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Stealth lipid coated aquasomes bearing recombinant human interferon-α-2b offered prolonged release and enhanced cytotoxicity in ovarian cancer cells
Biomedicine & Pharmacotherapy 69 (2015) 267–276
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Original Article
Stealth lipid coated aquasomes bearing recombinant human interferon-a-2b offered prolonged release and enhanced cytotoxicity in ovarian cancer cells Kamaljeet Kaur a, Preeti Kush a, Ravi Shankar Pandey b, Jitender Madan a,*, Upendra Kumar Jain a, Om Prakash Katare c a
Department of Pharmaceutics, Chandigarh College of Pharmacy, Mohali (Panjab) India SLT Institute of Pharmaceutical Sciences, Guru Ghasidas University, Bilaspur, India c University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 31 October 2014 Accepted 1st December 2014
Purpose: In present investigation, recombinant human interferon-a-2b (rhINF-a-2b) loaded aquasomes were prepared, optimized and overlaid with PEGylated phospholipid to offer prolong release and high therapeutic index against ovarian cancer, SKOV3 cells. Methods and results: Central Composite Design (CCD) and Response Surface Methodology (RSM) were employed to calculate the optimized conditions, 1:3 core to coat ratio, sonication power of 12.5 W and time of about 55 min for preparation of aquasomes. Consequently, rhINF-a-2b-Py-5-P-Aq.somes exhibited higher protein loading capacity and retained structural conformations of rhINF-a-2b, as compared to rhINF-a-2b-Cellob-Aq.somes, rhINF-a-2b-Tre-Aq.somes and rhINF-a-2b-Core (CaHPO4). Further, optimized rhINF-a-2b-Py-5-P-Aq.somes was superimposed with phospholipid-PEG2000 to prolong the release pattern of rhINF-a-2b from aquasomes. The rhINF-a-2b-core (CaHPO4) released 97.3% of protein in 1 h, while 95.3% of rhINF-a-2b was released by rhINF-a-2b-Tre-Aq.somes in 4 h. Concurrently, rhINF-a-2b-Cellob-Aq.somes and rhINF-a-2b-Py-5-P-Aq.somes released 96.2% and 97.8% of rhINF-a-2b respectively in 6 and 8 h. Ultimately, rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000 displayed evidence of its prolonged release pattern and released 98.1% of rhINF-a-2b in 336 h. FT–IR and XRD substantiated the involvement of vigorous intermolecular hydrogen bonding and amorphous geometry in rhINF-a-2b-Py-5-P-Aq.somes. In last, rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000 exhibited the 4.55, 1.92, 2.3, 2.8, and 3.84 fold reductions in IC50 as compared to free rhINF-a-2b, rhINF-a-2b-Py-5-PAq.somes, rhINF-a-2b-Cellob-Aq.somes, rhINF-a-2b-Tre-Aq.somes and rhINF-a-2b-Core (CaHPO4), respectively. Conclusion: Therefore, rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000 warrant further in depth in vitro and in vivo antitumor study to scale up the technology for clinical intervention. ß 2014 Elsevier Masson SAS. All rights reserved.
Keywords: rhINF-a-2b Aquasomes Trehalose Cellobiose Pyridoxal-5-phosphate Phospholipid-PEG2000 Cytotoxicity
1. Introduction Recombinant human interferon-a-2b (rhINF-a-2b), a single non-glycosylated USFDA approved cytokine is recommended to co-administer with cisplatin to elicit cytotoxicity against platinum resistant ovarian cancer cells [1]. In addition, rhINF-a-2b induces Abbreviations: rhINF-a-2b, Recombinant interferon-a-2b; Tre, Trehalose; Cellob, Cellobiose; Py-5-P, Pyridoxal-5-phosphate; PVA, Poly(vinyl) alcohol. * Corresponding author. Department of Pharmaceutics, Chandigarh College of Pharmacy, Mohali (Panjab) 140307, India. Tel.: +91 172 398 4209; fax: +91 172 398 4209. E-mail address: [email protected] (J. Madan). http://dx.doi.org/10.1016/j.biopha.2014.12.007 0753-3322/ß 2014 Elsevier Masson SAS. All rights reserved.
mitochondrial-mediated apoptosis in cancer cells, including activation of caspases-3 mediated pathway [2]. Chemically, rhINF-a-2b is a polypeptide chain of 165 amino acids [3]. However, short serum half-life (2–6 h), upregulation of P-glycoprotein mediated multi-drug resistance (P-gp-MDR), low therapeutic index, fluctuated plasma level, and rapid proteolytic degradation hamper the systemic drug delivery [2,4–6]. This necessitates the administration of rhINF-a-2b in a sustained release pattern for successive chemotherapy. Parenteral route of administration is the most common method for delivery of therapeutic proteins; however, it does not enhance the half-life of proteins in vivo [7]. Furthermore, poly(ethylene glycol) mediated stealth nanocomplex of rhINF-a-2b, despite
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excellent release profile is suffered with immunogenicity, antigenicity and low mean residence time [8,9]. Therefore, a stable drug delivery system is required for safe homing of rhINF-a-2b to gain maximum clinical benefits [2,6,10]. Several attempts have been made to vesiculize rhINF-a-2b for clinical intervention by using nanoparticles and microparticles [11–13]. Aquasomes are self-assembled ceramic nanoparticles whose surface can be non-covalently modified with carbohydrates [14]. Instrumentally, aquasomes congregate through non-covalent bonds, ionic bonds and Van der Waals forces [15]. Polysaccharide coating provides glassy molecular layer that adsorbs therapeutic protein without alteration in three-dimensional conformations. In addition, ceramic core superimposed with carbohydrates enhanced the cellular uptake in cancer cells [16,17]. Hence, aquasomes have been extensively investigated for delivery of both small and high molecular weight, pharmaceuticals [18,19]. However, aquasomes do not tender sustained release of proteins due to adsorption rather than encapsulation phenomena. Therefore, in present investigation, rhINF-a-2b was adsorbed on to the different polysaccharide coated self-assembled ultrafine ceramic nanoparticles. Process parameters that influence the synthesis of aquasomes were optimized by using Central Composite Design (CCD) [20] and Response Surface Methodology (RSM) computational techniques [21]. The optimized nanoformulation was then superimposed with phospholipid-PEG2000 (PEGylated phospholipid) to induce the sustained release pattern. It is reported that PEGylated phopholipid retains the pharmacological efficacy of protein, escapes macrophage uptake, and helps to attain high blood level [22,23]. The unique property of PEGylated phospholipid coating is that it allows release of therapeutic component from the drug delivery system through diffusion mechanism [23]. Aquasomes were characterized for particle size, zeta potential, protein loading capacity, in vitro release, protein stability and cytotoxicity against human ovarian cancer cell line, SKOV3.
2. Materials and methods 2.1. Materials Recombinant human interferon-a-2b (rhINF-a-2b, Molecular weight 17 Kda) was obtained as a gift sample from Reliance Life Sciences, Navi Mumbai, India. Trehalose (Tre) and cellobiose (Cellob) were obtained from Loba Chemie, New Delhi, India. Pyridoxal-5-phosphate (Py-5-P) was purchased from Central Drug House, New Delhi, India. All other chemicals used were of highest analytical grade. 2.2. Cell culture Human ovarian cancer cell line, SKOV3 was maintained in 95% air and 5% CO2 atmosphere at 37 8C using DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% fetal bovine serum. All experiments were performed with asynchronous cell populations in exponential growth phase (24 h after plating) [24]. 2.3. Experimental design for optimization The core (CaHPO4) to coat (polysaccharide) ratio was optimized by Central Composite Design (CCD) [20] to get Response Surface Methodology (RSM) [21] that selected an object, noticed the contributing factors and investigated the relationship between responses and factors. CCD enabled several independent variables to be investigated at the same time using a relatively small number of experiments, while RSM optimization analyzed the interactions between variables. Therefore, CCD-RSM defined the interactions
Table 1 Optimized conditions calculated by central composite design (CCD) and response surface methodology (RSM) for preparation of aquasomes. Runs
Core:coat (mg) (X1)
Sonication power (W) (X2)
Sonication time (min) (X3)
Particle size (nm)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1 3 3 1 5 3 3 –0.36 6.363 1 3 5 3 3 5 3 1 3 5 3
20 –0.113 12.5 5 20 12.5 12.5 12.5 12.5 20 12.5 5 12.5 12.5 5 25.11 5 12.5 20 12.5
20 55 55 90 90 55 -3.86 55 55 90 55 90 55 55 20 55 20 113.86 20 55
70.6 135.8 90.1 125.7 130.4 90.4 138.4 82.3 100.4 94.2 91.6 115.4 92.1 90.8 117.2 88.4 98.4 79.3 100.2 91.1
Core: CaHPO4; Coat: polysaccharide (Trehalose; cellobiose and pyridoxal-5phosphate).
between factors, avoided unwanted experiments and optimized the results [25]. Our preliminary investigation indicated that variables, such as core to coat (mg) ratio, sonication power (W) and sonication time (min) were the main factors that influenced the particle size of nanoformulations. A CCD model was used to statistically optimize the factors that affected the particle size of aquasomes. For each factor, experimental range was selected on the basis of previous investigations and probability of preparing the aquasomes at extreme values. The value range of variables were core:coat ratio (x1): 1:1 to 1:5 (mg), sonication power (x2): 5– 25.11 (W), and sonication time (x3): 20–113.86 (min). The design consisted of 20 runs (8 factorial points, 6 star points and 1 centre point) and 5 replicated runs (centre points) that yielded total 20 experiments (Table 1). The purpose of replication was to estimate the experimental errors and increase the accuracy. Each experimental run was repeated thrice (n = 3). Star points represented the extreme values (low and high) for each factor in the design and allowed estimation of second-order effects. Star points were at some distance, alpha, from the centre, based on the properties desired for design and number of factors in the design. Alpha in coded units was the axial distance from the centre point and made the design rotatable. A rotatable design provides equally good predictions at points equally distant from the centre, a very desirable property for RSM [21]. A design matrix comprising 20 experimental runs was constructed and responses were modelled by the following reduced linear model (Eq (1)): y ¼ b0 þ b1 x1 þ b2 x2 þ b3 x3 þ b11 x21 þ b22 x22 þ b33 x23 þ b12 x1 x2 þ b13 x1 x3 þ b
(1)
where y is the measured response associated with each factor level combinations; b0 is the intercept; bi’s (for i = 1, 2 and 3) are the linear effects, the b ii are the quadratic effects, the b ij’s (for i, j = 1, 2 and 3, i < j) are the interactions between the ith and ith variables. The statistical analysis was performed by using the software Design Expert (Version 8.0.7.1), where analysis of variance (ANOVA) was significant, when P < 0.05. An F-test was used to determine whether there was an overall regression
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Table 2 Various parameters used for preparation and characterization of aquasomes. Corea (CaHPO4):coata (oligomer)
Morphology
Size (nm)b
Zeta potentialb (–mV)
rhINF-a-2bb loading capacity (mg/mg of aquasomes)
1:0 1:3 1:3 1:3 1:3
Irregular crystals Spherical Spherical Spherical Spherical
90.1 2.3 98.5 4.3 101.9 6.4 104.4 5.9 125.3 3.2
–8.48 2.32 –15.6 1.15 –20.4 0.9 –23.2 1.26 –44.1 3.19
5.6 1.2mg/10 mg 20.4 3.1mg/10 mg 32.6 4.5mg/10 mg 48.3 2.3mg/10 mg 41.2 3.4mg/10 mg
(Tre) (Cellob) (Py-5-P) (Py-5-P-P-PEG2000)
Tre: trehalose; Cellob: cellobiose; Py-5-P: pyridoxal-5-Phosphate; P-PEG2000: PEgylated phospholipid. a Processing conditions: sonication power: 12.5 W and sonication time: 55 min. b Each experiment was performed in triplicate (n = 3)
relationship between the response variable ‘‘Y’’ and the entire set of ‘‘X’’ variables at 95% level of significance. 2.4. Preparation of rhINF-a-2b loaded aquasomes Ceramic core, CaHPO4 particles were prepared by precipitation method [18]. In brief, 0.75 M solution of disodium hydrogen phosphate (Na2HPO4) was gradually added to 0.25 M solution of calcium chloride (CaCl2) under sonication (Trans-O-Sonic, 15 sec/ cycle). The mixture was sonicated at 4 8C for 2 h. The precipitate, CaHPO4 was separated by centrifugation (REMI, Mumbai, India) at 15,000 rpm for 1 h. The product was washed five times with 50 mL of distilled water to remove any by-product. The precipitate was resuspended in distilled water and then passed through 0.22 mm membrane filter (MDI, Ambala, India) to collect the ceramic core particles of size below 200 nm. The ceramic core particles suspension was then dried overnight by using a lyophilizer (Lab India, Thane, India). Dried ultrafine ceramic core particles were coated separately with three polysaccharides, namely trehalose (Tre, distilled water), cellobiose (Cellob, distilled water) and pyridoxal-5-phosphate (Py-5-P, 0.1 M NaOH) under predetermined optimized conditions (Table 2) to impart the stability. Protein was adsorbed by incubating 1 mL of rhINF-a-2b (500 mg/ mL in sterile 0.9% w/v NaCl) solution with 50 mg/mL of respective aquasome sample at 4 8C for 24 h. Subsequently, aquasomes samples were centrifuged at 15,000 rpm for 1 h and supernatants were stored for further analysis. rhINF-a-2b loaded aquasomes were lyophilized (Lab India, Thane, India) to get the fine powder. 2.5. Phospholipid-PEG2000 coating over rhINF-a-2b-Py-5-P-Aq.somes To induce the sustained release pattern, rhINF-a-2b-Py-5-PAq.somes was superimposed with phospholipid-PEG2000. In brief, 40 mg of phospholipid-PEG2000 and 40 mg of rhINF-a-2b-Py-5-PAq.somes were suspended in 0.3% polyvinyl alcohol aqueous solution. This suspension was then stirred in an orbital shaker at 200 rpm for 60 min. The resultant suspension was then centrifuged at 15,000 rpm and 4 8C for 1 h and supernatant was preserved for further analysis. Finally, the pellet of aquasomes was dried in a lyophilizer (Lab India, Thane, India) to get the fine powder of rhINFa-2b-Py-5-P-P-PEG2000 nanoformulation [26]. 2.6. Characterization of aquasomes 2.6.1. Particle size and zeta potential Particle size and zeta potential of aquasomes were determined by zeta-sizer (Malvern Instruments, UK). In brief, 5 mg of each aquasome sample was suspended separately in 5 mL of sterile normal saline. The sample was then filled in a cuvette to determine the particle size and zeta potential. A 150 mV electric field was applied to measure the electrophoretic velocity of aquasomes. All measurements were performed at room temperature in triplicates (n = 3).
