1.
INTRODUCTION
Uncontrolled hemorrhage is the leading cause of preventable deaths in military conflicts,
traffic accidents, and surgical procedures. More than 90% of people die before reaching
the hospital. Not controlling hemorrhage at the place of injury makes it difficult to reach
the patient for definitive care. (1) Rapid, effective hemostatic material application in initial
hemorrhage phases can lead to an extended rescue time, resulting in a decline in high
mortality rates due to excessive bleeding. (2,3)
The loss of blood may lead to symptoms such as hypothermia, coagulopathy, and
functional organ failure. Hemostasis is a complex and continuous process that involves
blood vessels, platelets, and plasma coagulation factors. When the body's hemostatic
mechanisms fail to achieve hemostasis, hemostatic materials are necessary to quickly and
effectively stop bleeding while providing favorable conditions for wound repair.
Traditional hemostatic materials include tourniquets, bandages, hemostatic sponges,
gauze, and powder; however, achieving rapid and effective hemostasis in cases of internal
hemorrhage and non-compressible hemorrhages remains challenging. Additionally, the
removal of non-degradable bandages and gauze may result in secondary injury and
delayed healing. Therefore, innovative hemostatic materials that combine strong
hemostatic capability with a fast onset of action and excellent biocompatibility are
urgently required. (4,5)
Nanofibers based hemostats are being explored in recent times due to their advantages like
high surface area, structural similarity to natural fibers, and ability to encapsulate and deliver
bioactive agents (6,7) Due to these properties nanofibers dressing enhance blood absorption
and platelet activation, accelerating clot formation. Due to their structural similarity to the
natural extracellular matrix, they support cell adhesion and can be modified with hemostatic
agents to accelerates the hemostasis. (8–10). `These nanofibers can be produced using various
methods like electrospinning, self assembly, phase separation, template synthesis (11)Among
these techniques electrospinning stands out as one of the most practical approaches due to its
simple equipment needs, cost-effectiveness, and the capacity to generate continuous
nanofibers with exceptional characteristics such as diameters down to the nanometre range,
large specific surface area, and high porosity. (12,13). Electrospinning is a unique technique
that relies on the application of an electrostatic field to generate ultrafine fibres
(14)Generally, this process uses a straightforward setup consisting of four main components,
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as shown in Figure 1. Central to this process is the electrospinning machine, which typically
consists of three key components: a high-voltage power supply, a syringe pump with a
spinneret (needle), and a collector (14) The polymer solution is dispensed through the syringe
at a controlled rate, and when subjected to a high electric field, the repulsion of surface
charges in the droplet at the needle tip leads to the formation of a Taylor cone and subsequent
ejection of a fine polymer jet (15,16)As the jet accelerates toward the collector, it undergoes a
rapid stretching and whipping motion, resulting in the thinning of the jet and evaporation of
the solvent. This leads to the deposition of solid nanofibers on the collector surface. (17)
Fig no.1 electrospinning process
Cellulose derivatives, chitosan, gelatine, silk fibroin, collagen have been used to make
nanofibers. Often, a combination of polymers is necessary to make a good balance
between physical properties and biological activities. This study introduces electrospun
nanofibers composed of silk fibroin, nanocellulose, and collagen, engineered to enhance
hemostatic performance. (18) The synergistic combination of these natural biopolymers
offers excellent biocompatibility, mechanical strength, and structural similarity to the
native extracellular matrix (ECM), which promotes cellular adhesion. (19,20) Silk fibroin
contributes remarkable mechanical integrity and hemostatic potential, (19) while
nanocellulose enhances tensile strength and fluid uptake due to its nanostructure and
hydrophilicity. (20) Collagen, a key ECM component, supports cell attachment and wound
healing, further improving bioactivity. (21) Together, these materials form a biodegradable
and ECM-mimicking scaffold that can actively participate in initiating and stabilizing clot
formation, thereby accelerating the hemostatic process. (22)
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Collagen is known for its excellent biocompatibility and ability to initiate clot formation,
while silk fibroin provides hemostatic activity along with improved mechanical strength
and structural stability to the nanofibers. However, when used individually in electrospun
nanofibers, both materials exhibit certain functional and processing limitations, such as
inadequate mechanical stability and poor spinnability. These challenges can be effectively
overcome by combining these materials with nanocellulose and PEO.
