- Email: [email protected]
Drug delivery investigations of quaternised poly(propylene imine) dendrimer using nimesulide as a model drug
Colloids and Surfaces B: Biointerfaces 114 (2014) 121–129
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Drug delivery investigations of quaternised poly(propylene imine) dendrimer using nimesulide as a model drug E. Murugan ∗ , D.P. Geetha Rani, V. Yogaraj Department of Physical Chemistry, School of Chemical Sciences, University of Madras, Maraimalai Campus, Guindy, Chennai 600 025, TamilNadu, India
a r t i c l e
i n f o
Article history: Received 19 June 2013 Received in revised form 25 September 2013 Accepted 2 October 2013 Available online 12 October 2013 Keywords: Poly(propylene imine) dendrimer Drug carrier Nimesulide Solubility In vitro release Cytotoxicity
a b s t r a c t This study describes the demonstration of quaternized poly(propylene imine) dendrimer of generation3, QPPI (G3) as a drug carrier for poorly soluble drug nimesulide (NMD, an anti-inflammatory drug). QPPI (G3) was prepared by treating the surface amine groups of poly(propylene imine) dendrimer with glycidyltrimethyl ammonium chloride and it was characterized with FTIR, 1 H and 13 C NMR and MALDITOF mass spectral techniques. The drug carrying potential of QPPI (G3) was assessed by analyzing drug solubility, in vitro release and cytotoxicity studies. The observed results reveal that the aqueous solubility of NMD has been dramatically increased in the presence of QPPI (G3) and also can sustain the release of NMD. It is further noticed that the complexation of NMD with QPPI (G3) is responsible for increased solubility and sustained release. This complexation was evidenced through NMR (1 H & 2D) and UV–vis spectral techniques, DSC and DLS studies. Cytotoxicity study through MTT assay on Vero and HBL-100 cell lines reveal that this dendrimer increase the biocompatibility and the tolerance concentration of NMD in drug-dendrimer formulations. The observed results prove that the QPPI (G3) is one of the new promising candidate for effective delivery of NMD. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Dendrimers are outstanding candidates to act as drug carriers due to their well-defined nanostructures and chemical versatility [1,2]. As compared to the traditional polymeric drug vehicles, dendrimer shows excellent advantages more especially for drug delivery [3]. Their surface functionalities and monodispersity have enabled them as effective devices for pharmaceutical and biomedical applications [4–7]. Surface functionalities of dendrimers can be functionalized with drugs, targeting moieties and biologically active components [8]. Drugs or genes can be either encapsulated within the dendrimers through non-covalent strategies such as hydrophobic, ionic and hydrogen bond interactions or conjugated to the peripheral groups of dendrimers via covalent methods [9]. Poly(amidoamine) (PAMAM) and poly(propylene imine) (PPI) dendrimers are the extensively used ones among the numerous dendrimers used for drug delivery [10]. A bountiful reports are available which evidence for the drug carrying potential of dendrimers through properties like increased solubility of the hydrophobic drugs, sustained drug release behavior and increased efficiency of the drugs [11–13]. Dendrimer based drug-delivery
∗ Corresponding author. Tel.: +91 44 22202818/22202819; fax: +91 44 22352494. E-mail addresses: [email protected], emurugan [email protected] (E. Murugan). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.10.002
systems provide an attractive platform for loading and release of conventional drug molecules which improve the pharmacodynamic and pharmacokinetic behaviors of several families of drugs [14]. These in turn reveal the promising future of dendrimer based drug delivery systems. Dendrimers such as PAMAM and PPI possess cationic primary amine groups at the surface. Even though they showed excellent drug delivery efficacy, but their cytotoxicity is still a burning issue that limits the clinical applications of most these dendrimer based drug formulations [15,16]. They exhibited high cytotoxicity on numerous cell lines and serious hemolytic activity on red blood cells which is dependent on dendrimer generation, surface functionality/charge and concentration [17]. Cationic groups on dendrimer surface are the major determinant factor in generating cytotoxicity and hemolytic activity. These cationic charges interact with the phosphates on the cell membrane which leads to disturbance of lipid bilayers resulting in leakage of intracellular components [18]. In addition, the surface cationic dendrimers are rapidly cleared from the blood circulation systems which result in limited therapeutic efficacy and bioavailability of the administered drug-dendrimer formulation. In order to overcome this problem, the cationic charges on dendrimer surface have been neutralized through PEGylation, hydroxylation, glycosylation and acetylation and thereby reduced the cytotoxicity and hemolytic activity of cationic dendrimers [19–23]. Following the intensive research that has been conducted on the synthesis of dendrimeric compounds, several studies have been appeared by
122
E. Murugan et al. / Colloids and Surfaces B: Biointerfaces 114 (2014) 121–129
(a)
(b)
Fig. 1. (a) Synthetic route to get QPPI (G3) dendrimer from PPI (G3) dendrimer. (b) Structure of nimesulide (NMD).
focusing on the functionalization of the external groups of basic dendrimers. A diversity of functionalized dendrimers [24] has been prepared including those bearing carbohydrate moieties [25], chiral groups [26] and polymerizable [27] groups. The functionalized groups impart dendrimers the property to organize at the surfaces or to form organized supramolecular structures. Moreover, increased surface density and charge density provide potential applications by enhancing the solubility of low aqueous soluble drugs in aqueous medium and as delivery system for bioactive materials. Paleos et al. have modified the surface amine groups of poly(propyleneimine) dendrimer of generation 5 using glycidyltrimethyl ammonium chloride and thus obtained quaternised poly(propylene imine) (QPPI) dendrimer and this was in turn studied for the loading and release behaviors using pyrene as a guest molecule [28].Further it is worth to mention here that the QPPI dendrimer is expected to serve as an efficient drug delivery. Therefore to the best of our knowledge there are no studies devoted exclusively for the synthesis of dendrimer containing QPPI dendrimers of lower generation for the effective delivery of poorly soluble drugs in aqueous medium. This may emerge as promising candidate for safer and more effective compared to traditional drug delivery system. Nimesulide (NMD) is a non-steroidal anti-inflammatory drug (NSAID) with analgesic and antipyretic properties. It is chemically N-(4-nitro-2-phenoxyphenyl) methane sulfonamide, is a weakly acidic non-steroidal anti- inflammatory drug (Fig. 1b). It differs from other non-steroidal anti- inflammatory drugs (NSAIDs) in that its chemical structure contains a sulfoanilide moiety as the acidic group. Unlike other classical NSAIDs, it has high gastrotolerability due to its relatively high pKa value (6.5) and preferential cyclooxygenase (COX)-2 selectivity. It is mainly prescribed for the treatment of acute pain, the symptomatic treatment of osteoarthritis [29]. However, it’s poor solubility in aqueous medium (0.01 mg/ml) [30] gives rise to difficulties in the design of pharmaceutical formulations and leads to variable oral bioavailability. This restricts their use in topical and parenteral applications. Further, poorly
water-soluble drugs often require high doses in order to reach therapeutic plasma concentrations after oral administration. Hence to increase the therapeutic efficiency of poorly soluble drugs, its solubility in aqueous medium has to be increased. Previous reports say that in order to improve the aqueous solubility of NMD and for its effective delivery various methods were employed such as cyclodextrin-nimesulide complexation [31], micellar solubilization [32] and solid dispersion [33]. In this study, quaternised poly(propylene imine) dendrimer of generation 3, QPPI (G3) dendrimer was produced by the reaction of surface amine groups of poly(propylene imine) dendrimer of generation 3,PPI (G3) with glycidyltrimethyl ammonium chloride and the performance of this dendrimer was evaluated for its application as drug delivery system using nimesulide as a model drug. 2. Materials and methods 2.1. Materials PPI dendrimer Diamino cored (DAB) with terminal amino groups of generation 3 was purchased from Symo Chem (Netherlands) and used as received. Glycidyltrimethyl ammonium chloride was obtained from Sigma–Aldrich and methanol was obtained from SRL.MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) was purchased from Sigma–Aldrich, India. Dialysis membranes (Float-A-Lyzer) were obtained from Spectra Pore. Membrane filter of pore size 0.45 m was obtained from Himedia lab. Double distilled water was used for solubility and in vitro release studies. Vero cell lineage was isolated from kidney epithelial cells, extracted from an African green monkey. HBL-100 cell line was derived from human breast epithelial cells. Nimesulide was obtained as gift sample from Micro labs, Bangalore, India. UV–vis spectra were recorded on TECHCOMP 8500 instrument. Fourier Transform Infrared (FTIR) spectra were recorded on a Bruker Tensor-27 FTIR spectrometer. 1 H, 13 C and 2D NMR spectra
E. Murugan et al. / Colloids and Surfaces B: Biointerfaces 114 (2014) 121–129
were recorded with Bruker 500 MHz UltraShield Plus instrument. MALDI-TOF spectra were taken in perceptive bio systems voyagerDE instrument in negative ion mode taking Dithranol as the matrix. A Perkin-Elmer DSC model 7 was used for recording DSC thermograms. Dynamic Light Scattering (DLS) experiment was done using BI-200SM Goniometer with a Lexel laser model 95 argon laser (100 mW output power at a wavelength of 514.5 nm). Correlator, PD2000 was used and the scattering angle was 90◦ . Cytotoxicity study was done in King Institute of Preventive Medicine, Chennai, India.
123
equation, y = (15,602 ± 0.006)x (R2 = 0.999). The directional coefficient of the equation is equal to the absorption coefficient of NMD, εmax = 15,602 M−1 cm−1 . Using this calibration curve, the amount of NMD solubilized in each dendrimer solution was determined. The blank was performed on the same concentrations of dendrimer delivery solutions so as to cancel any absorbance that may be exhibited by the dendrimer molecules. The solubility experiments were conducted repeatedly three times so as to ensure the concordant values. From the results of phase solubility studies, phase solubility diagrams were constructed by plotting drug solubility versus molar concentration of each dendrimer solutions.
