Use of Spectroscopic Techniques for Evaluating the Coupling of

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The Use of Spectroscopic Techniques for Evaluating the Coupling of Porphyrins on Biocompatible Nanoparticles - a Potential System for Photodynamics, Theranostics and Nano-drug Delivery Applications Lais N. Magno, Fabio C. Bezerra, Luiz E. S. Freire, Rubens A. Guerra, Andris Figueiroa Bakuzis, and Pablo Jose Goncalves J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.6b10314 • Publication Date (Web): 16 Feb 2017 Downloaded from http://pubs.acs.org on February 21, 2017

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The Journal of Physical Chemistry

The Use of Spectroscopic Techniques for Evaluating the Coupling of Porphyrins on Biocompatible Nanoparticles - a Potential System for Photodynamics, Theranostics and Nanodrug Delivery Applications.

Lais N. Magno†, Fábio C. Bezerra†, Luiz Eduardo S. Freire†, Rubens A. Guerra†,‡, Andris F. Bakuzis† and Pablo J. Gonçalves†,•,* †

‡

Instituto de Física, Universidade Federal de Goiás, 74690-900, Goiânia, GO, Brazil. Faculdade Santa Rita de Cássia, Av. Adelina Alves Vilela, nº 393, Jd. Primavera, 75.524-680, Itumbiara-GO • Programa de Pós-graduação em Química, Universidade Federal de Goiás, 74690-900, Goiânia, GO, Brazil.

*To whom correspondence should be addressed. E-mail: [email protected]

Phone: +55 62 3521-1014, ext. 251, Fax: +55 62 3521-1014, ext. 247.

1 ACS Paragon Plus Environment


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Abstract Modern medicine has been searching for new and more efficient strategies for diagnostics and therapeutics applications. Considering this, porphyrin molecules have received great interest for applications in photodiagnostics and phototherapies, even as magnetic nanoparticles for drug-delivery systems and magnetic-hyperthermia therapy. Aiming to obtain a multifunctional system, which combines diagnostics with therapeutic functions on the same platform, the present study employed UV/Vis absorption and fluorescence spectroscopies to evaluated the interaction between mesotetrakis(p-sulfonatofenyl) porphyrin (TPPS) and maghemite nanoparticles (γ-Fe2O3). These spectroscopic techniques allowed us describe the dynamics of coupling porphyrins on nanoparticles and estimate the number of 21 porphyrins per nanoparticle. Also, the binding parameters, such as the association constants (Ka = 8.89×105 M-1) and bimolecular quenching rate constant (kq = 2.54×1014 M-1s-1) were obtained. These results suggest a static quenching process where the electrostatic attraction plays an essential role. The work shows that spectroscopic techniques are powerful tools to evaluate the coupling of organic molecules and nanoparticles. Besides, the system studied provides a relevant background for potential applications in bionanotechnology and

nanomedicine,

such

as:

1)

nano-drug

delivery

system,

2)

photodiagnostics/theranostics and/or 3) a combined action of Photodynamic and Hyperthermia Therapies, working in a synergetic way.


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1. Introduction Photodynamic Therapy (PDT) is a treatment modality of solid tumors based on the administration of a light-activated drug (photosensitizer) followed by the visible light illumination of the tumor and consequent formation of reactive oxygen species (ROS).1-3 Besides its success against cancer, the photodynamic action has also shown good results in treatments of psoriasis, retina disease, and against micro-organisms.4-7 In recent years, significant efforts have been made to develop: more efficient photosensitizer drugs (PS); new methodologies to characterize PS potential; new strategies for photodynamic action; new model systems to evaluate the photodynamic activity; and/or suitable nanotechnological platforms to improve drug delivery to tumor tissue.8-13 Among the PSs commonly studied, organic molecules such as porphyrins, phthalocyanines, chlorins, bacteriochlorins have generated considerable interest for uses in PDT.14,15 In particular, the anionic meso-tetrakis(p-sulfonatofenyl) porphyrin (TPPS) presents a potential for application in cancer therapy, and infectious diseases because of its photostability, high triplet states and singlet oxygen quantum yield.16-18 On the other hand, its high polarity may restrict the passive diffusion across the plasma membrane14 and the association with efficient drug delivery systems may be a powerful strategy to facilitate cellular uptake. Besides, some adverse effects of TPPS were reported19,20 which may be overcome by using targeted materials such as nano-vehicles for drug delivery systems.21 In order to improve efficacy and safety for drug delivery systems, different nanomaterials have emerged aiming at biomedical applications. Among them, magnetic nanoparticles (MNPs) have received close attention.22,23 MNPs obtained from oxides, such as magnetite and maghemite, exhibit particular features such as ultrafine size, 3 ACS Paragon Plus Environment


