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Surface modified PLGA nanoparticles for brain targeting of Bacoside-A
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S. Jose a,⇑, S. Sowmya a, T.A. Cinu a,b, N.A. Aleykutty a, S. Thomas c, E.B. Souto d,e,f,⇑ a
University College of Pharmacy, Mahatma Gandhi University, Cheruvandoor Campus, Ettumanoor, Kottayam 686631, Kerala, India Manipal College of Pharmaceutical Sciences, Manipal University, Manipal, Karnataka, India c Centre for Nanoscience and Nanotechnology, Mahatma Gandhi University, Kottayam, India d Research Centre in Biomedicine (CEBIMED), Fernando Pessoa University, Praça 9 de Abril, 349, P-4249-004 Porto, Portugal e Faculty of Health Sciences, Fernando Pessoa University, Rua Carlos da Maia, 296, P-4200-150 Porto, Portugal f Institute of Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes and Alto Douro, 5001-801 Vila-Real, Portugal b
a r t i c l e
i n f o
Article history: Received 13 April 2014 Received in revised form 3 June 2014 Accepted 28 June 2014 Available online xxxx Keywords: Nanoparticles Bacoside-A Poly(lactic-co-glycolic acid) Blood brain barrier
a b s t r a c t The present paper focuses on the development and in vitro/in vivo characterization of nanoparticles composed of poly-(D,L)-Lactide-co-Glycolide (PLGA) loading Bacoside-A, as a new approach for the brain delivery of the neuroprotective drug for the treatment of neurodegenerative disorders (e.g. Alzheimer Disease). Bacoside-A-loaded PLGA nanoparticles were prepared via o/w emulsion solvent evaporation technique. Surface of the nanoparticles were modified by coating with polysorbate 80 to facilitate the crossing of the blood brain barrier (BBB), and the processing parameters (i.e. sonication time, the concentration of polymer (PLGA) and surfactant (polysorbate 80), and drug-polymer ratio) were optimized with the aim to achieve a high production yield. Brain targeting potential of the nanoparticles was evaluated by in vivo studies using Wistar albino rats. The nanoparticles produced by optimal formulation were within the nanosized range (70–200 nm) with relatively low polydispersity index (0.391 ± 1.2). The encapsulation efficiency of Bacoside-A in PLGA nanoparticles was 57.11 ± 7.11%, with a drug loading capacity of 20.5 ± 1.98%. SEM images showed the spherical shape of the PLGA nanoparticles, whereas their low crystallinity was demonstrated by X-ray studies, which also confirmed no chemical interactions between the drug and polymer molecules. The in vitro release of Bacoside-A from the PLGA nanoparticles followed a sustained release pattern with a maximum release of up to 83.04 ± 2.55% in 48 h. When compared to pure drug solution (2.56 ± 1.23 lg/g tissue), in vivo study demonstrated higher brain concentration of Bacoside-A (23.94 ± 1.74 lg/g tissue) suggesting a significant role of surface coated nanoparticles on brain targeting. The results indicate the potential of surface modified PLGA nanoparticles for the delivery of Bacoside-A to the brain. Ó 2014 Published by Elsevier B.V.
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1. Introduction In the traditional Indian system of medicine ‘‘Ayurveda’’, Bacopa monniera (Linn) has been used as a potent nerve tonic (Gupta et al., 2014; Singh et al., 2014). It is thought to improve intelligence, memory and functioning of sense organs, and it has also been used to treat epilepsy, insomnia and asthma (Das et al., 2002; Deepak and Amit, 2013; Thomas et al., 2013). The nootropic activity of this plant extract has been attributed to the presence of two saponins, namely Bacoside-A and Bacoside-B, of which the former is the
⇑ Corresponding authors. Tel.: +91 9447600750 (S. Jose). Address: Faculty of Health Sciences of Fernando Pessoa University, Rua Carlos da Maia, 296, Office S.1, P-4200-150 Porto, Portugal. Tel.: +351 22 507 4630x3056; fax: +351 22 550 4637 (E.B. Souto). E-mail addresses: [email protected] (S. Jose), [email protected] (E.B. Souto).
most important which is a triterpenoid saponin. Bacoside-A has been reported to significantly improve acquisition, consolidation and retention of memory (Singh and Dhawan, 1982). The ethanolic extract of Bacopa has been found to increase the activity of antioxidative enzymes (e.g. superoxide dismutase (SOD), glutathione peroxidase and catalase) in the frontal cortex, striatum and hippocampus of rats (Gubbannavar et al., 2013). Several studies have been performed to prove its neuropharmacological effect (Pandey et al., 2010). Its cognitive enhancing property makes it a potential therapeutic for the treatment of neurodegenerative disorders (Limpeanchob et al., 2008). Bacoside-A has been tested for the treatment of neurodegenerative disorders, being Alzheimer disease one of the most common neurodegenerative disorders causing dementia among the elderly people. Oxidative stress and amyloid b are considered as the major etiological and pathological factors in the initiation and promotion of neurodegeneration in Alzheimer disease. However, the targeting
http://dx.doi.org/10.1016/j.ejps.2014.06.024 0928-0987/Ó 2014 Published by Elsevier B.V.
Please cite this article in press as: Jose, S., et al. Surface modified PLGA nanoparticles for brain targeting of Bacoside-A. Eur. J. Pharm. Sci. (2014), http:// dx.doi.org/10.1016/j.ejps.2014.06.024
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of therapeutics to the central nervous system (CNS) is limited by restrictive mechanisms imposed at the blood brain barrier (BBB). Opsonization by plasma proteins in the systemic circulation is also an impediment to cerebral drug delivery (Liu et al., 2009; Roney et al., 2005). The BBB, formed by the endothelial cells of the brain capillaries coupled together by tight junctions, inhibits the transport of 98% of all small drug molecules and 100% of large-molecule pharmaceutics into the brain, and thus drug delivery to the brain is restricted (Chen and Liu, 2012; Pardridge, 2003). Nanoparticles proved to be a potential drug targeting system to the brain with improved drug efficacy and reduced drug toxicity. Due to their unique features, such as large surface to mass ratio, their quantum properties and ability to carry and adsorb other particles such as drug, proteins and probes, nanoparticles are attractive for medical purposes. Among the various alternatives, polymeric nanoparticles demonstrate to be promising candidates as they are able of opening the tight junctions of the BBB, they effectively disguise the membrane barrier limiting characterizations of the drug molecule thus prolonging drug release and protecting drugs against enzymatic degradation (Chen and Liu, 2012). Biodegradable polymers are preferred as the matrix material for nanoparticles, including e.g. poly(lactic-co-glycolic acid) (PLGA), bovine serum albumin, and chitosan (Agyare et al., 2014). PLGA is one of the most successfully used biodegradable polymers because its hydrolysis leads to lactic acid and glycolic acid which are endogenous and easily metabolized by the body via the Krebs cycle. PLGA is approved by the US Food and Drug Administration (FDA) and European Medicine Agency (EMA) in various drug delivery systems in humans (Danhier et al., 2012; Semete et al., 2010). Polymeric nanoparticles in the size range of 100–200 nm are ideal for brain targeting (Olivier, 2005; Wohlfart et al., 2012). The extent of opsonization by organs with reticuloendothelial system (RES) is reported to be low in this size range. Such low and narrow size range also demonstrates high drug loading capacities and is able to protect the incorporated drug against degradation, thus increasing the chance of brain targeting and delivery (Modi et al., 2009; Pardridge, 2002; Peng et al., 2013). Surface modification of nanoparticles with certain surfactants, such as polysorbates, has shown to enhance their brain targeting potential (Kreuter et al., 1997; Wang et al., 2009). Polymeric nanoparticles coated with polysorbate 80 are reported to cross the BBB by mimicking the low density lipoproteins (LDL), enabling them to interact with the LDL receptor, resulting in the nanoparticles uptake by brain endothelial cells (Gelperina et al., 2002; Nagpal et al., 2013; Wohlfart et al., 2012).
