Thermogelling Platform for Baicalin Delivery for Versatile Biomedical

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Thermogelling Platform for Baicalin Delivery for Versatile Biomedical Applications Mohamed Haider, Mariame Ali Hassan, Iman S. Ahmed, and Rehab Shamma Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00480 • Publication Date (Web): 28 Jun 2018 Downloaded from http://pubs.acs.org on June 30, 2018

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Molecular Pharmaceutics

Thermogelling Platform for Baicalin Delivery for Versatile Biomedical

1

Applications

2 3

Mohamed Haidera,b,1*, Mariame A. Hassana,b,1, Iman S. Ahmeda, Rehab Shammab

4

a

5

Department of Pharmaceutics & Pharmaceutical Technology, College of Pharmacy, University of Sharjah, Sharjah

27272, United Arab Emirates

6

b

Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo11562, Egypt

7

These authors contributed equally to the work.

8

1

9 *Corresponding author:

10

Mohamed Haider PhD, MBA

11

Department of Pharmaceutics and Pharmaceutical Technology

12

Room M23-137, College of Pharmacy

13

University of Sharjah

14

Sharjah, UAE 27272

15

Office: +97165057414

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Fax: +97165585812

17

e-mail: [email protected]

18

ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Declaration of interest

1

The authors declare no conflict of interest.

2


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Abstract

1

Baicalin (BG) is a natural glycoside with several promising therapeutic and preventive

2

applications. However, its pharmaceutical potential is compromised by its poor water-solubility,

3

complex oral absorption kinetics and low bioavailability. In this work BG was incorporated in a

4

series of chitosan (Ch)/ glycerophosphate (GP)-based thermosensitive hydrogel formulations to

5

improve its water-solubility and control its release profile. Molecular interactions between BG

6

and GP were investigated using Fourier transform-infrared spectroscopy (FT-IR) and the ability

7

of GP to enhance the water-solubility of BG was studied in different release media. Drug-loaded

8

Ch/GP hydrogels were prepared and characterized for their gelation time, swelling ratio,

9

rheological properties in addition to surface and internal microstructure. Polyethylene glycol

10

(PEG) 6000 and hydroxypropyl methyl cellulose (HPMC) were incorporated in the formulations

11

at different ratios to study their effect on modulating the sol–gel behavior and the in vitro drug

12

release. In vivo pharmacokinetic (PK) studies were carried out using a rabbit model to study the

13

ability of drug-loaded Ch/GP thermosensitive hydrogels to control the absorption rate and

14

improve the bioavailability of BG. Results showed that the solubility of BG was enhanced in

15

presence of GP while the incorporation of PEG and/or HPMC had an impact on gelation time,

16

rheological behavior and rate of drug release in vitro. PK results obtained following buccal

17

application of drug-loaded Ch/GP thermosensitive hydrogels to rabbits showed that the rate of

18

BG absorption was controlled and the in vivo bioavailability was increased by 330% relative to

19

BG aqueous oral suspension. The proposed Ch/GP thermosensitive hydrogel is an easily

20

modifiable delivery platform that is not only capable of improving the solubility and

21

bioavailability of BG following buccal administration but also can be suited for various local and

22

injectable therapeutic applications.

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Keywords

1

Baicalin, thermosensitive hydrogels, chitosan, controlled release, bioavailability

2


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1. Introduction

1

Baicalin (BG; Baicalein-7-glucuronide; Figure 1a) is a glycoside extracted from the dried roots

2

of Scutellariae baicalensis Georgi, commonly known as Baikal skullcap. BG possesses a wide

3

range of therapeutic and preventive potential which has placed it recently in the center of focus

4

of interest as safe natural therapeutic ingredient. A PubMed search with the key word [Baicalin] -

5

restricted in the title - hits back ca. 700 articles from 2001 to date with 50% of them published in

6

the last five years (Figure 1b). As herbal medicine, the plant extract is officially listed in the

7

Chinese Pharmacopeia 1,2. Pharmacologically, BG is effective as antibacterial 3, antifungal 4,

8

antiviral 5, antiallergic, anti-inflammatory 6, antipyretic 7, antihypertensive, diuretic and

9

antithrombotic agent 8,9. In addition, BG has a sedative effect 10,11 and possesses antioxidant

10

activity and thus hepatoprotective 12. Recently, it has also been reported to exert anticancer effect

11

and neuroprotective effects 13,14.

12

Despite the medical benefits of BG, its therapeutic/clinical applications are still greatly limited

13

due to its poor water-solubility where BG is classified as class II drug in the Biopharmaceutical

14

Classification System (BCS) with an oral bioavailability as low as 2.2% of the administered dose

15

15

. Moreover, BG has very complex absorption kinetics due complex metabolic pathway.

