Compounds In Liposomes Research Articles

Inhibitory effect of Nigella sativa oil loaded to liposomal nanocarriers on Candida parapsilosis isolates.

Candida parapsilosis is the second most common species causing infectious diseases and can lead to biofilm resistance. This study aims to adjust and synthesize a liposomal compound of Nigella sativa and evaluate its antifungal properties against C. parapsilosis isolates. The liposomal formulation of N. sativa was optimized through the utilization of transmission electron microscopy (TEM), particle size analysis, zeta potential measurement, and UV-visible spectrophotometry. Furthermore, an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was conducted on peripheral blood mononuclear cells (PBMCs). The antifungal efficacy was evaluated in accordance with the M27-A3 guideline. The minimum inhibitory concentrations (MICs) of N. sativa oil and the liposomal formulation on C. parapsilosis isolates ranged from 128 to 8 µg/mL and from 250 to 31.25 µg/mL, respectively. The MIC50 and MIC90 values of N. sativa oil and the liposomal formulation were 125, 187, and 32, 96 µg/mL, respectively. The viability percentage of cells treated with the liposomal formulation and free N. sativa oil was 91% and 85%, respectively. The cytotoxicity of free N. sativa was significantly reduced when using nanoliposomes. The liposomal form of N. sativa showed greater antifungal properties compared to the free N. sativa extract against C. parapsilosis isolates.

CRISPR-Cas9 genome and long non-coding RNAs as a novel diagnostic index for prostate cancer therapy via liposomal-coated compounds.

CRISPR/Cas9 is a recently discovered genomic editing technique that altered scientist's sight in studying genes function. Cas9 is controlled via guide (g) RNAs, which match the DNA targeted in cleavage to modify the respective gene. The development in prostate cancer (PC) modeling directed not only to novel resources for recognizing the signaling pathways overriding prostate cell carcinoma, but it has also created a vast reservoir for complementary tools to examine therapies counteracting this type of cancer. Various cultured somatic rat models for prostate cancer have been developed that nearly mimic human prostate cancer. Nano-medicine can passively target cancer cells via increasing bioavailability and conjugation via specific legend, contributing to reduced systemic side-effects and increased efficacy. This article highlights liposomal loaded Nano-medicine as a potential treatment for prostate cancer and clarifies the CRISPR/Cas9 variation accompanied with prostate cancer. PC is induced experimentally in western rat model via ethinyl estradiol for 4 weeks and SC. dose of 3, 2'- dimethyl-4-aminobiphenyl estradiol (DAE) (50mg/kg) followed by treatment via targeted liposomal-coated compounds such as liposomal dexamethasone (DXM), liposomal doxorubicin (DOX) and liposomal Turmeric (TUR) (3mg/kg IP) for four weeks in a comparative study to their non-targeted analogue dexamethasone, doxorubicin and Turmeric. 3, 2'- dimethyl-4-aminobiphenylestradiol elicit prostate cancer in western rats within 5 months. Simultaneous supplementations with these liposomal compounds influence on prostate cancer; tumor markers were investigated via prostate-specific antigen (PSA), Nitric oxide (NOX) and CRISPR/Cas9 gene editing. Several long non-coding RNAs were reported to be deregulated in prostate cell carcinoma, including MALAT1. On the other hand, gene expression of apoptotic biomarkers focal adhesion kinase (AKT-1), phosphatidylinistol kinase (PI3K) and glycogen synthase kinase-3 (GSK-3) was also investigated and further confirming these results via histopathological examination. Liposomal loaded dexamethasone; doxorubicin and Turmeric can be considered as promising therapeutic agents for prostate cancer via modulating CRISPR/Cas9 gene editing and long non coding gene MALAT1.

Comparing the Effects of Free and Liposomal Indole Compounds on Bax and Bcl2 Gene Expression Changes in the KG-1 Cell Line

