INTRODUCTION:

Alzheimer's disease (AD) is a progressive neurodegenerative disorder¹. The degeneration of cholinergic neurons results in lower levels of acetylcholine². Symptoms include the gradual development of forgetfulness, confusion, language difficulties, poor judgement, and problems in moving¹. The USFDA has approved Cholinesterase inhibitors and N-methyl-D-aspartate (NMDA) receptor antagonists to treat Alzheimer's disease³.

Acetylcholine is a neurotransmitter which is broken down by cholinesterase. Inhibition of the cholinesterase results in more acetylcholine available for memory and cognitive functioning which is effective in treating AD⁴. Rivastigmine (RVT) is an acetylcholinesterase and butyrylcholinesterase inhibitor used to treat AD². It undergoes significant first-pass metabolism which results in lower oral bioavailability (36%) and a shorthalf-life of 1hr⁵. FDA has approved oral formulation of RVT for the treatment of mild to moderate Alzheimer's disease with an optimal therapeutic dose of 6-12mg per day⁶. The oral administration drug also leads to some adverse effects such as hepatotoxicity, renal failure and malaise which often lead to the termination of treatment by the patient. The transdermal route for drug delivery reduces the side effectsand provides easy access to therapeutically effective doses thus improving patient compliance^7-9.

Exelon® (5 and 10 cm² containing 9.0mg and 18mg of RVS) patch by Novartis contains Rivastigmine, applied once a day to treat mild, moderate and severe dementia associated with Alzheimer's disease. They are designed to release approximately 4.6mg (5cm²) and 9.5mg (10cm²) respectively per 24 h¹⁰. The major drawback of transdermal delivery is poor permeability of the skin which can be overcome by the use of permeation enhancers. But the use of permeation enhancers may cause skin irritation. The hydrophilic nature of drugs like RVT absorption through the skin may be incomplete or difficult due to limitations of partitioning into the stratum corneum. Therefore, it is necessary to formulate novel delivery systems such as polymeric nanoparticles¹¹, solid lipid nanoparticles, liposomes, transferosomes, ethosomesetc. loaded with RVT for improving the delivery of the drug through transdermal delivery.

Therefore,RVT-loaded polymeric nanoparticles (NPs) were opted to improve the permeation of the drug through the transdermal route.The NPs were optimized by using Design of Expert. The traditional pharmaceutical formulations are developed by the "one-factor-at-a-time approach"which is time-consuming, and moreover, the interactions between independent variables are not considered. Factorial designs or Response surface methodology (RSM), where all the factors are studied in all possible combinations, are considered the most efficient in estimating the influence of individual variables and their interactions can be identified with fewer experimental trials^12-14.

Hence the present study aimed to formulate and optimized RVT-loaded NPs of PLGA and chitosan by 3² factorial design and Box-Behnken designs of RSM, respectively for transdermal sustained delivery of a drug.

MATERIALS AND METHODS:

Material:

Rivastigmine Tartrate (RVT), Poly(lactic-co-glycolic) Acid (50:50) and Poloxamer 407 were purchased from Yarrow Chem Products Mumbai, India. Chitosan (CH) with medium molecular weight (Mw = 750,000 Da) was purchase from Hi-media Labs, India. Sodium tripolyphosphate, Acetic acid, were procured from Lobo Chennie, Mumbai, India.

Methodology:

Experimental design and optimization:

Based on the results obtained from the literature survey, polymer amount, STTP and surfactant concentration were found to be the major variables affecting particle size (PS), poly dispersive index (PDI) and percentage entrapment efficiency for polymeric NPs. Hence, 3² factorial designswere applied for PLGA NPs, where PLGA 50:50 (40, 70 and 100mg) and Poloxamer (0.5, 1.0 and 1.5%) were taken as independablevariables, whereas Box-Behnken designs were applied for CH NPs,by considering Chitosan (0.2, 0.5 and 0.8g), STTP (0.25, 0.5, and 0.75%) and Poloxamer 407(0.4, 0.7, and 1.0%) as independable variables to find the optimized goal with minimum PS and PDI and highest %EE. The responses obtained from the various runs of both the formulation were subjected to multiple regression analysis using "Design Expert" software (Stat-Ease, Inc., Minneapolis, MN) (version 11)¹⁵.

