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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
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Langmuir
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Biomedical Platform Development of Chlorophyll-based extract for Topic
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Photodynamic Therapy: Mechanical and Spectroscopic Properties
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Katieli da S.S. Campanholi*,a, Gustavo Bragaa, Jéssica B. da Silvab, Nicola L. da
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Rochad, Lizziane M. B. de Franciscob, Évelin L. de Oliveiraa, Marcos L. Bruschib,
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Lidiane V. de Castro-Hoshinoc, Francielle Satoc, Noboru Hiokaa and Wilker Caetanoa.
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a
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University of Maringá, Maringá, Paraná, Brazil. dInstitute of Chemistry, State University of
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Campinas.
Department of Chemistry, bDepartment of Pharmacy and cDepartment of Physics, State
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ABSTRACT
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Photodynamic therapy (PDT) is a therapeutic modality that has shown effectiveness in
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the inactivation of cancer cell lines and microorganisms. Treatment consists of
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activating the photosensitizer (PS) upon light irradiation of adequate wavelength. After
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reaching the excited state, the PS can handle the inter-system conversion through energy
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transfer to the molecular oxygen, generating reactive oxygen species. This especially
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applies to singlet oxygen (1O2), which is responsible for the selective destruction of the
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sick tissue. Photosensitizing compounds (Chlorophylls and derivatives) existing in the
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spinach extract have applicability for Photodynamic Therapy. This study aimed to
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develop and characterize the thermoresponsive bioadhesive system composed by
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Pluronic® F127 20.0% and Carbopol® 934P 0.2% (w/w) (FC) containing Chlorophylls-
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based extract 0.5% (w/w) (FC-Chl). Mechanical, rheological, in vitro release, sol-gel
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transition temperature and ex vivo permeability of spinach extract PS components
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(through pig ear skin) were investigated. Furthermore, photodynamic activity of the
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system was accessed through the uric acid and time-solved measures. The sol-gel
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transition temperature obtained for the FC-Chl system was 28.8 ± 0.3 °C. Rheological
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and texture properties of the platform were suitable for use as a dermatological system,
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exhibiting easy application and good characteristics of retention in the place of
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administration. In vitro release studies showed the presence of two distinct mechanisms
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that reasonably obey the zero-order and first-order kinetics models. PS components
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presented skin permeability and reached a permeation depth of 830 µm (between the
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epidermis and dermis). The photodynamic evaluation of the FC-Chl system was
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effective in the degradation of uric acid. The quantum yield (Φ∆1O2) and life time (τ1O2)
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of singlet oxygen showed similar values for the spinach extract and the isolated
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Chlorophyll a species in ethanol. These results allowed for the classification of the FC-
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Chl platform as potentially useful for delivery of Chlorophyll-based extract in the topic
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Photodynamic Therapy, suggesting that it is worthy for in vivo evaluation.
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INTRODUCTION
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Photodynamic therapy (PDT) consists of a minimally invasive therapeutic modality and
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has shown promising results for the treatment of primary and advanced carcinomas,
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metastases located in the head and neck regions, external lesions such as leukoplakia of
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the buccal cavity1, palliative treatment of inoperable cholangiocarcinoma2, urological
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neoplasms3, basal cell carcinoma, Kaposi's sarcoma (common in AIDS patients),
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melanoma, paramecia4, skin tumors4, viroses and fungal infections1,5. Compared with
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other medical modalities, including surgery, radiotherapy, chemotherapy and
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immunotherapy, PDT offers high selectivity. This culminates in minimal systemic
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toxicity, in addition to greater effectiveness on multiple drug resistant cells (MDR)6,7
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and the possibility of combining with other therapeutic modalities8,9.
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The treatment consists basically of the administration of a photosensitizer (PS), which is
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activated to the excited singlet state (1PSexc)8,10. The decay of the 1PSexc can occur in a
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radiative, a non-radiative or an inter-system crossing involving vibrational levels of the
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same energy and different multiplicities, whereas spin inversion occurs from 1PSexc to
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3
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neighboring molecules by Type 1 reactions, with formation of superoxide radical anion
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O2-*, hydroxyl radical OH*, hydrogen peroxide H2O2 and Type 2 reactions, where the
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drug in its excited triplet state can transfer its excess energy to the molecular oxygen
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(3O2), forming singlet oxygen (1O2), responsible for inciting oxidative damage to
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cells4,14–18.
