Sulfated Alginate for Biomedical Applications

2.1 Methods of Sulfation

Several methods of synthesizing S–Alg from either commercial or isolated Alg polymers have been reported. The methods utilize different sulfating agents including sulfur trioxide (SO₃) complexes,^[12-15] chlorosulfonic acid (HClSO₃),^[16-23] and N,N'-dicyclohexylcarbodiimide and sulfuric acid (DCC-H₂SO₄).^{[22, 24-27]} These syntheses are carried out in various solvents including water and dipolar aprotic solvents such as formamide, dimethylformamide (DMF), and dimethyl sulfoxide (DMSO). The sulfation reactions involve combining Alg with a sulfating agent for a set period of time, followed by adjusting the pH with aqueous NaOH to yield S–Alg with sulfate and carboxylate groups balanced by Na⁺ ions. Some approaches use Alg directly for the sulfation step (Scheme 2) while others involve the conversion of Alg from the sodium to a tetra- or tri-butylammonium salt (T-Alg) (Scheme 3) in order to improve the solubility in polar aprotic solvents. When sulfation of high MW Alg makes use of an Alg dispersion in a polar aprotic solvent in which Alg is poorly soluble,^[28] heterogeneous sulfation is likely to result. However, the use of T-Alg as the precursor overcomes this issue, at least to some extent, allowing for less heterogeneous sulfation. The majority of sulfating agents will introduce sulfate groups predominantly at the C2 and/or C3 hydroxyl groups, but some studies have investigated sulfation of the carboxylate C6 (Scheme 3).

Schweiger & Andrew (1972) initially investigated a method of sulfating cellulose^[12] and then Alg and pectin^[12] using a sulfur trioxide-dimethylformamide complex (SO₃·DMF), (Scheme 2, Reaction 1). This approach had previously been used for sulfation of chitosan (CS) as an early heparin-mimetic.^[29] This was the first report of successful sulfation of Alg without excessive degradation, and while a reduction in viscosity after sulfation was attributed to some degree of degradation, it was minor compared with previous methods used for other polysaccharides.^[12]

In 1979 Larm et al. described the conversion of Alg into a heparin analogue via multiple modification steps including sulfation.^[30] Firstly, a fraction of the carboxylate groups were reduced to hydroxyl groups at C6, followed by partial oxidation of C2/C3 to allow for reductive amination at these sites. The final sulfation step allowed for reaction at the remaining hydroxyl groups at C2/C3 as well as C6. This sulfation process was modified from the SO₃·DMF method where a sulfur trioxide-pyridine complex (SO₃·py) sulfating agent was first prepared and then added to the Alg/DMF solution (Scheme 2, Reaction 2). SO₃·py has been used by several other groups as a sulfating agent for Alg, sometimes with DMSO as the solvent,^[13] or with a first step of converting Alg to the tetrabutylammonium or tributylammonium salt (Scheme 3, Reaction 7).^{[15, 31]} Our recent study^[31] based on the hydrodynamic radius (r_H) before and after sulfation, as well as viscosity measurements, concluded that this reaction resulted in minor or no change to the chain length.

Huang et al. (2003) were the first to use a chlorosulfonic acid (HClSO₃) sulfating agent for the sulfation of Alg^[16] (Scheme 2, Reaction 3) as it had previously been successful at sulfating CS.^[32] This is now the most widely used method of sulfation of Alg.^{[17-21, 33]} In 2021, Mostafavi et al. used the HClSO₃ reagent to produce S–Alg, but rather than converting to the Na⁺ form they used tributylamine (TBA) to yield a tributylammonium salt of S–Alg (hydrophobized S–Alg).^[34] In 2014, Arlov et al. assessed the MW of Alg and S–Alg of different DS prepared by this reaction via size exclusion chromatography (SEC), and no depolymerization was observed as a result of the sulfation reaction.^[17]

Cong et al. (2014) adapted the above method to use a chlorosulfonic acid-pyridine complex (HClSO₃·py) for sulfation of Alg (Scheme 2, Reaction 4),^[35] which had been used for previous work on sulfation of other polysaccharides to fabricate heparin-mimetics^[36] and antitumor agents.^[37] This process was patented by GH Shi in 1997 for low MW polysaccharides.^[38]

Fan et al. (2011) detailed an uncommon sulfating agent trisulfamic acid, trisodium salt (N(SO₃Na)₃) prepared by adding a solution of sodium nitrite dropwise into sodium bisulfite (Scheme 2, Reaction 6).^[39] In this study the effects of reaction pH, temperature, time, and amount of sulfating agent on the resulting DS of S–Alg were investigated. S–Alg polymers of low MW in the range of 15 to 35 kg mol⁻¹ were obtained by oxidative degradation using hydrogen peroxide.