Preparation of RVT-loaded PLGA and CH NPs:

PLGA NPs were prepared by the Nano precipitation method with some modifications. The specified amount (Table 1) of PLGA was weighed and dissolved in 5ml of dichloromethane (organic phase). The above organic solution was added dropwise to the 10ml poloxamer 407solutions containing 10mg drug. The organic solvent was evaporated for 2h with continuous stirring on a magnetic stirrer (Remi). Further sonicated for 15 min by using a probe sonicator to reduce the size. The NPs suspension was centrifuged at 15,000rpm for 45 min at 4 ºC using a Cold centrifuge (Remi). The supernatant was analysed for free drug content, and sediment constituting NPs was freeze-dried¹⁶.

CH-NPs were prepared by the ionic gelation method. CH (Table 2) was dispersed in 10ml of 1% acetic acid solution. RVT was uniformly dissolved in CH solution. The STPP (45ml) solution was added dropwise using the syringe to the CH solution containing the drug under stirring at room temperature. 5ml of Polxamer 407 was added to the above solution to stabilize the NP and stirred for 30 minutes followed by sonication for 15 minutes using a probe sonicator. CH-NPs were collected by centrifugation at 15,000rpm for 45min at 4ºC and sediment constituting NPs was freeze-dried¹⁷.

Optimized of polymeric nanoparticle formulation:

The polymeric nanoparticles were optimized based on the desirability of the responses, i.e., minimum particle size and PDI and maximum entrapment efficiency. The optimized PLGS NP was formulated in a similar manner as the factorial batches with a concentration of 52.07mg of PLGA and 1.424% of poloxamer respectively. Whereas optimized CH NP was formulated with of 0.2g of Chitosan, 0.377% of STTP and 1.0% of poloxamer respectively.

Characterization of RVT-loaded polymeric nanoparticles:

Determination of particle size (PS), size distribution (PDI) and zeta potential:

The particle size, size distribution and zeta potential of nanoparticle were determined by dynamic light scattering by a zeta sizer (Nano ZS, Malvern Instrument, UK).

Percentage entrapment efficiency (%EE):

The nanosuspension was centrifuged by cold centrifugation at 10000rpm for 15mins at 4˚C and supernatant was collected and the concentration of RVT was analysed by UV spectroscopic method at 264nm¹⁸.

%EE = ((Total amount of drug – Amount of drug in supernatant) × 100))/((Total amount of drug))

Drug excipient compatibility studies by using Fourier transform infrared spectroscopy (FTIR) and

To determine whether there are any interactions between the drug and excipients, FTIR spectroscopic analysis was performed using the instrument BRUKER Alpha II.

Differential scanning calorimetry, Transmission Electron Microscopyand Scanning electron microscopy:

The physical state of RVT was characterized by DSC (DSC-60, Shimadzu, Japan). Each sample was sealed in standard aluminum pans with lids and purged with air at a flow rate of 40ml/minand the heat flow was recorded in the range of 30–300ºC under an inert nitrogen atmosphere.

The morphology of formulation was observed under TEM (TECNAI 200Kv TEM, Fei, Electron optics Oregon USA) using the negative staining method. The shape of vesicles can be determined by SEM(Zeis Sigma). In this method a drop of polymeric nanoparticle system was mounted on a clear glass stub, air dried and coated with Polaren E 5100 Sputter coater and visualised under SEM.

