**Figure 5.** Various approaches for preparing ALG‐based particulate carrier matrix. Below are a few vital employments of ALG‐mediated systems for enhanced actives delivery through various administration routes. *6.1. Oral Drug Delivery* Oral drug delivery is the first choice route of drug delivery for any therapeutic sub‐ stance due to its simplicity, patient compliance, cost‐effectiveness, easy bulk manufactur‐ ing, sustained and controlled release, and generation of immune response in case of vac‐ delivery through various administration routes. *6.1. Oral Drug Delivery* Oral drug delivery is the first choice route of drug delivery for any therapeutic sub‐ stance due to its simplicity, patient compliance, cost‐effectiveness, easy bulk manufactur‐ ing, sustained and controlled release, and generation of immune response in case of vac‐ cination [121–123]. However, some limitations exist, such as variable pH of the gastroin‐ testinal tract (the stomach is highly acidic while the small intestine is alkaline), first‐pass metabolism in the liver, the occurrence of hydrolytic enzymes, and an absorption barrier Oral drug delivery is the first choice route of drug delivery for any therapeutic substance due to its simplicity, patient compliance, cost-effectiveness, easy bulk manufacturing, sustained and controlled release, and generation of immune response in case of vaccination [121–123]. However, some limitations exist, such as variable pH of the gastrointestinal tract (the stomach is highly acidic while the small intestine is alkaline), first-pass metabolism in the liver, the occurrence of hydrolytic enzymes, and an absorption barrier in the liver intestine. This limits the extent of the therapeutic action of many drugs given by the oral route. Nevertheless, due to their non-toxicity, biocompatibility, and biodegradability, ALG hydrogels can be safely administered orally [124].

cination [121–123]. However, some limitations exist, such as variable pH of the gastroin‐ testinal tract (the stomach is highly acidic while the small intestine is alkaline), first‐pass metabolism in the liver, the occurrence of hydrolytic enzymes, and an absorption barrier in the liver intestine. This limits the extent of the therapeutic action of many drugs given by the oral route. Nevertheless, due to their non‐toxicity, biocompatibility, and biodegra‐ dability, ALG hydrogels can be safely administered orally [124]. in the liver intestine. This limits the extent of the therapeutic action of many drugs given by the oral route. Nevertheless, due to their non‐toxicity, biocompatibility, and biodegra‐ dability, ALG hydrogels can be safely administered orally [124]. Recently, Ilgin et al. used SA hydrogels as the carrier for diclofenac sodium to obtain controlled drug delivery at a specific pH. The porous structure made them valuable as a drug delivery system. The researchers prepared SA pH-responsive semi-interpenetrating hydrogels by altering the physical and biological properties of the biocompatible and biodegradable ALG via surface functionalization with the aid of monomers such as HEMA, MAPTAC, and MA. Polyvinyl alcohol (PVA) was employed to provide chemical stability,

and nMBA was the copolymer. The resultant hydrogels were evaluated for their swelling, drug loading, drug release, and antimicrobial activity. PVA and HEMA proved to be good polymeric systems because they enhanced biocompatibility and mechanical properties. Maximum swelling (38 gwater/ggel), drug loading (22.8%), and release profiles were observed at pH 7.0, where 95% release of drug was observed at the end of 2 h, as compared to 4.5% at pH 1.5. Antibacterial activity was observed against *E. coli*, *B. subtilis*, and *S. aureus* by the disk diffusion method, and it displayed excellent inhibition of the growth of all three microorganisms. Thus, ALG is confirmed to be a great, intelligent drug release platform for oral drug delivery, providing a controlled release [125].

To obtain colon-specific drug delivery for inflammatory bowel diseases such as Crohn's disease, it is challenging to move through the different pH conditions of the GI tract undisturbed. Hence, Ayub et al. tried to enhance the delivery of Paclitaxel to colonic cancer cells by formulating a self-assembled cysteamine-based disulfide cross-linked biodegradable thiolated SA-derived nanoparticles via a layer-by-layer assembly approach (as shown in Figure 6A). SA was oxidized using sodium periodate before adding cysteamine hydrochloride to alter its backbone. The disulfide bonds prevented the leakage of the drug before it reached its target site. The encapsulation efficiency of P3DL/PAH/PSCCMA was found to be 77.1%, with a cumulative-drug release of 45.1% after 24 h (as displayed in Figure 6B). The nanospheres demonstrated their maximum size at pH 7.0, thus signifying their efficacy in selectively delivering the actives to the colon. The MTT assay revealed the high viability (86.7%) of HT-29 cells (as shown in Figure 6C). More than 70% of the nanospheres were detected in human colon cancer adenocarcinoma HT-29 cells, indicating their high cellular uptake, as displayed in Figure 6D). Stability studies indicated that most nanospheres changed slightly in size and PDI but were stable with negligible differences in zeta potential. Thus, this approach could be employed for colon-targeted drug delivery with minimal toxic effects [126].

In order to furnish controlled drug delivery via the oral route, Cong et al. prepared an ALG hydrogel/CS micelle composite system encapsulating emodin. The research group prepared cross-linked micelles because, unlike conventional polymeric micelles, crosslinked micelles prevent drug leakage and protect them from dissociating quickly. Emodin was chosen as a model drug because it has poor water solubility and undergoes extensive first-pass metabolism. It was loaded onto CS micelles and then mixed with SA hydrogels developed by cross-linking with Ca2+ ions and β-GP to form hydrogel/micelle beads. Based on response surface methodology, the optimized biopolymer concentrations were determined. The 1:1 hydrogel/micelle beads showed sustained drug delivery, while the 3:1 ratio provided colon-specific delivery. The morphological, swelling, and degradation studies were carried out. The diameter of micelles increased from 80 nm in aqueous solution to 100–200 nm in the hydrogel, likely due to electrostatic interaction between the amino group of the CS chain and the carboxylate group of the ALG chain. The swelling of micelles was reduced at pH 1.2 due to SA hydrogel, whose swelling ratio is at acidic pH, thus preventing the release and degradation of CS chains. The initial release amount was 28% in SGF, and a final release amount of 85% for 1:1 micelle/hydrogel systems was reported. Thus, this pH-responsive hydrogel/micelle system could be an encouraging candidate for sustained-release or site-targeted actives delivery for unstable or hydrophobic actives [127].

Amphotericin-B is the first-choice drug for many fungal infections, including leishmaniasis. However, it has limited solubility, is susceptible to gastric pH, and its oral bioavailability is low; it is generally administered intravenously [128]. Hence, to overcome this issue, Senna et al. utilized nanostructured lipid carriers (NLC) combined with stimuli-sensitive ALG polymers using a high-pressure homogenization technique with calcium chloride as the cross-linker, providing a dual-benefit of oral drug delivery and protection from gastric pH without structural degradation and promoting drug release at intestinal pH. Nanostructured lipid carriers are systems in which hydrophilic drugs are dispersed in polymeric matrices, forming nanometric droplets, thus enhancing adsorption and preventing enzymatic degradation [129]. AmpB was solubilized by solid lipid glyceryl

monostearate (GSM). Studies using the trypan blue exclusion test showed that the NLCs showed low cytotoxicity on Vero cells (ATCC® no. CCL-81™), very high specificity, and their drug release profiles were equivalent to ALG swelling degree profiles, indicating that the drug delivery was primarily due to the polymer's swelling rate. In the responsive pH range, the carboxylic acid groups of ALGs became ionized and acquired a negative charge, resulting in electrostatic repulsion and allowing water molecules to enter. The NLC particles maintained their framework even after rehydration; thus, the aforementioned system proved promising for the delivery of AmpB orally [130]. their efficacy in selectively delivering the actives to the colon. The MTT assay revealed the high viability (86.7%) of HT‐29 cells (as shown in Figure 6C). More than 70% of the nano‐ spheres were detected in human colon cancer adenocarcinoma HT‐29 cells, indicating their high cellular uptake, as displayed in Figure 6D). Stability studies indicated that most nanospheres changed slightly in size and PDI but were stable with negligible differences in zeta potential. Thus, this approach could be employed for colon‐targeted drug delivery with minimal toxic effects [126].

Recently, Ilgin et al. used SA hydrogels as the carrier for diclofenac sodium to obtain controlled drug delivery at a specific pH. The porous structure made them valuable as a drug delivery system. The researchers prepared SA pH‐responsive semi‐interpenetrating hydrogels by altering the physical and biological properties of the biocompatible and bi‐ odegradable ALG via surface functionalization with the aid of monomers such as HEMA, MAPTAC, and MA. Polyvinyl alcohol (PVA) was employed to provide chemical stability, and nMBA was the copolymer. The resultant hydrogels were evaluated for their swelling, drug loading, drug release, and antimicrobial activity. PVA and HEMA proved to be good polymeric systems because they enhanced biocompatibility and mechanical properties. Maximum swelling (38 gwater/ggel), drug loading (22.8%), and release profiles were ob‐ served at pH 7.0, where 95% release of drug was observed at the end of 2 h, as compared to 4.5% at pH 1.5. Antibacterial activity was observed against *E. coli*, *B. subtilis*, and *S. aureus* by the disk diffusion method, and it displayed excellent inhibition of the growth of all three microorganisms. Thus, ALG is confirmed to be a great, intelligent drug release

To obtain colon‐specific drug delivery for inflammatory bowel diseases such as Crohn's disease, it is challenging to move through the different pH conditions of the GI tract undisturbed. Hence, Ayub et al. tried to enhance the delivery of Paclitaxel to colonic cancer cells by formulating a self‐assembled cysteamine‐based disulfide cross‐linked bio‐ degradable thiolated SA‐derived nanoparticles via a layer‐by‐layer assembly approach (as shown in Figure 6A). SA was oxidized using sodium periodate before adding cysteamine hydrochloride to alterits backbone. The disulfide bonds prevented the leakage of the drug before it reached its target site. The encapsulation efficiency of P3DL/PAH/PSCCMA was found to be 77.1%, with a cumulative‐drug release of 45.1% after 24 h (as displayed in Figure 6B). The nanospheres demonstrated their maximum size at pH 7.0, thus signifying

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 14 of 60

platform for oral drug delivery, providing a controlled release [125].

**Figure 6.** Biocompatible disulfide cross‐linked SA derivative nanoparticles for oral colon‐targeted drug delivery (**A**) P3DL/PAH/PSSCMA (a) TEM and (b) FeSEM pictures at magnifications of 110,000 and 100,000, respectively. (**B**) For 170 h in a simulated gastrointestinal medium, the cumulative % drug release of P3DL/PAH/PSSCMA was measured. \*\* means *p* < 0.01. (**C**) The effects of P3/PAH/PSSCMA, P3DL/PAH/PSSCMA, PCX and untreated P3/PAH/PSSCMA on (a) HT‐29 and (b) CRL 1790. Data marked with the same letters show significant difference between the samples. \* Indicates *p* < 0 .05 compared to the untreated samples. (**D**) P3DL/PAH/PSSCMA nanospheres tagged with rhodamine 110 are taken up by HT‐29 cells. Reproduced with permission from [126], copyright Taylor & Francis Online, 2019. **Figure 6.** Biocompatible disulfide cross-linked SA derivative nanoparticles for oral colon-targeted drug delivery (**A**) P3DL/PAH/PSSCMA (a) TEM and (b) FeSEM pictures at magnifications of 110,000 and 100,000, respectively. (**B**) For 170 h in a simulated gastrointestinal medium, the cumulative % drug release of P3DL/PAH/PSSCMA was measured. \*\* means *p* < 0.01. (**C**) The effects of P3/PAH/PSSCMA, P3DL/PAH/PSSCMA, PCX and untreated P3/PAH/PSSCMA on (a) HT-29 and (b) CRL 1790. Data marked with the same letters show significant difference between the samples.\* Indicates *p* < 0 .05 compared to the untreated samples. (**D**) P3DL/PAH/PSSCMA nanospherestagged with rhodamine 110 are taken up by HT-29 cells. Reproduced with permission from [126], copyright Taylor & Francis Online, 2019.

In order to furnish controlled drug delivery via the oral route, Cong et al. prepared an ALG hydrogel/CS micelle composite system encapsulating emodin. The research group prepared cross‐linked micelles because, unlike conventional polymeric micelles, cross‐linked micelles prevent drug leakage and protect them from dissociating quickly.

extensive first‐pass metabolism. It was loaded onto CS micelles and then mixed with SA hydrogels developed by cross‐linking with Ca2+ ions and β‐GP to form hydrogel/micelle beads. Based on response surface methodology, the optimized biopolymer concentrations were determined. The 1:1 hydrogel/micelle beads showed sustained drug delivery, while the 3:1 ratio provided colon‐specific delivery. The morphological, swelling, and degrada‐ tion studies were carried out. The diameter of micelles increased from 80 nm in aqueous solution to 100–200 nm in the hydrogel, likely due to electrostatic interaction between the amino group of the CS chain and the carboxylate group of the ALG chain. The swelling of micelles was reduced at pH 1.2 due to SA hydrogel, whose swelling ratio is at acidic pH, thus preventing the release and degradation of CS chains. The initial release amount was 28% in SGF, and a final release amount of 85% for 1:1 micelle/hydrogel systems was re‐ ported. Thus, this pH‐responsive hydrogel/micelle system could be an encouraging can‐ didate for sustained‐release or site‐targeted actives delivery for unstable or hydrophobic

actives [127].

