Consumption of HEMAs at later stages of reaction creates more distribution of HEMA towards the surface of the nanogel, thus after functionalization with IEM, methacrylate groups are more accessible for crosslinking. The reducing effect on modulus by addition of MEMA to nanogel formulation happened when PEG length increased. PEG₂oooDMA/HEMA/MEMA(10:45:45) (NG7) had significantly lower modulus than PEG2oooDMA/HEMA( 10:90) (NG3) (DK ~ 104 MPa), this is due to lower secondary crosslinking density in NG7 than in NG3. On the other hand, PEG2000DM A/HEM A/MEM A(50:25:25) (NGs) and PEG₂oooDMA/HEMA(50:50) (NG₄) had the same dry moduli (p- level = 0.52), which is due to higher density of former compared to latter by factor of 3. All samples showed decrease in modulus after immersion in water (day 3), although the difference between dry and wet modulus (AKdry-wet) was different for each group. FIG 5A does not show a difference in AK_dry-wet after PEG600DMA concentration increases from 10 to 50 mol%, even though water uptake is 20% higher in PEG_6OODM A/HEM A(50:50) (NG2) compared to PEG6ooDMA/HEMA( 10:90) (NGi), also the same trend was observed for PEG₆ooDMA/HEMA/MEMA(l 0:45:45) (NGs) and PEG_6OODMA/HEMA/MEMA(50:25:25) (NGe). This observation might be due to increase in v

(less packing), {f_ϊ '^ < 1 is also an important factor. More swelling decreases the value of

( 2 ) ^{1/ 3} but increases v, therefore several parameters such as <p₂, v ry, Vswoiien, p, and M_x have to change simultaneously to keep \ K_(jry-wct constant between two networks. On the other hand, when PEG length increased the \ K_{dry-w i} value increased for the networks with higher primary crosslink density. The AKdry-wet in PEG2oooDMA/HEMA( 10:90) (NG3) and PEG2_OOODMA/HEMA(50:50) (NG4) was 101.4 MPa and 153 MPa, respectively. Furthermore, the AKdry-wet of PEG₂oooDMA/HEMA/MEMA( 10:45:45) (NGv) and PEG2000DM A/HEM A/MEM A(50:25:25) (NGs) were 12 MPa and 152 MPa, respectively. This observation indicates that in longer PEG length, reduction of

i^s the dominant factor in changing K. The isotropic range of Poisson’s ratio is -1 < v < ½ at small strains, the upper limit v = ½ indicates that deformation causes no volume change and stress is a result of shape change. The lower limit v = -1 corresponds to network structure (i.e. foam, sponge) with high compressibility (Greaves, 2011). In all groups K_SWoiien remained relatively constant until the end of study.

EXAMPLE 16

Internally Degradable Networks

[00149] Referring to FIGS. 6A and 6B, graphs 600 of compressive modulus (K) vs. time for internally degradable nanogel networks (NG9-NG12) are shown. In internally degradable networks with PEG₆₀₀PLADMA as crosslinker, when the primary crosslinker concentration increased from 10 to 50 mol% there was no significant effect on dry compressive modulus (p- level = 0.98), as shown in FIG. 6A. The minimum difference between dry and wet modulus was observed for the network with lowest degree of swelling (PEG6ooPLADMA/HEMA( 10:90)) (NG9). In addition, K of values NG9 from day 0 until 47 are statistically the same (p- level between groups = 0.15). Based on this information and swelling trend for NG9, there was no significant degradation was happening during the time of experiment.

[00150] There was a reduction of 80.7 MPa in K for (PEG_6OOPLADM A/HEMA(50 : 50)) from day 0 to day 47 (p- level = 0.00024), and 21 MPa from day 4 to day 47 ip-level = 0.00146), therefore K reduction in NG10 was the result of both swelling and degradation simultaneously, although its swelling data showed a plateau throughout the swelling experiment (day 4 to day 56). The K values on day 0 and day 47 for PEG2OOOPLADMA/HEMA( 10:90) (NGii) were statistically the same as well (p-level = 0.33), similar to NG7 (FIG. 5D), a 6.6% deswelling was observed for this network, therefore trapped degraded species (lactic acid and PEG2000) not only reversed the osmosis effect but also were entangled inside the network and maintained the modulus. [00151] Comparing NG₁₁ and NG₇ in terms of possessing similar microstructure opens up a whole new topic, which is beyond the focus of our research. The Poisson’s ratio is defined as the ratio of transverse strain (e_t) to longitudinal strain (ei) (Eq 11), therefore when an isotropic polymer network swells, its behavior shifts toward incompressible rubber (v

0.5) with smaller longitudinal strain, yet any conclusion about changes in v for this case needs more pieces of information such as shear modulus or young’s modulus (Eq 3).

[00153] Keeping the primary crosslinker concentration constant at 10 mol% while PEG length increased from 600 to 2000 g/mol, decreased the dry compressive modulus from NG₉ to NG₁₁ (AK_dry = 140.7 MPa) t /7-level = 0.0013), although K_dry did not statistically changed for 50 mol% crosslinker while PEG length changed. This result shows that functional group density reduction of primary crosslinker has more detrimental effect on compressive modulus when primary crosslinker concentration is lower. Furthermore, in nanogels with PEG2000PLADMA as the primary crosslinker, when concentration increased from 10 to 50 mol%, dry compressive modulus increased about 50%. This implies that when the crosslinker is longer due to longer PEG, increasing the primary crosslink density is more dominant in increasing the mechanical property than the adverse effect of secondary crosslink density reduction. The maximum difference between dry and wet modulus was observed for PEG₂oooPLADMA/HEMA(50:50) (NG₁₂), which had the highest equilibrium water uptake, also its average K value on day 47 was 10.8 MPa lower than that of on day 4 with marginal p- level of 0.0499. The decrease in K indicates that M_x has increased due to hydrolytic degradation (shown by swelling data as well).

