Description:FIELD OF INVENTION
The present invention relates to a wound dressing composition comprising Curcuma
aromatica (CA) loaded silver nanoparticles (CAAgNPs). Also, the invention relates
to a process for the preparation of a wound dressing composition comprising
Curcuma aromatica (CA) loaded silver nanoparticles (CAAgNPs). The present
invention relates to a wound dressing composition comprising Curcuma aromatica
(CA) loaded silver nanoparticles (CAAgNPs) used in wound management.
Biological
Material
Source
Curcuma
aromatica (CA)
The dried rhizomes of CA were purchased from the local market
based in Pune, Maharashtra, India and identified by a botanist
from Botanical Survey of India (Western Regional Centre),
Pune, Maharashtra, India (Identification No. 1603220017147).
BACKGROUND OF INVENTION
Wound healing is a complex biological process involving inflammation,
proliferation, and remodelling phases. Chronic wounds, burns, and diabetic ulcers
remain a major global health concern due to delayed healing, microbial infections,
and scar formation. Conventional wound dressings such as cotton gauze or synthetic
films provide only passive protection and are often ineffective against bacterial
colonization, especially multidrug-resistant pathogens. Silver nanoparticles (AgNPs)
are well known for their broad-spectrum antimicrobial activity; however, their
synthesis using chemical methods raises toxicity and environmental concerns.
Green synthesis of AgNPs using plant extracts provides a biocompatible and eco
friendly alternative. Curcuma aromatica rhizome extract contains bioactive
phytoconstituents with anti-inflammatory, antimicrobial, and wound-healing
properties. When combined with a natural polymer such as chitosan, these bioactive
silver nanoparticles (CAAgNPs) can be incorporated into a wound dressing matrix
to provide antibacterial action, enhanced collagen deposition, cytokine regulation,
and accelerated scar-free healing. Thus, there is a need for a biocompatible,
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multifunctional wound dressing that addresses microbial resistance, inflammation,
and poor healing associated with conventional dressings.
IN201921026088A disclosed chitosan encapsulated curcumin-zinc nanoparticle
based wound healing formulation;
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IN202011004849A disclosed a wound dressing composition comprising a probiotic
and a curcumin or curcumin loaded solid lipid nanoparticles;
WO2020213002A1 disclosed a bio-compatible dressing matrix comprising: (a) a
scaffold comprising gelatin and chitosan; and (b) an active composition comprising
a combination of curcumin, Emblica officinalis extract, and Camellia sinensis
extract; and
IN202341090222A disclosed a process of preparation of gelatin alginate chitosan
composite film as a wound dressing material and product thereof.
Tawre, et al, 2024, in Carbohydrate Polymer Technologies and Applications 8,
100570 disclosed bioactive Curcuma aromatica-stabilized silver nanoparticles
embedded chitosan dressing with improved antibacterial, anti-inflammatory, and
wound healing properties.
The prior art lacks compositions that combine the broad-spectrum antibacterial
activity of silver, the bioactive phytoconstituents of Curcuma aromatica, and the
biocompatibility and film-forming ability of chitosan in a single multifunctional
dressing. Thus, the inventors of the present invention have successfully addressed
the drawbacks of existing wound dressings and formulated a novel, biocompatible,
and multifunctional wound dressing. This multifunctional dressing combines the
antibacterial activity of silver, the bioactive properties of Curcuma aromatica, and
the biocompatibility of chitosan, thereby enhancing wound healing through
regulation of inflammation, promotion of collagen deposition, and scar-free tissue
regeneration, effectively overcoming the limitations of conventional dressings.
OBJECTIVE OF INVENTION
An objective of the invention is to develop a wound dressing composition
comprising Curcuma aromatica (CA) loaded silver nanoparticles (CAAgNPs).
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Another object of the invention is to provide a biocompatible, stable, and eco
friendly alternative to chemically synthesized silver-based dressings.
Yet another object of the invention is to enhance dose-dependent antibacterial
activity.
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Yet another object of the invention is to regulate anti-inflammatory activity.
SUMMARY OF THE INVENTION
The present invention relates to a wound dressing composition comprising Curcuma
aromatica (CA) loaded silver nanoparticles (CAAgNPs) and other pharmaceutically
acceptable excipients thereof. Also, the invention relates to a process for the
preparation of a wound dressing composition comprising Curcuma aromatica (CA)
loaded silver nanoparticles (CAAgNPs) and other pharmaceutically acceptable
excipients thereof.
The present invention relates to the silver nanoparticle composition comprising
aqueous rhizome extract of Curcuma aromatica (CA) and silver nitrate as a
precursor.
The present invention relates to a wound dressing composition in the form of a
sponge, disc, film, composite, hydrogel, foam, powder, aerosolized powder, or
aerosolized liquid dressing.
Particularly, the present invention relates to the composition comprising Curcuma
aromatica (CA) loaded silver nanoparticles, which enhances the antibacterial and
anti-inflammatory activity.
More particularly, the present invention relates to a wound dressing composition
comprising Curcuma aromatica (CA) loaded silver nanoparticles used in wound
management.
DESCRIPTION OF THE DRAWINGS:
For a more complete understanding of the invention, reference should now be made
to the embodiments illustrated in greater detail in the accompanying drawings and
described by way of embodiments of the invention.