2.6.2. Transmission electron microscopy (TEM) Particle shape and surface topography were examined by using transmission electron microscope (Phillips Morgagani 268, Netherlands) maintained at the voltage of 80 KV. In brief, an aqueous dispersion of each sample of aquasomes was drop cast onto a carbon coated copper grid and grid was dried at room temperature before loading it into the microscope. 2.6.3. Fourier-Transforms–infrared (FT–IR) spectroscopy Fourier-Transforms–infrared (FT–IR) spectroscopy was exploited to record the spectrum of Core (CaHPO4), Tre, Cellob, Py-5-P, Tre-Aq.somes, Cellob-Aq.somes and Py-5-P-Aq.somes by using Spectrum BX (Perkin Elmer, Massachusetts, USA) infrared spectrophotometer. Samples were prepared in KBr disks by using a hydrostatic press at a force of 40 psi for 4 min. The scanning range employed was 400–4400 cm1 at a resolution of 4 cm1. 2.6.4. Powder X-ray diffraction (PXRD) pattern Powder X-ray diffraction (PXRD) patterns were recorded for core (CaHPO4), Tre-Aq.somes, Cellob-Aq.somes and Py-5-PAq.somes on a RIGAKU, Rotaflex RV 200 (Rigaku Corporation, Japan) PXRD using Ni-filtered, CuK a-radiation at a voltage of 60 Kv and current of 50 mA. The scanning rate employed was 18/min over the 108 to 608 diffraction angle (2u) range. 2.6.5. Protein loading capacity Protein loading capacity of aquasomes was determined as reported previously [27]. In brief, 50 mg sample of each aquasomes was suspended separately in Triton-X 100 (0.01%, w/v) solution and incubated in an orbital shaker for 1 h. The samples were then centrifuged at 15,000 rpm for 1 h and rhINF-a-2b concentration in the supernatant was determined by Bradford assay method [28] at 595 nm in an UV–Visible spectrophotometer (1800, Shimadzu, Kyoto, Japan).
Protein loading capacity ¼ Amount of rhINF-a-2b in supernatant Total rhINF-a-2b added=Total amount of aquasomes
2.6.6. In vitro protein release The in vitro release of rhINF-a-2b from tailored nanoformulation was determined by placing 50 106 U 0.19 mg of rINF-a-2b adsorbed on aquasomal nanoformulations (339.28 mg rhINFa-2b-Core (CaHPO4), 93.13 mg rhINF-a-2b-Tre-Aq.somes, 58.28 mg rhINF-a-2b-Cellob-Aq.somes, 39.33 mg rhINF-a-2bPy-5-P-Aq.somes and 46.11 mg rhINF-a-2b-Py-5-P-Aq.somesP.PEG2000) in 2 mL of phosphate buffer saline (pH 7.4) contained in vials. The vials were then fitted to an incubator shaker maintained at 37 8C and 100 rpm, as recommended for dissolution testing of parenteral products [29]. One millimeter sample was withdrawn at predetermined time intervals, centrifuged at 10,000 rpm for 10 min, supernatant was collected and pellet was resuspended again in
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dissolution medium along with 1 ml of fresh buffer (pH7.4) to maintain sink conditions. The rhINF-a-2b was quantified in all supernatants by using Bradford assay method [28] at 595 nm in an UV/Visible spectrophotometer (1800, Shimadzu, Kyoto, Japan). All measurements were performed in triplicates (n = 3). 2.6.7. Gel electrophoresis SDS-PAGE (Sodium dodecyl sulphate polyacrylamide gel electrophoresis) assay [30] was performed on 12.5% or 15% polyacrylamide gel. Naive rhINF-a-2b (100 mg/mL) and rhINF-a-2b isolated from rhINF-a-2b-Py-5-P-Aq.somes (100 mg/mL) were separately diluted in 50 mM Tris–HCl buffer (1:1, pH 6.8) containing 2% SDS, 11.6% (v/v) glycerol and 0.001% bromophenol blue with 1% b-mercaptoethanol. Both samples were warmed for few min in a boiling water-bath and loaded onto the gels. The protein bands were visualized by using coomassie brilliant blue dye. 2.6.8. Circular dichroism The far-ultraviolet circular dichroism (CD) spectrum of naive rhINF-a-2b (100 mg/mL) and rhINF-a-2b (100 mg/mL) isolated from rhINF-a-2b-Core (CaHPO4), rhINF-a-2b-Tre-Aq.somes, rhINF-a-2b-Cellob-Aq.somes and rhINF-a-2b-Py-5-P-Aq.somes were scanned from 180 to 260 nm at 25 8C with constant nitrogen flushing using instrument, Jasco-J-815 CD spectrometer in sterile normal saline (0.9% w/v NaCl). The specifications of instrument were 0.5 s scan speed; 200 nm/min sensitivity; 100-mdegree and 1 nm spectra bandwidth. Three scans per sample were taken and results were expressed as residual ellipticity [u] (deg cm2 dmol1).
½u ¼ u=10 C l MRW
uabs is the measured ellipticity in degrees, C is the concentration in mg/mL, l is the light path length in cm and MRW is the mean residual weight. 2.6.9. In vitro cell proliferation assay In vitro cell proliferation assay [31] was performed on human ovarian cancer cells, SKOV3. In brief, 6 103 SKOV3 cells were plated in 200 mL of DMEM medium. After 24 h of plating, the DMEM medium was removed and replaced with naive rhINF-a-2b, core (CaHPO4) particles, rhINF-a-2b-Core(CaHPO4), rhINF-a-2bTre-Aq.somes, rhINF-a-2b-Cellob-Aq.somes, rhINF-a-2b-Py-5-PAq.somes and rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000 at concentrations ranging from 200–1200 IU/mL equivalent of rINF-a-2b and incubated for 72 h. At the end of treatment, SKOV3 cells were treated with MTT (0.5 mg/mL) for 4 h at 37 8C. In last, DMEM was removed, cells were lysed and formazon crystals were dissolved by using 100 mL of DMSO. The absorbance was read at 570 nm using 630 nm as the reference wavelength in ELISA plate reader (Tecan, Switzerland). 2.7. Statistical analysis Statistical analysis was performed on the data by Student t-test, one and two-way analysis of variance (ANOVA) tests by using GraphPad04 Instat Tm (v, 3.10 Prism Software, CL) Software. P < 0.05 was considered significant. 3. Results 3.1. Design, synthesis and characterization of rhINF-a-2b loaded aquasomes Ultrafine ceramic core particles were synthesized by using precipitation phenomena [18]. Disodium hydrogen phosphate
(Na2HPO4) was used as a template for precipitating the ceramic core by using calcium chloride (CaCl2). The resultant calcium hydrogen phosphate (CaHPO4) particulate system was used as a core material for the preparation of aquasomes. Na2 HPO4 þ CaCl2 ! 2NaCl þ CaHPO4
(1)
Subsequently, ultrafine ceramic core particles were coated separately with trehalose (Tre), cellobiose (Cellob) and pyridoxal5-phosphate (Py-5-P) and incubated with rhINF-a-2b to generate rhINF-a-2b-Tre-Aq.somes, rhINF-a-2b-Cellob-Aq.somes and rhINF-a-2b-Py-5-P-Aq.somes. The rhINF-a-2b was also loaded onto the native core (rhINF-a-2b-Core) for comparative studies. 3.2. Optimization of experimental conditions that influenced the particle size Response Surface Methodology (RSM) [21] using the Central Composite Design (CCD) [20] model is a well-suited experimental design strategy that offers the possibility of investigating a large number of variables at different levels with only a limited number of experiments. The ranges for each variable were selected on the basis of our preliminary experiments. Experimental results concerning the tested variables on mean particle size of aquasomes are presented in Table 1. The response was individually fitted to a reduced linear model. For the response, the model, which generated a higher F value, was identified as the best-fitted model. Each obtained model was validated by ANOVA. Three-dimensional response surface plots were drawn for the optimization of ultrafine ceramic nanoparticles (Fig. 1A–D). These types of plots are useful for studying the effect of two factors on the response while the third factor was kept constant. Following reduced linear model equation (1) was derived by the best-fit method to describe the relationship between particle size (Y1) and ratio of core: coat (CaHPO4: polysaccharide), sonication power (W) and sonication time (min). Particle sizeðY 1 Þ ¼ 118:34455 1:37676 Sonication power
(1)
A positive value in regression equation for a response represents an effect that favours the optimization (synergistic effect); while a negative value indicates an inverse relationship (antagonistic effect) between the factors and the response [21]. The reduced linear model was found to be significant with F value of 4.65 (P < 0.0001), which indicates that response variable Y1 and the set of formulation variables were significantly related. The high R2 value indicated that 20.52% of variation in particle size could be explained by the regression on formulation factors. 3.3. Optimization by desirability function Optimization process was undertaken with desirability function to optimize the response. A high value of desirability coefficient d (0 d 1) indicates that the operating point can produce acceptable formulation results. The response particle size (Y1) was transformed into the desirability scale d1. The higher coefficient of determination and F value in terms of the reduced linear indicated the goodness of fit. The response surface plot for increasing desirability coefficient d with respect to changes in variables: core:coat (X1) and sonication power (X2) keeping the sonication time constant, has been shown in (Fig. 1A–D). The maximum value of desirability coefficient d = 0.701 was obtained at the conditions, core:coat ratio of 1:3, sonication power of 12.5 W and sonication time of about 55 min (Table 1). In order to evaluate the predictive power of this model and desirability coefficient, aquasomes were prepared under the optimized conditions (Table 2). Experimental values of aquasomes prepared using
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Fig. 1. Optimization of aquasomes by using Central Composite Design (CCD) and Response Surface Methodology (RSM), (A) influence of sonication power (W) and sonication time (min) on particle size (nm); (B) influence of sonication time (min) and core: coat (mg) ratio on particle size (nm); (C) influence of sonication power (W) and core:coat (mg) ratio on particle size (nm) and, (D) influence of sonication power (W) and core:coat (mg) ratio on desirability. The maximum value of desirability coefficient d = 0.701 was achieved at core:coat (mg) ratio of 1:3, sonication power (W) of 12.5 and sonication time of about 55 min.
optimal conditions were very close to the predicted values, with low percentage bias.
somes-P-PEG2000 as compared to –23.2 1.26 mV of rhINF-a-2bPy-5-P-Aq.somes confirmed the coating of PEGylated phospholipid (Table 2).
3.4. Particle size and zeta potential 3.5. Surface topography Particle size and zeta potential were determined to predict the therapeutic potential of protein loaded aquasomes. The mean particle size of rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000 was measured to be 125.3 3.2 nm, which was significantly (one way ANOVA test, P < 0.05) higher than 104.4 5.9 nm, 101.9 6.4 nm, 98.5 4.3 nm, and 90.1 2.3 nm, measured respectively for rhINFa-2b-Py-5-P-Aq.somes, rhINF-a-2b-Cellob-Aq.somes, rhINF-a-2bTre-Aq.somes and rhINF-a-2b-Core (CaHPO4) (Table 2 and Fig. 2A– E). In addition, zeta potential of rhINF-a-2b-Py-5-P-Aq.somes was found to be –23.2 1.26 mV which was significantly (one way ANOVA test, P < 0.05) higher than –8.48 2.32 mV, –15.6 1.15 mV and –20.4 0.9 mV measured respectively for rhINF-a-2b-Core (CaHPO4), rhINF-a-2b-Tre-Aq.somes and rhINF-a-2b-CellobAq.somes (Table 2). A significant (Unpaired t-test, P < 0.05) increase in zeta potential to –44.1 3.19 mV in rhINF-a-2b-Py-5-P-Aq.
