**1. Introduction**

Acute myocardial infarction (AMI) remains the primary cause of death worldwide. AMI occurs when blood flow to the coronary arteries is blocked, leading to necrosis, tissue remodeling and fibrosis that can cause progressive cardiac damage and heart failure [1]. A major problem in the recovery of AMI patients is the low proliferation percentage for heart cell regeneration [2]. For effective AMI treatment, it is necessary to prevent tissue remodeling, attenuate scar formation and promote cell proliferation and regeneration to replace damaged tissue. For this reason, tissue engineering emerges as a therapeutic alternative for the development of functional tissue that can be used for the regeneration of affected tissues. In cardiac tissue engineering, attempts are made to combine cells with biocompatible materials to generate a three-dimensional construct that can restore

**Citation:** Beltran-Vargas, N.E.; Peña-Mercado, E.; Sánchez-Gómez, C.; Garcia-Lorenzana, M.; Ruiz, J.-C.; Arroyo-Maya, I.; Huerta-Yepez, S.; Campos-Terán, J. Sodium Alginate/Chitosan Scaffolds for Cardiac Tissue Engineering: The Influence of Its Three-Dimensional Material Preparation and the Use of Gold Nanoparticles. *Polymers* **2022**, *14*, 3233. https://doi.org/10.3390/ polym14163233

Academic Editors: Antonia Ressler and Inga Urlic

Received: 16 June 2022 Accepted: 12 July 2022 Published: 9 August 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the damaged myocardium [3]. The ideal scaffolds for cardiac repair must have high porosity and biocompatibility as well as be permeable to nutrients and metabolic waste. In addition, they must have an adjustable degradation time to minimize the formation of fibrous capsules and promote incorporation into the host tissue to avoid a chronic inflammatory response [4]. Likewise, they must recreate the microenvironment, structure and three-dimensional organization of the myocardium; improve cell survival and promote cell adhesion, differentiation and maturation. In addition, the scaffolds must allow for vascularization to ensure the flow of oxygen and nutrients to the cells and favor the transmission of electrical and mechanical impulses for proper host-tissue coupling [5].

Natural polysaccharides such as alginate and chitosan have been widely used for tissue engineering because of their biocompatibility, biodegradability and structural similarity to the extracellular matrix components [6–10]. Chitosan, a polycationic polymer (the presence of positively charged amine groups), promotes the cell adhesion, proliferation and differentiation of different cell types [7,11]. Alginate, a polyanionic polymer (presence of negatively charged carboxyl groups), promotes regeneration and favors the vascularization and restoration of electrical conductivity and cell growth [6]. The chemical natures of alginate and chitosan polymers make them sensitive to pH (pKa = 3.4–3.7 and pKa = 6.3, respectively) of aqueous media, swelling in opposite directions [5].

Previous reports have shown that alginate/chitosan scaffolds improve mechanical and biological properties [8,9,12], in addition to promoting growth and maintaining cardiac cell viability [10,13–16]. They have a gradual degradation and favor cell retention, survival and migration to the affected area, allowing the formation of blood vessels, with reduction in fibrosis and hypertrophy area [14,17].

The addition or functionalization of biomaterials with metallic nanomaterials can improve the physical and electrical properties of the scaffolds [18–21]. Recently, the need and importance of designing and developing new cardiac patches based on conductive biomaterials for possible therapeutic application has been reported [22–24]. The properties of gold nanoparticles (AuNp) promote cardiac cell growth and contractility [25–28]. Biomaterials functionalized with AuNp favor cardiomyocyte elongation and alignment, with an increased expression of cardiac proteins and improved cell contraction [29–33]; however, the use of AuNp has not been studied in alginate/chitosan scaffolds.