2.2. Synthesis and characterization of QPPI (G3) dendrimer Quaternised poly(propylene imine) dendrimer, QPPI (G3) was synthesized by reacting the surface amine groups of generation 3 poly(propylene imine), PPI (G3) with glycidyl trimethyl ammonium chloride using the reported procedure [28]. Glycidyltrimethyl ammonium chloride is a simple, soluble, low molecular weight, quaternary ammonium salt. Further, since it exists as an epoxy hydro chloride it can easily react with primary amines of PPI (G3) through ring opening reactions. Even weak nucleophile is sufficient to open the epoxide ring compared to other leaving groups. More specifically, the epoxide ring opening in glycidyltrimethyl ammonium chloride may quickly occur even at room temperature than any other electrophilic carbon center. The typical procedure involves by taking PPI (G3) (0.1 g, 0.0601 mmol) in a 50 mL round bottom flask and was dissolved in water. To this an aqueous solution of glycidyltrimethyl ammonium chloride (0.049 g, 0.321 mmol) was added. The mixture was allowed to react for 24 h at room temperature and subsequently subjected to dialysis with the appropriate membrane in order to remove the excess of glycidyltrimethyl ammonium chloride and possibly unreacted dendrimers. The dialyzed solution was lyophilized to get quaternized poly(propylene imine) dendrimer QPPI (G3) (Fig. 1a). This product was characterized by FTIR, 1 H & 13 C NMR and MALDI-TOF spectral techniques. The degree of functionalization of the product was determined by 1 H NMR and MALDI-TOF spectral techniques and the newly prepared quaternised dendrimer was assessed for its drug carrying potential using NMD as a model drug. 2.3. Solubility study of NMD using QPPI (G3) dendrimer The solubility of NMD was quantitatively estimated by adopting the previously described phase solubility analysis method [34]. Both PPI (G3) and QPPI (G3) dendrimers were employed individually and studied the solubility of drug NMD. The degree of solubility of the drug was measured in aqueous solutions with increasing concentrations of both PPI (G3) and QPPI (G3) separately. That is, seven different concentrations of drug carriers PPI (G3) and QPPI (G3) were prepared in the range from 0.05 mM to 0.35 mM. Then from each concentration, 5 mL was taken in 20 mL vials and to which excess NMD (5 mg) was added. These vials were sonicated for 30 min and subsequently shaken mechanically for 24 h at 35 ◦ C. Parallely controlled experiment (without dendrimer) was also performed by taking 5 mg of drug in 5 mL of double distilled water. The vials were allowed to stand for 24 h to attain equilibrium. Then these solutions were centrifuged at 5000 rpm for 20 min and filtered using cellulose acetate membrane filter having pore size 0.45 m. The respective filtrate was diluted appropriately with double distilled water and analyzed using UV–vis spectrophotometer. The amount of NMD solubilized in aqueous medium in the presence of drug carriers viz., PPI (G3) and QPPI (G3) was estimated by measuring the characteristic absorbance observed at 397 nm (max ). The quantitative estimation of NMD was done using the calibration curve constructed within the drug concentration range of 3.47 M to 34.7 M which is described by the
2.4. NMR spectral studies about the interactions between NMD and QPPI (G3) dendrimer 2.4.1. 1 H NMR studies 1 H NMR experiments were conducted on a Bruker Advance 500.149 MHz NMR spectrometer at 298.2 K for QPPI (G3) dendrimer/NMD complex (2 mg of QPPI-(G3) and 1 mg of NMD dissolved in 1 mL of D2 O). The temperature was kept constant within (0.2 K) by the use of a Bruker temperature control unit. 2.4.2. 2D NMR studies NOESY experiments were obtained for a QPPI (G3) dendrimer/NMD solution (2 mg of QPPI (G3) and 1 mg of NMD dissolved in 1 mL of D2 O) acquired at 500.149 MHz, using a 300 ms mixing time and a 8.2 s 1 H 90◦ pulse width. The experiments were done with a 2 s relaxation delay and 205 ms acquisition time. Eight transients were averaged for each 400 × 1024 complex t1 increment. The data were processed with Lorentz–Gauss window function and zero filling in both dimensions to display data on a 2048 × 2048 2D matrix. All data were processed with NMRPipe software on a Linux workstation. 2.5. UV–vis spectroscopy, DSC and DLS studies about the interactions between NMD and QPPI (G3) dendrimer NMD in distilled water gives maximum absorbance at its characteristic wavelength (max 397 nm). UV–vis spectrometer was used to analyze the interaction between NMD and QPPI (G3)dendrimer. The saturated solutions obtained from the solubility studies were diluted to a proper concentration. Since the dendrimers in the diluted solutions give extremely low absorbance between 250 and 700 nm, the absorbance obtained from NMD-QPPI (G3) dendrimer solution would be solely from NMD. The absorbance of NMD at its characteristic wavelength was related with the amount/solubility of NMD in the dendrimer solutions or distilled water. The NMD-QPPI (G3)complex was prepared by freeze drying the saturated solutions of NMD obtained by solubility studies with 0.35 mM QPPI (G3). The interaction between NMD and QPPI (G3) was characterized by differential scanning calorimetry (DSC). DSC patterns of NMD, QPPI (G3) and NMD-QPPI (G3) complex were obtained. These samples (2–8 mg) were accurately weighed using electronic microbalance and heated in closed aluminum crimped cells at a rate of 10 ◦ C min−1 between 30 and 300 ◦ C temperature range under a nitrogen flow of 40 ml min−1 . The size of the dendrimers and their complexes with the drug were determined by DLS method. DLS was performed in triplicate with the sampling time set to automatic. NMD-QPPI (G3) complex was taken from the saturated solution of NMD obtained from the solubility studies with 0.35 mM QPPI (G3). Similarly, NMD-PPI (G3) complex was taken from the saturated solution of NMD obtained from the same concentration of PPI (G3). The size of the dendrimers without the drug was determined at the same concentration as in complex i.e., 0.35 mM.
124
E. Murugan et al. / Colloids and Surfaces B: Biointerfaces 114 (2014) 121–129
2.6. In vitro drug release studies
percentage of the untreated cell control. The obtained cell viability was plotted against the concentration of the dendrimer.
The extent of drug released from the dendrimer carriers i.e., PPI (G3) and QPPI (G3) in phosphate buffer solution (PBS) was studied by an equilibrium dialysis method under in vitro conditions [35]. The drug-dendrimer formulation for in vitro study was as follows, 2 mg of NMD was dissolved in 5 mL (0.2 mM) of each dendrimer carrier to get the corresponding formulation having drug concentration of 1.3 mM. Using this formulation, in vitro release study was performed by taking 5 mL of respective drug-dendrimer solution in dialysis bag (MW cutoff 1 k Da) and it was placed immediately in 150 mL beaker, containing 100 mL of PBS. The outer phase was stirred continuously to maintain sink conditions. At scheduled intervals, 3 mL of sample was withdrawn from outer phase and subsequently replenished with equal amount of fresh PBS. The withdrawn sample was analyzed spectrophotometrically monitoring the absorbance at characteristic wavelength of drug at 397 nm. The control experiment was also carried out by taking 2 mg of NMD in 5 mL of methanol to get the same drug concentration as in the drug-dendrimer formulations. This was also studied for in vitro release by usual UV–vis analysis. Three repeats were conducted for each sample. Irrespective of the dendrimer carrier and the control, plots were drawn for the percentage of drug released versus time. 2.7. Cytotoxicity assay The cytotoxicity of PPI (G3) and QPPI (G3) was evaluated by the well-established 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) colorimetric assay. Two cell lines viz., Vero (NCCS, passage numbers 14, 16 and 17) and HBL-100 (NCCS, passage numbers 4, 7 and 8) cell lines were chosen for the MTT colorimetric assay. The assay procedure involves the incubation of these two cell lines separately at 37 ◦ C in 5% CO2 and minimum essential medium (MEM) with Earle’s salts supplemented with 10% fetal calf serum. The cells were used when they reached 70–80% confluence. These cells were seeded in 96 well plates for 48 h before MTT assay to allow adherence of the cells to the plates. Then culture medium from the plates was replaced with fresh medium containing different concentrations 30 M to 1 mM of PPI (G3), QPPI (G3). The control study was performed by treating the cells only with the medium without the drug and the dendrimer samples. Following the standard protocol for MTT, the mediums were removed after 24 h from each of the well and the cells were incubated with MTT in PBS at a concentration of 1 mg/mL (100 L) for 4 h. After this, in each well the MTT containing medium was replaced with DMSO to dissolve the resulted purple formazan crystals which was generated from MTT by the living cells. Absorbance of the solutions in each well were recorded at 570 nm using a micro plate reader (Thermo multiscan EX). The viability of cells was calculated with regard to the untreated cell control, which was considered as 100% viability. The viability of cells treated with the different dendrimer is expressed as a
3. Results and discussion 3.1. Synthesis and characterization of QPPI (G3) dendrimer Earlier studies reveal that the commercial PAMAM and PPI dendrimers have been modified with surface functionalization using different moieties and explored the same for different applications and found convincing results. It is in this background, we have modified the commercial PPI (G3) in to QPPI (G3) via procedure followed in the earlier study. That is, the surface functionalization using glycidyltrimethyl ammonium chloride on PPI (G3) was performed by said procedure and known through characterization of the resultant product with spectral techniques like FTIR, 1 H and 13 C NMR and MALDI-TOF. The FTIR spectrum of QPPI (G3) dendrimer has characteristic peaks at 3398 cm−1 for O H (str) 2937 cm−1 and 2797 cm−1 for C H (str), 1488 cm−1 for C H (bend) and 1077 cm−1 for C N (str). 1 H NMR spectrum shows a sharp singlet at ␦ 3.270 with respect to N CH3 . 13 C NMR spectrum shows peaks at ␦ 47.283 corresponding to N CH3 . (All these spectra have been given in the supporting information as Figs. S1–S3.) MALDI-TOF spectrum shows characteristic peak corresponding to m/z value at 4458. All these results have confirmed the surface functionalization in PPI (G3) and thus prove that the QPPI (G3) containing quaternized poly(propylene imine) dendrimer and this was employed for drug delivery applications using drug NMD. 3.2. Solubility studies of NMD using QPPI (G3) dendrimer Drug solubility is one of the core factors, which influences the movement of a drug from the site of administration into the blood stream. It is widely acknowledged that insufficient drug solubility can lead to poor absorption [36]. The poor solubility of NMD can be addressed by employing PPI (G3) and QPPI (G3) dendrimers as solubility enhancers. Solubility studies were performed by keeping the seven different concentrations of PPI (G3) and QPPI (G3) individually in the range from 0.05 mM to 0.35 mM. NMD in double distilled water gives maximum absorbance at its characteristic wavelength (397 nm). On increasing the concentrations of PPI (G3) and QPPI (G3) the absorbance at characteristic peak were also observed to increase proportionately (Fig. 2a). The amount of solubilized drug was estimated in each concentration of dendrimer from the UV–vis absorption and the calibration curve. The results of solubility studies are given in Table 1. From the results of phase solubility studies, phase solubility diagrams were constructed by plotting drug solubility versus molar concentration of each dendrimer solution and shown in Fig. 2b. To record precisely, the solubility of drug in the presence of PPI (G3) dendrimer at the maximum concentration of 0.35 mM was increased up to 108 folds and
Table 1 Solubility of NMD in various concentrations of aqueous PPI (G3) and QPPI (G3) dendrimer solutions. S. no.
[Carrier] (×10−4 M)
1 2 3 4 5 6 7
0.50 1.00 1.50 2.00 2.50 3.00 3.50
[NMD] (×10−4 M)
Solubility increment, St /S0∗
Sol in mg/ml (St )
PPI (G3)
QPPI (G3)
PPI (G3)
QPPI (G3)
PPI (G3)
QPPI (G3)
6.84 9.51 13.5 20.9 25.5 29.3 35.2
5.854 8.048 10.45 12.47 16.22 19.89 23.78
0.211 0.292 0.416 0.643 0.785 0.902 1.084
0.180 0.248 0.322 0.384 0.499 0.613 0.733
21.1 29.2 41.6 64.3 78.5 90.2 108.4
18.03 24.79 32.19 38.40 49.96 61.29 73.26
S0∗ is the intrinsic solubility of NMD (0.01 mg/ml). Absorbance measured at max of NMD.
E. Murugan et al. / Colloids and Surfaces B: Biointerfaces 114 (2014) 121–129
125
Thereby, NMD gets internalized within the QPPI (G3) branches. This complexation substantially promoted the solubility of NMD. The increased solubility helps in increasing its bioavailability. On the other hand, Paleos et al. [28] have showed that the quaternised dendrimer has reduced the pyrene loading, as the dendrimer became hydrophilic and not to form the complex stably. We speculate the reason behind this might be due to the charged dendrimer and neutral pyrene molecule may have a poor interaction which in turn reflected in poor loading of pyrene in quaternized dendrimer. From the phase solubility diagram, it is understand that the aqueous solubility of NMD has been significantly increased in proportional to the concentration of both PPI (G3) and QPPI (G3). 3.3. NMR spectral studies about the interactions between NMD and QPPI (G3) dendrimer
Fig. 2. (a) UV–vis spectra of plain NMD (dotted line) and NMD with different QPPI (G3) concentrations (bold lines). (b) Solubility behavior of NMD at various concentrations of aqueous PPI (G3) and QPPI (G3) dendrimer solutions.
at the same concentration (0.35 mM) of the QPPI (G3), the solubility of NMD in aqueous medium has increased to the tune of 72 times as compared with its intrinsic solubility. This ensures that the water solubility of NMD has been significantly increased which was proportional to the concentration of both the dendrimer carriers. The dependence of NMD solubility on the dendrimer concentration in dendrimer solutions were described by the linear equations y = 9.81 ± 0.33x + 4.61 × 10−5 ± 7.01 × 10−5 (R2 = 0.9919) for PPI (G3) and y = 6.18 ± 0.22x + 4.76 × 10−5 ± 4.58 × 10−5 (R2 = 0.9913) for QPPI (G3). One can ascribe a physical sense to the coefficients of these straight line. The directional coefficient of these lines were respectively as 9.81 ± 0.33 and 6.18 ± 0.22 and these values in turn can be interpreted as number of drug molecules combined/bound by one molecule of dendrimer [37]. The number of drug molecules combined by a single PPI (G3) dendrimer was determined as 9.81 ± 0.33 and by QPPI (G3)dendrimer was determined as 6.18 ± 0.22. The drug loading can take place effectively due to sterically free surface interaction between the cationic NH2 group of PPI and the anionic part of drug (NMD) which in turn brings about more loading of drug or increased solubility of drug [38–40]. On the other hand, quite significant (but lesser than PPI (G3)) solubility increase in the presence of QPPI (G3) is due to (i) the availability of internal positive binding sites with neutral outer surface. (ii) The multi charged cations present in QPPI (G3) have attracted the anion of NMD whose formation is facilitated by the formation of HCl, a stronger acid in comparison with NMD and (iii) thus binds electrostatically with QPPI (G3) in aqueous medium. Therefore we hypothetically come to a conclusion that it formed corresponding complex viz., (NMD-N− SO2 CH3 )n (QPPI)n+ .