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Due to economic factors, PS obtained from abundant and natural raw materials attracts
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great interest19. The Chlorophylls, Pheophorbides and Pheophytins a and b, found in
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New Zealand spinach extract (Tetragonia expansa)20, are prominent in PDT and
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frequently reported in several studies involving glandular malignancies, human lung
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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
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(LoVo), in the evaluation of mutagenic and genotoxic effects, as well as studies of
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photodynamic inactivation of various microorganisms20–25. New Zealand Spinach is a
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grassy herbaceous species with dark green leaves rich in photosensitizing compounds
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(Chlorophylls, Pheophorbides and Pheophytins a and b). Its low cost combined with the
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wide cultivation and activity by PDT has aroused the interest of researchers. Thus,
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spinach extract has become a protagonist for several lines of scientific research24–28. The
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use of the extract in the PDT offers economic and environmental advantages over
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purified chlorophyll compounds, as it is obtained without the use of purification steps
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responsible for the generation of toxic wastes, which impact humans and the
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environment24,25. Furthermore, the use of extracts in PDT has offered advantages due to
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the different degrees of skin permeation of the PS compounds, distinct mechanisms of
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action and, above all, the synergic combined effect of the photoactive compounds
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present in the extract shown in recent studies29.
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The application of photoinduced treatment in skin lesions requires delivery systems
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displaying suitable physicochemical, mechanical and rheological characteristics.
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Moreover, the lipophilic nature of the PS demands the use of a platform capable of
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stabilizing the monomeric photoactive state of the chlorophylls. A topical
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administration system should have adequate consistency for manufacturing, storage and
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application. This means that the formulation should exhibit easy removal from the
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storage container, and also have adequate consistency when exposed to the skin to
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increase the permanence time of the drug at the place of action. Such properties are
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found in aqueous gels, which may be composed from the thermoresponsive copolymers
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of the Pluronic® class, responsible for the solubulization of lipophilic drugs30.
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F127 are thermoresponsive copolymers composed of polyethylene oxide (PEO)
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segments that give rise to the external hydrophilic shell and polypropylene oxide (PPO)
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responsible for the formation of the hydrophobic core31,32. In aqueous medium and with
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conditions of suitable concentration and temperature, the unimers associate themselves
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and give origin to copolymeric micelles, which can solubilize medicines of diverse
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chemical natures. These systems can be used as gelling agents in the concentration
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range of 20.0 % (w/w), giving semi-solid colloidal systems. When subjected to
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temperature increases (in addition to the Tsol/gel critical transition temperature) in the
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isotropic micellar structure, a cubic hexagonal core with more ordered segments is
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formed. This significantly increases the consistency of the biomimetic matrix. The high
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degree of structured water, and the quantity contained in the systems, allows for a
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smooth texture that ends up mimicking natural tissues. This increases the
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biocompatibility of the biomechanical systems32–35.
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The improved adhesiveness, stability, and consistency of drug delivery platforms can be
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achieved with the addition of carbomers. These are synthetic hydrophilic polymers
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derived from poly(acrylic acid) and feature high consistency gels at a pH higher than
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4.75 (pKa). The carbomer Carbopol® 934P, oral pharmaceutical grade polymer, features
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long, high molecular weight chains containing about 56-68% polyacrylic acid cross-
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linked with sucrose allyl esters35.
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The recognition of the advantageous properties of the polymer blends composed of
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different proportions and combinations, and types of Pluronic® with Carbopol®, aroused
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the interest of several researchers. These days, this constitutes a thermodependent
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biomedical matrix extensively explored and consolidated in the solubilization of
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drugs33,34,36–39. Therefore, this study aims to combine for the first time the consolidated
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activity of the natural PS inserted in the New Zealand Spinach matrix with the potential
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thermoresponsive pharmaceutical platform composed of Pluronic® F127 and Carbopol®
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934P, for the extensive treatment of primary or secondary morphological skin lesions.
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EXPERIMENTAL SECTION
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Materials. Spinach extract was obtained from New Zealand Spinach (Tetragonia
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tetragonioides) leaves (14.65±0.99 % w/w of mean yield±standard deviation), following
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the methodology previously reported40. F127 Pluronic® (poloxamer 407; molar mass=
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12600 g mol−1) and uric acid (UA) were purchased from Sigma-Aldrich (São Paulo,
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Brazil). Carbopol® C934P was purchased from Lubrizol Advanced Materials (São
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Paulo, Brazil), and Triethanolamine was bought at Galena (Campinas, Brazil).
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Membrane HAWP04700 MF in mixed ester cellulose (Millipore), 0.45 µm pore, 4.7 cm
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of diameter (white, smooth), was purchased from Merck (São Paulo, Brazil). All
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solvents employed were of analytical grade and used without further purification.
