2468-1873/XX $65.00+.00 © XXXX Bentham Science Publishers Pegylated Chitosan Biodegradable Nanoparticles Delivery of Salvia officinalis and Melissa officinalis for Enhanced Brain Targeting

Background:: Alzheimer's disease (AD) is a progressive neurodegenerative condition characterized by the gradual decline of cognitive abilities, primarily caused by impairments in the cholinergic system. AD is diagnosed based on the presence of specific pathological features, in-cluding senile plaques, neurofibrillary tangles, and the loss of neurons and synapses. Despite on-going efforts, the etiology of AD remains unclear, and there is a significant lack of effective treatments to meet the medical needs of affected individuals. The complex nature of AD, involv-ing multiple factors, presents challenges in the development of potential therapies. Numerous ob-stacles hinder the achievement of optimal pharmacological concentration of promising molecules for AD treatment. These obstacles include the presence of the blood-brain barrier (BBB), which restricts the entry of therapeutic agents into the brain, as well as issues related to poor bioavaila-bility and unfavorable pharmacokinetic profiles. Unfortunately, many therapeutically promising compounds have failed to overcome these hurdles and demonstrate efficacy in treating AD. background: Alzheimer’s disease (AD) is a progressive neurodegenerative disease that is manifested by depleted cognitive abilities resulted due to cholinergic impairments. AD is further diagnosed with pathological hallmarks including senile plaques, neurofibrillary tangles and neuronal and synaptic death. With constant efforts, few therapeutic targets and interventions have been identified but AD is still a disease with unclear etiopathology and unmet medical needs. The multifactorial nature of AD poses difficulties to develop a potential treatment. Unfortunately, large numbers of therapeutically efficient molecules for the treatment of AD failed to attain optimal pharmacological concentration due to numerous hurdles such as the presence of blood-brain barrier (BBB), poor bioavailability, or pharmacokinetic profile. Methods:: The PEGylated chitosan nanoconjugate was developed and evaluated for delivery of anti-Alzheimer natural extract of Salvia officinalis and Melissa officinalis to the brain. The nano-conjugates (S-PCN and M-PCN) were developed by ionic gelation technique. Result:: The nanoconjugates (S-PCN and M-PCN) were evaluated for various optical and in-vitro parameters. MTT assay on UCSD229i-SAD1-1 human astrocytoma cells indicated IC50 values of 0.42, 0.49, 0.67, and 0.75 μM for S-PCN, M-PCN formulations, and free Salvia officinalis and Melissa officinalis extracts, respectively. The In vitro assessments using cell lines have confirmed the improved uptake and distribution of nanoconjugates compared to free extracts. These findings were validated through confocal microscopy and apoptosis assays, revealing a substantial in-crease in the accumulation of nanoconjugates within the brain. The targeting potential OF M- PCN over S-PCN was found to be 2-fold significant. method: 1. Sample Preparation - Crude drug Salvia officinalis and Melissa officinalis, plants were collected from the botanical gardens of Warangal and Tirupathi and authenticated.The two plants, 1 g each, were crushed (using a lab mill) for 1 min, to obtain the corresponding powder. The extraction powder was performed as described in previous reports, via addition of 100 mL boiling water to 1 g of plant powder and after 5 min, the extract was filtered through a 0.45 mm filter. This procedure was optimized to obtain the highest potential activity of these plants. After the crude plant sedimentation, samples were filtered and maintained at 80 ͦ C, for freeze-drying procedures (Heto Holten A/S Drywinner, Allerød, Denmark). Then, solutions of 1% (w/v) of freeze-dried powder were dissolved in methanol for analytical evaluation other activity tests. Before injections, samples were filtered again through a 0.45 mm filter. 