3.3.1. 1 H NMR studies It is well known that the 1 H NMR spectroscopy is a useful technique to investigate the intermolecular interactions in solutions because it gives information pertinent to the formation of aggregates, ion pairing, encapsulation, and size variations [41]. The 1 H NMR technique was employed here to investigate the molecular interactions between QPPI (G3) dendrimer and NMD drug molecules. The chemical shift assignment for each proton in the dendrimer, NMD and dendrimer-NMD complex is so critical because the shift of each signal is helpful to define the zone of interaction between the dendrimer and NMD [42]. Fig. 3 shows the 1 H NMR spectra and chemical shift assignments for QPPI (G3)/NMD complex. The protons of NMD and QPPI (G3) dendrimer were labeled with numbers 1–8 and characters a–g, respectively. It can be observed that NMD has scattered NMR peaks in the range of 0.5–8.5 ppm and that QPPI (G3) dendrimer has seven NMR peaks in D2 O in the range of 1.0–4.0 ppm. Having assigned the chemical shifts of the QPPI (G3) dendrimer and NMD in D2 O, we can analyze the complexation of the dendrimer and NMD by 1 H NMR. The QPPI (G3)-NMD complex has shown two types of peaks which correspond to NMD molecule and dendrimer scaffold respectively. It is observed that significant changes were noticed in chemical shifts of methylene protons (d–f) in QPPI (G3) dendrimer induced by the addition of NMD. The downfield chemical shift of these protons localized at the outermost layer of the QPPI-G3) dendrimer proves the ionic interactions between quaternised ammonium groups of the dendrimer and deprotonated group of NMD [43,44]. 3.3.2. 2D-NOSEY analysis It is well known that 2D NOESY technique is proved to be an effective method for providing insights into the molecular features of host-guest interactions [45–47]. It is capable of revealing a spatial relationship between nuclei in a molecule or in a complex of molecules [45]. If the host and guest are bound, they should be in close proximity to each other and NOE cross- peaks should be seen in the corresponding spectral region. In other words, the absence of a NOE cross-peak in the region can be used to rule out the interaction between related nuclei [47]. In this study, 1 H–1 H NOESY experiments were performed to determine the dipolar contacts for both intermolecular and intramolecular interactions of QPPI (G3) dendrimer with NMD. The 1 H–1 H NOESY spectrum of a QPPI (G3)/NMD/D2 O solution at a mixing time of 300 ms is shown in Fig. 4a. Strong NOE cross-peaks are observed between the methyl protons of NMD and scaffold protons of the QPPI (G3) dendrimer, which indicates close proximity between these protons. The partial 1 H–1 H NOESY spectrum (the region of the dendrimer proton signals with chemical shifts ranging from 1.0 to 4.0 ppm) for QPPI (G3) dendrimer-NMD complex (Fig. 4b) shows 7 peaks corresponding to three CH2 protons (a–c) in the interior of the dendrimer, three CH2 protons (d–f) and one CH3 protons (g) in the
126
E. Murugan et al. / Colloids and Surfaces B: Biointerfaces 114 (2014) 121–129
Fig. 3.
1
H NMR spectrum of the QPPI (G3)-NMD complex.
outermost layer of QPPI (G3) dendrimer and these observations are in accordance with the 1 H NMR spectrum of the QPPI (G3)-NMD complex. Several cross peaks were obtained between the protons of QPPI (G3) and the protons of NMD. Intermolecular NOE peaks such as (7-c), (7-a) and (7-f) as well as intramolecular NOE peaks similarly (f-c), (f-d) and (c-d) were noticed. In addition to this, some intra molecular cross peaks (2–3) and (1–4) were also observed and this in turn indicates the interaction between the aromatic protons of NMD. Therefore, all these observations strongly confirm the interactions of NMD molecules with the scaffold protons of QPPI (G3) dendrimer.
3.4. UV–vis spectroscopy, DSC and DLS studies about the interactions between NMD and QPPI (G3) dendrimer The use of dendrimer as a drug delivery carrier depends on its ability to form a complex with the drug. The ability of complex formation depends on the interaction between them. Interactions between NMD and QPPI (G3) dendrimer molecules were determined by UV–vis spectroscopy. NMD in double distilled water gives maximum absorbance at its characteristic wavelength (397 nm). After the addition of increasing concentrations of QPPI (G3) dendrimers, red shifts were observed (Fig. 2a) (the max peak shifted about 10–12 nm from 397 nm). In general, any shift in max suggests the existence of interaction between the complex forming components [48]. In this study electrostatic interaction between the anionic group of NMD and the cationic groups of QPPI (G3) dendrimer can explain this red shift. The occurrence of shift noticed in (max ) supports the formation of complex through electrostatic interaction. The thermal behavior of pure drug NMD, QPPI (G3) and QPPI (G3)-NMD complex was studied using differential scanning calorimetry in order to assess the formation of complex. The DSC thermogram of NMD exhibited an endothermic peak at 148 ◦ C corresponding to its melting point (Fig. 5a). Highly hygroscopic QPPI (G3) dendrimer has shown a broad endothermic peak at 100 ◦ C (Fig. 5b) corresponding to its dehydration. The thermogram of the complex QPPI (G3)-NMD contained only the peak of QPPI (G3), whereas the drug peak has almost disappeared (Fig. 5c). Previous reports suggest that disappearance of endothermal peak corresponding to the drug is a proof of interactions between the
components of the complex [49,50]. This proves strongly that the drug has complexed completely. The interaction of NMD with dendrimer was further investigated by measuring the size of the dendrimers and their complexes with NMD using DLS experiments. The size of PPI (G3) was measured as 7.1 nm. Whereas, the size of PPI (G3)-NMD was increased from 7.1 nm to 90.1 nm.The size of QPPI (G3) was 215.8 nm and for QPPI (G3)-NMD complex it was 235.5 nm. From these results it reveal that in the case of PPI (G3)-NMD complex, NMD molecules have attached to the surface whereas in the case of QPPI (G3)-NMD complex, they have bound to the internal positive charges without contributing much to the increase in size forming a compact polyplex. This observation is in agreement with the previous findings [51]. All the DLS figures are given in the supporting information (Fig. S4). 3.5. In vitro drug release studies The in vitro release of NMD from QPPI (G3) and PPI (G3) was explored in PBS medium. We investigated the release profiles of NMD through dendrimer based carriers such as PPI (G3) and QPPI (G3) to study the extent of drug release from drug-dendrimer formulations. The release pattern was plotted as percentage of drug released versus time and the same was shown in Fig. 6. From the plots it was noticed that, for control formulation, 90% of NMD was released within 80 min whereas, after 1 h 40.25% of drug was released from PPI (G3)-NMD formulations. In the case of QPPI (G3)NMD formulations, the drug release gets sustained to 16.35% in the same span of time. After 5 h, 90.32% of drug was released from PPI (G3)-NMD formulations whereas 35.69% of drug was released from QPPI (G3)-NMD formulation. These results suggested that the QPPI (G3) dendrimer significantly contributed for the sustained release of NMD than their parent dendrimer and control formulation. The complex between the dendrimer and the drug was formed via electrostatic interaction, which depends on the ionic strength of the medium and thus the formed complex was structurally more stable in PBS which reflected in slower release of the drug. In general, the sustained/delayed release of drug by any carrier is an indication of effective internalization of drug with the carrier or formation of stable complex due to the interaction between carrier and drug. Hence this observation strongly proves that the QPPI (G3) dendrimer is a potential carrier for sustained release of the drug NMD.
E. Murugan et al. / Colloids and Surfaces B: Biointerfaces 114 (2014) 121–129
127
Fig. 6. Release behavior of NMD in presence of PPI (G3) and QPPI (G3) dendrimers. NMD and dendrimer concentrations were 1.3 mM and 0.2 mM in dialysis bag respectively.
3.6. Cytotoxicity assay
Fig. 4. (a) 1 H–1 H NOESY spectrum of the QPPI (G3)/NMD/D2 O solution at a mixing rate of 300 ms. (b) Expanded region of the 1 H–1 H NOESY spectrum shown in (a) showing the cross-peaks between protons of drug and QPPI (G3). The cross-peaks are indicated by rectangles.
Fig. 5. DSC thermograms of (a) plain NMD, (b) QPPI (G3) and (c) QPPI (G3)-NMD freeze dried inclusion complex.
The effect of PPI (G3) and QPPI (G3) dendrimers on the viability of Vero and HBL-100 cell lines was measured by the MTT assay. The variation of percentage cell viability incubated in Vero cell line and HBL-100 cell line with different concentrations from 30 M to 1 mM of PPI (G3) and QPPI (G3) were assessed. The relationships of cell viability with incubated PPI (G3) and QPPI (G3) dendrimer concentrations on the above mentioned two cell lines are shown in Supporting information (Figs. S5 and S6). At a concentration of 0.0312 mM, it can be observed that QPPI (G3) exhibited much higher cell viability (94% Vero and 88% HBL-100). In any cell line, under in vitro conditions, the degree of cytotoxicity of a particular carrier usually been evaluated based on the IC50 values (concentrations where 50% of cells are viable). IC50 of QPPI (G3) is 0.56 mM on Vero cell line and 0.42 mM on HBL cell line. It is understand that although a maximum concentration of 1 mM, QPPI (G3) carrier were taken for study, the IC50 values are drastically increased than its corresponding parent dendrimer irrespective of cell lines. It was observed that, QPPI (G3) dendrimer was proved to be significantly less cytotoxic than its respective parent dendrimer on both cell lines, which reveals that QPPI (G3) is much more biocompatible
Fig. 7. Cytotoxicity of PPI (G3) and QPPI (G3) dendrimers at different concentrations after 24 h of incubation as determined by MTT assay on HBL-100 cell lines. Each data point represents mean ± standard error (S.E.) (n = 3).
