Plant-Inspired Adhesive and Injectable Natural Hydrogels: In Vitro and In Vivo Studies

doi:10.21203/rs.3.rs-2484831/v1

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Research Article

Plant-Inspired Adhesive and Injectable Natural Hydrogels: In Vitro and In Vivo Studies

https://doi.org/10.21203/rs.3.rs-2484831/v1

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published 13 Jun, 2023

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The development of alternative therapeutic treatments based on the use of medicinal and aromatic plants (Juniper communis L.) has aroused interest in the medical field to find new alternatives to the conventional therapeutic treatments, which nowadays have shown problems related to bacterial resistance, high costs or sustainability in their production. The present work describes the use of hydrogels based on sodium alginate and carboxymethyl cellulose with combinations of juniperus leaves and berries extracts in order to characterize their chemical characteristics, antibacterial activity, tissue adhesion test, cytotoxicity in L929 cell line, and their effects on an in vivo model in mice in order to maximize the use of these materials in the healthcare field. It has been overall obtained an adequate antibacterial potential against S. aureus, E. coli, P. vulgaris with the use of doses above 100 mg. mL^− 1 of hydrogels, likewise a low cytotoxicity in hydrogels combined with extracts has been identified according with IC₅₀ value at 17.32 µg. mL^− 1 in comparison with the higher cytotoxic activity expressed by the use of control hydrogels with a value at 11.05 µg. mL^− 1. Besides, in general the observed adhesion was high to the different tissues, showing its adequate capacity to be used in different tissue typologies. Furthermore, the in-vivo results have not shown erythema and edema or other complications related with the use of the proposed hydrogels. These results suggest the feasibility of using these hydrogels in biomedical applications in reason of the observed safety.

Biological activity

Bioadhesive injectable hydrogel

In vivo skin irritation

Juniperus communis

Medicinal plants

Tissue engineering

The use of natural extracts (NE) has been part of the development of mankind throughout history in reason of their wide use in traditional medicine to recognize by means of empirical observation the positive effects of these in the evolution of certain diseases. Recently years has been rising the interest on traditional therapeutics, in the pharmaceutical industry, and the flavoring of beverages and food (Minero et al. 2017; Zheljazkov et al. 2017) by means of NE. In the great diversity of extracts in nature, those from the Juniper (Juniperus communis L. (common juniper, Fig. 1) family have attracted attention due to their different antibacterial, anti-inflammatory, and antifungal properties in applications such as traditional wound treatment, superficial treatment, and aroma therapy-based treatments. In addition, anticancer effects have been observed in extracts from juniper species due to the various effects against abnormal cell proliferation (Lantto et al. 2009). Different compositional profiles have been detected depending on the part juniper in which the extract is obtained, e.g., the parts of the plants that are generally of interest are the leaves, berries as well as parts of the trunk (Radaukova et al. 2018).These variations in the compositions obtained from the different parts of the plant can have diverse effects on the microbial activity, therapeutics, as well as their stability in the matrixes. It has been observed that juniper leaves extracts have an anti-inflammatory and antinociception action on nephrotic tissue in mice models (Al-Attar et al. 2017). However, this type of model presents the need to explore in depth the role of juniper leaves extracts in order to know their role in the prevention of cellular stress and their effect on the biological activity on tissues.

On the other hand, juniper berry-based extracts have been observed the influence on cellular activity in aspects such as gene expression, cellular stress, and induction of neuroblast cell death (Raasmaja et al. 2019). Moreover, it has identified a significant reduction on the activity of adenomas and adenocarcinomas in animal models (Yaman et al. 2021) with the use of juniper berries extracts. The main reason of the properties on berries is related with high content of α-pinene content, this can modulate global gene expression and could modify the metabolic action related to cancer cells in humans (Han and Parker 2017). Despite the extensive use of juniper berries since ancient times in culinary applications or traditional medicine, research on their activity is a topic that has aroused interest in order to increase their use in industrial production (Kacaniova et al. 2018) but the research in this area brings some challenges due to the compositional divergences that among the different species of the juniper family. In addition, interest in analyzing the role of berry-based extracts has intensified in the last decade by virtue of the anti-aging effects of these extracts, due that can prevent cellular oxidative stress in worms (Pandey et al. 2018).

