Evaluation of New Folate Receptor-mediated Mitoxantrone Targeting Liposomes In Vitro.

Background: Ligand-mediated liposomes targeting folate receptors (FRs) that are overexpressed on the surface of tumor cells may improve drug delivery. However, the properties of liposomes also affect cellular uptake and drug release.

Objective: Mitoxantrone folate targeted liposomes were prepared to increase the enrichment of drugs in tumor cells and improve the therapeutic index of drugs by changing the route of drug administration.

Methods: Liposomes were prepared with optimized formulation, including mitoxantrone folatetargeted small unilamellar liposome (MIT-FSL), mitoxantrone folate-free small unilamellar liposome (MIT-SL), mitoxantrone folate-targeted large unilamellar liposome (MIT-FLL), mitoxantrone folate-free large unilamellar liposomes (MIT-LL). Cells with different levels of folate alpha receptor (FRα) expression were used to study the differences in the enrichment of liposomes, the killing effect on tumor cells, and their ability to overcome multidrug resistance.

The results of the drug release experiment showed that the particle size of liposomes affected their release behavior. Large single-compartment liposomes could hardly be effectively released, while small single-compartment liposomes could be effectively released, MIT-FSL vs MIT-FLL and MIT-SL vs MIT-LL had significant differences in the drug release rate (P<0.0005). Cell uptake experiments results indicated that the ability of liposomes to enter folic acid receptor-expressing tumor cells could be improved after modification of folic acid ligands on the surface of liposomes and it was related to the expression of folate receptors on the cell surface. There were significant differences in cell uptake rates (p<0.0005) for cells with high FRα expression (SPC-A-1 cells), when MIT-FSL vs MIT-SL and MIT-FLL vs MIT-LL. For cells with low FRα expression (MCF-7 cells), their cell uptake rates were still different (p<0.05), but less pronounced than in SPC-A-1 cells. The results of the cell inhibition experiment suggest that MIT-FLL and MIT-LL had no inhibitory effect on cells, MIT-FSL had a significant inhibitory effect on cells and its IC50 value was calculated to be 4502.4 ng/mL, MIT-SL also had an inhibitory effect, and its IC50 value was 25092.1 ng/mL, there was a statistical difference (p<0.05), MIT-FSL had a higher inhibitory rate than MIT-SL at the same drug concentration. Afterward, we did an inhibitory experiment of different MIT-loaded nanoparticles on MCF-7 cells compared to the drug-resistant cells (ADR), Observing the cell growth inhibition curve, both MIT-FSL and MIT-SL can inhibit the growth of MCF-7 and MCF-7/ADR cells. For MCF- 7 cells, at the same concentration, there is little difference between the inhibition rate of MITFSL and MIT-SL, but for MCF-7/ADR, the inhibition rate of MIT-FSL was significantly higher than that of MIT-SL at the same concentration (P<0.05).

Conclusion: By modifying folic acid on the surface of liposomes, tumor cells with high expression of folic acid receptors can be effectively targeted, thereby increasing the enrichment of intracellular drugs and improving efficacy. It can also change the delivery pathway, increase the amount of drug entering resistant tumor cells, and overcome resistance.

Screening of Peptides that Specifically Binds to M3-M4 Extracellular Domain of Sodium Pump α1 Subunit and Analysis of Their Bioactivity In Vitro and In Vivo.

