Curcumin-loaded γ-cyclodextrin liposomal nanoparticles as delivery vehicles for osteosarcoma

The delivery of curcumin, a broad-spectrum anticancer drug, has been explored in the form of liposomal nanoparticles to treat osteosarcoma (OS). Curcumin is water insoluble and an effective delivery route is through encapsulation in cyclodextrins followed by a second encapsulation in liposomes. Liposomal curcumin's potential was evaluated against cancer models of mesenchymal (OS) and epithelial origin (breast cancer). The resulting 2-Hydroxypropyl-γ-cyclodextrin/curcumin - liposome complex shows promising anticancer potential both in vitro and in vivo against KHOS OS cell line and MCF-7 breast cancer cell line. An interesting aspect is that liposomal curcumin initiates the caspase cascade that leads to apoptotic cell death in vitro in comparison with DMSO-curcumin induced autophagic cell death. In addition, the efficiency of the liposomal curcumin formulation was confirmed in vivo using a xenograft OS model. Curcumin-loaded γ-cyclodextrin liposomes indicate significant potential as delivery vehicles for the treatment of cancers of different tissue origin. FROM THE CLINICAL EDITOR: Curcumin-loaded γ-cyclodextrin liposomes were demonstrated in vitro to have significant potential as delivery vehicles for the treatment of cancers of mesenchymal and epithelial origin. Differences between mechanisms of cell death were also evaluated.     

Efficient intradermal delivery of superoxide dismutase using a combination of liposomes and iontophoresis for protection against UV-induced skin damage

Superoxide dismutase (SOD) is a potent antioxidant agent that protects against UV-induced skin damage. However, its high molecular weight is a significant obstacle for efficient delivery into the skin through the stratum corneum and development of antioxidant activity. Recently, we developed a non-invasive transfollicular delivery system for macromolecules using a combination of liposomes and iontophoresis, that represents promising technology for enhancing transdermal administration of charged drugs (IJP, 403, 2011, Kajimoto et al.). In this study, in rats we attempted to apply this system to intradermal delivery of SOD for preventing UV-induced skin injury. SOD encapsulating in cationic liposomes was subjected to anodal iontophoresis. After iontophoretic treatment, the liposomes were diffused widely in the viable skin layer around hair follicles. In contrast, passive diffusion failed to transport liposomes efficiently into the skin. Iontophoretic delivery of liposomes encapsulating SOD caused a marked decrease in the production of oxidative products, such as malondialdehyde, hexanoyl lysine, and 8-hydroxi-2-deoxyguanosine, in UV-irradiated skin. These findings suggested that functional SOD can be delivered into the skin using a combination of iontophoresis and a liposomal system. In conclusion, we succeeded in developing an efficient intradermal SOD delivery system, that would be useful for delivery of other macromolecules.     

Human skin sandwich for assessing shunt route penetration during passive and iontophoretic drug and liposome delivery

This work explored the role of skin appendages (shunt route) in passive and iontophoretic drug and liposome penetration. The technique used an epidermis and stratum corneum sandwich from the same skin donor with the additional stratum corneum forming the top layer of the sandwich. Penetration was monitored during occluded passive and iontophoretic (0.5 mA cm(-2)) delivery of mannitol and estradiol solutions, and ultradeformable liposomes containing estradiol. The shunt route had a significant role during passive penetration of mannitol (hydrophilic compound), but was negligible during penetration of estradiol (lipophilic drug) and liposomes. In iontophoresis, the shunt route significantly contributed to the overall flux of all preparations, being highest for mannitol. However, shunts were not the only pathway for iontophoretic drug delivery and evidence was observed for the creation of new aqueous pathways via disorganization of the intercellular lipid domain of stratum corneum. The skin sandwich technique should prove valuable for general studies on routes of skin penetration.     