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Figure 1: Illustrates (a) Photographic images of the fabricated dressings and
morphological analysis using FESEM, (b) antibacterial activity of CAAgNPs/CS
against MDR pathogens;
Figure 2: Illustrates (a) photographic images of wound area on days 3, 7, and 10
after treatment, and (b) Percent wound contraction;
Figure 3: Illustrates (a) Histological imaging of the wound tissues stained with H
and E staining, (b) Microscopic imaging of the tissue sections for collagen
deposition;
Figure 4: Illustrates percentage-stained area of collagen and hydroxyproline
concentration; and
Figure 5: Illustrates (a) quantifying the levels of inflammatory cytokines, (b) percent
viability of HEK293 cells after treatment with CAAgNPs/CS dressings.
DETAILED DESCRIPTION OF THE INVENTION
In describing the embodiment of the invention, specific terminology is chosen for
the sake of clarity. However, it is not intended that the invention be limited to the
specific terms so selected, and it is to be understood that such specific terms include
all technical equivalents that operate in a similar manner to accomplish a similar
purpose. As used herein, reference to an element by the indefinite article “a” or “an”
does not exclude the possibility that more than one of the elements is present, unless
the context clearly requires that there is one and only one of the elements. The
disclosure of numerical ranges should be understood as referring to each discrete
point within the range, inclusive of endpoints, unless otherwise noted.
The term "about" used to qualify the quantities of ingredients, properties such as
concentration, and so forth, shall be interpreted to mean "approximately" or
"reasonably close to" and any statistically insignificant variations therefrom.
As used herein, the terms “comprising” “including,” “having,” “containing,”
“involving,” and the like are to be understood to be open-ended, i.e., to mean
including but not limited to.
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The terms “preferred” and “preferably” refer to embodiments of the invention that
may afford certain benefits, under certain circumstances. In an embodiment, the
aspects and embodiments described herein shall also be interpreted to replace the
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clause “comprising” with either “consisting of” or with “consisting essentially of” or
with “consisting substantially of”.
The terms and words used in the following description are not limited to the
bibliographical meanings, but, are merely used to enable a clear and consistent
understanding of the disclosure. Accordingly, it should be apparent to those skilled
in the art that the following description of exemplary embodiments of the present
disclosure are provided for illustration purpose only and not for the purpose of
limiting the disclosure as defined by the appended claims and their equivalents.
Features that are described and/or illustrated with respect to one embodiment may be
used in the same way or in a similar way in one or more other embodiments and/or
in combination with or instead of the features of the other embodiments.
The therapeutically effective amount administered to the patient, e.g., a mammal,
particularly a human, in the context of the present invention should be sufficient to
affect a therapeutic or prophylactic response in the patient over a reasonable time
frame. The dose can be readily determined using methods that are well known in the
art. One skilled in the art will recognize that the specific dosage level for any
particular patient will depend upon a variety of potentially therapeutically relevant
factors.
The term "subject" includes mammals (especially humans) and other animals, such
as domestic animals (e.g., household pets including cats and dogs) and non-domestic
animals (such as wildlife).
The term "Treating" or "treatment" of a disease includes (1) preventing the disease
from occurring in subject that may be predisposed to the disease but does not yet
experience or display symptoms of the disease, (2) inhibiting the disease, i.e.,
arresting its development, or (3) relieving the disease, i.e., causing regression of the
disease.
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"Pharmaceutically acceptable excipient" or "excipient" includes, but is not limited
to, a combination of drug compounds of the present invention to produce a dosage
form for topical administration. Any inert material that is combined is included. The
term "pharmaceutically acceptable excipient" refers to a pharmacologically active
ingredient by a regulatory authority; including but not limited to any
emollient/stiffening agent/ointment base, emulsifying agent/solubilizing agent,
humectant, thickening/gelling agent, preservative, permeation enhancer, chelating
agent, antioxidant, acidifying/alkalizing/buffering agent, vehicle/solvent, protective
agent, buffer, adjuvant, bioavailability promoter carriers, solubilizers (including
surfactants), wetting agents, dispersants, suspending agents and stabilizers shall be
included.
The term “Nanoparticles (NPs)” are tiny particles with dimensions typically ranging
from 1 to 100 nanometers (nm). Due to their small size and large surface area-to
volume ratio, they exhibit unique physical, chemical, and biological properties
compared to their bulk counterparts. The “Silver nanoparticles (AgNPs)” are
nanoscale particles of silver. The present invention discloses the Plant-based silver
nanoparticles (AgNPs) are silver nanoparticles synthesized using plant extracts.
The term ‘wound dressing’ refers to a biocompatible material applied to a wound to
protect it, prevent infection, absorb exudates, maintain moisture, and promote
healing. The dressing may be in the form of a film, hydrogel, membrane, foam, or
nanocomposite suitable for topical application.
"Pharmaceutical composition" of the present invention refers to a wound dressing
composition of Curcuma aromatica (CA), a vehicle generally accepted in the art
for topical administration of the combination of drug compound to a mammal, e.g., a
human. Such a medium includes all pharmaceutically acceptable excipients. For the
purposes of this disclosure, the phrase "pharmaceutical composition" is
interchangeable with the phrase "pharmaceutical formulation".
As used herein, the following terms shall have the meanings ascribed to them below,
unless the context indicates otherwise:
CS: Chitosan,
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CA: Curcuma aromatica,
AgNO3: silver nitrate,
AgNPs: silver nanoparticles,
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CAAgNPs: silver nanoparticles of Curcuma aromatica,
AA: Acetic acid,
NaOH: Sodium Hydroxide.
