Review

Near-infrared light-controllable on-dem

and
antibiotics release using thermo-sensiti
ve
hydrogel-based drug reservoir for com
bat-
Ge Gao1, Yao-Wen Jiang1, Hao-Ran Jia, Fu-Gen Wu
ing bacterial infection

Muhammad Arfan
23218110
 Outline
Background

Materials

Methods

Results

Summaries
Background
Backgrounds
MDR Bacteria Infections
Abscess Incission Drainage
Backgrounds
Prevention :
Treatment with proper dose and in the focal area

Treatments :
• Antibacterial agents
• High temperature (local heat by photothermal effect)
• Right amount of antibiotics
Backgrounds
Ideal Treatment

Drug release in an on-demand fashion in the focal area with antibiotics and local hyper
thermia.

Antiobiotic : Ciprofloxacin (potent)

High Temperature : < 50 °C (to reduce normal cells necrosis)
On-demand drug release : Stimuli-responsive component (Near-Infra red light)
Backgrounds
PDA NPs-Cip/GC Hydrogel
(Gel-Cip)

Ciprofloxacin (Cip)

Polydopamine Nanoparticle
(PDA NPs)

Glycol Chitosan
(GC)
Backgrounds

Ciprofloxacin
An antibiotic to treat a number of bacterial infection
Backgrounds

PDA Nps
Water dispersible, melanin-like material
(high compatibility).The aromatic ring enable
the adsorption of Cip via 𝜋- 𝜋 stacking
(attractive, non-covalent interactions)
& hydrogen bonding interaction
Backgrounds
GC
Its positive charge can be cross-linked with PDA NPs to form an injectable hydrogel
Materials
Materials
GC & Cip
DPA NPs & (3-(4,5-dimethyl-2-thiazolyl)-
2,5-diphenyl-2-H-tetra-Zolium bromide) (MTT)
Ethanol & Ammonium hydroxide solution
Staphylococcus aureus, Micrococcus luteus,
Escherichia coli & Proteus vulgaris
Human normal lung cells (AT II)
: Sigma Aldrich (St. Louis, USA)
: J&K Chemical Ltd (China)
: Aladdin Chemistry Co. Ltd (China)
: China Center of Industrial Culture
Collection (CICC, Beijing, China)
: Medical School of Southeast Univer-
sity
Methods
Methods
Antibacterial Effect
Plate method, live/dead staining assay, scanning electron microscopy, and bacteriostatic ring test.

Antibacterial Activity – Wound Healing

S. aureus infected mouse & mouse skin model

In vivo biosafety
Hispatological Examination & Blood Biochemistry Analysis Of mouse model
Methods
Methods
Methods
PDA NPs
420 μL ammonium hydroxide + 2.4 mL Ethanol in 5.4 mL Deionized Water (magnetic
Stirring).
30 mg Dopamine hydrochloride dissolved in 600 μL of DI Water was added into the above solut
ion.
After 12 H, was then purified by dialysis membrane for 48 H.
The morphology was investigated by an electron microscope.

Ammonium Ethanol
hydroxide

DI Water PDA NPs

Ethanol
Methods
PDA NPs/GC Hydrogel
10 mg GC & 4 mg PDA NPs were dissolved in 1 mL DI Water (5 mins stirring)
After 48 H, the above mixture turned into PDA NPs/GC hydrogel

GC

DI Water PDA NPs

PDA NPs
Methods
PDA NPs loaded with Cip
50, 100, or 200 μg of Cip + 2 mg/mL PDA in 1 mL DI Water.
After 24 H, centrifuged 14000 rpm (5 mins) then washed 3x in DI Water.
PDA NP-Cip was dissolved in water (till 50 μg/mL).
A Spectrofluorophotometer was used to quantify the Cip in the PDA Nps.
[𝑤𝑒𝑖𝑔ℎ𝑡 𝐶𝑖𝑝 𝑜𝑛 𝑃𝐷𝐴 𝑁𝑃𝑠]
The ratio was calculated : Cip Loading ratio = x 100%
[𝑤𝑒𝑖𝑔ℎ𝑡 𝐶𝑖𝑝 𝑜𝑛 𝑃𝐷𝐴 𝑁𝑃𝑠+𝑤𝑒𝑖𝑔ℎ𝑡 𝑃𝐷𝐴 𝑁𝑃𝑠]

