الفهرس 
Preparation and characterization of Plain Gelatin Nanoparticles (pGNPs) using Modified One-Step Desolvation MethodOnly 14 pages are availabe for public view
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Abstract
Intranasal administration of tacrine hydrochloride (TCR) is considered a practical and non-invasive route for enhancing its brain delivery. Moreover, this route offers the direct and fast transport of TCR to the brain, circumventing the blood brain barrier (BBB). However, the poor contact of formulations with the nasal mucosa and the low absorption of polar drugs are some of the major limitations which should be overcome for administration via this route. In addition, the surface charge and nature of nanoparticulate systems play an important role in the penetration of BBB. Polymeric nanoparticles (NPs) can greatly enhance the drug transportation across the nasal mucosa and BBB by virtue of their small particle size (PS) and also by using brain targeting-assisting additives. The incorporation of polymeric NPs into a mucoadhesive nasal gel provides a longer residence time to overcome the nasal mucocilliary clearance and thus offers a better opportunity for direct nose to brain transport. The objective of this study was to introduce modifications to the one-step desolvation method for preparation of small and monodisperse plain gelatin NPs (pGNPs) with high reproducibility and high colloidal stability. In addition, this study was aimed to prepare TCR-loaded GNPs (as hydrophilic nanosystems) and PLGA NPs (as hydrophobic nanosystems) coated with protamine (Pt) and incorporated into nasal gels for improving the drug bioavailability in brain and decreasing its systemic distribution, and also to study the influence of the nature of NPs on brain delivery.
The introduced modifications to the one-step desolvation method included the preparation of a freeze-dried high molecular weight gelatin (HMWG) and ionization of HMWG using the deionized water acidified at a predetermined pH. A full factorial design (FD) was constructed to study the influence of HMWG concentration, pH, and ratio of the aqueous to organic volume; volume ratio, on the characteristics of pGNPs prepared by the modified one-step desolvation method. Then, TCR-loaded GNPs were prepared by the modified one-step desolvation method and the effect of crosslinking on their characteristics was investigated. PLGA NPs were prepared by the nanoprecipitation method, and the type of used surfactant, drug to polymer ratio (DPR), and molecular weight (MW) of PLGA polymer were investigated in terms of their effects on characteristics of the PLGA NPs by the full FD analysis. The in-vitro degradation of PLGA NPs was studied by the turbidimetry method using a spectrophotometer. The selected TCR-loaded GNPs and PLGA NPs were coated with Pt and formulated in form of the nasal nanocomposite gels. Biological investigations including pharmacokinetic studies in plasma and brain, drug targeting efficiency, nose to brain direct transport (DTP%) and histopathological examinations were performed on Wister male albino rats for the selected nanocomposite gels of TCR-loaded GNPs and PLGA NPs coated with Pt.
The results showed that the suggested modifications for the one-step desolvation method have successfully produced small (56.03–253.9 nm) and monodisperse (PDI = 0.051 to 0.190) pGNPs with high reproducibility, compared to those prepared by the two-step desolvation method. In addition, their zeta potential (ZP) values ranged from 27.9 to 47.8 mV, indicating their good colloidal stability. The FD analysis showed that the tested factors directly affected PS and particle yield (Y%) of pGNPs, noting that the HMWG concentration had the most effect. TCR-loaded GNPs were also of small PS (100.47–187.31 nm) and narrow PDI values (0.055–0.112). The entrapment efficiency (EE%) ranged from 10.56 to 36.67%, where it was observed that the loading of TCR decreased the ZP values and increased Y%, compared to pGNPs. The crosslinking process significantly affected the characteristics of TCR-loaded GNPs, where the increase of glutaraldehyde concentration or crosslinking reaction time decreased PS significantly. However, only the increase of glutaraldehyde concentration significantly increased the rigidity of GNPs and only the increase of crosslinking reaction time decreased significantly the redispersion time of GNPs by sonication.