2. Preparation of PEGylated Chitosan Nanoparticles - Ionotropic gelation technique was employed for the synthesis of chitosan, encapsulating whole Salvia officinalis and Melissa officinalis extract separately. Accurately weighed 100 mg of Salvia officinalis and Melissa officinalis extract and 0.4% w/v of Chitosan were dissolved in 1% v/v aqueous glacial acetic acid (GAA) solution. Drop wise addition of 0.4% Sodium tripolyphosphate solution (TPP) was performed in drug polymer solution at the rate of 2ml/min (12 ml TPP in 20 ml drug polymer solution). The obtained particles dispersion were sonicated using a probe sonicator (S-4000; Misonix, Farmingdale, NY) at medium amplitude (50%) for 5 min to obtain nano sized particles. The dispersion was then filtered through a 0.2 um hydrophilic filter (Minsart, Sartorius) for isolation of smaller nanosize particles in order to achieve maximum transportation at targeted site. The nano sized particles, thus obtained were carefully purified by ultrafiltration (Amicon 8200 with a millipore PBMK membrane, MWCO 300000) against double distilled water at optimal temperature. The ultrafiltration facilitates elimination of residual of unreacted solvent and unbound drug. For the PEgylation process, accurately 50 mL of 0.3 % chitosan nanoparticles were added into polyethylene glycol (PEG) solution with a ratio of 3:1 and stirred at 500 rpm for 1 h. Further, dispersion was applied to the mixture for 60 seconds to produce homogeneous PEG-Chitosan nanoparticles. Result The formation of the PEGylated chitosan nanoparticles entrapping natural extract Salvia officinalis and Melissa officinalis ensued impulsively upon combination of the pawn anion TPP into the consistent Chitosan polymer solutions. Nanoparticle formation resulted from the ionic interactions between the negative charge ion TPP and the positively charged amino groups of Chitosan. The ratio of CS/TPP was optimized to attained stable dispersion and formation of nanosize particles. Preliminary experiments were performed in order to identify the optimal concentrations of CS and TPP for NP formation. The process parameters along with formulation parameters were thoroughly optimized for the achievement of physiochemical and thermal stable nanoparticles. The obtaining nano size particles were broadly characterized as either a clear solution, an opalescent suspension displaying a tyndall effect (NPs), or aggregate. 1 Particle size, Zeta Potential and Morphology The results achieved from the zeta sizer measurement displayed very distinct size of prepared S-PCN and M-PCN formulations ranging 150-250 nm (Figure 1- a & b). The nano size of the S-PCN, M-PCN formulations displayed decent encapsulation of extract in the polymer matrix due to the formulation and process optimization. The surface charge of both nanoformulation S-PCN, M-PCN was found to be -10.89 mV and -16.21 mV respectively (Figure 1- e & f) demonstrated negative charge nature of both formulation. The negative charge of formulation showed better stability and optimum candidature for enhance brain targeting. The pH of S-PCN, M-PCN formulations was measured as 6.9 ± 0.01 which play a vital role in nearly neutral microenvironment delivery for efficient brain targeting. The pH facilitate targeting mechanism act as the key element for the onsite degradation of the polymer matrix. This polymeric degradation activation mechanism enhanced the drug release at a controlled rate resulting into the desired therapeutic potential. 2 DLS Analysis The DLS outcomes again nanosize range dispersion of prepared S-PCN and M-PCN nanoformulation. The size distribution pattern of both nanoformulation is some identical to each other exhibiting size range of 160-240 nm for S-PCN and 150-230 nm for M-PCN formulation. The optimal nanosize range of both nanoformulation demonstrated the enhanced brain delivery and onsite targeting which efficiently comply the size of cells and its micro-environment. The DLS investigations showed diverse size distribution of and dispersion pattern. The PDI exhibited by S-PCN and M-PCN was found to be of 0.271 ± 0.08 and 0.259 ± 0.11. The DLS results showed enhance stability with even size distribution pattern of prepared nanoparticles between 100-500 nm (Figure 1-c & d). This nanosize stable pattern facilitates enhance diffusion of prepared nanoparticles across the blood brain barriers leading to optimal pharmacological potential during brain targeting. Therefore, it can be unswervingly state out that both the nanoformulations exhibited optimal and stable nano dispersal features for the operative brain targeting against Alzheimer management in clinical platform. 