Venlafaxine-Loaded Nanocomposite-Based Scaffolds: In Vitro, Ex Vivo and Cytotoxicity Evaluations

doi:10.21203/rs.3.rs-1436296/v1

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Venlafaxine-Loaded Nanocomposite-Based Scaffolds: In Vitro, Ex Vivo and Cytotoxicity Evaluations

https://doi.org/10.21203/rs.3.rs-1436296/v1

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Venlafaxine (VEN) is an antidepressant drug, but its delivery progress combined with a dependence on pathways and the incidence of toxicities that have infrequently led to FDA endorsement. This work focuses on developing novel electrospun polymeric nanofibers (NFs) for buccal delivery of VEN to avoid the hepatic metabolism, enzymatic degradation in the GIT, and develop an effective control of drug release. The optimized NFs were obtained by blending polylactic acid (PLA) and poly (ɛ-caprolactone) (PCL) and were characterized for morphology, drug-loading, FTIR, XRD, DSC, and in vitro drug release. Ex vivo permeability of the blend NFs was assessed using chicken pouch mucosa compared to VEN suspension followed by histopathological examination. Further, the cytotoxic effect was evaluated in three different cell lines using WST-1 assay. SEM morphologies refer to defect-free uniform NFs of PLA, PCL, and PLA/PCL mats. These fibers had a diameter ranging from 200–500 nm. The physico-thermal characterization of NFs depicted that the drug was successfully loaded and in an amorphous state in the PLA/PCL NFs. The cumulative release of NFs substantiated a bi-phasic profile with an initial burst release and a second sustained phase that reached 80% over 96 h following a non-Fickian diffusion mechanism. Ex vivo permeation emphasizes the major enhancement of the sustained drug release and the noticeable decrease in the permeability of the drug from NFs. Histopathology and cell toxicity studies demonstrated the preserved mucosal architecture and the preclinical safety. The developed PLA/PCL NFs can be promising drug carriers to introduce a step-change in improved psychiatric treatment healthcare.

Venlafaxine

electrospinning

polylactic acid

poly (ɛ-caprolactone)

sustained release

cytotoxicity.

Currently, the treatment of central nervous system (CNS) diseases, inclusive of main depression perseveres as a appall competition amidst the progress in therapeutic technology because of the presence of the blood-brain barrier (BBB) [1]. This barrier prohibits the permeability of therapeutic such as large molecules (e.g., proteins or antibodies) as well as low molecular weight ones. Minor molecules with a lipophilic feature can only pass it [2]. Venlafaxine (VEN) is used as a drug of choice for treating clinical depression even during pregnancy.

The dual antidepressant action of the drug has been ascribed to its suppression of neuronal reuptake of serotonin and norepinephrine with a subdued affinity for muscarinic cholinergic, histaminergic, or alpha-adrenergic receptors [3]. Unfortunately, VEN presents numerous side effects after oral administration like headache, nausea, drowsiness, constipation, weakness, tachycardia, insomnia, fatigue, and dry mouth. Moreover, P-glycoprotein (P-gp) behaves as a natural barrier to VEN transmission to the brain. In other words, VEN is a P-gp substrate, and P-gp can restrict the drug penetration into a specific site [4]. The daily dose of VEN varies from 75 to 350 mg/day. The drug is broadly metabolized in the liver via CYP2D6 and so has poor oral bioavailability (40–45%) [5]. VEN and its active metabolite (O-desmethylvenlafaxine (ODV)) have a highly efficient impact in improving efficacy, increasing the promptness of incipience of effect, and exhibiting the high integrity handling of depression symptoms. VEN administration reaches 2–3 times per day owing to its short steady-state elimination half-life of 3–4 h for VEN and 10 h for ODV, to keep adequate plasma levels of the drug [6]. It has extended and immediate formulations that have high efficacy in decreasing depression symptoms. It is very challenging to mature a therapeutic preparation with a gentle dissolution speed of freely soluble drugs. Therefore, to preserve a steady therapeutic level, multiple daily administrations of VEN-based forms are desirable. Many researchers strive to find better techniques to prepare controlled release formulations of VEN [7]. The extended-release formulations have a highly impactable effect in rising compliance and convenience of the patient as well as a superior benefit /risk ratio to the instant release of VEN [8]. The commonly used polymers such as pectin [9], chitosan [10], and polyvinylpyrrolidone (PVP) [11] have been claimed for irritation of gastric mucosa, uncontrolled fast release of a drug, an allergic reaction, respectively. Thereby limiting their usage, and none have received FDA approval yet. Nevertheless, some are considered suboptimal mainly because of either low drug loading efficiency or the stumpy dwelling time creating a discontinuity between formulation development and clinical manifestation. [7, 8, 12–15]. The developments in nanotechnology have given newfangled horizons in succeeding this objective since the nano-scaled systems can amend a desirable drug to have an extraordinary level of sensitivity to the physiological circumstances and specificity to reach the purposed organ. Innovative nanocarriers help to carry the drug introduced through various invasive or noninvasive directions in the body in a controlled way from the site of administration to the therapeutic target [16, 17]. Under these circumstances, the electrospinning process offers a great impact on drug loading efficiency, extended-release time, and integration into nanofibers. Electrospun nanofibers have gleaned special interest as drug delivery systems. Owing to their distinct functional feature such as eminently minor pore size, the enormous surface to volume ratio as well as the porous matrix that overcome the limitation of the poor drug payload [18]. Furthermore, increasing attention has focused on electro-spun nano scaffolds as witnessed related to their microporous architecture, the physicochemical properties of the utilized polymers, and modulating the formulation variables that offer ample flexibility to customize the drug release behaviors [18]. Also, their bioactivity, resorbability, biocompatibility, and degradability offer great benefits in drug delivery systems. Polylactic acid (PLA, an FDA-approved biopolymer) has been widely adopted for biomedical applications [19].