16

Intestinal enzymes and microbiota and extensive liver metabolism break the drug into its

17

aglycone form Baicalein (B); in turn, B is then glycosylated back rapidly to BG through

18

glucuronidation and sulfation in intestinal epithelia as well as in liver 16,17. Although the

19

metabolites are claimed to be relatively bioactive to varying extents, the glycosylated forms are

20

rapidly excreted accounting for the low bioavailability in part (Figure 1c). In addition, the natural

21

glycoside form has poor aqueous solubility that allows these complex metabolic pathways to

22

occur at full paces. Attempts to increase the solubility of BG and B majorly focused on particle

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size reduction techniques especially with B owing to its higher lipophilicity, smaller molecular

1

weight and ability to pass the intestinal epithelium passively. B was thus formulated into oral

2

chewable tablets 18,19, solid dispersions 20, microemulsions 21, nanoemulsions 22, self-assembled

3

nanoparticles 23, and nanocrystals 24. On the other hand, only few studies have succeeded to

4

increase the water-solubility of BG through inclusion in solid dispersions 25, liposomes 26 and

5

nanoliposomes 27. In this study, we present a novel delivery platform for BG that does not

6

depend on size reduction, but rather on solubilization of BG in the delivery system.

7

Chitosan (Ch) is a natural biodegradable linear amino-polysaccharide obtained by alkaline

8

deacetylation of crustacean shells and has been widely used in drug and protein delivery as well

9

as tissue engineering 28,29. Glycerophosphate (GP) is an organic compound naturally present in

10

the body as a source of phosphate and is administered for the treatment of phosphate metabolism

11

disorders 30. The aqueous mixture of Ch and GP responds non-linearly to temperature elevation

12

forming hydrogels and is classified as stimulus-responsive in situ forming gels 31. The

13

mechanism of gelation of the Ch/GP hydrogel system involves pH- and temperature-dependent

14

interactions. At temperatures lower than 37oC, the addition of GP increases the pH of Ch

15

solutions to around neutrality which would shield the electrostatic repulsion between adjacent Ch

16

chains and can theoretically result in pH-induced gel formation. However, due to electrostatic

17

attraction between the phosphate moieties of GP and protonated amine groups (–NH3+) of Ch,

18

the hydroxyl groups of GP increase stability and hydrophilicity in the Ch chains and thus

19

maintain the solubility of Ch for a period of time. An increase in temperature reduces the polarity

20

of both Ch chains and glycerol moiety of GP, causes Ch chain dehydration and reduces both the

21

Ch chain charge density and the attraction of Ch and GP. This will increase interchain

22


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hydrophobic attraction and hydrogen-bonding between chains and result in formation of

1

hydrogels 32–35.

2

The temperature-dependent sol-gel transition as well as the time of gelation can be adjusted to

3

occur at body temperature and at reasonable time frames through modifying the respective

4

proportions of the ingredients 36. This platform offers numerous advantages in drug delivery such

5

as biosafety and abundance of ingredients, versatility of applications, ability to control drug

6

release with proper inclusion of pharmaceutical additives, single step inclusion of drugs, and

7

ease of preparation and propensity for scaling up on industrial levels 37–39. Therefore, Ch/GP

8

combination is considered as a promising hydrogel platform for a variety of applications, such as

9

local drug delivery systems or injectable carriers for tissue engineering 30.

10

Nevertheless, Ch-based gels are limited by the intermolecular hydrogen bonds on Ch polymeric

11

chains which increase the rigidity of the hydrogel structure and reduce water permeability 40.

12

Several hydrophilic polymers have been added to Ch hydrogels to overcome these

13

limitations41,42. Poly(ethylene glycol) (PEG) is a highly hydrophilic, non-inflammatory, non-

14

immunogenic and biocompatible polymer commonly used as protective coating material for drug

15

delivery nanoparticles 43 and liposomes 44 and to provide similar protection when used as

16

conjugate to peptide and proteins 45. The addition of PEG to hydrogels renders them more

17

hydrophilic and biocompatible and alter their mechanical properties to become more flexible 42.

18

Hydroxy propyl methyl cellulose (HPMC), is another hydrophilic, biodegradable and

19

biocompatible naturally occurring polymer with several desirable properties. One of its most

20

important characteristics is the high swellability, which significantly affects the release kinetics

21

of an incorporated drug 46. The polymer is approved by FDA and is being used extensively in the

22

production of formulations with controlled release system 47. Thermosensitive Ch/HPMC

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hydrogels have been prepared where the addition of HPMC to Ch in presence of glycerol

1

facilitated the thermogelation at 32oC through large amounts of hydrophobic interactions. In

2

addition, it increased the compactness of the Ch hydrogels and improved their mechanical

3

strength 48,49.

4

The aim of this work is to develop and characterize Ch/ GP-based thermosensitive hydrogel

5

formulations that can improve the water-solubility and control the release of incorporated BG.

6

First, the ability of GP to enhance the water-solubility of the drug was investigated. Then, drug-

7

loaded Ch/GP hydrogels were prepared and characterized using different ratios of PEG and

8

HPMC to study their effect on modulating the sol–gel behavior and the in vitro drug release.

9

Finally, a rabbit model was used to study the ability of the drug-loaded Ch/GP thermosensitive

10

hydrogels to control the rate of absorption and improve the bioavailability of BG following

11

buccal administration.