Although there have been good results in the use of alginate/chitosan scaffolds for cardiomyocyte growth, it is interesting to test the use of star-type AuNp and tubular AuNp+Alg in this type of scaffold to favor cell adhesion and growth and increase cardiac protein expression, identifying the best conditions for scaffold fabrication for therapeutic purposes. Thus, the aim of this work was to compare different methods of fabrication of alginate/chitosan scaffolds to improve swelling percentages, permeability, porosity and degradation rate and to evaluate the effect of the functionalization of the proposed scaffolds with AuNp and AuNp+Alg on cardiomyocyte growth.

### **2. Materials and Methods**

### *2.1. Preparation of Alginate–Chitosan Scaffolds*

Sodium alginate (Sigma Aldrich, Mannheim, Germany, # 9005-38-3) and chitosan (medium molecular weight, Sigma Aldrich, Mannheim, Germany, # 448877) (0.75–1.25% *w*/*v*) powders were mixed and dissolved in ultrapure water (Milli-Q system, 18.2 M-cm) and acetic acid (1% *w*/*v*, Sigma Aldrich, Mannheim, Germany, #1005706). The pH was adjusted to be between 5 and 6 to favor the interaction between the biomaterials. The resulting mixture/solution was ready for undergoing the corresponding experimental method (1 to 4), see Figure 1. In all cases, the scaffold solutions placed within the 24-well box were frozen at −20 ◦C for 12 h and subsequently freeze-dried in a lyophilizer (Labconco Corporation, Kansas City, MO, USA) for 8 h at −49 ◦C under vacuum with a pressure of 0.100 mBar.

**Figure 1.** Four scaffold preparation methods: Method 1 (without sonication), Method 2 (with sonication), Method 3 (longer crosslinking time) and Method 4 (with sonication and longer crosslinking time). **Figure 1.** Four scaffold preparation methods: Method 1 (without sonication), Method 2 (with sonication), Method 3 (longer crosslinking time) and Method 4 (with sonication and longer crosslinking time).

Method 1 (without sonication). An alginate–chitosan solution of 0.5 mL was deposited in each well of a 24-well box. Then it was frozen, lyophilized and crosslinked with 1 mL of 1% *w*/*v* calcium gluconate in water for 15 min. Washes were performed with ultrapure water and again frozen at −20 °C for 12 h. Finally, they were lyophilized again for Method 1 (without sonication). An alginate–chitosan solution of 0.5 mL was deposited in each well of a 24-well box. Then it was frozen, lyophilized and crosslinked with 1 mL of 1% *w*/*v* calcium gluconate in water for 15 min. Washes were performed with ultrapure water and again frozen at −20 ◦C for 12 h. Finally, they were lyophilized again for 8 h.

8 h. Method 2 (with sonication). The alginate–chitosan solution was sonicated (Sonics Vibra Cell VCX 750, Newtown, CT, USA) for 5 min at 20 kHz and 750 W. Subsequently, the alginate–chitosan solution was deposited in each well of a 24-well box. After freezing and Method 2 (with sonication). The alginate–chitosan solution was sonicated (Sonics Vibra Cell VCX 750, Newtown, CT, USA) for 5 min at 20 kHz and 750 W. Subsequently, the alginate–chitosan solution was deposited in each well of a 24-well box. After freezing and lyophilization, crosslinking was performed with 1% calcium gluconate for 15 min. Subsequently, they were washed with ultrapure water, frozen and lyophilized for 8 h.

lyophilization, crosslinking was performed with 1% calcium gluconate for 15 min. Subsequently, they were washed with ultrapure water, frozen and lyophilized for 8 h. Method 3 (longer crosslinking time). The alginate–chitosan solution was deposited in each well of a 24-well box. After freezing and lyophilization, crosslinking was per-Method 3 (longer crosslinking time). The alginate–chitosan solution was deposited in each well of a 24-well box. After freezing and lyophilization, crosslinking was performed with 1% calcium gluconate for 30 min. Subsequently, washes were performed with ultrapure water, frozen and freeze-dried for 8 h.