Curcumin synergistically increases effects of β-interferon and retinoic acid on breast cancer cells in vitro and in vivo by up-regulation of GRIM-19 through STAT3-dependent and STAT3-independent pathways

Objective: The study aimed to investigate the effects of combination treatment of curcumin and β-interferon (IFN-β)/retinoic acid (RA) on breast cancer cells, including cell viability, apoptosis and migration, and to determine the mechanisms related to GRIM-19 through STAT3-dependent and STAT3-independent pathways.

Methods: The following groups were used for the in vitro experiment: control siRNA, GRIM-19 siRNA, IFN-β/RA and IFN-β/RA?+?curcumin. Cell viability is by the MTT method, cell apoptosis by flow cytometry and cell migration by wound healing experiment; GRIM-19, STAT3, survivin, Bcl-2, GADD153 and COX-2 expression was measured by Western blot. In vivo experiment, MCF-7 cells were subcutaneously injected into nude mice.

Results: GRIM-19 siRNA promoted MCF-7 cell proliferation and migration; inhibited cell apoptosis; and promoted the expression of STAT3, survivin, Bcl-2 and MMP-9. IFN-β/RA inhibited cell proliferation and migration; promoted cell apoptosis; up-regulated GRIM-19; and inhibited the expression of STAT3, survivin, Bcl-2 and MMP-9. Combination treatment of curcumin and IFN-β/RA had a stronger effect than that of the IFN-β/RA group. In addition, curcumin and IFN-β/RA combination inhibited the expression of COX-2 and up-regulated GADD153.

Conclusion: Curcumin synergistically increases the effects of IFN-β/RA on breast cancer cells. The mechanism may be related to the up-regulation of GRIM-19 through STAT3-dependent and STAT3-independent pathways.     

Biodistribution and improved anticancer effect of NIK-siRNA in combination with 5-FU for hepatocellular carcinoma

Increase of NF-κB inducing kinase (NIK) is known to promote the proliferation of the hepatitis B virus-derived hepatocellular carcinoma (HCC) cells. Previously, we have reported that NIK-specific siRNA in cationic liposomes was shown to suppress the expression of NIK and the proliferation of HCC cells (Cho et al., 2009). More improved suppression of NIK, followed by the improved antiproliferative effect on Hep3B cells, was achieved when 5-FU was cotreated with siRNA. Furthermore, biodistribution study after intravenous injection of siRNA into Hep3B-bearing Balb/c nude mice revealed that siRNA was highly accumulated in liver, followed by tumor, lung, spleen, kidney and heart. When encapsulated in cationic liposomes, larger amount of siRNA was found in tumor owing to the protection of siRNA from enzymatic degradation and enhanced permeability by liposome, suggesting a possible therapeutic modality of siRNA in liver-targeting cationic liposomal formulation for the treatment of hepatitis B virus-derived HCC.     

甘草次酸修饰姜黄素阳离子脂质体对肿瘤Walker256细胞的影响

目的：研究姜黄素及甘草次酸修饰姜黄素阳离子脂质体对Walker256细胞的影响。方法：不同浓度姜黄素与甘草次酸修饰姜黄素阳离子脂质体处理Walker256细胞后,采用CCK-8法检测细胞增殖抑制率;用流式细胞仪检测细胞吸收、细胞周期变化情况;Annexin V/PI双染法检测细胞凋亡;Western blot检测Wnt及β-catenin表达水平。结果：姜黄素和甘草次酸修饰姜黄素阳离子脂质体对肿瘤细胞Walker256均具有明显的抑制作用。与游离姜黄素相比,甘草次酸修饰姜黄素阳离子脂质体明显增强细胞对姜黄素的吸收,显著增强对Walker256细胞的增殖抑制、凋亡、细胞周期G2期的阻滞作用,明显下调Wnt及β-catenin的表达。结论：甘草次酸修饰姜黄素阳离子脂质体比游离姜黄素具有更强的抗肿瘤Walker256细胞的作用。     

Preparation and characterization of directly compactible layer-by-layer nanocoated cellulose

