Liposomes Containing Cationic Dimethyl Dioctadecyl Ammonium Bromide: Formulation,Quality Control,and Lipofection Efficiency

This article describes a novel, simple, and relatively inexpensive method to prepare cationic liposomes using an ethanol injection/pressure extrusion method. The study also demonstrated that binding erythrosine dye to cationic liposomes results in a shift of the absorption maximum of the dye from 528 nm to 549 nm at pH 4.25, allowing quantification and visualization of these vesicles. In addition, a relatively simple Ficoll-based gradient centrifugation method for separation of lipoplexes from unbound molecules is presented. Laboratory-formulated dimethyl dioctadecyl ammonium bromide (DDAB) containing liposomes were just as efficient in complexing nucleic acids as commercially available types, and binding increased as the positive to neutral lipid ratio was increased. Transfection efficiency of the DDAB-containing liposomes increased as the ratio of cationic to neutral lipid was increased from 1:1 to 4:1 with either PtdChol or DOPE as the neutral lipid. A concomitant increase in cytotoxicity of CSU-SA1 cancer cells was noted as the ratio of positive to neutral lipid of the liposomes was increased. Nevertheless, our present study showed that the 2:1 liposome is a good choice since it delivers functional plasmids at a comparable rate to commercial liposome formulations, has similar toxicities to the less harmful commercial liposomes, and is at least 1000-fold more economical to prepare inhouse, a major factor to be considered in preclinical and clinical studies with these carriers.     

Cationic Liposomes and Gene Therapy for Solid Tumors

Efforts in treatment have concentrated on the development of an ideal carrier for effective delivery of therapeutic agents into affected regions of a human body. Ideally, drugs would have to be delivered as close as possible, if not within, the affected cell. From its seminal production 30 years ago for the purpose of membrane research, the liposome has passed through various stages of development to become a vehicle of choice for numerous therapeutic applications. One category of these vesicles, positively charged or cationic liposomes, are commonly used for transfer of reporter and therapeutic genes into both mammalian and nonmammalian cells both in vitro and in vivo. While cationic liposomes have many advantages over other forms of delivery mechanisms, several problems hinder their efficient use. Development of a better liposomal transfection agent may indeed require a closer look at the present cationic vesicles, the biological milieu to which they are exposed, mechanisms of membrane breaching, and limitations to entry into the nucleus. This review discusses these limitations and suggests potential ways of improving liposomal delivery of genes in the context of solid tumors.     

Liposomes containing distamycins: preparation, characterization and antiproliferative activity

This article describes the production and characterization of two liposome formulations containing antitumor drugs, namely distamycin A (Dist) and a new alkyl derivative of distamycin A (C₁₆-Dist). Egg-PC/cholesterol liposomes (4:1 mol/mol) were prepared by reverse phase evaporation technique followed by extrusion through polycarbonate filters. The encapsulation efficiency was found to be almost complete for C₁₆-Dist (99.8%), while native distamycin A showed a lower yield (19.0%). The in vitro antiproliferative activity of the distamycins-containing liposomes determined on human leukaemic K562 cells, was 11-fold and 8-fold higher for native and alkyl derivative distamycin A, respectively, compared with that of the corresponding free drugs. Liposomal formulations show an increase in the activity and specificity of distamycins in experimental antitumor therapy.     

Receptor-mediated gene delivery to HepG2 cells by ternary assemblies containing cationic liposomes and cationized asialoorosomucoid

