Biodegradable Guar-Gum-Based Super-Porous Matrices for Gastroretentive Controlled Drug Release in the Treatment of Helicobacter pylori: A Proof of Concept
Abstract
An increase in resistance to key antibiotics has made the need for novel treatments for the gastric colonization of Helicobacter pylori (H. pylori) a matter of the utmost urgency. Recent studies tackling this topic have focused either on the discovery of new compounds to ameliorate therapeutic regimes (such as vonoprazan) or the synthesis of gastroretentive drug delivery systems (GRDDSs) to improve the pharmacokinetics of oral formulations. The use of semi-interpenetrating polymer networks (semi-IPNs) that can act as super-porous hydrogels for this purpose is proposed in the present work, specifically those displaying low ecological footprint, easy synthesis, self-floating properties, high encapsulation efficiency for drugs such as amoxicillin (AMOX), great mucoadhesiveness, and optimal mechanical strength when exposed to stomach-like fluids. To achieve such systems, biodegradable synthetic copolymers containing acid-labile monomers were prepared and interpenetrated with guar gum (GG) in a one-pot polymerization process based on thiol-ene click reactions. The resulting matrices were characterized by SEM, GPC, TGA, NMR, and rheology studies, and the acidic hydrolysis of the acid-sensitive polymers was also studied. Results confirm that some of the obtained matrices are expected to perform optimally as GRDDSs for the sustained release of active pharmaceutical ingredients at the gastrointestinal level, being a priori facilitated by its disaggregation. Therefore, the optimal performance of these systems is assessed by varying the molar ratio of the labile monomer in the matrices.
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Citation: Grosso, R.; Benito, E.; Carbajo-Gordillo, A.I.; García-Martín, M.G.; Perez-Puyana, V.; Sánchez-Cid, P.; de-Paz, M.-V. Biodegradable Guar-Gum-Based Super-Porous Matrices for Gastroretentive Controlled Drug Release in the Treatment of Helicobacter pylori: A Proof of Concept. Int. J. Mol. Sci. 2023,24, 2281. https://doi.org/ 10.3390/ijms24032281 Academic Editor: Silvana Alfei Received: 7 December 2022 Revised: 18 January 2023 Accepted: 19 January 2023 Published: 23 January 2023 Copyright: © 2023 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/). International Journal of Molecular Sciences Article Biodegradable Guar-Gum-Based Super-Porous Matrices for Gastroretentive Controlled Drug Release in the Treatment of Helicobacter pylori: A Proof of Concept Roberto Grosso 1, Elena Benito 1, Ana I. Carbajo-Gordillo 1, M. Gracia García-Martín1, Víctor Perez-Puyana 2, Pablo Sánchez-Cid 2and M.-Violante de-Paz 1,* 1Departamento de Química Orgánica y Farmacéutica, Facultad de Farmacia, Universidad de Sevilla, C/Prof. García González, n.º 2, 41012 Sevilla, Spain 2Departamento de Ingeniería Química, Facultad de Química, Universidad de Sevilla, 41012 Sevilla, Spain *Correspondence: [email protected] Abstract: An increase in resistance to key antibiotics has made the need for novel treatments for the gastric colonization of Helicobacter pylori (H. pylori) a matter of the utmost urgency. Recent studies tackling this topic have focused either on the discovery of new compounds to ameliorate therapeutic regimes (such as vonoprazan) or the synthesis of gastroretentive drug delivery systems (GRDDSs) to improve the pharmacokinetics of oral formulations. The use of semi-interpenetrating polymer networks (semi-IPNs) that can act as super-porous hydrogels for this purpose is proposed in the present work, specifically those displaying low ecological footprint, easy synthesis, selffloating properties, high encapsulation efficiency for drugs such as amoxicillin (AMOX), great mucoadhesiveness, and optimal mechanical strength when exposed to stomach-like fluids. To achieve such systems, biodegradable synthetic copolymers containing acid-labile monomers were prepared and interpenetrated with guar gum (GG) in a one-pot polymerization process based on thiol-ene click reactions. The resulting matrices were characterized by SEM, GPC, TGA, NMR, and rheology studies, and the acidic hydrolysis of the acid-sensitive polymers was also studied. Results confirm that some of the obtained matrices are expected to perform optimally as GRDDSs for the sustained release of active pharmaceutical ingredients at the gastrointestinal level, being a priori facilitated by its disaggregation. Therefore, the optimal performance of these systems is assessed by varying the molar ratio of the labile monomer in the matrices. Keywords: semi-IPN; super-porous hydrogels; guar gum; bioorthogonal chemistry; gastroretentive DDS; Helicobacter pylori; mucoadhesive polymers; amoxicillin; vonoprazan 1. Introduction In 2021, 26,560 new cases of gastric cancer were diagnosed in the United States [ 1 ]. This positions stomach neoplasms above larynx, ovary, and esophagus cancers in terms of incidence in the studied population. The most important risk factor reported for gastric cancer is infection by Helicobacter pylori (H. pylori), which is the pathogen that should be primarily addressed in gastric cancer prevention programs [2]. Currently, antibiotic-based therapies against this pathogen are not foolproof, especially considering the dramatic increase in antibiotic-resistant H. pylori strains. Thus, for example, some studies have pointed out that it is not possible to eradicate the bacterium in at least 2 out of 10 patients treated [ 3 ], leading to soaring resistance to antibiotics, probably due to the fact that combined antibiotic therapies are prescribed systematically when H. pylori is detected. In general terms, the ideal anti-H. pylori treatment is the combination of two to three antibiotics and a proton pump inhibitor [ 4 ]. Out of all the antibiotics analyzed previously, amoxicillin (AMOX) seems to have the lowest prevalence of resistance among H. pylori strains (except in Africa), therefore making it an ideal candidate for optimized Int. J. Mol. Sci. 2023,24, 2281. https://doi.org/10.3390/ijms24032281 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2023,24, 2281 2 of 22 dual therapy (i.e., amoxicillin and a proton pump inhibitor) to be studied as a first-line treatment for infections in most countries [4]. To overcome the drawbacks found in conventional therapies, improvements in anti-H. pylori therapies are required. Two main challenges have been identified [ 4 ]: (a) the amelioration of active pharmaceutical ingredient (API) combinations, for which the authors have recently proposed the use of vonoprazan fumarate (VONO, also known as vonoprazan) [ 5 ] as the optimal drug to be combined with AMOX in H. pylori infections [ 4 ], and (b) the development of ex-profeso-designed gastroretentive drug delivery systems (GRDDSs), capable of guaranteeing a prolonged and reliable performance of the antibiotic formulation. The development of buoyant systems is one of the main approaches to achieving effective GRDDSs [ 6 ]. The types of systems that have been designed as AMOX-GRDDSs vary from floating in-situ gelling systems [ 7 , 8 ] to floating beads [ 9 ], bilayer floating tablets [ 10 ], hollow tablets [ 11 ], floating capsular devices obtained by 3D printing [ 12 ], floating raft systems [ 13 ], and floating microballoons/spheres [ 14 ]. To the authors’ knowledge, all the formulations proposed for the manufacture of buoyant AMOX-loaded GRDDSs incorporate at least two or three swelling enhancers of effervescent excipients to guarantee such a feature during the expected release tempo. This current excipient-addition approach is costly and has an ecological impact that should be prevented in future GRDDS designs. It is of fundamental importance to develop systems that are capable of loading large quantities of AMOX, performing at different pHs (1.2 and higher), and are stable and capable of disintegration when the action is exerted. Additional properties, such as mechanical strength to keep them in the stomach when performing their therapeutic role, mucoadhesion, and the capability to encapsulate large drug loads, will be of great significance. The design of matrices with the above-mentioned properties for their use in suitable AMOX dosage forms is the main goal of the present work. Super-porous hydrogels (SPHs) are three-dimensional networks constituted by lightly cross-linked hydrophilic polymers that can absorb, swell, and retain aqueous solutions up to hundreds of times their own weight, within minutes, to reach the equilibrium swollen state regardless of their size in the dried state [ 15 ]. They display relevant advantages compared with other hydrogel systems [ 16 ], such as the presence of interconnected pores throughout the hydrogel matrices responsible for their superabsorbent properties. The general structure of the different types of SPHs is recorded in Figure 1. Int.J.Mol.Sci.2023,24,22812of23 amongH.pyloristrains(exceptinAfrica),thereforemakingitanidealcandidateforoptimizeddualtherapy(i.e.,amoxicillinandaprotonpumpinhibitor)tobestudiedasafirstlinetreatmentforinfectionsinmostcountries[4]. Toovercomethedrawbacksfoundinconventionaltherapies,improvementsinanti‐ H.pyloritherapiesarerequired.Twomainchallengeshavebeenidentified[4]:(a)theameliorationofactivepharmaceuticalingredient(API)combinations,forwhichtheauthors haverecentlyproposedtheuseofvonoprazanfumarate(VONO,alsoknownasvonoprazan)[5]astheoptimaldrugtobecombinedwithAMOXinH.pyloriinfections[4],and(b) thedevelopmentofex‐profeso‐designedgastroretentivedrugdeliverysystems (GRDDSs),capableofguaranteeingaprolongedandreliableperformanceoftheantibiotic formulation. ThedevelopmentofbuoyantsystemsisoneofthemainapproachestoachievingeffectiveGRDDSs[6].ThetypesofsystemsthathavebeendesignedasAMOX‐GRDDSs varyfromfloatingin‐situgellingsystems[7,8]tofloatingbeads[9],bilayerfloatingtablets [10],hollowtablets[11],floatingcapsulardevicesobtainedby3Dprinting[12],floating raftsystems[13],andfloatingmicroballoons/spheres[14].Totheauthors’knowledge,all theformulationsproposedforthemanufactureofbuoyantAMOX‐loadedGRDDSsincorporateatleasttwoorthreeswellingenhancersofeffervescentexcipientstoguaranteesuch afeatureduringtheexpectedreleasetempo.Thiscurrentexcipient‐additionapproachis costlyandhasanecologicalimpactthatshouldbepreventedinfutureGRDDSdesigns.It isoffundamentalimportancetodevelopsystemsthatarecapableofloadinglargequantitiesofAMOX,performingatdifferentpHs(1.2andhigher),andarestableandcapable ofdisintegrationwhentheactionisexerted.Additionalproperties,suchasmechanical