Thermogelling Platform for Baicalin Delivery for Versatile ... swelling ratio, and rheological properties in addition to surface and internal microstructure. Polyethylene glycol (PEG)

Thermogelling Platform for Baicalin Delivery for VersatileBiomedical ApplicationsMohamed Haider,*,†,‡,§ Mariame A. Hassan,†,‡,§ Iman S. Ahmed,† and Rehab Shamma‡

†Department of Pharmaceutics & Pharmaceutical Technology, College of Pharmacy, University of Sharjah, Sharjah 27272, UnitedArab Emirates‡Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt

*S Supporting Information

ABSTRACT: Baicalin (BG) is a natural glycoside with severalpromising therapeutic and preventive applications. However, itspharmaceutical potential is compromised by its poor water solubility,complex oral absorption kinetics, and low bioavailability. In this work,BG was incorporated in a series of chitosan (Ch)/glycerophosphate(GP)-based thermosensitive hydrogel formulations to improve its watersolubility and control its release profile. Molecular interactions betweenBG and GP were investigated using Fourier transform infraredspectroscopy (FT-IR), and the ability of GP to enhance the watersolubility of BG was studied in different release media. Drug-loaded Ch/GP hydrogels were prepared and characterized for their gelation time,swelling ratio, and rheological properties in addition to surface andinternal microstructure. Polyethylene glycol (PEG) 6000 and hydrox-ypropyl methyl cellulose (HPMC) were incorporated in the formulations at different ratios to study their effect on modulatingthe sol−gel behavior and the in vitro drug release. In vivo pharmacokinetic (PK) studies were carried out using a rabbit modelto study the ability of drug-loaded Ch/GP thermosensitive hydrogels to control the absorption rate and improve thebioavailability of BG. Results showed that the solubility of BG was enhanced in the presence of GP, while the incorporation ofPEG and/or HPMC had an impact on gelation time, rheological behavior, and rate of drug release in vitro. PK results obtainedfollowing buccal application of drug-loaded Ch/GP thermosensitive hydrogels to rabbits showed that the rate of BG absorptionwas controlled and the in vivo bioavailability was increased by 330% relative to BG aqueous oral suspension. The proposed Ch/GP thermosensitive hydrogel is an easily modifiable delivery platform that is not only capable of improving the solubility andbioavailability of BG following buccal administration but also can be suited for various local and injectable therapeuticapplications.

KEYWORDS: baicalin, thermosensitive hydrogels, chitosan, controlled release, bioavailability

1. INTRODUCTION

Baicalin (BG; baicalein-7-glucuronide; Figure 1a) is a glycosideextracted from the dried roots of Scutellariae baicalensis Georgi,commonly known as Baikal skullcap. BG possesses a widerange of therapeutic and preventive potential, which has placedit recently in the center of focus of interest as a safe, naturaltherapeutic ingredient. A PubMed search with the key word[Baicalin]restricted in the titlereturns ca. 700 articlesfrom 2001 to date with 50% of them published in the last fiveyears (Figure 1b). As herbal medicine, the plant extract isofficially listed in the Chinese Pharmacopeia.1,2 Pharmacolog-ically, BG is effective as an antibacterial,3 antifungal,4 antiviral,5

antiallergic, anti-inflammatory,6 antipyretic,7 antihypertensive,diuretic, and antithrombotic agent.8,9 In addition, BG has asedative effect10,11 and possesses antioxidant activity and thushepatoprotective activity.12 Recently, it has also been reportedto exert anticancer and neuroprotective effects.13,14

Despite the medical benefits of BG, its therapeutic/clinicalapplications are still greatly limited due to its poor water

solubility, where BG is classified as a class II drug in theBiopharmaceutical Classification System (BCS) with an oralbioavailability as low as 2.2% of the administered dose.15

Moreover, BG has very complex absorption kinetics due to acomplex metabolic pathway. Intestinal enzymes, microbiota,and extensive liver metabolism break the drug into its aglyconeform baicalein (B); in turn, B is then glycosylated back rapidlyto BG through glucuronidation and sulfation in intestinalepithelia as well as in liver.16,17 Although the metabolites areclaimed to be relatively bioactive to varying extents, theglycosylated forms are rapidly excreted, accounting for the lowbioavailability in part (Figure 1c). In addition, the naturalglycoside form has poor aqueous solubility that allows thesecomplex metabolic pathways to occur at full paces. Attempts to

Received: May 8, 2018Revised: June 26, 2018Accepted: June 28, 2018Published: June 28, 2018

Article

Cite This: Mol. Pharmaceutics 2018, 15, 3478−3488

© 2018 American Chemical Society 3478 DOI: 10.1021/acs.molpharmaceut.8b00480Mol. Pharmaceutics 2018, 15, 3478−3488