Unilamellar cationic liposomes have been prepared from an equimolar mixture of 3beta[N',N'-dimethylaminopropane)-carbomoyl] cholesterol (Chol-T), a higher homologue of 3beta[N',N'-dimethylaminoethane)-carbomoyl] cholesterol (DC-Chol), and dioleoylphosphatidyl-ethanolamine. The DNA binding capabilities of Chol-T and Chol-T/DOPE liposomes have been demonstrated in lipid impregnated paper-DNA binding assays and gel retardation experiments, respectively. These liposomes have been combined with pRSVL plasmid DNA and N-ethyl-N'-(3-trimethylpropylammonium) carbodiimide iodide modified asialoorosomucoid (Me+ CDI urea-AOM) to generate ternary electrostatic assemblies intended for selective entry into cells displaying the galactose-specific lectin. This effect has been evaluated in the human hepatocellular carcinoma cell line HepG2 in which high levels of luciferase activity were achieved (up to 1.84 x 10(7) relative light units/mg protein) after transfection with complexes containing liposomes (1-3 microg), Me+CDI urea-AOM (2 microg), and DNA (0.5 microg) in 0.5 mL culture medium. Transfections conducted in the presence of free asialoorosomucoid afforded much lower luciferase activity (up to 1.5 x 10(5) relative light units/mg protein) confirming that DNA uptake was predominantly via asialoorosomucoid receptor-mediated endocytosis. We concluded therefore that modular complexes used in our study display the carbohydrate moiety of the glycoprotein component prominently, thus permitting interaction of terminal galactose units with their cognate receptors on the cell membrane.     

Effect of the adjuvant dimethyl dioctadecyl ammonium bromide on the humoral and cellular immune responses to encephalomyocarditis virus

The effects of the adjuvant dimethyl dioctadecyl ammonium bromide (DDA) on the immune responses to encephalomyocarditis (EMC) virus were studied in mice. The humoral response, as measured by appearance of neutralizing antibodies, was slightly enhanced in mice immunized by the intraperitoneal route. Intracutaneously, DDA almost did not affect the humoral response but resulted in distinct enhancement of delayed type hypersensitivity (DH), as measured by the footpad swelling test. DH to EMC virus was found to be antigen-specific and could be passively transferred to normal mice with peritoneal exudate cells from immunized mice. Dose-response curves for DH and humoral antibody responses to EMC virus were not concordant. Low doses induced DH on day 6 without measurable circulating antibodies; high doses gave good antibody responses but suboptimal DH reactions. Immunization conferred a state of resistance to infection with virulent EMC virus. Protection seemed more related to DH than to the prevalence of specific antibodies at the time of infection.     

Preparation and Characterization of a Novel Nonviral Gene Transfer System: Procationic-Liposome-Protamine-DNA Complexes

Procationic-liposome-protamine-DNA (PLPD) vector, a novel nonviral gene delivery system, that may further adsorb transferrin (Tf) at its surface via electrostatic interactions to form Tf-PLPD, was prepared from soybean phosphatidylcholine (PC), cholesterol (Chol), and a kind of cholesterol derivative, CHETA(cholest-5-en-3-ol(3β)-[2-[[4-[(carboxymethyl)dithio]-1-iminobutyl] amino] ethyl] carba- mate) containing disulfide bond by film dispersion–filteration method. Central composite design was used to optimize the formulation. The presence of serum did not affect the transfection activity of PLPD or Tf-PLPD and the cell viability was not affected significantly when the cells were incubated with the complexes for 4 hr at 37°C. Compared with one kind of cationic liposomes(liposome-protamine-DNA), the PLPD had much less cytotoxicity to three hepar cell lines(including HepG2, SMMC7721, and Chang's normal heptocyte). The procationic lipoplex described here, combining the condensing effect of protamine and the targeting capability of Tf, was a perspective nonviral vector for gene delivery system.     

