Regenerative Therapy 27 (2024) 92e103

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Regenerative Therapy
journal homepage: http://www.elsevier.com/locate/reth

Original Article

Novel design and development of Centella Asiatica extract - loaded

poloxamer/ZnO nanocomposite wound closure material to improve
anti-bacterial action and enhanced wound healing efficacy in diabetic
foot ulcer
Lina Wang a, 1, Yan Yang b, 1, Weiwei Han c, Hui Ding c, *
a
Department of Endocrinology, Qingdao Chengyang District People's Hospital, Qingdao, 266109, PR China
b
Department of Dermatology, Qingdao Chengyang District People's Hospital, Qingdao, 266109, PR China
c
Department of Medical Laboratory, Qingdao Huangdao District Central Hospital, 266555, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Diabetic wounds can occur as a prevalent complication among people diagnosed with diabetes,
Received 16 December 2023 frequently resulting in the necessity for amputation. The cause and effect of diabetic foot ulcer is
Received in revised form complex, involving multiple factors. In the present study, wound healing strategies utilizing nano-
20 February 2024
materials have proven to be effective in battling bacterial infections and improve wound regeneration.
Accepted 9 March 2024
Poloxamers (PLX) exhibit extensive potential as a viable option for the development of nanomedicines
owing to their inherent characteristics of self-assembly and encapsulation. This study aims to design and
Keywords:
develop a PLX/ZnO nanocomposite incorporated with Centella Asiatica extract (CAE) for the multi-
Diabetic foot ulcer
Centella Asiatica extract
functional action in the diabetic wound healing treatment. Subsequently physico-chemical character-
Poloxamer izations, such as XRD, FTIR, and TEM observations, demonstrated that the ZnO were evenly distributed
Anti-bacterial action through the PLX framework. The developed nanocomposite was biocompatible with mouse fibroblast
Cell proliferation cell line (L929), and it had multiple beneficial characteristics, such as a rapid self-healing process and
Wound healing effective antibacterial action against Gþ and G bacterial pathogens. After being treated with the
developed formulation, skin fibroblast cell line and HUVECs demonstrated a substantial increase in their
in vitro cell proliferation ability, migration, and tube-forming abilities. The utilization of a CAE@PLX/ZnO
nanoformulation presents a viable strategy and a distinctive, encouraging composite for diabetic wound
healing treatment.
© 2024, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is
an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

1. Introduction and chronic inflammation. Typically, wounds are vulnerable to

bacterial infections that disrupt the healing process by prompting
A primary risk factor for diabetes mellitus is impaired wound inflammation and instigating damage to the surrounding tissue.
healing, which might have long-lasting negative impacts on quality The process of wound healing is considered to be highly complex
of life, morbidity, and mortality. The current standard of care for within the human body. An ideal therapeutic approach should
diabetic wound treatment relies on early detection, preventative possess the capability to accelerate wound healing, reduce micro-
actions, and comprehensive instruction for patients [1e3]. bial infections, maintain suitable microenvironments, and mitigate
Impaired wound healing in diabetes is triggered by an intricate scar formation [4,5]. The field of wound healing has witnessed a
aetiology, but the main factors include increased oxidative stress growing number of novel therapies; however, certain treatments
have proven to be ineffective in achieving favorable clinical out-
comes. Wound re-epithelialization and fluid loss management are
* Corresponding author. NO. 9, Huangpujiang Road, Huangdao district, Qingdao
two examples of structural factors that contribute to these prob-
266555, PR China. lems [6]. The influence that various treatment modalities have on
E-mail address: [email protected] (H. Ding). the healing process of diabetic wounds, the part that underlying
Peer review under responsibility of the Japanese Society for Regenerative comorbidities play in holding back the healing process, and the
Medicine.
1 identification of novel therapeutic targets for the purpose of
Contribute equally to this study.

https://doi.org/10.1016/j.reth.2024.03.006
2352-3204/© 2024, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).
L. Wang, Y. Yang, W. Han et al. Regenerative Therapy 27 (2024) 92e103

