Materials Science and Engineering C 63 (2016) 489–498
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Polyelectrolyte complex of vancomycin as a nanoantibiotic: Preparation, in vitro and in silico studies Dhiraj R. Sikwal, Rahul S. Kalhapure, Sanjeev Rambharose, Suresh Vepuri, Mahmoud Soliman, Chunderika Mocktar, Thirumala Govender ⁎ Discipline of Pharmaceutical Sciences, College of Health Sciences, University of KwaZulu-Natal, Private Bag X5400, Durban, South Africa
a r t i c l e
i n f o
Article history: Received 24 September 2015 Received in revised form 17 February 2016 Accepted 6 March 2016 Available online 9 March 2016 Keywords: Vancomycin Nanoplex Nanoantibiotics S. aureus Methicillin-resistant S. aureus Polyacrylic acid
a b s t r a c t Delivery of antibiotics by various nanosized carriers is proving to be a promising strategy to combat limitations associated with conventional dosage forms and the ever-increasing drug resistance problem. This method entails improving the pharmacokinetic parameters for accumulation at the target infection site and reducing their adverse effects. It has been proposed that antibiotic nanoparticles themselves are more effective delivery system than encapsulating the antibiotic in a nanosystem. In this study, we report on nanoparticles of vancomycin (VCM) by self-assembled amphiphilic–polyelectrolyte complexation between VCM hydrochloride and polyacrylic acid sodium (PAA). The size, polydispersity index and zeta potential of the developed nanoplexes were 229.7 ± 47.76 nm, 0.442 ± 0.075, −30.4 ± 5.3 mV respectively, whereas complexation efﬁciency, drug loading and percentage yield were 75.22 ± 1.02%, 58.40 ± 1.03% and 60.60 ± 2.62% respectively. An in vitro cytotoxicity study on three mammalian cell lines using MTT assays conﬁrmed the biosafety of the newly formulated nanoplexes. Morphological investigations using scanning electron microscope showed cube shaped hexagonallike particles. In vitro drug release studies revealed that the drug was completely released from the nanoplexes within 12 h. In silico studies revealed that the nano-aggregation was facilitated by means of self-association of VCM in the presence of the polymer. The supramolecular pattern of the drug self-association was found to be similar to that of the VCM dimer observed in the crystal structure of the VCM available in Protein Data Bank. In vitro antibacterial activity against susceptible and resistant Staphylococcus aureus proved that the potency of VCM was retained after being formulated as the nanoplex. In conclusion, VCM nanoplexes could be a promising nanodrug delivery system to treat infections of S. aureus origin. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The widespread use and abuse of antibiotics and their inadequate delivery to infection target sites due to pharmacokinetic constrains of available dosage forms, has contributed to the current serious issue of antimicrobial resistance (AMR) . Among various resistant infections, hospital acquired (HA) infections, which are responsible for approximate 6% of mortality (100,000 deaths per year), are considered a major concern , and are the sixth leading cause of death in developed countries, such as the United States . Until recently, Staphylococcus aureus was considered an important but infrequent cause of nosocomial pneumonia, especially in the elderly patients. However, in the previous two decades, there has been a dramatic increase in infections caused by
⁎ Corresponding author at: Private Bag X54001, Durban 4000, KwaZulu-Natal, South Africa. E-mail address: [email protected]
http://dx.doi.org/10.1016/j.msec.2016.03.019 0928-4931/© 2016 Elsevier B.V. All rights reserved.
methicillin-resistance S. aureus (MRSA), accounting for 20 to 40% of all HA pneumonia and ventilator associated pneumonia . Vancomycin (VCM), a glycopeptide antibiotic, is a drug of choice for treating pneumonia attributed to MRSA , and is generally administered by the intravenous route. However, studies have shown that systemic administration can cause toxicity and side effects, such as renaland nephro-toxicity . Preclinical [7,8] and clinical studies  have demonstrated the advantages of delivering VCM through the pulmonary route by achieving a high concentration in the lungs and bypassing high dose associated systemic side effects. AreoVanc, currently in phase II clinical trials, is the ﬁrst dry powder inhalation formulation of VCM to treat MRSA associated pneumonia in patients with cystic ﬁbrosis . Being a glycopeptide antibiotic, VCM is the ﬁrst line agent to treat MRSA caused by hospital and community acquired infections . While it has been extensively used since the late 1950s, and remains a gold standard for treating MRSA infections , there are concerns about the development of resistance to this drug by MRSA [13,14]. The current crisis of AMR could be overcome by developing nanoengineered drug delivery systems of current antibiotics [1,15]. Delivering
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antibiotics by various nanosized carriers is being proven as an efﬁcient strategy for improvements in the drug pharmacokinetic parameters, accumulation at target infection sites and reduction in their adverse effects as well as their ability to overcome microbial resistance mechanisms . It is proposed that nanoantibiotics enhance the clinical efﬁcacy of antibiotics in lung infections by accumulating in high concentration at the infection site and prolonging the residence time due to the efﬁcient bypass of the lungs' natural clearance mechanism compared to their free drug form . High localized antibiotic exposure to enhance clinical efﬁcacy and minimize AMR bacterial strains could be achieved successfully via nanodelivery systems . Nanoparticle sizes in the range of 100–400 nm can easily cross the thick mucosal layer of sputum in the lungs and thereby target antibiotics to bacterial colonies . Various nano-formulations of VCM, such as liposomes [19–21] and polymeric nanoparticles [22,23], have been formulated and proven to be superior in performance compared to the free drug. Most of these nanoformulations suffer from various drawbacks, such as complex and expensive preparation methods, toxicological issues (biocompatibility of polymers and surfactants, and traces of organic solvent), low drug