In 2008, Freeman et al. outlined the synthesis of S–Alg using DCC and H₂SO₄, with a first step of converting the Alg to the tributylammonium salt (T-Alg) (Scheme 3, Reaction 8).^[24] This method was adapted from a patent by Guo et al. (2002).^[40] Ma et al. (2016) conducted a study using this sulfation method directly for Alg as the starting material (Scheme 2, Reaction 5). They found that there was degradation of the Alg with this process, with more sulfating agents leading to a lower MW as determined via SEC measurements.^[25] This process was patented by Cohen et al. in 2007 for bioconjugates containing sulfated polysaccharides and bioactive peptides.^[41] Recently, we investigated in detail the product formed from this reaction and showed that a previously unacknowledged and unwanted adduct formed from the carbodiimide, resulting in an S–Alg product which is also functionalized with an N-acylurea.^[31]

Schmidt et al. (2016) used aqueous carbodiimide coupling chemistry to achieve sulfation of the Alg carboxylate at C6 with 2-aminoethyl hydrogen sulfate (Scheme 3, Reaction 9).^[42] The use of carbodiimide chemistry does not cause polymer degradation but is prone to the formation of an unwanted N-acylurea adduct from the rearrangement of the O-acylisourea intermediate.^{[43, 44]} Sulfation at the C6 carboxylate can introduce up to one sulfate per uronic acid residue. Furthermore, it results in the same net charge for the modified polymer in contrast to the other sulfation reactions of the C2/C3 hydroxyl groups where additional charges are introduced with the sulfate groups.

Of the eight approaches described above for sulfation of Alg, some reactions provide key advantages over others. In reactions where DCC-H₂SO₄ is used as a sulfating agent (Reactions 5 and 8), an unwanted N-acylurea adduct is formed during the sulfation process. Particularly for biomedical applications the purity of the S–Alg product is of high importance and these methods should be avoided for such applications. Some studies have analyzed Alg and S–Alg samples via SEC, viscosity measurements, or diffusion-ordered NMR to investigate the degradation of polymers during sulfation. It should be noted that viscosity measurements alone cannot be used to evaluate degradation, as the increase in the number of charged functional groups on the polymers leads to a reduction in viscosity, regardless of chain length. Some degree of depolymerization has been reported for Reaction 1 (viscosity measurements only) and significant degradation for Reactions 5 and 8 (SEC, NMR, and viscosity measurements). Additionally, for high MW Alg polymers which are poorly solubilized in solvents such as DMF, DMSO, and formamide, it is advantageous to first convert to a tri- or tetra-butylammonium salt form to improve solubility for a subsequent sulfation step, as in Reactions 7 and 8. Based on these considerations, Reactions 3 and 7 are recommended for producing S–Alg of high purity with minimal degradation of the polymers.

2.2 Evaluation of the Degree of Sulfation

The different approaches for the synthesis of S–Alg can achieve variable DS values or the number of sulfate groups per monosaccharide. While reactions that result in sulfation at C2 and C3 hydroxyl groups have a theoretical maximum DS of 2 if sulfation selectively at the C6 carboxylate occurs then the maximum DS can be 1. The determination and reporting of DS varies widely in the literature with some reporting the number of sulfate groups per monosaccharide and some per disaccharide. In this review, all values have been normalized to represent values per monosaccharide. Many studies fail to detail the characterization of S–Alg, including the DS value, which is a key parameter to quantify in order to reproduce the work, an important aspect recently highlighted for biomedical science.^[45] Figure 1A summarizes the range of DS values that have been reported for each synthetic method. Each method of sulfation yields a wide range of DS mainly depending on the ratio of sulfating agent to uronic acid monomer, while the MW of Alg and reaction conditions including temperature and reaction time may also impact the outcome. So far the highest DS reported was 1.9 using either the N(SO₃Na)₃ sulfating agent^[39] or SO₃·py.^[31] When using the N(SO₃Na)₃ sulfating agent,^[39] a reagent ratio of 2:1 (mol ratio of sulfating agent to Alg monomer) and a temperature of 40°C was found to be optimal. Furthermore, the highest DS of 1.9 was obtained at pH 9 with both lower and higher pH yielding lower DS. The method of C6 sulfation using 2-aminoethyl hydrogen sulfate was able to achieve a DS between 0.01 and 0.13 (with a theoretical maximum of 1).^[42]

Studies using the same sulfating agent, but with either Alg or T-Alg as a starting material, were compared to assess the effect of the starting material on the resulting DS. As displayed in Figure 1B,C, Reactions 2 and 7 (SO₃·py) were compared, as well as Reactions 5 and 8 (DCC-H₂SO₄). For S–Alg produced using the SO₃·py sulfating agent there is a general trend of a higher ratio of SO₃·py:Alg yielding a higher DS, regardless of whether the starting material was Alg or T-Alg. This trend is, however, not linear, and it appears that it is difficult to control DS if targeting DS values between 0.5 and 1.5. The highest DS achieved was for the T-Alg starting material which required a large excess of sulfating agent,^[31] however, only relatively lower ratios of sulfating agent have been used for Alg as a starting material. When using DCC-H₂SO₄ for synthesis of S–Alg, there has been very high variability in the resulting DS when T-Alg was used, with no clear trend for the effect of reagent ratio on DS. While it appears that better control of DS can be achieved using Alg as starting material, all data for this reaction were from two publications from a single research group and as such reproducibility cannot be assessed.^{[25, 46]}

To accurately determine the ratio of sulfating agent to Alg monomer, the degree of substitution and average monomer weight of T-Alg should be known and it should not simply be assumed that 100% conversion to the Bu₄N⁺ or Bu₃HN⁺ salt occurs. However, the characterization of T-Alg is rarely reported. Maatouk et al. (2021) reported the monomer weight of their T-Alg but did not detail how this was determined,^[47] while our recent study detailed the evaluation of T-Alg monomer weight from elemental analysis.^[31] The lack of this information in most reported studies means that it is presently not possible to accurately determine the effect of using T-Alg on the resulting DS from the current literature.

