Enhancing cytotoxicity of daunorubicin on drug-resistant leukaemia cells with microparticle-mediated drug delivery system.

Multidrug resistance is considered as a major obstacle for effective tumour chemotherapy. With the ability to deliver drugs into tumour cells, microparticles may act as a drug delivery vehicle to overcome drug resistance. In the present study, we developed an approach employing daunorubicin-loaded microparticles to surmount the drug resistance in leukaemia. The microparticles, derived from the drug-sensitive cells K562 and the drug-resistant cells K562/ADR, composed of cellular material, can effectively package drugs using intracellular and extracellular drug-loading method, respectively. The results demonstrated that the microparticles significantly improved the drug anti-tumour effect, which was influenced by the preparation methods and the source of donor cells. We further confirmed that the uptake of microparticles is mediated by an energy-driven endocytic process and mainly associated with clathrin-independent endocytosis and macropinocytosis. These results indicated that the microparticle could serve as a promising drug vehicle for the treatment of drug-resistant leukaemia.

Total Synthesis of Tanshinone I.

A novel total synthesis of tanshinone I (1) via the intermediate 3-hydroxy-8-methyl-1,4-phenanthrenedione (8) is described. The low overall yields and the use of expensive reagents in the synthesis process were minimized by the use of the Diels-Alder reaction to directly construct the 1,4-phenanthrenedione scaffold, providing tanshinone I (1) in only three steps.

[Preparation of CD123 mono-antibody modified tanshinone â ¡A loaded immunoliposome and its in vitro evaluation].

In the study, we developed a novel formulation, CD123 mono-antibody (mAb) modified tanshinone ⅡA loaded immunoliposome (CD123-TanⅡA-ILP) to achieve the targeted drug delivery for leukemia cells. Orthogonal test was used to optimize liposome preparation, and the TanⅡA-loaded PEGylated liposomes (TanⅡA-LP) of S100PC-Chol-(mPEG2000-DSPE)-TanⅡA at 19∶5∶1∶1 molar ratio were prepared by the thin film hydration-probe ultrasonic method. A post-insertion method was applied to prepare CD123-TanⅡA-ILP via thiolated mAb conjugated to the terminal of maleimide-PEG2000-DSPE. The cellular uptake assay was measured by flow cytometry, and the inhibitory effect of CD123-TanⅡA-ILP on NB4 cells proliferation was tested by using MTT assay. The results of cellular uptake assay showed that CD123-ILP could significantly increase the drug uptake of NB4 cells as compared with free drugs and LP. The IC50 values at 48 h incubation were 20.87, 11.71, 7.17 µmol•L⁻¹ respectively for TanⅡA，TanⅡA-LP and CD123-TanⅡA-ILP. CD123-ILP demonstrated a potential and promising targeted drug delivery strategy for acute myelogenous leukemia (AML) treatment.

[Preparation and antitumor effects of tanshinone â ¡A loaded albumin nanoparticles].

In this study, the tanshinone ⅡA loaded albumin nanoparticles were prepared by high pressure homogenization method. The formulation was optimized by central composite design-response surface method (CCD-RSM), with the particle size, encapsulation efficiency, and drug loading as indexes to investigate their in vitro anti-tumor effect. The results showed that the prepared nanoparticles had uniformly spherical morphology and uniform particle size distribution. The average particle size, encapsulation efficiency and drug loading of nanoparticles were about (175.7± 3.07) nm, 90.8%±1.47% and 5.52%±0.09%, respectively. Tanshinone ⅡA loaded albumin nanoparticles showed a more powerful antitumor effect than free tanshinone ⅡA for human promyelocytic leukemia NB4 cells. The preparation method of the drug-loaded albumin nanoparticles was simple and easy, and can significantly improve the solubility of tanshinone ⅡA, so it was helpful to extend its application in therapies against hematological malignancies.

Development of synthetic peptide-modified liposomes with LDL receptor targeting capacity and improved anticancer activity.

