Doxorubicin release from optimized electrospun polylactic acid nanofibers

Abstract

Electrospinning has been known as an efficient method for fabrication of polymer nanofibers. In this study, an electrospun nanofibrous mats based on polylactic acid with a defined release using doxorubicin was developed. The effects of process parameters, such as concentration, distance, applied voltage, temperature and flow rate on the mean diameter of electrospun doxorubicin-loaded polylactic acid nanofibers were investigated. The fiber morphology and mean fiber diameter of prepared nanofibers were investigated by scanning electron microscopy. Differential scanning calorimetry was employed to identify the presence of doxorubicin within nanofibers. Response surface methodology based on a five-level, five-variable central composite design was used to model the average diameter of electrospun polylactic acid/doxorubicin nanofibers. Mean fiber diameter was correlated to these variables by using a polynomial function at a 95% confidence level. The coefficient of determination of the model was found to be 0.93. The predicted fiber diameter was in good agreement with the experimental result. Differential scanning calorimetry results showed that the doxorubicin was loaded into the nanofibers successfully. In vitro drug release in phosphate-buffered solution and acetate buffer for the optimized and non-optimized samples demonstrated that diffusion is the dominant drug release mechanism for drug-loaded fibers. The initial burst release was observed for non-optimized nanofibers compared to optimized nanofibers. Optimized drug-loaded polylactic acid nanofibers could be good candidates for biomedical applications.

