In Vivo Observation of Lidocaine-Encapsulated Polymyxin/Glycol Nanoparticles Wound Dressing for the Treatment and Care of Abdominal Pain Incision in Intensive Care Unit

The developments of lidocaine chloride loaded nanoparticles are encouraging biomaterials, which could be used for wound healing applications for abdominal pain management. The current work presents the composition of wound dressings based on lidocaine chloride (LCH) (anesthetic drug)-loaded Polymyxin (PMN)/Glycol (GLY). The LCH nanocomposite (LCH@PMN/GLY) were fabricated by the LCH oxide solutions within the PMN/GLY matrix. The influences of different experimental limitations on PMN/GLY nanoparticles formations were examined. The PMN/GLY and LCH@PMN/GLY nanoparticle sizes were evaluated by high resolution-scanning electron microscopy (HR-SEM). Additionally, the antibacterial efficacy of PMN/GLY and LCH@PMN/GLY was developed for gram-positive and negative microorganisms. Moreover, we examined in vivo healing of skin wounds formed in mouse models over 20 days. In contrast to the untreated wounds, rapid healing was perceived in the LCH@PMN/GLY-treated wound with less damage. These findings indicate that LCH@PMN/GLY-based bandaging materials could be a potential innovative biomaterial for tissue repair and implantation and nursing care for wound healing applications for abdominal pain incision in intensive care unit (ICU) management in an animal model.


Introduction
Infections cause problems in the field of wound management because they form exudates and limit wound improvement, leading to the use of inappropriate sponges. Microorganisms are the main cause of infections [1][2][3][4]. The most common infection-producing microorganisms are E. coli and Klebsiella, which are gram-negative bacteria. On entering the human body, these microorganisms quickly establish colonies. These organisms can penetrate the cell nanocomposite in the body, and enter the actual fractions of tissues and cells, leading to internal infections. Therefore, wound dressings are required to prevent these bacterial infections [5][6][7][8].
Postoperative adhesion (POA) is a comprehensive inflection of various abnormal tissue hyperplasias, characterized by proliferated fibrous tissues sticking to the nearby normal organs [9][10][11]. The adhesion bands can take diversified phenotypes ranging from a thin layer of fibrous films between the adjacent tissues to a mixture of fibrous tissues, blood vessels, and nerves [12][13][14]. Depending on distinct positions and levels of the adhesion, POA may be accompanied with acute complications or be "silent" for several years. These complications, such as chronic pain, dysfunction of adjacent organs, and intestinal obstruction, can reduce the life quality of patients or even become life threatening [15][16][17]. Even non-sterilized wounds can increase the incidence of infections, leading to exudate formation around the edges. This leads to the use of various polymers, nanocomposite, and cryogels to improve wound dressings. However, previous studies have shown that these materials have limitations such as moisture entry, air passage, biocompatibility, environmental safety, and higher toxicity [18][19][20]. The use of 1 3 biological polymers, such as keratin, gelatin, heparin, hyaluronic acid, cellulose, chitosan, chitin, and alginic acid, for wound dressings is less economical. The use of Polymyxin B (PMN) and Glycol (GLY) nanocomposite formulations to heal serious burn and bone tissue injuries has been reported earlier [21][22][23]. The PMN/GLY nanocomposite is easy to deliver, biocompatible, and biodegradable; additionally, it has low toxicity. The nanocomposite compositions and thin films have antibacterial properties with low damage clearness capacity and high stability [24][25][26].
In the present study, we aimed to develop wound bandages based on PMN/GLY and LCH. The first step involved the fabrication of LCH inside the PMN/GLY matrix (LCH@ PMN/GLY) by PMN/GLY as important agents. Further, the surface of LCH was modified by adding PMN/GLY (LCH@ PMN/GLY). The GLY, PMN, and LCH@PMN/GLY were subsequently covered with cotton fabrics to generate the final composites. The wound bandaging studies were performed with the GLY, PMN and LCH@PMN/GLY composites utilizing antimicrobials in vitro and in vivo animal models.