In this study, we report an active targeting liposomal formulation directed by a novel peptide (AA13) that specifically binds to the low density lipoprotein receptor (LDLR) overexpressed on acute myeloid leukemia (AML) cells. The objectives of this study were to evaluate the in vitro and in vivo tumor drug targeting delivery of AA13-anchored liposomes on AML cells. AA13 conjugated to the distal end of DSPE-PEG2000-maleimide was incorporated into the liposomes via a postinsertion method. To study the effect of the peptide decoration and density on tumor cell targeting and internalization by AML cells (THP-1 and NB4), stealth liposomes bearing 3% (peptide/S100PC, molar ratio, LL) and 7% (peptide/S100PC, molar ratio, HL) AA13 were prepared, respectively. Higher uptake of LL (1.9- and 2.6-fold) and HL (2.3- and 3.6-fold) targeted liposomes occurred in THP-1 and NB4 cells, respectively, compared to untargeted liposomes. An LDLR inhibitor was used to confirm inhibition of the receptor-mediated cellular association of AA13 modified liposome in both cells. Daunorubicin (DNR) demonstrated a 2.2- and 3.5-fold higher cytotoxicity with the HL formulation and a 1.2- and 2.0-fold higher cytotoxicity with the LL formulation compared to the unmodified liposomal formulation in THP-1 and NB4 cells, respectively. Tumor drug accumulation of DNR-loaded HL was greater than that of the untargeted liposome in the biodistribution assay. The in vivo efficacy study in BALB/c nude mice bearing NB4 xenografts treated with DNR loaded HL also showed more tumor volume inhibition and a longer survival time compared to the untargeted formulation. In conclusion, the AA13-anchored liposomes demonstrated desirable potential as a promising vector for enhanced AML tumor drug targeting.

[Quality evaluation and stability investigation of asarone submicro emulsion injection].

The content of the asarone submicro emulsion injection was determind by HPLC method, and thereby a quality evaluation method was established based on indexes of pH value, particle size, peroxide value, methoxy aniline values, free fatty acid, lysophosphatidylcholine, visible foreign substances, insoluble particle, sterility, bacterial endotoxin and impurities, etc. The results showed that the injection exhibited uniform physical appearance and all the products were in milkwhite liquid. The content of the three batches products were respectively 102.9%, 100.8%, 97.70% of the labeled amount, with mean particle size of 210-250 nm, and other indexes all met with the standards. The reserved samples showed no obvious change in terms of detection indexes and indicated good stability after the accelerated stability test and long-term stability for 12 months. The quality evaluation method established in this study could be applied to quality control and stability investigation of asarone submicron emulsion injection, which laid a basis for further clinical research and application.

[Development of biphasic drug-loading lipid emulsion of Salvia miltiorrhiza and its quality evaluation].

The feasibility of simultaneously loading both liposoluble and water-soluble components of Salvia miltiorrhiza in emulsion was discussed, in order to provide new ideas in comprehensive application of effective components in S. miltiorrhiza in terms of technology of pharmaceutics. With tanshinone II (A) and salvianolic acid B as raw materials, soybean phospholipid and poloxamer 188 as emulsifiers, and glycerin as isoosmotic regulator, the central composite design-response surface method was employed to optimize the prescription. The coarse emulsion was prepared with the high-speed shearing method and then homogenized in the high pressure homogenizer. The biphasic drug-loading intravenous emulsion was prepared to investigate its pharmaceutical properties and stability. The prepared emulsion is orange-yellow, with the average diameter of 241 nm and Zeta potential of -35.3 mV. Specifically, the drug loading capacity of tanshinone II (A) and salvianolic acid B were 0.5 g x L(-1) and 1 g x L(-1), respectively, with a good stability among long-term retention samples. According to the results, the prepared emulsion could load liposoluble tanshinone II (A) and water-soluble salvianolic acid B simultaneously, which lays a pharmaceutical foundation for giving full play to the efficacy of S. miltiorrhiza.

Formulation optimization of prostaglandin E1-loaded lipid emulsion: enhanced stability and reduced biodegradation.

