Targeting compounds to the brain efficiently requires good permeation across the rigid blood-brain barrier and to retain in the brain for a longer time. Nanoparticles with a size below 200 nm can efficiently cross the blood-brain barrier and can also deliver the drug to the brain. Further, the presence of a hydrophilic coat on the surface of the nanoparticles can enhance the permeation across the blood-brain barrier. Coating the nanoparticles with polysorbate 80 is a well-established technique and has shown good promise in the delivery of numerous drugs across the blood-brain barrier. In this research work, an attempt has been made to check the effectiveness of polyethylene glycol surface-modified PLGA nanoparticles to enhance the concentration of RT in the brain. RT-loaded PEG-PLGA nanoparticles were prepared by the nanoprecipitation technique method. Optimization studies were performed to identify the effect of variables like the amount of polymer, the concentration of surfactant, and the volume of the organic phase used during the formulation of the nanoparticles.
Influence of parameters on size
Particle size plays an important role in determining the ability of the molecule to cross the brain barrier where in which, a particle size under 200 nm is necessary for crossing the blood-brain barrier [13]. Table 1 gives details about the size of various formulations. All the formulations were below 200 nm and the particle size of the formulations varied from a maximum of 179.60 ± 1.06nm (F25) to a minimum of 125.93 ± 0.55 (F3). The particle size of the formulations was influenced by the number of excipients used in the formulation, PEG- PLGA, Pluronic F68, and dichloromethane.
As observed from Table 1 when the volume of the organic phase and concentration of surfactant was kept constant, an increase in the concentration of the polymer increased the particle size. This increased particle size can be attributed to the improper dispersion of the polymer due to the increased viscosity of the solution. This increased viscosity will lead to improper separation and breakdown of the polymer. Thus, particle size increases with an increase in polymer concentration. Hence, it would be preferable to use lower amounts (10 mg) of polymer to obtain the least particle size.
Table 1
Rivastigmine loaded PEG-PLGA nanoparticulate formulations
Sl.No | Trial | Polymer | Volume of org. phase | Pluronic F68 (mg) | PS (nm) | PDI | ZP (MW) | DEE(%) |
1 | F1 | 10 | 1 | 0.5 | 134.00 ± 0.56 | 0.169 | -11 | 84 |
2 | F2 | 10 | 1 | 1 | 131.82 ± 0.82 | 0.147 | -12 | 72 |
3 | F3 | 10 | 1 | 1.5 | 125.93 ± 0.55 | 0.197 | -11 | 69 |
4 | F4 | 10 | 2 | 0.5 | 143.30 ± 0.62 | 0.184 | -10 | 79 |
5 | F5 | 10 | 2 | 1 | 137.17 ± 0.55 | 0.183 | -13 | 77 |
6 | F6 | 10 | 2 | 1.5 | 131.47 ± 1.50 | 0.198 | -14 | 76 |
7 | F7 | 10 | 3 | 0.5 | 162.37 ± 0.68 | 0.175 | -12 | 81 |
8 | F8 | 10 | 3 | 1 | 150.70 ± 1.05 | 0.217 | -14 | 78 |
9 | F9 | 10 | 3 | 1.5 | 146.13 ± 0.59 | 0.203 | -15 | 75 |
10 | F10 | 30 | 1 | 0.5 | 144.20 ± 0.60 | 0.169 | -11 | 77 |
11 | F11 | 30 | 1 | 1 | 141.70 ± 0.85 | 0.147 | -11 | 75 |
12 | F12 | 30 | 1 | 1.5 | 132.57 ± 1.10 | 0.197 | -12 | 71 |
13 | F13 | 30 | 2 | 0.5 | 151.07 ± 1.10 | 0.184 | -10 | 80 |
14 | F14 | 30 | 2 | 1 | 145.37 ± 0.67 | 0.183 | -15 | 78 |
15 | F15 | 30 | 2 | 1.5 | 140.73 ± 1.00 | 0.198 | -16 | 76 |
16 | F16 | 30 | 3 | 0.5 | 169.27 ± 1.21 | 0.175 | -12 | 83 |
17 | F17 | 30 | 3 | 1 | 158.87 ± 0.83 | 0.217 | -13 | 79 |
18 | F18 | 30 | 3 | 1.5 | 147.13 ± 0.83 | 0.203 | -14 | 73 |
19 | F19 | 50 | 1 | 0.5 | 153.37 ± 0.76 | 0.169 | -11 | 79 |
20 | F20 | 50 | 1 | 1 | 143.93 ± 0.49 | 0.147 | -13 | 76 |
21 | F21 | 50 | 1 | 1.5 | 141.57 ± 0.87 | 0.197 | -15 | 70 |
22 | F22 | 50 | 2 | 0.5 | 160.60 ± 0.89 | 0.184 | -11 | 81 |
23 | F23 | 50 | 2 | 1 | 157.70 ± 0.90 | 0.183 | -12 | 79 |
24 | F24 | 50 | 2 | 1.5 | 151.70 ± 0.95 | 0.198 | -14 | 75 |