Iontophoresis is a promising technique for enhancing transdermal administration of charged drugs. However, conventional iontophoresis is not sufficient for effective delivery of large, hydrophilic, or electrically neutral molecules. In this study, we utilized charged liposomes as carriers, focused on a transfollicular route for delivery of the liposomes, and optimized iontophoretic conditions and lipid composition for this method in both in vitro and in vivo conditions. As a result, we identified the optimum condition (lipid composition: DOTAP/EPC/Chol=2:2:1, current supply: 0.45mA/cm(2), duration: 1h) for effective iontophoretic delivery of aqueous solution, which cannot be transferred into the skin without charged liposomes. We also examined the pharmacological effects of iontophoresis of liposomes encapsulating insulin (INS-lipo) using a rat model of type I diabetes. Interestingly, iontophoresis of INS-lipo onto a diabetes rat skin resulted in a gradual decrease in blood glucose levels, with levels reaching 20% of initial values at 18h after administration. These lower blood glucose levels were maintained for up to 24h. Significant amount of insulin were also detected in plasma 18h after iontophoresis of INS-lipo. We succeeded in developing a non-invasive and persistent transfollicular drug delivery system that used a combination of liposomes and iontophoresis.     

Iontophoretic estradiol skin delivery and tritium exchange in ultradeformable liposomes

This work evaluated the in vitro transdermal iontophoretic delivery of tritiated estradiol from ultradeformable liposomes compared with saturated aqueous solution (control). Effects of current density and application time on tritium exchange with water were also determined. Penetration studies used three Protocols. Protocol I involved occluded passive steady state estradiol penetration from ultradeformable liposomes and control. The effect of current densities on drug penetration rates was also assessed (Protocol II). In Protocol III, three consecutive stages of drug penetration (first passive, iontophoresis and second passive) through the same human epidermal membranes were monitored. Such an experimental design investigated the possible effect of high current density (0.8 mA/cm²) on skin integrity. The tritium exchange study showed that extent of exchange correlated well with current density and time of application, with some shielding of estradiol by the liposomal structure. Liposomes enhanced estradiol passive penetration after occlusion. Protocol II showed that estradiol flux increased linearly with current density, although being delivered against electroosmotic flow. In Protocol III, reduction in flux of the second passive stage to near that of the first reflected a reversibility of the structural changes induced in skin by current.     

Cationic Liposomal Co-delivery of Small Interfering RNA and a MEK Inhibitor for Enhanced Anticancer Efficacy

Purpose

To test whether co-delivery of anticancer small interfering RNA (siRNA) and a chemical MEK inhibitor using cationic liposomes enhances anticancer activity in vitro and in vivo.

Method

MEK inhibitor PD0325901 was encapsulated in lipid layers of N',N''-dioleylglutamide-based cationic liposomes (DGL). Mcl1-specific siRNA (siMcl1) was complexed to DGL or PD0325901-loaded liposomes (PDGL). Efficiency of cellular siRNA delivery was tested using fluorescent double-stranded RNA. Silencing of target proteins was evaluated using Western blotting and real-time quantitative polymerase chain reactions. In vivo anticancer activity was tested using xenografted mice.

Results

Size and zeta potential of PDGL were similar to DGL. PDGL could deliver double-stranded RNA into cells with efficiencies comparable to DGL. Cellular co-delivery of siMcl1 and PD0325901 reduced expression of Mcl1 and pERK1/2 proteins and more effectively reduced tumor cell survival than other treatments. In mice, siMcl1 and PD0325901 co-delivered by PDGL inhibited growth of tumors 79%. Substantial apoptosis of tumor cells was observed following PDGL-mediated co-delivery of siMcl1, but not in other groups.

Conclusions

PDGL-mediated co-delivery of siMcl1 and MEK inhibitor, PD0325901, could serve as a potential strategy for combination chemogene anticancer therapy.     