Cip

DI Water
Spectrofluoro- Cip loading
photometer
ratio
PDA NPs
Methods
PDA NP-Cip/GC Hydrogel (Gel-Cip)
10 mg GC + 4 mg PDA NP-Cip (with different ratios) were dissolved in 1 mL DI Water.
Gel-Cip was formed after vortexing for 5 mins.

GC

DI Water Gel-Cip

PDA NP-Cip
Methods

Preparation and characterization of Gel, PDA NP-Cip, and Gel-Cip. (a) Photographs of GC solution (10 mg/mL),
PDA NP sol-ution (4 mg/mL), and Gel. (b) SEM images of Gel (left, scale bar: 40 μm) and the enlarged area (rig
ht, scale bar: 400 nm).
Methods(Antibacterial Effect)
MTT Assay
Was used to evaluate the in vivo cytotoxicity of Gel-Cip.
• 96 well-plate were seeded with AT II Cells (density of 5x104 cells/mL).
• After 24 H incubation, the medias were replaced with new culture medium with PDA NPs
(concentrations of : 0, 0.2, 0.5, 1, 2, or 4 mg/mL) or GC (0, 1, 3, 5, 10, or 15 mg/mL).
• After 24 H, washed with phosphate-buffered saline (PBS) 3x, then replaced with new
medium. Then, 10 μL of 5 mg/ml MTT solution was added each well.
• After 4 H, the original medium was removed and 150 μL of Dimethyl Sulfoxide (DMSO) w
as added into each well.
• Citotoxicity was evaluated after it was immersed in Complete Dulbecco’s Modified
Eagle’s Medium 1 mL overnight.
Methods(Antibacterial Effect)
AT II Cells
DMSO

Plates PDA NPs or

GC

MTT Solution
Methods(Antibacterial Effect)
Live/Dead Staining Assay
S. aureus was co-stained with green fluorescent nucleic acid (SYTO 9) & red fluorescent
Nucleic acid (propidium iodide, PI) for 20 mins.
After washing 3x with normal saline, the samples were recorded with a confocal microscope

SYTO 9

NaCl Confocal
Bacteria Samples
Solution Microscope

PI
Methods(Antibacterial Effect)
Morphology of Treated Bacteria Through Electron Microscope
Bacteria after different treatments were collected by centrifugation (6000 rpm, 5 mins) and
fixed with 2.5 % glutaraldehyde overnight.
After washing 3x with normal saline, bacteria were dehydrated through sequential treat-
ments (30%, 50%, 70%, 80%, 90%, and 100%) ethanol solutions (10 mins) and were centri-
fuged (5 mins).
The morphology was observed using a scanning electron microscope.

Glutaral-
dehide

Electron Bacteria
Bacteria Ethanol
Microscope Morphology
Methods(Antibacterial Effect)
Bacteriostatic Ring Test (Oxford Cup)
100 μL S. aureus was plate onto agar plate.
The oxfor cup containing 100 μL Gel-Cip was transferred to
the agar plates, followed by 808 NIR lase (0.5 W/𝑐𝑚2 ) 10 mins.
4 groups were compared (NIR, Gel, Gel+NIR, and Gel-Cip).
After 48 and 96 H, the diameters of the inhibition zone were
measured.

