Rifampicin-Loaded PLGA/Alginate-Grafted pNVCL-Based Nanoparticles for Wound Healing

Abstract

The topical therapy with rifampicin (RF)-based formulations is beneficial for treating postoperative wound infections and to accelerate healing. Despite recent research highlighting the antibiotic’s significant anti-inflammatory properties, limited topical wound healing products are currently available. The present study aimed to prove that the newly synthesized nanoparticles based on grafted alginate and poly(N-vinylcaprolactam) (pNVCL) and poly-lactic-co-glycolic acid (PLGA) contribute to the healing process of a wound. The methods used were at first the synthesis of the copolymer of alginate and pNVCL via grafting from technique and radical polymerization followed by water-in-oil-in water (W/O/W) emulsification; as oil phase PLGA dissolved in dichloromethane (DCM) was used. The formed nanoparticles were than characterized. The loaded RF was determined to be 160 µg/mL for a 20 mg formulation and within a four-hour time frame approximately 10% of the total loaded amount was released. The inhibitory concentrations (IC50) were 192.1 µg/mL for the nanoparticle, 208.8 µg/mL for pure rifampicin, and 718.1 µg/mL for the rifampicin-loaded nanoparticles. Considering the double role rifampicin was used for, the result was considered satisfactory in the way that these formulations could be used predominantly for postoperative wound irrigation in order to avoid infections and to improve healing.

1. Introduction

Wound care is becoming increasingly complex with the advent of advanced wound technology. However, the core of effective wound care can be distilled into five fundamental principles: wound assessment, wound cleansing, timely dressing changes, selection of appropriate dressings, and the use of antibiotics, where necessary. Surgical procedures treat a great variety of diseases or injuries when drug treatment is not effective. To maintain adequate wound management, the materials used in surgery (adhesives, sealants, hemostatic agents, wound dressings, absorbent sponges, and sutures) must meet a series of specific criteria [1]. They must be non-toxic, biocompatible, and support the cell proliferation necessary for tissue regeneration. At the same time, they must have good mechanical and physical properties to ensure durability.

Most postoperative wounds heal within an estimated time frame. The healing process is carried out in overlapping stages of hemostasis, inflammation, granulation, and epithelization [2]. This process can be accelerated by applying pharmaceutical products recommended for improving healing, disinfection, and skin regeneration.

There is a series of biocompatible and biodegradable biopolymers such as hyaluronic acid (HA), alginate (AgA), chitosan (Cs), collagen (Col), and silk fibroin (SF) which can help healing wounds faster. This is due to their similarity to macromolecules easily recognized by the human body. In the literature, several studies are based on developing new materials for wound repair, such as hydrogel dressings, gelatin sponges, bioactive glass, etc. Most of these studies based on biopolymers present a series of limitations and as a result, few products obtained from natural polymers have reached the stage of exploitation. That is why finding strategies to combine biopolymers would be of particular interest for wound healing. Naturally sourced biopolymers, which are biological macromolecules metabolized within the body, unlike synthetic polymers, do not cause chronic inflammatory or immunological reactions or toxicity due to their similarity to the extracellular matrix. A proper modification of structure or processing conditions towards functional biopolymers can alleviate some of their drawback such as poor solubility, lower producibility, poor mechanical properties, etc., but highlighting their advantages over synthetic polymers, including a well-defined and more intricate structure, biocompatibility, functionality, degradability, and renewability [3,4].

Among the biopolymers, alginic acid, a polysaccharide extracted from brown seaweeds, with distinct physical properties that make it valuable as a rheology modifier in various applications such as food products, paper consumables, printing inks, and biomaterials for medical and pharmaceutical preparations [5,6,7,8,9].

According to this research, sodium alginate can help treat wounds more effectively and reduce pain and inflammation. Sodium alginate is a polysaccharide formed by α-L-Mannuronic acid (M unit) and β-D-Guluronic acid (G unit) connected through a 1,4-glucosidic bond. This units are arranged irregularly as MM, GM/MG, and GG regions. Sodium alginate has a weak mechanical resistance and in order to overcome this problem, alginate has been chemically modified by reactions in the hydroxyl or carboxyl groups. One of the important strategies to improve and to design the characteristics of biopolymers is to modify them according to the desired features. More particularly, modification strategies have been employed to obtain a self-assembly behavior up to temperature or other external stimuli such as pH, glucose level, magnetic field, etc. A good strategy for biopolymer modification consists of chemical modification via graft copolymerization with synthetic monomers [10,11].