Preparation and characterization of a novel nonviral gene transfer system: procationic-liposome-protamine-DNA complexes

Procationic-liposome-protamine-DNA (PLPD) vector, a novel nonviral gene delivery system, that may further adsorb transferrin (Tf) at its surface via electrostatic interactions to form Tf-PLPD, was prepared from soybean phosphatidylcholine (PC), cholesterol (Chol), and a kind of cholesterol derivative, CHETA(cholest-5-en-3-ol(3β)-[2-[[4-[(carboxymethyl)dithio]-1-iminobutyl] amino] ethyl] carba- mate) containing disulfide bond by film dispersion-filteration method. Central composite design was used to optimize the formulation. The presence of serum did not affect the transfection activity of PLPD or Tf-PLPD and the cell viability was not affected significantly when the cells were incubated with the complexes for 4 hr at 37°C. Compared with one kind of cationic liposomes(liposome-protamine-DNA), the PLPD had much less cytotoxicity to three hepar cell lines(including HepG2, SMMC7721, and Chang's normal heptocyte). The procationic lipoplex described here, combining the condensing effect of protamine and the targeting capability of Tf, was a perspective nonviral vector for gene delivery system.     

A novel cationic cholesterol derivative,its formulation into liposomes,and the efficient transfection of the transformed human cell lines HepG2 and HeLa

A novel cationic cholesterol derivative, 3 β[N-(N',N',N'-trimethylaminopropane)-carbamoyl] cholesterol iodide (Chol-Q), has been formulated with equimolar amounts of dioleoyl phosphatidylethanolamine (DOPE) into stable unilamellar liposomes up to 100 nm in size for DNA delivery into mammalian cells. When compared with similarly constituted liposomes containing the tertiary analogue 3 β[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) in a band shift assay, liposomes displayed similar DNA binding affinities and appeared to afford complete protection to plasmid DNA against serum nuclease catalysed degradation at liposome:DNA ratios (w/w) of 2.5:1, 5:1, and 10:1 in incubation mixtures containing 5% fetal bovine serum at 37 C for 90 min. Chol-Q liposomes were, however, markedly less toxic to cells in culture over a wide range of concentrations with cells numbering 76% of untreated controls at 37.5 μg/mL complete medium in the human hepatocellular carcinoma line HepG2 and 75% at 30 μg/mL in cervical carcinoma HeLa cells. At these levels of Chol-T liposomes, cell numbers were 37% and 15%, respectively. Gene transfer experiments with pSV2CAT and pRSVCAT plasmids in HepG2 cells showed maximum efficiency at a Chol-Q liposome:DNA ratio of 5:1 (w/w) and at a Chol-T liposome:DNA ratio of 10:1. In HeLa cells, both liposome preparations performed best at a ratio of 2.5:1. Differences in transfection efficiencies over the liposome range of 5-20 μg/ mL were rather less pronounced with Chol-Q lipoplexes suggesting a greater versatility of this system.     

Liposomes in Pulmonary Applications: Physicochemical Considerations,Pulmonary Distribution and Antioxidant Delivery

Abstract

The application of liposomes for improved drug delivery to the lung is promising. Liposome-mediated pulmonary drug delivery promotes an increase in drug retention-time in the lung and more importantly, a reduction in extrapulmonary side-effects, invariably resulting in enhanced therapeutic efficacies. The engineering of an effective liposomal drug formulation for inhalation therapy must take into consideration the leakage problem associated with the nebulization process; vesicle stability and release kinetics within the pulmonary milieu; and, the altered pharmacokinetics of the entrapped drug. The delivery of liposome-entrapped antioxidants via the tracheobronchial route has been found to be very useful in increasing the half-times of the administered agents, thus providing a sustained release effect for prolonged drug action. The entrapment in liposomes of a-tocopherol, an extremely insoluble but highly effective antioxidant, has been shown to be very effective in ameliorating oxidant-induced injuries in the lung. The use of bifunctional liposomes containing two antioxidants have been determined to provide excellent resistance to an oxidative challenge and appears to hold promise for improved clinical applications in antioxidant therapy.     

