Liposome-enabled bufalin and doxorubicin combination therapy for trastuzumab-resistant breast cancer with a focus on cancer stem cells.

Breast cancer stem cells (BCSCs) play a key role in therapeutic resistance in breast cancer treatments and disease recurrence. This study aimed to develop a combination therapy loaded with pH-sensitive liposomes to kill both BCSCs and the okbulk cancer cells using trastuzumab-sensitive and resistant human epidermal growth factor receptor 2 positive (HER2+) breast cancer cell models. The anti-BCSCs effect and cytotoxicity of all-trans retinoic acid, salinomycin, and bufalin alone or in combination with doxorubicin were compared in HER2+ cell line BT-474 and a validated trastuzumab-resistant cell line, BT-474R. The most potent anti-BCSC agent was selected and loaded into a pH-sensitive liposome system. The effects of the liposomal combination on BCSCs and bulk cancer cells were assessed. Compared with BT-474, the aldehyde dehydrogenase positive BCSC population was elevated in BT-474R (3.9 vs. 23.1%). Bufalin was the most potent agent and suppressed tumorigenesis of BCSCs by ∼50%, and showed strong synergism with doxorubicin in both BT-474 and BT-474R cell lines. The liposomal combination of bufalin and doxorubicin significantly reduced the BCSC population size by 85%, and inhibited both tumorigenesis and self-renewal, although it had little effect on the migration and invasiveness. The cytotoxicity against the bulk cancer cells was also enhanced by the liposomal combination than either formulation alone in both cell lines (p < 0.001). The liposomal bufalin and doxorubicin combination therapy may effectively target both BCSCs and bulk cancer cells for a better outcome in trastuzumab-resistant HER2+ breast cancer.

Tumor-Targeted Delivery of Bufalin-Loaded Modified Albumin-Polymer Hybrid for Enhanced Antitumor Therapy and Attenuated Hemolysis Toxicity and Cardiotoxicity.

A novel albumin polymer hybrid with a core-shell structure was designed to target delivery of bufalin, which is an antineoplastic monomer with serious cardiotoxicity. The sheath layer was composed of ursodeoxycholic acid (UA)-modified bovine serum albumin (UA-BSA), while the stable core consisted of poly n-butyl cyanoacrylate (PBCA) nanoparticles. The UA-BSA was synthetized, and the substitution degree was characterized. The physical properties of bufalin-loaded UA-modified protein-PBCA nanocomplexes (BF-uPPNCs), such as morphology, particle size, and encapsulation efficiency, were evaluated. FTIR and DSC revealed the bufalin to be in an amorphous state. Furthermore, the in vitro release study indicated a sustained release profile of BF-uPPNCs. The MTT and cellular uptake study demonstrated that BF-uPPNCs significantly improved the inhibitory effect of the bufalin accompanied with an enhanced cell uptake capacity on HepG2 cells. In addition, in vivo research demonstrated that BF-uPPNCs had a better antitumor effect coupled with improved therapeutic effect, and reduced hemolysis, vascular irritation, and cardiotoxicity. This work therefore presented a novel albumin polymer hybrid with favorable stability, efficient tumor-targeted delivery potential, and side effect reduction ability, which can be a potential vehicle for an anticancer drug.

Surfactant Assisted Rapid-Release Liposomal Strategies Enhance the Antitumor Efficiency of Bufalin Derivative and Reduce Cardiotoxicity.

BACKGROUND: BF211, a derivative of bufalin (BF), shows significantly improved solubility and potent antitumor efficiency compared to BF. Unfortunately, the unwanted toxicity such as cardiotoxicity caused by unspecific distribution has hindered its clinical use. METHODS: PEGylated BF211 liposomes (BF211@Lipo) were designed and optimizely prepared based on the pre-prescription research. In vitro and in vivo cardiotoxicity was evaluated. In vivo pharmacokinetics and biodistribution of BF211@Lipo were investigated. In vivo antitumor activity and toxicity were evaluated in HepG2 cell xenograft models. The rapid-release triggered by Poloxamer 188 (P188) was assessed in vitro and in vivo. RESULTS: The optimized BF211@Lipo displayed a spherical morphology with a size of (164.6 ± 10.3) nm and a high encapsulation efficiency of (93.24 ± 2.15) %. The in vivo concentration-time curves of BF211 loaded in liposomes showed a prolonged half-life in plasma and increased tumor accumulation. No obvious abnormality in electrocardiograms was observed in guinea pigs even at 9 mg/kg. Moreover, to improve the efficient release of BF211@Lipo, a surfactant-assisted rapid-release strategy was developed, and the release-promoting mechanism was revealed by the fluorescence resonance energy transfer (FRET) and fluorescence nanoparticle tracking analysis (fl-NTA) technology. Sequential injection of BF211@Lipo and P188 could ignite the "cold" liposomes locally in tumor regions, facilitating the burst release of BF211 and enhancing the therapeutic index. CONCLUSION: Our progressive efforts that begin with preparation technology and dosage regimen enable BF211 to like a drug, providing a promising nano platform to deliver the cardiac glycosides and alleviate the side effects by decreasing unspecific biodistribution.

