Dose response and toxicity of doxorubicin microspheres in a rat tumor model.

The therapeutic response and toxic effects of chemotherapy using several doses of doxorubicin in conventional solution form or bound to an ion-exchange resin were compared in a rat tumor model, to assess the relationship of drug dose to therapeutic efficacy and associated toxicity. Single bolus injections of 3.0, 4.5, 6.0, 7.5 and 9.0 mg/kg were administered via the abdominal aorta to rats bearing hindlimb tumors. Tumor size was measured serially and the growth rates of treated groups were compared with a control growth curve. In addition, the effect of empty microspheres on tumor growth rate was assessed. The levels of circulating white blood cells were measured and compared to control levels to provide an indication of the severity of bone marrow toxicity experienced by each form of treatment. Finally, any difference in the distribution of doxorubicin to tumor, hindlimb and cardiac tissue following administration of doxorubicin as free drug or on microspheres was ascertained. Empty ion-exchange resin exerted a small although significant detrimental effect on tumor growth which may be explained by the embolization of microspheres in the precapillary blood vessels of the tumor resulting in a transient delay in tumor growth rate. The lowest dose of doxorubicin produced a significantly better therapeutic response when administered in the free drug form, but higher doses elicited an equivalent delay in tumor growth for both drug microsphere and free drug groups in a dose-dependent manner, with the maximum anti-tumor response occurring at the highest dose. Treatment with free doxorubicin at high doses resulted in significant reductions of circulating white blood cells suggesting the occurrence of bone marrow toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

肝癌特异性多柔比星免疫磷脂纳米粒的实验研究

目的:研制肝癌特异性多柔比星免疫磷脂纳米粒。方法:先制备出多柔比星聚氰基丙烯酸正丁酯毫微粒,再采用磷脂双分子层包裹,得到多柔比星磷脂纳米粒;将抗酸性铁蛋白单克隆抗体(McAb-PAF)与其相联,最后得到肝癌特异性多柔比星免疫磷脂纳米粒。并对其制备工艺、特性、细胞毒性、体内靶向性等进行了探讨。结果:制得的多柔比星免疫磷脂纳米粒粒径为97.2nm,抗体结合率78.8％,抗体活性保持良好;多柔比星免疫磷脂纳米粒对肝癌细胞的杀伤作用明显强于游离的多柔比星和普通脂质体;对于肝癌细胞荷瘤裸鼠,免疫磷脂纳米粒组较普通脂质体组抑瘤率有显著性差异。结论:肝癌特异性多柔比星免疫磷脂纳米粒,有可能成为一种新的药物靶向载体系统。     

Effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on allogeneic bone marrow transplantation in mice.

The effects of recombinant human granulocyte colony stimulating factor (rhG-CSF) on the survival rate and hemopoiesis after allogeneic bone marrow transplantation (BMT) was examined. Specific pathogen-free (SPF) C3H mice, kept in a flexible isolator for germ-free animals, received 10Gy of total body irradiation (TBI), and an allogeneic BMT 24 hours later. The 14 day survival rate was 33%. When the mice were received rhG-CSF (30 micrograms/kg/day subcutaneously) daily for 2 weeks following BMT, the 14 day survival rate rose to 79%. The effect of rhG-CSF treatment on hemopoiesis in BMT mice resulted in higher neutrophil counts in the peripheral blood by day 14 post-BMT. Administration of rhG-CSF after allogeneic BMT benefited survival rate and neutrophil recovery without any apparent complications.     

Sex differences in the pharmacokinetics of recombinant human granulocyte colony-stimulating factor in the rat.