Iontophoretic transdermal delivery of haloperidol

The in vitro iontophoretic transdermal delivery of haloperidol (HP) across pig skin was investigated. Anodal iontophoresis considerably increased HP skin penetration and accumulation as compared to the passive controls.The effect of NaCl and HP concentrations on the vehicle were also studied. As expected, HP iontophoretic transport decreased with NaCl content. On the other hand, HP concentration did not modify its electrotransport in the range of concentrations between 0.4 and 0.9 mg/mL, except at 24 hours. The influence of the current density (0.20-0.50 mA/cm2) was also investigated. The iontophoretic transport of HP tends to increase with current density. On the whole, this work shows that iontophoresis may be used to improve the topical application of HP for the treatment of chronic psychosis.     

Iontophoretic Transdermal Delivery of Haloperidol

The in vitro iontophoretic transdermal delivery of haloperidol (HP) across pig skin was investigated. Anodal iontophoresis considerably increased HP skin penetration and accumulation as compared to the passive controls.

The effect of NaCl and HP concentrations on the vehicle were also studied. As expected, HP iontophoretic transport decreased with NaCl content. On the other hand, HP concentration did not modify its electrotransport in the range of concentrations between 0.4 and 0.9 mg/mL, except at 24 hours. The influence of the current density (0.20–0.50 mA/cm²) was also investigated. The iontophoretic transport of HP tends to increase with current density. On the whole, this work shows that iontophoresis may be used to improve the topical application of HP for the treatment of chronic psychosis.     

Improving the direct penetration into tissues underneath the skin with iontophoresis delivery of a ketoprofen cationic prodrug

Current topical nonsteroidal anti-inflammatory drugs (NSAIDs) showed marginal efficacy in treatment of musculoskeletal disorders due to their fast clearance by skin blood flow and thus little direct penetration into the underlying muscle and joint tissues. Using ketoprofen (Kt) as a model NSAID and converting it to a cationic ester prodrug ketoprofen choline chloride (KCC), this study was to investigate the iontophoresis delivery of the prodrug KCC for improving the drug retention in the skin and the direct penetration into underlying tissues. From in vitro flux study, anodal iontophoresis of KCC showed 5 times higher flux than cathodal iontophoresis of Kt across human epidermis skin, and also 1.5 times higher across full thickness rat skin. From in situ dual agar gel model rat study, anodal iontophoresis of KCC showed 35 times more drug penetrating across the live skin into underlying agar gel and 22 times more drug retained in the skin than those from cathodal iontophoresis of Kt. Co-iontophoresis of a vasoconstrictor phenylephrine with KCC did not show better result than the iontophoresis of KCC alone. Overall, iontophoresis delivery of the cationic prodrug KCC showed great potential for direct penetration into local tissues underneath the skin.     

Targeting of liposome-associated calcipotriol to the skin: effect of liposomal membrane fluidity and skin barrier integrity

Many dermal diseases like psoriasis are characterized by major changes in skin barrier function, which challenge the reproducible delivery of drugs into specific layers of diseased skin. The purpose of this study was to elucidate how liposomal bilayer fluidity and barrier integrity affected the delivery of liposome-associated calcipotriol to the skin. Calcipotriol-containing gel state and liquid state dipalmitoylphosphatidyl-choline:dilauroylphosphatidylcholine liposomes were prepared by extrusion. Using Langmuir monolayers, calcipotriol was shown to affect the packing of the lipid membrane. The penetration of radioactively labeled lipid and calcipotriol into pig skin was examined using the Franz diffusion cell model, and tape stripping was applied to impose an impaired barrier. Distorting the skin barrier resulted in an enhanced penetration of lipid from both gel and liquid state liposomes. In addition, increased penetration of lipid from liquid state liposomes was observed compared to gel state liposomes into barrier-impaired skin. For barrier-impaired skin, an elevated calcipotriol-to-lipid ratio was found in the receptor fluid for both liposome compositions indicating that calcipotriol is released from the vesicles. This suggests that the liposome-mediated delivery of calcipotriol to the epidermis of diseased skin is affected by the fluidity of the liposomal membrane.     

