The Advantages of Polymeric Hydrogels in Calcineurin Inhibitor Delivery

: In recent years, polymeric hydrogels (PolyHy) have been extensively explored for their applications in biomedicine as biosensors, in tissue engineering, diagnostic processes, and drug release. The physical and chemical properties of PolyHy indicate their potential use in regulating drug delivery. Calcineurin inhibitors, particularly cyclosporine (CsA) and tacrolimus (TAC), are two important immunosuppressor drugs prescribed upon solid organ transplants. Although these drugs have been used since the 1970s to signiﬁcantly increase the survival of transplanted organs, there are concerns regarding their undesirable side e ﬀ ects, primarily due to their highly variable concentrations. In fact, calcineurin inhibitors lead to acute and chronic toxicities that primarily cause adverse e ﬀ ects such as hypertension and nephrotoxicity. It is suggested from the evidence that the encapsulation of calcineurin inhibitors into PolyHy based on polysaccharides, speciﬁcally alginate (Alg), o ﬀ ers e ﬀ ective drug delivery with a stable immunosuppressive response at the in vitro and in vivo levels. This not only may reduce the adverse e ﬀ ects but also would improve the adherence of the patients by the e ﬀ ective preservation of drug concentrations in the therapeutic ranges.


Introduction
Hydrogels represent a special class of polymers that are widely found in nature, fulfilling important functions in tissues such as elastin or collagen. Hydrogels are three-dimensional hydrophilic polymers forming cross-linked networks. They possess fascinating physical and chemical properties and adopt different shapes and sizes depending on the existing environment.
Recently, PolyHy have aroused great interest in different disciplines of science due to their biocompatibility and ability to absorb water. Relative to other polymeric materials, these properties make them excellent candidates for applications in biomedicine, such as the construction of diagnostic devices, cell culture substrates, blood detoxifiers, tissue engineering, drug delivery systems, etc. This review gives an overview of the classification and characterization of PolyHy, focusing on their physical and chemical properties for their application in controlled drug release.

Family of Hydrogels
Polymeric hydrogels can be classified based on different factors: i.
Types of bonds in the reticulated three-dimensional structure: • Physically cross-linked PolyHy: This hydrogel undergoes a transition from the three-dimensional state to the initial state of polymeric chains in solution and thus constitutes the so-called 'reversible ones'. Cross-links are formed via attractive forces or interactions between the polymeric chains ( Figure 1A), such as hydrogen bonding, hydrophobic and electrostatic interactions [1]. This group consists of multifunctional hydrogels based on the copolymers of polyacrylamide and tannic acid that are cross-linked through hydrogen bonds [2]. • Chemically cross-linked PolyHy: This type of hydrogel is more stable in comparison with the physically cross-linked one due to the involvement of covalent bonds ( Figure 1B). This indicates that they are irreversible, unless labile covalent bonds are intentionally introduced into the cross-linked structure. For instance, the chemical cross-linking of poly(vinyl alcohol) (PVA) hydrogels with citric acid has been successfully obtained to generate a controlled drug release system. Citric acid is a cheap and non-cytotoxic reagent compared to other crosslinkers that cannot be employed in biomedical applications due to their toxicity [3]. ii.
Stimuli-responsive hydrogels. The advances in chemical synthesis, availability of new precursors, and improvement in reaction mechanisms have resulted in the development of biomaterials that respond to environmental stimuli. This promotes the conversion of static hydrogels to dynamic ones that respond to generated changes or specific inputs in variables such as temperature and pH. Furthermore, the stimulus-sensitive PolyHy are known as 'smart hydrogels' because of their response to any slight changes in the environment, such as temperature, pH, ionic strength, light, electric field, and biomolecules, by changing the degree of swelling. The response of the smart hydrogels depends on the type of monomer or polymer used and/or structural modifications within the compound. The challenges in the development of smart hydrogels that exhibit a rapid response to the stimuli of the medium are primarily based on synthetic mechanisms [4][5][6][7][8][9]. iii.
PolyHy can also be classified based on their charge. Hydrogels are composed of molecular structures such as nonionic, cationic, anionic, amphoteric polyelectrolytes, or zwitterionic species. The development of zwitterionic hydrogels further confirmed their potential application in wound dressing [10,11]. The dual cross-linked networks composed of anionic and cationic monomers improve the mechanical properties of hydrogels and thus find application as injectable hydrogels [12]. On the other hand, new synthetic methodologies have been introduced in the field of cell adhesion to obtain composite hydrogels incorporating cationic groups in their structure to improve flexibility as well as cell adhesion [13]. iv.