EXAMPLE 17

Externally Degradable Networks

[00154] Referring to FIGS. 7A and 7B, graphs show compressive modulus (K) vs. time for externally degradable nanogel networks (NG13-NG16). In this example of an externally degradable nanogel network, the compressive modulus when dry is between about 100 MPa to about 250 MPa. The graph in FIG. 7A shows increasing the primary crosslinker (PEG600DMA) concentration from 10 to 50 mol% (decreased the dry compressive modulus (DK _{|ΐ n} = 113 MPa). This result confirms the effects of higher secondary crosslinking density and degree of entanglements in nanogels with lower concentration of primary crosslinker. The K_SWoiien from day 4 until the end of experiment (day 47) for PEG600DM A/HEM APL A/MEM A( 10:45:45) (NG13) (/7-level = 0.38) and PEG«x>DMA/HEMAPLA/MEMA(50:25:25) (NGw) (/7-level = 0.15) remained the same, therefore it may be concluded that the cleaved chains during this time were not able to change the integrity of the network due to the fact that cleaved nanogel particles in the bulk are trapped and not able to leave the network unless their neighboring nanogels have already dissociated themselves from the surface. The same result was also observed for PEG₂oooDMA/HEMAPLA/MEMA(10:45:45) (NGis) and PEG2000DM A/E1EM APLA/MEM A(50:25:25) (NGie). A graph 700 in FIG. 7B shows that when PEG length is larger (2000 g/mol), increasing the concentration of primary crosslinker, considerably increases the dry modulus.

[00155] The maximum difference between dry and swollen modulus was related to the networks with highest amount of water uptake, (PEG₂oooDMA/HEMAPLA/MEMA(50:25:25) (NGie) and

PEG₆ooDMA/HEMAPLA/MEMA(50:25:25)) (NGM), they also had the lowest K_SWoiien from day 4 until the end. The final K values of NGI₂ and NG_½, were compared and a 21.9 MPa difference (/7-level = 0.04) was observed with former having higher modulus, which indicates that cleavage of secondary crosslinks has more detrimental effect on decreasing the modulus compared to cleavage of primary crosslinks. This also indicates that most effective crosslinks including the entanglements to absorb elastic energy lies between the nanogels not within their core, which goes back to the concept of decreased heterogeneity in crosslinked nanogels due to absence of sol fraction, loops, and dangling nanogels. An important trend was revealed upon comparison of the final K values of internally degradable and externally degradable systems. This trend shows that compressive modulus of externally degradable networks on day 47 was significantly lower than final K values of internally degradable gels in the following fashion, the DK between NG9-NG13, NGio-NGw, NGn-NGis, and NGI₂-NGI₆ were 73.8 MPa (p- level = 0.0033), 104.3 MPa (/7-level = 0.00068), 13.7 MPa (/7-level = 0.017), and 29.0 MPa (/7-level = 0.00025), respectively. These results indicate: i) the most effective crosslinks including entanglements lie between the nanogels not in their cores, ii) the compressive modulus reduces more when the primary crosslinker concentration increases, or when the secondary crosslinker concentration decreases, which confirms the fact that entanglements in the overlapped volume contribute to modulus more effectively when the secondary crosslinking density is lower, iii) K reduction at constant crosslinker concentration is higher when PEG is shorter, and iv) K reduction is higher when concentration of secondary crosslinker is lower (the same as (ii)). The reason for conclusion (iii) is due to higher functional density of the crosslinker when PEG is shorter, and increased heterogeneity of crosslinks distribution.

EXAMPLE 18

Internally-Externally Degradable Networks

[00156] Referring to FIGS. 8A and 8B, graphs show compressive modulus (K) vs. time for intemally-externally degradable nanogel networks. In this example of an internally - extemally degradable nanogel network, the compressive modulus when dry is between about 50 MPa to about 150 MPa, which is a lower range than that for the externally degradable nanogel network. The internally-extemally networks had an overall lower compressive modulus compared to other three groups, as a result of increased flexibility and average mesh size, due to the addition of PLA (Clapper, 2007) to both primary and secondary crosslinkers. The maximum difference between dry and swollen modulus belonged to the network with the highest amount of water uptake (PEG₂oooPLADMA/HEMAPLA/MEMA(50:25:25)) (NG20) and faster degradation rate with no mechanical integrity after day 4. Looking back to equilibrium swelling results, this is the same hydrogel that turned into pieces after weighing on day 12 (FIG. 4). The compressive modulus results indicate that the network had started losing its effective crosslinks long before day 12. There was 70 MPa difference between K*_n (day 0) and Kswoiien (day 4) in PEG₆ooPLADMA/HEMAPLA/MEMA(50:25:25) (NGis), the K_SWoiien was statistically the same on day 4 and day 47 (p-level = 0.16). This is the network with declining water content. The degraded mass was still trapped in the gel and contributed to entanglement and deswelling. The K value of

PEG₆ooPLADMA/HEMAPLA/MEMA(l 0:45:45) did not change from day 0 to day 4 and onwards. This network had the lowest initial swelling as shown in the swelling observation in FIG. 4, yet water uptake linearly increased until the end of experiment. The degraded species were not able to leave the network due to tight secondary crosslinks. Now based on K evolution, M_x and consequently packing (Poisson’s ratio) have not changed in this network, which indicates no significant hydrolytic bond cleavage has happened in this network during swelling. The fact that water concentration is a crucial factor in defining hydrolysis reaction rate manifests itself in NG17 modulus plot. EXAMPLE 19

Non-Degradable Networks

[00157] The M_x values obtained from compressive modulus measurements and using Eq 4 for dry samples and Eq 5 for swollen samples were compared. There was an increase in M_x on day 3 for all the nanogels in this category. The lower M_x values associated with dry hydrogels is due to higher modulus in dry state, and it is related to the crosslinking density of each network and also entanglement effect in overlapped volume between nanogels. Crosslinking permanently traps the overlapped volume, which particularly behaves similar to trapped entanglements (temporary physical crosslinks) between single chains at low secondary crosslinking density and enhances the modulus. After swelling, polymer-polymer interactions in the overlapped regions are replaced by polymer-water (good solvent) interactions, as a result the equilibrium swollen modulus has much lower value compared to dry modulus even though M_x has not changed.