Keywords

Fibrous materials spinning processing

Introduction

Electrospinning is a simple and versatile technique of producing ultrafine fibers with micro- to nano-meter range diameters and with controlled surface morphology. The basis of electrospinning is application of a strong electric field. The polymer solution or melt is hosted in a syringe pump. When a high voltage is applied, the pendant drop of polymer solution will become highly electrified, and the induced charges are evenly distributed over the surface. When the voltage surpasses a threshold value, the electric force overcomes the surface tension of the droplet and one or multiple charged jets of the solution are ejected from the tip pf droplet. As the jet travels toward a collecting metal screen, solvent evaporates and a non-woven fabric mat is formed [1]. Polymer nanofibers have potential applications as membrane filters, scaffold for tissue engineering, wound dressing and drug delivery [2]. Several parameters affect electrospinning process such as polymer solution parameters (molecular weight, concentration, surface tension, viscosity, etc.), ambient conditions (temperature and humidity) and also processing parameters which include applied voltage, flow rate and tip to collector distance [3]. Affecting the characteristics of the final product such as physical, mechanical and electrical properties, fiber diameter is one of the most important structural features in electrospun nanofiber mats [4]. Therefore, controlling and optimizing the mean fiber diameter (MFD) which is a function of process parameters is crucial. Response surface methodology (RSM) is a collection of mathematical and statistical techniques for empirical modeling and has been widely used to optimize and design operating conditions. RSM has been used successfully for material and process optimization in numerous studies [5 –9]. Yordem et al. [7] investigated the effect of materials and process parameters on the diameters of polyacrylonitryl (PAN) electrospun fibers. The experimental design was carried out by RSM to set solution concentration, voltage, the distance between the syringe needle tip to the collector and solution flow rate. Sukigara et al. [8] have reported an experimental work via RSM, and shown that the effect of the applied voltage creating the electric field may be surprisingly small or expectedly significant depending on the solution concentration. Gu et al. [6], who also employed RSM, reported no significant effect of voltage on the processing of PAN nanofibers. They found that the concentration of solution played an important role in the average diameter of nanofibers. Zahedi et al. [9] prepared electrospun nanofibrous mats based on a poly(vinyl alcohol)/poly(ɛ-caprolactone) (80/20) hybrid with minimum fiber diameter and studied the drug release rate from nanofiners using tetracycline hydrochloride as a model drug. Several local delivery systems such as drug-loaded fibers, hydrogel and nanoparticles, have been studied for biomedical applications in recent years [10 –12]. Among these systems, biodegradable polymer electrospun nanofibers have attracted much attention because of their appealing features such as high surface to area ratio, high loading capacity and encapsulation efficiency [13,14]. Various anticancer drugs such as doxorubicin (DOX), paclitaxel (PTX), platinum complexes and dichloroacetate have been electrospun into fibers and used for postoperative local chemotherapy [15]. For example, Xu et al. reported preparation of ultrafine DOX-containing Polyethylene glycol (PEG)–Poly(L-lactic acid) (PLLA) fibers by electrospinning a water-in-oil emulsion, in which the aqueous phase contained the water-soluble drugs and the oily phase was a chloroform solution of PEG–PLLA. The results indicated that the DOX was entirely encapsulated inside the electrospun fibers [16]. Afterwards, they successfully loaded hydrophobic PTX and hydrophilic DOX simultaneously into PEG–polylactic acid (PLA) nanofiber mats by the emulsion-electrospinning method, and realized multidrug delivery [17]. Xie et al. fabricated cisplatin-loaded PLA/Poly(lactic-co-glycolic acid) (PLGA) (30/70) fibers for long-term sustained delivery of cisplatin to treat C6 glioma in vitro [14]. The drug encapsulation efficiency was more than 90%, and the cisplatin-loaded fibers showed sustained release for more than 75 days without initial burst release. Various materials including natural polymers, synthetic polymers and hybrid blends of the two have been used to obtain electrospun fibers. Synthetic polymers, especially biodegradable polymers, attracted special attention in electrospinning due to the elimination of a second surgery to remove the implanted carrier. In this study, PLA polymer solutions with different amounts of DOX were used to produce nanofiber mats by electrospinning. DOX is an anticancer chemotherapy drug. DOX is classified as an anthracycline antibiotic. DOX is commonly used to treat some leukemias and Hodgkin’s lymphoma, as well as cancers of the bladder, breast, stomach, lung, ovaries, thyroid, soft tissue sarcoma, multiple myeloma and others. The experimental parameters were DOX concentration in polymer solution, applied voltage, spinning distance, flow rate and temperature. The result of the experiment was average fiber diameter. In order to obtain a more systematic understanding of the process conditions and to establish a quantitative basis for the relationships between electrospinning parameters and fiber diameter, an empirical model of fiber diameter was constructed using RSM. Also, in vitro drug release characteristics, release mechanism and kinetics analysis using appropriate mathematical models were investigated in this study. To the best of our knowledge, no systematic study has been reported to establish a quantitative basis for the relationships between the electrospinning parameters and the diameter of DOX-loaded PLA nanofibers. Therefore, the main objective of the present work was to develop an empirical model within the context of RSM for statistically investigating the significance of the electrospinning factors and their interactions, as well as providing a prediction capability for the process conditions to achieve fibrous mats with minimum diameter. Also, DOX release from the optimized nanofibers was studied.

Materials and methods

Materials

PLA with molecular weight of 75 kDa was purchased from Sigma Aldrich. DOX (>99.0%) was supplied by Sigma Aldrich. Chloroform was purchased from Merck (Germany). All other chemicals and reagents were of analytical grade and used without further purification.

Preparation of electrospun nanofiberous scaffolds

Initially, DOX with different contents (0, 7.5, 15.0, 22.5, 30.0 w/w%) was add into 8 mL chloroform with stirring until a clear solution was obtained. In the second step, 1.0 g of PLA was added to the DOX/chloroform solution. This was followed by magnetic stirring until the polymer dissolved completely. PLA/DOX nanofiber mats were produced by an electrospinning machine (ANSTCO-RN/I, Asian Nanostructures Technology Co., Iran). Prepared solutions with different amounts of DOX were fed into a blunted needle by a syringe pump. The collector was a rotating cylindrical drum, which was placed at different distances (10–30 cm) from the needle. The humidity during the experiments was kept the same and it was about 65%.

Characterization of nanofibers morphology

The morphology of electrospun scaffolds was characterized by scanning electron microscopy (SEM; Vega ΙΙ XMU instrument Tescan, Czech Republic). Specimens were sputter-coated with gold for 20 s and imaged with a back-scattering detector. MFDs of scaffolds were calculated from their respective SEM images. For each experiment, average fiber diameter was determined from about 100 measurements of random fibers in four SEM micrographs taken from different areas of the mat.