Another classification involves natural and synthetic hydrogels. Due to the biocompatible, biodegradable, and non-toxic nature, natural hydrogels find potential biomedical applications. Based on the type of natural polymer, there are three biomaterials, namely protein-based, polysaccharide-based, and natural polyester-based hydrogels [14][15][16][17]. In this review, we are interested in focusing on the biomedical applications of hydrogels based on polysaccharides, mainly. Therefore, later, we dedicate a section to this type of biomaterial. On the other hand, synthetic hydrogels have favorable properties for industrial applications, such as superior mechanical properties compared to natural hydrogels. However, hybrid hydrogels with improved and intermediate properties between natural and synthetic hydrogels have been manufactured. Biocompatible hydrogels functionalized with carbon nanotubes have been successfully synthesized to stabilize hydroxyapatite. The cytotoxicity results of these materials show promising insights for their application in bone tissue engineering [18]. In addition, there is a group of biodegradable synthetic hydrogels, which have been widely used for biomedical applications. Principally, in this group we can mention polymers, such as poly(caprolactone), poly(glycolic acid), poly(lactic acid), poly(D,L-lactide-co-glycolide), and polyurethane [19].
The studies on fucoidan-modified PVA hydrogels have revealed substantial improvement in cell adhesion and hemocompatibility, suggesting the utility of these hybrid materials as implants or vascular devices [20]. In cell microenvironment engineering, these materials provide very thin platforms that are utilized to seed the endothelial cells. An attractive biocompatible scaffold based on modified gelatin and a biodegradable polyester of poly(D,L-lactic acid) (PDLLA) was developed. The combination of these two biomaterials provides the ideal environment for cytocompatibility and cellular interaction exhibited by gelatin. In addition, PDLLA provides appropriate mechanical resistance for the graft tissue in corneal endothelial transplant [21]. v.
Based on their size, hydrogels are classified into those containing macroscopic structures and those containing networks of smaller dimensions, such as microgels or nanogels. The conformation of hydrogels with micrometric precision at the level of individual cells has allowed their application as artificial cells [22,23], drug and cell carrier systems [24,25], and assembled elemental units in artificial tissue construction [26,27]. Polysaccharide-based microgels are one of the most commonly used hydrogels. Recent studies have focused on microgels that can stabilize and release lipophilic particles. Microgels were prepared from corn starch via an oxidized-annealing process. Further, the adsorption/release capacity of lysozyme by the microgels was evaluated. Studies showed that the oxidized-annealed microgels exhibit high charge and release capacities over a wide range of pH and ionic strength values. This is attributed to the electrostatic forces between the carboxyl groups generated by the oxidation-annealing process of the microgel and lysozyme [28]. The hydrogels embedded in microgels to form a hydrogel/microgel system have been utilized as wound dressings. The carboxymethyl chitosan-and oxidized carboxymethyl cellulose-based microgels are drug-loaded via the Schiff base reaction. The gel time, morphology, swelling ratio, weight loss ratio, mechanical properties, release profiles of pH-sensitive drugs, and antibacterial activities were analyzed. The addition of microgels was found to offer stability, improved mechanical performance, and sensitivity to drug release at different pH conditions for the hydrogels. The addition of Ag compounds to the hydrogel/microgel composites demonstrated desirable antibacterial properties, which ensured their possible application in wound dressing [9]. On the other hand, nanogels, which are also known as nano-sized hydrogels or hydrogel nanoparticles, are nanostructured three-dimensional networks composed of functional polymers, whose sizes are in the sub-micrometer region, typically from 20 to 200 nm. Hydrogel nanoparticles are formed by physical as well as chemical cross-linking methods. Nanogels offer several advantages, such as biocompatibility, high swelling capacity, and high-water solubility. In addition, they are classified as stimuli-responsive and nonresponsive hydrogels. Nanogels respond to fluctuations in environmental factors, such as temperature, pH, pressure, electric and magnetic fields, molecular species, ionic strength, or a combination of different factors. Therefore, nanogels are exceptional candidates for application in biomedicine, such as biosensors, drug release, tissue engineering, diagnosis, etc. [20,29,30].
Some of the examples of PolyHy are listed under the respective classifications in Table 1.