loading, short self-life of lipidic vesicles and physical instability leading to drug leakage [24,25]. Simple and cost effective formulation strategies with high payload and low toxicity are therefore needed. Instead of developing a nano-carrier and incorporating antibiotics into it, nano-particulate systems of antibiotics themselves could be more advantageous . A recent type of nano-particulate system by ionic interaction between oppositely charged polymers and peptides result in polyion complex micelles  or nanoplex formation . These complexes are capable of forming nano-sized aggregates by various factors, such as columbic, hydrophobic interactions and the conformational arrangement of polymers . Equivalent amounts of polyion units and monomer form an electro-neutral complex that results into water insoluble nano-complexes, while excess concentrations of one of the components makes them water soluble [27,28]. Nanoplexes represent a promising, simple, green and cost effective nanoformulation strategy that consists principally of the drug and the polyelectrolyte acting as a stabilizer [26,29,30]. Initially, nanoplexes formations were reported between oppositely charged peptide and a polymer [27,28]. The concept was later broadened, and nanoplexes were successfully formulated for antibiotics such as oﬂoxacin and levoﬂoxacin, using dextran sulfate as the complexing agent [26,30], and for other drugs, such as curcumin [29,31] and ibuprofen , using chitosan and poly(alkylamine hydrochloride) respectively. Despite the advantages offered by nanoplexes, there are very few reports on their preparation, mechanistic study of formation and applications for developing various classes of drugs, including antibiotics [26,29–31]. There is therefore the scope to identify combinations of polymers and drugs, especially antibiotics that could form nanoplexes, to obtain insight into the mechanism of their formation via molecular modeling studies, and to further explore their antibacterial performance. Although VCM has been encapsulated into various nanosystems as discussed earlier, there is no report on formation of nanoplexes of VCM despite numerous advantages offered by these nanosystems. As VCM is a glycopeptide antibiotic, we envisaged that it could form a nanoplex with an anionic polymer and could be a promising delivery system for pulmonary infections. Herein we report on the formulation development of nanoplexes from VCM hydrochloride, a potent antibiotic drug containing cationic group, with PAA as an oppositely charged polymer. We report on the effect of the charge ratio of VCM and PAA on the formation of the nanoplexes, and on their antibacterial potential. Furthermore, in silico studies were performed to investigate the system stability and estimate complexation efﬁciency (%CE). The optimized nanoplex system was in the nanometric size range with higher %CE, drug loading, nanoparticle recovery and was stable after lyophilization. Additionally, this optimized nanoplex system displayed superior antimicrobial activity as well as non-toxicity against the various cell lines studied. All these results proved its suitability as the nanoformulation for pulmonary delivery.
2. Experimental 2.1. Materials Polyacrylic acid sodium salt (PAA, MW ~ 2100) was purchased from Fluka (Germany). VCM hydrochloride was purchased from Sinobright Import and Export Co. Ltd. (China). Dialysis tubing (MWCO 14,000 Da) was purchased from Sigma-Aldrich (USA), 3-(4,5-dimethylthiazole-2-yl)-2,5diphenyltetrazolium bromide (MTT) was obtained from Merck Chemicals (Germany). Nutrient Broth, Mueller-Hinton Broth (MHB) and MuellerHinton Agar (MHA) were obtained from Biolab (South Africa). The bacterial cultures used were S. aureus ATCC 25923 and MRSA (S. aureus Rosenbach ATCC BAA 1683). Puriﬁed water used throughout the studies was produced in the laboratory with a Milli-Q water puriﬁcation system (Millipore corp., USA). All other chemicals and solvents used were of analytical grade and used without further puriﬁcation. 2.2. Methods 2.2.1. Preparation of VCM–PAA nanoplex Nanoplexes were prepared as per a modiﬁed literature reported method for the self-assembly of amphiphile and polyelectrolyte complexation [26,27]. In short, PAA solution (1% w/v) and VCM solution (1% w/v) were prepared separately in milli-Q water. At room temperature varying quantities of prepared PAA solution (1, 2, 4, 8 and 16 ml) were added drop wise to a VCM hydrochloride solution (20 ml) under magnetic stirring to obtain ﬁve different solutions forming VCM–PAA complexes. The formed complex solutions were further stirred for 3 h. 2.2.2. Characterization of VCM–PAA nanoplexes 220.127.116.11. Determination of size, polydispersity index (PDI) and zeta potential (ZP). The particle size, PDI and ZP of nanoplexes before and after lyophilization were determined by using a zeta sizer (Nano ZS, Malvern Instruments Corp, UK) at 25 °C in polystyrene cuvettes with a path length of 10 mm. All measurements were performed in triplicate by diluting 100 μl of the nanoplex suspension to 10 ml milli-Q water. 18.104.22.168. Fourier transform-infrared (FT-IR) analysis. The lyophilized VCM–PAA nanoplex, VCM and PAA were characterized by Fourier transform-infrared spectroscopy using a Bruker Alfa spectrophotometer (Germany) to conﬁrm complexation and formation of nanoplexes. 22.214.171.124. Determination of complexation efﬁciency (%CE). The %CE, which is the percentage amount of drug complexed in the nanoplex per amount of drug initially added, was determined by following a reported procedure . Nanoplex formulations were centrifuged (Beckman Coulter Optima™ MAX XP Centrifuge, USA) at 21,700 ×g for 60 min at 20 °C. The supernatant was collected and the non-entrapped drug measured by UV spectrophotometry at 280 nm (Schimadzu UV 1601, Japan). The %CE was calculated using the following equation. %CE ¼ ðTotal amount of VCM−amount of VCM in supernatantÞ=Total amount of VCM 100
126.96.36.199. Determination of percentage yield. The percentage yield refers to the mass of nanoplex recovered after freeze lyophilization. The optimized nanoplex (VNPX2) suspension was centrifuged to remove uncomplexed VCM and PAA followed by three washings with milli-Q water (10 ml), freeze dried, weighed and the percentage yield was determined by the following formula [31,32]. Percentage yield ð%Þ ¼ Total mass of nanoplex produced=Total mass of VCM and PAA added 100:
