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Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39256491
71525
10.1038/s41598-024-71525-w
Article
Design of multiple-function matrix encapsulated with Marjoram extract to support cellular functions, stimulate collagen synthesis and decrease infection in wound
Mohammadi Shahab
Khavarpour Maryam mkhavarpoor@yahoo.com

Ghadi Arezoo
grid.467532.1 0000 0004 4912 2930 Department of Chemical Engineering, Ayatollah Amoli Branch, Islamic Azad University, Amol, Iran
10 9 2024
10 9 2024
2024
14 2110917 3 2024
28 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
This study aimed to assess the role of the combination of design techniques of the engineered substrates, and the effect of encapsulating Marjoram (Origanum Majorana L.) into the matrix network was studied. To this end, PVA-PEG matrices were designed through 3 techniques of freeze–thaw (FT), the combination of both methods of freeze-drying and freeze-thawing(FT-FD), and ternary technique(freeze-drying,freeze-thawing,cross-linking(FT-FD/CL)), by combining equal volume ratios of both polymers. The results indicated the ternary technique can provide better physicochemical properties(porosity: 96%, lower degradation rate, higher modulus) compared to FT and FT-FD methods. Afterward, encapsulation of Marjoram-extracted bio-actives in the matrix network designed with the ternary technique demonstrated that the increase in the extract concentration up to 3% can increase encapsulation efficiency. The encapsulation also caused a more cohesive network by better bonding between functional groups in herbal biomolecules and polymer chains of the matrix. Mass transport mechanisms and release kinetics of matrix-encapsulated bio-actives indicated a deviation from Fickian diffusion and the release by diffusion and swelling process. Biologically, matrix-loaded herbal carbohydrate(Epi-alpha-Cadinol) improved fibroblast adhesion and distribution on the substrate surface, and led to the better synthesis of collagen fibers, especially in 3% herbal extract, and antibacterial activities owing to the controlled release of sesquiterpenoids and N-Acetyl-l-proline.

Keywords

Herbal carbohydrate polymers
Sesquiterpenoids
Origanum majorana L. (Marjoram)
Regenerative medicine
Medicinal herb-delivery systems
Polymeric matrix
Subject terms

Cell biology
Chemical biology
issue-copyright-statement© Springer Nature Limited 2024
==== Body
pmcIntroduction

In recent years, regenerative medicine has created a promising approach for treating diseases, improving cellular interactions, and subsequently regenerating damaged tissues1,2. In this field, the skin, as a main tissue with a protective role, is of special importance. This tissue can control body functions, like prevention of water loss, and protection from internal organs against mechanical stresses and entering bacteria3. Hence, failure to repair the dermal damage in the time (prompt and adequate repair) can cause serious problems for one health. In this matter, the skin can usually repair small injuries by promoting cell proliferation, while the self-healing process is hampered for large or extended wounds1,4,5.

In the past, substitutes such as allograft, autograft, and xenograft were considered as the main treatments, however, their problems like few donor sites, side effects, risk of immune rejection, and ethical restrictions have led to develop engineered substitutes and polymer-based matrices6–8. Such substrates can improve the cell-to-cell interactions and their functions (adhesion, proliferation, and migration) and result in synthesizing collagen fibers along with forming the regular extracellular matrix (ECM)4,9.

In this regard, many polymers can create proper conditions for improving cell behavior and increasing its proliferation10–15. Polyethylene glycol (PEG) is one of these polymers, that plays a crucial role in the imitation of the dermal behavior of the engineered substrates. This hydrophilic polymer can improve cellular adhesion to the surface of the matrix/substrate and increase cell signals and growth when loaded with biomaterials or herbal biomolecules such as protein, peptide, carbohydrate, or polysaccharide3,4,16,17. PEG also can act as the main factor in maintaining and stabilizing the moisture level in the designed matrix and lead to controlling the humidity in dry wounds and absorbing wound exudates in infection wounds2.

PVA is another polymer in this field that can facilitate the placement or removal of the matrix from the wound site due to the ability to create a plastic layer in the designed matrix structure. This polymer, as an absorbent material, prevents the re-deposition of pus at the wound site18,19 and can act as a suitable dressing owing to biocompatibility and non-toxicity. However, the use of this polymer as a single matrix is no suitable option for designing the engineered substrates due to their water solubility20. Hence, the combination of polymers to reach an integrated matrix plays an important role in mimicking the matrix from the native tissue behavior.

Based on the studies, the use of biomolecules such as herbal bio-actives in the structure of dermal substrates can also accelerate the wound healing process3,21–27 and eliminate the limitations of using traditional forms of these biomaterials (gel, ointment, cream, etc.) in infected open wounds4,28. Indeed, these herbal biomolecules due to their excellent multiple properties such as antibacterial, antioxidant, anti-inflammatory, and restorative features can promote cell-to-cell interactions and improve the biological properties of the matrix26,29–34. In this matter, Izadyari Aghmiuni et al. indicated that combining quince seed mucilage in the structure of dermal hybrid scaffolds can be effective in achieving smart substrates and imitating the biological behavior of skin3. However, it seems herbs that possess antibacterial properties along with protein derivatives can more effectively act to repair and regenerate damaged skin. Accordingly, the research of Iranian and Chinese traditional medicine has illustrated that Origanum Majorana L. (Marjoram) extract possesses a high antimicrobial activity which originates from its biomacromolecules such as polyphenols35–38. Nevertheless, the large number of these biomacromolecules in the aqueous and organic/alcoholic extracts of this medicinal herb such as carbohydrate polymers, diterpenoid, alkaloid, etc. may decrease therapeutic effects or product shelf-life, due to its adverse interactions with other components.

Hence, given the importance of the issue in designing dermis-like matrices with antibacterial properties and repairing effects, it seems that the formulations or networks loaded with these bio-actives as combination techniques (tissue engineering + drug delivery) can be a suitable approach for overcoming the mentioned problems.

Therefore, this study first aimed to fabricate a porous matrix as an infection-absorbing substrate with favorable mechanical properties. In the following, Marjoram-obtained herbal extract was encapsulated in the matrix network to assess antibacterial properties as well as the role of carbohydrate polymers of this extract in the adhesion and proliferation process of fibroblast cells and collagen synthesis and consequently skin regeneration. Accordingly, the novelty of this study is in a different attitude toward using medicinal herbs riched with carbohydrate polymers to increase their effectiveness, as well as to achieve a multiple-function matrix (in terms of antibacterial, antioxidant, and repairing properties) for supporting cellular functions and helping herbal biomolecules to avoid undesirable interactions. Moreover, we believe that such a matrix can effectively manage wound infection by the controlled and sustained release of matrix network-entrapped herbal bio-actives. To this end, PVA-PEG-based matrices, with a volume ratio of 1:1 (v/v), were separately prepared by the freeze–thaw process (three cycles), the combination of both methods of freeze-drying and freeze-thawing, and a ternary technique included freeze-drying, freeze-thawing, and cross-linking, to study the role of the processing techniques in mechanical, morphological, physicochemical properties of the matrix. In the following, the matrix that possessed more desirable features was selected to continue the study. Accordingly, the matrix loaded with 1%, 3%, and 5%w/v herbal extract was re-fabricated with the desired method, and the mentioned properties along with encapsulation efficiency percentage (EE%), the release behavior of herbal biomolecules from the matrix network and its Kinetics model, as well as biological properties (adhesion, proliferation) were studied. Finally, the better matrix was selected to determine of antibacterial activity of the biomolecules-loaded matrix against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria and to compare with discs containing gentamicin.

