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Biomaterials

Biomaterials Undergraduate Fire Talks

PEGylation of Cell Penetrating Peptide for Increased Serum Stability and CRISPR Efficiency

Saturday, October 11, 2025
9:20 AM - 9:25 AM PDT
Location: Room 32B

Introduction: : The use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) allows for the precise regulation of gene expression.[1] However, the clinical translation of this technology is constrained by delivery challenges. Compared to traditional viral vectors such as adenovirus or lentivirus, non-viral packaging is emerging as a low toxicity and nonintegrative alternative.[2] Cell penetrating peptides (CPPs), like the arginine-alanine-leucine-alanine (RALA) peptide, have shown improved CRISPR delivery efficiency and low cytotoxicity among other non-viral lipid or polymer-based vectors.[2] However, non-viral transfection vectors are frequently evaluated in serum-free conditions, which do not reflect in vivo environment where serum components can impact vector stability, nanoparticle aggregation, and cell uptake. Delivering CRISPR cargo using CPPs in serum presents additional challenges due to nonspecific CPP–serum interactions that reduce biodistribution, as well as proteolytic enzymes that compromise vector stability and circulation time.[2]
Polyethylene glycol (PEG) is an easily modified polymer with a binding affinity for serum albumins. PEGylation has shown promising results in improving the stability, lowering immunogenicity, and preventing aggregation of various biomolecules.[3-4] Methoxy polyethylene glycol (mPEG) contains a methoxy group cap, further reducing immunogenicity and aggregation compared to more reactive hydroxy groups found in PEG.[3] We hypothesize that PEGylation will achieve enhanced CRISPR delivery in serum conditions by improving stability and decreasing nanoparticle aggregation, while maintaining the low cytotoxicity and immunogenicity of RALA. Additionally, due to the hydrophilicity of PEG, we hypothesize that PEGylation will maintain RALA’s ability to complex and form stable nanoparticles with various CRISPR cargos and support efficient delivery to primary cells.

Materials and

Methods: : mPEG conjugated RALA (mPEG-RALA) was synthesized by reacting RALA with N-hydroxysuccinimide ester activated mPEG (mPEG-NHS) (Fig. 1).[4] mPEG-NHS was dissolved in DMSO at 10 mM with varying molecular weights (350, 1000, 2000, and 5000). Each mPEG-NHS solution was added to RALA in basic phosphate buffer saline (PBS) with pH 7.4 at 1 mg/mL and incubated on ice for 2 hours. The products (mPEG-RALA) were analyzed by mass spectroscopy and purified using HPLC. Nanoparticle characterization was performed using circular dichroism to assess structure and Zetasizer for particle size and zeta potential.
To assess serum stability, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was employed. mPEG-RALA nanoparticles of varying molecular weights were incubated in 10% fetal bovine serum (FBS) and removed at different timepoints (0, 15, 30, 60 min). A SDS negative gel was used to assess the degradation of mPEG-RALA nanoparticles based on band intensity. A SDS positive gel was used as a positive control to ensure nanoparticle encapsulation.

Results, Conclusions, and Discussions:: Mass spectroscopy of mPEG2k-RALA revealed polydispersed peaks separated by 44 Da, indicating the presence of PEG in the final product. A peak for lone RALA is not found, indicating that binding between PEG and RALA occurred (Fig. 1). Upon purification using HPLC, multiple fractions were collected due to the polydispersity of PEG, but the presence of RALA was verified by a UV absorption peak near 214 nm. Circular dichroism of mPEG-RALA revealed a similar ⍺-helical structure to unmodified RALA. Zetasizer of mPEG-RALA nanoparticles when complex to different CRISPR cargos showed similar particle size and zeta potential as unmodified RALA. SDS-PAGE of mPEG-RALA nanoparticles showed bands decreasing in intensity with longer FBS incubation times. Unmodified RALA had a faster drop in band intensity, indicating that mPEG-RALA achieves greater serum stability. Future studies will explore the efficacy of mPEG-RALA to delivery CRISPR to primary human mesenchymal stem cells (MSCs) in vitro, and the biodistribution of mPEG-RALA-CRISPR nanoparticles after in vivo injection in a mouse model. In conclusion, by enhancing serum stability through PEGylation, mPEG-RALA offers a more effective approach for CRISPR delivery in serum conditions, helping advance the clinical translation of CRISPR-based therapies.

Acknowledgements and/or References (Optional): : 1. Graham, J. P., Jose Gabriel Castro, Werba, L. C., Fardone, L. C., Francis, K. P., Anand Ramamurthi, Layden, M., McCarthy, H. O., & Gonzalez-Fernandez, T. (2024). Versatile Cell Penetrating Peptide for Multimodal CRISPR Gene Editing in Primary Stem Cells. BioRxiv (Cold Spring Harbor Laboratory). https://doi.org/10.1101/2024.09.23.614499

2. J. Graham, L. Werba, I. Federico, and T. Gonzalez-Fernandez, “CRISPR Strategies for Stem Cell Engineering: A New Frontier in Musculoskeletal Regeneration,” Eur. Cell. Mater., vol. 46, pp. 91–118, Nov. 2023, https://doi.org/10.22203/eCM.v046a05.

3. Guzman Gonzalez, V., Grunenberger, A., Nicoud, O., Czuba, E., Vollaire, J., Josserand, V., Le Guével, X., Desai, N., Coll, J.-L., Divita, G., & Faure, V. (2024). Enhanced CRISPR-Cas9 RNA system delivery using cell penetrating peptides-based nanoparticles for efficient in vitro and in vivo applications. Journal of Controlled Release, 376, 1160–1175. https://doi.org/10.1016/j.jconrel.2024.11.008

4. Ponnumallayan, P., & Fee, C. J. (2014). Reversible and rapid pH-Regulated self-assembly of a poly(ethylene glycol)–Peptide bioconjugate. Langmuir, 30(47), 14250–14256. https://doi.org/10.1021/la502360k