The pharmacokinetics of recombinant human granulocyte colony-stimulating factor (rhG-CSF) were studied in male and female rats. The serum concentration of rhG-CSF after iv and sc administration to male and female Sprague-Dawley rats at a dose of 5 and 100 micrograms/kg was investigated by a sandwich enzyme-linked immunosorbent assay. After iv administration, AUC and half-lives of rhG-CSF in female rats were smaller than those for male rats. The volume of distribution of rhG-CSF in female rats was not significantly different from that in male rats. After sc administration, AUC, mean residence time, and half-lives of elimination phase in female rats were smaller than those for male rats. The in vitro biological activities of rhG-CSF were investigated using [3H]thymidine uptake assay in cultures of bone marrow cells obtained from male and female rat femur. Female rat bone marrow cells showed a similar dose-response profile to rhG-CSF to that of male rat bone marrow cells. The effect of rhG-CSF administration in rats was a specific activity on the neutrophil lineage with an increase of neutrophils in peripheral blood. The in vivo effects of rhG-CSF after iv and sc administration to male and female rats at 5 and 100 micrograms/kg doses were determined. After 100 micrograms/kg administration, the neutrophil count in female rats was similar to that in male rats in the early period; however, the neutrophil count in female rats was lower than that in male rats 24 hr after administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro Evaluation of Doxorubicin‐Incorporated Magnetic Albumin Nanospheres

Magnetic albumin nanospheres that incorporate doxorubicin (M‐DOX‐BSA‐NPs) were prepared previously by our research group to develop magnetically responsive drug carrier system. This nanocarrier was synthesized as a drug delivery system for targeted chemotherapy. In this work, cytotoxic effects of doxorubicin (DOX)‐loaded/unloaded or magnetic/non‐magnetic nanoparticles and free DOX against PC‐3 cells and A549 cells were determined with the MTT test and the results were compared with each other. DOX‐loaded magnetic albumin nanospheres (M‐DOX‐BSA‐NPs) were found more cytotoxic than other formulations. The quantitative data obtained from flow cytometry analysis further verified the higher targeting and killing ability of M‐DOX‐BSA‐NPs than free DOX on both of the cancer cell lines. Additionally, the results of cell cycle analysis have showed that M‐DOX‐BSA‐NPs affected G1 and G2 phases. Finally, cell images were obtained using spin‐disk confocal microscopy, and cellular uptake of M‐DOX‐BSA‐NPs was visualized. The findings of this study suggest that M‐DOX‐BSA‐NPs represent a potential doxorubicin delivery system for targeted drug transport into prostate and lung cancer cells.     

Reduction of doxorubicin (adriamycin) bone marrow toxicity.

Doxorubicin (adriamycin), an antineoplastic antibiotic, is a potent suppressant of bone marrow. Previous studies on doxorubicin disposition indicated that its diversion from bone marrow in the first few minutes after administration should result in a marked decrease in total exposure to the drug (concentration X time) with a concomitant reduction in concentration-time-dependent toxicity. To test this hypothesis, the descending aorta of rabbits was occluded just proximal to the iliac bifurcation for 30 min to deprive bone marrow of blood flow. Both these rabbits and the control rabbits were given 5 mg/kg of doxorubicin intravenously, and the total white cell could in peripheral blood was monitored periodically for 15 days. The decrease in toxicity produced by the occlusion was quite evident by comparison of white cell counts and deaths in all groups. A possible mechanism of this effect was shown to be a decreased doxorubicin exposure of bone marrow tissue in the occluded animals as judged by relative doxorubicin concentration-time curves in rabbits with and without the aortic occlusion.     

骨髓靶向柔红霉素毫微粒的研究

采用乳液聚合法制备了柔红霉素聚氰基丙烯酸正丁酯毫微粒（１）,并对其形态,粒径分布,载药性,动物体内经时变化过程及骨髓靶向性进行了研究。结果表明１平均微粒径为７０ｎｍ,分布范围３０－２２０ｎｍ,包封率为９７．０％,载药一达５５．７％;小鼠胸脉注射相同剂量１及柔红霉素后,前者股骨内峰浓度提高到１．６２倍,ＡＵＣ提高到４．８７倍,总靶向效率从５．１７％提高到２４．１９％,表明１具骨髓靶向性。     

The long-circulating effect of pegylated nanoparticles revisited via simultaneous monitoring of both the drug payloads and nanocarriers