As mentioned in the previous classification summarized in Figure 2, PolyHy covers a wide range of materials and synthetic methods. In fact, these materials can be potentially applied in biomedicine due to their versatility to modify and adapt as required. This review focuses on naturally occurring, i.e., biodegradable, specifically polysaccharide-based hydrogels. Initially, the latest studies on natural hydrogels for application in tissue engineering are reviewed. The later sections present the studies on the controlled release of drugs by mainly focusing on calcineurin inhibitors (CNIs), which are common immunosuppressive drugs used in the treatment of autoimmune diseases and solid organ transplantation.
In addition, they are classified as stimuli-responsive and nonresponsive hydrogels. Nanogels respond to fluctuations in environmental factors, such as temperature, pH, pressure, electric and magnetic fields, molecular species, ionic strength, or a combination of different factors. Therefore, nanogels are exceptional candidates for application in biomedicine, such as biosensors, drug release, tissue engineering, diagnosis, etc. [20,29,30].

Tissue Engineering Applications of Natural Polymeric Hydrogels
The hydrogels consisting of natural polymers have been recognized as prime candidates for designing tissue scaffolds. This is attributed to the excellent characteristics such as cost efficiency, high compatibility, and biodegradability exhibited by this type of hydrogels. Natural polymeric hydrogels, such as polysaccharides and proteins, were similar to the natural extracellular matrix. Thus, the hydrogels are endowed with magnificent properties, such as cellular proliferation and survival, to overcome certain challenges in tissue engineering [58][59][60]. Recently, a microbial polysaccharide-based macroporous hydrogel was manufactured from salecan and gellan gum by a one-step method [58]. These were obtained via the association of polysaccharide chains by means of Van der Waals forces, hydrogen bonds, and hydrophobic interactions. It should be noted that the manufacturing methodologies did not require crosslinkers or toxic monomers. Instead, the salecan/gellan gum hydrogel that exhibits excellent biocompatibility and properties to mimic the native tissues were utilized. In this context, cardiac tissue engineering has emerged as a branch of tissue engineering, which uses combinations of cells, biological and/or synthetic materials, growth factors, differentiation factors, proangiogenic factors, and monitoring, as a promising and challenging approach to induce the regeneration of heart tissue [61]. This field of research gave rise

Tissue Engineering Applications of Natural Polymeric Hydrogels
The hydrogels consisting of natural polymers have been recognized as prime candidates for designing tissue scaffolds. This is attributed to the excellent characteristics such as cost efficiency, high compatibility, and biodegradability exhibited by this type of hydrogels. Natural polymeric hydrogels, such as polysaccharides and proteins, were similar to the natural extracellular matrix. Thus, the hydrogels are endowed with magnificent properties, such as cellular proliferation and survival, to overcome certain challenges in tissue engineering [58][59][60]. Recently, a microbial polysaccharide-based macroporous hydrogel was manufactured from salecan and gellan gum by a one-step method [58]. These were obtained via the association of polysaccharide chains by means of Van der Waals forces, hydrogen bonds, and hydrophobic interactions. It should be noted that the manufacturing methodologies did not require crosslinkers or toxic monomers. Instead, the salecan/gellan gum hydrogel that exhibits excellent biocompatibility and properties to mimic the native tissues were utilized. In this context, cardiac tissue engineering has emerged as a branch of tissue engineering, which uses combinations of cells, biological and/or synthetic materials, growth factors, differentiation factors, proangiogenic factors, and monitoring, as a promising and challenging approach to induce the regeneration of heart tissue [61]. This field of research gave rise to the study of biocompatible hybrid systems [62], such as polysaccharide-based PolyHy, for application in tissue engineering [63,64].
Alginate (Alg) and chitosan (CS) are natural polysaccharides that are widely used in biomedicine, as well as food and pharmaceutical industries [65,66]. This is attributed to the high biocompatibility and biodegradability of polysaccharides [15,67]. A recent study on the improvement of the mechanical tensile strength of decellularized extracellular cardiac matrix (ECM) using Alg/CS hydrogels [68] showed a high swelling capacity, good mechanical resistance, moderate biodegradable properties, and excellent cell viability. The results of the study reveal that CS-and Alg-based platforms find potential applications in cardiac tissue engineering. In addition, other natural polymers, such as collagen, gelatin (Gel), and fibrin, have been studied for myocardial tissue engineering. The collagen/CS composite scaffold has been widely used for myocardial tissue engineering [63]. Since collagen, which is the main component of the ECM within the myocardium, has a fast degradation rate and poor mechanical properties [69,70], it has been combined with other polysaccharides to improve the characteristics of the composite PolyHy.