The purpose of this study is to develop a new formulation for prostaglandin E1 (PGE1)-loaded lipid emulsion (Lipo-PGE1) with improved stability and reduced biodegradation. High-pressure homogenization was used to prepare the Lipo-PGE1, and high-performance liquid chromatography and accelerated test were used to evaluate its physicochemical stability. A tissue homogenate incubation test was firstly established and validated to assess its biodegradation. The factors influencing the stability of Lipo-PGE1, including oil phase, emulsifier, pH value, and drug concentration were systematically investigated. The optimized formulation consisting of poloxamer188 1.5% (w/v), egg lecithin 0.5% (w/v), soybean oil 10.0% (w/v), oleic acid 0.24% (w/v), and glycerol 2.2% (w/v), with the pH value at 4.0, was defined and characterized. When compared with the currently available commercial product of Lipo-PGE1, the degradation percentage of this optimized Lipo-PGE1 reduced by 47.1% after sterilization, the drug remaining percentage increased by 13.9% after storage at 4°C over 6 months. Also, a significant reduction in biodegradation of the optimized Lipo-PGE1 in comparison with the commercial Lipo-PGE1 was observed by a tissue homogenate incubation test. Overall, we provided a novel formulation for Lipo-PGE1 with a better physicochemical stability and a less biodegradation than the currently available commercial product of Lipo-PGE1, indicating its potential superiority in clinical application.

Growth-inhibitory and apoptosis-inducing effects of tanshinones on hematological malignancy cells and their structure-activity relationship.

This study has investigated the growth-inhibitory and apoptosis-inducing effects of dihydrotanshinone, tanshinone I, tanshinone IIA, and cryptotanshinone on hematological malignancy cell lines, aiming to explore their structure-activity relationship. The growth-inhibitory effects of the tanshinones on K562 and Raji cells were assessed using a modified MTT assay; the apoptosis-inducing effects were assessed by fluorescence microscopy and flow cytometry analysis. The changes in cellular morphology were observed using an inverted phase-contrast microscope. MTT results revealed that these tanshinones inhibited cell proliferation in a concentration-dependent and time-dependent manner. The IC50 values of dihydrotanshinone, tanshinone I, tanshinone IIA, and cryptotanshinone for K562 cells were 3.50, 13.52, 19.32, and 47.52 µmol/l at 24 h; 1.36, 4.70, 5.67, and 22.72 µmol/l at 48 h; and 1.15, 1.59, 2.82, and 19.53 µmol/l at 72 h, and the values for Raji cells were 3.30, 4.37, 12.92, and 52.36 µmol/l at 24 h; 1.55, 1.71, 6.54, and 25.45 µmol/l at 48 h; and 1.07, 1.38, 1.89, and 18.47 µmol/l at 72 h. The flow cytometry analysis demonstrated that these tanshinones induced apoptosis of K562 cells in a concentration-dependent manner, and dihydrotanshinone as well as tanshinone I were more potent than tanshinone IIA and cryptotanshinone. Some noticeable apoptotic morphologies could be observed by fluorescence microscopy on tanshinones-treated Raji cells. Collectively, these tanshinones caused growth inhibition and apoptosis in hematological malignancy cell lines, with dihydrotanshinone being the most potent, followed by tanshinone I, tanshinone IIA, and cryptotanshinone. These results suggested that the structure of aromatic ring A enhanced the cytotoxicity and the structure of ring C may have contributed to the cytotoxicity, but the mechanisms need to be further investigated.

Development of eugenol-loaded submicron emulsion and its antiepileptic effect through regulating the oxidative stress.

The purpose of this study was to develop an injectable submicron emulsion of eugenol (Eug-SE) and to investigate its antagonism on epilepsy. The formulation was optimized using a complete randomized design, comprising 5% (w/v) eugenol, 5% (w/v) soybean oil, 1.2% (w/v) egg phosphatidylcholine, 0.3% (w/v) poloxamer 188, and 0.03% (w/v) sodium oleate. The prepared Eug-SE was comprehensively evaluated in terms of its pharmaceutical characteristics, physicochemical stability, injection safety, antioxidant activity in vitro, and anti-epileptic effect in vivo. The mean particle size of Eug-SE was 176.1 ± 10.3 nm, the ζ-potential was -40.2 ± 1.8 mV, and the drug content was (95.3 ± 0.4) %. Moreover, the Eug-SE displayed excellent stability and improved safety compared to the eugenol solution. The Eug-SE (20 µg/mL) produced a significant neuroprotective effect against H2O2-induced oxidative damage in PC12 cells, which was attributed to the decrease of cellular reactive oxygen species level and mitochondrial damage. Besides, the in vivo test indicated that Eug-SE exerted an anti-epileptic effect in the PTZ treated mice. These results suggested that Eug-SE was a suitable dosage form of eugenol for injection, and displayed great therapeutic potential for neurological disease in the future.