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Instruments and Characterization. Studies of electronic absorption and fluorescence
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emission were performed in a UV-Vis spectrophotometer model Varian Cary 50
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(Agilent Technologies, São Paulo, Brazil) and Varian Cary Eclipse spectrofluorimeter
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(Agilent Technologies, São Paulo, Brazil), respectively.
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Rheological measurements were conducted on a MARS II rheometer (Thermo-Haake,
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Thermo Fisher Scientific Inc., Newington, Germany), using a 60 mm diameter steel
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cone plate geometry, separated by a gap of 0.052 mm. The FC and FC-Chl was
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carefully applied to the lower plate, allowing for the minimum shear and a minimum
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stabilization time of 1 minute before the determination. The RheoWin (Haake®)
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software version 4.10.0000 was used in oscillatory and continuous rheology.
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The texture profile analysis (TPA) was performed using a TA-XTplus texture analyzer
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(Stable Micro Systems, Surrey, UK), equipped with a polycarbonate analytical probe
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(10 mm diameter).
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The degradation profile of the UA by the PS contained in the FC-Chl platform was
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obtained in UV-Vis spectrophotometer, model Cary 50 (Agilent Technologies, São
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Paulo, Brazil), with 6 LEDs (9 mW, λmax= 636 nm) attached.
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The quantification of the quantum yield (Φ∆1O2) and lifetime (τ1O2) of the singlet
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oxygen was performed with a Near-Infrared fluorometer (NIR) (Edinburgh Analytical
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Instruments, Scotland, U.K.) coupled to a photomultiplier R5509 (Hamamatsu Co.,
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New Jersey, USA) and with a Hewlett Packard 54510B oscilloscope. The PS excitation
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was performed by a LASER Continuum Nd: YAG Sarulite III (Spectron Laser
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California, USA) at 661 nm (pulse of 5 ns and energy ≤ 5mJ/pulse). The electronic
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absorption spectra were acquired by Shimadzu UV-240 PC spectrophotometer (São
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Paulo, Brazil).
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The photoacoustic spectroscopy (PAS) measurements were performed using an
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assembled experimental setup, composed by a 1000-W Xenon model 68820 arc lamp
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(Oriel Instruments, California, USA) and a model 77250 monochromator (Oriel
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Instruments, California, USA), with 3.16-mm of input and output slits. Diffraction
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grating was performed with model 77296 (Oriel Instruments, California, USA), and
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model SR 540 (Stanford Research Systems, California, USA)41 was used for UV-Vis
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spectral and as a mechanical chopper. The spectra were recorded in intervals between
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200 and 800 nm, and the nominal power of the light source was 800 W with modulation
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frequencies of 15 Hz.
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The morphological analysis of the formulations was performed using a scanning
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electron microscope Quanta 250 (Thermo Fisher Scientific-FEI, Oregon, USA) for
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lyophilized samples in a Micromodulyo freeze dryer (Thermo Electron Corporation,
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Oregon, USA). The metallizer used for the preparation of scanning electron microscope
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samples was Sputter Coater, model SCD 050 (Bal-Tec, California, USA).
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Preparation of Thermoresponsive and Bioadhesive Platform
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The formulation was prepared by the addition of 150.0 mg of dried spinach extract and
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6.0 g of F127 (representing 0.5 % and 20.0 % w/w of the preparation, respectively) in
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ethanol, followed by evaporation of the solvent and formation of a solid thin film,
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which was kept in a desiccator for 24 h. Subsequently, the polymeric film was hydrated
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with 60.0 mg of C934P (0.2 % w/w) dispersed in 23.9 mL of water under vigorous
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stirring in an ice bath (obtaining a mixture of F127-C934P). The preparation had the pH
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neutralized with triethanolamine (pH 7). FC and FC-Chl samples were stored and
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protected from the light at 4.0 °C. The systems were kept at rest for 24 h prior to
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analysis38. The systems were named as FC-Chl, and they referred to the matrix
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composed of 20.0% F127, 0.2% C934P, and 0.5% w/w of the spinach extract (source of
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PS compounds). The gels obtained without addition of spinach extract were
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denominated FC and used as a reference in the analyses.