Elemental analysis has been the primary method used to determine DS of S–Alg. In addition, the sulfur or sulfate content of S–Alg can be determined through other techniques including ion chromatography^{[48, 49]} or nephelometry and turbidimetry measurements of BaSO₄,^{[14, 20, 39, 50]} as well as FTIR^[31] (see full description Section 2.3). Elemental analysis seems to be highly accessible these days and all recent studies (from 2017) that report DS values have used this method. It should be noted, however, that there have been different interpretations of elemental concentrations. Most research groups either use the S % only or S % and C % in formulas such as those listed in Table 1.

Equation 1 was first reported by Fan et al. (2011)^[39] and is derived from the theoretical S % being equal to the mass of sulfur (DS × 32 g mol⁻¹), divided by the monomer molar mass of Alg (C₆H₇O₆Na, 198 g mol⁻¹) plus the mass of SO₃Na groups present (DS × 103 g mol⁻¹) minus protons that are substituted for SO₃Na (DS × 1 g mol⁻¹), × 100%. A similar formula used by Xin et al. (2016) is Equation 2.^[53] The derivation of this formula is not explained in text, other than the assumption that the carboxylate is a Na⁺ salt which indeed is already taken into account in Equation 1. Therefore, Equation 2 will overestimate the DS from S % obtained from elemental analysis. Equation 3 was first reported by Park et al. (2018)^[26] and is derived from the of moles of sulfur (S % × 32 g mol-1) per 6 moles of carbon (6 × C % × 12 g mol⁻¹) as there are 6 carbon atoms per uronic acid monomer. This formula had previously been applied to the sulfation of other polysaccharides with 6 carbons per monomer using a variety of sulfating agents.^[56]

It is important to note that different groups make assumptions about the hydration and ionization state of S–Alg when calculating DS. Martinsen et al. conducted a study in 1991 determining MW of Alg polymers from two species of algae, including analyzing the water content by Karl Fischer titration.^[57] It was found that there were approximately two water molecules bound per uronic acid monomer. Subsequent papers referring to the Martinsen et al. study follow this thinking but only assume one water molecule per uronic acid monomer.^{[17, 58]} When determining DS from Equation 1 or 2, this does not take into account the presence of any water in the sample, so may lead to underestimating the DS. By using an element ratio such as S % : C % an accurate DS can be obtained without having to determine the amount of water present in the sample. It should be noted that for reactions that may result in the formation of an N-acylurea adduct (Reactions 5, 8, and 9), this method would underestimate DS.

It is often not explicitly stated, but most groups assume that each negative charge (sulfate and carboxylate) of S–Alg is in the sodium salt form,^{[16, 17]} and when using Equation 1 for DS calculation this is implicit. However, few studies actually measure the sodium content of S–Alg. Almost all sulfation methods have a final step where the pH is adjusted to above 8 with NaOH (aq), and considering the pK_a values of sulfate (0.5−1.5 for heparin)^[59] and carboxylate (3.38−3.65 for Alg),^[60] it is reasonable to assume the resulting S–Alg will have the charge on sulfate and carboxylate groups balanced by Na⁺ ions. However, what is often overlooked in the Fourier transform infrared (FTIR) spectra (described below) is the presence of a COOH shoulder at 1730 cm⁻¹. If this shoulder is present in the S–Alg spectrum, then there must be some monomers that are in the carboxylic acid form.^[57] This highlights the importance of using both S % and C % when calculating DS as it is not affected by the sodium content.

2.3 Spectroscopic Characterization

A common spectroscopic characterization technique used to verify the successful sulfation of Alg is FTIR. Figure 2A shows representative FTIR spectra for Alg, T-Alg, and S–Alg (DS = 1.8) synthesized by Reaction 7 (SO₃·py method via T-Alg), with relevant chemical structures shown in Figure 2B. Figure 2C lists the assignments of the main bands in the FTIR spectra based on previous literature.^{[16, 25, 61, 62]} Compared to the spectrum of Alg, the new bands from the tributylammonium ion in T-Alg appear between 2980 and 2830 cm⁻¹ due to the C–H stretches, as well as a C–N stretch at 1460 cm⁻¹ as previously reported.^{34, 62} Also present in the T-Alg spectrum is a small band at 1730 cm⁻¹ which results from the C═O stretch of –COOH groups as mentioned above. The most prominent change in the FTIR spectrum for S–Alg compared to Alg or T-Alg is the band at 1230 cm⁻¹ representing the S═O stretch. There is also a band at 1730 cm⁻¹ suggesting some of the carboxylate groups of this polymer are in the protonated form. Some literature spectra show a similar small shoulder to the one seen in Figure 2A, however, others have more pronounced bands at 1730 cm⁻¹ while others again have no such band present. We recently reported how FTIR can be used for estimating the DS of S–Alg.^[31] Thus, a linear correlation between the intensity of the absorbance of the S═O band (1230 cm⁻¹) relative to the Alg carboxylate band (1600 cm⁻¹) and DS determined from elemental analysis was established for four different types of alginates (different M/G ratio and MW).

