Mapua University

School of Chemical, Biological, and Materials Engineering and Sciences

Layer by Layer immobilization of amphoteric carboxymethyl

chitosan onto biocompatible silk fibroin nanofibrous mats

Hu tu, Guomin Wy, Yang Yi, Mengtian Huang , Rong Liu,

Xiaowen Shi
 Mapua University
School of Chemical, Biological, and Materials Engineering and Sciences
 Overview of this Report

• Layer by Layer immobilization of amphoteric carboxymethyl

chitosan onto biocompatible silk fibrion nanofibrous mats

• Conclusion and Recommendation

Objective of the Study
• To use CMC as the coated material for LBL, self assembled by
considering the excellent performance and characteristics.
• To characterize amphoteric LBL modified mats by various
technique including SEM, XPS, FT-IR, Contact Angle, Zeta-
Potential and Tensile Tester.
 Electrospinning
• It is a method to produce ultrafine fibers
by charging and ejecting polymer solution
through a spinneret under high voltage.

• Shares characteristics of both electro

spraying and conventional solution dry
spinning of fibers
Silk Fibroin
• A natural polymer consist of
layers of antiparallel beta sheets.
• Its primary structure mainly
consists of the recurrent amino
acid sequence .
• It is divided itself in three
structures Silk I, II and III
Carboxymethyl Chitosan
• Is a water soluble derivative of chitosan
which has lower immunogenicity, as well
as enhanced antimicrobial property.

• It is an amphoteric polysaccharide that

can display positively or negatively
charged.
METHODOLOGY
 Formation of SF nanofibrous mats
SF mats were
SF was fabricated in immersed into 75% The mats were
previous report v/v ethanol/ water characterize
solution
Formation of LBL-assembled nanofibrous
mats

The mats were

The mats were
Sf mats were immersed in
washed by 0.1 M The washing
immersed in 1 negatively charged
NaCl aqueous procedure were
mg/mL CMC CMC solution
solution for three repeated.
solution under neutral pH
times
for 20 mins
Characterization
• Zeta Potential were determined
• Malvern Nano 3690 (Malvern • FE-SEM, Zeiss, Germany
Instruments, UK)
 Characterization

• FT-IR, Thermo Nicolet, 170-SX, • X-ray photoelectron

USA Spectroscopy (XPS, Kratos, UK)
 Characterization
• Contact Angle Meter (CAST 3.0, USA • Thermal Analyzer (SDT Q600)
Kino Industry Co., LTD ., USA)

• Tensile Tester - ETM502A

In Vitro Antibacterial Activity Study
10x10 mm^2 matswere
sterilized and UV radiated

The mats were immersed in

5mL diluted bacteria
suspension containing E.
coli and S. aeureus

Incubated in a shaker and

diluted and transferred in
a Luria Bertanis medium

5mL without mats served

as control group

Scanning Electrron
Microscopy (SEM)
Cell Proliferation and Toxicity test
Murine Mc3T3 pre-
esteoblast cells were
cultured

Rounded LBL modified mats

were sterilized with ultraviolet
radiation

Rinsed with PBS and soaked in

growth medium

100𝜇𝐿 𝑀𝐶3𝑇3 were seeded

on the scaffolds and cultured

Cell counting Kit 8 and

Microplate Reader

Scanning Electron
Microscopy
RESULTS AND DISCUSSION
Morphology Investigation

Fig. 1. FE-SEM images of electrospun SF nanofibrous mats (a) and SF mats after physical crosslinking (b).
Morphology Investigation

Fig. 2. FE-SEM images of LBL modified nanofibrous mats with different bilayers: LBL0.5 (a), LBL1 (b), LBL5
(c), LBL5.5 (d), LBL10 (e) and LBL10.5 (f).
 Composition and
Morphology Investigation
Structural Analysis

Fig. 3. The FT-IR spectra of raw materials and various nanofibrous mats (a); the XPS narrow scans of LBL5.5
with the curve fit of C1s (b) and N1s (c).
 Morphology Composition and TGA data and mechanical
Investigation Structural Analysis properties of samples

Fig. 4. The thermal properties (a) and mechanical behavior (b) of various mats.
 Morphology Composition and TGA data and mechanical The Hydrophobicity of
Investigation Structural Analysis properties of samples our mats

Fig. 5. WCA data of SF and LBL modified nanofibrous mats over times.
Bacterial Inhibition
Activity

Fig. 6. Antibacterial activity of samples against E. coli and S. aureus. Error bars represented standard deviation
(n = 3).
Bacterial Inhibition
CCK-8 assay
Activity

Fig. 7. FE-SEM images of E. coli adhered on the SF mats and LBL modified mats: SF (a), LBL0.5 (b), LBL1 (c),
LBL5.5 (d), LBL10 (e), LBL10.5 (f).
 Bacterial Inhibition
CCK-8 assay
Activity

Fig. 8. FE-SEM images of SF mats and LBL modified mats with MC3T3 cells seeded on the surface for 48 h (a–g: SF
(a), LBL0.5 (b), LBL1 (c), LBL5 (d), LBL5.5 (e), LBL10 (f), LBL10.5 (g)) and cell viability results after 24 h’s,
48 h’s and 72 h’s cultivation (h). Error bars represented standard deviation (n = 3).
Conclusions
Conclusions
• LBL modified nanofibrous mats exhibited higher thermostability and
stronger mechanical properties after coating with CMC
• The surface of mats became rough and the hydrophily of mats enhanced
after the LBL procedure.
• The antimicrobial property of LBL structured mats increased a lot with the
introduction of CMC on the surface of the nanofibrous mats.

MYRDomino