However, the use of extracts is limited by their volatility and dispersion to the environment due to lose of their properties with time in the applications on tissues. Therefore, the knowledge about the type of medium that enables to extend the properties of the extracts as well as the identification of the best types of extracts from berries or leaves is a matter of interest in the scientific community. In this sense, the use of this type of extract has involved the experimentation of different vehicles for their transport from encapsulated model matrices (Bajac et al. 2022), impregnation in polymeric fibers (Bigdarvishi et al. 2021; Tyliszcak et al. 2019), polymer-ceramic composite materials (Zeng et al. 2014). Notwithstanding, the different methods of transport and source of extract present some challenges for their direct application on soft tissues such as skin, especially in the therapeutic treatment of cancer by virtue of the sensitivity of the components, hence a continuous study in the field is necessary in order to recognize the multiple using possibilities of these extracts and their combinations.

This study presents an overview of the functional characteristics, antibacterial activity, cytotoxicity, adhesion tissues response, and irritation evolution in in-vivo models of polymeric gels with combinations leaves and berries extracts of J. communis L. in order to identify their characteristics, increasing the current state of knowledge of this type of compositions to enhance the application in medicine as alternative material due to the economic feasibility, sustainability, and relative low cost production.

Materials and methods

Chemicals: Sodium alginate (SA, Mw: 4.2× 105, M/G = 1.52), Carboxymethyl cellulose sodium (CM-cellulose; CMC-Na, Viscosity: 1000–1400 mpa s, SD = 1.2), Glycerol (medical grade) and acetic acid, (CH₃COOH, glacial, ReagentPlus®, ≥ 99%) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Mineral water (Ca: 205.867 mg/L, Mg: 124.588 mg/L, K: 26.005 mg/L, Fe: 0.008 mg/L, Na: 138.181 mg/mL, S: 176.11 mg/L, F: 0.64 mg/L, Si: 48.21 mg/L, Cl: 21.94 mg/L, CO₃^− 2: 1433.5 mg/L) was obtained from local company in Türkiye. Ultrapure water was prepared by a Milli-Q50 SP Reagent Water System (Millipore Corporation, MA, USA) for the preparation of samples. Other reagents were of analytical grade.

Preparation of J. communis L. plant and extract: J. communis L. plant leaves and berries were collected above the village of İmaret, near Sivas, Türkiye, N39° 41' 54.3444'' E37°1' 5.6712'' at 1415 masl in 2022. The collected juniper leaves and berries were cleaned and spread out in a layer approximately 2 cm deep in a well-aerated shady place to dry for a month at RT before extracted. The extraction was carried out using soxhlet extractors in accordance with TAPPI standard (TAPPI, 1988). The subsamples for the extraction consisted of 50 g air-dried leaves and berries, which were grounded into fine powder immediately prior to the distillation. (The ratio of the biomass to solvent was 1:10) Grounded plant material was transferred to an extraction thimble which was placed in a Soxhlet apparatus with a connected reflux condenser. Appropriate solvent (ethanol and ethyl acetate) was then added to the flask and used as a solvent. After 6 h of extraction and complete exhaustion of the J. communis herbal material, the obtained extracts were evaporated to dryness under vacuum at 40°C and stored in sterile glass bottles at 4°C until further experiments.

Preparation and pharmaceutical compositions of sodium alginate/sodium-carboxymethylcellulose (SA/CMC) hydrogel

Hydrogel solution was prepared in the following manner: A homogeneous solution of mineral water was obtained by adding 0.5% v/v of CH₃COOH in 100 mL at RT under vigorous stirring (pH ⁓ 4.8-5). Next, 1.5%w/v CMC-Na and 1.5% w/v SA power were slowly added and stirred at 37°C until complete solubilization. To complete the carboxymethylation reaction, the reagents were mixed together by slowly adding the 6.5% v/v glycerol to form a uniform sticky solution at 37°C for 6 h in a water bath. To prepare J. communis plant leaves extract (designated as JL/SA-CMC) and berries extract (designated as JB/SA-CMC)-loaded SA/CMC hydrogels, leaf and berry extract (5%w/v) was added to the SA/CMC mixture respectively to obtain final composition of SA/CMC hydrogel. One set of 10 cc syringes was filled with SA/CMC hydrogel. Another sets of 10 cc syringes were filled with JL/SA-CMC and JB/SA-CMC hydrogels. The syringes were capped with a male Luer cap. The gel filled syringes were then placed into a Tyvek pouch and sterilized in an autoclave at 110°C for 30 minutes. The hydrogels were allowed to cool at room temperature (22°C ± 2°C) under an aseptic environment to achieve the desired hydrogels.