formed with 1% calcium gluconate for 30 min. Subsequently, washes were performed with ultrapure water, frozen and freeze-dried for 8 h. Method 4 (with sonication and longer crosslinking time). The alginate–chitosan solution was sonicated at 20 kHz and 750 W. The solution was then deposited in the 24-well box, frozen and lyophilized. Crosslinking was performed for 30 min with 1% calcium gluconate. Subsequently, washes were performed with ultrapure water, frozen and freeze-Method 4 (with sonication and longer crosslinking time). The alginate–chitosan solution was sonicated at 20 kHz and 750 W. The solution was then deposited in the 24-well box, frozen and lyophilized. Crosslinking was performed for 30 min with 1% calcium gluconate. Subsequently, washes were performed with ultrapure water, frozen and freeze-dried for 8 h.

### dried for 8 h. *2.2. Synthesis of Metallic Nanoparticles*

*2.2. Synthesis of Metallic Nanoparticles*  For the functionalization of alginate–chitosan scaffolds, metallic nanoparticles (Np) were prepared using a novel methodology with modifiable topography. This synthesis method is based on the preparation of citrate-stabilized gold nanoparticles [34], which are attached to the surface of a polymeric core consisting of Poly(D,L-lactide-co-glycolide) acid (PLGA) and stabilized with the copolymer Pluronic F-127. After the preparation of For the functionalization of alginate–chitosan scaffolds, metallic nanoparticles (Np) were prepared using a novel methodology with modifiable topography. This synthesis method is based on the preparation of citrate-stabilized gold nanoparticles [34], which are attached to the surface of a polymeric core consisting of Poly(D,L-lactide-co-glycolide) acid (PLGA) and stabilized with the copolymer Pluronic F-127. After the preparation of these metallic nanoprecursors, the growth of a gold shell is promoted on their surfaces, finally generating gold nanoparticles (AuNp).

these metallic nanoprecursors, the growth of a gold shell is promoted on their surfaces, finally generating gold nanoparticles (AuNp). The AuNp synthesis process is described below: first, the synthesis of PLGA (Sigma Aldrich, 102229183, Mannheim, Germany) cores was carried out. For this, a 10% *w*/*v* solution of PLGA in acetone (C3H6O) was prepared (Sigma Aldrich, #67641, Mannheim, The AuNp synthesis process is described below: first, the synthesis of PLGA (Sigma Aldrich, 102229183, Mannheim, Germany) cores was carried out. For this, a 10% *w*/*v* solution of PLGA in acetone (C3H6O) was prepared (Sigma Aldrich, #67641, Mannheim, Germany), which was drip-added to 38 mL of a 1% *w*/*v* aqueous solution of the triblock copolymer Pluronic F-127 (C3H6O-C2H4O)x (Sigma Aldrich, #9003116, Mannheim, Ger-