Preparation and evaluation of lyophilized liposome-encapsulated bufadienolides.

OBJECTIVE: The objective of this study was to prepare bufadienolides-loaded liposome (BU-lipo). METHODS: The BU-lipo was prepared by a thin-film hydration method involving sonication and lyophilization procedures. The lyophilized BU-lipo was characterized with regard to the appearance and particle size by scanning electron microscopy, transmission electron microscopy, and photon correlation spectroscopy. The entrapment efficiency (EE) of BU-lipo was evaluated by the microdialysis technique. RESULTS: In the optimal formulation, Lipoid E-80 and the mass ratio of cholesterol to lipid were fixed at 1.25% and 0.05. The media diameters of BU-lipo before and after lyophilization were about 100 nm, and the EEs of bufalin (B), cinobufagin (C), and resibufogenin (R) were 86.5%, 90.0%, and 92.1%, respectively. In the EE study, the probe recoveries of B, C, and R were 21.53 +/- 1.14%, 19.49 +/- 1.34%, and 20.19 +/- 1.25%, respectively, at a flow rate of 4 microL/min by the gain method. The EE of BU-lipo evaluated by microdialysis and ultrafiltration were equivalent. CONCLUSION: The lyophilized BU-lipo contained trehalose (10%) was stable up to 6 months in a desiccator under 2 degrees C-8 degrees C. The microdialysis technique has a wide application perspective in the investigation of the free-drug concentration of microcarrier systems.

Pluronic F127-based micelles for tumor-targeted bufalin delivery.

In this study, we developed novel thermal and redox-responsive micelles based on the Pluronic F127 tri-block copolymer and employed these for redox-responsive intratumor release of bufalin, an anti-cancer drug. Pluronic F127 was first functionalized with carboxylate groups, and then assembled into micelles. The HOOC-F127-COOH micelles are 20 ±â€¯4 nm in size at 37 °C, but expand to 281 ±â€¯5 nm when cooled to 4 °C. This allows for the free diffusion of bufalin into the micellar cores at low temperatures, while at 37 °C the micelles are much more compact and the drug molecules can be effectively held in their interiors. A high encapsulation efficiency and loading content were obtained via drug incorporation at 4 °C. The drug-loaded micelles were cross-linked with cystamine, which contains a disulfide bond responsive to the local cancer microenvironment. In vitro studies showed that drug release from the cross-linked micelles was low under normal physiological conditions, but markedly accelerated upon exposure to conditions representative of the intracellular tumor environment. Confocal microscopy revealed that the cross-linked micelles gave high levels of drug release inside the cells. In vivo studies in mice showed the drug-loaded cross-linked micelles have potent anti-tumor activity, leading to high levels of apoptosis of tumor cells and significant reductions in tumor volume. The drug-loaded cross-linked micelles did not significantly influence body weight, and there was no evidence for detrimental off-target effects. These results indicate that the Pluronic-based micelles developed in this work are promising drug delivery systems for the targeted treatment of cancer.

Systemic delivery of the anticancer agent arenobufagin using polymeric nanomicelles.

