Biomedical Platform Development of Chlorophyll-based extract for

Subscriber access provided by University of South Dakota

Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Biomedical Platform Development of Chlorophyll-based extract for Topic Photodynamic Therapy: Mechanical and Spectroscopic Properties Katieli da Silva Souza Campanholi, Gustavo Braga, Jéssica Bassi da Silva, Nicola Leone da Rocha, Lizziane Maria Belloto de Francisco, Évelin Lemos de Oliveira, Marcos Luciano Bruschi, Lidiane Hoshino, Francielle Sato, Noboru Hioka, and Wilker Caetano Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00658 • Publication Date (Web): 22 Jun 2018 Downloaded from http://pubs.acs.org on June 23, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 58 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

Langmuir

1

2

Biomedical Platform Development of Chlorophyll-based extract for Topic

3

Photodynamic Therapy: Mechanical and Spectroscopic Properties

4

Katieli da S.S. Campanholi*,a, Gustavo Bragaa, Jéssica B. da Silvab, Nicola L. da

5

Rochad, Lizziane M. B. de Franciscob, Évelin L. de Oliveiraa, Marcos L. Bruschib,

6

Lidiane V. de Castro-Hoshinoc, Francielle Satoc, Noboru Hiokaa and Wilker Caetanoa.

7

a

8

University of Maringá, Maringá, Paraná, Brazil. dInstitute of Chemistry, State University of

9

Campinas.

Department of Chemistry, bDepartment of Pharmacy and cDepartment of Physics, State

10

11

ABSTRACT

12

Photodynamic therapy (PDT) is a therapeutic modality that has shown effectiveness in

13

the inactivation of cancer cell lines and microorganisms. Treatment consists of

14

activating the photosensitizer (PS) upon light irradiation of adequate wavelength. After

15

reaching the excited state, the PS can handle the inter-system conversion through energy

ACS Paragon Plus Environment

Langmuir 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

16

transfer to the molecular oxygen, generating reactive oxygen species. This especially

17

applies to singlet oxygen (1O2), which is responsible for the selective destruction of the

18

sick tissue. Photosensitizing compounds (Chlorophylls and derivatives) existing in the

19

spinach extract have applicability for Photodynamic Therapy. This study aimed to

20

develop and characterize the thermoresponsive bioadhesive system composed by

21

Pluronic® F127 20.0% and Carbopol® 934P 0.2% (w/w) (FC) containing Chlorophylls-

22

based extract 0.5% (w/w) (FC-Chl). Mechanical, rheological, in vitro release, sol-gel

23

transition temperature and ex vivo permeability of spinach extract PS components

24

(through pig ear skin) were investigated. Furthermore, photodynamic activity of the

25

system was accessed through the uric acid and time-solved measures. The sol-gel

26

transition temperature obtained for the FC-Chl system was 28.8 ± 0.3 °C. Rheological

27

and texture properties of the platform were suitable for use as a dermatological system,

28

exhibiting easy application and good characteristics of retention in the place of

29

administration. In vitro release studies showed the presence of two distinct mechanisms

30

that reasonably obey the zero-order and first-order kinetics models. PS components

31

presented skin permeability and reached a permeation depth of 830 µm (between the

32

epidermis and dermis). The photodynamic evaluation of the FC-Chl system was

33

effective in the degradation of uric acid. The quantum yield (Φ∆1O2) and life time (τ1O2)

34

of singlet oxygen showed similar values for the spinach extract and the isolated

35

Chlorophyll a species in ethanol. These results allowed for the classification of the FC-

36

Chl platform as potentially useful for delivery of Chlorophyll-based extract in the topic

37

Photodynamic Therapy, suggesting that it is worthy for in vivo evaluation.

38

39

INTRODUCTION


Page 2 of 58

Page 3 of 58 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

Langmuir

40

Photodynamic therapy (PDT) consists of a minimally invasive therapeutic modality and

41

has shown promising results for the treatment of primary and advanced carcinomas,

42

metastases located in the head and neck regions, external lesions such as leukoplakia of

43

the buccal cavity1, palliative treatment of inoperable cholangiocarcinoma2, urological

44

neoplasms3, basal cell carcinoma, Kaposi's sarcoma (common in AIDS patients),

45

melanoma, paramecia4, skin tumors4, viroses and fungal infections1,5. Compared with

46

other medical modalities, including surgery, radiotherapy, chemotherapy and

47

immunotherapy, PDT offers high selectivity. This culminates in minimal systemic

48

toxicity, in addition to greater effectiveness on multiple drug resistant cells (MDR)6,7

49

and the possibility of combining with other therapeutic modalities8,9.

