(746f) Tumor Suppression Characteristics of Doxorubicin Encapsulated in Novel Peptidomimetic Nanoparticles

Introduction The use of nanoparticles (NPs) for targeted drug delivery has been considered extensively during the past years. Measurements have shown that tumor vasculature has a pore cutoff size of 400 nm and is highly permeable (EPR effect). Chemotherapeutic agents such as doxorubicin (Dox) and paclitaxel currently used for the treatment of tumors have little or no selectivity towards tumor cells and can diffuse out of the affected region, resulting in increased system toxicity. To circumvent these problems, a nanosized, biodegradable carrier that can provide a targeted sustained release is proposed. Since Dox could induce apoptosis [1], selectivity could be surmised by an increase in apoptotic signaling. Our laboratory has created poly(lactide acrylate) (PLAA) and poly(lactide-co-glycolide acrylate) (PLGAA) NPs for this purpose. To assist in self-assembly, the peptide sequence Cys-Val-Val-Val-Val-Val-Val-Lys-Lys (CV6K2) [2], known to self-assemble by itself into NPs of about 50 nm in aqueous environment, was conjugated to the PLAA and PLGAA macromers. The objective of this work was to load Dox into PLAA-CV6K2 and PLGAA-CV6K2 NPs, test the ability of these NPs to reduce the viability of tumor cells, and provide a simple overview of their mechanism of action.

Methods PLAA and PLGAA were synthesized as described [3]. The peptide sequence CVVVVVVKK was synthesized in solid phase using Fmoc-chemistry as shown in previous work [4]. 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 dialyzing a solution of the macromers in organic solvent against PBS. Dox (5%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 Dox and resuspend the NPs in cell culture media. Encapsulation efficiency and release of Dox from the NPs in PBS were determined by spectroflourometry. The NP size was determined by dynamic light scattering. For invasion and migrations studies, 4T1 (mouse breast tumor) cells were cultured at 1.5×104 cells/well on 24-well transwell inserts. In each study, cells were exposed to empty and Dox-loaded PLAA and PLGAA NPs (5µM). Free Dox was used as a positive control. The cells were allowed to migrate through the transwell membrane (8µm pore size) in the absence (migration) or presence (invasion, 5% wt gel in media) of MatrigelTM in the upper chamber. Cells were counted after 24 hours to determine migration extent. Flow cytometry (FCM) was also performed for the NPs and the free Dox. Cells were seeded at seeding density of 5×104 cells/cm2 in 24-well plates and treated for 24 hours. Afterwards, cells were marked with propidium iodide and annexin V for one FCM assay, to determine apoptosis or necrosis pathways, and in a second assay, they were treated for mitochondrial membrane potential and caspase activity. Both assays were done for a count of 1×104 cells.

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. Encapsulation of Dox in the NPs was 79 and 86% for the PLAA-CV6K2 and PLGAA-CV6K2, respectively. Release lasted for 24-35 days. Dox-loaded NPs showed significant decrease in migration compared with Dox alone, with 30% reduction for Dox and 69% and 75% reduction for PLAA and PLGAA NPs, respectively, compared to control. Invasion results showed a 78% reduction for Dox and 95% and 97% reduction for PLAA and PLGAA, respectively, compared to control. FCM measurements showed that Dox can induce both necrosis and apoptosis, while encapsulation of the Dox in the NPs increased the contribution of Dox to apoptosis for PLAA and PLGAA NPs, respectively. Encapsulation also showed a significant effect in caspase activity and reduced mitochondria potential compared to Dox by itself.

Discussion PLAA and PLGAA NPs were self-assembled by the amphiphilic-like properties of the CV6K2 peptide to a narrow distribution. The PLGAA, being more hydrophilic, allowed for higher water uptake, and thus a higher Dox diffusion and matrix degradation. Invasion and migration of the 4T1 cells indicates that enhanced uptake and continuous release of Dox from the NPs can increase cytotoxic activity of Dox. FCM results confirmed that encapsulation enhances apoptosis as compared to cell death by extrinsic effects. Since Dox acts by intercalating with the DNA structure and inducing apoptosis, the results suggest that the NPs enhanced uptake and apoptotic pathways.

References 1. Gewirtz, D.A., A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Biochem. Pharmacol., 1999. 57: p. 727-741. 2. Maltzahn, G.v., et al., Positively Charged Surfactant-like Peptides Self-assemble into Nanostructures. Langmuir, 2003. 19: p. 4332-4337. 3. He, X. 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. 4.He, X. and E. Jabbari, Solid-phase synthesis of reactive peptide crosslinker by selective deprotection. Protein and Peptide Letters, 2006. 13(7): p. 515-518.