many), under stirring (250 rpm) at a constant temperature of 10 ◦C. Subsequently, it was homogenized for 10 min in an ice bath with a sonic tip of 750 W, 20 kHz frequency and 40% amplitude and left in agitation for 4 h. At this stage of the AuNp synthesis, a modification to the PLGA nuclei preparation method was also carried out, which consisted of coating some of them with a sodium alginate solution. Briefly, 20 mL of PLGA nuclei was mixed with 1 mL of 1% (*w*/*v*) sodium alginate and the mixture was stirred (250 rpm) for 4 h at room temperature. PLGA nuclei were purified through three cycles of centrifugation (9000 rpm, 18 ◦C) and resuspension. The supernatant was discarded, and the pellets were dispersed in 30 mL of ultrapurified water. Subsequent to the PLGA cores preparation, gold nanoseeds were synthesized. These were obtained by mixing the following solutions: 0.125 mL of 0.01 M chloroauric acid (HAuCl4) (Sigma Aldrich, 1001642619, Mannheim, Germany), 10 mL of 0.0256 M trisodium citrate (Na3C6H5O7) (Sigma Aldrich, 1001851140, Mannheim, Germany) and 0.3 mL of 0.1 M sodium borohydride (NaBH4, Sigma Aldrich, 1002918750, Mannheim, Germany). The latter was added at a temperature of 4 ◦C. The formation of gold nanoprecursors was carried out by mixing both the PLGA cores and alginate-modified PLGA cores (PLGA+Alg) with the gold nanoseeds in a 1:1 ratio, under constant stirring at 250 rpm for 24 h, followed by centrifugation (7000 rpm, 18 ◦C) for 20 min. The supernatant was discarded and the pellets were dispersed in 30 mL of ultrapurified water. The gold nanoprecursors were sonicated for 10 min to avoid particle aggregation. Once the PLGA cores were coated with the gold nanoseeds, the metal shell was assembled on their surface by mixing 2.025 mL of AuNp nanoprecursors with 45 mL of a solution containing 3.69 mM potassium carbonate (K2CO3, Sigma Aldrich, 1002055627, Mannheim, Germany) and 0.025 M gold (III) chloride trihydrate (HAuCl4-3H2O) (Sigma Aldrich, 1001642619, Mannheim, Germany). The latter was carried out under stirring (250 rpm) at room temperature. After 5 min, 225 µL of fresh 0.5 M ascorbic acid was added to the mixture.

The above was performed for both PLGA cores and sodium-alginate-modified cores (PLGA+Alg). The functionalization of the alginate–chitosan scaffolds was carried out through the addition of AuNp in a calculated concentration range between 1 <sup>×</sup> <sup>10</sup>−<sup>12</sup> and <sup>3</sup> <sup>×</sup> <sup>10</sup>−<sup>9</sup> mg/mL. The AuNp concentration was calculated using the Lambert–Beer equation. For the above, the determination of the molar extinction coefficient was calculated based on the following equation:

$$
\varepsilon = \frac{N\_a \cdot \sigma}{2303} \tag{1}
$$

where *ε* is the molar extinction coefficient, *N<sup>a</sup>* is Avogadro's number, *σ* is the effective cross section in cm<sup>2</sup> and 2303 is (ln 10) <sup>×</sup> 1000.

### *2.3. Characterization of Alginate/Chitosan Scaffolds*

The alginate/chitosan scaffolds synthesized by the four methods and their subsequent modification with AuNp or AuNp+Alg were characterized by means of swelling, permeability, porosity and degradation tests in different aqueous media, recording their weight and thickness prior to their use. Fourier transform infrared spectroscopy (FTIR) was used as a chemical characterization technique.

### *2.4. Swelling Degree Studies*

For swelling measurements over time in a typical experiment, the scaffold (approximately 1.3 cm in diameter) is placed in contact with 2 mL of the aqueous medium at a constant temperature of 20 ◦C at different times, after which the sample is removed from the medium, weighed and placed back in the medium. The degree of swelling, *S*, in %, was calculated gravimetrically using Equation (2), where *W*<sup>s</sup> and *W*<sup>0</sup> are the weights of the swollen scaffold and dry scaffold (initial), respectively. The following aqueous media were used: ultrapure water (pH 7), PBS (phosphate buffer saline, pH 7.4) and buffers prepared

with mixtures of Na2HPO4/citric acid solutions to obtain pHs of 3, 5, 8 and 9 (measured with the Conductronic potentiometer model PC45 (Puebla, Mexico).

$$\text{Swelling\\_S. (\%)} = \left(\frac{W\_{\\$} - W\_0}{W\_0}\right) \times 100\tag{2}$$

### *2.5. Permeability Value*

The intrinsic permeability coefficient (*k*) was calculated according to Darcy's law:

$$k = K \frac{\mu}{\rho \mathcal{g}} \tag{3}$$

$$K = \frac{a}{A} \frac{L}{t} \ln \frac{H\_1}{H\_2} \tag{4}$$

where (*µ*) is the viscosity of the medium, (*ρ*) is the density of the medium, (*g*) is the gravity acceleration, (*a*) is the tube area, (*A*) is the cross-sectional area at the sample flow, (*L*) is the sample thickness (in this case of the scaffold) and (*H*1) and (*H*2) are the initial and final heights of the tube through which the medium passes. We used 27.5 cm for *H*2. A detailed description of the custom device is presented in the Supplementary Materials (Figure S1), which is similar to the other reported systems [35,36].

