Ph-responsive Hydrogel Loaded With Insulin As A Bioactive Dressing For Enhancing Diabetic Wound Healing

Presented by: –
Snehankit S.
Gurjar
Department: -
M. Pharm. IInd
year
(Pharmaceutics)
Guide: – Dr. V. P.
Wankhade

1. Introduction
• Diabetes mellitus (DM) is a growing public health concern throughout the world. Diabetic foot ulcers (DFUs)
are one of the most serious complications of diabetes, which occur in approximately 15 % of the diabetic
population.
• In the pathophysiological studies of DFUs, diabetic peripheral vascular disease and neuropathy are the two main
factors involved to affect the healing of DFUs. In these conditions, prolonged chronic inflammation plays an
inhibitory role in the repair of non-healing ulcers.
• Impaired granulocytic, chemotactic, and macrophage function, as well as the inhibited secretion of growth
factors and deregulated neovascularization occur.
• There is a risk of amputation in some extreme cases. The treatment of DFUs currently focuses on dressings that
prevent microbial infiltration and maintain a balanced moisture and gas exchange environment.
• For these chronic wounds, a bioactive dressing is required to be changed frequently, and various commercially
available dressings have not reached the desired anticipation.
• Therefore, more effective therapeutic approaches are urgently needed.
• Insulin, a hormone secreted by the pancreas, helps maintain blood glucose levels within the normal range.
Recently, increasing evidence has demonstrated that insulin contributes to wound healing. Insulin can promote
the re-epithelialization of damaged skin by stimulating the migration and proliferation of keratinocytes.
• In addition, insulin can stimulate the migration and tube formation of endothelial cells, which helps improve
angiogenesis during wound healing.

• As an ideal wound dressing, hydrogels are presented with many advantages including removing wound
exudate, providing a moist wound environment, preventing secondary infections, promoting cell proliferation
and differentiation, and accelerating tissue regeneration. These elements help create an optimal environment to
efficiently enhance wound healing. Previous studies also indicated that insulin had the ability to stimulate
collagen deposition and maturation with fibers organized like a basket weave (normal skin), rather than aligned
and cross-linked (scar tissue), which not only accelerated collagen deposition but also contributed to the quality
of healing.
• The major challenges of the topical administration of insulin are its short half-life and loss of bioactivity in the
peptidase-rich wound environment, especially in DFUs. An alternative strategy to overcome these issues is to
prepare biocompatible wound dressings for sustained delivery of insulin during the wound healing process.
• The controlled drugs delivery systems have widely used currently in the field of biomedical engineering, and
become a hotspot of research gradually.
• The study for smart drug delivery hybrid materials for healthcare applications mainly focus on developing
novel synthetic methodologies, precise physicochemical and biological characterization, greater understanding
of drugs release mechanism, tracking of new materials and products of their degradation in human body after
administration.
• In particular, the biocompatibility and biodegradability are critical for in vivo application, which ensure the
materials degradation inside the body and produce nontoxic natural byproducts can be easily eliminate.

• Hydrogels, whose structure is similar to the natural extracellular matrix, have been considered promising
biomaterials to deliver drugs or cells to promote healing.
• Researches have concerned with the loading of bioactive substances on hydrogel dressings to improve the
healing of DFUs, whereas few studies have taken the specific pathophysiological environment of the diabetic
wound into consideration, such as its acidic pH and high levels of glucose. Therefore, smart hydrogels
containing ‘‘sensor” moieties that can respond to environmental pH and regulate glucose concentration are ideal
choices for the development of wound dressings as medicated systems for DFUs healing.
• In addition, self-healing hydrogels have the ability to rapidly and autonomously self-recover following damage
induced by external forces, which benefits the maintenance of the integrity of network structures and
mechanical properties during the wound healing.
• Moreover, the majority of self-healing hydrogels are injectable, which is another attractive characteristic
favoring the nonsurgical treatment of patients using a mini-invasive medical approach, especially for irregular
trauma present in deeper regions.
• Thus, environmentally sensitive, self-healing, and injectable hydrogels formulated as drug-sustained release
systems can provide a promising therapy for DFUs treatment. In this study, we developed a self-healing and
injectable polysaccharide-based hydrogel characterized by pH-responsive long-term insulin release. The overall
synthetic process of the insulin-loaded hydrogel formulation is illustrated in Scheme 1A.

• The multifunctional hydrogel is composed of N-carboxyethyl chitosan (N-chitosan) and adipic acid
dihydrazide (ADH), which are crosslinked in situ by hyaluronic acid–aldehyde (HA-ALD).
• Such self-healing and injectable properties are particularly appealing for skin wound repair because they help
reduce gel fragmentation and integrate ruptured gels at the target site, even after external mechanical destruction,
and hence can continuously support skin wound healing.
• Although some self-healing and biocompatible hydrogels have been used for wound healing, they lack the
intelligent response to the specific microenvironment of diabetic wounds to release beneficial factors to optimize
wound healing.
• Herein, we expect that the self-healing hydrogel releases insulin with a pH-sensitive manner can decrease
glucose levels, promote wound healing, and improve peripheral neuropathy, which are highly attractive for
diabetic skin wounds (Scheme 1B).
• To the best of our knowledge, this is the first study to investigate the diabetic microenvironment responsive, self-
healing, and injectable hydrogels loaded with insulin as bioactive dressings for diabetic wound therapy.

Scheme 1. Schematic illustrating the hypothesis of the approach.
(A) The overall synthetic process of the insulin-loaded hydrogel.
(B) The hydrogel releases insulin in a pH-sensitive manner to promote wound healing and to improve peripheral
neuropathy, which is highly attractive for the repair of diabetic skin wounds.