The long-circulating effect is revisited by simultaneous monitoring of the drug payloads and nanocarriers following intravenous administration of doxorubicin (DOX)-loaded methoxy polyethylene glycol-polycaprolactone (mPEG-PCL) nanoparticles. Comparison of the kinetic profiles of both DOX and nanocarriers verifies the long-circulating effect, though of limited degree, as a result of pegylation. The nanocarrier profiles display fast clearance from the blood despite dense PEG decoration; DOX is cleared faster than the nanocarriers. The nanocarriers circulate longer than DOX in the blood, suggesting possible leakage of DOX from the nanocarriers. Hepatic accumulation is the highest among all organs and tissues investigated, which however is reversely proportionate to blood circulation time. Pegylation and reduction in particle size prove to extend circulation of drug nanocarriers in the blood with simultaneous decrease in uptake by various organs of the mononuclear phagocytic system. It is concluded that the long-circulating effect of mPEG-PCL nanoparticles is reconfirmed by monitoring of either DOX or the nanocarriers, but the faster clearance of DOX suggests possible leakage of a fraction of the payloads. The findings of this study are of potential translational significance in design of nanocarriers towards optimization of both therapeutic and toxic effects.     

Metabolism of doxorubicin in long-term bone marrow cultures and SR-4987 stromal established cell line

The metabolism of doxorubicin was studied in murine long-term bone marrow cultures (LTBMC) and in SR-4987 established stromal cells in comparison with primary cultures of murine and rat hepatocytes. The toxicity of metabolites was verified by testing their effects on the clonogenicity of granulo-macrophage progenitors. Metabolic activity was compared in subcellular fractions of SR-4987 cells and murine hepatocytes. Doxorubicin was transformed in long-term bone marrow cultures, SR-4987 cells and murine/rat hepatocytes to less toxic metabolites: 13-OH doxorubicin and a less polar metabolite which were non-toxic on granulo-macrophage progenitors. Among the hemopoietic compartments, stromal cells were responsible for the biotransformation of doxorubicin. The capability of the SR-4987 established stromal cell line to metabolize doxorubicin was higher than that of primary cultures of hepatocytes and bone marrow, and the highest activity was concentrated in the microsomes. These results suggest that in vitro models using primary cell cultures and established cell lines could be a useful tool for investigating the mechanisms underlying detoxification in the bone marrow stromal population.     

Improved targeting of antimony to the bone marrow of dogs using liposomes of reduced size

A novel liposomal formulation of meglumine antimoniate (MA), consisting of vesicles of reduced size, has been evaluated in dogs with visceral leishmaniasis to determine its pharmacokinetics as well as the impact of vesicle size on the targeting of antimony to the bone marrow. Encapsulation of MA in liposomes was achieved through freeze-drying of empty liposomes in the presence of sucrose and rehydration with a solution of MA. The resulting formulation, with a mean vesicle diameter of about 400 nm, was given to mongrel dogs with visceral leishmaniasis as an i.v. bolus injection at 4.2 mgSb/kg of body weight. The pharmacokinetics of antimony were assessed in the blood and in organs of the mononuclear phagocyte system and compared to those achieved with the free drug and the drug encapsulated in large sized liposomes (mean diameter of 1200 nm). The targeting of antimony to the bone marrow was improved (approximately three-fold) with the novel liposomal formulation, when compared to the formulation of MA in large sized liposomes. This study provides the first direct experimental evidence that passive targeting of liposomes to the bone marrow of dogs is improved by the reduction of vesicle size from the micron to the nanometer scale.     

