Alginate electrodeposition onto three-dimensional porous Co-Ni films as drug delivery platforms

Three-dimensional porous Co-Ni films/alginate hybrid materials have been successfully prepared by electrodeposition to be used as a steerable magnetic device for drug delivery. Firstly, 3D porous Co-Ni films were prepared as substrate for the subsequent eletrodeposition of the alginate biopolymer. Cyclic voltammetry, galvanostatic and potentiostatic studies were performed to establish the best conditions to obtain porous Co-Ni films. The electrochemical experiments were carried out in an electrolyte containing the metal salts and ammonium chloride at low pH’s. In a second stage, the electrochemical deposition of alginate as biocompatible polymer drug delivery carrier was performed. The characteristics of the alginate matrix were investigated in terms of electrochemical properties, morphology and drug release. The hybrid material obtained showed soft-magnetic behavior and drug release indicating its suitability to be used as a steerable magnetic drug delivery device.


3) Functional groups of the Alg films were not confirmed. They can be determined using a Fourier transform infrared (FTIR) spectroscopy.
We examined the litarature and some papers 1 clearly details that alginate is not modified by electrodeposition, reason why we did not analyze alginate by FTIR. We have modified the text accoring to the reviewer comment. The objective of the paper is the preparation of Co-Ni/alginate-hemoglobin hybrid materials as a steerable magnetic drug delivery platform. We used electrodeposition to prepare both Co-Ni and alginate films because it offers several advantages (low deposition times and low applied potentials) over other techniques like electrophoretic deposition 1 . We have clarified this point in the text according to the reviewer comment.
As it is clearly observed from the results, alginate cross-linking only takes place effectively onto the porous CoNi deposits and not onto the smooth CoNi films. This result indicates that alginate inserts into the porous structure and surrounds the magnetic film.

7)
The Authors co-deposited hemoglobin with Alg matrix, however there is no enough explanation about the electrodeposition of hemoglobin which presents difficulties related to the pH-dependent charge for this material. The electrokinetic properties of hemoglobin are strongly influenced by the nature of its fundamental building blocks, such as amino acids, containing cationic and anionic functional groups. As a result, hemoglobin shows pH dependent behavior with charge reversal at the isoelectric point, which was reported in literature to be pH = 6.8-7.0. Some comment is needed.
Here we would like to say that we used hemoglobin as a biomolecule test. In other words, we used hemoglobin to see if we could encapsulate any biomolecule into the alginate matrix and use the whole hybrid material as a steerable magnetic device. In view of the results, we can conclude saying that we have obtained an hybrid system able to be used as a drug delivery platform. On the other hand, the alginate matrix was obtained at slightly acidic pH's, so hemoglobin was below the isoelectric point. At this pH, hemoglobin is positively charges so it of hydrogen gas bubble on the electrode surface, which resulted in decreasing the adherence of Co-Ni films and even fell off. It is very necessary to provide more evidences and characterization for proving your viewpoint.
We agree with the reviewer in the sense that it is difficult to obtain Co-Ni films at high current densities. This is true from the first chloride bath tested in this study: quasi-pure nickel deposits were obtained when high negative current densities or potentials were applied. These deposits showed moderate adherence to the substrate, although the affinity of the seed layer of the substrate (Ni) with the very rich Ni electrodeposit favors the adherence, even when simulatenous and significant hydrogen evolution. However and after the addition of NH 4 Cl, we were able to increase cobalt incorporation into the films (as it can be observed in the composition analysis) as well as to improve the adherence even the simultaneous H 2 evolution. Porous deposits have been obtained at these conditions, but showing a correct adhesion to the substrate, even during their manipulation for the different characterization tests performed (SEM observation, cutting manipulation for magnetic properties characterization…) and the electrochemical retention of the alginate layer leading to the hybrid structures.