2. Materials and methods
2.1. Materials:
• Chitosan (degree of deacetylation 95%, viscosity 100–200 mps) was supplied by Aladdin (Shanghai, China).
• Sodium periodate (NaIO4) was purchased from Sigma-Aldrich.
• Hyaluronic acid (100– 200 k) and dipic acid dihydrazide (ADH) were supplied by Yuanye Biology (Shanghai,
China).
• The ultra-pure water used for all experiments was purified with a Milli-Q A10 filtration system (Millipore,
Billerica, MA, USA).
• All other chemicals were analytical grade and used without further purification.
• Insulin glargine was supplied by Sanofi-Aventis (France).
• Dulbecco’s Modified Eagle’s Medium (DMEM, low glucose), fetal bovine serum (FBS), and streptomycin-
penicillin were obtained from Gibco Life Technologies (USA).
• Paraformaldehyde solution (4%) and phosphate buffer saline (PBS) were purchased from Xilong Chemical Co.,
Ltd (Guangxi, China).
• The immortal human keratinocyte line HaCaT was supplied by ATCC (USA).
• The Cell Counting Kit-8 assay (CCK- 8) was purchased from Beibo (China) and the live/dead assay was supplied
by Biobe times Biotechnology Co., Ltd (Changsha, China).
• Streptozotocin (STZ) was obtained from Sigma-Aldrich (MO, USA), and the Bradford protein assay kit was
provided by Beyotime (CA, USA).
• Hematoxylin and Eosin (H&E), Masson’s trichrome stains were purchased from Thermo Fisher Scientific Co., Ltd
(Shanghai, China).
• Commercial antibodies were purchased from Abcam (MA, USA).

2.2. Hydrogel preparation and characterization
 N-chitosan was synthesized using the previously described Michael’s reaction.
• Chitosan (2.0 g, 12.4 mmol) and acrylic acid (3 mL, 12.7 mmol) were dissolved in (200 mL) deionized water
in a 500 mL three-necked round-bottom flask equipped with a stirrer, a condenser, and a nitrogen inlet.
• The reaction was stirred under a N2 atmosphere at 50 C for 72 h.
• After cooling down to room temperature, the mixture was kept at pH 11 by dropwise addition of a NaOH
solution (1 M).
• Afterwards, the solution was dialyzed (MWCO = 14000) against deionized water for 3 days with repeated
changes of water, freezing, and lyophilizing.
 HA-ALD was prepared as follows.
• Briefly, HA (1.0 g, 5 mmol) was dispersed in distilled water (100 mL).
• After that, sodium periodate (NaIO4) was added to the reaction flask and magnetically stirred in the dark at
room temperature for 3 h.
• The reaction was stopped by adding 10% (v/v) ethylene glycol.
• Afterwards, the products were dialyzed (MWCO = 3500) against water for 3 days, followed by freezing and
lyophilizing to obtain the HA-ALD powder.

 Finally, hydrogels could be prepared by in situ cross-linking of N-chitosan and ADH with HA-ALD.
• Briefly, 7.5 % N-chitosan (w/v) and 7.5% ADH (w/v) were dissolved in deionized water.
• Next, solutions of 5 % HA-ALD (w/v) were added into the above mixture.
• For hydrogel formation by imine and acyl-hydrazone bonds, the solution was stirred by Lab Dancer to obtain a
homogeneous hydrogel. The optical images of the hydrogel before and after gelatin formation were recorded
by a digital camera (Canon, Japan).
• Rheological data were collected using TA Instruments-Waters LLC.
• In addition, the infrared spectroscopy analysis was performed by Fourier Transform Infra-Red (FTIR)
spectrometer (BLUCK spectrophotometer) to confirm imine bonds and acyl-hydrazone bonds after freezing the
hydrogel at 40 C and lyophilizing.
• The hydrogel sample was freeze-dried in a freeze dryer, and the cross-section morphology of the hydrogels was
observed on a Hitachi S-4800 scanning electron microscope (Hitachi Science Systems, Japan).

3. Results and discussion
3.1. Synthesis and characterization of the hydrogel:
• The concept of moist healing has aroused great interest and hydrogel dressings can provide a moist environment to
help wound closure.
• Herein, we prepared a self-healing hydrogel, which can be formed easily by mixing dilute solutions of N-chitosan,
ADH, and HA-ALD (as the cross-linker).
• The sol-to-gel phase transformation process is illustrated in Fig. 1A.
• Mixed polymer solutions can easily be extruded through a 26-gauge needle without clogging, to form stable
hydrogels within 30 s at room temperature (Fig. 1B).
• As shown in Fig. 1C, the visual observation of a 0.3 cm central hole in the hydrogel diminished and finally
disappeared after 3 h, demonstrating the self-healing property of the polysaccharide hydrogel.
• The gelation kinetics of the self-healing hydrogels were recorded over time.
• As illustrated in Fig. 1D, the storage modulus (G0) and loss modulus (G00) gradually increased over time.
• The G0 was found to surpass the G00 at about 30 s, indicating the presence of sol–gel transformation.
• To quantitatively measure the self-healing behaviors of the polysaccharide hydrogel, we performed rheological
recovery tests.
• As shown in Fig. 1E, the storage modulus (G0) and the loss modulus (G00) curves intersect at the strain of 320 %,
indicating that the state of hydrogel is between solid and fluid.

