Production of Polycaprolactone/Atorvastatin Films for Drug Delivery Application

Developing biomaterials for tissue regeneration is a promising alternative for the recovery of various tissues, including bone. Atorvastatin (ATV) has a series of beneficial effects which includes bone anabolism, vasodilating


Introduction
Atorvastatin (ATV) belongs to the pharmacological class of statins and is an anti-cholesterol drug that inhibits the HMG-CoA reductase, an essential enzyme in cholesterol synthesis, and reduces cholesterol production by the human organism 1 .Traditionally, this drug is presented as a tablet.It has an absorbed fraction and absolute bioavailability of about 30% and 12%, respectively, due to a pre-systemic clearance in the gastrointestinal mucosa and its first-pass hepatic metabolism 2,3 .However, ATV has beneficial effects unrelated to lipid metabolism, the so-called pleiotropic effects.This includes bone anabolism, vasodilating, antithrombotic, antioxidant, anti-inflammatory, and immunosuppressive actions 4,5 .In 2002, Pasco et al. 6 , in a retrospective casecontrol study, Statins were associated with a 60% reduction in the risk of bone fractures when evaluating 1,375 women aged 50 to 95 years 6 .Some statins have been studied with polymers for controlled release 7,8 .However, for ATV, there is still no study available.
Biodegradable polymers have been investigated as raw materials to manufacture biomaterials applied to tissue regeneration and devices for drug delivery [9][10][11][12][13][14] .Due to Polycaprolactone (PCL) biocompatibility, and biodegradability, several applications in the medical field are found for it, such as drug delivery systems 9,15 ; films applied to tissue-engineered skin [16][17][18] ; coating for urethral stents in musculoskeletal tissue engineering 19 and scaffolds for supporting osteoblast and fibroblast growth 19,20 microspheres and nano formulations for drug delivery applied to cancer treatment 21 ; and resorbable scaffolds by injection molding for application in orbital eye repair 22 .
Mainly, PCL has been extensively studied to produce materials applied to both bone and cartilage repair 19,22,23 .In this context, different techniques such as electrospinning, solvent casting, and compression molding, among others 9,22 , have been used to produce biomaterials in different formats, such as films 9 , scaffolds 9 , among others 24 .
Biomaterials have played a prominent role in the development of tissue regeneration 1 .It can be used for local drug delivery as a promising alternative to recover various tissues, including bone 1 .In this context, two different technologies, the controlled release of drugs and tissue engineering, can be combined to provide efficient results in tissue repair.Polymeric materials in matrices, films, or scaffolds can guide and support tissue regeneration and work as vehicles for drug delivery [9][10][11][12][13] .The technology of controlled release of drugs can potentially overpass many problems related to the traditional administration of active pharmaceutical ingredients by regulating the rate and spatial localization of the released agent 14 .
Recent studies on metabolic abnormalities and obesity, particularly abnormal lipid metabolism, have been associated with diseases caused by cartilage degeneration, such as osteoarthritis 25 .Developing materials based on PCL and ATV can be an alternative to treat problems associated with bone, local cholesterol disorder, and cartilage diseases due to PCL 9,10 and ATV characteristics 25,26 .Thus, the objective of the present work was to produce and characterize PCL/ATV matrices with prolonged drug release.

. Materials
Atorvastatin calcium was kindly donated by the Oswaldo Cruz Foundation.PCL (average molar mass of 60,000g/mol) was supplied by Polymorph Ltd, China.Chloroform (PA) was obtained from Labsynth, Brazil.The manufacturer's name will not be disclosed for confidentiality reasons.

Production of PCL/ATV matrices
The polymeric matrices were prepared by solvent casting, where atorvastatin calcium and PCL were solubilized in 2 mL of chloroform at room temperature using room temperature the following quantity of atorvastatin 0; 0.1; 0.2; and 0.4 mg ATV/mg PCL.The values were based on the ATV pill formulations.The resulting mixtures were added to silicone molds (3 cm x 2 cm) and dried at room temperature for 1 hour.The matrices were named according to the amount of ATV in the sample: PCL (0 mg of ATV), PA20 (20 mg of ATV); PA40 (40 mg of ATV); PA60 (60 mg of ATV); PA80 (80 mg of ATV).

Fourier-Transform Infrared Spectroscopy (FTIR)
FTIR analysis was performed to investigate the possible interactions between ATV and PCL.The analysis was done using an Infrared Spectrometer (Bruker, model Vertex 7) with attenuated total reflectance (ATR) by recording measurements from 4000 to 400 cm −1 and 64 scans and compared with the raw materials (PCL and ATV).