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188.8.131.52. Drug loading. Dug loading is deﬁned as the percentage of drug complexing with polyelectrolyte to form nanoplexes . Percentage drug loading was determined UV spectrophotometrically at 280 nm (Shimadzu UV 1601, Japan) by measuring VCM concentration after dispersing the freeze dried VNPX2 nanoplex (10 mg) in PBS (10 ml) of pH 7.4. Drug loading was calculated using the following formula. Drug loading ð%Þ ¼ ðmass of VCM in nanoplex=mass of nanoplexÞ 100
184.108.40.206. Morphology. The surface morphology and shape of VCM–PAA nanoplex was examined using the Scanning Electron Microscope (SEM). Brieﬂy, samples were prepared by placing few drops of the nanoplex suspension on a cover slip placed on carbon tape, which were dried thoroughly and sputter coated with gold. The image was captured by ﬁeld-emission gun SEM (ZEISS FEGSEM Ultra Plus, Germany) at an accelerated voltage of 10 kV. For the estimation of particle size distribution, SEM images were analyzed using iTEM 5.0 image analyzer (Soft imaging system, Germany). 2.2.3. X-ray diffraction (XRD) XRD patterns of VCM, PAA and VCM–PAA nanoplex were obtained using a Bruker D8 advance diffractometer (Germany) equipped with a graphite monochromator operating at 40 kV and 40 mA. The radiation source was a CuKα X-ray source with λ = 1.5406 Å. Data was collected at a step of 0.021° and at a scanning speed of 0.454° s−1. The 2θ range covered was between 10° to 70°. The XRD analysis was also performed to evaluate physical stability of nanoplex formulation after storage at ambient temperature over a period of one month. 2.2.4. Differential scanning calorimetry (DSC) DSC experiments were performed to study the melting and crystallization behavior of VCM, PAA and VNPX2. Approximately 2 mg of sample was placed in aluminum pans and sealed by a crimper and then analyzed using DSC (Shimadzu DSC-60, Japan). Samples were heated up to 300 °C at a constant rate of 10 °C/min under constant nitrogen ﬂow of 20 ml/min. The stability of VNPX2 was also assessed by analyzing samples stored at ambient temperature for 30 days. 2.2.5. In-vitro drug release To evaluate the drug release pattern, in-vitro drug release studies of bare VCM and VCM–PAA nanoplex formulation were performed in PBS (pH 7.4). Brieﬂy, solutions of VCM and VCM–PAA nanoplex (1 ml) were added separately in dialysis tubing (MWCO 14,000 Da), diluted with PBS of pH 7.4 (1 ml) and dialyzed against 40 ml PBS (pH 7.4) at 37 °C in a shaking incubator at 100 rpm. To determine the amount of drug released, a ﬁxed quantity (3 ml) of samples were removed from the receiver solution at ﬁxed time intervals and equal amount of fresh PBS (pH 7.4) were added to maintain the sink conditions. The drug in samples was measured spectrophotometrically at 280 nm using a spectrophotometer (Schimadzu UV 1601, Japan) with PBS (pH 7.4) as a blank, and the experiments were performed in triplicate. Drug release data of the VCM and nanoplex were assessed kinetically by using several important mathematical models, such as zero order, ﬁrst order, Higuchi, Weibull, Hixson–Crowell and Korsmeyer–Peppas, by calculating parameters such as correlation coefﬁcient (R2), root mean square error (RMSE) and mean dissolution time (MDT) using an excel add-in DDSolver program (China) . 2.2.6. In vitro antibacterial activity The effect of complexation of VCM with PAA on its antimicrobial potential was investigated by performing in vitro antibacterial activity using a broth dilution technique. In brief, the target bacterial cultures (S. aureus and MRSA) were grown overnight in Nutrient Broth at 37 °C and then adjusted to 0.5 McFarland in MHB . A 2-fold serial dilution
of these bacterial cultures were performed in order to obtain a ﬁnal concentration of 2 × 105 colony forming units (cfu)/ml . Minimum inhibitory concentration values for VCM and the optimized VCM–PAA nanoplex formulation were determined against S. aureus and MRSA by following a previously reported procedure . Dilutions of the VCM, PAA and VCM–PAA nanoplexes were prepared in MHB and incubated with the bacterial cultures at 37 °C. Thereafter, at speciﬁed time intervals, 10 μl was spotted on MHA plates and incubated for 18 h at 37 °C to determine the MIC values. Experiments were performed in triplicate using PAA as a negative control. 2.2.7. In vitro cytotoxicity The cytotoxicity of the test materials was determined using the MTT assay after incubation with exponentially growing human breast adenocarcinoma (MCF 7), human cervix adenocarcinoma (HeLa) and human liver hepatocellular carcinoma (Hep G2) cell lines. The cells were maintained at 37 °C in a humidiﬁed atmosphere of 5% CO2 in air. Test materials were dissolved in milli-Q water as a stock solution, and further diluted in the culture medium to give ﬁnal concentrations of 20, 40, 60, 80 and 100 μg/ml . All three cell lines were seeded equivalently (2.5 × 103) into 96-well plates and incubated for 24 h to allow for cell adherence. Final treatment concentrations were achieved by replenishing the wells with fresh culture medium (100 μl per well) together with the appropriate concentration of the test solutions. The control wells were prepared by addition of culture medium only and the blank wells contained culture medium without cells. After the 48 h incubation, the culture medium and test material were removed and replaced with 100 μl of fresh culture medium and 100 μl of MTT solution (5 mg/ml in PBS) in each well. After 4 h of incubation, the media and MTT solution were removed and 100 μl of dimethylsulfoxide was added to each well to solubilize the MTT formazan. The optical density of each well was measured on a microplate spectrophotometer (Mindray MR-96A) at a wavelength of 540 nm (A540: absorbance at a wavelength of 540 nm) . All the experiments were performed with six replicates. The percentage cell viability was calculated as follows. Cell viability (%) = (A540 nm treated cells / A540 nm untreated cells) × 100. 2.2.8. Molecular modeling and in silico studies In silico modeling was performed to assess the stability and intermolecular forces required for molecular assembly, as well as to explain the mechanism of nanoplex formation. All the molecular modeling techniques were performed using Biovia Materials Studio (MS) 7.0  and installed on a remote Linux server at the Centre for High Performance Computing (CHPC), Cape Town, South Africa. 