Preparation and characterization of water-soluble prodrug, liposomes and micelles of Paclitaxel

Alternative formulations of paclitaxel were developed in order to improve its aqueous solubility, and characterized in vitro. A methacrylic acid based nanoconjugate of paclitaxel was synthesized by a simple esterification reaction with molecular weight of 1657 Da. The in vitro hydrolysis study on the prodrug of paclitaxel in presence of rat plasma has shown that the ester bond was quite stable (less than 1% of paclitaxel was liberated from prodrug in 24 h). This water-soluble prodrug was encapsulated into polyethylene glycol coated liposomes optimized with saturated lipids, to overcome the physical instability associated with paclitaxel. Under in vitro testing, prodrug liposomes seem very impressive with release of only 45% of payload in 180 h. Further, chemical as well as physical stability studies have shown that liposomes were stable without any signs of crystallization of paclitaxel. In addition, paclitaxel was covalently coupled to poloxamer via methacrylic acid linker to obtain a micelle forming conjugate. Evidence for self-assembly of this conjugate into micelles was provided by fluorescence spectroscopy, light scattering and differential scanning calorimetry techniques. Micellization of the conjugate was thermodynamically favored and the core of resulting micelles exhibited higher microviscosities (than poloxamer micelles). Release studies using dialysis technique along with high performance liquid chromatography revealed that paclitaxel is liberated from micelle in the form of methacrylic acid oligomer based prodrug in a gradual manner. These preliminary studies provided indication on the performance and feasibility of testing these carrier systems as a safer alternative to the Cremophor EL based paclitaxel formulation.     

In situ gel formulations for gene delivery: release and myotoxicity studies

The in vitro release of plasmid DNA and salmon sperm DNA from in situ gel formulations was investigated. Two in situ gel systems were studied: (a) an interpolymeric complex (IPC) of water-soluble polymers polymethacrylic acid (PMA) and polyethylene glycol (PEG) and (b) a hydroxypropylmethylcellulose-carbopol system (H:C). Two-way analysis of variance with replication demonstrated that both gel composition and medium pH influenced significantly the release of plasmid DNA from in situ gel formulations. When the release of both types of DNA was compared, higher release was observed for plasmid DNA compared to genomic salmon sperm DNA. Conformational analysis of the released plasmid DNA showed that DNA was released without degradation, but with remarkable conversion from supercoiled (SC) to open circular (OC). In addition, the tested in situ gel systems demonstrated protection from DNAse I degradation. The myotoxicity of the injectable gelling solutions was assessed by the cumulative release of creatine kinase (CK) over 120 min from the isolated rodent extensor digitorum longus (EDL) muscle. A higher level of cumulative CK was observed for IPC when compared to H:C (2:1). These results demonstrate that the in situ gelling systems can be considered as a valuable injectable controlled-delivery system for pDNA in their role to provide protection from DNAse degradation.     

Biodegradable polymeric microspheres for nalbuphine prodrug controlled delivery: in vitro characterization and in vivo pharmacokinetic studies

The objective of this work was to study the in vitro characteristics as well as in vivo pharmacokinetic performance of a series nalbuphine (NA) prodrug-loaded microspheres. An oil-in-water solvent evaporation method was used to incorporate the various NA prodrugs into poly(D,L-lactide-co-glycolide) (PLGA)-based microspheres. The morphology of microspheres under the scanning electron microscopy (SEM) revealed a spherical shape with smooth surface. Drug release rates for the microspheres were found to be a function of prodrug hydrophilicity, with higher drug release rates for microspheres loaded with more hydrophilic prodrugs. The release profiles fit well to the Baker and Lonsdale's spherical matrix model, suggesting the drug release from microspheres was consistent with a diffusion mechanism. The in vivo pharmacokinetic studies after s.c. injection of microspheres into rabbits showed sustained plasma NA-time profiles, with approximately 104.7, 67.2, and 41.0% relative bioavailability for microspheres loaded with nalbuphine propionate (NAP), nalbuphine pivalate (NPI), and nalbuphine decanoate (NDE), respectively. The in vitro release characteristics correlated well with the in vivo pharmacokinetic profiles. The results indicated that the prodrug hydrophilicity had significant effects on the in vitro as well as in vivo drug release kinetics. The present study demonstrates the feasibility of using biodegradable polymeric microspheres for controlled delivery of NA prodrugs.     