Fourier transform infra-red spectroscopy (FT-IR) analysis

The chemical structure of SA/CMC, JL/SA-CMC and JB/SA-CMC hydrogels was characterized by infrared (IR) spectroscopy. A sample of the 20 mg hydrogels were dried at 45°C for a period of 12 hours in order to evaluate the functional groups of the hydrogels. The spectra were measured on a Fourier transform (FT-IR) spectrophotometer (Bruker Alpha II spectrometer, Germany) in the wavelength range of 400–4000 cm^− 1 with a resolution of 4 cm^− 1.

Antimicrobial activity

The antimicrobial activity of the hydrogels was evaluated quantitatively by using the broth micro-dilution method according to the Clinical Laboratory Standard Institute (CLSI) guidelines (CLSI, 2020). In the antimicrobial activity study was used 3 indicator microorganisms including Staphylococcus aureus ATCC 292123, Escherichia coli ATCC 25922, and Proteus vulgaris ATCC 7829. The stock solution of hydrogels was prepared by dissolving 1 g of hydrogel in 2.5 ml sterile dH₂O and sterilized by autoclaving at 110°C for 5 min. Afterwards, by taking a sample from here, dilutions were prepared in various concentrations in the range of 300 mg/ml to 25 mg/ml in Mueller Hinton Broth (MHB). The study was briefly carried out as follows; after 18 ± 2 h incubation at 37°C in MHB medium, the densities of indicator microorganisms were adjusted McFarland standard No: 0.5 using the same medium. Firstly, dilution series of samples (100 µl) were transferred to microtiter plates and then adjusted indicator microorganism (100 µl) was added to the wells. Piperacillin/Tazobactam (8:1), indicator microorganism suspension and sterile MHB was used as the reference antimicrobial agents, negative control, and positive control, respectively. All assays were performed in triplicate. After the all-microtiter plates were incubated at 37°C for 24 h, the absorbance was recorded at 600 nm on microplate reader (Spectro Star Nano, Germany) and the lowest concentration that inhibits the indicator microorganism growth was determined as the minimum inhibitory concentration (MIC) value. Also, to evaluate the antibactericidal effect of sub-MIC concentrations, the inhibition ratio (%) was calculated by Eq. 1 (Kamoun et al. 2015):

$${I}{R}\left({\%}\right)=100-\left[\frac{{OD}_{GS}-{OD}_{B1}}{{OD}_{NC}-{OD}_{PC}}\right]*100$$

Where; the OD_B1 is the absorbance of medium containing only dilutions of the hydrogel samples, OD_GS is the absorbance of the bacterial solutions after incubating growth medium, OD_NC is the absorbance of the negative control and OD_PC is the absorbance of the positive control.

In vitro cytotoxicity assays

Mouse fibroblast L929 cells were used for the cytotoxicity assays of the hydrogels. Cells were grown in medium composed of DMEM (Gibco, Grand Island, NY, USA), 10% FBS (Capricorn, USA), 1% Penicillin-Streptomycin (100 IU/mL). For the study, 1 ml of each of the hydrogels (SA/CMC, JL/SA-CMC and JB/SA-CMC) were diluted in 5 ml DMEM vortexed for 5 minutes and incubated for 24 h. The cells were seeded on 96-well plates (100 µL/well), then the cells were exposed to dilutions of the hydrogels, from each of the hydrogel dilutions different volumes were extracted in order to achieve in the cell growth medium different concentrations of the hydrogels (100 µg. mL^− 1, 50 µg. mL^− 1, 25 µg. mL^− 1, 12. 5 µg. mL^− 1, 6.25 µg. mL^− 1, 3.125 µg. mL^− 1, 1.56 µg. mL^− 1, and 0.78 µg. mL^− 1), then were incubated at 5% CO₂ atmosphere and 37°C for 2 days. Cytotoxicity effects on L929 cell lines were determined by XTT assay 24 h after exposure to the different concentrations. Cell viability was measured in a microplate reader in the reference range of 475 nm. It was determined according to the observed orange color intensity at the end of the incubation period. All absorbance’s were compared with control samples (cells without any test concentration) representing 100% cell viability. The data were presented graphically and by regression was estimated the representative IC₅₀ for each of the hydrogels. All processes were accomplished in accordance with the ISO 10993-5 protocol.

