Poly(ε-caprolactone-b-ethylene oxide) (PCL_6kDa-b-PEO_5kDa) and poly(styrene-b-ethylene oxide) (PS_9.5kDa-b-PEO_18.0kDa) amphiphiles with carboxylic acid termination were purchased from Polymer Source (Montreal, Canada). Dexamethasone (DEX) (Cat No. BML-EI126-0001) was purchased from Enzo Life Sciences. Coumarin 6 dye (Cat No. 442631), Suberoylanilide hydroxamic acid (SAHA, also known as Vorinostat, Cat No. SML 0061), Lutein (Xanthophyll, Cat No. X6250), tetrahydrofuran (THF, Cat No. 401757), and dimethyl sulfoxide (DMSO, Cat No. 276855) were purchased from Sigma Aldrich. Hamilton™ metal hub blunt point needles (27 gauge, 410 µm o.d.; 210 µm i.d.) and glass Luer lock syringes (1 ml) were purchased from Fisher Scientific. Polytetrafluoroethylene heat shrink tubing (30 gauge, 0.006″ wall, shrink ratio 2:1) was purchased from Component Supply company (Lakeland, FL). For centrifugal filtration, Amicon Ultra centrifugal filter units (pore size: 100 kDa MWCO, Cat No. UFC910024) were purchased from Fisher Scientific.

In these experiments, we investigated the size distribution of empty micelles and BCP nanocarriers synthesized via EM-NP versus batch sonication. Scalable nanoprecipitation processes have been shown to yield smaller, more monodisperse nanoparticles than their batch counterparts (Lim et al., 2014) ¹ . We also studied the effect of drug encapsulation on resultant nanocarrier sizes. A fixed molar ratio of drug to mass of polymer was employed, yielding polymer:drug molar ratios of 0.18 and 0.072 for PCL-b-PEO and PS-b-PEO, respectively. These low ratios were employed as they represent high drug concentrations relative to polymer, useful to elucidate EE differences between drug molecular structures. To reduce variation resulting from solvent effects, THF was primarily employed with the exception of SAHA, which is only sparingly soluble in THF. For SAHA experiments, a THF/DMSO co-solvent system was employed, which also permitted preliminary investigation of the role of solvent selection in size and EE. For comparing processes, the same organic phases were employed, but mixing was provided by EHD flow or probe sonication for EM-NP and sonication, respectively.

First, empty PS-b-PEO and PCL-b-PEO control micelles were synthesized, evaluated via TEM, and their particle size distributions determined (Supplementary Figures S1, S2; Supplementary Table S2). For both BCPs, empty micelles synthesized via EM-NP had mean Feret lengths, L F , of ∼ 20 nm in THF, consistent with our prior results (Lee et al., 2019); however, micelles with PS BCPs were statistically smaller than those with PCL BCPs. Micelles synthesized in 1:1 THF:DMSO (i.e., used for SAHA synthesis) were ∼30 nm, which was statistically larger than those synthesized in THF alone (p < 0.0001). Polydispersity ranged from 0.13 to 0.23, with higher values in mixed solvent vs. pure THF (Supplementary Table S2). Nanoparticle size is determined by the ratio of mixing time (τ_mix) to BCP aggregation time (τ_agg). If τ _agg < τ_mix mass transfer limitations result in regional inhomogeneities that can broaden PSDs and lead to larger NPs (Johnson and Prud'homme, 2003a). When τ _agg > τ_mix a homogenous kinetics regime is obtained in which NP size is no longer correlated to mixing rate and uniform concentration profiles are obtained. NP size and polydispersity are minimized. DMSO is more viscous than THF at the concentrations employed, which would reduce mixing intensity and could explain the larger size and polydispersity observed here. Alternatively, DMSO could alter the magnitude of BCP supersaturation. According to δ_H and δ_tot HSPs (Supplementary Table S1), DMSO has lower compatibility (i.e., greater separation in values) than THF for the hydrophobic BCP blocks, which would increase BCP supersaturation compared to THF alone. Increased supersaturation lowers τ_agg, which would facilitate non-homogenous concentration profiles under identical mixing conditions. Both likely contribute to the observed increase in size and dispersity in mixed solvent.