Arenobufagin (ABG) is a major active component of toad venom, a traditional Chinese medicine used for cancer therapy. However, poor aqueous solubility limits its pharmacological studies in vivo due to administration difficulties. In this study, we aimed to develop a polymeric nanomicelle (PN) system to enhance the solubility of ABG for effective intravenous delivery. ABG-loaded PNs (ABG-PNs) were prepared with methoxy poly (ethylene glycol)-block-poly (d,l-lactic-co-glycolic acid) (mPEG-PLGA) using the solvent-diffusion technique. The obtained ABG-PNs were 105 nm in size with a small polydispersity index of 0.08. The entrapment efficiency and drug loading were 71.9% and 4.58%, respectively. Cellular uptake of ABG-PNs was controlled by specific clathrin-mediated endocytosis. In addition, ABG-PNs showed improved drug pharmacokinetics with an increased area under the curve value (a 1.73-fold increase) and a decreased elimination clearance (37.8% decrease). The nanomicelles showed increased drug concentrations in the liver and lung. In contrast, drug concentrations in both heart and brain were decreased. Moreover, the nanomicelles enhanced the anticancer effect of the pure drug probably via increased cellular uptake of drug molecules. In conclusion, the mPEG-PLGA-based nanomicelle system is a satisfactory carrier for the systemic delivery of ABG.

[Optimization preparation of chansu-loaded solid lipid nanoparticles by central composite design and response surface method].

OBJECTIVE: To optimize the formulation of chansu-loaded solid lipid nanoparticles (Cs-SLN). METHOD: Cs-SLN was prepared by cold homogenization technique. The effects of influence factors such as the weight of compritol 888 ATO, soybean lecithin and poloxamer 188, on mean diameter, entrapment efficiency, drug loading and overall desirability were investigated by using central composite design and response surface method. The data were imitated using multi-linear equation and second-order polynomial equation. RESULT: The latter was prior to the former considering from multiple correlation coefficients. Under the optimal conditions, the mean diameter, entrapment efficiency, drug loading of the Cs-SLN were 71.5 nm, 92.45% and 5.26%. CONCLUSION: The optimized preparation technique for Cs-SLN is stable, feasible and high inclusion rate. It can be used for production of Cs-SLN.

Polymeric prodrug of bufalin for increasing solubility and stability: Synthesis and anticancer study in vitro and in vivo.

Bufalin (BUF) exhibits promising potential for the treatment of various human cancers. However, its poor water solubility and unsatisfying stability in water limit its further clinical applications. In the current study, we fabricated a novel poly(ethylene glycol) (PEG)-based polymeric prodrug of BUF, PEGS-BUF, to improve its water solubility and stability at the prerequisite of maintaining its original anticancer activity. Water soluble and biocompatible PEG was firstly reacted with maleic anhydride (MAH) to afford carboxyl-terminated PEG, PEG-MAH. Then the double bond was reacted with n-propyl mercaptan via the Michael addition reaction to afford PEGS-COOH. At last, the 3α-hydroxyl group of BUF was reacted with the terminal carboxyl group of PEGS-COOH via esterification reaction to afford the final polymeric prodrug, PEGS-BUF, endowing BUF good water solubility, stability, and anticancer activity. It was demonstrated that the water solubility and stability of PEGS-BUF improved dramatically compared with that of its small molecular counterpart, BUF. Besides, both in vitro and in vivo experiments showed that PEGS-BUF exhibited comparable anticancer activity in comparison with that of free BUF.

Liver-targeting Resibufogenin-loaded poly(lactic-co-glycolic acid)-D-α-tocopheryl polyethylene glycol 1000 succinate nanoparticles for liver cancer therapy.

Liver cancer remains a major problem around the world. Resibufogenin (RBG) is a major bioactive compound that was isolated from Chansu (also called toad venom or toad poison), which is a popular traditional Chinese medicine that is obtained from the skin secretions of giant toads. RBG has strong antitumor effects, but its poor aqueous solubility and its cardiotoxicity have limited its clinical use. The aim of this study was to formulate RBG-loaded poly(lactic-co-glycolic acid) (PLGA)-D-α-tocopheryl polyethylene glycol 1000 succinate nanoparticle (RPTN) to enhance the treatment of liver cancer. RPTN, RBG-loaded PLGA nanoparticle (RPN), and RBG/coumarin-6-loaded PLGA-D-α-tocopheryl polyethylene glycol 1000 succinate nanoparticle (RCPTN) were prepared. The cellular uptake of RCPTN by HepG2 and HCa-F cells was analyzed using confocal laser scanning microscopy. Apoptosis was induced in HepG2 cells by RPTN, RBG solution (RS), and 5-fluorouracil solution (used as the negative controls), as assayed using flow cytometry. LD50 (median lethal dose) values were determined for RS and RPTN, and the liver-targeting properties were determined for RCPTN in intravenously injected mice. A pharmacokinetic study was conducted in rats, and the in vivo therapeutic effects of RPTN, RPN, and RS were examined in a mouse tumor model. The results showed that RCPTN simultaneously delivered both coumarin-6 and RBG into HepG2 and HCa-F cells. The ratio of apoptotic cells was increased in the RPTN group. The LD50 for RPTN was 2.02-fold higher than the value for RS. Compared to RS, RPTN and RPN both showed a significant difference in vivo not only in the pharmacodynamic study but also in anticancer efficacy, and RPTN performed much better than RPN. The detection indexes for drug concentration and fluorescence inversion microscopy images both demonstrated that RCPTN was much better at targeting the liver than RS. The liver-targeting RPTN, which displayed enhanced pharmacological effects and decreased toxicity for the loaded drug RBG, is therefore a promising intravenous dosage form that may be useful in the treatment of liver cancer.