Liposomes and niosomes as potential carriers for dermal delivery of minoxidil

The aim of this work was to formulate minoxidil loaded liposome and niosome formulations to improve skin drug delivery. Multilamellar liposomes were prepared using soy phosphatidylcholine at different purity degrees (Phospholipon 90, 90% purity, soy lecithin (SL), 75% purity) and cholesterol (Chol), whereas niosomes were made with two different commercial mixtures of alkylpolyglucoside (APG) surfactants (Oramix NS10, Oramix CG110), Chol and dicetylphosphate. Minoxidil skin penetration and permeation experiments were performed in vitro using vertical diffusion Franz cells and human skin treated with either drug vesicular systems or propylene glycol-water-ethanol solution (control). Penetration of minoxidil in epidermal and dermal layers was greater with liposomes than with niosomal formulations and the control solution. These differences might be attributed to the smaller size and the greater potential targeting to skin and skin appendages of liposomal carriers, which enhanced globally the skin drug delivery. The greatest skin accumulation was always obtained with non-dialysed vesicular formulations. No permeation of minoxidil through the whole skin thickness was detected in the present study irrespective of the existence of hair follicles. Alcohol-free liposomal formulations would constitute a promising approach for the topical delivery of minoxidil in hair loss treatment.     

Topical iontophoretic delivery

At present, transdermal iontophoresis is used in the topical delivery of local anesthetics and anti-inflammatory agents. The treatment of hyperhidrosis and the diagnosis of cystic fibrosis are other clinical applications of iontophoresis. Also, a glucose-monitoring device has been developed utilizing the principle of reverse iontophoresis. Commercial iontophoretic systems that would continuously deliver therapeutic agents into the systemic circulation, corresponding to the passive transdermal patches, do not exist at the moment; however, Alza Corporation has announced that it has received an approvable letter from the US FDA regarding a new drug application for Ionsys®, an iontophoretic, fentanyl-containing, transdermal analgesic. There is currently a lot of interest in the potential of closed-loop systems, which not only sense changes in the concentration of the analyte in the skin and in the subdermal tissues, but also administer a drug in response to the fluctuating concentration/need. Thus, self-regulated or patient-regulated systems that allow medication to be administered at home would enable controlled therapy, while accounting for the individual needs of the patient. Predictable and controlled non-invasive drug delivery on the one hand, and putative adverse effects on the other, determine the success of topical iontophoretic systems and methods. The often unavoidable skin sensitization/irritation and other adverse reactions have to be related to the therapeutic benefit(s) of (bio)molecule administration; for example, the potential for skin irritation associated with pain control medication is quite different (in terms of acceptability) to skin irritation associated with cancer treatment. An additional obstacle in the path to successful transdermal delivery is the stability issue of the (bio)molecule in the drug delivery system, skin, and target tissue.     

Studies on curcumin and curcuminoids. XXIII: Effects of curcumin on liposomal lipid peroxidation

The potential of curcumin as a photosensitzer, radical scavenger and iron-chelating agent within an artificial membrane (i.e., liposome) is discussed. Curcumin is found to have a dual effect on liposome stability, manifested as peroxidation of phospholipids in the membrane. Curcumin incorporated in the liposomes acts as a chelating agent of ferric and ferrous ions in the membrane.     

Targeted Delivery of Curcumin to Tumors via PEG-Derivatized FTS-Based Micellar System

Curcumin and S-trans, trans-farnesylthiosalicylic acid (FTS) are two promising anticancer agents. In this study, we demonstrated that the two agents exerted significant synergy in antitumor activity in various types of cancer cells with combination indices ranging from 0.46 to 0.98 (a value of less than unity indicates synergism). We have further shown that synergistic-targeted co-delivery of the two agents can be achieved via formulating curcumin in polyethylene glycol (PEG)-derivatized FTS-based nanomicellar system. Curcumin formulated in PEG-FTS micelles had small size of around 20 nm. The nanomicellar curcumin demonstrated enhanced cytotoxicity towards several cancer cell lines in vitro. Intravenous application of curcumin-loaded micelle (20 mg kg⁻¹ curcumin) led to a significantly more effective inhibition of tumor growth in a syngeneic mouse breast cancer model (4T1.2) than curcumin formulated in Cremophor/EL (P < 0.05).