Comparing samples synthesized using sonication to EM-NP, empty micelles synthesized via sonication in THF were larger (p < 0.0001) (Supplementary Figures S2A,C) with mean L F of ∼ 46 nm for PS-b-PEO and ∼ 35 nm for PCL-b-PEO and had larger polydispersities, 0.32 and 0.19 for each polymer, respectively, than EM-NP synthesis (Supplementary Table S2). These results suggest that τ_mix was lower in EM-NP and that a homogeneous kinetics regime with minimized size and polydispersity could be achieved. Interestingly, in sonication, micelles synthesized with PS-b-PEO were larger and more polydisperse than those synthesized using PCL-b-PEO, although size differences between these polymers could not be detected in EM-NP. Larger size can result from regional differences in concentration, particularly when τ _agg < τ_mix, and δ_H and δ_tot indicate less compatibility of PS blocks with the aqueous phase than PCL blocks (Table 1). Thus, it is possible that larger size resulted from increased supersaturation combined with insufficient mixing. It is also possible that low particle sizes in EM-NP result from kinetic trapping of metastable structures (Thanh et al., 2014; Johnson and Prud'homme, 2003b), whereas slower formation in sonication may permit greater access to thermodynamically favored structures. Sizes for empty micelles synthesized in TMF:DMSO mixed solvent via sonication could not be obtained because complex morphologies not indicative of micelles or NPs resulted (Supplementary Figures S2B,D). This further supports observations in EM-NP that DMSO either increases τ_mix through increased viscosity or decreases τ_agg by reducing hydrophobic BCP block compatibility.

Next, nanocarriers containing drug were synthesized via both processes and compared. In EM-NP, drug carrier sizes were statistically different for carriers synthesized with PS vs. PCL blocks (Figure 3; Supplementary Figure S3; Supplementary Table S3). Carriers were larger than empty micelles, except for DEX in PCL-b-PEO and SAHA in PS-b-PEO (Supplementary Table S2). Increased size with encapsulant loading can be attributed to nucleation of encapsulant aggregates that are stabilized by BCP absorption (Johnson and Prud'homme, 2003a; Nabar et al., 2018a). Size differences were also observed across drug types (Supplementary Table S3), with nanocarrier sizes generally increasing as coumarin 6 < DEX < lutein/SAHA. Coumarin 6 and DEX have similar, steroidal chemical structures (Figure 2) that are more compact than lutein and SAHA, which contain long aliphatic regions. Unfortunately, SAHA is not fully soluble in THF and was synthesized with DMSO as a co-solvent. As DMSO increases empty micelle sizes (Supplementary Table S2), comparison of the two nanocarriers is not appropriate and size correlation to aliphatic content was not attempted.

Comparing EM-NP nanocarrier sizes to those synthesized via sonication (Figures 3 vs 4, Supplementary Figures 3 vs 4, Supplementary Table S3), coumarin 6 and DEX displayed similar trends to those observed in empty micelles (Supplementary Table S2), with sizes increased for sonication processes compared to EM-NP (p < 0.001). However, polydispersities were relatively similar for both processes. Lutein and SAHA displayed no correlation between synthesis process and size. However, there was a correlation with polymer type; PCL-b-PEO lutein sonication samples were smaller than EM-NP samples (and vice versa for PS-b-PEO) (p < 0.001). Polydispersities between both processes also remained similar or showed no correlation. These results underscore the complexity of nanocarrier formation processes, which depend on several time scales, including τ_agg, τ_drug (hydrophobic drug aggregation times), and drug-polymer association times. τ_agg and τ_drug must be larger than τ_mix to ensure operation in the homogenous kinetics regime (Johnson and Prud'homme, 2003b). Additionally, drug-polymer association times will dictate whether nanocarriers form by drug-polymer association and then BCP aggregation or via drug aggregation followed by drug-polymer association. The latter can lead to significantly larger nanocarriers whose size is dictated by the drug aggregate size (Nabar et al., 2018b). The timescales τ_drug and τ_agg can be manipulated by changing the degree of drug and polymer supersaturation, respectively, to control encapsulation processes (Mahajan and Kirwan, 1994).