Among the monomers that can be used to induce thermoresponsive properties on the side chains of a biopolymer via grafting, there are the ones that exhibit lower critical solution temperatures (LCST) close to physiological conditions such as poly(N-isopropylacrylamide and poly(N-vinylcaprolactam) [12,13,14]. Ward et al. [15] reviewed the use of polymers that present an LCST and their applications in biomedical field.

Poly(N-vinylcaprolactam) (pNVCL) is a water-soluble polymer at room temperature that possesses a lower critical solution temperature (LCST) in the physiological range (32–38 °C), which extended its applications in the pharmaceutical industry. It is biocompatible, non-toxic, and the second most popular thermoresponsive polymer after poly(N-isopropylacrylamide). The biomedical applications of NVCL include improved drug delivery, tissue engineering, and wound healing [16]. Modification of alginate with a biocompatible monomer such as N-vinylcaprolactam can lead to functional biocompatible materials that import both the characteristics of alginate and NVCL.

The present research deals with the study of the copolymer potential of alginate with N-vinylcaprolactam synthesized via graft copolymerization and formulated as nanoparticles by coating with a poly(D, L-lactide-co-glycolide (PLGA) shell. The characteristics of the AgA-pNVCL matrix will be explored as sealant materials for surgical sutures. The motivation for formulating the copolymer with a shell of PLGA is to achieve a smaller burst release and prolonged cumulative release of rifampicin, a hydrophobic antibiotic with good antibacterial and anti-inflammatory properties [17,18]. PLGA offers the advantage of controlling the release of the incorporated drug along with having a very small particle size, and being at the same time a biocompatible material [19].

2. Materials and Methods

2.1. Chemicals

Alginic acid sodium salt (AgA) (CAS 9005-38-3) with a viscosity between 15 and 25 cP, N-vinylcaprolactam (NVCL) CAS 2235-00-9, the initiator system represented by ammonium persulfate (APS)—hydrogen peroxide—50% solution, and the poly(D,L-lactide-co-glycolide) 50:50 (PLGA) used to form the nano/microparticles were purchased from Sigma Aldrich, Saint Louis, MO, USA. The Milli-Q water and 0.5 wt.% acetic acid glacial solution were used as solvents. The product was dialyzed using a dialysis bag with a cut-off of 3.5 kDa produced by Scienova GmbH (Jena, Germany).

Rifampicin (RIF) with a 99% level of purity was purchased from S.C. Antibiotice S.A. (Iasi, Romania). All other chemicals were analytical grade. The reagents used in cytotoxicity tests and wound healing assays were obtained from Sigma-Aldrich (Hamburg, Germany) and used as received.

2.2. Preparation of Grafted Biopolymer

The grafted biopolymer i.e., alginate grafted with poli(N-vinylcaprolactam) (pNVCL) was synthesized by using an initiator system comprised of ammonium persulphate (APS, Sigma Aldrich, Saint Louis, MO, USA) and 50% hydrogen peroxide solution under nitrogen flow within a ratio of 0.1% against the monomer (NVCL).

2.3. Preparation of PLGA-Coated Alginate Grafted with pNVCL

The prepared biopolymeric matrix was used further to synthesize nano/microparticles containing and not containing rifampicin (the selected drug model for postsurgery wound healing).

An amount of prepared biopolymer was dissolved in water within a concentration of 0.1 wt.% and then rifampicin was added to the polymeric solution to ensure a good blending and formation of electrostatic bonds with the hydrophilic groups of the biopolymer. The theoretical amount used for the rifampicin loading within the matrix was 0.5 wt.% against the polymer amount. The mixture was stirred for a couple of hours then lyophilized.