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Physicochemical and Photodynamic Properties of the Thermoresponsive Drug
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Delivery Platform
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Interaction of the Chlorophyll-based extract with the F127-C934P Copolymer Blends:
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Spectroscopic Studies
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The interaction and solubility capacity studies of Chlorophyll-based extract (Chl) with
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the F127-C934P combination were performed by titration of aliquots of the solution
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composed of F127 4.0 % (w/v) and C934P 0.04 % (w/v) to a solution of 46.4 mg L-1 of
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spinach extract, both prepared in a Mcllvaine buffer (0.10 mol L-1; pH 7.4)42. The
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interaction was monitored by the acquisition of fluorescence emission spectra at 680 nm
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(excitation at 537 nm, Soret bands), obtained at 37.0 °C in a Cary Eclipse ACS Paragon Plus Environment
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spectrofluorimeter43. Furthermore, incorporation kinetics studies were conducted
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starting from 25.0 mg L-1 spinach extract solution in F127 4.0 % and C934P 0.04 %
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(w/v) mixed in Mcllvaine buffer (0.10 mol L-1; pH 7.4). The temporal effect of binding
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and monomerization of the main PS compounds contained in the spinach extract
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(Chlorophylls, Pheophorbides and Pheophytins a and b) was accompanied by the
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acquisition of fluorescence emission spectra in 680 nm (excitation at 498 nm, Soret
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bands). The kinetic curve obtained was fitted by the associative exponential model
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(Equation 1): = + − . + − .
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(1)
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where P corresponds to the values of the property (fluorescence emission intensity) at
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the time (t), k1obs and k2obs are the kinetics constants for the steps, Q and R are for the
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decay amplitudes of the property in the first and second phases, respectively44.
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Mechanical and Rheological Analyzes of the Thermoresponsive Drug Delivery
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Platform
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Continuous Shear (flow) Rheometry
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These measures were performed in continuous flow mode, at temperatures of 25.0 and
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37.0 °C. The flow curves for FC and FC-Chl were analyzed at several shear rates
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(between 10 to 2000 s-1) with cycles of increases and reductions over a period of 150 s,
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and then held at the maximum shear rate (upper limit) for 10 s. The flow properties of
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each formulation were investigated in five-fold replicates and the upward flow curves
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were modeled using the Power Law (Ostwald-de-Waele; Equation 2) and Herschel-
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Bulkley (Equation 3) equations:
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=
(2)
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= +
(3)
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where σ is the shear stress (Pa), σ0 is yield stress (Pa), k is the consistency index [(Pa
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s)n],
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The thixotropy and hysteresis areas were calculated using RheoWin (Haake®) software.
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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.
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Oscillatory Rheometry
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The analysis was performed in oscillatory mode, at 25.0 °C and 37.0 °C. The linear
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viscoelastic regions of each formulation (FC and FC-Chl) were previously determined.
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Afterwards, the frequency sweep analysis was performed from 0.1 to 10.0 Hz, in a state
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of fixed stress. The η' (dynamic viscosity), G' (elastic modulus), G" (loss modulus), and
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tan δ (loss tangent) were calculated using RheoWin (Haake®) software. The viscoelastic
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properties of at least three replicate samples were determined35,36,45,46.
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Determination of the Sol−Gel Transition Temperature
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The determinations of the sol-gel transition temperature (Tsol/gel) for the FC and FC-Chl
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platforms were conducted with the rheometer in oscillatory mode. The analysis was
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conducted in the linear viscoelastic region using the temperature ramp method. The
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temperature scanning analysis was performed with a heating rate of 10 °C min-1 and the
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temperature range was between 5.0 and 60.0 °C (frequency of 1.0 Hz). The temperature
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where G' was located was an intermediate region between the solution and gel states
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named Tsol/gel, for formulations in which dynamic viscosity significantly increased with
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increasing temperature. The determination of Tsol/gel was evaluated through the second
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derivative of the natural logarithm of the mean modulus of elasticity as a function of the
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mean temperature (∂2logG'/∂T2), where the abrupt change from positive to negative
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(passing through zero point) gives Tsol/Tgel for systems. Measurements were performed
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with at least five replicates for each formulation33,36.
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Texture Profile Analysis
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In the compression mode of the texture analyzer, the probe was compressed twice into
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each sample of FC and FC-Chl (stored in glass vials and held at rest), at a depth of 15
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mm, at a rate of 2 mm s-1, and with a permanence time of 15 s between the end of the
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first and the beginning of the second compression. The analysis was performed at 25.0
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°C and at 37.0 °C for at least five replicate samples. The parameters of compressibility,
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hardness, cohesiveness, elasticity, and adhesiveness were obtained from the results of
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the plot force by time and by distance34,36.
