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Biomaterials

Biomaterials - Poster Session D

Poster O1 - A scalable system for generating polymer lipid-based nanoparticle hydrogels to modulate the immune system

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  The immune system is highly adaptive in fighting against various diseases including cancer, chronic infections, and autoimmunity [1], [2], [3]. Thus, immunomodulatory treatments that leverage the immune system’s inherent sophistication can enable durable resistance against ever-changing and persistent diseases, as is the case for cancer immunotherapies [3]. To this end, lipid-based nanoparticles (LBNPs) including liposomes and lipid nanoparticles (LNPs) have improved delivery efficacy of many immunotherapeutics by providing tethering of or improving uptake of a variety of drug cargos – small molecules, nucleic acids, antibodies, and other proteins [4]. However, current LBNPs are delivered systemically and suffer from challenges in targeting and localized accumulation at specific sites of disease [5]. This is troublesome since the off-target delivery of immunotherapeutics can upset homeostasis and trigger severe adverse side effects [2], [5].

Meanwhile, hydrogels enjoy advantages in both fronts of – they offer localized and tunable drug release [6]. In particular, polymer—nanoparticle (PNP) hydrogels, in which the gel is dynamically crosslinked by its nanoparticle components, exhibit excellent shear-thinning/self-healing properties that enable their injection into the site of therapy [6], [7]. Incorporating LBNPs as the nanoparticle component of PNP hydrogels can help avoid the pitfalls of systemic injection while enjoying excellent modularity and tunability. Unfortunately this is tempered by challenges in scalability, which continues to limit their widespread utilization [6]. To address these challenges, we present here a scalable and modular strategy for the fabrication of polymer—LBNP (PLBNP) hydrogels.

Materials and

Methods:  Materials
Synthetic lipids 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) were obtained from Avanti Polar Lipids. DLin-MC3-DMA (MC3) as obtained from Cayman Chemical. Chemicals used in HPMC-C12 synthesis were obtained from Sigma Aldrich.

HPMC-C12 polymer synthesis
HPMC was dissolved in N-methyl-2-pyrrolidone, and dodecyl isocyanate and Hunig’s catalyst were added under anhydrous conditions at 50°C. The reaction was quenched with acetone and the product dialyzed and lyophilized.

LBNP preparation
Both liposomes and LNPs were synthesized via hydrodynamic flow focusing in a microfluidic system, which enables controlled lipid nanoprecipitation. This was performed in the Automated Nanoparticle System by Unchained Labs. For liposome synthesis, DOTAP or DSPG, DSPC, cholesterol, and DMG-PEG 2000 were dissolved in ethanol at ratio 12:46:40:2 and mixed with 1x PBS at ratio 1:4 in a 5-input 3D microfluidic chip (Dolomite). For LNP synthesis, MC3, DSPC, cholesterol, and DMG-PEG 2000 were dissolved in ethanol at ratio 50:10:38.5:1.5 and mixed with yeast RNA (N/P ratio of 1) in sodium acetate buffer (25 mM, pH 5) at ratio 1:3 in a chaotic MicroMixer chip (Dolomite).
The KrosFlo KR2i tangential flow filtration system was used to concentrate and buffer exchange LBNPs, using concentration/diafiltration/concentration followed by concentration schemes.
LBNPs were characterized by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA).

Rheological characterization
The rheological properties of PLBNP hydrogels were assessed on the Discovery Hybrid Rheometer 20 (TA Instruments). Frequency, amplitude, self-healing, and flow sweeps were performed on each hydrogel using a 20 mm cross-hatched geometry.

Results, Conclusions, and Discussions: Dodecyl-modified hydroxypropyl methylcellulose (HPMC-C12) polymers have been previously shown to produce biodegradable and tunable hydrogels when mixed with liposomes [7]. Therefore, we sought to validate our platform through developing a scalable fabrication strategy for HPMC-C12—LBNP hydrogels (Fig. 1A).Thus each of the process steps were made scalable: 1) synthesize HPMC-C12, 2) synthesize, concentrate, and diafilter LBNPs, and 3) mix components together via dual syringe mixing with an elbow connector to form a hydrogel (Fig. 1B).