Due to its simplicity, non-invasiveness, patient compliance, and economy, oral drug delivery is the most favored route. Particles having a size between 20–100 nm are readily absorbed from the cells while also avoiding renal clearance [131], while particles below 5 nm are quickly cleared via renal clearance. Thus, Thomas et al. prepared ALG-cellulose nanocrystal hybrid nanoparticles by a green method to achieve controlled-drug delivery of the drug rifampicin, an anti-tubercular agent with poor water solubility, which necessitates the search for alternate drug delivery routes. Cellulose nanocrystals (CNCs) were used to improve the mechanical stability, durability, and high diffusion rates due to ALG's highly porous structure. CNCs have been reported to reduce the voids in the gelatin structure [132]. In addition, CNCs have been shown to enhance the ALG bead's mechanical strength and improve drug release [133]. Ionotropic gelation was utilized to synthesize ALG-CNC NPs using water as the only solvent. The optimum nanoparticle preparation was found to be with 1% surfactant and a 1:6 ratio of ALG CNC. The drug entrapment efficiency (EE) was between 43–69%. The ALG-CNC-NPs swelled rapidly at pH 6.8 to 7.4. It showed 15% drug release in 2 h at pH 1.2, while at pH 7.4, almost 100% of the drug was released in 12 h, demonstrating its controlled-release profile. It has a negative zeta potential (−15 to −20 mV) due to free carboxylic groups, thus facilitating penetration into epithelial cells. CNCs and ALG-CNC NP's cytotoxicity was investigated via MTT assay using the L929 fibroblast. It showed 100% cell viability, thus suggesting that these nanoparticles could be a good candidate for drug delivery [134].

The researchers have prepared many formulations and systems to promote actives delivery efficiency via oral routes to beat oral drug delivery limitations. The recent literature includes preparations such as cationic cyclodextrin/ALG/CS nanoflowers/5 fluorouracil [135], hydroxyethylacryl CS/SA hydrogel/paracetamol [136], pH-sensitive nanocomposite using SA/pectin/tannic acid(TA)/silver(Ag)/propranolol [137], Cyperus esculentus starch-ALG/ibuprofen [138], calcium alginate (CA)/SWCNT-GI/curcumin [139], thiol-modified SA microspheres/bovine serum albumin [140], vitamin B12 modified amphiphilic SA nanoparticles/insulin [141], thiolated SA nanoparticles/docetaxel [142], CA beads/cetuximab/octreotide [143], ALG/barium ion/methotrexate [144], CS/CA/ liraglutide [145], nano pol cellulose (CMC)/ALG/CS [146].

#### *6.2. Ocular Drug Delivery*

Topical instillation in the eye is the most preferred non-invasive administration route of actives for various anterior and posterior segment diseases, for instance, glaucoma, uveitis, cataract, and age-related macular degeneration. It is best-loved due to its comfort in administration and being patient-friendly. Nevertheless, the eye has several protective anatomical barriers that limit drug absorption through this route. The lacrimal fluid drains the drug quickly from the ocular area. Hence, ocular drug delivery has the challenge of maintaining the conc. of the drug at the site of action for the necessary time [147,148]. To enhance the delivery of topical ocular therapeutics, researchers have principally highlighted two methods: (i) to improve the period of residence of the cornea by employing viscosity enhancers, mucoadhesive, particulate, and/or in situ gelling systems; and (ii) to enhance the permeability of the cornea using (a) penetration enhancers, (b) prodrugs, and (c) colloidal systems (like NPs and liposomes) [149].

There is a probability of permanent visual damage or blindness due to retinal diseases such as macular degeneration, uveitis, macular edema, etc., and hence, in such cases, immunosuppressant drugs must be provided on a long-term basis to sustain the functioning of the eye. Encapsulated-cell therapy, however, provides a modern approach with longlasting delivery of newly synthesized protein-rich drugs, eliminating the need for surgical treatment and is potentially removable by surgery. Some limitations pertaining to ECT use are its poor mechanical strength, inadequate biocompatibility, and lack of termination mechanisms [150]. Hence, Wong et al. synthesized an injectable ALG collagen (CAC) hydrogel with an inducible termination switch as the encapsulation matrix for glial-derived neurotrophic factor for safer ocular delivery. Here, collagen acts to enhance the cell viability

of ALG. The CAC ECT gels were developed by ionotropic gelation employing calcium chloride, where they used a Tet-on-pro-Casp8 switch mechanism for the effective termination of ECT systems. An oral DOX treatment was sufficient for the termination of the gel system. The therapeutic delivery was evaluated in pink-eyed dystrophic RCS/lav rats, a recessive RP model characterized by a progressive loss of photoreceptors and electrophysiological response. The retinal function of rats acquiring GDNF-secreting gels was found to be better than that of the control group. The ECT gel system was mechanically stable, viable, and functional in vivo. Stability studies revealed that following six months of implantation, the system was mechanically stable, strong, and assisted the growth of various types of cells, viz., HEK293 and ARPE-18 cells. Thus, this approach may be utilized to address a range of posterior eye disorders [151].

Nepafenac is a non-steroidal anti-inflammatory agent utilized to address post-surgery pain in the cornea, such as cataracts. Because of its water insolubility, it is only formulated as a suspension, which leads to irritation in the eye, severe lacrimation, and consequently decreased drug's residence time due to rapid drainage into the systemic circulation, limiting the conc. of the drug at the action site [152]. Hence, Shelley et al. utilized the ion-activated in-situ gel formation of ALG with Ca2+ ions in the lacrimal fluid to accomplish a sustained release of the actives. HPBCD was the actives solubilizer and permeation enhancer, and the retention time and permeation parameters were observed in the ex-vivo porcine perfusion eye model. The in-situ gel formulation showed a considerably increased diffusion rate and permeation rate than the placebo, with a sustained release of over 24 h with release kinetics following the Korsmeyer-Peppas equation [153].

In another investigation, Nagarwal et al. prepared CS-coated SA-CS nanoparticles for the intra-ocular delivery of 5-FU used for corneal carcinoma. It demonstrated an encapsulation efficiency (almost 27%) and drug loading capacity (around 19%). Moreover, the in-vitro and in-vivo drug-release profiles showed a sustained release (8 h) in contrast to the 5-FU solution and also showed good tolerability when tested by the Draize test on the rabbit eye [154].

Conventional ocular drug dosage forms, for example, solutions, suspensions, and ointments, possess certain disadvantages that cause poor absorption of the drug, such as the sweeping activity of eyelids, tears washing away the drug, impermeable endothelium, and blood barrier. In-situ gelling systems are an effective drug delivery and absorption method since the less viscous solution undergoes gelling in the presence of stimuli such as pH, ionic strength, and temperature. Hence, Noreen et al. developed ALG in situ gelling systems using gum obtained from Terminalia arjuna bark with moxifloxacin HCl as the drug for ophthalmic delivery. It undergoes a sol to gel transition at the pH of tear fluid. The preservative was methylparaben, and the osmolarity was adjusted with sodium chloride. The drug was stable and provided sustained release for 12 h. The ex vivo transcorneal penetration was found to be 4.76 ± 0.27%, and corneal hydration was 78.85 ± 0.19%. No ocular irritation was observed [155]. Thus, the overall results demonstrated the aforementioned system as a promising candidate for ocular drug delivery.

In another investigation, Polat et al. formulated nanofibrous ocular inserts for the therapy of bacterial keratitis incorporating the drug Besifloxacin HCl or BH-hydroxypropylbeta-cyclodextrin (HP-β-CD) complex comprising PCL/PEG fibrous inserts coated with mucoadhesive polymers such as SA or thiolated sodium alginate (TSA) based on the electrospinning method. The coating with SA and TSA increased the insert's bioadhesion. The preparation demonstrated an initial burst release followed by a slow release for two days, and the thickness and diameter of the inserts were comparable to commercial formulations. Drug loading efficiency was over 90%. Even after seven days of incubation, the inserts did not attain acidic pH values, indicating that the inserts did not cause eye irritation. In vitro studies on ARPE-19 cells on exposure to the ocular inserts demonstrated no cytotoxicity, and their antibacterial activity was comparable to that of commercial formulations. Ex vivo transport research showed that HP-β-CD enhanced solubility and corneal permeability, and

the actives delivery was equivalent to commercial formulations [156]. Hence, the overall results reported that the newly prepared system was suitable for ocular drug delivery.

Different researchers have studied various ALG-based preparations for enhanced ocular delivery of drugs. For example, CS/ALG multilayers/diclofenac [157], CS/ALG/ daptomycin [158], CS/ALG/acetamiprid [159], ALG/CS/levofloxacin [160], SA/glycerin/ flurbiprofen [161], ALG/peppermint phenolic extract [162], SA/methyl cellulose/ sparfloxacin [163], ALG/calcium gluconate(CaG)/tryptophan [164], SA dialdehyde/ carboxymethyl CS/limbal stem cells (LSC) [165], SA/methyl cellulose/CMC/carbopol/ pilocarpine [166], CS/SA/azelastine [167], SA/butyl methacrylate/lauryl methacrylate/ linezolid [168] are few of the current investigations which portrayed excellent ocular actives delivery.

## *6.3. Pulmonary Drug Delivery*

The lungs are an appealing site for the pulmonary administration of actives via diverse DDSs [169,170]. Moreover, the pulmonary route provides numerous benefits over traditional per oral administration, for instance, greater surface area with fast absorption owing to the high vascularization and evasion of the first-pass metabolism [171]. This selectivity enables targeted actives delivery and, therefore, diminishes the side effects [169]. Nevertheless, the pulmonary route is challenging to deliver the actives deep to the alveolar regions of the lung owing to diverse respiratory obstacles, including mechanical, chemical, pathological, and immunological hindrance [172]. The mechanical barrier employs mucociliary clearance to remove particles (having a mean diameter of >6 µm) [173]. When clearance takes place quicker than absorption, in particular of poorly soluble drugs, the availability of actives in the lungs might be restricted. Additionally, under pathologic states like asthma and COPD, excessive mucus is accumulated in the lung, which inhibits the deeper penetration of actives [173,174]. Furthermore, the chemical barrier containing proteolytic enzymes can degrade the breathed materials, which leads to the destruction of functions of actives and/or delivery carriers. Ultimately, the immunological barrier comprising primarily alveolar macrophages assembles the immunological reactions in the intense lung that eradicate all foreign materials with no difference between potential detrimental materials and advantageous ones. Thus, pulmonary bioavailability and systemic bioavailability for actives provided by maximum traditional pulmonary products are less. Therefore, the progress of unique, more effective DDSs is pivotal to alleviating the consequences of these obstacles [175].

The FDA-approved pulmonary dry powder inhalers are predominantly fast-releasing. To obtain sustained delivery to the lungs, Athamneh et al. developed an aerogel microsphere formulation for suitable delivery to the lower pulmonary tract using the ALG-HA hybrid system by the emulsion gelation method followed by drying with supercritical CO2. Combining these approaches resulted in highly porous Alg-HA microspheres with very low density and a high BET-specific surface area. The addition of HA to Alg successfully reduced the particle agglomeration and enhanced its biodegradation, possibly due to the formation of a hydrogen bond between ALG's carboxylate groups and N-acetylglucosamine amide in the hybrid aerogel. In addition, HA was employed to improve the physical characteristics of ALG, such as gelling efficiency and lung tolerance. This led to achieving the desired particle size for pulmonary deposition and improving biodegradability. However, more research using active pharmaceutical ingredients needs to be carried out in this area [100].

Drugs administered to the lungs to treat COPD and cancer suffer from low bioavailability due to distribution to other body tissues, thus requiring multiple dosing and increasing the risk of adverse effects [176]. Mahmoud et al. formulated microparticles based on ALG using the emulsified nano spray-drying process for the pulmonary carrier of roflumilast, a selective inhibitor of the phosphodiesterase-4 enzyme in lung cells. Isopropyl myristate was used as an oil, Tween 80 was used as a surfactant, and calcium beta-glycerophosphate was used as a cross-linking agent. The developed microparticles were assessed based on

encapsulation efficiency, particle size, and in vitro release of actives. The aerodynamic data showed that the actives could be deposited deep into the lungs at the bronchi or bronchial tube (as shown in Figure 7a). The formulation with the spherical-shaped microparticles swelled within 3 h at pH 7.4. Furthermore, it showed a remarkable cytotoxic effect on A-549 tumor cell lines (2.5-fold decrease in IC50 compared to the pure drug) (as demonstrated in Figure 7b) and reduced pro-inflammatory cytokines (TNF-alpha, IL-6, and IL-10) in contrast to pure actives (as exhibited in Figure 7c). In addition, CD-based microparticles showed higher sustained bronchodilation on human volunteers than marketed Ventolin®HFA (as shown in Figure 7d). Thus, CD-based microparticles could be a propitious approach for delivering roflumilast in humans [177]. assessed based on encapsulation efficiency, particle size, and in vitro release of actives. The aerodynamic data showed that the actives could be deposited deep into the lungs at the bronchi or bronchial tube (as shown in Figure 7a). The formulation with the spherical‐ shaped microparticles swelled within 3 h at pH 7.4. Furthermore, it showed a remarkable cytotoxic effect on A‐549 tumor cell lines (2.5‐fold decrease in IC50 compared to the pure drug) (as demonstrated in Figure 7b) and reduced pro‐inflammatory cytokines (TNF‐al‐ pha, IL‐6, and IL‐10) in contrast to pure actives (as exhibited in Figure 7c). In addition, CD‐based microparticles showed higher sustained bronchodilation on human volunteers than marketed Ventolin®HFA (as shown in Figure 7d). Thus, CD‐based microparticles could be a propitious approach for delivering roflumilast in humans [177].

HA hybrid system by the emulsion gelation method followed by drying with supercritical CO2. Combining these approaches resulted in highly porous Alg‐HA microspheres with very low density and a high BET‐specific surface area. The addition of HA to Alg success‐ fully reduced the particle agglomeration and enhanced its biodegradation, possibly due to the formation of a hydrogen bond between ALG's carboxylate groups and N‐acetyl‐ glucosamine amide in the hybrid aerogel. In addition, HA was employed to improve the physical characteristics of ALG, such as gelling efficiency and lung tolerance. This led to achieving the desired particle size for pulmonary deposition and improving biodegrada‐ bility. However, more research using active pharmaceutical ingredients needs to be car‐

Drugs administered to the lungs to treat COPD and cancer suffer from low bioavail‐ ability due to distribution to other body tissues, thus requiring multiple dosing and in‐ creasing the risk of adverse effects [176]. Mahmoud et al. formulated microparticles based on ALG using the emulsified nano spray‐drying process for the pulmonary carrier of roflumilast, a selective inhibitor of the phosphodiesterase‐4 enzyme in lung cells. Isopro‐ pyl myristate was used as an oil, Tween 80 was used as a surfactant, and calcium beta‐ glycerophosphate was used as a cross‐linking agent. The developed microparticles were

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 19 of 60

ried out in this area [100].