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http://pubs.acs.org/action/showCitFormats?doi=10.1021/acs.molpharmaceut.8b00480http://dx.doi.org/10.1021/acs.molpharmaceut.8b00480

increase the solubility of BG and B have majorly focused onparticle size reduction techniques, especially with B owing toits higher lipophilicity, smaller molecular weight, and ability topass the intestinal epithelium passively. B was thus formulatedinto oral chewable tablets,18,19 solid dispersions,20 micro-emulsions,21 nanoemulsions,22 self-assembled nanoparticles,23

and nanocrystals.24 On the other hand, only a few studies havesucceeded to increase the water solubility of BG throughinclusion in solid dispersions,25 liposomes,26 and nano-liposomes.27 In this study, we present a novel delivery platformfor BG that does not depend on size reduction but rather onsolubilization of BG in the delivery system.Chitosan (Ch) is a natural biodegradable linear amino-

polysaccharide obtained by alkaline deacetylation of crustaceanshells and has been widely used in drug and protein delivery aswell as tissue engineering.28,29 Glycerophosphate (GP) is anorganic compound naturally present in the body as a source ofphosphate and is administered for the treatment of phosphatemetabolism disorders.30 The aqueous mixture of Ch and GPresponds nonlinearly to temperature elevation forming hydro-gels and is classified as a stimulus-responsive in situ-forminggel.31 The mechanism of gelation of the Ch/GP hydrogelsystem involves pH- and temperature-dependent interactions.At temperatures lower than 37 °C, the addition of GPincreases the pH of Ch solutions to around neutrality, whichwould shield the electrostatic repulsion between adjacent Chchains and can theoretically result in pH-induced gelformation. However, as a result of electrostatic attractionbetween the phosphate moieties of GP and protonated aminegroups (−NH3+) of Ch, the hydroxyl groups of GP increasestability and hydrophilicity in the Ch chains and thus maintainthe solubility of Ch for a period of time. An increase intemperature reduces the polarity of both Ch chains and theglycerol moiety of GP, causes Ch chain dehydration, andreduces both the Ch chain charge density and the attraction ofCh and GP. This will increase interchain hydrophobicattraction and hydrogen bonding between chains and resultin the formation of hydrogels.32−35

The temperature-dependent sol−gel transition as well as thetime of gelation can be adjusted to occur at body temperature

and at reasonable time frames through modifying therespective proportions of the ingredients.36 This platformoffers numerous advantages in drug delivery such as biosafetyand abundance of ingredients, versatility of applications, abilityto control drug release with proper inclusion of pharmaceuticaladditives, single-step inclusion of drugs, and ease ofpreparation and propensity for scaling up on industriallevels.37−39 Therefore, the Ch/GP combination is consideredas a promising hydrogel platform for a variety of applications,such as local drug delivery systems or injectable carriers fortissue engineering.30

Nevertheless, Ch-based gels are limited by the intermo-lecular hydrogen bonds on Ch polymeric chains, whichincrease the rigidity of the hydrogel structure and reducewater permeability.40 Several hydrophilic polymers have beenadded to Ch hydrogels to overcome these limitations.41,42

Poly(ethylene glycol) (PEG) is a highly hydrophilic, non-inflammatory, nonimmunogenic, and biocompatible polymercommonly used as a protective coating material for drugdelivery nanoparticles43 and liposomes44 and is also used toprovide similar protection when used as a conjugate topeptides and proteins.45 The addition of PEG to hydrogelsrenders them more hydrophilic and biocompatible and alterstheir mechanical properties to become more flexible.42

Hydroxy propyl methyl cellulose (HPMC), is anotherhydrophilic, biodegradable, and biocompatible naturallyoccurring polymer with several desirable properties. One ofits most important characteristics is the high swellability, whichsignificantly affects the release kinetics of an incorporateddrug.46 The polymer is approved by the FDA and is being usedextensively in the production of formulations with a controlledrelease system.47 Thermosensitive Ch/HPMC hydrogels havebeen prepared where the addition of HPMC to Ch in thepresence of glycerol facilitated the thermogelation at 32 °Cthrough large amounts of hydrophobic interactions. Inaddition, it increased the compactness of the Ch hydrogelsand improved their mechanical strength.48,49

The aim of this work is to develop and characterize Ch/GP-based thermosensitive hydrogel formulations that can improvethe water solubility and control the release of incorporated BG.

Figure 1. (a) Structure of baicalin (BG) showing the aglycone baicalein (B) and glucuronic acid. (b) Growing interest in BG as a pharmaceuticalactive constituent from 2001 to date. Source: PubMedkey word: Baicalin [Title]. (c) Illustration of BG metabolic pathway.

Molecular Pharmaceutics Article

DOI: 10.1021/acs.molpharmaceut.8b00480Mol. Pharmaceutics 2018, 15, 3478−3488

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http://dx.doi.org/10.1021/acs.molpharmaceut.8b00480

First, the ability of GP to enhance the water solubility of thedrug was investigated. Then, drug-loaded Ch/GP hydrogelswere prepared and characterized using different ratios of PEGand HPMC to study their effect on modulating the sol−gelbehavior and the in vitro drug release. Finally, a rabbit modelwas used to study the ability of the drug-loaded Ch/GPthermosensitive hydrogels to control the rate of absorption andimprove the bioavailability of BG following buccal admin-istration.