The use of ¹H nuclear magnetic resonance spectroscopy (NMR) for the characterization of S–Alg is not commonly reported. This is likely due to the complex spectra where the chemical shifts for protons of the M and G residues depend on the neighboring monomers.^{[63, 64]} This is further complicated by the presence of water in the sample, as the anomeric peaks fall at a similar chemical shift to the HOD peak and can become completely obscured. However, we recently demonstrated the usefulness of ¹H NMR to evaluate if an N-acylurea adduct formed when carbodiimide chemistry is used in the sulfation reaction (Reaction 8).^[31]

Low MW (up to 14 kg mol⁻¹) sulfated poly(guluronic acid), sulfated poly(mannuronic acid), and S–Alg have been characterized using ¹³C NMR, allowing assignment of resonances arising from the C-atoms of each of the monomer units, and in addition for those C2 and C3 hydroxyl groups that are sulfated.^{[14, 17, 49, 65]} To assess regioselectivity in the sulfation reaction, studies have used ¹H,¹³C-heteronuclear single quantum coherence (HSQC) NMR for such low MW samples and observed preferential sulfation on C2 hydroxyl groups (compared to C3). This preference was observed for sulfated poly(guluronic acid), sulfated poly(mannuronic acid), and S–Alg produced using Reaction 3.^[17] The use of ¹³C NMR to characterize S–Alg of high MW is complicated by broad peaks and significant signal overlap for the resonances for C2, C3, C4 and C5 in the 60–80 ppm range. However, using ¹H,¹³C-HSQC NMR, we recently demonstrated successful evaluation of the regioselectivity of Reaction 7 applied to a high MW Alg polymer and found preferential C2 hydroxyl group sulfation.^[31] Other work, evaluating high M-content S–Alg prepared using Reaction 4, observed a weaker signal for C2 than C3 sulfated signals for M residues, indicating a preference for substitution of the C3 hydroxyl in M monomers.^[35] This was attributed to the hindrance of the axial C2 hydroxyl compared with the equatorial C3 hydroxyl in the M monomer.

Esposito et al. recently published their work utilizing multi-step strategies to achieve regioselective sulfation of Alg on the C2 or the C3 hydroxyl group as desired.^[55] For this process, low MW mannuronic acid enriched alginic acid (9 kg mol⁻¹, 83% M) was used as the starting material, and SO₃·py was the sulfating agent (Reaction 2), applied after different regioselective protection strategies. ¹H,¹³C-HSQC NMR was the primary characterization technique for confirming the site of sulfation on various S–Alg products. This is an important step toward the ability to tailor the sulfation pattern of S–Alg.

4.1 Cross–linking Strategies

S–Alg has been used to fabricate a variety of biomaterials including bulk hydrogel materials,^{[23, 33, 97-104]} particles,^{[27, 54, 102, 105-114]} and scaffolds,^{[22, 26, 115-125]} and has also been used as a component of composites and as a coating material^{[47, 52, 126-131]} as illustrated in Figure 4. To achieve cross-linking of the S–Alg polymers to fabricate hydrogels, two main cross–linking strategies have been used: ionic and covalent. Ionic cross–linking, predominantly using calcium ions, is the most commonly used method for the fabrication of S–Alg-based materials.^[8-10] It has been reported that S–Alg with a DS ≥ 0.5 cannot form a true hydrogel on the macroscale using calcium cross–linking and that the stiffness of hydrogels decreases with increasing DS.^[97] Two main strategies have been used to overcome this. One is to use a mixture of Alg and S–Alg to form hydrogels with better osmotic stability and stiffness.^{[97, 109]} The other is to use either barium or strontium salts, often in combination with a calcium salt.^{[23, 101, 109, 112]} Both barium and strontium form stronger ionic cross–links than calcium alone and can afford cross–linking not only of the G-units but also of the M-units, hence are able to improve material properties.^{[8-10, 132]} It should be noted, however, that barium is toxic with high doses causing paralysis or death in humans and in lower doses can be harmful in children and fetuses.^[132] It is expected that all ionically cross–linked alginate hydrogels will release the cross–linking ions over time, so care needs to be taken to adjust the total amount of barium used.

Covalent cross–linking of S–Alg polymers is often achieved by chemical modification to introduce additional chemical moieties such as vinyl groups for photo cross–linking^{[33, 98]} or tyramine for enzymatic cross–linking using tyrosinase in the presence of oxygen.^[100] Vinyl groups were introduced either using carbodiimide chemistry and reaction with 2-aminoethyl methacrylate (AEMA) (Scheme 4A) or using methacrylic anhydride (Scheme 4B), while tyramine was introduced using the coupling agent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) (Scheme 4C). When using carbodiimide chemistry, a reaction with the carboxylate groups of S–Alg reduces the overall charge of the polymer, while the use of an anhydride affords a reaction with the hydroxyl groups. In all cases, the synthetic strategy for producing these dual-modified Alg polymers has been to first introduce the sulfate groups (Scheme 4). In contrast, the use of boric acid as a covalent cross-linker does not require modification of S–Alg as it affords cross-linking between hydroxyl groups. Covalently cross–linked Alg and S–Alg hydrogels have higher stability and resistance to degradation under mild alkaline, low pH, or neutral conditions compared to ionically cross–linked hydrogels.^[133]