220.127.116.11. Electroneutral complex formation. A Blends module of MS 7.0 was used to calculate the thermodynamic parameters, such as mixing energy, binding energy and interaction parameter for the VCM–PAA electroneutral complex. Protein Data Bank (PDB) crystal structure coordinates for VCM monomer (PDB ID: 1SHO) and VCM-dimer (PDB ID: 1AA5) were used to construct respective 3D models [38,39]. A 3D structure of PAA was constructed using the polymer build tool in MS . The initial geometry of all the study models was optimized using Universal Force Field (UFF) . Mixing energy calculations were performed at 298 K as reference temperature. The energy of 100,000 pair conﬁgurations were evaluated to ﬁnd lowest one . A total of 10,000 cluster samples were chosen to calculate the coordination number. The lowest energy conﬁguration with the suggested coordination number was chosen to correlate with the experimental ﬁndings. 18.104.22.168. Nano-aggregation. The nanoplex unit model was constructed using an amorphous cell module of Materials Studio 7.0 . The nanoplex components of PAA sodium and VCM molecules were packed as per the optimized formulation molar ratio (1:0.07) inside a cubic cell
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(50 × 50 × 50 Å). The box was ﬁlled with water molecules to represent the solvent medium. The geometry of the packed molecules inside the cubic cell was then optimized using a UFF by employing a smart algorithm available in the forcite module of the software . Convergence tolerance criteria for the energy and force during the geometry optimization were set to 0.001 kcal/mol and 0.5 kcal/mol respectively. The maximum iterations to reach the convergence were ﬁxed at 500,000, and the optimization of cell parameters was allowed during energy minimization. The stability of the nanoplex unit system was assessed from its total heat of formation. A 3D model of the stable nanoplex unit was then analyzed by Discovery studio visualizer for intermolecular interactions, which are crucial for packing the polymer with drug molecules . 2.2.9. Statistical analysis All results obtained were reported as mean ± SD and the data analysis was performed using GraphPad Prism®5 (Graphpad Software Inc., USA). One way ANOVA and t-test was performed and the difference was considered signiﬁcant when p b 0.05. 3. Result and discussion 3.1. Preparation of VCM–PAA nanoplex Drug/polymer or charge ratios inﬂuence the formation and properties of nanoplexes. Charge ratio (Z+/−) can be deﬁned as the concentration of cationic units of peptide to the anionic units of polymer in a system . Therefore, an optimal charge ratio was determined by adding varying amounts (1–16 ml) of PAA solution (10 mg/ml) to a ﬁxed quantity (20 ml) of VCM solution (10 mg/ml) to obtain Z+/− of 0.16, 0.32, 0.64, 1.28 and 2.56. The addition of anionic PAA solution to the cationic VCM solution resulted in electrostatic adsorption of the VCM onto the PAA chains. At a certain critical concentration, there are enough drug-adsorbed-on-polymer molecules in the close proximity of each other to enable the formation of aggregates via hydrophobic interaction of the drug molecules. This critical concentration, at which aggregates are formed, depends on the hydrophobicity of a drug molecule . In the present study, this critical concentrations in terms of Z+/− were 0.64, 1.28 and 2.56, as observed visually by formation of the turbid solutions. There was no aggregate formation at Z+/− of 0.16 and 0.32, as the solutions were clear. The VCM precipitation into a nanoplex could have occurred due to the coupling of the hydrophobic interactions with the charge neutralization of the VCM as a result of the electrostatic interactions. In this nanoplex formation procedure, the PAA functioned as a polyelectrolyte that electrostatically stabilized the nanoplexes. The results are concordant with the previous ﬁndings, where nanoplex formation between the cationic drugs (oﬂoxacin and levoﬂoxacin) and dextran sulfate was observed . 3.2. Characterizations of VCM–PAA nanoplexes 3.2.1. Size, PDI, ZP and %CE At Z+/− of 0.64, 1.28 and 2.56, the particle sizes were 552.6 ± 93.37, 229.73 ± 47.76 and 262.2 ± 52.38 respectively, whereas the ZP increased gradually from − 39.03 ± 4.16 to − 18.53 ± 5.36 mV. The Malvern zeta sizer, Nano ZS works on the principle of dynamic light scattering which is also known as photon correlation spectroscopy (PCS). It has been reported previously that size measurements obtained by PCS is the size measured for nanoplex agglomerates and not of individual nanoparticles . This could be the reason for large size ranges obtained for all VNPX nanoplex formulations. The PDI values were 0.481 ± 0.055, 0.442 ± 0.075 and 0.374 ± 0.030 at Z+/− of 0.64, 1.28 and 2.56 respectively (Table 1). As discussed earlier, the high PDI values by PCS could be due to the fact that the size obtained for the nanoplex reﬂects the size of agglomerates and these agglomerates contributed to high PDI values. The %CEs at Z+/− of 0.64, 1.28 and 2.56 were
found to be 51.28 ± 2.21, 74.17 ± 1.2 and 39.2 ± 1.84% respectively (Table 1). There was an increase in %CE from Z+/− of 0.64 to 1.28, while the %CE decreased drastically at Z+/− of 2.56. This could be due to the insufﬁcient quantity of PAA to form aggregates via hydrophobic interaction of the drug molecules with the polymer, which is in line with the previously reported literature , and suggested the signiﬁcant inﬂuence of Z+/− on %CE and ZP (p b 0.5). The charge neutralizations at Z+/− of 1.28 favored the formation of VCM–PAA nanoplexes with increased hydrophobicity, ZP and %CE, and decreased particle size and PDI. Based on these results, the optimum condition and procedure to obtain an efﬁcient nanoplex system included the addition of a 2 ml solution of PAA (1% w/v) to 20 ml VCM hydrochloride solution (1% w/v) under magnetic stirring for 3 h at room temperature. The VCM–PAA nanoplex formulation, VNPX2, with a Z+/− of 1.28, was therefore considered an optimized formulation due to its lower size and PDI, an acceptable ZP value, and high %CE. Therefore, further studies were performed on this optimized formulation. This VNPX2 formulation was lyophilized using 5% glucose as cryoprotectant. The freeze dried formulation (10 mg) was redispersed in milli-Q water (10 ml) and analyzed for size, PDI and ZP in order to investigate the effect of lyophilization on its stability. The results are depicted in Table 2. Although, ZP of lyophilized VNPX2 (−25.7 ± 3.36 mV) after redispersion into milli-Q water shifted slightly towards the positive side, it was sufﬁciently large enough to keep the nanoparticles well dispersed in aqueous solution. This stability can be inferred by particle size and PDI of 201.8 ± 21 nm and 0.271 ± 0.001 respectively. Overall lowering of particle size and PDI of lyophilized VNPX2 was an indication of their stability in the dry form. These results strongly support the fact that greater stability can be achieved for pharmaceutical formulations if they are prepared in the dry form . 