Biodegradable Cationic Nanogels with Tunable Size, Swelling and pK_a for Drug Delivery

Abstract

Cationic polymers have garnered significant interest for their utility in intracellular drug delivery and gene therapy. However, due to their associated toxicities, novel synthesis approaches must be explored to develop materials that are biocompatible. The novel library of nanoparticles synthesized in this study exhibit tunable hydrodynamic diameters, composition and pH-responsive properties as a function of synthesis parameters. In addition, differences in the responsiveness of these nanoparticles under different pH conditions affords greater control over intracellular drug release.

1. Introduction

Nanomaterial size, surface charge, stealth-coating density, and responsive properties all play a major role in drug delivery performance. The ideal material parameters vary for disease targets.(Chacko et al., 2012; Petros and Desimone, 2010) Cationic nanomaterials have received considerable interest over the past several years because of their ability to transport fragile or cytotoxic therapeutic molecules into cells.(Blanco et al., 2015; Prabhu et al., 2015; Spencer et al., 2015; Suk et al., 2016; Whitehead et al., 2009) However, these nanomaterials with cationic surface charges have been correlated with increased levels of toxicity.(Fröhlich, 2012) As such, a general strategy to designing cationic nanomaterials is to have sufficient charge shielding or unionized species at the pH of the circulation (~7.4), and charge deshielding or ionization at the site of action. For example, acid labile PEG chains have been utilized to deshield nanoparticles.(Ko et al., 2016; Li et al., 2013) Chemically crosslinked nanogels are excellent delivery candidates and offer advantages including their resistance to degradation and protection of therapeutic cargo.(Chacko et al., 2012) The ability to tune size, charge, pK_a, and responsive properties allows for the rapid optimization of these nanomaterials for intracellular drug delivery.(Allen and Cullis, 2013; Kanasty et al., 2013)

The screening of large libraries of nanomaterials materials have elucidated valuable structure function relationships.(Akinc et al., 2008; Alabi et al., 2013; Green et al., 2008; Whitehead et al., 2014, 2012) For example, for a library of lipid nanoparticles, siRNA entrapment, pK_a, extracellular cellular stability, intracellular stability, uptake, hemolysis, and in vitro gene silencing were correlated to in vivo gene expression.(Alabi et al., 2013) The movement toward methodologies to predict performance based on material properties and computational methods could allow for the rapid generation of other non-lipid based high performing materials, such as the cationic nanomaterials utilized in this study.

To achieve this goal, an atom transfer radical polymerization (ATRP) synthesis approach is explored. Advances in atom transfer radical polymerization (ATRP) have reduced the amounts of transition metal needed to successfully conduct a controlled polymerization.(Matyjaszewski et al., 2006) Activators regenerated by electron transfer (ARGET) ATRP which is less sensitive to oxygen than other ATRP methods, allows the technique to be utilized in the presence of limited amounts of air, and can utilized to synthesize many formulations in parallel.(Jakubowski and Matyjaszewski, 2006; Matyjaszewski, 2012; Matyjaszewski et al., 2006)

Our laboratory has published on the development of cationic nanogels with responsive properties for drug delivery applications.(Fisher et al., 2009; Fisher and Peppas, 2009; Forbes et al., 2013; Forbes and Peppas, 2013; Knipe et al., 2014; Liechty et al., 2013) The goal of this work was to evaluate the major parameters that influence nanomaterial responsive properties. These include nanogel hydrodynamic size, nanogel pK_a, nanogel volume swelling ratio, and the pH range over which volume swelling occurs. In addition, we present the optimization of key reaction parameters that can be used to tailor nanomaterials size.