50

The treatment consists basically of the administration of a photosensitizer (PS), which is

51

activated to the excited singlet state (1PSexc)8,10. The decay of the 1PSexc can occur in a

52

radiative, a non-radiative or an inter-system crossing involving vibrational levels of the

53

same energy and different multiplicities, whereas spin inversion occurs from 1PSexc to

54

3

55

neighboring molecules by Type 1 reactions, with formation of superoxide radical anion

56

O2-*, hydroxyl radical OH*, hydrogen peroxide H2O2 and Type 2 reactions, where the

57

drug in its excited triplet state can transfer its excess energy to the molecular oxygen

58

(3O2), forming singlet oxygen (1O2), responsible for inciting oxidative damage to

59

cells4,14–18.

60

Due to economic factors, PS obtained from abundant and natural raw materials attracts

61

great interest19. The Chlorophylls, Pheophorbides and Pheophytins a and b, found in

62

New Zealand spinach extract (Tetragonia expansa)20, are prominent in PDT and

63

frequently reported in several studies involving glandular malignancies, human lung

64

adenocarcinoma tumor cell lines (A549), human breast (MCF-7), and human colon

PSexc11–13. The lifetime of the 3PSexc is long enough to allow its interaction with


Langmuir 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

65

(LoVo), in the evaluation of mutagenic and genotoxic effects, as well as studies of

66

photodynamic inactivation of various microorganisms20–25. New Zealand Spinach is a

67

grassy herbaceous species with dark green leaves rich in photosensitizing compounds

68

(Chlorophylls, Pheophorbides and Pheophytins a and b). Its low cost combined with the

69

wide cultivation and activity by PDT has aroused the interest of researchers. Thus,

70

spinach extract has become a protagonist for several lines of scientific research24–28. The

71

use of the extract in the PDT offers economic and environmental advantages over

72

purified chlorophyll compounds, as it is obtained without the use of purification steps

73

responsible for the generation of toxic wastes, which impact humans and the

74

environment24,25. Furthermore, the use of extracts in PDT has offered advantages due to

75

the different degrees of skin permeation of the PS compounds, distinct mechanisms of

76

action and, above all, the synergic combined effect of the photoactive compounds

77

present in the extract shown in recent studies29.

78

The application of photoinduced treatment in skin lesions requires delivery systems

79

displaying suitable physicochemical, mechanical and rheological characteristics.

80

Moreover, the lipophilic nature of the PS demands the use of a platform capable of

81

stabilizing the monomeric photoactive state of the chlorophylls. A topical

82

administration system should have adequate consistency for manufacturing, storage and

83

application. This means that the formulation should exhibit easy removal from the

84

storage container, and also have adequate consistency when exposed to the skin to

85

increase the permanence time of the drug at the place of action. Such properties are

86

found in aqueous gels, which may be composed from the thermoresponsive copolymers

87

of the Pluronic® class, responsible for the solubulization of lipophilic drugs30.

88

F127 are thermoresponsive copolymers composed of polyethylene oxide (PEO)

89

segments that give rise to the external hydrophilic shell and polypropylene oxide (PPO)


Page 4 of 58

Page 5 of 58 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

Langmuir

90

responsible for the formation of the hydrophobic core31,32. In aqueous medium and with

91

conditions of suitable concentration and temperature, the unimers associate themselves

92

and give origin to copolymeric micelles, which can solubilize medicines of diverse

93

chemical natures. These systems can be used as gelling agents in the concentration

94

range of 20.0 % (w/w), giving semi-solid colloidal systems. When subjected to

95

temperature increases (in addition to the Tsol/gel critical transition temperature) in the

96

isotropic micellar structure, a cubic hexagonal core with more ordered segments is

97

formed. This significantly increases the consistency of the biomimetic matrix. The high

98

degree of structured water, and the quantity contained in the systems, allows for a

99

smooth texture that ends up mimicking natural tissues. This increases the

100

biocompatibility of the biomechanical systems32–35.

101

The improved adhesiveness, stability, and consistency of drug delivery platforms can be

102

achieved with the addition of carbomers. These are synthetic hydrophilic polymers

103

derived from poly(acrylic acid) and feature high consistency gels at a pH higher than

104

4.75 (pKa). The carbomer Carbopol® 934P, oral pharmaceutical grade polymer, features

105

long, high molecular weight chains containing about 56-68% polyacrylic acid cross-

106

linked with sucrose allyl esters35.

107

The recognition of the advantageous properties of the polymer blends composed of

108

different proportions and combinations, and types of Pluronic® with Carbopol®, aroused

109

the interest of several researchers. These days, this constitutes a thermodependent

110

biomedical matrix extensively explored and consolidated in the solubilization of

111

drugs33,34,36–39. Therefore, this study aims to combine for the first time the consolidated

112

activity of the natural PS inserted in the New Zealand Spinach matrix with the potential

113

thermoresponsive pharmaceutical platform composed of Pluronic® F127 and Carbopol®

114

934P, for the extensive treatment of primary or secondary morphological skin lesions.


