|Year : 2016 | Volume
| Issue : 1 | Page : 40-48
Formulation and evaluation of liposomal transdermal patch for targeted drug delivery of tamoxifen citrate for breast cancer
Anjana Ashok Adhyapak, Babasaheb Gangadhar Desai
Department of Pharmaceutics, KLE University's College of Pharmacy, Belgaum, Karnataka, India
|Date of Web Publication||8-Jun-2016|
Anjana Ashok Adhyapak
Department of Pharmaceutics, KLE University's College of Pharmacy, Belgaum, Karnataka
Source of Support: None, Conflict of Interest: None
Background: In the present investigation, tamoxifen-loaded liposomes transdermal patch was formulated using eudragit-RL, hydroxypropyl methyl cellulose K-50, and ethyl cellulose.
Materials and Methods: Liposomes were formulated by solvent evaporation method using poly (sebacic acid-co-ricinoleic acid) in varying ratios and evaluated for particle size, drug loading, entrapment efficiency, transmission electron microscopy, differential scanning calorimetry, and X-ray diffraction. Formulated tamoxifen-loaded liposomes were finally incorporated into transdermal patch and evaluated for thickness drug content, moisture content, moisture uptake, folding endurance, tensile strength diffusion coefficient, permeability coefficient, in vitro permeation, and skin irritation. Optimized transdermal patches were tested for its pharmacokinetic and pharmacodynamics parameters
Results: Formulated transdermal patches showed improved bioavailability of tamoxifen when compared to its oral route.
Conclusion: Tamoxifen-loaded liposomal transdermal patches could serve as a better alternative to existing marketed formulation in terms of bioavailability.
Keywords: Evaluation, in vivo studies, liposomes, tamoxifen citrate, transdermal patch
|How to cite this article:|
Adhyapak AA, Desai BG. Formulation and evaluation of liposomal transdermal patch for targeted drug delivery of tamoxifen citrate for breast cancer. Indian J Health Sci Biomed Res 2016;9:40-8
|How to cite this URL:|
Adhyapak AA, Desai BG. Formulation and evaluation of liposomal transdermal patch for targeted drug delivery of tamoxifen citrate for breast cancer. Indian J Health Sci Biomed Res [serial online] 2016 [cited 2022 Jan 27];9:40-8. Available from: https://www.ijournalhs.org/text.asp?2016/9/1/40/183677
| Introduction|| |
Breast cancer is the most common malignancy among females affecting approximately one out of ten women. Aging of population in the industrialized world is the most obvious cause of increased breast cancer occurrence; indeed, the risk of developing breast cancer after 65 years of age is 5.8 times higher than before 65, and 150-fold higher than before 30 years of age. Breast cancer is one of the leading causes of cancer deaths among women. Nearly, 1 million new cases are diagnosed each year.
Oral administration of the nonsteroidal anti-estrogen such as tamoxifen is the treatment of choice for the patients with all stages of estrogen receptor positive breast cancer. Tamoxifen citrate (TC), 2-(4- [1,2-diphenyl-1-butenyl] phenoxy)-N, N-dimethyl ethanamine 2-hydroxy-1, 2, 3-propanetricarboxylate, the nonsteroidal anti-estrogen is a highly lipophilic drug. Currently, it is used as an endocrine therapeutic agent of choice for all stages of breast cancer and has also been approved in the USA for use as a chemo preventive agent in women at high risk for the disease. Oral tamoxifen undergoes extensive hepatic metabolism and the subsequent biliary excretion of metabolites. It can have harmful long-term side effects such as the development of endometrial cancer or an acquired tamoxifen resistance leading to further tumor progression. Other side effects include liver cancer, increased blood clotting, and ocular side effects such as retinopathy and corneal opacities. These effects were reported to be dose-dependent. To overcome these undesirable side effects, transdermal drug delivery (TDDS) is necessary to achieve optimum therapeutic outcomes for breast cancer for a prolonged period of time.,
TDDS have advantages such as attaining the required therapeutic concentration of antineoplastic agents at the tumor site and target the delivery of drug to tumor cells. Systemic side effects can be minimized by dose reduction, and frequent administrations of drug can also be eliminated. Finally, the proposed new extended drug delivery system may enhance the patient acceptability to treatment and reduce the inconvenience associated with conventional therapy.