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2. Materials and methods
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2.1. Materials
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Bacoside-A was received as a gift sample from Sami Labs Limited (Bangalore India). Poly (D,L-Lactide-co-Glycolide) [50:50] was purchased from Purac Biomaterials (Gorinchem, The Netherlands) and was used without further purification. Polyvinyl alcohol (PVA) was purchased from Research lab (Mumbai, India). Polysorbate 80 was procured from Nice Chemicals (Kochi, India). HPLC grade Acetonitrile and Water were purchased from CDH chemicals (New Delhi, India). All other chemicals and reagents used in this study were of analytical grade.
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2.2. Preparation of PLGA nanoparticles
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PLGA nanoparticles loaded with Bacoside-A were prepared by a modified version of the o/w emulsion solvent evaporation process
(Javadzadeh et al., 2010). PLGA polymer and Bacoside-A (drug polymer ratio 1:1, 1:2, 1:3, 1:5) were dissolved in methanoldichloromethane mixture (1:2) which formed the organic phase. The organic phase was emulsified with aqueous phase containing PVA 2% (m/V) using an ultraprobe sonicator (Sonics vibra cell, USA) at an output of 40 W for 5 min in an ice bath. To evaporate the organic solvent, the formed nanosuspension was kept overnight stirring in a magnetic stirrer. The resulting PLGA nanoparticle suspension was ultra-centrifuged (Fourtech, Mumbai, India) at 13,000 rpm for 30 min at 4 °C and the sediment obtained was washed with purified water and the washing process was repeated thrice in order to remove the adsorbed/non-loaded drug molecules. The washed nanoparticles were then freeze-dried using lyophilizer (Subzero lab instruments, Chennai, India).
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2.3. Coating of PLGA nanoparticles with polysorbate 80
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Coating of PLGA nanoparticles (drug-polymer ratio 1:2) was carried out by re-suspending nanoparticles in phosphate buffered saline at a concentration of 20 mg/ml under constant stirring. Relative to total suspension volume, polysorbate 80 was then added to give a final solution of 1% (m/V) polysorbate 80, the mixture was incubated for 30 min, and finally lyophilized. The surfactant coating was confirmed by FTIR analysis (Anand et al., 2010; Md et al., 2013; Mo et al., 2012).
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2.4. Optimization of process parameters
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2.4.1. Sonication time The energy input is a fundamental step in order to obtain emulsified systems. To study the influence of sonication time on nanoparticles size distribution, sonication time was varied between 2 and 7 min.
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2.4.2. PLGA content To verify the influence of the initial mass of polymer on the particles morphology, encapsulation efficiency (EE), and loading capacity (LC), PLGA content was varied at 10, 20, 30 and 50 mg/ml.
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2.4.3. Surfactant concentration Polyvinyl alcohol (PVA) concentration was varied from 1% to 3% (m/V) and the optimum concentration of PVA was evaluated to reach a monodispersed emulsion, and therefore a with a narrow particle size.
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2.5. Determination of process yield
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The process yield of nanoparticles was calculated by comparing quantity of polymer and drug initially taken for the production process, and the quantity of the nanoparticles finally obtained.
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2.6. Determination of encapsulation efficiency and loading capacity
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Encapsulation efficiency (EE) was calculated as the ratio of the drug content in the freeze dried powder to the initial drug amount added. The drug loading capacity (LC) was the ratio of the drug content to the freeze dried powder (Mohammadi et al., 2010; Narayanan et al., 2013). 5 mg of the freeze dried nanoparticles was vortexed with 5 ml of methanol for 1 h and was filtered through 0.22 lm membrane filter. Then, the drug content in the filtrate was analyzed by ultraviolet (UV) spectrophotometer at 278 nm (Mathew et al., 2010).
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3. Characterization
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3.1. Determination of particle size, zeta potential and morphology
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The particle size and zeta potential of the prepared nanoparticles (drug-polymer ratio 1:2) were analyzed by Zetasizer (Malvern, UK). The freeze dried powder samples were suspended in water and sonicated before measurement. The mean diameter and size distribution of the resulted homogeneous suspension were measured. Each value resulted from triplicate determinations. Morphology of the nanoparticles was analyzed by scanning electron microscopy (SEM) (JEOL JSM-6390, Tokyo, Japan). The nanoparticles were fixed on adequate supports and coated with platinum using platinum sputter module (JFC-1100, JEOL Ltd.), in a higher vacuum evaporator for 5 min at 20 mA. Observations under different magnifications were performed at 20 kv.
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3.2. X-ray diffraction analysis
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X-ray diffraction patterns were measured using hXh pert PRO, PANalytical instrument, using Cu Ka rays with a voltage of 40 kV and a current of 20 mA, over the 2h ranges 50 –600, with a step width 0.050 and a scan time of 2.0 s per step. X-ray diffraction pattern were determined for the pure Bacoside-A, polymer (PLGA), and for the optimized nanoparticles.
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3.3. In vitro release profile of Bacoside-A from nanoparticles
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The in vitro drug release from the surface modified nanoparticles was performed applying the dialysis technique. 1 ml of nanoparticle suspension (equivalent to 5 mg of Bacoside-A) was placed in a dialysis bag (molecular weight cut off 10,000–12,000, Hi-Media, India) sealed at both the ends and was soaked in 20 ml of phosphate buffer solution (pH 7.4) and maintained at 37 °C ± 0.5 °C with continuous stirring at 100 ± 5 rpm in a shaker. At predetermined time intervals, aliquots were withdrawn from the release medium and replaced with fresh phosphate buffer solution. The sample was assayed spectrophotometrically for Bacoside-A at 278 nm. The studies were performed in triplicate.
