Electrospinning of doxorubicin loaded silica/poly(ɛ-caprolactone) hybrid fiber mats for sustained drug release

Poly($\varepsilon$-caprolactone) (PCL with average molecular weight $80,000\,{\rm g}\,{\rm mo}{{{\rm l}}^{-1}}$) and polyethylene oxide (PEO) with molecular weight $900,000\,{\rm g}\,{\rm mo}{{{\rm l}}^{-1}}$) were bought from Sigma Aldrich. Doxorubicin hydrochloride (DOX, Mw  =  580) was secured from the Beijing Huafeng United Technology Co, Ltd. Chloroform purchased and tetraethyl orthosilicate (TEOS) were get from Sigma-Aldrich. Methanol, absolute ethanol and ammonia solution (25%) were obtained from El-Nasr Company.

2.2.1. Preparation of ${{\rm Si}{{{\rm O}}_{2}}}$ and ${{\rm DOX}@{\rm Si}{{{\rm O}}_{2}}}$.

2.2.2. Preparation of the electrospinning solution.

2.3.1. Microstructure characterizations.

2.3.2. In vitro drug release.

All the electrospun mats are cut into $1.0\times 1.0\,{\rm c}{{{\rm m}}^{2}}$ square pieces. The sample weight is recorded before dipping into a centrifuge tube filled with 10 ml of phosphate buffered saline solution (PBS). At time periods of 1 h, 2 h, 3 h, 1 d, 2 d, 6 d, 10 d, 14 d, 21 d and 33 d the amount of DOX released in the PBS is measured using UV-Vis spectrophotometer by measuring the absorbance at 480 nm. The experiments are processed in triplicate per sample. The percentage of released drug is calculated using the following equation:

$$\begin{align} \newcommand{\e}{{\rm e}} \displaystyle {\rm Released}\,{\rm drug }~ \left(\% \right)=\frac{{{M}_{t}}}{{{M}_{{\rm tot}}}}\times 100,\nonumber \end{align}$$

where ${{M}_{t}}$ is the mass of DOX released at time $\left(t \right)$ and ${{M}_{{\rm tot}}}$ is the total amount of DOX present on the fibrous mat [4].

Figure 1 shows the FESEM images of the electrospun PCL-PEO fibers loaded with DOX and ${\rm DOX@Si}{{{\rm O}}_{2}}$ via different methods at different magnifications. As can be seen in figures 1(A), (A1) and (A2), ${{M}_{1}}$ method leads to agglomeration of DOX particles on the surface of the fibers. This could be due to the diameter of PCL-PEO fiber which is smaller than DOX aggregates. The average diameter of PCL-PEO fiber is $1109.91{\rm nm}$ (figure 2(M₁)). The same agglomeration is also seen for ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ nanoparticles loaded on the fibers by the M₂ method as shown in (figures 1(B), (B1) and (B2)) with an average diameter of about $659.217\,{\rm nm}$ (figure 2(M₂)). On the other hand, the obtained ${{M}_{3}}$ method fiber shows less agglomeration of ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$. The dumbbell-shaped features of PCL-PEO suggest the encapsulation of drug particles inside the nanofiber shown in (figures 1(C), (C1) and (C2)). This could be due to utrasonication of the samples before electrospining. The average diameter of the obtained fibers is 1492.124 nm (figure 2(M₃)). However, the loading of ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ by the ${{M}_{4}}$ and ${{M}_{5}}$ method resulted to the insertion of the all particles inside the electrospun PCL-PEO nanofibers as shown in (figures 1(D), (D1), (D2), (E), (E2) and (E3)). The average diameter of obtained fibers is increased to 1880.713 nm for ${{M}_{4}}$ fibers and $2163.978\,{\rm nm}$ for ${{M}_{5}}$ method (figures 2(M₄) and (M₅)). Some of the ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ nanoparticles find in agglomeration inside the fibers caused the formation of the swollen beads on the nanofibers as shown by the dashed green circles. The above results focus on the effect of ultrasonic on morphology and nanoparticles distribution within the fiber mats, in which the loaded nanoparticles are highly agglomerate in absence of ultrasonic (${{M}_{2}}$) and less agglomeration distribution, is obtained with ${{M}_{3}}$ due to the sonication that separate agglomerated particles from each other. In this consequence, uniform distribution is achieved via sonication of ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ nanoparticles before and after the addition to the polymer solution, ${{M}_{4}}$ method. Finally, ${{M}_{5}}$ method provides an alternative approach for high drug encapsulation capability as presented by FESEM micrographs.