Liposomes are microscopic structures of one or more concentric spheres of lipid bilayers, enclosing aqueous compartment. It can be used as drug carrier for TDDS system.
Herein, in the present research investigation, we have formulated tamoxifen-loaded liposomes and characterized for particle size drug loading, entrapment efficiency, transmission electron microscopy (TEM), differential scanning calorimetry (DSC), and X-ray diffraction (X-RD). Formulated liposomes were incorporated in a transdermal patch and finally evaluated for thickness, drug content analysis, moisture content, moisture uptake, folding endurance, tensile strength, in vitro skin permeation study, in vitro release studies, and skin irritation studies followed by in vivo pharmacokinetic and pharmacodynamics studies.
| Materials and Methods|| |
TC was a gift sample from Dabur Pharmaceutical Ltd., Ghaziabad, India. Beta-cyclodextrin, eudragit-RL-100, ethyl cellulose, and hydroxypropyl methylcellulose (K-15) were procured from Lab Care Ltd., Bengaluru, Karnataka, India. All other chemicals and solvents were of analytical grade, purchased from Merck Pvt. Ltd., Bengaluru, Karnataka, India.
Preparation of tamoxifen citrate-containing liposomes
TC-loaded poly (sebacic acid-co-ricinoleic acid [SA: RA]) 50:50 (procured from Sigma Aldrich, Bengaluru, Karnataka, India) liposomes were prepared by solvent displacement method. Four different types of formulations, i.e., F1, F2, F3, and F4 were prepared, differing in the theoretical loading of TC by 5, 10, 20, and 30% w/w of polymer, poly (SA: RA) 50:50, respectively. The exact amount of polymer and drug used for the preparation of each type of system is indicated in [Table 1]. Known amount of poly (SA: RA) was dissolved in 10 ml acetone and magnetically stirred for 15 min separately, after which TC was dissolved in this organic phase and further stirred for 15 min. The 40 ml of ethanol and water mixture of 1:1 ratio was added to the organic phase containing drug and polymer and stirred at 1000 ± 5 rpm for 20 min. This system was stabilized for 30 min. The organic solvent from the tri-phasic system was removed by rapid evaporation using rotary flash evaporator; where drug and polymer displacement takes place in sequence as acetone, ethanol, and water. The resulting aqueous system was frozen in liquid nitrogen and lyophilized using Christ alpha 1–4 LD plus lyophilizer (Indian Institute of Science, IISc, Bengaluru, Karnataka, India) to obtain free flowing fluffy poly (SA: RA) 50:50 lipid nanoparticles of TC using glucose and mannitol (7% w/w of polymer and drug) as cryoprotectants, added prior to lyophilization.,
Evaluation of lyophilized liposomes
Prepared liposomes were evaluated for particle size using Malvern Laser Analyzer Instrument (IISc, Bengaluru, Karnataka, India), drug loading, entrapment efficiency, TEM, DSC, and X-RD.
Preparation of transdermal patch
Transdermal films of TC (5.0 mg/3.14 cm 2) containing different ratios of eudragit-RL, hydroxypropyl methyl cellulose (HPMC K-50), and ethyl cellulose were prepared on mercury surface. The required amount of drug and polymers were dissolved in methanol-dichloromethane (1:1) solvent system. Di-n-butyl phthalate (20 and 30% w/w of polymer) was used as plasticizer. Isopropyl myristate (IPM) and dimethyl sulfoxide (DMSO) were added to the polymer drug solution. The resultant homogeneous solution was poured into a circular plane with uniform surface on mercury substrate. The films were dried for a period of 24 h, and the rate of evaporation was controlled by inverting funnel over the Petri dish More Details. The dry films were wrapped in aluminum foil and kept in desiccators. Compositions of prepared formulations were tabulated in [Table 2], and photographs of the drug-containing patches are shown in [Figure 1].
|Figure 1: Photographs of tamoxifen citrate containing transdermal drug delivery systems|
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Evaluation of prepared transdermal patches
The thickness of film before and after the permeation study was determined using micrometer gauge (Mitoyoto, Japan). Film was measured at different places and mean value was calculated.