Langmuir 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

115

EXPERIMENTAL SECTION

116

Materials. Spinach extract was obtained from New Zealand Spinach (Tetragonia

117

tetragonioides) leaves (14.65±0.99 % w/w of mean yield±standard deviation), following

118

the methodology previously reported40. F127 Pluronic® (poloxamer 407; molar mass=

119

12600 g mol−1) and uric acid (UA) were purchased from Sigma-Aldrich (São Paulo,

120

Brazil). Carbopol® C934P was purchased from Lubrizol Advanced Materials (São

121

Paulo, Brazil), and Triethanolamine was bought at Galena (Campinas, Brazil).

122

Membrane HAWP04700 MF in mixed ester cellulose (Millipore), 0.45 µm pore, 4.7 cm

123

of diameter (white, smooth), was purchased from Merck (São Paulo, Brazil). All

124

solvents employed were of analytical grade and used without further purification.

125

Instruments and Characterization. Studies of electronic absorption and fluorescence

126

emission were performed in a UV-Vis spectrophotometer model Varian Cary 50

127

(Agilent Technologies, São Paulo, Brazil) and Varian Cary Eclipse spectrofluorimeter

128

(Agilent Technologies, São Paulo, Brazil), respectively.

129

Rheological measurements were conducted on a MARS II rheometer (Thermo-Haake,

130

Thermo Fisher Scientific Inc., Newington, Germany), using a 60 mm diameter steel

131

cone plate geometry, separated by a gap of 0.052 mm. The FC and FC-Chl was

132

carefully applied to the lower plate, allowing for the minimum shear and a minimum

133

stabilization time of 1 minute before the determination. The RheoWin (Haake®)

134

software version 4.10.0000 was used in oscillatory and continuous rheology.

135

The texture profile analysis (TPA) was performed using a TA-XTplus texture analyzer

136

(Stable Micro Systems, Surrey, UK), equipped with a polycarbonate analytical probe

137

(10 mm diameter).


Page 6 of 58

Page 7 of 58 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

Langmuir

138

The degradation profile of the UA by the PS contained in the FC-Chl platform was

139

obtained in UV-Vis spectrophotometer, model Cary 50 (Agilent Technologies, São

140

Paulo, Brazil), with 6 LEDs (9 mW, λmax= 636 nm) attached.

141

The quantification of the quantum yield (Φ∆1O2) and lifetime (τ1O2) of the singlet

142

oxygen was performed with a Near-Infrared fluorometer (NIR) (Edinburgh Analytical

143

Instruments, Scotland, U.K.) coupled to a photomultiplier R5509 (Hamamatsu Co.,

144

New Jersey, USA) and with a Hewlett Packard 54510B oscilloscope. The PS excitation

145

was performed by a LASER Continuum Nd: YAG Sarulite III (Spectron Laser

146

California, USA) at 661 nm (pulse of 5 ns and energy ≤ 5mJ/pulse). The electronic

147

absorption spectra were acquired by Shimadzu UV-240 PC spectrophotometer (São

148

Paulo, Brazil).

149

The photoacoustic spectroscopy (PAS) measurements were performed using an

150

assembled experimental setup, composed by a 1000-W Xenon model 68820 arc lamp

151

(Oriel Instruments, California, USA) and a model 77250 monochromator (Oriel

152

Instruments, California, USA), with 3.16-mm of input and output slits. Diffraction

153

grating was performed with model 77296 (Oriel Instruments, California, USA), and

154

model SR 540 (Stanford Research Systems, California, USA)41 was used for UV-Vis

155

spectral and as a mechanical chopper. The spectra were recorded in intervals between

156

200 and 800 nm, and the nominal power of the light source was 800 W with modulation

157

frequencies of 15 Hz.

158

The morphological analysis of the formulations was performed using a scanning

159

electron microscope Quanta 250 (Thermo Fisher Scientific-FEI, Oregon, USA) for

160

lyophilized samples in a Micromodulyo freeze dryer (Thermo Electron Corporation,

161

Oregon, USA). The metallizer used for the preparation of scanning electron microscope

162

samples was Sputter Coater, model SCD 050 (Bal-Tec, California, USA).


Langmuir 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

163 164

Preparation of Thermoresponsive and Bioadhesive Platform

165

The formulation was prepared by the addition of 150.0 mg of dried spinach extract and

166

6.0 g of F127 (representing 0.5 % and 20.0 % w/w of the preparation, respectively) in

167

ethanol, followed by evaporation of the solvent and formation of a solid thin film,

168

which was kept in a desiccator for 24 h. Subsequently, the polymeric film was hydrated

169

with 60.0 mg of C934P (0.2 % w/w) dispersed in 23.9 mL of water under vigorous

170

stirring in an ice bath (obtaining a mixture of F127-C934P). The preparation had the pH

171

neutralized with triethanolamine (pH 7). FC and FC-Chl samples were stored and

172

protected from the light at 4.0 °C. The systems were kept at rest for 24 h prior to

173

analysis38. The systems were named as FC-Chl, and they referred to the matrix

174

composed of 20.0% F127, 0.2% C934P, and 0.5% w/w of the spinach extract (source of

175

PS compounds). The gels obtained without addition of spinach extract were

176

denominated FC and used as a reference in the analyses.