Primary skin irritation test

The evaluation of skin irritation is one of the major index of dermal safety in Pharmaceutical/cosmetic applications. Skin compatibility of the hydrogels was conducted using the primary skin irritation test and can be considered as the first clinical step of the safety evolution. The study was carried out after review and approval of the research project by the Research Ethics Committee of Sivas Cumhuriyet University with approval number: 65202830-050.04.04-681. The dermal safety was conducted according to ISO-10993-10:2021 (Tests for irritation and skin sensitization) taking in account the procedure followed in previous works in the literature (Horn-Huey et al. 2013; Jibry et al. 2004). Briefly, six male Albino mice weighing approximately 20 g (5 weeks old) were obtained from animal hospital of Sivas Cumhuriyet University, Sivas, Türkiye. Mice were caged in a laboratory with standardized environmental conditions (20°C ± 2, 35–45% Relative humidity) and a constant day/night cycle. They were fed with a standard rat chow diet and water ad libitum in specific pathogen free laboratory for one week. 24-hr prior to the test, the hair from the back of each mouse were clean- shaved on both sides of the dorsal skin to expose adequately large test areas. The dorsal skin of mice was divided in four test sites (approximately 1.5 cm × 1.5 cm) for application and observation. The hairless dorsal skin was wiped with EtOH. Untreated control side was marked out on their upper backs. All mice were treated topically with their respective formulations once daily on day 1 of the 3-day study, with an application time of 24h, 48h, and 72h. 50 mg of SA/CMC, JL/SA-CMC and JB/SA-CMC hydrogel was applied onto the test area on the lower/upper back and carefully rubbed in. At the end of the each application time, the treated area was gently wiped with water-soaked gauze to remove any residual vehicle from the skin surface.

For the dermatologically correct assessment of differences in skin surfaces, each of the mice was kept in separate cages for the duration of the experiment. Moreover, all the visual assessments and the clinical evaluation was performed by a veterinary surgeon (not the investigator who applied the preparations). The skin irritation of hydrogel samples, such as erythema or edema, was evaluated by the scoring system of Draize dermal irritation test, including (0): no erythema or no edema, (1): very slight erythema or edema, (2) well-defined erythema or edema, (3): moderate erythema or edema, (4): severe erythema or edema.

Tissue adhesion of the hydrogels

Strong tissue adhesion is essential for in situ hydrogels in tissue bonding to resist mechanical forces during dynamic movements. In addition, adhesive hydrogels must be able to readily adhere to a variety of fresh living tissues, including pulmonary, gastric, spleen, cutis, muscle, adipocyte, and femur (Xiaoxuan et al. 2020). To demonstrate this in an experimental study, 100 mg of the freshly prepared hydrogel was applied to one side of the tissue of the mice. Then, the surface was lightly pressed manually, the pressing surface was covered with latex gloves in order not to interfere with the interaction of the hydrogels and the tissue. In order to vividly display the adhesive behavior of prepared hydrogels with various biological tissues was explored. The composition of the matrix hydrogel and the extracted combinations with matrix hydrogel were evaluated on each type of tissue.

Data analysis

All experimental results were analyzed using Origin Pro 20219.8.0.200 (Origin Lab, Northampton, MA, USA), Image Tool software and Sigma Plot for Windows Version 12.3 (Sigma Plot Software, Palo Alto, CA, USA). All data were expressed as mean ± standard deviations (SD) of the indicated number of experiments. Statistical analysis was performed using ANOVA followed by Student’s t-test (p < 0.05) for independent variables. Erythema/edema scores were evaluated by the Draize dermal irritation scoring system (DDISS). For all tests, a P value < 0.05 was considered statistically significant.