**Figure 7.** Design and characterization of emulsified spray‐dried ALG microparticles as a carrier for the dually acting drug roflumilast (**a**) In an ethanolic phosphate buffer saline solution (30% *v*/*v*; pH 7.4), release patterns of roflumilast and emulsified spray‐dried ALG microparticles were studied. **Figure 7.** Design and characterization of emulsified spray-dried ALG microparticles as a carrier for the dually acting drug roflumilast (**a**) In an ethanolic phosphate buffer saline solution (30% *v*/*v*; pH 7.4), release patterns of roflumilast and emulsified spray-dried ALG microparticles were studied. (**b**) The effects of roflumilast, a medicated CD formulation, and a non-medicated CD formulation on the proliferation of A-549 tumor cells. (**c**) TNF-alpha, interleukin-6, and interleukin-10 levels were reduced in A-549 tumor cells by roflumilast and CD formulation. (**d**) FEV1/FVC%—time curve in healthy human volunteers upon inhaling the chosen CD formulation vs. Ventolin® HFA. Reproduced with permission from [177], Copyright Elsevier 2018.

The pulmonary route has also administered anti-cancer drugs to treat lung cancer due to their faster onset of action and evasion of the first-pass metabolism. One of them, cisplatin, shows toxicity in rat liver, lungs, and kidneys, due to which Alsmadi et al. developed CS-ALG nanoporous aerogel carriers loaded with cisplatin for the treatment of lung cancer using an emulsion–gelation method followed by supercritical fluid extraction. The drug exhibited excellent drug loading (76%) while retaining its crystal structure and gave a sustained release of over 6 h. The cisplatin aerogels, thus prepared, decreased the lung toxicity in rats and prevented weight loss [178].

In another investigation, Iglesias et al. prepared ALG aerogel by thermal inkjet technology followed by supercritical drying to encapsulate salbutamol sulfate, which is used to treat asthma attacks and COPD. The current pulmonary drug delivery system suffers from limitations in particle size uniformity and the presence of surfactants. This can be potentially used for personalized medicine. However, the gel precursor concentrations severely restrict the printable region. The process was highly compatible with the active ingredient (salbutamol sulfate), having a homogenous texture in the nanoporous range. The SS-loaded aerogel was well deposited in the bronchi and bronchioles owing to their nano-particle size. Thus, these aerogels may pave the way for pulmonary drug delivery and personalized medicine, fulfilling the demands of nanostructured, miniaturized, highresolution product designs [179].

Liposomes, micelles, and polymeric drug particles have been delivered by pulmonary route using biocompatible, biodegradable, and flexible biopolymers such as CS, ALG, gelatin, and other natural polymers. Blends of CS-ALG nanoparticles and microparticles find their applications in controlled drug delivery systems, with calcium chloride often acting as the cross-linking agent. However, researchers have found that using calcium chloride for pulmonary particles induces inflammatory responses. Hence, Alnaief et al. formulated hybrid ALG-CS aerogels via the emulsion-gelation technique without any cross-linking agent. CS solution (2%) was put into the oil phase containing liquid paraffin and 4% surfactant (Span 80 or Span 85), followed by the addition of 1% ALG solution. The resultant hybrid particles were isolated from the oil phase by the process of centrifugation. The pore liquid was replaced with ethanol by a solvent exchange method. The prepared aerogels were later dried with supercritical carbon dioxide. The polymer addition order throughout the gelling procedure and the kind of surfactant employed greatly affected the properties of the hydrogel. Samples prepared using span 85 showed high positive zeta potentials (35.4 ± 5.37 mV), indicating that CS was surrounding the ALG core, whereas those prepared using span 80 showed high negative zeta potentials (−2.15 ± 3.70 to −5.98 ± 5.37 mV), suggesting that ALG was surrounding the CS core. The particle size ranged from 70 nm to 4.17 µm, whereas the aerodynamic diameter ranged from 0.17 to 2.29 µm [180]. Thus, the entire results revealed the developed system is a promising candidate for pulmonary actives delivery.

For the treatment of cystic fibrosis, a potentially life-threatening disease characterized by mutations in the cystic fibrosis transmembrane conductance regulator (CTFR), antibiotics (colistimethane sodium) are generally prescribed. Due to the loss of Cl-channel activity, the lungs become more susceptible to bacterial infections. Of these, Pseudomonas aeroginosa survives the antibiotic treatment and emerges as the predominant infecting organism over time [181–183]. Tobramycin is the drug of choice for such infections that are deteriorating with regular colistimethane sodium. One issue associated with tobramycin therapy is the non-compliance of patients due to twice-daily dosing, which leads to the failure of antibiotic treatment. Another concern is the potential for ototoxicity and nephrotoxicity associated with nearly all aminoglycosides, although there is less evidence of ototoxicity and nephrotoxicity in clinical trials with inhaled tobramycin [184,185]. Hence, Hill et al. developed ALG/CS particles using CaCl2 as the crosslinker. During the optimization studies, increasing the concentration of cations in the formulation resulted in a more significant aggregation of the particles. The optimal formulation was ALG: CS: tobramycin 9:1:1.5, giving high drug loading and narrow size distribution. Tobramycin release was evaluated, depicting a biphasic release with 18.9% of the entrapped drug released within 24 h. In vitro antibacterial studies showed that the drug-loaded optimal formulation showed a dosedependent activity against P. aeroginosa with a MIC of 6.25 µg/mL, while the unloaded formulation demonstrated no action. Tobramycin alone had a MIC of 1.5 µg/mL, possibly due to the drug diffusion rate. The Zeta potential was found to be 2.16 ± 0.07% mV and the %EE was 44.5%. SLPI conjugation enhanced the mucoadhesive properties [186]. Hence, the prepared system could help deliver drugs via the pulmonary route.

Moreover, investigators have developed various formulations to intensify pulmonary DDSs. For example, SA/CS/Tween 80/rifampicin [187], ALG/poly(N-isopropyl acryl amide (PNIPAAm)/theophylline [188], ALG/ciprofloxacin [189], ALG/CS/lapazine [190], ALG/CaCO3/levofloxacin/DNAse [191], ALG/HA/naproxen [99], ALG/CS/DOX/ paclitaxel [192], ALG modified PLGA nanoparticles/amikacin/moxifloxacin [193], ALG

particles encapsulating live Bacille Calmette– Guérin (BCG) and *Mycobacterium indicus pranii* (MIP) [194], CS/ALG/BSA gel/DOX [195] are some of the recent formulations showing excellent delivery of drugs via pulmonary delivery.

#### *6.4. Vaginal Drug Delivery*

Lately, the vagina has been considered a potential administration route replacing the parenteral route for actives delivery, bestowed with systemic effects that cannot be favorably administered per os due to the hepatic or GI degradation or to the commencement of adverse consequences in the GI. The significant constraint of such an administration route is described by the physiological elimination mechanisms, which are active in the vagina's lumen and are responsible for an unsatisfactory residence time of the conventional formulated system at the targeted site, following an unsteady actives dissemination onto the mucosa [196]. The vagina, a prominent female reproductive organ, allows for local and systemic actives delivery due to its large surface area and high blood supply. However, it is also associated with variations in the size of the endometrium during menstruation, which may alter the drug absorption properties [197,198].

Vaginal dosage forms should ideally be easy to administer, requiring fewer doses, and patient-compliant. However, it is challenging to formulate small water-soluble drugs through the vaginal route. Hence, Meng et al. prepared spray-dried microparticles (MPs) using thiolated-CS coated SA by the layer-by-layer method for the vaginal delivery of HIV microbicides. They were studied for their cytotoxicity and pre-clinical safety on human vaginal (VK2/E6E7) and endocervical (End1/E6E7) epithelial cell lines and in vivo on female mice. The outcomes showed that TCS-coated Ag-based multilayer microparticles had 20–50 fold more adhesion than native AGMPs [199]. Hence, the developed formulation could be useful for vaginal drug delivery.

Urogenital infections affect about a billion women worldwide. It is caused due to *Escherichia coli* and other enterobacteria commonly found in the vaginal tract. The chances of UTIs are increased in post-menopausal women, which account for 25% of all bacterial infections. Cystitis, the most common UTI, usually affects young women, with *E. coli* being the cause of most diseases. The treatment of UTIs is still a challenge because of frequent recurrence, the association of co-morbidities, and high prevalence. Cefixime, a third-generation cephalosporin, is usually given for such infections. However, its poor water solubility, limited oral bioavailability, and incomplete absorption limit its use. Hence, Maestrelli et al. developed a bioadhesive vaginal dosage form of cefixime that can overcome many drawbacks. They prepared CS-coated CA microspheres using an ionotropic gelation method, and their mean weight, diameter, %EE, and loading capacity were evaluated. All MS batches showed prolonged adhesion greater than 2 h on the excised porcine vaginal mucosa; %EE enhanced with increasing drug concentration. In vitro studies revealed the direct relationship between CFX drug release and % inhibition in *E. coli* metabolic activity [200]. Thus, the overall results demonstrated the developed system as an auspicious candidate for vaginal delivery of actives.

Despite having several benefits, for example, enormous surface area, evasion of the first-pass metabolism, and the ability to optimize drug absorption for systemic effects, it also has certain drawbacks, such as the restoration action on vaginal fluids and its acidic environment (pH 4.0–4.5), which hampers the local delivery of drugs due to low residence time and stability. Hence, Ferreira et al. formulated CA hydrogels based on the earlier formation of polyelectrolyte complexes (PECs) for the vaginal delivery of polymyxin B. First, PECs were formed between ALG and PMX, ensued by cross-linking with calcium chloride. The aforementioned system displayed a pore size of between 100–200 µm and adequate syringability; in vitro tests demonstrated mucoadhesiveness. The drug release was found to be pH-dependent with a sustained release of six days. A burst release was noticed at pH 7.4, and the drug was released by anomalous transport. At pH 4.5, actives release followed the Weibull model and actives transport was through Fick's diffusion [201]. Thus, the formulated system could be suitable for vaginal drug delivery.

Vulvovaginal candidiasis caused by the fungus Candida albicans is generally treated using azole antifungals like fluconazole given orally. However, fluconazole is reported to have severe side effects like nausea, vomiting, diarrhea, and abdominal pain [202,203]. Hence, Darwesh et al. developed vaginal inserts using PEC based on anionic ALG and cationic CS. The mucoadhesion was highest for Na–Alg-based vaginal inserts (pKa 3.21) as compared to carbopol (pKa 5.0) because the mucoadhesion depends on the no. of hydrogen bonds (-OH, -COOH) concerned in the mucoadhesion interaction. The ALG: CS (5:5) PEC revealed controlled release of fluconazole (RE6h ranged from 56.46 ± 3.42 to 79.38 ± 3.42%), good mucoadhesion, and therefore suitable vaginal retention. Moreover, it demonstrated excellent antifungal action against Candida albicans both in vitro (MIC for fluconazole vaginal inserts was 31 ± 0.4 mm and that of fluconazole solution was 22 ± 0.4 mm) and in vivo (complete healing after seven days in rats) with reduced inflammatory cells [204]. Thus, the overall results depicted that the formulated system could be a promising candidate for vaginal drug delivery.

In another investigation, Soliman et al. developed an in situ thermosensitive bioadhesive gel for the vaginal delivery of sildenafil citrate as a prospective therapy for endometrial thinning caused by the administration of clomiphene citrate for ovulation initiation in women with type II gonadotrophic anovulation. They were developed using various grades of Pluronic® (PF-68 and PF-127) grades, into which mucoadhesive polymers such as SA and hydroxyethylcellulose were incorporated in different concentrations. The thermosensitive gels were developed by the cold method. The acceptable range of Tsol-gel of 28–37 ◦C was achieved by decreasing PF-127 concentration and modulating the addition of PF-68. There was increased gel viscosity and mucoadhesive force when the concentration of Pluronic® was raised, but there was a reduction in drug release rate during in vitro evaluation in a standard semi-permeable cellophane membrane at pH 4.5 using citrate buffer to mimic the vaginal fluid. Due to its rapid swelling property, SA aided the formation of adhesive interaction between ALG and mucosa, which led to increased mucosal retention. The in situ gels substantially enhanced endometrial thickness and uterine blood flow, thus potentially improving conception chances in anovulatory women with clomiphene citrate failure [205].

Researchers also synthesized ALG/CS/P4/Pluronic® F-127/progesterone [206], CS/ SA/polycarbophil/metronidazole [207], SA/CS/*α*,*β*-glycerophosphate/Bletilla striata/ tenofovir [208], SA loaded with anise/fluconazole β-cyclodextrin inclusion complexes [209], CS/ALG/metronidazole [210], HPMC/SA/abacavir [211], dextran/ALG nanofibers/ clotrimazole [212], Polaxomer 407/SA/*Lactobacillus crispatus* [213], ALG/CMC/clove essential oil [214], and ALG/CS/metronidazole [215] formulations which enhanced the vaginal drug delivery.

#### *6.5. Nasal Drug Delivery*

Nasal actives delivery includes the inhalation of drugs into the extremely vascularized mucosal layer of the nasal epithelium, which ultimately reaches the systemic circulation [216]. The nose has an enormous surface area for actives absorption, quicker onset of action, and evasion of first-pass metabolism [217]. The curative action of the majority of the drugs is impacted owing to the BBB's existence. Therefore, nasal route delivery favors therapeutics straightaway reaching the central nervous system (CNS). Furthermore, nasal delivery of actives shows benefits from brain-targeting, fewer side effects, and easy administration [218].

Rhinosinusitis, inflammation of the nasal cavities characterized by nasal polyp growth, typically extending beyond the nasal valve, is generally treated using nasal corticosteroids since oral ones show systemic side effects. However, its long-term use causes an increase in intraocular pressure and changes in serum cortisol levels. Hence, Dukovski et al. formulated dexamethasone-loaded lipid/ALG nanoparticles dispersed in pectin solution based on an in-situ gelling process that undergoes a sol-gel transformation after contact with the Ca2+ ions in the nasal mucosa. The in vitro biocompatibility experiments utilizing colon

carcinoma Caco-2 cell lines suggested no effect on cell viability; however, in-vivo studies are required to confirm this observation [219].