Interaction between ouabain (OUA) and Na+/K+-pump remains in the current focus of hypertension research. This study aimed to find an oligopeptide that would antagonize the inhibitory effect of endogenous OUA on Na+/K+-pump and examine its activity at the cellular and organism levels. To this end, Phage Random 12 Peptide Library was employed to screen for specific polypeptide ligands that interact with M3-M4 extracellular domain of Na+/K+-pump α1 subunit known as OUA-binding site. Synthetic sequence ILEYTWLEAGGGS of extracellular domain M3-M4 of Na+/K+-pump α1 subunit was used as the target. The phage positive clones were screened and identified using the phage library and double sandwich ELISA. DNA was extracted and sequenced to synthesize 3 peptide ligands to Na+/K+-pump: P-A, P-B, and P-C. We also studied the effects of the short peptide with the highest potency for countering OUA on proliferation and apoptosis of EA.hy926 vascular endothelial cells and on systolic BP in spontaneously hypertensive rats (SHR). The effect of peptide P-A on proliferation (stimulation with physiological concentrations of OUA) and on apoptosis (stimulation with OUA in high concentrations) of EA.hy926 vascular endothelial cells was assessed by the MTT test and flow cytometry, respectively. In SHR rats, intravenous injection of P-A decreased systolic BP. Oligopeptide P-A competitively antagonized the inhibitory action of OUA on Na+/K+-pump, OUA-induced proliferation, and OUA-provoked apoptosis of cultured EA.hy926 cells. Our findings open vista for the emergence of novel hypertensive drugs.

Paris saponin II-induced paraptosis-associated cell death increased the sensitivity of cisplatin.

Paris Saponin II (PSII) has been regarded as an effective and imperative component isolated from Rhizoma Paridis saponins (RPS) and exhibited strong anti-tumor effects on a variety of cancer. Our results revealed that human non-small lung cancer cell lines NCI-H460 and NCI-H520 were exposed to 1 µM of PSII, which inhibited the proliferation of lung cancer cells and activated apoptosis, autophagy and paraptosis. PSII induced paraptosis-associated cell death prior to apoptosis and autophagy. It induced paraptosis based on ER stress through activation of the JNK pathway. Meanwhile, PSII increased the cytotoxicity of cisplatin through paraptosis-associated pathway. All in all, PSII induced paraptosis based on induction of non-apoptotic cell death, which would be a possible approach to suppress the multi-drug resistant to apoptosis.

Diagnostic Accuracy of Interleukin-27 in Bronchoalveolar Lavage Fluids for Pulmonary Tuberculosis.

BACKGROUND: The World Health Organization states that China had 0.9 million cases of tuberculosis in 2017, accounting for 9% of cases globally. Despite a decrease in the incidence and mortality of tuberculosis in China over time, development in choosing the appropriate prevention and control of TB is required. PURPOSE: The aim of this study was to evaluate the diagnostic significance of interleukin-27 in bronchoalveolar lavage fluids for pulmonary tuberculosis. MATERIALS AND METHODS: Eventually, 107 bronchoalveolar lavage fluids from patients were included in this study. The concentrations of interleukin-27 and adenosine deaminase were determined in bronchoalveolar lavage fluids using enzyme-linked immunosorbent assay. RESULTS: It was found that the concentrations of interleukin-27 in bronchoalveolar lavage fluids of sputum-positive pulmonary tuberculosis group were significantly higher than those in sputum-negative pulmonary tuberculosis, lung cancer, and previous pulmonary tuberculosis groups, respectively (all P<0.001). Interleukin-27 levels in bronchoalveolar lavage fluids could be used for diagnostic purpose for pulmonary tuberculosis, with the cutoff value of 7.867 pg/mL; interleukin-27 had a sensitivity of 68.8% and specificity of 100% for the differential diagnosis of pulmonary tuberculosis (sputum-negative and sputum-positive PTB) from lung cancer. And with the cutoff value of 6.012 pg/mL, IL-27 had sensitivity and specificity of both 100% for the differential diagnosis of PTB from previous PTB. The risk of pulmonary tuberculosis was positively associated with the concentrations of interleukin-27 and adenosine deaminase in bronchoalveolar lavage fluids. CONCLUSION: Interleukin-27 in bronchoalveolar lavage fluids is a sensitive and specific biomarker for the differential diagnosis of pulmonary tuberculosis from lung cancer and previous pulmonary tuberculosis.

Efficient Corrections for DFT Noncovalent Interactions Based on Ensemble Learning Models.