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3.4. Animal studies
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The targeting efficiency of polysorbate 80 coated Bacoside-A loaded PLGA nanoparticles (drug: polymer ratio 1:2) were compared with that of the free drug solution. Adult albino Wistar rats were procured from the Animal House, University College of Pharmacy, Cheruvandoor, M.G University, India after getting approval from the Institutional Animal Ethical Committee Board, (IAEC no: 021/MPH/UCP/CVR/12). Rats weighing approximately 200–250 g have been used for the in vivo study. The rats were maintained on pellet diet and water ad libitum. Selected rats were kept on a constant day and night cycle and fasted for 12 h before the study. The animals were divided into 2 groups of 6 rats each and were administered appropriate solution by intraperitoneal route. The group-1 was given Bacoside-A pure drug and group-2 was administered Bacoside-A bound with PLGA nanoparticles coated with 1% (m/V) polysorbate 80. For the in vivo experiments, formulations were resuspended in phosphate buffered saline. For the surfactant coating, a solution of 1% (m/V) polysorbate 80 was added, and the suspension was incubated for 30 min under stirring prior to administration. All the formulations were given in a dose equivalent to 20 mg/kg body weight. After 90 min of post injection, the rats were sacrificed by cervical dislocation. The brain, liver, spleen and kidneys were removed, weighed, and stored at
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20 °C. The organs of each animal were homogenized separately with 5 ml phosphate buffer containing methanol in the ratio (10:1), respectively, and centrifuged. The drug content in the supernatant was estimated using HPLC at 205 nm (EstellaHermoso de Mendoza et al., 2011; Srivastava et al., 2012; Wilson et al., 2008). The results were expressed as mean ± standard deviation (SD). Statistical analysis was carried out by Mann– Whitney t-test. Analysis was carried out using the trial version of Graph-PadÒ Prism v6 software. A difference was considered significant when P < 0.05.
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4. Results and discussion
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Bacoside-A-loaded PLGA nanoparticles were prepared by an o/w emulsion solvent evaporation method. The sonication time, PLGA and surfactant concentrations were optimized at 5 min, 20 mg/ml and 2%, respectively, based on the obtained particle size, encapsulation efficiency (EE%), particle morphology and zeta potential. When sonication time was increased from 2 to 7 min, particle size decreased from 288.3 ± 2.23 nm to 173 ± 2 nm. Nanoparticles prepared with PLGA concentration of 20 mg/ml depicted a spherical shape with minimal agglomeration and reaching an EE% of 37.23 ± 1.45%. PVA taken at a concentration of 2% developed particles with a mean size of 207.7 ± 1.56 nm and zeta potential of 20.03 ± 0.95 mV. Bacoside-A-loaded PLGA nanoparticles prepared at different drug-polymer ratio showed an EE% ranging between 19.48 ± 2.08% and 76.94 ± 4.00%, with a drug LC% varying from 9.74 ± 1.03% to 20.5±.98% (Table 1). The highest drug loading of 20.5 ± 1.98% was obtained for formulation containing drug and polymer in the ratio of 1:2, which was selected for further in vitro and in vivo characterization studies. PLGA concentration was fixed based on EE% and shape of the particles obtained by SEM images. Table 1 shows the effect of PLGA concentration on the encapsulation of the drug. The encapsulation of Bacoside-A increased with an increase in PLGA concentration. The EE% was found highest for F 7 (i.e. 50 mg/ml PLGA). The SEM images of the formulations containing different concentrations of PLGA were recorded. Nanoparticles prepared with 20 mg/ml of PLGA presented spherical shape without any agglomeration (Fig. 2 left). However, nanoparticles prepared with 50 mg/ml of PLGA presented a non-spherical shape with presence of agglomerates (Fig. 2, right) and the particle size was also found to increase. Spherical particles with smooth surface have a relatively lower surface to volume ratio, and thus a slower degradation rate when compared to non-spherical particles. From these results, F5 containing PLGA in the concentration of 20 mg/ml with an EE% of 37.23 ± 1.45 and spherical shaped particles with minimal agglomeration was selected over the other formulations. Formulations F6 and F7 showed higher EE% than F5 but exhibited a non-spherical shape and agglomeration. Thus, PLGA concentration of 20 mg/ml (F5) was selected. PVA concentration was fixed based on the obtained particle size and zeta potential. Formulations with different concentration of PVA were prepared, and it was observed that with increase in PVA concentration in the aqueous phase, the particle size decreased from 288.4 ± 3.5 nm (1% PVA) to 91.5 ± 1.36 nm (3% PVA). Table 2 shows that with the increase of PVA concentration, the zeta potential increased from 31.06 ± 1.55 mV for 1% (m/V) PVA to 6.65 ± 0.491 mV for 3% (m/V) PVA. The shift may be due to the fact that increasing the PVA concentration, the number of coating layers on the polymer surface will also increase, therefore shielding the negative charge on the surface of the particles. From the above results it was decided that 2% (w/v) PVA was best as a surfactant.
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Table 1 Process yield, encapsulation efficiency, and loading capacity of Bacoside A in nanoparticles composed of different drug to polymer ratio (Formulation code F11 stands for (1:1) drug–polymer ratio). Formulation code
Drug-polymer ratio
Process yield (%w/w)
Encapsulation efficiency (%w/w)
Loading capacity (%w/w)
F1 F2 F3 F4 F5
1:1 1:2 1:3 1:4 1:5
15.00 35.33 47.00 55.00 42.00
19.48 ± 2.08 57.11 ± 7.11 61.73 ± 2.85 66.70 ± 2.40 76.94 ± 4.00
9.74 ± 1.03 20.50 ± 1.98 15.25 ± 0.98 13.33 ± 0.47 12.76 ± 0.68
n = 3, ±SD
Table 2 Comparison between PVA concentration and the zeta potential of nanoparticles obtained with formulations F8, F9 and F10.