177

178

Physicochemical and Photodynamic Properties of the Thermoresponsive Drug

179

Delivery Platform

180

Interaction of the Chlorophyll-based extract with the F127-C934P Copolymer Blends:

181

Spectroscopic Studies

182

The interaction and solubility capacity studies of Chlorophyll-based extract (Chl) with

183

the F127-C934P combination were performed by titration of aliquots of the solution

184

composed of F127 4.0 % (w/v) and C934P 0.04 % (w/v) to a solution of 46.4 mg L-1 of

185

spinach extract, both prepared in a Mcllvaine buffer (0.10 mol L-1; pH 7.4)42. The

186

interaction was monitored by the acquisition of fluorescence emission spectra at 680 nm

187

(excitation at 537 nm, Soret bands), obtained at 37.0 °C in a Cary Eclipse ACS Paragon Plus Environment

Page 8 of 58

Page 9 of 58 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

Langmuir

188

spectrofluorimeter43. Furthermore, incorporation kinetics studies were conducted

189

starting from 25.0 mg L-1 spinach extract solution in F127 4.0 % and C934P 0.04 %

190

(w/v) mixed in Mcllvaine buffer (0.10 mol L-1; pH 7.4). The temporal effect of binding

191

and monomerization of the main PS compounds contained in the spinach extract

192

(Chlorophylls, Pheophorbides and Pheophytins a and b) was accompanied by the

193

acquisition of fluorescence emission spectra in 680 nm (excitation at 498 nm, Soret

194

bands). The kinetic curve obtained was fitted by the associative exponential model

195

(Equation 1): = + − . + − .

196

(1)

197

where P corresponds to the values of the property (fluorescence emission intensity) at

198

the time (t), k1obs and k2obs are the kinetics constants for the steps, Q and R are for the

199

decay amplitudes of the property in the first and second phases, respectively44.

200 201

Mechanical and Rheological Analyzes of the Thermoresponsive Drug Delivery

202

Platform

203

Continuous Shear (flow) Rheometry

204

These measures were performed in continuous flow mode, at temperatures of 25.0 and

205

37.0 °C. The flow curves for FC and FC-Chl were analyzed at several shear rates

206

(between 10 to 2000 s-1) with cycles of increases and reductions over a period of 150 s,

207

and then held at the maximum shear rate (upper limit) for 10 s. The flow properties of

208

each formulation were investigated in five-fold replicates and the upward flow curves

209

were modeled using the Power Law (Ostwald-de-Waele; Equation 2) and Herschel-

210

Bulkley (Equation 3) equations:

211

=

(2)

212

= +

(3)


Langmuir 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

213

where σ is the shear stress (Pa), σ0 is yield stress (Pa), k is the consistency index [(Pa

214

s)n],

215

The thixotropy and hysteresis areas were calculated using RheoWin (Haake®) software.

216

The yield value was obtained through the Casson model.

is the rate of shear (s-1), and n is the flow behavior index (dimensionless)33,36.

217

218

Oscillatory Rheometry

219

The analysis was performed in oscillatory mode, at 25.0 °C and 37.0 °C. The linear

220

viscoelastic regions of each formulation (FC and FC-Chl) were previously determined.

221

Afterwards, the frequency sweep analysis was performed from 0.1 to 10.0 Hz, in a state

222

of fixed stress. The η' (dynamic viscosity), G' (elastic modulus), G" (loss modulus), and

223

tan δ (loss tangent) were calculated using RheoWin (Haake®) software. The viscoelastic

224

properties of at least three replicate samples were determined35,36,45,46.

225 226

Determination of the Sol−Gel Transition Temperature

227

The determinations of the sol-gel transition temperature (Tsol/gel) for the FC and FC-Chl

228

platforms were conducted with the rheometer in oscillatory mode. The analysis was

229

conducted in the linear viscoelastic region using the temperature ramp method. The

230

temperature scanning analysis was performed with a heating rate of 10 °C min-1 and the

231

temperature range was between 5.0 and 60.0 °C (frequency of 1.0 Hz). The temperature

232

where G' was located was an intermediate region between the solution and gel states

233

named Tsol/gel, for formulations in which dynamic viscosity significantly increased with

234

increasing temperature. The determination of Tsol/gel was evaluated through the second

235

derivative of the natural logarithm of the mean modulus of elasticity as a function of the

236

mean temperature (∂2logG'/∂T2), where the abrupt change from positive to negative


Page 10 of 58

Page 11 of 58 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

Langmuir

237

(passing through zero point) gives Tsol/Tgel for systems. Measurements were performed

238

with at least five replicates for each formulation33,36.