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The chemical structure analyses of the obtained fiber mats and ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ nanoparticles are characterized by FTIR (figure 3). The FTIR spectrum of pure PCL-PEO nanofiber is shown in curve A of figure 3(a) through this spectrum the band at about, $1723.7\,{\rm c}{{{\rm m}}^{-1}}$ is associated with CO  −  stretching vibration of ester in PCL, the band occurred at $1237.7{\rm c}{{{\rm m}}^{-1}}$ is related to the O–C–O asymmetric stretching vibrations of PCL, and that of symmetric stretching occur at $1178.03\,{\rm c}{{{\rm m}}^{-1}}$, the bands at about $1104\,{\rm c}{{{\rm m}}^{-1}}$ and $1043.1\,{\rm c}{{{\rm m}}^{-1}}$ may be associated with the vibration of C–O present on PCL and the vibrational band at about $726.06\,{\rm c}{{{\rm m}}^{-1}}$ is related to ${\rm C}{{{\rm H}}_{2}}$ of PCL [18].

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The intense doublet band at about $2944.09\,{\rm c}{{{\rm m}}^{-1}}$ and $2868.8\,{\rm c}{{{\rm m}}^{-1}}$ assigned to asymmetric and symmetric vibration of ${\rm C}{{{\rm H}}_{2}}$ of PEO, respectively. The two bands at $1462.2\,{\rm c}{{{\rm m}}^{-1}}$ and $1290.2\,{\rm c}{{{\rm m}}^{-1}}$ are concerned with scissoring and twisting vibration of ${\rm C}{{{\rm H}}_{2}}$, respectively [19]. The band at $1365.4\,{\rm c}{{{\rm m}}^{-1}}$ is associated with wagging vibration of ${\rm C}{{{\rm H}}_{2}}$ belongs to PCL and PEO. Also the band at about $960.7\,{\rm c}{{{\rm m}}^{-1}}$ is assigned to rocking vibration of ${\rm C}{{{\rm H}}_{2}}$ found on PCL and PEO. On the other hand, there are some changes occur on the FTIR spectra of PCL-PEO fibers loaded with DOX or ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$. As illustrated in curves B, C and D of figure 3(a) for ${{M}_{1}},{{M}_{2}}$ and ${{M}_{3}}$ methods, respectively, we can see that the bands at about $1462.2\,{\rm c}{{{\rm m}}^{-1}}$, $1365.4\,{\rm c}{{{\rm m}}^{-1}}$, $1237.7\,{\rm c}{{{\rm m}}^{-1}}$, $1137.03\,{\rm c}{{{\rm m}}^{-1}}$, $1043.1\,{\rm c}{{{\rm m}}^{-1}}$ do not exist in the spectra. Also the intensity of the band at about $1723.7\,{\rm c}{{{\rm m}}^{-1}}$ and intensity of doublet bands at about $2944.09\,{\rm c}{{{\rm m}}^{-1}}$ and $2868.8\,{\rm c}{{{\rm m}}^{-1}}$ decrease in these spectra as compared to the spectrum of pure PCL-PEO. All these alterations could be related to the presence or adsorption of loaded particles on their surface. For the other methods ${{M}_{3}}$ and ${{M}_{4}}$, the spectra are resembled to the spectrum of pure PCL-PEO which may be because of ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ particles that are encapsulated by PCL-PEO fibers.

Figure 3(b) shows the FTIR spectra of pure SiO₂ and samples of DOX loaded on silica nanoparticles at different ratios 0.5, 2, 4% named ${{D}_{0.5}},{{D}_{2}},{{D}_{4}}$, respectively). The spectrum of pure ${\rm Si}{{{\rm O}}_{2}}$ nanoparticles illustrates the presence of absorption bands arising from asymmetric vibration of Si–O on the range of ($1046-1232\,{\rm c}{{{\rm m}}^{-1}}$), asymmetric vibration of Si–OH at ($945.3\,{\rm c}{{{\rm m}}^{-1}}$), and symmetric vibration of Si–O appeared at ($797.1\,{\rm c}{{{\rm m}}^{-1}}$).The intense characteristic absorption band at the range ($3095.7-3640\,{\rm c}{{{\rm m}}^{-1}}$) ascribed to O–H of stretching in H-bonded water. Also the band assigned at ($1629.2\,{\rm c}{{{\rm m}}^{-1}}$) is due to scissor bending vibration of molecular water. The loading of DOX into the silica nanoparticles led to obvious changes on the vibrational bands. The broadening of asymmetric vibration band of Si–O increased with the loading of drug into the silica nanoparticles. The intensity of vibration band of Si–OH decreases with the incorporation of DOX into the silica nanoparticles and shifts to higher wavenumbers. This may be due to: presence of drug into the silica nanoparticles increases their diameters which in turn decrease the length of the bond between Si–OH which cause its shift to higher wavenumbers.