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Formulation code
PVA % (w/v)
Particle size (nm)
Zeta potential (mV)
F8 F9 F10
1 2 3
288.4 ± 3.5 207.7 ± 1.56 91.5 ± 1.36
31.06 ± 1.55 20.03 ± 0.95 6.65 ± 0.49
The size of drug loaded nanoparticles (drug polymer ratio 1:2) was 77 ± 1 nm with a zeta potential of 19 ± 0.89 mV (Fig. 1). From the results obtained in Table 1, the formulation with the highest drug LC% was selected for in vivo studies. The nanoparticles (F12) showed an average particle size of 77.1 nm. Smaller particles below 100 nm can easily cross BBB by preventing spleen filtration. Zeta potential which reveals the physical stability of the formulation was 19 ± 0.89 mV. Surface charge on the particles could control the particles stability of the formulation through strong electrostatic repulsion of particles, being therefore an important factor to determine the in vivo interactions of nanoparticles with the cell membrane. Positively charged particles have a higher tendency to attach and internalize compared to negatively or neutrally charged particles. SEM analysis showed spherical shaped particles (Fig. 2). The images have been obtained using a scanning electron microscope
to determine the shape and surface morphology of the nanoparticles. SEM images have confirmed that the homogenous nature of particles, with a uniform distribution, without aggregation after lyophilisation. A spherical particle moving through a vessel does not deviate from its streamline motion unless it experiences an external force, which is not the case of non-spherical particles as they are susceptible to segregation. The cumulative percentage release of Bacoside-A from PLGA nanoparticles (drug-polymer 1:2) released up to 83.04 ± 2.55% within 48 h in phosphate buffer pH 7.4 (Fig. 3). The release rate of Bacoside-A loaded PLGA nanoparticles increased with increase of the drug-polymer ratio. Usually, the drug loaded carriers exhibit a biphasic release pattern. The diffractogram of nanoparticles (drug polymer 1:2) confirmed the drug loading into the formulated nanoparticles (Fig. 4). The diffractogram of Bacoside-A exhibited sharp peaks, indicating the crystalline nature of Bacoside-A, highlighting the presence of drug loaded nanoparticles in an amorphous and disordered-crystalline status or in solid state solubilized form in the polymer matrix of nanoparticles. The concentration of Bacoside-A in brain, liver, kidney and liver after intraperitoneal injection of polysorbate 80 coated nanoparticles and drug solution is shown in Fig. 5. The results indicate that only the polysorbate 80 coated nanoparticles were able to deliver Bacoside-A in the brain considerably, with the concentration of
Fig. 1. Particle size distribution (A, % intensity, average of 12 runs) and zeta potential (B, total counts, average of 3 runs) of the Bacoside-A loaded nanoparticles.
Please cite this article in press as: Jose, S., et al. Surface modified PLGA nanoparticles for brain targeting of Bacoside-A. Eur. J. Pharm. Sci. (2014), http:// dx.doi.org/10.1016/j.ejps.2014.06.024
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Fig. 2. SEM image of nanoparticles prepared with 20 mg/ml of PLGA (left, Formulation F5) and nanoparticles prepared with 50 mg/ml of PLGA (right, Formulation F7).
Counts (arbitrary units)
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(A)
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2 theta
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23.94 ± 1.74 lg/g, 90 min after administration. The results also indicate that only 2.56 ± 1.23 lg/g of the drug was found in the brain tissue of the group administered with drug solution. The concentration of Bacoside-A, after the intraperitoneal administration of pure drug solution, in the liver, spleen, and kidneys were 13.16 ± 2.02, 3.93 ± 0.85 and 14.24 ± 3.8 lg/g, respectively. However, when bound with polysorbate 80 coated PLGA nanoparticles, the concentration was 8.75 ± 2.77, 0.96 ± 0.132 and 7.66 ± 3.19 lg/g, respectively. Table 3 depicts the average percentage of dose reached in each organ comparing the values obtained with polysorbate 80 coated Bacoside-A-loaded nanoparticles (F12) and free Bacoside-A (S). Solvent evaporation method has been selected to obtain PLGA nanoparticles with homogenous size distribution. Nanoparticles coated with polysorbate 80 are thought to cross the BBB via plasma adsorption of apolipoproteins resulting in receptor mediated endocytosis by brain capillary endothelial cells, as apolipoproteins naturally cross the BBB. But another mechanism proposed is the nanoparticle adherence to the cell membrane with subsequent escape by the P-glycoprotein efflux system, and thus resulting in endothelial cell uptake of nanoparticles (Benvegnu et al., 2012; Gelperina et al., 2002). As the time of sonication increases, more energy is released during emulsification process, which leads to rapid dispersion of polymeric organic phase as nanodroplets of small size. This may be the reason for the decreased particle size observed with the increase of the sonication time. When sonication time of 7 min was set, metal contamination was observed in the nanosuspension (Table 4). With an increase in PLGA concentration, the viscosity of the organic phase increased, which caused the diffusion resistance for drug molecules from organic phase to the aqueous phase, thereby reducing the drug loss through diffusion and increasing
(B)
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10
20
30
40
2 theta 300
Counts (arbitrary units)
Fig. 3. Cumulative percentage of Bacoside A released in phosphate buffer pH 7.4.
Counts (arbitrary units)
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(C)
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20
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2-theta Fig. 4. XRD pattern of pure Bacoside A (A) and Bacoside A loaded nanoparticles (B) containing drug and polymer in the ratio 1:2.
the encapsulation (Song et al., 2008; Wang et al., 2013). But increase in PLGA concentration resulted in non-spherical particles with agglomeration, this may be due to the poor dispersion of PLGA solution into the aqueous phase as the viscosity of dispersed phase (polymer solution) increased. Coarse emulsions are obtained at higher polymer concentrations, which lead to the production of larger particles during the diffusion process. Thus, an optimum concentration of 20 mg/ml of PLGA was selected.
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Concentration of Bacoside A (µg/g)
formulation standard
25
20
10
5
brain
kidney
liver
spleen
Groups Fig. 5. Amount of drug per gram of brain, kidney, liver and spleen at 90 min after i.p. administration (20 mg/kg) of Bacoside A loaded formulation and Pure Bacoside A (S) (mean ± SD).