The biopolymeric matrix without rifampicin was dissolved in twice-distilled water within a concentration of 0.1 wt.%. The oil phase solution was prepared by dissolving 0.1 g PLGA (50/50) in dichloromethane (DCM) and Span 80 was added to ensure good stability of the emulsion. The oil phase was probe sonicated for 10 min then the water phase was added dropwise under strong stirring under vortex to prepare the water-in oil emulsion. The first emulsion was again probe sonicated and then added dropwise in the second water phase consisting of 5 wt.% polyvinyl alcohol low molecular weight, Tween 80, and salt to give the final water-in-oil-in water (W/O/W) dispersion. The solvent was evaporated at room temperature, and the system was kept under stirring overnight. The final dispersion was then centrifugated at 15,000 rpm under a temperature of 5 °C to collect the nano/microparticles of poly(N-vinylcaprolactam) grafted with alginate (AgA-g-pNVCL) coated with PLGA.

The formation of particles containing rifampicin was performed similarly except for the fact that the matrix loaded with rifampicin was dissolved in the water. The drug content was determined using high-performance liquid chromatography (HPLC) and it was reported in our previous work [4] as being 160 µg/mL in a 20 mg formulated sample.

2.4. Fourier Transform Infrared Spectroscopy (FTIR) Analysis

The structure was confirmed via FT-IR spectra of the copolymer films which were recorded with a Perkin-Elmer Spectrum-100 ATR-FTIR instrument (Shelton, CT, USA) scanning in the range of 600 to 4000 cm⁻¹.

2.5. Particle Size and Size Distribution of Nanoparticles

The particle size and particle size distributions of nanoparticles were determined by using a NanoZS model (Malvern Instruments, Grovewood, UK) zetasizer instrument. Each sample was prepared by dispersing nanoparticle suspension in MilliQ water (5000 times dilution for each sample) and added to a cuvette to be analyzed in triplicate (n = 3).

2.6. Differential Scanning Calorimetry (DSC)

The thermal properties of the samples were determined using a Perkin Elmer Di-amond DSC instrument (Grovewood Rd, Misterton, UK).

Approximately 5 mg of dried substances (RIF, NP, NP + RIF, and AgA-g-pNVCL) were placed in aluminum holders and heated between 20 and 250 °C, under a nitrogen atmosphere, at a heating rate of 10 °C/min.

2.7. Scanning Electron Microscopy (SEM)

The cross-sections of the lyophilized formulations were visualized via SEM studies using a Carl Zeiss 300 Sigma VP model equipped with Gemini Optical Technology (Los Angeles, CA, USA).

2.8. In Vitro Release Study of Formulations

The in vitro release studies of RF from lyophilized nanoparticles were conducted using the dialysis bag diffusion technique. The dried samples were loaded into the dialysis bag (cellulose membrane, molecular weight of 12,000 to 14,000 Da), and hermetically sealed via standard closure (Repligen, Waltham, MA, USA). The dialysis tube was immersed into 100 mL phosphate buffer (pH = 7) at 37 °C with a magnetic stirrer stirring at 300 rpm. At predetermined time intervals, 3 mL of the dissolution medium was withdrawn from the receptor compartment and replaced with an equal volume of fresh medium. The sample solution was assayed via HPLC for drug content. All the experiments were carried out in triplicate.

The release data obtained via the dialysis bag diffusion technique were fitted using the Korsmeyer-Peppas mathematical model (Equation (1)) [20].

$${\displaystyle \frac{M_{\mathrm{t}}}{M_{\infty }}}=k_{\mathrm{r} }{·t}^{{\mathrm{n}}_{\mathrm{r}}}$$

(1)

where $M_t$ is the drug mass released at time t; $M_{\infty }$ is the total drug mass released at time t and at equilibrium; $k_r$ is a rate constant dependent on the characteristics of the drug loaded system; and $n_r$ is the release exponent which defines the release mechanism. The value of the release exponent, $n_r$, suggests the nature of the release mechanism. If n = 0.5, we have a Fickian diffusion mechanism of the drug from the matrix, whereas if n > 0.5 an anomalous transport or non-Fickian behavior, and n < 0.5 a quasi-Fickian diffusion occurs.