PLA is either a semi-crystalline or amorphous biopolymer according to its relative ratio of PLA backbone chains synthesized in the chemical process. PLA has a decisive influence on the drug release systems known as a highly biocompatible, biodegradable, and easily spinnable polymer. Its biodegraded products are highly secure in the human body and considered as appropriate hydrophobic media for effective hydrophilic or hydrophobic drug loading [20]. Nonetheless, restrictions also occur for PLA NPs, such as non-specific uptake by the reticuloendothelial systems (RESs), besides minor drug loading capability and subdued encapsulation efficacy [21]. Besides, Poly (ɛ-caprolactone) (PCL) is a synthetic semicrystalline polymer that is commonly utilized in medical applications. Because of its good drug permeability, slow degradation rate, and extraordinary physicochemical properties for chemical modifications [22]. Upon the creation of PLA/PCL blend nanofibers, the resulting 3D nanofibers electrospun having a shorter time of degradation and higher mechanical features such as modulus and stiffness than the PCL nanofibers scaffolds and improving the bioactivity as well. PLA /PCL blend has impactable features and benefits of the physicochemical properties of polymeric materials different from those of individual virgin polymers [23, 24]. The blend formation is simpler and easier to be spinnable and capable of achieving the enhanced material properties by a suitable blend of individual polymeric components [25]. High drug concentration in the oral cavity is a requirement for sufficient drug absorption through buccal mucosa [26]. The buccal mucosa is a highly vascular tissue that authorizes direct systemic attainability through the internal jugular vein that bypassing the first-pass metabolism for different drugs, enhancing their systemic bioavailability. Also, buccal trajectory could be an alternative possibility for seizure dominance in epileptic unconscious patients [27].

Under the aforementioned umbrella, this study has adopted to inspect the effect of a novel PLA/PCL blend on the manufacturing of nanofibers electrospun (NFs). Then, the morphology of nanofibers was noticed by scanning electron microscope (SEM). Also, physicochemical, and thermal characterization of nanofibers were performed using Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), and differential scanning calorimetry (DSC). Finally, in vitro and ex vivo appraisal studies would be carried out compared with the corresponding drug solution. This study was implemented with the mathematical modeling for drug release kinetics in good accordance with the experimental release data. Histological examination was gauged to inspect the mucosal safety. In vitro cytotoxicity assay on different cell lines would be fulfilled. This approach we invented to offer a newfangled delivery system that sustains the release and prolongs the duration of action of VEN.

Materials

Venlafaxine hydrochloride (VEN) was kindly supplied as a gift sample from Pharaonia Pharmaceuticals (Alexandria, Egypt). Polylactic acid (PLA) (Mw 93,500 g/mol), was purchased from NatureWorks (Minnetonka, United States). Poly (ɛ-caprolactone) (PCL) (Mw 80,000 g/mol), Chloroform (CFM), and dimethylformamide (DMF) were purchased from Sigma-Aldrich (Steinheim, Germany). Liver cancers cells (Huh-7), Oral epithelial cells (OEC), Green monkey kidney cells (Vero) were garnered from Biomedical Research Section, Nawah Scientific Inc., (Mokatam, Cairo, Egypt). All other chemicals and solvents were of analytical grade and utilized as purchased.

Preparation of the composite NFs

A series of solutions with varied biodegradable polymers concentrations were prepared as listed in Table 1. A 10% (w/v) PLA solution was prepared by dissolving the proper amount of polymer in CFM/DMF binary solvent (80:20 v/v). Meanwhile, two different solutions of PCL (10 and 14% w/v) were prepared in binary solvent (70:30 v/v). The solution of combined biodegradable polymers composite of PLA and PCL at a 1:1 volume ratio was prepared in binary solvent (70:30 v/v). Then, based on preliminary studies, the fixed amount of VEN (20% w/v) was dissolved in the polymer solutions. All solutions were magnetically stirred for 3h at room temperature using a magnetic stirring (Hot Plate and Magnetic stirrer, Daihan Scientific Co., LTD, Korea). Plain NFs, as a control, were obtained with the identical composition without adding VEN. Meanwhile; cast films of utilized polymers were obtained for comparative study with the similar quantitative composition of NFs.

Table 1

Composition of different nanofibers.
Formulation	Polymer	Polymer concentration (%)	Polymer ratio (wt/wt)	Solvent ratio (v/v) (CFM: DMF)	Flow rate (mL/h)
F1	PLA	10	-	80:20	1.0
F2	PCL	14	-	70:30	0.6
F3	PLA:PCL	10	50:50	70:30	0.3

Electrospinning was carefully performed using NANON-01A electrospinner (MECC CO., LTD., Japan) combined with a high-voltage power supply, an infusion pump, a glass syringe attached to a stainless steel-smooth needle (outer diameter, 1.2 mm; inner diameter, 1.0 mm), and drum stainless-steel collector. During the electrospinning process, all prepared solutions were ejected at a constant voltage of 20 kV between the syringe needle and drum metal collector. The solutions of PLA, PCL, and PLA/PCL blend were sprayed at 1.0, 0.6, and 0.3 mL/h flow rates, respectively with a constant air gap of 15 cm straggled the needle tip and the collector. To fabricate the nonwoven and homogenous thick mesh, all resultant fibers were collected (diameter,40 mm; rotating speed, 200 rpm of a drum) on a flat aluminum foil sheet covering a drum collector. Furthermore, the electrospinning process was executed at 24°C and low humidity. The collected fibers were dried under vacuum till complete elimination of the residual solvents and moisture.

