Ex-vivo permeation study of chlorin e6-polyvinylpyrrolidone complexes through the chick chorioallantoic membrane model.

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Research Paper

Journal of Pharmacy And Pharmacology

Ex-vivo permeation study of chlorin e6-polyvinylpyrrolidone complexes through the chick chorioallantoic membrane model Romchat Chutoprapat, Lai W. Chan and Paul W. S. Heng GEA-NUS Pharmaceutical Processing Research Laboratory, Department of Pharmacy, National University of Singapore, Singapore

Keywords chick chorioallantoic membrane; chlorin e6; permeation study; polyvinylpyrrolidone Correspondence Paul W. S. Heng, GEA-NUS Pharmaceutical Processing Research Laboratory, Department of Pharmacy, National University of Singapore, No. 18 Science Drive 4, Block S4, Singapore 117543. E-mail: [email protected] Received October 23, 2013 Accepted January 1, 2013 doi: 10.1111/jphp.12222

Abstract Objective To investigate the influence of the hydrophilic polymer, polyvinylpyrrolidone (PVP) on the ex-vivo permeability of the poorly water-soluble photosensitizer, chlorin e6 (Ce6) using the chick chorioallantoic membrane (CAM) model. Methods The CAM was removed from the fertilized chicken egg at embryo age of 15 days. The permeation profiles of Ce6 and PVP complexes (Ce6-PVP) at 1 : 0, 1 : 1, 1 : 10, 1 : 50 and 1 : 100 w/w in different pH conditions were first studied using the CAM model with Franz diffusion cell over 8 h. The solution viscosity of the formulations and apparent solubility of Ce6 were also investigated. Key findings The permeability of Ce6 was found to be directly proportional to the amount of PVP used and the apparent solubility of Ce6. Permeability was only marginally affected by the solution viscosity of the formulations. The permeability of Ce6 was lowered in the acidic pH. Ce6-PVP at 1 : 100 w/w gave the highest percentage release of Ce6 across the CAM, with 23% at pH 3 and 55% at pH 7.4, after 8 h, respectively. Conclusions The present work suggests that PVP had served as penetration enhancer for the poorly water-soluble Ce6 and the CAM can serve as a useful biological membrane model for preclinical permeability study of biological and pharmaceutical substances. The Ce6-PVP formulation at 1 : 100 w/w can be applied for the further clinical investigation.

Introduction Over the past three decades, photodynamic therapy (PDT) has been widely developed as a treatment modality for cancer. It involves the administration of a photosensitizer which can absorb a specific wavelength of light and transforms to an active excited state, from which energy is transferred to oxygen to form reactive oxygen species that cause apoptosis or necrosis of the target tumour cells. Chlorin e6 (Ce6) is a pharmaceutical active drug substance that has gained considerable interest as a photosensitizer because of its low toxicity, rapid elimination from the body, fast and sufficiently selective accumulation in target tissues and tumouricidal properties.[1,2] Intravenous, oral and topical routes of Ce6 administration have been considered for the clinical application of PDT.[3,4] However, Ce6 in its original form is poorly soluble in water and unstable in acidic environment, leading to decreased photosensitizing efficacy. To

overcome this deficiency, polyvinylpyrrolidone (PVP) was used as a stabilizer and hydrophilic carrier for Ce6.[5] PVP is a synthetic, water-soluble, neutral pharmaceutical polymer with low toxicity and good biocompatibility. It is commonly used to improve drug solubility and protect drug against degradation in solution.[6] Chemical structures of Ce6 and PVP are shown in Figure 1. Photolon or Fotolon which consists of a mixture of Ce6 (MW = 596.67) and PVP (MW = 12000) at the ratio of 1 : 1 w/w has been approved for photodynamic diagnostics and topical treatment of malignant tumour of the skin and mucous membranes.[7] Several studies had shown the usefulness and safety of formulations containing Ce6 and PVP (Ce6-PVP) at 1 : 1 w/w in clinical use. Chin et al. demonstrated that Ce6-PVP (1 : 1, w/w) formulation had better selectivity in patients with angiosarcoma than Ce6 alone, with increased

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 943–953

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Permeation study of Ce6-PVP complexes

Romchat Chutoprapat et al.

eggshell. It serves as a support for respiratory and nutrient transport capillaries, and has been used to evaluate the ex-vivo and in-vivo activity of pharmaceutical substances,[11] wound healing,[12] angiogenesis and antiangiogenesis,[13] biocompatibility of biomaterial implants and biosensors.[14] In this study, the impact of PVP amount, viscosity and solubility on the ex-vivo permeability of Ce6 through non-keratinized biological membrane was investigated using the CAM with a Franz diffusion cell.