3.2.2. Fourier transform-infrared (FT-IR) analysis Polyionic complexation between VCM and PAA was conﬁrmed by FT-IR analysis of VCM, PAA and VNPX2. The \\NH\\ bending vibration at 1586 cm− 1 in the VCM was shifted towards 1453 cm− 1, and a shift in C\\O stretch of PAA from 1646 cm− 1 to 1549 cm− 1 was observed in the VNPX2 (Fig. 1). These changes in the position of the wavenumbers of the characteristic functional groups present in VCM and PAA conﬁrmed the physical interaction between them and the formation of the nanoplex. 3.2.3. Percentage yield The percentage yield or nanoparticle recovery of VNPX2 was found to be 60.60 ± 2.62%. This percentage yield of N50% was indicative of the fact that commercial industrial scale production of VNPX2 is possible. 3.2.4. Drug loading Drug loading was found to be 58.40 ± 1.03%. The high drug loading observed for VNPX2 nanoplexes was an indication of the formation of a stable polyionic complex in between VCM and PAA. 3.2.5. Morphology The SEM images (Fig. 2) showed that VNPX2 were of hexagonal cubic shape with a smooth surface and showed the lowest and highest particle size of 54.41 and 501.67 nm respectively. Particle size distribution by iTEM image analyzer showed that 41% of total population of nanoparticles was within the size range of 200–300 nm. Smaller particles below 100 nm could be the result of shrinkage and larger particles N320 nm due to agglomeration of nanoparticles during the drying step of the sample preparation method. These results corroborate well with previous results where smaller and larger particle sizes were observed due to shrinkage  and aggregation [44,45] of nanoparticles respectively.
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Table 1 Size, PDI, ZP and %CE of different formulations (n = 3). Formulation
VNPX0A VNPX0B VNPX1 VNPX2 VNPX3
0.16 0.32 0.64 1.28 2.56
– – 552.6 ± 93.37 229.7 ± 47.76 262.2 ± 52.38
– – 0.481 ± 0.055 0.442 ± 0.075 0.374 ± 0.030
– – −39.03 ± 4.16⁎ −30.46 ± 5.30⁎ −18.53 ± 5.36⁎
NA NA 51.28 ± 2.21⁎ 74.17 ± 1.2⁎ 39.20 ± 1.84⁎
NA — no aggregates formed. ⁎ Statically signiﬁcant (p b 0.05).
3.3. X-ray diffraction X-ray diffraction (XRD) is an important tool to detect any changes in the crystallinity of the formulation excipients to conﬁrm the formation of nanoparticles. Fig. 3i shows the XRD patterns of PAA, VCM, and lyophilized VNPX2 on day one and after one month. The XRD pattern of VCM did not show any noticeable peak, which may be due to the amorphous nature of its salt form. The two broad peaks were observed at 2θ values of 16.8° and 32.15° in PAA, indicating crystallinity of PAA. The peak at 16.8° completely disappeared whereas the one at 32.15° was suppressed and there was the appearance of a small diffused peak at 45.6° in VNPX2 nanoplex. This was an indication of transformation of crystalline PAA into the amorphous form after the complexation with VCM, and thus conﬁrms the formation of nanoplexes. Further, after a period of one month those broad amorphous halos at 32.15° and 45.6° were still present in the XRD pattern of VNPX2 nanoplex signifying its amorphous state stability. A combination of quick precipitation of VCM molecules upon charge neutralization, and their electrostatic interaction with PAA molecules, resulted in the formation of amorphous nanoplexes. The assembling of amorphous nanoplexes into crystalline structures is prevented by electrostatic interactions between the drug and polyelectrolyte . No change in the physical appearance of lyophilized VNPX2 after storage at ambient temperature for a month, and the similarity of XRD pattern of the freshly lyophilized VNPX2 with the stored (Fig. 3i. d), conﬁrmed the stability of VNPX2 nanoplex formulation. 3.4. Differential scanning calorimetry (DSC)
achieved at 8 h, whereas at the same time point from VNPX2, there was a 69.03 ± 7.17% release. At the end of 12 h, 95.42 ± 3.1% of the VCM was released from VNPX2. These results, showing a b 30% release from VNPX2 at the initial time periods, were indicative of sustained release proﬁle of the nanoplexes. The VCM release from the VNPX2 was found to follow Weibull model (R2 = 0.988), with a lower RMSE value of 3.59 over the period of 12 h. First order model, with an R2 0.985, was found to be closest to the best ﬁt Weibull model (Table 3). The value of the release exponent (n) obtained from the Korsmeyer– Peppas equation was 0.454, which indicates non-Fickian release mechanism. This suggests that drug release from hydrophilic polymer is a sequential process of solvent penetration, polymer hydration, drug dissolution and/or polymer erosion [47–49]. The mean dissolution time (MDT) values calculated for 95.42% release from VCM and VNPX2 were 3.15 and 5.61 respectively, which indicates that a slower release of VCM from VNPX2. 3.6. In-vitro antibacterial activity In vitro antibacterial activity of VNPX2 and bare VCM were compared by using broth dilution technique to conﬁrm that there was no change in potency of the VCM upon its transformation into nanoform. The MIC values for both bare VCM and VNPX2 against S. aureus and MRSA was 1.7 μg/ml at the end of the 18 h period. This conﬁrmed the preservation of antibacterial potency of VCM upon its complexation with PAA. The results are in good agreement with the previous study, where there was no change in antibacterial activity of oﬂoxacin and levoﬂoxacin after the complexation with dextran sulfate .