25 | F25 | 50 | 3 | 0.5 | 179.60 ± 1.06 | 0.175 | -10 | 85 |
26 | F26 | 50 | 3 | 1 | 163.57 ± 1.03 | 0.217 | -12 | 80 |
27 | F27 | 50 | 3 | 1.5 | 159.77 ± 1.00 | 0.203 | -15 | 76 |
PS-Particle Size, PDI-Poly Dispersibility Index, ZP-Zeta Potential, DEE- Drug Entrapment Efficiency, F1-F27- Formulations.
When variables like the amount of the polymer and Pluronic F68 were kept constant, an increase in the volume of organic solvent also led to an increase in the particle size. When the number of dichloromethane increases, the time is taken for it to evaporate also increases thereby increasing the particle size. Hence, lower amounts of organic phase would facilitate faster evaporation and better formation of nanoparticles.
Also, when the amount of polymer and organic phase was kept constant, increased levels of Pluronic F68 showed a decrease in the particle size. An increase in the Pluronic F68 concentration will reduce the surface tension of the solution leading to a better diffusion of the polymer from the organic solvent to the aqueous phase thereby producing smaller-sized particles.
So, to obtain particles with smaller size, it will be preferable to use higher amounts of Pluronic F68 with the least possible amounts of the organic phase and polymer, and this pattern hold for formulation F3 which has the least particle size of 125.93 ± 0.55 nm.
Zeta potential
The zeta potential of the formulation represents the surface change of the individual particles and it indirectly gives information regarding the ability of the particles to remain stable for a long time and to evade opsonization [14]. Neither change in the amounts of polymer and surfactant and volume of organic phase during the formulation had represented a significant effect on the zeta potential of the nanoparticles. The zeta potentials of the formulations were nearly neutral and varied from – 10.8 ± 0.87 mV to – 16.23 ± 1.06 mV.
The adsorption of the PEG block of the PEG PLGA core and polyethylene oxide (PEO) chain of Pluronic F68 act is the reason for obtaining low zeta potential values, around the neutral region. In general, it is hypothesized that zeta potential values above ± mV 30 mV would produce stable nanoparticle systems. But, Pluronic F68 is a stearic stabilizer that creates a shield by adhering around the surface of the nanoparticles thereby will prevent aggregation among the nanoparticles. Hence, these nanoparticles would remain sterically stable and will also evade phagocytosis by the macrophages in vivo.
Entrapment efficiency
RT is a hydrophilic drug and usually, nanoprecipitation method for hydrophilic drugs produces nanoparticles with low entrapment efficiency as the drug escapes from the organic phase to the aqueous. The entrapment efficiencies for the formulations were poor with the highest entrapment efficiency of 85% for formulation F25. A good relation was observed between entrapment efficiency and the excipients. With an increase in the amount of organic solvent and polymer, the entrapment efficiency of the formulations increased which can be attributed to an increase in the volume and space for the drug to get partitioned. Thus, the drug gets more room to enter either the polymer or organic phase leading to an increase in entrapment efficiencies. But, an increase in the amount of stabilizer, Pluronic F68, from 0.5 to 1.5% lead to a decrease in the entrapment efficiencies possibly because of enhanced solubility of RT in the aqueous phase. Thus, to have good entrapment efficiency, it would be better to use higher amounts of organic solvent and PEG- PLGA with lower amounts of stabilizer. Applying this same principle, it can be observed that formulation F25 with higher amounts of organic solvent (dichloromethane = 3 ml) and polymer (PEG- PLGA = 50 mg) and lower amounts of stabilizer (Pluronic F 68 = 0.5%) produced the highest entrapment efficiency values.