2. Materials and Methods

2.1. Materials

Poly(ethylene glycol) methyl ether methacrylate solution, with PEG with nominal Mn 2000 in 50 wt% water solution (PEGMA2k), 2-(diethylamino)ethyl methacrylate (DEAEMA), 2-(diisopropylamino)ethyl methacrylate (DPAEMA), methyl methacrylate (MMA), tert-butyl methacrylate (tBMA), benzyl methacrylate (BnMA), cyclohexyl methacrylate (CHMA), (trimethylsilyl)methacrylate (TMS-MA), 2-(Trimethylsilyloxy)ethyl methacrylate (TMS-HEMA), bis(2-methacryloyl)oxyethyl disulfide (DSDMA), myristyl trimethylammonium bromide (MyTAb), ethyl α-bromoisobutyrate (EBiB), L-ascorbic acid (AA), 6-(p-Toluidino)-2-naphthalenesulfonic acid sodium salt (TNS), dimethyl sulfoxide-d6 (DMSO-d6) and triethylamine were purchased from Sigma-Aldrich. Tris(2-pyrdiylmethyl)amine and hexyl methacrylate (HMA) were purchased from TCI America. Brij 30, Brij 35, copper (II)bromide (99+ %), acetone-d6 (99.8 % D), and deuterium oxide (99.8 % D) were purchased from Acros Organics. Tris(2-carboxylethyl)phosphine (TCEP) was purchased from EMD Millipore. Tetrahydrofuran, acetone, and hydrochloric acid were purchased from Fisher Scientific. All chemicals were used as received.

2.2. Nanogel synthesis and purification

Nanogels were synthesized via activators regenerated by electron transfer (ARGET) ATRP polymerization in an oil-in-water emulsion polymerization adapted from Forbes et al. (Forbes et al., 2013). The reactions were conducted in 2 mL batch sizes to enable the synthesis and screening of multiple formulations simultaneously as previously described (Spencer et al., 2018). Briefly, reactants were emulsified using a 24-tip microtip sonicator probe and the polymerizations were initiated by the addition of excess reducing agent in an inert environment. The reactions were stopped via exposure of the vials to air and addition of hydrochloric acid to a final concentration of 0.5N. Resulting nanogels were purified by the ionomer collapse method as described previously.(Fisher and Peppas, 2009). The nanogels were subsequently dialyzed against ultrapure deionized water for three days. Following dialysis samples were lyophilized and prepared for additional characterization.

2.3. Spectroscopic characterization

Nanogel composition was estimated using 1H Nuclear Magnetic Resonance (NMR) Spectroscopy. Spectra were obtained using a 400 MHz NMR (Varian Direct Drive 400 or Agilent MR 400) at 25°C. To determine composition, lyophilized nanogels were diluted in D₂O or DMSO-d6 at 10 – 15 mg/mL and disulfide crosslinks were degraded via addition of tris(2-carboxylethyl)phosphine to a final concentration of 10mM for 24 hours prior to acquisition. Spectra were analyzed on MestReNova 10.0 software.

Nanoparticle hydrodynamic diameters were measured using a Malvern ZetaSizer NanoZS with a MPT-2 multipurpose titrator. Nanogel samples were suspended at 0.5 mg/mL in 1x PBS and adjusted to pH 4.0. Hydrodynamic diameter was measured in triplicate at the initial pH and in increments of 0.3 up to pH 8.5. The titrator adjusted pH using 0.1 N NaOH, 0.1 N HCl, or 1 N HCl as needed. Each measurement was the average of a minimum of 12 tens second acquisitions.

2.4. 2-(p-toluidinyl) naphthalene-6-sulfonic acid (TNS) Assay

Nanoparticle solution (5 μL, 0.5mg/mL) and 10 μl of TNS at 120 μM in DMSO was added to 90μl of 150mM phosphate buffer at varying pH values in a 96-well microplate. Fluorescence intensity was measured at wavelengths of 325nm (excitation) and 425nm (emission) with a Biotek Cytation 3 Plate Reader. The pH value at which half of the maximum fluorescence intensity is obtained was determined to be the pK_a of the sample.