X-Ray Diffraction (XRD)
All the samples were analyzed on an X-Ray diffractometer (Rigaku, model Mini Flex II) operated with the Cukα source (λ = 1.5418Å).The scans were recorded over the 2θ = 6-60°, with a 2°/min scan speed.

Thermogravimetric analysis (TGA)
PCL, ATV, PA20, PA40, PA60, and PA80 samples were analyzed on a thermogravimetric analyzer (model Discovery 550, TA Instruments).The measurement was performed using platinum pans with about 5 mg of the sample at a heating rate of 20 °C/min under a nitrogen atmosphere, from room temperature to 700 °C.TGA curves and their derivative (DTG) were obtained.

Differential Scanning Calorimetry (DSC)
PCL, ATV, PA20, PA40, PA60, and PA80 samples were characterized on a differential scanning calorimeter (DSC model Discovery 250, TA Instruments).Samples with a mass of around 5 mg were heated in aluminum pans in the range from room temperature to 300°C, with a heating rate of 10°C/min under a flowing dry nitrogen atmosphere (20 cm 3 /min).

Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS)
To evaluate the morphology of the samples with different amounts of ATV, the raw materials, PA20, PA40, PA60, and PA80 samples were analyzed using a scanning electron microscope (JEOL, model JSM-6390LV) at an acceleration voltage of 15 kV.For this analysis, samples were recovered with gold.
The samples were vaporized with carbon to observe the drug distribution on the matrices, and an EDS spectrum was acquired.The spectra acquisition was made for 30 sec with a 15kV and 79 mA beam at 10 mm and a spot size of 71.For the acquisition of the EDS map, the exact condition of the beam was used, and it acquired 500 frames with a dead time between 17-19%.

In vitro release studies
To evaluate the capacity of the prepared films in releasing ATV, they were submitted to an in vitro release study using UV-Vis.A standard curve of ATV in a phosphate medium was obtained at 240 nm.Therefore, the in vitro release study was conducted using a Distek Evolution 6100 dissolution test system.The experiment was done in triplicate, using three vats for each sample.The samples (PA20 and PA40) were immersed in 900 mL of potassium phosphate buffer (pH = 7.4), at 37°C, under the agitation of 75 rpm, and aliquots were withdrawn at the following predetermined intervals: 5, 30, 60, 90, 120, 150, 180, 210, 240, 1440, 1800, 2880, 3180, 4320, 5760, 5940, 10080, 11520, 12960, 14400 and 15840 min.The drug concentrations were calculated using the equation (y = 33.195x+ 0.0176; with a correlation coefficient of 0.9603) obtained through the analytical curve of ATV in phosphate buffer pH 7.4.Drug releases were adjusted for the following models: zero-order kinetics, first order-kinetics, Hixson-Crowell, Higuchi, and Korsmeyer-Peppas 27,28 .The linearity of the results was evaluated by the determination coefficients (R 2 ).After the in vitro release study, the matrices morphology was analyzed by SEM as described in the above section.

FTIR analysis
FTIR analysis of PCL, ATV, and samples containing different amounts of ATV was performed, and the spectra are shown in Figure 1.The prominent bands of PCL and ATV are also presented in Figure 1.The results showed that ATV and PCL present bands with wavenumbers closer than those reported in the literature 19,[29][30][31] .
The spectra of PA20, PA40, PA60, and PA80 samples present the characteristic bands of both pure PCL and pure ATV.With the increase in the proportion of ATV in the samples, the ATV bands become more evident (Figure 1).The ATV characteristic bands around 1646 cm -1 for (C = O) and 1575 cm -1 for (C -N) can be observed in the region ranging from 1900 cm -1 to 1350 cm -1 .The strong carbonyl absorption of PCL overlaps the carbonyl absorption bands of ATV.Therefore, according to FTIR analyses, no chemical linkage was formed between ATV and PCL, and this is important as the primary objective is the release of ATV.The peaks related to PCL and ATV were observed in the samples PA20, PA40, PA60, and PA80.Peaks related to ATV, as in 2θ = 17.05° and 2θ = 19.57°are more clearly observed in samples with a greater amount of the drug 3,32 (See in detail on the right side in Figure 2).According to Figure 2, changes in PCL pick position occurred, probably due to the presence of ATV.