128
E. Murugan et al. / Colloids and Surfaces B: Biointerfaces 114 (2014) 121–129
than PPI (G3) (Fig. 7). These results show that QPPI (G3) dendrimer can effectively increase the tolerance concentration during their administration. These observations indicate that the quaternised dendrimer is proved to be better carrier to deliver NMD owing to its less cytotoxic effect/more biocompatibility. Previously reported studies reveal that electrostatic interaction occurs between positively charged polymers and negatively charged cell membranes have resulted in more cytotoxic effect [51]. Particularly, cationic dendrimers containing NH2 surface group, are prone to attract the negatively charged cell membrane that in turn damaged the cell lines and thus reflected more cytotoxic effect [19]. Therefore, in any carrier the cationic surface group of the polymer/dendrimer must be neutralized so as to eliminate the electrostatic interaction of surface group with the negatively charged cell membranes and this in turn reduced the cytotoxicity [52–56]. In that line of ideas, QPPI (G3) is containing neutralized surface group as the cationic primary amine groups in PPI (G3) were functionalized with glycidyltrimethyl ammonium chloride. Even though glycidyltrimethyl ammonium chloride have a positively charged quaternary ammonium groups, the methyl groups exposed at their surface are able to screen the internal positive charges and thus behaved as a neutral molecule [51,56] thereby their electrostatic interaction with cell lines Vero and HBL-100 were significantly reduced and hence lower cytotoxicity was noticed than its corresponding parent dendrimer. In general, as far as drug delivery study is concerned, even though any molecule is capable of increased drug loading efficiency, but their sustained release of drug and free/lower cytotoxicity is the most significant factor than the efficiency of drug loading. In view of this background, in the present study, although the commercial PPI (G3) gives 108 times increased drug loading, but its efficiency of drug release was observed with 90% in 300 min and also the cytotoxicity is higher. In contrast the quaternized dendrimer viz., QPPI (G3), though efficiency of drug loading is relatively lesser (72 times) than the commercial PPI (G3) (108 times), but its ability to release the drug was found to be 35% at 300 min with significantly reduced cytotoxicity. This proves that QPPI (G3) dendrimer provide a potential platform to reduce the cytotoxicity which gives the pathway to increase the biocompatibility.
4. Conclusions Dendrimer based drug delivery system provides an attractive platform to enhance the drug solubility and thus act as drug carrier. Quaternised poly(propylene imine) dendrimer loaded with a drug for its delivery purpose was reported for the first time. In the present study, we prepared a quaternised poly(propylene imine) dendrimer viz., QPPI (G3) and utilized as drug carrier of the drug NMD. QPPI (G3) prepared by reacting surface amine groups of PPI (G3) dendrimer with glycidyltrimethyl ammonium chloride and the modification was established through various spectral techniques. This dendrimer was explored to investigate the drug carrying potential of NMD. At a concentration of 0.35 mM QPPI (G3), the solubility of NMD has been increased up to 72 folds in comparison with its intrinsic solubility. Whereas, at the same concentration, the commercial PPI (G3) increases the solubility of NMD up to 108 folds. Based on these solubility results, it is concluded that the dendrimers have showed significant drug solubilizing potential which in turn proves that they are the promising candidate for NMD loading. 1 H NMR and 2D NOSEY NMR analyses have also shown the complexation of NMD with QPPI (G3) dendrimer. Various additional evidences strengthen the fact that the complexation of NMD with QPPI (G3) was responsible for the observed solubility enhancement. It is a matter to be considered here that under in vitro conditions, QPPI (G3) is able to sustain the release of NMD considerably. This reflected in the magnitude of interaction between the
dendrimer carrier and the drug molecule. The studies demonstrate the promising strategy for per oral delivery of NMD. QPPI (G3) dendrimer showed increased cell viabilities on Vero and HBL-100 cell lines which was correlated with their reduced cytotoxicity over the parent dendrimer and hence it is more biocompatible. The increase in IC50 value for QPPI (G3) can effectively increase its tolerance concentration during the preparation of NMD-QPPI (G3)dendrimer formulations. Further, it is worth to highlight here that though the drug loading potential for QPPI (G3) is relatively lesser than with the commercial PPI (G3) dendrimer, but its potential for sustained release of NMD drug with reduced toxicity prove that the QPPI (G3) is one of the potential candidate for NMD delivery. Therefore, the QPPI (G3) is a vital molecule in the design of polymeric drug delivery systems to enhance the bioavailability of the poorly soluble drugs. Acknowledgements The authors gratefully acknowledge CSIR, New Delhi, Government of India, for financial assistance. One of the authors D.P. Geetha Rani would like to thank University of Madras and Govt. of TamilNadu for providing financial support in the form of Junior Research Fellowship (TNJRF). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.colsurfb.2013.10.002. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33]
S. Svenson, D.A. Tomalia, Adv. Drug Deliv. Rev. 57 (2005) 2106. E. Roseita, D.A. Tomalia, Drug Discovery Today 6 (2001) 427. A.K. Patri, I.J. Majoros, J.R. Baker, Curr. Opin. Chem. Biol. 6 (2002) 466. G.R. Newkome, C.N. Moorefield, F. Vögtle, Dendrimers and Dendrons: Concepts, Syntheses, Applications, Wiley-VCH, Weinheim, Germany, 2001. J.M.J. Frechet, D.A. Tomalia, Dendrimers and Other Dendritic Polymers, Wiley, UK, 2001. E.R. Gillies, J.M.J. Fréchet, Drug Discovery Today 10 (2005) 35. M.A. Mintzer, M.W. Grinstaff, Chem. Soc. Rev. 40 (2011) 173. D.W. Grainger, T. Okano, Adv. Drug Deliv. Rev. 55 (2003) 311. A. D’Emanuele, D. Attwood, Adv. Drug Deliv. Rev. 57 (2005) 2147. A.R. Menjoge, R.M. Kannan, D.A. Tomalia, Drug Discovery Today 15 (2010) 171. U. Gupta, H.B. Agashe, A. Asthana, N.K. Jain, Biomacromolecules 7 (3) (2006) 649. Y. Cheng, J. Wang, T. Rao, X. He, T. Xu, Front. Biosci. 13 (2008) 1447. A.K. Patri, J.F. Kukowska-Latallo, J.R. Baker, Adv. Drug Deliv. Rev. 57 (2005) 2203. S. Svenson, Eur. J. Pharm. Biopharm. 71 (2009) 445. K. Jain, P. Kesharwani, U. Gupta, N.K. Jain, Int. J. Pharm. 394 (1–2) (2010) 122. R. Duncan, L. Izzo, Adv. Drug Deliv. Rev. 57 (15) (2005) 2215. R. Jeyprasesphant, J. Penny, R. Jalal, D. Attwood, N.B. Mckeown, A. D’Emanuele, Int. J. Pharm. 252 (1–2) (2003) 263. P.E. Smith, J.R. Brender, U.H. Durr, J. Xu, D.G. Mullen, M.M. BanaszakHoll, A. Ramamoorthy, J. Am. Chem. Soc. 132 (23) (2010) 8087. Y. Zhang, Y. Sun, X. Xu, X. Zhang, H. Zhu, L. Huang, Y. Qi, Y.M. Shen, J. Med. Chem. 53 (8) (2010) 3262. I.J. Majoros, T.P. Thomas, C.B. Mehta, J.R. Baker, J. Med. Chem. 48 (2005) 5892. R. Roy, M.G. Baek, J. Biotechnol. 90 (3–4) (2002) 291. R. Kolhatkar, K. Kitchens, P. Swaan, H. Ghandehari, Bioconjug. Chem. 18 (6) (2007) 2054. Y. Zhang, Y. Sun, X. Xu, H. Zhu, L. Huang, X. Zhang, Y. Qi, Y.M. Shen, Bioorg. Med. Chem. Lett. 20 (3) (2010) 927. D.K. Smith, F. Diederich, Chem. Eur. J. 4 (1998) 1353. N. Jayaranam, S.A. Nepogodiev, J.F. Stoddart, Chem. Eur. J. 3 (1997) 1193. J.F.G.A. Jansen, H.W.I. Peerlings, M.M. de Brabander-Van den Berg, E.W. Meijer, Angew. Chem. Int. Ed. Engl. 34 (1995) 1206. N. Moszner, T. Volkel, V. Rheinberger, Macromol. Chem. Phys. 197 (2) (1996) 621. Z. Sideratou, D. Tsiourvas, C.M. Paleos, Langmuir 16 (2000) 1766. N. Davis, R.N. Brogden, Drugs 48 (1994) 431. G. Piel, B. Pirotte, I. Delneuville, P. Neven, G. Llabres, J. Delarge, L. Delattre, J. Pharm. Sci. 86 (1997) 475. P.R. Vavia, N.A. Adhage, Drug Dev. Ind. Pharm. 25 (1999) 543. J. Jinno, D.M. Oh, J.R. Crison, G.L. Amidon, J. Pharm. Sci. 89 (2000) 268. S. Mutalik, Venkatesh, N. Udupa, Indian J. Physiol. Pharmacol. 46 (2002) 115.
E. Murugan et al. / Colloids and Surfaces B: Biointerfaces 114 (2014) 121–129 [34] T. Higuchi, K.A. Conners, Phase-solubility Techniques, Advances in Analytical Chemistry and Instrumentation, John Wiley, NY, 1965. [35] C. Kojima, K. Kono, K. Maruyama, T. Takagishi, Bioconjug. Chem. 11 (6) (2000) 910. [36] Y.H. Zhao, M.H. Abraham, J. Le, A. Hersey, C.N. Luscombe, G. Beck, B. Sherborne, I. Cooper, Pharm. Res. 19 (2002) 1446. [37] W. Yang, Y. Li, Y. Cheng, Q. Wu, L. Wen, T. Xu, J. Pharm. Sci. 98 (2009) 1075. [38] Y.Y. Cheng, Q.L. Wu, Y.W. Li, T.W. Xu, J. Phys. Chem. B 112 (2008) 8884. [39] P. Sandhya, J. Mareddy, D.P. Geetha rani, P.V. Gollapudi, S. Pal, J. Braz. Chem. Soc. 18 (2007) 384. [40] S.K. Kulkarni, Curr. Sci. 83 (2002) 1442. [41] E. Biosselier, C. Ornelas, I. Pianet, J.R. Aranzaes, D. Astruc, Chem. Eur. J. 14 (2008) 5577. [42] M. Santo, M.A. Fox, J. Phys. Org. Chem. 12 (1999) 293. [43] M.A.C. Broeren, B.F.M. de Waal, M.H.P. van Genderen, H.M.H.F. Sanders, G. Fytas, E.W. Meijer, J. Am. Chem. Soc. 127 (2005) 10334. [44] U. Boas, S.H.M. Sontjens, K.J. Jensen, J.B. Christensen, M.H. Chai, Y.H. Niu, W.J. Youngs, P.L. Rinaldi, J. Am. Chem. Soc. 123 (2001) 4670. [45] M.H. Chai, Y.H. Niu, W.J. Youngs, P.L. Rinaldi, Macromolecules 33 (2000) 5395.