3. Results and Discussion

3.1. Nanogel Synthesis

Three sets of nanogels were synthesized to investigate the effects of cationic monomer hydrophobicity, comonomer content, and crosslinking density on the responsive properties of the nanogels (Table 1). In set 1, the cationic monomer DEAEMA was exchanged with DPAEMA, a cationic monomer with increased hydrophobicity, at five ratios (0, 25, 50, 75, or 100 mol%). The five formulations were termed DP 0, DP 25, DP 50, DP 75, and DP 100 based on the mol% of DPAEMA incorporated during synthesis. In each case, the organic phase composition was held at a 3:1 ratio of cationic monomer to other uncharged comonomers. In set 2, nanogels were synthesized with increasing fractions of an uncharged comonomer. In this series, five ratios of DEAEMA to tBMA were investigated in which the organic phase was composed of either 0, 12.5, 25, 37.5, or 50 mol% tBMA with the remainder being DEAEMA. In set 3, the crosslinking density of the nanogels was modulated. In this series, crosslinking densities of 0.31, 0.63, 1.25, 2.5 and 5.0 mol% of DSDMA with respect to the total amount of monomer in the organic phase were utilized. All reactions were polymerized with a 100 mM concentration of PEGMA2k in the aqueous phase and were reacted using the heterogeneous ARGET ATRP emulsion polymerization under optimal conditions based on previous reports.(Forbes et al., 2013; Spencer et al., 2018) The chemical structures of the monomers used to attain the various nanogel formulations are shown in Figure 1.

Table 1.

Synthesis of nanogels with increasing ratios of DPAEMA. tBMA and DSD.V1A in the feed.

	Name	DEAEMAa	DPAEMAa	tBMAa	DSDMAb
Set 1: Cationic & Hydrophobic Comonomer	DP 0	75	0	25	1.25
	DP 25	56.2	18.8	25	1.25
	DP 50	37.5	37.5	25	1.25
	DP 75	18.8	56.2	25	1.25
	DP 100	0	75	25	1.25
Set 2: Hydrophobic Comonomer	tBMA 0	100	0	0	1.25
	tBMA	87.5	0	12.5	1.25
	12.5
	tBMA 25	75	0	25	1.25
	tBMA	62.5	0	37.5	1.25
	37.5
	tBMA 50	50	0	50	1.25
Set 3: Crosslinking Density	DSDMA	75	0	25	0.31
	0.31
	DSDMA	75	0	25	0.63
	0.63
	DSDMA	75	0	25	1.25
	1.25
	DSDMA	75	0	25	2.5
	2.5
	DSDMA	75	0	25	5
	5.0

Fig. 1.

Reagents used in the synthesis of cationic nanogels.

3.2. Nanogel Composition

Proton NMR spectroscopy was utilized to assess nanogel composition. Non-destructive techniques to assess nanogel composition such as ATR-FTIR did not provide adequate resolution. Similarly, ¹H NMR on the crosslinked nanogels risks underestimating hydrophobic content due to poor solvation of the nanogel core. As such, the nanogel disulfide crosslinks were degraded under acidic conditions with TCEP in deuterium oxide to dissolve the linear polymer fragments.

3.2.1. Composition of nanogels with DEAEMA/DPAEMA

Proton NMR spectroscopy on degraded nanogels was utilized to assess the ratio of cationic monomers (Figure S1) and to estimate overall composition. Proton NMR spectroscopy peaks of DEAEMA (δ 3.14, 4H) and DPAEMA (δ 3.64, 2H) demonstrate that cationic monomers were copolymerized at five different ratios. The DPAEMA fraction of cationic monomer incorporated into the nanogel was estimated to be 0, 14.6, 26.9, 42.7, and 67.4 mol % for DP 0, DP 25, DP 50, DP 75, and DP 100 respectively. The copolymerization ratio can also be observed at 1.1 – 1.3 ppm, DEAEMA (δ 1.18, 6H) and DPAEMA (δ 1.22, 12H), but is harder to distinguish due to overlapping peaks and the terminal methyl group proton peaks of tBMA (δ 1.27, 9H).

3.2.2. Composition of nanogels with increasing tBMA feed content

Composition was estimated using ¹H NMR based on a representative peak for DEAEMA (δ 1.18, 6H), tBMA (δ 1.27, 9H), and PEGMA2k (δ 3.55, 176H) as shown in Figure S2. Nanogel compositions are shown in Figure 2. Notably, the composition of tBMA increased with increasing tBMA feed content in the tBMA 0, tBMA 12.5, tBMA 25 and tBMA 37.5 formulations.