Enhancement of skin permeation of bufalin by limonene via reservoir type transdermal patch: formulation design and biopharmaceutical evaluation.

A reservoir-type transdermal delivery system (TDS) of bufalin was designed and evaluated for various formulation variables like different penetration enhancers, formulation matrix, rate controlling membranes as well as biopharmaceutical characteristics. Hairless mouse skin was used in permeation experiments with Franz diffusion cells. In vitro skin permeation study showed that terpenes, especially d-limonene was the most effective enhancer when ethanol and PG were used as the vehicle with a synergistic effect. Among different rate controlling membranes, ethylene vinyl acetate (EVA) membrane containing 19% vinyl acetate demonstrated a more suitable release rate for bufalin than the other membranes. In vivo pharmacokinetic study of the bufalin patch in rat showed steady-state of bufalin from 3h to 12 h. In vivo release rate and cumulative amount analyzed by deconvolution method demonstrated the sustained release of bufalin as long as the patch remained on the animal for at least 12 h. The MRT increased from 1h of IV administration to 9h of transdermal administration. In vitro permeation across mouse skin was found to have biphasic correlation with plasma AUC in the in vivo pharmacokinetic study. Current in vitro-in vivo correlation (IVIVC) enabled the prediction of pharmacokinetic profile of bufalin from in vitro permeation results. In conclusion, current reservoir transdermal patch containing 10% D-limonene as a permeation enhancer, 40% ethanol, 30% PG and 15% carbopol-water gel complex provided an improved sustained release of bufalin through transdermal administration. The bufalin patch was successfully applied to biopharmaceutical study in rats and demonstrated the feasibility of this transdermal formulation for future development and clinical trials.

Bufalin-loaded mPEG-PLGA-PLL-cRGD nanoparticles: preparation, cellular uptake, tissue distribution, and anticancer activity.

BACKGROUND: Recent studies have shown that bufalin has a good antitumor effect but has high toxicity, poor water solubility, a short half-life, a narrow therapeutic window, and a toxic dose that is close to the therapeutic dose, which all limit its clinical application. This study aimed to determine the targeting efficacy of nanoparticles (NPs) made of methoxy polyethylene glycol (mPEG), polylactic-co-glycolic acid (PLGA), poly-L-lysine (PLL), and cyclic arginine-glycine-aspartic acid (cRGD) loaded with bufalin, ie, bufalin-loaded mPEG-PLGA-PLL-cRGD nanoparticles (BNPs), in SW620 colon cancer-bearing mice. METHODS: BNPs showed uniform size. The size, shape, zeta potential, drug loading, encapsulation efficiency, and release of these nanoparticles were studied in vitro. The tumor targeting, cellular uptake, and growth-inhibitory effect of BNPs in vivo were tested. RESULTS: BNPs were of uniform size with an average particle size of 164 ± 84 nm and zeta potential of 2.77 mV. The encapsulation efficiency was 81.7% ± 0.89%, and the drug load was 3.92% ± 0.16%. The results of in vitro cytotoxicity studies showed that although the blank NPs were nontoxic, they enhanced the cytotoxicity of bufalin in BNPs. Drug release experiments showed that the release of the drug was prolonged and sustained. The results of confocal laser scanning microscopy indicated that BNPs could effectively bind to human umbilical vein endothelial cells. In the SW620 xenograft mice model, the BNPs could effectively target the tumor in vivo. The BNPs were significantly more effective than other NPs in preventing tumor growth. CONCLUSION: BNPs had even size distribution, were stable, and had a slow-releasing and tumor-targeting effect. BNPs significantly inhibited colon cancer growth in vitro and in vivo. As a novel drug carrier system, BNPs are a potentially promising targeting treatment for colon cancer.