strengthtokeeptheminthestomachwhenperformingtheirtherapeuticrole,mucoadhesion,andthecapabilitytoencapsulatelargedrugloads,willbeofgreatsignificance.The designofmatriceswiththeabove‐mentionedpropertiesfortheiruseinsuitableAMOX dosageformsisthemaingoalofthepresentwork. Super‐poroushydrogels(SPHs)arethree‐dimensionalnetworksconstitutedby lightlycross‐linkedhydrophilicpolymersthatcanabsorb,swell,andretainaqueoussolutionsuptohundredsoftimestheirownweight,withinminutes,toreachtheequilibriumswollenstateregardlessoftheirsizeinthedriedstate[15].Theydisplayrelevant advantagescomparedwithotherhydrogelsystems[16],suchasthepresenceofinterconnectedporesthroughoutthehydrogelmatricesresponsiblefortheirsuperabsorbentproperties.ThegeneralstructureofthedifferenttypesofSPHsisrecordedinFigure1. Figure1.Generalschemeofthedifferenttypesofsuper‐poroushydrogels. Today,uptothreegenerationsofSPHsareavailableforclinicaluse,butthird‐gener‐ ationSPHhybrids(SPHHs,alsocalledsuper‐poroushydrogelinterpenetratingnetworks orSPH‐IPNs)arepreferredbecausetheydisplaybettermechanicalstrength[17]. Figure 1. General scheme of the different types of super-porous hydrogels. Today, up to three generations of SPHs are available for clinical use, but thirdgeneration SPH hybrids (SPHHs, also called super-porous hydrogel interpenetrating networks or SPH-IPNs) are preferred because they display better mechanical strength [17]. SPH hybrids are prepared by adding a water-soluble or water-dispersible polymer (hybrid agent or Polymer 1) that can be cross-linked through chemical or physical crosslinking after the SPH is formed. Once the second network is formed (Polymer 2), the
Int. J. Mol. Sci. 2023,24, 2281 3 of 22 whole system becomes an interpenetrating polymer network [ 18 ]. If one of the constituent polymers is not cross-linked, the system is then known as a semi-IPN. For the eradication of H. pylori infection, the incorporation of mucoadhesive properties into the floating gastroretentive dosage forms is especially advantageous. They can adhere to the stomach wall, survive gastrointestinal motility for a longer period [ 19 ], and thus enhance the local action of the drug in the infected area [ 20 ]. Guar gum (GG) is a natural polysaccharide that displays interesting mucoadhesive properties; it is degradable under physiological conditions and can be used in GRDDSs [21,22]. The general objective of this work is to provide a new set of biocompatible materials for the development of streamlined, sustained-release GRDDSs of AMOX that, when administered with VONO in a combined oral formulation, can effectively eradicate H. pylori in the stomach of infected patients. These systems should have a low ecological footprint, be easy to synthesize, and possess mucoadhesive and floating properties by themselves, that is, without the need for any buoyancy-promoting excipients. Additionally, once the GRDDSs have exerted their function, the final material should be biodegradable and easily eliminated from the human body. To the authors’ knowledge, there is no precedent for the use of third-generation IPNs for the release of APIs in oral formulations based on thiol-ene click reactions. The use of orthogonal chemistry in the preparation of such materials will provide a wide range of possibilities in IPN-based matrices since innumerable types of natural polymers with unique properties can be combined with a variety of synthetic polymers prepared ex profeso for these systems. The degradability of the materials will be ensured not only by the hydrolysis of the chosen natural polysaccharide but also, and importantly, by the inclusion of labile segments in the second polymer that will form part of the semi-IPN-based matrices. To achieve this, several specific objectives have been set: - Prepare, characterize, and compare different semi-IPNs using GG as the natural polysaccharide due to its mucoadhesive properties. - To achieve this, it is necessary to first explore the preparation of several hydrophilic, biodegradable homo- and copolymers by polymerization procedures compatible with the chemical nature of GG. Due to the versatility of click chemistry (CC) and in search of an orthogonal chemical procedure in IPN formation, the thiol-ene click reaction is selected to prepare and characterize various homo- and copolymers. Several batches will be synthesized to optimize polymerization conditions and, hence, choose the polymeric systems with the best performance. This is the first time that this methodology for the preparation of IPNs has been investigated. - Synthesize a monomer that can enhance the biodegradability of the final matrices under the acidic conditions of the stomach. The compound, named acetaldehyde diallylacetal, is chosen as one of the diallyl monomers to be used in semi-IPN preparation so that the presence of the acetal group in its structure will be responsible for the increase in the biodegradability of the semi-IPN-based matrices. - Conduct a proof of concept on the ability of the new materials to behave as AMOX controlled-release matrices at pHs 1.2 and 5.0. 2. Results 2.1. Synthesis of Acetaldehyde Diallyl Acetal (B Monomer) The synthesis of the B monomer was conducted by a classic reaction of acetal formation [ 23 ] (Scheme 1), in which an equilibrium is generated, so working in the absence of moisture was essential. The title compound was isolated as a pure oil after distillation, as demonstrated by 1H-NMR (Figure 2) and 13C-NMR spectra.
Int. J. Mol. Sci. 2023,24, 2281 4 of 22 Int.J.Mol.Sci.2023,24,22814of23 2.Results 2.1.SynthesisofAcetaldehydeDiallylAcetal(BMonomer) ThesynthesisoftheBmonomerwasconductedbyaclassicreactionofacetalformation [23](Scheme1),inwhichanequilibriumisgenerated,soworkingintheabsence ofmoisturewasessential.Thetitlecompoundwasisolatedasapureoilafterdistillation, asdemonstratedby 1 H‐NMR(Figure2)and 13 C‐NMRspectra. Scheme1.Syntheticschemeofacetaldehydediallylacetal(Bmonomer). Figure2. 1 H‐NMRofacetaldehydediallylacetal(Bmonomer)at300MHzinCDCl 3 . ThebiodegradabilityoftheBmonomer(acetaldehydediallylacetal)inthepresence ofacidicfluids,suchasthosefoundinthestomach,hasbeenconfirmedandwillbediscussedbelow. 2.2.PreparationandCharacterizationofHomopolymersandCopolymersbyThiol‐EneReaction Forthepreparationof400mgofeachpolymer(repeatingunitsofsuchpolymersare recordedinFigure3),theappropriateamountofdithiolanddiallylmonomerswasmixed sothatthemmolesofthediallylmonomerswereidenticaltothoseof2,2’‐(ethylenedioxy)diethanethiol(EDDET)inthefeedforeverypolymerizationprocedure(Table1).Homopolymers(hom)andcopolymers(co)weresynthesizedusingdiallylcarbonate(CAR, commercial),(2R,3R)‐N,N’‐diallyltartramide(TAR,commercial),and/oracetaldehydediallylacetal(B,synthesizeddenovo)asdiallylmonomers,andEDDET(commercial)asthe dithiolmonomer.Polymerizationtookplaceintheabsenceofoxygenand,bymeansof thethiol‐eneclickreactions,initiatedphotolytically(photoinitiator:2,2‐dimethoxy‐2‐phe‐ nylacetophenone,DMPA).Forhomopolymerpreparation,onlyonediallylmonomerwas incorporatedinthefeed,leadingtoonlyonetypeofrepeatingunitinthepolymer(Figure 2),whereasinthecaseofthecopolymers,twodiallylmonomerswereusedand,hence, thecopolymerswereconstitutedbytwodifferentrepeatingunits(Figure2).Thereagents Scheme 1. Synthetic scheme of acetaldehyde diallylacetal (B monomer). Int.J.Mol.Sci.2023,24,22814of23 2.Results 2.1.SynthesisofAcetaldehydeDiallylAcetal(BMonomer) ThesynthesisoftheBmonomerwasconductedbyaclassicreactionofacetalformation [23](Scheme1),inwhichanequilibriumisgenerated,soworkingintheabsence ofmoisturewasessential.Thetitlecompoundwasisolatedasapureoilafterdistillation, asdemonstratedby 1 H‐NMR(Figure2)and 13 C‐NMRspectra. Scheme1.Syntheticschemeofacetaldehydediallylacetal(Bmonomer). Figure2. 1 H‐NMRofacetaldehydediallylacetal(Bmonomer)at300MHzinCDCl 3 . ThebiodegradabilityoftheBmonomer(acetaldehydediallylacetal)inthepresence ofacidicfluids,suchasthosefoundinthestomach,hasbeenconfirmedandwillbediscussedbelow. 2.2.PreparationandCharacterizationofHomopolymersandCopolymersbyThiol‐EneReaction Forthepreparationof400mgofeachpolymer(repeatingunitsofsuchpolymersare recordedinFigure3),theappropriateamountofdithiolanddiallylmonomerswasmixed sothatthemmolesofthediallylmonomerswereidenticaltothoseof2,2’‐(ethylenedioxy)diethanethiol(EDDET)inthefeedforeverypolymerizationprocedure(Table1).Homopolymers(hom)andcopolymers(co)weresynthesizedusingdiallylcarbonate(CAR, commercial),(2R,3R)‐N,N’‐diallyltartramide(TAR,commercial),and/oracetaldehydediallylacetal(B,synthesizeddenovo)asdiallylmonomers,andEDDET(commercial)asthe dithiolmonomer.Polymerizationtookplaceintheabsenceofoxygenand,bymeansof thethiol‐eneclickreactions,initiatedphotolytically(photoinitiator:2,2‐dimethoxy‐2‐phe‐ nylacetophenone,DMPA).Forhomopolymerpreparation,onlyonediallylmonomerwas incorporatedinthefeed,leadingtoonlyonetypeofrepeatingunitinthepolymer(Figure 2),whereasinthecaseofthecopolymers,twodiallylmonomerswereusedand,hence, thecopolymerswereconstitutedbytwodifferentrepeatingunits(Figure2).Thereagents Figure 2. 1H-NMR of acetaldehyde diallylacetal (B monomer) at 300 MHz in CDCl3. The biodegradability of the B monomer (acetaldehyde diallylacetal) in the presence of acidic fluids, such as those found in the stomach, has been confirmed and will be discussed below. 2.2. Preparation and Characterization of Homopolymers and Copolymers by Thiol-Ene Reaction For the preparation of 400 mg of each polymer (repeating units of such polymers are recorded in Figure 3), the appropriate amount of dithiol and diallyl monomers was mixed so that the mmoles of the diallyl monomers were identical to those of 2,2 0 -(ethylenedioxy) diethanethiol (EDDET) in the feed for every polymerization procedure (Table 1). Homopolymers (hom) and copolymers (co) were synthesized using diallyl carbonate (CAR, commercial), (2R,3R)-N,N 0 -diallyltartramide (TAR, commercial), and/or acetaldehyde diallylacetal (B, synthesized de novo) as diallyl monomers, and EDDET (commercial) as the dithiol monomer. Polymerization took place in the absence of oxygen and, by means of the thiol-ene click reactions, initiated photolytically (photoinitiator: 2,2-dimethoxy-2- phenylacetophenone, DMPA). For homopolymer preparation, only one diallyl monomer was incorporated in the feed, leading to only one type of repeating unit in the polymer (Figure 2), whereas in the case of the copolymers, two diallyl monomers were used and, hence, the copolymers were constituted by two different repeating units (Figure 2). The reagents and solvents used for each case and the polymerization conditions are summarized in Table 1.