(a)

NH

N HN

N

HO

O O

OH

O

OH

Materials and Methods

(b)

Materials N

O

n Figure 1 (a) The chemical structure of chlorin e6. (b) The chemical structure of polyvinylpyrrolidone.

therapeutic index in PDT without increased toxicity.[8] A very low toxicity in the dark, rapid clearance, high phototoxicity and selectivity of Ce6-PVP at 1 : 1, w/w for cancer cells in patient were demonstrated in clinical studies.[7,9] However, before human trials, additional preclinical evaluation studies such as the permeability of Ce6PVP formulations would be much warranted and it would also provide a better understanding of the impact of co-additives including PVP in the formulations. These preclinical studies would provide the basis for formulation optimization and where necessary, refinements in the formula to further improve the treatment protocol. Ultimately, efforts at the bench, such as this preclinical evaluation, would provide the means to ensure the best possible outcomes at the bedside. Mammalian models are usually used for preclinical evaluation of new drugs. However, mammalian models are expensive, subject to ethical and legal regulations, and the experiments involved are timeconsuming to conduct. The chick chorioallantoic membrane (CAM) provides an alternative model for preclinical study of biological and pharmaceutical substances. It offers the advantages of greater simplicity, rapidity, sensitivity, ease of performance and relatively lower cost, thus more suitable for large-scale screening than the mammalian model. Moreover, laws governing the use of animals for research in many countries permit the use of fertilized eggs without the need for authorization from animal experimentation committees, on condition that the experiments begin and end before the eggs hatch and the experiments employ adequate experimental designs to reduce the number of embryonated eggs needed.[10] The CAM is the outermost extra-embryonic membrane lining the inner surface of the 944

Freshly laid, fertilized specific pathogen-free chicken eggs of the White Leghorn strain were purchased from the Animal and Plant Health Center, Singapore. Ce6 (SPE Chemical, Shanghai, China), PVP (Kollidone 17 PF, BASF, Ludwigshafen, Germany), disodium hydrogen phosphate dihydrate, sodium chloride, potassium dihydrogen phosphate and potassium chloride (Merck, Darmstadt, Germany) and ammonium acetate (Sigma-Aldrich, Zwijndrecht, the Netherlands) were used as supplied. All other chemicals were of analytical reagent grade.

Preparation of Ce6 and PVP (Ce6-PVP) formulations Appropriate amounts of Ce6 were dissolved in phosphatebuffered solution (PBS) of pH 3 and pH 7.4 to produce Ce6 concentration of 5 mM. The respective Ce6 solutions were mixed with appropriate amounts of PVP and sonicated for 15 min to give Ce6: PVP ratios of 1 : 1, 1 : 10, 1 : 50 and 1 : 100 w/w. Maximum loading of PVP in Ce6-PVP formulations was 1 : 100 w/w.

Viscosity measurement The kinematic viscosities of the Ce6 and Ce6-PVP solutions were determined using an Ostwald viscometer (size C) at 37°C. The kinematic viscosity (v) of each test solution was calculated using the following equation:

v s v w = ts t w where vs and vw are the kinematic viscosities and ts and tw are the flow times of the test solution and water, respectively. For each test solution, the viscosity measurement was conducted in triplicate and the mean calculated.