Experimental design

Central composite design (CCD) was employed to study the effects of electrospinning parameters on the MFD of nanofibrous mats. The independent variables ^X₁, X₂, X₃, X₄, X₅ were as follows (low/high value): DOX/PLA concentration ((w/w%) 0/30; distance (cm) 10/30; voltage (kV) 8/22, flow rate (mL/h) 0.3/1.5 and temperature (℃) 25/45. The ranges of the variables were selected from trial experiments and represented the attainable limits for nanofiber formation and/or equipment operation. Each variable was coded at five levels: −2, −1, 0, +1 and +2. The selected factors and their levels are shown in Table 1. The coded values of these factors were obtained according to equation (1)

X_{i} = x_{i} - x_{0} / Δ x_{i}

(1)

where X_i is the coded value of the factor, x_i is the real value of the factor, x₀ is the real value of the factor at the center point, and Δx_i is the step change value of the factor. The design matrix is shown in Table 2.

Table 1.

Selected factors and their levels.

Variable	Low axial (−2)	Low factorial (−1)	Center (0)	High factorial (+1)	High axial (+2)
X₁ (A)	0.0	7.5	15.0	22.5	30.0
X₂ (B)	10.0	15.0	20.0	25.0	30.0
X₃ (C)	8.0	11.5	15.0	18.5	22.0
X₄ (D)	0.3	0.6	0.9	1.2	1.5
X₅ (E)	25.0	30.0	35.0	40.0	45.0

X₁ (A) = concentration (w/w%); X₂ (B) = distance (cm); X₃ (C) = voltage (kV); X₄ (D) = flow rate (mL/h); X₅ (E) = temperature (℃).

Table 2.

Experimental conditions and their responses.

Run no.	Coded value of X₁ (A)	Coded value of X₂ (B)	Coded value of X₃ (C)	Coded value of X₄ (D)	Coded value of X₅ (E)	Response MFD (nm)
1	1	−1	−1	−1	−1	198
2	0	0	2	0	0	328
3	0	0	0	0	0	254
4	0	0	0	0	−2	306
5	1	1	−1	−1	1	237
6	0	0	0	0	0	240
7	2	0	0	0	0	231
8	0	0	0	−2	0	267
9	−2	0	0	0	0	255
10	1	1	1	−1	−1	293
11	1	1	1	1	1	390
12	0	0	−2	0	0	142
13	1	−1	1	1	−1	373
14	−1	−1	1	−1	−1	189
15	−1	1	−1	1	1	224
16	−1	−1	−1	−1	1	177
17	−1	1	−1	−1	−1	256
18	0	0	0	0	0	243
19	0	2	0	0	0	241
20	0	0	0	2	0	275
21	−1	−1	−1	1	−1	192
22	0	0	0	0	2	267
23	−1	1	1	−1	1	296
24	0	0	0	0	0	251
25	0	0	0	0	0	263
26	0	0	0	0	0	252
27	0	−2	0	0	0	201
28	−1	1	1	1	−1	289
29	1	−1	−1	1	1	217
30	−1	−1	1	1	1	311
31	1	1	−1	1	−1	230
32	1	−1	1	−1	1	203

MFD: mean fiber diameter.

Thermal characterizaton

The thermal properties of the samples were determined using a differential scanning calorimetry (DSC; 200 F3 Maia®, Netzsch, Germany). The heating rate was controlled at 10℃/min. The starting and ending temperatures were 25 and 350℃, respectively.

Wet ability of nanofibrous scaffolds

The water contact angles of PLA and PLA/DOX nanofibrous scaffolds were measured by Sessile drop method with a G10 contact angle goniometer (Kruss, Germany) at room temperature. A water droplet was placed on the scaffold surface, and the contact angle was measured after 10 s.

In vitro DOX release

The cumulative DOX release from DOX-loaded nanofiber mat was investigated. Nanofibrous mats were immersed in 20 mL of phosphate-buffered solution (PBS) at pH = 7.4 and acetate buffer (pH = 4.8) under gentle shaking at 37℃. At predetermined time intervals, 4 mL of each extracted solution was analyzed by UV-vis spectroscopy at a wavelength of 480 nm. This amount of the solutions was immediately replaced with an equal volume of the dissolution medium to keep the volume constant.