Next, we evaluated drug EE for each carrier type synthesized via EM-NP and sonication (Figure 5, Supplementary Table S4). In EM-NP, PS-b-PEO generally displayed lower or statistically equivalent EE performance compared to PCL-b-PEO BCPs. It has been hypothesized that EE is driven largely by δ_H compatibility (Zhang et al., 2009), and the PS BCP block displays no hydrogen bonding capacity (i.e., δ _H = 0), which may explain its poorer performance. Remarkably, very high encapsulation efficiencies were observed for lutein and SAHA, which both contain long aliphatic regions (Figure 2), whereas minimal encapsulation in either polymer was observed for steroidal coumarin 6 and DEX compounds. These differences were abrogated in sonication synthesis, although PCL generally outperformed PS as observed in EM-NP. Lutein and SAHA PCL-b-PEO nanocarriers synthesized via EM-NP, but not sonication, also demonstrated significant size increases compared to coumarin 6 and DEX carriers (Supplementary Table S3). Previous studies have indicated an association between increased EE and larger NP size (Dai et al., 2015). These observations suggest a potential formation mechanism in which drug aggregates are passivated by PCL-b-PEO adsorption. The long aliphatic regions of lutein and SAHA likely promote slower aggregation (higher τ_drug) than that of steroidal compounds, which may stabilize rapidly through π-π stacking interactions (Akbulut et al., 2009). Growing aggregates can be stabilized by PCL adsorption through favorable drug-polymer interactions and/or slower τ_agg. With its low hydrogen bonding capacity, PS-b-PEO is unable to adequately stabilize growing aggregates of either type, resulting in its poor EE. This result suggests that the insufficient mixing observed in sonication processes, and the corresponding failure to achieve the homogenous kinetics regime, prevented efficient encapsulation of aliphatic hydrophobic compounds by sonication. Because of the complexity of EM-NP process, it is difficult to determine its associated τ_mix. In addition, determining τ_drug for each compound is beyond the scope of this work. However, these results suggest that EM-NP can enhance encapsulation efficiency, particularly for aliphatic compounds, by enhancing mixing.

Next, we compared performance of EM-NP to FNP processes in a micromixer [e.g., similar to (Ranadive et al., 2019; Johnson and Prud'homme, 2003b)] using a jet-mixing reactor (JMR) previously employed for silver nanoparticle synthesis (Holunga et al., 2005). The EM-NP system operates in semi-batch, whereas the JMR is a continuous flow system. Thus, these experiments assess the effect of semi-batch versus continuous operation. EM-NP operates at low organic:aqueous ratios, which decrease NP size in the homogenous kinetics regime and can potentially influence EE (Miladi et al., 2015). Thus, we utilized FNP conditions that matched organic:aqueous ratios of EM-NP at the conclusion of spray. These experiments were performed using coumarin 6 as a model encapsulant at drug: polymer ratios of 1.45 for PS-b-PEO and 3.64 for PCL-b-PEO. These ratios were much higher than those employed above and were increased to maximize EE for comparison to FNP systems.

Results for both processes were similar across polymer types with no statistical differences detected across any group (i.e., polymers or synthesis methods) (Figure 6, Supplementary Tables S5–S7). This encouraging result suggests similarity between these two processes. In EM-NP, EHD mixing is achieved in a large aqueous sink, whereas JMR achieves mixing in a confined cross-flow geometry. Even though the EM-NP reactor is ∼20x larger than the JMR, EHD mixing is rapid enough to produce NPs with high EE. Similarly, despite semi-continuous operation in which BCP and encapsulant supersaturation profiles are changing with time, results were not distinguishable from those obtained in the JMR. In FNP, relatively constant supersaturation is achieved after initial start-up, and here EM-NP was performed using short spray times approximating steady state conditions. It is unclear how longer EM-NP operation would influence products. Further refinement of EM-NP into a fully continuous process could reduce these disparities.

In addition to synthesis method (i.e., mixing effects), several variables affect the EE of hydrophobic compounds in polymer NPs, including the size of hydrophobic core, polymer-encapsulant, and polymer-solvent/anti-solvent compatibility (Liu et al., 2006). In this study, the collapsed hydrophobic core sizes were relative similar (i.e., R_g of PS and PCL in water estimated at ∼1.182 nm (Drenscko and Loverde, 2017) and ∼1.183 nm [Di Pasquale et al., 2014), respectively], thus differences in polymer, drug, and solvent compatibility would be expected to dominate any observed differences. Enhanced drug solubility in a polymer carrier can result from several different chemical interactions, but hydrogen bonding is the most frequently reported for hydrophobic drug carriers (Zhang et al., 2009). Additionally, solubility can be enhanced when drugs minimize unfavorable interactions with polymer, for example, by self-associate through π-π stacking (Akbulut et al., 2009). To understand the role of molecular compatibility, we evaluated EE in the context of several theoretical constructs, specifically HSPs (Table 1), including δ_tot and δ_H, Hansen solubility spheres (Ra) (Table 2), and Flory Huggins interaction parameters ( χ s p ) (Table 3). HSPs include components for dispersive δ_D, polar δ_P, and hydrogen bonding δ_H forces for each compound. HSPs that are similar and low Ra and χ s p values indicate greater compatibility. We evaluated correlations between our observed EE trends for each of these models across our four drug and two BCP panel for nanocarriers synthesized via EM-NP. EE results obtained through sonication were omitted from this analysis as EE might be impacted by poor mixing efficiency, whereas JMR results were incomplete.