Characterization techniques

Scanning electron microscope

The microscopic morphologies and size of the cast films and NFs were detected using a scanning electron microscope (SEM, JEOL JSM-6510LV, Oxford instruments, UK) at an acceleration voltage of 30 kV. The average diameter of these fibers was calculated using an image analysis tool in the Zeiss smart SEM® software for their quantification. Before the examination, the fibers were attached to an aluminum stub with double-sided adhesive tape and then sputter-coated with gold before images capturing to render them electrically conductive.

Attenuated total reflectance-Fourier infrared spectroscopy

Infrared spectra of the drug, polymers, and electrospun NFs were recorded on FT-IR spectra. It was fitted with an attenuated total reflectance mode (ATR) (Thermo-Fisher Scientific, Inc., Waltham, MA, USA) with a single-reflection diamond crystal using a Nicolet iS10 spectrometer. The resulting spectra were investigated over the range from 4000 to 550 cm^− 1 with a resolution set at 4 cm^− 1.

X-ray diffraction

The crystallinity of the pure drug and the formulations were inspected using X-ray diffraction (XRD, Shimadzu XRD-6000 diffractometer, Japan). The XRD was operated at 40 kV and 40 mA using Cu-Kα radiation in the range of (2θ) 5–55^◦ with a scanning rate of 0.05^◦/s at room temperature using the monochromatized diffractometer (λ = 0.154 Å) with graphite-sample monochromators. The crystallinity level was detected by applying the area integration method [28].

Differential scanning calorimetry

The thermal features of VEN, polymers and the NFs formulations recorded using differential scanning calorimetry (DSC, SII DSC 6200, Japan). Sealed samples heated in aluminum pans and an empty pan used as a reference. In a nitrogen atmosphere of 20 mL/ min flow rate, thermograms were recorded in the temperature range of 50–400°C with a heating rate of 10°C/min.

Evaluation of drug encapsulation efficiency

The drug entrapment efficiencies of the nanofiber mats were calculated as follows. Ten milligrams of dried drug-loaded nanofibers were cut and dissolved in 10 mL CFM for 5 min using a magnetic stirrer. The amount of the drug was analyzed by a UV/VIS spectrophotometer (Shimadzu UV-1601 PC, Japan) at wavelength of 285 nm and quantified using a previously constructed standard curve. The test was conducted in triplicate and the results were calculated as mean ± SD. The drug entrapment efficiency (EE %) of the process was evaluated using the following equation [29, 30]:

EE% = $\frac{{A}{m}{o}{u}{n}{t} {o}{f} {d}{r}{u}{g} {i}{n} {N}{F}{s}}{{D}{r}{u}{g} {i}{n}{i}{t}{i}{a}{l}{l}{y} {a}{d}{d}{e}{d}}$ X 100

In vitro drug release

In vitro release of VEN from the selected nonwoven nanofiber mats was studied using dialysis bags. NFs and casted films were divided into pieces holding 25mg of VEN and each was placed into a 50 mL PBS (phosphate-buffered saline, pH 7.4) underwent a rotary shaker incubation (GFL Gesellschaft für Labortechnik, Burgwedel, Germany) at 100 rpm and 37 ± 0.5 ^oC. At predetermined timed intervals (0.5, 1, 1.5, 2, 3, 4, 6, 24, 48, 72, and 96 h), samples were withdrawn and compensate with the dissolution medium. The collected samples were filtered through a 0.45 µm Teflon syringe filter and assayed spectrophotometrically at 226 nm for its drug content. All incubation processes were performed in triplicate and the results were expressed in terms of cumulative release as a function of time according to the following equation [31].

The cumulative drug release (%) =$\frac{{C}{t}}{{C}{\infty }} \times 100$

Where C_t is the amount of VEN released after time t and C_∞ refers to the total amount of drug-loaded theoretically into the NFs.

Mathematical models for drug release kinetics

To explain the mechanism of VEN release, release data were kinetically analyzed according to zero-order, first-order [32, 33], and Higuchi diffusion mechanism [34]. For further investigation of release mechanisms, the Korsmeyer-Peppas model was applied to determine the diffusion exponent (n) [35]. A model to which the release data were best fitted with the highest correlation coefficient (R²) was the one that termed the release of VEN.

Ex vivo assessment of buccal delivery systems

Ex vivo drug permeability test

The optimized formulation with promising in vitro results was exposed to drug permeation testing using chicken cyst tissues attached to modified Franz diffusion cells. Chicken cyst tissues were selected as a model membrane for the permeability study due to their histological similarity with buccal mucosa [36]. Chicken cyst tissues were freshly collected post-sacrifice from a local slaughterhouse. The tissues were firstly pre-treated by cleansing the loose connective fibers. Then it was eliminating from surface fats and cleaned with isotonic phosphate buffer (IPB) pH 7.4.

The washed tissues were instantaneously utilized in which their surface facing up the donor compartments contain 2 mL simulated saliva [37]. All receptor compartments were fulfilled by IPB pH 7.4, and the membrane was just below the surface of the recipient solution. All diffusion cells were placed in the rotary shaker incubation at 37^oC and 100 rpm. The samples were withdrawn at predetermined time intervals over 6 h, then filtered through a 0.45 µm Teflon syringe filter and were compensated by an equal volume of fresh IPB. The results were recorded using spectrophotometric measurements at 226 nm. The tests were conducted in triplicate (n = 3) and the average values of flux and permeability coefficient were calculated [38].