• With a further increase in the strain above the critical strain value, the hydrogel network is destroyed and
transitions to a solution state.
• Based on the strain-dependent oscillatory measurement results, the self-healing property of hydrogels was then
verified, and the effects of shear strain were determined by strain amplitudes of 300% and 0.1% at a fixed
frequency (1.0 Hz).
• As demonstrated in Fig. 1F, the hydrogel exhibited alternating liquid and solid behaviors with 90% recovery to
the original values of the moduli, which is completely reproducible upon additional strain cycles without any
external stimulus.
• Most importantly, this gel could become a sol under a greater strain (320%) and soon resume the gel state upon
strain removal.
• These shear-thinning and self-healing properties allow the injection of materials through a narrow needle, and an
instantaneous recovery of the initial mechanical strength of the hydrogels after injection in vitro.
• As drug delivery systems, conventional injectable hydrogels usually deteriorate or lose their mechanical strength
after injection, leading to delivery failure.
• However, our self-healing hydrogel could flow through a needle under force and maintain its stability after
injection.
• These properties are extremely desirable for many biological applications, which not only allow the encapsulation
and delivery of bioactive insulin to the target site, but also provide a three-dimensional microenvironment post-
injection for the growth and function of engineered cells for the healing of DFUs.

• The hydrogels are also characterized by attractive features owing to the reversible nature of dynamic bonds:
acylhydrazone and imine bonds derived from aldehyde groups at HA-ALD react with NH2NHCO– groups on
ADH and –NH2 groups on N-chitosan, respectively.
• These reversible dynamic bonds in hydrogels were confirmed by FTIR analysis in our previous study (Fig. S1).
Importantly, the existence of the acylhydrazone bond can release insulin encapsulated in hydrogels in a pH-
sensitive manner.
• In the acidic environment (especially at pH < 5.0), the release rates could increase notably as a result of the
broken acylhydrazone bond.
• These reversible dynamic bonds in hydrogels were confirmed by FTIR analysis in our previous study (Fig. S1).
• Importantly, the existence of the acylhydrazone bond can release insulin encapsulated in hydrogels in a pH-
sensitive manner.
• In the acidic environment (especially at pH < 5.0), the release rates could increase notably as a result of the
broken acylhydrazone bond.
• The morphology of the lyophilized polysaccharide hydrogel was characterized by SEM. As presented in Fig.
S2, the hydrogels had a porous structure.
• The pore sizes of hydrogels were about 100– 200 lm, which facilitated the migration of keratinocytes and
fibroblasts associated with skin healing, and promoted efficient exchange of nutrients and other waste.

Fig. 1. Characterization of hydrogel.
(A)The optical images of the gelation progress. The mixture of N-chitosan and ADH presented a sol state at room
temperature and underwent sol–gel transition after adding HA-ALD solution for about 30 s.
(B) The optical image of the injectable property.
(C) Visual observation of the self-healing process.

Fig. 1. Characterization of hydrogel
(D) Rheology profiles of the self-healing hydrogel (Temperature: 25 C, frequency: 10 rad s1, and strain: 0.1%).
(E) Strain amplitude sweep measurements (c = 0.1–1000%) of the hydrogel with a fixed angular frequency of 10 rad s1,
showing the gel-to-liquid transition at 320% of shear strain.
(F) Step strain measurements of the hydrogel with a fixed frequency of 10 rad s1. Each strain interval was kept as 150 s.

2.3. In vitro degradation studies
• In vitro degradation of the hydrogel at different pH values was evaluated in PBS (pH 7.4 and 6.5, 37 C) with
lysozyme (104 units/ mL).
• Hydrogel samples (1 mL) were placed in a glass vial with 2 mL PBS. Pre-weighed (Wi) hydrogel was
incubated at different time points. At predetermined time points, samples were taken out and washed with
deionized water and freeze-dried.
• Dry weights of the hydrogel were recorded as Wd. The degradation percentage was calculated by the
following equation:
% Degradation = (Wi- Wd)/Wi 100

3.2. pH response degradation and release of bioactive insulin
• In vitro degradation of hydrogel was explored in PBS containing lysozyme (104 units/mL).
• In the presence of lysozyme, after 2 and 12 days of degradation, about 88.20 ± 1.87 % and 26.30 ± 2.26 % of
the hydrogels remained at pH 7.4, while 79.47 ± 5.91 % and 3.40 ± 2.10 % remained at pH 6.5, respectively.
• As exhibited in Fig. 2A, pH decreased from 7.4 to 6.5 resulted in an accelerated degradation of the hydrogel.
• According to a previous study, the dynamic acylhydrazone bond is stable in neutral conditions.
• In contrast, it can undergo reversible reactions in a slightly acidic environment, which significantly affects the
hydrogel networks.
• This pH responsiveness is mainly owing to the swelling or hydrolysis of the hydrogel matrix because the
acylhydrazone bonds are labile at acidic pH.
• Based on the degradation profiles, the hydrogel can be almost completely degraded in 14 days.
• The rate of degradation of this hydrogel overlaps the rate of granulation tissue formation on the surface of
wounds, which would therefore avoid its adherence to wounds and interference with wound healing when
changing dressings.

Fig. 2. Bioactivity release and biocompatibility of the hydrogel.
(A)pH-sensitive hydrogels degrade with different pH values.