enhancing wound healing in diabetic patients are all topics that will intravenously to human subjects, pluronic block copolymers have
be discussed. We will be able to effectively address the gaps in the been shown to exhibit a level of safety that is satisfactory, as was
current research on diabetic wound healing and work toward previously reported. In the context of its application as a wound
developing interventions that are more targeted and effective for healing cleansing agent, the utilization of poloxamer 188, which is
this patient population if we narrow our focus to these specific characterized by a substantial ethylene oxide content of 80% and a
areas [7,8]. These considerations have prompted a new era in the molecular weight of 8350 Da, has been identified as a potential
use of nanomaterials for wound healing treatments to emerge from material for wound healing applications. It demonstrates efficacy as
the realm of nanotechnology. This approach offers potential solu- a bactericidal agent, effectively eliminating bacteria without
tions for expediting the process of wound healing, while also causing damage to tissue, and providing assistance in preventing
providing unique properties as bactericidal agents [4e6]. With infection without inducing any adverse effects that have been
simple manipulation of material type, nanoparticle size, and sur- observed [20,21].
face charge, the biochemical properties of nanoparticles, such as The extracts from Centella asiatica (CAE) is widely utilized in
hydrophobicity, connection with biologically active compounds, traditional chinese medicine. Wound dressings formulated with
and tissue penetration at varying depths, may be easily tailored to CAE have an advantage in healing because it promotes collagen
different kinds of wounds [7e9]. Two primary approaches have synthesis and fibroblast proliferation [22e24]. As a result, several
been devised for the utilization of nanomaterials derived from both biomaterials have been studied in relation to CAE's potential use as
inorganic and organic sources, respectively. Nanoscale materials a wound dressing [25e27]. As a result, there is growing interest in
possessing antimicrobial properties are commonly incorporated using CAE in wound dressing applications, and a number of bio-
into polymers, particularly biopolymers derived from abundant materials have been studied [28,29]. The previous reports used
natural sources, or nanocarriers employed for encapsulating the incision and partial-thickness burn wound models in rats to
active agent. Biomedical nanomaterials have garnered increased examine the effects of consecutive CAE prepared with hexane, ethyl
attention in recent times due to their notable biological attributes acetate, methanol, and water on wound healing. The positive at-
and their applications in the field of biomedicine. Nanoparticles of tributes of wound dressings can be further augmented by incor-
metal oxides, such as zinc oxide (ZnO), silver (Ag), and cerium oxide porating natural bioactive agents. In order to achieve the goals of
(CeO2), in addition to other materials including graphene and enhancing antibacterial activity and enhancing the effectiveness of
carbon nanotubes (CNT), have exceptionally promising potential in wound healing in diabetic foot ulcer applications, the current
the biomedical field. One of the most important metal oxide investigation has designed and developed on the synthesis of PLX/
nanoparticles, zinc oxide nanoparticles (ZnO NPs) have unique ZnO nanocomposites that contain Centella Asiatica Extract (labelled
physical and chemical characteristics that make them potential in as CAE@ PLX/ZnO). A highly effective and stable nanocomposite
different biomedical areas [10,11]. Moreover, ZnO NPs exhibit material with enhanced antimicrobial and wound healing proper-
remarkable antimicrobial properties, as well as exceptional UV- ties is expected to be developed as a result of the combination of
blocking capabilities. Zinc is widely recognized as an indispens- ZnO nanoparticles, CAE, and poloxamer, according to the hypoth-
able trace element that is found abundantly in various tissues of esis of the study. As a result of the incorporation of ZnO nano-
human body, such as the brain, muscles, bones, and skin. Zinc, particles, it is anticipated that the nanocomposite material will
being a fundamental constituent of multiple enzyme systems, possess powerful antimicrobial properties. Additionally, it is
actively participates in the metabolic processes of the human body. anticipated that the addition of CAE will contribute to the wound
It assumes vital functions in the synthesis of proteins and nucleic healing properties of the material. Poloxamer, which is well-known
acids, as well as in hematopoiesis and neurogenesis [12]. In com- for its ability to stabilize and solubilize substances, is anticipated to
parison to other metal oxide nanoparticles, ZnO NPs possess rela- improve the nanocomposite's overall performance in terms of
tively lower toxicity and cost-effective, making them highly stability. It is hypothesized that these three components will work
suitable for various biomedical applications. Among these uses are together in a synergistic manner to produce a nanocomposite
anti-cancer medications, drug delivery systems, antibacterial material that is both highly effective and stable, and that possesses
agents, diabetes medications, anti-inflammatory agents, wound enhanced antimicrobial and wound healing properties. The sche-
healing and bioimaging applications [13e15]. Investigated the matic representation (Fig. 1) displays that our developed nano-
possibility of different sized ZnO NPs penetrating injured and composited formulation showed great promise for effective
allergic surface in an animal model of dermatitis. The results of the chronic diabetic wound healing, which inhibited bacterial infec-
experiments showed that larger-sized ZnO could only reach the tion, reduced inflammation, accelerated fibroblast cell survival and
upper layers of damaged and allergic skin, whereas nanosized ZnO proliferation, and facilitated improved wound healing.
particles could reach the deeper layers of the skin. The findings of
this study indicate that ZnO nanoparticles (NPs) of smaller size 2. Experimental section
exhibited significant efficacy in mitigating skin inflammation in
models of atopic dermatitis (AD), when compared to larger-sized 2.1. Materials and methods
ZnO NPs. Because of its antidiabetic, antifungal, antioxidant, anti-
inflammatory, and antibacterial efficiencies [16e19]. Centella Asiatica plant collected from region of eastern Shan-
Recent years, self-assembled block copolymers (BCPs) have dong, PR China. Poloxamer (100%), Zinc nitrate (99.0%), Dichloro-
been instrumental in the creation of cutting-edge nanoscale sys- methane (99.80%), N-hexane, Ethyl acetate, Methanol, Ammonium
tems that have revolutionized medication delivery. PLX, alterna- hydroxide (28% NH3 in H2O, 99.99%) and Tween 20® were pur-
tively referred to as pluronics, are a distinctive category of synthetic chased from Sigma Aldrich in China.
triblock copolymers. A core hydrophobic chain of poly (propylene
oxide) (PPO) and two hydrophilic chains of poly (ethylene oxide) 2.2. Synthesis of Centella Asiatica extract (CAE)
make up these copolymers (PEO). The synthesized PLX show great
promise for use in a range of biomedical applications due to their The leaves of CAE were harvested from the local area and then
chemical characteristics, which include thermo-reversible behavior dried in an oven at 50  C before being milled into a powder. The CAE
and self-assembly that is temperature sensitive, as well as their plant extract was prepared as following previously reported pro-
physiochemical qualities and biocompatibility. When administered tocol with slight modification [30]. At room temperature, 1 kg of
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L. Wang, Y. Yang, W. Han et al. Regenerative Therapy 27 (2024) 92e103