Fabrication of LCH@PMN/GLY Nanocomposite Scaffolds
A polymer solution of PMN/GLY was prepared by mixing 10% w/v PMN and 20% w/v GLY in hexafluoroisopropanol (HFIP)/dichloromethane (DCM) solvent system for 6-12 h. For LCH loaded PMN/GLY, a 25% v/v 30 mM LCH solution was added to the polymer solution; whereas for control samples nanoparticle solution was replaced with similar amount of distilled water. The resulting nanocomposites were collected for characterisation and cell culture studies, respectively [35][36][37].

Characterization of PMN/GLY and LCH@PMN/GLY
The morphological properties were studied by scanning electron microscopy (HR-SEM) using a TECNAI-SEM (USA) instrument. In the HR-SEM analysis, one drop of the prepared PMN/GLY sample was placed on a carboncoated SEM grid at room temperature. Fifty-five readings of the SEM images were recorded to evaluate the ratio of the size and distribution. Dynamic light scattering (DLS, Beckmann-Coulter, Delsa-Nano) was used to analyze the particle size, size distribution, and polydispersity index of the nanoparticles [38][39][40].

Synthesis of PMN/GLY and LCH@PMN/GLY Nanocomposites for Bandaging
A nanocomposite of PMN/GLY and LCH@PMN/GLY for bandaging was fabricated by the DIP coating method. Briefly, cotton fabric was dished into the PMN/GLY and LCH@PMN/GLY solution, removed, and dehydrated at ~ 60 °C for 24 h. The moisture contents of the PMN/GLY and LCH@PMN/GLY matrix were examined. The samples were accurately weighed and subsequently dehydrated in a hot-oven for 5 h. The dehydrated fabrics of cotton were desiccated for 24 h to remove moisture and weighed again. The moisture contents of the PMN/GLY and LCH@PMN/ GLY were calculated using a previously reported protocol [41][42][43][44].

In vitro Biodegradation and Swelling Studies
To study the biodegradation of the LCH, PMN/GLY and LCH@PMN/GLY nanocomposite, 0.5 g (W i ) of LCH, PMN/ GLY and LCH@PMN/GLY nanocomposite were immersed in 50 mL of phosphate-buffered saline (PBS) and incubated at 37 °C for 7 days. Concurrently, a blank determination was performed in PBS on the same sample without the nanocomposite. After a period of 1, 4, and 7 days, the samples were washed several times with deionized water and dried in the oven at 40 ± 1 °C for 24 h.
The swelling ratio of the LCH, PMN/GLY and LCH@ PMN/GLY nanocomposite was measured according to the described methods [45][46][47]. To measure the water sorption potential of the LCH, PMN/GLY and LCH@PMN/ GLY nanocomposite, 0.1 g of dried powder of LCH, PMN/ GLY and LCH@PMN/GLY nanocomposite were immersed in 30 mL of PBS with the desired pH at room temperature for 16 h to achieve maximum swelling equilibrium, then removed and gently pressed between filter papers and weighed.

Antibacterial Properties of PMN/GLY and LCH@PMN/ GLY
The antibacterial properties of the synthesized PMN/GLY and LCH@PMN/GLY were examined using E. coli and S. aureus bacteria. The microorganism colony volumes were 10 4 percent/mL. The nanocomposite samples were added to the culture dishes and maintained at 37 °C for 24 h. The marked phases of the flexible microbes were quantified in the same manner as the microbes with the saline and agar plates. Bacterial growth rates were examined using statistical models. The cell viability in the synthesized nanocomposite was evaluated using the NIH-3T3 fibroblasts by Alamar Blue assay. The sterilized samples were placed in 96-well plates with the same fluency as the fibroblasts. The cells were marked for three different days. Cell viability was measured at 520 nm using a microplate reader. The experiments were repeated three times [48][49][50][51].