2.9. Cytotoxicity Studies

For the determination of the cytotoxicity and IC50 values of pure active substances and formulations, MTT assay was performed. The purpose of the tests was to evaluate the cytotoxic effect of different concentrations of active substances and formulations on 3T3-L1 cell lines; 3T3-L1 cells are derived from 3T3 cells and have a fibroblast-like morphology, but, under appropriate conditions, they differentiate into an adipocyte-like phenotype. The following solutions were prepared and used during the experiments:

Complete Medium (DMEM: F12 + 10%FBS + 1%Pen-Strep): 45 mL of DMEM: F12 (obtained from Gibco Cat No: 11320033), 5 mL of fetal bovine serum (FBS) (obtained from Gibco Cat No: 10500064), and 500 µL of Pen-Strep (obtained from Gibco Cat No: 15070063) were brought in a sterile flask in a laminar flow cabinet. In the experiments, the required quantity was freshly prepared.

A total of 171 µM Triton-X solution (positive control): 235 µL of 4263 mM stock Triton-X (obtained from Sigma-Aldrich Cat No: X100-100ML) solution was added to 765 µL medium. The concentration of the solution thus obtained was 1000 mM. Then, 1 µL of the 1000 mM intermediate stock was taken and added to 999 µL medium and a second 1000 µM intermediate stock was obtained. Finally, 171 μL of 1000 μM intermediate stock was taken and added to 829 μL medium, and 171 μM Triton-X solution was obtained. Finally, it was sterilized via filtration through a 0.22 μm membrane filter.

Sterile PBS: two of the PBS tablets (from Thermo Fisher Scientific, New York, NY, USA. Cat No: P4417-100TAB) were dissolved in 400 mL water and then sterilized in an autoclave device.

A total of 70% EtOH: this was prepared with the action of 96% EtOH to be used in routine cleaning processes. For this, it was prepared by mixing 365 mL of 96% EtOH with 135 mL of distilled water for 500 mL of solution.

Rifampicin Solutions: 16 mg of rifampicin was weighed and dissolved in 100 µL of DMSO, and the concentration of the resulting solution was found to be 160,000 µg/mL. By taking 5 μL of this solution and adding it to 995 μL medium, a solution with a concentration of 800 μg/mL (DMSO 0.5%) was obtained, and solutions with concentrations of 400, 200, 100, 50, 25, and 12.5 μg/mL were obtained with 2-fold serial dilutions.

Nanoparticles Solution: 20 mg of nanoparticles were weighed and dissolved in 100 µL of DMSO. The concentration of the obtained solution was obtained as 200,000 μg/mL. A total of 5 μL of this solution was added to 995 μL of medium and the concentration of the resulting solution was found to be 1000 μg/mL (0.5% DMSO). Based on this solution, solutions with concentrations of 500, 250, 125, 62.5, and 31.25 μg/mL were obtained with 2-fold serial dilutions.

Rifampicin-Loaded Nanoparticles Solution: 20 mg rifampicin-loaded nanoparticle was weighed and dissolved in 100 µL of DMSO. The concentration of the obtained solution was obtained as 200,000 μg/mL. A total of 5 μL of this solution was added to 995 μL of medium and the concentration of the resulting solution was found to be 1000 μg/mL (0.5% DMSO). Based on this solution, solutions with concentrations of 500, 250, 125, 62.5, and 31.25 μg/mL were obtained with 2-fold serial dilutions.

2.10. Scratch Assay

The in vitro scratch assay is an advantageous, accurate, and economical method to study cell migration [21,22]. The first step of the method involves the creation of a “scratch” on monolayer cells. In order to close the scratch, the cells on the edge will move toward its center until new cell-to-cell contacts are reestablished. Mouse fibroblast cells (cell line 3T3-L1) were used for this test. In the experiment, the cells were plated either in 10% FBS-containing cell culture medium or 1% FBS-containing cell culture medium. First, we compared the relevancy/appropriateness of these two different FBS concentrations. Second, the same test subjects in the former assay, rifampicin, nanoparticles, and rifampicin-loaded nanoparticles, were separately tested in the scratch assay in the presence of medium control to establish whether they altered the scratch closure rate compared to the control and to each other. This was achieved by capturing the image of the scratch in the beginning (0th h), and in the following 12th, 24th, and 30th h time points. Microscopic scratch images were used to calculate the closure of the gaps in time by using Image J software v1.54. The potential statistical significances between the percentages of different test subjects at the same time-points, and between the percentages of the same test subject at the different time-points were tested via two-way, and one-way ANOVA, respectively. The final statistical significance between the two test subjects in terms of wound-healing efficiency was tested again via one-way ANOVA. In this evaluation, Tukey’s multiple comparisons test was used to determine which groups were significantly different to each other.