[Comparison of kinetic behavior in both plasma and tissue after intravenous administration of alpha-asarone in lipid emulsion and aqueous solution in rats and mice].

OBJECTIVE: To compare the pharmacokinetics and tissue distribution of alpha-asarone in lipid emulsion and aqueous solution for injection and study the feasibility of lipid emulsion of alpha-asarone as the parenteral drug delivery system. METHOD: HPLC was used to determine the drug concentration in rat plasma and mice tissues after intravenous (i.v.) administration of lipid emulsion and aqueous solution of alpha-asarone at a single dose (40 mg x kg(-1)), respectively. RESULT: The plasma concentration-time profiles of lipid emulsion and aqueous solution of alpha-asarone after intravenous administration of them are similar and the drug concentration-time data were fitted to a two-compartment open model. The results of tissues distribution showed that distribution contents of alpha-asarone from lipid emulsion and aqueous solution in vivo are similar in lungs but lipid emulsion increased the uptake in livers and spleens, and decreased the uptake in hearts and kidneys for alpha-asarone. CONCLUSION: The plasma concentration-time profiles of alpha-asarone in lipid emulsion and aqueous solution are similar, but lipid emulsion significantly altered the tissue distribution of alpha-asarone, which may be beneficial to decrease its potential toxicity to heart and kidney.

[Preparation and quality evaluation of intravenous tanshinone II (A) emulsion].

OBJECTIVE: To prepare and evaluate an intravenous emulsion of tanshinone II(A). METHOD: Soybean phospholipid mixing with poloxamer 188 was used as emulsifier. Oleic acid and glycerol were used as co-emulsifier and isoosmotic adjusting agent, respectively. The coarse emulsion was first prepared and following homogenization was carried out for the coarse emulsion by using a high pressure homogenizer. RESULT: The average diameter of the prepared tanshinone II (A) emulsion was 211 nm with a zeta potential of -32. 1 mV. There had no changes of diameter, zeta potential, pH value, content and physical appearance for the tanshinone II (A) emulsion stored at 25 degrees C away from light during one year. CONCLUSION: The physicochemical properties of the prepared tanshinone II (A) emulsion was stable, which could meet the requirements of intravenous administration.

Preparation, characterization and uptake by primary cultured rat hepatocytes of liposomes surface-modified with glycyrrhetinic acid.

3-succinyl-30-stearyl glycyrrhetinic acid (Suc-GLAOSt) was synthesized as a targeting molecule, and incorporated in ordinary to liposomes (LP) to prepare a liposome surface-modified with glycyrrhetinic acid (LP-GLA), which could bind to the hepatocyte through the specific binding site of glycyrrhetinic acid (GLA) on the surface of rat cellular membrane. The maximal molar ratio of Suc-GLAOSt to total lipids in LP-GLA was 1:10. Calcein loaded liposome (Cal-LP) and calcein loaded LP-GLA (Cal-LP-GLA) were prepared by an ethanol injection method. The average diameter of Cal-LP and Cal-LP-GLA was 65 nm +/- 16 nm and 68 nm +/- 21 nm, respectively. The characteristics of cellular uptake of the two types of liposome were investigated through cellular uptake and competitive inhibition experiments. The uptake of Cal-LP-GLA by rat hepatocytes was markedly higher (3.3-fold) than that of Cal-LP (P < 0.01). The uptake of Cal-LP-GLA was inhibited, but the uptake of Cal-LP was not influenced by adding extraneous GLA. LP-GLA may be internalized by hepatocytes via the specific binding site, and can be used as a novel and promising carrier for targeting drug delivery to hepatocytes.

Anti-CD123 antibody-modified niosomes for targeted delivery of daunorubicin against acute myeloid leukemia.