Solubility determination The method employed was adapted from an earlier study.[15] Specific amounts of Ce6-PVP, at 1 : 0, 1 : 1, 1 : 10, 1 : 50 and 1 : 100 w/w, were added to the PBS of pH 3 and pH 7.4, respectively. The formulations in volumetric flasks were



agitated in a water bath (Thermo Scientific, Sunnyvale, CA, USA) at 100 rpm, room temperature (25°C) for 48 h. Solutions of Ce6 in PBS were stable throughout 48-h period at 25°C. The supernatants were separated by using centrifuge (Eppendorf 5418, Hamburg, Germany) at a speed of 14 000 rpm and centrifugal force of 16 873 g for 1 h. The concentrations of Ce6 in the supernatants were then determined by high-pressure liquid chromatography (HPLC; LC-2010C, Shimadzu Corporation, Kyoto, Japan) equipped with a C-18 column and detected at wavelength of 405 nm. Mobile phase consisted of 10%, v/v, of 0.5 M ammonium acetate solution (pH 5.5) and 90%, v/v, of methanol. The solubility of Ce6 in each formulation was determined in triplicates and the mean value reported.

Permeation Study Preparation of CAM The method employed was adapted from an earlier study.[16] The eggs were disinfected with 70%, v/v ethanol before placing them in an incubator equipped with an automatic rotator (Octagon 20, North Somerset, UK) at 37°C. After 7 days of incubation, corresponding to embryo age (EA) 7, an opening was made at the blunt end of the egg to detach the shell membrane from the developing CAM using sterilized forceps. The opening was then sealed with parafilm and egg returned to the incubator until EA 15. The eggshell was then cut into half along its length and the content of the egg emptied, leaving the CAM adhered to the underside of the shell beneath the inner shell membrane. The CAM was carefully washed with normal saline and separated from the inner shell membrane with a pair of forceps. The detached CAM was rinsed with normal saline and stored at −20°C until further use.


cumulative percentage of Ce6 permeated through the CAM was plotted against time. The permeation study for each Ce6-PVP formulation was carried out in quadruplicates and results averaged.

Quantification analysis of Ce6 The contents of Ce6 were determined by HPLC as described above for solubility with the flow rate and sample injection volume set at 0.5 ml/min and 20 μl, respectively.

Statistical analysis Statistical analyses of viscosity, solubility and permeation of Ce6 in different Ce6-PVP formulations were performed using one-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) test and Pearson’s correlation coefficient.[17] Statistical analysis difference yielding P < 0.05 was considered significant.

Results Viscosity The viscosities of Ce6-PVP at 1 : 0, 1 : 1, 1 : 10, 1 : 50 and 1 : 100 w/w in PBS of pH 7.4 at 37°C are shown in Figure 2a. The viscosities of Ce6-PVP at 1 : 0, 1 : 1, 1 : 10, 1 : 50 and 1 : 100 w/w were significantly higher than that of water by 1.1, 1.2, 1.3, 3 and 11 times, respectively (one-way ANOVA with Fisher’s LSD test, P < 0.05). As the concentration of Ce6 was kept constant (5 mM), the viscosity changes were attributed to the concentrations of PVP in Ce6-PVP at 1 : 1, 1 : 10, 1 : 50 and 1 : 100 w/w which corresponded to 0.3, 3, 15 and 30%, w/v, respectively. In this study, the relationship between the viscosities of Ce6-PVP formulations and concentrations of PVP in the formulations followed an exponential function (Figure 2b).

Permeation study by Franz diffusion cells Permeation studies of Ce6 in different Ce6-PVP formulations were conducted using the CAM with Franz diffusion cells (Hanson Research, Chatsworth, CA, USA) in a six-unit assembly (effective permeation area 1.77 cm2) for 8 h at 37°C. The receptor compartment consisted of 9.5 ml of isotonic PBS (pH 7.4), which was stirred at 900 rpm throughout the experiment. The CAM was thawed by pre-hydrating for 1 h with the pH 7.4 buffer solution. The CAM, with an underlying supporting cellulose membrane, was then placed between the donor and receptor compartments. An amount of 1 ml donor liquid was introduced into the donor compartment and covered with a rubber cap. At the predetermined interval (1, 2, 3, 4, 6 or 8 h), 1 ml sample was withdrawn from the receptor compartment and replaced with an equal volume of fresh PBS. The Ce6 concentration was determined by HPLC at wavelength of 405 nm. The