2. MATERIALS AND METHODS

2.1. Materials. Chitosan (Ch, medium molecular weight,70−85% deacetylation), disodium α,β-glycerophosphate (GP;glycerol-2-phosphate salt hydrate), baicalin (BG;(2S,3S,4S,5R,6S)-6-(5,6-dihydroxy-4-oxo-2-phenyl-chromen-7-yl) oxy-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid),polyethylene glycol (PEG) 6000, hydroxypropyl methylcellulose (HPMC), and a cellulose dialysis bag, molecularweight cutoff (MWCO) of 14 kDa were purchased from sigmaAldrich (St. Louis, MO, USA). Reagent grade acetic acid wasacquired from Merck & Co., Inc. (Darmstadt, Germany).2.2. Fourier Transform Infrared Spectroscopy. Pow-

ders of BG, GP, and Ch and a dry mixture of BG/GP (1:1molar ratio) were placed separately into transparent discsunder a pressure of 10 000−15 000 pounds per square inch.Similarly, aqueous solutions of BG/GP (1:1 molar ratio) andCh/GP were prepared and mounted on a transparent disc. Theinfrared spectra of the samples were recorded using a FourierTransform Infrared (FT-IR) spectrophotometer (JASCOFTIR 6300, Jasco, Easton, MD, USA). The set range for FT-IR was from 4000 to 400 cm−1 at a resolution of 4 cm−1. Thestretching modes and vibrational modes depict the chemicalbonding and other functional groups present in the material.2.3. Preparation of Thermosensitive Gels. Aqueous Ch

solution (1.8% w/v) was prepared by dissolving the polymer in0.1 M acetic acid and stirring overnight at room temperature toobtain a clear solution. The solution was added dropwise intoan equal volume of well-stirred 50% w/v GP solution indistilled water (pH = 7). The mixture was further stirred for 5min at room temperature to ensure homogeneous mixing. BG-loaded hydrogel formulation F1 was prepared by dissolving BGin GP solution to obtain a final concentration of 7.5 mg of BG/mL of the gel. Drug-loaded hydrogel formulations containingPEG-6000 alone (F2 and F4) or mixtures of PEG-6000 andHPMC (F3 and F5) were prepared by dissolving each polymerin GP solution at different required percentages prior to mixingwith Ch solutions. The composition of BG-loaded hydrogelformulations is summarized in Table 1. The preparedhydrogel-forming solutions were stored at 4 °C for furtherstudies.

2.4. Gelation Time. The time required for the sol-to-geltransformation was determined using the tube invertingmethod.50 Vials containing 1 mL of different formulations at4 °C were immersed in a water bath preheated at 37 °C. Thetime taken for the gel to show a lack of flowability tested every10 s was noted. To determine the reversibility of the system,gelled samples were returned onto ice to check their gel−soltransformation, and then, the time to gel was again observed.The test results are presented as mean value of threedeterminations ± standard deviation (SD).

2.5. Determination of Hydrogel Swelling Ratio. Equalvolumes of hydrogel-forming solutions were incubated at 37°C for 2 h. The formed hydrogels were placed in a freezer at−20 °C for 24 h. The frozen gels were then transferred to alyophilizer (Vir Tis Bench Top Pro, SP Scientific, USA) for 24h with a condenser temperature of −50 °C and a pressure of 7× 10−2 mbar. The dry weights (Wd) of the freeze-dried gelswere first determined, and then, the gels were submerged in 10mL of PB for 24 h at 37 °C followed by washing withdeionized water to remove any ions adsorbed on their surfacesand blotted dry using filter paper. The wet weights (Ws) wererecorded, and the swelling ratio (q) of the hydrogels was

calculated from the equation = ×−q 100.W WW

s d

dThe measure-

ments were performed in triplicate, and the swelling ratio (q)± SD for each gel preparation was recorded.

2.6. Rheological Studies. The rheological properties ofthe prepared thermosensitive hydrogels (F1 to F5) werecharacterized by generating complete rheograms at 37 °Cusing a Cone and Plate Brookfield Rheometer DV3THBequipped with Spindle CPR-40 and Rheocalc software(Brookfield Engineering Laboratories Inc., USA). The shearrate (s−1) was plotted as a function of shearing stress (N/m2),and coefficient of viscosity in centipoise (cp) along with otherparameters were calculated for the gels.

2.7. Hydrogel Microstructure Analysis. The surfacemorphology and cross sections of the freeze-dried hydrogelswere examined by field emission scanning electron microscope(SEM; VEGA XM variable pressure, Tescan AS, CzechRepublic). The samples were freeze-dried for 24 h as describedabove, and cross sections were prepared after the gels werefractured using liquid nitrogen before being fixed withconductive tape on a metal stub. The samples were sputter-coated with a gold−palladium (80−20%) target using a MiniSputter Coater (SC7620, Quorum Technologies, UK), and 1kV was applied for 2 min to have a thickness of 20 nm.

2.8. In Vitro Drug Release. The in vitro release of BGfrom its suspension in water, aqueous GP solution, and drug-loaded hydrogels (F1−F5) was studied using the dialysismethod.51 Cellulose dialysis bags with an MWCO of 14 kDawere used to retain the tested samples while allowing thesoluble BG to permeate into the release medium. Initially, 1

Table 1. Composition and Physical Properties of Drug-Loaded Thermosensitive Hydrogels

composition (% w/v)

formulation BG Ch GP PEG6000 HPMC gelation time (s) q (%)a coefficient of viscosity (cp)

F1 0.75 0.9 25 - - 47.33 ± 5.04 395.01 ± 2.10 718.9F2 0.75 0.9 25 10 - 43.75 ± 6.88 207.50 ± 4.31b 952.9F3 0.75 0.9 25 10 1 31.67 ± 8.78b 208.79 ± 7.63b 1051.4F4 0.75 0.9 25 20 - 40.50 ± 5.21 204.17 ± 7.90b 1365.4F5 0.75 0.9 25 20 2 20.25 ± 2.72b 204.44 ± 7.74b 3016.2

aData are mean values (n = 3) ± SD. bp < 0.05 versus F1. cq = swelling ratio.