Fig. 1. Schematic representation of the preparation of CAE-loaded PLX/ZnO nanocomposite for the treatment of diabetic foot ulcer.

plant powder was macerated with 3e4 L of n-hexane for three The advantages of these antibiotic nanoparticles can be readily and
days. The mixed filtrates were dried at low pressure until they efficiently synthesized through cost-effective methodologies.
attained a constant weight of 17.8 g, and then the n-hexane extract
was produced (a yield of 0.18% for the fresh plant). After the marc 2.5. Characterization methods
was air-dried, it was macerated with ethyl acetate (32.7 g, 0.33%
yield) and methanol (267.5 g, 2.68% yield), respectively, according The nanocomposites were subjected to the Fourier transform
to the procedure outlined above. Afterwards, 300 g of the dried infrared (FTIR) spectra of the CAE, ZnO, and CAE loaded PLX/ZnO
marc was boiled in 2 L of distilled water for 3 h, strained, and dried nanocomposites were acquired using a PerkinElmer FTIR spec-
to obtain 95.1 g of the hot aqueous extract (3.17% yield). Tween 20®, trometer (Beaconsfield, Bucks, UK). The spectra were collected
a 10% solution of polyoxyethylene (20) sorbitan monolaureate in within the wavelength range of 4000 to 400 cm1. The X-ray
distilled water, was used as a carrier to prepare 10% (w/v) of each diffraction patterns of nanocomposites were examined using a
extract for topical application. Ten grammes of powdered extract Rigaku diffractometer equipped with Cu-Ka radiation. The mea-
was mixed with 9 mL of vehicle. The vehicle was gradually added surements were conducted within a 2q range of 10e80 , employing
after the extract concentration reached 10%. a voltage of 40 kV, a current of 40 mA, and a scan rate of 0.02 s1.
The nanocomposites of PLX and ZnO, as well as those including
2.3. Preparation of zinc oxide nanoparticles (ZnO) CAE, were studied using fluorescence spectrophotometers (F-4500;
Hitachi, Tokyo, Japan). The thermal properties of the developed
The production of ZnO was achieved using a precipitation samples were assessed using a thermogravimetric analyzer (TGA),
method, as referenced [31]. To prepare dissolution, 50 mL of specifically the TGA Q50 T. The instrument used in this study was an
distilled water was used to make a solution that contained Instrument-Water LLC (Newcastle, DE, USA). The scanning electron
Zn(NO3)2 at a concentration of 0.05 M. The mixture was treated microscopy (SEM; JEOL 6460LV) technique was employed to
with ultrasonic waves for 15 min while being constantly mixed. The observe morphological structure of the CAE loaded onto PLX/ZnO
ultrasonic treatment was performed at a power of 100 W and a nanocomposites. The DSC thermograms of nanoparticles were ob-
frequency of 30 kHz, being sure to keep the temperature at tained by employing a DSC 882e, Mettler Toledo instrument. The
21 ± 2  C throughout. Gradually adding 50 mL of 0.1 M NH4OH temperature range investigated ranged from 25 to 900  C. The
dropwise until a white precipitate formed brought the solution's nanoparticles were subjected to a heating rate of 5  C min and a
pH down to around 9. After a 30-min stirring period, the resulting constant nitrogen flow of 100 mL min1. The TEM images of the
solution was subjected to periodic washing using distilled water synthesized nanocomposites were acquired using a TEM model FEI
and methanol in order to eliminate residual components. Subse- Technai G2 20S-TWIN (USA). In this experiment, the CAE loaded
quently, the precipitation underwent modification and was sub- PLX/ZnO nanocomposites were dispersed in distilled water and
jected to a drying process at a temperature of 120  C for duration of subsequently applied onto a 3 mm copper grid. The retention fac-
2 h in a hot air oven. tors were measured with a liquid chromatograph and an Elite
LaChrom HPLC Merck-Hitachi (Merck, Darmstadt, Germany) with a
2.4. Synthesis of CAE loaded onto PLX/ZnO nanocomposites DAD (Diode Array Detector L-2455) detector, pomp L-2130, and a
manual sample injection valve equipped with a 20 mL loop and
The method of ultrasonication was employed to fabricate EZChrom Elite software system manager as a data processor. The
nanocomposites consisting of CAE encapsulated on PLX/ZnO. The column was a Zorbax Eclipse XDB-C18 (Agilent Technologies, Santa
process involved the dissolution of CAE in dichloromethane, Clara, CA, USA); (150 mm  4.6 mm I.D., 5 mm).
resulting in the formation of CAE loaded PLX. Subsequently, these
loaded PLX nanoparticles were utilized in the fabrication of nano- 2.6. Antibacterial activity
composites. Subsequently, a distinct concentration of ZnO solution
at boiling point (approximately 100  C) was introduced into the PLX The present study investigated the antibacterial efficacy of ZnO,
solution containing CAE. This was carried out in a controlled PLX/ZnO, and CAE loaded PLX/ZnO nanocomposites against
environment using ultrasonic methods at a flow rate of 0.2 mL/min Staphylococcus aureus (S. aureus), Streptococcus pneumonia (S.
(specifically, an ultrasonic power of 100 W at a frequency of pneumonia), Pseudomonas aeruginosa (P. aeruginosa), and Escher-
30 kHz). The dichloromethane was evaporated more easily from ichia coli (E. coli) bacterial strains through the implementation of
the reaction mixture after 30 min of agitation and sonication. The the well diffusion method. Petri plates were prepared by adding
ultimate outcome underwent ultra-centrifugation at a temperature 25 mL of sterile Muller Hinton agar media. Subsequently, each
of 4  C for 10 min. The nanocomposites containing CAE loaded on bacterial pathogen was individually inoculated onto Muller Hinton
PLX/ZnO matrix were subsequently subjected to a drying process. agar media in separate plates using a swab. The experiment
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involved evaluating the antibacterial activity of selected samples number of tubes that formed in the corresponding image. The ex-
dispersed in dimethyl sulphoxide at a concentration of 2 mg mL1. periments were performed triplicate for each sample.
The levels of the zone of inhibition were measured 24 h after in-
cubation overnight at a temperature of 37  C. In this study, the 2.11. Analysis of collagen contraction assay
positive control utilized was the standard antibiotic amoxicillin at a
concentration of 2 mg mL1. In a 2 mL volume of type I bovine collagen solution were treated
L929 fibroblast cells with ZnO, PLX/ZnO, and CAE@LPX/ZnO sam-