3. Results and Discussions

3.1. Matrix Characterization

3.1.1. FTIR Spectroscopy

Spectroscopy tests were performed to detect either the appearance of new chemical bonds or the modification of existing ones, which can be attributed to possible interactions between grafted alginate (AgA-g-pNVCL) and polylactide-glycolide (PLGA) to form NPs compared with their pure constituents (AgA; PLGA and AgA-g-pNVCL). The obtained spectra are represented in Figure 1.

The purpose of the spectra analysis was to confirm the structural identity of the grafted copolymer and the nanoparticle formation of PLGA/AgA-g-pNVCL. The grafted copolymer’s structural identity has been reported in detail in our publication [4]. By combining the grafted copolymer of alginate with PLGA as a coating shell, it was expected that the spectra of the final nanoparticle would consist of both the specific vibration bands from the alginate-based copolymer and PLGA. The formation of new vibration bands and/or shifts to lower or higher peaks within the spectrum range were expected to occur. The spectra of PLGA contained a strong peak at 1094 cm⁻¹ specific for C–O–C stretching, as well as a peak at 1740 cm⁻¹ for C=O stretching. By analyzing the spectrum of the NP, the specific peak of C-O-C from PLGA was found at 1086 cm⁻¹ even though alginate-based matrix also contained a peak at 1020 cm⁻¹ thanks to the presence of -C-O- of the glucoside moieties indicating the presence of PLGA moieties. The peaks of CH, CH₂ stretching vibrations from 2900 to 2800 cm⁻¹, were observed to be more intense signaling than the conjugate formed between the PLGA and alginate matrix. Moreover, the peak at 3282 cm⁻¹ of OH groups increased in intensity and was wider, indicating the formation of the conjugate and the interactions between the two polymers via H-bonding.

3.1.2. Particle Size Analysis

The particle size of the formed nanoparticles was measured (Figure 2) and from the size distribution and intensity it was determined that the formed particles have nanometric size. It was found that the unloaded particles had a narrow distribution with an average size of 103 nm while the ones loaded with rifampicin (RF) showed a wider distribution but a unimodal peak with an average size distribution of 201 nm; this outcome confirmed the loading within the polymeric matrix.

3.1.3. DSC Analysis

Thermal properties of the polymeric nanoparticles with and without RF were tested to check the presence of the drug within the matrix and to determine the thermal stability and homogeneity of the prepared formulations. Figure 3 presents the DSC thermograms of pure RF and polymeric nanoparticles with and without RF.

The DSC thermogram of the pure drug (Figure 3) showed an endothermic peak at 189 °C corresponding to the melting point of RF. The thermal decomposition process of RF occurred in two stages: the first thermal decomposition occurred around 200 °C and the second one at 257 °C. The thermal events observed on the DSC curve are consistent with the reported literature [23]. On the DSC of NP + RF, we detected endothermic peaks around 189 °C and 250 °C. Liu et al. [24] also found out that RF had an exothermic crystallization peak at 213 °C and a degradation peak at 249 °C. The peak from 250 °C almost disappeared and overlapped with the degradation peak of the polymeric matrix, indicating the fact that Rf was encapsulated. When analyzing the thermogram of nanoparticles of AgA-g-pNVCL coated with PLGA (i.e., NPs), there were observed small peaks at 50 °C being assigned to the glass transition of PLGA and an endothermic peak around 217 °C being explained by the beginning of polymer degradation. Comparable results were found by Rao et al. [25]. They found for sodium alginate a broad endothermic peak around 100 °C and a sharp exothermic peak at 250 °C, the latter one being connected with its thermal decomposition. The copolymer AgA-g-pNVCL showed three thermal events at 80 °C, at 218 °C, and 250 °C. It seems that PLGA coating induced a less thermal stability of the molecule as the peak from 217 °C indicated the beginning of the nanoparticles’ degradation, especially the PLGA-attached moieties. A further degradation changed the macromolecular chains of AgA-g-PNVCL. The outcome from DSC analysis not only proved the complexity of the polymeric matrix but also confirmed the presence of RF and its thermal characteristics.