Formulation and characterization of transdermal patch loaded with optimized NPs:

Preparation of pressure sensitive adhesive (PSA) patch:

Pressure-sensitive adhesive patches were prepared by solvent casting method by using Eudragit E100 as polymer, Dibutyl sebacate as a plasticizer, and Succinic acid as a cross-linking agentin a ratio 10:5:1 as per the literature. 1g of Eudragit E100 was dissolved in 20ml of Isopropyl Alcohol with the help of a magnetic stirrer. 0.1g of Succinic acid was dissolved in small amounts of acetone and was added to the above solution. Later 0.5ml of Dibutyl sebacate was added. Then pure drug (PD) and polymeric nanoparticle i.e., PLGA NPs and CH NPs equivalent to 113mg of drug added with magnetic stirring to get pure drug loaded patch (PD), PLGA NPs loaded patch (PPN) and CH NPs loaded patch (PCN) of 1.77 mg/cm²drug concentration¹⁹.

Characterization of the PSA patches:

Drug content uniformity:

RVT content in the patches was estimated by placing a 1 cm² patch in 10ml volumetric flask and 5mL of methanol was added and sonicated for 10 min to extract the drug from patches and made up to 10mL volume with methanol. The samples were analysed by UV spectroscopic method at 264nm¹⁹.

In vitro permeation studies:

Excised hairless pork ear skin was used for the in vitro percutaneous absorption study because; it is readily accessible, and this animal skin has shown histological and biochemical properties similar to human skin¹⁹. The pig ear skin was collected from the slaughterhouse; hair was removed from the skin and with distilled water and forceps, blood stains and fatty tissues were removed from ears. Then, the skin sample was cut into the desired size and kept in the deep freezer (−20°C) until its use. The skin permeation studies used Franz Diffusion cell. The pig ear skin was mounted between donor and receptor cells stratum corneum surface towards donor cell and the patch of 2.25cm² was slicked to stratum corneum side. The surface of the skin was maintained at 32ºC using a circulating water bath. Reservoir compartment was filled with 12ml phosphate buffer of pH 6.4 which contained a small magnetic bead rotated at constant speed of 50rpm. Samples of 500μl were withdrawn at different time intervals and 500μl of fresh saline was replaced at each sampling point. The study was carried out over a 24h period. Later the collected samples were diluted suitably and absorbance was measured at 264nm²⁰.

Calculation of Skin Permeation Parameters:

The flux was determined from the linear portion of the slope. The slopes were obtained for all the formulations and the linearities were found to be between 0.5 h to 12 h for PD, between 0.5 h to 8 h for PPN and PCN. The RVT permeability coefficient (Kp) through pig skin was determined using the relationship established from the first law of Fick's diffusion, represented in the following equation:

𝐾𝑝 =𝐽/𝐶

Where 𝐽 is the flux and 𝐶 is the drug concentration in the donor compartment.

Enhancement ratio (ER) was calculated by following equation;

(Flux of RVT loaded in NPs patch)

ER = ----------------------------------------------

(Flux of RVT in conventional patch)

RESULTS AND DISCUSSION:

Formulation and characterization of polymeric nanoparticles:

Statistical analysis of experiment design of RVT loaded PLGA NPs:

RTV loaded PLGA NPs were successfully formulated by employing 3² factorial design. The results are shown in Table 1 and Fig 1a, 1b and 1c. All the responses studied were largely affected by the variables chosen as reflected by the results of regression analysis.

Particle size, PDI and %EE ofPLGA NPs:

The dimension of particles has a significant role in the delivery of drugs across cell membranes as per literature reported. The polymer and stabilizer effects on the particle size are shownin Table 1 and 1a. The increased amount of polymer (40 to 100mg), significantly increased the particle size. The reason could be that during emulsification, the viscosity of the organic phase increases due to a large amount of polymer and leads to the formation of nanosized droplets with a large surface area at the interface⁹. As stabilizer concentration increases, the particle size reduces and thereby, aggregation decreases. The formation of large size particle could be due to the reduced interfacial stability resulting from the insufficient amount of surfactant and finally led to the aggregation of nanoparticles. The average vesicle size of the polymeric nanoparticle prepared was found to range between 264.83 to 484.13 nm.