Thermal analysis -DSC, TGA and DTG
The samples PA20, PA40, PA60, PA80, PCL, and ATV were analyzed by TGA and DSC to evaluate the thermal properties of these materials.DTG results are presented in Table 1.DSC curves are shown in Figure 3 and the Endothermic events values in Table 2.The PCL thermal degradation occurred in a single stage of mass loss.The onset was 390°C and the maximum thermal degradation occurred at 448°C (Table 1).This stage corresponds to the depolymerization of PCL from the ends of the polymer chain, with the hydroxyl terminal groups forming ε-caprolactone 23 .These results agree with the literature that described that PCL is completely decomposed in a single stage, with a peak of maximum degradation around 430°C 9,33 .
As previously described 11,24 , a five-stage weight loss was recorded for TGA curves.According to Shete et al. 2 , the first phase of weight loss, around 80-140°C, is related to the loss of volatile that was absorbed on the surface.When adsorbed on the surface, water usually comes out at lower temperatures, around 60ºC, and, on the other hand, the connected water usually leaves around 100ºC.According to these authors, this result is consistent with the literature for ATV trihydrate 2 .In this work, stages of mass loss at 251ºC, 340ºC and 459ºC were observed for ATV.The theoretical value of mass loss for the trihydrate would be 4.46% and we observed a very close loss, 4.38%, indicating that this inference is probably correct 2 .It can also be observed in the TGA curve, that the greatest loss of volatile occurs after 200°C.
TGA analyze of PCL/ATV showed characteristics compatible with PCL and ATV.Table 1 shows the maximum temperatures observed in DTG curves (excluding the initial events at temperatures below 175°C) for both ATV, PCL, and PCL/ATV samples.These obtained results indicate that PCL/ATV samples showed intermediate characteristics between the curves of the drug and polymer in the thermal decomposition curves, indicating drug entrapment in the polymer matrix.
The DSC curves of ATV (Figure 4) present two endothermic events: one in the range of 80°C to 140ºC related to the loss of volatiles and the second around 160ºC due to the melting of ATV 32,34,35 .These results are consistent with the TGA data obtained in this work.The PCL matrix (Figure 3) showed a single endothermic peak that corresponds to the crystalline fusion at 52°C.A similar result was observed by Silva et al. 30 , Sekosan and Vasanthan 36 .
The peaks related to PCL fusion remained similar in the PCL/ATV samples.Nevertheless, since changes in the fusion enthalpy values were observed (Table 2) it is possible to infer   that ATV alters the crystallinity of PCL (Figure 4).The value of ΔH decreases with increasing ATV content.This result agrees with the XRD analysis (Figure 2) that showed changes in the characteristic peak of the PCL.The peaks related to the drug and the amount of energy involved in thermal transitions were also modified.Considering the matrices, the endothermic events related to ATV were observed in PA40, PA60, and PA80 samples.Although, probably, due to the recrystallization of ATV in PCL, these peaks were shifted to higher temperatures (Table 2).In the PA20 sample, the peaks related to ATV were not observed, perhaps due to the low concentration of drug present in the sample

SEM and EDS Analysis
SEM images of ATV particles, PCL matrix, PA20, PA40, PA60, and PA80 matrices are shown in Figure 5.The ATV micrograph (Figure 4a) shows agglomerates of needle structures with variable length and width.The pure PCL matrix (Figure 4b) presented a smooth surface.According to SEM images (Figure 4c, 4d, 4e, and 4f), the morphology of the matrices was affected by the amount of ATV in the sample.Structures smaller than 10 µm can be observed on the surfaces of PA40 and PA60 matrices.
As mentioned, in the present work, ATV calcium was used.EDS analysis was performed to evaluate the calcium distribution on PA20 and PA40 surfaces (Figure 5).The EDS calcium map allows concluding that the drug is dispersed in the matrices and the clusters at the PA40 matrix are constituted by the drug (Figure 5b).This allows the extrapolation for the clusters observed in the SEM images of PA60 and PA80 (Figure 4e-4f) and the conclusion that these samples have excess particles on the surface, probably, outside the polymeric structure.