3.1.4. SEM Observations

SEM was used to study the surface morphology of the polymeric nanoparticles with and without RF together with the pure constituents (lyophilized hydrogel of AgA-g-pNVCL and RF). The SEM images show that RF particles were smooth and rod-like (Figure 4d,e) whereas polymeric nanoparticles had spherical shapes and were irregular due to breakage after the lyophilization process (Figure 4b,c,f).

Even though the particles were collapsed due to the lyophilization, their spherical shape can be distinguished and in some marked places voids with rod-like shapes inside indicate the success of the encapsulation. The results from SEM were in line with DSC results confirming the expected outcome. Similar outcomes were obtained also by Snejdrova et al. [26] and by Rai et al. [27].

3.2. In Vitro Release Profile

Prior to evaluating the in vitro release behavior of rifampicin, the amount of drug trapped within the polymeric chains was determined via HPLC assay, and it was found out that 20 mg polymeric micro/nanoparticles consisted of 160 µg/mL of rifampicin as was reported within the previous study [4]. This outcome is useful for further studies such as cytotoxicity assessment, in vitro release, and wound closure capacities. Figure 5 describes the in vitro release profile of rifampicin within 7 h from the beginning of the experiment.

As shown in Figure 5, in the first 60 min from the administration of the formulation, there was an amount of about 1% of the total loaded rifampicin. As observed from the in vitro release profile, over the time interval release, the release capacity was studied and the low amount of rifampicin was determined via the HPLC method. More specifically, within the first hour from the administration, approximately 1% was released, followed by 1% more being further determined after another interval of 2 h. The outcome regarding the low release of RF from various matrices was previously reported. Bibire et al. [28] found out an amount of released RF within 4 h of 5.98 ppm while Parmar et al. [29] reported a delivered amount of RF of 10% within 10 h in the case of encapsulated RF within liquid-crystalline folate nanoparticles. Interestingly, they reported a self-assembly behavior to control the loading capacity of rifampicin and additionally its delivery profile. They found out that by decreasing the particle size of the particles encapsulating rifampicin, the loaded amount was lower. However, the low amount of rifampicin detected within the matrix did not show significant differences concerning its cytotoxicity activity or release profile. An additional factor reported which seemed to influence the release behavior was that rifampicin, when encapsulated within nanoparticles, was bound via its functional groups creating internal crosslinks which may have slowed down the release. Within the present study, rifampicin was encapsulated within the matrix of grafted alginate with poly(N-vinylcaprolactam) and protected by a shell of PLGA. The size of formed particles was determined as being around 100 without RF and 200 nm when RF was loaded, as presented within Figure 2. The explanation on the low efficacy regarding the loading amount but also the capacity of the nanoparticle to release the trapped RF was found to be on the base of created crosslinks between RF and polymeric matrix. Another reason was also the drug’s low solubility. Henwood et al. [30] reported the low solubility of rifampicin in water and buffer system up to the content of the amorphous moiety from the raw drug and the careful selection of the drug as a raw material is needed. The rifampicin used within the study was provided from a local medicine company and according to the DSC analysis (thermogram from Figure 3) a form II of rifampicin was determined so the polymorphism could influence the solubility in aqueous phases [31].

The drug release mechanism was assessed via the theoretical kinetic evaluation by applying the semi-empiric equation (1) of Peppas et al. [20] of the release profile within the first hour, which showed an abnormal non-Fickian mechanism (case II) and a matrix-dependent mechanism considering the values of the release rate of 0.958 min⁻¹ (R² = 0.9911) and n value of 0.7502 indicating that the release mechanism was case II which involved swelling and erosion of the matrix structure. Similar results for in vitro delivery of rifampicin were found by Hiremath et al. [32].

