Recombinant interferon-α-2b (rINF-α-2b), the first FDA-approved biotherapeutic, is recommended to be coadministered with an armamentarium of chemotherapeutic drugs at a dose of 50×106 IU once a week for 8 weeks in the treatment of ovarian cancer 1,2. It is a single, nonglycosylated and potent cytokine polypeptide, which contains 165 amino acids 3. However, short serum half-life (2–6 h), small therapeutic index and rapid proteolytic degradation by circulating enzymes cause fluctuations in plasma level and pose barriers in the development of a clinically viable sustained-release dosage form 4–6.
The parenteral route remains the most common method for delivery of proteins; however, it provides no stabilization to the shelf-life of proteins, nor does it increase the half-life of proteins in vivo7. Stealth nanoformulation of rINF-α-2b has been reported to offer prolonged release and improved biodistribution; however, it suffers from immunogenicity, antigenicity and low mean residence time 8,9. Moreover, stealth nanoformulation of rINF-α-2b induces more complications compared with native rINF-α-2b. Therefore, sustained drug delivery of rINF-α-2b is necessary to achieve the greatest therapeutic effects, with minimum side effects and need for repetitive dosing 4,5,10.
Our laboratory is actively engaged in the design and development of anticancer nanotherapeutics for tumour targeting 11–20. Microspheres can maintain adequate serum concentration for a long period of time, prevent degradation and increase the therapeutic index and half-life of drugs 4,5,10,21. Zinc complexes, poly(lactide-co-glycolide), gelatin, copolymerized gelatin, polycaprolactone and magnetite microspheres, have already gained attention for the delivery of rINF-α-2b 4,5,10,21–23. However, typical harsh manufacturing conditions influence the stability of protein and thereby biological activity.
Several stabilizing agents have also been impregnated over the surface of microspheres to augment the sustained-release pattern of proteins. Silk, serum albumin–alginate membrane and protamine sulphate (Pt) have been widely investigated to induce the desirable sustained release of drugs from microspheres 4,24,25. Compared with other coating agents, Pt is effective in protecting protein against environmental interruptions and extends the release rate both in vitro and in vivo4. Pt, a polycationic arginine-rich protein approved by FDA for human consumption, has unique properties and adsorbs on the microsphere’s surface by electrostatic interactions 4,26. The addition of cryoprotectants helps to prevent covalent aggregation of the encapsulated protein 27–29.
In our investigation, we have synthesized and optimized fluorescein isothiocynate (FITC)-tagged rINF-α-2b (*rINF-α-2b)-loaded stearic acid microspheres (*rINF-α-2b-SMs), pectin microspheres (*rINF-α-2b-PMs) and gelatin microspheres (*rINF-α-2b-GMs). The extended-release formulation was then impregnated with a gradient concentration of Pt to induce desirable sustained-release effects. The tailored nanoformulations were characterized for particle size, ζ potential, surface morphology, entrapment efficiency and in-vitro protein release. The post-translational stability and integrity of *rINF-α-2b in the optimized formulation was analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and circular dichroism (CD). Moreover, in-vitro cytotoxicity analysis of the optimized formulation was carried out on the human ovarian cancer cell line SKOV3.
Materials and methods
rINF-α-2b (5×107 IU∼0.19 mg, molecular weight∼17 kDa, purity∼98.0%) was obtained as a gift sample from Virchow Biotech Private Limited (Qutubullapur, Andhra Pradesh, India). Gelatin B (Type B; bloom strength∼225; 100–115 mmol/l of free carboxylic acid/100 g of protein; an isoelectric point of 4.5–5.2 and average molecular weight∼40 000–50 000 Da) and FITC (Isomer II) were purchased from Sigma Aldrich (St Louis, Missouri, USA). Stearic acid, pectin, Tween 20, span 80, span 85 and glutaraldehyde (25% v/v aqueous solution) were procured from S.D. Fine Chemicals (Mumbai, Maharashtra, India). All other chemicals used were of the highest analytical grade and used without further purification.