129
[46] M.H. Chai, A.K. Holley, M. Kruskamp, Chem. Commun. 168 (2007). [47] Y.Y. Cheng, Y.W. Li, Q.L. Wu, T.W. Xu, J. Phys. Chem. B 112 (2008) 41267. [48] Y. Cheng, H. Qu, M. Ma, Z. Xu, P. Xu, Y. Fang, T. Xu, Eur. J. Med. Chem. 42 (2007) 1032. [49] T. Prahlad, K. Rajendrakumar, J. Pharm. Biomed. Anal. 34 (2003) 333. [50] M. Panda, G. Patro, A. Malpani, M.E. Rao, Int. J. Pharm. Sci. Rev. Res. 9 (2011) 173. [51] J.H. Lee, Y.B. Lim, J.S. Choi, Y. Lee, T. Kim, H.J. Kim, J.K. Yoon, K. Kim, J.S. Park, Bioconjug. Chem. 14 (2003) 1214. [52] N. Malik, R. Wiwattanapatapee, R. Klopsch, K. Lorenz, H. Frey, J.W. Weener, E.W. Meijer, W. Paulus, R. Duncan, J. Control. Release 65 (2000) 133. [53] P.Y. Vuillaume, M. Brunelle, M.R. Van Calsteren, S. Laurent-Lewandowski, A. Begin, R. Lewandowski, B.G. Talbot, Y. Elazhary, Biomacromolecules 6 (3) (2005) 1769. [54] C.K. Poh, Z. Shi, T.Y. Lim, K.G. Neoh, W. Wang, Biomaterials 31 (2010) 1578. [55] R.B. Kolhatkar, K.M. Kitchens, P.W. Swaan, H. Ghandehari, Bioconjug. Chem. 18 (2007) 2054. [56] M.L. Patil, M. Zhang, S. Betigeri, O. Taratula, H.X. He, T. Minko, Bioconjug. Chem. 19 (2008) 1396.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Drug delivery investigations of quaternised poly(propylene imine) dendrimer using nimesulide as a model drug E. Murugan ∗ , D.P. Geetha Rani, V. Yogaraj Department of Physical Chemistry, School of Chemical Sciences, University of Madras, Maraimalai Campus, Guindy, Chennai 600 025, TamilNadu, India
a r t i c l e
i n f o
Article history: Received 19 June 2013 Received in revised form 25 September 2013 Accepted 2 October 2013 Available online 12 October 2013 Keywords: Poly(propylene imine) dendrimer Drug carrier Nimesulide Solubility In vitro release Cytotoxicity
a b s t r a c t This study describes the demonstration of quaternized poly(propylene imine) dendrimer of generation3, QPPI (G3) as a drug carrier for poorly soluble drug nimesulide (NMD, an anti-inflammatory drug). QPPI (G3) was prepared by treating the surface amine groups of poly(propylene imine) dendrimer with glycidyltrimethyl ammonium chloride and it was characterized with FTIR, 1 H and 13 C NMR and MALDITOF mass spectral techniques. The drug carrying potential of QPPI (G3) was assessed by analyzing drug solubility, in vitro release and cytotoxicity studies. The observed results reveal that the aqueous solubility of NMD has been dramatically increased in the presence of QPPI (G3) and also can sustain the release of NMD. It is further noticed that the complexation of NMD with QPPI (G3) is responsible for increased solubility and sustained release. This complexation was evidenced through NMR (1 H & 2D) and UV–vis spectral techniques, DSC and DLS studies. Cytotoxicity study through MTT assay on Vero and HBL-100 cell lines reveal that this dendrimer increase the biocompatibility and the tolerance concentration of NMD in drug-dendrimer formulations. The observed results prove that the QPPI (G3) is one of the new promising candidate for effective delivery of NMD. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Dendrimers are outstanding candidates to act as drug carriers due to their well-defined nanostructures and chemical versatility [1,2]. As compared to the traditional polymeric drug vehicles, dendrimer shows excellent advantages more especially for drug delivery [3]. Their surface functionalities and monodispersity have enabled them as effective devices for pharmaceutical and biomedical applications [4–7]. Surface functionalities of dendrimers can be functionalized with drugs, targeting moieties and biologically active components [8]. Drugs or genes can be either encapsulated within the dendrimers through non-covalent strategies such as hydrophobic, ionic and hydrogen bond interactions or conjugated to the peripheral groups of dendrimers via covalent methods [9]. Poly(amidoamine) (PAMAM) and poly(propylene imine) (PPI) dendrimers are the extensively used ones among the numerous dendrimers used for drug delivery [10]. A bountiful reports are available which evidence for the drug carrying potential of dendrimers through properties like increased solubility of the hydrophobic drugs, sustained drug release behavior and increased efficiency of the drugs [11–13]. Dendrimer based drug-delivery
∗ Corresponding author. Tel.: +91 44 22202818/22202819; fax: +91 44 22352494. E-mail addresses: [email protected], emurugan [email protected] (E. Murugan). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.10.002
systems provide an attractive platform for loading and release of conventional drug molecules which improve the pharmacodynamic and pharmacokinetic behaviors of several families of drugs [14]. These in turn reveal the promising future of dendrimer based drug delivery systems. Dendrimers such as PAMAM and PPI possess cationic primary amine groups at the surface. Even though they showed excellent drug delivery efficacy, but their cytotoxicity is still a burning issue that limits the clinical applications of most these dendrimer based drug formulations [15,16]. They exhibited high cytotoxicity on numerous cell lines and serious hemolytic activity on red blood cells which is dependent on dendrimer generation, surface functionality/charge and concentration [17]. Cationic groups on dendrimer surface are the major determinant factor in generating cytotoxicity and hemolytic activity. These cationic charges interact with the phosphates on the cell membrane which leads to disturbance of lipid bilayers resulting in leakage of intracellular components [18]. In addition, the surface cationic dendrimers are rapidly cleared from the blood circulation systems which result in limited therapeutic efficacy and bioavailability of the administered drug-dendrimer formulation. In order to overcome this problem, the cationic charges on dendrimer surface have been neutralized through PEGylation, hydroxylation, glycosylation and acetylation and thereby reduced the cytotoxicity and hemolytic activity of cationic dendrimers [19–23]. Following the intensive research that has been conducted on the synthesis of dendrimeric compounds, several studies have been appeared by
122
E. Murugan et al. / Colloids and Surfaces B: Biointerfaces 114 (2014) 121–129
(a)
(b)
Fig. 1. (a) Synthetic route to get QPPI (G3) dendrimer from PPI (G3) dendrimer. (b) Structure of nimesulide (NMD).
focusing on the functionalization of the external groups of basic dendrimers. A diversity of functionalized dendrimers [24] has been prepared including those bearing carbohydrate moieties [25], chiral groups [26] and polymerizable [27] groups. The functionalized groups impart dendrimers the property to organize at the surfaces or to form organized supramolecular structures. Moreover, increased surface density and charge density provide potential applications by enhancing the solubility of low aqueous soluble drugs in aqueous medium and as delivery system for bioactive materials. Paleos et al. have modified the surface amine groups of poly(propyleneimine) dendrimer of generation 5 using glycidyltrimethyl ammonium chloride and thus obtained quaternised poly(propylene imine) (QPPI) dendrimer and this was in turn studied for the loading and release behaviors using pyrene as a guest molecule [28].Further it is worth to mention here that the QPPI dendrimer is expected to serve as an efficient drug delivery. Therefore to the best of our knowledge there are no studies devoted exclusively for the synthesis of dendrimer containing QPPI dendrimers of lower generation for the effective delivery of poorly soluble drugs in aqueous medium. This may emerge as promising candidate for safer and more effective compared to traditional drug delivery system. Nimesulide (NMD) is a non-steroidal anti-inflammatory drug (NSAID) with analgesic and antipyretic properties. It is chemically N-(4-nitro-2-phenoxyphenyl) methane sulfonamide, is a weakly acidic non-steroidal anti- inflammatory drug (Fig. 1b). It differs from other non-steroidal anti- inflammatory drugs (NSAIDs) in that its chemical structure contains a sulfoanilide moiety as the acidic group. Unlike other classical NSAIDs, it has high gastrotolerability due to its relatively high pKa value (6.5) and preferential cyclooxygenase (COX)-2 selectivity. It is mainly prescribed for the treatment of acute pain, the symptomatic treatment of osteoarthritis [29]. However, it’s poor solubility in aqueous medium (0.01 mg/ml) [30] gives rise to difficulties in the design of pharmaceutical formulations and leads to variable oral bioavailability. This restricts their use in topical and parenteral applications. Further, poorly
water-soluble drugs often require high doses in order to reach therapeutic plasma concentrations after oral administration. Hence to increase the therapeutic efficiency of poorly soluble drugs, its solubility in aqueous medium has to be increased. Previous reports say that in order to improve the aqueous solubility of NMD and for its effective delivery various methods were employed such as cyclodextrin-nimesulide complexation [31], micellar solubilization [32] and solid dispersion [33]. In this study, quaternised poly(propylene imine) dendrimer of generation 3, QPPI (G3) dendrimer was produced by the reaction of surface amine groups of poly(propylene imine) dendrimer of generation 3,PPI (G3) with glycidyltrimethyl ammonium chloride and the performance of this dendrimer was evaluated for its application as drug delivery system using nimesulide as a model drug. 2. Materials and methods 2.1. Materials PPI dendrimer Diamino cored (DAB) with terminal amino groups of generation 3 was purchased from Symo Chem (Netherlands) and used as received. Glycidyltrimethyl ammonium chloride was obtained from Sigma–Aldrich and methanol was obtained from SRL.MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) was purchased from Sigma–Aldrich, India. Dialysis membranes (Float-A-Lyzer) were obtained from Spectra Pore. Membrane filter of pore size 0.45 m was obtained from Himedia lab. Double distilled water was used for solubility and in vitro release studies. Vero cell lineage was isolated from kidney epithelial cells, extracted from an African green monkey. HBL-100 cell line was derived from human breast epithelial cells. Nimesulide was obtained as gift sample from Micro labs, Bangalore, India. UV–vis spectra were recorded on TECHCOMP 8500 instrument. Fourier Transform Infrared (FTIR) spectra were recorded on a Bruker Tensor-27 FTIR spectrometer. 1 H, 13 C and 2D NMR spectra
E. Murugan et al. / Colloids and Surfaces B: Biointerfaces 114 (2014) 121–129
were recorded with Bruker 500 MHz UltraShield Plus instrument. MALDI-TOF spectra were taken in perceptive bio systems voyagerDE instrument in negative ion mode taking Dithranol as the matrix. A Perkin-Elmer DSC model 7 was used for recording DSC thermograms. Dynamic Light Scattering (DLS) experiment was done using BI-200SM Goniometer with a Lexel laser model 95 argon laser (100 mW output power at a wavelength of 514.5 nm). Correlator, PD2000 was used and the scattering angle was 90◦ . Cytotoxicity study was done in King Institute of Preventive Medicine, Chennai, India.
123
equation, y = (15,602 ± 0.006)x (R2 = 0.999). The directional coefficient of the equation is equal to the absorption coefficient of NMD, εmax = 15,602 M−1 cm−1 . Using this calibration curve, the amount of NMD solubilized in each dendrimer solution was determined. The blank was performed on the same concentrations of dendrimer delivery solutions so as to cancel any absorbance that may be exhibited by the dendrimer molecules. The solubility experiments were conducted repeatedly three times so as to ensure the concordant values. From the results of phase solubility studies, phase solubility diagrams were constructed by plotting drug solubility versus molar concentration of each dendrimer solutions.