Machine learning has exhibited powerful capabilities in many areas. However, machine learning models are mostly database dependent, requiring a new model if the database changes. Therefore, a universal model is highly desired to accommodate the widest variety of databases. Fortunately, this universality may be achieved by ensemble learning, which can integrate multiple learners to meet the demands of diversified databases. Therefore, we propose a general procedure for learning ensemble establishment based on noncovalent interactions (NCIs) databases. Additionally, accurate NCI computation is quite demanding for first-principles methods, for which a competent machine learning model can be an efficient solution to obtain high NCI accuracy with minimal computational resources. In regard to these aspects, multiple schemes of ensemble learning models (Bagging, Boosting, and Stacking frameworks), are explored in this study. The models are based on various low levels of density functional theory (DFT) calculations for the benchmark databases S66, S22, and X40. All NCIs computed by the DFT calculations can be improved to high-level accuracy (root-mean-square error RMSE = 0.22 kcal/mol in contrast to CCSD(T)/CBS benchmark) by established ensemble learning models. Compared with single machine learning models, ensemble models show better accuracy (RMSE of the best model is further lowered by ∼25%), robustness and goodness-of-fit according to evaluation parameters suggested by the OECD. Among ensemble learning models, heterogeneous Stacking ensemble models show the most valuable application potential. The standardized procedure of constructing learning ensembles has been well utilized on several NCI data sets, and this procedure may also be applicable for other chemical databases.

The synergistic anticancer effect of formosanin C and polyphyllin VII based on caspase-mediated cleavage of Beclin1 inhibiting autophagy and promoting apoptosis.

OBJECTIVES: Drug combination has a promising and potential development prospect in the treatment of various cancers. The objective of this study is to investigate the synergistic mechanisms of polyphyllin VII (PVII) and formosanin C (FC) in lung cancer. MATERIALS AND METHODS: The combination of FC and PVII influenced on the apoptosis, autophagy, and the relative signalling pathways were analysed in lung cancer cells. RESULTS: The combination of FC and PVII demonstrated a concentration- dependent growth inhibition in human lung cancer cells. The combination index (CI) obtained from four lung cancer cells was smaller than 1. This synergistic antitumour effect was based on the increase of their single proapoptotic effect but inhibiting FC-induced autophagy in NCI-H460 cells. FC and PVII activated proapoptotic elements like cleaved-caspase-3, -8, and -9 to induce Beclin1 cleaved into Beclin1-C which suppressed FC-triggered autophagy and enhanced apoptosis. CONCLUSIONS: Formosanin C and PVII showed a synergistic antitumour effect on lung cancer cells. The findings would provide the foundation for the use of combination drugs in the future.

Curcumin enhances the anti-cancer effects of Paris Saponin II in lung cancer cells.

OBJECTIVES: To investigate the synergistic mechanisms of Paris Saponin II (PSII) and Curcumin (CUR) in lung cancer. MATERIALS AND METHODS: The combination changed the cellular uptake of CUR and PSII, apoptosis, cell cycle arrest and cytokine levels were analysed on different lung cancer cells. RESULTS: The combination displayed a synergistic anti-cancer effect through promoting the cellular uptake of CUR on different lung cancer cells. Hoechst H33258 staining and FACS assay indicated that the combination of PSII and CUR induced cell cycle arrest and apoptosis. Western blot and cytokine antibody microarray suggested that the combination activated death receptors such as DR6, CD40/CD40L, FasL and TNF-α to induce cancer cells apoptosis, and up-regulated IGFBP-1 leading to inhibition of PI3K/Akt pathway and increase of p21 and p27, which therefore induced a G2 phase arrest in NCI-H446 cells. Meanwhile, the combination suppressed PCNA and NF-κB pathway in 4 kinds of lung cancer cells. They activated the phosphorylation of p38 and JNK, and inhibited PI3K in NCI-H460 and NCI-H446 cells, enhanced the phosphorylation of JNK in NCI-H1299 cells, and increased the phosphorylation of p38 and ERK, and suppressed PI3K in NCI-H520 cells. CONCLUSIONS: PSII combined with CUR had a synergistic anti-cancer effect on lung cancer cells. These findings provided a rationale for using the combination of curcumin and PSII in the treatment of lung cancer in future.

Antitumor and anti-metastatic mechanisms of Rhizoma paridis saponins in Lewis mice.