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biocompatibility and interesting magnetic properties for use in drug delivery application.22,23 In addition, several magnetic nanoparticle formulations have already been approved for clinical use as contrast agents for magnetic resonance imaging.24 Biocompatible magnetic fluids consist of ferrite nanoparticles dispersed as a magnetic colloid that may also be used for local magnetic Hyperthermia Therapy (HPT).25,26 HPT uses an external alternating magnetic field to promote an increase in local temperature (~42-44oC) and then cause the preferential destruction of cells and tissues.25-29 Recently it has been suggested that the combination of porphyrins with nanocomposites could potentially be used as diagnostic and/or therapeutic agents for tumors and other diseases.31,31 Nowostawska et al.32 synthesized a new multimodal (magnetic and fluorescent) porphyrin-magnetite nanoconjugate for biological imaging. Zhao et al.33-36 used a new nanocomposite of TPPS porphyrin with TiO2 nanowhiskers as effective bio-imaging and photodynamic therapeutic agents for rheumatoid arthritis theranostics. Besides, TPPS encapsulated into the ZnO nanomaterials have increased the generation of singlet oxygen37 and the photobactericidal activity of ZnO nanorods.38 A combined action of local HPT and PDT has been proposed over the last years.39-41 It was observed that for magnetic nanoemulsion containing maghemite nanoparticles and a PS drug, the photophysical properties and triplet quantum yield of PS does not change and was excellent for photodynamic activity and local hyperthermia therapy.42 Maghemite nanoparticles (γ-Fe2O3) also have a particular interest because they might be potential agents for cancer treatment by magnetic hyperthermia therapy.43 In addition, Kyoung et al.44 demonstrated the photodynamic/hyperthermia-combined therapeutic success of magnetite nanoparticles and photosensitizers conjugated with enhanced efficacy against cancer.


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Taking this into account, in the present work, a system that combines the actions of drug delivery, photodiagnostics and therapeutics on the same platform is proposed. In particular, we studied the coupling of TPPS porphyrin on γ-Fe2O3 nanoparticles aiming at applications in modern medicine as potential diagnostic and therapeutic agents. The results show that spectroscopic techniques are powerful tools to evaluate the coupling of organic molecules and nanoparticles. Using UV/Vis absorption and fluorescence spectroscopies, we were able to describe the dynamics of coupling porphyrins on nanoparticles, obtain the binding parameters (association constants and bimolecular quenching rate constant) and estimate the number of porphyrins per nanoparticle. Besides, the present system has great potential for use in drug delivery, and it can also use the fluorescent properties of TPPS porphyrin to be employed as a fluorescent probe agent for photodiagnostics, and/or in a combined action of PDT and HPT therapies. This combination can works in a synergetic way that could lead to an expected enhancement of the target tissue damage after minimal doses of heat dissipation and visible-light photosensitization.

2. Experimental Section Free-base TPPS molecules were purchased from Porphyrin Products Inc and their molecular structure is presented in Fig. 1.45 It is well known that hydrogen atoms in the central ring of TPPS porphyrin can be protonated in an acid environment.46-48 In particular, it has a pK value close to 5.0, i.e., it is in its non-protonated form at pH values > 6.0 and in a diprotonated one at pH < 4.5. The pH values and the protonation state can be adjusted by adding appropriate amounts of HCl or NaOH to the stock solution.