In vitro release study
PA60 and PA80 samples (Figure 6) were subjected to in vitro drug release studies.The films were produced in 3cm x 4cm molds and obtained thickness of 0.1cm.
ATV release was affected by its concentration in the sample as shown in Figure 7.During the studied period, it was observed that P60 (60 mg of ATV) and PA80 (80 mg of ATV) samples released 36,43% and 64,72% of ATV, respectively.The dissolution of the drug contained in the matrices can be considered prolonged because up to 15000 minutes it had not reached 100% release 37 .PCL is used in the development of controlled drug release systems due to its mechanical properties and these characteristics may have helped in the drug release behavior 9 .This result can be considered a positive one since one of the goals of this work was to produce ATV matrices with the prolonged release.
The experimental data were adjusted for the most used release models in the literature: zero-order kinetics, first-order kinetics, Hixson-Crowell, Higuchi, Korsmeyer-Peppas and the correlation coefficients were determined.The values of the adjusted coefficients as well as the R 2 are presented in Table 3.According to the data, PA60 samples can be explained by the Hixson-Crowell and Higuchi model, being the last one which fits best (Table 3).In the PA80 sample, the kinetic release could be explained by kinetics model of order zero, Hixson-Crowell, Higuchi and Korsmeyer-Peppas model, the last one with the higher correlation coefficient (Table 3).This shows that more than one model can explain the initial drug release behavior.However, it is important to highlight that only the Higuchi model can be adjusted for both samples, since the R 2 values were above 0.96 for both.Some considerations must be made for the application of the models.Higuchi's model, for example, is based   on Fick's law and in order to be used, it is necessary to consider different factors, such as: mass transport through the diffusion of the drug, being the limiting step; sink condition for ATV (which is poorly soluble in phosphate buffer pH 7.4 35 ); the device must not be eroded while the drug is being delivered; the diffusion coefficient of the species must be constant; the swelling of the device must be insignificant or happen quickly in order to achieve balance; among others 37 .The dissolution process can change the matrices morphology and, consequently, the release behavior 38 .In fact, it is not, immediately, the erosion of the PCL itself, but the erosion of the matrix due to the exit of ATV.This erosion can create preferential pathways for the fluid.
To understand the effect of ATV's release from the matrix, SEM analysis of PA60 and PA80 samples after the release study was made and showed that erosion of the matrix occurred (Figure 8).In the images, it was possible to observe the formation of pores when compared to the micrographs before the release (Figure 4e-4f) , possibly due to the exit of the drug particles that were well incorporated in the matrices (PCL degradation can take 2 to 4 years depending on the initial molecular weight of the device or implant 9 . Nevertheless, the formation of pores in the matrix structure for tissue engineering could be positive, since it allows the initial fixation of cells, serving as a structure and allowing tissue regeneration 9,39 .In this context, these matrices produced by PCL and ATV start as a matrix of tissue and, as the release proceeds, there is the formation of pores that could facilitate tissue growth.Similar behavior was also seen by Jiao et al. 40 where the pore structure in the nano-hydroxyapatite (HA)/PCL and micro-HA/PCL tissue engineering scaffolds produced by 3D printing facilitated the growth of blood vessels, the transport of nutrients, and provided a very favorable environment for the discharge of cellular metabolic waste 40 .Also, in the work of Liu D. the 3D printed PCL/Strontium-HA bone scaffold was prepared with the aim of simulating the natural bone components, exhibiting significant osteogenic activity in vitro and in vivo, being able to simultaneously release strontium and calcium ions to promote osteogenesis repair due to its porosity 41 .
According to the observation of SEM analysis about ATV release from matrices produced in this work, it is suggested that the mechanism of drug release from PA60 and PA80 samples occurs in the following steps: (1) Solubilization of the drug particles that were closest to the sample surface.
(2) Diffusion of the drug particles that were inside the matrices structure into the solution, forming pores where previously were drug particles, which may cause the erosion of the polymer.(3) Diffusion of the phosphate buffer solution into the matrix after the drug has started to dissolve, causing the swelling of the polymeric matrix and its erosion.Since PCL is hydrophobic, this process can be more difficult, being a factor with less impact on the release.These could explain the change in the release in the dissolution profile (Figure 8).

Conclusions
In the present work, PCL matrices containing ATV were obtained using the solvent casting method.The distribution of ATV in the matrices was uniform as observed in SEM analysis.All characterizations proved the incorporation of the drug in the matrices, maintaining its structure with minimal changes.It was observed that, at the end of the tests (after 15000 minutes), the obtained dissolution reached a maximum of 65%, indicating a prolonged release of the ATV, where the release process occurs in the following steps: solubilization of the drug on the matrix surface, diffusion of the drug from the matrices, and then erosion.The experimental data of the in vitro release analysis of the matrices were adjusted for different models and the one that showed the best fit for the two studied matrices was the Higuchi model, with a correlation coefficient above 0.95 for both.The conditions for the application of the Higuchi model were not completely satisfied in the present work, but, due to the obtained R 2 value, it is suggested that diffusion is an important step in the process of ATV release from PCL matrices produced by solvent casting.

Figure 6 .
Figure 6.(a) PA60 and (b) PA80.Both films were produced in 3cm x 4cm molds and obtained thickness of 0.1 cm.

Table 1 .
DTG maximum values for ATV, PCL, and PCL/ATV samples.

Table 2 .
Endothermic events values for ATV, PCL, and PCL/ATV samples.