The research in tissue engineering aims to manufacture materials that mimic native tissues of the human organs. The biomimetic materials capable of mimicking the structure and composition of the native extracellular matrices are essential for successful organ tissue repair and transplantation in humans. Similarly, fibrin-agarose tissue-like hydrogels (FATLHs) have allowed successful biofabrication of different biological substitutes, and thus, delivered promising ex vivo and in vivo results [71]. These fibrin-agarose-based materials biodegrade and integrate into implanted areas and vital organs without any histopathological alteration. Thus, FATLHs exhibit potential clinical uses in engineering applications due to their biosecurity and biocompatibility. Furthermore, these artificial tissues combine to be stronger, and thus offer a stable mechanical response along with favorable cytocompatibility [72].

Advantages of PolyHy in Controlled Drug Distribution
It is quite interesting that the number of papers published based on the application of PolyHy in the controlled release of drugs in the journals indexed within the WoS database exceeded eight hundred articles in the last five years (Figure 3). Polymers have been widely used as biomaterials for the manufacture of medical devices as well as tissue engineering platforms. In particular, hydrogels have aroused great interest in biomedicine, mainly focusing on the development of scaffolds that release therapeutic agents [73][74][75][76]. In this context, hydrogels based on natural polymers have been extensively studied for applications in biomedicine due to their intrinsic characteristics of biocompatibility, biodegradability, and similarities to the extracellular matrix [45,[77][78][79]. The physicochemical characteristics of biopolymeric hydrogels used in drug delivery systems, namely molecular weight, solubility, morphology, hydrophilicity, hydrophobicity, loading and release efficiency of the drug, surface energy, gelation process, etc., have been investigated [80][81][82].
Due to their natural abundance, biocompatibility, and physicochemical properties, hydrogels based on polysaccharides, specifically Alg and CS, are potentially used as drug delivery systems. Alg is a naturally abundant anionic polysaccharide extracted from brown algae, comprising two monomeric units, namely α-L-guluronic acid (G) and β-D-mannuronic acid (M) residues [83]. Due to the characteristic properties of Alg, such as affordability, non-toxicity, high adsorption capacity, and gelation capacity, these hydrogels are suitable for application in the food industry and biomedicine [84,85]. Alg hydrogels are prepared by physical or chemical cross-linking methods, as described above. However, the poor mechanical properties are a major drawback for the Alg hydrogels [86]. To resolve this limitation, previous studies reported the development of hybrid hydrogels. Jahanban-Esfahlan et al. [87] developed a hybrid hydrogel through the Schiff base con densation reaction of Alg with Gel, followed by the incorporation of Fe 3 O 4 magnetic nanoparticles as a strategy to improve the mechanical properties of the hybrid hydrogel ( Figure 4A). Scanning electron microscopy (SEM) was used to verify the microporous structure of Alg/Gel hydrogel without microphase separation ( Figure 4B). The hydrogel containing Fe 3 O 4 nanoparticles was loaded with an anti-cancer drug, namely doxorubicin hydrochloride. The in vitro study revealed that the doxorubicin-loaded Alg-Gel/Fe 3 O 4 magnetic hydrogel showed pH-responsive drug release behavior. Thus, the fabricated magnetic PolyHy has excellent potential as a drug delivery system for cancer chemotherapy.