Background: For patients with acute myeloid leukemia (AML), the long-term survival rate is still very low. This study examines the effects on AML cell lines of an indole chemical in its free and liposomal forms. Material and Method: In this experimental case control study, an AML-originated KG-1 cell line was cultured in RPMI 1640 medium. The cells were treated with the free and liposomal forms of an indole compound (C18H10N2F6O) at different concentrations of 20, 40, 100, 200, and 400 µg/mL after they attained the proper confluence. The cellular metabolic activity was examined by an MTT assay. The expression of BAX and BCL-2 genes was investigated by q-PCR to assess the apoptotic effect of that compound. The analysis was also done between each experimental group and the control group using t-test. P<0.05 was assumed significant. Results: Based on the MTT assay, the lethal effective dose of free indole was found to be 245.1 µg/ml and 164.8 µg/ml in 24 and 48 hours, respectively. The corresponding values for liposomal indole were 47.2 µg/ml and 40.6 µg/ml. Furthermore, treatment with free and liposomal forms of indole resulted in a decline in the expression level of the BCL-2 gene. However, in the case of the liposomal compound, this decrease was only statistically significant after 48 hours of treatment (P < 0.05). Furthermore, the expression of BAX gene increased after treatment with both free and liposomal forms of indole, but it significantly increased only after treatment with the liposomal compound (p < 0.05). Conclusion: These results suggest that an indole derivative, especially when liposomal, causes apoptosis in AML cells, hence exhibiting cytotoxic effects. To confirm the potential usefulness of this indole derivative as a therapeutic agent for inhibiting tumor progression in the setting of human malignancies, more studies on physiologically relevant models are necessary.

Effect of liposomal encapsulation of curcumin and α-tocopherol on sensory and physicochemical properties, and retention of antioxidant capacity of fortified cookies during baking

Curcumin and α-tocopherol, which are potent antioxidants usable for food fortification, are sensitive to heat and air, thus showing the need for protection. The aim of this study was to increase the antioxidant capacity retention of curcumin and α-tocopherol in fortified cookies via liposomal encapsulation. Liposomes were prepared using the homogenization method and the liposomes were multilamellar according to light microscopic images. Liposomal encapsulation exhibited encapsulation efficiencies greater than 90 %. The sensory tests, conducted using a 5-point hedonic scale for nine attributes, revealed that the incorporation of liposomal antioxidants did not compromise the sensory attributes of the cookies. The color of liposomal-curcumin incorporated cookie deviated greater than that of free-curcumin incorporated cookie from the regular cookie. Nevertheless, the color of the α-tocopherol incorporated cookies improved on using the liposomal compound as opposed to the free compound. The antioxidant capacity of the cookies, along with the controls, was measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. As expected, liposomal encapsulation of curcumin or α-tocopherol mitigated the reduction of antioxidant capacity of fortified cookies during baking. Briefly, liposomal encapsulation proves to be a potent method for retaining the antioxidant capacity of curcumin or α-tocopherol in cookies during baking.

Stability of bioactive compounds in liposomes after pasteurisation and storage of functional chocolate milk

SummaryIn the present study, liposomes with bioactive compounds (BC), vitamin E (VE) and vitamin C (VC), were characterised after pasteurisation and storage in chocolate milk. Liposomes were obtained using soy phosphatidylcholine (SPC), SPC with stearic acid (SA) or calcium stearate (CaS). Oxidative stability, size, shape and membrane packing were studied after pasteurisation and storage for 28 days at 4 °C. Oxidative stability was also studied after pasteurisation in extreme conditions: 6 months, 25 °C and light. The oxidative level determined in liposomes is much under the permitted limits, generating good quality food. SPC maintained stability in size, shape and membrane packing for 28 days at 4 °C. SPC:SA and SPC:CaS evidenced a decrease in liposomal aggregation and a higher membrane packing for 28 days at 4 °C, which favoured stability. These results were optimum because commercial functional milk must have a shelf life of 7 days at 4 °C. Pasteurised liposomes were incorporated in chocolate milk to study the sensory (after storage for 7 days at 4 °C) and microbiological stability. None of the liposomes contributed to the microbial load. SPC:SA presented high sensorial stability in the milk, being a potentially suitable medium to incorporate BC generating functional food.

HPLC-DAD-CAD-based approach for the simultaneous analysis of hydrophobic drugs and lipid compounds in liposomes and for cyclodextrin/drug inclusion complexes

In recent decades liposomes have become attractive carriers for hydrophobic drugs to enhance their solubility and improve their therapeutic application. For liposomal drug products, both drug and lipid quantification are required by regulatory authorities, making the implementation of precise quantification methods a step of crucial importance in formulation development and quality control. Therefore, the present study is focused on the development and validation of a simple and time-saving method for the simultaneous analysis of hydrophobic drugs and conventional liposomal components. The new HPLC method was established with a combined detection by a diode array detector (DAD) and a corona charged aerosol detector (CAD). As a wide calibration range of the liposomal components can be achieved (10−1000 μg/mL), the analysis of samples with different drug to lipid ratios is enabled. Moreover, an excellent precision including repeatability and low limits of detection (≤ 1.8 μg/mL) and limits of quantification (≤ 5.9 μg/mL) were accomplished for all analytes. The method was successfully applied to liposomes incorporating mitotane. Everolimus was additionally analyzed as hydrophobic model drug. Furthermore, cyclodextrin/mitotane inclusion complexes were investigated to proof a broad range of applications for the developed method.