Animal Experiments of the Wound Bandaging Model
The animal experiments were performed according to the guidelines of the 2 Department of Cardiology, Fuwai Central Cardiovascular Hospital, Henan Provincial People's Hospital, Zhengzhou 45,000, China. Sprague-Dawley rats aged 4 weeks and weighing 200 g were used. The rats were anesthetized by injecting ketamine (30 mg/kg) and xylazine (4 mg/kg). The upper phase of the rat's hair was detached and the specific sites were marked by skin hygienic through the soul. A 1.5 cm-wide skin damage was created in the dorsum of the rats using operating scissors and tongs. The wounds were covered with the baseline damage and used as the negative control (Group I), nanocomposite -loaded bandaging (Group II), PMN/GLY (Group III), and LCH@ PMN/GLY (Group IV). The extent of wound closure was analyzed by drawing the wound boundary at the end of 0, 4, 8 and 16 days. The wounds closure was expressed in mm 2 . The wounds area was calculated using previously reported methods [52][53][54].

Statistical Analysis
All inventory data are uttered as the mean ± standard deviations. Significant changes among groups were examined using one-way examination of modification, and changes for separate groups were dogged using Student's t tests. The outcomes were observed as a significant differences when p < 0.05.

Fabrication and Characterization
By employing an easy and effective method to fabricate LCH, the current study aimed at using PMN/GLY as a stabilizing agents as well as a nanocomposite system for the encapsulation of LCH. To avoid the clusters collisions and their growing into macro nanoparticle, the hydroxy fragments of PMN/GLY interact with the LCH and aid in steadying them and preventing their agglomeration and further proliferation. The graphical representation of the generation and anchoring of LCH within a PMN/GLY nanocomposite matrix is depicted in Fig. 1. Morphology analysis of PMN/ GLY and LCH@PMN/GLY was carried out by observing the nanocomposite using SEM. The SEM micrographs of PMN/GLY and LCH@PMN/GLY shown in Fig. 2a, b reveal isotropic and randomly oriented nanocomposite with a seemingly smooth morphology. The SEM images correspond to the DLS analysis results. The time-dependent difference in DLS analysis shows the particle size and diameters of the nanoparticles. The role of PMN/GLY is to stabilize the nanoparticle formation and provide the base matrix for the nanocomposites.

Fabrication of PMN/GLY and LCH@PMN/GLY Nanocomposites for Bandaging
Cotton fabrics were used as permeable, sustainable substrates for preparing PMN/GLY and LCH@PMN/GLY nanocomposites. It has been preferred over other kinds of cotton because of its intrinsic properties, natural abundance, comfort, hydrophilicity, and higher heat conduction. The cotton fabrics were dished into the PMN/GLY and LCH@ PMN/GLY solutions and dehydrated at 55 °C. The interactions amid the cotton fabrics and colloidal solutions resulted in physicochemical absorption of the gels on the surface of the cotton. The range of the cottons varied from 0.9 to 3.1% dips of coating enhanced from 1 to 4. It was witnessed that the bandaging showed a little improved flexibility with increase in the nanocomposite content.

LCH@PMN/GLY Surface Morphology
The morphology of the nanocomposite was observed by SEM. The SEM images in Fig. 2 show the uncovered fabric form as well as the smooth surface of the fabricated LCH@PMN/GLY. The additions of GLY/PMN-to-PMN/ GLY persuades significant differences in the superficial morphology of the covered fabrics with an intricate shell-like system on the surfaces. PMN/GLY enhances the rigidity and flexibility of the bandaging, though the tacky nature facilitates relaxed elimination from the wounded sites. After the releases of LCH, the morphological observations showed larger variations than those prior to release. The SEM images show the effectiveness of LCH on the wounded sites. After the release of LCH, the bandage surface became rigid, rough, and flexible [55][56][57].