It is quite interesting that the number of papers published based on the application of PolyHy in the controlled release of drugs in the journals indexed within the WoS database exceeded eight hundred articles in the last five years (Figure 3). Polymers have been widely used as biomaterials for the manufacture of medical devices as well as tissue engineering platforms. In particular, hydrogels have aroused great interest in biomedicine, mainly focusing on the development of scaffolds that release therapeutic agents [73][74][75][76]. In this context, hydrogels based on natural polymers have been extensively studied for applications in biomedicine due to their intrinsic characteristics of biocompatibility, biodegradability, and similarities to the extracellular matrix [45,[77][78][79]. The physicochemical characteristics of biopolymeric hydrogels used in drug delivery systems, namely molecular weight, solubility, morphology, hydrophilicity, hydrophobicity, loading and release efficiency of the drug, surface energy, gelation process, etc., have been investigated [80][81][82]. Due to their natural abundance, biocompatibility, and physicochemical properties, hydrogels based on polysaccharides, specifically Alg and CS, are potentially used as drug delivery systems. Alg is a naturally abundant anionic polysaccharide extracted from brown algae, comprising two monomeric units, namely α-L-guluronic acid (G) and β-D-mannuronic acid (M) residues [83]. Due to the characteristic properties of Alg, such as affordability, non-toxicity, high adsorption capacity, and gelation capacity, these hydrogels are suitable for application in the food industry and biomedicine [84,85]. Alg hydrogels are prepared by physical or chemical cross-linking methods, as described above. However, the poor mechanical properties are a major drawback for the Alg hydrogels [86]. To resolve this limitation, previous studies reported the development of hybrid hydrogels. Jahanban-Esfahlan et al. [87] developed a hybrid hydrogel through the Schiff base con densation reaction of Alg with Gel, followed by the incorporation of Fe3O4 magnetic nanoparticles as a strategy to improve the mechanical properties of the hybrid hydrogel ( Figure 4A). Scanning electron microscopy (SEM) was used to verify the microporous structure of Alg/Gel hydrogel without microphase separation ( Figure 4B). The hydrogel containing Fe3O4 nanoparticles was loaded with an anti-cancer drug, namely doxorubicin hydrochloride. The in vitro study revealed that the doxorubicin-loaded Alg-Gel/Fe3O4 magnetic hydrogel showed pH-responsive drug release behavior. Thus, the fabricated magnetic PolyHy has excellent potential as a drug delivery system for cancer chemotherapy.
(A) (B) The recent studies on Alg-based hydrogels primarily focused on improving the mechanical properties [58,88], manufacturing methods [89,90], payload of gels [21], and transport and release of hydrophilic and hydrophobic drugs [91,92] such as CNIs. These Alg-based PolyHy are good drug delivery systems for the brain, cancer, antibacterial activity, immunosuppressive therapies, etc. [93][94][95][96]. This review is specifically focused on the studies undertaken for the transport and release of CNIs to find the optimal systems in order to minimize the side effects on the human body.

Presentation, Classification, and Use
Calcineurin is a serine-threonine phosphatase with a calmodulin-binding catalytic subunit and a calcium-binding regulatory subunit. Due to its significance in cell development, this enzyme is The recent studies on Alg-based hydrogels primarily focused on improving the mechanical properties [58,88], manufacturing methods [89,90], payload of gels [21], and transport and release of hydrophilic and hydrophobic drugs [91,92] such as CNIs. These Alg-based PolyHy are good drug delivery systems for the brain, cancer, antibacterial activity, immunosuppressive therapies, etc. [93][94][95][96]. This review is specifically focused on the studies undertaken for the transport and release of CNIs to find the optimal systems in order to minimize the side effects on the human body.

Presentation, Classification, and Use
Calcineurin is a serine-threonine phosphatase with a calmodulin-binding catalytic subunit and a calcium-binding regulatory subunit. Due to its significance in cell development, this enzyme is expressed in most cells [97]; however, it has been better described in immune response. Particularly, the dephosphorylation of the transcription factor, namely the nuclear factor of activated T cells (NFAT), by calcineurin leads to translocation of the protein into the nucleus, which further activates different leukocyte proliferation and differentiation mediators [98]. This molecular mechanism is reported in T-lymphocytes, which are crucial for adaptive immune response and post-transplant organ rejection.
CNIs, as previously defined, are immunosuppressant drug-targeting calcineurins that are commonly used for the treatment of autoimmune diseases and subsequent to transplantation, including kidney transplantation. Furthermore, CNIs have been used for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [99]. CNIs include cyclosporine A (CsA) and tacrolimus (TAC), which binds cyclophilin and FK506 binding protein 12 (FKBP12), respectively [100]. These CNIs primarily inhibit the pathway of NFAT and thus prevent the transcription of interleukin-2 (IL-2), one of the most important cytokines for the growth and differentiation of T-helper lymphocytes.
Since their introduction in the 1970s, CNIs have significantly lower rejection rates, which further extends the time of graft survival. However, long-term side effects, such as nephrotoxicity [101] and hypertension [102], represent a current concern. On the other hand, CNIs were found to be safe in children diagnosed with glomerulopathy, which cannot be treated by steroids [103], another class of immunosuppressant drugs.

