**3. Results**

### *3.1. Fabrication, Characterization, and Stability Assessment of Poly(Lactic Acid) iNPs* **3. Results**

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 6 of 19

iNPs were prepared using PLA by the single emulsion-solvent evaporation method (Figure 1A) with two surfactants—PVA or PEMA. The iNPs produced were similar in size with diameters between 400–600 nm (Figure 1B) with low polydispersity indices (PDI) (Figure 1C). In contrast to size, the zeta potentials of iNPs were significantly different, where PLA-PVA were approximately −17 mV and PLA-PEMA were approximately −40 mV (Figure 1D). We performed additional studies aimed to determine the stability of iNPs following reconstitution in deionized water over 8 h under various storage temperatures [26]. Both PLA-PVA and PLA-PEMA showed less than 10% change in size (Figure 1E). Similarly, the zeta potential of iNPs remained stable with less than 10% variability over 8 h (Figure 1F). Both iNP formulations displayed similar stabilities independent of reconstitution and storage at room temperature or refrigeration. *3.1. Fabrication, Characterization, and Stability Assessment of Poly(Lactic Acid) iNPs*  iNPs were prepared using PLA by the single emulsion-solvent evaporation method (Figure 1A) with two surfactants—PVA or PEMA. The iNPs produced were similar in size with diameters between 400–600 nm (Figure 1B) with low polydispersity indices (PDI) (Figure 1C). In contrast to size, the zeta potentials of iNPs were significantly different, where PLA-PVA were approximately -17 mV and PLA-PEMA were approximately -40 mV (Figure 1D). We performed additional studies aimed to determine the stability of iNPs following reconstitution in deionized water over 8 h under various storage temperatures [26]. Both PLA-PVA and PLA-PEMA showed less than 10% change in size (Figure 1E). Similarly, the zeta potential of iNPs remained stable with less than 10% variability over 8 h (Figure 1F). Both iNP formulations displayed similar stabilities independent of reconstitution and storage at room temperature or refrigeration.

**Figure 1.** Physicochemical characterization of the synthesized iNPs. (**A**) Schema of the particle formulations utilized for this study. (**B**) Particle diameters were optimized to be in the range of 400-600 nm with (**C**) polydispersity indices in the range of 0.150-0.250. (**D**) Particles were also standardized across surface charge as represented by ζ potential. Additionally, particle stability following reconstitution in distilled water was determined at room temperature (20 °C) and refrigeration (4 °C) over a course of 8 h to confirm stability of particle size (**E**) and zeta potential (**F**). Schematic in (**A**) created with **Figure 1.** Physicochemical characterization of the synthesized iNPs. (**A**) Schema of the particle formulations utilized for this study. (**B**) Particle diameters were optimized to be in the range of 400–600 nm with (**C**) polydispersity indices in the range of 0.150–0.250. (**D**) Particles were also standardized across surface charge as represented by ζ potential. Additionally, particle stability following reconstitution in distilled water was determined at room temperature (20 ◦C) and refrigeration (4 ◦C) over a course of 8 h to confirm stability of particle size (**E**) and zeta potential (**F**). Schematic in (**A**) created with BioRender. Statistical differences between groups were determined by performing Student's *t*-test. Error bars represent SD. \*\* for *p* ≤ 0.01 and ns = not significantly different (*p* > 0.05).

#### *3.2. PLA iNPs Do Not Sequester PAMPs 3.2. PLA iNPs Do Not Sequester PAMPs*

SD. \*\* for *p* ≤ 0.01 and ns = not significantly different (*p* > 0.05).

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 7 of 19

One possible mechanism for iNP-mediated anti-inflammatory activity is through functioning as a sink to directly bind PAMPs to sequester them away from TLRs expressed on immune cells [10]. To evaluate the possibility of direct interactions between PAMPs and iNPs (Figure 2A), we incubated PLA-PVA or PLA-PEMA with fluorescein (FITC)-labeled LPS or CpG ODN. Following incubation, the samples were centrifuged to pellet the iNPs and the fluorescence intensity of the supernatant was measured. We tested direct iNP interactions with FITC-LPS and FITC-CpG ODN in PBS containing 10% FBS (Figure 2B,C, respectively). Compared to the FITC-LPS or FITC CpG ODN controls (dashed lines), no concentration-dependent reduction in FITC signal was observed for either iNP tested and the FITC signal variation was less than 20% from the control in all cases. These studies established that iNP sequestration of PAMPs is not a major mechanism by which iNPs elicit their inherent anti-inflammatory effects, warranting further investigation to understand if the protective mechanism is driven directly by iNP interaction with the immune cells of interest. One possible mechanism for iNP-mediated anti-inflammatory activity is through functioning as a sink to directly bind PAMPs to sequester them away from TLRs expressed on immune cells [10]. To evaluate the possibility of direct interactions between PAMPs and iNPs (Figure 2A), we incubated PLA-PVA or PLA-PEMA with fluorescein (FITC) labeled LPS or CpG ODN. Following incubation, the samples were centrifuged to pellet the iNPs and the fluorescence intensity of the supernatant was measured. We tested direct iNP interactions with FITC-LPS and FITC-CpG ODN in PBS containing 10% FBS (Figure 2B,C, respectively). Compared to the FITC-LPS or FITC CpG ODN controls (dashed lines), no concentration-dependent reduction in FITC signal was observed for either iNP tested and the FITC signal variation was less than 20% from the control in all cases. These studies established that iNP sequestration of PAMPs is not a major mechanism by which iNPs elicit their inherent anti-inflammatory effects, warranting further investigation to understand if the protective mechanism is driven directly by iNP interaction with the immune cells of interest.