A novel niosomal delivery system was designed and investigated for the targeted delivery of daunorubicin (DNR) against acute myeloid leukemia (AML). Anti-CD123 antibodies conjugated to Mal-PEG2000-DSPE were incorporated into normal niosomes (NS) via a post insertion method to afford antibody-modified niosomes (CD123-NS). Next, NS was modified with varying densities of antibody (0.5 or 2%, antibody/Span 80, molar ratio), thus providing L-CD123-NS and H-CD123-NS. We studied the effect of antibody density on the uptake efficiency of niosomes in NB4 and THP-1 cells, on which CD123 express differently. Our results demonstrate CD123-NS showed significantly higher uptake efficiency than NS in AML cells, and the uptake efficiency of CD123-NS has been ligand density-dependent. Also, AML cells preincubated with anti-CD123 antibody showed significantly reduced cellular uptake of CD123-NS compared to control. Further study on the uptake mechanism confirmed a receptor-mediated endocytic process. Daunorubicin (DNR)-loaded H-CD123-NS demonstrated a 2.45- and 3.22-fold higher cytotoxicity, compared to DNR-loaded NS in NB4 and THP-1 cells, respectively. Prolonged survival time were observed in leukemic mice treated with DNR-H-CD123-NS. Collectively, these findings support that the CD123-NS represent a promising delivery system for the treatment of AML.

[Tumor cell targetability of folate receptor-mediated mitoxantrone albumin nanoparticles].

OBJECTIVE: To study the tumor cell targetability of folate-conjugated mitoxantone-loaded albumin nanoparticles (MTO-BSANP-folate). METHODS: Bovine albumin nanoparticles were prepared by desolvation method. The activated folic acid (N-hydroxysuccinimide ester of folic acid) was conjuated to the surface of BSANP via the amino groups. The MTO-BSANP-folate was prepared by mixing folate-conjugated albumin nanoparticles with mitoxantrone and then cross-linked by glutaraldehyde. 3HTdR and flow cytometry were used to evaluate the targetability of MTO-BSANP-folate. RESULTS: The encapsulation rate of folate-conjugated mitoxantrone albumin nanoparticles was (96.55 +/- 0.96)% and the drug loading was (9.66 +/- 0.10)%. The results of 3HTdR showed that the efficacy of MTO-BSANP-folate in killing SKOV3 cells was higher than that of MTO-BSANP-folate, and the results of flow cytometry showed that the apoptosis-promoting effect of MTO-BSANP-folate was 3.5-4.5 times higher than that of MTO-BSANP. CONCLUSION: MTO-BSANP-folate could be targeted, via folate receptor, to the tumor cells rich in folate receptors.

[Studies on characteristics of absorption and separation of traditional Chinese medicine compound prescription by macroporous resin].

OBJECTIVE: Study the characteristics of absorption and separation of traditional Chinese medicine compound prescription using macroporous resin. METHOD: Study the techniquecs and characteristics of absorption and separation of a sample by macroporous resin, which is composed of coptis root, rhubarb and common anemarrhena rhizome, containing alkaloid, anthraquinone and saponin. RESULT: It is proved by qualitative and quantitative researches studies that after absorbed and separated by optimized technics process, most prime effective components or section fractions in traditional Chinese medicine compound prescription can be reserved maintained. CONCLUSION: If the techniquecs of separation is properly designed, the same kind of macropore resin can absorbd and separate various effective components or section in traditional Chinese medicine compound prescription which have with different chemical structures efficiently.

Uptake of albumin nanoparticle surface modified with glycyrrhizin by primary cultured rat hepatocytes.

AIM: To investigate the uptake difference between bovine serum albumin nanoparticle (BSA-NP) and bovine serum albumin nanoparticles with their surface modified by glycyrrhizin (BSA-NP-GL) and to develop a novel hepatocyte targeting BSA-NP-GL based on active targeting technology mediated by specific binding site of GL on rat cellular membrane. METHODS: Calcein loaded bovine serum albumin nanoparticles (Cal-BSA-NP) were prepared by desolvation process. Glycyrrhizin was conjugated to the surface reactive amino groups (SRAG) of Cal-BSA-NP by sodium periodate oxidization, which resulted in calcein-loaded bovine serum albumin nanoparticles with their surface modified by glycyrrhizin (Cal-BSA-NP-GL). The morphology of the two types of prepared nanoparticles (NP) was observed by transmission electron microscopy. The diameter of NP was measured with a laser particle size analyzer. The interaction between Cal-BSA-NP-GL and primary cultured hepatocytes was studied through cellular uptake experiments. The uptake amount of Cal-BSA-NP-GL and Cal-BSA-NP by rat hepatocytes was determined by fluorospectrophotometry. Uptake characteristics were investigated through experiments of competitive inhibition of specific binding site of GL. RESULTS: Both Cal-BSA-NP-GL and Cal-BSA-NP had regular spherical surfaces. The average diameter of Cal-BSA-NP-GL and Cal-BSA-NP was 77 and 79 nm respectively. The uptake amount of the two NP by hepatocytes reached its maximum at 2 h after incubation. The uptake amount of Cal-BSA-NP-GL by rat hepatocytes was 4.43-fold higher than that of Cal-BSA-NP. There was a significant difference in the uptake of Cal-BSA-NP-GL and Cal-BSA-NP by hepatocytes (P<0.01). The uptake of Cal-BSA-NP-GL was inhibited when GL was added previously to isolated rat hepatocytes, and the uptake of Cal-BSA-NP was not affected by GL. CONCLUSION: A binding site of GL is present on the surface of rat hepatocytes, BSA-NP-GL may be internalized via this site by hepatocytes and can be used as a drug carrier for active targeting of delivery drugs to hepatocytes.