Pharmacological Aspects of CNIs
CNIs are important for immunosuppression in solid organ transplanted patients. They have a narrow therapeutic index that promotes therapeutic drug monitoring (TDM) in order to avoid progressive toxic effects over time, such as nephrotoxic, cardiovascular, or neurological side-effects. In addition, the introduction of suboptimal concentrations of CNIs reduces the risk of organ rejection [104]. TDM is performed routinely by the immunoassay of whole blood samples due to the distribution of around 90% TAC and 50% CsA on erythrocytes [105]. A trough blood sample is required for the accurate estimation of the area under the curve for TAC, while a sample extracted two hours after oral administration (C2) is used to obtain the TDM of CsA.
Due to high inter-and intra-variabilities in pharmacokinetics (PK), a sensitive equilibrium needs to be maintained after the administration of a dose of CNI. This variability is mainly explained by the pharmacogenetic influence of the cytochrome P450 system (hepatic and intestinal polymorphisms in CYP3A4 and CYP3A5, respectively) that alters the pharmacokinetic metabolism of these drugs [106,107]. In addition, the intestinal efflux-pump P-glycoprotein contributes to the PK variability, resulting in the reduction in drug absorption and change in disposition [108]. This leads to significant drug interactions with other CYP450 inducers/inhibitors, giving rise to a complex therapy [105,109].
CsA and TAC are lipophilic and low solubility drugs with an oral bioavailability around 20-25%, which is variably influenced by food or fasting. The first-pass metabolism plays a key role in obtaining therapeutic blood plasma concentrations [110]. Thus, intravenous administration could improve the PK profile of the drugs. However, physiologically based PK models showed that different covariates, such as serum albumin level, hematocrit, CYP3A4 abundance, and CYP3A5 genotype status, are quantitatively responsible for PK variability, making it difficult to understand the drug disposition process [108,110]. Finally, it has been proposed that there is a requirement for pharmacogenetic screening prior to the administration of drugs in order to identify rapid or slow metabolizer patients and regulate the dosage to avoid adverse effects [111]. For instance, TAC is one of the drugs with available guidelines to support the necessity of pharmacogenetic evaluation before and after transplantation [106]. Nevertheless, it is an expensive drug without widespread routine examination to be used in every immunosuppressed patient. It requires the consultation of health professionals to determine the appropriate genotype-based dose adjustment [112].

The Adverse Effects of CNIs
Since the 1970s, the proven efficacy of CNIs in preventing rejections has been accompanied by undesirable side effects. Among the common and prominent side effects, hypertension appears to be a relevant one, resulting in an increase in graft failure as well as recipient mortality [113]. Thus, it has been observed in patients and experimental animals that the CNIs promote vasoconstriction, which is extended even to the arteriolar level in renal afferent arteriole [114] and promoted by sympatho-excitation [115]. This results from an imbalance between the pro-contractile (endothelin and thromboxane) and pro-dilative molecules (nitric oxide, prostaglandin E2, prostacyclin) and activation of the renin-angiotensin system after the administration of CNIs [116][117][118]. In addition, Hoorn et al. performed a study on mice that demonstrated the positive influence of TAC on increasing the blood pressure by the activation of the renal sodium chloride cotransporter [102], which is crucial for the reabsorption of sodium at the distal nephron. This may represent an additional mechanism for the induction of hypertension during the administration of CNIs.
Nephrotoxicity is another important adverse effect of the CNIs that leads to kidney failure. The acute CNI nephrotoxicity was reported as one of the first pieces of evidence for nephrotoxicity related to CNIs, with pivotal vasoconstriction mechanisms, as mentioned above. During acute toxicity, the CsA and TAC promote thrombotic microangiopathy [119] and isometric tubular vacuolization [120], augmenting the renal risk after organ transplantation. Since renal hemodynamic alterations are crucial mechanisms in CNI-induced nephrotoxicity, the diminution in renal blood flow [121] and increased renal vascular resistance in kidneys [120] also contribute to the acute toxicity levels. On the other hand, chronic CNI-related nephrotoxicity involves the alteration of the renal structure by inducing pro-fibrotic factors such as TGFβ [122] and promoting epithelial-mesenchymal transition, a major mechanism involved in the interstitial fibrosis of the kidneys [123]. Additionally, the inflammatory vasculopathy and endothelial dysfunction promoted by CNIs contribute to tissue remodeling [124]. All these effects are reinforced by the activation of the renin-angiotensin system, resulting in acute nephrotoxicity.