Lung cancer is one of the most common causes of death in the world. Rhizoma paridis saponins (RPS) have been found to show inhibition of pulmonary adenoma in previous research. However, the detailed mechanisms of RPS from a holistic view have not been established. In this study, Lewis pulmonary adenoma mice were successfully established to analyze the pathways involved in RPS intervening tumor formation and progression. As a result, RPS inhibited levels of cytokines or receptors such as VEGFD, VEGFR3, RAGE, IL6R, IL17BR, and CXCL16 which were regarded as the initiators induced tumor cell proliferation, adhesion, angiogenesis, and invasion. Meanwhile, RPS raised the content of SOD and CAT enzymes and thereby inhibited the aberrantly active NF-κB, and phosphorylation of PI3K/Akt and MAPK (including p38, Erk1/2, and JNK) signaling pathways. Soon after, RPS changed mRNA expression of nuclear factors containing NF-κB, HIF-1A, STAT3, and Jun, and consequentially suppressed the expression of angiogenesis, lymphangiogenesis, adhesion, inflammation, and invasion enzymes. In conclusion, this research provided a holistic view to understand the multi-target antitumor mechanisms of RPS which promoted the application of RPS in the future.

Systemic Perturbations of Key Metabolites in Type 2 Diabetic Rats Treated by Polyphenol Extracts from Litchi chinensis Seeds.

Our previous research obtained Litchi chinensis Sonn. seeds extract (LSE) which showed hypoglycaemic effects on type 2 diabetes (T2D) rats. In order to understand the detailed pathogenesis of diabetes intervened by LSE, the metabonomics strategy was used. As a result, LSE decreased the insulin resistance index and the levels of glucose in urine through elevating the mRNA level of insulin, while decreasing the expression of glucagon to enhance the function of the pancreas. Meanwhile, LSE regulated the glucose and fatty acid metabolisms via increasing the expression of glucose transporter (Glu) 2, Glu4, insulin receptor (IR), and IR substrate-2 (IRS2). LSE effectively restored the impairment of the IRS2/PI3K/Akt/mTOR insulin signaling in the livers. All in all, LSE played a pivotal role in the treatment of T2D through regulation of broad-spectrum metabolic changes and inhibition of the glycogenesis, proteolysis, and lipogenesis in T2D rats.

A cascaded QSAR model for efficient prediction of overall power conversion efficiency of all-organic dye-sensitized solar cells.

A cascaded model is proposed to establish the quantitative structure-activity relationship (QSAR) between the overall power conversion efficiency (PCE) and quantum chemical molecular descriptors of all-organic dye sensitizers. The cascaded model is a two-level network in which the outputs of the first level (JSC, VOC, and FF) are the inputs of the second level, and the ultimate end-point is the overall PCE of dye-sensitized solar cells (DSSCs). The model combines quantum chemical methods and machine learning methods, further including quantum chemical calculations, data division, feature selection, regression, and validation steps. To improve the efficiency of the model and reduce the redundancy and noise of the molecular descriptors, six feature selection methods (multiple linear regression, genetic algorithms, mean impact value, forward selection, backward elimination, and +n-m algorithm) are used with the support vector machine. The best established cascaded model predicts the PCE values of DSSCs with a MAE of 0.57 (%), which is about 10% of the mean value PCE (5.62%). The validation parameters according to the OECD principles are R(2) (0.75), Q(2) (0.77), and Qcv2 (0.76), which demonstrate the great goodness-of-fit, predictivity, and robustness of the model. Additionally, the applicability domain of the cascaded QSAR model is defined for further application. This study demonstrates that the established cascaded model is able to effectively predict the PCE for organic dye sensitizers with very low cost and relatively high accuracy, providing a useful tool for the design of dye sensitizers with high PCE.

Direct comparison of two albumin-based paclitaxel-loaded nanoparticle formulations: is the crosslinked version more advantageous?