Drug content analysis
The uniformity of drug distribution in the transdermal films was determined by taking known area of the films at different places of the film. The films were dissolved in 2 ml of methanol, sonicated for 10 min, and subsequently diluted with phosphate buffer saline (PBS) pH 7.4. After appropriate dilution, solutions were analyzed spectrophotometrically (ultraviolet [UV] Shimadzu-1700, Japan) for TC at 274 nm using solution of films prepared without drug as reference to neglect the absorption of components of the formulation, if any.
The prepared films were weighed individually and kept in desiccators containing activated silica at room temperature (30°C) for 24 h till a constant weight was attained. The percentage of moisture content was calculated as the difference between initial and final weight with respect to final weight.
A weighed film kept in desiccator at room temperature (30°C) for 24 h was taken out and exposed to 84% relative humidity (RH) in a stability chamber (Lab Care, Mumbai, Maharashtra, India) until a constant weight of film was obtained. The percentage moisture uptake was calculated as the difference between final and initial weight with respect to initial weight.
A strip of film (2 cm × 2 cm) was cut evenly and repeatedly folded at the same place till it broke. The number of times the film could be folded at the same place without breaking gave the value of the folding endurance.
Determination of tensile strength
The tensile strength was determined by using dynamic mechanical analyzer (computerized, EPLEXOR 500 N, IISC, Bengaluru, Karnataka, India). Exactly, 2 cm 2 patches of all the formulation were subjected and determined.
Determination of flux, diffusion coefficient, and permeability coefficient
Flux of drug permeated in case of in vitro was calculated from slope of the steady-state portion of permeation profile by linear regression analysis. Lag time was calculated from back extrapolation of steady-state portion of the graph. Diffusion coefficient (D/h 2) and permeability coefficient (K p) were also calculated for the in vitro studies using mentioned below equations, respectively, D/h 2 = 1/6 × T Lag and K p = Jss/CD, where, TLag is the lag time, JSS the flux at steady state, CD is concentration in donor compartment, D is the diffusion coefficient, and h is the diffusion path length.
In vitro skin permeation study
Female albino rats weighing 150–200 g were selected for permeation studies. The animals were sacrificed using anesthetic ether. The hair of the test animals was carefully trimmed short with a pair of scissors and the full thickness skin was removed from the abdominal region. The epidermis was prepared surgically by heat separation technique, which involved soaking of the entire abdominal skin in water at 60°C for 45 s, followed by careful removal of the epidermis. The epidermis was washed with water and used for permeability studies. Permeation studies were performed for different formulations across female rat skin in modified Keshary–Chien diffusion cell at 32 ± 0.5°C. The diameter of the donor compartment cell provides 3.14 cm 2 as effective constant areas. The films with area 3.14 cm 2 were applied to the skin using adhesive tape (cellophane) as backing layer. The phosphate buffer pH 7.4 (20 ml) was used as receptor compartment medium to ensure sink conditions and stability of the drug. This whole assembly was kept on a magnetic stirrer and the solution was stirred continuously using a magnetic bead. The samples were withdrawn at different time intervals and replaced with equal volume of diffusion medium. Samples were analyzed spectrophotometrically at 274 nm. To ascertain whether the components of the skin or other excipients of the film interfere in the drug analysis; blank experiment (films without drug) was run using skin as barrier membrane using PBS pH 7.4. When the solution was analyzed at 274 nm for any interfering constituents, the released constituents were amounting to an average of 0.04 ± 0.02%.
In vitro release studies
Release of TC from transdermal patch (10% w/w of DMSO as penetration enhancer) was measured using regenerated cellulose dialysis membranes (10 K MWCO, Himedia. Pvt. Ltd, Bengaluru, Karnataka, India). The membranes were washed and equilibrated with 0.1M PBS, and then mounted on Keshary–Chien diffusion cells (receptor volume 20 ml, permeation area 3.14 cm 2) by clamping them between the donor and receptor compartments. The receptor compartments were filled with phosphate buffer pH 7.4 maintained at 37 ± 0.5°C and constantly stirred at 100 rpm. Patch containing (10–60 mg of drug) was kept uniformly on the donor film and sampling ports were covered with Parafilm M ® (Fisher Scientific, Bengaluru, Karnataka, India). Samples were collected from the receptor fluid at predetermined time points and replaced with an equivalent amount of buffer. The drug content in the withdrawn samples was analyzed by UV-VIS method as described above. All release studies were conducted in triplicate.