4.2 Fabrication of S–Alg-based Bulk Materials

S–Alg-based bulk hydrogel materials have been used to encapsulate cells and/or proteins and the studies are summarized in Table 3 (entries 1–10). In all cases, the cargo was premixed with the polymer solution prior to cross–linking. In the cases of ionic cross–linking, various approaches were used. In one case, a calcium solution was added to the S–Alg solution with stirring until the gel formed.^[104] Other studies produced a pre-gel (viscous solution of polymer and cargo) which was either cast,^[97] cast and frozen,^[102] or cast in silicone mold that was sandwiched between dialysis membranes.^{[23, 101]} Cross–linking was then achieved either by immersion of the pre-gel into the cross–linking solution or by adding the cross–linking solution on top of the as-cast pre-gel. In one study by Gionet-Gonzales et al.,^[23] the S–Alg polymer was mixed with Alg modified by periodate oxidation and/or modified with an RGD-peptide sequence attached via carbodiimide chemistry. It was shown that the degree of oxidation affected the rate of degradation of the hydrogel disks and as such, could be used to tune the degradation rate where a higher degree of oxidation (5% compared to 1%) caused faster degradation. Furthermore, it was confirmed that the DS value did not affect the hydrogel properties such as elastic modulus and swelling ratio.

Two studies fabricated hydrogels by mixing two polymers with complementary functional groups. In one study, an S–Alg polymer modified with adamantane groups (Scheme 4C) was mixed with a poly-β-cyclodextrin (CD) to form a cross–linked hydrogel via host-guest interactions.^[103] It was demonstrated that disassembly of the hydrogel could be controlled by adding β-CD monomer which can compete with poly-β-CD for the adamantane groups. In another study, polyelectrolyte complexes formed between S–Alg and CS upon mixing, and these were further cross–linked with calcium ions.^[33] This study found that the degree of sulfation affected the compressive modulus of the gels, both as prepared and after partial degradation.

Formation of covalently cross–linked S–Alg-based hydrogel materials has been achieved using various synthetic strategies. Tyramine modified S–Alg (Scheme 4D) was cross–linked using tyrosinase and activation by oxygen.^[100] It was found that there was no difference in the compressive modulus compared to control hydrogels made from tyramine-modified Alg, nor was the swelling behavior different, which was not observed for control hydrogels prepared using ionic cross–linking where the swelling behavior was dependent on the DS. UV-cross–linking of methacrylated S–Alg was utilized in two studies, one which used S–Alg modified with AEMA through carbodiimide chemistry (Scheme 4A)^[98] and one which used S–Alg modified using methacrylic anhydride (Scheme 4B),^[33] in both cases for cell encapsulation.

In the study by Baei et al., two different approaches to the formation of S–Alg/CS hydrogels were explored.^[33] One approach used S–Alg modified with catechol groups through carbodiimide chemistry (Scheme 4B). This polymer was mixed with CS and cross–linking facilitated through oxidation with periodate with the intent to oxidize the catechol to o-quinone which would then react with the amine group of CS. It was acknowledged that periodate can also cause oxidation of the polysaccharides, potentially leading to Schiff base formation with the amine groups of CS. The second approach involved methacrylation of S–Alg followed by attachment of catechol groups (Scheme 4B) as well as methacrylation of CS. This allowed the formation of a dual-covalently cross–linked hydrogel with superior mechanical properties compared to the singly-covalently cross–linked hydrogel. In both cases, the hydrogels were additionally ionically cross–linked by calcium ions which affected their stiffness. The catechol-containing hydrogels were black in color, attributed to polymerized catechol.

4.3 Fabrication of S–Alg Particles

Various methods have been used for the fabrication of S–Alg-based particles for cell and drug delivery. To encapsulate cells or cell spheroids in S–Alg-based particles, premixing of the components was done prior to cross–linking in all cases.^{[109, 111, 112]} When encapsulating drugs, however, both a pre-mixing and a post-loading approach have been reported. Particle fabrication was achieved using nanoprecipitation,^{[54, 106-108, 110, 113, 114]} as well as spraying or injection into a cross–linker-containing bath.^{[27, 102, 105, 109, 111, 112, 134]} These studies are summarized in Table 3 (entries 10–22). In this review, the term nanoparticles will refer to particles up to 300 nm in diameter, while particles ranging from 301 to 999 nm will be termed sub-micron particles. Any particles larger than 1 µm will be classified as micron-sized particles. This follows the definition of polymer particle sizes set down by the National Science and Technology Council, USA.^[135]

Fabrication of submicron-sized particles has been achieved using a mixture of Alg and S–Alg to encapsulate the peptide calcitonin where the peptide–polysaccharide mixture was injected into a calcium bath.^[27] While pure peptide self-assembled into particles (680 nm), the presence of the Alg–S–Alg mixture greatly reduced the size to 320–380 nm at optimized reagent ratios.^[27] VEGF was loaded into particles prepared from a mixture of Alg and S–Alg by premixing and spraying the mixture into a calcium bath, achieving micron-sized particles.^[102] An alternative approach of post-loading was used to encapsulate bone morphogenetic protein 4 (BMP-4) into micron-sized particles produced by injecting S–Alg or mixtures of Alg and S–Alg into a calcium bath.^[105]