In one aspect, the invention provides a wound dressing composition comprising
Curcuma aromatica (CA) loaded silver nanoparticles (CAAgNPs).
In another aspect, the invention provides a process for the preparation of wound
dressing composition comprising Curcuma aromatica (CA) loaded silver
nanoparticles (CAAgNPs).
In one embodiment, the invention provides a wound dressing composition
comprising Curcuma aromatica (CA) loaded silver nanoparticles, polymer, and
other pharmaceutically acceptable excipients thereof.
In
another embodiment, wherein Curcuma aromatica (CA) loaded silver
nanoparticles comprises aqueous rhizome extract of Curcuma aromatica (CA) and is
present in the range of 0.1–3 g %w/v.
In another embodiment, wherein the other pharmaceutically acceptable excipients
are selected from solvent, crosslinker, neutralizing agent or a combination thereof.
In another embodiment, wherein the polymers are selected from but not limited to
sodium alginate, gelatin, carageenan, chitosan, chitin, fibrin, collagen, either alone
or in combination thereof. In a particular embodiment, wherein the polymer is
chitosan and is present in the range of 1-3% w/v.
In another embodiment, wherein the solvents are selected from but not limited to
acetic acid (AA), formic acid, lactic acid, citric acid, hydrochloric acid, sodium
hydroxide (NaOH), potassium hydroxide, ethanol, methanol, isopropanol, and
deionized water, either alone or in combination thereof. In a particular embodiment,
wherein the solvent is acetic acid and is in a range of 0.5-2% v/v.
In another embodiment, wherein neutralizing agents are selected from but not
limited to sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium
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hydroxide, ammonium hydroxide, and sodium carbonate, either alone or in
combination thereof. In a particular embodiment, wherein the neutralizing agent is
sodium hydroxide used to maintain the pH neutral.
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In another embodiment, wherein crosslinkers are selected from glutaraldehyde,
genipin, epichlorohydrin, formaldehyde, carbodiimides, sodium tripolyphosphate
(TPP), glyoxal, tannic acid, either alone or in combination thereof. In a particular
embodiment, wherein the crosslinker is glutaraldehyde and is present in the range of
0.01-0.05% w/v.
In another embodiment, wherein the silver nanoparticles comprise the rhizome
extract of Curcuma aromatica (CA) and a precursor.
In another embodiment, wherein the precursor is silver nitrate (AgNO3) and is
present in the range of 0.4–1 mM.
In another embodiment, wherein the plant extract of Curcuma aromatica in the
range of 0.1–3 g % w/v and silver nitrate (AgNO3) in the range of 0.4–1 mM are
combined to form Curcuma aromatica-mediated nanoparticles (CANP).
In another embodiment, wherein a wound dressing composition comprising,
(a) Curcuma aromatica loaded silver nanoparticles (CAAgNPs) present in
the range of 0.0032 to 0.1024% w/v;
(b) Chitosan present in the range of 1-3% w/v;
(c) Acetic acid present in the range of 0.5-2% v/v;
(d) Glutaraldehyde present in the range of 0.01-0.05% w/v;
(e) Sodium Hydroxide is in Q.S.; and
(f) Deionized water is in Q.S.
In another embodiment, wherein Curcuma aromatica loaded silver nanoparticles
(CAAgNPs) and chitosan are present in a ratio of 1:2 to 2:1.
In another embodiment, wherein the invention provides a process for the preparation
of the wound dressing, comprising the following steps:
(i) Mixing aqueous rhizome extract of Curcuma aromatica (CA) with silver
nitrate to form a solution and concentrate the synthesized CAAgNPs in a
freeze-drier;
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(ii) Dissolving chitosan in aqueous acetic acid (AA) under stirring for 2 h at
room temperature to form a solution;
(iii)
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Adding silver nanoparticles of Curcuma aromatica (CAAgNPs)
obtained in step (i) into a solution of step (ii) and kept at room temperature
with stirring for 2 h;
(iv) Adding crosslinker in the mixture obtained in step (iii) under constant
stirring for 4 h at room temperature;
(v) Pouring the mixture obtained in step (iv) into the petri plates and keeping
them overnight at -20 °C; and
(vi) Thawing the frozen samples obtained in step (v) and adding NaOH to
neutralize and form a hydrogel, further washing with deionized water, then
freezing and lyophilizing to obtain dressings.
In another embodiment, wherein the porosity of the wound dressing composition is
in the range of about 60% to 70%, thereby enhancing exudate absorption, gaseous
exchange, and cellular infiltration for wound healing applications. In a particular
embodiment, wherein the porosity of the wound dressing composition is 63.56%.
In another embodiment, wherein the degree of swelling of the dressing is in the
range of 55%–75% with a fluid retention time of 4-10 hours, thereby maintaining
wound moisture balance and reducing the need for frequent dressing replacement. In
particular embodiment, wherein the degree of swelling was 61.13% with a retention
time of 6-7 h.
In another embodiment, wherein the composition is in the form of a sponge, disc,
film, composite, hydrogel, foam, powder, aerosolized powder or aerosolized liquid
dressing. In a particular embodiment, wherein the composition is in the form of a
hydrogel.
In another embodiment, wherein the composition has antibacterial and anti
inflammatory activity.