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mL of the drug suspension in water, BG/GP aqueous solution,or drug-loaded hydrogel solutions was pipetted into a dialysisbag. Sealed bags were placed in 350 mL of release mediumstirred at 50 rpm using a USP-1 dissolution tester (Agilent708-DS, Agilent Technologies, NC, USA). To study thesolubilizing effect of GP on BG, the release pattern of BG fromits suspension in water, BG/GP aqueous solution, and F1 wasstudied in both distilled water and phosphate buffer (PB) (pH6.8). For investigating the ability of the prepared thermosensi-tive hydrogels (F1 to F5) to control the release of BG, only PB(pH 6.8) was used as release medium. At specific time intervals(0.25, 0.5, 0.45, 1, 2, 4, 6, 8, and 12 h), 5 mL of the releasemedium was collected and immediately replaced by an equalvolume of respective fresh medium. The absorbance of BG wasdetermined by UV spectroscopy at 276 nm. A standard curveof BG in PB (pH 6.8) was generated over the range of 0.1−50μg/mL and used to convert absorbance to concentration. Acumulative release profile was generated by normalizing thedata against the total amount of BG and reported as percentagedrug release. All release experiments were conducted intriplicates.The mechanism of drug release from thermosensitive

hydrogels was determined by applying zero order, first order,and second order kinetics and the Higuchi diffusion model.The following linear regression equation was employed forzero order kinetics: Ct = Co − kt; where Co is the zero-timeconcentration of the drug, Ct is the concentration of the drugat time t, and k is the apparent release rate constant. First orderkinetics was determined according to the equation LnCt =LnCo − kt. For second order kinetics, the following equationwas used: = + kt

C C1 1

t o. Drug release following Higuchi model

was determined using the equation Q = kt2 ; where Qrepresents the fraction of drug released in time t ,and k is theHiguchi dissolution constant.2.9. In Vivo Pharmacokinetic Study. 2.9.1. Study

Design. The in vivo studies were carried out to compare thebioavailability and pharmacokinetic (PK) parameters of BGfrom two different treatments in white New Zealand malerabbits (2.5−3 kg) using a nonblind, two-treatment,randomized, parallel design. Six rabbits were randomlyassigned to each treatment group (n = 6). Food was withdrawn10 h prior to the study with water ad libitum. Early morning,the assigned treatments were administered. No food wasallowed after dosing for 12 h. In group A; rabbits received theBG suspension through an intragastric tube. In group B;rabbits received a BG basic gel formulation (F1) with the helpof a gel applicator. The gel was placed onto the buccal regionof the rabbit, pressed for approximately 10 s, and then, theapplicator was removed. The BG dose was adjusted to be 10mg/kg.52 Blood samples (2 mL) were withdrawn from themiddle ear vein using a 26-gauge needle and syringe at 0(predose), 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, and 24 h afteradministration. Samples were collected into heparinized tubes,and plasma was obtained by centrifugation at 4000 rpm for 15min. The plasma was pipetted into glass tubes and frozen at−20 °C until analysis. All animal experiments were performedaccording to ethical principles and approved by the ResearchEthics Committee (REC) for Animal Subject Research at theFaculty of Pharmacy, Cairo University, Egypt, operatingaccording to the CIOMS and ICLAS international guidingprinciples for biomedical research involving animals of 2012.

Also, all animal experiments comply with Directive 2010/63/EU.

2.9.2. Chromatographic Conditions. Plasma concentra-tions of BG were determined using a selective, sensitive, andaccurate LC−MS/MS method that was developed andvalidated before use. All chemicals and reagents used were ofanalytical grade, and solvents were of HPLC grade.Toresemide stock solution was prepared and used as aninternal standard (IS) by dissolving 10 mg in methanol andserially diluting the mixture with mobile phase to give a finalworking concentration of 1 μg/mL. A Shimadzu (Shimadzu,Japan) series LC system equipped with a pump (LC-20AD)along with an autosampler (SIL-20A/HT) was used to inject20 μL aliquots of the processed samples on a reverse-phasemicroparticulate C18 Sunfire column (particle size = 5 μm, (50× 4.6) mm, Waters Corp., Milford, MA, USA). All analyseswere carried out at room temperature. The isocratic mobilephase was prepared by mixing 80% acetonitrile and 20% (0.1%formic acid in water), which was delivered at a flow rate of 1mL/min into the mass spectrometer’s electrospray ionizationchamber. Quantitation was achieved by MS/MS detection inpositive ion mode for both BG and IS, using an API-4000Triple Quadrupole LC−MS/MS Mass Spectrometer (ABSCIEX Instruments) equipped with a turbo ion spray interfaceat 400 °C. The ion spray voltage was 5000 V. The compoundparameters: declustering potential (DP), collision energy(CE), entrance potential (EP), and collision exit potential(CXP) were 106, 25, 10, and 6 V for BG and 30, 25, 10, and 10V for IS, respectively. Detection of ions was performed in themultiple reaction monitoring (MRM) mode, monitoring thetransition of the m/z 447.2 precursor ion to the m/z 271.1 forBG and the m/z 348.9 precursor ion to the m/z 263.9 for IS.Quadrupoles Q1 and Q3 were set on unit resolution. Theanalytical data were processed by Analyst software version 1.6(Applied Biosystems Inc., Foster City, CA). Plasma sampleswere spiked with BG acetonitrile solution to contain 0.1−10ng/mL. Aliquots of 0.5 mL of plasma samples were then spikedwith 10 μL of toresemide and mixed with 1 mL of acetonitrilein a 10 mL glass centrifuge tube. The tubes were shaken byvortex mixing for 30 s. After centrifugation for 10 min at 6000rpm, the supernatant was transferred to a small glass tube, and20 μL of the resulting supernatant was injected using theautosampler. All frozen plasma samples obtained from therabbits after receiving treatments A and B were thawed atambient temperature and assayed as described above withoutthe addition of BG.