粒细胞集落刺激因子在大肠杆菌中表达的研究进展

粒细胞集落刺激因子（granulocyte colony stimulating factors,G-CSF）是存在于正常人体、刺激骨髓细胞集落形成的集落刺激因子的一种。本文综述了G-CSF的发现和临床应用概况,重点调研其表达研究进展。     

MUC1 aptamer-targeted DNA micelles for dual tumor therapy using doxorubicin and KLA peptide

Targeted delivery of DNA nanoparticles is a promising approach in cancer therapy. Using aptamers, target specific delivery of DNA nanoparticles can be achieved. Further, aptamers can indirectly improve drug encapsulation efficiency of DNA nanoparticles for drugs intercalated within nucleic acid base pairs. Using DNA blocks, a micellar hybrid nanoparticle was prepared for the targeted co-delivery of doxorubicin and a pro-apoptotic peptide, KLA to tumor cells. Results demonstrated that anti-MUC1 aptamer could specifically deliver the synthesized DNA micelle into MCF-7 cells by improving its cellular uptake. Additionally, co-delivery of doxorubicin and KLA could significantly enhance the therapeutic efficacy of the construct resulting in reduction of required dose of doxorubicin that is a pivotal point in reducing chemotherapeutics side effects. Moreover, DOX–KLA–anti-MUC1–micelle remarkably inhibited tumor growth of tumor-bearing mice when compared with free drug. DOX–KLA–anti-MUC1–micelle also reduced toxic effect of free doxorubicin as determined by percent of body weight loss and survival rate in vivo.     

Polymeric nanoparticles of cholesterol‐modified glycol chitosan for doxorubicin delivery: preparation and in‐vitro and in‐vivo characterization

Objectives Polymeric nanoparticles have been extensively studied as drug carriers. Chitosan and its derivatives have attracted significant attention in this regard but have limited application because of insolubility in biological solution. In this work, we attempted to utilize cholesterol‐modified glycol chitosan (CHGC) self‐aggregated nanoparticles to increase aqueous solubility, and to reduce side effects and enhance the antitumour efficacy of the anticancer drug doxorubicin. Methods CHGC nanoparticles were loaded with doxorubicin by a dialysis method, and their characteristics were determined by transmission electron microscopy examination, light‐scattering study, in‐vitro drug‐release study, pharmacokinetic study in rats and in‐vivo antitumour activity in mice. Key findings The resulting doxorubicin‐loaded CHGC nanoparticles (DCNs) formed self‐assembled aggregates in aqueous medium. From the observation by transmission electron microscopy, DCNs were almost spherical in shape. The mean diameters of these nanoparticles determined by dynamic light scattering were in the range of 237–336 nm as the doxorubicin‐loading content increased from 1.73% to 9.36%. In‐vitro data indicated that doxorubicin release from DCNs was much faster in phosphate‐buffered saline at pH 5.5 than at pH 6.5 and 7.4, and the release rate was dependent on the loading content of doxorubicin in these nanoparticles. It was observed that DCN‐16 (drug loaded content: 9.36%) exhibited prolonged circulation time in rat plasma and showed higher antitumour efficacy against S180‐bearing mice than free doxorubicin. Conclusions These results indicated that CHGC nanoparticles had potential as a carrier for insoluble anticancer drugs in cancer therapy.     

Prolonged Circulation of Recombinant Human Granulocyte–Colony Stimulating Factor by Covalent Linkage to Albumin Through a Heterobifunctional Polyethylene Glycol

Purpose. Recombinant human granulocyte-colony stimulating factor (rhG-CSF) was covalently conjugated to both rat and human serum albumin (RSA and HSA respectively) to increases the circulating half life (t_1/2) of rhG-CSF. Methods. Conjugates of RSA (MW 67,000) and HSA (MW 66,000) were prepared by linking the two proteins through a heterobifunctional maleimido-carboxyl polyethylene glycol (PEG) and were tested in the rat. The conjugates were injected intravenously (IV) at the equivalent dose of 50 µg/kg of rhG-CSF, and white blood cell (WBC) counts and plasma concentrations of drug were determined. A comparison of pharmacokinetic parameters was made between rhG-CSF, the conjugates RSA-PEG-rhG-CSF and HSA-PEG-rhG-CSF, and a non-covalent mixture of rhG-CSF and HSA. Results. The albumin-rhG-CSF conjugates are eliminated more slowly from the circulation. The clearance values are reduced from 0.839 ± 0.121 ml/mm/kg for rhG-CSF to 0.172 ± 0.013 ml/min/kgfor RSA-PEG-rhG-CSF and 0.141 ± 0.005 ml/mm/kg for HSA-PEG-rhG-CSF. WBC counts increased in both absolute number and duration as compared to rhG-CSF alone. The albumin rhG-CSF conjugates had enhanced serum stability relative to free rhG-CSF. The rate of degradation of the albumin conjugates incubated in rat serum at 37°C decreased five fold. Conclusions. The results from the study show that specific conjugation of rhG-CSF to albumin decreases plasma clearance in vivo, causes increased WBC response, and increases serum stability as compared to free rhG-CSF.     