Results

Biomacromolecules study of Marjoram extract

The biomacromolecules and bioactive ingredients of Marjoram extract were analyzed by GC–MS device. The results indicated the identification of monoterpenoids [Linalool and Cis-alpha-bergamotene: 0.75%], phenol [Dinoterb: 0.97%], sesquiterpenoids [β-caryophyllene and Epi-alpha-Cadinol: 1.03%], alkaloid [3',8,8'-Trimethoxy-3-piperidyl-2,2'-binaphthalene-1,1',4,4'-tetrone: 2.09%], diterpenoid [1.07%], and protein [N-Acetyl-l-proline : 0.22%], as well as two compound of plant metabolite [Catechol: 39.05%] and 9,10-Secocholesta-5,7,10(19)-triene-3,24,25-triol, (3.beta.,5Z,7E) [53.93%] as main biomacromolecules (Table 1, Fig. 1a).Table 1 GC–MS analysis for Marjoram extract.

No	RT1	Compound name	peak areas (%)	(m/z)2	(g/mol)3	Molecular formula	Groups	
1	7.6	Linalool	0.52	55,69,93	154.25	C10H18O	Monoterpenoid	
2	8.86	Dinoterb	0.97	53, 131,225	240.21	C10H12N2O5	Phenols	
3	9.15	Catechol	39.05	63, 64,81, 109,110	110.11	C6H6O2	Plant metabolite	
4	11.79	N-Acetyl-l-proline	0.22	43,66,70, 94,113	157.17	C7H11NO3	A natural product found in plants as amino acid	
5	13.03	Cis-alpha-Bergamotene	0.23	93,94,119	204.35	C15H24	Bicyclic monoterpenoids	
6	13.58	β-caryophyllene	0.61	41,91,80, 121	204.35	C15H24	Bicyclic sesquiterpene	
7	18.53	Epi-alpha-Cadinol	0.42	81,105,121, 161	222.37	C15H26O	Sesquiterpenoids	
8	20.19	9,10-Secocholesta-5,7,10(19)-triene-3,24,25-triol, (3.beta.,5Z,7E)-	53.93	43, 44, 55, 211	416.6	C27H44O3	Secondary metabolite	
9	21.05	3',8,8'-Trimethoxy-3-piperidyl-2,2'-binaphthalene-1,1',4,4'-tetrone	2.09	57, 149,167, 184	487.5	C28H25NO7	Alkaloid	
10	23.52	Hexadeca-2,6,10,14-tetraen-1-ol, 3,7,11,16-tetramethyl-, (E,E,E)/[2-cis-Geranylgeraniol]	1.07	41, 69, 81, 165	290.5	C20H34O	Diterpenoid	
1Retention time.

2Main peaks.

3Molecular weight/mol.

Fig. 1 (a) GC–MS chromatogram for Marjoram extract consisting of 10 bioactive compounds, (b) FTIR spectra of matrices designed with 3 techniques of FT, FT-FD, and FT-FD/CL, (c) morphology of matrices in a scale bar of 5 µm along with histograms of pores size range on the matrix surface.

These results almost are in agreement with the reports of Bhardwaj et al. (2019) and Udaya Prakash et al.39,40.

The study of matrix characterizations

The interactions between polymeric chains of PVA and PEG were confirmed by studying the FTIR spectra of the designed matrices with techniques of FT, FT-FD, and FT-FD/CL (Fig. 1b). Accordingly, the shift in the intensities of the peaks related to O–H and C–H groups respectively, in the range of 3350–3500 cm−1 and 2800–3030 cm−1, for the studied samples than PVA and PEG spectra demonstrated the formation of an inter/intramolecular hydrogen bond between PVA and PEG. The shift in the position of the peaks in the range of 1650–1750 cm−1 (C=O), 1350–1500 cm−1 (O–H), 1050–1200 cm−1 (O–C–O), and 750–950 cm−1 (C–C) between two groups of FT and FT-FD shows that the use of a combination of two techniques can improve the interactions of polymers. It was also found that the cross-linker led to the shift in position and intensity of all functional groups, especially in C–H and O–C–O, in a matrix designed with the FT-FD/CL technique. It may provide more effective interactions between polymer chains and form a more integrated network compared to matrices designed with one or two techniques.

In the following, the morphological properties of matrices (the pore size, porosity, and distribution of pores on the matrix surface) were analyzed by studying SEM images. As observed in Fig. 1c and Table 2, although a porous surface with smaller pores was observed for the matrix made with the FT method compared to two other matrices, structurally, this matrix is similar to 2-dimensional (2D) substrates that can be led to a decrease in cellular proliferation, due to the increase of pro-death signals1. It can be related to the slow evaporation of the solvent at room temperature. Using the combination (FT-FD) technique provided a 55% increase in the porosity of this matrix rather than the FT method. In the following, the results indicated that the crosslinker plays an important role in the decrease of the pore size and their more uniform distribution on the surface as well as the increase of the porosity. Indeed, the crosslinker can create an internal network connection and lead to an interpenetrating polymer network due to partial interlacing on the molecular scale41. It was also found that combining 3 methods of FT, FD, and CL can improve the uniformity of pore size on the surface of this matrix (FT-FD/CL) compared to FT and FT-FD matrices, such that the pore size range in FT-FD/CL technique-designed matrix was 20% and ~ 72% less than matrices designed with FT method and FT-FD technique, respectively.Table 2 The morphological and physicomechanical parameters of matrices.

	FT	FT-FD	FT-FD/CL	
Mean pore size (μm)	0.51 ± 0.50	1.21 ± 0.37	0.66 ± 0.09	
Pore size range (μm)	0.24–0.67	0.64–1.85	0.49–0.83	
Porosity (%)	60 ± 1.81	93 ± 1.38	96 ± 1.14	
Swelling % (1 h)	47.92 ± 0.31	80.61 ± 0.36	85.86a ± 0.47	
Swelling % (72 h)	85.46b ± 0.16	303.27 ± 1.04	334.83 ± 0.36	
Degradation (%) (after 14 days)	100.00 ± 0.98	84.83 ± 1.11	76.27 ± 2.10	
Average maximum tensile strength (MPa)	9.81 ± 1.3	26.10 ± 0.9	25.06 ± 1.1	
Average modulus (MPa)	1.0 ± 0.15	1.5 ± 0.12	2.0 ± 0.20	
Average elongation (%)	56.1 ± 1.7	97.6 ± 1.18	98.8 ± 0.9	
aThe mean difference is significant at the 0.05 level.

bThe mean difference is significant at the 0.001 level.