The % cumulative amount of permeated drug per square centimeter was plotted versus time (h), and steady-state flux was calculated from the slope of the linear fraction of the plot as the following equation:

Flux = Jss=$\frac{{1ex}{${d}{Q}$}\!\left/ \!{-1ex}{${d}{t}$}\right.}{{A}}$

Where Jss is the steady-state flux; d_Q/dt is the permeation rate; A is the diffusion surface area (3.46 cm²).

The permeability coefficient P was calculated using the following equation:

$${P}=\frac{{J}{s}{s}}{{C}{d}}$$

Where P is the permeability coefficient and C_d is the initial drug amount in the donor side.

Histological examination

At the end of the permeability study, the chicken cyst tissues were histologically examined to investigate mucosa change, such as damage or irritation [36]. After careful removal of the pouch mucosa from the diffusion cells, it washed with IPB to remove any residual dosage forms. The tissues were fixed with 10% formalin and mounted in hard paraffin. It was successively partitioned and microscopically inspected after smearing with hematoxylin and eosin. Cellular stealth and tissue destruction were detected with a light microscope [21].

In vitro study of cell toxicity

The cytotoxic effects of pure VEN, plain NFs, and medicated NFs (F3) were studied in vitro using Liver cancers cells (Huh-7), Oral epithelial cells (OEC), Green monkey kidney cells (Vero) were garnered from Biomedical Research Section, Nawah Scientific Inc., (Mokatam, Cairo, Egypt). This work was approved by the Ethical Research Committee of the Faculty of Pharmacy, Mansoura University. Briefly, the cells were cultured in Dulbecco's modified eagle medium (DMEM) complemented with streptomycin (100 mg/mL), penicillin (100 units/mL), and 10% of heat-inactivated fetal bovine serum in a humidified atmosphere of 5% (v/v) CO₂ at 37°C. Cell viability was assessed by WST-1 (Water Soluble Tetrazolium Salts) assay. Aliquots of 50 µL cell suspension (3x10³ cells) were sown in 96-well plates and brooded in complete media for 24 h. All samples were sterilized by UV irradiation before use. Afterward, the cells were exposed to another aliquot of 50 µL media containing pure VEN at concentrations of 0.01, 0.1, 1, 10, and 100 µg/mL. VEN-equivalent amounts of plain and medicated NFs were used to prepare the above concentration series. After 48h of exposure, the cells were exposed to 10 µL WST-1 reagent and the absorbance was detected after 1 h at 450 nm (A₄₅₀) using a BMG Labtech®- Fluostar Omega microplate reader (Allmendgrün, Ortenberg, Germany). All the experiments were performed in triplicate. Representative phase-contrast images of cells were captured just before the addition of the WST-1 reagent (Olympus BX 41 microscope, Olympus, USA). The cytotoxic effect was expressed as the percent cell viability and was used to construct a dose-response graph. Then, the half-maximal inhibitory concentration (IC₅₀, the concentration that killed 50% of cells) in comparison with the untreated (control) cells was calculated from the dose-response curve. The percent cell viability was measured using the following equation.

$$\mathbf{C}\mathbf{e}\mathbf{l}\mathbf{l} \mathbf{V}\mathbf{i}\mathbf{a}\mathbf{b}\mathbf{i}\mathbf{l}\mathbf{i}\mathbf{t}\mathbf{y} \mathbf{\%}= \frac{{\mathbf{A}}_{450} \mathbf{o}\mathbf{f} \mathbf{t}\mathbf{r}\mathbf{e}\mathbf{a}\mathbf{t}\mathbf{e}\mathbf{d} \mathbf{c}\mathbf{e}\mathbf{l}\mathbf{l}\mathbf{s}}{{\mathbf{A}}_{450} \mathbf{o}\mathbf{f} \mathbf{c}\mathbf{o}\mathbf{n}\mathbf{t}\mathbf{r}\mathbf{o}\mathbf{l} \mathbf{c}\mathbf{e}\mathbf{l}\mathbf{l}\mathbf{s}} \times 100$$

Statistical analysis

The EE%, in vitro, and ex vivo experiments were all performed, and the data are reported as mean ± SD of at least three replicates. The results obtained were compared using one-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparisons using OriginPro® 2021 software (OriginLab Corporation, Northampton, MA, USA). Unpaired student t-test was used to compare the means of the ex vivo permeation study, with the ρ-value set as 0.05.

Characterization of NFs

Electrospinning conditions and morphology of various nanofibers

Figure 1 represented the morphology and sizes of all the different prepared samples of the cast films and NFs mats (F1, F2, and F3) as obtained from SEM examination. Figure 1a-b showed smooth fracture surfaces of plain and medicated PLA cast films, respectively. After the electrospinning process, Fig. 1c-d showed the smooth defect-free NFs with a narrow diameter distribution with an average size is about 500 nm referred to plain and medicated NFs of PLA, respectively. On the other hand, to optimize electrospinning conditions to fabricate well-defined nanofibers of PCL polymer, the concentration of PCL was varied from 10 to 14 wt%. As seen in Fig. 1e, 10% PCL NFs exhibits very narrowly, smooth, and beads-free NFs with average diameters ranging from 150 to 250 nm. The average diameter of 14% PCL NFs was increased to a predominant size of 300 nm owing to increasing PCL concentration from 10 to 14 wt% as demonstrated in Fig. 1f-g. Pure PCL NFs appeared smaller and much finer than the pure PLA embedded NFs and the same applies for both drug incorporated PCL and PLA NFs [39]. Meanwhile, PLA/PCL blend NFs produced smooth, very narrow, and uniform electrospun fibrous structures as can manifest in Fig. 1h-i.