In another embodiment, wherein the composition exhibits dose-dependent
antibacterial activity, with maximum inhibition observed for CAAgNPs1024/CS
against Pseudomonas aeruginosa NCIM 5029, Pseudomonas aeruginosa PAW1,
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Staphylococcus aureus NCIM 5021, and Staphylococcus aureus S8 thereby
demonstrating superior antibacterial efficacy compared to chitosan-only dressings
and commercial dressings.
In
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another embodiment, wherein the formulation is adapted for topical
administration.
In another embodiment, wherein the composition is used in wound management.
In another embodiment, wherein the composition accelerates wound healing by day
7 with 55–70% closure and collagen deposition of 50–60% stained area or 70–80
µg/mL hydroxyproline, achieves 90–95% closure by day 10 with smooth, scar-free
regenerated skin, and regulates inflammatory responses by reducing IL-6 levels to
about 15–30 pg/mL and TNF-α levels to about 70–90 pg/mL, thereby preventing
inflammation and promoting fibroblast proliferation, angiogenesis, and complete
tissue regeneration.
Other embodiments of the present disclosure will be apparent to those skilled in the
art
from consideration of the specification and practice of the disclosed
embodiments. The following examples should be considered as exemplary only,
with a true scope and spirit of the present disclosure being indicated by the claims.
Experimental
(I) Composition:
Ingredients
Example
1
Example
2
Example
3
Example
6
Example
4
CAAgNPs
(µg/mL)
32
64
128
256
Example
5
512
Chitosan (%
w/v)
2
2
2
2
1024
2
Acetic Acid (%
v/v)
1
1
1
1
2
1
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1
Glutaraldehyde
(% w/v)
0.025
0.025
0.025
0.025
0.025
Sodium
Hydroxide
Q.S.
Q.S.
Q.S.
Q.S.
0.025
Q.S.
Deionized water
Q.S.
Q.S.
Q.S.
Q.S.
Q.S.
(II) Procedure:
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Q.S.
Q.S.
The spherical-shaped CAAgNPs (13±5 nm) were synthesized using 0.5 g% (w/v) of
the aqueous rhizome extract of CA and 0.8 mM of AgNO3 (SRL, India) when
incubated at 60 °C for 144 h (Tawre, et al., 2022, Frontiers in Chemistry, 10, 1–17).
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The synthesized CAAgNPs were concentrated in a freeze-drier (Martin Christ
Alpha 1-2 LD plus, Germany) and a stock solution of 10 mg/mL of CAAgNPs was
prepared for further use. The minimum inhibitory concentration of the reported
CAAgNPs against the tested isolates ranged between 8–64 µg/mL. Therefore,
higher concentrations of CAAgNPs were used in the dressing preparation. Chitosan
(CS) (190–310 KDa; degree of deacetylation ≥75%) was purchased from Sigma
Aldrich, Germany, and used without any further purification. CAAgNPs/CS
dressings were fabricated as per the earlier studies with some modifications. Briefly,
CS powder weighing 2% (w/v) was dissolved in 1% (v/v) acetic acid solution (SRL,
India) with constant stirring for 2 h at room temperature. Then to this mixture,
CAAgNPs were added at concentrations 0, 256, 512, and 1024 µg/mL, respectively,
and kept at room temperature with stirring for 2 h. A crosslinker, glutaraldehyde
0.025% (w/v) (Sigma-Aldrich, Germany) was added to the mixture and kept with
constant stirring for 4 h at room temperature. The mixture was uniformly poured
into the petri plates and kept overnight at -20 ◦C. The frozen samples were thawed
by the addition of 0.1 M NaOH (HiMedia, India) solution and were neutralized by
rinsing with deionized water. The samples were frozen at -20 °C and further freeze
dried using a lyophilizer to obtain the dressing. They were designated CS,
CAAgNPs256/CS, CAAgNPs512/CS, and CAAgNPs1024/CS dressings. All the
dressings were sterilized by UV irradiation before use.
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(III) Evaluation: (Examples 4 to 6)
A. Physical and chemical characterization of CAAgNPs/CS dressing:
1. Structural characterization:
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The morphological analysis of the prepared CS and CAAgNPs/CS dressings was
performed using field emission scanning electron microscopy (FESEM) (FEI Nova
Nano SEM 450, Netherlands). The samples were coated with a thin layer of gold
using a sputter coater (Q150T ES Quorum, UK) and observed under FESEM at
500x and 5000x magnification. Fourier-transform infrared (FTIR) spectroscopy
(Opus, Bruker, Japan) was performed with CS powder, and CS, and CAAgNPs/CS
dressings to identify any of the possible interactions between the CAAgNPs, CS,
and the glutaraldehyde molecules. The infrared spectra of the samples were taken in
the range of 500–4000 cm-1 of the wavenumber with a resolution of 2 cm-1.
2. Porosity assessment
The porosity of the prepared CAAgNPs/CS dressing was evaluated by immersing
the dressings in absolute ethanol (SRL, India) until saturation. The weight of the
dressing was measured before and after the immersion. The percent porosity (P) was
determined using the following equation:
P (%) = [(w0 — w1) / ρV2] × 100
where, w0 and w1 are the weights of the dressings before and after immersion
respectively; V is the volume of the dressing before immersion = length x width x
height; ρ is the density of ethanol. The experiment was duplicated in triplicates.