The surface topography of aquasomes influences the stability and adsorption of protein. We observed an irregular crystalline shape of rhINF-a-2b-Core (CaHPO4), while somewhat spherical shape was observed from rhINF-a-2b-Tre-Aq.somes to rhINF-a2b-P-y-5-P-Aq.somes-P-PEG2000 (Fig. 2A–E and Table 2). 3.6. Fourier-Transforms–infrared (FT–IR) spectroscopy FT–IR spectrum was recorded to analyze the chemistry of adsorption of protein over the surface of aquasomes. In brief, Tre exhibited characteristics peaks at 1514 cm1 and 991.96 cm1 due to the presence of –OH groups. Continuation to this, Cellob also displayed the peaks at 1517.08 cm1 and 1036 cm1, indicating the presence of –OH groups. PO42 group may be marked at
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Fig. 2. Particle size distribution and transmission electron microscopy (TEM) of (A) rhINF-a-2b loaded core (CaHPO4) particles; (B) rhINF-a-2b loaded trehalose aquasomes (rhINF-a-2b-Tre-Aq.somes); (C) rhINF-a-2b loaded cellobiose aquasomes (rhINF-a-2b-Cellob-Aq.somes); (D) rhINF-a-2b loaded pyridoxal-5-phosphate aquasomes superimposed with PEGylated phospholipid (rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000). rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000 nanoformulation was appeared to be spherical in shape while rhINF-a-2b loaded core (CaHPO4) particles were irregular in appearance. Scale bar 100 nm in TEM.
1020.84 cm1 in addition to 1147.72 cm1, 942.14 cm1 and 851.48 cm1 for –OH groups in Py-5-P oligomer. The native core, CaHPO4 also indicated the presence of PO42 at 1048.05 cm1, however, distinct from Py-5-P. Upon coating of polysaccharides over the ceramic core particles, these peaks were shifted to either higher or lower wavelengths indicating the formation of intermolecular hydrogen bonds between ceramic core particles and polyoligomer coating (Table 3).
6 and 8 h (Fig. 4A and B). Thus, rhINF-a-2b-Py-5-P-Aq.somes offered slow release and designated as optimized nanoformulation. Next, to induce the sustained release pattern, rhINF-a-2bPy-5-P-Aq.somes was coated with PEGylated phospholipid (rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000). The rhINF-a-2b-Py-5P-Aq.somes-P-PEG2000 displayed evidence of its prolonged release pattern and released 98.1% of rhINF-a-2b in 336 h (Fig. 4B). 3.10. Gel electrophoresis
3.7. Powder X-ray diffraction (PXRD) pattern The crystallline lattice arrangement of aquasomes was elucidated by using PXRD technique. The PXRD pattern of core (CaHPO4) showed peaks that were intense and sharp indicating its crystalline state (Fig. 3). Upon the coating of core particles with Tre (TreAq.somes), Cellob (Cellob-Aq.somes) and Py-5-P (Py-5-PAq.somes) reduced the intensity of sharp peaks and deformed into amorphous structure. Maximal deformation was scanned in Py-5-P-Aq.somes. 3.8. Protein loading capacity Protein loading capacity depends on the experimental conditions acquired for aquasome preparation. Protein loading capacity was measured to be 48.3 2.3 mg/10 mg for rhINF-a-2b-Py-5-P-Aq.somes which was significantly (one way ANOVA test, P < 0.05) higher than 5.6 1.2 mg/10 mg, 20.4 3.1 mg/10 mg and 32.45 4.5 mg/10 mg, of rhINF-a-2b-Core (CaHPO4), rhINF-a-2b-Tre-Aq.somes and rhINF-a2b-Cellob-Aq.somes. In contrast, protein loading capacity was significantly (Unpaired t-test, P < 0.05) reduced to 41.2 3.4 mg/ 10 mg in rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000 as compared to 48.3 2.3 mg/10 mg of rhINF-a-2b-Py-5-P-Aq.somes due to loss of protein during coating of PEGylated phospholipid (Table 2). 3.9. In vitro protein release The potential of protein loaded aquasomes to prolong the release of rhINF-a-2b was examined in PBS, pH 7.4. The rhINF-a2b-core (CaHPO4) released 97.3% of protein in 1 h, while 95.3% of rhINF-a-2b was released by rhINF-a-2b-Tre-Aq.somes in 4 h. Concurrently, rhINF-a-2b-Cellob-Aq.somes and rhINF-a-2b-Py-5P-Aq.somes released 96.2% and 97.8% of rhINF-a-2b respectively in
The SDS-PAGE was used to authenticate the stability and integrity of rhINF-a-2b in optimized nanoformulation, rhINF-a2b-Py-5-P-Aq.somes. SDS-PAGE image of rhINF-a-2b isolated from optimized rhINF-a-2b-Py-5-P-Aq.somes and pure rhINF-a-2b under reduced conditions is shown in Fig. 5A. The rhINF-a-2b secondary structure contains two intramolecular disulphide bonds, Cys1-Cys98 and Cys29-Cys138 [32]. The molecular weight of rhINF-a-2b is reported to be 17 kDa [33]. Reducing agent, b-mercaptoethanol was employed to break any disulphide bonds, thus eliminated the presence of high molecular weight bands. The structural stability and integrity of the isolated rhINFa-2b was consistent with the pure rhINF-a-2b even after adsorption over the Py-5-P-Aq.somes. The molecular weight of rhINF-a-2b was observed to be 17 kDa as compared to the standard marker. 3.11. Circular dichroism (CD) Far-UV CD is a sophisticated approach for investigation of secondary structure of rhINF-a-2b that contains 5 a-helices. We observed that pure rhINF-a-2b and rhINF-a-2b isolated from aquasomes showed identical CD spectrum (Fig. 5B). Furthermore, analysis of secondary structure using CCA program suggested that 55% of rhINF-a-2b was comprised of a-helix, 22% of unorganized structure and 23% of others. However, we did not observe b structures. 3.12. In vitro cell proliferation assay In vitro therapeutic efficacy of rhINF-a-2b, rhINF-a-2b-Core (CaHPO4), rhINF-a-2b-Tre-Aq.somes, rhINF-a-2b-Cellob-Aq.somes,
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Table 3 Fourier-Transforms–infrared (FT–IR) spectroscopy assignment of aquasomes, scanned between 400 to 4400 cm1. Sample CaHPO4 Tre Cellob Py-5-P
Peaks (cm1)
Assignment
1048.05 650.29 1514.03 991.96 1517.08 1036.84 1020.84 1147.72 942.14 851.48
PO42 OH OH OH OH OH PO42 OH OH OH
Sample
Peaks (cm1)
Assignment
Tre-Aq.somes
1517.54 981.03 1515.92 1022.39 1025.12 1254.85 967.90 821.51
OH OH OH OH PO42 OH OH OH
Cellob-Aq.somes Py-5-P-Aq.somes
Tre: trehalose; Cellob: cellobiose; Py-5-P: pyridoxal-5-phosphate.
rhINF-a-2b-Py-5-P-Aq.somes and rhINF-a-2b-Py-5-P-Aq.somes-PPEG2000 was examined by standard cell proliferation assay by employing SKOV3, ovarian cancer cell line. The IC50 value (concentration of drug require to kill 50% of the cells) was used as an indicator to define the therapeutic efficacy of protein loaded aquasomes. The IC50 value for free rhINF-a-2b was measured to be 710 IU/mL at 72 h. Compared to this, the IC50 value was significantly (one way ANOVA test, P < 0.05) reduced to 156 IU/mL in rhINF-a2b-Py-5-P-Aq.somes-P-PEG2000, 300 IU/mL in rhINF-a-Py-5-PAq.somes, 360 IU/mL in rhINF-a-2b-Cellob-Aq.somes, 440 IU/mL in rhINF-a-2b-Tre-Aq.somes and 600 IU/mL in rhINF-a-2b-Core (CaHPO4) at the same incubation period (Table 4). However, core (CaHPO4) did not exert any cytotoxicity to ovarian cancer cells.
4. Discussion
effective and highly stable raw material for scaling the drug delivery cargos. Moreover, it is widely accepted for delivery of both low and high molecular weight therapeutic molecules [34]. The nanocrystalline, CaHPO4 ceramic core particles self-assemble during the reaction process under sonication due to augmentation of surface free energy [18]. Ultimately, ceramic core particles were coated with a thin film of polyhydroxy oligomer for preparing the aquasomes under optimized conditions, as calculated by CCD and RSM [20,21] (Tables 1 and 2). Polyhydroxy oligomer film stabilizes the ceramic core through ionic, non-covalent, and extropic forces. We observed that particle size of aquasomes increases as a function of concentration of core to coat ratio, whilst the other variables, such as sonication power and time were kept constant (Fig. 1A–D and Table 1). This may be attributed to availability of free surface of the core particles with the coating material [18]. Moreover, an enhancement in sonication power decreased the particle size of aquasomes, whereas other variables were kept
Proteins and peptides administered through parentral route of administration often require high stability, slow release and targeting at molecular receptors. Moreover, susceptibility of proteins to denaturation by proteolytic enzymes and other physicochemical limitations need special attention while encapsulating in micro- or nano-drug delivery systems. In present investigation, rhINF-a-2b loaded aquasomes were prepared to induce protein stability, sustained release and cytotoxicity in ovarian cancer cells. Calcium hydrogen phosphate (CaHPO4) was precipitated as fine powder to be used as a template for the preparation of aquasomes [18]. CaHPO4 is a biodegradable, cost
Fig. 3. Powder X-ray diffraction (PXRD) pattern of core (CaHPO4) particles, trehalose aquasomes (Tre-Aq.somes), cellobiose aquasomes (Cellob-Aq.somes), and pyridoxal5-phosphate aquasomes (Py-5-P-Aq.somes). Crystal lattice arrangement of core particles was deformed to amorphous structure upon coating with polysaccharides. Maximal amorphization was done by Py-5-P oligomer.
Fig. 4. A. In vitro release of rhINF-a-2b from aquasomes in PBS, pH 7.4. rhINF-a2b-core (CaHP4) released 97.3% of protein in 1 h, while 95.3% of rhINF-a-2b was released by rhINF-a-2b-Tre-Aq.somes in 4 h. Concomitantly, rhINF-a-2b-CellobAq.somes and rhINF-a-2b-Py-5-P-Aq.somes released 96.2% and 97.8% of rhINF-a2b, respectively in 6 and 8 h. Thus, rhINF-a-2b-Py-5-P-Aq.somes offered slow release and designated as optimized nanoformulation. B. rhINF-a-2b-Py-5-PAq.somes-P-PEG2000 offered 98.1% prolonged release in 336 h, carried out in PBS (pH 7.4). Each experiment was performed in triplicate (n = 3).
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Fig. 5. A. SDS-PAGE gel electrophoresis of naive rhINF-a-2b and rhINF-a-2b isolated from optimized rhINF-a-2b-Py-5-P-Aq.somes. The molecular weight of rhINF-a-2b was observed to be 17 kDa as compared to the standard marker, and (B) circular dichroism of naive rhINF-a-2b and rhINF-a-2b isolated from aquasomes that exhibited identical CD spectrum. The 55% of rhINF-a-2b was comprised of a-helix, 22% of unorganized structure and 23% of others. However, no b structure was observed. Three scans were taken for each sample (n = 3).
stable. We assumed that an enhancement in sonication power induced the segregation of self-assembled architecture of aquasomes and thus decreased the particle size (Fig. 1A–D and Table 1). Next, decrease in sonication time increased the particle size of aquasomes, though other variables, such as core to coat ratio and sonication power were kept steady. This could be attributed to aggregation of smaller particles [14] (Fig. 1A–D and Table 1). Experimentally, particle size analysis, zeta potential and TEM assured that all samples of aquasomes were near about 100 nm in diameter and almost spherical in shape (Table 2 and Fig. 2A–E). The Py-5-P-Aq.somes exhibited higher negative zeta potential due to the presence of most electronegative atoms in the chemical structure of Py-5-P oligomer, as compared to Tre- and Cellob polysaccharides (Fig. 2A–D and Table 2). Hence, rhINF-a-2b-Py-5P-Aq.somes was overlaid with PEGylated phospholipid. A significant (Unpaired t-test, P < 0.05) enhancement in negative zeta potential indicating the layering of PEGylated phospholipid over the surface of rhINF-a-2b-Py-5-P-Aq.somes (Table 2). This may be attributed to anionic nature of PEG chain that contributed in the enhancement in net negative charge of aquasomes. Poly vinyl alcohol (PVA), being a coating modifier helped in layering of PEGYlated phospholipid over the surface of optimized aquasomal nanoformulation. Next, FT–IR confirmed the formation of intermolecular hydrogen bonds between the ceramic core, CaHPO4 and polysaccharides (Tre, Cellob and Py-5-P) due to broadening and shifting of peaks of –OH groups either at higher or lower
wavelengths [35] (Table 3). PXRD was performed to elucidate the structural geometry of aquasomes (Fig. 3). XRD patterns substantiated the deformation of crystalline geometry of CaHPO4 to amorphous configuration after coating with polyhydroxy oligomer. Molecules, which form hydrogen bonds, are hydrophilic in nature and consequently confer a significant degree of organization of water molecules [14]. Hence, Py-5-P owing to the presence of electronegative atoms and charged group, PO42 facilitated the intermolecular hydrogen bond formation with CaHPO4 and believed to favour the surrounding of excess water molecules as compared to Tre and Cellob. Thus, Py-5-P-Aq.somes exhibited higher amorphized geometry as compared to Tre and Cellob. Typically, due to irregular structural geometry, the amorphous phase requires minimal energy and thus tenders the supreme solubility and bioavailability of drugs [36]. The higher protein loading capacity was displayed by rhINF-a-2b-Py-5-P-Aq.somes (Table 2). We propose that higher negative zeta potential and presence of excess electronegative atoms in the molecular structure of Py-5-P-Aq.somes would have allowed the formation of intermolecular hydrogen bonds with active site (tyrosine residue) of rhINF-a-2b. Consequently, these intermolecular hydrogen bonds have promoted the higher adsorption of protein over the surface of Py-5-P-Aq.somes. Next, in vitro release study was performed to test the release pattern of rhINF-a-2b from aquasomes in physiological milieu. The rhINF-a-2b-Py-5-PAq.somes exhibited the slow release of rhINF-a-2b as compared
Table 4 Percent cell viability and IC50 value of aquasomes, calculated by standard cell viability assay against SKOV3, ovarian cancer cell line at 72 h. IC50a (IU/ml)
Samplesa
% Cell viability (IU/ml) 0
200
400
600
800
1000
1200
rhINF-a-2b rhINF-a-2b-Core (CaHPO4) rhINF-a-2b-Tre-Aq.somes rhINF-a-2b-Cellob-Aq.somes rhINF-a-2b-Py-5-P-Aq.somes rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000 Core (CaHPO4)
99.7 0.1 98.2 1.2 97.4 1.4 98.3 1.9 99.1 0.3 98.6 1.4 99.6 0.1
80.5 3.6 78.4 4.3 72.5 5.6 69.1 4.7 56.4 3.6 22.4 3.1 98.1 0.5
65.8 6.2 62.4 3.1 53.2 4.1 45.5 3.9 42.4 3.8 15.2 2.8 98.6 0.2
55.8 4.9 50.3 4.7 40.3 6.6 36.4 4.1 32.4 3.2 12.6 4.0 99.1 0.3
44.9 5.2 38.6 2.9 34.5 3.7 28.8 2.0 22.6 3.4 8.5 1.4 97.9 0.4
40.8 2.8 30.9 6.2 24.3 3.3 26.7 2.9 15.3 3.1 5.8 2.3 97.2 0.8
38.4 6.3 26.4 2.1 18.5 2.9 15.4 3.7 12.4 2.9 2.4 0.1 98.3 0.2
710 600 440 360 300 156 –
rhINF-a-2b: recombinant interferon-a-2b; rhINF-a-2b-Core: rhINF-a-2b coated core (CaHPO4) particles; rhINF-a-2b-Tre-Aq.somes: rhINF-a-2b coated trehalose aquasomes; rhINF-a-2b-Cellob-Aq.somes: rhINF-a-2b coated cellobiose aquasomes; rhINF-a-2b-Py-5-P-Aq.somes: rhINF-a-2b coated pyridoxal-5-phosphate aquasomes; rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000: rhINF-a-2b coated pyridoxal-5-phosphate aquasomes superimposed with PEGylated phospholipid. IC50: concentration of the drug require to kill 50% of the cells. a All groups are statistically significant (P < 0.05, one way ANOVA test).