Solubility The solubilities of Ce6 in Ce6-PVP formulations at 1 : 0, 1 : 1, 1 : 10, 1 : 50 and 1 : 100 w/w in PBS of pH 7.4 and pH 3 are shown in Figure 3. At pH 7.4, Ce6-PVP at 1 : 10, 1 : 50 and 1 : 100 w/w enhanced the apparent solubility of Ce6 by 1.5, 2 and 2.2 times, respectively. However, Ce6-PVP at 1 : 1 w/w showed no significant difference in solubility of Ce6 when compared with the solubility of Ce6 in PBS (oneway ANOVA with Fisher’s LSD test, P < 0.05). The solubilities of Ce6 in Ce6-PVP at 1 : 50 and 1 : 100 w/w were also not significantly different. At pH 3, Ce6-PVP at 1 : 1, 1 : 10, 1 : 50 and 1 : 100 w/w enhanced the solubility of Ce6 by 1.5, 10, 71 and 238 times, respectively (one-way ANOVA with Fisher’s LSD test, P < 0.05). Nevertheless, the solubilities of Ce6 in all Ce6-PVP formulations at pH 3 were still lower than those of all formulations at pH 7.4.


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(a) 25

Kinematic viscosity (cSt)

20

15

10

5

0 Water

Ce6-PVP 1 : 0

Ce6-PVP 1 : 1

Ce6-PVP 1 : 10 Ce6-PVP 1 : 50 Ce6-PVP 1 : 100

(b) R2 = 0.9951

3.5

Ln kinematic viscosity (cSt)

3

2.5

2

1.5

1

0.5

0 0

5

10

15

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25

30

35

Concentration of PVP (%g/ml) Figure 2 (a) The viscosities of chlorin e6-polyvinylpyrrolidone (Ce6-PVP) at 1 : 0, 1 : 1, 1 : 10, 1 : 50 and 1 : 100 w/w in phosphate-buffered saline (PBS) of pH 7.4 measured by Ostwald viscometer at 37°C. Each value represents mean ± standard deviation (SD) and n = 3 (one-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) test, P < 0.05). (b) The exponential relationship between the viscosities of Ce6-PVP formulations and the concentrations of PVP in the formulations. Each value represents mean ± SD and n = 3 (one-way ANOVA with Fisher’s LSD test, P < 0.05).

Permeation study by Franz diffusion cell The effects of PVP concentration and pH on the permeation of Ce6 in different Ce6-PVP formulations were investigated by CAM as the permeation barrier in Franz diffusion cells. 946

Effects of PVP amounts on permeation of Ce6 The cumulative percentages of Ce6 released across CAM after 8 h from the different formulations in PBS of pH 7.4 are shown in Figure 4. At 8 h, Ce6-PVP at 1 : 100 and 1 : 50




Solubility of Ce6 in Ce6-PVP mixtures (mg/100 ml)

200 pH 3 180 pH 7.4

160 140 120 100 80 60 40 20 0

Ce6-PVP 1 : 0

Ce6-PVP 1 : 1


Cumulative percentages of Ce6 released across CAM for 8 h

Figure 3 The solubility of Ce6 in chlorin e6 (Ce6)-polyvinylpyrrolidone (PVP) at 1 : 0, 1 : 1, 1 : 10, 1 : 50 and 1 : 100 w/w in phosphate-buffered saline (PBS) of pH 3 and pH 7.4. Each value represents mean ± standard deviation (SD) and n = 3 (one-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) test, P < 0.05). +Significant difference with respect to solubility of Ce6 in PBS of pH 3. *Significant difference with respect to solubility of Ce6 in PBS of pH 7.4.

Ce6-PVP 1:0

60

1:1 1 : 10

50

1 : 50 1 : 100 40

30

20

10

0 0

1

2

3

4

5

6

7

8

9

Time (h) Figure 4 The cumulative percentages of chlorin e6 (Ce6) released across chick chorioallantoic membrane (CAM) from Ce6-polyvinylpyrrolidone (PVP) at 1 : 0 (△), 1 : 1 (■), 1 : 10 (□), 1 : 50 (●), 1 : 100 (○) in phosphate-buffered saline (PBS) of pH 7.4 by vertical Franz diffusion cells. Each value represents mean ± standard deviation (SD) and n = 4 (one-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) test, P < 0.05).