3. Swelling behavior and moisture retention capacity
The water uptake ability of the CAAgNPs/CS dressing was measured by immersing
it in deionized water and then allowing it to swell for 2 h. Subsequently, the
dressing was removed from the water, gently blotted, and weighed immediately.
The degree of swelling (DS) was calculated using the following equation:
Water Uptake (%) = [(w1 — w0) / w0] × 100
where, w0 and w1 are the weights of the dressings before and after immersion.
The moisture retention capacity of the dressing was quantified by placing the soaked
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dressing in a glass dryer at room temperature, and the DS was determined every
hour. The moisture retention time was recorded as the value of the DS reduced to
100%. The experiment was duplicated in triplicates.
Result:
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The fabricated CS, CAAgNPs256/CS, CAAgNPs512/CS, and CAAgNPs1024/CS
dressings were cut into pieces with a thickness of 2 mm and a size measuring 6 × 6
mm, for further application. The porosity of the dressing was estimated to be
63.56%. The degree of swelling was 61.13% with a retention time of 6–7 h. FESEM
displayed a network-like structure of the dressing, demonstrating its porous nature.
The distribution of CAAgNPs on the surface of the dressing was also evident
(Figure 1a). FTIR demonstrated a reduction in the peak intensity and shift in the
amide II band observed for CS powder, from 1589.06 to 1565.06 cm-1 and 1575.54
cm-1 in CS and CAAgNPs/CS dressing, respectively. These peaks were associated
with an ethylenic bond (C = C) confirming the crosslinking with glutaraldehyde. A
new peak at 2916.32 and 2916.89 cm-1 was observed in CS and CAAgNPs/CS
dressing corresponding to C–H stretching vibration frequency. Reduction of the
peak from 3354.59 cm-1 in CS powder to broadband at 3360.41 and 3358.49 cm-1 in
CS and CAAgNPs/CS dressing, corresponds to O–H stretching.
B. Antibacterial activity of CAAgNPs/CS dressing against MDR pathogens
Antibacterial efficacy of the prepared CS, CAAgNPs256/CS, CAAgNPs512/CS,
CAAgNPs1024/CS and commercial dressing was tested against P. aeruginosa NCIM
5029, P. aeruginosa PAW1 (clinical isolate), S. aureus NCIM 5021 and S. aureus
S8 (clinical isolate) using disk diffusion method followed as per the Clinical and
Laboratory Standards Institute guidelines (CLSI, 2021). Briefly, the O.D. of the
overnight grown culture in Luria Bertani (LB) (HiMedia, India) broth was adjusted
to 105 CFU/mL and 100 µL of the culture was spread on the Mueller Hinton (MH)
(HiMedia, India) agar plate using a sterile spreader. Cut pieces (6 × 6 mm) of the
CS, CAAgNPs256/CS, CAAgNPs512/CS, CAAgNPs1024/CS, and commercial (silver
based) dressings were placed on the MH agar plates. CS dressing served as the
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control. After 24 h of incubation at 37 °C, the plates were observed for the zone of
inhibition. The experiment was duplicated in triplicates.
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Dose-dependent antibacterial activity was observed for CAAgNPs256/CS,
CAAgNPs512/CS, and CAAgNPs1024/CS dressings. Maximum inhibition zone was
observed for CAAgNPs1024/CS dressing against P. aeruginosa NCIM 5029, P.
aeruginosa PAW1, and S. aureus NCIM 5021 compared to CS dressing. A
minimum zone of inhibition was observed for CAAgNPs512/CS, and CAAgNPs1024/
CS dressings against S. aureus S8. Commercial dressing demonstrated antibacterial
activity against S. aureus NCIM 5021 and S. aureus S8 with a maximum inhibition
zone, however, it did not display any antibacterial activity against P. aeruginosa
NCIM 5029 and P. aeruginosa PAW1. The antibacterial activity demonstrated by
CAAgNPs1024/CS dressing suggests their potential application as a wound dressing.
(Figure 1b).
C. In vivo studies:
1. Animals and experimental conditions:
All the animal experiments were performed at the animal housing facility of the
Agharkar Research Institute, Pune, India. The in vivo experiments were conducted
after seeking approval from the Institutional Animal Ethics Committee of Agharkar
Research Institute (Ethical approval: ARI/IAEC/2021/01). Animals of both sexes,
male and female, were used in the study. Wistar rats (6–8 weeks old), each weighing
180–200 g were selected for the study. During the study, animals were housed under
standard laboratory conditions (22 ± 2 ◦C, relative humidity of 55%, and 12–12 h of
light-dark cycle). The animals were fed with a standard chow diet and water ad
libitum. The animals were divided into six groups (n = 12 animals in each group)
namely, wound control (without any dressing), CS, CAAgNPs256/CS,
CAAgNPs512/CS, CAAgNPs1024/CS, and commercial (silver-based) dressing. Before
the creation of the wounds, the animals were anesthetized by intraperitoneal
injection of ketamine (80 mg kg-1) and xylazine (10 mg kg-1) (Themis Medicare
Limited, India). The fur from the dorsal neck region of each rat was trimmed with
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scissors and then shaved using a razor. Later, the skin was disinfected with 70%
ethanol. The full-thickness excisional wound of 6 mm diameter was created in the
dorsal thoracic region, 1 cm away from the vertebral column and 5 cm away from
the ear, as previously marked. The wounds were tightly covered with the dressings.