2.4. Insulin loading and bioactive release
• According to the protocol, 7.5 % N-chitosan (w/v) and 7.5% ADH (w/v) were dissolved in 1.5 mL deionized
water.
• Then, 1 U insulin glargine was encapsulated into the N-chitosan and ADH solution under stirring.
• Lastly, a 0.5 mL 5% HA-ALD (w/v) solution was added into the N-chitosan/ADH/insulin solution; the
solution was stirred gently and then incubated for 30 s to ensure full crosslinking of the hydrogels.
• Insulin-loaded hydrogels were soaked in PBS (0.01 M, 10 mL), by adding different doses of HCl/NaOH to
regulate the pH between 6.5 and 7.4 in a sterile glass bottle at 37 C.
• At the predetermined time points, a 5 mL volume of PBS was removed from the bottle and another 5 mL fresh
PBS was added, in order to keep the total medium volume in the bottle constant.
• The concentration of the released insulin was measured by Reversed-Phase High Performance Liquid
Chromatography (RP-HPLC, Thermo Scientific TM, USA) with a UV detector set at 227 nm.
• To evaluate the bioactivity of the released insulin, a cell scratch assay was conducted following the procedure
described previously.
• The immortal human keratinocyte cell line HaCaT was cultured in DMEM (low glucose) supplemented with
10% fetal bovine serum and 1% streptomycin-penicillin.
• The medium was replaced every 3 days, and cell cultures were maintained in a humidified incubator at 37 C
and 5 % CO2.

• After reaching 80 % confluence, cells were detached from the petri dish and seeded on 6-well plates at a
density of 2 105 cells/well in 1mL medium.
• Cells were allowed to reach confluency after 48 h, at which timepoint a single scratch was made using the tip
of a plastic disposable 20 mL pipette tip. All wells were washed twice with 1 mL of DMEM culture medium
to remove suspended cells and cellular debris.
• Next, three types of culture medium were used for further cell culture:
(a) without insulin,
(b) insulin solution from native insulin glargine at a concentration of 10-7 M, and
(c) supernatants with insulin released from hydrogel within 2 days at a concentration of 10-7 M.
• The distance across the scratch at 0 h (d0) and 24 h (d1) was measured under a phase contrast inverted
microscope (Olympus IX81, Japan).
• The relative migration distance (%) across the scratch was calculated as follows:
Relative migration distance (%) = (d0–d1)/d0 100.

3.2. pH response degradation and release of bioactive insulin
• Insulin is the most popular drug used in blood glucose control for DM.
• Herein, insulin glargine was adopted as a model drug and encapsulated into N-chitosan/HA-ALD hydrogels
at pH 7.4 and 6.5, respectively.
• The insulin release profile is demonstrated in Fig. 2B.
• Insulin was effectively encapsulated in the hydrogel and exhibited representative long-term pH-responsive
sustaine release behavior lasting for 14 days.
• Because of the existence of the acylhydrazone bond, decreasing the pH of the medium resulted in
accelerated insulin release from the hydrogels.
• To investigate the bioactivity of the released insulin, the cell scratch assay with HaCaT cells was performed.
• A scratch of approximately 800 lm in width was made on a confluent layer of HaCaT cells (Fig. 2C).

• The marginal cells migrated into the scratch to induce the gap closure.
• The scratch without insulin was closed by about 16.26 ± 1.57% after cell migration for 8 h, whereas the
scratch in the presence of native insulin or released insulin from the hydrogel was closed by about 41.52 ±
2.73% and 39.04 ± 2.57% after 8 h, respectively. It was indicated that the released insulin significantly
improved HaCaT migration and possessed similar bioactivity as that of native insulin (Fig. S3).
• The pH-sensitive hydrogel with self-healing features was conducive to the design of smart materials,
exhibiting vast potential for applications in which simultaneous multifunctionality is required.
• For instance, these pH-triggered bioactive release profiles of the hydrogel may have great significance in the
treatment of DFUs exposed to an acidic microenvironment.

Fig. 2. Bioactivity release and biocompatibility of the hydrogel
(B) Cumulative drug release from the insulin incorporated hydrogels with different pH values.

(C) Representative migration images at 0 and 8 h.

2.5. In vitro cell culture in hydrogels
• To evaluate the cell biocompatibility of the hydrogel and the bioactivity of the released insulin, the
proliferation rate of HaCaT was detected by the CCK-8 assay.
• Briefly, HaCaT was seeded on the hydrogel (hereinafter abbreviated to Gel) and on hydrogel with insulin (Gel
+ In) in 48-well plates at a density of 2 104 cells/well and incubated in 37 C and 5% CO2, and the control
group was without hydrogel (abbreviated to Con).
• After incubation for 1, 4, and 7 days, CCK-8 solution with a 10% volume of the medium was added into the
well after replacing the mediums.
• The samples were incubated at 37 C for 2 h. Then, 100 lL of the reaction solution was transferred into a new
96-well plate, and the optical density was measured at 450 nm using a microplate reader (Multiskan EX,
Thermo Fisher Scientific Inc., Shanghai, China).
• To evaluate the cell viability in the hydrogel, a live/dead assay was performed. On day 3 after HaCaT seeding,
the hydrogels were incubated with 1 mM calcein-AM for 15 min and then incubated with 1 lg/mL propidium
iodide (PI) for 5 min at 37 C.
• Next, the cell hydrogel constructs were imaged using fluorescence microscopy (Olympus IX71, Tokyo, Japan).

3.3. Biocompatibility of the hydrogel
• The biocompatibility and biological activity of the insulinloaded hydrogel on HaCaT proliferation were tested
by CCK-8 analysis.
• The results indicated that HaCaT treated with N-Chitoson/HAALD hydrogel exerted equal proliferative
capabilities to that of the control group; however, the sustained release of insulin from hydrogel significantly
promoted the proliferation of HaCaT cells (Fig. 2D).
• In addition, the cell viability evaluated by the live/dead assay indicated that the cells presented good viability
when cultured in the hydrogel matrix with or without insulin (Fig. 2E), which suggested that the hydrogels
possessed good biocompatibility and limited cytotoxicity.
• Moreover, the sustained release of bioactive insulin from the hydrogel could promote marked cell migration
and proliferation.
• A previous study confirmed that insulin could promote keratinocyte migration and proliferation in a dose- and
time-dependent manner through the insulin receptor (IR)/PI3K/Akt pathway.
• Herein, the hydrogel can maintain the bioactivity of released insulin and represent an excellent delivery system
for the sustained release of insulin.