Surfactant-polymer nanoparticles overcome P-glycoprotein-mediated drug efflux

Nanoparticles enhance the therapeutic efficacy of an encapsulated drug by increasing and sustaining the delivery of the drug inside the cell. We have previously demonstrated that Aerosol OT (AOT)-alginate nanoparticles, a novel formulation developed recently in our laboratory, significantly enhance the therapeutic efficacy of encapsulated drugs like doxorubicin in drug-sensitive tumor cells. The purpose of this study is to evaluate the drug delivery potential of AOT-alginate nanoparticles in drug-resistant cells overexpressing the drug efflux transporter, P-glycoprotein (P-gp). AOT-alginate nanoparticles were formulated using an emulsion-cross-linking process. Rhodamine 123 and doxorubicin were used as model P-gp substrates. Cytotoxicity of nanoparticle-encapsulated doxorubicin and kinetics of nanoparticle-mediated cellular drug delivery were evaluated in both drug-sensitive and -resistant cell lines. AOT-alginate nanoparticles enhanced the cytotoxicity of doxorubicin significantly in drug-resistant cells. The enhancement in cytotoxicity with nanoparticles was sustained over a period of 10 days. Uptake studies with rhodamine-loaded nanoparticles indicated that nanoparticles significantly increased the level of drug accumulation in resistant cells at nanoparticle doses higher than 200 microg/mL. Blank nanoparticles also improved rhodamine accumulation in drug-resistant cells in a dose-dependent manner. Nanoparticle-mediated enhancement in rhodamine accumulation was not because of membrane permeabilization. Fluorescence microscopy studies demonstrated that nanoparticle-encapsulated doxorubicin was predominantly localized in the perinuclear vesicles and to a lesser extent in the nucleus, whereas free doxorubicin accumulated mainly in peripheral endocytic vesicles. Inhibition of P-gp-mediated rhodamine efflux with AOT-alginate nanoparticles was confirmed in primary brain microvessel endothelial cells. In conclusion, an AOT-alginate nanoparticle system enhanced the cellular delivery and therapeutic efficacy of P-gp substrates in P-gp-overexpressing cells.     

The influence of recombinant human granulocyte colony-stimulating factor on granulopoiesis in mice recovering from cyclophosphamide treatment

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) was evaluated for its effects on granulopoiesis recovery in mice pretreated with cyclophosphamide. The cytotoxic agent was administered on days 0 and 7. rhG-CSF therapy (injected from days 8 to 28) accelerated the recovery and maintained an increased level of peripheral blood neutrophils. Marrow granuloid precursors depression, due the second dose of cyclophosphamide, was prevented by rhG-CSF, but this hemopoietic growth factor was unable to increase these cells in a similar way to that observed in normal mice. This suggests a decreased granuloid marrow proliferation capacity in cyclophosphamide treated mice. In contrast, in the spleen, rhG-CSF highly increased granuloid precursors. However, the contribution of spleen to granuloid recovery was scarce. Mice receiving rhG-CSF after cyclophosphamide demonstrated a biphasic recovery pattern in bone marrow GM-CFU population. A first rebound of GM-CFUs was followed by a nadir and then a second recovery where GM-CFU progenitor cells were significantly increased was observed. On the other hand, rhG-CSF therapy highly increased the spleen GM-CFU numbers at days 24 to 28. Although rhG-CSF increased splenic granulopoiesis, this result shows that the bulk of granulopoiesis recovery due to rhG-CSF therapy in cyclophosphamide pretreated mice occurred in the marrow.     