Determination of the anticancer properties of cis- and trans-diadamanthylcarboxylates of dirhenium(III)

The aim of the study. The aim of the work was to investigate in vivo anticancer activity of cis- and trans-diadamanthylcarboxylates of dirhenium(III) alone and together with cisplatin in form of nanobins.Materials and methods. Model of tumor growth, Guerin’s carcinoma; intraperitoneal administration of cisplatin, dirhenium(III) compounds in liposomes and of binary liposomes, containing both cytostatics; volumes and final weights of tumors were measured.Results. In vivo antitumor properties of two dirhenium(III) dicarboxylates with 1-adamantanecarboxylic acid moieties as ligands with cis- (I) and trans- (II) orientation of the carboxylic groups around a cluster fragment alone and together with cisplatin were presented; an attempt to understand differences in a possible mechanism of anticancer activity of the substances were undertaken. Antiradical and DNA-binding properties of I and II were the matter of consideration.Conclusions. Cis- and trans- compounds of dirhenium I and II had close antitumor activity in vivo with a little bit superiority of the cis- analog. Mechanisms of anticancer activity of I and II are different and may also include monofunctional adduct formation and subsequent interstrand cross-linking for the II substance, formation of protein-DNA cross-links, etc.

P217 - Physiologically-based pharmacokinetic (PBPK) modeling of liposomal compound x - deciphering the mystery of liposomal delivery

P217 - Physiologically-based pharmacokinetic (PBPK) modeling of liposomal compound x - deciphering the mystery of liposomal delivery

BETULIN DERIVATIVES. BIOLOGICAL ACTIVITY AND SOLUBILITY IMPROVEMENT

In the review the biological properties (antitumor, antiviral, hypolipidemic, anti-inflammatory, etc.) and bioavailability of betulin and betulinic acid derivatives were discussed. These compounds are isolated from various natural sources, including birch bark (Betula, Betulaceae). The structure-activity correlation was considered for well-known betulinic acid derivatives. The perspectivity of this compounds as active pharmaceutical ingredients was demonstrated by in vitro, in vivo, and ex vivo experiments. The type of antitumor actions, generally, depends on substituents at the C-3 and C-28 carbon atoms of the lupane skeleton. It is very important that the carboxyl group of betulinic acid in the C-28 position was present. In this case, the cytotoxicity of C-3 modified derivatives is extremely high for all tested cell lines. The use of these compounds in the medical practice is complicated because they have low bioavailability and poor water solubility (from 1 to 100 µg*l-1). The main chemical syntheses for solubility improvement of betulin derivatives by grafting of hydrophilic groups were discussed. Moreover, the colloid-chemical approaches for the bioavailability improving of triterpenoids include: 1) including of these compounds in liposomes, vesicles and other nanoparticles; 2) obtaining of micelles with high-molecular compounds; 3) colloid-chemical dissolution due to physico-mechanical action; 4) inclusion complexes formation; 5) using of polymers for triterpenoids grafting. Chemical modification of betulin and betulinic acid by polar groups, such as phosphate/phosphonate, sulfate, amino acids, etc. has been shown for bioavailability improving.

Simultaneous microencapsulation of hydrophilic and lipophilic bioactives in liposomes produced by an ecofriendly supercritical fluid process

Organic solvent residues are always a concern with the liposomes produced by traditional techniques. Our objectives were to encapsulate hydrophilic and lipophilic compounds in liposomes using a newly designed supercritical fluid process coupled with vacuum-driven cargo loading. Supercritical carbon dioxide was chosen as the phospholipid-dissolving medium and an ecofriendly substitute for organic solvents. Liposomal microencapsulation was conducted via a 1000-μm expansion nozzle at 12.41MPa, 90°C, and aqueous cargo loading rate of 0.25ml/s. Vitamins C and E were selected as model hydrophilic and lipophilic compounds encapsulated in the integrated liposomes. The average vesicle size was 951.02nm with a zeta potential of −51.87mV. The encapsulation efficiency attained was 32.97% for vitamin C and 99.32% for vitamin E. Good emulsion stability was maintained during storage at 4°C for 20days. Simultaneous microencapsulation in the liposomes was successfully achieved with this supercritical fluid process.