The human ovarian cancer cell line SKOV3 was maintained in 5% CO2 and 95% air at 37°C using Dulbecco’s modified Eagle’s medium (DMEM) (Biological Industries, Kibbutz Beit Haemek, Israel) supplemented with 5% fetal calf serum. All experiments were performed with asynchronous populations in exponential growth phase (24 h after plating) 11.
Analytical estimation of interferon-α-2b using fluorescein isothiocynate as a marker
The calibration curve of rINF-α-2b was prepared by conjugating FITC on to the protein macromolecule 30. Briefly, rINF-α-2b was dissolved in 1 ml of borate buffer (pH∼8.5) followed by the addition of 1% FITC (1 : 26 FITC to rINF-α-2b). This was designated as *rINF-α-2b. The conjugation reaction was then completed by incubating the solution at 25°C in an orbit shaker for 3 h. Different aliquots (10–200 μg/ml) were prepared from the stock solution. A standard curve was prepared by measuring the fluorescence intensity using a fluorimeter (spectra fluor; Tecan, Mannedorf, Switzerland; λexe∼485 nm, λemi∼535 nm).
Preparation of *rINF-α-2b-bearing stearic acid microspheres
Stearic acid microspheres encapsulating *rINF-α-2b (*rINF-α-2b-SMs) were prepared by the solvent diffusion method with slight modification, estimated to be safe for protein encapsulation 31. Briefly, *rINF-α-2b (20 mg/ml) was dissolved in 5 ml of distilled water containing Tween 20 (0.25 ml). Accurately weighed 1 g of stearic acid was dissolved in 10 ml of ethanol and 10 ml of acetone. The aqueous phase was added dropwise in the organic phase with continuous stirring at 1000 rpm for 30 min. The resulting microspheres were lyophilized using a freeze dryer (Lab India, Thane, Maharashtra, India) and collected.
Preparation of pectin microspheres incorporating interferon-α-2b
The *rINF-α-2b-loaded pectin microspheres (*rINF-α-2b-PMs) were prepared by the emulsion dehydration technique found to be optimum for protein encapsulation 32. Briefly, 400 mg of pectin and *rINF-α-2b (20 mg/ml) were dissolved in 20 ml of distilled water and stirred overnight to solubilize completely. The mixture was dispersed in 50 ml of isooctane containing 1.25% (w/v) of span 85 and stirred continuously to fabricate a stable emulsion. The emulsion was rapidly cooled at 15°C followed by the addition of 50 ml of acetone to dehydrate the pectin droplets. This emulsion was stirred at 1000 rpm for 30 min at room temperature to allow complete solvent evaporation of the organic phase. The microspheres were washed with acetone and lyophilized using a freeze dryer (Lab India).
Preparation of interferon-α-2b-loaded gelatin microspheres
The gelatin microspheres incorporating *rINF-α-2b (*rINF-α-2b-GMs) were prepared by the emulsion polymerization technique using a chemical crosslinker, glutaraldehyde 22. A mixture of 25 ml of toluene and 25 ml of chloroform containing 2.5 g of span 80 was mixed in a sampling tube, and then 2 ml of gelatin aqueous solution (20% w/v) incorporating 20 mg/ml of *rINF-α-2b was added to the organic mixture followed by sonication (10 W, TOS Sonicator; Soniweld India Ltd, Mumbai, Maharashtra, India) for 10 min. The resulting emulsion was quickly poured into 40 ml of a precooled mixture of 25% chloroform and 75% toluene containing 2 g of span 80. The gelatin emulsion was crosslinked with glutaraldehyde-saturated toluene (10 : 10 v/v). The crosslinking reaction was carried out at 0°C for 3 h by stirring at 600 rpm. The resulting microspheres were successively washed with isopropanol and dried overnight using a lyophilizer (Lab India).
Protamine sulphate-impregnated gelatin microspheres
The coating of Pt over *rINF-α-2b-GMs nanoformulation was achieved by the addition of Pt aqueous solution (5–20 mg/ml) to 50 mg of *rINF-α-2b-GMs and stirred at 1000 rpm for 30 min at room temperature. The resulting microspheres were centrifuged at 10 000 rpm for 10 min and the pellet was dried in a dessicator and collected.