[00158] Referring to FIGS. 9A and 9B, graphs 900 show molecular weight between crosslinks (M_x) vs. time for non-degradable nanogel networks; M_x calculated based on Rubber Elasticity Theory (Eq 4 and Eq 5) and Gaussian distribution assumption for polymer chain conformations. FIG. 9 A and 9B show graphs illustrating molecular weight between crosslinks (M_x) vs. time for non-degradable nanogel networks; M_x calculated based on Rubber Elasticity Theory (Eq 4 and Eq 5) and Gaussian distribution assumption for polymer chain conformations. DM_C between dry and wet state is larger for networks with lower secondary crosslink density and higher concentration of more hydrophilic segments. Higher hydrophilicity increases swelling, as a result polymer chains in the overlapped area become more disentangled. At higher concentration of secondary crosslinking, the effect of overlapping on increasing the modulus was diminished (similar to the effect of trapped entanglements in high crosslinked chains). PEG₂oooDMA/HEMA(50:50) (NG4) and PEG_2OOODMA/HEMA/MEMA(50:25:25) (NG_S) had highest values of M_x at equilibrium swollen state due to higher degree of swelling, and consequently less polymer-polymer interactions. The M_x values of NG2 on day 3 and day 57 are statistically the same ( -level = 0.066). The average M_x for all the networks remained constant after day 3, due to absence of hydrolytic degradation. All the M_x values were less than 2500 g/mol.

[00159] Referring to FIGS. 10A and 10B, graphs 1000 show molecular weight between crosslinks (M_x) vs. time for non-degradable nanogel networks; M_x calculated based on Equilibrium Swelling Theory (Eq 10) and Gaussian distribution assumption for polymer chain conformations.

[00160] FIGS. 10A and 10B show M_x values evaluated based on swelling equilibrium theory (Eq 10). Since the dry values ofM_x are irrelevant due to lack of fundamental theory supporting Eq 10, only the swollen values ofM_x were considered, which are dependent on f₂ and polymer density (assignedM_x = 0 for dry state). The higher the p_p and/or the lower the f₂ are, the higher the M_x will be. The density of PEG₂oooDMA HEMA(10:90) (NG3) calculated based on dry mass and mold volume had the highest value among all four networks in this plot (FIG. 10A). The higher value of NG3 density compared to that of NG4 contradicts the theoretical expectation, since the higher density monomer (PEG2000) is more concentrated in NG4. The reason behind this discrepancy may be due to non-homogeneous mixture of nanogel and solvent (acetone), which can be the result of solvent-solute incompatibility. In addition, any small changes in f₂ is able to cause a much larger impact on M_x in Eq 10 than in Eq 5, therefore Flory-Rehner equation tends to overestimate theM_x if only f₂ is concerned. The basis of this overestimation is the contribution of free energy of mixing forM_x evaluation in Eq 10. FIG. 10B, on the other hand, shows that theM_x at equilibrium swollen state is higher for PEG_2OOODMA/HEMA/MEMA(50:25 :25) (9000 g/mol) than

PEG2_OOODMA/HEMA MEMA(10:45:45) (4000 g/mol), which was expected. The discrepancy between the values ofM_x based on Eq 5 and Eq 10 may also be related to: i) invalid assumption of Gaussian chain behavior, ii) different dry and swollen Poisson’ s ratios for each network, and iii) different c values for each nanogel-water system. The M_x values in FIG. 10A are all less than 1400 g/mol, but in FIG 10B are less than 7000 g/mol.

EXAMPLE 20

Internally Degradable Networks

[00161] Referring to FIG. 11A and 1 IB, graphs 1100 show molecular weight between crosslinks (M_x ) vs. time for internally degradable nanogel networks based on: (A) Rubber Elasticity Theory, and (B) Equilibrium Swelling; All chains are assumed to possess Gaussian Distribution of Conformations.

[00162] The trends observed for M_x based on compressive modulus data show an initial increase in M_x values from day 0 to 4 in internally degradable networks due to the coupled effect of decreased level of chain entanglements in the overlapped volume (similar to non-degradable networks) and also the cleavage of ester bonds. The greater increase in M_x from dry to swollen state (day 4) belonged to PEG₂oooPLADMA/HEMA(10:90) (NGn) and PEG_2OOOPLADMA/HEMA(50:50) (NG12), which had the higher level of water uptake compared to their PEG600 counterparts. There was no statistical difference between M_x of PEG_6OOPLADMA/HEMA(10:90) (NG9) on day 4 and day 47 (/-level = 0.96). In NG10 network, there was a 64 g/mol difference (/- level = 0.00085) between M_x on day 4 and day 47, with latter having higher value. The final values ofM_x on day 47 were different for each network (/2-level between groups « 0.05), in the following order: PEG₂oooPLADMA/HEMA(50:50) > PEG2000PL ADM A/HEM A( 10:90) > PEG₆ooPLADMA/HEMA(50:50) >

PEG_6OOPLADMA/HEMA(10:90), that follows the exact same order as water uptake values. TheM_x s on day 4 and day 47 for NGn and NG12 were statistically the same with /-level values of 0.093 and 0.069, respectively. Although, there was a marginal difference between compressive modulus of NG12 on day 4 and day 47, yet based on evaluated M_x, it was concluded that there was no significant degradation in this network. All the M_x values in FIG. 11A were less than 1000 g/mol.