Influence of liposome encapsulated essential oils on properties of chitosan films

AbstractThe effect of the encapsulation of eugenol and cinnamon leaf essential oil (CLEO) in lecithin liposomes on the losses of these compounds during the chitosan film formation process by casting was evaluated. Film‐forming dispersions and films with eugenol or CLEO (either free or encapsulated) were obtained and characterized. The content of eugenol in active films was quantified by means of solvent extraction and gas chromatograph analysis. The encapsulation of eugenol or CLEO in lecithin liposomes led to the films retaining 40% − 50% of the incorporated eugenol, whereas only 1% − 2% was retained when eugenol was incorporated by direct emulsification. Films with liposomes exhibited a lamellar microstructure which improved film extensibility and increased water vapour barrier capacity with respect to those with free emulsified compounds. Liposomes also modified the optical properties of the films, reducing their gloss, increasing colour saturation and making them redder in colour. The encapsulation of volatile active compounds in liposomes appears to be a good strategy for obtaining antimicrobial films with essential oils. © 2016 Society of Chemical Industry

HPLC analysis as a tool for assessing targeted liposome composition

Functionalized phospholipids are indispensable materials for the design of targeted liposomes. Control over the quality and quantity of phospholipids is thereby key in the successful development and manufacture of such formulations. This was also the case for a complex liposomal preparation composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), Cholesterol (CHO), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000). To this end, an RP-HPLC method was developed. Detection was done via evaporative light scattering (ELS) for liposomal components. The method was validated for linearity, precision, accuracy, sensitivity and robustness. The liposomal compounds had a non-linear quadratic response in the concentration range of 0.012–0.42mg/ml with a correlation coefficient greater than 0.99 with an accuracy of method confirmed 95–105% of the theoretical concentration. Furthermore, degradation products from the liposomal formulation could be identified. The presented method was successfully implemented as a control tool during the preparation of functionalized liposomes. It underlined the benefit of HPLC analysis of phospholipids during liposome preparation as an easy and rapid control method for the functionalized lipid at each preparation step as well as for the quantification of all components.

HPLC method for quantification of Camellia sinensis catechins entrapped in liposomal carriers

Many natural products of commercial importance in food and pharmaceutical sectors, are complex mixtures of substances. Examples of herbal substances with claimed health benefits include the phenolic compounds. Camellia sinensis (green tea) is a plant with a high concentration of polyphenols with several biological properties such as antioxidant, antitumor, antimicrobial, etc. However, the biological effect can be drastically diminished or lost after oral administration, due to low solubility of the compound in the gastrointestinal tract (low absorption), and the effect of presystemic metabolism. The entrapment of bioactive compounds in liposomes has been proposed as a promising method to overcome bioavailability restrictions. However, depending on the properties of the phenolic substance, a strong physicochemical interaction with the lipid bilayer can emerge, which makes the quantification of the entrapment efficiency difficult. The aim of this work was to develop and validate a HPLC method for quantification of green tea (GT) catechins epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) encapsulated in liposomal lipid carriers. Liquid, spray dried and freeze dried compositions containing lecithin:cholesterol: GT lyophilized extract:surfactant (8:1:2:2) were prepared and used for validation of the HPLC method. Samples were extracted with ultrapure water and with a mixture of water plus triton X-100 (in order to open the lipid vesicles), and analysed using a reversed phase C-18 column. The results were compared with the non encapsulated lyophilized extract of GT and standard substances. A significant change in the HPLC profiles was observed for the encapsulated samples, which was linked to the strong molecular interaction between the GT polyphenols with the phosphatidylcholine lipid bilayers. High encapsulation efficiencies were observed for liquid and dried formulations, which were dependent of the polyphenol moiety (EGCG > ECG).