Particle size and ζ potential
Particle size and ζ potential of microspheres were analysed using a zeta sizer (Malvern Instruments, Worcestershire, UK). Briefly, 5 mg sample of each type of microsphere was dispersed separately in 5 ml of PBS (pH∼7.4), and the samples were filled in a cuvette to determine the particle size and ζ potential. A 150 mV electric field was applied to measure the electrophoretic velocity of the microspheres. All measurements were taken at room temperature in triplicate (n≥3).
Scanning electron microscopy and fluorescence microscopy
The shape and surface morphology of tailored microspheres were observed by scanning electron microscopy (SEM). The powdered samples were loaded on to the aluminium stubs with double-sided tape and coated with gold vapour. The analysis was carried out at 5–10 kV on the SEM (JSM-6100 scanning microscope; JEOL, Tokyo, Japan). Fluorescence microscopy was used to analyse the distribution of *rINF-α-2b in microspheres. The analysis was carried out using a fluorescence microscope (Leica Biomed, Wetzlar, Germany).
Determination of encapsulation efficiency and protein loading capacity
The encapsulation efficiency and protein loading capacity were determined by dissolving 10 mg sample of each type of microsphere in 1 ml of 0.1 mol/l sodium citrate solution (pH∼8.0) and incubating for 16 h to hydrolyse the stearic acid, pectin and gelatin polymeric membrane 33. The samples were centrifuged at 15000 rpm to remove the polymeric clumps. Subsequently, the supernatant solution of each sample was analysed for *INF-α-2b using a fluorimeter (λexe∼485 nm, λemi∼535 nm; Tecan). The per cent encapsulation efficiency was calculated as:
In-vitro release of *interferon α-2b from microspheres formulations
The in-vitro release of *rINF-α-2b from microsphere formulation was determined by suspending 50×106 IU∼0.19 mg of *rINF-α-2b (encapsulated in 1.11 mg of *rINF-α-2b-SMs, 0.283 mg of *rINF-α-2b-PMs, 0.199 mg of *rINF-α-2b-GMs and *rINF-α-2b-Pt-GMs) in 1 ml of PBS (pH∼7.4). The sampling tubes were then fitted to an orbit shaker maintained at 100 rpm (as recommended for dissolution testing of parenteral products) 23. The temperature was set at 37°C. The samples were withdrawn at predetermined time intervals and replaced with fresh buffer (PBS; pH∼7.4) to maintain sink conditions. The quantity of *rINF-α-2b was determined using a fluorimeter (λexe∼485 nm, λemi∼535 nm; Tecan).
Stability study of *rINF-α-2b in optimized formulation using gel electrophoresis and circular dichroism
Gel electrophoresis of *rINF-α-2b isolated from optimized formulation
The SDS-PAGE assay was performed on 12.5 or 15% polyacrylamide gel 34. The optimized formulation (*rINF-α-2b-Pt-GMs-15) was degraded until the concentration of *rINF-α-2b reached 100 μg/ml. The pure rINF-α-2b and isolated *rINF-α-2b in equal concentrations were separately diluted in 50 mmol/l Tris HCl buffer (1 : 1; pH∼6.8) containing 2% SDS, 11.6% (v/v) glycerol and 0.001% bromophenol blue with 1% β-mercaptoethanol. Both samples were heated in a boiling water bath for 3 min and loaded on to the gels. After electrophoresis, proteins were visualized using Coomassie brilliant blue dye.
Circular dichroism of *rINF-α-2b isolated from optimized formulation
The far-ultraviolet (UV) CD spectrum of pure rINF-α-2b and isolated *rINF-α-2b was measured at a concentration of 100 μg/ml from 180 to 260 nm at 25°C with constant nitrogen flushing using an instrument (Jasco-J-815 CD spectrometer; Jasco Analytical Instruments, Easton, Maryland, USA) in PBS (pH∼7.4). After 10 min of sample preparation, all measurements were taken with the following instrument settings: 0.5 s scan speed, 200 nm/min sensitivity, 100 millidegree and 1 nm spectra bandwidth. An average of three scans were taken. The results were expressed as residual ellipticity [θ] (deg cm2/dmol).
where θ is measured ellipticity in degrees, C is concentration in mg/ml, l is light path length in cm and MRW is mean residual weight.