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to rhINF-a-2b-Cellob-Aq.somes, rhINF-a-2b-Tre-Aq.somes and rhINF-a-2b-Core (CaHPO4) (Fig. 4A). This may be donated by relatively higher intermolecular hydrogen bond formation in rhINF-a-2b-Py-5-P-Aq.somes that sluggish the release pattern of protein in physiological buffer. As expected, rhINF-a-2b-Py-5-PAq.somes-P-PEG2000 exhibited the prolonged release of rhINF-a2b up to 336 h due to the presence of layering of PEGylated phospholipid (Fig. 4B). PEGylated phospholipid behaves like a surfactant in aqueous phase, hence, aggregated structure of PEGylated phospholipid formed boundary over the surface of aquasomes that induced the prolonged release of rhINF-a-2b. Moreover, PEGylation also enhances the blood circulation time of drug delivery system by preventing RES uptake and makes it long circulating [26]. The stability and integrity of adsorbed protein was investigated by using gel electrophoresis and CD (Fig. 5A–B). The stability assessment study showed that the structural integrity of the protein did not appear to be compromised by the preparation technique. Polyhydroxy oligomer film prevents peptide secondary structure from changing shape and being damaged by generating a glossy film that acts as dehydroprotectant. Hydrogen bond helps in base pair matching and stabilization of secondary structure of protein, such as a-helices and b-sheets [14]. Thus, aquasomes provided conformational stabilization as well as a high degree of surface exposure to protein. In last, in vitro cytotoxicity assay was performed on SKOV3, ovarian cancer cells to test the therapeutic efficacy of protein loaded aquasomes (Table 4). The rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000 exhibited the 4.55, 1.92, 2.3, 2.8, and 3.84 fold reductions in IC50 as compared to respectively free rhINF-a-2b, rhINF-a-2bPy-5-P-Aq.somes, rhINF-a-2b-Cellob-Aq.somes, rhINF-a-2b-TreAq.somes and rhINF-a-2b-Core (CaHPO4). Cancer cells generally bear net negative charge at physiological milieu [37]. Hence, rhINFa-2b-Py-5-P-Aq.somes-P-PEG2000 offered maximal steric repulsion owing to higher negative zeta potential (Table 2), preventing the cellular uptake by SKOV3 cells and thereby prolonged the cytotoxic effect and decreased the IC50 value. Therefore, rhINF-a2b-Py-5-P-Aq.somes-P-PEG2000 enhanced the stability of protein in its amorphous geometry and tendered sustained cytotoxic effect against ovarian cancer cells. 5. Conclusion In conclusion, narrow particle size distribution of aquasomes may be achieved at 1:3 core to coat ratio, sonication power and time of about 12.5 W and 55 min. The rhINF-a-2b-Py-5-PAq.somes-P-PEG2000 offered prolonged release and enhanced cytotoxicity of rhINF-a-2b against SKOV3, ovarian cancer cells. Moreover, secondary structure of protein was also preserved by Py-5-P-Aq.somes. Therefore, rhINF-a-2b-Py-5-P-Aq.somes-PPEG2000 warrant further in depth in vitro and in vivo preclinical investigations to scale up the technology for clinical translation. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. References [1] Ingersoll SB, Ahmad S, Finkler NJ, Edwards JR, Holloway RW. Cellular therapy for ovarian cancer: experimental and clinical perspectives. Curr Med Chem 2012;19:3787–93. [2] Willemse PH, deVries EG, Mulder NH, Aalders JG, Bouma J, Sleijfer DT. Intraperitoneal human recombinant interferon a-2b in minimal residual ovarian cancer. Eur J Cancer 1990;26:353–8.
275
[3] Sanchez A, Tobio M, Gonzalez L, Fabra A, Alonso MJ. Biodegradable micro- and nanoparticles as long-term delivery vehicle for interferon-a. Eur J Pharm Sci 2003;18:221–9. [4] Frank MH, Pomer S. Interferon alpha-2b differentially affects proliferation of two human renal cell carcinoma cell lines differing in the P-glycoproteinassociated multidrug-resistant phenotype. J Cancer Res Clin Oncol 1999;125: 117–20. [5] Jiang Y, Shi K, Xia D, Piao H, Quan P, Song T, et al. Protamine modified metal ion-protein chelate microparticles for sustained release of interferon. Int J Pharm 2011;407:31–7. [6] Santana H, Martı´nez E, Sanchez JC, Moya G, Sosa R, Hardy E, et al. Molecular characterization of recombinant human interferon a-2b produced in Cuba. Biotecnol Aplicada 1999;16:154–9. [7] Li Z, Li L, Liu Y, Zhang H, Li X, Luo F, et al. Development of interferon a-2b microspheres with constant release. Int J Pharm 2011;410:48–53. [8] Fishburn CS. The pharmacology of PEGylation: balancing PD with PK to generate novel therapeutics. J Pharm Sci 2008;97:4167–83. [9] Langer R, Cleland JL, Hanes J. New advances in microsphere-based single-dose vaccines. Adv Drug Deliv Rev 1997;28:97–119. [10] Hong H, Jo JR, Yeon JH, Hong JT, Jung KH, Yoo SK, et al. Preparation of branched dextran microspheres of soluble interferon-alpha and its activity in vitro and in vivo. J Microbiol Biotechnol 2011;21:176–82. [11] Alejandro S, Maria T, Libia G, Angels F, Maria JA. Biodegradable micro- and nanoparticles as long term delivery vehicles for interferon-alpha. Eur J Pharm Sci 2003;18:221–9. [12] Gulia M, Rai S, Jain UK, Katare OP, Katyal A, Madan J. Sustained release protamine sulphate impregnated microspheres may reduce the frequent administration of recombinant interferon-a-2b in ovarian cancer: in vitro characterization. Anticancer Drugs 2014;25:63–71. [13] Nitta SK, Numata K. Biopolymer-based nanoparticles for drug/gene delivery and tissue engineering. Int J Mol Sci 2013;14:1629–54. [14] Kossovsky N, Gelman A, Sponsler EE, Hnatyszyn HJ, Rajguru S, Torres M, et al. Surface-modified nanocrystalline ceramics for drug delivery applications. Biomaterials 1994;15:1201–7. [15] Pandey RS, Sahu S, Sudheesh MS, Madan J, Kumar M, Dixit VK. Carbohydrate modified ultrafine ceramic nanoparticles for allergen immunotherapy. Int Immunopharmacol 2011;11:925–31. [16] Kanwar JR, Mahidhara G, Kanwar RK. Novel alginate-enclosed chitosancalcium phosphate-loaded iron-saturated bovine lactoferrin nanocarriers for oral delivery in colon cancer therapy. Nanomedicine 2012;7: 1521–50. [17] Singh D, Singh S, Sahu J, Srivastava S, Singh MR. Ceramic nanoparticles: recompense, cellular uptake and toxicity concerns. Artif Cells Nanomed Biotechnol 2014;17:1–9. [18] Cherian AK, Rana AC, Jain SK. Self-assembled carbohydrate-stabilized ceramic nanoparticles for the parenteral delivery of insulin. Drug Develop Indust Pharm 2000;26:459–69. [19] Rojas-Oviedo I, Salazar-Lopez RA, Reyes-Gasga J, Quirino-Barreda CT. Elaboration and structural analysis of aquasomes loaded with indomethacin. Eur J Pharm Sci 2007;32:223–30. [20] Bei YY, Zhou XF, You BG, Yuan ZQ, Chen WL, Xia P, et al. Application of the central composite design to optimize the preparation of novel micelles of harmine. Int J Nanomed 2013;8:1795–800. [21] Yiran M, Xinyi Z, Jian L, Qi S. The comparison of different daidzein-PLGA nanoparticles in increasing its oral bioavailability. Int J Nanomed 2012;7: 559–70. [22] Lim SB, Rubinstein I, Sadikot RT, Artwohl JE, Onyuksel H. A novel peptide nanomedicine against acute lung injury: GLP-1 in phospholipid micelles. Pharm Res 2011;28:662–72. [23] Gill KK, Nazzal S, Kaddoumi A. Paclitaxel loaded PEG(5000)-dspe micelles as pulmonary delivery platform: formulation characterization, tissue distribution, plasma pharmacokinetics, and toxicological evaluation. Eur J Pharm Biopharm 2011;79:276–84. [24] Ma L, Lai D, Liu T, Cheng W, Guo L. Cancer stem-like cells can be isolated with drug selection in human ovarian cancer cell line SKOV3. Acta Biochim Biophys Sin 2010;42:593–602. [25] Loveymi BD, Jelvehgari M, Zakeri-Milani P, Valizadeh H. Statistical optimization of oral vancomycin-eudragit RS nanoparticles using response surface methodology. Iranian J Pharm Res 2012;11:1001–12. [26] Vij N, Min T, Marasigan R, Belcher CN, Mazur S, Ding H, et al. Development of PEGylated PLGA nanoparticle for controlled and sustained drug delivery in cystic fibrosis. J Nanobiotechnol 2010;8:22–40. [27] Goyal AK, Rawat A, Mahor S, Gupta PN, Khatri K, Vyas SP. Nanodecoy system: a novel approach to design hepatitis B vaccine for immunopotentiation. Int J Pharm 2006;309:227–33. [28] Kang J, Lambert O, Ausborn M, Schwendeman SP. Stability of proteins encapsulated in injectable and biodegradable poly (lactide-co-glycolide)-glucose millicylinders. Int J Pharm 2008;357:235–43. [29] Anand O, Yu LX, Davit BM. Dissolution testing for generic drugs: an FDA perspective. AAPS J 2011;3:328–35. [30] Berg K, Heron I. SDS-polyacrylamide gel electrophoresis of purified human leucocyte interferon and the antiviral and anticellular activities of the different interferon species. J Gen Virol 1980;50:441–6. [31] Mosmann T. Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65: 55–63.
276
K. Kaur et al. / Biomedicine & Pharmacotherapy 69 (2015) 267–276
[32] Bodo G, Maurer-Fogy I. In: Dian-zani F, Rossi GB, editors. The interferon system, vol. 24. New York: Serono Symposium Publication, Raven Press; 1986. p. 23–7. [33] Bordens R, Grossberg SE, Trotta PP, Nagabhushan TL. Molecular and biological characterization of recombinant interferon-a-2b. Seminal Oncol 1997;24. S941-S9-51. [34] Jain SS, Jagtap PS, Dand NM, Jadhav KR, Kadam VJ. Aquasomes: a novel drug carrier. J Appl Pharm Sci 2012;2:184–92.
[35] Rogez G, Ribera BF, Credi A, Ballardini R, Gandolfi MT, Balzani V, et al. A molecular plug socket connector. J Am Chem Soc 2007;129:4633–42. [36] Kaur M, Bhatia RK, Pissurlenkar RR, Coutinho EC, Jain UK, Katare OP, et al. Telmisartan complex augments solubility, dissolution and drug delivery in prostate cancer cells. Carbohydrate Polym 2014;101:614–22. [37] Abercrombie M, Ambrose EJ. The surface properties of cancer cells. A review. Cancer Res 1962;22:525–48.
