Development of Collagen-Binding EGCG-Encapsulated PNIPAAm

Results

Materials and Methods

Development of Collagen-Binding EGCG-Encapsulated PNIPAAm Nanoparticles for Applications to Strain Injury Chinwendu Chukwu1, Joseph Freeman PhD1

1Rutgers, the State University of New Jersey, Piscataway NJ

Abstract

Background

Future Experimentation

References

Biomedical Engineering

Tendon and ligament (T/L) are dense connective tissuescomposed of cells and a dense extracellular matrix (ECM).Within the ECM, collagen is the most abundant protein,comprising the main structural component of the connectivetissue. The prime treatment strategy to repair sub-failure T/Linjuries is rest, ice, compression, and elevation. This can beslow and insufficient for restoration of mechanical properties.During T/L wound healing, new collagen and crosslinks aredeposited to the wound bed and form a matrix for ECMremodeling and new tissue growth. That said, the formationand organization of the collagen matrix is essential forsuccessful wound healing and regeneration of the scar tissue’smechanical strength. This study focuses on the development ofan injectable treatment to strengthen the ligament to preventfurther injury during the proliferative phase of wound healing.The development of an injectable thermoresponsive implant isfurther explained.

Components of the Collagen-Binding System :

• Biocompatible thermoresponsive polymer

• Induces mechanical strain response of collagen fibers

Poly(N-isopropylacrylamide) (PNIPAAm)

• A major polyphenol derived from green tea

• Assist in modulating inflammation & clearance of NPs

Epigallocatechin Gallate (EGCG)

• Chitosan and the carbodiimide coupling agents, EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide] and NHS (N-hydroxysuccinimide), facilitate the formation of covalent bonds between tissue proteins

• The delivery system is primed with these agents to achieve adhesion between the nanoparticles and collagen

Chitosan and Carbodiimide Coupling Agents

1. PNIPAAm NPs attach to collagen fibers

2. NPs contract upon heating, pulling the collagen

3. EGCG is absorbed into the ECM

4. Cross-linking of collagen network proceeds

Figure 1. Mechanism of action of collagen-binding nanoparticle drug delivery system.

Synthesis of the PNIPAAm Nanoparticles:

Mix NIPAAm, BIS, and SDS in

DI water

Stir mix under nitrogen gas for 30 min

Add initiator solution to the reaction flask

Stir reaction solution for 4

hr at 70C

Dialyze the nanoparticle

solution for 5d

Lyophilize the solution

Collect the nanoparticles

Figures 2-5. Synthesis process of the PNIPAAm nanoparticles. The nanoparticles were synthesized via free-radical polymerization of N-isopropylacrylamide (NIPAAm) monomer with potassium persulfate (KPS) initiator. The reaction solution was stirred at 70C for 4hr (Fig. 2) and left to cool to room temperature (Fig. 3). Once at room temperature, the solution was dialyzed (Fig. 4) for 5 to 7 days, followed by lyophilization. The nanoparticles are collected following lyophilization (Fig. 5).

Acronyms: BIS - N,N′-Methylenebisacrylamide, SDS – Sodium Dodecyl Sulfate, DI - deionized

• Prime the PNIPAAm nanoparticles with chitosan and carbodiimide coupling agents to create an adhesive nanoparticle surface

➢ Measure the binding affinity of the modified nanoparticles to collagen I fibers

• Encapsulate EGCG on adhesive nanoparticles ➢ Measure EGCG load and release properties

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4. Kapoor, M., et al (2004). The American Journal of Pathology, 165(1), 299–307. https://doi.org/10.1016/s0002-9440(10)63297-x

5. Lipman, K., et al (2018). Drug Design, Development and Therapy, Volume 12, 591–603. https://doi.org/10.2147/dddt.s154660

Figure 6. Microscopic images of the PNIPAAm nanoparticles. Images were captured at a micron scale through the Leica Application Suite (LAS) software. Based on formulation, the nanoparticles were expected to be 100 nm in size; however, the images indicate that the synthesized nanoparticles are larger in size. This may be due to aggregation of the particles or may indicate incorrect concentrations of the monomer or initiator measured during the particle synthesis.

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