(D) Population of cells cultivated in the hydrogel and insulin-loaded hydrogel for 1, 4, and 7 days.

(E) Fluorescence microscope images of HaCaT cells cultivated in the hydrogels for 1 and 4 days (*p < 0.05).

2.6. Diabetic rat model and wound healing examination
• Animal experiments were conducted according to the National Institutes of Health’s Guide for the Care and
Use of Laboratory Animals (NIH Publications No.8023, revised 1978).
• The animal handling protocol was approved by the Animal Care and Use Committee of The Second Hospital
of Jilin University. Adult male prague-Dawley (SD) rats (250 g, 8-weeks-old) were obtained rom the Animal
Center, College of Basic Medical Sciences, Jilin University.
• All rats were housed in standard plastic rodent cages under an artificial controlled environment (25 C; 12-h
light/dark cycle, humidity 50–70% and ad libitum).
• Type 1 diabetic rats were induced by a single intraperitoneal injection of streptozotocin (STZ, 65 mg/kg).
Rats were regarded as diabetic when their blood glucose level exceeded 16.7 mmol/L one week post-STZ
administration.
• The blood glucose level of the rats was recorded throughout the study using a glucose meter (Johnson, China)
from the tail vein.
• A total of 48 rats were chosen to establish the diabetic wound models, 3 weeks after DM induced by STZ.
Under anesthesia with intraperitoneal pentobarbital sodium (35 mg/kg), a 5-mm diameter full thickness foot
skin wound was made using a punch biopsy device.
• The wounds were washed with saline, and then divided into three different treatment groups: control group
(untreated), Gel group (200 lL hydrogel), and Gel + In group (200 lL hydrogel containing 0.1 U insulin).
• The dosage of insulin used was based on previous studies.

• Each wound was covered with sterile gauze and fixed with an elastic adhesive bandage.
• Each rat was individually housed in a cage. At 0, 4, 8, 12, and 16 days post-operatively the wounds were
recorded using a digital camera and measured by Image J software (National Institutes of Health, Bethesda,
MD, USA).
• The residual wound area was calculated using the following formula: Residual wound area (%) = Sn/So
100% (S0: initial wound area, Sn: wound area at different time points).

3.4. Insulin-loaded hydrogel enhanced DUFs healing
• The insulin-loaded hydrogel dressings were injected to cover the wounds of STZ-induced diabetic rats to
investigate the effects of released insulin on chronic wound healing in vivo.
• After treating the wounds with hydrogel or insulin-loaded hydrogel, the blood glucose level decreased
significantly, especially when incorporated with insulin (Fig. S4).
• Representative wound closure images of each group at different time points are shown in Fig. 3A.
• In order to observe the rate of wound healing, the residual wound area was quantified by Image J software.
• Consistent with the gross observation, the insulin-loaded hydrogel group presented a smaller residual wound
area than that of the other groups during the entire healing process.
• At 16 days after treatment, the wound appearance of the Gel + In group was almost completely healed, while
the
• residual areas of the Con and Gel groups were 31.91 ± 4.91% and 9.30 ± 3.61%, respectively (Fig. 3B).
• In addition, the healing rate of the wound covered with hydrogel alone was also significantly faster than that
of the control group (p < 0.05).
• These results indicated that the insulin-loaded hydrogel could also promote the wound healing process
through sustained release of insulin.
• The histological results observed in our models were consistent with the wounds contraction observed in the
optical images.

• The H&E staining revealed that the insulin-loaded hydrogel dressing significantly enhanced well organized
granulation tissue forming at the wound site on day 4.
• On day 12, more granulation tissue was formed in the insulin-loaded hydrogel group, and the structure became
denser.
• Without the protection of the hydrogel and the beneficial effect of insulin, necrotic tissue, foreign bodies, and
inferior-quality granulation tissue were observed at the wound site both on days 4 and 12 in the control group
(Fig. 3C).
• Histologically, wound healing is a complex process which includes several overlapping phases: hemostasis,
inflammation, proliferation, re-epithelialization, and remodeling.
• Firstly, inflammatory cells migrate to the wound bed because of growth factors and cytokines released by the
aggregated platelets.
• The aggregation and activation of inflammatory cells are crucial to the transition from inflammation to repair
phases.
• Topical insulin treatment increased the inflammatory cell infiltrate at 4 days; however, 12 days post-operatively,
there was a sharp drop in the number of inflammatory cells in the insulin-loaded hydrogel group, which was
significantly different than the control group (p < 0.05).
• In contrast, a delay in the peak inflammatory cell infiltrate was observed in the control group, which experienced
a sharp rise in the number of inflammatory cells after 12 days, which was significantly higher than the Gel group
and Gel + In group (Fig. 3D, p < 0.05).
• The regulation of oxidative and inflammation responses is one of the mechanisms on promoting wound healing
after topical insulin application.