[00163] The trends in M_x from equilibrium swelling data followed the same trends observed based on K data except that it predicted much higher values for swollen M_x for networks with higher <|)2S (NGn and NG12) (FIG. 11B). Furthermore, it showed that the M_x increased 868 g/mol for NG12 and decreased 276 g/mol for NGn from day 4 to day 47 with /- levels of 0.007 and 0.02, respectively. The reduction in M_x in PEG2000PL ADM A/HEM A( 10:90) is due to the increase in f₂ during this time (recall from swelling data) and entanglement of degraded species (PEG2000). Also, low crosslinking density in internally degradable nanogels, leads to release of longer chains by only cleaving few crosslinks, therefore released chains spend more time inside the network before they diffuse to the surrounding solvent. The surprising observation was that Eq 8 predicted higherM_x on day 47 for NG12 than on day 4, which is an indication of degradation and mesh size increase, although Eq 5 did not show the same result.

EXAMPLE 21

Externally Degradable Networks

[00164] Referring to FIG. 12A and 12B, graphs 1200 show molecular weight between crosslinks (M_x) vs. time for externally degradable nanogel networks based on: (A) Rubber Elasticity Theory, and (B) Equilibrium Swelling; All chains are assumed to possess Gaussian Distribution of Conformations. [00165] The observation highlights in M_x trends based on rubber elasticity theory (Eq 4-5) in externally degradable networks are as follows: the initial increase in M_x value is a combination of decreased entanglement in the overlapped area along with hydrolytic degradation of PLA linkages surrounding the nanogels. The initial increase in M_x is greater for networks with lower secondary crosslinking densities and higher content of hydrophilic crosslinker, similar to what was observed for non-degradable networks. The M_x values on day 4 and day 47 were statistically the same for PEG₆ooDMA/HEMAPLA/MEMA( 10:45:45) (NG13) (p-level = 0.38), PEG₆ooDMA/HEMAPLA/MEMA(50:25:25) (NGM) (p-level = 0.15), and PEG₂oooDMA HEMAPLA/MEMA( 10:45:45) (NGis) (p-level = 0.32) networks, yet it increased for PEG₂oooDMA/HEMAPLA/MEMA(50:25:25) (NGie) (p-level = 0.04), which is an indication of enlarging secondary mesh size. The NG13 and NG15 both had the lowest M_x value (480 g/mol) (FIG. 12A), with W of 16.7% and 52%, respectively. The highest M_x (4393 g/mol) belonged to NG_½, which had the highest water content (80%).

[00166] The M_x values based on swelling equilibrium (FIG. 12B) were higher for NG₁₆ (7642 g/mol) and NG₁₄ (1725 g/mol), and it remained constant from day 4 until day 47. NGi₃ (66 g/mol) and NG₁₅ (824 g/mol) had the lowest M_x. Eq 10 tends to evaluate relatively larger values for M_x except for NG₁₃.

EXAMPLE 22

Internally-Externally Degradable Networks

[00167] FIGS. 13A, 13B, and 13C illustrate graphs 1300 showing the M_x based on compressive modulus results for this category. Graphs 1300 show molecular weight between crosslinks M_x vs. time for intemally-externally nanogel networks based on: (A-B) Rubber Elasticity Theory, and (C) Equilibrium Swelling; all chains are assumed to possess Gaussian Distribution of Conformations.

[00168] The sharp increase of M_x to 35,700 g/mol in PEG2OOOPLADMA/HEMAPLA/MEMA(50:25 :25) (NG20) is an indication of rapid hydrolytic degradation (FIG. 13A), which was previously predicted based on swelling result as well. The PEG600PLADM A/HEM APLA/MEMA( 10:45:45) (NG₁₇) and

PEG2000PLADM A/HEMAPLA/MEMA( 10:45 :45) (NG₁₉) had slight initial increase in M_x, yet there was no change until the end, also their final M_x values were 396 and 604 g/mol, respectively. The M_x for PEG₆ooPLADMA/HEMAPLA MEMA(50:25:25) (NGis) remained statistically the same on day 4 and day 47 (p-level = 0.24). [00169] A one-order of magnitude lower M_x for NG20 was observed on day 4, evaluated based on swelling data than from compressive modulus data (FIG. 13C), which is surprising. As it was shown in swelling results, the intact swollen mass was measured up until day 12 but then after that on day 15 the gel turned into pieces while trying to measure its weight. This discrepancy between these results as it was predicted before in external compressive modulus analysis section may arise from the fact that majority of effective crosslinks reside in the outer layer of nanogels (secondary crosslinks), where they also share a high level of entanglements, therefore losing simultaneous connectivity from outer and inter layers has detrimental effect on sustaining mechanical integrity, even though the gel keeps swelling without dissolving. This is an evidence for systematic degradation even when the network can no longer store elastic energy. Another conclusion is rubber elasticity seems to be able to more accurately predict the events at molecular level during degradation, when degraded chains are no longer entrapped or entangled inside the network.

EXAMPLE 23

Evaluation of M_v based on Non-Gaussian Chain Assumption

[00170] Based on M_x values of all networks in dry state evaluated by Eq 4, they were all less than 140 g/mol. For Gaussian distribution assumption to be valid, the minimum number of bonds per repeat unit should be N ~ 10 (Hiemenz, 2007), in addition, the minimum number of such units has to be about 100 (Pohorecki, 2010), therefore Gaussian M_x has to be at least equal to or greater than 10 x 14 g/mol x 100 = 14,000 g/mol (molecular weight of the repeat unit C¾ in M_x = 14 g/mol). Another definition of Gaussian chain was introduced by Tobita (1992):“In order for the conformation of the chain to be Gaussian, at least 50 carbon atoms between crosslinking points would be necessary.”

