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Formulation and Delivery - Biomolecular

(W1230-01-03) Cellular Uptake of Bare and Corona Coated PEO-b-PCL and PEO-b-PBCL Micelles by HCT116 and SW620 Cell Lines

Wednesday, October 23, 2024
12:30 PM – 1:30 PM MT

    Presenting Author(s)

  • SM

    Sirazum Munira

    Graduate Research Assistant
    University of Alberta
    Edmonton, Alberta, Canada

    • Main Author(s)

  • SM

    Sirazum Munira

    Graduate Research Assistant
    University of Alberta
    Edmonton, Alberta, Canada

    • Co-Author(s)

  • ID

    Igor De Paiva, Ph.D.

    Medical Science Liaison
    AstraZeneca
    Edmonton, Alberta, Canada

  • PP

    Prashant Pandey

    Visiting Research Assistant
    University of Alberta
    Edmonton, Alberta, Canada

  • AA

    Aliakbar Ashkarran

    Assistant Professor
    Michigan State University
    East Lansing, Michigan, United States

  • SS

    Shahriar Sharifi

    Post Doctoral Fellow
    Michigan State University
    EAST LANSING, Michigan, United States

  • ST

    Soheyl Tadjiki

    Managing Director
    Postnova Analytics
    Salt Lake City,, Utah, United States

  • HG

    Hassan Gharibi

    Post Doctoral Fellow
    Karolinska Institute
    NA, Blekinge Lan, Sweden

  • AA

    Amir Ata Saei

    Assistant Professor
    Karolinska Institutet
    123, Stockholms Lan, Sweden

  • MM

    Morteza Mahmoudi

    Professor
    Michigan State University
    EAST LANSING,, Michigan, United States

  • AL

    Afsaneh Lavasanifar, PhD

    Professor
    University of Alberta
    Edmonton, Alberta, Canada

Purpose: After nanocarriers are administered in the biological environments, they are exposed to high-concentration proteins forming a protein layer around the nanocarrier, which is known as protein corona (PC)1. The formation of the protein corona can modify diverse properties of the nanocarriers like size, surface properties, giving them a new biological identity2. This new biological identity can influence interactions between nanoparticles and biological systems such as bioavailability, cellular uptake, intracellular trafficking and toxicity3. Polymeric micelles (PMs) are promising nano-carriers for poorly water-soluble chemotherapeutics. There is, however, little known about their fate following administration in the biological system. This research aims to study the protein corona profiles of different PMs self-assembled from poly(ethylene oxide)-b-poly(Ɛ-caprolactone) (PEO-b-PCL) and PEO-b-poly(α-benzyl carboxylate-ε-caprolactone) (PEO-b-PBCL) with varying degrees of polymerization (DP) following their incubation with human plasma (HP). Furthermore, our objective is to explore the influence of different characteristics of the block copolymers and the micelles formed from them, and the impact of the protein corona formed around these micelles on their cellular uptake by HCT116 and SW620 colorectal cancer cells.

Methods: Seven di-block copolymers of different DPs were synthesized by ring-opening polymerization of either Ɛ-caprolactone (Ɛ-CL) or α-benzyl carboxylate-ε-caprolactone (BCL) by methoxy-polyethylene oxide (m-PEO) (MW 5000g/mol) to form PEO-b-PCL (DP 19, 43, 64, 79) and PEO-b-PBCL (DP 19, 35, 60) block copolymers, respectively. The average molecular weights (MW) and polydispersity of the synthesized block polymers were analyzed by 1H NMR and GPC. Near-infrared fluorophore Cy5.5 was conjugated to the hydrophobic blocks to make the PMs traceable. PMs were prepared by the cosolvent evaporation method, and their size and morphology were characterized by Zetasizer and transmission electron microscopy (TEM), respectively. For the analysis of the biological identity of the PMs, they were incubated with cell culture media supplemented with 55% human plasma to form protein corona around them. To separate the unbound and loosely bound proteins from protein corona-coated PMs, both bare and corona-coated PMs were passed through the Asymmetrical Flow Field-Flow Fractionation (FFF) (AF4) system. To elucidate the composition of the protein corona on the surface of the protein corona-coated PMs, samples from the initial, middle, and final elution time scale of the FFF histogram were analyzed by liquid chromatography-mass spectrometry (LC-MS). To study the effect of the protein corona composition on the cellular uptake of the PMs, HCT116 and SW620 cells were incubated with the PMs for 6 and 24 h in the presence and absence of human plasma and analyzed using flow cytometry.

