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Synthesis and Optimization of Novel Poly-(β-amino ester) Synthesis and Optimization of Novel Poly-( -amino ester) Polymer for Gene-Delivery Polymer for Gene-Delivery

Maria Montes

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Synthesis of PBAE-cationic polymer and optimization of

molecular weight for linkage of PBAE polymer to cell

penetrating peptide for further cell transfections.

Bossmann Lab Group,

Bridges to the Future Program,

Developing Scholars Program (DSP),

Johnson Cancer Research Center,

Funding provided by the National

Institutes of Health/National Institute

of General Medical Sciences Award

5R25GM119968-02.

Synthesis and Optimization of Novel Poly-(β-amino ester) Polymer for Gene-

Delivery. Maria Montes-Gonzalez, Obdulia Covarrubias Zambrano, Tej Shrestha, and Stefan H. Bossmann

Kansas State University Manhattan, KS

Department of Chemistry

• Polymer was successfully synthesized and characterized.

•Zeta potential experiments demonstrated optimal conditions to obtain

a positive zeta potential charge, which is ideal for cellular uptake

during a transfection.

•Cell experiments demonstrated successful transfection for 150µM

PBAE incubated for 30 min with GFP plasmid DNA.

•WTAS successfully synthesized and loaded onto cells to determine

cell penetration and toxicity.

1Kim, J.; Kang, Y.; Green, J. Stable and effective poly(ethylene glycol)-

poly(beta-amino ester) nanoparticles for gene delivery. September 1, 2018.

293.301. 2Mangraviti, Antonella, et al. “Polymeric Nanoparticles for Nonviral Gene

Therapy Extend Brain Tumor Survival in Vivo.” ACS Nano, vol. 9, no. 2, 2

Feb. 2015, pp. 1236–1249., doi:10.1021/nn504905q.3Smith, Tyrel T., et al. “In Situ Programming of Leukaemia-Specific T Cells

Using Synthetic DNA Nanocarriers.” Nature Nanotechnology, 17 Apr. 2017,

pp. 1–8., doi:10.1039/NNANO.2017.57.4Wang, Hongwang, et al. “Rationally Designed Peptide Nanosponges for Cell-

Based Cancer Therapy.”

24 hours at 90ºC

+

1,4-butanediol diacrylate 3-amino-1-propanol

Step 1

tris(2-aminoethyl) amine

Polymer Synthesis

+

Gene delivery is one of the most promising methods to treat

multiple diseases through the alteration of a genetic code to

enhance or inhibit gene expression (NIH). The potential for gene

delivery to be used as a treatment for cancerous cells is the

beginning for advanced personalized healthcare. Current methods

for cancer are costly, nonspecific, and come with major side effects

that lowers quality of life in cancer patients. Previous studies have

demonstrated Poly-(β-amino ester) (PBAE) to be biodegradable,

non-toxic, and capable to deliver payloads in a targeted manner.

For this study, PBAE was synthesized and characterized by

Nuclear Magnetic Resonance, Dynamic Light Scattering, and Zeta-

Potential. Cell experiments were conducted to determine

transfection efficiencies. Also, conjugation of PBAE with a cell

nucleus penetrating peptide (WTAS) will be investigated to

develop a more effective gene delivery system, composed of two

nanocarriers. Specifically, focus is emphasized on the advancement

of non-viral gene delivery systems that can have a general

application towards resolving current barriers in gene therapy.

WTAS Live Confocal StudiesStep 2

• Non-toxic,

• Cell penetration to cell nuclei within a couple minutes after exposure.

PBAE Polymer : WTAS : EDC : DMAP

1 : 4 : 4 : 4

2 hours at r.t.

Intermediate 1

50 µM WTAS loaded into B16F10 cells

Peptide Synthesis

(W)TAS : PLKWPGKKKKGKPGKRKEQEKKKRRTR

Fmoc

Fmoc

Fmoc

Positive zeta potential charge is ideal for

cellular uptake through endocytosis, which

was obtained only these PBAE concentrations

and incubation time with GFP plasmid DNA

Linking PBAE to WTAS

T=2 hrsT=0 hrs

Acknowledgements

Abstract

Objective

Transfection experiments Conclusion & Next Steps

References

Poly(β-amino ester)

THF

Incubation of PBAE with DNA at various concentrations and time

Cell transfection experiments in Neural stem cells (NSC)

150µM PBAE (1.05:1) incubated with 500 ng pGFP DNA for 30 min

GFP fluorescent

cells transfected

Non-fluorescent

cells

Merged images

PBAE polymer WTAS C-terminal side

Synthesis and Optimization of Novel Poly-(β-amino ester) Polymer for Gene-DeliveryRecommended Citation

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