Indeed, in terms of the process, crosslinking led to chemical or physical bonds between polymer chains in the material before the freeze-thawing and freeze-drying processes, which subsequently can ensure a more consistent structure throughout the material due to helping to stabilize the polymer network. This pre-treatment can also ensure that the polymer chains are uniformly distributed and connected, and result in a uniform template for subsequent processes. In these circumstances, during the freezing process in cycles of the freeze-thawing process, ice crystals within the polymer network were more uniformly formed and led to a stronger network. In the following, the removal of water by sublimation, during freeze-drying, provided a porous structure. While freeze-thawing alone created a structure with irregular size and distribution of the pores due to variations in the freezing process. Likewise, although freeze-drying preserves the pore structure created during freeze-thawing, if the initial pore formation is irregular, the resulting pore structure will also be irregular (Fig. 1c). In this field, crosslinking as a pre-treatment method helps to mitigate this by providing a more uniform initial structure. Hence, the synergistic effect of these three methods includes pre-freeze-thawing stability, controlled pore formation during freeze-thawing, and maintaining the uniformity of the pores after freeze-drying. Such that each method addresses different aspects of pore formation and stabilization:Crosslinking provides a uniform and stable starting structure.

Freeze-thawing creates the pores within this uniform structure.

Freeze-drying preserves these pores without introducing irregularities.

Finally, this integrated approach resulted in a matrix with a significantly improved uniformity in pore size compared to using freeze-thawing alone and a combination of freeze-thawing and freeze-drying.

The study of the swelling behavior of all three matrices illustrated that the combination of two or more methods (FT-FD or FT-FD/CL) to fabricate the matrix can significantly increase the swelling (%) compared to the design with one method (FT) (Fig. 2a and Table 2). It can be due to two factors: (1) how the solvent is evaporated which is related to the uniformity of the substrate network; (2) the reduction of the simple hydrolyzed process of polymer chains in the swelling medium due to better networking of polymer chains subsequently reduction of the degradation rate of the substrate. In this field, although a minor difference was observed between the swelling (%) of matrices at first hours, in the following, the FT matrix indicated lower swelling (%) owing to the dispersion of pores on the surface and formation of the uniform network (p < 0.001). Such that this feature can result in slower absorption of wound infections on this matrix42.Fig. 2 (a) Swelling (%) (during 72 h), (b) water contact angle with matrix surface, (c) degradation rate (during 14 days), *p < 0.1 and **p < 0.05.

In the following, interactions between the matrix surface and water were assessed by the WCA test to understand how wound exudate was absorbed across the matrix (Fig. 2b). In this assay, the WCA values on matrices were calculated by depositing a water droplet on the scaffold surface and defining the angle of the tangent to the droplet surface at the point of contact with the scaffold and the scaffold surface itself, at the first moment of drop contact with the surface (by capturing an image of the droplet, and measuring the angle formed at the contact point using with specialized software that can accurately determine the angle based on the droplet’s shape such as a goniometer). In this regard, measurements were taken from both sides of the droplet to ensure accuracy (Right and Left Contact Angle: measurement of the angle on the right and left side of the droplet, respectively). The results indicated although all matrices possessed a contact angle of 55° < θ < 90° and a proper wettability on different points of their surface (almost equal wettability based on the area of the heat map, especially for FT-FD and FT-FD/CL), the FT-FD/CL technique-prepared matrix demonstrated better wettability along with more symmetric spreading of the drop onto the surface, due to the formation of more integrated network on the surface. The results of the swelling test and SEM images confirm this matter.

The degradation profile study of polymeric matrices also confirms when a matrix has a more uniform and integrated network, its degradation rate is decreased. Accordingly, although the degradation (%) of all three matrices increased after 14 days (Fig. 2c, Table 2), in the first 3 days lower degradation rate was assigned to the FT-FD/CL matrix (lower slope or gradient of lines). Hence, it seems that the combined techniques can play a crucial role in physicochemical properties such as the degradation behavior of substrates due to the creation of more complex structures compared to the single method. It was also found that the use of a crosslinker to design a matrix can significantly decrease the degradation rate compared with an FT-FD matrix. It can confirm that the enzymatic degradation first led to the breaking of the simple bonds on the polymer network, so that significant differences were observed in degradation (%) between FT-FD/CL with FT-FD and FT, after 14 days (p < 0.1 and p < 0.05, respectively).

The study of the mechanical properties is also illustrated when the crosslinker is added to the design process of the matrix, minor differences are observed in the maximum tensile strength and elongation between matrices designed with FT-FD/CL and FT-FD (Table 2). However, it was found FT-FD/CL-designed matrix in terms of the modulus value possesses a significant difference from the two other matrices (Fig. 3). It can be owing to the crosslinker function to form covalent bonds and/or a relatively short sequence of chemical bonds for more connection of chains of two polymers and a more cohesive network. Such that it can link linear molecules of polymer with each other and create a new structure formed multi-disciplinary or multi-modal. The results of SEM images and porosity confirm this data. Accordingly, the use of a crosslinker led to a 1.33-fold and twofold increase in the modulus of the FT-FD/CL matrix compared to FT-FD and FT matrices. Moreover, the combination of FT and FD methods played an important role in improving mechanical properties so that the elongation of the FT-FD matrix was about 74% more than the elongation of FT, along with a 1.5-fold increase in modulus.Fig. 3 Stress–strain curves of the designed matrices along with their mechanical behavior [maximum tensile strength, modulus, and elongation at break].

Based on the results, it seems that the matrix designed with FT-FD/CL technique can act as suitable substrate/dressing to cover exuding wounds due to physicochemical, mechanical and morphological properties. Hence, this matrix (FT-FD/CL) was selected to continue the study.

Characterizations of matrices loaded with herbal biomolecules

To assess the role of herbal biomolecules in cellular behavior and interactions, the Marjoram extracts with weight ratios of 1%, 3%, and 5% (wt%), were encapsulated into matrices designed with FT-FD/CL technique. Based on the results, increasing the concentration of extract led to an increase in encapsulation efficiency percentage (EE%) (Fig. 4a), although there was no significant difference between weight ratios of 3% and 5% (p > 0.001). This can be related to the capacity of the matrix for the encapsulation of herbal bio-actives so that an excessive increase in the weight percentage of extract had no significant effect on its EE%. Namely, weight ratios of > 3% led to wasting herbal extract in the medium and its dissolution in the solvent. When the weight ratio is less than 3%, the EE% decreases due to a lower concentration of herbal extract (HE).Fig. 4 (a) The encapsulation efficiency percentage (EE%) for matrices containing herbal bio-actives, (b) FTIR spectra for designed matrices, (c) the cumulative release (%) of the herbal extract from matrix network.