The characterisation,pharmacokinetic and tissue distribution studies of TPGS modified myricetrin mixed micelles in rats

This study was designed to investigate the bioavailability and targeting of myricetrin-loaded ternary micelles modified with and without TPGS. The particle diameters of myricetrin-loaded micelles and myricetrin-loaded-TPGS micelle were 30.93?±?1.34?nm and 26.42?±?0.89?nm, respectively, while their respective encapsulation efficiencies (m/m, %) were 83.3?±?1.08 and 93.8?±?1.18. The release rate of myricetrin in the micellar system clearly exceeded the free myricetrin in the three media (pH 6.8 phosphate buffer, pH 1.2 HCl solution and double distilled water). In vivo studies displayed that the bioavailability of myricetrin mixed micelles was remarkably improved than the free drug after oral administration. Moreover, the results of tissue distribution showed that myricetrin-loaded-TPGS micelles accumulated well in the liver tissue. Based on these results, it was speculated that myricetrin-loaded-TPGS micelles might act as a promising carrier for liver targeting with improved hepatic concentration of myricetrin compared with the myricetrin-loaded micelles.     

Galactosylated bovine serum albumin nanoparticles for parenteral delivery of oridonin: tissue distribution and pharmacokinetic studies

Abstract

Bovine serum albumin (BSA) nanoparticle is a promising drug carrier system. Oridonin (ORI)-loaded galactosylated BSA nanoparticle (ORI-GB-NP) was prepared for liver targeting delivery of ORI. This work was designed to investigate the in vitro release, in vivo pharmacokinetics and tissue distribution of ORI-GB-NP. ORI-GB-NP was prepared by the desolvation method. The particle size of ORI-GB-NP was 172.0?±?8.3?nm with narrow size distribution. The in vitro release of ORI-GB-NP exhibited biphasic drug release pattern with an initial burst release and consequently sustained release. Pharmacokinetic analysis displayed that ORI-GB-NP and ORI-loaded BSA nanoparticle (ORI-BSA-NP) could enhance the drug plasma level and prolong the circulation time in contrast with ORI solution. Meanwhile, compared with ORI-BSA-NP, ORI-GB-NP could deliver more ORI to liver and simultaneously reduce the toxicity of ORI to heart, lung and kidney. In conclusion, ORI-GB-NP could be a promising drug delivery system for liver cancer therapy.     

Gels and liposomes in optimized ocular drug delivery: studies on ciprofloxacin formulations

Ciprofloxacin (CPFX) containing therapeutic systems were developed using gel- and liposome-based formulations to minimize tear-driven dilution in the conjunctival sac, a long-pursued objective in ophthalmology. Physicochemical properties (pH, osmolarity, viscosity, expansivity, membrane fluidity and in vitro CPFX release rate) of the preparations were studied by the appropriate methods. For gel preparation, the bio-adhesive poly(vinyl alcohol) and polymethacrylic acid derivatives were applied in various concentrations. In our liposome-supported carrier systems, multilamellar vesicles from lecithin and alpha-L-dipalmithoyl-phosphatidylcholine provided the encapsulating agent. Electron paramagnetic resonance (EPR) spectroscopy was applied to study the molecular interactions in the ophthalmic formulations. The polymer hydrogels used in our preparations ensured a steady and prolonged active ingredient release. In addition, encapsulation of the CPFX into liposomes prolonged the in vitro release of the antibacterial agent depending on the lipid composition of the vesicles.     