Int. J. Mol. Sci. 2023,24, 2281 5 of 22 Int.J.Mol.Sci.2023,24,22815of23 andsolventsusedforeachcaseandthepolymerizationconditionsaresummarizedin Table1. Figure3.Schematicrepresentationofconstitutionalrepeatingunitsforhom‐pCAR,hom‐pTAR,and hom‐pB.Inthecaseofthecopolymers,therepeatingunitswerethosefoundinhom‐pCARand hom‐pBinvariedratios,dependingonthefeed. Table1.Preparationofhomopolymersandcopolymers. PolymerCAR (μL) TAR (mg) B (μL) DiallylMonomers MolarRatio EDDET (μL) Initiator (mg) Solvent (mL) hom‐pCAR176.8‐ ‐ ‐ 200.731.62(EtOAc) hom‐pTAR‐222.4‐ ‐ 158.6252(MeOH) hom‐pB‐‐202.2‐ 200.731.62(EtOAc) co‐p(CAR 80 B 20 )141.5‐ 40.480:20211.231.62(EtOAc) co‐p(CAR 50 B 50 )88.4‐ 101.150:50211.231.62(EtOAc) co‐p(CAR 20 B 80 )35.36‐ 161.720:80211.231.62(EtOAc) Polymerizationconditions:[Diallylmonomers]and[EDDET]inthisreactionwereboth0.487M. Reactionconditions:[diallylmonomers]=0.616M;[EDDET]=0.616M;solvent=2mLofeither EtOAcorMeOH;initiator=DMPA10%;T=18°C;UVradiation=365nm,180W,10min;stirred for48h. Theresultinghomopolymersandcopolymerswereanalyzedbynuclearmagnetic resonance(NMR),thermogravimetricanalysis(TGA),andgelpermeationchromatography(GPC).TheNMRspectrawereusedtoconfirmthecompositionofthepolymers andcalculatetheratiooftheconstitutionalrepeatingunits(CRUs)inthecopolymers(Figure2).GPCandTGAdatafromthecopolymersaresummarizedinTable2. Table2.GPCandTGAdataforsynthesizedhomopolymersandcopolymers. Polymer Diallyl Monomer andMolar Percentage a M wb M nb M w /M nb TGA c 𝑻 𝒅 𝟎 (°C)T d (°C)ΔW(%) hom‐pCARCAR42,60033,4001.3268.5340.698.9 hom‐pTARTAR21,90018,0001.2277.9351.388.7 hom‐pBB19,80016,5001.2226.5350.699.4 co‐p(CAR 80 B 20 )CAR‐B80:2021,50018,0001.2253.4339.9100.0 co‐p(CAR 50 B 50 )CAR‐B50:5011,20088001.3230.9334.098.3 co‐p(CAR 20 B 80 )CAR‐B20:80810069001.2236.4342.298.9 a EachpolymerusesEDDETasthedithiolmonomer. b DeterminedbyGPC. c Onsetdecomposition temperaturecorrespondingto10%ofweightloss(𝑇 ),maximumratedecompositiontemperatures (T d ),andweightlossattherespectivedecompositionstep[ΔW(%)]determinedbyTGA. Figure 3. Schematic representation of constitutional repeating units for hom-pCAR, hom-pTAR, and hom-pB. In the case of the copolymers, the repeating units were those found in hom-pCAR and hom-pB in varied ratios, depending on the feed. Table 1. Preparation of homopolymers and copolymers. Polymer CAR (µL) TAR (mg) B (µL) Diallyl Monomers Molar Ratio EDDET (µL) Initiator (mg) Solvent (mL) hom-pCAR 176.8 - - - 200.7 31.6 2 (EtOAc) hom-pTAR - 222.4 - - 158.6 25 2 (MeOH) hom-pB - - 202.2 - 200.7 31.6 2 (EtOAc) co-p(CAR80B20) 141.5 - 40.4 80:20 211.2 31.6 2 (EtOAc) co-p(CAR50B50) 88.4 - 101.1 50:50 211.2 31.6 2 (EtOAc) co-p(CAR20B80) 35.36 - 161.7 20:80 211.2 31.6 2 (EtOAc) Polymerization conditions: [Diallyl monomers] and [EDDET] in this reaction were both 0.487 M. Reaction conditions: [diallyl monomers] = 0.616 M; [EDDET] = 0.616 M; solvent = 2 mL of either EtOAc or MeOH; initiator = DMPA 10%; T = 18 ◦C; UV radiation = 365 nm, 180 W, 10 min; stirred for 48 h. The resulting homopolymers and copolymers were analyzed by nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and gel permeation chromatography (GPC). The NMR spectra were used to confirm the composition of the polymers and calculate the ratio of the constitutional repeating units (CRUs) in the copolymers (Figure 2). GPC and TGA data from the copolymers are summarized in Table 2. Table 2. GPC and TGA data for synthesized homopolymers and copolymers. Polymer Diallyl Monomer and Molar Percentage aMwbMnbMw/MnbTGA c T0 dTd(◦C) ∆W(%) hom-pCAR CAR 42,600 33,400 1.3 268.5 340.6 98.9 hom-pTAR TAR 21,900 18,000 1.2 277.9 351.3 88.7 hom-pB B 19,800 16,500 1.2 226.5 350.6 99.4 co-p(CAR80B20) CAR-B 80:20 21,500 18,000 1.2 253.4 339.9 100.0 co-p(CAR50B50) CAR-B 50:50 11,200 8800 1.3 230.9 334.0 98.3 co-p(CAR20B80) CAR-B 20:80 8100 6900 1.2 236.4 342.2 98.9 a Each polymer uses EDDET as the dithiol monomer. b Determined by GPC. c Onset decomposition temperature corresponding to 10% of weight loss ( T0 d ), maximum rate decomposition temperatures (T d ), and weight loss at the respective decomposition step [∆W(%)] determined by TGA. The NMR spectra of the synthesized homopolymers confirmed that they possess the expected chemical structure according to their repeating unit. The 1 H and 13 C-NMR peaks for each homopolymer are recorded below:
Int. J. Mol. Sci. 2023,24, 2281 6 of 22 Hom-pCAR 1H-NMR (300 MHz, CDCl 3 ) δ ppm: 4.23 (t, 4H, H-a, J a,f = 6.4 Hz), 3.65 (t, 4H, H-b, J b,d = 6.9 Hz), 3.62 (s, 4H, H-c), 2.72 (t, 4H, H-d), 2.65 (t, 4H, H-e, J e,f = 7.2 Hz), 2.02–1.89 (m, 4H, H-f). 13 C-NMR (75 MHz, CDCl 3 ,hom-pCAR) δ ppm: 155.1 (C=O), 71.9, 71.0 (C-b), 70.6, 70.3 (C-c), 66.5, 66.3 (C-a), 31.7, 31.5 (C-d), 29.4, 28.8, 28.7 (C-e, C-f). Hom-pTAR 1H-NMR (300 MHz, (CD 3 ) 2 SO) δ ppm: 7.73 (t, 2H, H-a, J a,f = 5.7 Hz), 5.47 (d, 2H, H-b, J b,c = 7.2 Hz), 4.22 (d, 2H, H-c), 3.55 (t, 4H, H-d, J d,g = 7.0 Hz), 3.53 (s, 4H, H-e), 3.28–3.05 (m, 4H, H-f), 2.64 (t, 4H, H-g), 2.57–2.40 (m, 4H, H-h), 1.68 (q, 4H, H-i, J f,i =J h,i = 7.2 Hz). 13 C-NMR (75 MHz, (CD 3 ) 2 SO, hom-pTAR) δ ppm: 172.4 (C=O), 73.0 (C-c), 70.6 (C-d), 70.0 (C-e), 38.0 (C-f), 31.1 (C-g), 29.9 (C-i), 29.4 (C-h). Hom-pB 1H-NMR (300 MHz, CDCl 3 ) δ ppm: 4.77–4.58 (m, 1H, H-a), 3.80–3.42 (m, 12H, H-b, H-c, H-d), 2.80–2.57 (m, 8H, H-e, H-f), 1.85 (q, 4H, H-g, J d,g =J f,g = 6.3 Hz), 1.34–1.24 (m, 3H, H-h). 13 C-NMR (75 MHz, CDCl 3 ,hom-pB) δ ppm: 99.9, 99.4 (C-a), 71.6, 71.2, 71.0, 70.8, 70.3, 63.8, 63.3 (C-c, C-d), 61.5 (C-b), 32.2, 32.1, 30.1, 30.0 (C-g), 31.5, 31.4 (C-e), 29.3, 29.2 (C-f), 19.8, 19.5 (C-h). Regarding the CAR/B copolymers, they also displayed the peaks corresponding to their expected structure. Their quantitative compositions (ratios of CRUs) were determined by 1 H-NMR spectra analyses, comparing the integrals of peaks corresponding to protons C H2 -OCO- from the CAR moiety (4.25 ppm, 4 protons per CRU) with those of the methyl protons from the B monomer (1.28 ppm, 3 protons per CRU). These data are recorded in Table 3. Table 3. Experimental molar ratios of constitutional repeating units for the synthesized CAR/B copolymers. Copolymer Ratio Diallyl Monomers in the Feed (in Molar Percent) 1H-NMR Integrals for the Peaks at δ(ppm) 4.25 (from CAR Moiety: 4 H) and 1.28 (from B Moiety: 3 H) 1H-NMR Integrals for the Peaks at δ(ppm) 4.25 (from CAR Moiety: 4H) and 1.28 (from B Moiety: 3 H) Divided by the n.