Introduction
Three dimensional nano-structured architectures have been explored for new devices such as sensors, supercapacitors, batteries, and fuel cells 1,2 because of their great potential for rapid electrochemical reactions arising from the extremely large specific surface areas for charge and mass (gas) transport. Although different techniques have been employed for the preparation of these 3D networks like sol-gel chemistry, templating or de-alloying, 3,4 electrodeposition has been revealed as a very effective method to fabricate them. The reason is that the hydrogen bubbles originated from the cathode reaction create a continuous path from the substrate to the electrolyte-air interface during the deposition process allowing creating this 3D porous structure. 5,6 Different metallic materials and alloys have been used to successfully prepare these 3D porous materials (gold, copper, silver, copper-tin or gold-platinum, among others) mainly for electrocatalytic applications or electrode materials in batteries. [5][6][7][8] However, magnetic 3D porous metallic materials have never been prepared by electrodeposition with drug delivery applications. Magnetic materials have received increased attention during the last years as microrobotic systems which are wirelessly steered by a magnetic manipulation system 9,10 for biomedical applications such as temporary diagnosis and treatment.
The intelligence of these devices can be defined by choosing the appropriate combination of materials and methods. In this sense, Co-Ni system has been selected for the preparation of the magnetic 3D porous materials because they show soft-magnetic properties to be wirelessly controlled and corrosion resistance to be used in body fluids. 11,12 On the other hand, alginate, a naturally occurring biopolymer, is finding increasing applications in the biotechnological industry, mainly due to the unique properties to be used as a matrix for the entrapment and/or delivery of a variety of proteins, cells, drugs,… These properties include: (i) a relatively inert Magnetic measurements were taken in a SQUID magnetometer at 300 K in helium atmosphere.
The magnetization-magnetic field curves were recorded maintaining the samples parallel to the applied magnetic field.

Co-Ni 3D porous films preparation and characterization
Firstly, a cyclic voltammetric study of the electrolyte was performed in order to establish the potential and intensity range were the different either reduction or oxidation processes of both metallic ions take place. The electrolyte composition was selected taking into consideration not only the standard potentials of each metal (E Co The scan was initiated at a controlled potential were no current was detected and towards negative potentials. A continuous current increase was observed at -0.7 V corresponding to simultaneous cobalt and nickel reduction because of the closeness of their reductions potentials.
On the other hand, a clear reduction peak was not observed because of hydrogen evolution took place at very close potential due to the electrocatalytic properties of the deposited metals.
During the positive scan, just one oxidation peak was recorded attributed to cobalt and nickel oxidation as a result of a Co-Ni alloy formation. This result was expected in view of the Co-Ni

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Physical Chemistry Chemical Physics phase diagram 17 where Co and Ni are miscible. From these experiments, the potential window at which each process took place has been determined owing to establish potential ranges for film's electrodeposition. The reduction and oxidation reactions are depicted in Fig. 1.  Fig. 2 shows some representative current density-time (j-t) transients recorded during Co-Ni electrodeposition process. Under quiescent conditions and at low applied potentials (Fig. 2, Curve a), j-t transients show a first peak attributed to the alloy nucleation process, followed by a progressive current increase related to the alloy growth. As the applied potential was made more negative (Fig. 2, Curve b), the nucleation process is not observed showed an increased porosity regarding those obtained potentiostatically, they were characterized by poor adherence and poor uniformity.

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In order to try to improve the porosity, homogeneity and adherence of the films, ammonium chloride was used as additive. By adding NH 4 + ions to the electrolyte, the amount of H + can be increased. It was also shown in Ref. 19 that at high current densities the discharge of NH 4 + on the electrode results in the production of atomic hydrogen adsorbed to the electrode, which should produce molecular hydrogen. Moreover, pH of the electrolyte was also modified in order to favor hydrogen evolution. the deposit to the substrate, even for the deposits obtained at pH=3. On the other hand, the pH decrease led to an increase in the film porosity as a consequence of a higher hydrogen evolution.
The porosity of the films was higher as the applied potential was more negative (Fig. 6). Thus, the best conditions that allowed obtaining 3D porous Co-Ni films were from the electrolyte 0.2 M CoCl 2 + 0.9 M NiCl 2 + 0.5 M H 3 BO 3 + 0.4 M NH 4 Cl at pH = 1. Moreover, highly negative applied current densities (> |3 A cm -2 |) were needed to allow both Co-Ni codeposition and hydrogen evolution, the last acting as template.