Liposomes incorporating sodium deoxycholate for hexamethylmelamine (HMM) oral delivery: Development,characterization, and in vivo evaluation

Liposomes incorporating sodium deoxycholate (NaDC) were prepared by the method of reverse phase evaporation and used for drug delivery by the oral route. Hexamethylmelamine (HMM), an anti-tumor agent, was chosen as a model drug and encapsulated into liposomes incorporating NaDC (NaDC-Lip). Several properties of NaDC-Lip containing HMM (HMM NaDC-Lip), such as particle size, entrapment efficiency, pinacyanol chloride (PIN) spectral characteristics with various molar ratio of NaDC/PC, as well as the vesicle stability measurements with calcein were evaluated. In vivo, the area under the plasma concentration–time curve obtained from the pharmacokinetics study of HMM NaDC-Lip was found to be ~?9.76- and 1.21-fold higher than that of HMM solution and HMM Lip, respectively, indicating that NaDC-Lip can be used as a potential carrier for oral drug administration.     

Gemcitabine cationic polymeric nanoparticles against ovarian cancer: formulation,characterization, and targeted drug delivery

This study focused on gemcitabine (GTB) delivery of cationic polymeric nanoparticles to treat ovarian cancer in order to promote effective localized delivery and drug retention during biological discharge. To begin, four GTB-loaded polymer nanoparticles were prepared: chitosan nanoparticles (CS-NPs), polysarcosin nanoparticles (PSar-NPs), poly-l-lysine & polysarcosin nanoparticles (PLL-PSar-NPs), and chitosan & polysarcosin nanoparticles (CS-PSar-NPs). Based on preliminary particle size, zeta potential, encapsulation efficiency, DSC, surface morphology, release profiling, and cellular internalization studies using rhodamine 123 and Nile red fluorescent markers, it was hypothesized that CS-PSar-NPs could be the best cationic formulation with strong biocompatibility and anticancer activity against the OVCAR-8 ovarian cancer cell line. To improve effective targeting, cellular penetration, and in vitro cytotoxicity, epidermal growth factor receptor variation III (EGFRvIII) is attached over all four polymeric nanoparticles. Confocal imaging revealed that EGFRvIII-conjugated cationic GTB polymeric nanoparticles had a greater cellular uptake and double internalization capabilities than unconjugated nanoparticles, as well as time-dependent cell entrance. GTB and EGFRvIII-conjugated polymer nanoparticles would have a stronger potential to infiltrate ovarian cancer cells during the first hour of incubation. According to TEM and FTIR findings, EGFRvIII conjugation across the non-target CS-PSar-NP surface was successful, making CS-PSar-NPS-EGFRvIII more target-specific and thus a safer drug delivery candidate for ovarian cancer treatment.

Highlights

• GTB loaded non-target CS-PSar-NPs & active targeted CS-PSar-NPs-EGFRvII developed.
• SEM, AFM, DSC, particle size, zeta potential, internalization performed for CS-PSar-NPs.
• MTT & CLSM study confirmed CS-PSar-NPS-EGFRvII was binding specific to OVCAR-8 cells
• Fabrication of EGFRvII over nanoparticles confirmed by TEM.
• CS-PSar-NPS-EGFRvII safer candidate for ovarian cancer.

     

Novel topical formulation for ischemic chronic wounds. Technological design,quality control and safety evaluation

Ulceration of the foot in diabetes is common and disabling, and frequently leads to amputation of the leg. The pathogenesis of foot ulceration is complex, clinical presentation variable and management requires early expert assessment. Despite treatment, ulcers readily become chronic wounds. Chronic wounds are those that remain in a chronic inflammatory state failing a normal healing process patterns. This is partially caused by inefficient eradication of opportunistic pathogens like Pseudomonas aeruginosa. We propose its control or eradication will promote wound healing. Lactobacillus plantarum cultures supernatants (LAPS) shows antipathogenic and pro-healing properties. The main objective was to design two pharmaceutical dosage forms by using LAPS as active pharmaceutical ingredient and to perform its quality control, in vitro activity conservation tests and human trials (safety evaluation). Both selected formulations reach the technological quality expected for 120 days, shows adequate occlusive characteristics and proper adhesion to human skin. From the in vitro release assays were found that LAPS shows adequate release from matrix and maintain its antimicrobial and anti-biofilm activity. First human trials were developed and neither edema nor erythema on healthy skin voluntaries was found. We conclude that C80 and C100 are adequate for their use in future clinical trials to demonstrate a comprehensive therapeutic effectiveness in ischemic chronic wounds.     