º of H for each Peak Experimental CAR/B Molar Ratios (in %) CAR B 4.25 ppm 1.28 ppm 4.25 ppm 1.28 ppm CAR B co-p(CAR80B20) 80 20 4 a0.6494 1 0.2165 82.1 17.9 co-p(CAR50B50) 50 50 4 a2.7339 1 0.9113 52.4 47.6 co-p(CAR20B80) 20 80 4 a10.6580 1 3.5527 21.9 78.1 aThe integral for the peak at 4.2 ppm was fixed to 4.0. 2.3. Degradability Studies of the B Monomer and co-p(CAR50B50) under Acidic Aqueous Conditions Hydrolytic degradation studies were followed by 1 H-NMR spectroscopy. To simulate the acidic aqueous environment of the stomach, the samples were dissolved in a mixture of DMSO-d 6 /D 2 O/TFA- and the experiments conducted at 37 ◦ C. The integral of the peak corresponding to the methyl group linked to the acetal moiety (either in B, or in cop(CAR 50 B 50 )) was compared with that from the methyl group of the degradation product, i.e., acetaldehyde. Figure 4shows the 1 H-NMR spectra of both the degradation studies of the B monomer (Figure 4a) and of the copolymer co-p(CAR 50 B 50 ) (Figure 4b) at fixed times: 0, 10, 30, 60, and 90 min. The results obtained in the hydrolytic degradation trials are shown in Figure 5.
Int. J. Mol. Sci. 2023,24, 2281 7 of 22 Int.J.Mol.Sci.2023,24,22817of23 (a)(b) Figure4. 1 H‐NMRstudiesofthehydrolyticdegradationof(a)Bmonomerand(b)co‐p(CAR 50 B 50 ) withtime. Figure5.EvolutionofhydrolyticdegradationoftheBmonomerandco‐p(CAR 50 B 50 )overtime. Figure 4. 1 H-NMR studies of the hydrolytic degradation of ( a ) B monomer and ( b )co-p(CAR 50 B 50 ) with time. Int.J.Mol.Sci.2023,24,22817of23 (a)(b) Figure4. 1 H‐NMRstudiesofthehydrolyticdegradationof(a)Bmonomerand(b)co‐p(CAR 50 B 50 ) withtime. Figure5.EvolutionofhydrolyticdegradationoftheBmonomerandco‐p(CAR 50 B 50 )overtime. Figure 5. Evolution of hydrolytic degradation of the B monomer and co-p(CAR50B50) over time. 2.4. Preparation of Guar-Gum-Based Semi-IPNs Once the versatility of the polymerization process was optimized and the degradability properties of the new materials confirmed, five types of semi-IPNs containing GG (Polymer 1) and the thiol-ene polymer (Polymer 2) were prepared. The cross-linked Polymer 2 was grown in a colloidal aqueous suspension of Polymer 1 so that the final materials possessed Polymer 1 and Polymer 2 molecular chains that were tightly intertwined. The optimal concentration of GG in this kind of network has been established to be ca. 4% [ 13 ].
Int. J. Mol. Sci. 2023,24, 2281 8 of 22 The degree of cross-linking for Polymer 2 was fixed to 8% based on the percentage of allyl groups joined to the cross-linker. Tritiol trimethylolpropane tris(3-mercaptopropionate) (TriT) was used as a covalent cross-linker. The amounts of EDDET, CAR, TAR, and/or B added to form each semi-IPN, as well as that of the covalent cross-linker TriT (dissolved in MeOH; concentration: 368.5 mg/mL), are recorded in Table 4. All the samples displayed a Polymer 1/Polymer 2 ratio of 1:1 in weight, and they were bench-stable for weeks. Table 4. Preparation of guar-gum-based semi-IPNs. semi-IPN CAR (µL) TAR (mg) B (µL) Diallyl Monomers Molar Ratio EDDET (µL) TriT (mg) Initiator (mg) GG-p(TAR50B50) - 122 87.7 50:50 160.1 22.7 27.4 GG-pB - - 198.1 100 180.9 25.7 31 GG-p(CAR50B50) 86.6 - 99.1 50:50 180.9 25.7 31 GG-p(CAR80B20) 138.6 - 39.6 80:20 190.4 27 31 GG-p(CAR20B80) 34.35 - 158.5 20:80 190.4 27 31 Reaction conditions: initiator = DMPA 10%; methoxypolyethyleneglycol-350 (MeO-PEG 350) = 1 g; GG = 400 mg dissolved in 9 mL of distilled water; T = 18 ◦C; UV radiation: 365 nm, 180 W, 10 min; reaction time: 24 h. 2.5. Characterization of Guar-Gum-Based Semi-IPNs The rheological properties of the selected semi-IPNs (GG-p(TAR 50 B 50 ), GG-p(CAR 50 B 50 ), and GG-pB) were evaluated. The rheological characterization encompassed strain and frequency sweep tests. Firstly, strain sweep tests (0.002–0.1% at 1 Hz) were carried out to determine the linear viscoelastic range and critical strain of the samples. Then, frequency sweep tests (0.02–10 Hz, at a constant strain within the linear viscoelastic range) were performed to analyze the stability of the samples. In these tests, the values of the viscoelastic moduli are collected (Table 5, G 0 and G 00 corresponding to the elastic and viscous moduli, respectively) as well as the relationship between them (tan ( δ ) = G 00 /G 0 ) and the specific viscosity (η*). Table 5. Critical strain, elastic modulus (G 0 ), and tan ( δ ) values at 0.02, 1.00, and 10.0 Hz for the different systems evaluated. Systems Critical Strain (%) G0(Pa) tan (δ) 0.02 Hz 1.00 Hz 10.0 Hz 0.02 Hz 1.00 Hz 10.0 Hz GG-p(CAR50B50) 0.012 ±0.003 183 ±34 681 ±51 1012 ±22 0.81 ±0.09 0.35 ±0.02 0.23 ±0.01 GG-p(TAR50B50) 0.012 ±0.003 133 ±24 574 ±48 886 ±21 0.88 ±0.06 0.39 ±0.03 0.25 ±0.01 GG-pB 0.086 ±0.020 120 ±20 495 ±40 765 ±20 0.85 ±0.05 0.38 ±0.03 0.24 ±0.01 The morphologies of these three semi-IPNs (GG-p(TAR 50 B 50 ), GG-p(CAR 50 B 50 ), and GG-pB) were studied by scanning electron microscopy (SEM). Before SEM observations, the samples underwent critical point drying to prevent the alteration of their surface topography [24]. The porosity of the systems and their free volume were evaluated from the resulting SEM images. In this way, a digital processing free software, FIJI Image-J (National Institutes of Health, Bethesda, MD, USA), was used to determine the mean pore size of the selected hydrogels. Pore diameters were calculated manually by measuring 50 pores in each sample, and they were consistently identical for all of them (diameters ca. 0.290 µm). 2.6. AMOX Loading and In Vitro Drug Release Studies A preliminary dissolution study using a co-p(CAR 50 B 50 )-based semi-IPN was performed as a proof of concept. AMOX was loaded into the matrix (10% of the total weight of the formulation). The release studies were conducted in simulated gastric fluid sine pepsin at pH 1.2 (SGF-1.2) and pH 5.0 (SGF-5) at 37 ◦C.