Co-electrodeposition of alginate and hemoglobin. Characterization of the hybrid material.
Once the conditions of the preparation of 3D porous Co-Ni films were optimized, the electrodeposition of alginate onto the Co-Ni films was studied. We selected electrodeposition rather than electrophoretic deposition because of faster deposition rates and lower applied potentials are needed to prepare the alginate films. The electrolyte employed to perform the electrodeposition is: 1 wt.% sodium alginate, 0.1 M K 2 SO 4 and 0.5 wt.% CaCO 3 (in suspension). The mechanism responsible for the anodic electrodeposition of alginate starts with the generation of protons at the anode surface due to water electrolysis (Eq. 1). These protons immediately react with the suspended CaCO 3 particles releasing Ca 2+ ions (Eq. 2). Finally, these locally generated Ca 2+ ions interact with alginate chains inducing their cross-linking onto the substrate (Eq. 3). 21 The reactions taking place during this process are:

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Once the conditions for alginate deposition were controlled, hemoglobin was added to the previous electrolyte as a test drug. Hemoglobin, a globular protein present in our blood and responsible to transport oxygen throughout the body, was selected because of its easy detection.
The same chronoamperometric experiments than those previously performed from the hemoglobin-free electrolyte were made. The j-t transients showed the same profile and the alginate cross-linking was achieved. Moreover, it could also be observed that hemoglobin was retained into the alginate matrix because it acquired a pink color. The reason for its retention is that the electrodeposition of alginate is carried out at a pH below the isoelectric point of hemoglobin (pH= 6.8-7). 22 Thus, hemoglobin is positively charged being able to migrate towards the working electrode (negative electrode).
In order to study the viability of the Co-Ni/alginate-hemoglobin hybrid material as a microrobotic system wirelessly steered by a magnetic manipulation system, the magnetic properties of the hybrid material were examined. Fig. 10 shows the normalized magnetizationmagnetic field curves of the Co-Ni porous films (Fig. 10 Oe for the Co-Ni and Co-Ni/alginate-hemoglobin materials, respectively (Inset Fig. 10). From the magnetic results one can infer that although some surface oxidation of the Co-Ni porous films may occur during alginate deposition as a consequence of the positive potentials applied, the Co-Ni films are not greatly affected showing magnetism. Thus, the hybrid materials could be used as microrobotic system manipulated by a magnetic field. On the other hand and based on the H C values, the authors confirm that the crystalline structure of the Co-Ni films is facecentered cubic. Co-Ni films obtained from chloride-based electrolytes can show different crystalline structures depending on the electrodeposition conditions. [23][24][25] While hcp (hexagonalclose packed) structure is obtained when very low deposition potentials are applied; fcc structure is observed at high potential values. The H C values of hcp Co-Ni films are clearly higher than those of fcc Co-Ni. Finally, the study of hemoglobin release from the alginate matrix was performed to evaluate the suitability of the hybrid material as a drug delivery carrier. Samples were immersed in 0.1 M NaCl solution at 37 ºC and allowed to swell and release hemoglobin. At defined times the solvent volumes were collected and analyzed by the luminol test. This test is based on the chemiluminescence reaction of luminol (3-Aminophthalhydrazide) with an oxidizing agent in a basic media and under the presence of oxygen. 26 In our study, the iron from hemoglobin serves as a catalyst for the chemiluminescence reaction that causes luminol to glow. Moreover, the blue glow is iron concentration time-dependent. Thus, the concentration of hemoglobin can be monitored through the chemiluminescence time before fading. The release profiles of hemoglobin from alginate matrixes are shown in Fig. 11. The chemiluminescence time at fading against sample collection time are represented for the hybrid materials obtained at different applied potentials for alginate-hemoglobin codeposition. As it can be observed, in all cases hemoglobin release is higher as immersion time is higher, indicating that the release of hemoglobin is continuous with time. However, different profiles and kinetic of hemoglobin release are observed. While at low applied potentials (Fig. 11, curves a and b) the release profile follows an exponential behavior; at higher applied potentials the release profile follows a logarithmic equation (Fig. 11, curve c). The reason could be the more porous structure of the alginate matrix obtained at the most positive potential allowing the release of hemoglobin at shorter times.

Conclusions
The authors report the preparation of Co-Ni/Alginate-hemoglobin hybrid materials for drug delivery applications and wireless controlled. The optimization of the electrodepostion conditions has allowed obtaining 3D porous Co-Ni films with high adherence and uniformity.
Low pH values and NH 4 Cl presence into the electrolyte are beneficial to obtain a porous structure. After that, alginate was electrodeposited onto the previously prepared Co-Ni films by

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Physical Chemistry Chemical Physics applying positive potential. Hemoglobin was retained inside the biopolymer matrix during the electrodeposition process. The magnetic properties of the hybrid material showed a softmagnetic behavior indicating its viability as a steerable magnetic device. Moreover, the release of hemoglobin from alginate with time indicates its suitability as a drug delivery platform.