3.3. Cytotoxicity Studies

Cytotoxicity is a crucial factor in wound dressing fabrication. More importantly, it can support skin tissue restoration. The first step of the assessment of the cytotoxicity of rifampicin was to calculate the IC50 values. The cytotoxicity data about rifampicin were limited. In a study by Saravanan et al., the IC50 value of rifampicin was found to be 87 µg/mL. For the assessment, human monocyte cells (THP-1) were used; the cells were incubated with rifampicin for 72 h and then measured for their cytotoxicity with Alamar blue [33]. Within the present study, the IC50 value for rifampicin was found to be 208.8 µg/mL for 3T3-L1 cell line. Cells were incubated with rifampicin for 24 h and cytotoxicity was measured via MTT assay. The differences between the two tests were primarily that the cell type and then also incubation time obviously resulted in different IC50 values. As the proposed formulations are intended to be used for external use on wounds, the most relevant cell type to be tested was decided to be 3T3-L1. Figure 6 describes the cytotoxicity results and the IC50 values calculated for all studied samples.

IC (inhibitory concentration) curves are dose/response curves used to determine the specific drug concentration required to reduce the viable cell population by a certain percentage compared to cells grown without drug exposure. The IC50, or inhibitory concentration at 50%, represents the concentration of a compound needed to inhibit a biological process by half. IC50 values are crucial in cytotoxicity assessments as they indicate the amount of a drug required to achieve this inhibition. The IC curves demonstrate changes in the population due to increased cell death or decreased cell proliferation [34]. Determining the IC50 value has significant implications beyond merely inhibiting cell growth or increasing cell death. In cancer treatment, using certain drugs at the IC50 concentration can reduce tumor growth by half. If IC50 is identified at a lower concentration, it indicates that the drug will be effective at lower doses, thereby reducing systemic toxicity in patients. Utilizing the IC50 concentration can effectively kill cancer cells and halt their growth while minimizing the toxic effects on healthy cells in the body. The values found for the prepared formulations were 192.1 µg/mL for the nanoparticle, 208.8 µg/mL for pure rifampicin, and the nanoparticle loaded with rifampicin had a IC50 value of 718 µg/mL. As observed, the IC50 value of the conjugate (i.e., nanoparticle of AgA-g-pNVCL/PLGA loaded with RIF) had a higher value than its constituents itself. Within the literature this phenomenon is defined as “mixture effect” [35]. From a toxicological point of view, the study of the combined effects of substances is crucial because organisms are often exposed to complex mixtures of chemicals rather than single substances. When the combined effect of chemicals is greater than the sum of their individual effects, a synergetic effect of the mixture is reported. It seems that it is also the case of the present formulation; the loaded nanoparticle with rifampicin presented a value of IC50 three times higher than its constituents. The reason behind this phenomenon needs to be investigated in further investigations up to the concentration dose and time of exposure.

3.4. Cell Scratch Assay (Wound Healing)

A cell scratch assay was used to assess the wound-healing potential of the nanoparticles-based formulations. Lower concentrations than the MTT assay were tested to avoid cytotoxic effects. During a period of 30 h, wound closure was monitored at 0-, 12-, 24- and 30-h intervals. A relative wound area (%) was determined. Figure 7 describes the development of scratches in the presence of tested materials.

As shown within Figure 7, the microscope images were taken at 0, 12, 24, 30 h and % wound area was determined by using Image J software, a Java-based image processing program developed at the National Institutes of Health and the Laboratory for Optical and Computational Instrumentation. The percentage of wound area data were normalized via proportioning according to hour 0. The statistical significance of the difference between different groups at the same hours of the data showing % wound area against time were shown via two-way ANOVA test and the statistical significance of the difference between different hours of the same groups was shown via one-way ANOVA test. The ANOVA test was used to investigate whether the difference between the groups in % wound healing calculated according to the formula specified in the procedure was statistically significant. Tukey’s multiple comparisons test was used to determine between which groups there was a significant difference. Figure 8 described the closure activity in time and the wound area was determined after each time interval. As observed, the activity of the rifampicin loaded within the nanoparticles was more obvious after 24 h from the application of the nanoparticle’s solution containing RF.