[00171] Based on this statement, M_x should be equal to or greater than 50 x 14 g/mol = 700 g/mol, thus based on either of these cutoffs, the assumption of Gaussian distribution of conformations for the backbone chain segment between crosslinks in nanogel networks seems invalid. To derive a relationship between compressive modulus and M_x in a crosslinked network of non-Gaussian chains, expanded version of Kuhn and Griin distribution function can be used, which the Gaussian result is the first term in the series (Treloar, 1954; Hiemenz, 2007)

(Eq 12-13).

11 , . V₃^

[00173]

175 N 2 ) (0 ) (13)

[00174] Furthermore, by applying Flory-Rehner equation, M_x = 14 iV_x can be determined based on equilibrium swelling data for non-Gaussian network (Treloar, 1954) (Eq 14).

EXAMPLE 24

Non-Degradable Networks

[00176] There was no difference in M_x trends between Gaussian and non-Gaussian assumptions in by rubber elasticity and equilibrium swelling theories for non-degradable networks. Swelling equilibrium theory with Gaussian and non-Gaussian assumptions predicted the same values for M_x (HG. 10A and 10B, and FIG. 15A and 15B), although rubber elasticity predicted lower values for M_x with non-Gaussian assumption compared to Gaussian (FIG. 9A and 9B, and FIG. 14A and 14B).

EXAMPLE 25

Internally Degradable Networks

[00177] The same trends and values were observed for M_x (< 3500 g/mol) based on equilibrium swelling theory with both assumptions, Gaussian and non-Gaussian (FIG. 1 IB and FIG. 16B), although rubber elasticity theory predicted lower M_x, when non-Gaussian assumption was applied (< 500 g/mol) compared to values with Gaussian assumption (< 1000 g/mol) (FIG. 11A and FIG. 16A).

EXAMPLE 26

Externally Degradable Networks

[00178] Once more, rubber elasticity theory predicted lower M_x with non-Gaussian assumption (< 3500 g/mol), as opposed to Gaussian assumption (< 5000 g/mol), also the trends were identical based on both assumptions (FIG. 12A and FIG. 17A). The equilibrium swelling theory predicted the same trends and values with both assumptions (FIG. 12B and FIG. 17B). EXAMPLE 27

Internally-Externally Degradable Nanogel Networks

[00179] In this category, the results followed the same conclusions as the other networks. The rubber elasticity theory one more time predicted lower M_x with non-Gaussian assumption with a factor of 2 in the denominator. The non-Gaussian assumption for short polymer chains is applied when the end-to-end distance of the chain under deformation is approaching its maximum value (N_xb), where chain can no longer extend beyond it. The fact that swelling equilibrium predicted same values for M_x with either assumption indicates that the extent of chain deformation during swelling did not approach the contour length, therefore non-Gaussian treatment was not necessary, although the same argument does not apply to rubber elasticity predictions. Implementing non-Gaussian distribution evaluated lower M_x value for the same compressive modulus, which is a more precise assessment of a macroscopic nanogel network.

EXAMPLE 28

Mass Loss Analysis

[00180] Hydrolytic degradation is a process that starts with water uptake by amorphous regions within the network followed by hydrolytic cleavage of hydrolytically labile bonds (e.g. ester), which finally transforms the polymer into oligomers and monomers (Fu, 2010). For further investigation of macroscopic degradation in a more reasonable time scale, PEG₄₆₀₀ was chosen as crosslinker core segment, a substantially hydrophilic oligomer relative to PEG₆₀₀ and PEG₂ooo in high concentration (50 mol%). The same four categories of nanogel structures: non-degradable, internally degradable, externally degradable, and internally - extemally degradable were prepared with PEG₄₆₀₀ in their backbones. The hydrolysis of hydrophobic PLA segments begins as soon as they become in contact with water at a rate governed by hydrolysis kinetic (Eq 15), (Metters, Polym 2000):

(15)

[00182] In this equation [E] is the concentration of ester bonds, [¾0] is the concentration of water within the swollen network, which can be assumed constant when considering highly swollen gels (volumetric swelling ratio Q_v > 4) (FIGS. 24A-D) (Metters, Polym 2000), and [H⁺] is the concentration of hydronium ion in the surrounding water, which in this case is the hydronium ion concentration associated with DI water plus the acidic species leaching out from the degradable sample (hydrolysis product) during the course of degradation, therefore [H⁺] is not constant, since buffer was not used here.

[00183] Eq 15 can be simplified to Eq 16, a pseudo second-order kinetic rate law, by combining constant values. After integration [E]/[H⁺] was obtained as an exponential function of time (Eq 17) (Rawlings, 2015).

[00185] k' is the new rate constant that has the water concentration value lumped into it, [E]_o and [H⁺]₀ are initial concentrations of ester groups and hydronium ion, respectively. Measuring swollen weights of PEG4600 hydrogel series was not feasible due to their highly swollen character and sticking to Kimwipes during excess water removal from the surface, therefore water uptake data for these hydrogels is not available. By keeping Eq 17 in mind and following the macroscopic behavior of the networks including rate of mass loss, pH of the surrounding water, and also visually following the samples during degradation, may try to explain the microscopic behavior in nanogel networks during degradation.

[00186] Eq 17 indicates: i) [E]/[H⁺] and rate of [E]/[H⁺] reduction decreases with time, and ii) higher [H⁺], increases the rate of [E] reduction due to autocatalysis. Based on this kinetic information, a decreasing trend is expected for mass loss vs. time if degradable macromers (PEGPLADMA, HEMAPLA) were unconnected chains. From the mass loss data (Figure 23) and the pH values during degradation (Table 10), it is evident that mass loss increases with time, and consists of two distinct stages, first stage is from day 0 to 60, and second stage is from day 60 to 90.