12 13

2. Materials and methods

14

2.1 Materials

15

Chitosan (Ch, medium molecular weight, 70-85% deacetylation), disodium α, β-

16

Glycerophosphate (GP; glycerol-2-phosphate salt hydrate), Baicalin (BG; (2S,3S,4S,5R,6S)-6-

17

(5,6-dihydroxy-4-oxo-2-phenyl-chromen-7-yl) oxy-3,4,5-trihydroxy-tetrahydropyran-2-

18

carboxylic acid), polyethylene glycol (PEG) 6000, hydroxypropyl methyl cellulose (HPMC) and

19

cellulose dialysis bag, molecular weight cut-off (MWCO) of 14 kD were purchased from sigma

20

Aldrich (St Louis, USA). Reagent grade acetic acid was acquired from Merck & Co., Inc.

21

(Darmstadt, Germany).

22 23


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2.2 Fourier transform infrared spectroscopy

1

Powders of BG, GP and Ch, dry mixture of BG/GP (1:1 molar ratio) were placed separately into

2

transparent discs under pressure of 10,000–15,000 pounds per square inch. Similarly, aqueous

3

solutions of BG/GP (1:1 molar ratio) and Ch/GP were prepared and mounted on transparent disc.

4

The infrared spectra of the samples were recorded using Fourier-Transform Infrared (FT-IR)

5

spectrophotometer (JASCO FTIR 6300, Jasco, Easton, MD, USA). The set range for FT-IR was

6

from 4000 to 400 cm-1 at a resolution of 4 cm-1. The stretching modes and vibrational modes

7

depict the chemical bonding and other functional groups present in the material.

8 9

2.3 Preparation of thermosensitive gels

10

Aqueous Ch solution (1.8% w/v) was prepared by dissolving the polymer in 0.1M Acetic acid

11

and stirring overnight at room temperature to obtain a clear solution. The solution was added

12

dropwise into an equal volume of well-stirred 50% w/v GP solution in distilled water (pH = 7).

13

The mixture was further stirred for 5 min at room temperature to ensure homogenous mixing.

14

BG-loaded hydrogel formulation F1 was prepared by dissolving BG in GP solution to obtain a

15

final concentration of 7.5 mg BG/ml of the gel. Drug-loaded hydrogel formulations containing

16

PEG-6000 alone (F2 and F4) or mixtures of PEG-6000 and HPMC (F3 and F5) were prepared by

17

dissolving each polymer in GP solution at different required percentages prior to mixing with Ch

18

solutions. The composition of BG-loaded hydrogel formulations is summarized in Table 1. The

19

prepared hydrogel forming solutions were stored at 4oC for further studies.

20 21



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2.4 Gelation time

1

The time required for sol-to-gel transformation was determined using the tube inverting method

2

50

. Vials containing 1 ml of different formulations at 4oC were immersed in water bath preheated

3

at 37oC. The time taken for the gel to show lack of flowability tested every 10 sec was noted. To

4

determine the reversibility of the system, gelled samples were returned onto ice to check their

5

gel-sol transformation and then the time to gel was again observed. The test results are presented

6

as mean value of three determinations ± SD.

7 8

2.5 Determination of hydrogel swelling ratio

9

Equal volumes of hydrogel-forming solutions were incubated at 37 °C for 2 h. The formed

10

hydrogels were placed in a freezer at -30 oC for 24 h. The frozen gels were then transferred to a

11

lyophilizer (Vir Tis Bench Top Pro, SP Scientific, USA) for 24 h with a condenser temperature

12

of -50°C and a pressure of 7 × 10-2 mbar. The dry weights (Wd) of the freeze-dried gels were first

13

determined and then the gels were submerged in 10 ml PB for 24 h at 37 °C followed by washing

14

with deionized water to remove any ions adsorbed on their surfaces and blotting dry using filter

15

paper. The wet weights (Ws) were recorded and the swelling ratio (q) of the hydrogels was

16

calculated from the equation: =

× 100. The measurements were performed in

triplicates and the swelling ratio (q) ± SD for each gel preparation were recorded.

17 18 19

2.6 Rheological Studies

20

The rheological properties of the prepared thermosensitive hydrogels (F1 to F5) were

21

characterized by generating complete rheograms at 37°C using Cone and Plate Brookfield

22

Rheometer DV3THB equipped with Spindle CPR-40 and Rheocalc software (Brookfield

23


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Engineering Labs Inc., USA). The shear rate (sec-1) was plotted as a function of shearing stress

1

(N/m2) and coefficient of viscosity in centipoise (cp) along with other parameters were

2

calculated for the gels.

3 4

2.7 Hydrogel microstructure analysis

5

The surface morphology and cross-sections of the freeze-dried hydrogels were examined by field

6

emission scanning electron microscope (SEM; VEGA XM variable pressure, Tescan AS, Czech

7

Republic). The samples were freeze-dried for 24 h as described above and cross-sections were

8

prepared after fracturing the gels using liquid nitrogen before being fixed with conductive tape

9

on a metal stub. The samples were sputter-coated with a Gold-Palladium (80-20%) target using

10

Mini Sputter Coater (SC7620, Quorum Technologies, UK) and applied 1kV for 2 min to have a

11

thickness of 20 nm.