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w/w gave the highest cumulative percentages of Ce6 diffused across the CAM, which were higher than those from Ce6-PVP at 1 : 10, 1 : 1 and 1 : 0 w/w by 1.4, 2.5 and 3.7 times, respectively (one-way ANOVA with Fisher’s LSD test, P < 0.05). The permeated concentrations of Ce6 from Ce6PVP at 1 : 100 and 1 : 50 w/w at 8 h were not significantly different. However, Ce6-PVP at 1 : 100 w/w was the optimal formulation for enhancing the permeation of Ce6 because of its consistent exhibition of the highest permeation rate of Ce6 throughout experimental period. The correlation between the solubility of Ce6 in the different Ce6-PVP formulations and the permeation kinetics of Ce6 across the CAM at 8 h is shown in Figure 5a. There was a high statistically significant linear relationship between the solubility and permeation of Ce6 in the formulations (Pearson’s correlation coefficient = 0.912, P < 0.05). In contrast, the permeation of Ce6 was related with the viscosity of formulations in non-linear manner (Pearson’s correlation coefficient = 0.572, P < 0.05) (Figure 5b). Increasing viscosity of Ce6-PVP formulation up to 6 cSt led to a significant increase in permeation of Ce6.

Effects of pH on permeation of Ce6 The solubility of Ce6 was significantly decreased in acidic pH 3, which was less than that of Ce6 in pH 7.4 by 240 times (Figure 3). In this study, the effects of pH on the permeation across the CAM of Ce6 from Ce6-PVP formulations at 1 : 0, 1 : 1, 1 : 10, 1 : 50 and 1 : 100 w/w were investigated. Figure 6 shows the cumulative percentages of Ce6 released across the CAM at 8 h from Ce6-PVP at 1 : 0, 1 : 1, 1 : 10, 1 : 50, 1 : 100 w/w in PBS of pH 3 and pH 7.4. The permeations of Ce6 from Ce6-PVP at 1 : 0, 1 : 1, 1 : 10, 1 : 50 and 1 : 100 w/w at pH 7.4 were higher than those at pH 3 by 1.4, 1.5, 2, 2.6 and 2.4 times, respectively (one-way ANOVA with Fisher’s LSD test, P < 0.05).

Discussion

Solubility Solubility of the drug is recognized as one of the most important factors affecting the permeability of the drug. For Ce6, its solubility is mainly affected by the pH environment because of the presence of three carboxyl groups in molecule. It exists in different ionic forms depending on pH.[21] In this study, the solubilities of Ce6 and Ce6-PVP formulations were evaluated in PBS of pH 7.4, simulating blood, and pH 3, simulating gastric conditions. The solubilities of Ce6 from all Ce6-PVP formulations at pH 3 were lower than those of all formulations at pH 7.4. Ce6 was practically insoluble in acidic pH due to protonation of the carboxyl groups, leading to increased hydrophobicity and insolubility of Ce6 in acidic pH.[5] Whereas, the ionization of carboxylic acid groups of Ce6 in the alkaline pH increases solubility of Ce6 with decreases in hydrophobic interaction, leading to lower aggregation.[22] Although the increase of pH value is able to enhance Ce6 solubility, the pH of Ce6 formulation should not exceed physiological pH value of 7.4 for minimizing incidences of drug precipitation at injection sites.[23,24] Therefore, the adding of a hydrophilic polymer such as PVP in Ce6 formulation is required for the enhancement of Ce6 solubility. Addition of PVP to Ce6 solutions at pH 3 and pH 7.4 resulted in increased solubility of Ce6, which could be attributed to the interaction between PVP and the carboxyl groups of Ce6 via hydrogen bond formation. In the previous study on Ce6-PVP interaction, Chin et al. had demonstrated the existence of hydrogen bonding interaction between Ce6 and PVP by a steadystate fluorescence technique, resulting in the formation of a soluble complex.[25] The binding interaction between Ce6 and PVP had been further investigated in our laboratory using a direct thermodynamic approach.[22] The results had indicated that Ce6 could interact with PVP via hydrophobic bonding, leading to disaggregation of Ce6 in aqueous solution. This study suggested that PVP was able to enhance the aqueous solubility of Ce6 not only in pH 7.4 but also in pH 3 at a high Ce6-PVP ratio of 1 : 100 w/w.