2.9.3. Pharmacokinetic Analysis. Data from the plasmaanalysis were analyzed for each rabbit using WinNonlin(version 1.5, Scientific Consulting, Inc., Cary, NC). Non-compartmental analysis was pursued, and the pharmacokineticvariables; Cmax (maximal drug concentration; ng/mL), Tmax(time for maximal drug concentration; h) and AUC(0−t) (areaunder the curve; ng·h/ml) were generated. The area under thecurve from zero to infinity AUC(0−∞) was calculated from the

equation = +−∞ −AUC AUC tCk(0 ) (0 )

t ; where Ct is the last

measured concentration at 24 h, and k is the elimination rateconstant estimated from the slope of the terminal log−linearphase. The elimination half-life (t1/2) was calculated as t1/2 =Ln2/k. The mean transit time (MTT) was calculated fromAUMC/AUC, where AUMC is the area under the firstmoment curve. The relative bioavailability ( f rel) was calculatedfor a BG F1 hydrogel formulation relative to the BG aqueous



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suspension as AUCF1/AUCsusp. All results were expressed asmean ± SD.2.10. Statistical Analysis. All in vitro data points are the

average of three independent experiments performed and areexpressed as mean ± SD. Statistical significance betweenresults was assessed by Student’s t test (two-tailed; p < 0.05).For in vivo studies, statistical inferences were based onuntransformed values for Cmax and AUC variables andobserved values for t1/2. The nonparametric Signed RankTest (Mann−Whitney’s test) was used to compare Tmaxbetween the two treatment groups. The one-way analysis ofvariance (ANOVA) F-test was used for testing the equality ofseveral means. For multiple comparisons, the procedure usedwas the least significant difference (LSD).

3. RESULTS AND DISCUSSION

3.1. FT-IR Spectrophotometric Analysis. BG showedvery poor solubility in water and acidic Ch solution at the doseused. In contrast, the inclusion of BG in aqueous GP solutionresulted in yellowish, clear, relatively thick liquid that remainedclear after the addition of Ch. The FTIR spectrophotometricanalysis (Figure 2) shows the characteristic absorption bandsof BG, Ch, GP, and their mixtures. BG is characterized bypeaks at 1726 cm−1 (for −COOH), 1660 cm−1 (for C=O),1065 cm−1 (C−O in ether and hydroxyl groups), and 1608,1573, and 1498 cm−1 (for aromatic C=C). On the other hand,GP has a characteristic peak at 900 cm−1 (aliphatic phosphate)and 3650−3250 cm−1 (C−OH). Most of these characteristicpeaks were retained at weak intensities in both the physicalmixture of dry powders of BG and GP and in their aqueoussolution. The broad peak of BG in the range of 3650−3250cm−1 (for −OH) appeared very weak in its mixtures with GP.This suggests intermolecular interaction (e.g., strong inter-

molecular hydrogen bonding) between molecules.53 Thisintermolecular H-bond between BG and GP might accountin part for the increased solubility of the drug. Worth notinghere that the intense peaks in the aqueous mixture at 3200 and1640 cm−1 pertain to water absorption.54 Similarly, thespectrum of the GP and Ch aqueous mixture showed a similarretention of characteristic peaks of each component at lowerintensities indicative of interaction (intermolecular hydrogenbonding) with more intense absorption for water at 1640cm−1.

3.2. Determination of Gelation Time. The gelation timeof the prepared formulations was measured at physiologictemperature (37 °C). As shown in Table 1, the addition of 10and 20% PEG, in F2 and F4, respectively, slightly reduced thegelation time when compared to the F1 hydrogel. A significantdecrease in gelation time was observed when HPMC wasincorporated in F3 and F5, where the gelation time wasreduced by 34 and 57%, respectively, relative to F1 hydrogel (P< 0.05). These results demonstrated that the addition ofHPMC into the Ch/GP hydrogel could increase the rate ofgelation and that the higher the concentration of HPMC inhydrogel, the shorter is the gelation time. The results alsoshowed that the composition of the hydrogel did not affect thereversibility of the system, as evidenced by the low SD for eachsample (Table 1).The addition of PEG to Ch/GP maintains a homogeneous

solution state at neutral pH and room temperature becausehydrogen bonding between PEG and water moleculesdominates. Upon heating toward the gelation temperature(37 °C), the mobility of polymer chains increases. PEG followsan inherent tendency toward dehydration, the hydrogen bondsweaken, and strong interactions between water and both Chand PEG are lost.55,56 Hydrophobic interactions among Ch

Figure 2. FTIR spectra of (a) Ch, (b) GP, (c) Ch/GP aqueous mixture (Ch/GPaq), (d) BG, (e) BG/GP dry mixture (BG/GPdry), and (f) BG/GPaqueous mixture (BG/GPaq). Square brackets indicate regions with characteristic peaks and their repetition in the mixtures. Dotted boxes indicatethe regions of water absorption interference.