2.2. Synthesis and characterization of QPPI (G3) dendrimer Quaternised poly(propylene imine) dendrimer, QPPI (G3) was synthesized by reacting the surface amine groups of generation 3 poly(propylene imine), PPI (G3) with glycidyl trimethyl ammonium chloride using the reported procedure [28]. Glycidyltrimethyl ammonium chloride is a simple, soluble, low molecular weight, quaternary ammonium salt. Further, since it exists as an epoxy hydro chloride it can easily react with primary amines of PPI (G3) through ring opening reactions. Even weak nucleophile is sufficient to open the epoxide ring compared to other leaving groups. More specifically, the epoxide ring opening in glycidyltrimethyl ammonium chloride may quickly occur even at room temperature than any other electrophilic carbon center. The typical procedure involves by taking PPI (G3) (0.1 g, 0.0601 mmol) in a 50 mL round bottom flask and was dissolved in water. To this an aqueous solution of glycidyltrimethyl ammonium chloride (0.049 g, 0.321 mmol) was added. The mixture was allowed to react for 24 h at room temperature and subsequently subjected to dialysis with the appropriate membrane in order to remove the excess of glycidyltrimethyl ammonium chloride and possibly unreacted dendrimers. The dialyzed solution was lyophilized to get quaternized poly(propylene imine) dendrimer QPPI (G3) (Fig. 1a). This product was characterized by FTIR, 1 H & 13 C NMR and MALDI-TOF spectral techniques. The degree of functionalization of the product was determined by 1 H NMR and MALDI-TOF spectral techniques and the newly prepared quaternised dendrimer was assessed for its drug carrying potential using NMD as a model drug. 2.3. Solubility study of NMD using QPPI (G3) dendrimer The solubility of NMD was quantitatively estimated by adopting the previously described phase solubility analysis method [34]. Both PPI (G3) and QPPI (G3) dendrimers were employed individually and studied the solubility of drug NMD. The degree of solubility of the drug was measured in aqueous solutions with increasing concentrations of both PPI (G3) and QPPI (G3) separately. That is, seven different concentrations of drug carriers PPI (G3) and QPPI (G3) were prepared in the range from 0.05 mM to 0.35 mM. Then from each concentration, 5 mL was taken in 20 mL vials and to which excess NMD (5 mg) was added. These vials were sonicated for 30 min and subsequently shaken mechanically for 24 h at 35 ◦ C. Parallely controlled experiment (without dendrimer) was also performed by taking 5 mg of drug in 5 mL of double distilled water. The vials were allowed to stand for 24 h to attain equilibrium. Then these solutions were centrifuged at 5000 rpm for 20 min and filtered using cellulose acetate membrane filter having pore size 0.45 m. The respective filtrate was diluted appropriately with double distilled water and analyzed using UV–vis spectrophotometer. The amount of NMD solubilized in aqueous medium in the presence of drug carriers viz., PPI (G3) and QPPI (G3) was estimated by measuring the characteristic absorbance observed at 397 nm (max ). The quantitative estimation of NMD was done using the calibration curve constructed within the drug concentration range of 3.47 M to 34.7 M which is described by the
2.4. NMR spectral studies about the interactions between NMD and QPPI (G3) dendrimer 2.4.1. 1 H NMR studies 1 H NMR experiments were conducted on a Bruker Advance 500.149 MHz NMR spectrometer at 298.2 K for QPPI (G3) dendrimer/NMD complex (2 mg of QPPI-(G3) and 1 mg of NMD dissolved in 1 mL of D2 O). The temperature was kept constant within (0.2 K) by the use of a Bruker temperature control unit. 2.4.2. 2D NMR studies NOESY experiments were obtained for a QPPI (G3) dendrimer/NMD solution (2 mg of QPPI (G3) and 1 mg of NMD dissolved in 1 mL of D2 O) acquired at 500.149 MHz, using a 300 ms mixing time and a 8.2 s 1 H 90◦ pulse width. The experiments were done with a 2 s relaxation delay and 205 ms acquisition time. Eight transients were averaged for each 400 × 1024 complex t1 increment. The data were processed with Lorentz–Gauss window function and zero filling in both dimensions to display data on a 2048 × 2048 2D matrix. All data were processed with NMRPipe software on a Linux workstation. 2.5. UV–vis spectroscopy, DSC and DLS studies about the interactions between NMD and QPPI (G3) dendrimer NMD in distilled water gives maximum absorbance at its characteristic wavelength (max 397 nm). UV–vis spectrometer was used to analyze the interaction between NMD and QPPI (G3)dendrimer. The saturated solutions obtained from the solubility studies were diluted to a proper concentration. Since the dendrimers in the diluted solutions give extremely low absorbance between 250 and 700 nm, the absorbance obtained from NMD-QPPI (G3) dendrimer solution would be solely from NMD. The absorbance of NMD at its characteristic wavelength was related with the amount/solubility of NMD in the dendrimer solutions or distilled water. The NMD-QPPI (G3)complex was prepared by freeze drying the saturated solutions of NMD obtained by solubility studies with 0.35 mM QPPI (G3). The interaction between NMD and QPPI (G3) was characterized by differential scanning calorimetry (DSC). DSC patterns of NMD, QPPI (G3) and NMD-QPPI (G3) complex were obtained. These samples (2–8 mg) were accurately weighed using electronic microbalance and heated in closed aluminum crimped cells at a rate of 10 ◦ C min−1 between 30 and 300 ◦ C temperature range under a nitrogen flow of 40 ml min−1 . The size of the dendrimers and their complexes with the drug were determined by DLS method. DLS was performed in triplicate with the sampling time set to automatic. NMD-QPPI (G3) complex was taken from the saturated solution of NMD obtained from the solubility studies with 0.35 mM QPPI (G3). Similarly, NMD-PPI (G3) complex was taken from the saturated solution of NMD obtained from the same concentration of PPI (G3). The size of the dendrimers without the drug was determined at the same concentration as in complex i.e., 0.35 mM.
124
E. Murugan et al. / Colloids and Surfaces B: Biointerfaces 114 (2014) 121–129
2.6. In vitro drug release studies
percentage of the untreated cell control. The obtained cell viability was plotted against the concentration of the dendrimer.
The extent of drug released from the dendrimer carriers i.e., PPI (G3) and QPPI (G3) in phosphate buffer solution (PBS) was studied by an equilibrium dialysis method under in vitro conditions [35]. The drug-dendrimer formulation for in vitro study was as follows, 2 mg of NMD was dissolved in 5 mL (0.2 mM) of each dendrimer carrier to get the corresponding formulation having drug concentration of 1.3 mM. Using this formulation, in vitro release study was performed by taking 5 mL of respective drug-dendrimer solution in dialysis bag (MW cutoff 1 k Da) and it was placed immediately in 150 mL beaker, containing 100 mL of PBS. The outer phase was stirred continuously to maintain sink conditions. At scheduled intervals, 3 mL of sample was withdrawn from outer phase and subsequently replenished with equal amount of fresh PBS. The withdrawn sample was analyzed spectrophotometrically monitoring the absorbance at characteristic wavelength of drug at 397 nm. The control experiment was also carried out by taking 2 mg of NMD in 5 mL of methanol to get the same drug concentration as in the drug-dendrimer formulations. This was also studied for in vitro release by usual UV–vis analysis. Three repeats were conducted for each sample. Irrespective of the dendrimer carrier and the control, plots were drawn for the percentage of drug released versus time. 2.7. Cytotoxicity assay The cytotoxicity of PPI (G3) and QPPI (G3) was evaluated by the well-established 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) colorimetric assay. Two cell lines viz., Vero (NCCS, passage numbers 14, 16 and 17) and HBL-100 (NCCS, passage numbers 4, 7 and 8) cell lines were chosen for the MTT colorimetric assay. The assay procedure involves the incubation of these two cell lines separately at 37 ◦ C in 5% CO2 and minimum essential medium (MEM) with Earle’s salts supplemented with 10% fetal calf serum. The cells were used when they reached 70–80% confluence. These cells were seeded in 96 well plates for 48 h before MTT assay to allow adherence of the cells to the plates. Then culture medium from the plates was replaced with fresh medium containing different concentrations 30 M to 1 mM of PPI (G3), QPPI (G3). The control study was performed by treating the cells only with the medium without the drug and the dendrimer samples. Following the standard protocol for MTT, the mediums were removed after 24 h from each of the well and the cells were incubated with MTT in PBS at a concentration of 1 mg/mL (100 L) for 4 h. After this, in each well the MTT containing medium was replaced with DMSO to dissolve the resulted purple formazan crystals which was generated from MTT by the living cells. Absorbance of the solutions in each well were recorded at 570 nm using a micro plate reader (Thermo multiscan EX). The viability of cells was calculated with regard to the untreated cell control, which was considered as 100% viability. The viability of cells treated with the different dendrimer is expressed as a
3. Results and discussion 3.1. Synthesis and characterization of QPPI (G3) dendrimer Earlier studies reveal that the commercial PAMAM and PPI dendrimers have been modified with surface functionalization using different moieties and explored the same for different applications and found convincing results. It is in this background, we have modified the commercial PPI (G3) in to QPPI (G3) via procedure followed in the earlier study. That is, the surface functionalization using glycidyltrimethyl ammonium chloride on PPI (G3) was performed by said procedure and known through characterization of the resultant product with spectral techniques like FTIR, 1 H and 13 C NMR and MALDI-TOF. The FTIR spectrum of QPPI (G3) dendrimer has characteristic peaks at 3398 cm−1 for O H (str) 2937 cm−1 and 2797 cm−1 for C H (str), 1488 cm−1 for C H (bend) and 1077 cm−1 for C N (str). 1 H NMR spectrum shows a sharp singlet at ␦ 3.270 with respect to N CH3 . 13 C NMR spectrum shows peaks at ␦ 47.283 corresponding to N CH3 . (All these spectra have been given in the supporting information as Figs. S1–S3.) MALDI-TOF spectrum shows characteristic peak corresponding to m/z value at 4458. All these results have confirmed the surface functionalization in PPI (G3) and thus prove that the QPPI (G3) containing quaternized poly(propylene imine) dendrimer and this was employed for drug delivery applications using drug NMD. 3.2. Solubility studies of NMD using QPPI (G3) dendrimer Drug solubility is one of the core factors, which influences the movement of a drug from the site of administration into the blood stream. It is widely acknowledged that insufficient drug solubility can lead to poor absorption [36]. The poor solubility of NMD can be addressed by employing PPI (G3) and QPPI (G3) dendrimers as solubility enhancers. Solubility studies were performed by keeping the seven different concentrations of PPI (G3) and QPPI (G3) individually in the range from 0.05 mM to 0.35 mM. NMD in double distilled water gives maximum absorbance at its characteristic wavelength (397 nm). On increasing the concentrations of PPI (G3) and QPPI (G3) the absorbance at characteristic peak were also observed to increase proportionately (Fig. 2a). The amount of solubilized drug was estimated in each concentration of dendrimer from the UV–vis absorption and the calibration curve. The results of solubility studies are given in Table 1. From the results of phase solubility studies, phase solubility diagrams were constructed by plotting drug solubility versus molar concentration of each dendrimer solution and shown in Fig. 2b. To record precisely, the solubility of drug in the presence of PPI (G3) dendrimer at the maximum concentration of 0.35 mM was increased up to 108 folds and
Table 1 Solubility of NMD in various concentrations of aqueous PPI (G3) and QPPI (G3) dendrimer solutions. S. no.
[Carrier] (×10−4 M)
1 2 3 4 5 6 7
0.50 1.00 1.50 2.00 2.50 3.00 3.50
[NMD] (×10−4 M)
Solubility increment, St /S0∗
Sol in mg/ml (St )
PPI (G3)
QPPI (G3)
PPI (G3)
QPPI (G3)
PPI (G3)
QPPI (G3)
6.84 9.51 13.5 20.9 25.5 29.3 35.2
5.854 8.048 10.45 12.47 16.22 19.89 23.78
0.211 0.292 0.416 0.643 0.785 0.902 1.084
0.180 0.248 0.322 0.384 0.499 0.613 0.733
21.1 29.2 41.6 64.3 78.5 90.2 108.4
18.03 24.79 32.19 38.40 49.96 61.29 73.26
S0∗ is the intrinsic solubility of NMD (0.01 mg/ml). Absorbance measured at max of NMD.
E. Murugan et al. / Colloids and Surfaces B: Biointerfaces 114 (2014) 121–129
125
Thereby, NMD gets internalized within the QPPI (G3) branches. This complexation substantially promoted the solubility of NMD. The increased solubility helps in increasing its bioavailability. On the other hand, Paleos et al. [28] have showed that the quaternised dendrimer has reduced the pyrene loading, as the dendrimer became hydrophilic and not to form the complex stably. We speculate the reason behind this might be due to the charged dendrimer and neutral pyrene molecule may have a poor interaction which in turn reflected in poor loading of pyrene in quaternized dendrimer. From the phase solubility diagram, it is understand that the aqueous solubility of NMD has been significantly increased in proportional to the concentration of both PPI (G3) and QPPI (G3). 3.3. NMR spectral studies about the interactions between NMD and QPPI (G3) dendrimer
Fig. 2. (a) UV–vis spectra of plain NMD (dotted line) and NMD with different QPPI (G3) concentrations (bold lines). (b) Solubility behavior of NMD at various concentrations of aqueous PPI (G3) and QPPI (G3) dendrimer solutions.
at the same concentration (0.35 mM) of the QPPI (G3), the solubility of NMD in aqueous medium has increased to the tune of 72 times as compared with its intrinsic solubility. This ensures that the water solubility of NMD has been significantly increased which was proportional to the concentration of both the dendrimer carriers. The dependence of NMD solubility on the dendrimer concentration in dendrimer solutions were described by the linear equations y = 9.81 ± 0.33x + 4.61 × 10−5 ± 7.01 × 10−5 (R2 = 0.9919) for PPI (G3) and y = 6.18 ± 0.22x + 4.76 × 10−5 ± 4.58 × 10−5 (R2 = 0.9913) for QPPI (G3). One can ascribe a physical sense to the coefficients of these straight line. The directional coefficient of these lines were respectively as 9.81 ± 0.33 and 6.18 ± 0.22 and these values in turn can be interpreted as number of drug molecules combined/bound by one molecule of dendrimer [37]. The number of drug molecules combined by a single PPI (G3) dendrimer was determined as 9.81 ± 0.33 and by QPPI (G3)dendrimer was determined as 6.18 ± 0.22. The drug loading can take place effectively due to sterically free surface interaction between the cationic NH2 group of PPI and the anionic part of drug (NMD) which in turn brings about more loading of drug or increased solubility of drug [38–40]. On the other hand, quite significant (but lesser than PPI (G3)) solubility increase in the presence of QPPI (G3) is due to (i) the availability of internal positive binding sites with neutral outer surface. (ii) The multi charged cations present in QPPI (G3) have attracted the anion of NMD whose formation is facilitated by the formation of HCl, a stronger acid in comparison with NMD and (iii) thus binds electrostatically with QPPI (G3) in aqueous medium. Therefore we hypothetically come to a conclusion that it formed corresponding complex viz., (NMD-N− SO2 CH3 )n (QPPI)n+ .