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Original Article
Stealth lipid coated aquasomes bearing recombinant human interferon-a-2b offered prolonged release and enhanced cytotoxicity in ovarian cancer cells Kamaljeet Kaur a, Preeti Kush a, Ravi Shankar Pandey b, Jitender Madan a,*, Upendra Kumar Jain a, Om Prakash Katare c a
Department of Pharmaceutics, Chandigarh College of Pharmacy, Mohali (Panjab) India SLT Institute of Pharmaceutical Sciences, Guru Ghasidas University, Bilaspur, India c University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 31 October 2014 Accepted 1st December 2014
Purpose: In present investigation, recombinant human interferon-a-2b (rhINF-a-2b) loaded aquasomes were prepared, optimized and overlaid with PEGylated phospholipid to offer prolong release and high therapeutic index against ovarian cancer, SKOV3 cells. Methods and results: Central Composite Design (CCD) and Response Surface Methodology (RSM) were employed to calculate the optimized conditions, 1:3 core to coat ratio, sonication power of 12.5 W and time of about 55 min for preparation of aquasomes. Consequently, rhINF-a-2b-Py-5-P-Aq.somes exhibited higher protein loading capacity and retained structural conformations of rhINF-a-2b, as compared to rhINF-a-2b-Cellob-Aq.somes, rhINF-a-2b-Tre-Aq.somes and rhINF-a-2b-Core (CaHPO4). Further, optimized rhINF-a-2b-Py-5-P-Aq.somes was superimposed with phospholipid-PEG2000 to prolong the release pattern of rhINF-a-2b from aquasomes. The rhINF-a-2b-core (CaHPO4) released 97.3% of protein in 1 h, while 95.3% of rhINF-a-2b was released by rhINF-a-2b-Tre-Aq.somes in 4 h. Concurrently, rhINF-a-2b-Cellob-Aq.somes and rhINF-a-2b-Py-5-P-Aq.somes released 96.2% and 97.8% of rhINF-a-2b respectively in 6 and 8 h. Ultimately, rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000 displayed evidence of its prolonged release pattern and released 98.1% of rhINF-a-2b in 336 h. FT–IR and XRD substantiated the involvement of vigorous intermolecular hydrogen bonding and amorphous geometry in rhINF-a-2b-Py-5-P-Aq.somes. In last, rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000 exhibited the 4.55, 1.92, 2.3, 2.8, and 3.84 fold reductions in IC50 as compared to free rhINF-a-2b, rhINF-a-2b-Py-5-PAq.somes, rhINF-a-2b-Cellob-Aq.somes, rhINF-a-2b-Tre-Aq.somes and rhINF-a-2b-Core (CaHPO4), respectively. Conclusion: Therefore, rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000 warrant further in depth in vitro and in vivo antitumor study to scale up the technology for clinical intervention. ß 2014 Elsevier Masson SAS. All rights reserved.
Keywords: rhINF-a-2b Aquasomes Trehalose Cellobiose Pyridoxal-5-phosphate Phospholipid-PEG2000 Cytotoxicity
1. Introduction Recombinant human interferon-a-2b (rhINF-a-2b), a single non-glycosylated USFDA approved cytokine is recommended to co-administer with cisplatin to elicit cytotoxicity against platinum resistant ovarian cancer cells [1]. In addition, rhINF-a-2b induces Abbreviations: rhINF-a-2b, Recombinant interferon-a-2b; Tre, Trehalose; Cellob, Cellobiose; Py-5-P, Pyridoxal-5-phosphate; PVA, Poly(vinyl) alcohol. * Corresponding author. Department of Pharmaceutics, Chandigarh College of Pharmacy, Mohali (Panjab) 140307, India. Tel.: +91 172 398 4209; fax: +91 172 398 4209. E-mail address: [email protected] (J. Madan). http://dx.doi.org/10.1016/j.biopha.2014.12.007 0753-3322/ß 2014 Elsevier Masson SAS. All rights reserved.
mitochondrial-mediated apoptosis in cancer cells, including activation of caspases-3 mediated pathway [2]. Chemically, rhINF-a-2b is a polypeptide chain of 165 amino acids [3]. However, short serum half-life (2–6 h), upregulation of P-glycoprotein mediated multi-drug resistance (P-gp-MDR), low therapeutic index, fluctuated plasma level, and rapid proteolytic degradation hamper the systemic drug delivery [2,4–6]. This necessitates the administration of rhINF-a-2b in a sustained release pattern for successive chemotherapy. Parenteral route of administration is the most common method for delivery of therapeutic proteins; however, it does not enhance the half-life of proteins in vivo [7]. Furthermore, poly(ethylene glycol) mediated stealth nanocomplex of rhINF-a-2b, despite
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excellent release profile is suffered with immunogenicity, antigenicity and low mean residence time [8,9]. Therefore, a stable drug delivery system is required for safe homing of rhINF-a-2b to gain maximum clinical benefits [2,6,10]. Several attempts have been made to vesiculize rhINF-a-2b for clinical intervention by using nanoparticles and microparticles [11–13]. Aquasomes are self-assembled ceramic nanoparticles whose surface can be non-covalently modified with carbohydrates [14]. Instrumentally, aquasomes congregate through non-covalent bonds, ionic bonds and Van der Waals forces [15]. Polysaccharide coating provides glassy molecular layer that adsorbs therapeutic protein without alteration in three-dimensional conformations. In addition, ceramic core superimposed with carbohydrates enhanced the cellular uptake in cancer cells [16,17]. Hence, aquasomes have been extensively investigated for delivery of both small and high molecular weight, pharmaceuticals [18,19]. However, aquasomes do not tender sustained release of proteins due to adsorption rather than encapsulation phenomena. Therefore, in present investigation, rhINF-a-2b was adsorbed on to the different polysaccharide coated self-assembled ultrafine ceramic nanoparticles. Process parameters that influence the synthesis of aquasomes were optimized by using Central Composite Design (CCD) [20] and Response Surface Methodology (RSM) computational techniques [21]. The optimized nanoformulation was then superimposed with phospholipid-PEG2000 (PEGylated phospholipid) to induce the sustained release pattern. It is reported that PEGylated phopholipid retains the pharmacological efficacy of protein, escapes macrophage uptake, and helps to attain high blood level [22,23]. The unique property of PEGylated phospholipid coating is that it allows release of therapeutic component from the drug delivery system through diffusion mechanism [23]. Aquasomes were characterized for particle size, zeta potential, protein loading capacity, in vitro release, protein stability and cytotoxicity against human ovarian cancer cell line, SKOV3.
2. Materials and methods 2.1. Materials Recombinant human interferon-a-2b (rhINF-a-2b, Molecular weight 17 Kda) was obtained as a gift sample from Reliance Life Sciences, Navi Mumbai, India. Trehalose (Tre) and cellobiose (Cellob) were obtained from Loba Chemie, New Delhi, India. Pyridoxal-5-phosphate (Py-5-P) was purchased from Central Drug House, New Delhi, India. All other chemicals used were of highest analytical grade. 2.2. Cell culture Human ovarian cancer cell line, SKOV3 was maintained in 95% air and 5% CO2 atmosphere at 37 8C using DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% fetal bovine serum. All experiments were performed with asynchronous cell populations in exponential growth phase (24 h after plating) [24]. 2.3. Experimental design for optimization The core (CaHPO4) to coat (polysaccharide) ratio was optimized by Central Composite Design (CCD) [20] to get Response Surface Methodology (RSM) [21] that selected an object, noticed the contributing factors and investigated the relationship between responses and factors. CCD enabled several independent variables to be investigated at the same time using a relatively small number of experiments, while RSM optimization analyzed the interactions between variables. Therefore, CCD-RSM defined the interactions
Table 1 Optimized conditions calculated by central composite design (CCD) and response surface methodology (RSM) for preparation of aquasomes. Runs
Core:coat (mg) (X1)
Sonication power (W) (X2)
Sonication time (min) (X3)
Particle size (nm)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1 3 3 1 5 3 3 –0.36 6.363 1 3 5 3 3 5 3 1 3 5 3
20 –0.113 12.5 5 20 12.5 12.5 12.5 12.5 20 12.5 5 12.5 12.5 5 25.11 5 12.5 20 12.5
20 55 55 90 90 55 -3.86 55 55 90 55 90 55 55 20 55 20 113.86 20 55
70.6 135.8 90.1 125.7 130.4 90.4 138.4 82.3 100.4 94.2 91.6 115.4 92.1 90.8 117.2 88.4 98.4 79.3 100.2 91.1
Core: CaHPO4; Coat: polysaccharide (Trehalose; cellobiose and pyridoxal-5phosphate).
between factors, avoided unwanted experiments and optimized the results [25]. Our preliminary investigation indicated that variables, such as core to coat (mg) ratio, sonication power (W) and sonication time (min) were the main factors that influenced the particle size of nanoformulations. A CCD model was used to statistically optimize the factors that affected the particle size of aquasomes. For each factor, experimental range was selected on the basis of previous investigations and probability of preparing the aquasomes at extreme values. The value range of variables were core:coat ratio (x1): 1:1 to 1:5 (mg), sonication power (x2): 5– 25.11 (W), and sonication time (x3): 20–113.86 (min). The design consisted of 20 runs (8 factorial points, 6 star points and 1 centre point) and 5 replicated runs (centre points) that yielded total 20 experiments (Table 1). The purpose of replication was to estimate the experimental errors and increase the accuracy. Each experimental run was repeated thrice (n = 3). Star points represented the extreme values (low and high) for each factor in the design and allowed estimation of second-order effects. Star points were at some distance, alpha, from the centre, based on the properties desired for design and number of factors in the design. Alpha in coded units was the axial distance from the centre point and made the design rotatable. A rotatable design provides equally good predictions at points equally distant from the centre, a very desirable property for RSM [21]. A design matrix comprising 20 experimental runs was constructed and responses were modelled by the following reduced linear model (Eq (1)): y ¼ b0 þ b1 x1 þ b2 x2 þ b3 x3 þ b11 x21 þ b22 x22 þ b33 x23 þ b12 x1 x2 þ b13 x1 x3 þ b
(1)
where y is the measured response associated with each factor level combinations; b0 is the intercept; bi’s (for i = 1, 2 and 3) are the linear effects, the b ii are the quadratic effects, the b ij’s (for i, j = 1, 2 and 3, i < j) are the interactions between the ith and ith variables. The statistical analysis was performed by using the software Design Expert (Version 8.0.7.1), where analysis of variance (ANOVA) was significant, when P < 0.05. An F-test was used to determine whether there was an overall regression
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Table 2 Various parameters used for preparation and characterization of aquasomes. Corea (CaHPO4):coata (oligomer)
Morphology
Size (nm)b
Zeta potentialb (–mV)
rhINF-a-2bb loading capacity (mg/mg of aquasomes)
1:0 1:3 1:3 1:3 1:3
Irregular crystals Spherical Spherical Spherical Spherical
90.1 2.3 98.5 4.3 101.9 6.4 104.4 5.9 125.3 3.2
–8.48 2.32 –15.6 1.15 –20.4 0.9 –23.2 1.26 –44.1 3.19
5.6 1.2mg/10 mg 20.4 3.1mg/10 mg 32.6 4.5mg/10 mg 48.3 2.3mg/10 mg 41.2 3.4mg/10 mg
(Tre) (Cellob) (Py-5-P) (Py-5-P-P-PEG2000)
Tre: trehalose; Cellob: cellobiose; Py-5-P: pyridoxal-5-Phosphate; P-PEG2000: PEgylated phospholipid. a Processing conditions: sonication power: 12.5 W and sonication time: 55 min. b Each experiment was performed in triplicate (n = 3)
relationship between the response variable ‘‘Y’’ and the entire set of ‘‘X’’ variables at 95% level of significance. 2.4. Preparation of rhINF-a-2b loaded aquasomes Ceramic core, CaHPO4 particles were prepared by precipitation method [18]. In brief, 0.75 M solution of disodium hydrogen phosphate (Na2HPO4) was gradually added to 0.25 M solution of calcium chloride (CaCl2) under sonication (Trans-O-Sonic, 15 sec/ cycle). The mixture was sonicated at 4 8C for 2 h. The precipitate, CaHPO4 was separated by centrifugation (REMI, Mumbai, India) at 15,000 rpm for 1 h. The product was washed five times with 50 mL of distilled water to remove any by-product. The precipitate was resuspended in distilled water and then passed through 0.22 mm membrane filter (MDI, Ambala, India) to collect the ceramic core particles of size below 200 nm. The ceramic core particles suspension was then dried overnight by using a lyophilizer (Lab India, Thane, India). Dried ultrafine ceramic core particles were coated separately with three polysaccharides, namely trehalose (Tre, distilled water), cellobiose (Cellob, distilled water) and pyridoxal-5-phosphate (Py-5-P, 0.1 M NaOH) under predetermined optimized conditions (Table 2) to impart the stability. Protein was adsorbed by incubating 1 mL of rhINF-a-2b (500 mg/ mL in sterile 0.9% w/v NaCl) solution with 50 mg/mL of respective aquasome sample at 4 8C for 24 h. Subsequently, aquasomes samples were centrifuged at 15,000 rpm for 1 h and supernatants were stored for further analysis. rhINF-a-2b loaded aquasomes were lyophilized (Lab India, Thane, India) to get the fine powder. 2.5. Phospholipid-PEG2000 coating over rhINF-a-2b-Py-5-P-Aq.somes To induce the sustained release pattern, rhINF-a-2b-Py-5-PAq.somes was superimposed with phospholipid-PEG2000. In brief, 40 mg of phospholipid-PEG2000 and 40 mg of rhINF-a-2b-Py-5-PAq.somes were suspended in 0.3% polyvinyl alcohol aqueous solution. This suspension was then stirred in an orbital shaker at 200 rpm for 60 min. The resultant suspension was then centrifuged at 15,000 rpm and 4 8C for 1 h and supernatant was preserved for further analysis. Finally, the pellet of aquasomes was dried in a lyophilizer (Lab India, Thane, India) to get the fine powder of rhINFa-2b-Py-5-P-P-PEG2000 nanoformulation [26]. 2.6. Characterization of aquasomes 2.6.1. Particle size and zeta potential Particle size and zeta potential of aquasomes were determined by zeta-sizer (Malvern Instruments, UK). In brief, 5 mg of each aquasome sample was suspended separately in 5 mL of sterile normal saline. The sample was then filled in a cuvette to determine the particle size and zeta potential. A 150 mV electric field was applied to measure the electrophoretic velocity of aquasomes. All measurements were performed at room temperature in triplicates (n = 3).