Surface morphological properties
The morphology of the nanoparticles observed using SEM as shown in Fig. 1, indicated uniformly distributed spherical shaped particles with a smooth texture. There was no aggregation among the particles due to the presence of PEG coating around the nanoparticle’s surface. This PEG coat can be confirmed by the presence of a dark layer around the bright nanoparticles.
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Figure 1. SEM image of RT PEG- PLGA nanoparticles
Compatibility study using DSC
The Thermograms of RT, PEG- PLGA, Pluronic F 68 showed a sharp endothermic peak at 129.31 ºC, 63.92 ºC, 56.55 ºC respectively, corresponding to the melting points of the individual compounds as shown in Fig. 2. The thermogram obtained for the nanoparticles did not show the melting curve of RT which indicates that the drug was well encapsulated in the nanoparticles in a molecular state.
Drug release kinetics
In vitro, drug release studies were carried out for 24 hours using the dialysis bag method in phosphate buffer saline of pH 7.4 as the medium. The cumulative percentage release of RT from RT PEG-PLGA was found to be a maximum of 61.33 ± 3.78% (formulation F3) over 24 hours. The drug release profile in Fig. 3 indicates a biphasic release pattern of the drug from the nanoparticles. Initially, around 20% of the drug was released from the nanoparticles and by the 48th hour, nearly 65% of the drug was released in a sustained manner. The initial burst release may be attributed to the drug present in the aqueous phase and adsorbed on the surface of nanoparticles.
Pharmacokinetic and tissue distribution studies
After intravenous (I.V) administration, the concentration of RT in blood Fig. 4 and brain Fig. 5 over some time was estimated in male Wistar rats after administering RT PBS and RT PEG- PLGA. The pharmacokinetic parameters were calculated by the noncompartmental model using WinNonlin (version 5.1). I.V administration of RT PEG- PLGA sustained the release of RT from the nanoparticles which resulted in enhanced and prolonged residence of RT in the body. The Cmax of RT after administration of RT PEG-PLGA (1625 ng/ml) was comparatively higher than the Cmax obtained after administration of RT PBS (1754 ng/ml). An increase in T1/2 of RT in plasma from 5.8 to 14.6 h after administration through nanoparticles indicates the sustained release nature of PEG-PLGA and the additional benefit incurred by the hydrophilic PEG part which helps evade opsonization. Further, with an increase in molecular weight of PEG, a slower release pattern can be obtained and the stealth nature of PEG-PLGA can be varied. Though RT PEG-PLGA showed better pharmacokinetic parameters when compared to RT PBS, it is of minor importance as RT needs to enter and remain in the brain for a longer time.
Brain distribution
The Cmax of RT in the brain achieved after i.v administration of RT PEG-PLGA was more (1058 vs 421 ng/ml) when compared to the concentration RT in the brain achieved after i.v administration of RT PBS. After being encapsulated in the PEG- PLGA polymer, higher concentration (Cmax= 1058 ng/ml) and longer circulation time of RT (t1/2= 17.7 h) was observed than the free form (Cmax= 421 ng/ml and t1/2= 5.70 h) which might be possible because of the stealth effect provided by PEG. Opsonin proteins are present in the bloodstream and bind with conventional nonstealth nanoparticles allowing macrophages of the reticuloendothelial system to easily recognize and remove the drug before the therapeutic concentration is achieved [21]. To prevent opsonization hydrophilic coatings have been widely used as it prevents the adsorption of opsonin protein via steric repulsion force thereby promoting longer circulation. Since PEG is a hydrophilic surfactant, it inhibits the P-glycoprotein efflux pump and also, enhances uptake by brain endothelial cells by covalently coupling with apolipoprotein E, A-1. Thus, it is evident that the hydrophilic PEG corona present attached to the PLGA has enhanced the permeation of RT across the blood-brain barrier. Secondly, the PEG coat can solubilize the endothelial cell membrane lipids and increase its membrane fluidization thereby enhancing the passage of RT PEG- PLGA. Further, the small size of the nanoparticles and the existence of a high concentration gradient across the blood capillaries could have also enhanced the permeation of RT PEG- PLGA across the blood-brain barrier.