Bacteria

Diameter of
NIR Agar Plate
Inhibition Zone
Methods(Antibacterial Activity)
S. Aureus Infected Mouse Model (in vivo)
6 weeks old female mice were injected with 100 μ L
S. Aureus in the right back. After 2 days infection,
divided into 11 groups and treated with :
PBS (60 μL, 2 groups), Cip (60 μL, 2 groups), Gel-Cip
(60 μL, 2 groups), PDA-NP-Cip (60 μL, 1 group).
Then each group was radiated with 808 nm NIR laser (10 mins).
After 24 H, mice were photograpped and sacrificed.
The infected tissues were collected and homogenized with dilution factor of : 10, 100, 1000,
and 10000) and plate on agar plate.
After incubation overnight, colonies were counted to be evaluated.
Methods(Antibacterial Activity)
S.aureus

Right back
of the mice

PBS, 2
Cip, 2 Colonies
Gel-Cip, 2
PDA Np-Cip, 1 counted

NIR
Methods(Antibacterial Activity)
Mouse Skin Defect Model (wound healing)
1 day before the infection, the mice were disinfected with
75% Ethanol solution on the right back, and shaved (1 x 1 𝑐𝑚2 ).
S.Aureus cells were diluted 10x and washed 3x with normal
saline. The wounds inoculated with 100 μL S.aureus (1 x 106
CFU/mL). After D-1 infection, mice treated with NIR, PDA NPs,
PDA NPs+NIR, Gel, Gel+NIR, Cip, Cip+NIR, Gel-Cip, PDA NP-Cip+NIR, or Gel-Cip+NIR.
The wounds observed everyday.
Methods(Antibacterial Activity)

Ethanol
NIR
PDA NPs
PDA NPs+NIR,
Gel,
Right back of
Gel+NIR, Wound
S.aureus mice
Cip,
Cip+NIR, Observation
Gel-Cip,
PDA NP-Cip+N
IR, or
Gel-Cip+NIR
Methods(In vivo biosafety)
Hemanalysis & Biochemical Analysis
Blood samples of mice after treatments were evaluated
Using an automatic hematology analyzer (BC-2800Vet,
Mindray, China) and biochemical analysis using an automated
biochemical analyzer (SMT-100 V, Seamaty, China)
(White Blood Cells, Neurophillic Granulocyte, Red Blood Cells,
Hemoglobin, Mean Cell Volume, Mean Corpuscular Hemoglobin, Mean Corpuscular Hemogl
obin Concentration, Blood Platelet, Liver Function Parameter, Aspartate Transferase, Renal
Function Parameters, and Creatinine.
Methods(In vivo biosafety)

WBC,
Gran,
automated RB,
biochemical HGB,
analyzer MCV,

Blood samples
MCH, Blood
NCHC,
BP, Analysis
LVP,
automatic hematology AT,
analyzer RFP,
Creatin,
Methods(In vivo biosafety)
Histological Analysis
After in vivo wound healing treatments, the mice were sacrificed,
and the skin tissues were collected, fixed with 4%paraformaldehyde
Solution, embedded into paraffin, and stained with hematoxylin
and eosin (H&E).
Major organs were also collected and compared with the normal
uninfected mice as a control.
Methods(In vivo biosafety)

Paraformal
dehyde

Skin tissues/
Paraffin Major organs Comparation

H&E
Results
Results

(c) UV–vis spectra and (d) zeta potentials of PDA NPs loaded with different quantities of Cip. The drug (Cip) loading ratio = WCip/(WCip + WPDA NPs
) × 100% (W denotes weight). (e) Frequency-dependent shear rheology of Gel-Cip. (f) Shear rate-dependent viscosity changes of Gel and Gel-Cip. In
set: The photograph of the hand-written letters “gel” on a slide using a needle containing Gel-Cip. (g) Temperature change of Gel-Cip irradiated with a
n 808 nm laser at different power densities (1.6, 1.0, or 0.5 W/cm2) for 10 min (h) NIR light-triggered Cip release from Gel-Cip. Gel-Cip was irradiated
with an 808 nm NIR laser (0.5 W/cm2) for 10 min followed by an interval of 30 min each time.
Results

(a) Photographs of the agar plates and (b) the corresponding statistical histogram of S. aureus colonies without treatment (Control)
and with different treatments (NIR, PDA NPs, PDA NPs + NIR, Gel, Gel + NIR, Cip, Cip + NIR, Gel-Cip, or Gel-Cip + NIR). Data are
presented as mean values ± SD from three parallel experiments per group (n = 3).
Results