Mechanical Properties
The mechanical properties of nanocomposite and nanocomposite are critical for successful dressing applications in order to confirm the integrity of the nanocomposite [58][59][60]. The mechanical properties for pure materials (LCH@PMN/GLY), as prepared PMN/GLY and LCH@ PMN/GLY are shown in Fig. 3a. The addition of LCH to the LCH@PMN/GLY had a variable influence on tensile  strength and the overall elongation. The PMN/GLY and LCH@PMN/GLY exhibited a tensile strength of 2.2 ± 0.6 and 2.9 ± 0.4 MPa, respectively, which was sufficient for tissue covering the wound. (Fig. 3b). The increased compressive strength means that the samples are more fragmented. These findings have shown that the composites PMN/GLY has strong surface strength suited for therapeutic use [61][62][63]. The break elongation values represent the LCH and PMN/GLY bandage versatility. Pure LCH, PMN/GLY and LCH@PMN/GLY, the as-prepared PMN/ GLY exhibited an elongation in the range from 20 to 50% at the fracture points (Fig. 3c). Figure 3 also shows that the tensile strength increased with an increase in LCH concentration. The results of the tensile modulus (Fig. 3d) was consistent with the stress test. Overall, PMN/GLY demonstrated higher tensile modulus and elongation than LCH, PMN/GLY and LCH@PMN/GLY, however it exhibited lower maximum strength. Therefore, flexibility would be an important consideration for the application of LCH@ PMN/GLY nanocomposites for various types of wound surface [35][36][37][38].

Biodegradation and swelling studies
Nanocomposites have become the subject of improvement for use in many applications [64][65][66]. The percentage weight loss (Fig. 4A) revealed limited degradation of the LCH@ PMN/GLY bandages. All the as-fabricated bandages showed a degradation of 24 to 27% (1 day), 39 to 48% (4 days) and 75 to 84% (7 days) after immersion in PBS medium, respectively. The presence of LCH reduced the degradation rate in the composite bands. The interaction of LCH, PMN/GLY and LCH@PMN/GLY bandages can be due to this. However the as-manufactured bandage has been in vitro deteriorated although its features and shape have remained unaltered even after the 7th day. After 1, 4 and 7 days of incubation in PBS medium, swellings of the PMN/GLY and LCH@ PMN/GLY dressings were examined (Fig. 4b). Figure 4b suggested that LCH@PMN/GLY exhibited higher swelling capacity compared to pure PMN/GLY nanocomposite [50][51][52]. Furthermore, even after LCH were introduced, the nGel and LCH@PMN/GLY bands displayed similar swelling behaviour. The existence of LCH nanoparticles of

Antibacterial and Cell Viability Analyses
The antibacterial properties of the fabricated PMN/GLY and LCH@PMN/GLY dressings were investigated using S. aureus (gram-positive microbe) and E. coli (gram-negative microbe), and the results are shown in Fig. 5a, b. In our previous studies, the LCH@PMN/GLY dressings exhibited higher antibacterial activity with both microbes compared to LCH@PMN/GLY. The bacterial property of LCH@PMN/ GLY was reduced by the presence of free LCH@PMN/GLY. The outcomes of cell viability established that LCH (positive control) and PMN/GLY (control) did not demonstrate any harm on days 1, 3, and 7 in development with NIH-3T3 fibroblasts (Fig. 6a, b). The synthesized LCH@PMN/ GLY dressings showed ~ 75% viability after day 1 of culture, which further increased up to ~ 93% after 3 and 7 days of development. The reduced cell viability on day 1 was due to the presence of free PMN/GLY with the NIH3T3 fibroblasts. After day 1, the remaining viable fibroblasts multiplied, consequently improving the viability.