PDI value considers that particles are homogeneous and provide stability to the formulation. The polymer and stabilizer effects on the PDI are shown in Table 1 and 1b. It indicates that as amount of polymer (40 to 100mg) increased, the PDI also increased, but with the stabilizer, it was deceased. As the stabilizer concentration increases, the particles have less tendency for aggregation and leave the particles in a more homogeneous nature in the formulation.

The effect of PLGA and poloxamer concentration on the % entrapment efficiency of formulations obtained from the 3² full factorial design showed that with the increase in polymer concentration, the entrapment efficiency of polymeric nanoparticles formulations significantly increased shown in Table 1 and 1c. This is due to increased polymer concentration increasing the viscosity of the organic phase which will resist the diffusion of drug into aqueous phase leading to the incorporation of more drugs inside the nanoparticle²¹.The % entrapment efficiency was found to be increases till 1 % of stabilizer further increasing the concentration of stabilizer leads to decrease in the entrapment efficiency. Poloxamer stabilize the PLGA nanoparticle at the interface by diffusing out the water molecules. The increasing concentration of poloxamer favours the aqueous solubility of drug which in turn leads to the increased partition of a drug in water thereby decreasing entrapment efficiency.

The effect of formulation variables on particle size, PDI and %EE can simultaneously be studied by applying regression analysis. Quadratic model implied significant with model f-value of 30.72 and 20.59 respectively with particle size and %EE, whereaslinear model implied significantly with model f-value of 50.67 with PDI. The Predicted R² is in reasonable agreement with the Adjusted R²; i.e., the difference is less than 0.2.The following equation computed from the analysis results given as follows:

Particle Size (Y1)= +382.34+49.74 (A)* -56.78 (B)* -2.63 (AB) +9.14 (A²)-19.61 (B²)*(1)

PDI (Y2) =+0.3876+0.069(A)* -0.0517 (B)* (2)

% EE (Y3) = +88.31+9.75 (A)* +3.63 (B)* -2.28 (AB) -1.10 (A²) -10.41 (B²)* (3)

Where A, B are the polymer and stabilizer concentration respectively, the coefficient in this equation represents the 'standardized beta coefficient' and the variable significance was indicated by asterisk sign (*). The positive value before the factor indicates positive effect and negative value indicates negative effect on the particle size. The developed regression model was found to be significant (p<0.05) statistically with a high adjusted R² value of 0.9884, 0.9169.and0.9158 respectively for particle size, PDI and %EE. The model has a curvature at higher levels of formulation variables indicates the significance of quadratic term B² on particle size and %EE. Simultaneously, the response surface also represented the effect of interaction as well as linear term on the particle size.

Table1: Effect of independent process variables on responses of PLGA NPs as per 3² Full Factorial Designs

Form. Code	A: PLGA 50:50(mg)	B: Poloxamer 407 (%)	Y1: Particle Size (nm)	Y2 PDI	Y3 EE (%)	Zeta potential mV
FP1	70	1	384.3	0.391	89.98	-30.7
FP2	70	0.5	412	0.431	72.3	-21.6
FP3	40	1.5	264.83	0.251	71.23	-32.6
FP4	70	1.5	313.5	0.372	82.03	-27.7
FP5	100	1	442.5	0.426	93.25	-24.9
FP6	40	0.5	380.65	0.372	60.64	-32.1
FP7	100	0.5	484.13	0.528	87.67	-29.1
FP8	100	1.5	357.8	0.398	89.14	-21.8
FP9	40	1	340.5	0.312	79.7	-20.3
FP10	70	1	380.34	0.395	88.12	-23.7

Fig. 1: Response surface curve representing the effect of Poloxamer and PLGA on a) the particle size of PLGA PNs b) the PDI of PLGA NPs c) % EE of PLGA NPs