3 Transmission Electron Microscopy (TEM) The TEM analysis showed very discrete particles size exhibiting oval shape nanoparticles of both nanoformulation. The size revealed by TEM analysis for S-PCN and M-PCN was ranging 100-250 nm validating DLS measurement zeta sizer analysis (Figure 2- a & b). The formation of nanoparticles by entrapping natural extract showed better crosslinking between polymer and cross linker avoiding unwanted leakage. Also the aggregation of nanoparticles was found negligible showing better PEGylation process of chitosan boundaries. The TEM outcomes displayed suitable nano carrier system for the effective brain delivery, revealing decent BBB infiltration appearance of both nanoformulation. 4 Scanning Electron Microscopy (SEM) The SEM analysis significantly the results obtained by zeta sizer and TEM assay showing fine particles formation with spherical shape and smooth morphology. The SEM images noteworthy validates the sharp oval boundaries of both nanoformulation exhibiting better PEGylation process. The SEM images also clarifies no sign of clusters formation of agglomeration of particles showing significant PEG outer layer. The SEM analysis exhibiting size range of 150-250 nm again qualitatively validating the TEM, and zeta-sizer analysis and confirming the ideal brain targeting delivery characteristics of both S-PCN and M-PCN (Figure 2 – c & d). 5 In-vitro drug release studies In vitro drug release data of Salvia officinalis and Melissa officinalis extract associated with PEGylated nanoformulations is demonstrated in figure 3- a & b. The drug release pattern from both the nanoformulation S-PCN and M-PCN at different pH (4.0 & 7.4) exhibited a non-linear release profile characterized by a relatively faster initial drug release during the first 3-4 h, followed by slower release in later period. The two pH range was provided to deeply evaluate the effect of nanoformulations for better brain targeting and onsite delivery. The biphasic drug release pattern was observed by both nanoformulation with initial bursting of nanoparticles in early 1-8 h followed by slow release in 24 h. The in-vitro drug release studies suggested that initially both S-PCN and M-PCN provided burst release of drug extract at pH 4.0. The drug release was found to be 89.45 ± 3.67 % at 6h, 91.42 ± 2.11 at 8 h, 90.26 ± 1.84 % at 6 h and 95.67 ± 2.20 % at 8 h for S-PCN and M-PCN, respectively. On the contrary at pH 7 the drug release was significantly (P < 0> S-PCN. 6 In vitro cellular uptake The capacity of cellular targeting and intracellular transport of developed nanoformulation S-PCN and M-PCN evaluated and measured by using UCSD229i-SAD1-1 human astrocytoma cells line. The human astrocytoma cells line are imperative part of BBB and broadly engaged for the examination of brain delivery. The developed S-PCN and M-PCN showed noteworthy cellular acceptance and circulation compared to the free drug extract of Salvia officinalis and Melissa officinalis when evaluated by CLSM analysis. The CLSM signals for the developed S-PCN and M-PCN were resilient and sharp with enhance absorbance when treated with Rhodamine B isothiocyanate (RITC) compared to the free drug extract of Salvia officinalis and Melissa officinalis suspension on incubation for 12 h (Figure 4). In addition, the confocal laser scanning microscopic intense fluorescence signals displayed by nanoformulations showed the clear sign of vesicular localization of nanoparticles demonstrating enhance endocytic pathway progression. The CLSM signals showed by M-PCN samples treated UCSD229i-SAD1-1 human astrocytoma cells showed sharp red fluorescence signal around the cell nucleus when compared to the cells treated S-PCN incubated at 4 h and 12 h of time periods which is found enhanced and significant. The results of CLSM intensity examination showed 2 folds enhance cellular uptake and resilience in-vitro by M-PCN compared to S-PCN on the brain cell membranes. The S-PCN and M-PCN treated cells were also quantitatively observed inductively attached with the plasma optical emission (ICP-OE) spectrometry for 12 h of incubation. The results efficiently inveterate that the around ~45% of M-PCN and ~33% of S-PCN nanoformulation have pointedly traversed into the BBB layer, validated by the transwell assay at basolateral side. The free drug extracts showed scanty diffusion across BBB via UCSD229i-SAD1-1 human astrocytoma cells of ~16% signifying non-significant intracellular transport and penetrating efficiency due to early adsorption at cell membrane restricting direct diffusion to the cells (Figure 3c). Overall, at different incubation time interval, the cell uptake and transportation capability of M-PCN was remarkable compared to S-PCN with strong fluorescent adverts bereft of any morphological difference in cell lines, resulting in enhanced brain targeting efficiency. 