Fig. 2.

Composition of nanogels synthesized with varying rations of DPAEMA, tBMA and DSDMA in the feed. Analysis was performed using proton NMR spectroscopy.

The tBMA 50 formulation appeared to have a decreased ratio of tBMA with respect to DEAEMA (see Figure S2). This result was suspected to be a result of poor solvation of the tBMA 50 formulation in water. Upon analysis of the degraded tBMA 50 formulation in DMSO-d6 the molar ratio of tBMA to DEAEMA was higher indicating poor solvation or self-assembly that resulted in an apparent decrease in tBMA content (see Figure S3).

3.2.3. Composition of nanogels with increasing crosslinking density

The nanogels in set 3 were synthesized with a disulfide crosslinking agent, DSDMA, with increasing mol fraction (0.31 – 5 mol %) with respect to organic phase monomer. Proton NMR of the crosslinking series (Figure S4) demonstrated increasing PEG (δ 3.55, 176H) content, while the ratio of DEAEMA (δ 1.18, 6H) to tBMA (δ 1.27, 9H) remained constant (Figure 2). This set of reactions indicated that the small changes to the organic phase can have dramatic effects on the nature of the polymerization.

The composition of nanogels followed feed ratios for the organic phase monomers for all three sets of formulations. However, the degree of PEGMA2k incorporated into nanogels increased with increasing the hydrophobicity of the organic phase monomers (hydrophobic comonomer or crosslinker). The observation of increased PEG content in nanogels with an increasing hydrophobic organic phase gives additional insight into tuning the degree of PEG on the surface of nanogels for specific formulations as a function of the composition of the organic phase of the polymerization.

3.3. Nanogel hydrodynamic size and critical swelling pH

The cationic nanogels described here are intended to be used as intracellular drug delivery vehicles. Based on previous work in the field, a successful intracellular carrier must be able to circulate in the bloodstream in the presence of serum proteins, reach the target site in the body, be endocytosed by cells and reach the cytosol for the delivery of therapeutic cargo. The use of poly(electrolyte) nanogels in this work was designed such that nanogels would have a collapsed conformation at physiological pH and a swollen conformation at slightly acidic pH values that are commonly associated with tumor tissues.(Zhang et al., 2010) To test the responsive nature of the nanogels, hydrodynamic size as a function of pH was determined using dynamic light scattering in 1x PBS. The tertiary amines on the cationic nanogels ionize as a function of pH and give rise to pH dependent swelling. In this section, size and swelling behavior was related to cationic monomer, comonomer content and the degree of crosslinking.

3.3.1. Critical swelling pH and cationic monomer

Increasing the hydrophobicity of the pendant group on the cationic monomer shifted the nanogel pK_a to lower values. The tertiary amine methacrylate monomers 2-(dimethylamino)ethyl methacrylate, 2-(diethylamino)ethyl methacrylate, and 2-(diisopropylamino)ethyl methacrylate have been investigated for drug delivery applications due to pK_a values near physiological pH. (Agarwal et al., 2012; Amalvy et al., 2004; Lee et al., 2013; Peng et al., 2010; Plamper et al., 2007)

In set 1, the hydrophobicity of the cationic monomer was increased by substituting a fraction of the DEAEMA with DPAEMA in nanogels copolymerized with tBMA and PEGMA2k. In Figure 3A the hydrodynamic size of the nanogels as a function of pH for the set of formulations is shown. Nanogels exhibit a volume swelling transition dependent on the hydrophobicity of the cationic monomer in the nanogels.

Fig. 3.

Dynamic light scattering as a function of pH for A) formulations with increasing ratios of DPAEMA to DEAEMA. Symbols designate DP 100 (●), DP 75 (♦), DP 50 (▴), DP 25 (■), and DP 0 (×); B) formulations with increasing ratios of tBMA to DEAEMA. Symbols designate tBMA 0 (●), tBMA 12.5 (♦), tBMA 25 (▴), tBMA 37.5 (■), and tBMA 50 (×); and C) formulations with increasing crosslinking densities of DSDMA. Symbols designate DSDMA 0.31 (●), DSDMA 0.63 (♦), DSDMA 1.25 (▴), DSDMA 2.5 (■), and DSDMA 5.0 (×).