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Sonication time (min)
Mean diameter (nm)
F1 F2 F3
2 5 7
288.3 ± 2.2 207.7 ± 1.5 173.0 ± 2.0
n = 3, ±SD
15
0
394
Formulation code
As the surfactant concentration increased, the zeta potential moved towards higher positive charge, and positively charged particles have a greater tendency to attach and internalize compared to negatively charged particles; therefore, based on this fact surfactant concentration was fixed at 2%. A successful nanoparticle system should have a high loading capacity to reduce the quantity of carrier required for administration. The drug loading capacity of nanoparticles was found to be 14.33 ± 4.00% against a theoretical drug loading of 29.97 ± 13.11% depending on drug-polymer ratio. As mentioned above, Bacoside-A loaded nanoparticles showed an average particle size of 77 ± 1 nm. Smaller particles (
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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps 4 5
Surface modified PLGA nanoparticles for brain targeting of Bacoside-A
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S. Jose a,⇑, S. Sowmya a, T.A. Cinu a,b, N.A. Aleykutty a, S. Thomas c, E.B. Souto d,e,f,⇑ a
University College of Pharmacy, Mahatma Gandhi University, Cheruvandoor Campus, Ettumanoor, Kottayam 686631, Kerala, India Manipal College of Pharmaceutical Sciences, Manipal University, Manipal, Karnataka, India c Centre for Nanoscience and Nanotechnology, Mahatma Gandhi University, Kottayam, India d Research Centre in Biomedicine (CEBIMED), Fernando Pessoa University, Praça 9 de Abril, 349, P-4249-004 Porto, Portugal e Faculty of Health Sciences, Fernando Pessoa University, Rua Carlos da Maia, 296, P-4200-150 Porto, Portugal f Institute of Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes and Alto Douro, 5001-801 Vila-Real, Portugal b
a r t i c l e
i n f o
Article history: Received 13 April 2014 Received in revised form 3 June 2014 Accepted 28 June 2014 Available online xxxx Keywords: Nanoparticles Bacoside-A Poly(lactic-co-glycolic acid) Blood brain barrier
a b s t r a c t The present paper focuses on the development and in vitro/in vivo characterization of nanoparticles composed of poly-(D,L)-Lactide-co-Glycolide (PLGA) loading Bacoside-A, as a new approach for the brain delivery of the neuroprotective drug for the treatment of neurodegenerative disorders (e.g. Alzheimer Disease). Bacoside-A-loaded PLGA nanoparticles were prepared via o/w emulsion solvent evaporation technique. Surface of the nanoparticles were modified by coating with polysorbate 80 to facilitate the crossing of the blood brain barrier (BBB), and the processing parameters (i.e. sonication time, the concentration of polymer (PLGA) and surfactant (polysorbate 80), and drug-polymer ratio) were optimized with the aim to achieve a high production yield. Brain targeting potential of the nanoparticles was evaluated by in vivo studies using Wistar albino rats. The nanoparticles produced by optimal formulation were within the nanosized range (70–200 nm) with relatively low polydispersity index (0.391 ± 1.2). The encapsulation efficiency of Bacoside-A in PLGA nanoparticles was 57.11 ± 7.11%, with a drug loading capacity of 20.5 ± 1.98%. SEM images showed the spherical shape of the PLGA nanoparticles, whereas their low crystallinity was demonstrated by X-ray studies, which also confirmed no chemical interactions between the drug and polymer molecules. The in vitro release of Bacoside-A from the PLGA nanoparticles followed a sustained release pattern with a maximum release of up to 83.04 ± 2.55% in 48 h. When compared to pure drug solution (2.56 ± 1.23 lg/g tissue), in vivo study demonstrated higher brain concentration of Bacoside-A (23.94 ± 1.74 lg/g tissue) suggesting a significant role of surface coated nanoparticles on brain targeting. The results indicate the potential of surface modified PLGA nanoparticles for the delivery of Bacoside-A to the brain. Ó 2014 Published by Elsevier B.V.
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1. Introduction In the traditional Indian system of medicine ‘‘Ayurveda’’, Bacopa monniera (Linn) has been used as a potent nerve tonic (Gupta et al., 2014; Singh et al., 2014). It is thought to improve intelligence, memory and functioning of sense organs, and it has also been used to treat epilepsy, insomnia and asthma (Das et al., 2002; Deepak and Amit, 2013; Thomas et al., 2013). The nootropic activity of this plant extract has been attributed to the presence of two saponins, namely Bacoside-A and Bacoside-B, of which the former is the
⇑ Corresponding authors. Tel.: +91 9447600750 (S. Jose). Address: Faculty of Health Sciences of Fernando Pessoa University, Rua Carlos da Maia, 296, Office S.1, P-4200-150 Porto, Portugal. Tel.: +351 22 507 4630x3056; fax: +351 22 550 4637 (E.B. Souto). E-mail addresses: [email protected] (S. Jose), [email protected] (E.B. Souto).
most important which is a triterpenoid saponin. Bacoside-A has been reported to significantly improve acquisition, consolidation and retention of memory (Singh and Dhawan, 1982). The ethanolic extract of Bacopa has been found to increase the activity of antioxidative enzymes (e.g. superoxide dismutase (SOD), glutathione peroxidase and catalase) in the frontal cortex, striatum and hippocampus of rats (Gubbannavar et al., 2013). Several studies have been performed to prove its neuropharmacological effect (Pandey et al., 2010). Its cognitive enhancing property makes it a potential therapeutic for the treatment of neurodegenerative disorders (Limpeanchob et al., 2008). Bacoside-A has been tested for the treatment of neurodegenerative disorders, being Alzheimer disease one of the most common neurodegenerative disorders causing dementia among the elderly people. Oxidative stress and amyloid b are considered as the major etiological and pathological factors in the initiation and promotion of neurodegeneration in Alzheimer disease. However, the targeting
http://dx.doi.org/10.1016/j.ejps.2014.06.024 0928-0987/Ó 2014 Published by Elsevier B.V.
Please cite this article in press as: Jose, S., et al. Surface modified PLGA nanoparticles for brain targeting of Bacoside-A. Eur. J. Pharm. Sci. (2014), http:// dx.doi.org/10.1016/j.ejps.2014.06.024
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of therapeutics to the central nervous system (CNS) is limited by restrictive mechanisms imposed at the blood brain barrier (BBB). Opsonization by plasma proteins in the systemic circulation is also an impediment to cerebral drug delivery (Liu et al., 2009; Roney et al., 2005). The BBB, formed by the endothelial cells of the brain capillaries coupled together by tight junctions, inhibits the transport of 98% of all small drug molecules and 100% of large-molecule pharmaceutics into the brain, and thus drug delivery to the brain is restricted (Chen and Liu, 2012; Pardridge, 2003). Nanoparticles proved to be a potential drug targeting system to the brain with improved drug efficacy and reduced drug toxicity. Due to their unique features, such as large surface to mass ratio, their quantum properties and ability to carry and adsorb other particles such as drug, proteins and probes, nanoparticles are attractive for medical purposes. Among the various alternatives, polymeric nanoparticles demonstrate to be promising candidates as they are able of opening the tight junctions of the BBB, they effectively disguise the membrane barrier limiting characterizations of the drug molecule thus prolonging drug release and protecting drugs against enzymatic degradation (Chen and Liu, 2012). Biodegradable polymers are preferred as the matrix material for nanoparticles, including e.g. poly(lactic-co-glycolic acid) (PLGA), bovine serum albumin, and chitosan (Agyare et al., 2014). PLGA is one of the most successfully used biodegradable polymers because its hydrolysis leads to lactic acid and glycolic acid which are endogenous and easily metabolized by the body via the Krebs cycle. PLGA is approved by the US Food and Drug Administration (FDA) and European Medicine Agency (EMA) in various drug delivery systems in humans (Danhier et al., 2012; Semete et al., 2010). Polymeric nanoparticles in the size range of 100–200 nm are ideal for brain targeting (Olivier, 2005; Wohlfart et al., 2012). The extent of opsonization by organs with reticuloendothelial system (RES) is reported to be low in this size range. Such low and narrow size range also demonstrates high drug loading capacities and is able to protect the incorporated drug against degradation, thus increasing the chance of brain targeting and delivery (Modi et al., 2009; Pardridge, 2002; Peng et al., 2013). Surface modification of nanoparticles with certain surfactants, such as polysorbates, has shown to enhance their brain targeting potential (Kreuter et al., 1997; Wang et al., 2009). Polymeric nanoparticles coated with polysorbate 80 are reported to cross the BBB by mimicking the low density lipoproteins (LDL), enabling them to interact with the LDL receptor, resulting in the nanoparticles uptake by brain endothelial cells (Gelperina et al., 2002; Nagpal et al., 2013; Wohlfart et al., 2012).