Kinetics and mechanism of drug release

Korsmeyer et al. derived a simple relationship which described the drug release from a polymeric system (equation (1)) [18,19]. In equation (2), M_t/M_∞ is a fraction of drug released at time t, k is the release rate constant and n is the release exponent. The n value is used to characterize different release kinetics

M_{t} / M_{\infty} = {kt}^{n}

(2)

In this model, the value of n characterizes the release mechanism of the drug. In the case of cylindrical tablets, 0.45 ≥ n corresponds to a Fickian diffusion mechanism, 0.45 < n < 0.89 to the non-Fickian transport, n = 0.89 to the case II (relaxational) transport and n > 0.89 to the super case II transport.

Results and discussion

Construction of model equations

Thirty-two experiments were designed using CCD methodology. The experimental process conditions and their responses are presented in Table 2. SEM images of prepared nanofibers are shown in Figure 1. Design Expert 7 Software (trial version, Stat-Ease Inc., USA) applied to analyze the results. A polynomial model for the average variations of DOX/PLA nanofibers was chosen and fitted to the results. Equation (3) found to be adequate for the MFD prediction of DOX/PLA elctrospun nanofibers.

MFD = 249.92 + 18.12 B + 41.04 C + 16.37 D - 17.18 BD + 24.18 CD

(3)

Figure 1.

SEM images of prepared nanofibers at different conditions (Table 2).

A, B, C, D and E are the coded values for DOX concentration, tip to collector distance, applied voltage, temperature and flow rate, respectively. Table 3 shows the results of analysis of variance (ANOVA) of the developed model. It implies that the fitted model is significant in 95% confidence level (P-value < 0.05). Values of R² and adjusted R² for the model are 0.93 and 0.88, respectively. The high value of R² indicates that the polynomial equation is capable of representing the system under the given experimental domain. The contribution of the model was 92.5%, and the lack of fit value was not significant, which imply that the model is accurate. Figure 2 represents predicted against actual values for size.

Figure 2.

Predicted vs. actual values of nanofiber size.

Table 3.

The ANOVA analysis using coded values.

Source	Sum of square	DOF	Mean square	F	P > F
Model	81,984.28	20	4099.21	6.75	0.0012
Concentration	1053.38	1	1053.38	5.73	0.0271
Distance	7884.38	1	7884.38	12.98	0.0042
Voltage	4042.04	1	4042.04	6.53	0.0151
Temperature	6435.38	1	6435.38	1059	0.0077
Distance × temperature	4726.56	1	4726.56	7.78	0.0176
Voltage × temperature	9360.56	1	9360.56	15.41	0.0024
Lack of fit	44.34	1	44.34	0.66	0.4545
Pure error	337.5	5	67.5
Total	88,668.2	31

Note: Significant at 5% level (P < 0.05).

The effects of electrospinning parameters on MFD of nanofibers

DOX/PLA concentration

The effect of concentration on MFD is shown in Figure 3(a) to (d). Figure 3(a) shows that at constant temperatures, MFD increased with increasing concentration. As shown in Figure 3(e), an increase in fiber diameter with the increase in solution concentration was observed, which agrees with the other studies [20 –22]. At higher concentrations, however, there are extensive chain entanglements, resulting in higher viscoelastic forces which tend to resist against the electrostatic stretching force. Mo et al. [23], Ryu et al. [24] and Katti et al. [25] also reported a significant relationship between fiber diameter and solution concentration in electrospinning process.

Figure 3.

Contour and surface plots of variables: (a) concentration vs. temperature, (b) distance vs. voltage, (c) concentration vs. flow rate.

Applied voltage

Figure 3(c) and (d) shows the effect of voltage on the MFD. As shown, MFD increased initially and then decreased by increasing applied voltage. Increasing the applied voltage will increase the electric force and cause the jet to extend more in the electric field and produces fibers with smaller diameter. But it also draws more solution out of the capillary. If increasing the electrostatic force draws much more solution out of the capillary, the fiber diameter will be increased with the increase in applied voltage as reported by Zhang et al. [26], Demir et al. [27] and Baker et al. [28]. The balance between these two effects will determine the final diameter of electrospun fibers.