7 In vitro cytotoxicity assay The MTT assay was employed for the investigation of developed M-PCN and S-PCN toward UCSD229i-SAD1-1 human astrocytoma cells. The MTT assay qualitatively showed significant anti-proliferation capability of nanoformulations in 24h of incubation. The investigations showed sharp cell viability of 100% and 10% by control Normal control (saline solution) and negative control group (Triton X 100 surfactant solution) respectively. The developed S-PCN and M-PCN showed notable cell viability of 96%, 89%, 76% & 65% and 98%, 90%, 80% & 71% at different concentration (0.1, 1, 10 and 20 μg/mL of individual concentration) on 24 h of incubation (Figure 3d). Whereas free drug extract of Salvia officinalis and Melissa officinalis showed cell viability of 96%, 88%, 68%, & 48% and 95%, 86%, 69% & 52% respectively on 24 h of incubation. The MTT investigation established non-significant cell cytotoxicity by different samples in 24h of incubation showing nonlinear relationship between incubation time and anti-proliferation efficiency. The MTT results clearly displayed significant cell viability of nanoformulation over free drug extract in 24 h of incubation expressing biologically safe brain targeting efficiency with negligible toxicity on human astrocytoma cells. The enhance cell viability showed by developed S-PCN and M-PCN is due to better physiochemical compatibility between nanocomposite resulting in efficient cellular transport and brain delivery. On inter-comparison of nanoformulation the cell viability of M-PCN is greater than S-PCN with less cell cytotoxicity at higher concentration. The inter-comparison results showed better endocytosis and resilience of M-PCN which is found statistically significant when analyzed by student’s T test. Overall the cell toxicity examinations clearly expounds that the developed nanocomposite may be used as novel drug carrier encapsulating natural extract for the treatment of brain diseases as targeted delivery system. 8 Apoptosis assay The Apoptosis investigation showed by free drug extract, S-PCN and M-PCN and verified striking apoptosis at all concentrations. The developed S-PCN and M-PCN showed inherent apoptosis compared to the free drug extract. It has been noted out that both S-PCN and M-PCN showed mitochondrial apoptosis phenomenon or death activator by provoking cell surface receptor. By activating cell surface receptor the activation of caspase cascade establishes optimum cell death which results in desired apoptosis process. The apoptosis index of free drug were found to be 0.39 and 0.42 for Salvia officinalis and Melissa officinalis respectively whereas the S-PCN and M-PCN showed apoptotic index of 0.66 and 0.79 respectively. The nanoformulation showed significant apoptosis action compared to plain free natural extract which is nearly two folds more and found significant (*P<0.01) (Figure 5). The chief cause for better apoptosis of nanoformulation over free drug extract is the nanosized particles, causing quick onsite drug transportation, sufficient distribution and better release. On inter-comparison of S-PCN and M-PCN the apoptosis potential is significant showed by m-PCN compared to S-PCN when analyzed by student T test. Overall PEgylation of chitosan nanoparticles facilitates better circulation of nanoparticles in brain microenvironment causing extended release and negligible drug toxicity resulting in better brain targeting against Alzheimer disease. Conclusion:: Based on the findings, it can be inferred that biodegradable PEGylated chitosan nanoconjugates hold promise as effective nano-targeting agents for delivering anti-Alzheimer drugs to the brain. The incorporation of PEGylated chitosan nanoparticles in this approach