It becomes feasible to administer vaccines in dry powder form during influenza outbreaks due to their ease of transport, patient compliance, and stability. As compared to conventional vaccines, dry powder vaccines provide both humoral and cellular immunity adequately. Thus, Dehghan et al. formulated ALG NPs using the ionotropic gelation method encapsulating whole inactivated influenza viruses and administered them to rabbits, which showed significantly higher IgG titers when short single-stranded cytosine triphosphate and guanine triphosphate-containing synthetic oligodeoxynucleotides (CpG ODN) were used as the adjuvant (as exhibited in Figure 8c,d). ALG NPs released 65.87% of CpG ODN, 35.68% of QS, and 34.00% of influenza antigen within 4 h (as displayed in Figure 8a). In addition, the XTT test demonstrated safety for in-vivo applications (as shown in Figure 8b), making the formulation useful for nasal drug delivery [220]. *Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 24 of 60

**Figure 8.** Preparation, characterization, and immunological evaluation of ALG nanoparticles loaded with whole inactivated influenza virus: Dry powder formulation for nasal immunization in rabbits (**a**) % of viral protein, QS, and CpG ODN released in vitro from ALG NPs over four hours. (**b**) After 2‐ and 24‐h exposure with varying concentrations of each formulation, the effect of ALG NPs and influenza virus suspension on cell viability in Calu‐6 cell lines (**c**) HAI antibody titers in each vac‐ cination group on day zero (control), day 45 (after prime dose), day 60 (after the second dose), day 75 (after the third dose), and day 90 (after the final booster) (**d**) IgG titers in blood samples taken from each vaccinated cohort on days zero (negative control), 45 (prime dose), 60 (second dose), 75 (third dose), and 90 (after the final booster). \* means *p* < 0.05, \*\* means *p* < 0.01, \*\*\* means *p* < 0.001. Reproduced with permission from [220], Copyright Elsevier 2019. In a different study, Rao et al. formulated the anti‐Parkinson drug Ropinirole as a **Figure 8.** Preparation, characterization, and immunological evaluation of ALG nanoparticles loaded with whole inactivated influenza virus: Dry powder formulation for nasal immunization in rabbits (**a**) % of viral protein, QS, and CpG ODN released in vitro from ALG NPs over four hours. (**b**) After 2- and 24-h exposure with varying concentrations of each formulation, the effect of ALG NPs and influenza virus suspension on cell viability in Calu-6 cell lines (**c**) HAI antibody titers in each vaccination group on day zero (control), day 45 (after prime dose), day 60 (after the second dose), day 75 (after the third dose), and day 90 (after the final booster) (**d**) IgG titers in blood samples taken from each vaccinated cohort on days zero (negative control), 45 (prime dose), 60 (second dose), 75 (third dose), and 90 (after the final booster). \* means *p* < 0.05, \*\* means *p* < 0.01, \*\*\* means *p* < 0.001. Reproduced with permission from [220], Copyright Elsevier 2019.

thermoreversible in situ nasal gel. The rationale for the nasal delivery of Parkinson's pa‐ tients is that it becomes difficult forthem to swallow oral solid dosage forms due to muscle rigidity. It was prepared by the cold method using PF 127 and HPMC K4M as the ther‐ moreversible polymer. They observed an astounding five‐fold enhancement in the bioa‐ vailability of ropinirole in contrast to intravenous drug delivery. Furthermore, in vitro evaluation of sheep nasal mucosa demonstrated that the in situ gel had a more protective impact on nasal mucosa than the plain drug, which caused mucosal damage [221]. In a different study, Rao et al. formulated the anti-Parkinson drug Ropinirole as a thermoreversible in situ nasal gel. The rationale for the nasal delivery of Parkinson's patients is that it becomes difficult for them to swallow oral solid dosage forms due to muscle rigidity. It was prepared by the cold method using PF 127 and HPMC K4M as the thermoreversible polymer. They observed an astounding five-fold enhancement in the bioavailability of ropinirole in contrast to intravenous drug delivery. Furthermore, in vitro

In another investigation, Youssef et al. developed SA nanoparticles for the anti‐mi‐ graine drug Almotriptan utilizing the w/o/w double emulsion solvent evaporation proce‐ dure. It is a water‐soluble drug; hence, SLNs were prepared to help pass the drug through

dium CMC, and carbopol. In addition, the gelling temperature, gelling time, viscosity, gel strength, pH, %EE, and in vitro mucoadhesion were evaluated [222]. The overall results

Vaccines typically include adjuvant substances to enhance the humoral or cellular response to an antigen. The most common antigen, aluminum, instigates a Th2 antibody response; hence, aluminum‐based vaccines are unsuitable for intracellular pathogens and

demonstrated the prepared formulation to be suitable for nasal drug delivery.

evaluation of sheep nasal mucosa demonstrated that the in situ gel had a more protective impact on nasal mucosa than the plain drug, which caused mucosal damage [221].

In another investigation, Youssef et al. developed SA nanoparticles for the antimigraine drug Almotriptan utilizing the w/o/w double emulsion solvent evaporation procedure. It is a water-soluble drug; hence, SLNs were prepared to help pass the drug through the lipophilic BBB. Poloxamer 407 was used as a stabilizer and evaluated for its physicochemical properties by combining various mucoadhesive polymers, for example, SA, sodium CMC, and carbopol. In addition, the gelling temperature, gelling time, viscosity, gel strength, pH, %EE, and in vitro mucoadhesion were evaluated [222]. The overall results demonstrated the prepared formulation to be suitable for nasal drug delivery.

Vaccines typically include adjuvant substances to enhance the humoral or cellular response to an antigen. The most common antigen, aluminum, instigates a Th2 antibody response; hence, aluminum-based vaccines are unsuitable for intracellular pathogens and chronic diseases. Currently, usable vaccines through the intramuscular (i.m.) or subcutaneous (s.c.) routes fail to elicit a mucosal immune response. Nasal immunization, a relatively simple, non-invasive route, can provide mucosal immunity [223–227]. However, an acceptable mucosal immune response depends on the mucociliary clearance, the tolerogenic character of the mucosal epithelium, and the huge size of the antigen. To circumvent this problem, the antigens can be incorporated into mucoadhesive polymeric NPs, extending the duration of antigen residence and conferring protection against enzymes [228,229]. Hence, Mosafer et al. developed SA-coated CS and trimethyl CS nanoparticles incorporated with the PR8 influenza virus for nasal administration. The zeta potential was −29.6 mV. The vaccine was evaluated in BALB/c mice, wherein the PR8-TMC-ALG formulation manifested a more excellent IgG2a/IgG1 ratio than PR8-TMC, PR8-CHT, and PR8-ALG. Thus, ALG-NPs could be used as immunoadjuvants for nasal immunization. The ALG-coated NPs generated an excellent immune reaction compared to uncoated NPs [230].

The recent nasal formulations include SA/Polaxomer 407/gellan gum/timosaponin BII [231], SA in situ gels based on agomelatine (AGM) [232], ALG based magnetic short nanofibres 3D composite hydrogel encapsulating human olfactory mucosa stem cells [233], OA-dopamine conjugate [234], ALG nanoparticles/venlafaxine (VLF-AG-NPs) [235], *Mycobacterium bovis* Bacille Calmette-Guérin (BCG)-loaded microparticles using ALG/CS [236], ALG/CS/attenuated Androctonus australis hector (Aah) venom [237], CS/ALG nanoparticles/SpBMP-9 (growth factor) [238], ALG/trimethyl CS liposomes/lipopeptide subunit vaccine [239], SA microspheres/*Lactobacillus casei* [240].

#### *6.6. Transdermal Drug Delivery*

Transdermal drug delivery relates to the delivery of actives via the skin for systemic or local absorption. They are advantageous over the conventional administration routes due to their capability to deliver controlled release of drugs, reducing the first-pass metabolism, less systemic side effects, effective control of drug plasma profile, and patient compliance [241]. However, its applications are limited owing to the extensive skin hindrance, particularly the stratum corneum. Consequently, few actives can penetrate the skin and reach the blood at a therapeutic concentration [242].

Insulin is usually given hypodermically via s.c. injection for the therapy of diabetes mellitus. But it generates biohazardous waste; furthermore, patient compliance is also a challenge, especially in children. Several approaches have come into the picture to resolve the issue, such as inhalational microparticles, oral administration of nanoparticles, and needle-free high-pressure injection systems [243–245]. Among these, transdermal patches have attracted much attention owing to minimal pain and tissue injury [246,247]. In this context, Yu et al. fabricated a dissolving polymer microneedle patch comprising 3-aminophenyl boronic acid-modified ALG (ALG-APBA) and HA that can quickly dissolve in the interstitial fluid of the skin. Alginate was chemically modified by adding 3-aminophenyl boronic acid to form ALG-APBA, and HA was crosslinked to form MNs in the presence of Ca2+ ions. The MNs thus prepared were evaluated for their mechanical strength, degradation, ex vivo skin insertion, stability of insulin, in vivo transdermal delivery to SD rats, and pharmacokinetic and pharmacodynamic activity in SD rats. In addition, the resistance of MNs to static and dynamic forces was calculated, and it was found that no change in the tips was observed even after the addition of 10–100 g weight on the microneedle patch, with no breaks in the needle after the addition of 500 g, demonstrating its excellent mechanical strength. The encapsulated insulin has comparable pharmacological activity to an s.c. injection with the same insulin dose, with RPA and RBA at 90.5 ± 6.8% and 92.9 ± 7.0%, respectively [248]. Overall results made the aforementioned system advantageous for nasal drug delivery.

In a different investigation, Lefnaoui et al. fabricated transdermal films based on ALG-CS PECs for the antiasthmatic drug ketotifen fumarate (KF). Polyethylene glycol was used as a plasticizer, and Span 20, and Tween 80 were utilized as permeability enhancers. The films were prepared by the film casting method and evaluated for their uniformity in weight, thickness, folding endurance, loss of moisture, and absorption of moisture. Furthermore, actives release and permeation through the rat abdomen mounted on the Franz diffusion cell were characterized. The transdermal films made by a 1:1 ratio of CS and ALG yielded smooth, flexible, strong, bioadhesive, biocompatible films. The drug release research showed that the KF was released in a controlled way over a long period (99.88% release after 24 h) to treat asthma, allergic rhinitis, and conjunctivitis [249]. The results revealed that the prepared formulation is favorable for transdermal drug delivery.

In another study, Abebeet et al. developed a self-adhesive hydrogel for strainresponsive transdermal delivery using gallic acid (GA) modified ALG as the mucoadhesive polymer. The model drug, caffeine, was encapsulated in the hydrogels prepared by the one-pot synthesis method. The diffusion kinetics were controlled by Fickian diffusion. The developed hydrogels had a 25% increase in tensile strength and twice the transdermal release of the drug. A powerful adhesion of 100 kPa was reported on a glass substrate. A close to 800% strain was observed, and it was attributed to the free movement and adhesion of ALG and polyacrylic acid. It could withstand body movement as well as skin stretching when tested on human skin. Thus, GA hydrogel seems to be a promising route for strain-controlled TDD [250].

In another investigation, Abnoos et al. prepared a CS-ALG nanocarrier for the transdermal delivery of pirfenidone (PFD) in idiopathic pulmonary fibrosis, a disease characterized by progressive dyspnea and pulmonary function loss [251,252]. CS-SA nanoparticles were developed by the pre-gelation technique, and the drug Pirfenidone, an anti-inflammatory and antifibrotic agent, was encapsulated with 94% efficiency. The prepared nanoparticles were evaluated using SEM, TEM, and DLS. These studies showed that particle morphology was spherical with an average size of 80 nm. FTIR spectra observed the complex formation between CS and ALG. The drug release studies show that PFD had undergone an initial burst release of 12% after 5 h and was later released in a sustained manner for 24 h. Ex vivo studies revealed that skin permeation of PFD was improved by using CS-SA nanoparticles compared to standard PFD solution. The permeation was also observed through fluorescent microscopic images labeled with FITC. SEM studies reveal that the nanoparticles could remain stable for six months [253]. Thus, the results proved that the prepared formulation is useful for transdermal drug delivery.

In another investigation, Anirudhan et al. formulated bio-polymer matrix films acquired from CMC, SA, and PVA to transdermally deliver diltiazem, a calcium channel blocking drug used for cardiac failure. The oral bioavailability of DTZ is only 30–40%. Hence, alternative routes of drug delivery are being explored. Since diltiazem (DTZ) is hydrophilic in nature, a hydrophilic matrix consisting of polyethylene glycol coated vinyl trimethoxy silane-g-CS (PEG@VTMSg-CS) with matrices like Na-ALG, CMC, and PVA was developed. A dispersion of the matrix was prepared to avoid the undesirable effect of shrinkage of the polymer network when the drug is being eluted out of the matrix. The drug release studies were based on Franz diffusion cells. SA films showed more significant swelling than PVA films. The ALG films displayed a thickness value of

0.052 ± 0.01 mm and a water permeation of 0.07 g cm2/24 h. DTZ permeation was less due to less film thickness. The in vitro skin penetration research of DTZ on rat skin revealed the effectiveness of the films with more than 49% viability in HaCaT and PBMC cell lines with no histological alterations on the skin. The present formulation could deliver 70% of the drug in 24 h [254]. Thus, the overall results demonstrated that the system is promising for transdermal drug delivery.

The currently investigated formulations include ALG/PVA/ciprofloxacin electrospun composite nanofibers [255], SA/propylene glycol/metoclopramide films [256], SA/ glucosamine sulphate [257], ALG/maltose composite microneedles/insulin [258], ALG/CS/ rabeprazole [259], oleic acid/SA/Na CMC/2,3,5,40-tetrahydroxystilbene 2-O-β-D-glucoside (THSG) [260], SA/CS/piplartine [261], SA/polyvinyl alcohol/quercetin [262], ALG/Hidrox-6/Hydroxytyrosol [263], sodium L-cysteine ALG/isopropyl myristate/ropinirole hydrochloride [264], ALG coated aminated nanodextrin/CS coated folate decorated aminated β-CD nanoparticles/curcumin/5-flurouracil [265] which intensified transdermal drug delivery.