Electrosteric stealth Rivastigmine loaded liposomes for brain targeting: preparation,characterization, ex vivo,bio-distribution and in vivo pharmacokinetic studies

Being one of the highly effective drugs in treatment of Alzheimer’s disease, Rivastigmine brain targeting is highly demandable, therefore liposomal dispersion of Rivastigmine was prepared containing 2?mol% PEG-DSPE added to Lecithin, Didecyldimethyl ammonium bromide (DDAB), Tween 80 in 1:0.02:0.25 molar ratio. A major challenge during the preparation of liposomes is maintaining a stable formulation, therefore the aim of our study was to increase liposomal stability by addition of DDAB to give an electrostatic stability and PEG-DSPE to increase stability by steric hindrance, yielding what we called an electrosteric stealth (ESS) liposomes. A medium nano-sized liposome (478?±?4.94?nm) with a nearly neutral zeta potential (ZP, ?8?±?0.2?mV) and an entrapment efficiency percentage of 48?±?6.22 was prepared. Stability studies showed no major alteration after three months storage period concerning particle size, polydispersity index, ZP, entrapment efficiency and in vitro release study confirming the successful formation of a stable liposomes. No histopathological alteration was recorded for ESS liposomes of the sheep nasal mucosa. While ESS liposomes showed higher % of drug permeating through the sheep nasal mucosa (48.6%) than the drug solution (28.7%). On completing the in vivo pharmacokinetic studies of 36 rabbits showed 424.2% relative bioavailability of the mean plasma levels of the formula ESS compared to that of RHT intranasal solution and 486% relative bioavailability of the mean brain levels.     

Etoposide encapsulated in positively charged liposomes: pharmacokinetic studies in mice and formulation stability studies.

Etoposide is an antineoplastic agent which acts by forming a ternary complex with topoisomerase II and DNA, causing DNA breaks and cell death. In recent studies we have demonstrated that encapsulation in liposomes increases the antitumour efficacy and reduces the adverse effects associated with etoposide. The present study was thus conducted to evaluate whether encapsulation in cationic liposomes altered the pharmacokinetics of etoposide and to study the effect of cholesterol incorporation on the stability of the liposomes. Etoposide-encapsulated unilammellar liposomes were synthesized by thin film hydration followed by extrusion. The drug was administered to Swiss albino mice at a dose of 10 mg kg(-1). The concentration of the drug in plasma was analysed at different time points till 360 min after injection, using a h.p.l.c. method. The terbium chloride-dipicolinic acid interaction method was applied to study the stability of the formulation in mouse serum and also following storage at 0( composite function)C over a period of time. The effect of the free and liposomal drug on myelosuppression was evaluated at 10 mg m(-2)and 40 mg m(-2)dose levels by quantifying blood cell counts on day 15 and day 21 following a 5 day course of therapy. Encapsulation in cationic liposomes increased the area under the concentration vs time curve to 42.98 microghml(-1)from 24.18 microghml(-1)in the case of the free drug. Half-life (beta) was 58. 62 and 186 min in the case of free and liposomal etoposide, respectively. In the stability studies, incorporation of cholesterol progressively stabilized the formulation in serum. The use of sucrose at increasing concentrations as a cryoprotectant also increased the shelf stability of the formulation at 0( composite function)C. Toxicity studies using a dose of pure drug revealed that though myelosuppression was evident in both liposomal- and free drug-treated groups on day 15 it was reversed by day 21 following initiation of therapy. The present findings suggest that liposomes could serve as an alternative mode of delivery for etoposide.     

Establishment and validation of an ex vivo human cervical tissue model for local delivery studies.

The objective of this study was to establish and validate an ex vivo human cervical tissue model appropriate for transport studies of molecular and especially nucleic acid based drugs. For that purpose conditions had to be established for a standardized tissue handling and preparation following hysterectomy to allow an immediate experimental use of fresh tissue samples. Samples of the ectocervical, endocervical and the transition zone representing the entire cervix organ were characterized in Franz diffusion cells by the determination of the in vitro permeation of low and high molecular weight markers (propanolol, mannitol, dextran 4000, 10,000, 20,000 and 40,000Da). Additionally, the permeability of mannitol and dextran 4000 across fresh and frozen cervical tissue was compared. The apparent permeability coefficients (P(app)) of the various markers demonstrated (i) that with increasing molecular weight the marker permeability decreases, (ii) an upper permeability limit between 10,000 and 20,000Da, (iii) no significant difference of the permeability across the three cervical tissue zones, (iv) a statistically significant but effectively small variation of the permeability among different patient samples. A continuous difference of approximately two log values between the P(app) values of mannitol and dextran 4000 makes them suitable as an internal marker control pair for each biopsy. Moreover, the P(app) values of both markers across fresh and frozen tissue are comparable. According to the presented data we conclude that the human cervical tissue model has been well characterized and is therefore suitable for local delivery and permeation studies.     