EXAMPLE 29

Non-Degradable Networks

[00187] No mass loss and no degradation by-products were predicted for this system due to lack of hydrolytically labile groups. FIG. 19A and 19B show schematic stmctures of non-degradable active nanogel and macroscopic network created by polymerizing overlapped nanogel particles. Oligomeric poly (methacrylate) chains are connected to each other via primary non-degradable crosslinks (PEGDMA) and polymeric poly(methacrylate) chains connect nanogel particles together and create the final network. The pH value of surrounding water remained constant at 4.5 during the 90-day study (Table 10).

EXAMPLE 30

Internally Degradable Networks

[00188] In degradable nanogel based networks, based on the location of the cleavable linkages, different mass loss behaviors were predicted. In internally degradable system, the PLA linkages were attached to both sides of the PEG block, and next to PLA chains on both sides, methacrylate functionalities were covalently attached, which all together assembled the degradable primary crosslink. The oligomeric polymethacrylate chains (non- degradable), were created during nanogel synthesis through the connection of these methacrylate functional groups to active chains, the degradable network was then formed through the connection of these short poly(methacrylate) chains via the degradable crosslinks. In addition, non-degradable side chains (HEMAs) were also attached to the oligomeric poly(methacrylate) chains in a random fashion, where the most accessible ones were post- functionalized with methacrylate groups. In the larger scale the overlapping nanogels were crosslinked during photopolymerization by connecting polymeric poly(metacrylate) chains, which had been originated from reaction between post-nanogel dangling methacrylate groups. At the molecular level, as soon as water (acidic water in this case) is in contact with the hydrolyzable PLA linkages, the acid-catalyzed hydrolytic bond cleavage starts either on one side or both sides of the PEG blocks, followed by the release of PEG units and lactic acid from the interior regions of nanogels, and start diffusing out into the free volume between the nanogels, and ultimately to the surrounding water. The rate of pH reduction in surrounding water is governed by the rate of degradation and the rate of diffusion, on the other hand, the rate of degradation is dictated by Eq 17 and microstructure of the network. The rate of diffusion according to reptation model developed by de Gennes for a single, flexible chain trapped in a permanent network, is dependent on diffusion path length, degree of entanglement, friction factor of each segment, average mesh size, and temperature. When enough crosslinks are cleaved and diffused into the solvent, then the free oligomeric poly(methacrylate) chains with their dangling side chains (or dangling one sided crosslink) that are not connected to polymeric poly(methacrylate) chains start diffusing out, but since their molecular weight is not large due to their short length, this transition should be smooth without a significant effect on the rate of mass loss. Based on Table 10, the pH of water gradually reduced from 4.5 (day 30) to 3.5 (day 90), so it appears that this was accelerated hydrolysis period, which manifested itself in FIG. 23 by increased rate of mass loss after day 60. At later stages of degradation when enough number of nanogels are degraded, then the polymeric poly(methacrylate) chains can release themselves from the network either as a plain chain or with dangling oligomeric poly(methacrylate) chains attached to it. Even under accelerated condition, on day 90 with average mass loss of 92%, 2/3 of samples still had undissolved residual pieces. This observation confirms the systematic degradation and delaying the microscopic reverse gelation (primary burst effect), which is a characteristic of heterogeneous degradable networks, the microscopic reverse gelation in conventional heterogeneous networks starts at 78% mass loss.

[00189] FIGS. 22A-C illustrate (A) Intemally-externally degradable active nanogel; (B) Macroscopic network of polymerized overlapped nanogel; (C) Schematic stmctures of hydrolysis degradation by-products. FIGS. 22A and 22B show the structure of internally degradable active nanogel and the macroscopic network formed by crosslinking the overlapped nanogels. FIG. 22C shows schematic structure of degradation by-products of internally degraded nanogel network. Based on Eq 17, [E]/ [H⁺] ratio decreases with higher rate in an exponential decay regime, when the difference the number in the parenthesis ([H⁺]o - [E]o) becomes larger. The pH of the surrounding solution remained constant at 4.5 from day 0 until day 30 (Table 10). FIG. 23 is a graph of mass loss (%) vs. time in DI water and ambient condition for non-degradable, internally degradable, externally degradable, and intemally- externally degradable nanogel networks of PEG4600 series. FIG. 23 shows a linear mass loss of 14% during this time. The mass loss continued with the same rate until day 60, and then it became much faster with a rate of 1.98% per day due to increased [H⁺]o. As shown in Fig. 23, the linear mass loss of the externally degradable nanogel network NG23 was about 10% over 90 days, showing a slow rate of degradation of the externally degradable nanogel compared to the internally degradable nanogel.

EXAMPLE 31

Externally Degradable Networks

[00190] FIG. 21 shows the structure of externally degradable active nanogel and the macroscopic network formed after photopolymerization. In this system the degradable PLA linkages are attached to HEMA, which are randomly attached to the oligomeric polymethacrylate chains in the nanogel structure, and some of them are also connected to the final network through polymeric poly(methacrylate) chains. In this type of polymer , at early stages of degradation only low molecular weight acidic species are released. There was no observation of significant mass loss from day 0 to day 60. The effect of released acidic molecules manifested itself one more time through decreased pH value of the surrounding water from day 30 to 90, and accelerated hydrolysis effect after day 60. Since other structural elements in the nanogel and the polymeric poly(methacrylate) chains connecting the nanogels are all non-degradable, it was predictable to have a prolonged stable mass until majority of the degradable side chains were cleaved. The network of attached nanogels may be transformed into cleaved nanogels trapped within the secondary network. From this stage onwards, there may be a rise in the rate of mass loss due to losing individual cleaved nanogels from the surface, since the other cleaved nanogels inside the bulk of the samples are still trapped. This stage of erosion is followed by the release of individual polymeric poly(methacrylate) chains free of dangling nanogels and this cycle continues systematically until the entire mass is eroded (surface erosion). Final pH on day 90 was 4.0, which is higher than that of internally degradable samples on this day, which is due to less PLA content. This gear shifting in degradation behavior from bulk erosion to surface erosion happened only by changing the location of the labile linkages and keeping other parameters the same. In this type of network, the primary and secondary reverse gelations may be completely by-passed and the entire sample is eroded layer by layer.