Nanoparticles have been produced using nanoprecipitation which involves the mixing of precursor solutions with agitation. Work by the group of Cohen, encapsulated oligonucleotides using nanoprecipitation with vortexing and stirring.^{[106, 107]} In one study, the effect of the order of mixing S–Alg, calcium, and small interfering ribonucleic acid (siRNA) solutions demonstrated no difference in particle size which was 130–140 nm.^[107] Encapsulation of plasmid deoxyribonucleic acid (pDNA) in calcium cross–linked S–Alg resulted in a particle size of 270 nm which was significantly larger than those encapsulating siRNA related to the different sizes of the oligonucleotide.^[106] Our recent work evaluated the encapsulation of lactoferrin in S–Alg particles using sonication in combination with nanoprecipitation.^[54] A reduction in size was found for particles prepared using S–Alg (175 nm) compared to those made from Alg (245 nm), and while similar particle sizes were observed using S–Alg with DS values of 0.8 and 1.1, a significantly larger particle size was found using S–Alg with a DS of 1.7. Nanoprecipitation was also used to form core–shell nanoparticles from PSS with the anticancer drug doxorubicin (DOX) covalently attached through an imide bond (Scheme 4D). Particle fabrication was achieved by dropwise addition of an organic solution of the PSS-DOX conjugate mixed with a second anticancer drug CXB into an aqueous solution of borax acting as the cross-linking agent. This allowed the formation of particles <150 nm in size.^[114]

The requirement for ionic cross–linking using calcium ions was explored in our study where it was demonstrated that the HBP lactoferrin could form S–Alg-based nanoparticles in the absence of calcium ions.^[54] Direct assembly of nanoparticles without the use of a cross–linker was likewise achieved using proteins with high binding affinity for S–Alg.^[110] Specifically, a small matrix MMP-7 inhibitor protein, C9, modified with heparin-binding protein domains was mixed with S–Alg, vortexed, and incubated overnight to form spherical nano-sized (≈10 nm) complexes. Another example encapsulating the HBPs hepatocyte growth factor (HGF) and insulin-like growth factor 1 (IGF-1) in S–Alg particles using nanoprecipitation with vortex and no calcium cross–linker formed particles of 50–100 nm in size.^[113] These studies demonstrate that polyelectrolyte complex formation between high pI proteins and S–Alg can facilitate particle formation.

4.4 Fabrication of S–Alg Particles

This section includes hydrogel scaffolds that incorporate S–Alg. In some cases, S–Alg is a major component while S–Alg has also been used to prepare hydrogel scaffolds where the major component is for example Alg, gelatin methacrylate (GelMA) or polyvinyl alcohol (PVA). These studies are summarized in Table 3 (entries 23–35). Scaffolds have been made using bioprinting,^{[26, 115, 116, 120, 125]} electrospinning,^{[22, 118, 119, 121, 122]} particle assembly,^[117] or freeze drying.^{[123, 124]} Cross–linking of the S–Alg component in the scaffolds was done mainly using a calcium source, although other strategies have been used with examples included below.

Bioprinting of hydrogel scaffolds involves the fabrication of a bioink that is printed onto a platform and subsequently or simultaneously, the bioink is cross–linked. An important factor for the printability of a bioink is its rheological properties. While some studies have reported that the addition of S–Alg into Alg bioinks causes a decrease in the viscosity of the bioink^[115] or a decrease in scaffold stiffness,^[120] other studies have not observed such effects.^[26] The reduction in viscosity of S–Alg compared to Alg has been demonstrated to depend both on the MW and DS of S–Alg.^[31] Therefore, it is important to consider the DS and the amount of S–Alg included in bioinks.

The use of bioprinting allows for the incorporation of cells within the scaffold as demonstrated in some studies.^{[26, 116, 120, 125]} An interesting approach to bioprinting was demonstrated using S–Alg (DS = 0.8) combined with Alg, where selective printing of primary neurons in the S–Alg gel within an Alg carrier restricted the mobility of the neurons.^[120] Another example of compartmentalizing different components using bioprinting is the encapsulation of cardiac cells in an RGD-modified Alg matrix which was surrounded by an outer shell of S–Alg encapsulating extracellular vesicles (EVs).^[116] A study making use of Alg and S–Alg in GelMA bioinks and creating scaffolds through dual ionic and UV-cross–linking demonstrated that the presence of S–Alg improved the compressive properties of cell and protein-laden scaffolds.^[125]

Studies that have prepared fibrous scaffolds using electrospinning have in all cases used a combination of S–Alg and PVA. Different cross–linking strategies were used including ionic,^[22] thermal,^[121] and covalent using glutaraldehyde as the cross–linker.^{[118, 119, 122]} When using high weight ratios of S–Alg (DS = 0.9) (PVA:S–Alg from 7:3 to 5:5), nanofibers with diameters ranging from 75 to 280 nm could be produced.^[22] Furthermore, it was shown that increasing the S–Alg content caused less bead formation on the nanofibers. Another study found that using S–Alg (30 wt.%) rather than pure PVA enhanced nanofiber uniformity and reduced the fiber diameter significantly from 450 to 185 nm.^[119] Similar observations of lower average fiber diameter and higher uniformity when fibers are produced with S–Alg have likewise been made in other studies.^{[118, 121, 122]} It was demonstrated that increasing the S–Alg content caused an increase in swelling and degradation rate and furthermore, a reduction in mechanical properties.^[122]

4.5 Fabrication of S–Alg Particles

For applications of S–Alg in composites and as coatings, S–Alg does not form the main component and is combined with materials such as thermoplastic polymers,^{[47, 52, 128-131]} polyurethane elastomers^[34] or metal oxide particles,^[126] while some studies used a model gold substrate for fundamental studies.^{[127, 136]} These studies are summarized in Table 3 (entries 36–45).