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Ionic magnetic fluids containing maghemite nanoparticles dispersed in water were prepared from the oxidation of magnetite, which were synthesized by the coprecipitation method.49 Maghemite magnetic nanoparticles (MNPs) are an ionic magnetic fluid, whose superficial charge is defined by the pH of a solution. In the present work, acid pH (close to 4.0) was chosen as the ionic MNPs are highly stable at this pH value resulting in a cationic surface charge density.50 -

SO 3-

O3S

~0.6 nm N NH ~0.7 nm

HN N

-

O3 S

SO 3-

Figure 1. Molecular structure of TPPS.45

The MNPs were prepared by the chemical co-precipitation method.51 The characterization of MNPs was performed by X-Ray Diffraction (XRD) and Vibrating Sample Magnetometer (VSM). These measurements were performed in Lab X XRD6000 equipment, from Shimadzu and ADE Magnetics, EV9 System model, respectively. The interaction between TPPS porphyrins with biocompatible MNPs were monitored through the UV/Vis absorption and fluorescence emission spectroscopic techniques as a function of its relative concentrations. The UV/Vis absorbance spectra were obtained using a Beckmann DU640 UV-Vis spectrophotometer, and the fluorescence emission spectra were measured using a Fluorolog-3 spectrofluorometer 6 ACS Paragon Plus Environment

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(Horiba Jobin Yvon Inc). All measurements were performed at room temperature (~27oC). In order to avoid the inner filter effect, associated to absorption of a molecule (or a system) at the excitation and emission wavelengths,52,53 the fluorescence intensity values used to obtain the binding constant were corrected using the following equation: = 10 / where Fcorr and Fm are the corrected and measured fluorescence, respectively. Aexc and Aem are the absorbance of the sample at excitation and emission wavelengths, and dexc and dem the cuvette pathlength in the excitation and emission direction (in cm units), respectively.

3. Results & Discussion 3.1. Photophysical characterization of TPPS porphyrin The nitrogens of the porphyrin central pyrrole ring may be diprotonated, which cause changes in its photophysical properties.46 Fig. 2a presents the absorption and Fig. 2b the emission spectra of TPPS porphyrin in non-protonated and diprotonated forms. The absorption spectrum of TPPS is well-characterized presenting two mains bands at the UV-Vis region: the B-band (Soret band) located between 380 and 450 nm, and the Q-bands between 500 and 650 nm. The protonation of the central ring of TPPS causes a red shift in the Soret band and the Q-bands are drastically modified. Besides, the two peaks observed in the fluorescence emission spectra for the nonprotonated species (642 and 700 nm) are reduced to just a single broad structureless band (672 nm) and a small shoulder located at the red part of the spectrum. These results are in agreement with the literature46 and can be interpreted in accordance to Gouterman´s four-orbital model, where the reduction of the Q-band structuration is an indicative of the porphyrin ring



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symmetry increase.54 Protonation of porphyrins causes significant changes in the photophysical and nonlinear properties46-48,55 and can be considered the first step towards molecular aggregation.56

Figure 2. (a) Absorption spectra and (b) fluorescence emission spectra of TPPS porphyrin in diprotonated (doted) and non-protonated (solid line) forms. The inset in (a) shows the Q bands of both forms in more details.


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3.2. Nanoparticles characterization XRD is an analytical technique primarily used for phase identification of a crystalline material and can provide information on unit cell dimensions. The XRD pattern of the maghemite nanoparticles used in this work is shown in Fig. 3a. The pattern illustrates that the maghemite sample used matches the standard maghemite found in the literature.50 The six diffraction peaks: (220), (311), (400), (422), (511), and (440) suggest that the obtained XRD pattern corresponds to γ-Fe2O3.50,57 The main peak of the cubic maghemite structure is centered at 2θ = 35.8 and corresponds to the crystalline plane with Miller indices of (311). Besides, the XRD peaks are characteristic of the iron oxide spinel structure, and can be attributed to γ-Fe2O3.57 The broadening of the most intense diffraction peak of the stock maghemite powder sample provided the average diameter of 4.8 nm of the nanocrystalline domain49. VSM systems are used to provide magnetic properties of materials as a function of the magnetic field, temperature and time. They are ideally suited for research and development, production testing, quality and process control. Fig. 3b shows the magnetization curve obtained by VSM measurement, from which one can obtain the saturation value of the colloid (0.15 emu/g) and the magnetic moment (0.0082 emu). The low value of the magnetization reveals the magnetic particle concentration of the stock sample. From those data, it was possible find out the number of particles in fluid (3.6 × 1015 MNPs/cm3).



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Figure 3. (a) X-ray diffraction pattern and (b) magnetization curve of maghemite fluid used.