HPMC-C12 was synthesized as previously described in Appel et al. [8], which enables parallelized batchwise fabrication and a product that can be lyophilized for long-term storage. Three LBNPs were synthesized and used: anionic DSPG-based liposomes, cationic DOTAP-based liposomes, and MC3-based LNPs. Both the anionic liposomes and MC3 LNPs were used to make a 2 wt% LBNP with 5 wt% HPMC-C12 (2:5) hydrogel, while the cationic liposomes were used to make a 1:5 hydrogel.

Lipid-based nanoparticle quality remains good during synthesis and processing
LBNPs were synthesized in a microfluidic system. Since microfluidics generates a dilute product (0.1 wt%) and contains organic phase solvents, ultrafiltration/concentration and diafiltration in a tangential flow filtration system were performed to bring LBNPs to higher concentrations necessary for mixing (>7.5 wt%) and remove organics. Anionic liposomes, cationic liposomes, and MC3 LNPs formed monodisperse particles less than 100 nm in diameter after synthesis and remained less than 210 nm after processing (Fig. 2A,C). The trends in charge characteristics were consistent with the charged or ionizable lipid used (Fig. 2B).

Polymer-LBNP hydrogels exhibit shear-thinning and self-healing properties
Using two luer-lock syringes, LBNPs were mixed with redissolved HPMC-C12 in an elbow connector for about 15 minutes. The 2:5 anionic liposomal and LNP hydrogels exhibited stiffnesses around 100-150 Pa at 10 rad/s and the 1:5 cationic liposomal hydrogel at 20 Pa. All hydrogels exhibited shear thinning properties and remained in the gel phase and in the tested angular frequency range from 10e-1 rad/s to 10e2 rad/s (Fig. 2E-G).

Since hydrogel production scale-up suffers setbacks from [6], we expect to further test our PLBNP hydrogels for batch-to-batch variability and perform functional assessments on drug delivery efficacy.

Acknowledgements (Optional):  J.S.W. and S.K.N. are thankful for the National Science Foundation Graduate Research Fellowship.

References
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[2] Q. Jin, Z. Liu, and Q. Chen, “Controlled release of immunotherapeutics for enhanced cancer immunotherapy after local delivery,” Journal of Controlled Release, vol. 329, pp. 882–893, Jan. 2021, doi: 10.1016/j.jconrel.2020.10.019.
[3] A. D. Waldman, J. M. Fritz, and M. J. Lenardo, “A guide to cancer immunotherapy: from T cell basic science to clinical practice,” Nat Rev Immunol, vol. 20, no. 11, pp. 651–668, Nov. 2020, doi: 10.1038/s41577-020-0306-5.
[4] Z. Gu, C. G. Da Silva, K. Van der Maaden, F. Ossendorp, and L. J. Cruz, “Liposome-Based Drug Delivery Systems in Cancer Immunotherapy,” Pharmaceutics, vol. 12, no. 11, Art. no. 11, Nov. 2020, doi: 10.3390/pharmaceutics12111054.
[5] S. Lim et al., “Recent advances and challenges of repurposing nanoparticle-based drug delivery systems to enhance cancer immunotherapy,” Theranostics, vol. 9, no. 25, pp. 7906–7923, Oct. 2019, doi: 10.7150/thno.38425.
[6] S. Correa et al., “Translational Applications of Hydrogels,” Chem. Rev., vol. 121, no. 18, pp. 11385–11457, Sep. 2021, doi: 10.1021/acs.chemrev.0c01177.
[7] S. Correa, A. K. Grosskopf, J. H. Klich, H. Lopez Hernandez, and E. A. Appel, “Injectable liposome-based supramolecular hydrogels for the programmable release of multiple protein drugs,” Matter, vol. 5, no. 6, pp. 1816–1838, Jun. 2022, doi: 10.1016/j.matt.2022.03.001.
[8] E. A. Appel, M. W. Tibbitt, M. J. Webber, B. A. Mattix, O. Veiseh, and R. Langer, “Self-assembled hydrogels utilizing polymer–nanoparticle interactions,” Nat Commun, vol. 6, no. 1, p. 6295, Feb. 2015, doi: 10.1038/ncomms7295.