In this field, the analysis of the FTIR spectra of the HE and matrix with and without HE indicated that the spectrum of the matrix loaded with 1% HE was more similar to the FT-FD/CL matrix spectrum (HE-free matrix) (Fig. 4b, Table 3), while the increase in the weight ratio of HE led to the change in the intensity of O–H, C–H, and C=O functional groups (stretching vibrations). It can be explained by better interactions of herbal carbohydrates with the matrix when the polymer chains are networked by the crosslinker. Indeed, we believe that several types of interactions can be enhanced in a crosslinked polymer network. In this field, a more detailed discussion of these interactions has been referred which is related to hydrogen bonding, van der Waals forces, and covalent bonds. Generally, Herbal carbohydrates often contain multiple hydroxyl (–OH) groups. Hence, in a crosslinked polymer network, these hydroxyl groups can form hydrogen bonds with other components of the matrix, including the polymer chains and any other functional groups present. In this regard, crosslinking can promote the density and stability of these hydrogen bonds and lead to better overall interaction and integration of the carbohydrates within the matrix. Crosslinking can promote the density and stability of these hydrogen bonds and lead to better overall interaction and integration of the carbohydrates within the matrix. Moreover, van der Waals forces that occur weak intermolecular forces, when polymer chains are crosslinked, can optimize the physical proximity and orientation of the chains and allow herbal carbohydrates to interact more effectively. The crosslinked network provides a stable environment that can enhance these interactions. Moreover, in some cases, the crosslinker itself might react with functional groups on the herbal carbohydrates, and lead to the formation of covalent bonds. This creates a very stable interaction, effectively integrating the carbohydrates into the polymer matrix network. Hence, it seems that the benefits of crosslinking can directly affect the main parameters of a designed matrix such as structural stability (by creating a more stable and consistent environment for the herbal carbohydrates, enhancing their functional properties, and ensuring they remain evenly distributed within the matrix), mechanical properties, controlled release of herbal carbohydrates (by interactions within the network and consequently slower release along with sustained delivery of active ingredients), and swelling or absorption (by improving the interaction of the matrix with aqueous environments).Table 3 Absorption bands observed in the matrices containing herbal bio-actives.

Wavenumber (cm−1)	Functional groups	Groups (HE-loaded matrix)	
HE1%	HE3%	HE5%	
3500–3550	O–H	Almost the same as the spectrum of the FT-FD/CL matrix	The decrease in the intensity of peaks compared with the FT-FD/CL matrix	
2800–3030	C–H	The presence of a stronger peak than two other groups	The better interactions of polymer chains and herbal bio-actives, subsequently the shift in intensity and position of peaks	
1650–1750	C=O	The presence of a weak peak than two other groups	The change in intensity and position of peaks due to the increase in interactions of HE and matrix network during the crosslinking process	
1350–1500	O–H	The change in intensity and position of peaks than pure HE; however, the FTIR spectra, in this range are almost similar to each other for matrices loaded with 1% and 5% HE	
1050–1200	O–C–O	
750–950	C–C	

Moreover, it was found that the fingerprint region of the FTIR spectra, typically ranging from approximately 1400 to 500 cm−1, is almost similar to each other for matrices loaded with 1% and 5% HE. It can be related to a lower dosage of HE in the matrix network (for 1%) and a larger amount of the trapped HE for 5%. Indeed, when the amount of HE in the matrix network is low, fewer interactions occur between the HE and polymeric structure. Therefore, here, except for the O–C–O group (1000–1200 cm−1), no significant change was observed in the fingerprint region for the 1% HE-loaded matrix than the empty matrix. Accordingly, most interactions between 1% HE and matrix polymers are related to O–H and C–H functional groups which are identified in changes in the intensity of the peaks. Likewise, it seems that an increase in HE concentration (up to 5%) can act similarly to 1% HE. Indeed, a 5% HE-loaded matrix may possess a larger amount of HE that has not interacted with the matrix and led to faster release in aqueous medium. This resulted in the similarity of two 1% and 5% spectra in the mentioned region, especially for the O–C–O group.

Release profiles of HE and their kinetics

The controlled release of materials (drugs, biomolecules, bio-active, etc.) not only can maintain their effective dosages in the body, but also provide a well-defined behavior of its kinetics27. Therefore, to assess how to release herbal bio-actives from the matrix network, HE release profiles and their kinetics were studied over 72 h. Accordingly, a lower release percentage was related to the matrix containing 3% HE (93.21%), while two other matrices (1% and 5%) respectively possessed the release percentage of 100% and 98%, after 72 h. As observed in Fig. 4c, although release profiles of all three matrices possessed the same trend, the matrix containing 3% HE resulted in a more stable release of herbal bio-actives so that the HE release slowly and continuously continued until day 3. This can be related to better formation of possible bonds between polymeric chains and herbal biomolecules/biomacromolecules as well as the difference in EE (%) of each sample, resulting in a decrease in the matrix degradation (%) and subsequently lower release of HE for matrix loaded with 3% HE. Such that it can play an important role in reducing the growth of bacteria at the wound site. Indeed, we believe that the reduction in loading HE or its excessive increase within the matrix network affects the interaction of the matrix polymers and HE. In this field, the matrix with less loaded HE, due to the low amount of extract in its network, led to the release of all the HE in a short period of time. Similarly, the matrix with an excess amount of HE resulted in a greater release of HE not interacting with the matrix polymers, especially in the first hours.

In this regard, it was found that the matrix containing 5% HE, due to the larger amount of the trapped HE, possessed a higher release value, especially at hours 5–10 and 36–48; after 48 h, the release rate was decreased owing to the significant decrease of HE content in matrix network (Table 4). Notably, the increase in release (%) of herbal bio-actives between hours 5–10 and 36–48, for all three samples, can also be related to the degradation behavior of the matrix network which is caused by a two-stage degradation process in the first three days.Table 4 The amount of HE released from the polymer matrix after 72 h along with its kinetics models.

	HE (%)	Time interval (h)	
0–1	1–3	3–5	5–10	10–24	24–36	36–48	48–72	
HE (mg)	1	9.34	3.98	4.35	19.12	9.68	6.93	18.89	1.35	
3	24.69	10.98	16.32	38.37	33.57	41.25	69.24	15.52	
5	46.25	24.90	25.45	90.80	57.20	61.71	122.05	16.35	
	HE%	Kinetics models			
Parameters	Zero-order	First-order	Korsmeyer–Peppas	Higuchi	Hixson–Crowell			
Matrix	1	R2	0.88	0.71	0.97	0.96	0.57			
K	1.02	0.06	5.15	9.33	− 0.02			
n	–	–	0.52	–	–			
3	R2	0.93	0.80	0.98	0.97	0.63			
K	3.50	0.07	13.16	31.19	− 0.47			
n	–	–	0.57	–	–			
5	R2	0.91	0.77	0.98	0.97	0.60			
K	6.22	0.06	25.84	56.10	− 0.05			
n	–	–	0.56	–	–			
Significant values are given in bold.