Table 2: Results of Responses of CH NPs as per Box-Behnken Design

Form. Code	X1 Chitosan (g)	X2 STTP %	X3 Poloxamer 407 (%)	Y1 Particle Size (nm)	Y2 PDI	Y3 EE (%)	Zeta Potential mV
FC1	0.8	0.75	0.7	473	0.491	87.23	-24.1
FC2	0.2	0.75	0.7	171.9	0.281	72.67	-30.8
FC3	0.8	0.5	1	445	0.351	85.34	-29.7
FC4	0.5	0.5	0.7	357	0.372	79.94	-30.6
FC5	0.5	0.25	0.4	449	0.326	72.63	-32.9
FC6	0.2	0.25	0.7	321	0.282	78.87	-25.9
FC7	0.8	0.5	0.4	461	0.528	82.19	-33.2
FC8	0.5	0.75	1	324.9	0.348	75.14	-31.8
FC9	0.5	0.25	1	421	0.332	87.12	-26.9
FC10	0.8	0.25	0.7	481.8	0.495	81.15	-31.6
FC11	0.5	0.75	0.4	298.9	0.412	80.98	-27.3
FC12	0.2	0.5	0.4	202.9	0.325	70.12	31.7
FC13	0.2	0.5	1	325.7	0.258	85.12	22.5

Fig. 2: Response surface curve representing the effect of STTP and Chitosan on a) the particle size b)the PDI C)% EE of CH NPs

Zeta potential of NPs:

PLGA being a negatively charged polymer imparts anionic nature to PLGA NPs, whereas anionic nature of CH NPs is due to STTP negatively charged ionizing agent. The zeta potential values were found in the range from −20.3mV to −32.1mV and −24.1mV to −33.2mV for PLGS NPs and CH NPs respectively, as shown in Tables1 and 2.

Optimization of polymeric nanoparticles:

The experimental value of PS, PDI and %EE of optimized PLGA NPs showed 291.3nm, 0.296 and 75.31%, whereas optimized CH NPS showed 339.3nm, 0.264, and 83.91%, respectively. That is in good agreement with the predicted values generated by the RSM, i.e., within 5% error, and the result assures the validity of RSM model.

Drug excipient compatibility studies by using Fourier transform infrared spectroscopy:

FTIR analysis confirmed the chemical interaction. The chemical structure of the Rivastigmine, which possesses absorption bonds close to 1660 cm^–1 and 1406 cm^–1is, corresponded to the ester functional group and C=C bands, C-H stretching in RVT appeared at 2872.21cm⁻¹ and N-H stretching at 3424.11cm^-1 which appeared to remain the same in the nanoparticulate system indicating no existence of the different association form of RVT with the NPs²².

Differential scanning calorimetry (DSC), TEM), and SEM:

As shown in Fig 3, the drug RVT had three endothermic peaks; in that two was sharp peak at 106 and 130.16ºC and one broad and diminished peak 218.51ºC. The PLGA NPs and CH NPs showed exothermic peak at 241.2ºC and 249.9ºC respectively; instead of 218.51ºC endothermic peak ofdrug and the shifting of broad endothermic peaks 97.81 and 92.63ºC respectively to the lower side. The result indicated that the nanoparticles consisted of a homogeneous amorphous drug polymer matrix. The sharp peak at 130.16ºC exhibited by RVT was not visible in both the RVT-loaded NPs, indicating that the polymers encapsulated RVT in the NPs^16,17.

The SEM photomicrograph (Fig.4A and 4B) revealed that the lyophilized polymeric nanoparticles were non-spherical, smooth, amorphous and in the nanometre range. The morphological characteristics of the NPs were observed using TEM, shown in Fig. 5A and 5B., respectively. The images reveal that both NPs were non-aggregated, uniform and spherical in shape and particle size in the range of 200nm.

Fig. 3: DSC thermograms of drug and NPs

Fig. 4: SEM image of optimized A) PLGA NPs B) CH NPs

Fig. 5: TEM image of optimized A) PLGA NPs B) CH NPs

Formulation and characterization of transdermal PSA patch loaded with optimized NPs:

Prepared patches were smooth in appearance and uniform in thickness.

Drug content uniformity:

The drug content was found to be 1.66±0.04, 1.58±0.03, and 1.56±0.05mg/cm2 respectively PD, PPN and PCN patches.