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chains prevail above the gelation temperature, creating physicaljunction zones of polymer chain segments.56 A further decreasein sol-to-gel time in the presence of an increasing amount ofHPMC may be a result of the ability of the polymer to act as aviscosity-enhancing and gel-promoting agent.57

3.3. Determination of Swelling Ratio. The swelling ratio(q) is defined as the fractional increase in the weight of thehydrogel resulting from water absorption. Swelling is the resultof an interaction between the hydrogel matrix and aqueousenvironment and can be used as a tool to measure the averagefree volume within the matrix as well as the cross-linkingdensity.58 Hydrogel swelling can be divided into three phases.In the initial phase, water molecules interact with polar andhydrophilic groups, and hydrogels start swelling. This increasein volume leads to the second phase, where the hydrophobicnetworks are exposed and bonds with a hydrophobic natureare formed. In the last phase, the gel starts to absorb extrawater because of osmotic pressure. The elasticity of the matrixis attributed to retraction forces, which oppose the extraswelling, and the hydrogel will attain its optimum swellingpoint.59

The addition of PEG to Ch/GP (F2−F5) has significantlydecreased the swelling ratio of the hydrogel (Table 1) byapproximately 48%, while the addition of HPMC apparentlyhad no significant effect. This decrease in water content ispartially a result of the increase in the amount of solid contentdue to polymer addition, which impedes water penetration.The reduced swelling may also be an indication of a decreasein flexibility of the polymer chains and formation of a morerigid hydrogel network especially in the presence of PEG,which is known for its tendency toward dehydration, leading toan increase in hydrophobic interactions and physical cross-linking of the Ch chains in the hydrogel matrix. It should benoted that a reduction in swelling due to the addition of PEGcan be manipulated to maintain the integrity of the hydrogelfor longer periods and control gel erosion following differenttopical or internal applications.3.4. Determination of Rheological Properties. Rheo-

logical studies demonstrated that the five hydrogel formula-tions (F1 to F5) exhibited non-Newtonian behavior charac-terized by shear thinning as shown by a drop in viscosity atincreasing rate of shear (Figures 3). F1 and F2 gel formulationsdisplayed mainly pseudoplastic flow typical of polymericdispersions, with their curves beginning very close to theorigin at low rates of shear. F3 and F4 hydrogel formulationsexhibited plastic flow with yield values amounting to 6.5 and35 N/m2, respectively (Figure 3a). A plastic system does notbegin to flow until a shear stress corresponding to the yieldvalue is exceeded, which could be a result of an increasedpercentage of polymers added to F3 and F4 compared to F1and F2. The F5 hydrogel, a more substantially structuredsystem containing the highest percentage of PEG and HPMCamong the five formulations, showed a highly bulged curvewith a spur-like protrusion (Figure 3b). The high spur value(47.5 N/m2) that traces out a bowed upward curve could befrom the breakdown of the three-dimensional structure of thegel in the viscometer. Table 1 shows the coefficients ofviscosity of the five hydrogel formulations determined at 10rpm and a torque of 10. These results are consistent withresults obtained from gelation time and swelling studies, inwhich more viscous gels showed shorter gelation times and lessswelling compared to F1. The rheological behavior and theviscosity of hydrogels are of great importance, since they can

affect viscoelasticity, spreadability, injectability, bioadhesion,tolerability, and in vitro drug release. The results suggest thatthe rheological properties of the different gel formulations canbe tailored for a wide range of applications. For instance, an F5gel demonstrated that it can be used in intramuscular depotinjection for slow drug release with prolonged action, while F3and F4 gels are expected to adhere properly to biologicalmembranes because of their yield value. The flow behavior ofless viscous gels such as F1 and F2 on the other hand could bemore appropriate to apply to sensitive areas such as inintranasal delivery or injectable scaffolds.

3.5. Microstructure Analysis. As a drug delivery platform,the microstructure of a hydrogel is an important parameter,because the pore size, shape, and free volume directly affect thedrug entrapment and release.60 SEM micrographs of thesurface and cross section views of thermosensitive hydrogelsF1, F4, and F5 are shown in Figure 4. The F1 hydrogel (Ch/GP) micrograph showed a diversified, highly porous micro-structure with surface irregularities and a high degree ofinterconnectivity. The micrograph of the F4 hydrogel showedthat the addition of 20% PEG to F1 resulted in a comparativelysmoother surface, but the gel was less porous with occasionallylarger pores compared to F1. The micrograph of they F5hydrogel containing 20% PEG and 2% HPMC showed a highlycompact structure with fibrous surface where no pores orcracks were detected; however, the cross section view showedan inner structure characterized by uniform small porescompared to F1 and F4, which is in accordance with gelationtime, swelling, and rheology results. SEM results indicate thatthe addition of PEG and HPMC to the basic gel may greatlyaffect the inner structure of the gel with subsequent impact ongelation time, swellability, rheological properties, and drugrelease.

Figure 3. Rheograms of (a) F1 → F4 hydrogel formulations at 37 °Cshowing pseudoplastic (F1 and F2) or plastic flow with a yield value(F3 and F4). (b) Rheogram of F5 hydrogel formulation at 37 °Cshowing a spur value.



