Bupivacaine HCl (1-butyl-2',6'-pipecoloxylidide hydrochloride), an amide local anesthetic compound, is a local anesthetic drug utilized for intraoperative local anesthesia, post-operative analgesia and in the treatment of chronic pain. However, its utility is limited by the relative short duration of analgesia after local administration (approximately 9 h after direct injection) and risk for side effects. This work is aimed to develop a nanoemulsion of bupivacaine HCl with sustained local anesthetics release kinetics for improved pain management, by exhibiting extended analgesic action and providing reduced peak levels in the circulation to minimize side effects. Herein, biodegradable oils were evaluated for use in nanoemulsions to enable sustained release kinetics of bupivacaine HCl. Only with castor oil, a clear and stable nanoemulsion was obtained without the occurrence of phase separation over a period of 3 months. High loading of bupivacaine HCl into the castor oil-based nanoemulsion system was achieved with about 98% entrapment efficiency and the resulting formulation showed high stability under stress conditions (accelerated stability test) regarding changes in visual appearance, drug content, and droplet size. We show herein that the in vitro release and in vivo pharmacokinetic profiles as well as pharmacodynamic outcome (pain relief test) after subcutaneous administration in rats correlate well and clearly demonstrate the prolonged release and extended duration of activity of our novel nanoformulation. In addition, the lower C value achieved in the blood compartment suggests the possibility that the risk for systemic side effects is reduced. We conclude that castor oil-based nanomulsion represents an attractive pain treatment possibility to achieve prolonged local action of bupivacaine HCl.
Superoxide dismutase (SOD) is an antioxidant protein. When administered orally, it has low bioavailability due to its low permeation. In a previous study we fused gliadin peptide P51 (LGQQQPFPPQQPYPQPQPF) and gliadin peptide P61 (QQPYPQPQPF) with SOD Citrus limon (SOD_Cl), namely GliSOD_P51 and GliSOD_P61 to increase permeation of SOD_Cl through intestine. In this work, the permeation of fluorescein isothiocyanate (FITC)-Dextran 10 kDa, FD10 and 40 kDa, FD40 as paracellular transport markers across excised rat intestinal wall was investigated with the presence of GliSOD_P51 and GliSOD_P61. A permeability study was performed using non-everted rat intestine by incubating FD10 or FD40 with SOD_Cl, and GliSOD_P61. The presence of SOD_Cl, GliSOD_P51 or GliSOD_P61 inside intestine (apical) and outside intestine (basolateral) was analyzed by protein electrophoresis. The concentration of FD that penetrated to the basolateral solution was analyzed by spectrofluorometry. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed the presence of GliSOD_P51 and GliSOD_P61 but not SOD_Cl in basolateral compartment. The percentage of FD10 but not FD40 and SOD_Cl that penetrated to the basolateral solution significantly increased with the presence of gliadin in GliSOD_P51 and GliSOD_P61. GliSOD_P51 and GliSOD_P61 are able to penetrate the rat intestinal epithelial membrane and the gliadin peptides facilitate FD10 to penetrate the epithelial.
The physical interaction between 2 substances frequently occurs along the mixing and manufacturing of solid drug dosage forms. The physical interaction is generally based on coarrangement of crystal lattice of drug combination. The cold contact method has been developed as a simple technique to detect physical interaction between 2 drugs. This method is performed by observing new habits of cocrystal that appear on contact area of crystallization by polarization microscope and characterize this cocrystal behavior by melting point determination. Has been evaluated by DSC, this method is proved suitable to identify eutecticum interaction of pseudoephedrine HCl-acetaminophen, peritecticum interaction of methampyrone-phenylbutazon, and solid solution interaction of amoxicillin-clavulanate, respectively.
Background: Lipopeptide-based gene carriers have shown low cytotoxicity, are capable in cell membrane penetration, are easy to manufacture and therefore are great potential candidates for gene delivery applications. Objectives: This study aims to explore a range of short synthetic lipopeptides, (Lau: Lauryl; Pal: Palmitoyl) consisting of an alkyl chain, one cysteine (C), 1 to 2 histidine (H), and lysine (K) residues by performing in-silico molecular interaction and in-vitro evaluation. Methods: The molecular interactions between the lipopeptides and Importin-α receptor were performed using AutoDock Vina and Amber14. The lipopeptide/DNA complexes were evaluated in-vitro for their interactions, particle size, zeta potential and transgene expression. Transfection efficiency of the lipopeptides and Pal-CKKHH-derived liposome was carried out based on luciferase transgene expression. Results: The in-silico interaction showed that Lau-CKKH and Pal-CKKHH hypothetically expedited nuclear uptake. Both lipopeptides had lower binding energy (-6.3 kcal/mol and -6.2 kcal/mol, respectively), compared to the native ligand, viz, nuclear localization sequence (-5.4 kcal/mol). The short lipopeptides were able to condense DNA molecules and efficiently and formed compacted nanoparticles. Based on in-vitro evaluation on COS-7, Pal-CKKHH was found to be the best transfection agent amongst the lipopeptides. It transfection efficiency (ng Luc/mg total protein) increased up to ~3-fold higher (1163 + 55) as it was formulated with helper lipid DOPE (1:2). The lipopeptide-based liposome (Pal-CKKHH: DOPE=1:2) also facilitated luciferase transgene expression on human embryonic kidney cells (293T) and human cervical adenocarcinoma cells (HeLa) with transfection efficiency 1779 +52 and 260 + 22, respectively. Conclusion: Our study for the first time has shown that the fully synthesized short lipopeptide Pal-CKKHH is able to interact firmly with the Importin-α he lipopeptide is able to condense DNA molecules efficiently, facilitate transgene expression, expedite nuclear uptake process, and hence has the characteristics of a potential transfection agent.
The main requirement of transfection agent has to condense DNA in nanoparticle size, protect the DNA from nucleases and other degrading enzymes during its transport in cell cytoplasm and nucleus and should not toxic to target cells. In this research, lipopeptide composed of palmitoyl (C-16) and short peptide sequence have been designed fully synthesized and tested to DNA condensation capability and toxicity. The DNA condensation study was performed using EtBr exclusion assay and cytotoxicity determination was carried out by colorimetric MTT assay. It was revealed that lipopeptide-based transfection agent of Pal-CKKHH and Pal-CKKHH-YGRKKRRQRRR-PKKKRKV condensed DNA molecules efficiently. The lipopeptide was less toxic compared to Lipofectamine and Poly-L-Lysine, that shown by 90% of CHO-K1 cells remained viable when they were treated with 4.36 µM Pal-CKKHHYGRKKRRQRRR-PKKKRKV. Meanwhile, there were only ~75% and 80% of CHO-K1 viable cells when it was treated with PLL and Lipofectamine®2000, respectively. Moreover, cell viability of HepG2 was ~ 75% after treated with 2.18 µM of Pal-CKKHH-YGRKKRRQRRR-PKKKRKV and decreased to ~65% when the lipopeptide concentration increased to 8.72 M. In summary, the synthesized lipopeptide condenses DNA molecules efficiently, less toxic than Lipofectamine®2000 and PLL and has possibility to be explored as a non-viral gene delivery vehicle.
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