When the closure activity is compared between the tested samples (Figure 9) it can be seen that the activities of the distinct solutions of nanoparticles and rifampicin are much higher than the complex formulation of Rf loaded inside the nanoparticles.

A total closure effect of 60% was determined in the case of the RF-loaded nanoparticles while separate solutions of rifampicin and nanoparticles showed around 80% (Figure 10).

Previously reported results [28] demonstrated the capacity to proliferate the cells when hydrogels composed of the similar polymeric carrier were loaded with rifampicin. The scratch assay was performed to test the migration and proliferation of Normal Human Dermal Fibroblasts (NHDF) within a maximum time frame of 72 h. The RF-loaded hydrogels demonstrated a synergetic effect between polymeric matrix and drug, the wound closure activity being more evident after 24 h from exposure, while RF itself showed a decreased activity after 24 h. This outcome may be explained by the advantage that RF was trapped inside the polymeric matrix and the closure activity had a sustained characteristic. However, many studies found a poor closure activity of rifampicin-based polymeric systems. Most of the studies associating the activity of rifampicin with an antibacterial one [36] in topical treatment of surgical wounds and rifampicin-based systems may be recommended for wound site irrigation [37] as an antiseptic agent rather than purely for wound healing. As reported, wound irrigation supposes the usage of the material as an antiseptic solution to sanitize the postoperative wound and to remove the elements that affect the healing process. When performed correctly, it helps removing any extra cellular debris, surface bacteria, wound exudate, dressing residue, and residual topical agents [38].

4. Conclusions

In the specialized literature, there are not enough formulations that can be used as postoperative wound sealants. Starting from this fact, the authors of this study considered that it is useful to develop a nanoparticulate formulation based on alginate grafted with poly(N-vinylcaprolactam) and coated with PLGA loaded with rifampicin (RF). Rifampicin (RF) is an antimicrobial drug which manages and treats diverse mycobacterial infections and gram-positive bacterial infections present at wounds after surgeries. However, the topical therapy with rifampicin-based formulations seemed to be efficient not only for treating infections but also to contribute to the healing process. The newly synthesized nanoparticles based on grafted alginate and poly(N-vinylcaprolactam) and PLGA (AgA-g-PNVCL/PLGA) contribute to the healing process of a wound. The methods used were at first the synthesis of the copolymer of alginate and pNVCL via grafting from technique and radical polymerization followed by W/O/W emulsification by use as an oil phase PLGA dissolved in dichloromethane (DCM). The formed nanoparticles were characterized by means of their particle size showing a size of 200 nm for those particles loaded with RF; FT-IR spectroscopy proved the chemical identity of the copolymer and the conjugate. The presence of bands of each pure substance within the new product together with the broadening of OH- and COOH-showing interactions led to the substance identification. Morphological observation performed via SEM revealed the presence of the RF particles within the polymeric network; the thermal characteristics determined via DSC indicated the presence of a form II RF which is less susceptible to dissolution, explaining its low drug loading and in vitro release profile. It seemed that due to the low solubility of the drug together with the susceptibility to be strongly attached to the polymeric matrix via H bonds, a low released amount of RF was determined as approximatively 10% within 4 h from the administration. However, the analysis of the kinetics of the release profile showed that the delivery process was based more on a non-Fickian mechanism up to the erosion of the polymeric matrix. The cytotoxicity of the tested formulations via MMT assay (pure RF, unloaded nanoparticles, and RF-loaded nanoparticles) revealed the non-toxic character of the formulations, and the inhibitory concentrations (IC50) were determined as 192.1 µg/mL for the nanoparticles, 208.8 µg/mL for pure rifampicin, and 718.1 µg/mL for the nanoparticles loaded with rifampicin. Wound closure capacity was assessed via in vitro scratch assay and a total of 60% closure for the prepared RF-loaded nanoparticles was found. Considering the double role rifampicin was used for, the result was considered satisfactory in the way that these formulations could be used more with the wound irrigation postsurgery to avoid infections and to contribute to the healing.

The main limitation of the study consisted of not having enough case studies regarding the usage of rifampicin entrapped within polymeric nanoparticles to compare the results and to make a fair opinion. However, the obtained in vitro results for the delivery capacity and the wound healing ability will be studied further in an in vivo environment.