Viscosity

Permeation study by Franz diffusion cell

The relationship between the viscosities of Ce6-PVP formulations in PBS of pH 7.4 at 37°C and concentrations of PVP in the formulations was found to follow an exponential function. This result is in good agreement with a report by Buhler that mentioned that the viscosity of aqueous solutions with PVP increased exponentially with PVP concentration.[18] The exponential relationship between concentration and apparent viscosity of dispersion is generally described for colloidal systems.[19,20] This suggests that Ce6-PVP formulations as well as the aqueous solution of PVP might be more accurately described as colloidal dispersions.

The effects of PVP amount and pH on the permeation of Ce6 in different Ce6-PVP formulations were investigated by using CAM as the biological membrane in Franz diffusion cells. Previous studies in our laboratory had shown that CAM of EA 15 was the most suitable for use as permeation biological membrane model.[16,26] The CAM of an incubated chicken egg at EA 15 and schematic representation of the mature CAM are shown in Figure 7a and 7b, respectively. Histologically, CAM consists of three layers, ectoderm, mesoderm and endoderm. The outer ectoderm cells are flat and aligned in a single layer. The mesoderm is an embryonic connective tissue containing scattered fibroblasts, col-

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Cumulative percentages of Ce6 released across CAM at 8 h

(a) 70 r = 0.912 60

50

40

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20

10

0 60

80

100

120

140

160

180

200

Solubility of Ce6 in Ce6-PVP mixtures at pH 7.4 (%mg/ml)


(b) 70 r = 0.572 60

50

40

30

20

10

0 0

5

10

15

20

25

Viscosity of Ce6-PVP mixtures in PBS of pH 7.4 (cSt) Figure 5 (a) The correlation between the solubility of chlorin e6 (Ce6) in the Ce6-polyvinylpyrrolidone (PVP) formulations at pH 7.4 and the cumulative percentages of Ce6 released across chick chorioallantoic membrane (CAM) at 8 h (Pearson’s correlation coefficient, P < 0.05). (b) The correlation between the viscosity of Ce6-PVP formulations in phosphate-buffered saline (PBS) of pH 7.4 and the cumulative percentages of Ce6 released across CAM at 8 h (Pearson’s correlation coefficient, P < 0.05).

lagen fibres and small- to medium-sized blood vessels. The inner endoderm consists of the single layer of cells in contact with allantoic fluid, an embryonic excretory product.[27] Moreover, the surface membrane of CAM con-

tains protein, phospholipid and cholesterol, which are similar to those found in mammalian cell membranes.[28] Transcellular pathway is a common route of drug transport through CAM.[29]


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60 pH 3 pH 7.4

50

40

30

20

10

0 Ce6-PVP 1 : 0

Ce6-PVP 1 : 1


Figure 6 The cumulative percentages of chlorin e6 (Ce6) released across chick chorioallantoic membrane (CAM) at 8 h from Ce6polyvinylpyrrolidone (PVP) at 1 : 0, 1 : 1, 1 : 10, 1 : 50, 1 : 100 w/w in phosphate-buffered saline (PBS) of pH 3 and pH 7.4 by vertical Franz diffusion cells. Each value represents mean ± standard deviation (SD) and n = 4 (one-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) test, P < 0.05).

(a)

(b)

Trancellular route

Ectoderm Mesoderm Endoderm

Drug diffusion Figure 7 (a) Chick chorioallantoic membrane (CAM) of an incubated chicken egg at embryo age (EA) 15. (b) schematic representation of three layers of chick mature CAM.