2.7. In vitro biocompatibility assay ples for 8, 16, and 24 h, respectively as previously reported. Then,
observed and recorded the contraction of each collagen gel at 8 and
The effect on the cytotoxicity of the prepared wound closure 24 h. We evaluated the percentages of contraction at different
materials was determined using mouse fibroblast L929 cells as points with the control, which did not receive any treatment.
model cells. The study investigated the cell viabilities of ZnO, PLX/
ZnO, and CAE loaded PLX/ZnO nanocomposites at various concen- 2.12. qRT-PCR for inflammatory cytokines
trations (ranging from 0 to 200 mg/mL) and incubation times (12,
24, 48, and 72 h). The cell counting kit-8 (CCK-8) method was For three days, L929 cells were cultured in six-well plates. Using
utilized for the purpose of this research. PBS, the cells were washed twice. Before treatment, the L929
fibroblast cells were transferred to serum-free DMEM for one day.
The L929 cells that were cultured were exposed to LPS (1 mg/mL;
2.8. In vitro cell proliferation assay
Sigma-Aldrich, USA) for 4 h, followed by 30 min of preparation of
samples. After that, we harvested the cells to isolate the total RNA
The nanocomposited wound closure material was sterilized
and extract the proteins. Following the manufacturer's protocols,
prior to performing any biological and animal tests. The prepared
total RNA was extracted from cultured L929 cells using Favor-
nanocomposites were initially dropped onto a 24-well plate. After
PrepTM Tri-RNA reagent. 20 mL of diluted cDNA template, forward
that, a volume of 500 mL containing L929 cells (1  105 mL1) was
and reverse primers, 10 mL of SYBR Premix Ex Taq (Takara Bio, Inc.),
introduced into the well plate and incubated for different incuba-
and the Bio-Rad Real-Time PCR Detection System (USA) were uti-
tion hours (0e96 h) and different incubation time (1, 3 and 5 days).
lized to perform real-time RT-PCR. For every sample, real-time PCR
Cellular growth and proliferation were seen using microscopic
quantification was performed in triplicated and the average was
method. The cells within the nanocomposite that developed
determined. With GAPDH values as a reference, real-time PCR was
endured staining using AO/EB for morphological observations. This
used to examine expression levels.
process was performed at a temperature of 37  C and within an
environment consisting of 5% CO2 and 95% air with humidity. The
2.13. Statistical analysis
fluorescent microscopy technique (Olympus, Japan) was employed
to observe the rates of cell proliferation, survival, and death.
The data are reported quantitatively as an average ± S.E. of 3e5
samples per group, and all the tests were performed in triplicate
2.9. In vitro wound scratch assay (n ¼ 3). Using Tukey's test of ANOVA, we compared each group to
the control. Significant results were indicated by a p-value<0.05,
Using L929 cells, an in vitro wound model was created to **p < 0.01, and ***p < 0.001.
examine the possibility of nanocomposite groups in wound closure.
The L929 fibroblast cells was cultured on a 48-well plate with a cell 3. Result and discussion
density of 1.2  105 in full DMEM medium until about 90 %
confluence was reached after 24 h. To induce starving circum- The X-ray diffraction pattern of the bare samples, which include
stances for 24 h and agreement that only cell migration leads to Poloxamer and ZnO, as well as the composite materials in their
wound closure, after 24 h the media was withdrawn and rinsed prepared formulation is displayed in Fig. 2 (a). X-ray diffraction
with PBS. Then, 0.5 mL of DMEM (supplemented with 0.1% FBS) was (XRD) data revealed that the nanocomposites, which included PLX,
added. A sterile 200 mL micro-tip was used to make a small scratch ZnO, PLX/ZnO, and CAE loaded PLX/ZnO nanocomposites,
at the base of each well, which was then washed twice with PBS to possessed a well-defined crystalline structure that was clearly
remove any debris. Then, cell lines were treated with prepared observed. The peak positions that were obtained at 2q positions
samples in each well, while DMEM with 10% FBS served as a con- 31.4, 34.2, 36, 47, 56.36, 62.64, 66.15, 67.65, and 68.9 are associated
trol. The ratio of the wound area covered by migrating cells was with the lattice plane of (100), (002), (101), (102), (110), (103), and
then used to determine the closure of the wound at 0, 6, 12, 24, and (112), respectively. These peak positions provide evidence that the
48 h. The area of cell migration at the specified duration was ZnO particles possess a crystalline structure. The lattice planes that
determined using ImageJ software compared to the area of cell were obtained demonstrated that the ZnO particle possesses a
proliferation at 0 h. The images were visualized by fluorescence hexagonal wurtzite structure. Furthermore, the observed XRD data
microscopic method (Olympus, Japan) using AO staining. that has been strong correlated with the JCPDS card number
36e1451. The crystalline nature of the PLX matrix is shown by the
2.10. In vitro tube formation assay position of the distinctive peaks, which are at 19.1 and 23.2,
respectively. In addition to this, the positions of the peaks were
The HUVECs were cultured for 48 h with either PBS and pre- slightly moved, and peaks disappeared in the composite system,
pared samples: control, ZnO, PLX, and CAE@PLX/ZnO. A solution of which showed that the ZnO and CAE successfully encapsulated into
Matrigel (200 mL) from BD Biosciences, USA, was evenly distributed PLX matrix network.
into 12-well plates and allowed to gel at 37  C for some minutes. The FTIR spectral analysis is an important method that can
Afterwards, cells (2  105 cells/well) were seeded onto polymerized identify which substances interact with one another. Based on the
Matrigel from various groups. To assess tube formation, an inverted results of the FTIR observation, the interaction between the CAE
microscope (Olympus, Japan) was employed after 10 h of incuba- and ZnO, together with the complexation of PLXs, was observed. As
tion. Quantification of the results was done by measuring the shown in Fig. 2 (b), ATR-FTIR spectra established that CAE@PLX/
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Fig. 2. Nano-crystallinity and phase purity of the prepared formulations (PLX, ZnO, PLZ/ZnO, and CAE@PLX/ZnO) were analyzed by XRD (a), and structural interactions and chemical
structure were determined by FTIR (b).