• These outcomes suggested that topical insulin administration could induce inflammatory cell infiltration and
shorten the inflammation stage in diabetic rats.
• In our studies, the topical administration of insulin and consequent shortening of the local inflammatory
response phase led to faster wound contraction and re-epithelialization.
• Possible mechanisms involved in the therapeutic effects of topical insulin administration in wound healing
include inhibition in the production of reactive oxygen species (ROS), protection from oxidative damage to
proteins, and promotion of early macrophage infiltration and inflammation resolution at the injured site by
regulating wound monocyte chemoattractant protein-1 (MCP-1) expression.
• At the maturation process of wound repair, collagen deposition plays a significant role in scar formation, and
wound contraction is a result of fibroblasts cross-linking with collagen fibers.
• Masson staining was performed to detect the newly formed collagen deposition in the regenerated skin tissue.
• Although limited collagen was observed at the edge of the wound on day 4, collagen deposition was more
significant in the Gel + In group than in the Con group.
• After 12 days, the collagen deposition was further increased and developed at the center of the wounds treated
with insulin-loaded hydrogel.
• In contrast, the collagen was still limited to the periphery of the wounds in the Con group (Fig. 3E).
• The results indicated that more collagen deposition occurred on the regenerated tissues in the insulin-loaded
hydrogel group than in the control or hydrogel groups.

• In terms of quantitative analysis (Fig. 3F), after 12 days, 42 ± 7.0 % of collagen deposition was detected in the
wounds of the Gel + In group, which was 2.0-fold and 1.3-fold higher than that observed in the control group
(21.3 ± 4.2 %) and hydrogel group (33.3 ± 5.8 %), respectively.
• It has been reported that a hyperglycemic environment in the local wound interferes with wound healing by
inhibiting cell proliferation and hindering collagen synthesis and accumulation.
• In addition, insulin has been reported to be a strong and selective stimulator of collagen synthesis in skin
fibroblasts.
• Thus, the dressings loaded with hydrogel containing insulin could effectively improve the formation of
collagen to recover DFUs, which could be hindered by diabetic condition.

Fig. 3. General appearance and histology of wound healing.
(A)Representative images of the wound area were photographed on day 0, 4, 8, and 12.
(B)Residual area of wounds was measured on day 0, 4, 8, and 12 by Image J software.

(C) Inflammatory infiltrate and granulation tissue formation were observed by H&E staining at 4 and 12 days
after the operation.
(D) Quantitative analysis of the number of inflammatory cells by counting the number of cell nuclei per field that
had highly condensed chromatin.

(E) Collagen deposition was improved with hydrogels containing insulin. The wound tissues were observed by
Masson trichrome staining on day 4 and 12 post-operatively. The blue area in the wounds showed newly synthesized
collagen fibers.
(F) Quantitative analysis of the collagen deposition in the wound sites conducted by Image J software (W: Wound
area, N: Normal tissue, *p < 0.05, **p < 0.01, ***p < 0.001 compared with control group; #p < 0.05 compared with
the hydrogel group). (For interpretation of the references to color in this figure legend, the reader is referred to the
web version of this article.)

2.7. Histology and immunohistochemical staining
• At 4 and 12 days post-operatively, the wound and surrounding tissues were collected and fixed in 4%
paraformaldehyde solution and embedded in paraffin for routine histological processing.
• Five-micron thickness sections of the tissues were prepared.
• According to standard protocols, samples were stained with H&E, Masson trichrome, anti-K14 antibodies
(clone MiB-1; Dako), and anti-CD 31 antibody (ARG52748, Arigo).
• In addition, on the 32nd day after operation, staining was performed to evaluate the regeneration of hair follicles
and sebaceous glands.

3.5. Insulin-loaded hydrogel promoted re-epithelialization and neovascularization
• Re-epithelialization and neovascularization are critical issues for advanced wound regeneration.
• Re-epithelialization includes the reconstruction of the epithelium on the newly formed wound tissue;
otherwise, wounds would exit ever without undergoing re-epithelialization.
• Based on the H&E staining results, a neo-epidermis layer was observed 4 days post-operatively in theinsulin-
loaded hydrogel group, whereas no epidermis formation could be observed in the control group.
• On day 12, regular epithelium was formed in the Gel + In group, as well as in the Gel group, but only a
limited namount of epithelium was observed along the edge of the wound in the Con group. In the process of
reepithelialization, keratinocyte proliferation and differentiation play an important role.
• Therefore, immunohistochemical staining targeting K14 was performed to detect the re-epithelialization
status of the wound (Fig. 4A).
• The quantitative analysis indicated that the mean density of K14-positive cells in the newly formed epithelial
layer of Gel + In group was 2.5-fold higher than that of the Con group after 4 days, and was 2.3-fold and 1.6-
fold higher than that observed in the Con and Gel groups after 12 days (p < 0.05), which suggested that
topical insulin delivery stimulated keratinocyte migration and proliferation at the wound sites (Fig. 4B).

Fig. 4. Immunohistochemical staining of K14 and
CD31 in wound tissues.
(B) Quantification analysis of K14-positive cells
calculated by Image J.
Fig. 4. Immunohistochemical staining of K14
and CD31 in wound tissues.
(A)Immunohistochemical staining of K14 on
day 4 and 12. The neo-epidermis above the
wound area was shown by brownish
staining (Purple arrows).

2.8. Motor nerve conduction velocity measurement
• After anesthesia by intraperitoneal pentobarbital sodium (35 mg/kg), motor nerve conduction velocity (MNCV)
was measured in the wounded sciatic tibial nerve site.
• The whole process was carried out on a temperature-controlled mat in order to maintain the body temperature at
37 C.
• The right sciatic nerve distal to the knee was stimulated using bipolar electrodes of an electromyograph
machine, Endeavor CR (Natus Neurology Incorporated, US).
• The action potential of the muscle was monitored from the abductor hallucis via unipolar pin electrodes.
• The MNCV was calculated on days 0, 8, 16, and 32 after treatment.