Spinning distance

The effect of spinning distance on MFD is shown in Figure 3(c) and (d). As shown, increasing distance has two different effects on the MFD. The effect of spinning distance is not always the same. Varying the distance has a direct influence on the jet flight time as well as electric field strength. Increasing the spinning distance means that the electric field strength (E = V/d) will decrease, resulting in less acceleration, which leads to fibers with larger diameters. On the other hand, longer spinning distance will provide more time not only to stretch the jet in the electric field but also to evaporate the solvent, thereby encouraging formation of thinner fibers. The balance between these two effects will determine the final fiber diameter. Decrease in fiber diameter [29] as well as increase in fiber diameter [30 –32] with increasing spinning distance was reported in the literature. There were also some cases in which spinning distance did not have a significant influence on fiber diameter [33,34].

Flow rate

As shown in Figure 3(e) and (f), MFD increased with increasing the flow rate of solution, which agrees with the previous studies [35,36]. Increasing the flow rate, more amount of solution is delivered to the tip of the needle enabling the jet to carry the solution away faster. This could bring about an increase in the jet diameter, favoring thicker fiber formation.

Temperature

Among the electrospinning parameters such as concentration, applied voltage, spinning distance and feeding rate, the temperature of the spinning process is another significant factor that influences the MFD of electrospun fibers as it could change the elastic properties of the polymer solution. Figure 3(a) and (b) shows the effect of temperature on the MFD. As can be seen, for all concentrations, fiber diameter decreased by increasing the temperature. Similar result was reported by Wang et al. [37] in the electrospinning of PAN fibers. According to the their report, fibers with a diameter of 65–85 nm were prepared by electrospinning at 88.7℃ with a 6 wt.% solution, whereas larger fibers with a diameter of 190–240 nm were frequently obtained at room temperature. In consequence, the temperature facilitates the electrical stretching of the whipping jet by providing an adequately elastic response of the solution.

Optimization

After analyzing the parameters and their interactions with each other for individual response (MFD), determining the best electrospinning conditions, paying attention to the point that nanofibers diameters of nanofibrous mats must have minimum values, the best amounts of each effective parameter along with the response for the electrospinning conditions are as follows: 11.5 kV voltage, 22.4 (w/w%) concentration, 15 cm distance, 30℃ temperature and 1.2 mL/h flow rate. According to the above conditions, the minimum average diameter of DOX/PLA nanofibrous mats was about 124 nm. In order to investigate the reliability of the fibers produced from the electrospinning process, a test was conducted to measure the fiber diameter from the given set of process parameters. SEM image of optimum conditions and fiber diameter distribution is shown in Figure 4. The MFD was about 112 ± 14 nm. Comparing the experimental result with the response provided by the model shows that the difference is less than 10% and they are close to each other.

Figure 4.

SEM image of nanofibers and fiber diameter distribution at optimum condition.

DSC analysis

DSC curves provided information on the thermal transformations in the nanofibers and the state of the drug after loading process. Figure 5 shows DSC thermograms of DOX, optimized PLA nanofibers and corresponding nanofibers containing DOX. The glass transition temperature (T_g) values of the neat and encapsulated PLA nanofibers were relatively about 55 and 50℃, respectively. By adding drug into the electrospinning polymer fibers, the small molecule drug acted on the molecular chains and made the molecular chains move easily, leading to a lower T_g. DOX had a melting point (T_m) about 220℃ [38] resulting in an endothermic peak in the DSC curve and giving the evidence for crystal structures of the drug. The absence of a characteristic melting peak of DOX in the DSC curve of DOX -loaded PLA nanofibers represented that the drug in the nanofibers were converted to amorphous phase and that the entire added drug was bound to the polymer. In other words, the loading process was appreciable.

Figure 5.

DSC thermograms of (a) DOX (b) PLA nanofibers and (c) DOX-loaded PLA nanofibers.