12 13

2.8 In vitro drug release

14

The in vitro release of BG from its suspension in water, aqueous GP solution and drug-loaded

15

hydrogels (F1-F5) was studied using the dialysis method 51. Cellulose dialysis bags with MWCO

16

of 14 kD were used to retain the tested samples while allowing the soluble BG to permeate into

17

the release medium. Initially, 1 ml of the drug suspension in water, BG/GP aqueous solution or

18

drug-loaded hydrogel solutions was pipetted into a dialysis bag. Sealed bags were placed in 350

19

ml of release medium stirred at 50 rpm using USP-1 dissolution tester (Agilent 708-DS, Agilent

20

Technologies, NC, USA). To study the solubilizing effect of GP on BG, the release pattern of

21

BG from its suspension in water, BG/GP aqueous solution and F1 was studied in both distilled

22

water and phosphate buffer (PB) (pH 6.8). For investigating the ability of the prepared

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thermosensitive hydrogels (F1 to F5) to control the release of BG, only PB (pH 6.8) was used as

1

release medium. At specific time intervals (0.25, 0.5, 0.45, 1, 2, 4, 6, 8 and 12 h), 5 ml of the

2

release medium were collected and immediately replaced by equal volume of respective fresh

3

medium. The absorbance of BG was determined by UV spectroscopy at 276 nm. A standard

4

curve of BG in PB (pH 6.8) was generated over the range of 0.1 – 50 µg/ml and used to convert

5

absorbance to concentration. A cumulative release profile was generated by normalizing the data

6

against the total amount of BG and reported as percentage drug release. All release experiments

7

were conducted in triplicates.

8

The mechanism of drug release from thermosensitive hydrogels was determined by applying

9

zero order, first order and second order kinetics and Higuchi diffusion model. The following

10

linear regression equation were employed for zero order kinetics = − ; where Co is the

11

zero-time concentration of the drug, Ct is the concentration of the drug at time t and k is the

12

apparent release rate constant. First order kinetics was determined according to the equation

13

= − . For second order kinetics, the following equation was used: = + .

14

Drug release following Higuchi model was determined using the equation = ; where Q

15

represents the fraction of drug released in time t and k is the Higuchi dissolution constant.

16 17

2.9 In vivo pharmacokinetic study

18

2.9.1 Study design

19

The in vivo studies were carried out to compare the bioavailability and pharmacokinetic (PK)

20

parameters of BG from two different treatments in white New Zealand male rabbits (2.5-3 kg)

21

using a non-blind, two-treatment, randomized, parallel design. Six rabbits were randomly

22

assigned to each treatment group (n = 6). Food was withdrawn 10 h prior to the study with water

23


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ad libitum. Early morning the assigned treatments were administered. No food was allowed after

1

dosing for 12 h. In group A; rabbits received BG suspension through an intragastric tube. In

2

group B; rabbits received BG basic gel formulation (F1) with the help of gel applicator. The gel

3

was placed onto the buccal region of the rabbit, pressed for approximately 10 sec, and then the

4

applicator was removed. The BG dose was adjusted to be 10 mg/kg 52. Blood samples (2 ml)

5

were withdrawn from the middle ear vein using a 26-gauge needle and syringe at 0 (predose),

6

0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12 and 24 h after administration. Samples were collected into

7

heparinized tubes and plasma was obtained by centrifugation at 4000 rpm for 15 min. The

8

plasma was pipetted into glass tubes and frozen at −20°C until analysis. All animal experiments

9

were performed according to ethical principles and approved by the Research Ethics Committee

10

(REC) for Animal Subject Research at the Faculty of Pharmacy, Cairo University, Egypt,

11

operating according to the CIOMS and ICLAS international guiding principles for biomedical

12

research involving animals 2012. Also, all animal experiments comply with Directive

13

2010/63/EU.

14 15

2.9.2 Chromatographic conditions

16

Plasma concentrations of BG were determined using a selective, sensitive and accurate LC-

17

MS/MS method that was developed and validated before use. All chemicals and reagents used

18

were of analytical grade and solvents were of HPLC grade. Toresemide stock solution was

19

prepared and used as internal standard (IS) by dissolving 10 mg in methanol and serially diluted

20

with mobile phase to give a final working concentration of 1µg/mL. A Shimadzu (Shimadzu,

21

Japan) series LC system equipped with a pump (LC-20AD) along with an auto-sampler (SIL-

22

20A/HT) was used to inject 20 µL aliquots of the processed samples on a reverse-phase micro-

23



Page 14 of 47

particulate C18 Sunfire column, particle size 5 µm, (50 x 4.6) mm (Waters Corp., Milford,

1

MA). All analyses were carried out at room temperature. The isocratic mobile phase was

2

prepared by mixing 80% acetonitrile and 20% (0.1% formic acid in water) which was delivered

3

at a flow rate of 1 ml/min into the mass spectrometer’s electrospray ionization chamber.