### *6.7. Mucosal Drug Delivery*

In order to evade parenteral routes and beat biological impediments, one more DDS, specifically the mucosal delivery system, appeared in the limelight. Mucus gel layers line the vagina, lungs, and gastrointestinal tract (GIT). GIT represents a complicated condition, including several digestive enzymes and a changeable pH (acidic pH-stomach and basic pH-intestine). Different drugs necessitate being guarded against the circumstances referred to above to evade degradation by proteolytic enzymes. These pharmaceuticals exhibit inadequate permeability in gastric mucosa and also bear the first-pass effect. To preserve the actives of interest facing such conditions, ALG serves as one of the most satisfactory polymers with mucoadhesive characteristics and improved permeation results, and acts as a defensive envelope for such actives through mucosal delivery [266]. Mucoadhesion refers to the capability of materials to bind to the mucosal membrane and offer some retention. Mucoadhesive drug delivery provides various benefits over other routes of administration, for example, faster distribution to the local blood vessels, avoidance of first-pass metabolism, reduced dose frequency, and rapid relief [267]. Thus, the formulated system might be helpful for mucoidal drug delivery.

Oral cancer, one of the most common cancers, is usually treated with surgery and chemotherapy [268], but they have the drawback of alteration of facial morphology followed by surgical procedures. Hence, to provide an alternative approach, Shtenberg et al. formulated a mucoadhesive ALG/liposome paste. DOX-loaded liposomes were prepared, and ALG-fluorescein was synthesized using FITC and ethylenediamine; on homogenization, a cross-linked hybrid paste was formed. It showed the liposome's sustained release from ALG cross-linked paste (release index of 8). Furthermore, in vitro toxicity studies on human cell lines acquired from tongue SCC (CAL 27) showed viability of 15% after 48 h. Thus, polymers released from the liposomes were active and effective. Therefore, this approach could be used for potential oral cancer therapy [269].

Oral mucosal vaccine delivery has the disadvantage of significant enzymatic degradation of the antigen in the GI tract, and these have difficulty in uptaking from the intestine. To overcome this issue, HbsAg-loaded CS nanoparticles with an ALG coating were prepared by Saraf et al., in which lipopolysaccharides acted as an adjuvant. It showed elevated levels of sIgA in intestinal (3.32 mIU/mL) secretions compared to non-ALG coated HB-CNPs (as displayed in Figure 9c). Thus, ALG-coated CS NPs (CNPs) could entrap HbsAg effectively (as shown in Figure 9b). The release profile of protein showed that 84% of the drug was released at 0.5 h. owing to CNPs' loose binding that led to increased desorption at the acidic pH, but 95.5% of the drug was released at 48 h (as displayed in Figure 9a). Coating with ALG stabilized increased the stability of CNPs and provided a sustained release of 51.42% after 48 h. In addition, the ALG-coated NPs adhered better than uncoated NPs. The cell viability of RAW 264.7 cell lines was 70.45%; nevertheless, the cell viability decreased

to 66.45% after 48 h. This time-dependent decrease in cytotoxicity could be due to contact between positively charged nanoparticles and negatively charged membranes. Mucosal M-cell-specific ALG-coated CS nanoparticles elicited a significant immunological response in mice (as demonstrated in Figure 9d). Hence, overall results showed the aforementioned system is favorable for mucosal drug delivery [270]. *Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 28 of 60

**Figure 9.** Lipopolysaccharide‐derived ALG coated Hepatitis B antigen‐loaded CS nanoparticles for oral mucosal immunization (**a**) Release profile of produced nanoparticles in vitro. (**b**) HBsAg release as determined by SDS‐PAGE: Lane 1: Molecular weight markers; Lane 2: HBsAg solution; Lane 3: HBsAg loaded CS nanoparticles, Lane 4: HBsAg‐loaded ALG coated CS nanoparticles produced from LPS. (**c**) The levels of sIgA in the fluid secretions of mice immunized with different formula‐ tions. (**d**) Anti‐HBsAg IgG levels in mice inoculated orally with various formulations. Reproduced with permission from [270], Copyright Elsevier, 2020. **Figure 9.** Lipopolysaccharide-derived ALG coated Hepatitis B antigen-loaded CS nanoparticles for oral mucosal immunization (**a**) Release profile of produced nanoparticles in vitro. (**b**) HBsAg release as determined by SDS-PAGE: Lane 1: Molecular weight markers; Lane 2: HBsAg solution; Lane 3: HBsAg loaded CS nanoparticles, Lane 4: HBsAg-loaded ALG coated CS nanoparticles produced from LPS. (**c**) The levels of sIgA in the fluid secretions of mice immunized with different formulations. (**d**) Anti-HBsAg IgG levels in mice inoculated orally with various formulations. Reproduced with permission from [270], Copyright Elsevier, 2020.

In another investigation, Ghumman et al. utilized linseed mucilage‐ALG mucoad‐ hesive microspheres loaded with Metformin HCl developed by an ionotropic gelation procedure for mucosal actives delivery. Drug encapsulation efficiency was up to 92% with sustained release for 12 h. The results revealed that an optimized formulation (FM‐4) could furnish sustained release for up to 12 h and hold the level of blood glucose by reg‐ ulating and enhancing the absorption of metformin systemically. Therefore, LSM, a natu‐ ral emerging mucoadhesive agent, proved to be preferable for controlled release mucoad‐ hesive microspheres designed for oral utilization [271]. In periodontitis, there is inflammation and destruction of tooth tissues, and the path‐ In another investigation, Ghumman et al. utilized linseed mucilage-ALG mucoadhesive microspheres loaded with Metformin HCl developed by an ionotropic gelation procedure for mucosal actives delivery. Drug encapsulation efficiency was up to 92% with sustained release for 12 h. The results revealed that an optimized formulation (FM-4) could furnish sustained release for up to 12 h and hold the level of blood glucose by regulating and enhancing the absorption of metformin systemically. Therefore, LSM, a natural emerging mucoadhesive agent, proved to be preferable for controlled release mucoadhesive microspheres designed for oral utilization [271].

ogen forms a biofilm around the inflamed tissues. Currently, treatment approaches in‐ clude mechanical removal of the biofilm aided by antibiotics whose systemic side effects limit the effectiveness of treatment. Hence, Kilicarslanet al. used clindamycin phosphate‐ loaded ALG/CS PEC film to overcome this issue. CS and ALG, being oppositely charged biopolymers, form a PEC by cross‐linking with each other. It was observed that increased ALG concentrations in the polymer mixture increased adhesiveness, making it valuable for mucosal drug delivery [272]. In another investigation, Gonçalves et al. developed highly porous ALG/carrageenan In periodontitis, there is inflammation and destruction of tooth tissues, and the pathogen forms a biofilm around the inflamed tissues. Currently, treatment approaches include mechanical removal of the biofilm aided by antibiotics whose systemic side effects limit the effectiveness of treatment. Hence, Kilicarslanet al. used clindamycin phosphateloaded ALG/CS PEC film to overcome this issue. CS and ALG, being oppositely charged biopolymers, form a PEC by cross-linking with each other. It was observed that increased ALG concentrations in the polymer mixture increased adhesiveness, making it valuable for mucosal drug delivery [272].

aerogel nanoparticles for drug delivery of powdered model drugs quercetin and keto‐ profen. Biopolymer aerogels have low toxicity and are biocompatible, with large surface In another investigation, Gonçalves et al. developed highly porous ALG/carrageenan aerogel nanoparticles for drug delivery of powdered model drugs quercetin and ketoprofen.

areas with accessible pores or drug loading. The emulsion gelation method was used to produce the hybrid biopolymer nanoparticles, followed by drying using supercritical

Biopolymer aerogels have low toxicity and are biocompatible, with large surface areas with accessible pores or drug loading. The emulsion gelation method was used to produce the hybrid biopolymer nanoparticles, followed by drying using supercritical CO2. Pectin and ALG had better interaction, with an increased degree of cross-linking between the two polysaccharides giving NPs of higher specific surface area (>300 m2 g –1) and lower shrinkage. The drug-loading capacity was 17 ± 2% for ketotifen and 3.8% for quercetin. The cytotoxicity test using the MTS assay on human carcinoma Caco-2 cell line showed almost 100% cell viability, thus demonstrating the prepared nanoparticles to be highly non-toxic. The drug release from ALG/κ-carrageenan aerogel was quicker than from ALG/pectin [273]. Thus, the developed system was found to be suitable for drug delivery.

In another research, Martín et al. developed effective antifungal mucoadhesive drug delivery systems to treat oral candidiasis using ALG microspheres containing the drug nystatin. Oral candidiasis is caused by the opportunistic pathogen Candida albicans, especially in immunocompromised, diabetic patients undergoing chemotherapy. Nystatin, a polyene antifungal antibiotic, is indicated to treat mucocutaneous fungal infections caused by C. albicans. However, nystatin possesses a huge lactone ring and numerous double bonds, which render it amphiphilic and amphoteric and thus render its formulation difficult. Consequently, it has been formulated as micellar gels, liposomes, intralipids, niosomes, and various other dosage forms [274–276]. Thus, Martin et al. developed nystatin microspheres based on the emulsification/internal gelation procedure with some modifications [277]. Evaluation of particle size, zeta potential, swelling behavior, loading content, encapsulation efficiency, rheology, mucoadhesive force, drug release studies, antimicrobial activity, and in vivo studies were carried out. It was found that the mean size of the microspheres was 85–135 um, and the viscosity was that of a Newtonian fluid. The drug release was in two steps; first, an initial burst release was followed by a more sustained drug release, with about 60% of the drug released in the first 2 h. The prepared microspheres showed marked antifungal activity with a drastic reduction in C. albicans colonies. In vivo research on the buccal mucosa of experimental animals demonstrated that the retained amount, which is 4 to 6 times higher than the MIC, was enough to elicit the therapeutic response [278].

Researchers have also formulated lysozyme mucoadhesive tablets/Ca2+ cross-linked ALG with HPMC [279], silybin/nanocrystals-in-microspheres PEC/ALG/CS [280], co-delivery of ketorolac and lidocaine/polymeric wafers/2:1 SA:PVP −25 and 10% glycerol [281], HPMC/ SA/nicotine [282], ALG modified with maleimide-PEG/ibuprofen sodium [283], silicone sheet/dexamethasone/ALG [284], ALG/sterculia gum/citicoline [285], ALG/ghatti gum/ montmorillonite/flubiprofen [286], pectin/ALG/repaglinide [66], polyacrylamide-g/locust bean gum/ALG/ketoprofen [287], CS/CA/*Lactobacillus casei* [288] for improved mucosal actives delivery.

#### *6.8. Intravenous Drug Delivery*

ALG, owing to its biocompatibility and safety in vivo, has been explored for intravenous drug delivery in order to increase the bioavailability of drugs that the conventional oral route cannot deliver.

Hydrophobic medicines can be entrapped in hydrophilic nanoparticles like CA and delivered to their site of action. Curcumin and resveratrol are both polyphenolic molecules found in nature that have anti-cancer properties. Their low water solubility and bioavailability unfortunately limit their therapeutic utility. In a study by Saralkar et al., the emulsification and cross-linking procedures were used to make curcumin and resveratrol-based CA nanoparticles. Particle size, zeta potential, moisture content, physicochemical stability, and %EE were all measured in the nanoparticles. For the combined estimation of curcumin and resveratrol, the UPLC methodology was designed and validated. The in vitro efficiency of ALG nanoformulation on DU145 prostate cancer cells was investigated. Curcumin and resveratrol had entrapment efficiencies of 49.3 ± 4.3% and 70.99 ± 6.1%, respectively. In 24 h, resveratrol had a greater release than curcumin (87.6 ± 7.9% against 16.3 ± 3.1%). Curcumin, both in solution and as nanoparticles, was found to be taken up by cells. Resveratrol

was poorly absorbed by cells. On DU145 cells, the drug-loaded nanoparticles cause cytotoxicity. The drug solution was more hazardous than nanoparticles at high concentrations. The intravenous administration of the ALG nanoformulation was proven to be safe [289].

Over the last 10 years, multifunctional theranostics have created some intriguing novel possibilities for chemotherapy and tumor detection. In a study by Yang et al., the photosensitizer chlorin e6 (Ce6) and the anticancer medication DOX were subsequently adsorbed onto the magnetic mesoporous silica nanoparticles (M-MSNs) to produce a pH-sensitive drug release and for adsorbing P-glycoprotein short hairpin RNA (P-gp shRNA) for preventing multidrug resistance, ALG/CS polyelectrolyte multilayers (PEM) were constructed on the M-MSNs (M-MSN(DOX/Ce6)/PEM/P-gp shRNA). Upon laser illumination, the nanoparticles with a mean diameter of 280 nm showed a pH-responsive drug release profile and increased singlet oxygen formation in tumor cell lines. The delivery mechanism only released 12 percent Ce6 after 30 h at pH 7.4 but >95 percent after 36 h at pH 4.0. In terms of DOX release, roughly 30% of it was released from M-MSN(DOX/Ce6)/PEM nanocomposites in 32 h at physiological pH (7.4), compared to a 46% release of DOX at pH 4.0. The multifunctional nanocomplexes greatly boosted apoptosis in vitro, as demonstrated by the CCK-8 assay and calcein-AM/PI co-staining. Using cancerous Balb/c mice for the animal studies, researchers used a combination of photodynamic treatment and chemotherapy to achieve a synergistic anti-cancer activity in vivo. Additionally, the cores of the bifunctional Fe3O4-Au nanoparticles in the multifunctional nanocomplexes allowed dual-modal MR and CT imaging, revealing high uptake into tumor-bearing animals via intravenous injection. This study demonstrates the excellent performance of magnetic mesoporous silica nanocomposites as a multipurpose delivery system for imaging-guided cancer synergistic therapy [290].