[Preparation of bovine serum albumin nanoparticles surface-modified with glycyrrhizin].

AIM: To study the preparation of bovine serum albumin nanoparticles surface-modified with glycyrrhizin(BSA-NP-GL) targeting to hepatocytes. METHODS: The bovine serum albumin nanoparticles (BSA-NP) were prepared by desolvation process. Glycyrrhizin (GL) was oxidized by sodium periodate to be conjugated to surface reactive amino groups (SRAG) of the BSA-NP. The SRAG were quantified by spectrophotometric method using 2, 4, 6-trinitrobenzenesulfonic acid(TNBS). Glycyrrhetinic acid(GA) hydrolyzed from GL, which was on the surface of BSA-NP-GL was assayed by HPLC after isolation by sephadex G-50. Both methods were used to verify the conjugation achieved. HPLC was used to determine surface density of GL on BSA-NP-GL. RESULTS: The amount of SRAG of the BSA-NP-GL decreased by 19.6% compared with normal BSA-NP. The amount of GL molecule was 9.2% of the total determined SRAG of BSA-NP. The mean diameter of the BSA-NP-GL was 73 nm with round shape. The stability of BSA-NP-GL was constant when it was stored at 25 degrees C and 37 degrees C during 10 days. CONCLUSION: BSA-NP-GL was successfully prepared, which is considered to establish an experimental foundation for further research on its ability for targeting to hepatocytes.

[Study on the tumor cell targetability of folate-conjugated albumin nanoparticles].

OBJECTIVE: To study folate-conjugated albumin nanoparticles targeting to tumor cells via folate receptor-mediated endocytosis. METHODS: The activated folic acid (N-hydroxysuccinimide ester of folic acid) was conjuated to the surface of bovine serum albumin nanoparticles (BSANP) via the amino groups. The extent of the influence that concentration, incubation time and free folate exerted on the uptake of Folate-BSANP by ovarian cancer cells (SKOV3) was determined using fluorescence spectrophotometer. RESULTS: Folate-conjugated BSANP (Folate-BSANP) was successfully achieved. Uptake of Folate-BSANP by cancer cells was gradually increased with the extension of incubation time or the increase of Folate-BSANP concentration, and the uptake could be competitively inhibited by excess free folate. CONCLUSION: Folate-BSANP could be delivered into tumor cells via folate receptor-mediated endocytosis and significantly targeted to tumor cells with rich folate receptors.

[Preparation of liposomes surface-modified with glycyrrhetinic acid targeting to hepatocytes].

OBJECTIVE: To study the preparation of liposomes surface-modified with glycyrrhetinic acid targeting to hepatocytes. METHOD: 3-succinic-30-stearyl glycyrrhetinic acid(Suc-GAOSt), one of the amphiphilic glycyrrhetinic acid derivatives, was synthesized as targeting molecules, liposomes surface-modified with glycyrrhetinic acid has been produced with ethanol injection method. RESULT: Targeting molecules can be mixed into the liposomal membrane. It was confirmed that the targeting molecules is 9% of the total lipids at the most in the liposomes. CONCLUSION: Liposomes surface-modified with glycyrrhetinic acid was successfully prepared, which is considered to be a potential approach targeting to hepatocytes.