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3.3. Coupling of porphyrins on nanoparticles

Fig. 4 presents the effect of maghemite nanoparticles on the fluorescence spectra of diprotonated TPPS porphyrin excited at two different wavelengths. Fig. 4(a) shows the fluorescence spectra upon excitation which corresponds at main Q band of the diprotonated form (640 nm), while in Fig. 4(b), it shows the excitation at main Q band of the non-protonated form (515 nm). The insets show the fluorescence intensity as a function of nanoparticle concentration. The results show that adding nanoparticles causes a significant decrease in the fluorescence of TPPS porphyrins dipronated until their extinction (inset of Fig 4a). On the other hand, a new emission band is formed when the sample is excited at 515 nm, which can be attributed to deprotonation of TPPS (Fig. 2b).

Fig. 5 shows the change in the absorption spectra during the titration process of MNPs (from 0 to 0.59 µM) in the presence and in the absence of TPPS porphyrins (10

µM), Fig. 5a and the inset of 5a, respectively. Fig. 5a shows that the addition of MNPs causes an increase in absorption in the range of 480 – 750 nm, except for a slight reduction of the Q band (close to 650 nm). However, there are no appreciable spectral changes in the profiles of the UV-visible spectra of TPPS, which shows that the interaction between the MNPs and porphyrins does not cause the formation of molecular aggregates.56 Self-aggregation induces new vibration modes, compared with the monomeric species, which are associated with relative motions of monomers in the aggregate structure.58 This effect increases the probability of non-radiative energy dissipation reducing the generation of triplet states and singlet oxygen, the efficiency of photodynamic

action

and,

consequently,

is

undesirable

for

photodynamic

applications.56,58,59



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Figure 4. Emission spectra of TPPS porphyrin in the presence of magnetite nanoparticles in two different wavelengths of excitation: (a) at 640 nm; (b) at 515 nm. The inset shows the fluorescence intensity as a function of nanoparticle concentration.

Fig. 5b shows a subtraction of the MNPs absorption spectra (inset of Fig. 5a) from the solution spectrum containing TPPS + MNPs (Fig. 5a). This set of data was called [(MNP + TPPS) - MNP]. The [(MNP + TPPS) – MNP] spectra show a strong


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reduction of the Q band (centered at 644 nm) and four new bands centered in 520, 557, 593, 650 nm (black line). It is important to observe that the profile of these new bands is similar to the non-protonated TPPS form (Fig. 2a). It is in accordance with the fluorescence measurement confirming the deprotonation of TPPS porphyrin in the presence of MNPs. Fig. 5c summarizes the changes observed in the absorption spectra of TPPS in the interaction with MNPs. Fig. 5c shows the TPPS in the presence of MNPs (TPPS + MNP) and absence of MNPs (TPPS), the MNP solely (MNP) and the subtraction [(MNP + TPPS) - MNP] shown in Fig. 5b. The combination fluorescence quantum yield (ΦF) and intersystem crossing yield (ΦT) indicates the potential of a molecule for theranostics applications, which combines photodiagnostics and photherapeutics.60 It was previously reported that nonprotonated TPPS shows a smaller fluorescence quantum yield (0.16) than its diprotonated form (0.37).61 Although this reduction occurs, the fluorescence signal is a considerable value for photodiagnostic applications. On the other hand, non-protonated TPPS presents a higher triplet formation (0.77) than the protonated one (0.37), showing that it is more efficient for PDT applications. The non-protonated and diprotonated TPPS present ΦF + ΦT values 0.93 and 0.74, respectively, showing that for nonprotonated TPPS, virtually all the photons absorbed lead either to fluorescence or to intersystem crossing to the triplet state.60 Thus, non-protonated TPPS in the MNPs presents a greater potential for theranostic applications than the diprotonated one.