3.5. Insulin-loaded hydrogel promoted re-epithelialization and neovascularization
• Many important substances for wound healing (e.g., growth factors, oxygen, nutrients) are delivered and
infiltrated into the wound beds via the blood vessels.
• Therefore, neovascularization plays an essential role in accelerating tissue remodeling process and wounds
may fail to heal due to inadequate local vascular supply.
• The immunohistochemical staining with CD31, which is a marker of endothelial cells, indicated more
neovascularization had occurred during wound healing when treated with Gel + In than Con or Gel groups at
both days 4 and 12 (Fig. 4C).
• Furthermore, the number of newly formed blood vessels observed in each field in the Con, Gel, and Gel + In
groups was 2.7 ± 1.2, 3.7 ± 1.2, and 7.7 ± 1.5 at 4 days, and 11.3 ± 3.1, 15.0 ± 2.6, and 21.7 ± 3.1 per field at
12 days, respectively (Fig. 4D).
• These results revealed that hydrogel coated with insulin can efficiently enhancing wound healing by
increasing neovascularization around the wound under diabetic condition.

Fig. 4. Immunohistochemical staining of K14 and
CD31 in wound tissues.
(D) Quantification analysis of new blood vessels
(W: Wound area, N: Normal tissue, *p < 0.05, **p <
0.01).
Fig. 4. Immunohistochemical staining of K14
and CD31 in wound tissues.
(C) Immunohistochemical staining of CD31
observed on day 4 and 12. Black arrows
indicated new blood vessels.

2.9. Western blotting analysis
• To determine the expression levels of growth factors in the wound tissues, western blotting was conducted to
evaluate the expression of TGF-b1 and VEGF in wound tissues.
• Briefly, the wound tissues were excised after 4 and 12 days of treatment, and lysed in 1 mL RIPA
supplemented with proteinase inhibitors to extract total protein from the tissues.
• The mixture was incubated on ice for 30 min and centrifuged at 12,000g for 10 min.
• The protein concentration was determined using a Bradford protein assay kit. For western blotting analysis,
equal amounts of protein (100 lg) were loaded onto 10% SDS-PAGE gels and transferred to PVDF
membranes.
• The membranes were probed with the specific primary antibodies after being blocked with 5% nonfat milk at
room temperature for 1 h.
• All antibodies were purchased from Abcam as follows: VEGF (anti-VEGF antibody, ab53465, dilution
1:1000), TGF-b1 (anti-TGF-b1 antibody, ab179695, dilution 1:1000) at 4 C overnight and subsequently
incubated with a secondary goat-anti-rabbit antibody (ab97051, dilution 1:5000) at 37 C for 45 min.
• The target bands were then scanned using enhanced chemiluminescence (Bio-Rad), and TGF-b1, and VEGF
expression was normalized to b-actin.

3.6. Insulin-loaded hydrogel induced TGF-b1 and VEGF expression
• To evaluate the growth factors secreted around the wound, we next determined the expression of TGF-b1 and
VEGF in wound tissues.
• When wounds were treated with the insulin-loaded hydrogel, protein levels of TGF-b1 and VEGF in the wound
tissues were significantly upregulated compared with those in the hydrogel and control groups.
• Meanwhile, the expression of TGF-b1 and VEGF in the hydrogel group were also slightly higher than those in
the control group (Fig. 5A).
• Quantitative analysis revealed that compared with the control group, the expression of TGF-b1 in the hydrogel
group and the insulin-loaded hydrogel group was 1.1- fold and 1.4-fold higher at day 4, and 1.7-fold and 1.9-
fold higher at day 12, respectively (Fig. 5B).
• The expression of VEGF in the hydrogel group and insulin-load hydrogel group was 1.1-fold and 1.4-fold
higher than control group at day 4, and 1.2-fold and 1.4-fold higher than control group at day 12 (Fig. 5C).
• These results indicated that the hydrogel dressing created a favorable microenvironment, which was conducive
to the secretion of growth factors for diabetic wound healing, and insulin loading could further promote the
effects.
• A series of growth factors provide the cellular and molecular signals necessary for the wound healing process.
TGF-b1 has been recognized as being involved in the regulation of the reepithelialization process by promoting
keratinocyte migration.

• Furthermore, VEGF is a key regulatory growth factor in wound healing directly involved in angiogenesis.
• Enhanced concentrations of TGF-b1 and VEGF at the wound edges are responsible for promoting epithelial
cell migration and angiogenesis .
• Combining the results, the histological observations and the immunohistochemistry findings clearly revealed
that the bioactive insulin-load hydrogel stimulated re-epithelialization and neovascularization, which was
closely related to the stimulation of keratinocytes and endothelial cell migration and proliferation.

Fig. 5. Western blotting detection of growth factors in wound tissues.
(A)Representative blots demonstrated the protein levels of TGF-b1 and VEGF in wound tissues.

The quantitative analysis of
(B) TGF-b1
The quantitative analysis of
(C) VEGF were normalized to b-actin expression (*p < 0.05, ** p <
0.01).

2.10. Statistical analysis
• Data were expressed as mean ± standard deviation (SD) and the statistical analysis was carried out using
ANOVA with Tukey’s posthoc analysis (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered a
statistically significant difference.

