(236c) Uptake of Fluorescently-Labeled Peptidomimetic Nanoparticles by Tumor Cells

Introduction Nanoparticles (NPs) are increasingly being used for a variety of drug delivery applications. However, the size of the NPs is a relevant design parameter that should be considered when producing them. For example, NPs of less than 400 nm in diameter are selectively uptaken by tumor tissue due to their increased permeability (EPR effect). Antitumor drugs, such as Paclitaxel and Doxorubicin, can have enhanced effectiveness when uptaken by cells. It is thus necessary that NPs have a small diameter and a narrow distribution so that selectivity by size can be achieved. In our lab, we have synthesized poly(lactide acrylate) (PLAA) and poly(lactide-co-glycolide acrylate) (PLGAA) NPs of about 50-150 nm. The PLGAA macromer has a peptide sequence, Cys-Val-Val-Val-Val-Val-Val-Lys-Lys (CV6K2) [1], grafted to their acrylate group that enables self-assembly into narrowly distributed NPs when dialyzed against aqueous solution. The objective of this work was to encapsulate a fluorescein isothiocyanate (FITC) marker into PLGAA NPs and measure their uptake into 4T1 cells. Uptake was measured by spectrofluorometry and verified with fluorescent microscopy as a function of time.

Methods PLAA and PLGAA were synthesized by ring opening polymerization of lactide and glycolide units and functionalized with acryloyl chloride as described [2]. The peptide sequence CVVVVVVKK was synthesized in solid phase using Fmoc-chemistry as previously described [3]. Conjugation was performed by coupling the cysteine residue of the peptide sequence to the acrylate group of the macromers. After conjugation, 50 mg of PLAA-CV6K2 or PLGAA-CV6K2 NPs were self-assembled by dialysis of the macromers in organic solvent against PBS. FITC (2%wt loading) was added to the organic solution to allow encapsulation on the NPs. After self-assembly, the NP suspensions were centrifuged to remove the unencapsulated FITC. Encapsulation was determined to be >98%, and the solution of the FITC NPs were stored for later use. NP size was determined by dynamic light scattering. For uptake studies, 4T1 cells (mouse breast tumor) were seeded at a density of 5×104 cells/well in 96-well plates. NPs (2 mg/mL) were incubated with the cells for 24 hours, with time points every two hours. At every time point, the supernatant and cells of the corresponding groups were collected and analyzed. Cells were also seeded at 5×104 cells/cm3 in culture plates, allowed to reach 70% confluence, and exposed to the FITC NPs under the same conditions for 24 hours. Cells were washed and fixed at 1, 2, 6, 12, and 24 hours and subsequently observed by fluorescent microscopy.

Results When conjugated, the peptide reduced the size of the PLAA NPs from 290 nm to 110 nm, and the PLGAA NPs from 320 to 145 nm. Distribution of the NPs was narrow, with >95% below the tumor pore cutoff size of 400 nm. When cells were cultured with the NPs, a gradual increase in the NP uptake was seen for both PLAA and PLGAA, with a 72% and 69%, respectively, of the total NPs uptaken by cells after 24 hours. We accounted for more than 95% of the initial loading of NPs in solution. This trend was confirmed by fluorescent microscopy. After 1 and 2 hours, no significant uptake could be seen for the NPs, but a fraction could be observed in close proximity to the cytoskeleton. After 12 and 24 hours, uptake was considerably more significant than the initial time points. Many of the NPs could be seen in the same location as the cytoskeleton of the cell, surrounding the nucleus. Few particles remained out of the area occupied by cells.

Discussion NPs with a very small diameter and a narrow distribution could be self-assembled by the amphiphilic-like properties of the CV6K2 peptide. Grafting of the peptide allowed for macromer assembly into a spherical organized structure, with the more hydrophobic polymer in the inside and the charged peptide on the outer surfaces of the NPs. After 24 hours, around 70% of the NPs were taken up, most likely due to endocytosis of the NPs after interaction with the cells. The positively-charged amino acids can facilitate interaction of the NPs with the negatively charged cell membrane. After a period of 24 hours, the presence of NPs inside and in close proximity to the cells indicated that uptake could be achieved by the use of these small carriers and used for delivery applications in tumor targeting.

References 1. Maltzahn, G.v., et al., Positively Charged Surfactant-like Peptides Self-assemble into Nanostructures. Langmuir, 2003. 19: p. 4332-4337. 2. He, X.Z. and E. Jabbari, Material properties and cytocompatibility of injectable MMP degradable poly(lactide ethylene oxide fumarate) hydrogel as a carrier for marrow stromal cells. Biomacromolecules, 2007. 8(3): p. 780-792. 3. He, X.Z. and E. Jabbari, Solid-phase synthesis of reactive peptide crosslinker by selective deprotection. Protein and Peptide Letters, 2006. 13(7): p. 515-518.