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2. Assessment of wound contraction rate:
The area of the wound was measured and photographed on days 3, 7, and 10 post
wounding. The dressings were changed every alternate day. Gradual changes in
wound healing were evaluated by tracing the wound area on transparent tracing
paper on days 0, 3, 7, and 10 post-wounding. A transparent sheet was placed over
the wound and the wound boundaries were traced using a marker. The tracing paper
was then overlaid on graph paper and the number of squares within the wound area
were counted on days 3, 7, and 10. The percentage of wound contraction (WC) was
estimated using the following equation:
WC (%) = [ At0 — At3/7/10) / At0] × 100
where, At0, is the wound area in mm at the time of injury; At3/7/10 is the wound area
in mm at day 3, 7, and 10 post-injuries.
Wound healing ability of CAAgNPs/CS dressing:
Compared to the wound control groups, wounds treated with CAAgNPs1024/CS and
commercial dressing showed an enhanced wound healing effect on day 7 reaching
its maximum on day 10 (Fig. 2a). Besides, the regenerated skin was smooth like
normal skin without scar formation. On day 7, contraction of the edges was
observed in the wounds treated with CAAgNPs1024/CS and commercial dressings.
Until day 10, the wounds treated with CAAgNPs1024/CS and commercial dressings
were completely dry and covered with granulation tissue with no apparent swelling.
There were no adverse effects such as scar formation, necrosis, and inflammation.
Comparatively, the surface of the wound control group was covered with a pale and
hard crust. Wound closure analysis revealed there was no significant difference
between wound control and other groups on day 3 (Fig. 2b). On day 7, healing
enhanced significantly in the wounds treated with CAAgNPs1024/CS (62.50%; p <
0.01) and commercial dressings (66.67%; p < 0.001) as compared to the wound
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control (37.50%).
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Almost complete epithelialization and closure of the wound occurred on day 10. On
day 10, healing improved further in wounds treated with CAAgNPs512/CS (78.33%;
p < 0.05), CAAgNPs1024/CS (93.75 %; p <0.0001), and commercial (92.08%; p
<0.0001) dressings as compared to wound control (58.33%). The findings suggest
that the CAAgNPs1024/ CS dressing accelerates the wound healing process at early
stages similar to the commercial dressings.
3. Histological analysis of the wounded skin tissue:
For histological analysis, the wounded skin tissues (3 × 3 mm) were excised at
different intervals on days 3, 7, and 10 and fixed in 10% formaldehyde (SRL, India)
solution at 4 ◦C for 24 h. The excised wound tissues were processed, embedded in
paraffin, sectioned, and stained with hematoxylin and eosin (H and E) (HiMedia,
India). Briefly, the prepared tissue blocks were sliced into 5 μm sections using a
microtome (Hoverlabs, India). The sections were deparaffinized, rehydrated, and
stained with H and E for histological examination. The stained sections were
observed under an optical microscope (Nikon Eclipse E200, Japan). Wound healing
was qualitatively studied by analyzing the presence of inflammatory infiltrate,
vascularization, tissue granulation, epithelization, and the amount of collagen
deposition. A comparative analysis between the control and treated groups was
carried out.
H and E staining confirmed the presence of inflammatory cells with eosinophils in
the epidermis, in all the wound control groups, and wounds treated with CS, and
commercial dressings, on day 3. A mild inflammation was observed in the
epidermis of wounds treated with CAAgNPs256/CS, CAAgNPs512/CS, and
CAAgNPs1024/CS dressings, on day 3. On day 7, incomplete epithelialization of the
epidermis with necrotic and inflammatory cells was detected in the wound control
groups.
The wounds treated with CAAgNPs256/CS, CAAgNPs512/CS,
CAAgNPs1024/CS, and commercial dressings did not show the presence of
inflammatory cells. Instead, irregular granulation and fibrogenesis were detected in
treated wounds. On day 10, wound control showed irregular re-epithelialization and
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healing. Treated wounds showed a uniform fibroblast layer along with uniform
granulation and epidermis. The formation of hair follicles, and endothelial
cells/blood vessels was evident in treated wounds, on day 10. Thus, complete
regeneration of tissue around the wound site was observed on day 10. Complete
wound healing was observed in the wounds treated with CAAgNPs512/ CS,
CAAgNPs1024/CS, and commercial dressings.
4. Qualitative and quantitative estimation of collagen content:
Direct red-80 stain was purchased from Sigma-Aldrich, Germany. The excised
wound tissues were processed as mentioned above and the sections were stained
with direct red 80 for the qualitative estimation of collagen deposition. The sections
were observed under an optical microscope and the percentage of collagen was
analyzed using ImageJ software (Version 1.5a Java 1.8).
Hydroxyproline content determination is an indirect method for the detection of
collagen. Blood was drawn from the retro-orbital plexus of rats on days 3, 7, and 10
to obtain serum. The serum samples collected on the respective days 3, 7, and 10
were used for the estimation of hydroxyproline using a colorimetric assay kit
(Elabscience, USA) by the alkali hydrolysis method. The protocol was followed as
per the manufacturer’s instructions. The absorbance was recorded colorimetrically
at 550 nm. The assays for each group were carried out twice, in triplicates.
Hydroxyproline content was calculated using the following equation:
Hydroxyproline (μg / mL) = (ΔA1 / ΔA2) × c × f × VTotal / VSample)
where, ΔA1 = ODSample – ODBlank; ΔA2 = ODStandard – ODBlank; c =
concentration of standard (5 μg/mL); f = dilution factor of the sample before the
test; VTotal = total volume of hydrolysis solution (10 mL); VSample = total volume of
sample (mL).