Hydrophilic properties of optimized nanofibers

The water contact angles for PLA and PLA/DOX scaffolds were 105 ± 4° and 67 ± 3°, respectively, which imply that PLA/DOX scaffold was more hydrophilic than PLA nanofibrous scaffold. The incorporation of DOX into PLA mat decreased the hydrophobicity of the PLA membrane. It could be due to the addition of the water-soluble DOX with the hydrophilic hydroxyl groups and the increase of the hydrophilicity of the composite membranes.

In vitro drug release from the optimized nanofibrous mats

Figure 6 shows accumulative release of DOX from nanofibrous mats under optimized and non-optimized conditions at different pH. As shown, the cumulative release amount from optimized nanofibers was only about 15% in PBS 7.4 in 10 h. For the lower pH, a faster DOX release from the fibers was observed with about 40% in 10 h. It is reasonable that the solubility of DOX increases under pH 7.4, leading to a faster DOX diffusion from the fibers into medium. It is well known that diffusion is the typical drug release mechanism for drug-loaded fibers in the early stage owing to the slow degradation of PLA, thus the mobility of polymer chains seems plays the dominant role [17]. In order to investigate the effect of fiber diameter optimization on DOX release from PLA nanofibers, non-optimized nanofibers prepared at experiment No. 11 (MFD ∼ 400 nm), which have the same amount of drug with optimized nanofibers (22.5 (w/w) %), were examined. As shown in Figure 6, the amount of DOX release was higher than optimized nanofibers at pH 4.8 and 7.4. The initial burst release was observed for non-optimized nanofibers compared to optimized ones. Results showed that larger nanofibers release DOX faster than smaller ones. These differences in drug release as a function of fiber diameter may be a result of swelling behavior and drug diffusion in the different fiber diameters. Similar result was reported by Xie et al. [39] for chlortetracycline release from PLA nanofibers. However, drug release mechanisms are often very complex and formulation properties such as drug, polymer and solvent choice can directly affect electrospinning process and resulting drug dispersion in the fibers [40]. Therefore, formulation properties and processing parameters must be considered simultaneously when developing drug-loaded electrospun fibers. In order to evaluate the release mechanism of DOX from drug-loaded samples the resultant data were fitted to the Korsmeyer–Peppas model. The data obtained from in vitro drug release studies were plotted as log cumulative percentage of drug release versus log time, to study the release kinetics. The results are presented in Table 4. It shows that in PBS media the drug was released by a non-Fickian diffusion mechanism, but in buffer with pH 4.8, the drug was released by a Fickian diffusion kinetics.

Figure 6.

Cumulative release of DOX from optimized and non-optimized nanofibrous mats in different buffers.

Table 4.

DOX release kinetic parameters for nanofibrous mats in different media.

Nanofibrous mat	R²	Mechanism	n	k	Release medium
Optimized	0.95	Fickian	0.45	0.14	PBS
Optimized	0.99	Non-Fickian	0.61	0.07	Acetate buffer
Non-optimized	0.94	Non-Fickian	0.54	0.04	PBS
Non-optimized	0.96	Non-Fickian	0.67	0.11	Acetate buffer

PBS: phosphate-buffered solution.

Conclusion

DOX-loaded PLA nanofiber mats were prepared in this study. Process optimization of the MFD was performed using RSM with CCD. DOX concentration, spinning distance, voltage, temperature and flow rate were the studied factors for this purpose. A polynomial equation for MFD was developed. Afterwards, in order to show the generalized ability of the model for predicting new conditions, a set of experiments was carried out. The small differences of experimental data with predicted values, indicating the good prediction ability of the model. In vitro drug release from nanfibrous mats and the release mechanism were investigated. Results showed that the diffusion mechanism is dominant. To conclude, electrospinning was shown to be very promising approach to the formulation of DOX in order to enhance its release in a sustained and prolonged manner, and therefore higher in vitro antitumor efficacy. Thus, the prepared PLA/DOX nanofibers at optimum conditions are highly promising as local implantable scaffolds for the treatment of a tissue defect after tumor resection.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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