239 240

Texture Profile Analysis

241

In the compression mode of the texture analyzer, the probe was compressed twice into

242

each sample of FC and FC-Chl (stored in glass vials and held at rest), at a depth of 15

243

mm, at a rate of 2 mm s-1, and with a permanence time of 15 s between the end of the

244

first and the beginning of the second compression. The analysis was performed at 25.0

245

°C and at 37.0 °C for at least five replicate samples. The parameters of compressibility,

246

hardness, cohesiveness, elasticity, and adhesiveness were obtained from the results of

247

the plot force by time and by distance34,36.

248 249

Release and Cutaneous Permeation of Chlorophyll-based Extract

250

In vitro Spinach Extract Release

251

The release studies were performed at 37.0 °C using the FC-Chl (30.0 mg) platform

252

inserted in a dialysis bag formed by Millipore HAWPO4700 MF smooth membrane.

253

The receptor solution medium was composed of F127 4.0 % (w/v) in an aqueous

254

medium, ensuring the sink conditions (F127 concentration equals at most 10 %

255

saturation concentration). The system was prepared by adding 3.00 ml of the receptor

256

medium in a quartz cell, with magnetic stirring. The FC-Chl dialysis bag was immersed

257

in the upper region of the receptor solution, freeing the location of the light beam

258

incident for kinetic measurements through fluorescence emission at 680 nm by time.

259

The adjustment of the kinetic profile of fluorescence emission to obtain the order of

260

reaction was performed by Equation 4:


Langmuir 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

261

= −

Page 12 of 58

(4)

262

where P∞ is the fluorescence property at infinity, Pt is fluorescence property at time (t),

263

with k as the first-order kinetic constant47.

264

265

Ex vivo Study of Permeation and Skin Retention

266

Preparation of Pig Skin

267

As it resembles human skin in terms of morphology, pig skin was used as an ex vivo

268

model system in gel permeation studies48. The pig skin segments were supplied by the

269

Experimental Farm of Iguatemi (EFI), which belongs to the State University of

270

Maringá, Brazil. Samples were taken from young, white animals, submitted to recent

271

slaughter. After selecting regions of skin with absences of wounds, warts, and bruises,

272

segments of the dorsal side of the auricle were excised using a surgical scalpel, with a

273

subsequent removal of subcutaneous fat from the skin samples. The partitions were

274

stored at -18.0 ° C until the date of use.

275

Permeation Studies of FC-Chl: PAS Technique

276

After the thermal equilibrium of the pig ear in the environment, 30.0 mg of FC-Chl

277

platform was spread over an area of 1 cm2 on the corneal side of the cutaneous segment

278

of the pig ear. The photoacoustic measurements were performed after 30 and 240

279

minutes, on both sides of the segment (epidermis and dermis)41,49. After the samples

280

analyzes had thickness measured with a digital micrometer, depth values of drug

281

penetration into the skin were calculated using Equation 5:

282

#

! = "$.%

(5)


Page 13 of 58 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

Langmuir

283

where µ is the thermal diffusion length (m), D is the thermal diffusivity of the skin

284

(3.10-8 m2 s-1), and f is the light modulation frequency (Hz)41,49.

285 286

Photophysical studies

287

Φ∆1O2 and τ1O2 evaluations of Chlorophyll-based Extract

288

The photodynamic evaluation of the FC-Chl drug delivery platform was estimated by

289

the adaptation of the indirect method proposed by Fischer, et al.50, Rabello, et al.51, and

290

Gerola et al.52 At the bottom of a cuvette of polished faces with 1.00 cm optical length,

291

30.0 mg was added to the FC-Chl system. After the addition of a FC-Chl biomedical

292

platform, UA 0.5 µmol L-1 solution (strong singlet oxygen sequestrant and reactive

293

oxygen species) prepared in Mcllvaine buffer 0.10 mol L-1 (pH 7.4) was deposited

294

above the gel inserted into the bottom of the cuvette. The system was adapted to a UV-

295

Vis spectrophotometer, where 6 LEDs were coupled. This equipment works with

296

modulated phase radiation, allowing the LED light to not influence the measurements.

297

The kinetics of UA degradation were accompanied by the acquisition of electronic

298

absorption spectra, at 30.0 °C. The decay of the band at 293 nm for UA was fitted by

299

the bi-exponential kinetics model (Equation 6)51,53:

300

& = & + '( ) *+, + '- ) *.,

301

(6)

302

A1 and A2 are the decay amplitudes of the absorption in the first and second stages,

303

respectively, while k1 and k2 are the degradation rate constants for their respective

304

steps51.