Paclitaxel loaded PEG5000–DSPE micelles as pulmonary delivery platform: Formulation characterization, tissue distribution, plasma pharmacokinetics, and toxicological evaluation

The objective of the present study was to evaluate the potential of paclitaxel loaded micelles fabricated from PEG₅₀₀₀–DSPE as a sustained release system following pulmonary delivery. PEG₅₀₀₀–DSPE micelles containing paclitaxel were prepared by solvent evaporation technique followed by investigation of in vitro release of paclitaxel in lung simulated fluid. Tissue distribution and plasma pharmacokinetics of the PEG–lipid micelles after intratracheal and intravenous administrations were investigated in addition to intratracheally administered taxol. Finally, toxicological profile of PEG₅₀₀₀–DSPE was investigated. Paclitaxel was successfully formulated in PEG–lipid micelles with encapsulation efficiency of 95%. The PEG–lipid micelles exhibited a sustained release behavior in the simulated lung fluid. Intratracheally administered polymeric micellar paclitaxel showed highest accumulation of paclitaxel in the lungs with AUC_0–12 in lungs being 45-fold higher than intravenously administered formulation and 3-fold higher than intratracheally delivered taxol. Paclitaxel concentration in other non-targeted tissues and plasma were significantly lower as compared to other groups. Furthermore, toxicity studies showed no significant increase in levels of lung injury markers in PEG₅₀₀₀–DSPE treated group as compared to saline-treated group. PEG₅₀₀₀–DSPE micelles delivered intratracheally were able to sustain highest paclitaxel concentrations in lungs for long periods of time, thus apprehending their suitability as pulmonary drug carriers.     

Low molecular weight chitosan-coated liposomes for ocular drug delivery: in vitro and in vivo studies

In this study, low molecular weight chitosan coated liposomes (LCHL) were designed and prepared for ocular drug delivery, the coating mechanism was studied, and in vitro and in vivo characterization was conducted. The effects of molecular weight and concentration of low molecular weight chitosan on the liposomal coating were studied. The numeric relations between coating variables and coating efficiency were established using a mathematical model. Morphology of LCHL was examined by transmission electron microscopy (TEM). Cytotoxicity and cell internalization of FITC-BSA labeled LCHL in a rabbit conjunctival epithelium (RCE) cell line were studied. Cyclosporin A (CsA) was encapsulated as a model drug, and in vitro drug release and in vivo drug absorption were investigated. LCHL demonstrated low toxicity to RCE cells. In vitro drug release measurement showed that LCHL had a delayed release profile compared with non-coated liposomes. In vivo study in rabbits showed that the concentrations of CsA in cornea, conjunctiva, and sclera were remarkably increased by LCHL. In conclusion, LCHL might be a potential ocular drug carrier with characteristics such as prolonged drug retention, enhanced drug permeation, and biocompatibility.     

In vivo tissue distribution and efficacy studies for cyclosporin A loaded nano-decorated subconjunctival implants