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Effects of PVP amounts on permeation of Ce6 The permeation of Ce6 increased as PVP concentration increased. This is postulated to be due to several possibilities. Firstly, the inherent property of PVP as a solubilizer increased the solubility of Ce6 in the formulation, enhancing the driving force for Ce6 diffusion. Furthermore, the enhancement of solubility of drug increases thermodynamic activity by converting the crystalline drug into amorphous state, which generally possesses a high-energy state that facilitates the permeation rate of drug through the membrane.[30,31] Several studies indicated that the viscosity of drug formulations had an effect on the permeation and diffusion of drug into the biological membrane. Melis Decerf and Ooteghem demonstrated that the diffusion velocity into the surface of rabbit cornea of drug containing in eye drops decreased with increasing viscosity of formulation.[32] Moreover, El Maghraby proved that a decrease in viscosity of microemulsion enhanced its permeation flux.[33] Therefore, the viscosity of formulation can be one of most important factors affecting the permeability of drug. Increasing viscosity of Ce6-PVP formulation up to 6 cSt led to a significant increase in permeation of Ce6. It is expected that the high viscosity of formulation enabled the drug to adhere to the surface of membrane, hence enhancing the drug permeation. However, the permeation enhancement effect reached maximum at a certain viscosity and then remained rather constant despite further increases in the viscosity. This is not in agreement with some of the findings reported, where the increased viscosity of drug solution was found to decrease drug permeation and prolong drug release.[34] The anti-aggregation and plasticizer properties of PVP could also possibly enhance Ce6 molecules transport across the CAM. As already well known, one potential disadvantage of photosensitizer drugs is the molecular aggregation which reduces the sensitizing efficiency during PDT.[35] For Ce6, there are several alkyl groups at one end of the molecule, which render the molecule hydrophobic. Three peripherally attached carboxylic groups are present at the other end, which exist in different ionic forms, depending on the pH conditions. The amphiphilic Ce6 molecule has an inherent tendency to aggregate by molecular stacking.[36] The aggregates will grow in size and decrease in molecular mobility, leading to reduced permeability of drug.[37] A previous study in our laboratory had revealed that complete disaggregation of Ce6 was achieved by adding PVP at Ce6-PVP ratio of 1 : 100 w/w.[22] Moreover, the polymeric plasticizer may interact and disrupt the organized protein and lipid structures and thereby increase the flexibility of the CAM, resulting in a higher permeation of drug. Therefore, many plasticizers such as propylene glycol, dibutyl phthalate, diethyl phthalate, hydroxypropyl


methylcellulose and PVP have been used to improve percutaneous absorption of drugs.[38–40] Lastly, the hydration effect of PVP on CAM membrane could have occurred. The increased membrane hydration could modify partitioning of drug from the formulation into the membrane with the swelling and opening of the structural components of the membrane, leading to an increased diffusivity and penetration of drug. The interaction between the carbonyl group in PVP and hydroxyl group in water was found to contribute to the hydration capability of PVP.[41] However, PVP was expected to be retained by the CAM owing to its large molecular size. Basically, the penetration enhancement of drug across a biological membrane can be categorized into three strategies: increasing drug solubility in the formulation, increasing drug diffusivity and enhancing drug solubility in the membrane.[42,43] This study had indicated that PVP served as a penetration enhancer for the poorly watersoluble drug by enhancing its solubility, disaggregation and diffusivity.

Effects of pH on permeation of Ce6 The permeations of Ce6 from Ce6-PVP formulations (1 : 0, 1 : 1, 1 : 10, 1 : 50 and 1 : 100 w/w) at pH 7.4 were higher than those at pH 3. As previously discussed, pH environment had an effect on the solubility of Ce6 and the solubility of Ce6 in Ce6-PVP formulation was important for permeation through the CAM membrane. A decreased solubility of Ce6 in acidic pH 3 had resulted in reduced permeability of Ce6. Therefore, Ce6 itself may not effectively permeate the gastrointestinal membrane or into cancer cells surrounded by an acidic environment.[44] Although the acidic pH had lowered the permeability of Ce6, permeation of Ce6 increased as the PVP concentration was increased. This suggests that PVP is a useful penetration enhancer for poorly water-soluble drug in not only neutral pH but also in acidic pH conditions. This study had demonstrated that there was a potential for using CAM in permeation screening of formulation candidates for drug permeability via non-keratinized biological barriers. This application can be established as an alternative to preclinical mammalian models.