The ratio of RT concentration in brain and blood was calculated at different time points following i.v administration of RT PEG-PLGA and RT PBS. At all time points, the concentration of RT in the brain was high after administration of RT PEG- PLGA. Apart from calculating the brain/ blood ratio, the percentage change (increase) in RT brain- blood ratio (α) was calculated at various times intervals as shown in Fig. 6. The α values indicate an initial increase from 36% (0.25 hr) to ~ 56% (1hr) possibly due to the entry of higher amounts of RT PEG- PLGA. The α value decreased from ~ 56% (at 1 hr) to ~ 41% (at 2 hr). But again, the, α value gradually increased from ~ 41% to ~ 61% (8 hr) indicating the increased T1/2 of RT and redistribution of RT from the body to the brain. Hence, it can be confirmed that the PEG coat, apart from increasing the permeability to the brain, would also promote redistribution to the brain and increase its retention in the brain by providing a stealth nature to the nanoparticles. Though it is a well-established fact that tween 80 coated nanoparticles have shown promise in enhancing the permeation of various drugs across the blood, it has not shown industrial applicability citing the presence of tween 80 in the formulations. As the usage of PEG has wide industrial applicability, PEG conjugated polymers like PEG- PLGA, PEG- PCL, etc can hold good promise in enhancing the concentration of drugs across the blood-brain barrier.
Pharmacodynamic studies
It is purported that this positive interaction of nanoparticles with neuronal and astrocyte membranes would not only provide integrity to the membrane but would also help restore the disrupted BBB.
Scopolamine, a muscarinic cholinergic antagonist, is known to cause cognitive impairments. Studies have recommended that the administration of scopolamine specifically impairs the development of spatial navigation strategies, hence affecting acquisition rather than recall or memory consolidation. Scopolamine effects central brain mechanisms underlying spatial learning, impairment of inhibitory avoidance behavior following disturbed amyloid cholinergic functions. Owing to these rationales and to assess the effectiveness of RT PEG-PLGA, screening of learning and memory was done through measuring transfer latency in elevated plus maze and acquisition time in Morris water maze test in scopolamine challenged rats [18]. An elevated plus-maze consisting of two open and two enclosed arms was used for an evaluation of memory. Time taken for rats in the plus-maze escaped from the open arm to the enclosed arm was recorded. Rats often feel uncomfortable in open and elevated spaces. The Morris water maze is one of the most effectively used screening models in behavioral neuroscience to investigate spatial learning and memory. Morris water maze learning is thought to rely extensively on the hippocampus and involves several major neurotransmitter systems. One such major neurotransmitter system of great importance is the cholinergic system. There was a significant decrease in learning and memory function after cognitive impairment through dysfunctioning of cholinergic transmission by scopolamine which was evidenced by increased transfer latency and acquisition trial in an elevated plus-maze and Morris water maze test. RT at a dose of 2 mg/kg significantly improved both learning and memory was concluded by observing a reduction in transfer latency and acquisition time after cognitive impairment that was induced by 0.5 mg/kg scopolamine. But when compared to RT PBS, RT PEG- PLGA showed better-improved learning and memory enhancement skills. Further, the acetylcholinesterase levels were found to be highest for group 4 which served as scopolamine administered positive group, and least for group 2 which was administered with RT PEG- PLGA. Hence it can be strongly concluded that RT PEG- PLGA are highly efficient in permeating the blood-brain barrier and delivering the drug, RT in a controlled fashion to increase memory learning skills.
Histopathological analysis
The histology of the sections was examined using a trinocular light microscope. The histology of group 4 which received scopolamine showed marked alterations in terms of increased neuronal loss, vacuolated cytoplasm, ghost cells, and hemorrhages (Fig. 7c). The photomicrographs of rats which were treated with RT PBS (Fig. 7a) and RT PEG-PLGA (Fig. 7b) did not show any signs of neuronal degeneration, vacuolization/ spongiosis, and inflammation. This indicates that RT PBS and RT PEG- PLGA not only reduce the scopolamine-induced neuronal degeneration but also don’t induce inflammation. The group of rats which served as control (Group 3) did not show any neuronal degeneration or any inflammation (Fig. 7c). Thus, it can be envisaged that RT PEG- PLGA can effectively reverse scopolamine-induced neuronal damage and does not induce any inflammation in the brain.