Improvement of the Pharmacokinetics and In Vivo Antibacterial Efficacy of a Novel Type IIa Topoisomerase Inhibitor by Formulation in Liposomes

Several useful properties of liposome-based formulations of various existing antibacterial drugs have been reported. These properties include lower MICs, improved pharmacokinetics, lower toxicity, selective distribution to infected tissues, and enhanced in vivo efficacy. Here we report in vivo studies of a liposomal formulation of a member of a novel class of antibacterial type II topoisomerase inhibitors, others of which have progressed to early phases of clinical trials. The free (i.e., nonliposomal) compound has broad-spectrum MICs but suboptimal pharmacokinetics in rats and mice, characterized by a high volume of distribution and rapid clearance. The liposomal formulation of the compound had essentially unchanged MICs but greatly reduced volume of distribution and clearance in rats and mice. In an in vivo mouse model of Staphylococcus aureus infection of one thigh, the liposomal compound localized preferentially to the infected thigh, whereas the free compound showed no preference for the infected versus the uninfected thigh. Most importantly, the liposomal compound had enhanced efficacy at clearing the infection compared with the free compound. Delivery of this class of compounds as liposomal formulations may offer clinical advantages compared with free compounds.

Tumor growth suppression by gadolinium-neutron capture therapy using gadolinium-entrapped liposome as gadolinium delivery agent

Neutron capture therapy (NCT) is a promising non-invasive cancer therapy approach and some recent NCT research has focused on using compounds containing gadolinium as an alternative to currently used boron-10 considering several advantages that gadolinium offers compared to those of boron. In this study, we evaluated gadolinium-entrapped liposome compound as neutron capture therapy agent by in vivo experiment on colon-26 tumor-bearing mice. Gadolinium compound were injected intravenously via tail vein and allowed to accumulate into tumor site. Tumor samples were taken for quantitative analysis by ICP-MS at 2, 12, and 24h after gadolinium compound injection. Highest gadolinium concentration was observed at about 2h after gadolinium compound injection with an average of 40.3μg/g of wet tumor tissue. We performed neutron irradiation at JRR-4 reactor facility of Japan Atomic Energy Research Institute in Tokaimura with average neutron fluence of 2×1012 n/cm2. The experimental results showed that the tumor growth suppression of gadolinium-injected irradiated group was revealed until about four times higher compared to the control group, and no significant weight loss were observed after treatment suggesting low systemic toxicity of this compound. The gadolinium-entrapped liposome will become one of the candidates for Gd delivery system on NCT.

In vitro phototoxicity of ultradeformable liposomes containing chloroaluminum phthalocyanine against New World Leishmania species

The use of photodynamic therapy (PDT) against cutaneous leishmaniasis (CL) based on chloroaluminum phthalocyanine (ClAlPc) is a promissory alternative therapy. The main purpose of this article was to assess the internalization and in vitro phototoxic activities of ClAlPc encapsulated in ultradeformable liposomes (UDL-ClAlPc) in Leishmania parasites and mammalian cells. Cell internalization was determined by fluorescence microscopy, cell and parasite damage by standard MTT or direct microscopic analysis and a phototoxic index (PI) was calculated as the compound activity (IC50) at 0J/cm2/IC50 at 17J/cm2. Liposomal and free ClAlPc were internalized by infected and non-infected THP-1 cells and co-localized in the mitochondria. Treatment of UDL-ClAlPc was almost 10 times more photoactive than free ClAlPc on THP-1 cells and promastigotes and intracellular amastigotes of Leishmania chagasi and Leishmania panamensis. Liposomal compounds were active on non-irradiated and irradiated cells however PI higher than 50 were calculated. PI for amphotericin B referential drug were lower than 1.2. Empty liposomes tested at the same lipid concentration of active ClPcAl-liposomes were non-toxic. Upon photodynamic treatment a nonselective-parasite activity against intracellular amastigotes were observed and loss of membrane integrity resulting in a release of parasites was detected. Further studies oriented to evaluate both the state of infection after PDT and the effectiveness of UDL as delivery vehicles of ClAlPc in CL experimental models are required.

PHOTODEGRADATION OF NIMODIPINE AND FELODIPINE IN MICROHETEROGENEOUS SYSTEMS

The photochemical behavior of nimodipine (NIMO) and felodipine (FELO), photolabile drugs widely used as antihypertensive calcium channel blockers, is studied in constrained media. Specifically, we are interested in the kinetic analysis of 4-aryl-1,4-dihydropyridine photodegradation processes when they are incorporated in biological-mimicking systems like micelles or liposomes. In order to establish if the nature of the head of surfactant (ionic or nonionic) could be important modulating the photo-reactivity of these drugs, we studied the photodegradation of NIMO and FELO incorporated in micelles formed with sodium dodecyl sulfate (SDS, anionic), dodecyl-pyridinium chloride (DPC, cationic) and mono lauryl sucrose ester (MLS, nonionic) as surfactants. Additionally, the results of the photodegradation of these compounds in liposomes were also included. The results clearly indicate that both dihydropyridines studied, NIMO and FELO, are located near to the interface, but the surface charge of micelles does not affect neither, the photodegradation rate constant nor the photodegradation products profile. The absence of singlet oxygen generation in micellar media is consistent with the proposition of these 4-aryl-1,4-dihidropyridines located near to the interface of the micelle, where a polar environment is sensed. In addition, the ethanol preferential location on membranes and dihydropyridine enhanced photodegradation by alcohol presence are interesting results to consider in future research.