Nanoparticles using albumin as particle matrix have entered the mainstream of drug delivery. It was reported that non-crosslinked albumin nanoparticles were unstable in circulation and could deliver drugs into tumor through gp60/SPARC pathway; in contrast, the delivery of drugs with stable nanoparticles was dependent on enhanced permeability and retention effect. Thus, it is questionable which kind of nanoparticles was more advantageous. Two versions of albumin-bound paclitaxel nanoparticles were prepared. In vitro, the non-crosslinked particles could rapidly disintegrate and the crosslinked was stable. The pharmacokinetics of both formulations was different especially at early time and the non-crosslinked particles were cleared rapidly. After non-crosslinked particle treatment paclitaxel had a tendency to accumulate into heart and kidney and following therapy with the crosslinked particles, paclitaxel was liable to be delivered into lung, spleen and liver. The delivery efficiency of paclitaxel into tumor following the non-crosslinked particle treatment was greater than that of the crosslinked (p<0.05), thus resulting in a considerably improved antineoplastic activity. Moreover, the non-crosslinked formulation was only slightly more toxic. It was concluded that the non-crosslinked formulation was more advantageous for the delivery of paclitaxel and our conclusion might be generalized to other lipophilic drugs delivered with albumin nanoparticles.

Pegylated liposomal mitoxantrone is more therapeutically active than mitoxantrone in L1210 ascitic tumor and exhibits dose-dependent activity saturation effect.

Our previous studies have proved that encapsulation of mitoxantrone into pegylated SUVs (plm60-s) could enhance its antineoplastic efficacy (Li et al., 2008b). However, why plm60-s is more therapeutically active than free mitoxantrone (MIT), and whether pharmacokinetics and activity of plm60-s exhibits dose-dependency are left unknown. In studies with L1210 ascitic tumor-bearing mice in which the dose of MIT was elevated from 2 to 8mg/kg, a saturation of antineoplastic efficacy was observed after plm60-s, and not after free MIT therapy. Total MIT concentrations in plasma, liver and ascitic fluids after plm60-s increased linearly with escalated doses. The released MIT concentrations in ascitic fluid increased continuously before reaching the peak at a dose of 6mg/kg and then decreased. In vitro release experiments using ascitic fluid as release medium revealed that at high concentrations of plm60-s the release of drug was inhibited. At a dose of 4mg/kg, the areas under the curve (AUC) of released MIT in ascitic fluid after plm60-s were higher than those after free MIT, which might be responsible for the enhanced efficacy of plm60-s. These observations may be used to choose a dose regimen of plm60-s to ensure optimal efficacy and to expound the reasons why plm60-s was more therapeutically active.

Prolongation of time interval between doses could eliminate accelerated blood clearance phenomenon induced by pegylated liposomal topotecan.

Repeated injection of pegylated liposomes could elicit the disappearance of long-circulating characteristic, referred to as "accelerated blood clearance phenomenon." ABC phenomenon typically occurs when entrapped drugs are not cytotoxic, but recently it was reported that multiple doses of pegylated liposomal topotecan, a cytotoxic drug, could also induce the phenomenon in rats. To reveal whether the phenomenon could be induced in dogs and the effect of time interval between doses on the magnitude of ABC, pLT was repeatedly injected into beagle dogs with a time interval of 7, 21 and 28 days. The anti-PEG Ig M levels were detected using ELISA. It was found that ABC phenomenon could be induced in dogs by pLT. Inter-individual difference in anti-PEG antibody production could be observed, and antibody levels were directly correlated with the magnitude of ABC. Furthermore, time interval between doses had marked effect on the magnitude of ABC phenomenon. When the time interval was prolonged from 1 week to 4 weeks, ABC phenomenon could be eliminated. By comparing the pharmacokinetic profiles of lipid vesicles and entrapped topotecan, it was found that "empty pegylated vesicles" be formed in circulation, which might be responsible for the occurrence of ABC phenomenon.

Accelerated blood clearance of pegylated liposomal topotecan: influence of polyethylene glycol grafting density and animal species.