In this field, the study of the mass transport mechanisms and the release kinetics of matrix-encapsulated bioactives indicated that the Korsmeyer–Peppas and Higuchi models possess the most consistent with this polymer system and the better mathematical models to describe the HE release profile (Table 4). It suggests that the primary mechanism of release is controlled by diffusion, and seems that the bioactive molecules have been uniformly dispersed in the matrix structure, which is related to the designed technique of matrix. Although, for the matrix containing 1% HE, the release mechanism is suggested in which the release rate is less dependent on the matrix-entrapped HE concentration such that there is relatively little correlation between HE release and matrix network (R2 in Hixson–Crowell model: 0.57). Likewise, the zero-order model also showed a good fit with HE released from the matrices containing 3% and %5 HE (R2 = 0.91 and R2 = 0.94, respectively), caused by the interaction between HE and the polymer network during cross-linking, due to greater amount of HE than matrix containing 1% HE.

Hence, the release rate in these two matrices was more stable and regular, compared to the matrix loaded with 1% HE, at a period of time. Moreover, it was found that the designed polymer substrate has a deviation from Fickian diffusion (0.43 < n43), and the release mechanism is more complex than simple diffusion. This non-Fickian or anomalous mechanism implies that both diffusion and polymer swelling contribute to the HE release. In this field, "n" values in the range of 0.43–0.85 can confirm this mechanism44.

Based on the results, the matrices containing 3% and 5% HE were selected to continue the study (biocompatibility, cellular adhesion, and cell proliferation) due to EE% > 85% and more stable release during 72 h.

The study of biological properties

After 5 h of cell culture on matrices, the SEM images of substrates along with HSF cells were observed to study how cells adhere and distribute on the surface (Fig. 5a). The comparison of the matrices containing HE with the control group (without HE) indicated that HE due to existing N-Acetyl-l-proline (a natural product and protein derivatives) plays an important role in improving interactions of cell–matrix. Moreover, the results illustrated that the matrix containing 3% possessed higher adhesion along with a more uniform distribution of cells and could decrease cell colony formation on the surface. It can be related to the controlled release of herbal biomolecules (such as carbohydrate polymers: Linalool, Epi-alpha-Cadinol, diterpenoid) and biological signals created by them. However, when HE content increased to more than 3%, the cell adhesion reduced and distribution occurred at the edges of the matrix surface. It can be due to the higher content of matrix-entrapped HE so that the greater content is released into the culture medium when the matrix network is swelled and leads to culture medium turbidity and cytotoxicity45.Fig. 5 (a) SEM images of designed matrices along with fibroblasts (cellular adhesion) with a scale bar of 50 µm, (b) biocompatibility of HSF cells cultured on matrices (during 72 h), (c) the inverted microscope images (200× magnification) to study the proliferation and distribution of fibroblast cells as well as their collagenization on the matrix after 72 h, (d) the effect of matrix-encapsulated bio-actives on the growth inhibition of E.coli and S.aureus bacteria using the disc diffusion method.

Biocompatibility of HSF cells on the studied matrices (with and without HE) and control group was assessed by the MTT assay at 24, 48, and 72 h (Fig. 5b). The comparison of cell viability (%) for all studied groups indicated that the matrix itself is compatible with cells, so that, matrices with and without HE led to the promotion of cell growth, during 72 h. However, for the 5% HE-loaded matrix, this increasing trend was reported up to 48 h and then the cell growth reduced. This can be attributed to culture medium turbidity due to the higher release of HE. In the following, it was found that content of 3% HE possessed more compatibility compared to other groups, so that, there was no significant difference in cell viability (%) between this matrix and the control group (p > 0.01). Notably, given that porous scaffolds/matrices have a high surface area that can adsorb the formazan dye and complicate the quantification of formazan crystals formed by viable cells, formazan adsorption on such substrates can lead to false negative results since not all the formazan-produced by viable cells is accurately measured. Moreover, DMSO used to extract formazan crystals from cells, might not completely remove formazan adsorbed onto the matrix and be led to false positive cytotoxicity results. Hence, to overcome these challenges, here, calibration along with validation with imaging techniques (SEM and inverted microscope) was carried out to visually confirm cell viability and distribution within the matrix. In this field, calibration involves creating a calibration curve that accounts for the unique dye absorption characteristics of each matrix. To this end, a series of known concentrations of formazan dye solutions were prepared, and a calibration curve was plotted as concentration vs absorbance. Each matrix was then incubated with the prepared formazan dye solutions for a fixed period, allowing the dye to interact with the matrices as it would in an actual assay. After incubation, the formazan dye was extracted from the matrices using the DMSO, and the amount of dye was quantified by a spectrophotometer and calibration curve to determine absorbance and concentration.

In the following, the HSF cells were cultured on the matrices, and after 72 h observed under an inverted microscope (200X magnification). The matrix without HE was also used as the control group to study the effect of the HE on proliferation of cells. Based on the results, the matrix containing 3% HE led to a higher proliferation of fibroblast cells along with a more uniform cell distribution on the matrix surface, after 72 h. This creates better conditions to synthesize collagen from fibroblasts such that, on the 3rd day, stronger collagen fibers were observed on the matrix surface containing 3% HE than the 5% HE (Fig. 5c). It can be due to protein and carbohydrate derivatives in the HE (respectively, N-Acetyl-l-proline and sesquiterpenoids like Epi-alpha-Cadinol) that improve the functions of fibroblast cells to synthesize collagen fibers46.

Hence, it can be concluded that biomolecules extracted from Marjoram play a crucial role in the timely production of collagen fibers and subsequently the regeneration of the native ECM. However, matrices containing 3% of these herbal bio-actives can provide the best cellular function (cell adhesion, proliferation, and distribution). Hence, the matrices containing 3% HE were selected to assess the role of bioactive molecules in inhibiting the growth of bacteria.

To assess the antibacterial activities of Marjoram-extracted biomolecules on the E.coli and S.aureus bacteria, the disc diffusion assay was carried out with the matrix containing 3% HE (i.e. 6 μg) (M+), empty matrix (M−), blank sterilized discs (negative control: C−), and discs containing Gentamicin at a concentration of 10 μg (positive control: C+). Based on the results, the creation of the clear zone of 23.1 mm and 26.8 mm, for both plates containing S.aureus and E.coli, respectively, around M+ indicated more antimicrobial activity than C+ (Fig. 5(d)). Such that a 19% and 20% increase in inhibiting the growth of S.aureus and E.coli was observed on the M + , compared to C+. Based on GC–MS analysis, the 9,10-Secocholesta-5,7,10(19)-triene-3,24,25-triol, (3.beta.,5Z,7E), and catechol the main carbohydrate polymers can play an important role in this property (antibacterial). The reports also confirm that these polymers, along with other carbohydrates such as terpenoids (Linalool), as well as alkaloids can disrupt the growth of bacteria47,48.