TEM images of pure ${\rm Si}{{{\rm O}}_{2}}$ and ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ nanoparticles are indicated in figures 4(A), (B), (B1) and (B2). As can be seen, the as prepared pure silica nanoparticles are spherical in shape that agglomerated to form clusters. The loading of DOX into the silica nanoparticles leads to formation of uniform spheres larger than pure silica. The diameter of ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ nanoparticles are ranged from $30-50\,{\rm nm}$. In addition, figure 4(C) shows the XRD spectra of the synthesized pure silica nanoparticles and DOX loaded silica nanoparticles. The XRD pattern of pure SiO₂ exhibits a broad peak at $2\theta ={{22}^{\circ }}$, which reveals the amorphous nature of the silica nanoparticles. The broadening of this peak is obvious owing to the smaller grain size effect. The absence of other peaks indicates that there are no impurities in the prepared silica nanoparticles [20]. For ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ sample, there is a small shift of $2\theta$ to ${{22.5}^{\circ }}$, as indicated from the XRD pattern in figure 4(C). In addition, the broadening of the peak decreases slightly with the loading of DOX into the silica nanoparticles. These changes reflect the incorporation of DOX within the structure of silica nanoparticles.

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For investigating the amount of encapsulated DOX into the ${\rm Si}{{{\rm O}}_{2}}$ nanoparticles excluding the interference of the drug content changes into the measurement, the TGA thermal analysis of pure silica nanoparticles and loaded with doxorubicin is investigated. Figures 5(A)–(C) illustrate the stages of weight loss. The TGA analysis of pure silica nanoparticles exhibited four stages of weight loss which could be indicated as follow: first stage of weight loss occurred at around $30{{\,}^{\circ }}{\rm C}-249.1{{\,}^{\circ }}{\rm C}$, in which the mass loss during this stage is about 0.38%. This is related to the vaporization of physically adsorbed water on the silica surface. Second stage of weight loss happens during the temperature range about $249{{\,}^{\circ }}{\rm C}-508.2{{\,}^{\circ }}{\rm C}$ which contributes to a weight loss of about 3.87%. The loss in weight for this stage is related to the escape of entrapped water and apportion of residual solvent (ethanol) in the porous structure of silica matrix. The third stage of weight loss covers the range of temperature ($508.66{{\,}^{\circ }}{\rm C}-688.56{{\,}^{\circ }}{\rm C}$). During this stage the weight loss (2.3%) may be due to the dehydroxylation of the silanol group and the degradation of residual reactant (TEOS). The fourth stage $688.56{{\,}^{\circ }}{\rm C}-990.02{{\,}^{\circ }}{\rm C}$ is suggested to be due to the degradation of residual TEOS and dehydroxylation of silanol groups. The weight loss at this stage is about 1.44% [21]. Therefore, the total weight loss of silica nanoparticles along all range of temperature ${{10}^{\circ }}{\rm C}-{{1000}^{\circ }}{\rm C}$ is about 7.986% of the total mass. This means that the prepared silica nanoparticles are thermally stable up to $1000{{\,}^{\circ }}{\rm C}$. The observation of TGA thermal analysis of ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ shows that there are apparently two stages of weight loss: (i) first one occurred over the temperature range of $10.0{{\,}^{\circ }}{\rm C}-579.7{{\,}^{\circ }}{\rm C}$, the weight loss in this case is about 53.22%, which may due to loss of water, large portion of solvents and also the decomposition of all present DOX; (ii) second stage appears to cover the temperature range of $579.7{{\,}^{\circ }}{\rm C}-987.7{{\,}^{\circ }}{\rm C}$, the weight loss is about 8.69%, this may be due to the escape of the residual solvents. The overall amount of loaded DOX is estimated by TGA. In the TGA measurement, the drug desorbs after decomposing, which is detected as a temperature dependent weight reduction. However quantification of the loaded-drug fraction in the pores of material is not as simple as just quantifying the total drug content of the sample [22]. The loading fractions are estimated from the mass loss ratio between $10{{\,}^{\circ }}{\rm C}$ and $1000{{\,}^{\circ }}{\rm C}$ compared to the total initial weight. Since the total weight loss of pure silica is about 7.986%, while that for ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ is about 61.91%, therefore the loading of DOX into the silica nanoparticles is about 53.92%.