DSC is an important tool to understand melting behavior and crystallinity of materials in formulation system . The thermal behavior of PAA, VCM and lyophilized VNPX2 are presented in Fig. 3ii. PAA and VCM exhibited endothermic peaks at 156 °C and 110 °C respectively whereas there was no noticeable endothermic peak in the thermogram of lyophilized VNPX2 nanoplex. This indicated complexation of VCM and PAA to a nanoplex and its conversion in to the amorphous form. There was no change in thermal behavior of lyophilized VNPX2 nanoplex after storage at room temperature for 30 days conﬁrming its stability. The obtained DSC results are in line with XRD results. 3.5. In vitro drug release In-vitro drug release patterns of the bare VCM and nanoplex formulation in PBS (pH 7.4) are shown in Fig. 4. The percentage release from bare VCM was 50.94 ± 4.07%, whereas from VNPX2 it was 28.90 ± 7.15% at the end of 2 h. The 100% drug release for the bare VCM was Table 2 Size, PI and ZP of VNPX2 nanoplex before and after lyophilization (n = 3). Parameter
Size (nm) PDI ZP (mv)
229.7 ± 47.76 0.442 ± 0.075 −30.46 ± 5.30
201.8 ± 21 0.271 ± 0.001 −25.7 ± 3.36
Fig. 1. FT-IR spectra for (a) VCM, (b) PAA and (c) VNPX2.
D.R. Sikwal et al. / Materials Science and Engineering C 63 (2016) 489–498
Fig. 2. SEM images of VNPX2 nanoplexes, scale bar (a) 1 μm and (b) at 200 nm (inset showing particle size distribution).
3.7. In vitro cytotoxicity In order to determine the biosafety and to establish nontoxic dosages for biomedical applications of newly synthesized nanomaterials, formulation scientists employ cytotoxicity studies . The viability of cells after exposure to the test material is quantiﬁed using cytotoxicity assays. The MTT assay is based on the reduction of MTT by viable cells into a crystalline blue formazan. The resultant formazan crystal formation is proportional to the number of viable cells . An in vitro cell culture system using the MTT assay was utilized to determine the biosafety of i) the optimized nanoplex formulation VPNX2, ii) VCM and iii) PAA. The results displayed a high percentage of cell viability of 78.14 to 91.67% and 76.24 to 82.45% at all concentrations for VCM and PAA respectively, across all the cell lines studied Figs. S1–S3 (Supplementary information). These ﬁndings conﬁrm the nontoxic nature of these materials to eukaryotic cells. The percentage cell viability obtained for VPNX2 was between 78.04 and 81.42% for MCF 7 cells, 78.07 to 83.61% for HeLa cells and 76.98 to 84.98% for Hep G2 cells for all concentrations (Fig. 5). There were no dose dependent trends observed in the percentage cell viability for any of the test materials, across all cell lines, within the concentration range studied. Test materials displaying cell viabilities N75% can be considered to be of low toxicity and biologically safe [52,53]. Since VPNX2 displayed cell viabilities N75% across the various cell lines it can therefore be considered as nontoxic and safe to mammalian cells. The optimized VPNX2 formulation displays the capability of being a promising nanoantibiotic for therapeutic applications within the
biomedical and pharmaceutical domain as it possesses superior antimicrobial activity as well as non-toxicity against the cell lines studied. 3.8. In silico studies 3.8.1. Electroneutral complex formation The nanoplex drug delivery system is often formed by neutralizing polyionic units in polymer in the presence of drug molecules with equivalent amount of counter charge units. To produce an electroneutral complex in the current nanoplex system, the equivalent amount of VCM required to neutralize the anion units in PAA would be 22. As each VCM molecule possess two cation units in the form of ammonium ions, as shown in Fig. 6, the single VCM molecule can neutralize two anionic units of PAA. Thus to form an electroneutral complex, 11 VCM molecules per PAA are needed. Mixing energy calculations can predict the number of VCM molecules that can interact with PAA with favorable enthalpy and entropy. Therefore, we calculated thermodynamic parameters for mixing of PAA and VCM based on the Flory-Huggins model , that calculate the free energy of mixing, using interaction parameter, χ, coordination number (Z) and binding energy (E) . In our mixing energy calculations, we found that approximately 6 VCM molecules were able to interact with one PAA, as observed from the coordination number (Zij) 5.69. However, this can produce a half neutralized complex that could not support nano-aggregation. The
Fig. 3. i) XRD pattern and ii) DSC thermograph of (a) PAA, (b) VCM, and lyophilized VNPX2 at (c) day 1 and (d) day 30.
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Fig. 4. Percentage cumulative drug release from bare VCM and VNPX2 (n = 3).
presence of more VCM molecules is therefore required to compensate for the excess anions in the PAA and enable the formation of nanoaggregates. As the PAA volume in the current conﬁguration did not facilitate more than 6 VCM molecules, their addition for neutralization may be drawn by their self-association as dimers. Therefore, 6 VCM (dimers) can explain the uncertainty in the complete neutralization of the PAA to produce an electroneutral complex. This was supported by the subsequent results obtained from the second Blends calculations using VCM (dimer) with PAA. The occurrence of VCM (dimer) at room temperature was already reported with experimental evidence and theoretical support [56,57]. In our alternate calculation, the coordination number for the VCM (dimer) was Zij = 5.665, which is equivalent to approximately 11 VCM molecules and 22 cationic units combined. Finally, based on the electroneutral complex obtained in the Blends calculation, as shown in Fig. 7, we predict that a dimerized VCM molecule could neutralize 4 units of PAA, and thus an electroneutral complex was obtained with 11 VCM molecules, which were constituted by the dimerization of approximately 6 VCM molecules. The interaction parameter, mixing energy and binding energy, as shown in Table 4, strongly favored the mixed state for VCM–PAA over the pure components. Though, these values were more favorable for VCM (monomer) over VCM (dimer) (Table 4), the nano-aggregation is only possible with completely neutralized VCM (dimer)–PAA complex. 3.8.2. Nano-aggregation It is observed that the concentration of VCM in the optimized formulation was more than that of the VCM in electroneutral complex. This indicates that the molecular aggregation that occurred after neutralization entrapped a few more VCM molecules by supramolecular selfassociation. In order to understand the formation of nanosized aggregates via electroneutral complex, the components as per the optimized formulation ratio were allowed to aggregate in the solvent medium, the amorphous cell module being used for this packing task. This produced a 3D cubic cell with three PAA sodium and 42 VCM molecules, which is equivalent to the molar ratio of the study formulation. During the geometry optimizations process, the molecules stufﬁng and cell parameters were
Fig. 5. Cytotoxicity assay of VNPX2 displaying percentage cell viability after exposure to various concentrations of VPNX2 on MCF 7, HeLa and Hep G2 cells. Results are presented as mean ± SD (n = 6).