EXAMPLE 32

Internallv-Externallv Degradable Networks

[00191] In this system internal and external degradable linkages are cleaved simultaneously, initially releasing lactic acid and PEG segments followed by oligomeric poly(methacrylate) chains and further the release of plain polymeric poly (methacrylate) chains, as shown in FIG. 22C. The rate of mass loss in internally-extemally degradable samples followed the same trend as internally degradable gels, with accelerated hydrolysis effect showing after day 60, and one out of three samples had still residual pieces. The final conclusion is that the overall mass loss rate is governed by internal degradability rather than external. The pH of water remained constant at 4.5 from day 0 until day 30 and decreased to 3.0 (Table 10) between day 30 and 90. The higher [H⁺] on day 90 in this case is due to higher concentration of PLA in internally-extemally degradable nanogels. The average mass loss on day 90 was 85%, and there was no statistical difference between the final mass loss of NG22 and NG24 ip- level = 0.68). [00192] Table 10 discloses pH values of surrounding water solution during hydrolysis of PEG4600 hydrogel series; t=0 shows pH value of neat DI water.

TABLE 10

pH

PH PH pH

Time Internally-

Non- Internally Externally

Externally

[Day] Degradable Degradable Degradable

Degradable

Network Network Network

Network

0 4.5 4.5 4.5 4.5

2 4.5 4.5 4.5 4.5

30 4.5 4.5 4.5 4.5

90 4.5 3.5 4 3

EXAMPLE 33

Visual Analysis

[00193] The degradation process for 20 days in all four nanogel networks in PEG4600 series was observed. The photopolymerized discs had white color before being submerged in water, which is an indication of semi-crystallinity and it is due to micro scale alignment of PEG4600 segments (the critical lowest molecular weight for PEG to crystallize in crosslinked networks is 1000) (Qiao, 2004). In all samples the diameter of the discs almost doubled after spending 1 day in DI water and the swollen network had opaque (white) color. In non- degradable samples, the swollen gel lost a slight degree of opacity due to water penetration into the network but overall stayed white during the 20 days of observation. In internally degradable case, the samples became transparent after spending 20 days in DI water, which is an indication of increased mesh size that allows more water penetrating into the bulk and as a result less scattering. The addition of PLA to both sides of the PEG increases the crosslinker length and consequently opens up the primary mesh size. In externally degradable networks, the samples were initially opaque but the opacity started to slightly decrease after day 1 and remained the same until day 20, which confirms that mesh size does not increase further and sample lose mass by free nanogels leaving the network surface. In internally-extemally degradable samples, opacity decreased dramatically between day 1 and day 7, which shows a larger mesh size created. Between day 7 and day 20 the sample was extremely swollen, had a macroscopic burst effect, and it turned into pieces while trying to take it out of the vial.

EXAMPLE 34

Biocompatibilitv of Model Nanogel

[00194] In one example, internally degradable nanogel PEG_46OOPLADMA/HEMA/MEMA(50:25:25) (NG22) was selected for a cell biocompatibility study due to its intermediate content of PLA compared to externally and internally-extemally degradable nanogels of the PEG4600 series. The cytocompatibility of NG22 at different concentrations in cell culture media was evaluated by the direct contact test with a monolayer of L929 mouse fibroblast cells according to ISO standards (ISO 10993-5, 1999). Briefly, L929 cells were sub-cultured and seeded into six-well tissue culture plates at a concentration of 50,000 cells per well. L929 cells were incubated for 24 h at 37 °C in 5% carbon dioxide atmosphere. Dry nanogel powder at a concentration of 10 ug/mL, 20 ugmL, 50 ug/mL, 100 ug/mL or 200ug/mL was added and the cells were incubated for another 48 h. L929 cells were examined microscopically for cellular response using a phase contrast inverted microscope (Leica, WLD MPS32, Germany). The morphology of the L929 cells was assessed in comparison with a control (media only).

[00195] The L929 mouse fibroblast cells after 48 h of treatment were analyzed for changes in their spindle morphology and adherence to the culture plate. The results demonstrate there was no change in the spindle shape of cells cultured with up to 50 pg/mL of nanogel and cells strongly adhered to the culture plate. The cell morphology was comparable to that of the control. However, at higher nanogel concentrations, the cells showed signs of toxic effects, with some loss of the spherical morphology and an increase in detachment from the culture plate. L929 cells treated with 100 pg/mL nanogel concentration appear mildly affected and at 200 pg/mL, the nanogel was severely toxic to L929 mouse fibroblast cells.

EXAMPLE 35

Nanogel for Slow-Release Drug Delivery

[00196] In one example, a nanogel disclosed herein may be used for slow-release delivery of a small hydrophobic drug. To achieve desired nanogel properties for slow-release drug delivery, an externally degradable nanogel may be used. In this example, the selected nanogel has a hydrophobic core in contact with the hydrophobic drug molecules, and a hydrophilic outer layer in contact with the surrounding water. This exemplary nanogel is amphiphilic, with the direction of amphiphilicity from a hydrophobic core to a hydrophilic outer layer.

[00197] For the exemplary slow-release delivery nanogel, a nonpolar monomer is selected for side chains (mono vinyl monomer) of the nanogel and a hydrolytically labile linkage (PLA) is used at the point of the attachment of the side chain to the nanogel structure. The hydrophobic side chains including the hydrophobic labile linkage align themselves towards the core (away from surrounding water). The semi-hydrophilic nature of the crosslinker (which, in this example, determines the overall amphiphilic property of the nanogel) controls the rate of water diffusion. Subsequently, a layer by layer degradation of the side chains dictates the slow release of the drug cargo.