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2. Materials and methods
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2.1. Materials
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Bacoside-A was received as a gift sample from Sami Labs Limited (Bangalore India). Poly (D,L-Lactide-co-Glycolide) [50:50] was purchased from Purac Biomaterials (Gorinchem, The Netherlands) and was used without further purification. Polyvinyl alcohol (PVA) was purchased from Research lab (Mumbai, India). Polysorbate 80 was procured from Nice Chemicals (Kochi, India). HPLC grade Acetonitrile and Water were purchased from CDH chemicals (New Delhi, India). All other chemicals and reagents used in this study were of analytical grade.
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2.2. Preparation of PLGA nanoparticles
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PLGA nanoparticles loaded with Bacoside-A were prepared by a modified version of the o/w emulsion solvent evaporation process
(Javadzadeh et al., 2010). PLGA polymer and Bacoside-A (drug polymer ratio 1:1, 1:2, 1:3, 1:5) were dissolved in methanoldichloromethane mixture (1:2) which formed the organic phase. The organic phase was emulsified with aqueous phase containing PVA 2% (m/V) using an ultraprobe sonicator (Sonics vibra cell, USA) at an output of 40 W for 5 min in an ice bath. To evaporate the organic solvent, the formed nanosuspension was kept overnight stirring in a magnetic stirrer. The resulting PLGA nanoparticle suspension was ultra-centrifuged (Fourtech, Mumbai, India) at 13,000 rpm for 30 min at 4 °C and the sediment obtained was washed with purified water and the washing process was repeated thrice in order to remove the adsorbed/non-loaded drug molecules. The washed nanoparticles were then freeze-dried using lyophilizer (Subzero lab instruments, Chennai, India).
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2.3. Coating of PLGA nanoparticles with polysorbate 80
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Coating of PLGA nanoparticles (drug-polymer ratio 1:2) was carried out by re-suspending nanoparticles in phosphate buffered saline at a concentration of 20 mg/ml under constant stirring. Relative to total suspension volume, polysorbate 80 was then added to give a final solution of 1% (m/V) polysorbate 80, the mixture was incubated for 30 min, and finally lyophilized. The surfactant coating was confirmed by FTIR analysis (Anand et al., 2010; Md et al., 2013; Mo et al., 2012).
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2.4. Optimization of process parameters
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2.4.1. Sonication time The energy input is a fundamental step in order to obtain emulsified systems. To study the influence of sonication time on nanoparticles size distribution, sonication time was varied between 2 and 7 min.
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2.4.2. PLGA content To verify the influence of the initial mass of polymer on the particles morphology, encapsulation efficiency (EE), and loading capacity (LC), PLGA content was varied at 10, 20, 30 and 50 mg/ml.
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2.4.3. Surfactant concentration Polyvinyl alcohol (PVA) concentration was varied from 1% to 3% (m/V) and the optimum concentration of PVA was evaluated to reach a monodispersed emulsion, and therefore a with a narrow particle size.
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2.5. Determination of process yield
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The process yield of nanoparticles was calculated by comparing quantity of polymer and drug initially taken for the production process, and the quantity of the nanoparticles finally obtained.
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2.6. Determination of encapsulation efficiency and loading capacity
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Encapsulation efficiency (EE) was calculated as the ratio of the drug content in the freeze dried powder to the initial drug amount added. The drug loading capacity (LC) was the ratio of the drug content to the freeze dried powder (Mohammadi et al., 2010; Narayanan et al., 2013). 5 mg of the freeze dried nanoparticles was vortexed with 5 ml of methanol for 1 h and was filtered through 0.22 lm membrane filter. Then, the drug content in the filtrate was analyzed by ultraviolet (UV) spectrophotometer at 278 nm (Mathew et al., 2010).
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Please cite this article in press as: Jose, S., et al. Surface modified PLGA nanoparticles for brain targeting of Bacoside-A. Eur. J. Pharm. Sci. (2014), http:// dx.doi.org/10.1016/j.ejps.2014.06.024
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3. Characterization
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3.1. Determination of particle size, zeta potential and morphology
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The particle size and zeta potential of the prepared nanoparticles (drug-polymer ratio 1:2) were analyzed by Zetasizer (Malvern, UK). The freeze dried powder samples were suspended in water and sonicated before measurement. The mean diameter and size distribution of the resulted homogeneous suspension were measured. Each value resulted from triplicate determinations. Morphology of the nanoparticles was analyzed by scanning electron microscopy (SEM) (JEOL JSM-6390, Tokyo, Japan). The nanoparticles were fixed on adequate supports and coated with platinum using platinum sputter module (JFC-1100, JEOL Ltd.), in a higher vacuum evaporator for 5 min at 20 mA. Observations under different magnifications were performed at 20 kv.
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3.2. X-ray diffraction analysis
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X-ray diffraction patterns were measured using hXh pert PRO, PANalytical instrument, using Cu Ka rays with a voltage of 40 kV and a current of 20 mA, over the 2h ranges 50 –600, with a step width 0.050 and a scan time of 2.0 s per step. X-ray diffraction pattern were determined for the pure Bacoside-A, polymer (PLGA), and for the optimized nanoparticles.