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-014-9595-6) contains supplementary material, which is available to authorized users.KEY WORDS: curcumin, dual–functional carrier, micelles, synergy, S-trans, trans-farnesylthiosalicylic acid     

Native and β-cyclodextrin-enclosed curcumin: entrapment within liposomes and their in vitro cytotoxicity in lung and colon cancer

With a view to improving the solubility and delivery characteristics of poorly water-soluble drugs, we prepared β-cyclodextrin-curcumin (βCD-C) inclusion complexes (hydrophilic curcumin) and entrapped both native curcumin (hydrophobic) and the complexes separately into liposomes; these were then assessed for in vitro cytotoxicity in lung and colon cancer cell lines. Optimization of curcumin entrapment within βCD was achieved, with the resultant βCD-C complexes prepared by methanol reflux. Inclusion complexes were confirmed using UV spectroscopy, Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction. The water solubility of βCD-C complexes improved markedly (c.f. native curcumin) and successful entrapment of complexes into liposomes, prepared using a thin-film hydration approach, was also achieved. All the liposomal formulations were characterized for curcumin and βCD-C complex entrapment efficiency, particle size, polydispersity and stability at 2–8°C. Curcumin, βCD-C complex and their optimized liposomal formulations were evaluated for anticancer activity in lung (A-459) and colon (SW-620) cancer cell lines. All curcumin-containing formulations tested were effective in inhibiting cell proliferation, as determined via an MTT assay. The median effective dose (EC₅₀) for all curcumin formulations was found to be in the low µM range for both lung and colon cancer cell lines tested. Our results confirm that βCD inclusion complexes of poorly water soluble drugs, such as curcumin can be entrapped within biocompatible vesicles such as liposomes, and this does not preclude their anticancer activity.     

可共载基因与敏化剂用以增强多西他赛抗肿瘤效果的脂质纳米载药系统的制备与评价

本研究制备了一种纳米载药系统,通过对几种化学或基因药物的同时递送,实现多种药物的协同效应或降低肿瘤对药物的耐药性,从而达到提高肿瘤治疗的目的。在本研究中,通过考察粒径分布及载药量对不同的阳性脂材进行筛选,成功制备可以同时传递阴离子小干扰RNA（siRNA）和化疗药物多西他赛的阳性纳米脂质载体（cNLC）。同时,利用能与肝癌细胞表面特异性结合的新型肽SP94对cNLC的表面进行修饰,最终制得具有主动靶向功能的cNLC。琼脂糖凝胶色谱结果显示制得的cNLC可以有效的装载siRNA。超滤离心法去除游离药物后,HPLC图谱显示cNLC能够高效的包载多西他赛。与市售制剂泰索帝相比,制得的SP94-cNLC显示出更强的细胞毒性,这表明SP94修饰的cNLC能更高效的递送多西他赛进入肝癌细胞。而在姜黄素和多西他赛共载的NLC中,通过姜黄素对多西他赛的敏化作用提高多西他赛在侵袭性癌细胞中的细胞毒性,这种共载系统能够改善化疗药物单独使用时疗效较差的缺点。     

塞来昔布脂质体凝胶的研制及其体外经皮渗透动力学考察

目的研制塞来昔布脂质体凝胶,并对其体外经皮渗透动力学进行考察。方法采用薄膜分散法制备塞来昔布脂质体,均匀设计筛选最佳处方及制备工艺,并以卡波姆940为基质制成脂质体凝胶;用Franz扩散池研究塞来昔布脂质体凝胶与塞来昔布普通凝胶的经皮渗透规律。结果塞来昔布脂质体凝胶的平均粒径为(369.5±10.8)nm,平均包封率为(81.6±2.2)%(n=3);体外透皮试验表明塞来昔布脂质体凝胶的累积透过量显著大于普通凝胶(P<0.05),药物透皮速率与皮肤蓄积量显著大于普通凝胶(P<0.01)。结论塞来昔布脂质体凝胶制备简单,能促进药物透皮吸收,值得进一步研究。