3.7. Insulin-loaded hydrogel improved the quality of the healed wounds and peripheral neuropathy
• High quality healing is beneficial to the recovery of skin function in the injured areas.
• The sub-epithelial matrix of the wounds was evaluated by considering granulation tissue, inflammation,
fibroblasts, collagen deposition, and neovascularization into the scoring system (Table S1).
• DFUs treated with the insulin loaded hydrogel showed higher maturation scores than the control group at both
days 4 and 12 (p < 0.01 and p < 0.001, respectively), as well as for the hydrogel group at day 12 (p < 0.05).
• In addition, treatment with hydrogel alone also achieved a higher maturation score than the control group after
12 days (p < 0.05) (Fig. 6A).
• On day 32 of treatment, all wounds healed completely and scar tissue formed.
• As illustrated in Fig. 6B, the distribution of regenerative hair follicles (black arrows) and sebaceous glands
(red arrows) were used to evaluate the quality of the healed wounds by H&E staining.
• In the Gel + In group, new hair follicles and sebaceous glands in the wounds were located at the center of the
regenerated skin, and were almost the same as that of the surrounding normal skin tissue.
• These skin appendages were relatively sparse at the edge of the regenerative skin in the Gel group, while there
was almost no new appendage observed at the center of the regenerative skin in the Con group.
• The specific statistical analysis showed that the number of regenerated hair follicles was 1.00 ± 0.89, 2.67 ±
1.21, and 6.17 ± 1.47 (Fig. S5), the number of new sebaceous glands were 1.17 ± 0.75, 2.50 ± 1.09, and 5.33 ±
1.63 (Fig. S6), in the Con, Gel, and Gel + In groups, respectively.

• DM impairs wound healing by hindering collagen synthesis and accumulation, inhibiting cell migration and
proliferation, prolonging inflammation, and interfering with re-epithelialization and neovascularization.
• Insulin plays a crucial role in a series of cellular processes and serves as a chemoattractant and mitogen for
cells undergoing wound healing.
• Although direct topical application of insulin has achieved some positive results, topical application of insulin
peptides presents disadvantages in terms of their short half-life and loss of bioactivity in the chronic
inflammation phase in diabetic wounds.
• In this study, we prepared a novel injectable and self-healing hydrogel that delivered insulin to the wound site
and released bioactive insulin steadily and in a pH-dependent manner for 14 days.
• Herein, the insulinloaded hydrogel shortened the inflammation phase, enhanced granulation tissue formation,
collagen deposition, reepithelialization, neovascularization, and achieved higher subepithelial matrix
maturation scores, thus promoting DFUs healing.
• Interestingly, treating DFUs with hydrogel alone could also enhance the wound healing.
• These effects may be because the
• hydrogels create a moist wound environment, provide protection from secondary infections, absorb wound
exudate, as well as have the blood-glucose-lowering effect from the chitosan.
• Previous studies have described how insulin binds to the IR and Insulin like growth factor (IGF) receptors,
and then activates downstream signaling pathways.
• When insulin is incorporated in hydrogel, its sustained release in the wound produces beneficial effects on
cell differentiation and glucose transport.

• Accelerating and regulating collagen formation during wound healing repair are key issues for skin
regeneration. Insulin can promote the healing of damaged skin by stimulating cellular migration and
angiogenesis.
• Insulin not only stimulates the proliferation of keratinocytes and endothelial cells, but also increases the
production of matrix proteins.
• Moreover, skin fibroblasts involved in the development of granulation tissue express the IR and their growth
rate is promoted by insulin.
• In general, topical insulin administration enhances wound healing by lowering the hyperglycemic
environment in the wound and by stimulating keratinocyte, fibroblast, and endothelial cell migration and
proliferation.
• In order to evaluate the function of the peripheral nerve, we examined the sciatic nerve MNCV.
• As shown in Fig. 6C, the conduction function of the sciatic nerve in the control group and hydrogel group
declined with time.
• Conversely, the insulin-loaded hydrogel group improved the sciatic nerve function, especially on day 16 and
32 (p < 0.05).
• It has been reported that DM peripheral neuropathy is related to metabolic abnormalities caused by
hyperglycemia, as well as ischemia and hypoxia in nutrient vessels in the nerves.
• In this study, amelioration of MNCV may have benefited from lower blood glucose levels and higher local
concentrations of growth factors, such as TGF-b and VEGF in the wound’s microenvironment, which is
beneficial to neurovascular regeneration.

Fig. 6. Evaluation of wound healing quality.
(A)Sub-epithelial matrix maturation scores on day 4 and 12.
(B) H&E staining of the distribution of new sebaceous glands (red arrows) and hair follicles (black arrows) in the wound after 32
days of treatment.
(*p < 0.05, ** p < 0.01, ***p < 0.001 compared with control group; #p < 0.05 compared with the hydrogel group).
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(C) Improvement of diabetic peripheral neuropathy following treatment of insulin-load hydrogel in DFUs
(*p < 0.05, ** p < 0.01, ***p < 0.001 compared with control group; #p < 0.05 compared with the hydrogel
group).
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of
this article.)

4. Conclusion
• Our findings show that self-healing, injectable, and pHresponsive hydrogels could be synthesized through the
formation
• of the reversible dynamic bonds, acylhydrazone and imine bonds, which are derived from aldehyde groups at
HA-ALD.
• These are reactive with NH2NHCO– groups on ADH and –NH2 groups on Nchitosan.
• Insulin can be encapsulated in the hydrogels during the in situ cross-linking process.
• The sustained and pH-triggered drug release system showed excellent biocompatibility and the released
insulin had good bioactivity.
• Additionally, in vivo studies confirmed that the insulin-loaded hydrogel dressing shortened the inflammation
phase, enhanced granulation tissue formation, promoted collagen deposition, accelerated re-epithelialization
and neovascularization, and thus significantly benefited DFUs healing.
• Moreover, these dressings postponed or even improved diabetic peripheral neuropathy. Thus, the insulin-
loaded hydrogels have highly promising therapeutic potential for DFUs.

Ph-responsive Hydrogel Loaded With Insulin As A Bioactive Dressing For Enhancing Diabetic Wound Healing

Ph-responsive Hydrogel Loaded With Insulin As A Bioactive Dressing For Enhancing Diabetic Wound Healing

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