(T1430-01-06) Fabrication of pH-Responsive Acetalated Dextran Nanoparticles Using Flash Nanoprecipitation for Intracellular Delivery of Payloads

Purpose: Flash nanoprecipitation (FNP) employs rapid turbulent millisecond mixing of an organic solvent phase (usually containing amphipathic polymer and hydrophobic payload) and a miscible aqueous solvent phase to produce monodispersed nanoparticles.¹ pH-responsive delivery systems that are sensitive to acidic conditions not only enable the precise release of payloads from endolysosomes into the cytoplasm but also prevent leakage of drugs at neutral pH (blood or extracellular spaces). Acetalated dextran (Ac-Dex) nanoparticles have been shown to exhibit higher sensitivity at acidic pH due to the presence of acid-labile acetal groups. Furthermore, Ac-Dex nanoparticles are synthesized using a naturally occurring polymer, dextran, which has greater biodegradability and biocompatibility.² However, the current formulation strategy used to fabricate Ac-Dex nanoparticles requires the use of a multi-step, exothermic emulsion process, and toxic organic solvents such as chloroform or dichloromethane. Polyvinyl alcohol is widely used to stabilize Ac-Dex nanoparticles, but the incorporation of non-ionic surfactants such as Pluronic F-127 (F-127) or D-α-tocopheryl polyethylene glycol succinate (TPGS) as stabilizers may enhance the circulation times of Ac-Dex nanoparticles due to the presence of a polyethylene glycol component. We hypothesize that Ac-Dex nanoparticles could be efficiently stabilized using amphiphilic polymers with a rapid, scalable nanoprecipitation approach such as FNP. Here, we utilized a confined impingement jet (CIJ) mixer to fabricate Ac-Dex nanoparticles using simple, organic-solvent ethanol and F-127 or TPGS as stabilizers. We have then evaluated the encapsulation efficiency of a wide range of payloads and demonstrated pH-responsive release and intracellular uptake and release using model fluorophores.

Methods: The Ac-Dex polymer was synthesized as reported elsewhere.³ For the fabrication of nanoparticles using FNP, Ac-Dex polymer or hydrophobic payloads were dissolved in ethanol (organic phase) while 5% w/v TPGS or 5% w/v F-127 or hydrophilic payloads were dissolved in deionized water (aqueous phase). Both organic and aqueous phases impinged against each other in a CIJ mixer and the mixture was collected into a quencher reservoir containing deionized water. Ethanol was evaporated overnight at room temperature followed by centrifugation to remove excess surfactant and unencapsulated payloads. Physicochemical characteristics of Ac-Dex nanoparticles were measured including size via dynamic light scattering (DLS), morphology through transmission electron microscopy (TEM), and surface charge using electrophoretic light scattering (ELS). Ac-Dex nanoparticles were lyophilized using 10% w/v mannitol as a cryoprotectant and the stability of the formulation was measured over 30 days. Time-dependent cellular uptake of DiD-loaded nanoparticles in RAW 264.7 macrophages was determined through flow cytometry, and the uptake mechanism was verified using chlorpromazine (CPZ). Finally, the intracellular release of model payload, rhodamine from Ac-Dex nanoparticles after 24h incubation with RAW 264.7 macrophages was confirmed using live-cell confocal microscopy.