4

Quantitation was achieved by MS/MS detection in positive ion mode for both BG and IS, using

5

an API-4000 Triple Quadrupole LC-MS/MS Mass Spectrometer (AB SCIEX Instruments)

6

equipped with a Turbo ionspray interface at 400 °C. The ion spray voltage was 5000 V. The

7

compound parameters: declustering potential (DP), collision energy (CE), entrance potential

8

(EP) and collision exit potential (CXP) were 106, 25, 10 and 6 V for BG and 30, 25, 10, and 10

9

V for IS, respectively. Detection of ions was performed in the multiple reaction monitoring

10

(MRM) mode, monitoring the transition of the m/ɀ 447.2 precursor ion to the m/ɀ 271.1 for BG

11

and m/ɀ 348.9 precursor ion to the m/ɀ 263.9 for IS. Quadrupoles Q1 and Q3 were set on unit

12

resolution. The analytical data were processed by Analyst software version 1.6 (Applied

13

Biosystems Inc., Foster City, CA). Plasma samples were spiked with BG acetonitrile solution to

14

contain 0.1- 10 ng/mL. Aliquots of 0.5 mL of plasma samples were then spiked with 10

15

µL toresemide and mixed with 1 mL acetonitrile in a 10-mL glass centrifuge tube. The tubes

16

were shaken by vortex-mixing for 30 sec. After centrifugation for 10 min at 6000 rpm, the

17

supernatant was transferred to a small glass tube and 20 µL of the resulting supernatant were

18

injected using the auto-sampler. All frozen plasma samples obtained from the rabbits after

19

receiving treatment A and B were thawed at ambient temperature and assayed as described above

20

without the addition of BG.

21 22 23

2.9.3 Pharmacokinetic analysis


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Data from plasma analysis were analyzed for each rabbit using WinNonlin (version 1.5,

1

Scientific consulting, Inc., Cary, NC). Non-compartmental analysis was pursued and the

2

pharmacokinetic variables; Cmax (maximal drug concentration; ng/ml), Tmax (time for maximal

3

drug concentration; h) and AUC(0-t) (area under the curve; ng.h/ml) were generated. The area

4

under the curve from zero to infinity AUC (0-∞) was calculated from the equation:

5

("∞) = (" ) + ; where Ct is the last measured concentration at 24 h, and k is the $

6

elimination rate constant estimated from the slope of the terminal log-linear phase. The

7

elimination half-life (t1/2) was calculated as t1/2 = Ln2/k. Mean Transit Time (MTT) was

8

calculated from AUMC/AUC where AUMC is the area under the first moment curve. The

9

relative bioavailability (frel) was calculated for BG F1 hydrogel formulation relative to BG

10

aqueous suspension as AUCF1/AUCsusp. All results were expressed as mean ± SD.

11 12

2.10

13

Statistical analysis

All in vitro data points are the average of three independent experiments performed expressed as

14

means ± SD. Statistical significance between results was assessed by Student t-test (two-tailed;

15

p< 0.05). For in vivo studies, statistical inferences were based on untransformed values for Cmax

16

and AUC variables and observed values for t1/2. The nonparametric Signed Rank Test (Mann-

17

Whitney’s test) was used to compare Tmax between the two treatment groups. The one-way

18

analysis of variance (ANOVA) F-test was used for testing the equality of several means. For

19

multiple comparison the procedure used was the Least Significant Difference (LSD).

20 21 22



Page 16 of 47

3. Results and discussion

1

3.1 FT-IR spectrophotometric analysis

2

BG showed very poor solubility in water and acidic Ch solution at the dose used. In contrast, the

3

inclusion of BG in aqueous GP solution resulted in yellowish clear relatively thick liquid that

4

remained clear after the addition of Ch. The FTIR spectrophotometric analysis (Figure 2) shows

5

the characteristic absorption bands of BG, Ch, GP and their mixtures. BG is characterized by

6

peaks at 1726 cm-1 (for -COOH), 1660 cm-1 (for C=O), 1065 cm-1 (C-O in ether and hydroxyl

7

groups), and 1608, 1573 and 1498 cm-1 (for aromatic C=C). On the other hand, GP has

8

characteristic peak at 900 cm-1 (aliphatic phosphate) and 3650–3250 cm-1 (C-OH). Most of these

9

characteristic peaks were retained at weak intensities in both the physical mixture of dry powders

10

of BG and GP and in their aqueous solution. The broad peak of BG in the range of 3650–3250

11

cm-1 (for -OH) appeared very weak in its mixtures with GP. This suggests intermolecular

12

interaction (e.g. strong intermolecular hydrogen bonding) between molecules 53. This

13

intermolecular H-bond between BG and GP might account in part for the increased solubility of

14

the drug. Worth noting here that the intense peaks in the aqueous mixture at 3200 and 1640 cm-1

15

pertain to water absorption 54. Similarly, the spectrum of GP and Ch aqueous mixture showed

16

similar retention of characteristic peaks of each component at lower intensities indicative of

17

interaction (intermolecular hydrogen bonding) with more intense absorption for water at 1640

18

cm-1.