Mucoadhesive buccal tablets containing silymarin Eudragit-loaded nanoparticles: formulation,characterisation and ex vivo permeation

Eudragit-loaded silymarin nanoparticles (SNPs) and their formulation into buccal mucoadhesive tablets were investigated to improve the low bioavailability of silymarin through buccal delivery. Characterisation of SNPs and silymarin buccal tablets (SBTs) containing the optimised NPs were performed. Ex vivo permeability of nominated SBTs were assessed using chicken pouch mucosa compared to SNPs and drug suspension followed by histopathological examination. Selected SNPs had a small size (<150?nm), encapsulation effciency (>77%) with drug release of about 90% after 6?h. For STBs, all physicochemical parameters were satisfactory for different polymers used. DSC and FT-IR studies suggested the presence of silymarin in an amorphous state. Ex vivo permeation significantly emphasised the great enhancement of silymarin permeation after NPs formation and much more increase after formulating into BTs relative to the corresponding drug dispersion with confirmed membrane integrity. Incorporation of SNPs into BTs could be an efficient vehicle for delivery of silymarin.     

A quality by design (QbD) case study on liposomes containing hydrophilic API: II. Screening of critical variables, and establishment of design space at laboratory scale

Two statistical designs were used in this case study as part of an investigation into the feasibility and the advantages of applying QbD concepts to liposome-based complex parenteral controlled release systems containing a hydrophilic active pharmaceutical ingredient (API). The anti-viral drug Tenofovir was used as a model compound. First design (Plackett-Burman) was used to screen eight high-risk variables obtained from risk analysis and assess their impact on liposome characteristics (drug encapsulation efficiency, particle size, and physical stability). It was discovered that out of eight high-risk variables only lipid and drug concentration had significant effects on the drug encapsulation efficiency. This allowed the use of a central composite design (CCD) (with more predictive capability) to fully elucidate the relationship between lipid concentration, drug concentration and encapsulation efficiency. On comparing the CCD model generated response surface with additional data points, the accuracy and robustness of the model was confirmed. Using this developed model, the design space for Tenofovir liposomes preparation has been established in a laboratory setting, within which the preparation variability is minimized. With regard to sample storage stability, it was shown that at 4 °C the prepared Tenofovir liposomes, dispersed in aqueous phase, achieved stability for at least 2 years. These principles can be applied to liposomes containing other hydrophilic APIs, and can provide time and cost saving to industrial formulation scientists, and result in a more robust liposome preparation process.     

New synthetic glycolipids for targeted gene transfer: synthesis, formulation in lipoplexes and specific interaction with lectin

Nonviral gene delivery systems are a promising approach for gene therapy applications, despite their low in vivo gene transfer efficiency. One approach to enhance this efficiency is to incorporate targeting elements into cationic lipid/DNA complexes (lipoplexes). Ligand-containing lipoplexes have to retain their efficiency while exposing accessible ligand on their surface. Physicochemical properties (particle size, surface charge, and efficacy of DNA complexation) of the lipoplexes largely determine their gene transfer efficiency. We synthesized glycolipids with various galactosylated head ligand and incorporated them into lipoplexes. We showed that incorporation of up to 33% mol of glycolipid did not change the physicochemical properties of lipoplexes. Some of our glycolipids yielded lipoplexes whose galactosyl heads were well exposed on the surface as demonstrated by a strong interaction with Ricinus communis agglutinin. Glycolipid-containing lipoplexes gave an efficient gene transfer on hepatocytes, although no ligand-targeted transfection could be observed.     