Improved antitumor efficacy and reduced toxicity of liposomes containing bufadienolides.

Long-circulating liposomes are used extensively nowadays for enhancing the therapeutic effect and reducing the toxicity of anticancer drugs. In this paper, a traditional Chinese medicine, toad venom, which has long been used in the clinic for tumor therapy with unpleasant side effects, was incorporated into poloxamer modified liposomes to increase its antitumor effect and reduce its toxicity. Our preparation of bufadienolides liposomes had a particle size of around 70 nm and an entrapment efficiency of about 87.6%. Lyophilized liposomes well retained their appearance, particle size and encapsulation efficiency for 3 months. The in vitro release results verified the sustained release properties of the bufadienolides liposomes. The concentration of bufadienolides in modified liposomes that caused 50% cell killing was much lower than that of free drug for both Lovo cells and NCI-H157 cells. Compared to the bufadienolides solution and the unmodified liposomes, the bufadienolides liposomes significantly prolonged the retention time and increased the area under the curve in vivo. The antitumor efficiency of the bufadienolides liposomes against mice bearing H22 liver cancer cells and Lewis pulmonary cancer cells were 2.15 and 2.96, respectively, times that of a bufadienolides solution at the same toxicity. The safety test results demonstrated that the bufadienolides liposomes had an LD(50) that was 3.5 times the LD(50) of bufadienolides solution and caused no allergen-related or blood vessel irritation effects. All these results proved that poloxamer modified bufadienolides liposomes have improved antitumor efficacy and safety.

Wheat germ agglutinin-grafted lipid nanoparticles: preparation and in vitro evaluation of the association with Caco-2 monolayers.

A bioadhesive drug delivery system, wheat germ agglutinin (WGA)-grafted lipid nanoparticles, was developed for the oral delivery of bufalin (a hydrophobic active component extracted from the traditional Chinese medicine Chan'su). The lipid nanoparticles associated with poly(vinyl alcohol) (PVA) were prepared by high-pressure homogenization. WGA was coupled to lipid nanoparticles by activating the hydroxyl group using glutaraldehyde, and then conjugating the nanoparticles with WGA. WGA-grafted lipid nanoparticles with a mean particle size of 164 nm and zeta potential of -10.6 mV were obtained with bufalin encapsulation of 68.2%. The amount of bound WGA was approximately 28.9% of the amount of WGA initially added. The association study between fluorescent 6-coumarin-loaded WGA-grafted lipid nanoparticles and Caco-2 monolayers showed that WGA enhanced the cellular uptake of nanoparticles compared with WGA-free lipid nanoparticles. These results suggest that WGA-grafted lipid nanoparticles could be a promising carrier to enhance cellular uptake. They could also improve drug bioavailability through the oral route.

The efficacy and safety of bufadienolides-loaded nanostructured lipid carriers.

Bufadienolides-loaded nanostructured lipid carriers (BU-NLC) were prepared for parenteral application using glyceryl monostearate as solid core, medium-chain triglyceride and oleic acid as liquid lipid material, and Lipoid E-80, sodium deoxycholate and pluronic F68 as stabilizers. In this study, the in vitro cytotoxicity, pharmacokinetics, biodistribution, antitumor efficacy and safety of BU-NLC were evaluated. Against human astrocytoma cell line (U87-MG) and human gastric carcinoma cell line (HGC-27) BU-NLC exhibited cytotoxicity that was similar to that of the free drug, and superior to that of the commercially available fluorouracil injection. BU-NLC exhibited a linear pharmacokinetic behavior at doses ranging from 0.25 to 1.0 mg/kg. The improved pharmacokinetic profile of bufadienolides when formulated in BU-NLC resulted in a higher plasma concentration and lower clearance after intravenous administration compared with bufadienolides solution (BU-S). A biodistribution study indicated that bufadienolides were mainly distributed in the lung, spleen, brain and kidney, and the longest retention was observed in the brain. A sarcoma-180 tumor model further confirmed the advantages of BU-NLC versus BU-S. Hemolysis and acute toxicity investigations showed that BU-NLC was safe when given by intravenous injection with reduced toxicity. In conclusion, the NLC system is a promising approach for the intravenous delivery of bufadienolides.