ATR-FTIR analysis

As shown in Fig. 2a, the spectrum of VEN has characteristic peaks at 3347 cm^− 1 that attributed to ‒O-H (stretch), 2937, 2858, and 2578 cm^− 1 attributed to ‒C-H (stretch), 1613 cm^− 1 that exhibited from ‒C-C (stretch) and 1153 cm^− 1 that refers to = C-N (stretch). In Fig. 2b, the PLA film spectrum showed the absorption peaks of the reduced –OH group at 3381 cm^− 1, the C = O stretching of the carboxyl groups that were assigned at 1752 cm^− 1, and ether bond (C–O stretching) ascribed at 1183,1130, and 1086 cm^− 1. Similar characteristic peaks had shown in Fig. 2c of PCL film that has also exhibited a strong peak of C = O stretching at 1723 cm^− 1 credited to carboxyl groups of PCL. As evident in Fig. 2e-g, there are no significant differences between the spectra of both PLA and PCL cast films and their plain NFs. The spectrum of the PLA/PCL blend (Fig. 2d) showed assignable peaks of both PLA and PCL, and no new peaks are noticed in its spectrum. A significant broad peak at 3347 cm^− 1 was noticed (Fig. 2f&h&j) that referred to O-H (stretch) of VEN. Meanwhile, the FT-IR spectrum of F3 NFs (Fig. 2j), presented sharp absorption peaks at 1754 and 1726 cm^− 1 of C = O stretching of the carboxyl groups of PLA and PCL, respectively.

Crystallinity evaluation of NFs

XRD patterns of VEN, cast films, and NFs are evident in Fig. 3. As shown in Fig. 3a, VEN is a highly crystalline powder with characteristic reflections at 2θ of 6.7°, 8.3°, 12.7°, 13.6°, 16.4°, 18.9°, 20.3°, 21.3°, 21.5°, 25.1°, 28.6° and 35.1° (Bragg angles) well supported by the literature [40–42]. As seen in Fig. 3b, PLA cast film has exhibited strong Bragg reflection occurred at 2θ values of 16.8 and 19.5^o that is ascribed to (200)/ (110) and (203) crystallographic planes of a-form PLA crystal, respectively. Meanwhile, the sharp diffraction peaks appeared at 21.3^o and 23.8^o, corresponding to (110) and (200) crystal plane diffraction of PCL, respectively (Fig. 3c). Figure 3d showed crystalline peaks of both PLA and PCL polymers in their blend. After electrospinning, typical amorphous structures of both F1 and F2 and their plains exhibited small intensities peaks at the similar positions of PLA and PCL reflections.

Thermal analysis of NFs

DSC has been conducted to be a fast method for appraising physicochemical interactions between constituents of the formulation via the comparative studies of the thermal curves of pure substances, the polymeric NFs, and their drug-loaded fibers. Exemplary DSC heating thermograms are represented in Fig. 4. DSC thermogram of VEN, Fig. 4a, displayed a sharp melting endotherm at 211.87°C exhibiting its crystalline nature [43]. Figure 4b showed a cast PLA film manifests both a glass transition temperature (T_g) at 65°C and an endothermic melting peak (T_m) at 154°C [44]. Whereas the cold crystallization exotherm exhibited a crystallization temperature (Tc) at 94°C. Figure 4c showed the exhibited temperature values of pure PCL (T_m = 60. and T_g = − 60°C) [45, 46]. Meanwhile, the T_m values in both PLA and PCL NFs (F1 and F2) and their plains are almost unchanged compared to their film casts thermograms that shown in Fig. 3(e&h).

Encapsulation efficiency of electrospun NFs

The entrapment efficiency is an indispensable criterion for the evaluation of the drug amount incorporated in NFs. All PLA, PCL, and PLA/PCL composite nanofibers have been delineated to have adequate encapsulation efficiency (≥ 95%).

In vitro drug release study

In vitro release study of VEN from PLA, PCL, and PLA/PCL blend was conducted in a phosphate buffer solution at pH 7.4 for 96 h to evaluate the potential application of the drug-loaded NFs as a drug delivery system. As shown in Fig. 5, all the drug-loaded NFs scaffolds have two stages (bimodal) of drug-release patterns. The first stage shows an initial burst release with a significant amount of drug released in the first eight hours, followed by the incrementing increase.

Figure 5 presented the cumulative amount of drug released within two days was 37 ± 7.7, 65 ± 8.6, 80 ± 3.3, and 100 ± 1.7% for PCL, PLA, PLA/PCL blend (F1) NFs, and the drug alone, respectively. Whereas F3 exhibited the highest release rate reached 80% over four days of both PLA and PCL nanofibers.

Mathematical models for drug release kinetics

The selection of an acceptable model for fitting drug release data is worthy for defining the release characteristics using model-dependent slants. The initial “burst release” was taken into account for adjusted mathematical equations. The coefficient of determination values (R²) was calculated corresponding to four mathematical models. As illustrated in Table 2, the four formulations did not obey either zero-order or first-order kinetics. The best explanation of the release obeys to Higuchi and Korsmeyer-Peppas models, as their plots showed high linearity [47]. Besides, for the implementation of the release mechanism explanation, the release mechanism is represented as a function of the diffusion exponent (n). Both VEN-release and F1 kinetic models at pH 7.4 fitted well with Fickian-diffusion (n < 0.5). However, both medicated F2 and F3 followed the non-Fickian diffusion (anomalous) model (0.5 < n < 1).