Fourier transform infrared spectroscopy (FTIR)

Figure 2 shows the FTIR spectra obtained for pure SA/CMC, JL/SA-CMC and JB/SA-CMC hydrogels in the wavenumber range from 500 to 4000 cm^− 1.

Stretching vibrations of the O‒H bonds are observed at 3270 cm^− 1 in all cases, in the bands 2933 cm^− 1 and 2884 cm^− 1 are observed symmetric and asymmetric vibrations of aliphatic chains of sodium alginate and carboxymethylcellulose. The peak at 1599 cm^− 1 is associated with vibrations of the carbonyl group (‒C = O) in alginate. The bands at 1411 cm^− 1 and 1027 cm^− 1 are considered characteristic vibrations of ‒COO and ‒C‒O bonds in alginate and carboxymethylcellulose. The bands at 1250 cm^− 1 and 1213 cm^− 1 are associated with the vibration of ‒CO groups (Deepa et al. 2016; Huq et al. 2012). Moreover, at 1107 cm^− 1 is observed the characteristic bands of carboxymethylcellulose which is related to vibrations of the C‒OH groups (Habibi et al. 2014). The peak at 1323 cm^− 1 is associated with stretching vibrations of the COO functional group in sodium alginate (Lan et al. 2018). In addition, the associated bands between 993 cm^− 1 to 607 cm^− 1 are considered characteristic of alginates in reason of its structure dominated by pyranose rings, which usually present vibrational bands in this region (Larosa et al. 2018). It is noted that in this region the signals around 993 cm^− 1 are linked to deformation vibrations of C₃‒O₃H₃ bonds (Hou et al. 2018), at 921 cm^− 1 is associated with deformation vibrations of C‒C‒H bonds, at 850 cm^− 1 is associated with deformation vibrations of C‒O‒C (glycosidic linkage), at 817 cm^− 1 is related with deformation vibrations of the ring in the C‒C‒O groups. Lastly, the signals at 597 cm^− 1 are related to ring breathing vibrations (Cardenas-Jiron et al. 2011). Nonetheless, it is detected with the incorporation of the juniper extracts a shift to 599 cm^− 1, as well as a peak with lower intensity than in the matrix hydrogel. The reason of the shift is explained by an interaction of the terpene groups of the extracts that slightly modify the electronegative activity of the carbon bonds of the alginate in the matrix hydrogel.

Evaluation of antimicrobial activity of hydrogels

The MIC values of hydrogel compositions against indicator microorganisms are shown in the Table 1. It is observed that the applications of the hydrogels can provide an adequate degree of bacterial inhibition, showing a minimum antibacterial activity with the use of concentrations at 100 mg. mL^− 1 against the inhibition of S. aureus, it is not observed that the incorporation of extracts of leaves and berries decreases the dose required to generate inhibition. It is identified that in the case of E. coli, the use of a dose of 150 mg. mL^− 1 allows the inhibition of this pathogen; it was not observed that the incorporation of extract modifies this tendency. In the case of inhibition against P. vulgaris, inhibition was observed with the hydrogel without extract with the use of 150 mg. mL^− 1, this tendency was modified with the incorporation of leaves extracts, which reduced the concentration necessary for inhibition to 100 mg. mL^− 1, in the case of the combination with berries extracts has been observed inhibition at 150 mg. mL^− 1.

Table 1

Minimum Inhibition Concentrations (MIC, mg. mL^-1) of different hydrogel compositions against indicator microorganisms
Composition	Minimum Inhibition Concentrations (mg.mL^− 1)
Composition	S. aureus	E. coli	P. vulgaris
SA/CMC	100	150	150
JL/SA-CMC	100	150	100
JB/SA-CMC	100	150	150
Ciprofloxacin (C₁₇H₁₈FN₃O₃)	˂0.0019	˂0.0019	˂0.0019