Skin irritation studies
The skin irritation test was carried out on male Wistar albino rats weighing 200–225 g. The animals were kept under standard laboratory conditions, with temperature of 25 ± 1°C and RH of 55 ± 5%. The animals were housed in polypropylene cages, six per cage, with free access to standard laboratory diet and water ad libitum. The hair on the dorsal side of the rats was removed with an electric hair clipper on the previous day of the experiment (32). The rats were divided into three groups (n = 6). Group I served as control, without any treatment. Group II received topical 5 mg selected TC liposomal patch formulation and Group III received 0.8% v/v aqueous solution of formalin as a standard irritant (33). The animals were applied with new TC-transdermal patch or new formalin solution, each day up to 6 days. Finally, the application sites were graded according to a visual scoring scale, always by the same investigator. The mean erythemal scores were recorded (ranging from 0 to 4) depending on the degree of erythema as follows: No erythema = 0, slight erythema (barely perceptible-light pink) =1, moderate erythema (dark pink) = 2, moderate to severe erythema (light red) = 3, and severe erythema (extreme redness) = 4.
The animals were restricted by hands for 5 min after application and then the rats were placed in individual cages. Blood samples of 500 µl were collected, at several predefined intervals after dosing (1, 2, 4, 6, 8, 12, and 24 h) by 250 µl heparinized glass capillary tubes into 1.5 ml vials. Following centrifugation at 4000 rpm for 10 min, plasma was separated and then frozen immediately at −20°C until assayed. Before detection, the samples were thawed and then 50 µl of n-hexane, 200 µl of acetonitrile, and 200 µl methanol were added. The mixture was centrifuged for 10 min at 4000 rpm. About 50 µl of supernatant were injected into high-performance liquid chromatography. Phenomex C8 (250 × 4.6 mm) 5 µ column was used for the analysis. The samples were then prepared for analysis as described above. The other five (Group IV) rats received an oral dose of 4 mg/kg TC aqueous solution by gavage needle. The AUC0–24 was calculated by the trapezoidal rule for the time interval 0 to the last measurable point, 24 h. The peak plasma concentration Cmax and time to reach the maximum drug plasma concentration tmax were obtained from the concentration time plot.
Tumor was induced in dorsal right flank of all animals by subcutaneous injection of 4 × 106 MCF-7 cell suspension prepared in plain Dulbecco's modified eagle medium on day 0. When tumors reached a minimum size of approximately 80–90 mm 3 on day 19, animals were randomly divided into four groups, Group I – control (no treatment), Group II – TC patch (wherein mice received TC transdermal patch 5 mg), Group III – TC liposome patch (wherein mice received TC liposomes transdermal patch containing drug equivalent to 5 mg), and Group IV – oral dose of 4 mg/kg TC. Each group consisted of five mice. Group II, III, and IV received TC at dose of 5 mg/kg. Tumors were measured every day after first dose administered on day 20, till day 28. The tumor volumes were calculated by the following formula as reported earlier: Tumor volume ¼ 0:5 ab 2 where a = length of tumor mass and b = breadth of tumor mass growing on flank of animal. Results were evaluated for statistical significance employing one-way ANOVA (Bonferroni test). The differences in tumor volume were considered statistically significant at P < 0.05.
| Results and Discussion|| |
Preparation and evaluation of TC liposomes
TC-loaded poly (SA: RA) liposomes were prepared by solvent displacement method. Polyanhydride-based polymer poly (sebacic-co-ricinoleic acid) 5:5 was used as carrier. Poly (SA: RA) 7:3 used in this study is a hydrophobic polymer, built of natural fatty acids, which may be used for release of both hydrophobic and hydrophilic drugs. Glucose and mannitol were used as cryoprotectants that are added prior to lyophilization to facilitate the particle formation and prevent shrinkage of lipid particles on lyophilization. Cryoprotectants used were polysaccharides and are nontoxic to the human body that metabolizes within the body by glycolysis followed by Krebs cycle to produce carbon dioxide and water to generate a form of usable energy.