19 20

3.2 Determination of gelation time

21

Gelation time of the prepared formulations was measured at physiologic temperature (37 °C). As

22

shown in Table 1, the addition of 10% and 20% PEG, in F2 and F4 respectively, slightly reduced

23


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the gelation time when compared to F1 hydrogel. A significant decrease in gelation time was

1

observed when HPMC was incorporated in F3 and F5, where the gelation time was reduced by

2

34% and 57%, respectively, relative to F1 hydrogel (P< 0.05). These results demonstrated that

3

the addition of HPMC into Ch/GP hydrogel could increase the rate of gelation and that the higher

4

the concentration of HPMC in hydrogel, the shorter is the gelation time. The results also showed

5

that the composition of the hydrogel did not affect the reversibility of the system, as evidenced

6

by the low SD for each sample (Table 1).

7

The addition of PEG to Ch/GP maintains a homogenous solution state at neutral pH and room

8

temperature because hydrogen bonding between PEG and water molecules dominates. Upon

9

heating towards the gelation temperature (37oC), the mobility of polymer chains increases. PEG

10

follows an inherent tendency towards dehydration, the hydrogen bonds weaken and strong

11

interactions between water and both Ch and PEG are lost 55,56. Hydrophobic interactions among

12

Ch chains prevail above the gelation temperature, creating physical junction zones of polymer

13

chain segments 56. Further decrease in sol-to-gel time in presence of increasing amount of HPMC

14

may be due to the ability of the polymer to act as viscosity-enhancing and gel-promoting agent57.

15 16

3.3 Determination of swelling ratio

17

The swelling ratio (q) is defined as the fractional increase in the weight of the hydrogel due to

18

water absorption. Swelling is the result of interaction between the hydrogel matrix and aqueous

19

environment and can be used as a tool to measure the average free volume within the matrix as

20

well as the crosslinking density 58. Hydrogel swelling can be divided into 3 phases. In the initial

21

phase, water molecules interact with polar and hydrophilic groups and hydrogels start swelling.

22

This increase in volume leads to the second phase where the hydrophobic networks are exposed

23



Page 18 of 47

and bonds having hydrophobic nature are formed. In the last phase, the gel starts to absorb extra

1

water because of osmotic pressure. The elasticity of the matrix is attributed to retraction forces

2

which opposes the extra swelling and the hydrogel will attain its optimum swelling point 59.

3

The addition of PEG to Ch/GP (F2-F5) has significantly decreased the swelling ratio of the

4

hydrogel (Table 1) by approximately 48% while the addition of HPMC apparently had no

5

significant effect. This decrease in water content is partially due to the increase in the amount of

6

solid content due to polymer addition which impedes water penetration. The reduced swelling

7

may also be an indication to decrease in flexibility of the polymer chains and formation of more

8

rigid hydrogel network especially in the presence of PEG known for its tendency towards

9

dehydration leading to an increase in hydrophobic interactions and physical crosslinking of the

10

Ch chains in the hydrogel matrix. It should be noted that a reduction in swelling due to addition

11

of PEG can be manipulated to maintain the integrity of the hydrogel for longer periods and

12

control gel erosion following different topical or internal applications.

13 14

3.4 Determination of rheological properties

15

Rheological studies demonstrated that the five hydrogel formulations (F1to F5) exhibited Non-

16

Newtonian behavior characterized by shear thinning as shown by a drop in viscosity at

17

increasing rate of shear (Figures 3). F1 and F2 gel formulations displayed mainly pseudoplastic

18

flow typical of polymeric dispersions with their curves beginning very close to the origin at low

19

rates of shear. F3 and F4 hydrogel formulations exhibited plastic flow with yield values

20

amounting to 6.5 and 35 N/m2, respectively (Figure 3a). A plastic system does not begin to flow

21

until a shear stress corresponding to the yield value is exceeded which could be due to increased

22

percentage of polymers added to F3 and F4 compared to F1 and F2. F5 hydrogel, a more

23


Page 19 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


substantially structured system containing the highest percentage of PEG and HPMC among the

1

five formulations, showed a highly bulged curve with a spur like protrusion (Figure 3b). The

2

high spur value (47.5 N/m2) that traces out a bowed upcurve could be due to the breakdown of

3

the three-dimensional structure of the gel in the viscometer. Table 1 shows the coefficients of

4

viscosity of the five hydrogel formulations determined at rpm 10 and torque 10. These results

5

are consistent with results obtained from gelation time and swelling studies in which more

6

viscous gels showed shorter gelation times and less swelling compared to F1. The rheological

7

behavior and the viscosity of hydrogels are of great importance since they can affect

8

viscoelasticity, spreadability, injectability, bioadhesion, tolerability and in vitro drug release. The

9

results suggest that the rheological properties of the different gel formulations can be tailored for

10

a wide range of applications. For instance, F5 gel demonstrate that it can be used in

11

intramuscular depot injection for slow drug release with prolonged action while F3 and F4 gels

12

are expected to adhere properly to biological membranes because of their yield value. The flow

13

behavior of less viscous gels such as F1 and F2 on the other hand could be more appropriate to

14

apply to sensitive areas such as in intranasal delivery or injectable scaffolds.