Int. J. Mol. Sci. 2023,24, 2281 9 of 22 Drug release was determined by UV spectroscopy at 229 nm using an ex-profeso- prepared calibration curve. The release of AMOX was measured over 7.5 h, finding that there was no burst release in the first minutes, which supports the adequacy of the encapsulation of the antibiotic; on the other hand, the release of AMOX was dependent on the pH of the medium. 3. Discussion The target of the current work is the development of alternative polymeric materials that possess optimal tailor-made properties to be used as matrices in GRDDS-based formulations. Thus, since high mechanical strength is required to keep them intact throughout their stay in the stomach [ 25 ], the preparation of tightly intertwined semi-IPNs was addressed, in which GG was one of the polymers of the dual systems. The tightly intertwined structure at the molecular level of the semi-IPNs will also ensure the porosity of the final materials and, therefore, their capability to remain buoyant in gastric fluids [ 6 ], a key feature for matrices used in GRDDSs since floating systems are less likely to be expelled from the stomach compared to others [ 26 ]. Moreover, such porosity will guarantee the large encapsulation capacity of APIs (a necessary property when the controlled release of drugs at high doses is required, as is the case of AMOX) and high swelling capacities [27], which ensure the flow of gastric fluids within them. It has also been demonstrated that drug release is dependent on the swellable/erodible properties of the matrices [ 12 ]. The permanence in the stomach of these semi-IPN-based formulations can be boosted by enhancing their bioadhesive properties [ 21 ]. GG, a natural biodegradable polysaccharide, has been demonstrated to display high bioadhesive properties [ 22 ], which will impart this feature to the final matrices formed. There are two main strategies that have been tested for the formation of second- and third-generation IPNs. Relevant examples, prepared for potential use in drug delivery systems, are exemplified in Table 6. In general terms, the formation of second-generation IPNs (with poorer mechanical properties than those of the third generation) is usually conducted either by the mixture of two natural polymers [ 28 , 29 ] or by the mixture of a natural polymer and a hydrophilic synthetic macromolecule such as poly(vinyl alcohol) (PVA) [28,30], which cross-links reversibly.
Int. J. Mol. Sci. 2023,24, 2281 16 of 22 25 and 37 ◦ C and the formulation of AMOX + GG-p(CAR 50 B 50 ), respectively) and showed no significant differences. Int.J.Mol.Sci.2023,24,228117of23 Whensubmittingthesamehydrogeltobothstrainandfrequencytestsat25and37 °C,similarprofileswereobserved,showingnosignificantdifferencesbetweenthem(Figure9a,b).Thisprovedthegoodresistanceofthesystemtobodytemperatureand,therefore,itspossibleapplicationforcontrolleddrugreleasetreatments,astherheological propertieswereverysimilarat25and37°C.Itisworthmentioningthateventhoughthe incorporationofAMOXreducedG’andG’’values,possiblyduetoaslightmodification ofthemicrostructure,thehydrogelmaintaineditssolid‐likebehavior,whichisfundamentalfortheapplication.Thisfactcouldbeprovedbycomparingthevaluesoftan(δ)obtainedat1Hzforeachsystem,whichwereverysimilar(0.35,0.31,and0.36forGG‐ p(CAR50B50),at25and37°CandtheformulationofAMOX+GG‐p(CAR50B50),respectively)andshowednosignificantdifferences. (a)(b) Figure9.(a)Strainsweeptestsofthedifferentsystemsevaluated:GG‐p(CAR50B50),at25and37°C, andtheformulationofAMOX+GG‐p(CAR50B50).(b)Frequencysweeptestsofthedifferentsystems evaluated:GG‐p(CAR50B50),at25°Cand37°C,andtheformulationofAMOX+GG‐p(CAR50B50). SEMobservationsofthenetworksformedconfirmedthesuper‐porousstructureof thethreesystems,ascanbeobservedinFigure10.AnalysiswithFIJIImage‐Jsoftware revealedthateverysamplehadasimilarpercentageofporosity(between22%and29%), notfindingsignificantdifferences.Althoughporesizewasheterogeneouswithinevery sample,theirmeanporediameterswereca.0.290μmforallthesamples(0.290μmfor [GG‐p(TAR50B50)],0.294μmfor[GG‐p(CAR50B50)],and0.288μm[GG‐pB]). (a)(b) 10 -3 10 -2 10 -1 10 2 10 3 G' G'' GC-p(CAR 50 B 50 ) 25 ºC GC-p(CAR 50 B 50 ) 37 ºC GC-p(CAR 50 B 50 ) 37 ºC + AMOX G', G'' / (Pa) Strain / (%) 10 -2 10 -1 10 0 10 1 10 2 10 3 G' G'' GC-p(CAR 50 B 50 ) 25 ºC GC-p(CAR 50 B 50 ) 37 ºC GC-p(CAR 50 B 50 ) 37 ºC + AMOX G', G'' / (Pa) Frequency / (Hz) Figure 9. ( a ) Strain sweep tests of the different systems evaluated: GG-p(CAR 50 B 50 ), at 25 and 37 ◦ C, and the formulation of AMOX + GG-p(CAR 50 B 50 ). ( b ) Frequency sweep tests of the different systems evaluated: GG-p(CAR 50 B 50 ), at 25 ◦ C and 37 ◦ C, and the formulation of AMOX + GG-p(CAR 50 B 50 ). SEM observations of the networks formed confirmed the super-porous structure of the three systems, as can be observed in Figure 10. Analysis with FIJI Image-J software revealed that every sample had a similar percentage of porosity (between 22% and 29%), not finding significant differences. Although pore size was heterogeneous within every sample, their mean pore diameters were ca. 0.290 µ m for all the samples (0.290 µ m for [GG-p(TAR50B50)], 0.294 µm for [GG-p(CAR50B50)], and 0.288 µm [GG-pB]). Int.J.Mol.Sci.2023,24,228117of23 Whensubmittingthesamehydrogeltobothstrainandfrequencytestsat25and37 °C,similarprofileswereobserved,showingnosignificantdifferencesbetweenthem(Figure9a,b).Thisprovedthegoodresistanceofthesystemtobodytemperatureand,therefore,itspossibleapplicationforcontrolleddrugreleasetreatments,astherheological propertieswereverysimilarat25and37°C.Itisworthmentioningthateventhoughthe incorporationofAMOXreducedG’andG’’values,possiblyduetoaslightmodification ofthemicrostructure,thehydrogelmaintaineditssolid‐likebehavior,whichisfundamentalfortheapplication.Thisfactcouldbeprovedbycomparingthevaluesoftan(δ)obtainedat1Hzforeachsystem,whichwereverysimilar(0.35,0.31,and0.36forGG‐ p(CAR50B50),at25and37°CandtheformulationofAMOX+GG‐p(CAR50B50),respectively)andshowednosignificantdifferences. (a)(b) Figure9.(a)Strainsweeptestsofthedifferentsystemsevaluated:GG‐p(CAR50B50),at25and37°C, andtheformulationofAMOX+GG‐p(CAR50B50).(b)Frequencysweeptestsofthedifferentsystems evaluated:GG‐p(CAR50B50),at25°Cand37°C,andtheformulationofAMOX+GG‐p(CAR50B50). SEMobservationsofthenetworksformedconfirmedthesuper‐porousstructureof thethreesystems,ascanbeobservedinFigure10.AnalysiswithFIJIImage‐Jsoftware revealedthateverysamplehadasimilarpercentageofporosity(between22%and29%), notfindingsignificantdifferences.Althoughporesizewasheterogeneouswithinevery sample,theirmeanporediameterswereca.0.290μmforallthesamples(0.290μmfor [GG‐p(TAR50B50)],0.294μmfor[GG‐p(CAR50B50)],and0.288μm[GG‐pB]). (a)(b) 10 -3 10 -2 10 -1 10 2 10 3 G' G'' GC-p(CAR 50 B 50 ) 25 ºC GC-p(CAR 50 B 50 ) 37 ºC GC-p(CAR 50 B 50 ) 37 ºC + AMOX G', G'' / (Pa) Strain / (%) 10 -2 10 -1 10 0 10 1 10 2 10 3 G' G'' GC-p(CAR 50 B 50 ) 25 ºC GC-p(CAR 50 B 50 ) 37 ºC GC-p(CAR 50 B 50 ) 37 ºC + AMOX G', G'' / (Pa) Frequency / (Hz) Int.J.Mol.Sci.2023,24,228118of23 (c) Figure10.(a)SEMphotofromGG‐p(TAR 50 B 50 ).(b)SEMphotofromGG‐pB.(c)SEMphotofrom GG‐p(CAR 50 B 50 ). 3.4.AMOXLoadingandInVitroDrugReleaseStudies ThereleasestudieswereconductedinsimulatedgastricfluidsinepepsinatpH1.2 (SGF‐1.2)andpH5.0(SGF‐5)at37°C.ThedrugreleasewasdeterminedbyUVspectroscopy(Figure11).Theseassaysdemonstratedthat(a)thesynthesizedsystemscouldencapsulatethedesireddrugeffectivelysinceburstreleasewasprevented,and(b)AMOX couldbereleasedfromitinacontrolled,sustainedmanner,itsdeliverybeingmorerapid atpH5.0thanatpH1.2.Infact,cumulativedrugreleasedataapproached55%atpH5.0 after7.5h,whileitbarelyreached30%atpH1.2. PreviousscientificstudiesregardingthereleaseofAMOXfromgastroretentivesystemsweregenerallyperformedatpH1.2asthiscorrespondstothenormalacidityfound inahealthy,emptystomach;hence,theassayswereconductedatthatpHforcomparative reasons.Nonetheless,itcannotbeforgottenthattherapeuticapproachesforthetreatment ofH.pyloriinfectionstendtoincludeanacidic‐secretion‐blockingcompound,suchasregularprotonpumpinhibitorsorthemorenoveldrugvonoprazan.Therefore,theexpected gastricpHwouldinevitablybelessacidic,whichwouldjustifythestudyofAMOXrelease atpH5.0andtheconsiderationofthesedataasoptimalonesinthetreatmentofH.pylori infections.Additionalstudiesthathavefocusedontheanalysesofotherparametersrelatedtodrugreleasefromthegeneratedsemi‐IPNswillbepublished. Figure11.CumulativedrugreleaseforGG‐p(CAR 50 B 50 ),10%ofloadedamoxicillin,attwodifferent pHlevels. Figure 10. ( a ) SEM photo from GG-p(TAR 50 B 50 ). ( b ) SEM photo from GG-pB. ( c ) SEM photo from GG-p(CAR50B50).