Formulations of F3 and F4 were finally selected for DSC and X-RD studies due to their prominent results in respect to particle size, entrapment efficiency, and dispersion of these nanoparticles.
To identify the mechanism of sustained drug release, we first characterized the physical state of the drug within the nanoparticles. The DSC thermograms shown in [Figure 2] indicates melting peak of TC was absent in DSC thermograms of nanoparticles containing TC, reveals that the drug was dispersed as an amorphous form or dissolution state. X-RD peaks [Figure 3] revealed the crystalline form of drug.
|Figure 2: Differential scanning calorimetry thermograms of (a) pure tamoxifen citrate (b) pure polymer (c) physical mixture (d) F3 and (e) F4|
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|Figure 3: X-ray diffraction pattern of tamoxifen citrate, pure polymer, physical mixture, F3 and F4, respectively|
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The percentage yield of liposomal formulations shown in [Table 2] was found to be in the range of 61–73% w/w. Comparative loss in F1 is more than F4 which might be due to the presence of comparatively large quantity of polymer in F1. The loss of yield might be due to recovery problem and adherence of formulation due to sticky nature of lipid polymer. Four different ratios of drug to polymer were used to prepare liposomes. The entrapment efficiency and drug loading of TC in the poly (SA: RA) of 7:3 based nanoparticles was found in the range of 90–93% w/w. The large entrapment efficiency can be explained by the TC hydrophobicity and insolubility in water, which minimizes its loss into external water phase. Polyanhydrides are relatively hydrophobic polymers; therefore, improved incorporation of drugs having low water solubility is expected.
The particles of size range in between 400 and 600 nm were obtained. Particle size was increased slightly with increasing TC content. The particle size (mean diameter) variation observed in the TC loaded poly (SA: RA) nanoparticles might be due to the variations in drug and polymer concentration and also the method of preparation. [Figure 4] shows the scanning electron microscope studies of lipid nanoparticles and typical F3 drug-loaded formulation. Results of drug release studies in all four formulations are summarized in [Figure 5].
|Figure 4: The scanning electron microscope studies of lipid nanoparticles (a) and typical F3 (b) drug-loaded nanoparticle in patch formulation|
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The results of changes in mean particle sizes are presented in [Table 3]. It was worth noting that the TC liposomes did not show any significant changes in either drug content or particle size.
|Table 3: Stability studies of prepared liposomes showing particle size and drug content over 90 days|
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Preparation and evaluation of transdermal patch
The formulations were subjected to physical examination; films appeared to be slightly translucent suggesting that the drug was not completely solubilized rather dispersed/suspended in the matrix. The studies revealed that addition of di-n-butyl phthalate at 30% w/w was fixed and standardized for formulation F1-F4 and incase of F5 and F6, 20% w/w, respectively. All the developed and prepared formulations of polymer were smooth, uniform, and flexible films were obtained [Table 4].
|Table 4: Formulation composition of tamoxifen citrate containing matrix transdermal systems|
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Thickness and uniformity of weight
Thickness of films varied between 0.106 and 0.127 mm [Table 5], suggesting that formulation variables used in the study did not produce any significant effect on the thickness of films. The uniformity of weight varied between 135.7 ± 0.7 and 202.2 ± 0.9, as the Eudragit concentration decreased, decrease in the weight was obtained.
|Table 5: Determination of flux, diffusion coefficient, and permeability coefficient|
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Drug content analysis
The drug content of all the formulations was in between 97.15 and 99.62 with a low standard deviation (≤0.61). The results of drug content analysis have shown that the method employed to prepare films in this study was capable of giving films with uniform drug distribution with an insignificant batch variability (P > 0.001).