For cancer treatment, nanocarrier drug delivery systems (NDDSs) have received more attention than traditional drug delivery methods. The rapid evacuation of activated macrophages from the bloodstream, however, hinders efficacy. In a study by Wang et al., glycyrrhizin (GL) was loaded into ALG nanogel particles (NGPs) to create a versatile delivery mechanism to reduce activated macrophage clearance and improve anticancer activity with GL and DOX combined therapy. GL-ALG NGPs could not only prevent eliciting macrophage immuno-inflammatory responses, but they could also reduce macrophage phagocytosis. DOX/GL-ALG NGPs increased DOX bioavailability by 13.2 times compared to free DOX in the blood. The use of mice with normal immune systems instead of nude mice in the construction of tumor-bearing mice further revealed that NGPs are biocompatible. In vitro and in vivo, GL-mediated ALG NGPs had an exceptional hepatocellular carcinoma targeting effect [291].

Arsenic trioxide (ATO) is efficacious in managing acute promyelocytic leukemia (APL) and late-stage primary hepatic cancer, although it has serious adverse effects. Hence, Lian et al., in order to address these issues, synthesized red blood cell membrane-camouflaged ATO-loaded SA nanoparticles (RBCM-SA-ATO-NPs, RSANs). Ion crosslinking was used to make ATO-loaded SA nanoparticles (SA-ATO-NPs, SANs), wherein RBCM was deposited over the surface to make RSANs. RSANs had a mean particle size of 163.2 nm with an entire shell-core bilayer arrangement and a 14.31 percent encapsulation efficiency. When relative to SANs, a decreased phagocytosis in RAW 264.7 macrophages was observed RSANs by 51%, and the in vitro cumulative release rate was 95% at 84 h, indicating a notable sustained release. Moreover, RSANs were found to have reduced cytotoxicity than natural 293 cells and to have anti-cancer activity on both NB4 and 7721 cells. In vivo investigations also revealed that ATO can induce minor organ damage, whereas RSANs can minimize toxicity and boost anti-tumor efficacy [292].

Curcumin administration by nanocarriers is an appealing strategy for overcoming its limited bioavailability and rapid metabolism in the liver. Karabasz et al. developed AA-Cur, a blood-compatible ALG-curcumin combination that produced colloidally stable micelles of around 200 nm and displayed high cytotoxic effects against mouse cancer cell lines, as previously demonstrated. In their study, they investigated AA-toxicity and

anticancer efficacy in two different animal tumor models. In the first study, C57BL/6 mice were administered with colon cancer MC38-CEA cells subcutaneously. Breast tumor 4T1 cells were administered orthotopically, that is, into the mammary adipose tissue of BALB/c mice in the second study. Investigations of blood biochemistry, histology, morphology, DNA integrity (comet assay), and cytokine screening were used to assess the toxicity of intravenously injected AA-Cur (flow cytometry). The anticancer effects of AA-Cur were determined by comparing the development of colon MC38-CEA- or orthotopically injected breast 4T1 tumor cells in untreated and AA-Cur-treated mice. Four injection dosages of AA-Cur revealed no toxicity, showing that the conjugate is safe to use. The anti-cancer efficacy of AA-Cur was moderate in colon MC38-CEA and breast 4T1 carcinomas [293].

Other applications of ALG in intravenous drug delivery include ALG/deferoxamine conjugates [294], ALG Microparticles/Amphotericin B [295], ALG/poly(amidoamine)/ MC3T3-E1 pre-osteoblasts hybrid hydrogel [296], octanol grafted ALG nanoparticles/ propofol [297], pH-Responsive ALG/CS multilayer coating on Mesoporous Silica Nanoparticles encapsulating DOX [298], ALG-glycyl-prednisolone conjugate nanogel [299].

## *6.9. Others*

The buccal mucosa is an extremely appealing route of actives delivery for actives with low bioavailability, poor gastric stability, and vulnerability to first-pass metabolisms such as proteins and peptides, by carrying the actives straightaway into the bloodstream. The oral mucosa also lacks Langerhan cells, making the oral mucosa tolerant to various allergens [300]. Aphthous stomatitis, a type of inflammation in the intraoral cavity whose causative agent is still unknown, is currently treated symptomatically using corticosteroids. Ambroxol is gaining popularity as an upcoming agent to treat chronic inflammation. Laffleur and Küppers developed a buccal dosage form by anchoring sulfhydryl groups of the amino acid cysteine onto the ALG adhesive backbone, incorporating ambroxol as the antisecretory drug. Mucoadhesive studies show that ALG-SH had an 11.56-fold increase in adhesion time due to the binding of the sulfhydryl group to the cysteine-rich mucus glycoprotein, leading to prolonged residence time in comparison to the weak van der Waals and hydrogen bonding in native polymers. Permeation studies on freshly excised buccal mucosa showed a 1.89-fold increase in permeation of ALG-SH as compared to native ALG. The mechanism of permeation enhancement was by tyrosine kinase inhibition by disulfide bond formation between the sulfhydryl group of the polymer and the cysteine group of the protein. Ambroxol release from ALG-SH showed a 1.4-fold-controlled release compared to native ALG, possibly due to inter and intra-crosslinking formation, thus providing stability. Therefore, sulfhydryl-anchored ALG could be used for the effective therapy of aphthae [301].

Periodontal diseases affect the gums and bones of the teeth due to bacterial infections. The desirable properties of drug delivery systems for periodontal illnesses include low toxicity, biodegradability, and the ability to treat bacterial infections. Hence, Prakash et al. utilized a controlled-release formulation of amoxicillin using PVA/ALG/hydroxyapatite (HAp) films by wet precipitation to treat periodontal infections. SEM studies show HAp NPs were effectively blended and embedded with amoxicillin irrespective of the annealing temperature. The in-vitro analysis, cell viability assay (70%), fluorescent staining, and hemolysis assay provided conclusive evidence for the suitability of this composite film in treatment. It gave a sustained release, with 87% of the actives released by day 10. The swelling ratio was almost 80% for all films annealed at different temperatures. The tensile strength was more significant than the standard PVA/SA patch, and it increased with increasing annealing temperature. The fabricated films exhibit high anti-bacterial action against *Escherichia coli*, *Staphylococcus aureus*, *Enterococcus faecalis*, and *Pseudomonas aeruginosa*. The fabricated films are also highly biocompatible and hemocompatible, as evidenced by in vitro analysis, cell viability studies, fluorescent staining, and hemolysis assay. Annealing at different temperatures ranging from 300, 500, and 700 ◦C, respectively, gave films that can encapsulate and release the drug at different

extents. HAp also helps in the regeneration of damaged bone segments, in addition to its drug matrix properties. Thus, PVA/SA/HAp/amoxicillin films are good candidates for treating periodontal defects, orthopedic implants, and bone grafting [302].

The sublingual route of vaccine delivery is attracting a lot of attention because of the ease of self-administration of vaccines as well as an abundance of antigen-presenting cells in the sublingual mucosa. In contrast to intravenous and subcutaneous vaccines, sublingual vaccines confer mucosal immunity as well against pathogens like SARS, HIV, and HPV [303]. However, they suffer from a lack of adhesion and absorption through the sublingual epithelium. Hanson et al. prepared a biopolymer platform based on mucoadhesive ALG and CMC polymer wafers loaded with HIV gp14 protein for the delivery via sublingual route and protection of protein vaccines. The wafers were prepared by dissolving ALG:CMC polymers along with NaCl in deionized water. Microstructural analysis of the wafers revealed that CMC wafers had huge pores with thick strands, while the pores of ALG were smaller and smoother. This may be due to CMC being phased out of water during freezing while amorphous ALG chains were more flexible, hence more extensive entanglement. In addition, it was found that wafers with high ALG content showed high mechanical stability as well as protection from impairment owing to lyophilization and exorbitant heat, partly due to their network-forming capability and poor crystallinity. In contrast, a large number of CMC incorporated wafers were extremely mucoadhesive to sublingual mucosa tissue and could endure washing, leading to enhanced protein permeation into the tissue. Compared to liquid gp140 solution, the vaccines produced comparable T-cell and B-cell mediated immune reactions in mice and comparable IgA and IgG levels in blood, vagina, and saliva. The optimum formulation (CMC:ALG ratio of 1:1) could be safely stored and carried without a cold chain while also preserving its immunogenicity following vaccination in mice via a sublingual route. Thus, this novel platform could be used for the potential delivery of sublingual vaccines [304].

ALG-based formulations are presently in the limelight owing to their exceptional characteristics. They are researched meticulously to obtain the utmost benefits of drug delivery (shown in Tables 2 and 3).

#### **7. Recent Advances in ALG Formulations in Wound Healing**

Skin represents the largest human organ that functions as a protective barrier against dehydration, pathogens, environmental stresses, etc. [335]. Skin injuries can be acute or chronic and can occur in arterial insufficiency, diabetes mellitus, immunological disorders, and other infections. Rarely does complete re-epithelialization happen in repairing skin defects; hence, increased attention is diverted to preparing wound dressings to promote wound healing and reduce scars while also protecting from microbial infections and dehydration at the wound site [336,337].

Wound dressings play a vital part in wound management as they are applied to a variety of burns and wounds to aid in the repair and renewal of injured tissues, as well as to encourage healing and minimize the infection risk. The optimum wound dressing should decrease the recovery period and discomfort, increase tissue regeneration and recovery, absorb excess exudate from the wounds, encourage healing, and prevent infectious complications. For several decades, scientists have aspired to develop a specialized dressing to treat injuries. Traditionally, dressings are made from plant-based fibers, honey, animal fats, etc. Currently, biopolymeric materials are used as wound dressings as they offer unique properties, such as antibacterial, re-epithelializing, antioxidizing, and antiinflammatory properties, that significantly promote wound healing [338,339].

Recent developments in wound dressings also enable the release of therapeutic agents to restore skin homeostasis and integrity [336]. Biopolymers are attractive wound healing materials as they can maintain a moist wound bed due to water sorption; they can also absorb any tissue exudates and allow oxygen permeation across the wound [340]. Furthermore, because of the hydrophilic nature and structural features of these wound dressings, a sustained release of the encased bioactive substances could be achieved [341]. Both synthetic and natural polymers are useful for wound dressing, including heparin, CS, HA, dextrans, ALGs, and β-glucans, because of their desirable physical and biochemical properties [338,342].

ALG is a biopolymer widely used for tissue engineering applications, particularly in wound healing [343]. It has been used as a food additive for ages; hence, it is considered a biocompatible polymer. It has found applications in tissue regeneration and bioactive delivery because of its biodegradability and slow dissolution in the biological fluids when cross-linked with exchangeable cations. This rate could be adjusted by controlling the oxidation [344] and reducing ALG's molecular weight [345]. ALG composites are used for wound healing and soft tissue regeneration to strengthen the capacity and material characteristics of native ALG to adapt to different biomedical applications [346–348]. Calcium ions are released when water-insoluble CA encounters wound exudates as a function of calcium ions being replaced by sodium ions in bodily fluids, which can operate to achieve hemostasis. The SA-based fibers absorb a massive amount of exudates and transform into a gel-like substance, which maintains the moist barrier on the wound surface [349,350]. Some recent investigations of ALG composites in wound healing applications have been discussed in Table 4.

Diabetes-induced wounds currently have no effective treatment and thus represent a challenge in wound healing. Reports suggest that bacteria-caused inflammations due to the alkaline pH of ulcer wounds and incomplete blood flow to the wounds due to slow angiogenesis may be responsible for delayed diabetic wound healing. Hence, Wang et al. developed a novel, pH-sensitive CA-based hydrogel loaded with protamine nanoparticles and hyaluronan oligosaccharides (protamine NP/HAO CA hydrogel) by the ionotropic gelation method. The loading efficiency of HAO was 85.4 ± 6.25%, and 44.5% of the drug was released in 8 h at pH 3.0. At pH 8.0, which mimics the diabetic wound state, CA hydrogel swelled by the absorption of water, and a faster drug release of NPs and HAO took place. Compared to plain CA hydrogels, the protamine-loaded ones showed improved antibacterial activity against *E. coli* and *S. aureus*, enhanced adherence to wound tissue sites, and absorption of wound fluid into the hydrogels. Wounds closed with rates of up to 96.8% after 14 days without ulceration and festering at the wound site. Re-epithelialization was

observed, indicated by the formation of fibroblasts and collagen. Moreover, the composite hydrogels reduced inflammation and encouraged angiogenesis [351].

PECs produced from the polysaccharides ALG and CS at defined flow rates, pH, and agitation speeds give rise to slender, stable, and transparent sponges or films possessing excellent swelling ability in physiological medium. Unfortunately, these PECs have inadequate mechanical properties, which may negatively affect their performance in articulated regions such as knees, shoulders, and elbows. To overcome this, Pires et al. fabricated CS-ALG-based wound dressings with the addition of poly (dimethylsiloxane) to improve the flexibility and sorption capacity in contact with biological fluids. The optimum amount of poly(dimethylsiloxane) per gram of PEC was found to be 0.1 g, wherein the formulation exhibited high stability, a tensile strength of 12 MPa, non-hemolysis, and induction of thrombus formation. Moreover, when beta-carotene and thymol were loaded onto the formulations via the supercritical carbon dioxide impregnation/deposition (SSI/D) technique at 45 ◦C and 250 bar for 14 h, higher bioactive loading capacities were observed using SSI/D and a high depressurization rate (10 bar/min). A substantial amount of Thy and Bc were preserved in the matrix structure with the SSI/SD method, which functioned as a reservoir system. Thus, the combination of ALG, CS, and silicone gel could be a potentially successful wound dressing in the case of less articulated body sites and low extruding wounds [352].

The combination of ALG with proteins holds a lot of promise in enhancing the cellular interactions of ALG and for tailoring the biodegradation of the composite materials in tissue regenerating applications. Elastin, a highly flexible protein abundant in the extracellular matrix, is a good candidate for composites because it renders the tissues elastic enough to withstand continuous cycles of deformation/recovery without rupturing. Hence, Bergonzi et al. fabricated ALG/human elastin-like polypeptide (HELP) hybrid films by the solvent casting method with loaded curcumin to provide antioxidant activity. Strong intermolecular hydrogen bonding between the N-H amide bonds of HELP and the carboxylate group of ALG leads to close network formation due to molecular associations, thus giving higher tensile strength and Young's modulus, which is representative of its higher elasticity. Swelling capacity was higher in HALCur than in ALCur (as shown in Figure 10a). The incorporation of HELP was not only instrumental for obtaining a controlled release of curcumin (as depicted in Figure 10b), which helped in a higher antioxidant effect (as illustrated in Figure 10d), but also in enhancing the cytocompatibility of the final biomaterial, as shown in Figure 10c). With more in vivo studies, it may be possible to design customized bioplatforms for biomedical applications [353].