Int. J. Mol. Sci. 2023,24, 2281 17 of 22 3.4. AMOX Loading and In Vitro Drug Release Studies The release studies were conducted in simulated gastric fluid sine pepsin at pH 1.2 (SGF-1.2) and pH 5.0 (SGF-5) at 37 ◦ C. The drug release was determined by UV spectroscopy (Figure 11). These assays demonstrated that (a) the synthesized systems could encapsulate the desired drug effectively since burst release was prevented, and (b) AMOX could be released from it in a controlled, sustained manner, its delivery being more rapid at pH 5.0 than at pH 1.2. In fact, cumulative drug release data approached 55% at pH 5.0 after 7.5 h, while it barely reached 30% at pH 1.2. Int.J.Mol.Sci.2023,24,228118of23 (c) Figure10.(a)SEMphotofromGG‐p(TAR 50 B 50 ).(b)SEMphotofromGG‐pB.(c)SEMphotofrom GG‐p(CAR 50 B 50 ). 3.4.AMOXLoadingandInVitroDrugReleaseStudies ThereleasestudieswereconductedinsimulatedgastricfluidsinepepsinatpH1.2 (SGF‐1.2)andpH5.0(SGF‐5)at37°C.ThedrugreleasewasdeterminedbyUVspectroscopy(Figure11).Theseassaysdemonstratedthat(a)thesynthesizedsystemscouldencapsulatethedesireddrugeffectivelysinceburstreleasewasprevented,and(b)AMOX couldbereleasedfromitinacontrolled,sustainedmanner,itsdeliverybeingmorerapid atpH5.0thanatpH1.2.Infact,cumulativedrugreleasedataapproached55%atpH5.0 after7.5h,whileitbarelyreached30%atpH1.2. PreviousscientificstudiesregardingthereleaseofAMOXfromgastroretentivesystemsweregenerallyperformedatpH1.2asthiscorrespondstothenormalacidityfound inahealthy,emptystomach;hence,theassayswereconductedatthatpHforcomparative reasons.Nonetheless,itcannotbeforgottenthattherapeuticapproachesforthetreatment ofH.pyloriinfectionstendtoincludeanacidic‐secretion‐blockingcompound,suchasregularprotonpumpinhibitorsorthemorenoveldrugvonoprazan.Therefore,theexpected gastricpHwouldinevitablybelessacidic,whichwouldjustifythestudyofAMOXrelease atpH5.0andtheconsiderationofthesedataasoptimalonesinthetreatmentofH.pylori infections.Additionalstudiesthathavefocusedontheanalysesofotherparametersrelatedtodrugreleasefromthegeneratedsemi‐IPNswillbepublished. Figure11.CumulativedrugreleaseforGG‐p(CAR 50 B 50 ),10%ofloadedamoxicillin,attwodifferent pHlevels. Figure 11. Cumulative drug release for GG-p(CAR 50 B 50 ), 10% of loaded amoxicillin, at two different pH levels. Previous scientific studies regarding the release of AMOX from gastroretentive systems were generally performed at pH 1.2 as this corresponds to the normal acidity found in a healthy, empty stomach; hence, the assays were conducted at that pH for comparative reasons. Nonetheless, it cannot be forgotten that therapeutic approaches for the treatment of H. pylori infections tend to include an acidic-secretion-blocking compound, such as regular proton pump inhibitors or the more novel drug vonoprazan. Therefore, the expected gastric pH would inevitably be less acidic, which would justify the study of AMOX release at pH 5.0 and the consideration of these data as optimal ones in the treatment of H. pylori infections. Additional studies that have focused on the analyses of other parameters related to drug release from the generated semi-IPNs will be published. 4. Materials and Methods 4.1. Chemicals Chemicals used in this work were acquired from different suppliers and used as received. Guar gum (product reference: G4129-250G), diallyl carbonate (CAR), 2,2 0 - (ethylenedioxy)diethanethiol (EDDET), trimethylolpropane tris(3-mercaptopropionate) (TriT), and methoxypolyethyleneglycol-350 (MeO-PEG-350) were acquired from Sigma- Aldrich (Madrid, Spain). Calcium chloride (CaCl 2 ) was provided by Fisher Scientific (part of Thermo Fisher Scientific; Waltham, MA, USA). Alfa Aesar (part of Thermo Fisher Scientific; MA, USA) was the supplier of (2R,3R)-N,N 0 -diallyltartramide (TAR) and allyl alcohol. 2,2-Dimethoxy-2-phenylacetophenone (DMPA) and acetaldehyde were purchased from Acros Organics (part of Thermo Fisher Scientific; MA, USA). 4.2. Preparation and Characterization of Homopolymers and Copolymers B monomer (acetaldehyde diallylacetal) was prepared following the recipe described by Verdugo-Fernández [ 23 ]. The other monomers were dissolved in polymerization solvent, either ethyl acetate (EtOAc, 2 mL) or methanol (MeOH, 2 mL), depending on their
Int. J. Mol. Sci. 2023,24, 2281 18 of 22 solubilities. Next, the initiator (dissolved in MeOH, concentration: 340 mg/mL) was added to the monomer mixtures, and the final solutions were immediately exposed to UV light (365 nm, 180 W) for 10 min while stirred. After that, the UV light was turned off, and the reactions proceeded for 48 h at 18 ◦ C. Once the reaction time was over, the solvent was removed under reduced pressure, yielding a transparent uncolored syrup in every case except for hom-pTAR, for which the new polymeric material was a white solid. The yields of the polymeric syntheses were quantitative in all cases. Additionally, NMR spectra demonstrated that there were no unreacted monomers in the polymerization media (Figure 6). Experiments of nuclear magnetic resonance (NMR), electrospray ionization mass spectrometry (ESI-MS), and scanning electron microscopy (SEM), as well as lyophilization procedures, were conducted at the CITIUS Service (University of Seville). 1 H-NMR and 13 C-NMR spectra were recorded at 300 K with a Bruker—AV NEO 300 MHz for solutions in CDCl 3 or DMSO-d 6 . Chemical shifts ( δ ) are reported as parts per million downfield from Me 4 Si and Jin Hz. Jis assigned and not repeated. All the assignments were confirmed by COSY and HSQC experiments. The mass spectrum of the B monomer was obtained using a Kratos MS80RFA instrument. The thermogravimetric analyzer was a TA Instruments Q-600 SDT (New Castle, DE, USA). Platinum pans containing approximately 5 mg of each sample were used. Trials were conducted under a nitrogen atmosphere (flow rate: 100 mL/min) at a heating rate of 10 ◦C/min (from 25 to 600 ◦ C), and the thermal decomposition pattern was studied. Gel permeation chromatography analyses were carried out using a Waters apparatus equipped with a Waters 2414 refractive index detector and two Styragel ® HR columns ( 7.8 ×300 mm2 ) linked in series, thermally stabilized at 40 ◦ C, using N,N-dimethylformamide (DMF) containing LiBr 5.8 mM as the mobile phase (flow rate: 1.0 mL/min ). Molecular weights were estimated against polystyrene standards. 4.3. Preparation and Characterization of Guar-Gum-Based Semi-IPNs The systems were prepared under an inert atmosphere. To do so, 400 mg of GG was mixed with 9 mL of deoxygenated distilled water, and the content was stirred thoroughly until a homogeneous mixture of GG and water was achieved. Next, 1 g of PEG and the corresponding amount of EDDET, CAR, TAR, and/or B (Table 4) were added to each sample, as well as the appropriate volume of a solution of the covalent cross-linker TriT (in MeOH; concentration: 368.5 mg/mL) to reach a degree of covalent cross-linking of 8%. The systems were gently mixed at 18 ◦ C, and then the initiator was charged (dissolved in MeOH; concentration: 340 mg/mL) and homogenized. The vials were placed in a roller for 10 min while simultaneously being exposed to UV light (365 nm, 180 W). Polymerizations were allowed to proceed overnight at 18 ◦ C in an orbital stirrer at 50 rpm. The final GG concentration that was aimed in every experiment was 4%, identical to that of the polymer synthesized de novo (Polymer 2). The presence of PEG was removed by the critical point drying method [ 24 ]; consecutive water–acetone/water–ethanol baths would ensure not only the removal of the biocompatible, non-toxic PEG but also any remaining unreacted monomer left, if necessary. All the rheological measurements were carried out in an AR2000 rheometer (TA Instruments) using plate–plate geometry (diameter 40 mm) with a rough surface to avoid the slipping of the sample. The temperature was maintained at 25 ◦ C using a Peltier heating system. Images were recorded by means of a field emission scanning electron microscope, FEI Teneo, at an accelerating voltage of 5 kV using secondary electrons. The samples were dried by the critical point drying method in order to prevent the alteration of their surface topography. This protocol is widely applied for the analysis of biological tissues and was followed according to a previous study [24].