TCR-loaded PLGA NPs had small PS (85.09–237.67 nm) and narrow PDI (0.075–0.224). EE% ranged from 4.35 to 33.78%, where it was observed that the loading of TCR increased the negativity of PLGA NPs, compared to the plain PLGA NPs. Through the FD analysis, the type of surfactant had the most effect on the PS and ZP of PLGA NPs, followed by the effect of DPR. On the contrary, all tested factors affected significantly EE%, where DPR had the most effect, followed by type of surfactant and then the MW of PLGA. The in-vitro degradation study was carried out for two selected formulae; P15 and P18, where the degradation of PLGA NPs was slow process, as about 72 and 66% of NPs were degraded during 37 days, respectively.
The coating of the selected TCR-loaded GNPs and TCR-loaded PLGA NPs with Pt increased their PS from 120.47 to 141.87 and from 165.93 to 196.43 nm, respectively. In addition, the particles acquired permanent positive surface charges in the physiological pH; 21.80 and 22.53 mV, respectively.
Pt-coated GNPs and Pt-coated PLGA NPs reduced the plasma bioavailability of TCR compared to the uncoated NPs, indicating the ability of Pt-coated NPs to reduce TCR biodistribution into the non-targeted sites. Regarding the brain pharmacokinetic parameters, groups received the Pt-coated GNPs or Pt-coated PLGA NPs showed the highest brain pharmacokinetic parameters compared to those received the uncoated NPs, as well as to the control groups. The Cmax and AUC0-12h for the Pt-coated GNPs were 9.3 and 3 times greater than those of the group received the uncoated GNPs, respectively. Also, the Cmax and AUC0-12h for the Pt-coated PLGA NPs were 2.6 and 2.8 times greater than those of the group that was given the uncoated PLGA NPs, respectively. In regard to the brain targeting efficiency, the results showed that Pt-coated GNPs and Pt-coated PLGA NPs transported 60.55% and 68.4% of TCR, respectively, via the direct nose-to-brain pathway. Additionally, the parameter of plasma-to-brain distribution ratio (Kp) for Pt-coated GNPs and Pt-coated PLGA NPs was 2.5 and 3.2 times higher, respectively, than that of the control group that received IV TCR solution. Moreover, the brain targeting efficiency (DTE%) of Pt-coated GNPs and Pt-coated PLGA NPs increased to 253.51 and 316.53%, respectively. By comparing the selected Pt-coated GNPs (hydrophilic nanosystem), to Pt-coated PLGA NPs (hydrophobic nanosystem), the Pt-coated GNPs had higher brain Cmax (777 ng/g) and shorter tmax (15 min), due to their faster enzymatic degradation, leading to the burst release of TCR. On the contrary, the slow biodegradation of Pt-coated PLGA NPs resulted in lower brain Cmax (369 ng/g) and longer tmax (60 min). However, due to the hydrophobic nature of Pt-coated PLGA NPs, their brain targeting efficiency was better than that of Pt-coated GNPs, where its AUC% and DTE% values were 1.26 and 1.27 times higher than those of Pt-coated GNPs.
Finally, the histopathological study showed that the selected Pt-coated GNPs and Pt-coated PLGA NPs in the form of nanocomposite gels were safe on nasal mucous membranes and different brain tissues, showing only minor congestion in some blood vessels.
Therefore, this study revealed that the enhanced transport of TCR following IN administration of the nanocomposite gels of Pt-coated GNPs or Pt-coated PLGA NPs may help in decreasing the dose and frequency of the drug administration and possibly maximizing the therapeutic benefit of TCR and minimizing its systemic side effects, which are highly required for the chronic treatments.
Keywords: Intranasal administration; hydrophilic nanosystems; hydrophobic nanosystems; Tacrine hydrochloride; Protamine sulfate; Positive surface charge; Brain targeting efficiency; Nose to brain direct transport.