Biodegradable implants are promising drug delivery systems for sustained release ocular drug delivery with the benefits such as minimum systemic side effects, constant drug concentration at the target site and getting cleared without surgical removal. Dry eye syndrome (DES) is a common disease characterized with the changes in ocular epithelia surface and results in inflammatory reaction that might lead to blindness. Cyclosporin A (CsA) is a cyclic peptide that is frequently employed for the treatment of DES and it needs to be applied several times a day in tear drops form. The aim of this study was to evaluate in vivo behavior and efficacy of the developed nano-decorated subconjunctival implant systems for sustained release CsA delivery. Biodegradable Poly-?-caprolactone (PCL) implant or micro-fiber implants containing CsA loaded poly-lactide-co-glycolide (85:15) (PLGA) or PCL nanoparticles were prepared in order to achieve sustained release. Two of the formulations PCL–PLGA–NP-F and PCL-PCL-NP-I were selected for in vivo evaluation based on their in vitro characteristics determined in our previous study. In this study, formulations were implanted to Swiss Albino mice with induced dry eye syndrome to investigate the ocular distribution of CsA following subconjunctival implantation and to evaluate the efficacy. Tissue distribution study indicated that CsA was present in ocular tissues such as cornea, sclera and lens even 90 days after the application and blood CsA levels were found lower than ocular tissues. Efficacy studies also showed that application of CsA-loaded fiber implant formulation resulted in faster recovery based on their staining scores.     

齐多夫定棕榈酸酯脂质体的制备及在小鼠体内的药物分布

目的制备齐多夫定棕榈酸酯脂质体并考察其在小鼠体内的分布情况。方法采用乙醇注入法制备齐多夫定棕榈酸酯脂质体;采用HPLC法测定静脉注射后小鼠体内齐多夫定的质量浓度。结果脂质体包封率为95.2%,粒径为(75.6±42.2)nm。小鼠尾静脉分别注射齐多夫定棕榈酸酯脂质体30.0mg.kg-1和齐多夫定溶液15.85mg.kg-1后,体内消除半衰期脂质体组为16.4min,溶液组为5.74min;30min内脂质体组肝、脾、肾和脑(15min内)中齐多夫定的AUC值分别为溶液组的1.6倍、1.19倍、80%、1.8倍。结论采用乙醇注入法制备的齐多夫定棕榈酸酯脂质体包封率高,粒径均匀;可延长药物体内消除半衰期,提高了其在肝和脑的靶向性。齐多夫定棕榈酸酯脂质体有望成为一种理想的抗艾滋病制剂,值得进一步深入研究。     

Baclofen-loaded microspheres in gel suspensions for intrathecal drug delivery: In vitro and in vivo evaluation

Severe spasticity is a very disabling disorder treated by continuous baclofen intrathecal infusion which unfortunately remains an expensive and uncomfortable treatment. In order to address these issues, new sustained release formulations designed for intrathecal baclofen delivery were sought with the aim of minimising the burst effect of baclofen which can lead to toxicity.Baclofen was encapsulated in poly(lactide-co-glycolide) (PLGA) microspheres which were then dispersed in chitosan thermosensitive gels, Pluronic^® PF-127 gels, carboxymethylcellulose solutions or Ringer lactate solution. The release rate was assessed in vitro using continuous flow cells and in vivo after intrathecal injection in goats: baclofen was quantified in cerebrospinal fluid (CSF) and plasma, and the associated pharmacological effect was evaluated. The results showed that the burst effect was reduced by at least a factor of 2 in vitro, after microsphere dispersion in viscous media. In vivo, PF-127 gel was found to be the best vehicle to reduce the burst effect by a factor of 10 in CSF, and by a factor of 2 in plasma. The toxic effect of baclofen due to the burst effect was reduced by the dispersion in PF127 gels. Therapeutic levels of baclofen in CSF were maintained during at least 1 month.     

In vitro and in vivo methotrexate disposition in alveolar macrophages: comparison of pharmacokinetic parameters of two formulations