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3.3. In vitro release profile of Bacoside-A from nanoparticles
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The in vitro drug release from the surface modified nanoparticles was performed applying the dialysis technique. 1 ml of nanoparticle suspension (equivalent to 5 mg of Bacoside-A) was placed in a dialysis bag (molecular weight cut off 10,000–12,000, Hi-Media, India) sealed at both the ends and was soaked in 20 ml of phosphate buffer solution (pH 7.4) and maintained at 37 °C ± 0.5 °C with continuous stirring at 100 ± 5 rpm in a shaker. At predetermined time intervals, aliquots were withdrawn from the release medium and replaced with fresh phosphate buffer solution. The sample was assayed spectrophotometrically for Bacoside-A at 278 nm. The studies were performed in triplicate.
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3.4. Animal studies
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The targeting efficiency of polysorbate 80 coated Bacoside-A loaded PLGA nanoparticles (drug: polymer ratio 1:2) were compared with that of the free drug solution. Adult albino Wistar rats were procured from the Animal House, University College of Pharmacy, Cheruvandoor, M.G University, India after getting approval from the Institutional Animal Ethical Committee Board, (IAEC no: 021/MPH/UCP/CVR/12). Rats weighing approximately 200–250 g have been used for the in vivo study. The rats were maintained on pellet diet and water ad libitum. Selected rats were kept on a constant day and night cycle and fasted for 12 h before the study. The animals were divided into 2 groups of 6 rats each and were administered appropriate solution by intraperitoneal route. The group-1 was given Bacoside-A pure drug and group-2 was administered Bacoside-A bound with PLGA nanoparticles coated with 1% (m/V) polysorbate 80. For the in vivo experiments, formulations were resuspended in phosphate buffered saline. For the surfactant coating, a solution of 1% (m/V) polysorbate 80 was added, and the suspension was incubated for 30 min under stirring prior to administration. All the formulations were given in a dose equivalent to 20 mg/kg body weight. After 90 min of post injection, the rats were sacrificed by cervical dislocation. The brain, liver, spleen and kidneys were removed, weighed, and stored at
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20 °C. The organs of each animal were homogenized separately with 5 ml phosphate buffer containing methanol in the ratio (10:1), respectively, and centrifuged. The drug content in the supernatant was estimated using HPLC at 205 nm (EstellaHermoso de Mendoza et al., 2011; Srivastava et al., 2012; Wilson et al., 2008). The results were expressed as mean ± standard deviation (SD). Statistical analysis was carried out by Mann– Whitney t-test. Analysis was carried out using the trial version of Graph-PadÒ Prism v6 software. A difference was considered significant when P < 0.05.
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4. Results and discussion
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Bacoside-A-loaded PLGA nanoparticles were prepared by an o/w emulsion solvent evaporation method. The sonication time, PLGA and surfactant concentrations were optimized at 5 min, 20 mg/ml and 2%, respectively, based on the obtained particle size, encapsulation efficiency (EE%), particle morphology and zeta potential. When sonication time was increased from 2 to 7 min, particle size decreased from 288.3 ± 2.23 nm to 173 ± 2 nm. Nanoparticles prepared with PLGA concentration of 20 mg/ml depicted a spherical shape with minimal agglomeration and reaching an EE% of 37.23 ± 1.45%. PVA taken at a concentration of 2% developed particles with a mean size of 207.7 ± 1.56 nm and zeta potential of 20.03 ± 0.95 mV. Bacoside-A-loaded PLGA nanoparticles prepared at different drug-polymer ratio showed an EE% ranging between 19.48 ± 2.08% and 76.94 ± 4.00%, with a drug LC% varying from 9.74 ± 1.03% to 20.5±.98% (Table 1). The highest drug loading of 20.5 ± 1.98% was obtained for formulation containing drug and polymer in the ratio of 1:2, which was selected for further in vitro and in vivo characterization studies. PLGA concentration was fixed based on EE% and shape of the particles obtained by SEM images. Table 1 shows the effect of PLGA concentration on the encapsulation of the drug. The encapsulation of Bacoside-A increased with an increase in PLGA concentration. The EE% was found highest for F 7 (i.e. 50 mg/ml PLGA). The SEM images of the formulations containing different concentrations of PLGA were recorded. Nanoparticles prepared with 20 mg/ml of PLGA presented spherical shape without any agglomeration (Fig. 2 left). However, nanoparticles prepared with 50 mg/ml of PLGA presented a non-spherical shape with presence of agglomerates (Fig. 2, right) and the particle size was also found to increase. Spherical particles with smooth surface have a relatively lower surface to volume ratio, and thus a slower degradation rate when compared to non-spherical particles. From these results, F5 containing PLGA in the concentration of 20 mg/ml with an EE% of 37.23 ± 1.45 and spherical shaped particles with minimal agglomeration was selected over the other formulations. Formulations F6 and F7 showed higher EE% than F5 but exhibited a non-spherical shape and agglomeration. Thus, PLGA concentration of 20 mg/ml (F5) was selected. PVA concentration was fixed based on the obtained particle size and zeta potential. Formulations with different concentration of PVA were prepared, and it was observed that with increase in PVA concentration in the aqueous phase, the particle size decreased from 288.4 ± 3.5 nm (1% PVA) to 91.5 ± 1.36 nm (3% PVA). Table 2 shows that with the increase of PVA concentration, the zeta potential increased from 31.06 ± 1.55 mV for 1% (m/V) PVA to 6.65 ± 0.491 mV for 3% (m/V) PVA. The shift may be due to the fact that increasing the PVA concentration, the number of coating layers on the polymer surface will also increase, therefore shielding the negative charge on the surface of the particles. From the above results it was decided that 2% (w/v) PVA was best as a surfactant.
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Table 1 Process yield, encapsulation efficiency, and loading capacity of Bacoside A in nanoparticles composed of different drug to polymer ratio (Formulation code F11 stands for (1:1) drug–polymer ratio). Formulation code
Drug-polymer ratio
Process yield (%w/w)
Encapsulation efficiency (%w/w)
Loading capacity (%w/w)
F1 F2 F3 F4 F5
1:1 1:2 1:3 1:4 1:5
15.00 35.33 47.00 55.00 42.00
19.48 ± 2.08 57.11 ± 7.11 61.73 ± 2.85 66.70 ± 2.40 76.94 ± 4.00
9.74 ± 1.03 20.50 ± 1.98 15.25 ± 0.98 13.33 ± 0.47 12.76 ± 0.68
n = 3, ±SD
Table 2 Comparison between PVA concentration and the zeta potential of nanoparticles obtained with formulations F8, F9 and F10.