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1. EXPERIMENTAL SECTION
2.1Materials
Nanocellulose and marine collagen were procured from Ottokemi Chemicals, Mumbai,
and Vedayukt India Pvt. Ltd., respectively. Silk fibroin (SF) was obtained from Fibroheal
Pvt. Ltd., while polyethylene oxide (PEO) was sourced from Loba Chemicals. All
chemicals used in the study were of analytical reagent (AR) grade.
2.1 Preparation of electrospinning solution
Electrospinning solutions were prepared by dissolving each polymer component
separately, followed by blending in specific ratios. Collagen was dissolved in 0.5 M acetic
acid at concentrations ranging from 10% to 18% (w/v), while PEO was dissolved in
deionized water at concentrations between 2% and 15% (w/v). Silk fibroin (SF) was used
at 5% (w/v), and nanocellulose (NC) was dispersed in water at concentrations of 2% to 5%
(w/v). All solutions were stirred on a magnetic stirrer under mild heating to ensure
complete dissolution. The components were then combined to prepare final formulations
with total polymer concentrations of 12%, 15%, or 20% (w/v) in 10 mL volumes. Each
blend was evaluated based on extrudability, spinnability, and mat integrity. Formulations
exhibiting optimal performance in all three parameters were selected for further
characterization via SEM. The summary of electrospinning solutions and their process
performance is shown in Table.1
2.2 Fabrication of nanofibers by electrospinning
Nanofibers were fabricated using an electrospinning apparatus (E-SPIN ES2
NANOTECH). A 5 mL plastic syringe fitted with a 22-gauge needle (outer diameter: 0.8
mm, inner diameter: 0.6 mm) was used to load all electrospinning solutions. The collecting
drum was covered with an aluminum foil and rotated at a speed between 1,000 and 1,500
rpm. The flow rate was maintained at 1.0 mL/h, with an applied voltage of 24 kV, and the
needle was positioned 7 cm away from the drum. UV illumination was maintained
throughout the electrospinning process to aid in the sterilization of the nanofiber mats.
Electrospinning was conducted at ambient temperature.
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Table.1 Summary of Electrospinning Solutions and Their Process Performance
Batch Concentration Extrud Spinn Mat Comments
(Total solids in 10 ability ability Integr
mL) ity
F1 12%w/v (Collagen ++++ ++ + Easy to extrude, fragile fibers,
10%, PEO 2%) risk of beading
F2 15%w/v (Collagen +++ +++ ++ Good spinning and moderate
13%, PEO 2%) mat morphology.
F3 20%w/v (Collagen ++ +++ +++ Good fiber formation and mat
18%, PEO 2%) + strength, slower extrusion.
F4 12%w/v (SF 5%, +++ +++ ++ Smooth spinning, easy
PEO 7%) extrusion, fair mat formation
F5 15%w/v (SF 5%, ++ +++ +++ Thicker mat, extrusion is
PEO 10%) + moderate.
F6 20%w/v (SF 5%, + + + Unstable jet due to high
PEO 15%) viscosity, hard to extrude.
F7 12%w/v (Collagen +++ +++ +++ Optimal, smooth fibers
3%, SF 5%, PEO + +
Slight clogging,
2%, NC 2%)
dense mat
F8 15%w/v ++++ +++ +++ Best-performing blend with
(collagen5%, + + excellent spinning, extrusion,
5%SF, 2%PEO, and mat integrity.