Table 2

Release kinetics of *in vitro* release data
Model	Parameter	VEN	F1	F2	F3
Zero-order	R ²	0.898	0.755	0.721	0.772
	K_o	1.346	0.434	0.411	1.295
	Q_o	0.372	0.371	0.121	0.536
First-order	R ²	0.798	0.798	0.795	0.700
	K₁	0.316	0.243	1.190	0.364
	Q_o	1.354	0.755	0.133	1.416
Higuchi	R ²	0.985	0.974	0.850	0.966
	K_H	1.868	0.834	0.034	2.001
	Q_o	0.057	0.109	1.108	0.085
Korsmeyer-Peppas	R ²	0.951	0.995	0.991	0.986
	n	0.420	0.346	0.624	0.514
	K_KP	0.179	0.241	1.430	0.263
Where;
R² is correlation coefficient; while, Ko, K₁, K_H and K_KP are zero-order release, first order release, Higuchi, and Korsmeyer constants, respectively. Qo and n are the initial release amount (µg/mL) and diffusion or release exponent, respectively.

Ex vivo drug permeability through chicken pouch mucosa

The chicken cyst is regarded as the best fit convenient model mucosa for this study; it is widely available and presents an alternative to partially keratinized rabbits’ mucosa and the keratinized mucosa of hamsters/ rats. Otherwise, dogs, monkeys, and pigs are also considered as non-keratinized mucosa but they have been restricted in this study to have more superfine and highly porous mucosa than that of humans [37]. On comparing F3 with control VEN solution, the rate of permeation through the NFs system significantly decreased (ρ < 0.0001) relative to the corresponding drug solution from 76.348 ± 1.04 to 56.812 ± 1.02 µg h^− 1, respectively. Figure 6 displayed that, at 6 h, the quantity of VEN infiltrated from a drug solution exceeds that permeated from the nanofiber system. Regarding permeation flux (J), which is defined as the drug amount infiltrated per centimeter square per hour [48], combined nanofiber decreased the flux of VEN significantly from 22.063 ± 0.8 to 16.419 ± 0.5 µg cm^− 2 h^− 1 (ρ = 0.0005). Another parameter of permeation coefficient (P) that is used to measure the movement rates of drug molecules across a convoluted polymer was also predestined. Data in Table 3 displayed that the permeation coefficient of F3 was diminished compared to the control; however, it was not significantly different (ρ = 0.0653).

Table 3

*Ex vivo* permeation parameters of VEN solution and VEN-loaded PLA/PCL composite NFs (F3) through chicken cyst mucosa determined at 37˚ C, data defined as average ± SD (n = 3).
Parameter Formulation	Rate of permeation (dQ/dt)	Permeation flux (J) (µg cm^− 2 h^− 1)	Permeation coefficient (P) (cm h^− 1)
Pure VEN	76.348 ± 1.04	22.063 ± 0.8	0.011 ± 0.002
F3	56.812 ± 1.02	16.419 ± 0.5	0.008 ± 0.0005
Unpaired t-test	(ρ < 0.0001)	(ρ = 0.0005)	(ρ = 0.0653)
ρ value that represents the outcome of the unpaired t-test analysis (at the level of ρ < 0.05).

Histological examination

Figure 7 represented four H&E-stained sections of the mucosa viz., fresh (control), positive control-treated (VEN), negative control-treated (plain), and mucosa treated with VEN NFs (F3). The microscopic pictures of the fresh group manifest normal non-keratinized stratified squamous epithelial lining (EP) resting on folded basement membrane with normal lamina propria underneath composed of highly vascular connective tissue (CT) with no signs of cellular injury, degeneration, or inflammatory. However, as shown in (Fig. 7.i and Fig. 8.i), the mucosa treated with VEN reveals a marked reduction in mucosal thickness and extensive damage of the underlying CT (ρ < 0.0001). Negative control (plain, Fig. 7.ii and Fig. 8.ii) treated mucosa was found to be intact with preserved structure reveals a slight reduction in mucosal thickness and loosening of the underlying CT (ρ < 0.001). After treating mucosa with VEN composite NFs (F3, Fig. 7.iii and Fig. 8.iii), neither cell necrosis nor structural damage was noticed.

In vitro cytotoxicity study

A WST-1 assay was applied to study in vitro cell viability as an indicator of cell damage or cytotoxicity. The cytotoxic effects of VEN, plain NFs, and medicated NFs (F3) were evaluated in three different cell lines e.g., Huh-7, OEC, and Vero cells. The cell viability plot (Fig. 9) showed that more than 80% of cells were viable after 48 h of incubation even in the existence of very high concentrations of VEN or F3 (100 µg/ml) as seen in Table 4. Furthermore, demonstrative phase-contrast images (Fig. 10) were encouraging cell viability results with similar absorbance of tested samples indicating no harmful effect.

Table 4

IC₅₀ of VEN, plain, and combined samples in the studied cell lines after 48 h.
Formula	IC₅₀ (µg/ml)
Formula	Vero	Huh-7	OEC
VEN	993.15 ± 67.95	596.08 ± 34.26	440.48 ± 60.52
Plain	182.74 ± 17.49	229.88 ± 22.27	217.55 ± 10.96
F3	498.58 ± 61.35	337.04 ± 45.07	250.62 ± 11.90
IC₅₀; the concentration that killed 50% of cells, Vero; Green monkey kidney, Huh-7; Liver cancer, OEC; Oral epithelial cell (mean ± SD; n = 3).