demonstrates enhanced delivery capabilities, ultimately leading to improved therapeutic out-comes. other: Characterization 1 Particle size, Zeta potential, pH and Morphology The developed S-PCN, M-PCN particle size and surface charge was measured by Malvern Zetasizer 3000 particle size and zeta potential analyzer (Malvern Instruments, Bedfordshire, UK). The Zeta potential of S-PCN, M-PCN was examined by smearing the principle of electrophoretic movement of particles in an applied electrical field. The concentration of both S-PCN, M-PCN formulation was attuned at 0.01% w/v by distilled water or in 0.01 M sodium chloride solution for potential assessment. The pH was calculated by using a digital pH meter (HI-TECH WATER TECH. New Delhi, India). The pH meter was first calibrated using buffer tablet, the pH meter was dipped in a beaker comprising S-PCN and M-PCN nanoformulations on post calibration. The nanoformulations, evaluation was triplicated and the measurement was repeated thrice with an average value along with SD was reported. 2 Dynamic Light Scattering (DLS) The S-PCN and M-PCN nanoformulations was examined for the Dynamic Light Scattering (DLS) investigating mean diameter and PDI by employing Brook-heaven BI 9000 AT instrument (Brookheaven Instrument Corporation, USA). The DLS examination was measured for the more distinct and significant evaluation of both S-PCN and M-PCN nanoformulations. The DLS evaluation were done at wavelength 417 and 215 nm for S-PCN and M-PCN nanoformulations receptively at temperature of 25°C. 3.3 Transmission Electron Microscopy (TEM) The TEM of both S-PCN and M-PCN nanoformulations was measured by using Hitachi H-7500 TEM analyzer. TEM metaphors were obtained to visualize the shape and structure of nanoformulaion. The S-PCN and M-PCN nanoformulations were coated with 2.5% w/v of phosphor-tungstic acid (PTA) solution and placed in a copper disc grid. The grid was then desiccated in 60 watt LED lamp (Philips, India Ltd) and was finally placed into the disc holder and scanned for TEM evaluation. 4 Scanning Electron Microscopy (SEM) The morphology and structure of prepared S-PCN and M-PCN nanoformulations were analyzed by SEM, Nova Nano SEM 450, Germany. Before the SEM assessment, the formulations were lyophilized by using freeze dry lyophilizer (REMI, New Delhi, India). The dried formulations were then placed on a SEM stub by using dual adhesive tape at 50mA 5-10 minutes via sputter (KYKY SBC-12, Beijing, China). A SEM aided with secondary electron detector was engaged to get the digital images of the developed S-PCN and M-PCN nanoformulations. 5 Entrapment Efficiency (EE): EE plays essential part in transporting the bioactive to the targeted site at detailed therapeutic dose in order to get the anticipated therapeutic value. To measure the EE, both the nanoformulations were centrifuged at 10000 rpm for 5 minutes to obtain the pellets. The collected supernatant was carefully diluted with PBS of pH 7 and the drug content was determined spectrophotometrically by using UV spectrophotometer (Schimadzu, Japan) at 317 nm and 215 nm for S-PCN and M-PCN nanoformulation respectively against a blank solvent. The EE can be measured by using the following formula: EE= weight of drug in nanoformulation / initial weight of drug taken x 100 6 In vitro Drug Release studies The release of from both S-PCN and M-PCN nanoformulations was tracked to predict the diffusion and kinetic behavior of the nanosystems for desired therapeutic efficiency. For release studies, both S-PCN and M-PCN obtained after centrifugation were suspended in 10 mL of a phosphate buffered saline (PBS) solution, pH 7.4. This nanoparticle suspension was transferred to clean Eppendorf’s tube and placed in a water bath at 37 °C under stirring. After 0.5, 1, 2, 4, 6, and 24 h, samples were collected from the bath and centrifuged at 14 000 rpm for 5 min (BOECO, Hamburg, Germany). Supernatants were analyzed by UV spectroscopy and used to calculate the amount of drug released from the nanoparticles over the specified time. Triplicate samples were analyzed at each time. 7 Cell Line studies 7.1 Cell Culture and Seeding The Human UCSD229i-SAD1-1 human astrocytoma cells line was obtained from NCCs Pune and was conserved in Dulbecco’s modified Eagles Medium. The cell line was then supplemented with 10