الفهرس 
Preparation and evaluation of in-situ forming vesicles encapsulating a model drug
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Abstract
Intra-articular injection is becoming of major interest nowadays due to the poor blood supply at the targeted site of action which minimizes the effect of the orally administered moiety. Based on the integral role of non-steroidal anti-inflammatory drugs (NSAIDS) in the long-term treatment of Rheumatoid Arthritis (RA), there is an ongoing effort to develop novel drug delivery systems like in-situ forming nanoparticles (ISNs) to increase the bioavailability of non-steroidal anti-inflammatory drugs and minimize their side effects through intra-articular injection at the site of inflammation. Hence, the aim of this work was to develop, characterize and optimize targeted in-situ forming nanoparticles (ISNs) for intra-articular delivery of piroxicam as a model NSAID for rheumatoid arthritis treatment. A 33 full factorial experimental design was conducted to assess the significance levels of the investigated independent factors on the dependent variables. Also, morphological characterization, injectability and rheological studies were conducted to evaluate the optimized ISNs. Furthermore, the interaction between the drug and other formulation components was monitored by FTIR. Additionally, in-vivo evaluation of the biological performance and histopathological examination were studied to determine the efficacy of the optimized ISNs for treatment of RA using arthritic adult male Sprague–Dawley rats. Formulations showing significant histological improvements were further biochemically studied.
The current investigations together with the obtained results and attained conclusions can be summarized as follows:
1. The wavelength of maximum absorbance (λmax) for PX was 252 nm in phosphate buffer solution (pH=7.4).
2. PX-loaded ISNs were prepared using a 33 full factorial experimental design to investigate the joint influence of formulation variables and experimental conditions on the obtained formulations using Design-Expert® software.
3. In-situ forming nanoparticles could be prepared using synthetic polymers like: PDLG in combination with gelatin to increase the viscosity at the site of injection.
4. In the 33 full factorial experimental design:
a. Three factors were evaluated as independent variables at three levels each.
b. The independent variables were: (A) percentage of internal phase relative to the total formulation (internal and external phases) at three levels (10% w/v, 20% w/v, 30% w/v), (B) percentage of gelatin at three levels (2.5%w/v, 5% w/v, 10% w/v) and (C) percentage of PDLG at three levels (2.5% w/v, 5% w/v, 10% w/v).
c. All formulations compromised the same concentration of Brij® 52 and Eudragit RL (2.5% each).
d. Experimental trials were performed at all the 27 possible combinations.
e. Particle size, PDI, release rate constant, T50%, T25% and MDT were chosen as the dependent variables.
f. Results showed that only gelatin percentage had a significant (p < 0.0001, p =0.0004, p= 0.0065p =0.0003) effect on T25%, T50%, MDT and K-values, respectively.
g. The two-factor combination AC had a significant (p=0.0032, p < 0.0001, p=0.0044, p=0.0009) effect on T25%, T50%, MDT and K-values, respectively.
h. The two-factor combination AB had a significant (p = 0.0184) effect on T25% value.
5. The obtained particles had a PS ranging from 146.5 ± 4.95 to1310 ± 130.10 nm
6. ISN-14 with 20% internal phase and gelatin and PDLG at 5% level showed the smallest PS whereas ISN-27 compromising 30% internal phase and gelatin and PDLG at 10% level showed the largest one.
7. Using self-emulsifying oils as Captex® improved the physicochemical properties of the obtained ISNs.
8. Optimum target release pattern was observed in ISN-26 comprising 30% internal phase, gelatin 10% and PDLG at 5% level, in addition to Brij® 52 2.5% and Eudragit RL 2.5% dissolved in DMSO as the internal phase and emulsified into Captex® stabilized with Tween® 80 as the external oily phase where an initial release of 22 % was observed in the first hour followed by a sustained release pattern for 7 days.
9. The kinetics of the drug release from all investigated ISNs was fitted to zero and Higuchi models and the obtained results were confirmed by fitting into the Korsmeyer-Peppas equation. All formulation exhibited a diffusion release pattern following the Higuchi model.
10. TEM Images for the selected formulation (ISN-26) revealed the formation of spherical nanoparticles with a dense core and a less dense outline
11. Farrow’s constant of the selected formulation (ISN-26) was found to be 1.1048 which confirmed the formulation pseudoplastic properties.
12. The selected formulation (ISN-26) succeeded to flow rapidly through the 19-gauage needle when compared to the market product which indicates good injectability of the prepared formulation.
13. FT-IR examination showed disappearance of the N-H peak of gelatin as well as the O-H peak of Brij® 52 and the C=O peak of the drug indicating the formation of hydrogen bonds between the drug and both the gelatin and the Brij that may have improved the encapsulation of the drug during the deposition of the ISNs.
14. The biological performance of the optimized formulation was studied on thirty six adult male Sprague–Dawley rats (mean body weight of 200 ± 50 g). The results of the in-vivo study can be summarized as follows:
• A comparative study was conducted to compare the efficacy of the IA optimum formulation (ISN-26), its drug-free analogue (placebo), oral drug solution and intra-articular drug suspension.
• The optimized formulation succeeded to restore the joints histological architecture as evidenced by the accelerated chondrogenic maturity and significant restoration of articular cartilage thickness.
• ISN-26 exhibited a marked anti-inflammatory potential related to suppressing the pivotal chemokines and downregulating the fundamental protein expression of STAT-3 and RANKL.
The above-mentioned data would, definitely, point out the potentiality of formulating PX in the form of ISNs using PDLG as a synthetic polymer in combination with gelatin for the formulation of ISNs. In addition, the use of self-emulsifying oils as Captex® improved the characteristics of the obtained particles. All of this could successfully achieve an acceptably rapid initial drug release that provides a suitable onset of action as well as a sustained drug release over 7 days, thus, reducing dosing frequency.