Moreover, the antibacterial effect of sub-MIC concentrations, it was observed that a difference in antimicrobial activity between matrix hydrogel and compositions with extracts. On evaluating the Minimum Inhibition Concentrations values and the inhibition ratio obtained for this type of combinations, it is observed that hydrogel with juniper berries extract shows a 73.9 ± 3.1% bacterial inhibition against E. coli in comparison with hydrogels based on leaves extracts, in which have observed a 90.08 ± 3.5% ration of inhibition (data not shown here). Pathogenic microorganisms that colonize the skin surface, such as S. aureus, which cause atopic dermatitis, adversely affect the quality of life. In addition, some pathogenic microorganisms cause serious infections when they penetrate the skin barrier and proliferation in the cell. In this context, it is important that the applications of theses hydrogels to the skin surface to provide an alternative to the use of drugs in reason that the current treatment can generate bacterial resistance. Similarly, for P. vulgaris, have been observed a value of 25.8 ± 4.5% bacterial inhibition in the matrix at 100 mg. mL^− 1 concentration, while 90 ± 3.2% bacterial inhibitions were observed in the hydrogel with juniper extracts. Therefore, it is possible considered antimicrobial activity in the hydrogel matrix at high concentrations.

The present study has also observed the antibacterial activity of combinations of carboxymethylcellulose and sodium alginate-based hydrogels. Despite previous study observations of the components separately showing low antimicrobial activity (Asadpoor et al. 2021; Kumar et al. 2019; Puscalescu et al. 2020), their combination by other side can increase their antimicrobial activity (Han et al. 2017; Shao et al. 2015). The antimicrobial activity of combinations of SA and CMC are based on different parameters like the used amount, other components in matrix, pH of the medium, and temperature of incubation (Abdellatif Soliman et al. 2021; Han et al. 2017). Therefore, in this study, the presence of antimicrobial activity in the prepared hydrogel matrix is basically attributed to the its intrinsic composition. Hitherto it is seen that the antimicrobial activity is generally determined by the agar disc diffusion method (Han et al. 2017; Kamoun et al. 2015; Kumar et al. 2019). Nevertheless, this methodology presents disadvantages to evaluate the antimicrobial activity of hydrogels or aqueous compounds due to interaction of the components and their viscosity may interfere with bacterial growth or may provide certain in homogeneities in the dispersion. This study shows a view of the antimicrobial activity that can result from hydrogels based on SA and CMC and their combinations with juniper extracts, showing a potential use in dermal treatments and thus being an alternative to the use of drugs and ointments of high economic value.

In vitro cytotoxicity evolution of hydrogels

Figure 3 shows the dose-response curves of the different hydrogels combinations, based on the regression of the points of this curve has been established the value of half maximal inhibitory concentration (IC₅₀) on the cell development of fibroblast cells of L929 mice. An IC₅₀ were calculated value of 11.05 µg. mL^− 1 in the hydrogel matrix, in the combination with juniper leaves extracts a value of 15.19 µg. mL^− 1, and the hydrogel containing berry extracts a value of 17.32 µg. mL^− 1. The use of quantities of a couple of micrometers thick is commonly used in cosmetology, thus, the tissue exposure is usually a couple of micrograms per volume of hydrogel, emulsion or ointment. In this sense, the IC₅₀ provides a first overview of the possible toxic effects at the microscopic level on cell development, then a product with a low IC₅₀ has a high probability of promoting cellular toxicity. In the case of hydrogels with combinations of extracts, it has been observed that their incorporation increases cell tolerance to combinations of alginate and carboxymethylcellulose hydrogels matrices. It has been observed that hydrogels constituted by SA and CMC have different behaviors in relation to cell viability; alginates generally have poor characteristics related to cell development (Hurtada et al. 2022). Hence, the combination with other components can increase their ability to enrich cell development, as it is the case of the combination with CMC, since it has been observed that this promotes the stability of the cell medium to increase its viability over time (Jeong et al. 2018).