[00198] The above specification, examples, and data provide a complete description of the structure, features and use of exemplary implementations of the invention. Since many implementations of the invention can be made without departing from the spirit and scope of the invention, the invention resides in the claims hereinafter appended. Furthermore, structural features of the different implementations may be combined in yet another implementation without departing from the recited claims.

Claims

WHAT IS CLAIMED IS:

1. A nanogel comprising:

at least one mono vinyl monomer;

at least one divinyl monomer;

a chain transfer agent; and

an initiator;

wherein the at least one mono vinyl monomer comprises a degradable linkage, the degradable linkage positioned at a location within the nanogel selected to control at least one of a rate and a route of degradation of the nanogel.

2. The nanogel of claim 1, wherein the at least one mono vinyl monomer forms a side chain and the at least one di vinyl monomer forms a crosslinker.

3. The nanogel of claim 2, wherein the degradable linkage is positioned at a point of attachment of the at least one mono vinyl monomer side chain to the nanogel.

4. The nanogel of any one of claim 1 to claim 3, wherein the degradable linkage includes a hydrolytically labile linkage.

5. The nanogel of any one of claim 1 to claim 3, wherein the degradable linkage comprises polylactide (PLA).

6. The nanogel of any one of claim 1 to claim 3, wherein the at least one divinyl monomer comprises an other degradable linkage.

7. The nanogel of claim any one of claim 1 to claim 3, wherein the at least one divinyl monomer is selected from polyethylene dimethacrylate (PEGDMA) and polyethylene glycol-co- polylactide dimethacrylate (PEGPLADMA).

8. The nanogel of any one of claim 1 to claim 3, wherein a cargo molecule is bound to the nanogel and controlling at least one of the rate and the route of degradation results in controlled release of the cargo.

9. The nanogel of any one of claim 1 to claim 3, wherein the at least one mono vinyl monomer includes an external degradable moiety on a surface of the nanogel.

10. The nanogel of any one of claim 1 to claim 3, wherein the at least one mono vinyl monomer is selected from 2-methoxyethyl methacrylate (MEMA) and 2-hydroxyethyl methacrylate (HEMA).

11. The nanogel of any one of claim 1 to claim 3, wherein the at least one mono vinyl monomer is a degradable copolymer of HEMA and polylactide (HEMAPLA).

12. The nanogel of claim 2, comprising a hydrophobic core in contact with a hydrophobic cargo molecule, wherein degradation of the at least one mono vinyl monomer side chain via the degradable linkage controls a release of the hydrophobic cargo molecule.

13. The nanogel of any one of claim 1 to claim 3 or claim 12, wherein the nanogel has a weight- averaged molecular weight less than 65 kg/mol.

14. The nanogel of claim 2, wherein the crosslinker is present at a concentration of from about 10.0 mol to about 90.0 mol .

15. The nanogel of claim 2, wherein the side-chain is present at a concentration of from about 10.0 mol to about 90.0 mol .

16. The nanogel of claim 1, wherein the nanogel has glass transition temperature of about -45 °C to about 25 °C.

17. The nanogel of claim 6, wherein the nanogel has a glass transition temperature of about - 45 °C to about 5 °C.

18. The nanogel of any one of claim 1, 2, 3, 12, 14, 15, or 16, wherein the nanogel has equilibrium swelling of about 5.0 wt% to about 90.0 wt%.

19. The nanogel of claim 1, wherein the nanogel comprises an externally degradable nanogel network having a compressive modulus when dry that is between about 100 MPa to about 250 MPa.

20. The nanogel of claim 6, wherein the nanogel comprises an internally-extemally degradable nanogel network having a compressive modulus when dry that is between about 50 MPa to about 150 MPa.

21. The nanogel of any of claim 1, 2, 3, 12, 14, 15, 16, or 19, wherein the route of degradation begins at a surface of the nanogel where the degradable linkage is positioned.

22. The nanogel of claim 1, wherein the rate of degradation produces a mass loss of about 10% over 90 days.

23. A method of synthesizing a nanogel comprising:

combining at least a di- vinyl monomer, a mono-vinyl monomer, an initiator, and a chain transfer agent to form a mixture, wherein at least one of the di-vinyl monomer and mono-vinyl monomer comprises a degradable linkage; and

initiating a free -radical polymerization reaction of the mixture to form the nanogel, wherein the degradable linkage is selectively positioned within the nanogel to achieve a desired degradation rate of the nanogel.

24. The method of claim 23, wherein a drag is bound to the nanogel, the degradable linkage is positioned on an external surface of the nanogel, and the desired degradation rate is useful for slow-release delivery of the dmg.

25. The method of any one of claim 23 to claim 24, wherein the mono-vinyl monomer comprises the degradable linkage.

26. The method of claim 25, wherein the mono-vinyl monomer forms a side-chain of the nanogel.

27. The method of any one of claim 23 to claim 24, wherein the di- vinyl monomer forms a crosslinker of the nanogel and comprises the degradable linkage.

28. The method of claim 25, wherein the di-vinyl monomer comprises an other degradable linkage, the degradable linkage is positioned in a side-chain of the nanogel and the other degradable linkage is positioned in a crosslinker of the nanogel.

29. The method of any one of claim 23 to claim 24, wherein the degradable linkage is selectively positioned within the nanogel to achieve a desired hydrophilicity or hydrophobicity of the nanogel.

30. The method of any one of claim 23 to claim 24, wherein the degradable linkage is selectively positioned within the nanogel to achieve either bulk or surface degradation of the nanogel.

31. The method of claim 26, comprising degrading the nanogel in water, wherein a pH of a surrounding aqueous medium changes from about 4.5 to about 4.0.

32. The method of claim 28, comprising degrading the nanogel in water, wherein a pH of a surrounding aqueous medium changes from about 4.5 to about 3.0.

33. The method of any one of claim 23 to claim 24, comprising degrading the nanogel by adjusting at least one of pH, temperature, and light.