Electrospun NFs have received special attention as drug delivery systems owing to their distinct functional feature and easy fabrication techniques [18]. The polymeric system for this study was carefully selected following considerations of the several FDA approved. They are biodegradable poly (α-hydroxy ester) based polymer family like poly (lactic acid) and poly(ε-caprolactone). This work focuses on developing novel electrospun polymeric NFs for buccal delivery of VEN to avoid the hepatic metabolism, enzymatic degradation in the GIT and develop an effective control of drug release. VEN-loaded NFs were successively fabricated by the electrospinning method using biocompatible polymers. All the parameters involved in the electrospinning process were carefully optimized, such as polymer concentration, polymer ratio in case of composite fibers, flow rate, applied voltage, and tip-to-collection distance. In the present study, the polymer solution (PLA, PCL and PLA/PCL blend) and VEN in their respective solvents were electrospun under the applied electrical potential of 20kV over a tip-to-collector distance of 15cm and flow rates of 1.0, 0.6, and 0.3 mL/h, respectively.

The morphological studies and size of the cast films and NFs were evaluated by SEM. The smooth fracture surface of the cast film of PLA and PCL is attributed to its relatively brittle fracture manner in the two films before the electrospinning process. While electrospinning of 10%PLA yielded optimum fibers with an average diameter of 400–500 nm under well-defined conditions. Their morphology may be due to the selected binary solvent system of CHM: DMF (80:20) that has low surface tension, suitable viscosity, high miscibility, and high conductivity which aid in the production of nanofibers [49]. Similarly, the boiling point of each solvent in the binary system plays a significant role in NFs formation in which DMF has a higher boiling point than CHM that offers enough time for the spinning process completion correctly without any beads formation [50]. As the increment of PCL concentration from 10–14%, there was a gradual increase in the fiber diameter. The observation indicated that with an increase in the PCL concentration, there is an increase in the electrical force exerted on the jet, which in turn increases the mass throughput. That results in chain entanglement and consequently increasing the average diameter size of NFs [51–53]. Representative SEM images of plain and medicated NFs revealed smooth and regular NFs with no drug crystals identified on/or outside the surface. Additionally, the film with blend fiber at PLA/PCL ratio of 80:20 is characterized with the smoothest surface and the highest orientation. These observations confirmed the high compatibility of the drug with a polymer-solvent matrix that belongs to a homogeneously distributed drug within the fiber preparation. Also, it was clear that the integration of VEN in the polymer solutions has no significant effect on either the viscosity of the polymeric solutions or the average diameter size of NFs. We focused our attention on the PLA/PCL binary system to obtain a narrow and smooth diameter of VEN-NFs.

FTIR spectra manifest that the assignable peaks of the drug are visible in all the drug-loaded NFs, in addition to the lack of novel absorption peaks. These outcomes confirm that no chemical interaction occurs between the drug and the utilized polymers during the preparation of NFs [39].

The XRD patterns of the prepared NFs may be inferred to the less perfect crystalline structure indicating a subdued degree of the crystallinity of these NFs. During the electrospinning process, the fast solidification of the fibers restricted the order of three-dimensional polymer lattice and retards the crystallization process [54]. These results are in agreement with the literature [42, 55].

DSC thermograms confirms a fact of the semi-crystalline nature of PLA polymer. PCL accelerates the crystallization rate of PLA with a small effect on its final crystallinity degree [56, 44]. These PCL crystals designed in the boundaries may supply the nucleating sites for PLA to crystallize. An overall decrease was revealed in the crystallinity of both PCL and PLA configuration in NFs composite in correlation with XRD analysis [56]. Of these, blending PLA and PCL can overcome the shortcomings of both PLA and PCL [57, 58].

The heat flow value of VEN in blends decreased indicating that the drug-loaded fibers samples contain free amorphous regions. It may be attributed to the extremely rapid vaporization of the solvent from the NFs during the electrospinning process, leading to the failure of the drug molecules to form a complete crystalline lattice within the NFs [59]. DSC studies confirmed that the drug molecules were evenly distributed at the molecular level in the nanofibers matrix and were existent in an amorphous state, hence favor the adequate compatibility and the stability of the composite NFs [59]. Further, the drug is not influencing much the melting and glass transition temperatures of the blend constituents that are in good agreement with FTIR and XRD spectra.

All prepared nanofibers (F1, F2, and F3) revealed the immense encapsulation efficiency percentage owing to the exceptional high surface area of NFs and the absence of drug loss during NFs preparation [60].

In vitro release study of VEN from nanofibers represented as sustained/controlled release of drug as drug disperses to the release medium across the carrier regularly [39]. The cumulative amount of drug released manifested the fast drug release from the buffer solution may be ascribed to the high solubility of VEN in water and its availability to contact directly with the diffusion cellulose membrane [61]. Many endeavors have evidenced that hydrophilic drugs commonly tend to migrate to the surface of NFs when they were implanted in hydrophobic polymers [39]. Meanwhile, PCL NFs revealed the lowest release rate attributed to the partial crystallinity of PCL and the presence of five hydrophobic -CH₂ moieties in its repeating units [62]. Thus, the limited diffusion of the drug from the nanofibers occurred. Otherwise, PLA NFs showed a higher cumulative release rate that referred to the high amorphous PLA in nature. The degradation of PCL does not create acidic byproducts, unlike the degradation of PLA. It makes PCL more adequate nanomaterial for the improvement of long-term implantable devices for its sluggish degradation rate [63]. F3 drug release profile confirmed the enhancement of the blend features due to the presence of both polymers in the same matrix. These explanations affirmed that the NFs effectively sustainably deliver the drug and thereby enhancing patient compliance. Generally, the drug release from NFs mats was modulated by a mixed mechanism of drug diffusion from nanopores of the NFs and degradation of the polymeric matrix [39]. Besides, there are insufficient physical and chemical interactions between the hydrophilic drug molecules and the hydrophobic polymers matrix, as evident in the study of ATR- FTIR spectra.