Results: The size of Ac-Dex nanoparticles fabricated using TPGS and F-127 was found to be 173 ± 56 nm and 168 ± 57 nm, respectively. TEM images confirm the spherical morphology of Ac-Dex nanoparticles (Figure 1A and B). The surface charge of both TPGS and F-127 stabilized Ac-Dex nanoparticles were neutral. There was no size difference observed after immediate reconstitution of lyophilized cake of TPGS-Ac-Dex as compared to the pre-lyophilization sample. While F-127-Ac-Dex showed an increase in size to 292 nm post lyophilization as compared to the pre-lyophilization size of 168 nm. Furthermore, after 30 days of storage of lyophilized cake, TPGS-Ac-Dex showed a slight increase in size to 220 nm whereas F-127-Ac-Dex demonstrated a greater increase in size to 609 nm. Ac-Dex nanoparticles were able to encapsulate a wide range of payloads that are hydrophilic and hydrophobic in nature, as well as a macromolecule (Figure 2A). TPGS-Ac-Dex nanoparticles demonstrated a pH-dependent release of a model molecule doxorubicin, with a ~7-fold change in release at pH 4 as compared to pH 7.4 over 48 hours (Figure 2B). TPGS-Ac-Dex nanoparticles were non-cytotoxic and flow cytometry analysis demonstrated a time-dependent cellular uptake of TPGS-Ac-Dex nanoparticles, which plateaued at 6 h. The addition of CPZ showed significant inhibition of cellular uptake of TPGS-Ac-Dex nanoparticles as compared to non-CPZ treated cells (Figure 3A). The merged confocal image shows diffused red fluorescence inside cells, confirming the cytosolic release of rhodamine dye from TPGS-Ac-Dex nanoparticles (Figure 3B).

Conclusion: Ac-Dex nanoparticles fabricated via flash nanoprecipitation with TPGS and F-127 as stabilizers consistently formed monodispersed nanoparticles and allowed efficient encapsulation of payloads. The use of TPGS increased the stability of Ac-Dex nanoparticles after lyophilization as compared to F-127. TPGS-Ac-Dex nanoparticles were effectively taken up by the cells with a clathrin-dependent cellular internalization mechanism and were able to release payloads into the cytosol. Overall, the FNP is a promising method to fabricate pH-responsive Ac-Dex nanoparticles for intracellular delivery of payloads.

References: 1. Johnson, B. K., & Prud'homme, R. K. (2003). Flash nanoprecipitation of organic actives and block copolymers using a confined impinging jets mixer. Australian Journal of Chemistry, 56(10), 1021. https://doi.org/10.1071/ch03115
2. Maia, João & Evangelista, Marta & Gil, Helena & Ferreira, Lino. (2014). Dextran-based materials for biomedical applications.
3. Bachelder, E. M., Beaudette, T. T., Broaders, K. E., Dashe, J., & Fréchet, J. M. (2008). Acetal-derivatized dextran: An acid-responsive biodegradable material for therapeutic applications. Journal of the American Chemical Society, 130(32), 10494–10495. https://doi.org/10.1021/ja803947s

Acknowledgments: This work was made possible by an NIEHS-funded predoctoral fellowship to KH (T32 GM133369-02)

Physiochemical characteristics of Ac-Dex nanoparticles formulated with stabilizers (A) TPGS and (B) and F-127. The size of Ac-Dex particles formulated with TPGS and F-127 was measured using dynamic light scattering and reported as d. nm, Positive stain TEM images of TPGS or F-127 stabilized Ac-Dex nanoparticles. Scale bar is 200 nm.



(A) Encapsulation efficiency of hydrophilic and hydrophobic small molecules, and a macromolecule in Ac-Dex particles made with either TPGS or F-127. (B) In vitro doxorubicin release from Ac-Dex TPGS particles in pH 4 and pH 7.4 over 4, 24, and 48 hours. Data are represented as mean ± SD (n=3).



Intracellular uptake of Ac-Dex nanoparticles stabilized with TPGS in RAW 264.7 cells. (A) Time-dependent cellular uptake of DiD-encapsulated nanoparticles measured through flow cytometry. For inhibition studies, cells were treated with chlorpromazine (CPZ) for 6 hours prior to nanoparticle addition. Data are represented as mean ± SD (n=3). Significance was determined using 1-way ANOVA and Tukey’s multiple comparison test **** p < 0.0001. (B) A merged confocal image showing the cytosolic release of rhodamine-dye (red color) from Ac-Dex nanoparticles after 24 h incubation with RAW 264.7 cells. Blue color represents the nuclei stain. Scale bar is 15 um.