In the case of the leaves extracts of juniper species have shown positive effects on preventing the cell deterioration due to that the extracts from these species usually have a low tendency to cell degradation, becoming a feasible candidate in therapeutic treatments in reason of its preventive effect on healthy tissues during chemotherapies (Och et al. 2015), especially when combined with polymeric matrices as medium of transport. The behaviors have observed with the incorporation of juniper berry extracts is related to its high content of polyphenols that increase antioxidant activity and protect cell deterioration (Ivanova et al. 2018), implying that to promote cell deterioration at macroscopic level it is necessary to use high concentrations in order to decrease cell activity, especially in the treatment of cancer cells (Ivanova et al. 2021)

Primary skin irritation test

The in vivo safety of the prepared hydrogels with respect to skin irritation was tested in mice (Fig. 4). The skin sites of hydrogels application of all mice were assessed at 24, 48, and 72 h after removing hydrogels visually before and after applications, as shown in Fig. 5. It is observed that after the study period there is no skin irritation, inflammation, so that according to the ISO-10993-10:2021 classification an indication of “0” is presented by virtue of the absence of edema or erythema after evaluation for 24, 48 and 72 hours (Fig. 5). In addition, during the period no abnormal capillary growth factors were observed, suggesting that the use of this type of hydrogels does not generate significant alterations in the development of the epithelial tissue during their use. The observed results can be related to the high antioxidant capacity offered by juniper extracts (Al-Attar et al. 2017; Pandey et al. 2018), despite the knowledge of the antioxidant activity, the possible effects of their use based on polymeric vehicles such as present in this study were not known on epithelial tissue until now.

Tissue adhesion test

Tissue adhesion is a major factor in the practical application of tissue adhesives, wound dressings/patches, and wearable medical devices (Ghobril et al. 2015; Qu et al. 2018). The adhesion properties of produced hydrogels with various biological tissues were investigated, and the results are shown in Fig. 6. Biological tissues, which are derived from mice including the pulmonary, gastric, spleen, cutis, muscle, could easily adhere to the SA/CMC, JL/SA-CMC, and JB/SA-CMC hydrogels. Adequate adhesion of the hydrogels to the biological tissues was observed in all cases, despite the incorporation of the extracts were observed no significant changes to their adhesive behavior. The observed performance may be related to the functional groups that compose the hydrogel, as well as the interactions between the tissues. It has been observed that the adhesion of hydrogels to tissues is related to the interactions that are established between the carboxylic groups of the hydrogel matrices and the interactions with amide formation, which they usually cover part of the biological material. This adhesion is usually mediated by the formation of covalent bonds between the carboxyl-amide groups (Sun et al. 2021). This suggests that the use of the present matrix is feasible for its extensive use on a variety of tissues as it guarantees the adherence and interaction of the components during the treatment. Likewise, one of the factors that provides a positive value to the adherence is that it allows to prevent changes or disruptions during the treatment that could affect the final activity as well as the biological effects that this type of hydrogels offer on the tissues.

Different hydrogels have been studied based on sodium alginate and carboxymethylcellulose combined with extracts of juniper leaves and berries. It has been found that the combination at structural level is dominated by a strong relationship of the functional groups of the hydrogel, increasing their compatibility and homogeneity. Moreover, the antibacterial activity of hydrogels with berry extracts has a high antibacterial potential against S. aureus, and the combinations with leaves extracts have shown a high inhibition potential against P. vulgaris. For other side, it has been observed with the combination of extracts a decreasing of the toxicity of the hydrogels, showing their safety with values of up to 17.32 µg. mL^− 1 as a safe dose when occurs direct cell exposure. It has been identified that the adhesion of this type of hydrogels in a variety of tissues is feasible, since there are no contraindications to be applied in case it is required in any of the tissues evaluated. The in-vivo model has shown adequate results demonstrating the safety in the use of these compounds, thus the future and scale-up of these hydrogels combinations are promising for future research in human models to further explore and expand the development as an alternative therapeutic treatment or as a scientific basis for future applications in medicine.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author contributions

Kerim Emre Öksüz and Cristian Daniel Bohórquez-Morenoconducted the biomaterial synthesis and chemical characterization of the materials. İlker Şen performed the animal experiments with the help of Kerim Emre Öksüz and Cristian Daniel Bohórquez-Moreno.Ceylan HepokurandEmine Dinçer analyzed the in vitro and in vivo experiments with the help of İlker Şen contributed to data interpretation, discussion, and critical comments on shaping the manuscript. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Sivas Cumhuriyet University (26.10.2022/65202830-050.04.04-681).

Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper

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Plant-Inspired Adhesive and Injectable Natural Hydrogels: In Vitro and In Vivo Studies

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