305

The quantifications of the Φ∆1O2 and τ1O2 were performed by the direct method, for

306

time-solved measurements. The Chlorophyll-based extract and Chlorophyll a, in the


Langmuir 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

Page 14 of 58

307

purified form, both in ethanol, were subjected to excitation at 661 nm at 30.0 °C. The

308

1

309

(1270 nm). The τ1O2 was determined by applying the first-order exponential kinetics

310

model to the phosphorescence decay curve. The absorbance at 661 nm was maintained

311

at 0.3 for the Chlorophyll-based extract, Chlorophyll a, and for the standard

312

(Pheophorbide, Φ∆1O2= 0.60 in ethanol)54,55. For the quantification of Φ∆1O2, Equation

313

7 was applied:

O2 generated was identified by the decay of near-infrared phosphorescence intensity

314

φ ∆. PS = 315

A I φ A I Std PS

2

PS

Std

∆ . Std

n τ n τ Std 2

∆ , Std

PS

∆ , PS

(7)

316 317

A is the intensity of absorption at λ661 nm of the PS (photosensitizer) or Std (Standard

318

Pheophorbide in ethanol), τ is the lifetime of singlet oxygen, and I is the area of the

319

emission spectra of the singlet oxygen.

320 321

Morphological Analysis of Systems

322

The FC and FC-Chl platforms (mantained at 25.0 ºC) were submitted to instant freezing

323

using liquid nitrogen at -196 ° C for 20 min. Afterwards, the frozen samples were

324

lyophilized for 48 h56. Segments of the dried samples were carefully deposited on the

325

surface of the stub containing double sided adhesive carbon tape. Subsequently, the

326

samples were metallized by deposition of a thin layer of gold and evaluated by scanning

327

electron microscopy.

328

329

Statistical Analysis


Page 15 of 58 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

Langmuir

330

Statistical analyzes were performed by the t-test using Microsoft Office Excel software,

331

2013 version (Microsoft, 2013, USA) to evaluate the effect of adding Chlorophyll-based

332

extract to the Tsol/gel. Furthermore, the effects of the presence of the active principle and

333

temperature changes in the mechanical and rheological properties (TPA, continuous

334

rheology, and oscillatory rheology in six of the most representative frequencies 0.681,

335

2.144, 4.664, and 10.000 Hz) were compared statistically using a t-test. The level of

336

significance used for rejection of the null hypothesis was 5% (p < 0.05).

337

338

RESULTS

339

Biocompatible drug solubilization systems based on triblock copolymers have been

340

explored in many studies aiming for the development of an ideal topical biomedical

341

platform33. They are advantageous because they have hydrophilic (shell) and

342

hydrophobic (core) microenvironments, capable of solubilizing and stabilizing the

343

monomeric state of several PS34,57.

344

The interaction studies of the polymers mixed with the PS help the evaluation of

345

solubility and stabilization capacity of the PS in the polymer blend. This is important

346

because the state of PS aggregation and nanostructural organization is a fundamental

347

condition for PDT in drug delivery studies33.

348 349

Interaction of the Chlorophyll-based Extract with the F127-C934P Copolymer

350

Blends: Spectroscopic Studies

351

The aim of this analysis was to evaluate the effect of hydrophobic drug diffusion to the

352

micellar interface, and also its influence on the monomerization of the compounds. An

353

effective PS in the PDT must present a relatively high singlet oxygen quantum yield, a


Langmuir 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

354

condition closely linked to the presence of monomerized species in the system.

355

Although the spinach extract is predominantly composed of Chlorophylls,

356

Pheophorbides and Pheophytins a and b (degradation product of Chlorophylls a and b

357

with photodynamic activity), carotenoids, vitamins, lipids, minerals, and flavonoids,26

358

the fluorescence emission (620-800 nm) is selective and demonstrates the partitioning

359

behavior of PS (Chlorophylls, Pheophorbides and Pheophytins a and b) in the

360

microenvironments of the F127-C934P micellar copolymer nanostructures. Figure 1A-

361

B and 1C show two-step studies. In the first one (Figure 1A-B), the effect of the initial

362

interaction of the PS compounds with the increase of the F127 and C394P concentration

363

was evaluated. In this case, fluorescence measurements were taken three minutes after

364

the addition of each aliquot of the polymer solution (fixed stabilization time). In the

365

second one (Figure 1C), the temporal effect of solubilization and redistribution of PS

366

(obtaining the kinetic profile of incorporation) in the combined polymeric system, with

367

PS and copolymer maintained in fixed concentration, was evaluated.