Upon repeated administration, empty pegylated liposomes lose long-circulating characteristics, referred to as accelerated blood clearance (ABC) phenomenon. However, pegylated liposomal cytotoxic drug formulations could not elicit the phenomenon. In the study, it was found that repeated injection of pegylated liposomal topotecan could induce ABC phenomenon in Wistar rats, beagle dogs, and mice, which might be associated with the formation of empty liposomes in circulation because of the rapid drug release rate. In rats, the 9% polyethylene glycol (PEG) formulation induced more severe ABC phenomenon than 3% PEG formulation despite the similar anti-PEG immunoglobulin M (IgM) levels following the first dose. Antibody neutralization experiments revealed that high PEG formulation was easily neutralized by IgM. Repeated administration of 3% PEG formulation in dogs could result in more severe ABC phenomenon. It seems that slow infusion was liable to cause ABC phenomenon. In all animal species, considerable intraindividual variability of IgM levels could be observed. Our observations may have important implications for the development, evaluation, and therapeutic use of pegylated liposomal cytotoxic drug formulations because using the current drug loading technology, most of the cytotoxic drugs could not be stably loaded in liposomes and rapid drug leakage from liposomes might occur in circulation.

Liposomal topotecan formulation with a low polyethylene glycol grafting density: pharmacokinetics and antitumour activity.

OBJECTIVES: PEGylated liposomes could evade recognition by the reticulo-endothelial system and prolong the circulation time of vesicles, resulting in enhanced targeting efficiency and antitumour effect. Typically, vesicles are modified with distearoylphosphatidylethanolamine (DSPE)-polyethylene glycol (PEG) at a high PEG grafting density. However, long circulation time and slow drug release rate might induce severe hand-foot syndrome in clinical practice. In this study, a liposomal topotecan formulation with a low PEG grafting density was prepared and its pharmacokinetics, acute toxicity and antitumour effect were investigated. METHODS: Topotecan was loaded into liposomes using an ammonium sulfate gradient. The resulting formulation was injected to healthy Wistar rats at different dose levels to investigate whether its clearance followed linear kinetics. Biodistribution was performed in Lewis lung cancer-bearing mice. The acute toxicity was evaluated in healthy mice and beagle dogs. To compare the antitumour effects of different formulations and dose schedule, RM-1 prostate, Lewis lung, H446 and L1210 cancer models were used. KEY FINDINGS: Topotecan could be encapsulated into low DSPE-PEG liposomes with ∼100% loading efficiency. The clearance of the liposomal formulation followed linear kinetics at a dose level ranging from 0.5 to 4 mg/kg despite the fact that the vesicles were coated at a low PEG density. Compared with free topotecan the liposomal formulation preferentially accumulated into tumour zones instead of normal tissues. Both formulations could rapidly accumulate into liver and tumour, but the liposomal formulation was cleared from tissues at a slow rate relative to the conventional formulation. In rats and beagle dogs, liposomal formulations could not induce skin toxicity. In all the tumour models, smaller split doses were more therapeutically active than larger doses when the overall dose intensity was equivalent. CONCLUSIONS: This has been the first report that plasma kinetics of a liposomal formulation with a low PEG density followed linear kinetics. Moreover, due to its short circulation half-life, the formulation did not induce skin toxicity. Our data revealed that the dose schedule of liposomal drugs should be adjusted in accordance with the biophysical and biological properties of the formulations to achieve the optimal therapeutic efficacy.

Sulfosalicylate mediates improved vinorelbine loading into LUVs and antineoplastic effects.