adjusted simultaneously to minimize the energy and produce a stable system. The total enthalpy of the optimized system was found to be −133.350827 kcal/mol that include the non-bond energy contribution of −198.537 kcal/mol. The ﬁnal stable system, as shown in Fig. S4 (Supplementary information), has unsymmetrical sides with hexagonal cell like geometry [A = 47.42 Å B = 48.0 Å C = 52.15 Å; α = 78.27° β = 89.21° γ = 86.74°] where the polymer and drug molecules were packed by means of ionic and hydrophobic forces. The optimized model, as shown in Fig. S5 (Supplementary information), suggest that the PAA chain was exposed to the solvent medium by means of ionic interactions and held the VCM to the interior by forming hydrogen bonds. The PAA bound VCM further built the hydrophobic network cooperatively with several other VCM molecules to form molecular aggregates. Most of the cell boundary, as displayed in Fig. S5 (Supplementary information), was occupied by ionic functional groups that can facilitate the aggregation of these units in multiple to form particles. The ﬁnal cell model, with dimensions similar to hexagonal cell, also suggest that the sequential arrangement of this unit could result in the formation of hexagonal like particles, as observed in the SEM study (Fig. 2).
Table 3 Mathematical models for drug release from VNPX2. Sr. no.
1 2 3 4 5 6
Zero order First order Higuchi Korsmeyer–Peppas Weibull Hixson–Crowell
Q = k·t + Q0 Q = Q0 ekt Q = k·t1/2 Q = k·tn Q = 1exp [−(t)b/a] Q1/3 = kt + Q1/3 0
0.404 0.985 0.912 0.952 0.988 0.968
24.89 3.99 9.66 7.30 3.59 5.65
Where, Q0 and Q are the start value of Q and the amount of drug released in time t respectively, k, a and b represent the rate constant, the time constant, and the shape parameter respectively, n is the diffusional exponent which is an indicative of drug release mechanism.
Fig. 6. A 2D structure of VCM. Atoms that are labelled with numbers are the important interaction sites delivered for packing.
D.R. Sikwal et al. / Materials Science and Engineering C 63 (2016) 489–498 Table 5 The geometry of the various hydrogen bonds observed in VCM–PAA (1:0.07) nanoplex unit.
Fig. 7. A 3D structure of optimized conﬁguration of the electroneutral complex displaying 6 VCM-dimers (each dimer was shown with different color) surrounding one PAA molecule (ball and stick model). (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)
Table 4 Thermodynamic parameters calculated for mixing the components at 298 K. Mixing Components
VCM (dimer)-PAA VCM–PAA
χ = Interaction parameter; Emix = Mixing energy; Eij avg = Binding energy average; Zij = Coordination number.
The cooperative packing of the VCM molecules displayed unique hydrophobic wadding pattern among the VCM molecules, as shown in Fig. 8. The hydrophobic interactions, such as π–π T-shaped, π–alkyl and alkyl–alkyl interactions, were found to be the signiﬁcant forces of molecular aggregation. Interestingly, the same forces were reported by Loll et.al in the crystal structure of the VCM-dimer . A typical hydrogen bond between the amide and carboxylic acid functional groups also contributed to packing the VCM molecules, as shown in Fig. S6 (Supplementary information).
Distance Å (D-H–A)
VCM:H111 VCM:H111 VCM:H150 VCM:H150 VCM:H151 VCM:H151 VCM:N-H55 VCM:N-H55 VCM:N-H55 VCM:N-H55 VCM:N-H55 VCM:N-H55 VCM:N-H55 VCM:N-H56 VCM:N-H56 VCM:N-H56 VCM:N-H56 VCM:N-H56 VCM:N-H56 VCM:N-H56 VCM:N-H56 VCM:N-H56 VCM:O-H131 VCM:O-H131
PAA:O PAA:O PAA:O PAA:O PAA:O PAA:O VCM:O123 VCM:O27 VCM:O27 VCM:O5 VCM:O5 VCM:O27 VCM:O53 VCM:O124 VCM:O123 VCM:O5 VCM:O5 VCM:O124 VCM:O58 VCM:O5 VCM:O5 VCM:O27 VCM:O53 VCM:O53
2.52035 2.17992 2.45267 2.47147 2.60807 2.3816 2.56961 2.49102 2.66929 2.58839 2.42924 2.89709 2.61032 2.50356 2.544 2.58839 2.99245 2.90929 2.99323 2.69116 2.70208 2.39474 2.32739 2.64999
129.603 133.808 119.197 137.523 154.9 131.863 95.382 108.605 141.794 96.064 141.202 124.149 161.509 129.002 96.85 96.064 98.774 114.146 95.701 98.936 133.286 140.314 102.286 114.829
169.868 124.832 130.092 101.117 161.395 121.008 143.94 132.525 108.872 159.792 142.442 90.72 120.237 92.293 152.681 159.792 143.825 95.592 142.006 112.003 142.861 121.279 132.895 111.319
The intermolecular interactions were analyzed in detail to understand the process of aggregation. To begin with, the interaction of PAA with VCM, as shown in Fig. S6 (Supplementary information), took place by means of hydrogen bonding between the carboxyl of the PAA and the hydroxyl groups (OH111, OH150 and OH151) of VCM, as labelled in Fig. 6. The average hydrogen bond distances were calculated from the VCM–PAA H-bond geometry, as indicated in Table 5, and optimum distance was found to be 2.4357 Å. As part of neutralization, a salt bridge interaction was also observed between the ammonium ion of VCM and the carboxylate ion of PAA, as shown in Fig. S6 (Supplementary information). Intermolecular interactions among the VCM molecules were majorly hydrophobic and partially ionic. The partial ionic interaction was due to hydrogen bonding between the donor and acceptor functional groups that are present in VCM molecules. Amide and carboxyl groups are the two most important functional groups in the VCM molecule that can be considered for potential hydrogen bond interaction sites. The amide and carboxylic acid functional groups, either of which can act as proton donor and acceptor, are well known supramolecular synthons that were shown to be important in the formation
Fig. 8. A 3D structure of unique hydrophobic interaction pattern observed during the aggregation of VCM molecules.