Cytotoxicity analysis of optimized gelatin microsphere formulation
Colorimetry-based MTT assay was used to study the per cent cell viability 17. Briefly, 5×103 SKOV3 cells were plated in 200 μl of DMEM medium. After 24 h of plating, the DMEM medium was replaced with rINF-α-2b, *rINF-α-2b-GMs, *rINF-α-2b-Pt-GMs-15 and respective blank GMs at concentrations ranging from 200 to 1200 μg/ml equivalent of rINF-α-2b and incubated for 24, 48 and 72 h. Then, cells were treated with MTT (0.5 mg/ml) for 4 h at 37°C. Finally, the medium was removed, cells were lysed and formazon crystals were dissolved using 100 μl of dimethyl sulfoxide The absorbance was read at 570 nm using 630 nm as the reference wavelength in an enzyme-linked immunosorbent assay plate reader (Tecan).
Statistical analysis was performed on the data using one-way and two-way analysis of variance using GraphPad04 Instat (v, 3.10; GraphPad Software Inc., La Jolla, California, USA) Software. P values less than 0.05 were considered significant.
Preparation and characterization of *rINF-α-2b-loaded microspheres
Microencapsulation is a promising approach for protein drug delivery, which avoids proteolysis and improves the release profile. The *rINF-α-2b-loaded microspheres were successively prepared using stearic acid (*rINF-α-2b-SMs), pectin (*rINF-α-2b-PMs) and gelatin (*rINF-α-2b-GMs). To avoid denaturation of *rINF-α-2b during processing, emulsification was carried out in the cold with minimum use of organic solvents to avoid harsh manufacturing conditions, as shown in Fig. 1. The formulation *rINF-α-2b-GMs that offered extended release was impregnated with Pt to further induce the sustained-release pattern.
Characterization of microspheres
Particle size and ζ potential analysis
The implications of particle size and ζ potential analysis have been considered vital, as the size of the microspheres may affect the protein encapsulation efficiency, syringeability and rate of protein release 5,33. The particle size and ζ potential of *rINF-α-2b-SMs (4.1±2.1 μm, −34.0±0.04 mV), *rINF-α-2b-PMs (13±4 μm, −29±0.02 mV), *rINF-α-2b-GMs (8.3±2.1 μm, −7.7±0.04 mV) and *rINF-α-2b-Pt-GMs (12.7±2.5 μm, 15.7±0.05 mV) were found to be optimum for parenteral administration (Fig. 2 and Table 1). The microspheres that are intended for parenteral administration in the range of 40–120 μm generally lead to severe complications like pulmonary embolism and stroke 34.
The surface morphology of *rINF-α-2b-loaded microspheres was examined by SEM. The formulations *rINF-α-2b-SMs, *rINF-α-2b-PMs and *rINF-α-2b-GMs were spherical in shape with a slightly rough surface, whereas *rINF-α-2b-Pt-GMs were found to be smooth and spherical. Fluorescence microscopy also provided compelling evidence that *rINF-α-2b was homogeneously distributed in all batches of microsphere formulations (Fig. 2).
Analysis of encapsulation efficiency and protein loading capacity
The encapsulation efficiency and protein loading capacity depend on the experimental conditions acquired for microsphere formulations. The encapsulation efficiency of *rINF-α-2b-SMs, *rINF-α-2b-PMs and *rINF-α-2b-GMs was found to be 10.2±3, 50.2±5 and 76.0±7.4%, respectively (Table 1).
In-vitro protein release
We have examined the potential of microspheres for sustained release of *rINF-α-2b in physiological conditions by in-vitro dissolution testing. The percentage release of *rINF-α-2b from *rINF-α-2b-SMs, *rINF-α-2b-PMs and *rINF-α-2b-GMs under sink conditions is presented in Fig. 3a–b. The release of *rINF-α-2b from *rINF-α-2b-SMs was 94.5±4% in 24 h, whereas it was 96.0±3.2% in 168 h from *rINF-α-2b-PMs and 97.4±2.1% in 288 h from *rINF-α-2b-GMs. The *rINF-α-2b-GM formulation that offered maximum prolonged release was considered optimum and coated with a gradient concentration (5–20 mg/ml) of Pt to further extend the release rate (Fig. 3a). The formulation *rINF-α-2b-Pt-GMs-15 released 95.0±4.2% of *rINF-α-2b over 2 weeks and suppressed the initial burst by inducing the desirable sustained release.