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Figure 5. Absorption spectra of: (a) diprotonated TPPS porphyrin (10 µM) in the presence and absence of MNPs (inset of (a)); (b) subtraction of MNPs absorption spectrum from TPPS porphyrin + MNPs solution spectrum and (c) comparative spectra of TPPS porphyrin and NMPs and the interaction between them. 14 ACS Paragon Plus Environment

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3.4. Binding parameters It is well accepted that the quenching mechanisms are usually classified into static and dynamic ones. Dynamic quenching process results from the collision between the fluorophore and quencher, which can affect the excited states of fluorophore and its fluorescence emission spectrum. On the other hand, the static quenching process is caused by the formation of a ground-state complex resulting in changes in the absorption spectrum.62 The evaluation of the affinity between TPPS molecules and MNPs can be performed by the Stern–Volmer quenching constant of the accessible fraction (Ka) obtained from the modified Stern–Volmer equation,62,63

1 1 1 = = + − ∆ where F0 and F are the fluorescence intensities of fluorophore (TPPS) in the absence and presence of the quencher (MNPs), respectively; [Q] is the concentration of the quencher and parameter fa is the fluorophore fraction accessible to the quencher. If Ka is known, we can also determine the bimolecular quenching rate constant by kq = Ka/ , where is the fluorescence lifetime of the fluorophore in the absence of the quencher. The ratio between the fluorescence lifetime of the fluorophore in the absence and presence of the quencher () is a standard method to indicate whether the quenching in the system is static or dynamic.52,61 A constant plot of / as a function of [Q], suggests that the interaction does not affect the excited state of the fluorophore and the quenching process is attributed to a static process. Furthermore, they reported that in systems where some type of binding interaction occurs, kq generally displays values



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much larger than 1 × 1010 M-1s-1 (characteristic value for quenching controlled by diffusion).62-67 The values of Ka, fa and kq were obtained from the fitting of the plot F0/∆F as a function of 1/[Q] (Fig. 6), resulting in Ka = 8.89 × 105 M-1, fa = 4.35 and

!

= 2.54

×1014 M-1s-1. In the literature, comparable values of Ka for the interaction of porphyrins and nanoparticles63,66-68 were reported The value kq obtained is obviously much greater than

!

≅ 1010 M-1s-1, suggesting that the quenching mechanism observed is associated

to a static process. The fluorescence lifetime of TPPS used was = 3.5 ns, in accordance with Ref. (18). Borissevitch et al.63 asserted that a fa value > 1 can mean that a single quencher molecule is able to quench more than one fluorophore molecule. In fact, it is in accordance with the results which will be presented in the next section.

Figure 6. Modified Stern–Volmer dependence of TPPS luminescence quenching by MNPs.


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3.5. Estimation of number of porphyrins per MNP The emission band generated by the excitation at 515 nm increased linearly with the concentration of the nanoparticles until reaching its saturation, at the concentration at 0.47 uM (inset of Fig. 4b). From that point onwards, all the diprotonated porphyrins that were free in the solution were linked to the nanoparticles. Owing to the fact that the molar concentration of TPPS porphyrin is known (10 µM), a value of approximately 21 porphyrins per MNP can be estimated. This value seems reasonable, considering the characteristics of MNP and TPPS. Maghemite nanoparticles have a diameter of about 4.8 nm,49 which provides a superficial area close to 7.24 × 10-17 m². Based on the TPPS dimensions presented in Fig. 1, the TPPS can be considered as a square with a diagonal close to 1.9 nm, which provides an area of around 1.8 × 10-18 m². Thus, we were able to obtain a maximum of 40 porphyrins/MNPs, when the whole area of MNPs is covered by porphyrins. However, a smaller number of porphyrins per MNP is expected due to the electrostatic repulsion between the anionic porphyrins. The strategy adopted to estimate the number of porphyrins attached to each nanoparticle seems quite reasonable for the results found in the literature, for example, Shaikh et al.66 found a ratio of ∼4 porphyrin molecules per Au-NPs. The TPPS-MNP system studied herein presents potential for applications in bionanotechnology and nanomedicine described as follows: 1) Fluorescent properties of porphyrins can be used as a fluorescent probe of the nanoparticles for photodiagnostics and/or theranostics applications – photodiagnosis based on fluorescence imaging have become important methods to investigate clinical practice, particularly in oncology by identifying and locating cancerous lesions.33,60,70