The side effects of CsA and TAC pose limitations on the long-term survival of the transplant and overall prognosis of the patient receiving the organ. The non-selective inhibition induced by the CNIs on the immune system may further reduce the resistance to infections and augment the potential spread of malignant conditions [125]. In addition, the CNIs may increase the risk of cardiovascular and renal diseases through dyslipidemia and hyperglycemia [126].
The majority of the acute side effects result from the high variability in the plasma concentrations of the CNIs, depending on the dosage. This is an important issue to be considered during the determination of the appropriate dosage in patients. Drug formulation based on the technological aspects provides new perspectives in drug delivery and disposition, which could possibly optimize the stable therapeutic plasma concentration, avoid high or low exposure to the drug (wide peak-to-trough plasma fluctuations), and improve the quality of life after immunosuppressive therapy.

Encapsulation of the Calcineurin Inhibitors
CsA presents limitations in oral and injectable administration, where its low aqueous solubility and hydrophobic nature have encouraged the development of drug delivery systems with maximum bioavailability. Thus, natural PolyHy have been proposed as local delivery systems for the sustained release of CsA, which may be an effective way to resolve this problem [127]. Sang Shin et al. [95] proposed a formulation based on injectable hydrogels composed of calcium hyaluronic acid (HA-Ca) and sodium Alg (Alg-Na) for the sustained release system of CsA. The injectable HA-Ca-Alg hydrogels were successfully prepared at different values of gel time and volume ratios without the external addition of calcium salts. The morphology of PolyHy was characterized and analyzed by scanning electron microscopy, which showed the existence of irregular pores. This feature offers suitable microenvironments for the transport of large-sized immunosuppressive drugs, such as CsA. The results reveal effective immunosuppressive activity for prolonged duration without any effects on blood clotting and normal cells. This study suggested the effective applicability of injectable HA-Ca-Alg PolyHy for the sustained delivery of hydrophobic immunosuppressive drugs. Nanostructured poly(2-hydroxyethyl methacrylate) hydrogels (p-HEMA) containing microemulsions have been developed for the prolonged ophthalmic administration of CsA [128], which yielded promising results. However, p-HEMA lenses cannot be worn for long duration due to their low oxygen permeability.
Studies were performed on different delivery systems for the administration of TAC, such as mPEG-poly(lactic acid) nanoparticles [83], poly (lactide-co-glycolide) microspheres [129], and triglycerol monostearate hydrogels [130]. Recently, a mixed PolyHy system based on thermosensitive polypeptides and poloxamer was proposed to develop a sustained release reservoir for the delivery of subcutaneously administered TAC in an in vivo model [131]. In addition, the rational design of two hydrogelators was reported to establish an easy method for the release of TAC that induces an immune response to overcome the organ transplant rejection in an animal model [132]. The in vitro cell experiments showed that the amount of TAC released from the hydrogel was logarithmically proportional to the number of activated T-cells, demonstrating a better cell-inhibition effect than the free TAC drug. The in vivo liver transplantation trials indicated that the smearing of PolyHy on the graft surfaces after surgery enhanced the survival rate. This could be a potential alternative to ensure therapeutic and stable concentrations of TAC during the first months of organ transplantation that exhibit critically high rates of rejection due to the inadequate plasma concentration of the immunosuppressants [112]. Another study was focused on the development of immunosuppressive prodrugs, such as TAC modified with dibenzocyclooctin (DBCO) and its conjugation to Alg PolyHy modified with an azido group via click chemistry ( Figure 5) [133].