Moisture content and moisture uptake
Moisture content and moisture uptake studies provide information regarding stability of the formulation. The results revealed that the moisture content and moisture uptake were found to increase with increasing concentration of hydrophilic polymer (HPMC). The presence of penetrations enhancers DMSO and IPM did not show any major changes in moisture content and moisture uptake values. In case of DMSO, slight increment in both parameters was observed. This may be due to the water affinity of DMSO. The small moisture content in the formulation helps them to remain stable and from being a completely dried and brittle films, and low moisture uptake protects the material from microbial contamination and bulkiness of the films. Thus, the results of physicochemical studies conducted on different polymeric films containing tamoxifen favored the combination of these polymers for preparation of transdermal films.
Determination of tensile strength and folding endurance
All the formulations showed very good tensile strength and folding endurance values ranging in between 12.91 ± 0.15–13.07 ± 0.09 kg/cm 2 and 38.50 ± 1.29–46.00 ± 2.16 kg/cm 2, respectively.
Determination of flux, diffusion coefficient, and permeability coefficient
The in vitro release profile is an important tool that predicts in advance how a drug will behave in vivo. The results of in vitro skin permeation studies of TC from transdermal patches are shown in [Figure 6] and [Table 6]. The cumulative amount of drug release from (2.0 cm 2, area of 3.14 cm 2), formulation from F3, F4, F5, and F6 was (4.278, 4.56, 4.224, and 4.665 mg) high when compared to other formulations, this phenomenon attributed due to the amount of combination hydrophilic and hydrophobic polymer used in the formulations. When the cumulative amount of drug permeated with an area of 3.14 cm 2, patches through rat skin was plotted against time. The Flux, permeation coefficient and diffusion coefficient of formations F3, F4, F5, and F6 were high when compared to other two formulations. The reason for increase in these parameters could be due to the hydrophilic nature of the polymer and its swelling properties.
|Figure 6: Cumulative amount of tamoxifen citrate permeation through female rat skin|
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Skin irritation studies
Skin irritation studies reveals that transdermal patches consisting of eudragit-RL, HPMC K-50, and ethyl cellulose are nontoxic to skin. There were no signs of erythema or edema on skin surface suggesting that the formulated patches are safe for therapeutic use.
Results of pharmacokinetic studies are shown in [Table 7], when administered transdermal, the drug was present in rat plasma for a much longer period compared to the oral administration, 18 h versus 4 h, respectively. By providing a nonfluctuated and continuous delivery of TC into the bloodstream, transdermal administration may offer sustained efficacy with reduced side effects. A relative bioavailability of formulation ranges in between 0.56 and 0.74 was estimated for transdermal versus oral administration. The obtained pharmacokinetic parameters are shown in [Table 7].
|Table 7: Pharmacokinetic parameters after tamoxifen transdermal and oral single dose administration to rats|
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In vivo studies in nude mice tumors latency period were observed to be 16 days postsubcutaneous injection of MCF-7 cells. Once the tumors reached an approximate size of 80–90 mm 3 as observed on day 19, they were randomly divided into four groups as mentioned earlier. Animals were treated with transdermal patch of TC liposomes equivalent to 5 mg, TC oral formulation and TC transdermal patch. Group III receiving TC liposome patch demonstrated significantly reduced tumor volumes (P < 0.05) as compared to tumors of mice from control group as well as mice receiving TC oral formulation after 23 days. On day 25, mice treated with TC transdermal patch and TC oral formulation showed significant reduction [Figure 7] in tumor volumes compared to control group. However, on day 26 and thereafter, TC liposomes transdermal patch treated mice presented statistically reduced tumor volumes (P < 0.05) in comparison to TC oral formulation and TC patch formulation.
| Conclusion|| |
Tamoxifen-loaded liposomes were formulated by using solvent evaporation method using poly (SA: RA) where the optimized liposomal formulation F3 showed particle size 418 ± 0.07, drug loading 18.78 ± 2.84%, and entrapment efficiency 90.32 ± 2.30%. Optimized liposomes were formulated into finalized form as transdermal patch using a blend of polymers such as eudragit-RL, HPMC K-50, and ethyl cellulose. Optimized patch formulation F3 was found to have the best release profile when compared to other patches with no skin irritation. Pharmacokinetic and pharmacodynamics profile of formulated patch confirms its advantage over other conventional existing formulations.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]