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Formulation code
PVA % (w/v)
Particle size (nm)
Zeta potential (mV)
F8 F9 F10
1 2 3
288.4 ± 3.5 207.7 ± 1.56 91.5 ± 1.36
31.06 ± 1.55 20.03 ± 0.95 6.65 ± 0.49
The size of drug loaded nanoparticles (drug polymer ratio 1:2) was 77 ± 1 nm with a zeta potential of 19 ± 0.89 mV (Fig. 1). From the results obtained in Table 1, the formulation with the highest drug LC% was selected for in vivo studies. The nanoparticles (F12) showed an average particle size of 77.1 nm. Smaller particles below 100 nm can easily cross BBB by preventing spleen filtration. Zeta potential which reveals the physical stability of the formulation was 19 ± 0.89 mV. Surface charge on the particles could control the particles stability of the formulation through strong electrostatic repulsion of particles, being therefore an important factor to determine the in vivo interactions of nanoparticles with the cell membrane. Positively charged particles have a higher tendency to attach and internalize compared to negatively or neutrally charged particles. SEM analysis showed spherical shaped particles (Fig. 2). The images have been obtained using a scanning electron microscope
to determine the shape and surface morphology of the nanoparticles. SEM images have confirmed that the homogenous nature of particles, with a uniform distribution, without aggregation after lyophilisation. A spherical particle moving through a vessel does not deviate from its streamline motion unless it experiences an external force, which is not the case of non-spherical particles as they are susceptible to segregation. The cumulative percentage release of Bacoside-A from PLGA nanoparticles (drug-polymer 1:2) released up to 83.04 ± 2.55% within 48 h in phosphate buffer pH 7.4 (Fig. 3). The release rate of Bacoside-A loaded PLGA nanoparticles increased with increase of the drug-polymer ratio. Usually, the drug loaded carriers exhibit a biphasic release pattern. The diffractogram of nanoparticles (drug polymer 1:2) confirmed the drug loading into the formulated nanoparticles (Fig. 4). The diffractogram of Bacoside-A exhibited sharp peaks, indicating the crystalline nature of Bacoside-A, highlighting the presence of drug loaded nanoparticles in an amorphous and disordered-crystalline status or in solid state solubilized form in the polymer matrix of nanoparticles. The concentration of Bacoside-A in brain, liver, kidney and liver after intraperitoneal injection of polysorbate 80 coated nanoparticles and drug solution is shown in Fig. 5. The results indicate that only the polysorbate 80 coated nanoparticles were able to deliver Bacoside-A in the brain considerably, with the concentration of
Fig. 1. Particle size distribution (A, % intensity, average of 12 runs) and zeta potential (B, total counts, average of 3 runs) of the Bacoside-A loaded nanoparticles.
Please cite this article in press as: Jose, S., et al. Surface modified PLGA nanoparticles for brain targeting of Bacoside-A. Eur. J. Pharm. Sci. (2014), http:// dx.doi.org/10.1016/j.ejps.2014.06.024
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Fig. 2. SEM image of nanoparticles prepared with 20 mg/ml of PLGA (left, Formulation F5) and nanoparticles prepared with 50 mg/ml of PLGA (right, Formulation F7).
Counts (arbitrary units)
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(A)
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2 theta
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23.94 ± 1.74 lg/g, 90 min after administration. The results also indicate that only 2.56 ± 1.23 lg/g of the drug was found in the brain tissue of the group administered with drug solution. The concentration of Bacoside-A, after the intraperitoneal administration of pure drug solution, in the liver, spleen, and kidneys were 13.16 ± 2.02, 3.93 ± 0.85 and 14.24 ± 3.8 lg/g, respectively. However, when bound with polysorbate 80 coated PLGA nanoparticles, the concentration was 8.75 ± 2.77, 0.96 ± 0.132 and 7.66 ± 3.19 lg/g, respectively. Table 3 depicts the average percentage of dose reached in each organ comparing the values obtained with polysorbate 80 coated Bacoside-A-loaded nanoparticles (F12) and free Bacoside-A (S). Solvent evaporation method has been selected to obtain PLGA nanoparticles with homogenous size distribution. Nanoparticles coated with polysorbate 80 are thought to cross the BBB via plasma adsorption of apolipoproteins resulting in receptor mediated endocytosis by brain capillary endothelial cells, as apolipoproteins naturally cross the BBB. But another mechanism proposed is the nanoparticle adherence to the cell membrane with subsequent escape by the P-glycoprotein efflux system, and thus resulting in endothelial cell uptake of nanoparticles (Benvegnu et al., 2012; Gelperina et al., 2002). As the time of sonication increases, more energy is released during emulsification process, which leads to rapid dispersion of polymeric organic phase as nanodroplets of small size. This may be the reason for the decreased particle size observed with the increase of the sonication time. When sonication time of 7 min was set, metal contamination was observed in the nanosuspension (Table 4). With an increase in PLGA concentration, the viscosity of the organic phase increased, which caused the diffusion resistance for drug molecules from organic phase to the aqueous phase, thereby reducing the drug loss through diffusion and increasing
(B)
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20
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2 theta 300
Counts (arbitrary units)
Fig. 3. Cumulative percentage of Bacoside A released in phosphate buffer pH 7.4.
Counts (arbitrary units)
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2-theta Fig. 4. XRD pattern of pure Bacoside A (A) and Bacoside A loaded nanoparticles (B) containing drug and polymer in the ratio 1:2.
the encapsulation (Song et al., 2008; Wang et al., 2013). But increase in PLGA concentration resulted in non-spherical particles with agglomeration, this may be due to the poor dispersion of PLGA solution into the aqueous phase as the viscosity of dispersed phase (polymer solution) increased. Coarse emulsions are obtained at higher polymer concentrations, which lead to the production of larger particles during the diffusion process. Thus, an optimum concentration of 20 mg/ml of PLGA was selected.
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Concentration of Bacoside A (µg/g)
formulation standard
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20
10
5
brain
kidney
liver
spleen
Groups Fig. 5. Amount of drug per gram of brain, kidney, liver and spleen at 90 min after i.p. administration (20 mg/kg) of Bacoside A loaded formulation and Pure Bacoside A (S) (mean ± SD).
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Sonication time (min)
Mean diameter (nm)
F1 F2 F3
2 5 7
288.3 ± 2.2 207.7 ± 1.5 173.0 ± 2.0
n = 3, ±SD
15
0
394
Formulation code
As the surfactant concentration increased, the zeta potential moved towards higher positive charge, and positively charged particles have a greater tendency to attach and internalize compared to negatively charged particles; therefore, based on this fact surfactant concentration was fixed at 2%. A successful nanoparticle system should have a high loading capacity to reduce the quantity of carrier required for administration. The drug loading capacity of nanoparticles was found to be 14.33 ± 4.00% against a theoretical drug loading of 29.97 ± 13.11% depending on drug-polymer ratio. As mentioned above, Bacoside-A loaded nanoparticles showed an average particle size of 77 ± 1 nm. Smaller particles (