Hence, it seems that this designed matrix not only can act as an alternative substrate or promising dressing to manage infected wounds and decrease bacterial drug-resistance, but also increase the repairing process of damaged skin by promoting the proliferation of fibroblast cells and synthesizing collagen fibers due to carbohydrate and protein derivatives existed in the matrix network.

Discussion

This research aimed to study the effect of the combination of techniques, used to design polymeric substrates, on the properties of the engineered matrix, as well as to achieve a multiple-function matrix (in terms of antibacterial, antioxidant, and repairing properties) via encapsulating herbal bio-actives (such as carbohydrate polymers and proteins) in matrix network for supporting cellular functions and helping these bio-actives to avoid undesirable interactions. To this end, the PVA-PEG matrices with the equal volume ratio of both polymers were separately designed through 3 techniques of the freeze–thaw process (FT, three cycles), the combination of both methods of freeze-drying and freeze-thawing (FT-FD), and a ternary technique included freeze-drying, freeze-thawing, and cross-linking (FT-FD/CL). Based on the results, the use of the ternary technique led to better physicochemical properties compared with two other methods. Such that the use of crosslinker in this method improved the uniformity of pore size on the surface of the matrix, and led to a 20% and ~ 72% decrease in pore size range than matrices designed with the FT method and DT-FD technique, respectively. The matrix fabricated with this technique also provided a proper swelling behavior along with a more uniform network rather, a more controlled degradation rate (due to the lower nomber of the simple bonds on the polymer network1), and a 1.33-fold and twofold increase in modulus compared with FT-FD and FT matrices. In this regard, several reports have indicated that crosslinking polymer chains in designed scaffolds can also lead to the regulation of mechanical properties49. In these conditions, mechanical properties can affect cellular responses and their interactions with the substrate (such as cell attachment, proliferation, or migration) due to controlling or modulating the elastic modulus, according to the intended applications of the substrate50. Such substrates can also maintain their structural stabilities by integrating the substrate network51. The studies of Izadyari Aghmiuni et al., on combining techniques to improve the mentioned properties can also confirm our findings1. According to this research team., controlling these features can result in better and faster absorption of wound infections on the matrix42. The results of interactions between the matrix surface and water supported other reports3. Accordingly, the FT-FD/CL technique-prepared matrix demonstrated better wettability along with more symmetric spreading of the drop onto the surface, due to the formation of more integrated network on the surface. This feature can provide more uniform and stable absorption of wound infections3. The reportes also demonstrate when a matrix has a more uniform and integrated network, its degradation rate is decreased, namely, the increase in the days in which cells need a suitable substrate for proliferation27,52.

Given that the modulus of native skin is usually between 2 and 20 MPa, depending on its position in the body53, and the comparison of results in this study, the FT-FD/CL matrix was selected to continue the study.

In the following, encapsulation of bio-actives extracted from Origanum Majorana L. (Marjoram) in the matrix network designed with ternary technique indicated that the increase in the concentration of extract (from 1 to 5%) led to an increase in encapsulation efficiency percentage (EE%), although there was no significant difference between weight ratios of 3% and 5% due to capacity of the matrix network for the encapsulation process. Moreover, the results of FT-IR demonstrated that the encapsulation process with 3% herbal extract can cause a more cohesive network by better bonding between functional groups of bio-actives and polymer chains of the matrix. The study of the mass transport mechanisms and the release kinetics of matrix-encapsulated bioactives also illustrated that the Korsmeyer–Peppas and Higuchi models possess the most consistent with this polymer system and the better mathematical models to describe the release profile of herbal carbohydrate polymers and proteins. However, the designed polymer matrix with this technique possessed a deviation from Fickian diffusion (0.43 < n)43, and so the herbal extract release happened by diffusion and swelling. According to Saurí et al., it suggests that the primary mechanism of release is controlled by diffusion. Hence, the bioactive molecules have been uniformly dispersed in the matrix structure and the designed network possesses a homogeneous form along with the same surface thickness54, which is related to the designed technique of matrix. In the following, polymer swelling contribute to the HE release. "n" values in the range of 0.43–0.85 can confirm this mechanism44. Hence, the polymer matrix swells upon contact with the release medium, allowing more HE to diffuse out. As the matrix swells and eventually erodes, the HE release is facilitated further, integrating both diffusion and erosion processes.

The study of the biological properties of this matrix also indicated that carbohydrates and protein derivatives loaded in the matrix network play an important role in adhering to and distributing on human skin fibroblasts on the matrix surface. Based on the reports, N-Acetyl-L-proline is an acetylated form of L-proline which plays an important role in treating skin ulcers. Indeed, the fibroblast cells need a group of amino acids such as proline and its derivatives to create the main structure of collagen fibers46. The presence of sesquiterpenoids, alkaloids, phenols, and monoterpenoids in the extract of this herb can also confirm the effects of antioxidant, anti-inflammatory, and wound healing50,51. Based on the reports, these properties can play an important role in the reduction of wound infection and inflammation, the proliferation rate of fibroblasts, and skin regeneration3,52. In this matter, the matrix containing 3% herbal extract possessed higher adhesion along with a more uniform distribution of cells owing to the controlled release of herbal biomolecules (such as sesquiterpenoids and N-Acetyl-l-proline) and biological signals created by them. Based on the research, the controlled content of bioactives can reduce culture medium turbidity and cytotoxicity45. It can confirm a higher proliferation of fibroblasts along with collagen synthesis and formation of stronger fibers in this matrix compared to the 5% herbal extract.

The studies on the bioactive compounds of Marjoram extract also illustrate that this herb-obtained extract possesses antimicrobial potency. In this field, Chirumamilla et al. (2022) and Mohammed et al. stated that 9,10-Secocholesta-5,7,10(19)-triene-3,24,25-triol, (3.beta.,5Z,7E) can act as an antibacterial agent in the many medicinal herbs59,60. Catechol also is another bioactive in this matter that possesses intrinsic antimicrobial properties and can create an antibacterial efficiency of more than 99%61. Hence, it seems that Marjoram extract due to having a high percentage of these two bio-actives can be a promising alternative for antibiotics and decrease bacterial drug-resistance.

Accordingly, the study of the antibacterial activity of Marjoram extract on the E.coli and S.aureus bacteria, vs discs containing gentamicin (at a concentration of 10 μg) as positive control indicated that the mentioned matrix creates a clear zone of 23.1 mm and 26.8 mm, for both plate containing S. aureus and E. coli, respectively than positive control (namely, a 19% and 20% increase in inhibiting the growth of S. aureus and E. coli compared to positive control). As anticipated, such the results are related to the main carbohydrates of this extract (9,10-Secocholesta-5,7,10(19)-triene-3,24,25-triol, (3.beta.,5Z,7E), and catechol), which can play an important role in this property (antibacterial). The reports of Shareef et al. and Altameme et al. also confirm that these carbohydrates, along with other biomolecules like terpenoids (Linalool), and alkaloids can disrupt the growth of bacteria47,48.