To quantify the critical swelling pH for each formulation, a logistic curve was fit to each pH-dependent hydrodynamic size profile with four parameters, Equation 1.

$$D_h=\frac{A-D}{1+\text{exp}\left(B*\left(pH-C\right)\right)}+D$$

This fit was specifically chosen because the four parameters in Equation 1 corresponded to material properties of specific interest to nanoparticle function. Parameter A is the swollen hydrodynamic diameter in 1x PBS, B describes the slope of the swelling transition, C is the critical swelling pH, and D is the collapsed hydrodynamic diameter in 1x PBS. Least squares regression was utilized to determine the best fit parameters for each formulation. Data points at pH values greater than the critical swelling pH for formulations that demonstrated aggregation were not included in the sigmoidal curve fitting. The fitting parameters for each formulation are compiled in Table S1.

The swelling transition shifts to lower pH values with increasing copolymerization ratios of the more hydrophobic comonomer (7.10, 7.02, 6.67, 6.54, and 6.30 for DP 100, DP 75, DP 50, DE 25, and DE 0 respectively). Nanogels were approximately 130 nm swollen and 75 nm collapsed and did not trend with cationic monomer feed ratio. However, as the fraction of DPAEMA and the hydrophobicity of the formulations increased, nanogels in the collapsed state began to aggregate. In the collapsed state nanogels are primarily stabilized by the degree of surface PEGylation. However, for the DP 100, DP 75, and DP 50 formulations, the increased hydrophobic content could not be stabilized by the PEGMA2k in the nanogel. Increasing alkyl chain length on a tertiary amine will result in a weaker base due to the steric hindrance associated with the attack of the proton. For these nanogels, increasing DPAMEA content results in more steric hindrance of the amine, thereby shifting pK_a to lower values.

Importantly, the use of the more hydrophobic tertiary amine resulted in the ability to shift critical swelling response while maintaining a similar width of the volume swelling transition range. Copolymerizaiton of nanogels with DPAEMA is a way to shift pK_a while maintaining sharp pH responsiveness of the nanomaterial. However, there is a limit to the amount of DPAEMA that can be copolymerized for nanogels intended for intravenous delivery due to the tradeoff with colloidal stability at neutral pH values.

3.3.2. Critical Swelling pH and hydrophobic monomer content

The pH dependent hydrodynamic size of nanogels with increasing concentrations of hydrophobic comonomer was determined with dynamic light scattering (Figure 3B). The tBMA 0, tBMA 12.5, and tBMA 25 formulations were colloidally stable across the pH range tested in 1x PBS. The tBMA 37.5 and tBMA 50 formulations tailed toward higher hydrodynamic diameters and large polydispersity indices above pH 7.6 and 7.3 respectively, characteristic of aggregation. This aggregation is a consequence of the higher tBMA composition in the hydrogel network and is observed as a deviation from the swelling behavior predicted by Equation 1 (Figure 3B). Analogous to the trend observed for increased DPAEMA content in set 1, there is a tradeoff with the amount of hydrophobic comonomer that can be stabilized by the PEG content in the nanogels. Increasing the hydrophobic comonomer content in the nanogels resulted in a shift in the critical swelling pH from 7.76 to 6.55. The range of the swelling transition broadened with increasing tBMA content from a range of about 0.4 pH units to 2.0 pH units for the tBMA 0 and tBMA 50 formulations respectively This phenomenon can be described by a combination of increased polymer-polymer interactions and the reduction of ionizable groups in the polymer network. Increasing the hydrophobic content of the nanogels reduces solvation of the network and results in increased steric hindrance. Additionally, the decrease in ionizable functional groups reduces the osmotic pressure gradient of salts diffusing into the nanogel network. The combination of these factors results in the need for lower pH values to achieve swelling.(Liechty et al., 2013; Siegel and Firestone, 1988; van de Wetering et al., 1999) Overall, these data demonstrate how hydrophobic comonomers can be incorporated into nanogel networks to shift critical swelling pH and control the range of pH responsive behavior.