Results: The average sizes of the PMs were between 44±0.38 nm and 99 ±0.79 nm. Zeta potential values of the PMs were slightly negative. Among the seven polymers, PCL64, PCL79 and PBCL19 were chosen for protein corona analysis due to their spherical shapes shown in both TEM imaging and FFF. A deeper analysis of proteins coronas in the averaged middle fractions shows that the 20 top proteins from the three PM coronas comprised 23 unique proteins, among which, 18 proteins were shared. There were a few proteins that were specific to one or two PMs, which might account for variabilities noted in the cellular uptake of PMs. Orthogonal Partial Least Squares discriminant analysis (OPLS-DA), a supervised multivariate data analysis algorithm, was employed to analyze the corona proteomes of different PMs. OPLS-DA models highlighted the proteins most specifically enriched or depleted in each PM corona. The result reveals a high similarity between PCL64 and PCL79 compared to PBCL19. For example, APOA4 and CFI were enriched in the corona of PBCL19 vs coronas of PCL64 and PCL79, while RPL9 and ACTB were depleted. Therefore, the uniqueness of each corona composition could underline the differences noted in PM uptake. The cellular uptake study showed that PEO-PCL PMs (PCL64 and PCL79) had higher cellular uptake compared to PEO-PBCL (PBCL19) in both cell lines under study. The presence of HP showed a significant reduction in cellular uptake of PEO-PCL nanoparticles (PCL64 and PCL79) in 24 h study in both HCT116 and SW620 cell lines. However, PBCL19 micelles did not show any significant difference in uptake irrespective of the presence or absence of HP. Overall, uptakes in 24 h treatments were higher than 6 h treatments. This may be because of increased adsorption of unique proteins on the PM surface due to prolonged exposure to the different proteins of the HP attributing to the morphology of the micelles made from different molecular weight polymers.

Conclusion: The results reflect the critical role of the structure, morphology surface properties and other characteristics of the block copolymers and PMs formed from them on their protein corona profiles. It also highlights the potential influence of protein corona profiles on the cellular uptake of the PMs. Overall, PMs formed from PBCL19 showed significantly different zeta potential, protein coronal profile and cellular uptake compared to PEO-PCL micelles.

References: 1. Walczyk D, Bombelli FB, Monopoli MP, Lynch I, Dawson KA. What the Cell “Sees” in Bionanoscience. J Am Chem Soc. 2010;132(16):5761-5768. doi:10.1021/ja910675v
2. Nguyen VH, Lee BJ. Protein corona: a new approach for nanomedicine design. Int J Nanomedicine. 2017;12:3137-3151. doi:10.2147/IJN.S129300
3. Rahman M, Laurent S, Tawil N, Yahia L, Mahmoudi M. Nanoparticle and Protein Corona. In: Rahman M, Laurent S, Tawil N, Yahia L, Mahmoudi M, eds. Protein-Nanoparticle Interactions: The Bio-Nano Interface. Springer; 2013:21-44. doi:10.1007/978-3-642-37555-2_2

Acknowledgements: This research is funded by NSERC Discovery and NSERC CREATE PoND. The manuscript of the research might be submitted for journal publication before the AAPS-2024 PHARMSCI 360 meeting in October.


Figure: (A) Characteristics of the used PEO-PCL and PEO-PBCL di-block copolymers and their micelles in this study; (B) Morphology of PMs by transmission electron microscopy (TEM) images


Figure: A) The overlap between the top 20 most abundant proteins in NP coronas. B) KEGG pathways enriched for the top 23 unique most abundant proteins found in the corona of three NP types. C) The top 10 most abundant proteins for each NP corona are shown in the order of abundance in LC-MS analysis. (D) OPLS-DA models highlighting the enriched and depleted proteins in NP protein corona.


Figure: MFI of (A) HCT116 cells and (B) SW620 cells after 6 hours (solid bar) and 24 hours (checkered bar) treatment with Cy5.5-labelled PEO114-PCL64, PEO114-PCL79 and PEO114-PCL19 polymeric micelles in the presence (red) and absence (green) of human plasma.