2.6.2. Transmission electron microscopy (TEM) Particle shape and surface topography were examined by using transmission electron microscope (Phillips Morgagani 268, Netherlands) maintained at the voltage of 80 KV. In brief, an aqueous dispersion of each sample of aquasomes was drop cast onto a carbon coated copper grid and grid was dried at room temperature before loading it into the microscope. 2.6.3. Fourier-Transforms–infrared (FT–IR) spectroscopy Fourier-Transforms–infrared (FT–IR) spectroscopy was exploited to record the spectrum of Core (CaHPO4), Tre, Cellob, Py-5-P, Tre-Aq.somes, Cellob-Aq.somes and Py-5-P-Aq.somes by using Spectrum BX (Perkin Elmer, Massachusetts, USA) infrared spectrophotometer. Samples were prepared in KBr disks by using a hydrostatic press at a force of 40 psi for 4 min. The scanning range employed was 400–4400 cm1 at a resolution of 4 cm1. 2.6.4. Powder X-ray diffraction (PXRD) pattern Powder X-ray diffraction (PXRD) patterns were recorded for core (CaHPO4), Tre-Aq.somes, Cellob-Aq.somes and Py-5-PAq.somes on a RIGAKU, Rotaflex RV 200 (Rigaku Corporation, Japan) PXRD using Ni-filtered, CuK a-radiation at a voltage of 60 Kv and current of 50 mA. The scanning rate employed was 18/min over the 108 to 608 diffraction angle (2u) range. 2.6.5. Protein loading capacity Protein loading capacity of aquasomes was determined as reported previously [27]. In brief, 50 mg sample of each aquasomes was suspended separately in Triton-X 100 (0.01%, w/v) solution and incubated in an orbital shaker for 1 h. The samples were then centrifuged at 15,000 rpm for 1 h and rhINF-a-2b concentration in the supernatant was determined by Bradford assay method [28] at 595 nm in an UV–Visible spectrophotometer (1800, Shimadzu, Kyoto, Japan).
Protein loading capacity ¼ Amount of rhINF-a-2b in supernatant Total rhINF-a-2b added=Total amount of aquasomes
2.6.6. In vitro protein release The in vitro release of rhINF-a-2b from tailored nanoformulation was determined by placing 50 106 U 0.19 mg of rINF-a-2b adsorbed on aquasomal nanoformulations (339.28 mg rhINFa-2b-Core (CaHPO4), 93.13 mg rhINF-a-2b-Tre-Aq.somes, 58.28 mg rhINF-a-2b-Cellob-Aq.somes, 39.33 mg rhINF-a-2bPy-5-P-Aq.somes and 46.11 mg rhINF-a-2b-Py-5-P-Aq.somesP.PEG2000) in 2 mL of phosphate buffer saline (pH 7.4) contained in vials. The vials were then fitted to an incubator shaker maintained at 37 8C and 100 rpm, as recommended for dissolution testing of parenteral products [29]. One millimeter sample was withdrawn at predetermined time intervals, centrifuged at 10,000 rpm for 10 min, supernatant was collected and pellet was resuspended again in
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dissolution medium along with 1 ml of fresh buffer (pH7.4) to maintain sink conditions. The rhINF-a-2b was quantified in all supernatants by using Bradford assay method [28] at 595 nm in an UV/Visible spectrophotometer (1800, Shimadzu, Kyoto, Japan). All measurements were performed in triplicates (n = 3). 2.6.7. Gel electrophoresis SDS-PAGE (Sodium dodecyl sulphate polyacrylamide gel electrophoresis) assay [30] was performed on 12.5% or 15% polyacrylamide gel. Naive rhINF-a-2b (100 mg/mL) and rhINF-a-2b isolated from rhINF-a-2b-Py-5-P-Aq.somes (100 mg/mL) were separately diluted in 50 mM Tris–HCl buffer (1:1, pH 6.8) containing 2% SDS, 11.6% (v/v) glycerol and 0.001% bromophenol blue with 1% b-mercaptoethanol. Both samples were warmed for few min in a boiling water-bath and loaded onto the gels. The protein bands were visualized by using coomassie brilliant blue dye. 2.6.8. Circular dichroism The far-ultraviolet circular dichroism (CD) spectrum of naive rhINF-a-2b (100 mg/mL) and rhINF-a-2b (100 mg/mL) isolated from rhINF-a-2b-Core (CaHPO4), rhINF-a-2b-Tre-Aq.somes, rhINF-a-2b-Cellob-Aq.somes and rhINF-a-2b-Py-5-P-Aq.somes were scanned from 180 to 260 nm at 25 8C with constant nitrogen flushing using instrument, Jasco-J-815 CD spectrometer in sterile normal saline (0.9% w/v NaCl). The specifications of instrument were 0.5 s scan speed; 200 nm/min sensitivity; 100-mdegree and 1 nm spectra bandwidth. Three scans per sample were taken and results were expressed as residual ellipticity [u] (deg cm2 dmol1).
½u ¼ u=10 C l MRW
uabs is the measured ellipticity in degrees, C is the concentration in mg/mL, l is the light path length in cm and MRW is the mean residual weight. 2.6.9. In vitro cell proliferation assay In vitro cell proliferation assay [31] was performed on human ovarian cancer cells, SKOV3. In brief, 6 103 SKOV3 cells were plated in 200 mL of DMEM medium. After 24 h of plating, the DMEM medium was removed and replaced with naive rhINF-a-2b, core (CaHPO4) particles, rhINF-a-2b-Core(CaHPO4), rhINF-a-2bTre-Aq.somes, rhINF-a-2b-Cellob-Aq.somes, rhINF-a-2b-Py-5-PAq.somes and rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000 at concentrations ranging from 200–1200 IU/mL equivalent of rINF-a-2b and incubated for 72 h. At the end of treatment, SKOV3 cells were treated with MTT (0.5 mg/mL) for 4 h at 37 8C. In last, DMEM was removed, cells were lysed and formazon crystals were dissolved by using 100 mL of DMSO. The absorbance was read at 570 nm using 630 nm as the reference wavelength in ELISA plate reader (Tecan, Switzerland). 2.7. Statistical analysis Statistical analysis was performed on the data by Student t-test, one and two-way analysis of variance (ANOVA) tests by using GraphPad04 Instat Tm (v, 3.10 Prism Software, CL) Software. P < 0.05 was considered significant. 3. Results 3.1. Design, synthesis and characterization of rhINF-a-2b loaded aquasomes Ultrafine ceramic core particles were synthesized by using precipitation phenomena [18]. Disodium hydrogen phosphate
(Na2HPO4) was used as a template for precipitating the ceramic core by using calcium chloride (CaCl2). The resultant calcium hydrogen phosphate (CaHPO4) particulate system was used as a core material for the preparation of aquasomes. Na2 HPO4 þ CaCl2 ! 2NaCl þ CaHPO4
(1)
Subsequently, ultrafine ceramic core particles were coated separately with trehalose (Tre), cellobiose (Cellob) and pyridoxal5-phosphate (Py-5-P) and incubated with rhINF-a-2b to generate rhINF-a-2b-Tre-Aq.somes, rhINF-a-2b-Cellob-Aq.somes and rhINF-a-2b-Py-5-P-Aq.somes. The rhINF-a-2b was also loaded onto the native core (rhINF-a-2b-Core) for comparative studies. 3.2. Optimization of experimental conditions that influenced the particle size Response Surface Methodology (RSM) [21] using the Central Composite Design (CCD) [20] model is a well-suited experimental design strategy that offers the possibility of investigating a large number of variables at different levels with only a limited number of experiments. The ranges for each variable were selected on the basis of our preliminary experiments. Experimental results concerning the tested variables on mean particle size of aquasomes are presented in Table 1. The response was individually fitted to a reduced linear model. For the response, the model, which generated a higher F value, was identified as the best-fitted model. Each obtained model was validated by ANOVA. Three-dimensional response surface plots were drawn for the optimization of ultrafine ceramic nanoparticles (Fig. 1A–D). These types of plots are useful for studying the effect of two factors on the response while the third factor was kept constant. Following reduced linear model equation (1) was derived by the best-fit method to describe the relationship between particle size (Y1) and ratio of core: coat (CaHPO4: polysaccharide), sonication power (W) and sonication time (min). Particle sizeðY 1 Þ ¼ 118:34455 1:37676 Sonication power
(1)
A positive value in regression equation for a response represents an effect that favours the optimization (synergistic effect); while a negative value indicates an inverse relationship (antagonistic effect) between the factors and the response [21]. The reduced linear model was found to be significant with F value of 4.65 (P < 0.0001), which indicates that response variable Y1 and the set of formulation variables were significantly related. The high R2 value indicated that 20.52% of variation in particle size could be explained by the regression on formulation factors. 3.3. Optimization by desirability function Optimization process was undertaken with desirability function to optimize the response. A high value of desirability coefficient d (0 d 1) indicates that the operating point can produce acceptable formulation results. The response particle size (Y1) was transformed into the desirability scale d1. The higher coefficient of determination and F value in terms of the reduced linear indicated the goodness of fit. The response surface plot for increasing desirability coefficient d with respect to changes in variables: core:coat (X1) and sonication power (X2) keeping the sonication time constant, has been shown in (Fig. 1A–D). The maximum value of desirability coefficient d = 0.701 was obtained at the conditions, core:coat ratio of 1:3, sonication power of 12.5 W and sonication time of about 55 min (Table 1). In order to evaluate the predictive power of this model and desirability coefficient, aquasomes were prepared under the optimized conditions (Table 2). Experimental values of aquasomes prepared using
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Fig. 1. Optimization of aquasomes by using Central Composite Design (CCD) and Response Surface Methodology (RSM), (A) influence of sonication power (W) and sonication time (min) on particle size (nm); (B) influence of sonication time (min) and core: coat (mg) ratio on particle size (nm); (C) influence of sonication power (W) and core:coat (mg) ratio on particle size (nm) and, (D) influence of sonication power (W) and core:coat (mg) ratio on desirability. The maximum value of desirability coefficient d = 0.701 was achieved at core:coat (mg) ratio of 1:3, sonication power (W) of 12.5 and sonication time of about 55 min.
optimal conditions were very close to the predicted values, with low percentage bias.
somes-P-PEG2000 as compared to –23.2 1.26 mV of rhINF-a-2bPy-5-P-Aq.somes confirmed the coating of PEGylated phospholipid (Table 2).
3.4. Particle size and zeta potential 3.5. Surface topography Particle size and zeta potential were determined to predict the therapeutic potential of protein loaded aquasomes. The mean particle size of rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000 was measured to be 125.3 3.2 nm, which was significantly (one way ANOVA test, P < 0.05) higher than 104.4 5.9 nm, 101.9 6.4 nm, 98.5 4.3 nm, and 90.1 2.3 nm, measured respectively for rhINFa-2b-Py-5-P-Aq.somes, rhINF-a-2b-Cellob-Aq.somes, rhINF-a-2bTre-Aq.somes and rhINF-a-2b-Core (CaHPO4) (Table 2 and Fig. 2A– E). In addition, zeta potential of rhINF-a-2b-Py-5-P-Aq.somes was found to be –23.2 1.26 mV which was significantly (one way ANOVA test, P < 0.05) higher than –8.48 2.32 mV, –15.6 1.15 mV and –20.4 0.9 mV measured respectively for rhINF-a-2b-Core (CaHPO4), rhINF-a-2b-Tre-Aq.somes and rhINF-a-2b-CellobAq.somes (Table 2). A significant (Unpaired t-test, P < 0.05) increase in zeta potential to –44.1 3.19 mV in rhINF-a-2b-Py-5-P-Aq.