**Figure 2.** Under typical in vitro serum conditions, both particle types fail to sequester FITC-tagged TLR agonists. (**A**) Particles and FITC-conjugated TLR agonists were co-incubated at 37 °C and 5% CO2 for 1 h and then pelleted to determine direct interactions between particles and TLR agonists. When co-incubated with PBS containing 10% FBS, both (**B**) FITC-LPS and (**C**) FITC-CpG ODN fail to interact with particles alone as signified by the dashed line representing 100% FITC signal of FITC-LPS (**B**) or FITC-CpG ODN (**C**) alone. Schematic in (**A**) created with BioRender. Statistical differences between groups were determined by performing Student's *t*-test. Error bars represent SD. \* for *p* ≤ 0.05 and \*\*\*\* for *p* ≤ 0.0001. *3.3. BMMΦs Associate with and Internalize PLA-PEMA More Extensively Than PLA-PVA*  **Figure 2.** Under typical in vitro serum conditions, both particle types fail to sequester FITC-tagged TLR agonists. (**A**) Particles and FITC-conjugated TLR agonists were co-incubated at 37 ◦C and 5% CO<sup>2</sup> for 1 h and then pelleted to determine direct interactions between particles and TLR agonists. When co-incubated with PBS containing 10% FBS, both (**B**) FITC-LPS and (**C**) FITC-CpG ODN fail to interact with particles alone as signified by the dashed line representing 100% FITC signal of FITC-LPS (**B**) or FITC-CpG ODN (**C**) alone. Schematic in (**A**) created with BioRender. Statistical differences between groups were determined by performing Student's *t*-test. Error bars represent SD. \* for *p* ≤ 0.05 and \*\*\*\* for mboxemphp ≤ 0.0001.

#### As iNPs do not directly interact with PAMPs, we aimed to further understand the *3.3. BMMΦs Associate with and Internalize PLA-PEMA More Extensively Than PLA-PVA*

differences in cellular interactions and uptake between various iNPs. To assess iNP-cell interactions, we prepared Cy5.5-conjugated versions of iNPs with similar physicochemical characteristics as unlabeled PLA-PEMA and PLA-PVA (Supplemental Figure S1). As iNPs do not directly interact with PAMPs, we aimed to further understand the differences in cellular interactions and uptake between various iNPs. To assess iNP-cell interactions, we prepared Cy5.5-conjugated versions of iNPs with similar physicochemical characteristics as unlabeled PLA-PEMA and PLA-PVA (Supplemental Figure S1). Fluorescence microscopy showed that BMMΦs displayed a higher propensity to associate with

PLA-PEMA compared to PLA-PVA (Figure 3A), which was also seen using RAW 264.7 cells (Supplemental Figure S2). Flow cytometry was further used to quantitatively measure cell uptake of particles and confirmed that PLA-PEMA associated more rapidly with BMMΦs than PLA-PVA. Within 1 h of iNP incubation with BMMΦs, approximately 75% of BMMΦs were PLA-PEMA-Cy5.5<sup>+</sup> while only 30% of BMMΦs were PLA-PVA-Cy5.5<sup>+</sup> (Figure 3B). Since the formulations differed mainly in the surfactant choice and the resultant zeta potential of the iNP, these results suggest that the choice of the negatively charged PEMA drives the propensity of BMMΦs to preferentially interact with iNPs compared to those prepared using PVA. Fluorescence microscopy showed that BMMΦs displayed a higher propensity to associate with PLA-PEMA compared to PLA-PVA (Figure 3A), which was also seen using RAW 264.7 cells (Supplemental Figure S2). Flow cytometry was further used to quantitatively measure cell uptake of particles and confirmed that PLA-PEMA associated more rapidly with BMMΦs than PLA-PVA. Within 1 h of iNP incubation with BMMΦs, approximately 75% of BMMΦs were PLA-PEMA-Cy5.5+ while only 30% of BMMΦs were PLA-PVA-Cy5.5+ (Figure 3B). Since the formulations differed mainly in the surfactant choice and the resultant zeta potential of the iNP, these results suggest that the choice of the negatively charged PEMA drives the propensity of BMMΦs to preferentially interact with iNPs compared to those prepared using PVA.