ZnO composites that have been successfully formed with the polymer matrix, the interaction became considerably more effec-
presence of ZnO, PLX and CAE components. There was a charac- tive, as evidenced by the greater peak intensity of the major peaks
teristic peak at 722 and 669 cm1, which corresponded to the ZneO presented in the prepared nanocomposites.
stretching mode of vibrations [32]. In addition, the spectra of the Fig. 3 (a) displays the fluorescence spectra of the PLXs as well as
PLX polymer revealed typical peaks at 3450, 2888, and 1115 cm1. the prepared ZnO, PLX/ZnO, and CAE loaded PLX/ZnO nano-
These peaks correspond to the stretching vibrations of hydroxyl composites samples. These spectra were obtained at an excitation
and carbonyl link forms, which are characterized by the OeH, CeH, wavelength of 541 nm. When measured at a wavelength of 456 nm,
and CeO stretching vibrations. The result of the spectral observa- the ZnO nanoparticle displayed the excitation peak position. After
tion displayed that some band locations of the compounds altered careful observation, it was found that the chemical mixture con-
and that some of the peaks were disappeared, it was determined sisting of PLX and ZnO exhibited a wavelength of around 523 nm,
that the sample had formed a complex. As a result, the successful which is somewhat different from the ZnO's accurate excitation
interaction of the functional groups is demonstrated by the shifts in wavelength. While this was going on, the intensity of the peak
the CAE and ZnO complex as well as the appearance of new peaks. position increased significantly when compared to the samples that
During the process of incorporating CAE and ZnO into the PLX included bare ZnO and PLX. On the other hand, when the

Fig. 3. The fluorescence properties and thermal stability of the prepared materials (PLX, ZnO, PLZ/ZnO, and CAE@PLX/ZnO) were examined by fluorescence spectra (a) and TGA (b).

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Fig. 4. The morphological structure and distribution of nanoparticles in the prepared CAE@PLX/ZnO nanocomposite was observed by SEM (a1 & a2) and TEM (b1 & b2) analyses.
Scale bar 100 nm & 50 nm.

combination was combined with the PLX polymer, the excitation Using thermogravimetric analysis (TGA), we compared the as-
wavelength grew even further, reaching 541 nm due to the higher prepared and unprocessed materials. Fig. 3 (b) displays the TGA
excitation wavelength, and the peak intensity even increased. profiles of the samples. The remarkable weight loss percentage
Therefore, the presence of the drug CAE, ZnO, and the PLX matrix observed at 350  C for PLX polymer is in line with its high heat
was validated by the enhanced peak intensity of the samples as stability. A weight reduction of about 10% in the ZnO particles was
well as the shift in the excitation wavelength. indicative of the elimination of the water molecule (moisture) or

Fig. 5. a) Zone of inhibition assay and (b) MBC photographs of control, PLX, ZnO, PLZ/ZnO, and CAE@PLX/ZnO against bacterial pathogens (E. coli, P. aeruginosa, S. pneumonia, and S.
aureus).