3.3.1. 1 H NMR studies It is well known that the 1 H NMR spectroscopy is a useful technique to investigate the intermolecular interactions in solutions because it gives information pertinent to the formation of aggregates, ion pairing, encapsulation, and size variations [41]. The 1 H NMR technique was employed here to investigate the molecular interactions between QPPI (G3) dendrimer and NMD drug molecules. The chemical shift assignment for each proton in the dendrimer, NMD and dendrimer-NMD complex is so critical because the shift of each signal is helpful to define the zone of interaction between the dendrimer and NMD [42]. Fig. 3 shows the 1 H NMR spectra and chemical shift assignments for QPPI (G3)/NMD complex. The protons of NMD and QPPI (G3) dendrimer were labeled with numbers 1–8 and characters a–g, respectively. It can be observed that NMD has scattered NMR peaks in the range of 0.5–8.5 ppm and that QPPI (G3) dendrimer has seven NMR peaks in D2 O in the range of 1.0–4.0 ppm. Having assigned the chemical shifts of the QPPI (G3) dendrimer and NMD in D2 O, we can analyze the complexation of the dendrimer and NMD by 1 H NMR. The QPPI (G3)-NMD complex has shown two types of peaks which correspond to NMD molecule and dendrimer scaffold respectively. It is observed that significant changes were noticed in chemical shifts of methylene protons (d–f) in QPPI (G3) dendrimer induced by the addition of NMD. The downfield chemical shift of these protons localized at the outermost layer of the QPPI-G3) dendrimer proves the ionic interactions between quaternised ammonium groups of the dendrimer and deprotonated group of NMD [43,44]. 3.3.2. 2D-NOSEY analysis It is well known that 2D NOESY technique is proved to be an effective method for providing insights into the molecular features of host-guest interactions [45–47]. It is capable of revealing a spatial relationship between nuclei in a molecule or in a complex of molecules [45]. If the host and guest are bound, they should be in close proximity to each other and NOE cross- peaks should be seen in the corresponding spectral region. In other words, the absence of a NOE cross-peak in the region can be used to rule out the interaction between related nuclei [47]. In this study, 1 H–1 H NOESY experiments were performed to determine the dipolar contacts for both intermolecular and intramolecular interactions of QPPI (G3) dendrimer with NMD. The 1 H–1 H NOESY spectrum of a QPPI (G3)/NMD/D2 O solution at a mixing time of 300 ms is shown in Fig. 4a. Strong NOE cross-peaks are observed between the methyl protons of NMD and scaffold protons of the QPPI (G3) dendrimer, which indicates close proximity between these protons. The partial 1 H–1 H NOESY spectrum (the region of the dendrimer proton signals with chemical shifts ranging from 1.0 to 4.0 ppm) for QPPI (G3) dendrimer-NMD complex (Fig. 4b) shows 7 peaks corresponding to three CH2 protons (a–c) in the interior of the dendrimer, three CH2 protons (d–f) and one CH3 protons (g) in the
126
E. Murugan et al. / Colloids and Surfaces B: Biointerfaces 114 (2014) 121–129
Fig. 3.
1
H NMR spectrum of the QPPI (G3)-NMD complex.
outermost layer of QPPI (G3) dendrimer and these observations are in accordance with the 1 H NMR spectrum of the QPPI (G3)-NMD complex. Several cross peaks were obtained between the protons of QPPI (G3) and the protons of NMD. Intermolecular NOE peaks such as (7-c), (7-a) and (7-f) as well as intramolecular NOE peaks similarly (f-c), (f-d) and (c-d) were noticed. In addition to this, some intra molecular cross peaks (2–3) and (1–4) were also observed and this in turn indicates the interaction between the aromatic protons of NMD. Therefore, all these observations strongly confirm the interactions of NMD molecules with the scaffold protons of QPPI (G3) dendrimer.
3.4. UV–vis spectroscopy, DSC and DLS studies about the interactions between NMD and QPPI (G3) dendrimer The use of dendrimer as a drug delivery carrier depends on its ability to form a complex with the drug. The ability of complex formation depends on the interaction between them. Interactions between NMD and QPPI (G3) dendrimer molecules were determined by UV–vis spectroscopy. NMD in double distilled water gives maximum absorbance at its characteristic wavelength (397 nm). After the addition of increasing concentrations of QPPI (G3) dendrimers, red shifts were observed (Fig. 2a) (the max peak shifted about 10–12 nm from 397 nm). In general, any shift in max suggests the existence of interaction between the complex forming components [48]. In this study electrostatic interaction between the anionic group of NMD and the cationic groups of QPPI (G3) dendrimer can explain this red shift. The occurrence of shift noticed in (max ) supports the formation of complex through electrostatic interaction. The thermal behavior of pure drug NMD, QPPI (G3) and QPPI (G3)-NMD complex was studied using differential scanning calorimetry in order to assess the formation of complex. The DSC thermogram of NMD exhibited an endothermic peak at 148 ◦ C corresponding to its melting point (Fig. 5a). Highly hygroscopic QPPI (G3) dendrimer has shown a broad endothermic peak at 100 ◦ C (Fig. 5b) corresponding to its dehydration. The thermogram of the complex QPPI (G3)-NMD contained only the peak of QPPI (G3), whereas the drug peak has almost disappeared (Fig. 5c). Previous reports suggest that disappearance of endothermal peak corresponding to the drug is a proof of interactions between the
components of the complex [49,50]. This proves strongly that the drug has complexed completely. The interaction of NMD with dendrimer was further investigated by measuring the size of the dendrimers and their complexes with NMD using DLS experiments. The size of PPI (G3) was measured as 7.1 nm. Whereas, the size of PPI (G3)-NMD was increased from 7.1 nm to 90.1 nm.The size of QPPI (G3) was 215.8 nm and for QPPI (G3)-NMD complex it was 235.5 nm. From these results it reveal that in the case of PPI (G3)-NMD complex, NMD molecules have attached to the surface whereas in the case of QPPI (G3)-NMD complex, they have bound to the internal positive charges without contributing much to the increase in size forming a compact polyplex. This observation is in agreement with the previous findings [51]. All the DLS figures are given in the supporting information (Fig. S4). 3.5. In vitro drug release studies The in vitro release of NMD from QPPI (G3) and PPI (G3) was explored in PBS medium. We investigated the release profiles of NMD through dendrimer based carriers such as PPI (G3) and QPPI (G3) to study the extent of drug release from drug-dendrimer formulations. The release pattern was plotted as percentage of drug released versus time and the same was shown in Fig. 6. From the plots it was noticed that, for control formulation, 90% of NMD was released within 80 min whereas, after 1 h 40.25% of drug was released from PPI (G3)-NMD formulations. In the case of QPPI (G3)NMD formulations, the drug release gets sustained to 16.35% in the same span of time. After 5 h, 90.32% of drug was released from PPI (G3)-NMD formulations whereas 35.69% of drug was released from QPPI (G3)-NMD formulation. These results suggested that the QPPI (G3) dendrimer significantly contributed for the sustained release of NMD than their parent dendrimer and control formulation. The complex between the dendrimer and the drug was formed via electrostatic interaction, which depends on the ionic strength of the medium and thus the formed complex was structurally more stable in PBS which reflected in slower release of the drug. In general, the sustained/delayed release of drug by any carrier is an indication of effective internalization of drug with the carrier or formation of stable complex due to the interaction between carrier and drug. Hence this observation strongly proves that the QPPI (G3) dendrimer is a potential carrier for sustained release of the drug NMD.
E. Murugan et al. / Colloids and Surfaces B: Biointerfaces 114 (2014) 121–129
127
Fig. 6. Release behavior of NMD in presence of PPI (G3) and QPPI (G3) dendrimers. NMD and dendrimer concentrations were 1.3 mM and 0.2 mM in dialysis bag respectively.
3.6. Cytotoxicity assay
Fig. 4. (a) 1 H–1 H NOESY spectrum of the QPPI (G3)/NMD/D2 O solution at a mixing rate of 300 ms. (b) Expanded region of the 1 H–1 H NOESY spectrum shown in (a) showing the cross-peaks between protons of drug and QPPI (G3). The cross-peaks are indicated by rectangles.
Fig. 5. DSC thermograms of (a) plain NMD, (b) QPPI (G3) and (c) QPPI (G3)-NMD freeze dried inclusion complex.
The effect of PPI (G3) and QPPI (G3) dendrimers on the viability of Vero and HBL-100 cell lines was measured by the MTT assay. The variation of percentage cell viability incubated in Vero cell line and HBL-100 cell line with different concentrations from 30 M to 1 mM of PPI (G3) and QPPI (G3) were assessed. The relationships of cell viability with incubated PPI (G3) and QPPI (G3) dendrimer concentrations on the above mentioned two cell lines are shown in Supporting information (Figs. S5 and S6). At a concentration of 0.0312 mM, it can be observed that QPPI (G3) exhibited much higher cell viability (94% Vero and 88% HBL-100). In any cell line, under in vitro conditions, the degree of cytotoxicity of a particular carrier usually been evaluated based on the IC50 values (concentrations where 50% of cells are viable). IC50 of QPPI (G3) is 0.56 mM on Vero cell line and 0.42 mM on HBL cell line. It is understand that although a maximum concentration of 1 mM, QPPI (G3) carrier were taken for study, the IC50 values are drastically increased than its corresponding parent dendrimer irrespective of cell lines. It was observed that, QPPI (G3) dendrimer was proved to be significantly less cytotoxic than its respective parent dendrimer on both cell lines, which reveals that QPPI (G3) is much more biocompatible
Fig. 7. Cytotoxicity of PPI (G3) and QPPI (G3) dendrimers at different concentrations after 24 h of incubation as determined by MTT assay on HBL-100 cell lines. Each data point represents mean ± standard error (S.E.) (n = 3).