Accordingly, our result indicates that such a matrix not only can act as a promising dressing to control infected wounds due to its desirable properties, but also decreases bacterial drug-resistance, and increases the repairing process of wounds by promoting the proliferation of fibroblasts and stimulating collagen fiber synthesis due to carbohydrate polymers and protein derivatives loaded in the matrix network.

Materials

Polyethylene glycol (PEG, Mn: 4000), polyvinyl alcohol (PVA, MW: 8500–124,000, hydrolyzed 99%), borax (hydrated or anhydrous borate of sodium), tetrazolium salt 3-(4, 5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT, 2.5 mg/ml), dimethyl sulfoxide (DMSO), and ethanol 96% were purchased from Sigma (Sigma-Aldrich Inc., St. Louis, MO, USA). Origanum Majorana L. (Marjoram) was obtained from Glass Garden Company (Tehran, Iran). Dulbecco’s modified eagle’s medium–low glucose (DMEM-LG), fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were purchased from Gibco (Massachusetts, USA). Human skin fibroblast cells (HSF cells) with NCBI Code C646 were also purchased from the Iranian Pasture Institute cell bank (Tehran, Iran). Discs containing gentamicin at a concentration of 10 μg and blank sterilized discs were respectively obtained from Farazmed Company and Padtan Teb Company. Gram-positive and negative bacteria [i.e. Staphylococcus aureus (S. aureus, PTCC No: 1917) and Escherichia coli (E-coli, PTCC No: 1276), respectively] were purchased from the Iranian Research Organization for Science and Technology (IROST)-Persian Type Culture Collection.

Methods

Preparation of herbal extract

Origanum Majorana L. (Marjoram) leaves (Glass Garden Company, Tehran, Iran) were first washed with double-distilled water (ddH2O) for removal of possible dust, then dried in the dark until (25 °C). Afterward, leaves were ground and the aqueous extract was prepared by macerating 20 g of herb powder in 200 ml of ddH2O for (50 °C, 48 h). Finally, the obtained extract was filtered, frozen (− 20 °C, 24h), and then dried by the freeze-dryer under a vacuum at – 80 °C (Martin Christ, Gamma 2–16 LSC), for 48h. The lyophilized extract was stored at 4–8 °C, for analysis of its active ingredients and other tests.

Notably, Origanum Majorana L. plant samples were of commercial origin and were obtained from Glass Garden Company (Tehran, Iran). This plant is cultivated at appropriate yield and collected, stored and controlled by the respectable company.

Preparation of PVA-PEG matrix with 3 techniques

PVA (10 g) and PEG (5 g) were separately dissolved in ddH2O to prepare the 10 wt% (w/v) PVA and 5 wt% PEG solutions, respectively (25 °C, 24 h). In the following, the PVA-PEG hybrid solution with a volume ratio of 1:1 (v/v) was prepared by adding PEG solution into PVA solution (70 °C, 6 h).

Afterward, the mentioned solution was cast into wells of 24-well culture plates, frozen at – 20 °C (24 h), and then thawed at 25 °C (4 h). The freeze–thaw process was repeated for three cycles. After thawing in cycle 3, the solvent was evaporated at 25–30 °C for 48 h, and prepared matrices, with the abbreviated name of FT, were saved at 4–8 °C to perform future tests.

In the following, to assess the effect of manufacturing technique on the physicochemical and mechanical properties of the matrix, two other substrates were designed. To this end, the other plate containing PVA-PEG solution was lyophilized for 48h, after thawing in cycle 3. The obtained matrices in this step were called FT-FD.

In the other group, before the freeze–thaw process, one cross-linking process was carried out by adding 1 ml of borax solution (2 wt%) to 10 ml of PVA-PEG polymer solution. This solution was stirred at 50 °C for 3 h, then plated, and dried with a freeze–thaw process (3 cycles) and freeze-drying. These matrices were called FT-FD/CL, as well.

Finally, the morphologic, mechanical, and physicochemical properties of designed matrices were assessed to select a better matrix and continue the study.

Encapsulation of herbal extract into the selected polymer matrix

The herbal dried powder was dissolved in 10 ml of PVA-PEG polymer solution (1:1 v/v) at 50 °C for 2 h, to prepare 1%, 3%, and 5%w/v HE-polymer solutions. Afterward, 1 ml of borax solution (2 wt%) was separately added to each HE-polymer solution. The solutions were then mixed at 50 °C for an additional 2h and sonicated by a bath sonicator at 35 °C (10 min). Finally, HE-polymer solutions were cast into wells of 24-well culture plates to prepare the matrix with the selected technique.

The study of characterizations herbal extract and polymer matrix

Phytochemical analysis

The bio-macromolecules or bioactive ingredients determination of marjoram extract was performed with a phytochemical assessment of herbal dried extract by Gas Chromatography-Mass Spectrometry (GC–MS: 6890, coupled with Mass Spectrometry, N-5973 Model, Agilent). The equipment included an HP5-MS column (high: 15 m, inner diameter: 0.25 mm) and the injection method of splitless (injection volume:1 µl; extraction solvent: chloroform). Moreover, the detector temperature was set to 280 °C (retention time: 7 min) and the oven temperature was programmed from 60 to 250 °C (ramp: 10 °C/min). After dissolving the sample into the solvent, the process was run in the range of 30–290 m/z and the data were compared to Willey and NIST libraries.

Characterization of polymer matrices with/without HE

Physicochemical, morphological, and mechanical properties

Fourier Transform Infrared Spectroscopy (FTIR) analysis: the interactions between polymeric chains and surface chemistry in all designed matrices were studied by FTIR spectrometer (ALPHA, Bruker, Germany) at the region of 400–4000 cm−1 and the resolution of 4 cm−1.

Morphology analysis: the morphology of designed matrices was analyzed with a scanning electron microscope (SEM, XL30 ESEM, Philips, Germany) at an accelerating voltage of 20kV. Finally, the mean pore size along with the pore size range was determined by Clemex vision software 3.5 by choosing 20 pores on each sample, randomly.

Porosity study: the porosity (%) of matrices was determined by a liquid displacement method4. In this method, dry matrices were separately weighed (Wd) then immersed in 96% ethanol for 30 min and reweighed. Finally, the porosity (%) was measured with Eq. (1). (n = 3).1 Porosity(%)=Ww-WdDe×Vs×100Wd:theweightofdrymatrixWd:theweightofswollenmatrixVs:thevolumeofswollenmatrixDe:densityofethanolat25∘C

Water-uptake assay: the swelling (%) of the studied matrices was measured by immersing each substrate (diameter: 1.2 mm, thickness: 4.5–5 mm) in PBS solution (pH = 7.2, 37 °C). To this end, the dry weight (Wd) of matrices was determined; they were then immersed in PBS for specified time intervals (1, 3, 7, 14, 24, 48, and 72 h), and their weight was recorded as Wt (wet weight), at the end of each period. Finally, the swelling (%) was measured by the following equation2.2 Swelling%=Ww-WdWd×100

Water contact angle (WCA) measurement: to this end, the 2 μl ddH2O water drops as reference fluid were placed on the surface of matrices (10 × 10 mm, the minimum of 3 droplets) and their WCAs were calculated with a semi-automatic contact angle goniometer (CAG-10, JIKAN)62. The images were recorded by a digital camera.