3%NC)
F9 20%w/v ++ +++ +++ Strong and dense mat; reduced
extrudability due to high
(Collagen8%, + +
viscosity.
5%SF, 2%PEO,
5%NC)
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2.3 Fourier-transform infrared spectroscopy (FTIR)
This study employed the KBr pellet method with a Jasco FTIR 6x instrument. The FTIR
spectra of silk fibroin, Nanocellulose, collagen, PEO and fabricated nanofibers were
recorded over the wavelength range 4000- 650 cm-1,with a wavelength resolution of 4 cm-
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, and 40 scans.
2.4 Field Emission Scanning Electron Microscopy (FESEM)
A Field Emission Scanning Electron Microscope (FEI Nova Nano SEM 450) was used to
evaluate the shape and diameter of Nanofiber threads. The assembled Nanofibers were air-
dried for a whole night before being scanned. The specimens were seen at room
temperature and at an accelerating voltage of 15 kV.
2.5 XRD
X-ray diffraction measurements were carried out using Bruker AXS D2 Phaser(Germany)
instrument with CuK as source (0.154nm) and detector ventec1. The scanning range was 2
and 80, and the step size and count time per step were 0.023851 and 0.5s.
2.6 Mucoadhesion studies
The mucoadhesive properties of the nanofiber dressing were evaluated using a Brookfield
texture analyser (CT-3). A piece of rat skin was fixed on the bottom platform of the
machine using double-sided adhesive tape. Likewise, the nanofiber mat of 14 mm
diameter was attached to the top platform using double-sided tape. The Nanofiber mat was
allowed to attach to the skin with a preload of 5 N for 60 s. Subsequently, the top platform
was lifted at a 2 mm/min speed until separation. The maximum adhesive strength of the
samples was calculated using the maximum adhesive force at the time of separation.
2.7 Disintegration test
The disintegration behaviour of the nanofiber mat formulation was evaluated in triplicate
using a modified Petri dish assay method. Approximately 4 mg of the nanofiber mat was
placed in 8 mL of pre-warmed phosphate-buffered saline (PBS, pH 7.0) to facilitate the
complete disintegration of the fibres. The experiment was carried out in an incubator
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maintained at 37 °C. The results were recorded as the mean ± standard deviation (SD) of
three independent measurements.
2.8 In Vitro Blood Clotting
Anticoagulated whole blood was collected from a blood bank.10 mL of blood was taken in
three test tubes. Small pieces (0.5 cm × 0.5 cm) of nanofiber dressing samples were added
individually to the test tubes. These tubes were then placed in a water bath maintained at
37°C for 10 minutes to allow the samples to equilibrate with the [Link] this period, 2
mL of 0.2 M calcium chloride solution was added to each tube to recalcify the blood. This
procedure was repeated for all test tubes. Once recalcification was complete, the test tubes
were returned to the 37°C water [Link] 10-second intervals, one test tube from each
group was removed, tilted, and observed for blood flow. The remaining tubes were left in
the water bath. This step was repeated at 10-second intervals until no blood flow was
observed and clotting was complete. The time taken for complete coagulation in each test
tube was recorded.
2.9 Hemolysis assay
Human Blood was procured from the blood bank of Sassoon Hospital, Pune. The blood
was directly drawn into ethylenediaminetetraacetic acid (EDTA)- coated blood collection
tubes. Subsequently, the blood was centrifuged at 3000 rpm for 10 min. The supernatant
was discarded, and the intact red blood cells (RBCs) at the bottom of the tube were re-
dispersed in 1 mL of sterile PBS. The RBCs were incubated with nanofiber mats of
different combinations for 90 min. After incubation, the samples were centrifuged at 3000
rpm for 10 min to collect the supernatant. The absorbance of the supernatant was
spectrophotometrically recorded at 490 nm. Distilled water was used as the positive
control (As positive control), while sterile PBS was the negative control (Abs negative
control). % Hemolysis was calculated as shown in Eq. 1.