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Release and Cutaneous Permeation of Chlorophyll-based Extract
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In vitro Spinach Extract Release
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The release studies were performed at 37.0 °C using the FC-Chl (30.0 mg) platform
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inserted in a dialysis bag formed by Millipore HAWPO4700 MF smooth membrane.
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The receptor solution medium was composed of F127 4.0 % (w/v) in an aqueous
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medium, ensuring the sink conditions (F127 concentration equals at most 10 %
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saturation concentration). The system was prepared by adding 3.00 ml of the receptor
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medium in a quartz cell, with magnetic stirring. The FC-Chl dialysis bag was immersed
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in the upper region of the receptor solution, freeing the location of the light beam
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incident for kinetic measurements through fluorescence emission at 680 nm by time.
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The adjustment of the kinetic profile of fluorescence emission to obtain the order of
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reaction was performed by Equation 4:
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= −
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(4)
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where P∞ is the fluorescence property at infinity, Pt is fluorescence property at time (t),
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with k as the first-order kinetic constant47.
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Ex vivo Study of Permeation and Skin Retention
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Preparation of Pig Skin
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As it resembles human skin in terms of morphology, pig skin was used as an ex vivo
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model system in gel permeation studies48. The pig skin segments were supplied by the
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Experimental Farm of Iguatemi (EFI), which belongs to the State University of
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Maringá, Brazil. Samples were taken from young, white animals, submitted to recent
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slaughter. After selecting regions of skin with absences of wounds, warts, and bruises,
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segments of the dorsal side of the auricle were excised using a surgical scalpel, with a
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subsequent removal of subcutaneous fat from the skin samples. The partitions were
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stored at -18.0 ° C until the date of use.
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Permeation Studies of FC-Chl: PAS Technique
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After the thermal equilibrium of the pig ear in the environment, 30.0 mg of FC-Chl
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platform was spread over an area of 1 cm2 on the corneal side of the cutaneous segment
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of the pig ear. The photoacoustic measurements were performed after 30 and 240
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minutes, on both sides of the segment (epidermis and dermis)41,49. After the samples
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analyzes had thickness measured with a digital micrometer, depth values of drug
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penetration into the skin were calculated using Equation 5:
282
#
! = "$.%
(5)
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where µ is the thermal diffusion length (m), D is the thermal diffusivity of the skin
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(3.10-8 m2 s-1), and f is the light modulation frequency (Hz)41,49.
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Photophysical studies
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Φ∆1O2 and τ1O2 evaluations of Chlorophyll-based Extract
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The photodynamic evaluation of the FC-Chl drug delivery platform was estimated by
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the adaptation of the indirect method proposed by Fischer, et al.50, Rabello, et al.51, and
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Gerola et al.52 At the bottom of a cuvette of polished faces with 1.00 cm optical length,
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30.0 mg was added to the FC-Chl system. After the addition of a FC-Chl biomedical
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platform, UA 0.5 µmol L-1 solution (strong singlet oxygen sequestrant and reactive
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oxygen species) prepared in Mcllvaine buffer 0.10 mol L-1 (pH 7.4) was deposited
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above the gel inserted into the bottom of the cuvette. The system was adapted to a UV-
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Vis spectrophotometer, where 6 LEDs were coupled. This equipment works with
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modulated phase radiation, allowing the LED light to not influence the measurements.
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The kinetics of UA degradation were accompanied by the acquisition of electronic
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absorption spectra, at 30.0 °C. The decay of the band at 293 nm for UA was fitted by
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the bi-exponential kinetics model (Equation 6)51,53:
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& = & + '( ) *+, + '- ) *.,
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(6)
302
A1 and A2 are the decay amplitudes of the absorption in the first and second stages,
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respectively, while k1 and k2 are the degradation rate constants for their respective
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steps51.
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The quantifications of the Φ∆1O2 and τ1O2 were performed by the direct method, for
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time-solved measurements. The Chlorophyll-based extract and Chlorophyll a, in the
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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
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surface of the stub containing double sided adhesive carbon tape. Subsequently, the
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samples were metallized by deposition of a thin layer of gold and evaluated by scanning
327
electron microscopy.
328
329
Statistical Analysis
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Statistical analyzes were performed by the t-test using Microsoft Office Excel software,
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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
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condition closely linked to the presence of monomerized species in the system.
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Although the spinach extract is predominantly composed of Chlorophylls,
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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).
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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
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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
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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,
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or 0.25% (w/w)] combined with methylene blue [0.25, 0.50, or 0.75 % (w/w)] for
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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
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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