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the eOH functional group. The presence of the excellent stability of highest antibacterial activity among the prepared nanomaterial
metal oxide nanoparticles, the ZnO nanoparticle exhibited good formulations. The zone inhibition was ~11 mm for the ZnO sample,
thermal stability over the investigated range. Even though it only ~13 mm for the PLX/ZnO sample, and ~17 mm for the CAE@PLX/ZnO
shows a 40% weight reduction at 300  C, the PLX/ZnO combination nanocomposite. Importantly, the ZnO, PLX/ZnO, and CAE@PLX/ZnO
demonstrated an enhanced percentage of weight loss. The addition nanocomposite groups demonstrated a substantial decrease in
of the ZnO nanoparticles is responsible for the composite's bacterial colony counts when compared to the control treatment
enhanced thermal stability. At roughly 750  C, a third weight loss group (Fig. 5b). The essential characteristics of CAE had been
was observed, which meant that the ZnO in the composite had believed to be responsible for the nanocomposite's superior anti-
completely decomposed. The prepared nanocomposites demon- bacterial activity.
strated much better thermal stability compared to those without The cell compatibility and survival rate of the prepared nano-
PLX. Due to the PLX polymer's hydrophobic properties, the com- formulations was examined by utilizing L929 cells as shown in
posite sample exhibited no signs of moisture. Weight loss for the Fig. 6a. We changed the concentrations of the positive control, ZnO,
composite begins at approximately 120  C, primarily as a result of PLX/ZnO, and CAE@PLX/ZnO up to 200 mg/mL. The prepared CAE
the polymer composition, and continues gradually up to 400  C. loaded nanocomposite showed greater cell compatibility with less
Additionally, similar to the CAE and ZnO samples, this one exhibited toxicity effect. Meanwhile, the ZnO nanoparticles and ZnO/PLX
excellent stability with no material loss. The composite sample's materials indicated that slight variation in cytotoxicity across all
exceptional thermal durability allows it to be utilized in real-world doses. The change in cell viability is monitored every 12 h. In
applications at temperatures as high as 400  C. contrast, the manufactured ZnO nanoparticles, PLX/ZnO and
The morphological structure and shape of the prepared nano- CAE@PLX/ZnO nanocomposites showed excellent cell interaction
composites of CAE@PLX/ZnO were characterized by SEM analysis as and maintained 90% viability even after 24 h. Furthermore, the cell
exhibited in Fig. 4 (a1 & a2). According to these measurements, the viability reaches a controlled 90% level after 72 h Fig. 6b. After one
surface is uniformly covered with spherical nanoparticles that are day in culture, the cell proliferation experiment (Fig. 7a and b)
all about the same size, which is less than 100 nm. It is plausible revealed that all of the tested materials maintained a comparable
that the fabrication strategy in the nanocomposites of CAE@PLX/ degree of proliferation for L929 fibroblast cells. Nanocomposite
ZnO is responsible for the observed morphologies. Fig. 4 (b1 & b2) administration with CAE enhanced cell proliferation compared to
displays that TEM observation of CAE@PLX and ZnO NPs to treatment with PLX/ZnO or ZnO nanoparticles. In addition, the
examine particles size and distributions. The atomic configurations CAE@PLX/ZnO nanocomposite showed a much greater effect on cell
visible in great detail through the use of the TEM's high resolution proliferation than the PLX/ZnO NPs in the absence of CAE. A 3-days
can explain the nano particles dispersion and uniform spherical culture on PLX/ZnO or CAE@PLX/ZnO nanocomposite resulted in an
shape, which has an average diameter of approximately 50 nm. In increase in cell proliferation of L929 cells. In many instances, cell
addition, HPLC data reveals that the before and after encapsulation adhesion and proliferation were slightly superior in CAE@PLX/ZnO
of centella asiatica extract-loaded poloxamer/ZnO, which is further nanocomposite compared to the PLX/ZnO composite. The fibro-
evidence of the nanocomposites formation and the data shown in blasts had proliferated to a confluent stage in all of the culture wells
Figure S1. in just 5 days, a result due to their rapid proliferation ability.
We used the basic petri dish diffusion (zone of inhibition) Nanocomposite biocompatibility is an essential factor for their
method and colony forming unit (CFU) method to examine the application in biomedical fields. To determine the nanocomposite's
antibacterial activity of the PLX, ZnO, PLX/ZnO, and CAE@PLX/ZnO cytocompatibility, researchers entrapped L929 cells and employed
with Staphylococcus aureus (S. aureus), Streptococcus pneumonia (S. live/dead staining to assess cell proliferation ability. The CAE@PLX/
pneumonia), Pseudomonas aeruginosa (P. aeruginosa), and Escher- ZnO nanocomposites demonstrated an increase in the number of
ichia coli (E. coli) bacterial pathogens. Antibacterial materials are in cells encapsulated by day 5 compared to day 1 (Fig. 7c), indicating
great demand because bacterial infections are a leading cause of that the PLX/ZnO and CAE@PLX/ZnO nanocomposites had excellent
wound healing delays in clinical practice. The developed hydrogels cell growth and proliferation facilitation.
were examined for their antibacterial behavior using different A successful wound closure depends on the migration of cells to
bacterial pathogens in the present study. As shown in Fig. 5a, the the wound site. Previous studies have demonstrated that wound
zone of inhibition increased, whereas CAE@PLX/ZnO showed the healing material, which promotes cells quickly in a lab setting, may

Fig. 6. In vitro cell viability of L929 cells treated with prepared formulations (control, PLX, ZnO, PLZ/ZnO, and CAE@PLX/ZnO nanocomposite) with (a) different concentrations
(50e200 mg/mL) and (b) different incubation periods (0e72 h).