128
E. Murugan et al. / Colloids and Surfaces B: Biointerfaces 114 (2014) 121–129
than PPI (G3) (Fig. 7). These results show that QPPI (G3) dendrimer can effectively increase the tolerance concentration during their administration. These observations indicate that the quaternised dendrimer is proved to be better carrier to deliver NMD owing to its less cytotoxic effect/more biocompatibility. Previously reported studies reveal that electrostatic interaction occurs between positively charged polymers and negatively charged cell membranes have resulted in more cytotoxic effect [51]. Particularly, cationic dendrimers containing NH2 surface group, are prone to attract the negatively charged cell membrane that in turn damaged the cell lines and thus reflected more cytotoxic effect [19]. Therefore, in any carrier the cationic surface group of the polymer/dendrimer must be neutralized so as to eliminate the electrostatic interaction of surface group with the negatively charged cell membranes and this in turn reduced the cytotoxicity [52–56]. In that line of ideas, QPPI (G3) is containing neutralized surface group as the cationic primary amine groups in PPI (G3) were functionalized with glycidyltrimethyl ammonium chloride. Even though glycidyltrimethyl ammonium chloride have a positively charged quaternary ammonium groups, the methyl groups exposed at their surface are able to screen the internal positive charges and thus behaved as a neutral molecule [51,56] thereby their electrostatic interaction with cell lines Vero and HBL-100 were significantly reduced and hence lower cytotoxicity was noticed than its corresponding parent dendrimer. In general, as far as drug delivery study is concerned, even though any molecule is capable of increased drug loading efficiency, but their sustained release of drug and free/lower cytotoxicity is the most significant factor than the efficiency of drug loading. In view of this background, in the present study, although the commercial PPI (G3) gives 108 times increased drug loading, but its efficiency of drug release was observed with 90% in 300 min and also the cytotoxicity is higher. In contrast the quaternized dendrimer viz., QPPI (G3), though efficiency of drug loading is relatively lesser (72 times) than the commercial PPI (G3) (108 times), but its ability to release the drug was found to be 35% at 300 min with significantly reduced cytotoxicity. This proves that QPPI (G3) dendrimer provide a potential platform to reduce the cytotoxicity which gives the pathway to increase the biocompatibility.
4. Conclusions Dendrimer based drug delivery system provides an attractive platform to enhance the drug solubility and thus act as drug carrier. Quaternised poly(propylene imine) dendrimer loaded with a drug for its delivery purpose was reported for the first time. In the present study, we prepared a quaternised poly(propylene imine) dendrimer viz., QPPI (G3) and utilized as drug carrier of the drug NMD. QPPI (G3) prepared by reacting surface amine groups of PPI (G3) dendrimer with glycidyltrimethyl ammonium chloride and the modification was established through various spectral techniques. This dendrimer was explored to investigate the drug carrying potential of NMD. At a concentration of 0.35 mM QPPI (G3), the solubility of NMD has been increased up to 72 folds in comparison with its intrinsic solubility. Whereas, at the same concentration, the commercial PPI (G3) increases the solubility of NMD up to 108 folds. Based on these solubility results, it is concluded that the dendrimers have showed significant drug solubilizing potential which in turn proves that they are the promising candidate for NMD loading. 1 H NMR and 2D NOSEY NMR analyses have also shown the complexation of NMD with QPPI (G3) dendrimer. Various additional evidences strengthen the fact that the complexation of NMD with QPPI (G3) was responsible for the observed solubility enhancement. It is a matter to be considered here that under in vitro conditions, QPPI (G3) is able to sustain the release of NMD considerably. This reflected in the magnitude of interaction between the
dendrimer carrier and the drug molecule. The studies demonstrate the promising strategy for per oral delivery of NMD. QPPI (G3) dendrimer showed increased cell viabilities on Vero and HBL-100 cell lines which was correlated with their reduced cytotoxicity over the parent dendrimer and hence it is more biocompatible. The increase in IC50 value for QPPI (G3) can effectively increase its tolerance concentration during the preparation of NMD-QPPI (G3)dendrimer formulations. Further, it is worth to highlight here that though the drug loading potential for QPPI (G3) is relatively lesser than with the commercial PPI (G3) dendrimer, but its potential for sustained release of NMD drug with reduced toxicity prove that the QPPI (G3) is one of the potential candidate for NMD delivery. Therefore, the QPPI (G3) is a vital molecule in the design of polymeric drug delivery systems to enhance the bioavailability of the poorly soluble drugs. Acknowledgements The authors gratefully acknowledge CSIR, New Delhi, Government of India, for financial assistance. One of the authors D.P. Geetha Rani would like to thank University of Madras and Govt. of TamilNadu for providing financial support in the form of Junior Research Fellowship (TNJRF). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.colsurfb.2013.10.002. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33]
S. Svenson, D.A. Tomalia, Adv. Drug Deliv. Rev. 57 (2005) 2106. E. Roseita, D.A. Tomalia, Drug Discovery Today 6 (2001) 427. A.K. Patri, I.J. Majoros, J.R. Baker, Curr. Opin. Chem. Biol. 6 (2002) 466. G.R. Newkome, C.N. Moorefield, F. Vögtle, Dendrimers and Dendrons: Concepts, Syntheses, Applications, Wiley-VCH, Weinheim, Germany, 2001. J.M.J. Frechet, D.A. Tomalia, Dendrimers and Other Dendritic Polymers, Wiley, UK, 2001. E.R. Gillies, J.M.J. Fréchet, Drug Discovery Today 10 (2005) 35. M.A. Mintzer, M.W. Grinstaff, Chem. Soc. Rev. 40 (2011) 173. D.W. Grainger, T. Okano, Adv. Drug Deliv. Rev. 55 (2003) 311. A. D’Emanuele, D. Attwood, Adv. Drug Deliv. Rev. 57 (2005) 2147. A.R. Menjoge, R.M. Kannan, D.A. Tomalia, Drug Discovery Today 15 (2010) 171. U. Gupta, H.B. Agashe, A. Asthana, N.K. Jain, Biomacromolecules 7 (3) (2006) 649. Y. Cheng, J. Wang, T. Rao, X. He, T. Xu, Front. Biosci. 13 (2008) 1447. A.K. Patri, J.F. Kukowska-Latallo, J.R. Baker, Adv. Drug Deliv. Rev. 57 (2005) 2203. S. Svenson, Eur. J. Pharm. Biopharm. 71 (2009) 445. K. Jain, P. Kesharwani, U. Gupta, N.K. Jain, Int. J. Pharm. 394 (1–2) (2010) 122. R. Duncan, L. Izzo, Adv. Drug Deliv. Rev. 57 (15) (2005) 2215. R. Jeyprasesphant, J. Penny, R. Jalal, D. Attwood, N.B. Mckeown, A. D’Emanuele, Int. J. Pharm. 252 (1–2) (2003) 263. P.E. Smith, J.R. Brender, U.H. Durr, J. Xu, D.G. Mullen, M.M. BanaszakHoll, A. Ramamoorthy, J. Am. Chem. Soc. 132 (23) (2010) 8087. Y. Zhang, Y. Sun, X. Xu, X. Zhang, H. Zhu, L. Huang, Y. Qi, Y.M. Shen, J. Med. Chem. 53 (8) (2010) 3262. I.J. Majoros, T.P. Thomas, C.B. Mehta, J.R. Baker, J. Med. Chem. 48 (2005) 5892. R. Roy, M.G. Baek, J. Biotechnol. 90 (3–4) (2002) 291. R. Kolhatkar, K. Kitchens, P. Swaan, H. Ghandehari, Bioconjug. Chem. 18 (6) (2007) 2054. Y. Zhang, Y. Sun, X. Xu, H. Zhu, L. Huang, X. Zhang, Y. Qi, Y.M. Shen, Bioorg. Med. Chem. Lett. 20 (3) (2010) 927. D.K. Smith, F. Diederich, Chem. Eur. J. 4 (1998) 1353. N. Jayaranam, S.A. Nepogodiev, J.F. Stoddart, Chem. Eur. J. 3 (1997) 1193. J.F.G.A. Jansen, H.W.I. Peerlings, M.M. de Brabander-Van den Berg, E.W. Meijer, Angew. Chem. Int. Ed. Engl. 34 (1995) 1206. N. Moszner, T. Volkel, V. Rheinberger, Macromol. Chem. Phys. 197 (2) (1996) 621. Z. Sideratou, D. Tsiourvas, C.M. Paleos, Langmuir 16 (2000) 1766. N. Davis, R.N. Brogden, Drugs 48 (1994) 431. G. Piel, B. Pirotte, I. Delneuville, P. Neven, G. Llabres, J. Delarge, L. Delattre, J. Pharm. Sci. 86 (1997) 475. P.R. Vavia, N.A. Adhage, Drug Dev. Ind. Pharm. 25 (1999) 543. J. Jinno, D.M. Oh, J.R. Crison, G.L. Amidon, J. Pharm. Sci. 89 (2000) 268. S. Mutalik, Venkatesh, N. Udupa, Indian J. Physiol. Pharmacol. 46 (2002) 115.
E. Murugan et al. / Colloids and Surfaces B: Biointerfaces 114 (2014) 121–129 [34] T. Higuchi, K.A. Conners, Phase-solubility Techniques, Advances in Analytical Chemistry and Instrumentation, John Wiley, NY, 1965. [35] C. Kojima, K. Kono, K. Maruyama, T. Takagishi, Bioconjug. Chem. 11 (6) (2000) 910. [36] Y.H. Zhao, M.H. Abraham, J. Le, A. Hersey, C.N. Luscombe, G. Beck, B. Sherborne, I. Cooper, Pharm. Res. 19 (2002) 1446. [37] W. Yang, Y. Li, Y. Cheng, Q. Wu, L. Wen, T. Xu, J. Pharm. Sci. 98 (2009) 1075. [38] Y.Y. Cheng, Q.L. Wu, Y.W. Li, T.W. Xu, J. Phys. Chem. B 112 (2008) 8884. [39] P. Sandhya, J. Mareddy, D.P. Geetha rani, P.V. Gollapudi, S. Pal, J. Braz. Chem. Soc. 18 (2007) 384. [40] S.K. Kulkarni, Curr. Sci. 83 (2002) 1442. [41] E. Biosselier, C. Ornelas, I. Pianet, J.R. Aranzaes, D. Astruc, Chem. Eur. J. 14 (2008) 5577. [42] M. Santo, M.A. Fox, J. Phys. Org. Chem. 12 (1999) 293. [43] M.A.C. Broeren, B.F.M. de Waal, M.H.P. van Genderen, H.M.H.F. Sanders, G. Fytas, E.W. Meijer, J. Am. Chem. Soc. 127 (2005) 10334. [44] U. Boas, S.H.M. Sontjens, K.J. Jensen, J.B. Christensen, M.H. Chai, Y.H. Niu, W.J. Youngs, P.L. Rinaldi, J. Am. Chem. Soc. 123 (2001) 4670. [45] M.H. Chai, Y.H. Niu, W.J. Youngs, P.L. Rinaldi, Macromolecules 33 (2000) 5395.
129
[46] M.H. Chai, A.K. Holley, M. Kruskamp, Chem. Commun. 168 (2007). [47] Y.Y. Cheng, Y.W. Li, Q.L. Wu, T.W. Xu, J. Phys. Chem. B 112 (2008) 41267. [48] Y. Cheng, H. Qu, M. Ma, Z. Xu, P. Xu, Y. Fang, T. Xu, Eur. J. Med. Chem. 42 (2007) 1032. [49] T. Prahlad, K. Rajendrakumar, J. Pharm. Biomed. Anal. 34 (2003) 333. [50] M. Panda, G. Patro, A. Malpani, M.E. Rao, Int. J. Pharm. Sci. Rev. Res. 9 (2011) 173. [51] J.H. Lee, Y.B. Lim, J.S. Choi, Y. Lee, T. Kim, H.J. Kim, J.K. Yoon, K. Kim, J.S. Park, Bioconjug. Chem. 14 (2003) 1214. [52] N. Malik, R. Wiwattanapatapee, R. Klopsch, K. Lorenz, H. Frey, J.W. Weener, E.W. Meijer, W. Paulus, R. Duncan, J. Control. Release 65 (2000) 133. [53] P.Y. Vuillaume, M. Brunelle, M.R. Van Calsteren, S. Laurent-Lewandowski, A. Begin, R. Lewandowski, B.G. Talbot, Y. Elazhary, Biomacromolecules 6 (3) (2005) 1769. [54] C.K. Poh, Z. Shi, T.Y. Lim, K.G. Neoh, W. Wang, Biomaterials 31 (2010) 1578. [55] R.B. Kolhatkar, K.M. Kitchens, P.W. Swaan, H. Ghandehari, Bioconjug. Chem. 18 (2007) 2054. [56] M.L. Patil, M. Zhang, S. Betigeri, O. Taratula, H.X. He, T. Minko, Bioconjug. Chem. 19 (2008) 1396.