### *2.6. Porosity*

The scaffolds' porosity was determined by the liquid displacement method and ethanol was used as the penetrating medium because it does not induce shrinkage or swelling, is not a solvent for polymers and is able to easily penetrate the pores. Each scaffold was placed in a cylinder with a known volume of ethanol, in which it was left for 48 h, the scaffold was removed, and the final volume was recorded. Finally, the following equation was used:

$$\text{Porosity } \left( \% \right) = \left( \frac{\mathcal{W}\_{\text{S}} - \mathcal{W}\_{\text{0}}}{\rho V} \right) \times 100 \tag{5}$$

where (*W*S) is the weight of the saturated scaffold, (*W*0) is the initial weight of the scaffold, (*ρ*) is the ethanol density and (*V*) is the volume of liquid displaced.

### *2.7. Degradation*

Degradation studies were divided into two main studies. The first study was performed at conditions similar to cell cultures in which the scaffolds (5 mm in diameter approximately) were immersed in M199 medium (11150067, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with fetal bovine serum (A4766801, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) to a pH of 7.4 and were placed in an incubator at 37 ◦C for 17 days to evaluate their degradation degrees at the following times: days 1, 2, 3, 6, 10 and 17. At all times, as much water as possible was removed from the container with the scaffold and weighed, and new medium was placed before returning to the incubator. For the second study, the material degradation (approximately 1.3 cm in diameter) exposed to different aqueous media for 7 days at laboratory conditions (20 ◦C) was calculated. In a typical experiment, for the scaffold immersed in the medium, 2 mL was used with each wash and proceeded with the following times and number of washes: twice for 15 min, once for 16 h and twice for 15 min. Finally, as much water as possible was removed from the container with the scaffold, frozen at −20 ◦C for at least 12 h and freeze-dried in a lyophilizer at −49 ◦C and a pressure of 0.09 mBar for 6 h (followed gravimetrically until there was no weight change). The degradation degree, *D*, in %, was calculated according to Equation (5), where *W*<sup>F</sup> and *W*<sup>0</sup> are the final weight of the dry scaffold and exposed

to either degradation condition and the weight of the initial dry scaffold (before being exposed to either degradation condition), respectively.

$$\text{Depradation degree} = n \,\, \text{D} \,\, \text{(\%)} = \left(\frac{\text{W}\_0 - \text{W}\_\text{F}}{\text{W}\_0}\right) \times 100\tag{6}$$

## *2.8. Infrared*

Fourier transform infrared spectra with attenuated total reflectance (FTIR-ATR) were taken from 650 cm−<sup>1</sup> to 4000 cm−<sup>1</sup> using a Perkin–Elmer model 100 spectrometer (Waltham, MA, USA) equipped with a diamond tip.

### *2.9. Characterization of the Metallic Nanoparticles*

The particle sizes, their distributions and the zeta potentials of AuNp and AuNp+Alg were analyzed by dynamic light scattering using a Nanosizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). Samples were diluted 1:100 in ultrapure water and analyzed at 25 ◦C, with a scattering angle of 90.

The Np morphology was determined by scanning electron microscopy, using a TM3030PLUS scanning electron microscope (Hitachi, Germany) with an operating voltage of 15 kV; and moreover, the AuNp morphology was examined using a JEM-1010 transmission electron microscope (Jeol, Tokyo, Japan) with a voltage of 60 kV. The AuNp aqueous dispersion was diluted 1:100 and 15 µL was deposited on a copper grid (200 mesh). The grid was allowed to dry at room temperature before analysis. UV-Vis spectroscopy analysis was conducted in order to visualize the plasmon of the gold nanoparticles.