Stability analysis of *rINF-α-2b in optimized formulation (*rINF-α-2b-Pt-GMs-15)
SDS-PAGE was used to ascertain the stability and molecular mass of *rINF-α-2b in optimized formulation. The SDS-PAGE results of isolated *rINF-α-2b from the optimized formulation and free rINF-α-2b under reduced conditions are shown in Fig. 4. The secondary structure of rINF-α-2b can be characterized by two intramolecular disulphide bonds Cys1–Cys98 and Cys29–Cys138 35. It comprises molecular masses from 17 to 28 kDa 36. The reducing agent β-mercaptoethanol was used to break any disulphide bonds, and thus eliminate the presence of high-molecular-weight bands. We observed that the structural integrity of the isolated *rINF-α-2b did not appear to be compromised by the emulsification technique. The molecular weight of rINF-α-2b was observed to be 17 kDa as represented by the standard marker.
Far-UV CD is an effective approach for the investigation of the secondary structure of rINF-α-2b, which contains five α-helices. Pure rINF-α-2b and isolated *rINF-α-2b from *rINF-α-2b-Pt-GMs showed a similar UV CD spectrum (Fig. 5). Deconvolution of the secondary structure using the Convex Constraint Algorithm programme indicates that 55% corresponds to α-helix, 22% to unorganized structure and 23% to others. However, we did not notice any β-structures.
Cytotoxicity analysis in the ovarian cancer cell line
The in-vitro therapeutic efficacy of free rINF-α-2b, *rINF-α-2b-GMs, *rINF-α-2b-Pt-GMs and respective blank formulations was examined by cytotoxicity assay on SKOV3, the ovarian cancer cell line. The IC50 value (concentration of drug required to kill 50% of the cells) was used as an indicator to define the therapeutic efficacy of tailored microsphere formulations. The IC50 value for free rINF-α-2b appeared to be in the order of 757.1 IU/ml>714.3 IU/ml>628.6 IU/ml at 24, 48 and 72 h. Compared with this, it was found to be 914.3 IU/ml>742.85 IU/ml>514.3 IU/ml for *rINF-α-2b-GMs and 1142.85 IU/ml>914.3 IU/ml>414.3 IU/ml for *rINF-α-2b-Pt-GMs at the same time interval (Fig. 6a–c). Blank microspheres did not exert any cytotoxicity to ovarian cancer cells.
Delivery of chemotherapeutic drugs or proteins through the parenteral route of administration often required a long circulation profile, safe homing and targeting at molecular receptors. Susceptibility of proteins to denaturation by circulating proteolytic enzymes and other environmental conditions needs special attention while encapsulating in microdrug or nanodrug delivery systems. In the present investigation, our aim was to sustain the release of *rINF-α-2b from the microparticulate system to reduce the frequent parenteral administration in ovarian cancer. Unlike synthetic polymers, lipid, carbohydrate and protein-based polymers of biodegradable nature were selected to synthesize the *rINF-α-2b-loaded microspheres. Cold homogenization/emulsification techniques with minimum use of organic solvents were used to encapsulate *rINF-α-2b in stearic acid (*rINF-α-2b-SMs), pectin (*rINF-α-2b-PMs) and gelatin (*rINF-α-2b-GMs) microspheres (Fig. 1). Particle size analysis, SEM and fluorescence microscopy informed that the microspheres were optimum for parenteral administration, spherical in shape with slightly rough surface and have homogeneous distribution of *rINF-α-2b (Fig. 2a–c). Therapeutic efficacy-determining parameters such as encapsulation efficiency, protein loading capacity and in-vitro release profile were determined to follow the optimization (Table 1 and Fig. 3). We have reported that *rINF-α-2b-GMs have shown excellent potential to encapsulate a maximum number of rINF-α-2b molecules with prolonged-release effect. All the batches of protein-loaded microspheres have shown an initial burst followed by a slow release in the order of 88.72>85.26>60.61%, respectively, by *rINF-α-2b-SMs, *rINF-α-2b-PMs and *rINF-α-2b-GMs at 6 h time interval. This necessitates that the extended-release formulation *rINF-α-2b-GMs be coated with a suitable stabilizing agent to suppress the initial burst in order to induce the sustained-release pattern both in vitro and in vivo. This may help us to regulate the plasma fluctuations in in-vivo investigations.