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3.6. In Vitro Drug Release. Drug release is essential forthe efficacy of the treatment and for the optimum patientcompliance. The effect of GP on the solubility of BG wasstudied by mixing the drug with an aqueous solution of GP(50% w/v) and comparing the drug release with the BGdispersion in water and F1 gel formulation (Figure 5a). Whendistilled water was used as release medium, only 43% of thedrug was released from its aqueous dispersion after 12 h. Incontrast, the drug release was significantly enhanced in thepresence of GP or when the drug was incorporated in Ch/GPhydrogels F1 (p ≤ 0.0001), although the latter showed that thepresence of Ch played a role in slowing down the release overthe first 4 h.When the in vitro release studies were carried out in PB (pH

6.8), the release of the drug from its aqueous dispersionsignificantly increased and was similar to that of BG in GPaqueous solution (p = 0.085), where all of the drug wasreleased after just 4 h (Figure 5b). These results confirm thoseobtained from the FT-IR analysis, suggesting the presence ofinteraction between GP and BG, where, apparently, thephosphate groups in GP (or PB) play an essential role inincreasing the drug water solubility and hence its release/dissolution. Figure 5b also shows that the drug release from F1was, yet again, delayed owing to the Ch matrix. However, itwas noticed that the BG release from F1 in PB exhibited adelay in rate compared to its release in distilled water. Forinstance, the average percentage drug release from F1 after 2 hin distilled water was about 78% compared to only 60% in PB.This could be explained by improved gelation of Ch in thepresence of PB, thus decreasing the gel porosity and increasingits mechanical resistance against degradation.61 Likewise, thecomplex of BG-GP is assumed to gain more stability in thepresence of PB, and thus, the resulting viscous solution wouldresist dilution in release buffer, resulting in slower drug release.The effect of hydrogel composition on the in vitro release of

BG from the prepared thermosensitive hydrogels is shown inFigure 6. All hydrogel formulations controlled the release ofBG for at least 8 h before reaching a plateau. The

Figure 4. SEM micrographs of thermosensitive hydrogels (a) in surface views and (b) in cross section views.

Figure 5. In vitro release of BG in (a) distilled water and (b) PB (pH6.8). Data points are mean ± SD (n = 3).



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incorporation of 10% (F2) and 20% (F4) PEG 6000 to F1 didnot significantly affect the rate of drug release relative to F1 (p= 0.817 and p = 0.392, respectively). However, the addition ofHPMC,considerably retarded the release of BG (Figure 6b),where the time required to release 50% of the drug from F3and F5 increased significantly by 46 and 60%, respectively (p ≤0.0001 for both F3 and F5), when compared to F1 containingCh and GP only (Figure 7).Drug release from hydrogels depends on the dissolution of

the drug, its diffusion out of the hydrogel, and the matrixswelling and erosion or degradation. The mechanism of drugrelease from thermosensitive hydrogels F1 → F5 followed adiffusion-controlled pattern (Table 2), which correlates withprevious studies using other drug molecules showing a similarmechanism of drug release from Ch/GP thermosensitivehydrogels.30,62 The slower rate of drug release in the presenceof HPMC, especially when 2% of the polymer wasincorporated (F5) is probably because of decreased matrixporosity and increased viscosity as confirmed by SEM, gelationtime, swelling and rheological studies. PEG, on the other hand,is known as a good solubilizing agent for class II drugs, so inabsence of HPMC, F2 and F4 may have facilitated thesolubilization of BG and enhanced its diffusion out of the

polymer despite of the decrease in swelling and porosity of thematrix when compared to F1.

3.7. In Vivo Study. To assess the impact of loading BGinto thermosensitive gels on the PK and in vivo bioavailabilityof BG, rabbits were administered a dose of 10 mg/kg of F1hydrogel through buccal application, and the results werecompared to an equal dose of BG oral suspension. Remarkabledifferences in the rate and extent of drug absorption from thetwo treatments were observed. The mean AUC0−24 estimatefrom F1 hydrogel group was 18.5 ± 2.9 ng·h/mL whichrepresented 330% of the mean AUC0−24 estimate from the oralsuspension group (5.6 ± 1.8 ng.h/mL). The statisticallysignificantly higher bioavailability of BG from the F1 hydrogel(p ≤ 0.0001) could be a result of the elimination of BG initialhepatic degradation through the buccal route. The increase inbioavailability could be also a result of improved solubilizationof the drug in the hydrogel formulation. The mean Cmaxestimate from the oral suspension group (2.98 ng/mL) washigher relative to the mean Cmax estimate from F1 hydrogelgroup (2.6 ng/mL); however, the difference was notstatistically different (p = 0.541). This further confirmed theefficient absorption of BG from the buccal route. The meanTmax estimate of BG from the suspension group (0.08 ± 0.14h) was statistically significantly shorter (p ≤ 0.001) comparedto that of the F1 hydrogel group (1.0 ± 0.46 h), indicating veryrapid absorption of BG from the suspension. The delayed Tmaxof BG from F1 formulation on the other hand is consistentwith the slow release of BG from the hydrogel owing to thepresence of Ch. There was no significant difference betweenthe mean t1/2 estimates of the two groups (p = 0.7), which isconsistent with the pharmacokinetic theory, in which anincrease in absorption should not alter elimination.63 Themean MTT estimate calculated from the F1 hydrogel group(8.2 ± 0.5 h) was higher relative to the suspension group (7.5± 0.7 h), which could be a result of a higher mean absorptiontime (MAT) taken by the drug molecules to be absorbed into

Figure 6. In vitro release of BG from thermosensitive hydrogels.Effects of adding (a) PEG (F2 and F4) and (b) HPMC (F3 and F5)on the release of BG from Ch/GP (F1) hydrogels. Release wasperformed in PB (pH 6.8). Data points are mean ± SD (n = 3).