Int. J. Mol. Sci. 2023,24, 2281 19 of 22 4.4. AMOX Loading and In Vitro Drug Release Studies A preliminary dissolution study using the co-p(CAR 50 B 50 ) semi-IPN was performed as a proof of concept. AMOX was loaded into the matrix by dissolving it in DMSO (concentration: 500 mg mL −1 ) and then dropping it over a 50 mg bead of the lyophilized system. The concentration for AMOX loading was chosen to be 10% of the total weight of the loaded sample. Then, the drug-loaded material was placed into dialysis tubes (1 kDa cut-off) and submerged in vessels containing simulated gastric fluid sine pepsin (prepared according to [ 42 ]) either at pH 1.2 (SGF-1.2) or pH 5.0 (SGF-5), depending on the sample. All the vessels were then placed inside an orbital incubator at 37 ◦ C and stirred gently (100 rpm) throughout the release experiments. At each predetermined time interval, 5 mL of the medium was withdrawn from each vessel, and its absorbance was measured at 229 nm. Dilutions of the samples were conducted when necessary. An equal volume of prewarmed, freshly prepared medium was added to the dissolution vessels after each withdrawal. The amount of AMOX released by the system was calculated by means of the calibration curve. 5. Conclusions In this study, the preparation of novel semi-IPNs for their future use as matrices in GRDDSs was successfully addressed using guar gum (GG, Polymer 1) and a biodegradable synthetic copolymer (Polymer 2) as the unique components of super-porous matrices. The biodegradable Polymer 2 was synthesized by means of a new procedure, thiolene click reactions, in the design of semi-IPNs as DDSs. This polymerization process was initially tested by the preparation of different linear homopolymers using labile monomer acetaldehyde diallylacetal (B monomer), diallyl carbonate (CAR), and (2R,3R)- N,N 0 -diallyltartramide (TAR) as the diallyl monomers; 2,2 0 -(thylenedioxy)diethanethiol (EDDET) was the selected dithiol monomer in every case. The resulting polymers were characterized by GPC and NMR studies, confirming the adequacy of the process. All the evaluated macromolecules were demonstrated to be thermally stable below 200 ◦ C (TGA analysis), while their weight average molecular weight ranged from 42,600 to 19,800, showing that CAR was the diallyl monomer that polymerized more efficiently under the tested conditions. The procedure to generate copolymers with varied potential degradability was investigated next. Three combinations of the diallyl monomers CAR and B were chosen (molar ratios 80:20, 50:50, and 20:80). The experimental compositions of the copolymers fitted perfectly well with the theoretical monomer ratios, as confirmed by 1 H-NMR spectra. The acidic hydrolyses of the B monomer and the copolymer co-p(CAR 50 B 50 ) (to be used as Polymer 2 in semi-IPNs) were investigated to confirm their hydrolysis and, hence, the future degradability of the semi-IPNs to be prepared. In total, 67% of acetal moieties from polymer co-p(CAR 50 B 50 ) were degraded under acidic conditions in 4 hours, as corroborated by NMR studies. Next, semi-IPNs were synthesized using the mucoadhesive polysaccharide guar gum and different combinations of CAR/B diallyl monomers. Trimethylpropane tris(3- mercaptopropionate) (TriT) was the cross-linking agent in every case (degree of crosslinking: 8%). The rheological properties of the resulting networks were evaluated. Every system showed similar values for tan ( δ ) ( ≈ 0.37 at 1.00 Hz) and similar behavior (enhanced solid-like features) when the frequency of stimuli increased. Moreover, SEM observations confirmed the super-porous morphology of the systems, with a mean pore diameter of 0.290 ± 0.004 µ m. Based on the overall results presented, semi-IPN formulations with CAR and B as diallyl monomers are expected to perform best as polymeric matrices for the sustained release of APIs at the gastrointestinal level. Preliminary loading and release assays of AMOX from the designed semi-IPNs demonstrated that AMOX could be released from it in a controlled, sustained manner. These results pave the way for the preparation of GRDDSs for the treatment of H. pylori infections as well as other pathologies. These mechanically strong, well-structured, mucoadhesive,
Int. J. Mol. Sci. 2023,24, 2281 20 of 22 super-porous semi-IPN matrices may be of use in oral drug administration as they will be capable of controlling the release of selected APIs, such as AMOX, while disintegrated under the mildly acidic conditions of the stomach of patients treated. Author Contributions: Conceptualization, M.-V.d.-P. and R.G.; methodology: M.-V.d.-P., R.G., E.B. and M.G.G.-M.; validation: (a) synthesis, polymerization methods, formation of IPN and NMR characterization of homo- and copolymers: R.G. and M.-V.d.-P.; (b) GPC and TGA studies: E.B. and M.G.G.-M.; (c) rheological studies: R.G., V.P.-P. and P.S.-C.; (d) hydrolytic studies: A.I.C.-G.; investigation, R.G., M.-V.d.-P., A.I.C.-G., E.B. and M.G.G.-M.; resources, M.-V.d.-P., M.G.G.-M. and E.B.; writing—original draft preparation, R.G., M.-V.d.-P., V.P.-P. and A.I.C.-G.; writing—review and editing, M.-V.d.-P., M.G.G.-M. and E.B.; supervision, M.-V.d.-P., M.G.G.-M. and E.B.; project administration, M.-V.d.-P.; funding acquisition, M.-V.d.-P. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Ministerio de Ciencia e Innovación-Agencia Estatal de Investigación (MCI/AEI/FEDER, EU), grant number PID2020-115916GB-I00, the Fondo Europeo de Desarrollo Regional (FEDER), and la Consejería de Economía y Conocimiento (Junta de Andalucía), grant number US-1380587. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. References 1. American Cancer Society: Cancer Facts & Figures. Atlanta Am. Cancer Soc. 2021, 1–72. 2. Fernández-Navarro, P.; Roquette, R.; Nuñez, O.; de Sousa-Uva, M.; García-Pérez, J.; López-Abente, G.; Nunes, B.; González- Sánchez, M.; Dinis, J.; Carmona, R.; et al. Atlas of Cancer Mortality in Portugal and Spain (2003–2012). Natl. Inst. Health Doutor Ricardo Jorge (Port.) Natl. Inst. Health Carlos III (Spain) 2021. [CrossRef] 3. Lee, Y.-C.; Dore, M.P.; Graham, D.Y. Diagnosis and Treatment of Helicobacter pylori Infection. Chin. J. Gastroenterol. 2022 ,73, 183–195. [CrossRef] 4. Grosso, R.; De-Paz, M.V. Scope and Limitations of Current Antibiotic Therapies against Helicobacter pylori: Reviewing Amoxicillin Gastroretentive Formulations. Pharmaceutics 2022,14, 1340. [CrossRef] 5. Kiyotoki, S.; Nishikawa, J.; Sakaida, I. Efficacy of vonoprazan for helicobacter pylori eradication. Intern. Med. 2020 ,59, 153–161. [CrossRef] 6. Iglesias, N.; Galbis, E.; Romero-Azogil, L.; Benito, E.; Lucas, R.; García-Martín, M.G.; De-Paz, M.V. In-depth study into polymeric materials in low-density gastroretentive formulations. Pharmaceutics 2020,12, 636. [CrossRef] [PubMed] 7. Rajinikanth, P.S.; Balasubramaniam, J.; Mishra, B. Development and evaluation of a novel floating in situ gelling system of amoxicillin for eradication of Helicobacter pylori. Int. J. Pharm. 2007,335, 114–122. [CrossRef] [PubMed] 8. Patel, D.M.; Patel, D.K.; Patel, C.N. Formulation and Evaluation of Floating Oral In Situ Gelling System of Amoxicillin. ISRN Pharm. 2011,2011, 8. [CrossRef] [PubMed] 9. Rajinikanth, P.S.; Mishra, B. Stomach-site specific drug delivery system of clarithromycin for eradication of Helicobacter pylori. Chem. Pharm. Bull. 2009,57, 1068–1075. [CrossRef] 10. Ranade, A.N.; Wankhede, S.S.; Ranpise, N.S.; Mundada, M.S. Development of bilayer floating tablet of amoxicillin and aloe vera gel powder for treatment of gastric ulcers. AAPS Pharm. Sci. Tech. 2012,13, 1518–1523. [CrossRef] [PubMed] 11. Rossi, A.; Conti, C.; Colombo, G.; Castrati, L.; Scarpignato, C.; Barata, P.; Sandri, G.; Caramella, C.; Bettini, R.; Buttini, F.; et al. Floating modular drug delivery systems with buoyancy independent of release mechanisms to sustain amoxicillin and clarithromycin intra-gastric concentrations. Drug Dev. Ind. Pharm. 