%Hemolysis=¿ ¿
2.10 Zeta potential measurement
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The zeta potential was measured using a Zetasizer (Malvern Instruments). All sample
solutions were prepared in double-distilled water.
2.11 Platelet Adhesion test
Platelet-rich plasma (PRP) collected by centrifugation of whole blood. An amount of
100 µL PRP was added to each well containing nanofiber film samples and incubated at
37 °C for 2 hours. After incubation, the samples were gently washed twice with pre-
warmed 1× PBS to remove non-adherent platelets. The samples were then fixed,
dehydrated through a ethanol, freeze-dried, and finally analyzed using scanning electron
microscopy (SEM).
2.12 MTT Cytotoxicity Assay
The cytotoxicity of the nanofiber dressing was evaluated using the MTT assay. HaCaT
(human keratinocyte) cells were cultured in DMEM medium supplemented with 10% fetal
bovine serum and 1% antibiotics. Around 10,000 cells were cultured in each well of a 96-
well plate and incubated for 24 hours at 37°C in 5% CO₂.Sterilized nanofiber samples (0.5
cm × 0.5 cm × 0.5 cm) were then added. The plates were incubated for 1, 4, and 7 days. At
each time point, MTT reagent was added and incubated for 2 hours to allow formation of
purple formazan crystals. After removing the media, 100 µL of DMSO was added to
dissolve the crystals.
Absorbance was measured at 540 nm and 660 nm using a microplate reader, and cell
viability was calculated by comparing to control wells without nanofibers.
( Sample 540 −Sample 660 )
Cell Viability (%)= X 100
( Control 540 −Control660 )
2.13 In vivo studies
Wistar rats weighing in the range of 180 – 200 gm were used for the in vivo studies. The
experimental protocol was approved by the Institutional Animal Ethics Committee,
AISSMS College of Pharmacy, Pune (Approval no. CPCSEA/IAEC/ ). The 48 albino
Wistar rats, were divided into six groups, with eight animals in each group. Ketamine (10
mg per 1 kg of weight) was administered to anaesthetize the rats.
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2.13.1 Rat femoral artery model
A rat femoral artery incision model was used to assess hemostatic performance under in
vivo conditions. The medial surface of the rat’s thigh was gently shaved, and the
superficial femoral artery was identified. (23) A skin incision was made parallel to the
femoral sheath, followed by careful dissection of the surrounding soft tissues to expose the
femoral neurovascular bundle. Using a dissector, the femoral sheath was opened to clearly
expose the artery. (24) Once visualised, the femoral artery was punctured with a 24-gauge
needle and allowed to bleed freely for 15 seconds. Bleeding severity was assessed using a
validated scale ranging from 0 to 4: 0 = no bleeding, 1 = intermittent bleeding, 2 =
continuous bleeding, 3 = controllable spurting, 4 = life-threatening bleeding. (25,26) If the
initial injury did not result in scale 4 bleeding, the site was either manipulated or an
additional puncture was made to ensure standardized bleeding severity across all animals.
Three types of dressings—control (no treatment), standard commercial dressing, and
nanofiber-based dressing—were applied to the wound site. The time required to achieve
stable clot formation was then recorded for each group to compare hemostatic
effectiveness. (26)
2.13.2 Rat liver incision model
Upon the onset of anaesthesia, the abdominal area of Wistar rats was shaved and cleaned.
A 3 cm midline incision was made through the skin and abdominal wall by using dissector.
The liver was gently exposed, and a 1 cm longitudinal incision was made on one liver
lobes. (27)liver allowed to bleed for 15 seconds, with the injury standardized to severity
scale 4 (severe bleeding). Three dressings were then applied to the wound: dressing
(control), a standard commercial dressing, a nanofiber-based hemostatic dressing. The
time to achieve stable clot formation was recorded for each group. (28) This procedure
aligns with established rat liver hemorrhage models used to evaluate topical hemostatic
agent (29)
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