The surface topography of aquasomes influences the stability and adsorption of protein. We observed an irregular crystalline shape of rhINF-a-2b-Core (CaHPO4), while somewhat spherical shape was observed from rhINF-a-2b-Tre-Aq.somes to rhINF-a2b-P-y-5-P-Aq.somes-P-PEG2000 (Fig. 2A–E and Table 2). 3.6. Fourier-Transforms–infrared (FT–IR) spectroscopy FT–IR spectrum was recorded to analyze the chemistry of adsorption of protein over the surface of aquasomes. In brief, Tre exhibited characteristics peaks at 1514 cm1 and 991.96 cm1 due to the presence of –OH groups. Continuation to this, Cellob also displayed the peaks at 1517.08 cm1 and 1036 cm1, indicating the presence of –OH groups. PO42 group may be marked at
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Fig. 2. Particle size distribution and transmission electron microscopy (TEM) of (A) rhINF-a-2b loaded core (CaHPO4) particles; (B) rhINF-a-2b loaded trehalose aquasomes (rhINF-a-2b-Tre-Aq.somes); (C) rhINF-a-2b loaded cellobiose aquasomes (rhINF-a-2b-Cellob-Aq.somes); (D) rhINF-a-2b loaded pyridoxal-5-phosphate aquasomes superimposed with PEGylated phospholipid (rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000). rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000 nanoformulation was appeared to be spherical in shape while rhINF-a-2b loaded core (CaHPO4) particles were irregular in appearance. Scale bar 100 nm in TEM.
1020.84 cm1 in addition to 1147.72 cm1, 942.14 cm1 and 851.48 cm1 for –OH groups in Py-5-P oligomer. The native core, CaHPO4 also indicated the presence of PO42 at 1048.05 cm1, however, distinct from Py-5-P. Upon coating of polysaccharides over the ceramic core particles, these peaks were shifted to either higher or lower wavelengths indicating the formation of intermolecular hydrogen bonds between ceramic core particles and polyoligomer coating (Table 3).
6 and 8 h (Fig. 4A and B). Thus, rhINF-a-2b-Py-5-P-Aq.somes offered slow release and designated as optimized nanoformulation. Next, to induce the sustained release pattern, rhINF-a-2bPy-5-P-Aq.somes was coated with PEGylated phospholipid (rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000). The rhINF-a-2b-Py-5P-Aq.somes-P-PEG2000 displayed evidence of its prolonged release pattern and released 98.1% of rhINF-a-2b in 336 h (Fig. 4B). 3.10. Gel electrophoresis
3.7. Powder X-ray diffraction (PXRD) pattern The crystallline lattice arrangement of aquasomes was elucidated by using PXRD technique. The PXRD pattern of core (CaHPO4) showed peaks that were intense and sharp indicating its crystalline state (Fig. 3). Upon the coating of core particles with Tre (TreAq.somes), Cellob (Cellob-Aq.somes) and Py-5-P (Py-5-PAq.somes) reduced the intensity of sharp peaks and deformed into amorphous structure. Maximal deformation was scanned in Py-5-P-Aq.somes. 3.8. Protein loading capacity Protein loading capacity depends on the experimental conditions acquired for aquasome preparation. Protein loading capacity was measured to be 48.3 2.3 mg/10 mg for rhINF-a-2b-Py-5-P-Aq.somes which was significantly (one way ANOVA test, P < 0.05) higher than 5.6 1.2 mg/10 mg, 20.4 3.1 mg/10 mg and 32.45 4.5 mg/10 mg, of rhINF-a-2b-Core (CaHPO4), rhINF-a-2b-Tre-Aq.somes and rhINF-a2b-Cellob-Aq.somes. In contrast, protein loading capacity was significantly (Unpaired t-test, P < 0.05) reduced to 41.2 3.4 mg/ 10 mg in rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000 as compared to 48.3 2.3 mg/10 mg of rhINF-a-2b-Py-5-P-Aq.somes due to loss of protein during coating of PEGylated phospholipid (Table 2). 3.9. In vitro protein release The potential of protein loaded aquasomes to prolong the release of rhINF-a-2b was examined in PBS, pH 7.4. The rhINF-a2b-core (CaHPO4) released 97.3% of protein in 1 h, while 95.3% of rhINF-a-2b was released by rhINF-a-2b-Tre-Aq.somes in 4 h. Concurrently, rhINF-a-2b-Cellob-Aq.somes and rhINF-a-2b-Py-5P-Aq.somes released 96.2% and 97.8% of rhINF-a-2b respectively in
The SDS-PAGE was used to authenticate the stability and integrity of rhINF-a-2b in optimized nanoformulation, rhINF-a2b-Py-5-P-Aq.somes. SDS-PAGE image of rhINF-a-2b isolated from optimized rhINF-a-2b-Py-5-P-Aq.somes and pure rhINF-a-2b under reduced conditions is shown in Fig. 5A. The rhINF-a-2b secondary structure contains two intramolecular disulphide bonds, Cys1-Cys98 and Cys29-Cys138 [32]. The molecular weight of rhINF-a-2b is reported to be 17 kDa [33]. Reducing agent, b-mercaptoethanol was employed to break any disulphide bonds, thus eliminated the presence of high molecular weight bands. The structural stability and integrity of the isolated rhINFa-2b was consistent with the pure rhINF-a-2b even after adsorption over the Py-5-P-Aq.somes. The molecular weight of rhINF-a-2b was observed to be 17 kDa as compared to the standard marker. 3.11. Circular dichroism (CD) Far-UV CD is a sophisticated approach for investigation of secondary structure of rhINF-a-2b that contains 5 a-helices. We observed that pure rhINF-a-2b and rhINF-a-2b isolated from aquasomes showed identical CD spectrum (Fig. 5B). Furthermore, analysis of secondary structure using CCA program suggested that 55% of rhINF-a-2b was comprised of a-helix, 22% of unorganized structure and 23% of others. However, we did not observe b structures. 3.12. In vitro cell proliferation assay In vitro therapeutic efficacy of rhINF-a-2b, rhINF-a-2b-Core (CaHPO4), rhINF-a-2b-Tre-Aq.somes, rhINF-a-2b-Cellob-Aq.somes,
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Table 3 Fourier-Transforms–infrared (FT–IR) spectroscopy assignment of aquasomes, scanned between 400 to 4400 cm1. Sample CaHPO4 Tre Cellob Py-5-P
Peaks (cm1)
Assignment
1048.05 650.29 1514.03 991.96 1517.08 1036.84 1020.84 1147.72 942.14 851.48
PO42 OH OH OH OH OH PO42 OH OH OH
Sample
Peaks (cm1)
Assignment
Tre-Aq.somes
1517.54 981.03 1515.92 1022.39 1025.12 1254.85 967.90 821.51
OH OH OH OH PO42 OH OH OH
Cellob-Aq.somes Py-5-P-Aq.somes
Tre: trehalose; Cellob: cellobiose; Py-5-P: pyridoxal-5-phosphate.
rhINF-a-2b-Py-5-P-Aq.somes and rhINF-a-2b-Py-5-P-Aq.somes-PPEG2000 was examined by standard cell proliferation assay by employing SKOV3, ovarian cancer cell line. The IC50 value (concentration of drug require to kill 50% of the cells) was used as an indicator to define the therapeutic efficacy of protein loaded aquasomes. The IC50 value for free rhINF-a-2b was measured to be 710 IU/mL at 72 h. Compared to this, the IC50 value was significantly (one way ANOVA test, P < 0.05) reduced to 156 IU/mL in rhINF-a2b-Py-5-P-Aq.somes-P-PEG2000, 300 IU/mL in rhINF-a-Py-5-PAq.somes, 360 IU/mL in rhINF-a-2b-Cellob-Aq.somes, 440 IU/mL in rhINF-a-2b-Tre-Aq.somes and 600 IU/mL in rhINF-a-2b-Core (CaHPO4) at the same incubation period (Table 4). However, core (CaHPO4) did not exert any cytotoxicity to ovarian cancer cells.
4. Discussion
effective and highly stable raw material for scaling the drug delivery cargos. Moreover, it is widely accepted for delivery of both low and high molecular weight therapeutic molecules [34]. The nanocrystalline, CaHPO4 ceramic core particles self-assemble during the reaction process under sonication due to augmentation of surface free energy [18]. Ultimately, ceramic core particles were coated with a thin film of polyhydroxy oligomer for preparing the aquasomes under optimized conditions, as calculated by CCD and RSM [20,21] (Tables 1 and 2). Polyhydroxy oligomer film stabilizes the ceramic core through ionic, non-covalent, and extropic forces. We observed that particle size of aquasomes increases as a function of concentration of core to coat ratio, whilst the other variables, such as sonication power and time were kept constant (Fig. 1A–D and Table 1). This may be attributed to availability of free surface of the core particles with the coating material [18]. Moreover, an enhancement in sonication power decreased the particle size of aquasomes, whereas other variables were kept
Proteins and peptides administered through parentral route of administration often require high stability, slow release and targeting at molecular receptors. Moreover, susceptibility of proteins to denaturation by proteolytic enzymes and other physicochemical limitations need special attention while encapsulating in micro- or nano-drug delivery systems. In present investigation, rhINF-a-2b loaded aquasomes were prepared to induce protein stability, sustained release and cytotoxicity in ovarian cancer cells. Calcium hydrogen phosphate (CaHPO4) was precipitated as fine powder to be used as a template for the preparation of aquasomes [18]. CaHPO4 is a biodegradable, cost
Fig. 3. Powder X-ray diffraction (PXRD) pattern of core (CaHPO4) particles, trehalose aquasomes (Tre-Aq.somes), cellobiose aquasomes (Cellob-Aq.somes), and pyridoxal5-phosphate aquasomes (Py-5-P-Aq.somes). Crystal lattice arrangement of core particles was deformed to amorphous structure upon coating with polysaccharides. Maximal amorphization was done by Py-5-P oligomer.
Fig. 4. A. In vitro release of rhINF-a-2b from aquasomes in PBS, pH 7.4. rhINF-a2b-core (CaHP4) released 97.3% of protein in 1 h, while 95.3% of rhINF-a-2b was released by rhINF-a-2b-Tre-Aq.somes in 4 h. Concomitantly, rhINF-a-2b-CellobAq.somes and rhINF-a-2b-Py-5-P-Aq.somes released 96.2% and 97.8% of rhINF-a2b, respectively in 6 and 8 h. Thus, rhINF-a-2b-Py-5-P-Aq.somes offered slow release and designated as optimized nanoformulation. B. rhINF-a-2b-Py-5-PAq.somes-P-PEG2000 offered 98.1% prolonged release in 336 h, carried out in PBS (pH 7.4). Each experiment was performed in triplicate (n = 3).
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Fig. 5. A. SDS-PAGE gel electrophoresis of naive rhINF-a-2b and rhINF-a-2b isolated from optimized rhINF-a-2b-Py-5-P-Aq.somes. The molecular weight of rhINF-a-2b was observed to be 17 kDa as compared to the standard marker, and (B) circular dichroism of naive rhINF-a-2b and rhINF-a-2b isolated from aquasomes that exhibited identical CD spectrum. The 55% of rhINF-a-2b was comprised of a-helix, 22% of unorganized structure and 23% of others. However, no b structure was observed. Three scans were taken for each sample (n = 3).
stable. We assumed that an enhancement in sonication power induced the segregation of self-assembled architecture of aquasomes and thus decreased the particle size (Fig. 1A–D and Table 1). Next, decrease in sonication time increased the particle size of aquasomes, though other variables, such as core to coat ratio and sonication power were kept steady. This could be attributed to aggregation of smaller particles [14] (Fig. 1A–D and Table 1). Experimentally, particle size analysis, zeta potential and TEM assured that all samples of aquasomes were near about 100 nm in diameter and almost spherical in shape (Table 2 and Fig. 2A–E). The Py-5-P-Aq.somes exhibited higher negative zeta potential due to the presence of most electronegative atoms in the chemical structure of Py-5-P oligomer, as compared to Tre- and Cellob polysaccharides (Fig. 2A–D and Table 2). Hence, rhINF-a-2b-Py-5P-Aq.somes was overlaid with PEGylated phospholipid. A significant (Unpaired t-test, P < 0.05) enhancement in negative zeta potential indicating the layering of PEGylated phospholipid over the surface of rhINF-a-2b-Py-5-P-Aq.somes (Table 2). This may be attributed to anionic nature of PEG chain that contributed in the enhancement in net negative charge of aquasomes. Poly vinyl alcohol (PVA), being a coating modifier helped in layering of PEGYlated phospholipid over the surface of optimized aquasomal nanoformulation. Next, FT–IR confirmed the formation of intermolecular hydrogen bonds between the ceramic core, CaHPO4 and polysaccharides (Tre, Cellob and Py-5-P) due to broadening and shifting of peaks of –OH groups either at higher or lower
wavelengths [35] (Table 3). PXRD was performed to elucidate the structural geometry of aquasomes (Fig. 3). XRD patterns substantiated the deformation of crystalline geometry of CaHPO4 to amorphous configuration after coating with polyhydroxy oligomer. Molecules, which form hydrogen bonds, are hydrophilic in nature and consequently confer a significant degree of organization of water molecules [14]. Hence, Py-5-P owing to the presence of electronegative atoms and charged group, PO42 facilitated the intermolecular hydrogen bond formation with CaHPO4 and believed to favour the surrounding of excess water molecules as compared to Tre and Cellob. Thus, Py-5-P-Aq.somes exhibited higher amorphized geometry as compared to Tre and Cellob. Typically, due to irregular structural geometry, the amorphous phase requires minimal energy and thus tenders the supreme solubility and bioavailability of drugs [36]. The higher protein loading capacity was displayed by rhINF-a-2b-Py-5-P-Aq.somes (Table 2). We propose that higher negative zeta potential and presence of excess electronegative atoms in the molecular structure of Py-5-P-Aq.somes would have allowed the formation of intermolecular hydrogen bonds with active site (tyrosine residue) of rhINF-a-2b. Consequently, these intermolecular hydrogen bonds have promoted the higher adsorption of protein over the surface of Py-5-P-Aq.somes. Next, in vitro release study was performed to test the release pattern of rhINF-a-2b from aquasomes in physiological milieu. The rhINF-a-2b-Py-5-PAq.somes exhibited the slow release of rhINF-a-2b as compared
Table 4 Percent cell viability and IC50 value of aquasomes, calculated by standard cell viability assay against SKOV3, ovarian cancer cell line at 72 h. IC50a (IU/ml)
Samplesa
% Cell viability (IU/ml) 0
200
400
600
800
1000
1200
rhINF-a-2b rhINF-a-2b-Core (CaHPO4) rhINF-a-2b-Tre-Aq.somes rhINF-a-2b-Cellob-Aq.somes rhINF-a-2b-Py-5-P-Aq.somes rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000 Core (CaHPO4)
99.7 0.1 98.2 1.2 97.4 1.4 98.3 1.9 99.1 0.3 98.6 1.4 99.6 0.1
80.5 3.6 78.4 4.3 72.5 5.6 69.1 4.7 56.4 3.6 22.4 3.1 98.1 0.5
65.8 6.2 62.4 3.1 53.2 4.1 45.5 3.9 42.4 3.8 15.2 2.8 98.6 0.2
55.8 4.9 50.3 4.7 40.3 6.6 36.4 4.1 32.4 3.2 12.6 4.0 99.1 0.3
44.9 5.2 38.6 2.9 34.5 3.7 28.8 2.0 22.6 3.4 8.5 1.4 97.9 0.4
40.8 2.8 30.9 6.2 24.3 3.3 26.7 2.9 15.3 3.1 5.8 2.3 97.2 0.8
38.4 6.3 26.4 2.1 18.5 2.9 15.4 3.7 12.4 2.9 2.4 0.1 98.3 0.2
710 600 440 360 300 156 –
rhINF-a-2b: recombinant interferon-a-2b; rhINF-a-2b-Core: rhINF-a-2b coated core (CaHPO4) particles; rhINF-a-2b-Tre-Aq.somes: rhINF-a-2b coated trehalose aquasomes; rhINF-a-2b-Cellob-Aq.somes: rhINF-a-2b coated cellobiose aquasomes; rhINF-a-2b-Py-5-P-Aq.somes: rhINF-a-2b coated pyridoxal-5-phosphate aquasomes; rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000: rhINF-a-2b coated pyridoxal-5-phosphate aquasomes superimposed with PEGylated phospholipid. IC50: concentration of the drug require to kill 50% of the cells. a All groups are statistically significant (P < 0.05, one way ANOVA test).