MTX-liposomes, prepared with a polymerised core (LSP), were administered in anaesthetised rats by pulmonary instillation versus free drug. No toxicological effects were macroscopically observed. After each time point: 15, 30, 60 and 90 min, animals were humanely killed and analyses of radio-signal were done. This approach allowed recovery of MTX or breakdown products within biological samples. Previously, kinetics of MTX cellular uptake was performed to identify the cytotoxic concentration of drug formulation for human macrophage. Flow cytometry was set-up to characterise liposomal uptake by ex vivo pulmonary macrophage. Cells were isolated by bronchioloalveolar washes from animals. Results have shown clear different pharmacokinetic parameters between free MTX and the liposomal form of MTX. Unlike classical liposomes, which are mainly taken up by the reticulo-endothelial system, LSP-MTX was not targeted to spleen or kidney. The route of administration could be an explanation of this phenomenon. In addition, LSP-MTX was more retained by the lung tissue. Moreover, free form of the drug reaches easily lymph node. This latest result should be taken into consideration for neoplasic disease and more specifically when lymph nodes are a way for pulmonary metastasis. Finally, LSP-MTX should be tested in physio-pathological model of lung cancer to evaluate the influence of the variation of liposomal formulation pharmacokinetic parameters on the drug efficacy.     

肺靶向阿奇霉素脂质体的制备及其在小鼠体内的分布

目的研究肺靶向阿奇霉素阳离子脂质体的制备方法并考察其在小鼠体内的分布。方法利用旋转薄膜-冻融法制备肺靶向阿奇霉素脂质体。用高效液相色谱法测定给药后小鼠体内各组织中的药物浓度。结果制得的脂质体平均粒径为6.582 μm，表面电荷为+19.5 mV，包封率大于75%，稳定性好。药物体外释药符合Higuchi方程。小鼠尾静脉给药后，阳离子脂质体主要被肺摄取，在肺部的滞留时间明显延长，AUC值约为阿奇霉素溶液的8.4倍。结论采用薄膜-冻融法，添加十八胺可制得具有较高包封率及稳定性的阿奇霉素阳离子脂质体，在小鼠肺部的分布优于注射液，能达到肺靶向目的。     

肺靶向羟基喜树碱脂质体的制备及其在小鼠体内的组织分布研究

目的： 研究羟基喜树碱脂质体的制备方法并考察其肺靶向及在小鼠体内的分布。方法： 采用薄膜分散－冻融法制备,添加D-甘露糖和十八胺修饰可得到肺靶向羟基喜树碱脂质体;用HPLC法测定给药后小鼠体内不同组织中的药物浓度。结果： 制得的脂质体平均粒径大于2 μm,表面电荷为+21.5 mV,包封率大于65%,稳定性好,符合要求。羟基喜树碱脂质体和注射液经小鼠尾静脉给药后,脂质体主要被肺摄取,在肺部停留的时间较普通注射剂显著延长,其相对摄取率r_e为60.72,脂质体组的肺靶向效率t_e为17.57。结论： 本实验制得羟基喜树碱脂质体具有较高包封率及稳定性,在小鼠肺部浓度高、滞留时间长,能达到肺靶向目的。     

In vitro and in vivo evaluation of WGA-carbopol modified liposomes as carriers for oral peptide delivery

Surface modification of liposomal nanocarriers with a novel polymer-lectin conjugate was proposed for enhancing the systemic uptake of encapsulated peptide and protein therapeutics after oral administration. Wheat germ agglutinin (WGA) was covalently attached to carbopol (CP) using the carbodiimide method. The prepared WGA-CP conjugate retained the biological cell binding activity of WGA without any evidence of cytotoxicity to Caco-2 monolayers. Cationic liposomes in the size range of 100 nm were prepared by the lipid film hydration method followed by probe sonication and surface modification with negatively charged WGA-CP. The uptake of WGA-CP liposomes by Caco-2 cells was significantly higher than that of non-modified or CP liposomes. The uptake was dependent on the surface concentration of WGA, temperature, and incubation period and was significantly inhibited in the presence of chlorpromazine and 10-fold excess of free WGA. These results suggest the involvement of active transport mechanism for the cellular uptake of the modified liposomes, mediated mainly by binding of WGA to its specific cell membrane receptors. Dual channel confocal microscopy confirmed the simultaneous association and internalization of the polymer conjugate and the liposomal carrier by Caco-2 cells and intestinal membrane of rats. In addition, the pharmacological efficacy of calcitonin, a model peptide drug, was enhanced by more than 20- and 3-fold following peroral administration of calcitonin-loaded WGA-CP liposomes when compared to non-modified and CP liposomes, respectively.