盐酸西替利嗪滴剂的制备、质量控制及稳定性考察

目的制备盐酸西替利嗪滴剂,建立其质量控制方法并考察其稳定性。方法采用β-环糊精包合技术制备盐酸西替利嗪滴剂,高压液相色谱法测定有关物质和含量,影响因素试验、加速试验和长期试验考察其稳定性。结果本制剂口感适宜,较好地掩盖主药的苦涩味;盐酸西替利嗪检测浓度在25.1～251.0μg.mL-1与峰面积线性关系良好（r=0.999 9）,其平均回收率为99.9%,RSD为0.86%;恒温加速试验6个月和长期留样试验12个月,其性状、pH值、溶液的颜色、有关物质、含量以及微生物限度等均未见明显改变。结论该制剂处方工艺合理可行,质量可控,稳定性良好。     

l-Cysteine conjugated poly l-lactide nanoparticles containing 5-fluorouracil: formulation,characterization, release and uptake by tissues in vivo

Abstract

Context: Targeted delivery of drugs is still a therapeutic challenge and numerous methods have been reported for the same.

Objective: In this study, emphasis was placed on developing nanoparticles loaded with 5-fluorouracil (FU) and modifying the surface of the nanoparticles by conjugation with amino acid, to improve the distribution of 5-FU in the lungs.

Methods: An emulsion solvent evaporation technique was used to formulate nanoparticles of FU using Poly l-lactide and Pluronic F-68. The nanoparticles were conjugated with l-Cysteine using EDC as the activator of COOH group and were evaluated for product yield, particle size, surface morphology, amount of conjugation by Ellman’s method and in vitro drug release study.

Results and conclusion: The results indicated 60–65% yield with an average particle size of 242.7?±?37.11?nm for the cysteine conjugated nanoparticle (CNP) formulation and more than 70% conjugation of cysteine. The cumulative percentage of drug released over a period of 24?h was found to be 58%. An increase in distribution of the delivery system in lungs (11.4% ID after 1?h) in mice was found indicating the role of l-Cysteine in the transport mechanism to the lungs. In vivo kinetic studies in rats revealed higher circulation time of CNP as compared to pure FU solution. The study helps in designing a colloidal delivery system for increased distribution of drugs to the lungs and may be helpful in delivery of drugs in conditions like non-small cell lung carcinomas.     

注射用右丙亚胺制备、质量控制及稳定性考察

目的制备注射用右丙亚胺,建立其质量控制方法并考察其稳定性。方法制备注射用右丙亚胺;采用高效液相色谱法测定其含量及有关物质,并对制剂进行稳定性考察。结果本品处方以100mg/瓶甘露醇作为赋形剂,用0.1mol/L盐酸调节pH为1.9;右丙亚胺在4.4～44μg/ml浓度范围内线性关系良好,r=0.9999（n=5）,恒温加速试验6个月及长期留样24个月时主药含量及有关物质未见明显变化。结论该制剂处方工艺可行,质量可控,稳定性良好;所建立的含量测定方法重复性好,专属性强,结果准确可靠。     

注射用盐酸帕洛诺司琼的制备、质量控制及稳定性考察

目的:制备注射用盐酸帕洛诺司琼,建立其质量控制方法并考察其稳定性.方法:制备注射用盐酸帕洛诺司琼;采用高效液相色谱法测定其含量及有关物质,并对制剂进行稳定性试验.结果:本品处方以10%甘露醇作为填充剂,用磷酸二氢钠调节溶液pH值为4.5～6.0;盐酸帕洛诺司琼在 12.00 μg·mL-1～150.01 μg·mL-1浓度范围内线性关系良好(r＝0.999 9),恒温加速试验6个月及长期留样18个月时主药含量及有关物质未见明显变化.结论:该制剂处方工艺可行,质量可控,稳定性良好;所建立的含量测定方法重复性好,专属性强,结果准确可靠.