368

Figure 1

369

Figure 1A shows the variation in fluorescence emission intensity with the F127-C934P

370

copolymer concentration. The low intensity of fluorescence emission for the

371

Chlorophyll-based extract in aqueous medium is related to the formation of non-

372

fluorescent aggregates40,58 and the occurrence of thermal relaxation, produced by

373

collision of chlorophyll chromophore with water molecules (internal conversion)10,42,59.

374

Subsequent addition of the polymer blend resulted in initial interaction and

375

monomerization effects of the Chlorophyll-based extract with the nanostructured

376

copolymer microenvironment. This fact allowed for the increase of the maximum of

377

fluorescence emission at 675 nm with the increase of the concentration of the

378

copolymers until reaching the saturation (constant maximum fluorescence emission).


Page 16 of 58

Page 17 of 58 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

Langmuir

379

The data can be better visualized from the isotherm provided in Figure 1B, which

380

demonstrates the interaction and monomerization maximum42 after addition of 3.0 g L-1

381

(0.2 mmol L-1) of F127 and 0.03 g of L-1 of C934P. However, the subsequent addition

382

of the polymers does not result in significant spectrophotometric variations.

383

On the other hand, the kinetic profile presented in Figure 1C demonstrates that in

384

addition to the effects of marked initial interaction, the PS goes through processes of

385

temporal redistribution, capable of stabilizing the Chlorophyll-based extract in the

386

nanostructured copolymer microenvironment in the following hours60. This kinetic

387

profile of drug binding (Figure 1C) was fitted by the associative exponential model (R-

388

Square 0.976), showing two distinct interaction phases obtained in 0.8 (initial

389

accentuated bond) and 369.5 minutes (PS dynamics in the micellar microenvironment

390

with redistribution effects).

391

The interactions of the F127 nanocarrier system with Chlorophyll a (magnesium

392

Chlorophyll) and other isolated derivatives (Pheophorbide, Pheophytin, Zinc-

393

Chlorophyll, and Zinc-Chlorophyllide) were conducted by Gerola et al40,52,61. In general,

394

the authors observed the effective monomerization of the hydrophobic PS in the F127

395

micellar system. The location of the PS in the F127 copolymer was influenced by the

396

chemical structure of the compounds, concerning the presence (and identity) of the

397

metal and the phytyl chain61. Similarly, Campanholi et al62 developed studies that aimed

398

to obtain micellar formulations composed of F127 that allowed for two treatment steps,

399

one dependent on light (photodynamic therapy) and another independent of light (action

400

of naphthoquinones). For this, the authors developed formulations composed by the

401

simultaneous combination of a photosensitizer (derived from purified Chlorophyll a,

402

Pheophorbide and Zn-Pheophorbide) with a Naphthoquinone (purified Lapachol and β-

403

lapachone). The authors noted the maintenance of the monomeric profile and the ability


Langmuir 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

404

to generate singlet oxygen by the photosensitizers after the simultaneous formulation.

405

These results are in accordance with the data presented in this manuscript, which

406

likewise showed pronounced effects of the photosensitizers when inserted in a complex

407

matrix composed of more than one element.

408 409

Mechanical and Rheological Analyzes of the Bioadhesive Thermoresponsive

410

System

411

The rheological studies of flow behavior and textural parameters allow for the

412

prediction of the suitability of the thermodependent biomedical platform in vivo, under

413

physiological conditions. The rheological profiles present important properties to be

414

considered at the time of manufacture, storage, application, and stability of the final

415

product.

416 417

Preparation of Thermoresponsive Bioadhesive Platform

418

The in vitro study of the thermoresponsive bioadhesive properties of the binary system

419

consisting of FC-Chl is relevant for providing predictions regarding the handling,

420

manufacture, administration, and potential clinical use of the formulations.

421

Formulations that present the easy application in cutaneous systems and adherence for

422

prolonged periods in the epidermis are advantageous, since they allow for longer drug

423

permanence with adequate concentration in the active site, thus guaranteeing greater

424

therapeutic effect. In this perspective, determination of the sol-gel transition temperature

425

is an important parameter during the development of topical delivery systems, since

426

formulations with Tsol/Tgel between 25-37 °C behave like liquid at room temperature,

427

allowing for easy application and gel in the body temperature, increasing the time of

428

permanence in the place of performance due to the low fluidity63. Evaluations involving


Page 18 of 58

Page 19 of 58 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

Langmuir

429

oscillatory rheology are important because they allow for the determination of the

430

gelation temperature (Tsol/Tgel transition) from the analysis of the values of the elastic

431

modulus (G') (Figure 2A and 2B) as a function of the temperature increase64.