In the prior experiments, EE was evaluated for a fixed polymer:drug molar ratio, selected to be low to enable differences between drug, polymer, and solvent interactions to be assessed. However, it is recognized that increasing drug concentration in the organic solution can dramatically enhance EE. Thus, we next evaluated the effect of increasing polymer:drug molar ratio on EE using coumarin 6 as a model drug. In these experiments, coumarin 6 concentration was held constant at 0.1 µmol, whereas polymer concentration was varied to generate different molar ratios ranging from 0.05–7.28 for PCL and 0.02–2.91 for PS (Figure 7; Supplementary Tables S6, S7). For comparison, values of 0.18 and 0.072 were used in our prior EM-NP vs. sonication experiments, and values of 3.64 and 1.45 were used for our EM-NP vs. FNP experiments, for PCL and PS, respectively.

First, we note that in the absence of polymer stabilizer (i.e., polymer:drug ratio = 0), coumarin-6 aggregates form that are sufficiently large to be entirely removed during purification steps. Thus, BCP is required for drug stabilization. At low polymer to coumarin 6 ratios of ∼0.02–0.09 (i.e., drug in excess), EE was very low (i.e., from 0.3–4%). However, EE steadily increased to a saturation value at polymer:drug ratios of ∼3.6 for PCL-b-PEO (EE: 51.3 ± 8.49%) and ∼1.5 for PS-b-PEO (EE: 43.9 ± 7.14%), representing a breakpoint. After this point, higher polymer:drug ratios did not yield a statistically significant increase in EE.

This response resembles a Langmuir adsorption processes, further supporting a formation mechanism in which drug aggregates are stabilized by BCP adsorption. As the drug concentration in these experiments is fixed, τ_drug should remain relatively constant (i.e., baring changes in solution viscosity), whereas drug-polymer association time and τ_agg would be decreased at increasing ratio [i.e., higher polymer concentration and supersaturation (Johnson and Prud’homme, 2003)]. At low polymer:drug ratios, insufficient polymer is present to stabilize the growing drug aggregate, which will eventually precipitate from solution. We observed this phenomenon during centrifugal purification of these samples, with large losses on the filter membrane filters. As polymer:drug ratio increases, polymer chains are increasingly able to stabilize and ultimately saturate the coumarin 6 aggregate surface. At the breakpoint, the growth of coumarin 6 aggregates is quenched by adsorption of BCPs on aggregate surface, representing a condition in which τ_drug and τ_agg are approximately equivalent (Johnson and Prud'homme, 2003b).

Both polymers exhibited similar EE maxima, although as predicted by compatibility factors, PCL was slightly higher than PS (∼51 vs. ∼44%). However, at these high polymer:drug ratios, differences between polymer-drug compatibility were largely minimized, enabling high EE to be obtained. This encouraging result suggests greater latitude in nanocarrier BCP selection is possible if high polymer concentrations are employed relative to drug. However, although similar EE maxima were observed, the breakpoints at which these behaviors occurred differed. τ_agg is reduced with increasing BCP hydrophobicity (i.e., PS or PCL) (Johnson, 2003), and is dependent on molecular weight of the hydrophobic block. Since the molecular weight of the PS BCP block was ∼1.5 times that of the PCL block and PS has less compatibility with the water anti-solvent (i.e., greater differences in δ_tot and δ_H), PS-b-PEO τ_agg will be lower, consistent with its breakpoint positioned at a lower polymer:drug ratio. Thus, whereas high and relatively equivalent EE can be obtained for either polymer, this occurs at a lower polymer:drug ratio for more hydrophobic polymers. However, result should be interpreted with some caution as different polymer:drug ratios were employed for PS and PCL studies. Further experiments at different ratios would enhance these findings.

Because increased drug loading can also affect NP size (Johnson and Prud'homme, 2003a; Nabar et al., 2018a), we also characterized NP sizes at the Langmuir polymer:drug molar ratio breakpoints (i.e., 1.45 for PS-b-PEO and 3.64 for PCL-b-PEO) (Figures 7C,D; Table 4). For PS BCPs, nanocarrier sizes were statistically larger to those observed at low polymer:drug ratio (Supplementary Table S3), whereas for PCL BCP nanocarriers sizes at the break point were slightly smaller (p < 0.001). This may result from faster stabilization of drug aggregates limiting their size. However, despite these encouraging results, we did observe a greater number of worm-like structures (Figures 7E,F), particular for PS BCPs. Thus, morphology should be considered in optimizing polymer:drug molar ratios. It is likely that an optimum occurs near the breakpoint in which high EE with spherical morphology can be obtained. We also note that although a particular polymer: drug ratio may be employed during synthesis, the nanocarriers produced can be concentrated or diluted as needed. Drug release and toxicity behaviors would have to be further explored at the concentration of intended use.