3.3.3. Critical swelling pH and crosslinking density

Dynamic light scattering as a function of pH was used to measure volume swelling for the formulations with different degrees of crosslinking. The volume swelling ratio of the nanogels was calculated as the fraction of the swollen hydrodynamic diameter (pH 4.0) relative to the collapsed hydrodynamic diameter (pH 8.0) to the power of 3. Nanogel volume swelling ratio ranged from 250% for the most crosslinked formulation (DSDMA 5.0) to 740 % for the least crosslinked formulation (DSDMA 0.31). Notably, the critical swelling pH was consistent among formulations with different crosslinking agent feed ratios. This indicates that volume swelling can be tuned independently of critical swelling pH (Figure 3C), Table S1.

For intracellular drug delivery of nanomaterials, efficient endosomal escape is important for therapeutic efficacy. Two of the proposed mechanisms are membrane destabilization and the proton sponge effect. Liposomes are commonly reported to disrupt endosomes by the membrane destabilization method while polymeric materials such as polyethylenimine (PEI) are reported to be primarily driven by the proton sponge mechanism.(Evans et al., 2013) As such, a nanogel characteristic that is likely to have a major impact on endosomal disruption ability is the degree of volume swelling. The protonation of the tertiary amine in the nanogels will result is a large osmotic pressure gradient of salts. Larger degrees of volume swelling could be associated with larger gradients and result in more potent endosomal disruption.

The three sets of nanogels detailed here allow for control over nanogel pH responsive swelling properties. Cationic monomer hydrophobicity was utilized to shift the critical swelling pH to lower values, the addition of increased amounts of hydrophobic comonomers broadens the pH dependent swelling transition, and decreasing crosslinking density allows for control over volume swelling with limited impact on the critical swelling pH.

3.3.4. Critical Swelling pH and hydrophilic monomer content

Hydrophobic functional groups are readily compatible with the emulsion polymerization utilized herein, and copolymerization with increasing hydrophobic content shifts the swelling onset to lower pH values. As such, copolymerization of nanogels with hydrophilic functional groups should shift the critical swelling pH to higher values. However, the use of emulsion polymerization limits the hydrophilicity of monomers that be effectively copolymerized into the core of the nanogel. Notably, the use of hydrophilic monomers in emulsion polymerization commonly results in microscale polymer networks or polymerization of the monomer in the aqueous phase. One approach to incorporating hydrophilic groups into the core of the nanogel is to utilize hydrophobic protecting groups. Here, hydroxyl ethyl methacrylate protected with a trimethylsilyl group was copolymerized with nanogels in partial amounts with tBMA, to test the ability to shift pK_a to higher values (Table S2). The trimethylsilyl protecting group was removed during nanogel purification in presence of 0.5 N HCl.

Dynamic light scattering was utilized to measure hydrodynamic diameter as a function of pH for the formulations with increasing feed ratios of TMS-HEMA. Figure 4 shows a shift in critical swelling pH for formulations with higher substitution of the protected monomer. Through this approach, a variety of monomers can be effectively incorporated into nanogels including hydroxyl, carboxyl, and primary amine functionalities and resulting nanogels can have enhanced diversity for drug delivery applications.

Fig. 4.

Dynamic light scattering as a function of pH for nanogel formulations polymerized with increasing degrees of substitution of TMS-HEMA for tBMA. Symbols designate 0% OH (●), 20% OH (▴), and 40% OH (■).

3.4. TNS Assay for determining pK_a

The determination of critical swelling pH based upon dynamic light scattering is both time and labor intensive. As such, an alternative method to determine nanogel pK_a that was amenable to analyzing multiple formulations simultaneously was desired. In the literature, a microwell plate based pK_a assay based upon TNS has been utilized to determine pK_a for lipid nanoparticles.(Alabi et al., 2013) This assay uses an anionic fluorophore that interacts with cationic charges. As such, minimal fluorescence was observed above the pK_a when nanogels were in the uncharged state and fluorescence reached a maximum when cationic charges and hydrophobic interactions dominate.