432

Figure 2

433

Figures 2A and 2B show the sigmoidal curve obtained by the natural logarithm (log G')

434

of the mean elastic modulus G' as a function of the mean temperature, where the

435

inflection point provides the Tsol/Tgel value. Figures also show the ∂2loG'/∂T2 of the

436

natural logarithm of G' by the mean temperature, where the abrupt change from positive

437

to negative (or vice versa), through point zero, gives Tsol/gel for systems. The data shows

438

low values for the elastic modulus at temperatures below the gelation temperature

439

(Tsol/gel), characterizing elastic liquid. This observation is linked to the nature of the

440

intermolecular forces, since the polymer chains under conditions below the Tsol/gel are

441

water-soluble. The elevation of temperature allows for the reorganization of the

442

polymeric micelles, constituted of microenvironments of different polarities. The outer

443

hydrophilic region (shell), composed predominantly of PEO segments, involves the

444

hydrophobic core composed of the PPO groups, with lower water content. The process

445

of reorganization and alignment of micelles results in an abrupt increase in logG', and

446

the sample is classified as a gel or viscous solid. After the constitution of the gel state,

447

G' showed to be independent of the temperature35,63,65. Tsol/gel was obtained by the

448

second derivative of the logarithmic sigmoid of the modulus of elasticity by

449

temperature, reaching values of temperature and standard deviation of 27.9 ± 0.5 °C for

450

FC and 28.8 ± 0.3 °C for FC-Chl, with the coefficient of variation lower than 2% in all

451

cases, and the insignificant variation by t-test (p= 0.1935).

452

Similar data was verified by Junqueira et al33, when studying formulations composed of

453

different ratios of Pluronic® F127 [15.0, 17.5, or 20.0% (w/w)] and C934P [0.15, 0.20,


Langmuir 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

454

or 0.25% (w/w)] combined with methylene blue [0.25, 0.50, or 0.75 % (w/w)] for

455

controlled release in PDT. The authors verified (by oscillatory rheology) measurements

456

in a range of Tsol/gel between 25-37.0 °C for the respective proportions of biomedical

457

platforms. Concordant data was noted by Borghi-Pangoni et al66 when studying the

458

mechanical and rheological properties of the same polymeric blends containing

459

hypericin. The development of platforms suitable for dermatological purposes was also

460

the target of studies proposed by De Souza Ferreira et al35, which explored several

461

proportions of Pluronic® F127 and polycarbophil. From the temperature ramp generated

462

in oscillatory rheology analyzes, the authors noted Tsol/gel values in the range of 27-39

463

°C.

464

As discussed recently in the literature, thermo-dependent changes in the flow of

465

polymer blend formed by F127-C934P point to advantageous properties in terms of

466

manufacture, handling, and administration, since the dermatological preparation

467

presents itself as a liquid of easy manipulation and potting at room temperature. The

468

higher viscosity acquired in contact with body temperature allows for the active

469

principle to remain in the desired place for longer, optimizing the release and promoting

470

greater photodependent therapeutic effects.

471

472

Continuous Shear (flow) Rheometry

473

The evaluation of the flow properties of bioadhesive drug delivery platforms provides

474

the understanding of the structure of the system against the various continuous tensions

475

whose formulations are submitted during the preparation, storage, and removal of

476

administration steps. The rheograms of flow shown in Figure 3 are related to the force

477

required to allow shearing in lamellar layers of the gel as a function of the velocities of


Page 20 of 58

Page 21 of 58 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

Langmuir

478

displacement of the flow layers. The analysis of the upward and downward flow curves

479

of the platforms provide information on the behavior of the fluid with regard to changes

480

in viscosity with applied tension, suspension stability, consistency index with

481

temperature and the addition of active principle. Furthermore, the analysis provides a

482

prediction of the biomedical platform's ability to recover to its initial condition after the

483

applied stress ceases.

484

Figure 3

485

Figure 3 shows the rheogram of flow of the polymer formulations in the absence (FC)

486

and presence of Chlorophyll-based extract (FC-Chl) at 25.0 and 37.0 °C. The verified

487

upward and downward curves are related to two study steps, one reporting the increase

488

(ascending) and the other for the (descending) reductions of the shear rate. It is verified

489

that most of the flow curves presented do not overlap, characterizing different degrees

490

of thixotropy34, a property related to the occurrence of viscosity variation with time.

491

This is due to Brownian movements that lead to structural rearrangements. In addition,

492

the rheograms obtained do not start from the origin indicating a plastic behavior, where

493

the yield value can be obtained by extrapolating the tangent line to the linear region of

494

the curve33,34,67. For statistical comparisons of the effect of the temperature changes and

495

addition of Chlorophyll-based extract in the flow properties of the formulations, the

496

outward curve of each rheogram was defined by the Power Law model, which allows

497

for obtaining the consistency index (k) and flow behavior (n) (Table 1).

498

Table 1

499

The formulations presented in Table 1 demonstrated flow behavior indices below unity,

500

which characterizes a non-Newtonian shear-thinning behavior (n

Biomedical Platform Development of Chlorophyll-based extract for

Recommend Documents