Conclusion The viscosity of Ce6 was significantly increased by hydrophilic polymer PVP at mass ratio of 1 : 50 and 1 : 100. The highest formulation viscosity was found in Ce6-PVP at 1 : 100 w/w. The viscosity profile of Ce6-PVP had indicated that the Ce6-PVP formulations were colloidal systems. The optimal ratio of Ce6-PVP at 1 : 100 w/w enhanced the aqueous solubility of Ce6 in pH 7.4 and pH 3 by 2.3 and 238 times, respectively. Ce6-PVP at 1 : 100 w/w gave the highest cumulative percentages of Ce6 released across the


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CAM at 8 h with 55% at pH 7.4 and 23% at pH 3. Ce6-PVP formulations at pH 7.4 gave higher release of Ce6 across CAM compared with formulations at pH 3 due to the higher solubility of Ce6 at pH 7.4. The viscosity of formulation had slightly affected the permeability of Ce6. This study demonstrated that the permeation of Ce6 across CAM was directly proportional to the solubility of Ce6 in the formulation, which could be enhanced by PVP and pH of the formulation. The Ce6-PVP formulation at 1 : 100 w/w can be applied for the further clinical study.

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Declarations Conflict of interest The Author(s) declare(s) that they have no conflicts of interest to disclose.

Funding This work was supported by the National Medical Research Council, NMRC (R-148-000-114-213), Singapore.

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Chick Chorioallantoic Membrane Assay: A 3D Animal Model for Study of Human Nasopharyngeal Carcinoma.

Application of the chick embryo chorioallantoic membrane in neurosurgery disease.

Demonstration of synergistic effects of hyperthermia and photodynamic therapy using the chick chorioallantoic membrane model.

Angiogenic Potential of Human Neonatal Foreskin Stromal Cells in the Chick Embryo Chorioallantoic Membrane Model.

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma.

3D chick embryo chorioallantoic membrane model as an in vivo model to study morphological and histopathological features of feline fibrosarcomas.

The chick chorioallantoic membrane (CAM) as an in vivo model for photodynamic therapy.

Chick chorioallantoic membrane model for in ovo evaluation of timolol maleate-brimonidine tartrate ocular inserts.

The Chick Embryo Chorioallantoic Membrane as an In Vivo Assay to Study Antiangiogenesis.

Feasibility Study of Ex Ovo Chick Chorioallantoic Artery Model for Investigating Pulsatile Variation of Arterial Geometry.

Angiostatic activity and metabolism of cortisol in the chorioallantoic membrane (CAM) of the chick embryo.

The chicken chorioallantoic membrane model in biology, medicine and bioengineering.

Acute inflammatory response to cowpox virus infection of the chorioallantoic membrane of the chick embryo.

The chick chorioallantoic membrane promotes survival of co-transplanted rat carotid bodies and nodose ganglia.

Dynamics of the Developing Chick Chorioallantoic Membrane Assessed by Stereology, Allometry, Immunohistochemistry and Molecular Analysis.

A tool for the quantification of radial neo-vessels in chick chorioallantoic membrane angiogenic assays.

Calcium-binding protein of the chick chorioallantoic membrane. II. Vitamin K-dependent expression.

Angiostatic activity of anticancer agents in the chick embryo chorioallantoic membrane (CHE-CAM) assay.

Calcium-binding protein of the chick chorioallantoic membrane. I. Immunohistochemical localization.

The interrelationships between sodium ion, calcium transport and oxygen utilization in the isolated chick chorioallantoic membrane.

The human carotid atherosclerotic plaque stimulates angiogenesis on the chick chorioallantoic membrane.

Calcium-stimulated respiration and active calcium transport in the isolated chick chorioallantoic membrane.

Angiogenesis is associated with collagenous protein synthesis and degradation in the chick chorioallantoic membrane.

Stretch-induced intussuceptive and sprouting angiogenesis in the chick chorioallantoic membrane.