Pt was used to shield the *rINF-α-2b-GMs that aided the stability of therapeutic protein and regulated the sustained-release pattern (Fig. 3). We observed the in-vitro release profile of *rINF-α-2b-GMs impregnated with a gradient concentration of Pt (5–20 mg/ml) and reported that 15 mg/ml of Pt was the optimum concentration for coating *rINF-α-2b-GMs. Further, enhancement in the Pt concentration did not extend the release rate in vitro. Hence, *rINF-α-2b-Pt-GMs-15 nanoformulation was designated as the optimized formulation. The basis for this optimization may be the complete neutralization of negative charge of *rINF-α-2b-GMs by positively charged Pt that made changes in the surface chemistry of GMs by electrostatic interaction. The negative charge in gelatin is induced by the terminal carboxylic end of the gelatin monomers (Table 1). Thus, in-vitro release profile of optimized nanoformulation substantiates the possible use of *rINF-α-2b-Pt-GMs-15 as a sustained-release carrier. However, Pt does not alter the principal dissolution characteristics of the protein-loaded microspheres 4. The stability assessment study showed that the structural integrity of the protein did not appear to be compromised by the preparation technique, as evidenced by CD and gel electrophoresis (Figs 4 and 5). Consistent with the in-vitro release data, the sustained-release pattern of the optimized formulation was also measured by cytotoxicity assay using the SKOV3 cell line in terms of IC50 value. The IC50 value of *rINF-α-2b-Pt-GMs-15 was lower than that of free rINF-α-2b and *rINF-α-2b-GMs because of its prolonged-release delivery at 72 h (Fig. 6). However, we could not correlate the results of in-vitro release with the cytotoxicity assay (Fig. 3). The surface charge on the cell membrane of mammals is negative at pH∼7.4. However, it increases during tumorigenesis and decreases during necrosis. Therefore, the neoplastic cells are different in their surface charge from their normal counterparts. There is an increase in the positive surface charge of cancer cells at low pH until a plateau is reached. At high pH, the negative surface charge of the cells also increases, reaching a plateau 37. Therefore, we propose that *rINF-α-2b-Pt-GMs-15 because of their positive charge would adhere to the negatively charged SKOV3 cells and release *rINF-α-2b by polymerization and diffusion so that a high and effective concentration is reached to induce apoptosis. As the cell line does not contain any phagocytes, which can engulf microspheres, the formulations would remain more stable in the culture media that show prolonged cytotoxic effect.
We investigated the encapsulation mechanism of *rINF-α-2b in lipid, carbohydrate and protein-based microspheres to achieve a high encapsulation efficiency and protein loading capacity. *rINF-α-2b is a fragile molecule and encapsulation requires, in addition, the preservation of its structural integrity and functionality. We estimated that *rINF-α-2b-Pt-GMs-15 offered sustained release of protein over 2 weeks and released 95.01±4.23% of *rINF-α-2b. The stability study assessment by gel electrophoresis and CD confirmed the post-translational structural integrity of *rINF-α-2b in *rINF-α-2b-Pt-GMs-15 nanoformulation. In-vitro therapeutic efficacy testing of the optimized formulation further provided compelling evidence with enhanced cytotoxicity and low IC50 in SKOV3 cells. Therefore, the Pt-coated microspheres may potentially be used for sustained delivery of *rINF-α-2b and warrant further in-depth in-vivo study to scale up the technology for clinical intervention.
Conflicts of interest
There are no conflicts of interest.
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Keywords:© 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
cytotoxicity; gelatin; microspheres; pectin; protamine; rINF-α-2b; stearic acid; sustained release