Figure 7. Time required for 50% drug release (T50%) fromthermosensitive gels F1−F5. Data points are mean ± SD (n = 3).

Table 2. Kinetics of in Vitro Release of BG fromThermosensitive Hydrogels

formulation zero first second diffusion

F1 0.7779 0.642 0.447 0.918F2 0.747 0.589 0.387 0.895F3 0.832 0.665 0.418 0.947F4 0.795 0.613 0.363 0.929F5 0.892 0.676 0.364 0.981



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the systemic circulation from the hydrogel compared to thesuspension. However, this difference in MTT was notstatistically different (p = 0.09), which indicates that the F1hydrogel was able to increase the bioavailability of BG withoutprolonging the blood circulation time of the drug.The mean PK parameters, Cmax, AUC0−t, Tmax, t1/2, and

MTT, of the two treatment groups are reported in Table 3.

The qualitative visual examination of the plasma profilesindicates that most of the rabbits retained the reported doublepeaks for BG (Figure 8). Flavonoids are known to exhibit a

double peak in their plasma-time profile owing to theenterohepatic cycle.64 The double peaks, however, were notobserved in two of the rabbits. Lu T. et al. explained thebimodal profile of BG by the presence of double-siteabsorption, namely, the duodenum and colon.65 Therefore, itmight be assumed that some of the gel has been ingested bymost of the rabbits upon application.66 This emphasizes theimpact of BG solubilization on enhancing the intestinalabsorption from the gut even upon unintentional ingestionof the gel.

4. CONCLUSIONIn this study, BG was loaded into the Ch/GP thermosensitivehydrogels. The initial mixing of BG with GP resulted incomplete solubilization of the drug probably because of ionicinteraction. The incorporation of PEG and HPMC into thehydrogels resulted in manipulation of the sol-to-gel transition,gelation time, rheology, and the in vitro release of the drug.Drug-loaded Ch/GP thermosensitive hydrogels were alsoeffectively absorbed into the bloodstream upon buccal

application with significantly higher bioavailability comparedto an oral BG suspension. To our knowledge, this is the firststudy on the feasibility of buccal administration of BG. Inaddition, the proposed formulation has been shown to be aneasily modifiable delivery platform that can be suited forvarious local and injectable therapeutic applications throughdifferent routes of administration.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.molpharma-ceut.8b00480.

(1) The composition and results for hydrogelscontaining PEG 4000; (2) A figure showing the unstablevs stable formulation to support the finding; and (3)Statistical analyses and p-values for Figures 5−8 (PDF)

■ AUTHOR INFORMATIONCorresponding Author*Office: +97165057414; Fax: +97165585812; E-mail:[email protected] (M.H.)ORCIDMohamed Haider: 0000-0003-3150-9275Iman S. Ahmed: 0000-0002-2676-9446Author Contributions§M.H. and M.A.H. contributed equally to the work.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was funded in part by Boehringer Ingelheim(FOAPAL:4004-1201/120102) and by the University ofSharjah (V.C./G.R.C./S.R. 83/2015 To MAH). The releasestudies have been carried out with the help of the researchstudents Sahar Abdelmoniem, Iman Hakmi, and Hazem Issaand the teaching assistant Ahd Bakri Al Nosh.

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Table 3. Mean PK Parameters of BG following OralAdministration of 10 mg/kg of BG in Suspension orHydrogel Formulation (F1) to Rabbits

parameter BG suspension F1 p-value

Cmax (ng·mL−1) 2.98 ± 1.12 2.6 ± 0.75 0.541AUC0−t (ng·h·ml−1) 5.60 ± 1.83 18.5 ± 2.9

b 0.0001Tmax (h) 0.08 ± 0.14 1 ± 0.46

b 0.0001t1/2 (h) 8.1 ± 2.0 7.6 ± 2.8 0.700MTT (h) 7.5 ± 0.7 8.2 ± 0.5 0.090f rel - 330.3%

aData are mean values (n = 6 for each treatment) ± SD. bP < 0.05versus BG suspension. cf rel = AUCF1/AUCsuspension

Figure 8. Mean (±SD) plasma BG concentrations following oraladministration of 10 mg/kg of BG suspension or F1 hydrogel inrabbits (n = 6).



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http://pubs.acs.orghttp://pubs.acs.org/doi/abs/10.1021/acs.molpharmaceut.8b00480http://pubs.acs.org/doi/abs/10.1021/acs.molpharmaceut.8b00480http://pubs.acs.org/doi/suppl/10.1021/acs.molpharmaceut.8b00480/suppl_file/mp8b00480_si_001.pdfmailto:[email protected]://orcid.org/0000-0003-3150-9275http://orcid.org/0000-0002-2676-9446http://dx.doi.org/10.1021/acs.molpharmaceut.8b00480

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Thermogelling Platform for Baicalin Delivery for Versatile ... swelling ratio, and rheological properties in addition to surface and internal microstructure. Polyethylene glycol (PEG)