(c) Confocal fluorescence images of S. aureus stained with SYTO 9/PI dyes after different treatments (NIR, PDA NPs, PDA NPs +
NIR, Gel, Gel + NIR, Cip, Cip + NIR, Gel-Cip, or Gel-Cip + NIR). Scale bar: 25 μm.
Results

(c) Confocal fluorescence images of S. aureus stained with SYTO 9/PI dyes after different treatments (NIR, PDA NPs, PDA
NPs + NIR, Gel, Gel + NIR, Cip, Cip + NIR, Gel-Cip, or Gel-Cip + NIR). Scale bar: 25 μm.
Results

(a) SEM images of S. aureus cells without treatment (control) and with different treatments (NIR, PDA NPs, PDA NPs + NIR, Gel,
Gel + NIR, Cip, Cip + NIR, Gel-Cip, or Gel-Cip + NIR). Scale bar: 1 μm.
Results

(b) Photographs and (c) the corresponding statistical histogram of the inhibition zones of S. aureus, M. luteus, E. coli, and
P. vulgaris cells with different treatments (A: NIR, B: Gel, C: Gel + NIR, D: Gel-Cip, and E: Gel-Cip + NIR) after 48 h of culture.
Data are presented as mean values ± SD from three parallel experiments per group (n = 3)
Results

In vivo antibacterial tests. (a) Protocol for the establishment of bacteria-infected model (including bacterial inoculation and different t
reatments before harvest). (b) Photographs of S. aureus-infected mice without treatment (control) and with different treatments as i
ndicated at day 4 (the frst row), and photographs of bacterial colonies derived from the homogenized tissue dispersions (with differe
nt dilution factors) of the infected sites of mice without treatment (control) or with different treatments as indicated. (c) The correspo
nding statistical histogram of bacterial colonies in (b). Data are presented as mean values ± SD from three parallel experiments per
group (n = 3).
Results

In vivo antibacterial tests. (a) Protocol for the establishment

of bacteria-infected model (including bacterial inoculation an
d different treatments before harvest). (b) Photographs of S.
aureus-infected mice without treatment (control) and with di
fferent treatments as indicated at day 4 (the frst row), and p
hotographs of bacterial colonies derived from the homogeni
zed tissue dispersions (with different dilution factors) of the i
nfected sites of mice without treatment (control) or with diffe
rent treatments as indicated. (c) The corresponding statistic
al histogram of bacterial colonies in (b). Data are presented
as mean values ± SD from three parallel experiments per gr
oup (n = 3).
Results

In vivo antibacterial tests. (a) Protocol for the establishment

of bacteria-infected model (including bacterial inoculation an
d different treatments before harvest). (b) Photographs of S.
aureus-infected mice without treatment (control) and with di
fferent treatments as indicated at day 4 (the frst row), and p
hotographs of bacterial colonies derived from the homogeni
zed tissue dispersions (with different dilution factors) of the i
nfected sites of mice without treatment (control) or with diffe
rent treatments as indicated. (c) The corresponding statistic
al histogram of bacterial colonies in (b). Data are presented
as mean values ± SD from three parallel experiments per gr
oup (n = 3).
 Results