Here, the TNS assay was utilized to estimate the pK_a of nanogels and was compared to the critical swelling pH determined from dynamic light scattering as a function of pH (Figure 5). The TNS assay captured the same trend as observed from pH-dependent DLS, and only demonstrated minor deviations from the critical swelling pH. The TNS represents a method for rapid determination of pK_a and allows for direct comparison among formulations. For the TNS assay, all formulations were compared in a single assay in a matter of hours. However, for DLS, the generation of each curve required several hours and was subject to day to day variations in pH monitoring.

Fig. 5.

Comparison of the critical swelling pH determined by dynamic light scattering (●) and pKa determined by TNS assay (♢).

The most notable difference elucidated by the TNS assay was a trend toward lower pK_a values for formulations with increased crosslinking densities. This trend is consistent with the trends observed for increasing the hydrophobicity of the nanogel networks.

Furthermore, it is important to note that TNS fluorescence reached a maximum and then began to decrease as pH decreased. This result is attributed to the increased hydrophilicity of the nanogels at low pH. For hydrophilic networks, this could limit the utility of the TNS assay. However, the trends observed here demonstrate that the TNS assay provides an excellent first estimate of nanogel pK_a.

3.5. Synthesis of a library of formulations

A small library of formulations were polymerized simultaneously utilizing six comonomers, two PEGMA2k feed concentrations and two crosslinking ratios (Table 2). The monomer feed ratios were selected to give nanogels with responsive properties that spanned the physiologically relevant region for drug delivery applications. Nanogel properties were analyzed by the TNS assay and dynamic light scattering.

Table 2.

Polymerization of nanogels with varying comonomers, crosslinking densities, and PEGMA2k feed concentrations.

Entry	[PEG]	% DSDMA	Comonomcr
1	60	1	DPAEMA
2	90	1	DPAEMA
3	60	2.5	DPAEMA
4	90	2.5	DPAEMA
5	60	1	MMA
6	90	1	MMA
7	60	2.5	MMA
8	90	2.5	MMA
9	60	1	tBMA
10	90	1	tBMA
11	60	2.5	tBMA
12	90	2.5	tBMA
13	60	1	BnMA
14	90	1	BnMA
15	60	2.5	BnMA
16	90	2.5	BnMA
17	60	1	CHMA
18	90	1	CHMA
19	60	2.5	CHMA
20	90	2.5	CHMA
21	60	1	HMA
22	90	1	HMA
23	60	2.5	HMA
24	90	2.5	HMA

The pK_a of the nanogels decreased with increasing hydrophobicity of comonomers (Figure 6A). This trend matches that of increasing hydrophobicity associated with increasing ratios of hydrophobic comonomers and hydrophobic cationic monomer. A slight decrease in pK_a was also observed with an increase in crosslinking density which is consistent with the earlier observation.

Fig. 6.

Nanogel pKa determined by the TNS assay (A) and hydrodynamic diameter of nanogels at swollen (pH 4.0) (○) and collapsed pH (pH 7.5) (●) by dynamic light scattering (B).

Dynamic light scattering was used to determine hydrodynamic sizes of the nanoparticles at swollen (pH 4) and physiological pH (pH 7.4) (Figure 6B). It is important to note that as a result, the nanogels copolymerized with methyl methacrylate may not have assumed a completely collapsed conformation under the physiological pH conditions. A few formulations exhibited larger hydrodynamic diameters at the physiological pH compared to the low pH condition. These results were attributed to aggregation. As expected, formulations polymerized with lower concentrations of crosslinker displayed larger swollen diameters.

Six formulations were further analyzed for pH dependent hydrodynamic size with dynamic light scattering. The formulations were synthesized with 2.5% DSDMA and 90 mM PEG monomer and were copolymerized with tBMA, BnMa, CHMA, HMA, and DPAEMA (Figure 7). The methyl methacrylate formulations aggregated over the pH range and was not included. The critical swelling pH followed the results of the TNS assay with DPAEMA > tBMA > BnMA ~ CHMA > HMA. Furthermore, the DPAEMA had the narrowest volume swelling transition range. The volume swelling transition range increased and the maximum volume swelling decreased with an increase in side chain hydropobicity.