Collagen deposition was quantified qualitatively using direct red-80 dye. Compact
collagen bundles in the dermis were evident in the wounds treated with
CAAgNPs1024/CS and commercial dressing. Wound control showed randomly
oriented collagen fibrils. The percent-stained area for collagen was calculated and a
dose-dependent increase was observed for all the CAAgNPs/CS dressing treatment
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groups from day 3, reaching its maximum at day 10. On day 7, CAAgNPs1024/CS
(52.04%; p < 0.001) treated wounds showed maximum collagen deposition which
was comparatively higher than the control wounds (26.03%). Maximum collagen
deposition was observed for wounds treated with CAAgNPs1024/CS (58.02%; p <
0.05) followed by CAAgNPs512/CS (52.58%; p < 0.01) on day 10 as compared to
the wound control (34.35%). Increased collagen deposition indicates that
CAAgNPs1024/CS dressing enhance the wound healing process.
Quantitative estimation performed using the hydroxyproline assay confirmed the
maximum collagen deposition for all the groups on day 7 compared to day 3.
Elevated levels of hydroxyproline were detected in wounds treated with
CAAgNPs512/CS (71.68 µg/mL; p <0.05), CAAgNPs1024/CS (76.51 µg/mL; p <
0.01), and commercial (77.45 µg/mL; p < 0.01) dressing as compared to the wound
control (51.57 µg/mL), on day 7. This suggests that maximum collagen formation
was achieved in wounds treated with CAAgNPs1024/CS dressing on day 7.
Subsequently, a decrease in the hydroxyproline content was observed in wounds
treated with CAAgNPs512/CS, CAAgNPs1024/CS, and commercial dressing, on day
10, indicating that equilibrium was achieved between the rate of collagen formation
and degradation. (Figure 3 and 4)
5. Quantitative estimation of inflammatory cytokine (IL-6 and TNF-α) levels:
Rat interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) ELISA kits were
purchased from Elabscience, USA. Rat serum samples collected on days 3, 7, and
10 respectively were evaluated for the levels of pro-inflammatory cytokines such as
IL-6 and TNF-α using ELISA assay. The protocols were followed as per the
manufacturer’s instructions. The absorbance was recorded at 450 nm and
concentrations were interpolated using the standard curves. The assays for each
group were carried out twice, in triplicates.
Inflammatory responses:
When pro-inflammatory cytokines were monitored, high levels of IL- 6 were
observed in all the groups on day 3 compared to days 7 and 10. On day 3, the IL-6
levels increased significantly in wounds treated with CAAgNPs512/CS (39.59
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pg/mL; p < 0.01), CAAgNPs1024/CS (45.94 pg/mL; p < 0.01) and the commercial
(47.29 pg/mL, p < 0.001) dressing compared to the wound control (22.15 pg/mL).
On day 7, there was a significant reduction in the IL-6 levels in wounds treated with
CAAgNPs1024/CS (27.82 pg/mL, p < 0.01) dressings compared to wound control
(42.29 pg/mL). Subsequently, IL-6 levels subsided by day 10 in wounds treated
with CAAgNPs1024/CS (17.38 pg/mL, p < 0.001) and commercial (20.43 pg/mL, p <
0.01) dressings. However, maximum IL-6 levels were observed in the wound
control and wound treated with CS dressing on day 10. The results indicate that
CAAgNPs1024/CS and commercial dressing enhance the wound healing process
without scar formation compared to the control groups. Increase in the TNF-α levels
was noted on day 3 compared to days 7 and 10. On day 3, TNF-α levels elevated
significantly in the wounds treated with CAAgNPs1024/CS (263.29 pg/mL, p <
0.001) dressing as compared to wound control (133.09 pg/mL). On day 10, there
was a decrease in TNF-α levels in the wounds treated with CAAgNPs1024/CS (80.38
pg/mL; p < 0.05) dressing as compared to wound control (109.83 pg/mL). Low
levels of TNF-α observed on day 10 indicate that the CAAgNPs1024/CS dressing
contributes to the formation of fibroblasts and the expression of growth factors,
thereby, aiding the wound healing process. (Figure 5a)
D. Biocompatibility of CAAgNPs/CS dressings with HEK293 cells
Human embryonic kidney (HEK) 293 cell line was purchased from the National
Centre for Cell Sciences, Pune, Maharashtra, India. Dulbecco’s modified Eagle’s
media (DMEM), penicillin and streptomycin solution (100 µg/mL), trypsin-EDTA
0.25, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye and
sodium dodecyl sulfate (SDS) were purchased from HiMedia, India; fetal bovine
serum (FBS) was purchased from Sigma-Aldrich, Germany. HEK293 cells were
grown until complete confluency in DMEM containing 10% FBS and 1% Penicillin
Streptomycin solution for 24 h at 37 °C in a humidified atmosphere of 5% CO2. The
cells were trypsinized and a cell density of 1×104 cells/well was seeded in a 24-well
plate, incubated for 24 h at 37 ºC with 5% CO2. Subsequently, sterile CS,
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CAAgNPs256/CS, CAAgNPs512/CS, CAAgNPs1024/CS, and commercial dressings
were soaked in DMEM for 1 h and placed in a 24-well plate. The wells without any
dressing served as a positive control (Liang et al., 2016). The plate was incubated
for 24 h at 37 ºC with 5% CO2. After that, the medium from the wells was aspirated
and 5 mg/mL of MTT solution was added, followed by incubation at 37 ºC for 4 h.