In Vivo Examination of LCH
The results of the in vivo investigation of the PMN/GLY and LCH@PMN/GLY are shown in Fig. 7. The PMN/GLY and LCH@PMN/GLY dressings displayed outstanding wound healing properties after 0, 4, 8 and 16 days, compared to saline treatment (Fig. 7a). The reduction in wound damage in mouse models at different periods (0, 4, 8, 16 and 20 days) can be seen in the Fig. 7. The treatment with fabric control displayed ~ 50% wound reduction after day 16, while coating with nanocomposite bandaging resulted in 70% reduction. The LCH@PMN/GLY -treated wound attained 80% closure on day 16. LCH@PMN/GLY treated mice had almost complete wound closure after day 20. This improved rate of wound closure was attributed to the rapid healing properties of the LCH released from the bandaging in the presence of GLY and PMN (Fig. 7b). The LCH released from the nanocomposites slowly changed the physiological conditions and interacted with specific wound sites. Further, LCH has been gambled to decrease the period for fibroblast to enter wounds. It also has excellent anti-inflammatory properties, which may enhance the re-epithelialization rate [67].

Histological Evaluation
Histological study is a powerful tool for observing improvement in cure and tissue regeneration. The histological based on a specified on the healing potential of damaged mouse tissue caused by the effect on the PMN/ GLY and LCH@PMN/GLY (Fig. 8) by hematoxyl and eosin (H&E). After 0, 4, 8, 16 and 20 days, the LCH@ PMN/GLY displayed excellent healing as compared to the naked wound. Figure 8 shows that compact keratinocytes in the epidermis are indicated for PMN/GLY and LCH@PMN/GLY wounds relative to the bare wound in histological parts. Furthermore, LCH@PMN/GLY also increased the healing rate of GLY/PMN-coated wounds as shown in the histological micrograms. LCH nanoparticles have biocompatible and anticoagulant properties, making it an excellent candidate for wound cure, as shown in the study in vitro and in vivo. In comparison, a segment light microscopy quantitated absolute new subcutaneous tissue growth. The degree of wound closure was macroscopically calculated (Fig. 8). Therefore, in vivo study of Sprague-Dawley rats confirmed the increased wound healing ability of the as-fabricated LCH@PMN/GLY. The results (Fig. 8) suggested that the LCH@PMN/GLY nanocomposite treatment promotes significant healing through the migration of fibroblasts and appropriate development of epithelial cells, and the restoration of blood flow via training of new blood vessels [68]. In our effort to introduce a tissue treating method, we have developed LCH@ PMN/GLY bandage material based on PMN/GLY conjugated with LCH, which was first tested for tissue regeneration care management, to the best of knowledge.

Conclusion
Our study aimed at developing novel wound bandages by covering double nanocomposite loaded with LCH on the surface of cotton fabrics. Herein, cotton acts as a supporting layer for the nanocomposite. Well-dispersed LCH are generated with the stabilizing effect of PMN/GLY as the nanocomposite medium. The nanocomposite thus formed had a size in the series of ~ 10-50 nm. Together PMN/ GLY and LCH@PMN/GLY nanocomposite bandages showed significant antibacterial properties even at low absorptions. A slow rate of releases were detected for the nanocomposites bandages with ~ 2 µg/cm 2 of LCH leaking out in 48 h. These low attentions of LCH are very nontoxic for the human body. The LCH@PMN/GLY bandaging displayed 100% closure of full width wound by day 20, which may be because the occurrence of PMN/GLY quickens in the medium, therefore assisting wounds closure. The wound bandages might be effortlessly exposed off from the wounds sites without affecting the tissues. The results of these investigations suggest that LCH@PMN/GLY could be a promising wounds care management composition having effective damage and scar prevention.  Author Contributions ZP, JZ, and YM assisted with NP synthesis and characterization; ZJ, and YS assisted with molecular and biochemical analysis; WW assisted with data curation, formal analysis, and validation; LC assisted with supervised the research.

Compliance with Ethical Standards
Competing interests The authors declare that they have no competing interests.
Ethical Approval and Consent to Participate All animal experiments were approved by the Ethics Committee of the Emergency Intensive Care Unit, Wenling First People's Hospital, Wenling 317500, China in accordance with the guidelines on animal care and use (File No: 2019-10).

Consent for Publication
Not applicable.