In some composites, the different components of the composite are fabricated separately before final assembly. One example is ketogenic-loaded PLGA nanoparticles inside a calcium cross–linked Alg–S–Alg hydrogel which was placed between poly(ε-caprolactone) (PCL)–gelatin electrospun mats to enhance the mechanical performance.^[130] A similar material was made from a calcium Alg–S–Alg hydrogel sandwiched between PCL–gelatin electrospun mats for cell delivery.^[128] This study found that increasing the number of electrospun mat layers reduced the degradation rate and this was attributed to decreasing the ion exchange inside the composite. Another approach is to add a mixture of Alg, S–Alg, and decellularized extracellular matrix inside a 3D printed PCL scaffold prior to freeze-drying and cross–linking the hydrogel with calcium solutions.^[131] Alternatively, a single-step fabrication approach was used to make core-shell particles with PCL forming the shell and S–Alg loaded with GFs forming the core via a water-in-oil-in-water double emulsion method.^[47] Covalent attachment of S–Alg to the end-groups of NCO-terminated cationic polyurethane allowed fabrication of supramolecular polyurethanes that were ionically cross–linked, facilitated by the sulfate and carboxylate groups of S–Alg, with the amines of the polyurethane.^[34]

There are a few studies that have coated S–Alg or a mixture of Alg and S–Alg onto scaffolds. One approach was to covalently cross–link Alg and S–Alg with the cross–linker adipic acid dihydrazide using carbodiimide chemistry (Scheme 4A) as a coating on a poly(lactide-co-ε-caprolactone) (PLCL) scaffold.^[129] This study found that the presence of Alg was required to increase the solution viscosity. A different approach to coating was applied to PCL nanofiber scaffolds that were surface-activated by cold atmospheric plasma to facilitate direct coating with S–Alg.^[52] S–Alg coatings have also been applied to Fe₃O₄ particles.^[126] The use of dopamine-conjugated S–Alg facilitated not only electrostatic attraction between S–Alg and the Fe₃O₄ particles but also afforded covalent attachment of the mussel-inspired adhesive macromolecule. The coated particles had a negative zeta potential (−48 mV) while the parent particles were positively charged (+ 52 mV). Based on a reduction in mean particle size after applying the coating (from 930 to 350 nm) it was concluded that the S–Alg coating suppressed particle aggregation. Unfortunately, these studies do not evaluate the longevity of the coating which is a parameter that is difficult to predict and even some covalently attached coatings have shown poor adhesion stability after immersion in simple solutions.^[137]

5.1 Binding affinity of S–Alg to Proteins and Other Therapeutics

One important property of S–Alg, is the ability to bind HBPs with high binding affinity similar to heparin and heparan sulfate. Earlier work evaluated a range of GFs including EGF and IGF and the cytokine interleukin 6 (IL-6).^{[24, 54, 140]} In comparison, Alg does not show specific binding affinity to HBPs and this has been verified in recent works. Evaluation of the binding affinity is achieved using either surface plasmon resonance (SPR) or a quartz crystal microbalance with dissipation (QCM-d). In both techniques, the S–Alg polymer is often attached to a surface using various conjugation chemistries and as such, the biomolecule may be attached in different conformations.

In recent years a number of additional HBPs have been evaluated. Itzhar et al. produced heparin-binding peptides and used SPR to evaluate the affinity of three such peptides to S–Alg. All demonstrated high binding affinity (K_A = 2.6 × 10⁶–5× 10⁷ M⁻¹) to S–Alg polymer, as well as heparin.^[110] Anitha et al., determined the binding affinity of lactoferrin to S–Alg (DS = 0.6) using SPR to be K_A = 2.0 × 10⁷ M⁻¹ which was similar to that reported for heparin.^[54] SPR has also been used to evaluate binding of EVs to S–Alg where very high binding affinity (K_A = 3 × 10¹⁴ M⁻¹) was similar to that of heparin (K_A = 1 × 10¹⁴ M⁻¹).^[116] A study by Ma et al., investigated the binding affinity between PSS and FGF-2 as well as VEGF165.^[68] It was observed that FGF-2 had a similar binding affinity to PSS (K_A = 3.6 × 10⁷ M⁻¹) as to heparin (K_A = 3.6 × 10⁷ M⁻¹), while VEGF165 showed specific binding to heparin (K_A = 1.2 × 10⁶ M⁻¹) and only weak binding to PSS (K_A = 5.6 × 10³ M⁻¹).