15 16

3.5 Microstructure analysis

17

As a drug delivery platform, the microstructure of a hydrogel is an important parameter because

18

the pore size, shape and free volume directly affect the drug entrapment and release 60. SEM

19

micrographs of surface and cross-section views of thermosensitive hydrogels F1, F4 and F5 are

20

shown in Figure 4. F1 hydrogel (Ch/GP) micrograph showed a diversified, highly porous micro-

21

structure with surface irregularities and a high degree of interconnectivity. The micrograph of F4

22

hydrogel showed that the addition of 20% PEG to F1 resulted in a comparatively smoother

23



Page 20 of 47

surface but the gel was less porous with occasional larger pores compared to F1. Micrograph of

1

F5 hydrogel containing 20% PEG and 2% HPMC showed a highly compact structure with

2

fibrous surface where no pores or cracks were detected however, the cross-section view showed

3

an inner structure characterized by uniform small pores compared to F1 and F4 which is in

4

accordance with gelation time, swelling and rheology results. SEM results indicate that the

5

addition of PEG and HPMC to the basic gel may greatly affect the inner structure of the gel with

6

subsequent impact on gelation time, swellability, rheological properties and drug release.

7 8

3.6 In vitro drug release

9

Drug release is essential for the efficacy of the treatment and for the optimum patient

10

compliance. The effect of GP on solubility of BG was studied by mixing the drug with an

11

aqueous solution of GP (50% w/v) and comparing the drug release with BG dispersion in water

12

and F1 gel formulation (Figure 5a). When distilled water was used as release medium only 43%

13

of the drug was released from its aqueous dispersion after 12 h. In contrast the drug release was

14

significantly enhanced in presence of GP or when the drug was incorporated in Ch/GP hydrogels

15

F1 (p≤ 0.0001), although the latter showed that the presence of Ch played a role in slowing down

16

the release over the first 4 h.

17

When the in vitro release studies were carried out in PB (pH 6.8), the release of the drug from its

18

aqueous dispersion significantly increased and was similar to that of BG in GP aqueous solution

19

(p=0.085), where all the drug was released after just 4 h (Figure 5b). These results confirm those

20

obtained from the FT-IR analysis suggesting the presence of interaction between GP and BG

21

where, apparently, the phosphate groups in GP (or PB) play an essential role in increasing the

22

drug water-solubility and hence its release/dissolution. Figure 5b also shows that the drug release

23


Page 21 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


from F1 was, yet again, delayed owing to Ch matrix. However, it was interestingly noticed that

1

BG release from F1 in PB exhibited a delay in rate compared to its release in distilled water. For

2

instance the average percentage drug release from F1 after 2 h in distilled water was about 78%

3

compared to only 60% in PB. This could be explained by improved gelation of Ch in presence of

4

PB thus decreasing the gel porosity and increasing its mechanical resistance against degradation

5

61

6

. Likewise, the complex of BG-GP is assumed to gain more stability in presence of PB and thus

the resulting viscous solution would resist dilution in release buffer resulting in slower drug

7

release.

8

The effect of hydrogel composition on the in vitro release of BG from the prepared

9

thermosensitive hydrogels is shown in Figure 6. All hydrogel formulations controlled the release

10

of BG for a least 8 h before reaching a plateau. The incorporation of 10% (F2) and 20% (F4)

11

PEG 6000 to F1, did not significantly affect the rate of drug release relative to F1 (p=0.817 and

12

p=0.392 respectively). However, the addition of HPMC, considerably retarded the release of BG

13

(Figure 6b) where the time required to release 50% of the drug from F3 and F5 increased

14

significantly by 46% and 60%, respectively (p≤0.0001 for both F3 and F5), when compared to

15

F1 containing Ch and GP only (Figure 7).

16

Drug release from hydrogels depends on the dissolution of the drug, its diffusion out of the

17

hydrogel and the matrix swelling and erosion or degradation. The mechanism of drug release

18

from thermosensitive hydrogels F1→F5 followed a diffusion-controlled pattern (Table 2) which

19

correlates with previous studies using other drug molecules showing similar mechanism of drug

20

release from Ch/GP thermosensitive hydrogels 30,62. The slower rate of drug release in presence

21

of HPMC especially when 2% of the polymer was incorporated (F5) is probably due to decrease

22

in matrix porosity and increased viscosity as confirmed by SEM gelation time, swelling and

23



Page 22 of 47

rheological studies. PEG on the other hand, is known as a good solubilizing agent for class II

1

drugs so, in absence of HPMC, F2 and F4 may have facilitated the solubilization of BG and

2

enhanced its diffusion out of the polymer despite of the decrease in swelling and porosity of the

3

matrix when compared to F1.

4 5

3.7 In vivo study

6

To assess the impact of loading BG into thermosensitive gels on the PK and in vivo

7

bioavailability of BG, rabbits were administered a dose of 10 mg/kg of F1 hydrogel through

8

buccal application and results were compared to an equal dose of BG oral suspension.