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This method uses non-invasive technology with a high spatial resolution and real-time display, and can use the fluorescence of porphyrins for a more effective treatment.2,60 2) A combined action of photodynamic therapy (PDT) and Hyperthermia (HPT) therapies – PDT is a well-established technology based on retaining a photosensitizer by tumor cells which are photoactivated by visible light. Likewise, HPT promotes a rise in temperature at the biological target, treated with magnetic nanoparticles, for externally applied AC magnetic fields. The presence of the porphyrin/MNP system in target tissues enables HPT and PDT therapies to be used simultaneously with synergetic effects. The combined action of these therapies can lead to an enhancement of tumor damage after minimal doses of heat dissipation and visible-light photosensitization.39-41 3) Nanoparticles can be used as a nano-drug delivery system for photosensitizers - this system can be tailored to be highly specific to achieve the desired concentration and safe drug release in the target tissue. In treatments involving a drug delivery system, the therapeutic agent is incorporated into a macromolecule or a nanoparticle and circulates in the blood stream, accumulating inside the tumor, through the Enhanced Permeability and Retention effect (EPR effect).69 The EPR effect is the property by which molecules tend to accumulate preferably in tumor tissue than in normal tissue. This property is essential for the proper functioning of drug delivery systems using magnetic nanoparticles.69 In order to obtain an effective nano-drug delivery system, properties such as loading rate, release time, targeting and biocompatibility tests need to be better investigated. In principle, the loading rate might be controlled tuning the pH of the media, since this affects the surface density of the nanoparticle. The amount of photosensitizers covering the nanoparticle surface can be adjusted by changing the


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experimental conditions, as for instance through the ratio of MNP to photosensitizer species. In addition, one can use MNP of distinct size and enhance the number of photosensitizer per particle, among other factors. Besides, in a system that contains MNPs and photosensitizers, the release time can also be in principle controlled through magnetic hyperthermia, which will result in an increase of MNP temperature that could release the therapeutic cargo. However, one should keep in mind that the nanocomplex system presented in this work is not yet perfect for such application. In order to achieve this goal one need to couple to the nanoparticle surface with polyethylene glycol (PEG) molecules to prolong the circulation time.71,72 On the other hand, it is fair to say, that our model system is crucial because one can firstly understand how to monitor and control the number of TPPS molecules attached to the surface and then later couple molecules for long circulation and active targeting. Independent of this the nanoparticle presented here could be used for intratumoral delivery maintaining several biomedical functionalities. Free base porphyrins can chelate transition metals, which can affect their photophysical characteristics and photodynamic efficiency. For example, Pavani et al.73 showed that the chelation of a porphyrin with zinc is a good strategy to help the maintenance of the photophysical⁄photochemical properties of the photosensitizers in the intracellular environment. On the other hand, the chelation with paramagnetic metal ions, such as Mn3+ and Fe3+, increases the spin-orbit coupling in TPPS porphyrin and reduces the excited state lifetimes. It results in a nonfluorescent complex with a low intersystem crossing and consequently generation of reactive oxidative species reduced.74



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The incorporation reaction of a metal ion in the porphyrin ring can be very slow with a low yield.75 Besides, the insertion of a metal ion into tetrapyrrole macrocycles depends of the availability of metal ions in the environment. The slow reaction rate for insertion of metals into porphyrins can be attributed to the difficulty in deforming of porphyrin ring.75 In this sense, the coupling of TPPS on nanoparticles also could be an interesting strategy to avoid an undesirable chelation.

4. Conclusion In the present work, we described the coupling of anionic porphyrins on maghemite nanoparticules using UV/Vis absorption and fluorescence spectroscopic techniques. These techniques are powerful tools to evaluate the coupling of organic molecules and nanoparticles. They allowed us to describe the dynamics of coupling porphyrins on nanoparticles, obtain the binding constants, the bimolecular quenching rate constant and estimate the number of porphyrins per each MNP. Moreover, the absorption and fluorescence measurements revealed that the binding between them can be associated to a static process, where electrostatic attraction plays essential role. The development

of

the

present

system

provides

a

relevant

background

for

bionanotechnology and nanomedicine applications as a multifunctional system with combined diagnostics and therapeutic functions, such as a nano-drug delivery system, photodiagnostics/theranostics and for PDT therapy.

Acknowledgements The Brazilian funding agencies that sponsored the work were Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG and the Conselho Nacional de 20 ACS Paragon Plus Environment

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Desenvolvimento Científico e Tecnológico - CNPq. The authors are very grateful to the Grupo de Física dos Materiais (IF⁄UFG) for allowing us to access their spectrofluorometer (Fluorolog FL3-221; Horiba Jobin Yvon Inc.).

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Use of Spectroscopic Techniques for Evaluating the Coupling of

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