Full-thickness healing requires extensive healing time, which increases the risks of infections, wound ulcers, necrosis, and even fatal complications. To tackle this, a hybrid hydrogel composed of amine-functionalized fish collagen and OA was prepared by Feng et al. by a simple Schiff base reaction, and antimicrobial peptides bacitracin and Polymyxin B (AC/OSA-PB) were loaded into the hydrogels without the need for catalysts. These hydrogels illustrated modifiable gelation time, stable rheology, and a strain resembling that of human skin. Moreover, they could effectively cause inhibition of *S. aureus* and *E.coli* growth, promoting angiogenesis and cell proliferation in vitro. Similar results were observed in vivo too, with enhanced full-thickness wound healing ability by promoting granular tissue formation and deposition of collagen and accelerating neovascularization and re-epithelialization [354].

In a similar study related to full-thickness wound healing by Chaudhary et al., CA nanoparticles (CA-NPs) were used as hemostatic agents along with antimicrobial AgNPs in a CS-based hydrogel network. Herein, the fresh blood of the subjects was used in the hydrogels to substitute the growth factors required for wound healing. The CA-NPs had mean hydrodynamic sizes of 1037 nm and 120.56 nm, respectively, and a zeta potential of less than −30 mV indicated their negligible electrostatic repulsion tendency. The prepared hydrogels exhibited good spreadability and viscoelasticity. Moreover, they showed bacterial inhibition against both gram-positive and gram-negative strains and also

illustrated remarkable scar-free healing in vivo for up to 15 days by aiding in collagen deposition and acting as a protective sheath against microbial contamination for diabetesinduced wounds. Thus, the proposed composite films could be an exciting wound dressing for patients who have chronic diabetes [355]. *Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 39 of 60

**Figure 10.** ALG/human elastin‐like polypeptide composite films with antioxidant properties for potential wound healing applications. (**a**) swelling (A) and stability (B) graphs of ALCur, HALCur films encapsulating 0.1% curcumin. (**b**) % curcumin release characteristics of ALCur, HALCur films encapsulating 0.1% curcumin. (**c**) Cell viability assay of ALG/HELP composites on human fibroblast cell lines. (**d**) DPPH assay of ALCur and HALCur films indicating antioxidant activities. # *p* <0.05. Reproduced with permission from [353], copyright Elsevier 2020. **Figure 10.** ALG/human elastin-like polypeptide composite films with antioxidant properties for potential wound healing applications. (**a**) swelling (A) and stability (B) graphs of ALCur, HALCur films encapsulating 0.1% curcumin. (**b**) % curcumin release characteristics of ALCur, HALCur films encapsulating 0.1% curcumin. (**c**) Cell viability assay of ALG/HELP composites on human fibroblast cell lines. (**d**) DPPH assay of ALCur and HALCur films indicating antioxidant activities. # *p* <0.05. Reproduced with permission from [353], copyright Elsevier 2020.

Full‐thickness healing requires extensive healing time, which increases the risks of infections, wound ulcers, necrosis, and even fatal complications. To tackle this, a hybrid hydrogel composed of amine‐functionalized fish collagen and OA was prepared by Feng et al. by a simple Schiff base reaction, and antimicrobial peptides bacitracin and Poly‐ myxin B (AC/OSA‐PB) were loaded into the hydrogels without the need for catalysts. These hydrogels illustrated modifiable gelation time, stable rheology, and a strain resem‐ bling that of human skin. Moreover, they could effectively cause inhibition of *S. aureus* and *E.coli* growth, promoting angiogenesis and cell proliferation in vitro. Similar results were observed in vivo too, with enhanced full‐thickness wound healing ability by pro‐ moting granular tissue formation and deposition of collagen and accelerating neovascu‐ larization and re‐epithelialization [354]. In a similar study related to full‐thickness wound healing by Chaudhary et al., CA nanoparticles (CA‐NPs) were used as hemostatic agents along with antimicrobial AgNPs in a CS‐based hydrogel network. Herein, the fresh blood of the subjects was used in the hydrogels to substitute the growth factors required for wound healing. The CA‐NPs had In another study, Sharma et al. loaded rifampicin into ALG-gelatin fibers through a physical cross-linking reaction by the extrusion-gelation method and then embedded it into transdermal films for wound healing applications. The tensile strength of the fibers was between 2.32 ± 0.45 to 14.32 ± 0.98 N/mm2 , and the extensibility was between 15.2 ± 0.98% to 30.54 ± 1.08%. The range of moisture absorption was low (up to 14.68%), which is essential for transdermal films. Other parameters such as the swelling ratio and water vapor transmission rate demonstrated the extensive gelation properties of the polymer and the reduced channel pore size. Antibacterial activities against *E. coli* and *S. aureus* were observed for the transdermal films. In vivo animal studies exhibited close to 83 degrees of contraction of the wound, marginally less than the commercial formulation (91.87 ± 3.72%). The drug release from the films followed sustained release; this could be due to sufficient contact of the wound dressing with the wound layer with hair growth signs evident from the 10th day onwards. Thus, the proposed fiber-in-films could be excellent carriers for drug delivery and wound healing purposes [356].

mean hydrodynamic sizes of 1037 nm and 120.56 nm, respectively, and a zeta potential of less than −30 mV indicated their negligible electrostatic repulsion tendency. The prepared hydrogels exhibited good spreadability and viscoelasticity. Moreover, they showed bac‐ terial inhibition against both gram‐positive and gram‐negative strains and also illustrated remarkable scar‐free healing in vivo for up to 15 days by aiding in collagen deposition Other studies where ALG composites showed promising results in wound healing include SA/HA films/sulfadiazine/silver nanoparticles [357], ALG/gelatin fibers/ curcumin [358], SA/xanthan gum film/pycnogenol [359], ALG/CS/maltodextrin/pluronic F127/pluronic P123/tween 80/curcumin polymeric micelles [360], SA/pectin/cefazolin nanoparticles [361], CA/ibuprofen hydrogels [362], ALG/cannabidiol [363], SA/polyvinyl

and acting as a protective sheath against microbial contamination for diabetes‐induced wounds. Thus, the proposed composite films could be an exciting wound dressing for alcohol/curcumin/graphene [364], ALG/polyhexanide/AgNPs [365], ALG/carboxymethyl CS/Kangfuxin sponges [366].

**Table 4.** Recent investigations of ALG-based composites for wound healing applications.

#### **8. Conclusions**

ALG emerges as a prospective naturally derived biomaterial in NDDSs due to the biocompatible, degradable characteristics and gel-forming capability of ALG. Controlled and targeted actives delivery via ALG carrier can be accomplished via well-created formulation as well as accurate parameters of synthesis concerned with the process of fabrication. Hence, this article provides an extensive review of the recent advances of ALG and its advancement in actives delivery. The most significant characteristics of ALG encompass safety, biocompatibility, and ease of preparation. Due to its biocompatible, biodegradable, and non-toxic characteristics, it is employed in diverse drug-delivery technologies. A significant challenge that persists is the preparation of environmentally friendly procedures for the NPs formation having a narrow size distribution, high mechanical and chemical stability, and practicality to scale up to the volumes of industrial-scale production. Moreover, it is critical to assess the toxic effects on cells, immune response, and biodegradability of such formulations in drug delivery.

As we look towards the future, the ALG-based composites utilized in pharmaceutical applications are possibly going to develop significantly. Although ALG composites are already used clinically for wound healing, they perform quite a passive function. Forthcoming dressings will probably perform a considerably more active role. One or more bioactives that assist wound healing can be loaded into ALG-based dressings, as such gels have displayed usefulness in preserving local conc. of biological factors (for example, proteins) for a prolonged period. In wound healing, and more usually, actives delivery, accurate control

over the single vs. multiple drugs delivery or drug release in sustained vs. sequential manner in response to exterior environmental modifications is immensely advisable.

Therefore, investigators are required to modernize the ALG-associated composite's advancement, and this review is the origin of advice for forthcoming investigation.

**Author Contributions:** Conceptualization and supervision: M.A.S.A., A.S., M.J.A. and S.P.; Resources: M.A.S.A., M.J.A., R.R.R., R.M. and S.P.; Literature review and writing—original draft preparation: M.A.S.A., R.R.R., A.S., S.P., P.S., R.M. and M.J.A.; writing—review and editing: M.A.S.A., R.R.R., A.S., L.S.A., A.D., M.J.A. and S.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** MA would like to thank the Deanship of Scientific Research at Umm Al-Qura University for supporting this work by Grant Code: (22UQU4290565DSR53).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** MA would like to thank the Deanship of Scientific Research at Umm Al-Qura University for supporting this work by Grant Code: (22UQU4290565DSR53). Author RR would like to acknowledge the financial support by the Department of Mechanical Engineering and Mechanics at Lehigh University. He would also like to thank his mentor, Arindam Banerjee, for continuous support and motivation. SP would like to thank Ramesh Parameswaran and Vignesh Muthuvijayan for their guidance in preparing the manuscript. Author SP would like to thank the Indian Institute of Technology Madras, Ministry of Human Resource Development for providing financial assistantship. Author MJA acknowledges the support of the Deanship of Scientific Research at Prince Sattam bin Abdulaziz University.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

## **References**

## *Article* **Valorization of Berries' Agro-Industrial Waste in the Development of Biodegradable Pectin-Based Films for Fresh Salmon (***Salmo salar***) Shelf-Life Monitoring**

**Janira Romero 1 , Rui M. S. Cruz 2,3, Alexandra Díez-Méndez 1 and Irene Albertos 4,\***

1 Faculty of Sciences and Art, Universidad Católica de Ávila (UCAV), Calle Canteros s/n, 05005 Ávila, Spain

2 Department of Food Engineering, Institute of Engineering, Campus da Penha, Universidade do Algarve, 8005-139 Faro, Portugal

**Abstract:** The healthy properties of berries are known; however, red fruits are very perishable, generating large losses in production and marketing. Nonetheless, these wastes can be revalued and used. The main objective of this study was the development of biodegradable pectin films with berry agro-industrial waste extracts to monitor salmon shelf-life. The obtained extracts from blueberries, blackberries, and raspberries wastes were evaluated in terms of flavonols, phenols and anthocyanins contents, and antioxidant capacity. Then, pectin films with the extracts of different berries were developed and characterized. The results showed that the blueberry extract film was thicker (0.248 mm), darker (L\* = 61.42), and opaquer (17.71%), while the highest density (1.477 g/cm3 ) was shown by the raspberry films. The results also showed that blueberries were the best for further application due to their composition in bioactive compounds, antioxidant capacity, and color change at different pHs. The salmon samples wrapped in blueberry films showed lower values of pH and deterioration of fish during storage compared to the control and pectin samples. This study contributes to the valorization of berries agro-industrial waste by the development of eco-friendly films that can be used in the future as intelligent food packaging materials contributing to the extension of food shelf-life as a sustainable packaging alternative.

**Keywords:** biopolymers; intelligent food packaging; bioactive natural compounds; biodegradable; shelf-life; sustainability

## **1. Introduction**

Food packaging is an essential part of the food sector as it guarantees the quality and safety of products, helps in the transport process, provides stable storage, prevents damage and losses, and ensures greater safety for the consumer [1,2]. Traditionally, the packaging is made from petroleum derivatives; however, they generally contain chemicals considered harmful, such as bisphenol and phthalates [3]. In addition, the long life and resistance of plastics to degradation have generated an accumulation of waste that has a great negative impact on the environment [4]. For this reason, we are currently looking for plastic reduction systems and the development of sustainable alternatives. Some of these alternatives are bio-based polymers, e.g., pectin [1] that can generate biodegradable packaging materials. Likewise, these new materials can be incorporated with different compounds, improving characteristics such as permeability or optical features, which allow the production of smart packaging systems [5].

Intelligent packaging is one type of smart packaging and is defined as one capable of making decisions to increase the shelf-life of the food, inform the consumer, and improve

**Citation:** Romero, J.; Cruz, R.M.S.; Díez-Méndez, A.; Albertos, I. Valorization of Berries' Agro-Industrial Waste in the Development of Biodegradable Pectin-Based Films for Fresh Salmon (*Salmo salar*) Shelf-Life Monitoring. *Int. J. Mol. Sci.* **2022**, *23*, 8970. https://doi.org/10.3390/ijms23168970

Academic Editors: Valentina Siracusa and Swarup Roy

Received: 20 July 2022 Accepted: 9 August 2022 Published: 11 August 2022

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

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

the quality of the product through various methods such as pH indicators through color changes [6]. The containers with pH indicators may incorporate extracts of various fruits or vegetables that have different properties and allow color changes associated with the variation in the pH of the product. Some of these extracts can be obtained from berries such as blueberries, raspberries, and blackberries, which possess antioxidant capacity thanks to their bioactive compounds, such as phenolic compounds and anthocyanins [7]. Berries are a product with increasing attractiveness, and their consumption has increased in recent years due to their beneficial properties for health such as antioxidants, anticancer substances, vitamins, minerals, fibers, and other essential nutrients [8,9]. However, berries are very perishable products since they have a high water content, which makes them susceptible to mechanical damage, contamination, freezing, or dehydration, generating large losses [10]. These losses generate serious nutritional, economic, and environmental problems [11]. To circumvent these problems, the fruits are normally processed, for example, for obtaining juices, from which numerous by-products can be achieved [12]. Food waste is already a global problem that endangers the long-term food supply chain, with 1.300 million tons being discarded every year [13]. For this reason, the Sustainable Development Goals (SDGs) require, by 2030, a halving of food waste per capita in the supply and consumption chain [13]. More than 1.748 million tons of wasted food correspond to fruits and vegetables [13], so there are already several reviews on the possible applications of these residues. Bayram et al., 2021 [14] studied the possible use of biopolymers, biocomposites, smart packaging, and edible films or coatings. In the processing of fruits and vegetables, by-products consisting of seeds and shells are produced in large quantities, which present a large concentration of bioactive components such as antioxidants, pigments, proteins, essential oils, enzymes, and dietary fibers [13].