The Group led by Mhanna has used QCM-d to study the binding of the HBP FGF-2 to S–Alg in different ways. In their 2017 study, layer-by-layer assemblies of collagen and S–Alg were created on the gold-coated QCM crystal, and it was shown that higher binding affinity was achieved when using S–Alg with higher DS (DS = 0.4, 1.0, 1.3).^[136] In their 2019 study, the S–Alg polymers were attached to the QCM crystal via end-group functionalization. A similar finding was made in that the DS (0.4, 1.0, 1.3) of S–Alg was found to affect the binding of FGF-2 with an increased binding affinity for the polymers with higher DS.^[127] Their 2020 study evaluated the effect of the concentration of S–Alg used for attachment to the QCM crystal and found higher FGF-2 and β-NGF binding affinity (both HBPs) when a higher concentration of S–Alg had been used.^[120]

5.2 Application of S–Alg to Sustain the Release of Therapeutic Agents

Encapsulation of a range of pharmaceutical compounds in a hydrogel-based material fabricated from or incorporating S–Alg has been demonstrated in numerous studies. This includes small molecule drugs, HBPs, other proteins, oligonucleotides, EVs, as well as nanoparticles encapsulating drugs. In the area of wound healing (a total of four articles identified) various approaches have been taken including encapsulation of platelet-derived growth factor (PDGF) for enhanced vascularization^[98] and encapsulation of Prussian blue nanozymes for enhanced would closure and macrophage phenotype regulation.^[104] In bone tissue engineering, strategies include delivery of the single-chain 32 amino acid polypeptide hormone salmon calcitonin (sCT)^[27] and co-encapsulating BMP-2 with MC3T3-E1 cells to promote osteogenesis.^[26]

The presence of S–Alg can effectively regulate both the EE and release profile of many therapeutic compounds. Enhanced EE has been demonstrated for transforming growth factor-beta 1 (TGF-β1) in a Ca²⁺-cross–linked S–Alg scaffold where a higher amount was encapsulated in the S–Alg materials compared with the Alg control (151 ng and 128 ng, respectively),^[141] and for the positively charged small molecule drug tetracycline hydrochloride with EE of 35% in S–Alg particles, compared to 1% in Alg particles.^[108] The EE of the low pI protein BSA can also be increased when using S–Alg compared to Alg in double emulsion PCL particles (94 versus 88%, respectively). However, the presence of S–Alg rather than Alg increased the rate of release of BSA in PBS in accordance with higher electrostatic repulsion for the S–Alg containing particles.^[47] In contrast, Munarin and colleagues found no to a minor decrease in EE upon encapsulating VEGF in S–Alg compared to Alg control in Ca²⁺ cross–linked materials (EE of 66 and 70%, respectively). The S–Alg containing materials were, however, able to attain a better controlled and sustained release of VEGF in PBS compared to the Alg control.^[102]

Many studies have observed a similar ability of S–Alg to control and prolong the release of HBP which has been attributed to the higher binding affinity of S–Alg compared to Alg for HBPs as discussed in Section 5.1. Studies include PDGF-BB encapsulated in photo-cross–linked S–Alg based materials,^[98] BMP-4 in Ca²⁺-cross–linked particles,^[105] TGF-β1 in thermally cross–linked PVA/S–Alg nanofibrous scaffolds,^[121] BMP-2 in Ca²⁺-cross–linked 3D printed scaffolds.^[26] In the study by Baei et al., there was a direct correlation between the rate of release of TGF-β1 encapsulated in an interpenetrating network (IPN) scaffold and DS (0–0.67) with the total amount released over 14 days, reducing from 83% to 42% with increasing DS.^[33] In addition to its high binding affinity to HBPs, the negative charges of S–Alg also contribute to slowing down the release of the positively charged polypeptide hormone sCT when encapsulated in Ca²⁺-cross–linked particles where the presence of S–Alg reduced the burst release compared to the Alg control and furthermore, extended the overall release.^[27]

It should be noted that in the studies described above, PBS was used as the release medium. The name PBS is used for buffers of different compositions (with or without added Ca²⁺ and Mg²⁺, different ionic strength) which is not always explicitly reported. The importance of this is that PBS has a significantly higher phosphate content than serum and that PBS (without added calcium ions) will superficially enhance the release rate of cargo from Ca²⁺-cross–linked Alg-based materials due to the high affinity between Ca²⁺ and phosphate ions.^[142]

Many studies have used tissue culture medium to study the release of HBP for S–Alg-based materials and in these studies, the presence of S–Alg again controlled and prolonged the rate of release. The cumulative release over 7 days of TGF-β3 from IPN constructs was 66% for control Alg materials compared to 42% for the S–Alg containing materials, and sustained release of TGF-β3 over 7 days was observed for the S–Alg containing IPN.^[139] The same lab reported an initial burst release of TGF-β3 from Alg-containing scaffolds (55% in 3 days) while the sustained release was observed from S–Alg-containing scaffolds.^[129] The release of TGF-β1 from a Ca²⁺-cross–linked S–Alg scaffold and Alg control displayed a burst release of 20 and 56%, respectively, and while the HBP was released (≈100%) from the Alg control in 3 days, the S–Alg containing scaffold afforded release (≈92%) over 7 days.^[123]

An interesting observation was made in the study by Gionet-Gonzales et al. who investigated the binding and release of IGF-1, a non-HBP high pI protein (pI = 8.5^[143]), and the HBP HGF from ionically cross–linked disks in serum-free media.^[23] They observed higher burst release for the disks produced using S–Alg with a high DS compared to Alg and S–Alg with a low DS for IGF-1, whereas a direct correlation between DS and EE and release rate was observed for HGF, with faster release from the Alg control. This demonstrates the specific affinity of S–Alg for HBPs.