### *2.10. Primary Culture of Chicken Embryonic Cardiomyocytes*

Cardiomyocytes were obtained from chicken embryos after 7 days of incubation. Detailed information on cardiomyocytes isolation and culture is presented in the Supplementary Materials. For each scaffold, 1 <sup>×</sup> <sup>10</sup><sup>6</sup> cells were cultured and incubated for 7 days at 37 ◦C in supplemented medium 199. Animal use protocols and study procedures were based on the Official Mexican Standard (NOM-062-ZOO-1999). The project was approved by the research, ethics and biosafety committees of the Hospital Infantil de México Federico Gómez (HIM-2020-059).

### *2.11. Indirect Cytotoxicity Assay*

To demonstrate that the different elaborated scaffolds did not generate cytotoxic particles, an indirect cytotoxicity assay was performed using the MTT method. The viability of cells cultured with medium that had contact with scaffolds functionalized with AuNp and AuNp+Alg was also compared. Indirect cytotoxicity assays were performed using a monolayer of cardiomyocytes by triplicate.

### *2.12. Scanning Electron Microscopy*

Scaffolds and constructs were fixed with glutaraldehyde (4%) and dehydrated through a series of graded ethanol concentrations (50◦ to absolute). They were critical-pointdried (Samdri 789A, Tousimins Research Co., Rockville, MD, USA) and coated with gold film (Denton Vacuum Desk 1A, Cherry Hill Industrial Center, Moorestown, NJ, USA). The samples were observed under a JEOL JSM 5300 (Tokyo, Japan) scanning electron microscope, and the accelerating voltage was 15 kV.

### *2.13. Histological Analysis*

Constructs were fixed with paraformaldehyde (4%) and processed according to standard histological technique. Transverse sections of 3 µm thickness were made and stained with hematoxylin–eosin (H&E) and scanned and digitized with Aperio CS2 equipment (Leica Biosystems, Deer Park, IL, USA).

### *2.14. Immunohistochemical Analysis*

The sections were deparaffinized and subjected to antigenic recovery in sodium citrate buffer (pH 6). Endogenous peroxidase blocking was performed with hydrogen peroxide (3%) for 30 min. A nonspecific binding blockade was performed for 3 h. The sections were incubated with the primary antibodies anti-tropomyosin (SC:74480, 1:500) and anti-PCNA (AB-2426, 1:1000) at room temperature overnight. They were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 30 min. The antigen–antibody complex was revealed with an immunodetection kit (Vector Laboratories, Inc. Cat # 30026, Berlingame, CA, USA) and counterstaining was performed with hematoxylin. Quantitative analysis (Intensity (Int)) was performed by digital pathology.

## *2.15. Western Blot*

Total protein extraction from the constructs was performed with lysis buffer (T-PER, Thermo Fisher Scientific, Waltham, MA, USA) and added to protease inhibitor (Sigma Aldrich, Mannheim, Germany). Protein concentration was quantified using the direct microdrop method (NanoDrop lite, Thermo Fisher Scientific, Waltham, MA, USA). A total of 30 µg of total protein was subjected to 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Bio-Rad). Antibodies were used at the following dilutions: primary antibodies troponin I (SC-365446 1:1000), MYH (SC-376157, 1:1000) and GAPDH (SC-48167), followed by incubation with HRP-coupled secondary antibodies in blocking solution for 1 h at room temperature, antimouse (SC-516102, 1:10,000) and anti-goat (SC-2020, 1:20,000). Finally, immunodetection by chemiluminescence (Super Signal® West Femto, Thermo Scientific, Waltham, MA, USA) was performed with autoradiographic plates. Densitometry analysis was performed with Image J software, version 1.45 (National Institute of Health, Bethesda, MD, USA).