The release mechanism of VEN and F1 is fitted well with the Fickian mechanism indicated that the release of the drug is mainly mediated by its diffusion across the polymeric matrix [64]. While the release of F2 and F3 followed an anomalous non-Fickian one signified that the release was controlled by drug diffusion and polymer erosion [65]. It consists of two dominant driving forces, namely drug desorption and diffusion-controlled release kinetics [39]. When we took all the obtained outcomes together, it was concluded that F3 was a promising delivery system of VEN, and hence it deserved to undergo further studies.

The ex vivo results could emphasize the enhancement of the sustained drug release and the noticeable decrease in the permeability of the drug from NFs (ρ < 0.05) compared to the dug solution. VEN is a BCS class I drug that suffers from a rapid absorption leading to a short duration of action [66]. These results pointed that the prepared NF formulation could be a facile and green approach to be used for buccal administration with minimal drug permeation, less expected systemic absorption, and thereby convenience for patients.

Buccal histopathology is useful to study the toxicity effects of the selected formulation on the integrity of buccal mucosa. The exhibited observations are ascribed to the pH value of VEN NFs (6.58 ± 0.32), which was within the pH range of human buccal mucosa (6-7.5), reflecting its safety for buccal administration [67]. Additionally, it may be related to the good biocompatibility, favorable mechanical properties, low immunogenic reactions, and chemical versatility of the utilized alloplastic (PLA and PCL) materials [68].

In vitro cytotoxicity is referred to as safe and cytocompatible electrospun NFs. These may be owing to its highly biocompatible composition and mild formulation procedures undertaken. According to these preliminary toxicity results, the WST-1 assay judged that F3 is non-toxic and cytocompatible as exhibited values of IC₅₀ indicating too high viability of the exposure cells up to 2 days. These results propose that the VEN scaffold has the potential for biomedical applications.

Electrospinning is a highly sophisticated, robust, and applicable technique used in the fabrication of ultrafine fibers from different electrostatic fluids. The optimized formula was successfully fabricated by blending PLA and PCL at a ratio of (1:1) fixed at 10% w/v. Morphological analysis refers to free-beads, smooth, very narrow, and uniform electrospun fibrous structures of PLA, PCL, and PLA/PCL NFs mats. Spectral and thermal patterns are matched with the drug release profiles that affirm the PLA is more amorphous than PCL and manifest an enhanced sustained release profile with a decline in the permeability of the drug from the new blend at pH 7.4. Also, highly fitting models suggest the drug release mechanism is close to Higuchi and Korsmeyer-Peppas models. Histopathology and cytotoxicity studies showed no evidence of any noticeable histological effects, confirming the safety of buccal VEN delivery. In a nutshell, PLA/PCL blend NFs may be deemed to be a prospecting and safe controlled-release strategy for buccal delivery of antidepressant drugs. To make a complete judgment about the developed nanosystem, further studies including experimental animals are beyond the scope of this paper and will be investigated in our future work.

Author Contributions

Heba M. Hashem: Conceptualization, Methodology, Formal analysis, Data curation, Writing - original draft. Amira Motawea: Conceptualization, Methodology, Formal analysis, Data curation,Writing - review & editing. Ayman H. Kamel: Supervision, Conceptualization, Writing - review & editing. E.M. Abdel Bary: Supervision, Re-sources, Formal analysis. Saad S.M. Hassan: Supervision, Writing - review & editing. All authors read and approved the final manuscript.

Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Acknowledgements

None. All authors had full access to all the data in the study and took responsibility for the integrity and the accuracy of the data.

Competing interest

The authors declare no competing interests.

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Venlafaxine-Loaded Nanocomposite-Based Scaffolds: In Vitro, Ex Vivo and Cytotoxicity Evaluations

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Introduction

Materials And Methods

Materials

Preparation of the composite NFs

Characterization techniques

Scanning electron microscope

Attenuated total reflectance-Fourier infrared spectroscopy

X-ray diffraction

Differential scanning calorimetry

Evaluation of drug encapsulation efficiency

EE% = \(\frac{{A}{m}{o}{u}{n}{t} {o}{f} {d}{r}{u}{g} {i}{n} {N}{F}{s}}{{D}{r}{u}{g} {i}{n}{i}{t}{i}{a}{l}{l}{y} {a}{d}{d}{e}{d}}\) X 100

The cumulative drug release (%) =\(\frac{{C}{t}}{{C}{\infty }} \times 100\)

Mathematical models for drug release kinetics

Flux = Jss=\(\frac{{1ex}{${d}{Q}$}\!\left/ \!{-1ex}{${d}{t}$}\right.}{{A}}\)

Histological examination

Statistical analysis

Results

Characterization of NFs

Electrospinning conditions and morphology of various nanofibers

ATR-FTIR analysis

Crystallinity evaluation of NFs

Thermal analysis of NFs

Encapsulation efficiency of electrospun NFs

Mathematical models for drug release kinetics

Histological examination

Discussion

Conclusion

Declarations

References

Supplementary Files

Status:

Version 1