9

Remarkable differences in the rate and extent of drug absorption from the two treatments were

10

observed. The mean AUC0-24 estimate from F1 hydrogel group was 18.5±2.9 ng.h/mL which

11

represented 330% of the mean AUC0-24 estimate from the oral suspension group (5.6±1.8

12

ng.h/mL). The statistically significantly higher bioavailability of BG from F1 hydrogel

13

(p≤0.0001) could be due to elimination of BG initial hepatic degradation through the buccal

14

route. The increase in bioavailability could be also due to improved solubilization of the drug in

15

the hydrogel formulation. The mean Cmax estimate from the oral suspension group (2.98 ng/mL)

16

was higher relative to the mean Cmax estimate from F1 hydrogel group (2.6 ng/mL) however the

17

difference was not statistically different (p=0.541). This further confirm the efficient absorption

18

of BG from the buccal route. The mean Tmax estimate of BG from the suspension group

19

(0.08±0.14 h) was statistically significantly shorter (p≤ 0.001) compared to F1 hydrogel group

20

(1.0±0.46 h) indicating very rapid absorption of BG from the suspension. The delayed Tmax of

21

BG from F1 formulation on the other hand is consistent with the slow release of BG from the

22

hydrogel owing to the presence of Ch. There was no significant difference between the mean t1/2

23


Page 23 of 47 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


estimates of the two groups (p=0.7) which is consistent with the pharmacokinetic theory in

1

which an increase in absorption should not alter elimination 63. The mean MTT estimate

2

calculated from F1 hydrogel group (8.2±0.5 h) was higher relative to the suspension group

3

(7.5±0.7 h) which could be due to higher mean absorption time (MAT) taken by the drug

4

molecules to be absorbed into the systemic circulation from the hydrogel compared to the

5

suspension. However, this difference in MTT was not statistically different (p=0.09) which

6

indicate that F1 hydrogel was able to increase the bioavailability of BG without prolonging the

7

blood circulation time of the drug.

8

The mean PK parameters, Cmax, AUC0-t, Tmax, t1/2 and MTT of the two treatment groups are

9

reported in Table 3. The qualitative visual examination of the plasma profiles indicates that most

10

of the rabbits retained the reported double peaks for BG (Figure 8). Flavonoids are known to

11

exhibit double peak in their plasma-time profile owing to enterohepatic cycle 53. The double

12

peaks, however, were not observed in two of the rabbits. Lu T. et al. explained the bimodal

13

profile of BG by the presence of double-site absorption; namely; duodenum and colon 54.

14

Therefore, it might be assumed that some of the gel has been ingested by most of the rabbits

15

upon application 55. This emphasizes the impact of BG solubilization on enhancing the intestinal

16

absorption from the gut even upon unintentional ingestion of the gel.

17 18

4. Conclusion

19

In this study, BG was loaded into Ch/GP thermosensitive hydrogels. The initial mixing of BG

20

with GP resulted in complete solubilization of the drug probably due to ionic interaction. The

21

incorporation of PEG and HPMC into the hydrogels resulted in manipulation of the sol-to-gel

22

transition, gelation time, rheology and the in vitro release of the drug. Drug-loaded Ch/GP

23



Page 24 of 47

thermosensitive hydrogels were also effectively absorbed into the blood stream upon buccal

1

application with significantly higher bioavailability compared to an oral BG suspension. To our

2

knowledge, this is the first study on the feasibility of buccal administration of BG. Besides, the

3

proposed formulation has been shown to be an easily modifiable delivery platform that can be

4

suited for various local and injectable therapeutic applications through different routes of

5

administration.

6

Acknowledgement

7

This work was funded in part by Boehringer Ingelheim (FOAPAL:4004-1201/120102) and by

8

University of Sharjah (V.C./G.R.C./S.R. 83/2015 To MA). The release studies have been carried

9

out with the help of the research students Sahar Abdelmoniem, Iman Hakmi and Hazem Issa, and

10

the teaching assistant Ahd Bakri Al Nosh.

11 12

Supporting Information

13

The following are supporting information to this article: 1) The composition and results for

14

hydrogels containing PEG 4000 2) A figure showing the unstable vs. stable formulation to

15

support the finding and 3) Statistical analyses and p-values for figures 5-8.

16 17


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Page 34 of 47

Table 1. Composition and physical properties of drug-loaded thermosensitive hydrogels Formulation F1 F2 F3 F4 F5

BG

Composition (% w/v) Ch GP PEG6000 HPMC

Gelation time (sec)*

q (%)*

0.75 0.75 0.75 0.75 0.75

0.9 0.9 0.9 0.9 0.9

47.33 ± 5.04 43.75 ± 6.88 31.67 ± 8.78** 40.50 ± 5.21 20.25 ± 2.72**

395.01 ± 2.10 207.50 ± 4.31** 208.79 ± 7.63** 204.17 ± 7.90** 204.44 ± 7.74**

25 25 25 25 25

10 10 20 20

1 2

*

1

Coefficient of viscosity (cp) 718.9 952.9 1051.4 1365.4 3016.2

2 3 4

Data are mean values (n=3) ± SD p

Thermogelling Platform for Baicalin Delivery for Versatile Biomedical

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