Processes 2020, 8, x FOR PEER REVIEW 10 of 18 exhibit critically high rates of rejection due to the inadequate plasma concentration of the immunosuppressants [112]. Another study was focused on the development of immunosuppressive prodrugs, such as TAC modified with dibenzocyclooctin (DBCO) and its conjugation to Alg PolyHy modified with an azido group via click chemistry ( Figure 5) [133].  The Alg hydrogels were found to be non-immunogenic and showed the controlled local release of immunosuppressive drugs, such as TAC. This strategy demonstrated the intravenous injection of fresh prodrugs into the hydrogels. Therefore, there is no need to replace the implanted gels within the tissues as the drug levels drop to the subtherapeutic levels, which avoids the trauma and inflammation resulting from repeated gel injection. This alternative plays a key role in long-term adherence to the treatment. The risk factors for organ rejection include polypharmacy, adverse effects, and lack of the patient's adherence to the chronic health conditions [112]. Thus, any new technology for the reduction in drug administration and preservation of stable drug concentrations in the therapeutic range is an important advancement in therapeutics. CS, another natural polysaccharide, is also widely used and studied as a drug delivery system [134]. The chemicallymodified CS compounds containing hydrophilic and hydrophobic groups (HGC) were synthesized and loaded with TAC to be directed toward the kidney [135]. In this study, HGC-TAC showed no toxicity toward HK-2 cells and enhanced the cellular uptake. The in vivo biodistribution tests showed high renal accumulation of HGC. The in vivo TAC release profile demonstrated a higher renal concentration and lower plasma profile, indicating the suitability of HGC-TAC as a safe TAC delivery system for the kidneys while maintaining low plasma concentrations of TAC. Finally, type I collagen-= tacrolimus Figure 5. Schematic illustration of the binding of DBCO-drug (e.g., tacrolimus) prodrugs to the alginate-N3 gels. The schemes were extracted from reference [133]. PubChem Compound Summary for CID 445643, Tacrolimus. Retrieved 19 August 2020 from https://pubchem.ncbi.nlm.nih.gov/compound/ Tacrolimus.
The Alg hydrogels were found to be non-immunogenic and showed the controlled local release of immunosuppressive drugs, such as TAC. This strategy demonstrated the intravenous injection of fresh prodrugs into the hydrogels. Therefore, there is no need to replace the implanted gels within the tissues as the drug levels drop to the subtherapeutic levels, which avoids the trauma and inflammation resulting from repeated gel injection. This alternative plays a key role in long-term adherence to the treatment. The risk factors for organ rejection include polypharmacy, adverse effects, and lack of the patient's adherence to the chronic health conditions [112]. Thus, any new technology for the reduction in drug administration and preservation of stable drug concentrations in the therapeutic range is an important advancement in therapeutics. CS, another natural polysaccharide, is also widely used and studied as a drug delivery system [134]. The chemically-modified CS compounds containing hydrophilic and hydrophobic groups (HGC) were synthesized and loaded with TAC to be directed toward the kidney [135]. In this study, HGC-TAC showed no toxicity toward HK-2 cells and enhanced the cellular uptake. The in vivo biodistribution tests showed high renal accumulation of HGC. The in vivo TAC release profile demonstrated a higher renal concentration and lower plasma profile, indicating the suitability of HGC-TAC as a safe TAC delivery system for the kidneys while maintaining low plasma concentrations of TAC. Finally, type I collagen-based PolyHy have also been used to encapsulate primary immunosuppressive agents, such as TAC. The hydrogels were found to exhibit adequate porosity, degree of swelling, drug release, blood compatibility, and cell proliferation [136].

Conclusions
The PolyHy represent a special class of polymers that are extensively found in nature. These cross-linked networks possess fascinating physical and chemical properties and adopt different shapes and sizes depending on the existing environment. Compared to other polymers, these hydrogels possess the unique characteristic of water absorption and arouse great scientific interest due to their biocompatibility and versatility. As discussed in this review, the PolyHy are used as carriers for the drug delivery of certain medications that present low bioavailability and tissue distribution, variable plasma therapeutic levels, high first-pass metabolism, or even low compliance of the patient to the treatment. All of these limitations affect the pharmacological therapy and can be resolved by improving the delivery system. The CNIs, namely TAC and CsA, constitute a drug family that was proposed to be encapsulated in PolyHy for the treatment of autoimmune diseases but are more relevant for immunosuppressive treatment after solid organ transplant. The first in vitro and in vivo assays of CNIs indicated effective immunosuppressive activity and therapeutic concentrations in the target organ. The unique in vivo results were not only observed in experimental animals but also in pilot clinical organ transplant protocols, which is beneficial for the development of solid therapeutics. Despite the development of other immunosuppressants, the CNIs are potentially used for maintenance therapies after solid organ transplants due to their excellent cost-efficiency, representing the first line of drugs to prevent acute and chronic rejections [137].
Author Contributions: C.S.-Y., L.E. and C.A.A. have contributed to writing the manuscript, its review and editing. All authors have read and agreed to the published version of the manuscript.
Funding: This research was supported by Fondecyt #1201251.