2016,42, 332–339. [CrossRef] [PubMed] 12. Charoenying, T.; Patrojanasophon, P.; Ngawhirunpat, T.; Rojanarata, T.; Akkaramongkolporn, P.; Opanasopit, P. Fabrication of floating capsule-in-3D-printed devices as gastro-retentive delivery systems of amoxicillin. J. Drug Deliv. Sci. Technol. 2020 ,55, 101393. [CrossRef] 13. Kamsali, A.; Eranti, B.; Ch, M.; Manne, R.; Barghav, G.C.; Reddy, P.S. Development and Optimization of Amoxicillin Floating Raft System to effectively treat Helicobacter pylori infection. Ars Pharm. 2020,61, 163–168. [CrossRef] 14. Awasthi, R.; Kulkarni, G.T.; Pawar, V.K.; Garg, G. Optimization studies on gastroretentive floating system using response surface methodology. AAPS Pharm. Sci. Tech. 2012,13, 85–93. [CrossRef] [PubMed] 15. Gils, P.S.; Ray, D.; Sahoo, P.K. Characteristics of xanthan gum-based biodegradable superporous hydrogel. Int. J. Biol. Macromol. 2009,45, 364–371. [CrossRef]
Int. J. Mol. Sci. 2023,24, 2281 21 of 22 16. Gemeinhart, R.A.; Park, H.; Park, K. Pore structure of superporous hydrogels. Polym. Adv. Technol. 2000 ,11, 617–625. [CrossRef] 17. Wang, B. Super Porous Hydrogel: A Promising Gastroretentive Drug Delivery System. World J. Pharm. Pharm. Sci. 2017 ,6, 129–159. [CrossRef] 18. Omidian, H.; Park, K.; Rocca, J.G. Recent developments in superporous hydrogels. J. Pharm. Pharmacol. 2007 ,59, 317–327. [CrossRef] [PubMed] 19. Zhao, S.; Lv, Y.; Zhang, J.B.; Wang, B.; Lv, G.J.; Ma, X.J. Gastroretentive drug delivery systems for the treatment of Helicobacter pylori. World J. Gastroenterol. 2014,20, 9321–9329. [CrossRef] 20. Mandal, U.K.; Chatterjee, B.; Senjoti, F.G. Gastro-retentive drug delivery systems and their in vivo success: A recent update. Asian J. Pharm. Sci. 2016,8, 575–584. [CrossRef] 21. Amin, M.L.; Ahmed, T.; Mannan, M.A. Development of floating-mucoadhesive microsphere for site specific release of metronidazole. Adv. Pharm. Bull. 2016,6, 195–200. [CrossRef] 22. Sharma, B.; Sandilya, A.; Patel, U.; Shukla, A.; Sadhu, S.D. A bio-inspired exploration of eco-friendly bael gum and guar gum-based bioadhesive as tackifiers for packaging applications. Int. J. Adhes. Adhes. 2021,110, 102946. [CrossRef] 23. Verdugo-Fernández, P.M. Stimuli Cleavable ABA Block Copolymers: Synthesis of Degradable Thermoplastic Elastomers from Renewable Resources [Dissertation]; Universitat Rovira i Virgili: Tarragona, Spain, 2020. 24. Bray, D. Critical Point Drying of Biological Specimens for Scanning Electron Microscopy. Supercritical Fluid Methods and Protocols. Methods In Biotechnology; Williams, J.R., Clifford, A.A., Eds.; Humana Press: Totowa, NJ, USA, 2000; pp. 235–243. [CrossRef] 25. Tripathi, J.; Thapa, P.; Maharjan, R.; Jeong, S.H. Current State and Future Perspectives on Gastroretentive Drug Delivery Systems. Pharmaceutics 2019,11, 193. [CrossRef] 26. Jafar, M.; Salahuddin, M.; Khan, M.S.A.; Alshehry, Y.; Alrwaili, N.R.; Alzahrani, Y.A.; Imam, S.S.; Alshehri, S. Preparation and in vitro - in vivo evaluation of luteolin loaded gastroretentive microsponge for the eradication of helicobacter pylori infections. Pharmaceutics 2021,13, 2094. [CrossRef] 27. Carvalho, I.C.; Medeiros Borsagli, F.G.L.; Mansur, A.A.P.; Caldeira, C.L.; Haas, D.J.; Lage, A.P.; Ciminelli, V.S.T.; Mansur, H.S. 3D sponges of chemically functionalized chitosan for potential environmental pollution remediation: Biosorbents for anionic dye adsorption and ‘antibiotic-free’ antibacterial activity. Environ. Technol. 2021,42, 2046–2066. [CrossRef] 28. Biswas, A.; Mondal, S.; Das, S.K.; Bose, A.; Thomas, S.; Ghosal, K.; Roy, S.; Provaznik, I. Development and Characterization of Natural Product Derived Macromolecules Based Interpenetrating Polymer Network for Therapeutic Drug Targeting. ACS Omega 2021,6, 28699–28709. [CrossRef] 29. Hadke, J.; Khan, S. Preparation of sterculia foetida-pullulan-based semi-interpenetrating polymer network gastroretentive microspheres of amoxicillin trihydrate and optimization by response surface methodology. Turk. J. Pharm. Sci. 2021 ,18, 388–397. [CrossRef] 30. Raghu Kiran, C.V.S.; Gopinath, C. Development and evaluation of interpenetrating polymer network based superporous hydrogel gastroretentive drug delivery systems (SPH IPN-GRDDS). Mater. Today Proc. 2021,46, 3056–3061. [CrossRef] 31. Panahi, Y.; Gharekhani, A.; Hamishehkar, H.; Zakeri-Milani, P.; Gharekhani, H. Stomach-specific drug delivery of clarithromycin using a semi interpenetrating polymeric network hydrogel made of montmorillonite and chitosan: Synthesis, characterization and in vitro drug release study. Adv. Pharm. Bull. 2019,9, 159–173. [CrossRef] 32. Majeed, A.; Pervaiz, F.; Shoukat, H.; Shabbir, K.; Noreen, S.; Anwar, M. Fabrication and evaluation of pH sensitive chemically cross-linked interpenetrating network [Gelatin/Polyvinylpyrrolidone-co-poly(acrylic acid)] for targeted release of 5-fluorouracil. Polym. Bull. 2022,79, 1–20. [CrossRef] 33. Cardos, I.A.; Zaha, D.C.; Sindhu, R.K.; Cavalu, S. Revisiting therapeutic strategies for h. Pylori treatment in the context of antibiotic resistance: Focus on alternative and complementary therapies. Molecules 2021,26, 6078. [CrossRef] 34. Vukovi´c, J.S.; Filipovi´c, V.V.; Babi´c Radi´c, M.M.; Vukomanovi´c, M.; Milivojevic, D.; Ilic-Tomic, T.; Nikodinovic-Runic, J.; Tomi´c, S.L. In Vitro and In Vivo Biocompatible and Controlled Resveratrol Release Performances of HEMA/Alginate and HEMA/Gelatin IPN Hydrogel Scaffolds. Polymers 2022,14, 4459. [CrossRef] 35. Tuan, H.N.A.; Nhu, V.T.T. Synthesis and properties of pH-thermo dual responsive semi-IPN hydrogels based on N,N 0 - diethylacrylamide and itaconamic acid. Polymers 2020,12, 1139. [CrossRef] [PubMed] 36. Dragan, E.S.; Dinu, M.V.; Ghiorghita, C.A.; Lazar, M.M.; Doroftei, F. Preparation and Characterization of Semi-IPN Cryogels Based on Polyacrylamide and Poly(N,N-dimethylaminoethyl methacrylate); Functionalization of Carrier with Monochlorotriazinylβ - cyclodextrin and Release Kinetics of Curcumin. Molecules 2021,26, 6975. [CrossRef] [PubMed] 37. Ferris, C.; de Paz, M.V.; Galbis, J.A. L-arabinitol-based functional polyurethanes. J. Polym. Sci. Part A Polym. Chem. 2011 ,49, 1147–1154. [CrossRef] 38. Ferris, C.; de Paz, M.V.; Galbis, J.A. Synthesis of Functional Sugar-Based Polyurethanes. Macromol. Chem. Phys. 2012 ,213, 480–488. [CrossRef] 39. Ferris, C.; de Paz, M.V.; Aguilar-de-Leyva, A.; Caraballo, I.; Galbis, J.A. Reduction-sensitive functionalized copolyurethanes for biomedical applications. Polym. Chem. 2014,5, 2370–2381. [CrossRef] 40. Iglesias, N.; Galbis, E.; Valencia, C.; Díaz-Blanco, M.J.; Lacroix, B.; De-Paz, M.-V. Biodegradable double cross-linked chitosan hydrogels for drug delivery: Impact of chemistry on rheological and pharmacological performance. Int. J. Biol. Macromol. 2020 , 165, 2205–2218. [CrossRef]
Int. J. Mol. Sci. 2023,24, 2281 22 of 22 41. Iglesias, N.; Galbis, E.; Valencia, C.; De-Paz, M.-V.; Galbis, J.A. Reversible pH-sensitive chitosan-based hydrogels. Influence of dispersion composition on rheological properties and sustained drug delivery. Polymers 2018,10, 392. [CrossRef] 42. Brüning, J.; Petereit, A.C.; Alig, E.; Bolte, M.; Dressman, J.B.; Schmidt, M.U. Characterization of a new solvate of risedronate. J. Pharm. Sci. 2011,100, 863–873. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.