The formed formazan crystals were dissolved by adding a solubilizing buffer (10%
SDS in 0.01N HCl) and incubated overnight. The absorbance was read at 570 nm
using an ELISA plate reader (Hidex, Finland) and the percent cell viability was
calculated using the following equation.
�
�𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 (%) = (𝐴T/𝐴C) × 100
where AC and AT were the absorbance (570 nm) of the control and test respectively.
More than 90% of HEK293 cell viability was observed in cells treated with the
CAAgNPs1024/CS dressing that contained the highest dose of CAAgNPs.
CAAgNPs/CS dressings showed no significant reduction in HEK293 cell viability
compared to the control. HEK293 cells treated with commercial dressing showed a
significant difference in cell viability (79.71%, p ≤ 0.01) compared to the control.
CAAgNPs1024/CS dressing demonstrated increased cell viability than commercial
dressing, suggesting less toxicity towards HEK293 cells (Figure 5b).
E. Statistical analysis:
Statistical analysis was performed using GraphPad Prism 9.0. The data was
expressed as the average ± standard deviation. Any significant differences between
the control and test groups were performed using one-way and two-way ANOVA,
followed by Bonferroni’s multiple comparison test. All statistical tests were set at a
significance level α of 0.05 and considered significant at * p < 0.05, ** p < 0.01,
*** p < 0.001, or **** p < 0.0001. ImageJ software was used to process and
analyze the images.
➢ Technological Advancements of the Invention
• Develops a multifunctional wound dressing that integrates antimicrobial,
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anti-inflammatory, and regenerative properties in a single composition.
• Achieves superior biocompatibility compared to chemically synthesized
AgNP dressings, minimizing cytotoxicity risks.
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• Provides formulation flexibility – can be prepared as films, sponges, foams,
hydrogels, powders, or aerosolized dressings.
• Offers a sustainable, cost-effective, and scalable approach suitable for
medical and commercial applications. , C , Claims:We Claim:
1. A wound dressing composition comprising Curcuma aromatica (CA) loaded
silver
nanoparticles (CAAgNPs), polymer, and other pharmaceutically
acceptable excipients thereof.
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2. The wound dressing composition as claimed in claim 1, wherein the other
pharmaceutically acceptable excipients are selected from solvent, crosslinker or
a combination thereof.
3. The wound dressing composition as claimed in claim 1, wherein the polymers
are selected from sodium alginate, gelatin, carageenan, chitosan, chitin, fibrin,
and collagen, either alone or in combination thereof.
4. The wound dressing composition as claimed in claim 2, wherein the solvents are
selected from acetic acid, formic acid, lactic acid, citric acid, hydrochloric acid,
sodium hydroxide, potassium hydroxide, ethanol, methanol, isopropanol, and
deionized water, either alone or in combination thereof.
5. The wound dressing composition as claimed in claim 2, wherein crosslinkers are
selected
from glutaraldehyde, genipin, epichlorohydrin, formaldehyde,
carbodiimides, sodium tripolyphosphate (TPP), glyoxal, and tannic acid, either
alone or in combination thereof.
6. The wound dressing composition as claimed in claim 2, wherein neutralizing
agents are selected from sodium hydroxide, potassium hydroxide, calcium
hydroxide, magnesium hydroxide, ammonium hydroxide, and sodium carbonate,
either alone or in combination thereof.
7. The wound dressing composition as claimed in claim 1, wherein the wound
dressing composition comprising,
(a) Curcuma aromatica loaded silver nanoparticles (CAAgNPs) present in
the range of 0.0032 to 0.1024% w/v;
(b) Chitosan present in the range of 1-3% w/v;
(c) Acetic acid present in the range of 0.5-2% v/v;
(d) Glutaraldehyde present in the range of 0.01-0.05% w/v;
(e) Sodium Hydroxide is in Q.S.; and
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(f) Deionized water is in Q.S.
8. The process for preparation of the wound dressing comprises following steps of:
(i) Mixing aqueous rhizome extract of Curcuma aromatica (CA) with silver
nitrate to form a solution and concentrate the synthesized CAAgNPs in a
freeze-drier;
(ii) Dissolving chitosan in aqueous acetic acid under stirring for 2 h at room
temperature to form a solution;
(iii) Adding silver nanoparticles synthesized using Curcuma aromatica
(CAAgNPs) obtained in step (i) into a solution of step (ii) and kept at room
temperature with stirring for 2 h;
(iv) Adding crosslinker in the mixture obtained in step (iii) under constant
stirring for 4 h at room temperature;
(v) Pouring the mixture obtained in step (iv) into the petri plates and keeping
them overnight at -20 °C; and
(vi) Thawing the frozen samples obtained in step (v) and adding NaOH
solution to neutralize and form a hydrogel, further washing with deionized
water, then freezing and lyophilizing to obtain dressings.
9. The wound dressing composition of claim 1, wherein the composition is in the
form of a sponge, disc, film, composite, hydrogel, foam, powder, aerosolized
powder or aerosolized liquid dressing.
10. The wound dressing composition of claim 1, wherein the composition has
antibacterial and anti-inflammatory activity and is used in wound management.
Dated this on 20th day of September 2025