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to rhINF-a-2b-Cellob-Aq.somes, rhINF-a-2b-Tre-Aq.somes and rhINF-a-2b-Core (CaHPO4) (Fig. 4A). This may be donated by relatively higher intermolecular hydrogen bond formation in rhINF-a-2b-Py-5-P-Aq.somes that sluggish the release pattern of protein in physiological buffer. As expected, rhINF-a-2b-Py-5-PAq.somes-P-PEG2000 exhibited the prolonged release of rhINF-a2b up to 336 h due to the presence of layering of PEGylated phospholipid (Fig. 4B). PEGylated phospholipid behaves like a surfactant in aqueous phase, hence, aggregated structure of PEGylated phospholipid formed boundary over the surface of aquasomes that induced the prolonged release of rhINF-a-2b. Moreover, PEGylation also enhances the blood circulation time of drug delivery system by preventing RES uptake and makes it long circulating [26]. The stability and integrity of adsorbed protein was investigated by using gel electrophoresis and CD (Fig. 5A–B). The stability assessment study showed that the structural integrity of the protein did not appear to be compromised by the preparation technique. Polyhydroxy oligomer film prevents peptide secondary structure from changing shape and being damaged by generating a glossy film that acts as dehydroprotectant. Hydrogen bond helps in base pair matching and stabilization of secondary structure of protein, such as a-helices and b-sheets [14]. Thus, aquasomes provided conformational stabilization as well as a high degree of surface exposure to protein. In last, in vitro cytotoxicity assay was performed on SKOV3, ovarian cancer cells to test the therapeutic efficacy of protein loaded aquasomes (Table 4). The rhINF-a-2b-Py-5-P-Aq.somes-P-PEG2000 exhibited the 4.55, 1.92, 2.3, 2.8, and 3.84 fold reductions in IC50 as compared to respectively free rhINF-a-2b, rhINF-a-2bPy-5-P-Aq.somes, rhINF-a-2b-Cellob-Aq.somes, rhINF-a-2b-TreAq.somes and rhINF-a-2b-Core (CaHPO4). Cancer cells generally bear net negative charge at physiological milieu [37]. Hence, rhINFa-2b-Py-5-P-Aq.somes-P-PEG2000 offered maximal steric repulsion owing to higher negative zeta potential (Table 2), preventing the cellular uptake by SKOV3 cells and thereby prolonged the cytotoxic effect and decreased the IC50 value. Therefore, rhINF-a2b-Py-5-P-Aq.somes-P-PEG2000 enhanced the stability of protein in its amorphous geometry and tendered sustained cytotoxic effect against ovarian cancer cells. 5. Conclusion In conclusion, narrow particle size distribution of aquasomes may be achieved at 1:3 core to coat ratio, sonication power and time of about 12.5 W and 55 min. The rhINF-a-2b-Py-5-PAq.somes-P-PEG2000 offered prolonged release and enhanced cytotoxicity of rhINF-a-2b against SKOV3, ovarian cancer cells. Moreover, secondary structure of protein was also preserved by Py-5-P-Aq.somes. Therefore, rhINF-a-2b-Py-5-P-Aq.somes-PPEG2000 warrant further in depth in vitro and in vivo preclinical investigations to scale up the technology for clinical translation. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. References [1] Ingersoll SB, Ahmad S, Finkler NJ, Edwards JR, Holloway RW. Cellular therapy for ovarian cancer: experimental and clinical perspectives. Curr Med Chem 2012;19:3787–93. [2] Willemse PH, deVries EG, Mulder NH, Aalders JG, Bouma J, Sleijfer DT. Intraperitoneal human recombinant interferon a-2b in minimal residual ovarian cancer. Eur J Cancer 1990;26:353–8.
275
[3] Sanchez A, Tobio M, Gonzalez L, Fabra A, Alonso MJ. Biodegradable micro- and nanoparticles as long-term delivery vehicle for interferon-a. Eur J Pharm Sci 2003;18:221–9. [4] Frank MH, Pomer S. Interferon alpha-2b differentially affects proliferation of two human renal cell carcinoma cell lines differing in the P-glycoproteinassociated multidrug-resistant phenotype. J Cancer Res Clin Oncol 1999;125: 117–20. [5] Jiang Y, Shi K, Xia D, Piao H, Quan P, Song T, et al. Protamine modified metal ion-protein chelate microparticles for sustained release of interferon. Int J Pharm 2011;407:31–7. [6] Santana H, Martı´nez E, Sanchez JC, Moya G, Sosa R, Hardy E, et al. Molecular characterization of recombinant human interferon a-2b produced in Cuba. Biotecnol Aplicada 1999;16:154–9. [7] Li Z, Li L, Liu Y, Zhang H, Li X, Luo F, et al. Development of interferon a-2b microspheres with constant release. Int J Pharm 2011;410:48–53. [8] Fishburn CS. The pharmacology of PEGylation: balancing PD with PK to generate novel therapeutics. J Pharm Sci 2008;97:4167–83. [9] Langer R, Cleland JL, Hanes J. New advances in microsphere-based single-dose vaccines. Adv Drug Deliv Rev 1997;28:97–119. [10] Hong H, Jo JR, Yeon JH, Hong JT, Jung KH, Yoo SK, et al. Preparation of branched dextran microspheres of soluble interferon-alpha and its activity in vitro and in vivo. J Microbiol Biotechnol 2011;21:176–82. [11] Alejandro S, Maria T, Libia G, Angels F, Maria JA. Biodegradable micro- and nanoparticles as long term delivery vehicles for interferon-alpha. Eur J Pharm Sci 2003;18:221–9. [12] Gulia M, Rai S, Jain UK, Katare OP, Katyal A, Madan J. Sustained release protamine sulphate impregnated microspheres may reduce the frequent administration of recombinant interferon-a-2b in ovarian cancer: in vitro characterization. Anticancer Drugs 2014;25:63–71. [13] Nitta SK, Numata K. Biopolymer-based nanoparticles for drug/gene delivery and tissue engineering. Int J Mol Sci 2013;14:1629–54. [14] Kossovsky N, Gelman A, Sponsler EE, Hnatyszyn HJ, Rajguru S, Torres M, et al. Surface-modified nanocrystalline ceramics for drug delivery applications. Biomaterials 1994;15:1201–7. [15] Pandey RS, Sahu S, Sudheesh MS, Madan J, Kumar M, Dixit VK. Carbohydrate modified ultrafine ceramic nanoparticles for allergen immunotherapy. Int Immunopharmacol 2011;11:925–31. [16] Kanwar JR, Mahidhara G, Kanwar RK. Novel alginate-enclosed chitosancalcium phosphate-loaded iron-saturated bovine lactoferrin nanocarriers for oral delivery in colon cancer therapy. Nanomedicine 2012;7: 1521–50. [17] Singh D, Singh S, Sahu J, Srivastava S, Singh MR. Ceramic nanoparticles: recompense, cellular uptake and toxicity concerns. Artif Cells Nanomed Biotechnol 2014;17:1–9. [18] Cherian AK, Rana AC, Jain SK. Self-assembled carbohydrate-stabilized ceramic nanoparticles for the parenteral delivery of insulin. Drug Develop Indust Pharm 2000;26:459–69. [19] Rojas-Oviedo I, Salazar-Lopez RA, Reyes-Gasga J, Quirino-Barreda CT. Elaboration and structural analysis of aquasomes loaded with indomethacin. Eur J Pharm Sci 2007;32:223–30. [20] Bei YY, Zhou XF, You BG, Yuan ZQ, Chen WL, Xia P, et al. Application of the central composite design to optimize the preparation of novel micelles of harmine. Int J Nanomed 2013;8:1795–800. [21] Yiran M, Xinyi Z, Jian L, Qi S. The comparison of different daidzein-PLGA nanoparticles in increasing its oral bioavailability. Int J Nanomed 2012;7: 559–70. [22] Lim SB, Rubinstein I, Sadikot RT, Artwohl JE, Onyuksel H. A novel peptide nanomedicine against acute lung injury: GLP-1 in phospholipid micelles. Pharm Res 2011;28:662–72. [23] Gill KK, Nazzal S, Kaddoumi A. Paclitaxel loaded PEG(5000)-dspe micelles as pulmonary delivery platform: formulation characterization, tissue distribution, plasma pharmacokinetics, and toxicological evaluation. Eur J Pharm Biopharm 2011;79:276–84. [24] Ma L, Lai D, Liu T, Cheng W, Guo L. Cancer stem-like cells can be isolated with drug selection in human ovarian cancer cell line SKOV3. Acta Biochim Biophys Sin 2010;42:593–602. [25] Loveymi BD, Jelvehgari M, Zakeri-Milani P, Valizadeh H. Statistical optimization of oral vancomycin-eudragit RS nanoparticles using response surface methodology. Iranian J Pharm Res 2012;11:1001–12. [26] Vij N, Min T, Marasigan R, Belcher CN, Mazur S, Ding H, et al. Development of PEGylated PLGA nanoparticle for controlled and sustained drug delivery in cystic fibrosis. J Nanobiotechnol 2010;8:22–40. [27] Goyal AK, Rawat A, Mahor S, Gupta PN, Khatri K, Vyas SP. Nanodecoy system: a novel approach to design hepatitis B vaccine for immunopotentiation. Int J Pharm 2006;309:227–33. [28] Kang J, Lambert O, Ausborn M, Schwendeman SP. Stability of proteins encapsulated in injectable and biodegradable poly (lactide-co-glycolide)-glucose millicylinders. Int J Pharm 2008;357:235–43. [29] Anand O, Yu LX, Davit BM. Dissolution testing for generic drugs: an FDA perspective. AAPS J 2011;3:328–35. [30] Berg K, Heron I. SDS-polyacrylamide gel electrophoresis of purified human leucocyte interferon and the antiviral and anticellular activities of the different interferon species. J Gen Virol 1980;50:441–6. [31] Mosmann T. Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65: 55–63.
276
K. Kaur et al. / Biomedicine & Pharmacotherapy 69 (2015) 267–276
[32] Bodo G, Maurer-Fogy I. In: Dian-zani F, Rossi GB, editors. The interferon system, vol. 24. New York: Serono Symposium Publication, Raven Press; 1986. p. 23–7. [33] Bordens R, Grossberg SE, Trotta PP, Nagabhushan TL. Molecular and biological characterization of recombinant interferon-a-2b. Seminal Oncol 1997;24. S941-S9-51. [34] Jain SS, Jagtap PS, Dand NM, Jadhav KR, Kadam VJ. Aquasomes: a novel drug carrier. J Appl Pharm Sci 2012;2:184–92.
[35] Rogez G, Ribera BF, Credi A, Ballardini R, Gandolfi MT, Balzani V, et al. A molecular plug socket connector. J Am Chem Soc 2007;129:4633–42. [36] Kaur M, Bhatia RK, Pissurlenkar RR, Coutinho EC, Jain UK, Katare OP, et al. Telmisartan complex augments solubility, dissolution and drug delivery in prostate cancer cells. Carbohydrate Polym 2014;101:614–22. [37] Abercrombie M, Ambrose EJ. The surface properties of cancer cells. A review. Cancer Res 1962;22:525–48.