Liposomal vinorelbine formulation is desirable, as it might improve the therapeutic activity of vinorelbine. However, because of its lipophilic and membrane-permeable properties, vinorelbine is hard to be formulated into liposomes using conventional drug-loading technologies. To improve vinorelbine retention, ammonium salts of several anionic agents were employed to prepare liposomal vinorelbine formulations. It was found that 5-sulfosalicylate (5ssa) could form stable complexes with vinorelbine and stabilize entrapped vinorelbine. The resultant vesicles had an in vitro release t(1/2) of ~12.49 hours in NH(3)-containing media, which is longer than those of sulfate and phytate vesicles (~0.57 hours). The circulation half-life of vinorelbine after the injection of 5ssa vesicles into normal mice was ~13.01 hours, accounting for ~2-fold increase relative to that of sulfate vesicles. Improved drug retention correlated with enhanced antitumor efficacy. In the RM-1/c57 model, 5ssa vesicles were more efficacious than sulfate vesicles (P < 0.05). In RM-1/BDF1 and Lewis lung cancer/c57 models, antitumor efficacy was also considerably improved after vinorelbine encapsulation into 5ssa vesicles. For instance, in the RM/BDF1 model, liposomal vinorelbine was at least 4-fold more therapeutically active than free vinorelbine. Our results demonstrated that 5ssa could stabilize vinorelbine relative to other anions, resulting in the formulation with improved drug retention and efficacy. Improved vinorelbine retention might be associated with the formation of insoluble precipitate, which could be proved by precipitation study and decreased drug-release rate at a high D/L ratio.

Encapsulation of vinorelbine into cholesterol-polyethylene glycol coated vesicles: drug loading and pharmacokinetic studies.

OBJECTIVES: Pegylated liposome formulations of vinorelbine with prolonged circulation half-life (t½) are desirable. However, DSPE-PEG could affect vinorelbine loading into vesicles due to electrostatic interactions. To resolve this problem, chol-PEG was used to prepare pegylated liposomal vinorelbine and the factors affecting drug loading and plasma pharmacokinetics were investigated. METHODS: Vinorelbine was loaded into liposomes using a novel triethylamine 5-sulfosalicylate gradient. The effects of cholesterol and chol-PEG on drug loading were investigated. Pharmacokinetic studies were performed in normal KunMing mice treated with different liposomal vinorelbine formulations. To clarify the effects of chol-PEG on membrane permeability, drug release experiments were performed based on the fluorescence dequenching phenomenon of a fluorescence marker. KEY FINDINGS: In contrast to DSPE-PEG, even at high PEG grafting density (∼ 8.3 mol%), chol-PEG had no effect on vinorelbine loading into HSPC/cholesterol (3 : 1, mass ratio) vesicles. However, for the formulations with low cholesterol content (HSPC/cholesterol 4 : 1), loading efficiency decreased with increasing chol-PEG content. In vivo, the vinorelbine t½ of low cholesterol formulations decreased with increasing chol-PEG content, but for high cholesterol liposomes, the maximum vinorelbine t½ was achieved at ∼ 3 mol% chol-PEG grafting density. The resulting vinorelbine circulation t½ was ∼ 9.47 h, which was greater than that of non-pegylated liposomes (∼ 5.55 h). Drug release experiments revealed that chol-PEG might induce membrane defects and concomitant release of entrapped marker, especially at high chol-PEG density. CONCLUSIONS: Through the investigation of the effects of chol-PEG and cholesterol, an optimum pegylated liposomal vinorelbine formulation with prolonged t½ was achieved. In plasma, the membrane defect induced by chol-PEG may counteract the long circulation characteristics that chol-PEG afforded. When these two opposite effects reached equilibrium, the maximum vinorelbine t½ was achieved.

Novel sulfobutyl ether cyclodextrin gradient leads to highly active liposomal irinotecan formulation.