In vivo wound healing study. (a) In vivo wound healing experimental outline. (b) Photographs of S. aureus-infected wounds of mice ta
ken at different time points after various treatments as indicated and H&E-stained images of wound tissue sections in the mice at day
4 after different treatments (yellow arrows: blood vessels, green arrows: hair follicles). For clearer images, please refer to the enlarge
d ones in Fig. S17. (c) The corresponding statistical diagram of wound area healing rates of mice in (b). Data are presented as mean
values ± SD from three parallel experiments per group (n = 3). ***P < 0.001, one-way ANOVA. (For interpretation of the references to
color in this fgure legend, the reader is referred to the Web version of this article.)
Results
In vivo wound healing study.
(a) In vivo wound healing ex-
perimental outline. (b) Photog
raphs of S. aureus-infected w
ounds of mice taken at differ-
ent time points after various
treatments as indicated and
H&E-stained images of woun
d tissue sections in the mice
at day 4 after different treatm
ents (yellow arrows: blood ve
ssels, green arrows: hair follic
les). For clearer images, plea
se refer to the enlarged ones
in Fig. S17. (c) The correspon
ding statistical diagram of wo
und area healing rates of mic
e in (b). Data are presented a
s mean values ± SD from thre
e parallel experiments per gro
up (n = 3). ***P < 0.001, one-
way ANOVA. (For interpretati
on of the references to color i
n this fgure legend, the reade
r is referred to the Web versio
n of this article.)
Results
In vivo wound healing study.
(a) In vivo wound healing ex-
perimental outline. (b) Photog
raphs of S. aureus-infected w
ounds of mice taken at differ-
ent time points after various
treatments as indicated and
H&E-stained images of woun
d tissue sections in the mice
at day 4 after different treatm
ents (yellow arrows: blood ve
ssels, green arrows: hair follic
les). For clearer images, plea
se refer to the enlarged ones
in Fig. S17. (c) The correspon
ding statistical diagram of wo
und area healing rates of mic
e in (b). Data are presented a
s mean values ± SD from thre
e parallel experiments per gro
up (n = 3). ***P < 0.001, one-
way ANOVA. (For interpretati
on of the references to color i
n this fgure legend, the reade
r is referred to the Web versio
n of this article.)
Results
Biosafety evaluation. (a–h) Blood
hemanalysis of white blood cells
(WBC), neutrophilicgranulocyte
(Gran), red blood cells (RBC),
hemoglobin (HGB), mean
cell volume (MCV), mean corpus-
cular hemoglobin (MCH), mean
corpuscular hemoglobin concen-
tration (MCHC), and blood platelet
(PLT). (i–l) Biochemical analysis
of alanine transferase (ALT), as-
partate transferase (AST), blood
urea nitrogen (BUN), and creatin-
ine (CRE). Data are presented as
mean values ± SD from three
parallel experiments per group
(n = 3). (m) H&E-stained tissue
slices of major organs (hearts, live
rs, spleens, lungs, and kidneys) in
normal uninfected mice (control)
and infected mice after treatment
of Gel-Cip + NIR. Scale bar: 200 μ
m.
Results
Biosafety evaluation. (a–h) Blood
hemanalysis of white blood cells
(WBC), neutrophilicgranulocyte
(Gran), red blood cells (RBC),
hemoglobin (HGB), mean
cell volume (MCV), mean corpus-
cular hemoglobin (MCH), mean
corpuscular hemoglobin concen-
tration (MCHC), and blood platelet
(PLT). (i–l) Biochemical analysis
of alanine transferase (ALT), as-
partate transferase (AST), blood
urea nitrogen (BUN), and creatin-
ine (CRE). Data are presented as
mean values ± SD from three
parallel experiments per group
(n = 3). (m) H&E-stained tissue
slices of major organs (hearts, live
rs, spleens, lungs, and kidneys) in
normal uninfected mice (control)
and infected mice after treatment
of Gel-Cip + NIR. Scale bar: 200 μ
m.
Summaries
Summaries
1. Gel-Cip+NIR succeed in killing bacteria (colonies reduced-in
vivo & in vitro)
2. Photothermal effect didn’t cause a necrosis to cells and organs
3. Wound treated with Gel-Cip healed faster compared to another
treatments
4. The Gel-Cip treatment was safe for in vivo (blood and organ
analysis)
Weakness
1. Treatment on the surface area only
2. Not implemented on the animals who are identically closer to
human (chimpanzee)
3. Not implemented on the MDR bacteria strains.
Potentials
1. Used to treat wounds on Diabetes Mellitus patient.
2. Used on an organ (not in the surface of skin)
3. Used to kill MDR bacteria.
Thank you