The residues resulting from processing, called pomace, also in the case of berries can be reused in the food industry as ingredients or natural additives due to the bioactive compounds [11]. As previously referred to, the waste generated represents a great loss of valuable nutrients. For this reason, the biotransformation of waste is receiving increasing attention since it can be used as a resource to obtain useful products with added value [8]. There are numerous biopolymers used in the development of packaging materials, with poly (lactic acid) (PLA), cellulose, starch, and chitosan being more widely used [15]. PLA is a biodegradable polymer with mechanical properties very similar to those of thermoplastics. Starch is also biodegradable and is a polymer that is easily found; however, it has a strong hydrophilic behavior, making it sensitive to moisture [15]. On the other hand, cellulose is one of the most abundant renewable materials being used as a filler or host polymer in packaging [15]. Chitosan contains antimicrobial properties, so it can be used as a host and antimicrobial agent [15]. As previously referred to, another polymer that can be used in the development of packaging films is pectin, in which plant extracts can be easily added to generate active ingredients [16]. Pectin is a good component thanks to its ability as a gelling and emulsifying agent, generating water-soluble films with low opacity [16]. Essential oils can be added to pectin films, the most common being cinnamon, rosemary, oregano, cloves, thyme, lemon, and orange. Other elements that can be incorporated into pectin are agricultural residues such as banana, orange, and lemon peels among others [16]. Plant extracts rich in phenolic compounds are also added to pectin to increase the antioxidant capacity of the films, although there is less research in this area compared to in essential oils [16].

On the other hand, blueberry residues have multiple uses such as biofuel, biogas, biochar, or biocrude oil production [17]. There are also various uses in food as additives; for example, Rai et al., 2021 [18] observed that blueberry residues produce certain ferments that help to improve intestinal microbiota and intestinal function, corroborating its potential use as a functional food [18–20]. Different studies used blueberry residues to develop smart packaging as films based on different biopolymers such as chitosan, starch, and gelatin [21–27]. However, no subsequent application was carried out as smart packaging on food products such as meat or fish.

Fresh fish is considered key in the diet as it provides 17% of the animal protein ingested [28]. Specifically, salmon (*Salmo salar*) is one of the most consumed products worldwide, representing 93% of production [29]. Its consumption has been increasing thanks to the fact that it contains beneficial components for health such as omega-3 longchain polyunsaturated fatty acids (LC-PUFA) [28] as well as its color, taste, protein content, vitamins, and antioxidants [30]. However, salmon and other fishery products are very perishable due to their high-water activity, almost neutral pH, and other specific components that favor biochemical, physical, and microbial deterioration during the production chain, specifically the deterioration that begins immediately after capture [28].

To our knowledge, there is little information on the use of residues of other berries such as raspberries and blackberries for the development of smart packaging materials, as well as its comparison with blueberries and their subsequent use in fish products, particularly in salmon. In addition, few studies in the literature reported the use of pectin as a matrix to develop intelligent packaging. For this reason, this study aimed to develop and characterize biodegradable pectin films with the incorporation of blueberry, raspberry, and blackberry waste extracts and to study their effect on fresh salmon shelf-life.

#### **2. Results and Discussion**

#### *2.1. Characterization of the Extracts*

The results (Table 1) indicated significant differences (*p* < 0.05) in flavonol content among the different extracts. The blueberry extract was the one with the highest flavonols content (13.65 ± 0.01 mg of quercetin equivalents per g of extract), followed by the blackberry extract. Similar results were obtained in other studies [31,32].

**Table 1.** Characterization of the antioxidant properties of the extracts.

The values (mean ± standard deviation) followed by different lowercase letters in the same column, for each parameter, are significantly different (*p* < 0.05).

Blackberry and blueberry extracts showed the highest level of anthocyanins. The raspberry extract (Table 1) presented the lowest level of anthocyanins compared with the blackberry extract (*p* < 0.05). Different studies reported significant differences between anthocyanins obtained from blackberries and raspberries, being the latter of lower content [32,33]. Sariburun et al., 2010 [32] reported that the content of anthocyanins in extracts using water was more effective as they have a high solubility in water. In a variety of raspberry called "Rubin", anthocyanins obtained from an extract with water were 60.3 ± 0.7 mg GAE/100 g, while in an extract with methanol they were 24.1 ± 0.4 mg GAE/100 g. In the case of the blackberries, extraction with water proved to be more effective, showing values of 54.8 ± 0.8 mg GAE/100 g while with methanol the values were lower (41.3 ± 0.3 mg GAE/100 g) [32]. In short, a higher anthocyanin content was shown in blackberries, as in our study.

Blueberry extract showed the highest content of total polyphenols, differing significantly from raspberry and blackberry extracts, although their levels of polyphenols were also high. Other authors reported a higher polyphenol content in blackberry extract (24.85 ± 0.11 mg g−1 ) while that of cranberry extract was significantly lower (6.08 ± 0.04 mg g −1 ) [23] due to the extraction method in water to ensure food and environmental safety. In addition, phenols are an important indicator of antioxidant capacity [23]. In our study, the

highest antioxidant capacity in both methods was obtained in blueberry extract (Table 1), while no significant differences (*p* > 0.05) were observed between the antioxidant capacity of blackberry and raspberry extracts. This shows the expected positive correlation between total polyphenol content and antioxidant capacity. The use of two different methods to measure the antioxidant capacity under different reaction conditions allowed us to establish a more precise view of the antioxidant capacity for the developed films. Günde¸sli et al., 2019 [34] reported the highest antioxidant capacity in blackberries, followed by raspberries. Unlike other berries, blueberries have the greatest antioxidant capacity in the early stages of ripening, which is related to the high levels of flavonols and hydroxycinnamic acids in the pre-ripening stage [35].

On the other hand, the antioxidant capacity of raspberries was 40% lower than that of blueberries.

#### *2.2. Characterization of the Developed Films*

#### 2.2.1. Thickness, Density, and Hardness

The results for thickness, density, and hardness are presented in Table 2. Significant differences (*p* < 0.05) were obtained in the thicknesses of all films, with the pectin films being thinner while the blueberry films were thicker. In either case, it is shown that the addition of fruit powder significantly increased the thickness of the films compared to the control (pectin). For blueberries, similar values were recorded in the literature in the studies reported by Luchese et al., 2018 [24] and Andretta et al., 2019 [26] in cassava starch films. However, in other studies, Luchese et al., 2018 [25] and Jamróz et al., 2019 [36] obtained significantly lower thickness values, even at higher percentages of blueberry and yerba mate extracts. This decrease in thickness is possibly due to the particle size of the powder, which is within 100–300 µm. In the study by Luchese et al., 2018 [25], a lower thickness was observed in the films made from blueberry powder with smaller particle sizes. On the other hand, similar results to those obtained in the thickness of the films with blackberry extract (0.2 mm) were observed in the studies of Gutiérrez, 2018 [37] and Sganzerla et al., 2021 [38]. However, the films made with raspberry powder were thicker than those reported in Yang et al., 2021 [39], possibly due to differences in powder concentration.

**Table 2.** Thickness, density, and hardness of the developed films.

The values (mean ± standard deviation) followed by different superscript letters in the same column, for each parameter, are significantly different (*p* < 0.05).

Table 2 shows also the density in the different films; the film with raspberry extract was the one with the highest value (1.477 g/cm3 ) followed by the film with blueberry extract. However, the results obtained were lower than those of Yang et al., 2021 [39], possibly due to the different matrices used. In our study, the films were produced with pectin while Yang et al., 2021 [39] used a matrix comprised of pectin, sodium alginate, and xanthan gum. This could be due to the intermolecular interactions between anthocyanins and the matrix, directly influencing their properties and, therefore, the films that contain them [40], and thus, hydrogen bonds benefit from a regular arrangement of the matrix chain in the film, generating a higher density [41–43].

On the other hand, there was also a trend of increasing hardness from pectin films to the films incorporated with the berries' extracts. The film with the blackberry extracts presented the highest hardness while the film with the raspberry extracts the lowest. Probably, the intermolecular hydrogen bonds were formed between the hydroxyl groups

of the raspberry extract and the matrix, reducing the crosslinking of water and the matrix, thus decreasing the hardness of the film [39].

#### 2.2.2. Color

The color and opacity of the films are shown in Table 3. Significant differences were observed in all the color parameters between the different films, except for the L\* parameter, where there were no significant differences (*p* > 0.05) between the film with blackberries and the film with blueberries. However, the pectin film showed the highest luminosity (L\*) and hue (h\*). It is worth noting a clear tendency h\* decrease from pectin film (85.19) to the film with blackberries (26.19). Despite the absence of differences in the L\* value between the films with blackberry and blueberry extracts, these presented different results in the other parameters. The film with blackberry extract was more reddish compared will all the other films. This may be due to the different composition of anthocyanins in the different extracts. The trend to the red/purple is the result of the presence of cyanidin, which is purple at neutral pH [23]. The results of the parameters a\* and b\* coincide with the ones given in the study of Kurek et al., 2018 [23], where the same trend was observed among films developed with blackberries and blueberry extracts. Of the films incorporated with berry extracts, the film with raspberry extract was the one that presented the greatest luminosity. The luminosity of the films with raspberry extract (74.75) was similar (77.76) to the raspberry films (0.5 g/L raspberry films) reported by Yang et al., 2021 [39].

**Table 3.** Color and opacity of the developed films.

The values (mean ± standard deviation) followed by different superscript letters in the same column, for each parameter, indicate significant differences (*p* < 0.05).

#### 2.2.3. Opacity

The protective action against light is an important feature in the packaging of food since UV radiation and light are powerful lipid oxidizers [44]. Regarding opacity, there was a clear trend of increase in the incorporation of extracts (Table 3). The film with blueberry extract showed higher opacity than the film with blackberry extract, followed by the raspberry one.

Conversely, Kurek et al., 2018 [23] reported that blackberry extract films turned out to be opaquer than blueberry extract films.

### 2.2.4. Color Changes at Different pH

The color parameters were evaluated in solutions with different pHs to determine whether color changes at an environmental pH can be shown. The highest color variability according to pH changes was presented in the films with blueberry and raspberry extracts (Figure 1).

In the case of blueberries, significant differences (*p* < 0.05) were determined in the R value from pH 2 to 12. Yellowish brown hues of pH 2–5 were obtained, turning to a slightly darker brown up to pH 12. However, the film with raspberry extract showed very few color variations at different pHs, apart from some points showing significant differences (*p* < 0.05) in R, G, and B values between pH 2 and 6. In fact, these changes were not noticeable in the visual assessment (Figure 1).

**Figure 1.** Films color changes at different pH value. RB (Raspberry), BK (Blackberry), and BB (Blueberry). Values followed by different superscript letters in the same row, for each parameter, indicate significant differences (*p* < 0.05). **Figure 1.** Films color changes at different pH value. RB (Raspberry), BK (Blackberry), and BB (Blueberry). Values followed by different superscript letters in the same row, for each parameter, indicate significant differences (*p* < 0.05).

In the case of blueberries, significant differences (*p* < 0.05) were determined in the R value from pH 2 to 12. Yellowish brown hues of pH 2–5 were obtained, turning to a slightly darker brown up to pH 12. However, the film with raspberry extract showed very few color variations at different pHs, apart from some points showing significant differences (*p* < 0.05) in R, G, and B values between pH 2 and 6. In fact, these changes were not noticeable in the visual assessment (Figure 1). Other authors such as Kurek et al. 2018 [23] determined color parameters L, a, and b in chitosan films with blueberry and blackberry residue in a pH range of 2–12. Red colors from pH 2 to 4; blue/green at pH 5, 6, and 7; and dark green at pH 10 and 12 were observed for blueberries [23]. Separately, a red color was displayed at pH 2 and 4; violet at pH 5, 6, and 7; and dark blue at pH 10 and 12 for blackberries. In contrast, in a film of carboxymethylcellulose (CMC) and blackberries, the colors obtained in a pH range of 1 to 13 varied from pink in an acid medium to yellowish green in a basic medium [38], possibly due to the influence of the CMC matrix. Luchese et al. 2017 [22] determined the color changes in chitosan films with blueberries, on a pH scale of 2–12, obtaining results like those of Kurek et al. 2018 [23]. In addition, the process of prior blanching of the fruits caused the luminosity values to be lower, obtaining darker films [22]. This procedure was also performed in our study, which is why darker films were obtained such as those of Other authors such as Kurek et al., 2018 [23] determined color parameters L, a, and b in chitosan films with blueberry and blackberry residue in a pH range of 2–12. Red colors from pH 2 to 4; blue/green at pH 5, 6, and 7; and dark green at pH 10 and 12 were observed for blueberries [23]. Separately, a red color was displayed at pH 2 and 4; violet at pH 5, 6, and 7; and dark blue at pH 10 and 12 for blackberries. In contrast, in a film of carboxymethylcellulose (CMC) and blackberries, the colors obtained in a pH range of 1 to 13 varied from pink in an acid medium to yellowish green in a basic medium [38], possibly due to the influence of the CMC matrix. Luchese et al., 2017 [22] determined the color changes in chitosan films with blueberries, on a pH scale of 2–12, obtaining results like those of Kurek et al., 2018 [23]. In addition, the process of prior blanching of the fruits caused the luminosity values to be lower, obtaining darker films [22]. This procedure was also performed in our study, which is why darker films were obtained such as those of Luchese et al., 2017 [22]. This fact is because in the production of films with unblanched fruits there is a greater degradation of anthocyanins [22]. Andretta et al., 2019 [26] observed in their study with blueberries a color variation from red/orange at acid pH and green/ yellowish at basic pH. Yun et al., 2019 [45] developed films with starch-based blueberries, obtaining colors from pale violet to intense red when exposed to hydrogen chloride. However, in exposure to ammonia, the violet turned blue and then green.