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of the acetic acid dimer and the biological conformation of bilirubin . Their signiﬁcance in the crystal packing of organic molecules was also well documented [58,59]. The average hydrogen bond distances, as calculated from the inter VCM H-bond geometry and indicated in Table 5, was found to be 2.6417 Å. Furthermore, as compared to VCM, the interaction of PAA with the solvent was more facilitated because of its ionic nature due to the presence of carboxylate function. Thus, the PAA played an interfacial role to stabilize the hydrophobic aggregation of VCM in the presence of polar solvent-like water. As described previously, the hydrophobic attraction among the VCM molecules was attributed to the aromatic rings and the isopropyl moiety of the VCM structure. While packing, the VCM adopted a conformation that facilitated the vertical interaction of the aromatic ring π orbital of one molecule with that of the other. This T-shaped π–π interaction, as shown in Fig. 8, was typically observed in several biological systems, including the drug-receptor complex, and was found to be the most favorable interaction among the π–π stacking interactions [60,61]. As evidenced from the hydrophobic interactions data in Table 6, the highly abundant π–alkyl, and relatively less abundant alkyl–alkyl interactions, are the other hydrophobic forces that held the VCM molecules together for aggregation. In the current model system, the chlorine substituent on the aromatic ring was assigned in the class of the alkyl interaction group, as the halogen atoms are considered hydrophobic. The alkyl groups of isopropyl moiety and the chlorine atoms from the aromatic ring were found to be the prominent acceptors for interacting with the π orbitals of aromatic ring, as shown in Fig. 8. The interaction of halogen and alkyl groups with π orbitals was clearly emphasized in the organic crystal packing . Hydrophobic interaction among the hydrocarbons is also well known in lipids and non-polar organic substances [63,64]. A similar attraction, in the form of alkyl interaction, was observed between the methyl groups of isopropyl moiety of the VCM molecules, as shown in Fig. 8. The demonstration of a large number of hydrophobic forces among the VCM molecules clearly indicates that the VCM structure was highly repellent to the polar surface, and its aggregation with other VCM molecules was supported by the ionic interaction with PAA at the solvent interface. Overall, the molecular modeling study of the nanoplex unit demonstrated the formation of the VCM–PAA electroneutral complex with favorable thermodynamic parameters. The study also conﬁrmed the ability of self-association of VCM to form molecular aggregates by means of hydrophobic attraction. Our modeling of the electroneutral complex, followed by self-associated aggregation in the presence of PAA at the solvent interface, clearly supported the concept behind the construction of VCM–PAA nanoplex drug delivery system.
Table 6 The geometry of various hydrophobic interactions observed in VCM–PAA (1:0.07) nanoplex unit. Types
π–π T-shaped π–π T-shaped π–π T-shaped π–Alkyl π–Alkyl π–Alkyl π–Alkyl π–Alkyl π–Alkyl π–Alkyl π–Alkyl π–Alkyl π–Alkyl π–Alkyl π–Alkyl π–Alkyl π–Alkyl π–Alkyl π–Alkyl Alkyl Alkyl Alkyl Alkyl Alkyl Alkyl Alkyl Alkyl
VCM VCM VCM VCM VCM VCM VCM VCM VCM VCM VCM VCM VCM VCM VCM VCM VCM VCM VCM VCM:Cl101 VCM:Cl101 VCM:C162 VCM:C8 VCM:C9 VCM:Cl34 VCM:C162 VCM:C9
VCM VCM VCM VCM:C8 VCM:C9 VCM:Cl101 VCM:C162 VCM:C162 VCM:Cl34 VCM:C162 VCM:Cl34 VCM:Cl34 VCM:C8 VCM:C9 VCM:C8 VCM:C9 VCM:Cl101 VCM:C9 VCM:C9 VCM:C8 VCM:C9 VCM:C8 VCM:C8 VCM:Cl101 VCM:C162 VCM:C162 VCM:C162
5.29518 5.1919 5.15836 5.2575 5.38409 4.57499 4.54983 5.46543 3.75787 4.19218 5.36947 5.10009 5.43642 4.69629 3.61166 3.91387 5.25081 3.27596 5.01365 3.76306 3.92789 3.8989 3.85826 4.43414 3.30472 3.69152 4.06476
would certainly result in a novel and efﬁcient pulmonary drug delivery system of nano-VCM for clinical trials. Acknowledgement The authors acknowledge the University of KwaZulu-Natal (UKZN) and the National Research Foundation (NRF) of South Africa for ﬁnancial support (Grant No. 87790 and 88453), Microscopy and Microanalysis Unit (MMU) (UKZN) for SEM analysis and Carrin Martin for proof reading the manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2016.03.019. References
4. Conclusion An optimized nanoplex formulation, VNPX2, with a particle size in the nanometric range and a higher %CE, ZP, drug loading and nanoparticle recovery was successfully prepared using a self-assembly drugpolyelectrolyte complexation method. The VNPX2 nanoplexes were stable after lyophilization and storage at ambient temperature. Molecular modeling studies demonstrated the stability of the nanoplex system and the intermolecular forces involved in molecular assembly. The study was able to explain the mechanism behind achieving an electroneutral complex and identiﬁed the key features that were responsible for nanoaggregation and drug complexation. This nanoantibiotic system can be a promising inhalation delivery system. Further investigations into the preservation of antibacterial potency of VCM in VNPX2 suggest that the dry powder form of VNPX2 can be effectively used for combating lung infections caused by S. aureus and MRSA. This could be an effective delivery system for patients with cystic ﬁbrosis suffering from lung infections by actively delivering the drug in the nanoform at the target site and avoiding the side effects associated with intravenous delivery. Future research on the formulation development of powder aerosol of VNPX2
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