OBJECTIVES: Liposomal delivery of irinotecan could provide protection against drug hydrolysis, deliver more active lactone form to tumours and prolong irinotecan exposure time. Nevertheless, conventional drug-loading technologies have typically resulted in undesired drug retention properties. To resolve the problem, a modified gradient loading method was developed and the resulting formulations were evaluated in a systemic manner. METHODS: Irinotecan was loaded into liposomes using a novel sulfobutyl ether beta-cyclodextrin (sbe-CD) gradient. The effect of drug-to-lipid ratio (D/L) and polyethylene glycol (PEG) grafting density were investigated. Drug release experiments were performed in ammonium-containing medium based on the fluorescence dequenching phenomenon of irinotecan. Pharmacokinetic studies were performed in normal balb/c mice treated with different formulations. To compare the anti-tumour effect of different formulations, an RM-1 prostate cancer model was used. Acute toxicity studies were performed in healthy female c57 mice. KEY FINDINGS: Irinotecan could be encapsulated into liposomes with >90% loading efficiency at a high drug-to-lipid mass ratio (>0.5). In-vitro release experiments revealed that sbe-CD anion was more able to retain irinotecan than sulfate. Moreover, the elevated D/L ratio elicited decreased drug release kinetics. Both trends had also been observed when the effects of anions and D/L ratio on half-life of irinotecan were assessed. Pegylated liposomal irinotecan loaded with sbe-CD/triethylammonium gradient had irinotecan half-life values ranging from 9.4 to 13.1 h, surpassing vesicles prepared by the triethylammonium sulfate method (∼4.5 h). In the RM-1 tumour model, all the liposomal irinotecan formulations were more therapeutically active than free irinotecan and the formulation with a high D/L ratio was the most efficacious. Moreover, the high D/L formulation might be less toxic than free irinotecan based on acute toxicity studies. CONCLUSIONS: The novel sbe-CD gradient could mediate effective irinotecan loading and improve irinotecan retention, thus resulting in highly active liposomal irinotecan formulations. The improvement in drug retention might be associated with the formation of complicated aggregates inside vesicles.

Development of pegylated liposomal vincristine using novel sulfobutyl ether cyclodextrin gradient: is improved drug retention sufficient to surpass DSPE-PEG-induced drug leakage?

The purpose of this study is to develop novel stable PEGylated liposome vincristine formulations with optimal antitumor efficacy. Vincristine could interact with negatively charged distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), leading to rapid drug release from vesicles. To improve drug retention, vincristine was loaded into vesicles using sulfobutyl ether cyclodextrin (sbe-CD) as trapping agent. Despite that, vincristine could not form a precipitate with sbe-CD; the aggregation status of vincristine/sbe-CD inside vesicles must be complicated because drug retention was considerably improved in vivo. Theoretical consideration revealed that the release constant K equals to pA(m)k(1)k(2)/([H(+)](i)[sbe(-)](i)V(i) ), which can be used to expound why increasing drug/lipid ratio induced decreased vincristine circulation half-life. The stabilization effect afforded by sbe-CD was sufficient to surpass DSPE-PEG-induced drug leakage, so PEGylated liposomal vincristine formulations with prolonged circulation half-life (t(1/2): from 43.6 to 70.0 h) could be achieved, of which the formulation pLV-c-2.9-3 exhibited optimal antitumor effects and reduced toxicity. The strategy might be used to load other vinca alkaloids into PEGylated liposomes and improve their retention inside vesicles.

Nigericin-mediated liposome loading of topotecan: is nigericin a potential drug release regulator?

Topotecan was loaded into large unilamellar vesicles using nigericin-generated pH gradient or with triethylammonium (TA) ion gradient. Despite that in both loading methods, the encapsulated counter ion was 5-sulfosalicylate (5ssa), the resultant formulations exhibited distinct properties. In NaCl-containing release buffer, vesicles prepared with nigericin/Na(+) system could release topotecan at a faster rate than 5ssa-TA vesicles, whereas in the absence of Na(+), there was no difference in the drug release kinetics. In plasma, both formulations could prolong the circulation halftime of topotecan, but 5ssa-TA vesicles were more able to stabilize the encapsulated topotecan, as evidenced by the increased t(1/2) and decreased conversion of lactone to carboxylate form. However, the improved drug retention did not mean the elevated safety and efficacy. In L1210 ascitic tumor model, the administration of 5ssa-TA vesicles at 10mg/kg induced the early death of ∼60% mice at 6-7 days. In contrast, the treatment with nigericin/Na(+) vesicles at the same dose level resulted in a mean survival time of ∼18.0 days, ∼1.38-fold of that of free topotecan. In addition, the formulation was safer than free topotecan. The results indicated that nigericin could be used as release regulator to optimize drug release kinetics, resulting in safe and efficacious liposome-based topotecan formulation. The results were promising since no liposome topotecan formulations with reduced toxicity were reported.