Fig. 7.

Dynamic light scattering as a function of pH for formulations with different comonomers. Symbols designate tBMA (●), BnMA (♦), CHMA (▴), HMA (■), and DPAEMA (×).

The formulations described in this paper demonstrate how nanogel responsive properties can be tailored using monomer selection and feed ratios. However, nanogels can also be tuned using a breadth of reaction parameters. Properties of specific interest for drug delivery applications include size and polydispersity. As such, a series of polymerizations focused on the parameters that influence nanogel size and polydispersity index were evaluated.

3.6. Tailoring Nanogel Size and polydispersity

The ability to tune nanomaterials size is paramount in achieving partitioning into tissues of interest and maintaining the desired circulation half-life. Previously we have demonstrated the ability to tune PEG surface density.(Spencer et al., 2018) A multitude of reaction parameters can be varied to adjust nanomaterials properties in miniemulsion polymerization including surfactant concentration and structure, temperature, pressure, pH, ratio of continuous and dispersed phases, viscosity, and organic phase composition.

3.6.1. Effect of Organic Phase volume % on nanogel size

The effect of monomer loading in the polyermization was evaluated by varying the volume fraction of the organic phase of the emulsion polymerization between 2.5 and 7.5 vol %. Here, reactions were conducted and nanogel size was measured at a swollen (pH 4) and collpased (pH 8) state (Figure 8). Lowering the organic phase volume from 7.5 to 2.5% resulted in a decrease in the swollen hydrodyanmic diameter from 143 to 62 nm. In addition, the collapsed hydrodyamic diameter decreased slightly with with decreasing volume fraction of the organic phase. Decreasing the loading of monomer in the polymerization provides a straightforward way to reduce particle size. However, the tradeoff is in the nanogel yielded per volume of the reaction.

Fig. 8.

Hydrodynamic diameter of nanogels at swollen (pH 4.0) (○) and collapsed pH (pH 7.5) (●) as determined by dynamic light scattering for a series of nanogels polymerized with increasing organic phase volume percent.

3.6.2. Hexadecane as a Costabilizer to Control Nanogel Size

The use of a small fraction of costabillizer in miniemulsion has been utilized to prevent Ostwald ripening. Here n-hexadecane was substituted for a fraction of the organic phase volume to investgate the effect of the costabilizer on the nanogels formed in this emulsion scheme. A range of n-hexadecane volumes between 0 and 20 % of the organic phase was tested. The organic phase volume was kept constant at 7.5 vol %.

The swollen and collapsed diameters of nanogels were measured with dynamic light scattering. (Figure 9). Low percentages of n-hexadecane (< 4%) resulted in nanogels with larger collapsed sizes and higher polydispersity. However, as the volume fraction of hexadecane increased form 4 – 20 vol%, nanogels decreased in both swollen and collapsed hydrodynamic size and maintained very low polydispersity indices. The use of a costabilizer represents an additional tool for optimizing size.

Fig. 9.

Hydrodynamic diameter and polydispersity index of nanogels polymerized with increasing ratios of hexadecane in the organic phase as determined by dynamic light scattering.

4. Conclusions

In this work, the ability to finely tune pH responsive properties of nanogels was described. In summary: 1) Increasing the hydrophobicity of monomers shifts pK_a to lower pH values 2) increasing the ratio of non-responsive monomers to ionizable monomer broadens the swelling transition 3) Decreasing crosslinking agent feed ratios increases the degree of volume swelling. Furthermore, increasingly hydrophobic monomers can be utilized to lower pK_a while maintaining the steepness of the swelling transition and protected hydrophilic monomers can be used in the emulsion and then deprotected to increase the critical swelling pH.

The effect of initiator concentration, the use of a costabilizer, and organic phase volume on the size of nanogels was demonstrated. Nanogel swollen size decreased substantially with lower organic phase volumes and the use of a costabilizer at 5 – 15 % of the organic phase decreased polydispersity index and nanogel size.

Overall, this work related material properties to nanogel function to allow for the predictable synthesis of nanogels with desired size, swelling, and pH responsive properties.