**Figure 3.** PLA-PVA and PLA-PEMA particle formulations show similar behavior when interacting with TLR agonists and cells with the exception being the increased propensity of PLA-PEMA particle uptake by cells. (**A**) Fluorescence microscopy establishes that PLA-PEMA-Cy5.5 interact to a greater extent with BMMΦs than PLA-PVA-Cy5.5. (**B**) Quantification with flow cytometry after 1-hr co-incubation at 37 °C and 5% CO2 confirms cells associate to a greater extent with PLA-PEMA-Cy5.5 particles (30 µg/mL) than PLA-PVA-Cy5.5 particles (30 µg/mL). Both PLA-PVA (300 µg/mL) and PLA-PEMA (300 µg/mL) treatments result in dramatic decreases in BMMΦ cellular uptake of (**C**) FITC-LPS (100 ng/mL) and (**D**) FITC-CpG ODN (100 ng/mL) following 18-hr incubation at 37 °C and 5% CO2. Statistical differences between groups were determined by performing a Student's *t*-test. Error bars represent SD. \* for *p* ≤ 0.05, \*\*\* for *p* ≤ 0.001, and \*\*\*\* for *p* ≤ 0.0001. *3.4. PLA Particles Hinder LPS and CpG ODN Interaction with BMMΦs*  **Figure 3.** PLA-PVA and PLA-PEMA particle formulations show similar behavior when interacting with TLR agonists and cells with the exception being the increased propensity of PLA-PEMA particle uptake by cells. (**A**) Fluorescence microscopy establishes that PLA-PEMA-Cy5.5 interact to a greater extent with BMMΦs than PLA-PVA-Cy5.5. (**B**) Quantification with flow cytometry after 1-hr co-incubation at 37 ◦C and 5% CO<sup>2</sup> confirms cells associate to a greater extent with PLA-PEMA-Cy5.5 particles (30 µg/mL) than PLA-PVA-Cy5.5 particles (30 µg/mL). Both PLA-PVA (300 µg/mL) and PLA-PEMA (300 µg/mL) treatments result in dramatic decreases in BMMΦ cellular uptake of (**C**) FITC-LPS (100 ng/mL) and (**D**) FITC-CpG ODN (100 ng/mL) following 18-hr incubation at 37 ◦C and 5% CO<sup>2</sup> . Statistical differences between groups were determined by performing a Student's *t*-test. Error bars represent SD. \* for *p* ≤ 0.05, \*\*\* for *p* ≤ 0.001, and \*\*\*\* for *p* ≤ 0.0001.

#### differentially with BMMΦs (Figure 3A,B). Collectively, this suggests that the immuno-*3.4. PLA Particles Hinder LPS and CpG ODN Interaction with BMMΦs*

modulatory activity of iNPs is dependent on their interactions with BMMΦs. To assess this, we first treated BMMΦs with either PLA-PEMA or PLA-PVA followed by incubation with either FITC-LPS (Figure 3C,D) or FITC-CpG ODN (Figure 3A–D). Qualitatively it was observed that despite the greater interaction of PLA-PEMA with BMMΦs, both iNP formulations decreased the association of FITC-CpG ODN with the BMMΦs (Figure 3A). For both LPS and CpG ODN, flow cytometry shows quantitatively that iNP pre-treatment significantly decreased the overall interaction of BMMΦs with LPS and CpG ODN (Figure 3C,D, respectively). These decreases in PAMP interactions with the cells occurs regardless of iNP type, suggesting that iNP-mediated interruption of BMMΦ and LPS or CpG ODN As described above, iNPs do not sequester LPS or CpG ODN (Figure 2), but interact differentially with BMMΦs (Figure 3A,B). Collectively, this suggests that the immunomodulatory activity of iNPs is dependent on their interactions with BMMΦs. To assess this, we first treated BMMΦs with either PLA-PEMA or PLA-PVA followed by incubation with either FITC-LPS (Figure 3C,D) or FITC-CpG ODN (Figure 3A–D). Qualitatively it was observed that despite the greater interaction of PLA-PEMA with BMMΦs, both iNP formulations decreased the association of FITC-CpG ODN with the BMMΦs (Figure 3A). For both LPS and CpG ODN, flow cytometry shows quantitatively that iNP pre-treatment significantly decreased the overall interaction of BMMΦs with LPS and CpG ODN (Figure 3C,D, respectively). These decreases in PAMP interactions with the cells occurs regardless of iNP type, suggesting that iNP-mediated interruption of BMMΦ and LPS or CpG ODN interactions is a process independent of iNP

As described above, iNPs do not sequester LPS or CpG ODN (Figure 2), but interact

uptake and surfactant composition. Flow cytometry studies (Supplemental Figure S3) revealed reduced CD14 and TLR4 surface molecule expression in response to iNP treatment, suggesting that iNP-mediated disruption of BMMΦ-PAMP interactions may be influenced by the reductions in CD14 and TLR4 surface expression.