Degradation rate: for this study, a medium containing 13 µg/ml lysozyme and 1 ml PBS were used as wound exudate-like medium, and matrices with the initial weight of Wi were immersed into the mentioned medium (37 °C, 14 days)4. Notably, the sampling was carried out on days 1, 3, 6, 10, and 14. To this end, matrices were taken out from the degradation medium and reweighed, after freeze-drying (Wt). Afterward, the degradation (%) of substrates was measured by Eq. (3).3 Degradation%=Wi-WtWi×100

Mechanical properties: in this method, 5 parallel tests were carried out, according to ASTM D3039 standard3, and matrices in size of 10 × 5 × 2 mm3 were prepared. In the following, the mechanical stability and elasticity behavior of each matrix were analyzed by the tensile tester instrument (Zwick-Roell, 1446) with a strain rate of 2 mm/min.

Encapsulation efficiency (EE%): The 50 mg/ml HE solution was prepared by dissolving dried extract powder in PVA-PEG solution and then diluted (50,000, 25,000, 12,500, 6250, 3125, 1562.5, 781.25, 390.625, 195.312, and 97.656 ppm). Afterward, the absorbance value of solutions was obtained at a wavelength of 525 nm by a spectrophotometer (Shimadzu UV-1601, Kyoto, Japan), to draw a standard curve [concentration (ppm)-optical density (OD)]. The HE-encapsulated matrices (with the weight ratio of 1, 3, 5 wt%) were then immersed in PBS for 20 min. In the following, the samples were centrifuged and the concentration of the matrix-released extract was calculated by studying the absorbance of the supernatants obtained by centrifuging at 525 nm and then placing it in the standard curve equation. Notably, to remove the effect of the PVA-PEG for studying the absorbance value of HE, the PBS containing empty matrix (PVA-PEG) was selected as a control sample. Finally, the EE (%) was measured by Eq. (4)27.4 EE%=Ctotal-CentrappedCtotal×100

The HE release behavior and its kinetics study: this test was carried out to analyze the matrix-released HE during 72 h. To this end, matrices containing loaded HE and empty matrix (as a control sample) were dialyzed through a dialysis sac (Mw: 14,000 Da) at 37 °C for 72 h, while immersed in PBS (20 ml). Notably, sampling intervals were considered 1, 3, 5, 10, 24, 48, and 72 h. At specified time intervals, PBS solution (2 ml) was replaced with fresh PBS (2 ml); the HE concentrations in the solutions removed from the system were determined by measuring the absorbance at 525 nm and the standard curve equation. Finally, the cumulative release (%) of the HE was determined by Eq. (5). In the following, release data for all three matrics containing HE was studied by kinetics models to analyze the release kinetics [Eq. (6)]63.5 CumulativeRelease%=Wentrapeddrug(t)Wencapsulateddrug×100

6 Higuchi:Mt=KH·t0.5Zero - order:Mt=C0+KZ·tFirst - order:Mt=C0·expKF·t2.303Korsmeyer - Peppas:MtM∞=K×tnHixson - Crowell:M013-Mt13=KHC·tKH:the Higuchi dissolution constantKZand KF:release constantMtM∞:fraction of drug released at time tK:the rate constant,n:the release exponentM0:the initial amount of medicineKHC:the constant incorporating the surface-volume relationMt:the amount of drug released at the time tC0:the initial amount of drug

Notably, the matrices that possessed better physicochemical and mechanical properties along with EE% of more than 85% and more stable release were selected to continue the study.

Biological properties of herbal extract-loaded matrix

Cell adhesion: Matrices with and without herbal extract (HE) were sterilized by UV (45 min) and placed in a 24-well plate. Afterward, HSF cells (1 × 106 cells/well) were cultured on these matrices supplemented with DMEM-LG medium and FBS (15%). After 5 h of incubation, the matrices were observed under SEM to assess the cellular adhesion and distribution on the matrix.

MTT assay: This test was carried out to study the biocompatibility of designed matrices [empty matrix (PVA-PEG, as control group) and matrices containing HE]. To this end, HSF cells were cultured on the sterilized matrices (96-well plate, 1 × 104 cells/well) and incubated in a medium containing DMEM-LG and 15% FBS, for 24, 48, and 72 h. The media were replaced with 100 µl of the fresh medium along with 20 µl of MTT, at the end of each time; then cells were incubated for 4 h. Finally, after studying the absorbance value at 570 nm, the cell viability (%) was calculated by Eq. (7)3.7 Cell viability(%)=ODsampleODcontrol×100

The proliferation rate of skin cells: HSF cell growth on the designed matrices (with and without HE) was studied for 72 h. At the end of this time, the matrices containing skin cells were collected and observed under an inverted microscope (Nikon Corporation, Japan) with a magnification of 200×.

Notably, the matrices that possessed better biological properties were selected to continue the study and perform antimicrobial tests.

Antimicrobial properties of HE-loaded matrix

To determine of antibacterial activity of the HE-loaded matrix, Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria as the test strains were initially incubated in luria broth (LB) medium at 37 °C (24 h). In the following, The antibacterial activity of the samples was assessed by the disc diffusion method64. To this end, the blank sterilized discs and discs containing Gentamicin at a concentration of 10 μg were respectively used as negative and positive control groups. Afterward, both discs along with the HE-loaded matrix and empty matrix (as a control group) were placed on the surface of the plates covered with Mueller Hinton Agar (MHA) medium, which was incubated and cultured with the mentioned bacteria, separately, (i.e. each plate consisted of 4 discs). The plates were then incubated at 37 °C for 24 h; finally, the diameter of the inhibition zone around the discs was measured.

Statistical analysis

Except for the mechanical test (n = 5), Tests were carried out in triplicate (n = 3). All data were reported as mean ± standard deviation (SD). Statistical analyses were also performed using one-way ANOVA (LSD), two-way ANOVA ((Tukey), and t-test by Graph Pad Prism 8 Demo software, with P-values of < 0.001, < 0.01, < 0.05, and < 0.1.

Acknowledgements

We would like to express our deep appreciation to Dr. Azadeh Izadyari Aghmiuni for all her guidance.

Author contributions

Sh. M. and M.Kh conceived the idea and developed the study framework. A.Gh created the figures. M.Kh. supervised the project. All authors carried out the tests, analyzed the results and contributed to writing the manuscript.

Data availability

The datasets generated and/or analyzed during the current study are not publicly available due [REASON WHY DATA ARE NOT PUBLIC] but are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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