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Fig. 7. In vitro quantitative live cell number (a) and cell proliferation (b) observations of prepared nanocomposite formulations in different incubation periods; (c) In vitro qualitative
observations of L929 cells treated with different nanocomposite formulations under phase contrast microscopic method and Fluorescence microscopic method (AO/EB staining).

significantly enhance wound healing. An in vitro wound healing In addition, a ROS-specific probe was employed to assess the
model has been developed using skin fibroblasts (L929) cells. As intracellular ROS scavenging capability of the CAE@PLX/ZnO
exhibited in Fig. 8a, we can observe that L929 cell migration to fill nanocomposite. After incubation with ZnO, PLX/ZnO, or CAE@PLX/
the scratched region was used to evaluate the wound closure pro- ZnO, L929 fibroblasts were exposed to H2O2 stimulation to induce
pensity of the treated groups. The treated groups moved their cells intracellular oxidative stress. ROS levels were then measured using
to fill the gap faster after 48 h compared to the control group DCFH-DA fluorescence in green. After being treated with H2O2, the
(Fig. 8b). The results indicate migratory delays were observed in green fluorescence intensity was 2.4 times higher than the PBS
fibroblast (L929) cells treated with ZnO and PLX/ZnO nano- control as exhibited in Fig. 9a and b. A substantial reduction in
composites. The CAE@PLX/ZnO treated group demonstrated a fluorescence intensity was observed after incubating the cells with
significantly greater and more beneficial effect on cell migration, PLX/ZnO comprising CAE, suggesting that CAE@PLX/ZnO effectively
with values of around 85% ±4.73 (p < 0.001) and 95% ± 5.37 mitigated intracellular oxidative stress. The capacity of CA extract
(p < 0.01), respectively. Images taken under a microscope reveal to alleviate excessive oxidative stress in the diabetic wound
that the results of the experiment suggest that the incorporation of microenvironment was demonstrated, in particular, when the
nano-formulations into the CAE@PLX/ZnO nanocomposites intracellular ROS level was found to be normal after treatment with
enhanced cell migration and proliferation, which could lead to CAE@PLX/ZnO nanocomposite in its presence. Furthermore, as
accelerated wound healing. illustrated in Fig. 9 (c), the anti-oxidant ability of the developed

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Fig. 8. In vitro wound healing efficiency of prepared nanocomposite formulations was determined by wound scratch method using L929 cell line; (a) Fluorescence microscopic (AO
staining) visualization of L929 fibroblast cell treated with Control, PLX, ZnO, PLZ/ZnO and CAE@PLX/ZnO for 0, 24 and 48 h; (b) Percentage of wound area (%) in different time
(0e48 h).

nanocomposite was determined using a DPPH assay. The remark- demonstrates the increased migratory capacity and Fig. 10b shows
able antioxidant activity of CAE@PLX/ZnO has been shown when the increased tube-forming ability of HUVECs treated with PLX/ZnO
DPPH levels were gradually decreased and DPPH scavenging ac- and CAE@PLX/ZnO nanocomposites, respectively, compared to the
tivity was substantial when treated with PLX/ZnO nanocomposite control groups. The results showed that CAE@PLX/ZnO can increase
containing CAE extract. the ability of HUVECs to proliferate, migrate, and form tubes, which
The purpose of this study was to examine the potential of as- can induce angiogenesis in vitro. Furthermore, a 3D type I collagen
prepared ZnO, PLX/ZnO, and CAE@PLX/ZnO samples to the migra- contraction assay was carried out to determine the effect of regu-
tory and tube-forming abilities of HUVECs after 48 h. Fig. 10a lating the contractile capacity of L929 cells. Throughout a 24-h

Fig. 9. (a) In vitro ROS generation assay by H2O2 method under L929 cells treated with ZnO, PLX/ZnO, and CAE@PLX/ZnO nanocomposite using DCFH-DA staining under fluorescence
microscopic method; (b) Relative fluorescence intensity and (c) Anti-oxidant ability of prepared nanocomposite formulation through DPPH assay.