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Figure 5(D) shows the DSC thermal analysis of the pure silica nanoparticles and ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$. For pure silica nanoparticles, there is an endothermic peak centered at $88.1{{\,}^{\circ }}{\rm C}$. This is attributed to removal of physically adsorbed water molecules, ethanol and ammonia at the surface of silica. The endothermic peak at about $695{{\,}^{\circ }}{\rm C}$ may be due to removal of residual water present on pores and vaporization of other solvents present due to the dehydroxylation process (release of water molecules due to condensation of silanol groups and formation of siloxane linkage) [21–23]. For ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$, the first endothermic peak centered for the pure silica and ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ at the same temperature is due to dehydration. Doxorubicin has a melting point (${{T}_{{\rm m}}}$) at about $230{{\,}^{\circ }}{\rm C}$ as illustrated in literature [24], resulting in an endothermic peak in the DSC curve giving the evidence for crystal structure of the drug. The absence of native doxorubicin ${{T}_{{\rm m}}}$ peak on DSC curve of ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ nanoparticles shows that the drug in nanoparticles is in an amorphous phase. In other words, encapsulation process disrupts the native doxorubicin crystals, indicating that the encapsulation process is appreciable. However on the DSC curve of ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$, there is an exothermic peak centered at about $350{{\,}^{\circ }}{\rm C}$. This may refer to the process of condensation of silanol (−Si–OH) groups that occurred at the surface of the silica particles and accompanied with the elimination of water molecule. This process supports the gathering of silica particles that cause the formation of new siloxane (−Si–O–Si−) bond at the interface. The presence of siloxane permits the more ordering of the product which demonstrate the appearance of the exothermic and peak negative enthalpy change [25]. This means that loading of DOX into silica nanoparticles lead to occurrence of dehydroxylation process at a lower temperature. The endothermic peak at about $695{{}^{\circ }}{\rm C}$ may be due to removal of residual water molecules and other solvents present in pores.

The in vitro release of DOX using the electrospun mats is studied by the immersion of the fibrous mat in PBS solution ($pH=7.4$) at $37{{}^{\circ }}{\rm C}$. For comparison, the release of DOX by PLC-PEO/DOX and PLC-PEO/${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ in which ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ is loaded by different methods; ${{M}_{2}}, {{M}_{3}}, {{M}_{4}}$ and ${{M}_{5}}$ are also investigated. As indicated in figure 6, all the electrospun mats show gradual increase of DOX release with time. The sample contains DOX loaded directly into the nanofiber. Curve in figure 6 indicates three stages of release profile when compared to other samples which exhibit two stages of release profile. The release profile using PLC-PEO/DOX indicates at first an initial rapid release within the first period of incubation, followed by a moderate increase in release with time and then almost constant release during the last periods of incubation. However the total amount of DOX release at the last period of incubation is 91.77%. This fast initial release could be attributed to the presence of DOX agglomerated on the surface of PCL-PEO nanofiber which leads to the direct exposure of DOX to the PBS that result in initial rapid release of DOX.

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For the other samples in which ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ is loaded into the fiber by different methods, the release profile exhibits two stages of release profile; initial, slightly fast release, and secondly sustained release profile. The inhibition of the proliferation of the tumor cells through the early periods of implantation of fibrous mats loaded with anticancer drug is an important demand. This can be satisfied via the rapid initial drug release from the implanted mat. Also the sustained release of drug which follows initial rapid release provides an adequate concentration of anticancer drug over an extended period, this beneficial to avoid repeated administration of drug [3]. For the delivered samples, the DOX release rate is slower and the maximum amount of DOX release is less than that in case of using PLC-PEO-DOX [26].

The release profile of ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ loaded into the nanofiber affected by the method of loading. As shown in curves B, C, D and E of figure 6, the release profile is higher (for both initial, and sustained stages) for sample in which ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ is loaded by M₂ method as compared with M₃, M₄, M₅ methods of loading. For example, the release of DOX at 33rd day (last day of incubation) is about 68.64% for this method and about 58.02%, 59.71%, 37.03% for the other three used methods respectively. M₂ method results in the presence of ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ agglomerated on the surface of the nanofiber (as illustrated in FESEM micrographs) while the other methods of loading incorporate ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ inside the nanofiber. This may be due to, DOX is released from ${\rm Si}{{{\rm O}}_{2}}$ to the medium when present on the surface while when incorporated into the fiber, tends to be first released from the SiO₂ matrix. Subsequently release is occurred from the polymer matrix to the medium [4]. While both M₄ and M₅ methods load ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ into the nanofiber, the release profile of DOX from core shell is slower. This may be due to the preparation by core shell which results in the formation of core which contains dispersed ${\rm DOX}@{\rm Si}{{{\rm O}}_{2}}$ and also thick sheath formed of polymer which means that DOX undergoes more difficulties in steps when released to the medium [27].