In vitro permeation study of PSA patches of RVT:

In vitro permeation profile of RVT from PSA patches contains pure drug and drug-loaded NPS; shown in Fig 6. The total quantity of RVT delivered from PPN and PCN patches was found to be 2210.25 and 2710.82µg, respectively, which was substantially higher than from convention patch PD, which was 741.40µg (p < 0.0001). This might be due to the hydrophobicity of PLGA and CH, which are known to be permeable to the skin. Therefore, the permeation of drug from patch containing drug loaded in NPs was enhanced due to changing the physicochemical properties of drug i.e., the hydrophilic nature of drug and increasing skin partitioning and drug flux the skin. Also, the surface charge of NPs interacts with opposite charge site of cell membrane and the tight junction of mucosal epithelial cells to allow opening of the tight junctions further allow the permeation of drug in deeper site²³. Parameter of permeability, as shown in Table 3, the steady-state flux was more in the case of patches embedded with PNs, than the PD. The steady-state flux and permeability coefficient of PPN and PCN was found to be 223.64µg/cm2.h and 61.43 × 10 -3 cm/h and 265.18µg/cm2.h and 73.25× 10-3 cm/h, respectively whereas for PD was shown to be 55.01µg/cm2.h and 14.75× 10-3 cm/h respectively after 24hrs. Results indicated that the flux of PPN and PCN was 4.06 and 4.82-fold higher than PD²⁴. The results conclude that,the nanosized system of NPs could improve drug delivery efficacy by enhancing drug penetration to the entire depth of the skin through hair follicle route. The surfactant in NPs swells the stratum corneum, and the intact vesicle can penetrate through the intact skin cells ²⁵.

Table 3: In Vitro Permeation Parameters of RVT From Different PSA Patches

Form. Code	Permeated amount at 24 h (µg/cm²)	Flux (µg/cm².h)	Permeability constant(Kp) ×10−3 (cm/h)	Enhancement ratio
PD	741.40	55.01	14.75	-
PPN	2210.25	223.64	61.43	4.06
PCN	2710.82	265.18	73.25	4.82

Fig. 6: In vitro permeation s of RVT from different PSA patches

Comparative studies of developed patches with marketed patches:

The Exelon® patch (9mg)has in vivodrug release rate of 4.6mg/5cm²/24 h, whereas the in vitro permeation rate of developed PPN and PCN patches were 2.21 mg/2.25 cm2/ 24 h and 2.71 mg/2.25 cm2/ 24 h respectively. The PD, PPN and PCN patches should release the same amount of RVT with a patch area of 23.18, 4.68 cm2 and 3.82 cm2 through the skin. The developed PPN and PCN patches containdrugs in NPs which helps the drug permeate more than the Exelon® and conventional PD patches, it may be due to changing the physicochemical properties of drug.Itconcludes that the developed PSA patch of drug loaded in a polymeric nanoparticle can be a potential alternate for Exelon® patch.

CONCLUSION:

RVT-loaded PLGA and CH NPs were prepared using modified nanoprecipitation method and ionic gelation method, respectively, with nano size distribution (<400 nm) and higher entrapment efficiency.In vitro release was found to follow first-order Higuchi model kinetics in both PLGA and CH NPs. The release of RVT from NPs was slower than RVT solution, which may be attributed to higher molecular weight of polymer. In vitro permeation studies conclude that developed patch of drug loaded in polymeric nanoparticle could be a potential alternate for exiting marketed Exelon® patch as a generic product.

ACKNOWLEDGEMENTS:

The authors are grateful to Vision Group on Science and Technology, KSTePs, Karnataka, for providing financial support (Under VGST scheme: KSTePS/VGST-RES/F/GRD Mo.707/2017-18) to conduct research work.Authors also thank STIC, Kochi for performing SEM and TEM analysis.

CONFLICT OF INTERESTS:

The authors declared no conflict of interest.

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Received on 17.01.2022 Modified on 04.07.2022

Research J. Pharm. and Tech 2023; 16(7):3175-3182.

DOI: 10.52711/0974-360X.2023.00522