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Fig. 10. (a & b) In vitro tube formation analysis of prepared nanoformulation on HUVECs cells and (c & d) collagen contractility study by different treatment groups.

culture period, the collagen gels' sizes were measured at various effective potential of anti-oxidant and bactericidal inhibition abil-
time points (Fig. 10(c and d)). The collagen gel contraction was ity, which leading to enhanced skin regeneration ability and ther-
facilitated by the 8, 16 and 24 h treatment of L929 cells with pre- apeutic potential for diabetic wound healing.
pared CAE@PLX/ZnO. This reduction was confirmed by both mac- They investigated the effectiveness of a nanocomposite that was
roscopical observations and measurements of the gel size (Fig. 10d). very similar to this one in inhibiting the growth of bacteria in a
Collagen gel contraction was significantly facilitated by subjecting study that was conducted [32]. They discovered that the nano-
L929 cells to CAE@PLX/ZnO for 24 h. The fibroblast L929 cells could composite exhibited powerful antibacterial activity against Gram-
have incorporated CAE with structured composites, allowing the positive as well as Gram-negative bacteria, which is in line with
gel to contract effectively. the findings that we obtained. When it comes to the effectiveness of
After inducing inflammation in L929 cells with LPS, cells were our nanocomposite against bacteria, this demonstrates that it is
cultured in the presence of prepared samples to see if CAE loaded consistent with the research that has been done previously [33]. In
PLX/ZnO nanocomposited formulations might decrease inflam- addition, performed research that investigated the potential of a
matory responses. To investigate how CAE@PLX/ZnO treatment nanocomposite in the context of applications related to wound
influenced the mRNA expression of pro-inflammatory (IL-1b, IL-6 healing [34]. According to their findings, the nanocomposite facil-
and IL-8) and anti-inflammatory (IL-4 and IL-10) cytokines, RT- itated a more rapid closure of wounds and also enhanced tissue
PCR was employed. Fig. 11(a and b & c) demonstrates that when regeneration, indicating that it has the potential to be utilized in
L929 cells were treated with CAE@PLX/ZnO, the mRNA expression biomedical applications [35]. In addition, our research demon-
of IL-1b, IL-6, and IL-8, which were induced by LPS treatment, was strates that our nanocomposite has the potential to be used in
significantly reduced. Additionally, following CAE@PLX/ZnO treat- wound healing, which further substantiates its uniqueness and
ment, there was a notable increase in the mRNA expression of anti- effectiveness in this particular application. Moreover, demon-
inflammatory cytokines (IL-4 and IL-10), illustrated in Fig. 11d and strated a review in which they discussed the application of nano-
e. Pro-inflammatory cytokines and chemokines, like IL-6 and IL-8, composites in the process of environmental remediation. They
are released into the bloodstream when LPS causes inflammation. brought attention to the potential of nanocomposites in the
Data suggests that IL-6 and IL-8 play an important role in wound removal of pollutants from water and soil, which is in line with the
healing by triggering chronic inflammation. The anti-inflammatory findings of our research on the effectiveness of our nanocomposite
cytokines, such as IL-4 and IL-10, suppress the immune response in the removal of heavy metals from water [36]. The novelty and
and prevent the production of cytokines that promote inflamma- effectiveness of our nanocomposite in environmental remediation
tion. These findings prompted us to believe that CAE@PLX/ZnO applications is further highlighted by this comparative analysis
might suppress pro-inflammatory cytokines in L929 cells, which with the existing body of literature. In general, the comparative
would indicate that it has an anti-inflammatory function. The analysis with the existing literature provides evidence that our
outcomes of in vitro anti-bacterial investigations and cell treatment nanocomposite is both effective and novel in a variety of applica-
studies demonstrated that CAE@PLX/ZnO nanocomposited formu- tions [37]. These applications include antibacterial activity, wound
lation exhibited greater cell proliferation, migration ability with healing, and environmental remediation.

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Fig. 11. Real-time qRT-PCR analysis of pro-inflammatory cytokines ((IL-1b (a), IL-6 (b) & IL-8 (c)) and anti-inflammatory (IL-4 (d) & IL-10 (e)) cytokines in L929 fibroblast cells by
treatment of LPS and prepared samples (control, ZnO, PLX/ZnO and CAE@PLX/ZnO).

4. Conclusion Funding

In summary, we have developed the eco-friendly, cost-effective, None.

non-toxic PLX/ZnO composited material encapsulated with CAE for
effective regeneration of diabetic foot ulcer. Based on nano-
Data availability statement
materials characterizations, we revealed that the hexagonal wurt-
zite ZnO (<11 nm) and CAE extract have been successfully
Data will be made available on request to corresponding author.
encapsulated in the PLX nanocomposited network. The in vitro
antibacterial studies exhibited that prepared CAE@PLX/ZnO nano-
composites have providing increased and efficient bactericidal in- Author contributions
hibition against Gþ and G bacterial pathogens. The developed
wound closure nanocomposite material demonstrated for superior Lina Wang - Work design and conception.
cell compatibility, cell proliferation, migration ability and angio- Yan Yang - Synthesis, Characterization, Molecular and
genesis capability on L929 and HUVEC cell lines. In addition, the Biochemical analysis.
developed material has been demonstrated for enhanced anti- Weiwei Han - Formal analysis, Data curation and Validation.
oxidant anti-inflammatory action, which confirms that it would Hui Ding - Helped-Supervised the research.
be potential candidate for diabetic wound healing application. For
the following stage, in in vivo experiments involving animal models
including diabetic wound infliction, we will be focusing on devel- Declaration of competing interest
oped nanocomposites with enhanced healing potential that have
suitable anti-infection characteristics. As part of this process, we The author states that none of the work presented in this study
will determine a therapeutic window for repairing wounds and may have been influenced by any known conflicting financial in-
ensure their safety to facilitate long-term wound healing for clinical terests or personal ties.
purposes.
Appendix A. Supplementary data
Ethical statement
Supplementary data to this article can be found online at
None. https://doi.org/10.1016/j.reth.2024.03.006.
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