New potential antitumoral fluorescent tetracyclic thieno[3,2-b]pyridine derivatives: interaction with DNA and nanosized liposomes

Fluorescence properties of two new potential antitumoral tetracyclic thieno[3,2-b]pyridine derivatives were studied in solution and in liposomes of DPPC (dipalmitoyl phosphatidylcholine), egg lecithin (phosphatidylcholine from egg yolk; Egg-PC) and DODAB (dioctadecyldimethylammonium bromide). Compound 1, pyrido[2',3':3,2]thieno[4,5-d]pyrido[1,2-a]pyrimidin-6-one, exhibits reasonably high fluorescence quantum yields in all solvents studied (0.20 ≤ ΦF ≤ 0.30), while for compound 2, 3-[(p-methoxyphenyl)ethynyl]pyrido[2',3':3,2]thieno[4,5-d]pyrido[1,2-a]pyrimidin-6-one, the values are much lower (0.01 ≤ ΦF ≤ 0.05). The interaction of these compounds with salmon sperm DNA was studied using spectroscopic methods, allowing the determination of intrinsic binding constants, Ki = (8.7 ± 0.9) × 103 M-1 for compound 1 and Ki = (5.9 ± 0.6) × 103 M-1 for 2, and binding site sizes of n = 11 ± 3 and n = 7 ± 2 base pairs, respectively. Compound 2 is the most intercalative compound in salmon sperm DNA (35%), while for compound 1 only 11% of the molecules are intercalated. Studies of incorporation of both compounds in liposomes of DPPC, Egg-PC and DODAB revealed that compound 2 is mainly located in the hydrophobic region of the lipid bilayer, while compound 1 prefers a hydrated and fluid environment.

Role of phospholipids in the oral and parenteral delivery of poorly water soluble drugs

This paper reviews the role of phospholipids, as excipients for oral and parenteral delivery of poorly water soluble compounds and as natural constituents of many biological systems. The nomenclature of phospholipids and aspects of their physico-chemical, toxicological, biochemical properties and regulatory status, which are of importance for pharmaceutical scientist are reviewed. The oral administration of poorly water soluble compounds in phospholipid based formulations and latest developments in dissolution testing using phospholipid/bile salt containing biorelevant media are discussed. The intravenous administration of poorly water soluble compounds in liposomes, mixed micelles, oil-in-water emulsions and their dissolution mechanism in blood are discussed. In this context, the extemporaneously loading of the lipid vehicles with poorly water soluble compounds is described.

Optimalized transient transfection of chondrogenic primary cell cultures

Abstract We aimed to find a transfection method which provides high efficiency with minimal cytotoxic and/or apoptotic effects for gene transfer into multilayer primary chondrogenic cell cultures. The pEGFP-C1 plasmid was introduced into the cell culture and the efficiency of transformation quantified by GFP fluorescence; the resulting nucleofection was effective but resulted in severe apoptosis. Two liposomal reagents designed to allow transfection into adherent cells did not deliver the plasmids sufficiently and cartilage formation did not occur. In addition, a third liposomal compound, recommended for transfection into either adherent or suspension cell cultures, lead to acceptable transfection efficiency but no cartilage formation. When an amphiphilic reagent was used however, there was acceptable transfection efficiency as well as cartilage formation. The viability of the cells which were transfected using the amphiphilic reagent remained unaffected but proliferation was severely diminished, particularly in the presence of GFP. In addition, the amount of cartilage decreased when GFP was expressed, despite unchanged levels of mRNAs of sox9 and aggrecan core protein, factors reflecting on the efficiency of chondrogenesis. Overexpression of both the constitutively active delta and gamma isoforms of catalytic subunit of calcineurin, a protein phosphatase described as a positive regulator of chondrogenesis, decreased protein level of Sox9 and subsequent cartilage formation. In conclusion, we found that amphiphilic reagent applied prior to the adhesion of cells provides a useful means to transfer plasmids to primary differentiating chondrogenic cells.