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Purpose

Recent studies have shown cell-penetrable lysine dendrimers as promising carriers for targeted anti-cancer drug delivery due to their unique internalization mechanism and biodegradability [1]. In this project, the internalization of dendrimer-doxorubicin conjugates was visualized through confocal microscopy.

Introduction

Experimental Methods

References

Results & Discussions

Conclusions

Acknowledgement

 

Cellular Internalization of G3 Pegylated Poly-L-Lysine Dendrimer with Doxorubicin

Joanna Lum1, Yewon Pak2, Peter W. Swaan21. UMBC, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250

2. Department of Pharmaceutical Sciences, Bio- and Nanotechnology Center, University of Maryland, Baltimore, MD 21201

VISUALIZATION OF G3 PLL-DOX UPTAKE

Confocal laser scanning microscopy of MCF-7 cells after incubation with free-DOX (a, b, c), G3 PLL_DOX nanoparticles (d, e ,f). The scale bars correspond to 25 μm in all images.

Cytotoxicity of free-DOX and G3 PLL-DOX

• Doxorubicin (DOX) is an anti-cancer drug that exerts its function by inhibiting the progression of the enzyme topoisomerase II, which relaxes supercoils in DNA for transcription.

• The penetration into cancer cells is usually limited due to drug resistance and low circulating half-life of chemotherapeutic drugs [2].

• Targeted drug delivery can improve drug efficacy and minimize undesired side effects.

• Dendrimers are favored over traditional linear polymers because of their:• Biodegradability• Branched structure which facilitates

endocytosis• Lower toxicity

• PLL dendrimer, shown in figure 1, was synthesized by reacting L-Lysine methyl ester with ONP-Lys-(BOC)2 through a process of deprotection and addition by previous work of graduate student, Yewon Pak.

CELL CULTUREBreast Cancer MCF-7 cell line was obtained and cultured in media supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and streptomycin in a humidified incubator with 5% CO2 at 37oC.CYTOTOXICITY ASSAYThe cellular toxicity of free doxorubicin and lysine-doxorubicin conjugates was determined by measuring the inhibition of cell growth using a WST-1 assay. The percentage cell viability was then determined.CONFOCAL LASER SCANNING MICROSCOPY (CLSM) STUDY MCF-7 cells were seeded onto clean and sterile coverslips placed in a 4-chamber slide at an initial density of 2 x 105 cells/well and cultured overnight. The cells were then treated with free-DOX and Lysine-DOX conjugates for 4 and 8 hours (equivalent DOX concentration: 30 μg/ml), respectively. After incubation, the cells were fixed, stained and observed by a confocal microscope.

Fig. 3: Confocal laser scanning microscopy of MCF-7 cells after incubation with HBSS buffer (A,B) free-DOX (C, D), G3 PLL-DOX nanoparticles (E,F). All cells were fluorescently stained with DAPI.

• G3 Pegylated PLL Dendrimer-DOX showed greater inhibition at lower concentrations in MCF-7 cells relative to free DOX

• G3 Pegylated PLL Dendrimer-DOX was endocytosed into small and punctate vesicles. The vesicles were shown to traffic towards the nucleus where they could eventually inhibit DNA replication of tumor cells.

• Poly-L-Lysine dendrimer can be used a prospective vehicle for sustained drug release targeted to cancer cells.

[1] Zhao, J., Zhou, R., Fu, X., Ren, W., Ma, L., Li, R., Zhao, Y., Guo, L. (2000). Cell-penetrable lysine dendrimers for anti-cancer drug delivery: synthesis and preliminary biological evaluation. AAPS PharmSciTech. [2] Sun, Y., Zhang, J., Han, J., Tian, B., Shi, Y., Ding, Y., . . . Han, J. (2016). Galactose-Containing Polymer-DOX Conjugates for Targeting Drug Delivery. AAPS PharmSciTech.

• Fig. 2: The half maximal inhibitory concentrations (IC50) showed the effectiveness of G3 PLL-DOX and DOX with increasing concentration. This measurement indicated that 1.30 μM of DOX was needed, while only 0.87 μM G3 PLL-DOX was needed to achieve the same level of inhibition.

• Fig. 1: Final Structure of G3 Pegylated PLL Dendrimer with DOX

IC50: 0.87μM IC50: 1.30μM

This work was supported by a funding from the UMBC Office of Academic and Pre-Professional Advising, and a UMB-UMBC partnership.

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DOXHBSS G3 PLL-DOX

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