多柔比星纳米载药系统逆转肿瘤多药耐药的研究进展

多柔比星是治疗乳腺癌、肺癌的1种有效的化疗药物。现存的主要问题是临床使用时肿瘤细胞产生多药耐药。为克服多药耐药,研究者们报道了一些利用纳米载药系统传递多柔比星的方法。纳米载药系统生物相容性好,稳定性高,具有药物控释和靶向性的优点,在药物传递中应用广泛。纳米载药系统可分为无机物纳米系统、基于脂质的纳米系统和聚合物纳米系统3种类型,在多柔比星载药中均有应用。综述近年来有关纳米载药系统最新研究文献,对其研究进展作了分析,并展望了其发展前景。     

人血浆蛋白多烯紫杉醇纳米粒子的自组装及对肿瘤的靶向性

目的：提高多烯紫杉醇的溶解度,提高其靶向性能。方法用二硫键断裂法制备人血清白蛋白多烯紫杉醇纳米粒子,并用电镜进行观察。对粒子载药量、稳定性及粒子对肿瘤部位趋向性进行表征。结果二硫键断裂法成功组装了白蛋白?多烯紫杉醇纳米粒子。电镜观察该纳米粒子外貌为80～100 nm左右的球形粒子,高效液相和蛋白质定量检测多烯紫杉醇载药量可达21．5％。结果白蛋白多烯紫杉醇纳米制剂可显著提高多烯紫杉醇的溶解度,提高对肿瘤靶向性。结论白蛋白?多烯紫杉醇纳米粒子对肿瘤细胞具有良好的靶向作用,具有临床运用的潜在价值。     

Effect of cross-linking on the in vitro release kinetics of doxorubicin from gelatin implants

Doxorubicin is one of the most potent anti-tumor agents used generally in the treatment of bone cancer. Like other cancer chemotharepeutics, it produces undesirable side effects such as cardiotoxicity, which is especially severe when administrated via the conventional intravenous route. In order to minimize the systemic toxicities and to make this drug more suitable for the treatment of bone cancer, an implantable delivery system with cross-linked gelatin as the biodegradable matrix material was developed. This delivery system could possibly improve targeting of the drug as well as sustain the rate of release of the drug to the tumor. Glutaraldehyde was used as a cross-linking agent. Incorporation of glutaraldehyde in the matrix was needed to maintain the mechanical strength of the implant and to sustain the rate of release of the drug from the implant. Besides cross-linking the gelatin matrix, glutaraldehyde was found to cross-link the free amino group of doxorubicin. The effect of cross-linker concentration on the stability of the drug in the implant and on the rate and extent of release were also evaluated. In conclusion, cross-linked gelatin implants were developed for the local delivery of doxorubicin.     

Surfactant-polymer Nanoparticles: A Novel Platform for Sustained and Enhanced Cellular Delivery of Water-soluble Molecules

Purpose Nanoparticles, drug carriers in the sub-micron size range, can enhance the therapeutic efficacy of encapsulated drug by increasing and sustaining the delivery of the drug inside the cell. However, the use of nanoparticles for small molecular weight, water-soluble drugs has been limited by poor drug encapsulation efficiency and rapid release of the encapsulated drug. Here we report enhanced cellular delivery of water-soluble molecules using novel Aerosol OT™ (AOT)-alginate nanoparticles recently developed in our laboratory. Materials and Methods AOT-alginate nanoparticles were formulated using emulsion-crosslinking technology. Rhodamine and doxorubicin were used as model water-soluble molecules. Kinetics and mechanism of nanoparticle-mediated cellular drug delivery and therapeutic efficacy of nanoparticle-encapsulated doxorubicin were evaluated in two model breast cancer cell lines. Results AOT-alginate nanoparticles demonstrated sustained release of doxorubicin over a 15-day period in vitro. Cell culture studies indicated that nanoparticles enhanced the cellular delivery of rhodamine by about two–tenfold compared to drug in solution. Nanoparticle uptake into cells was dose-, time- and energy-dependent. Treatment with nanoparticles resulted in significantly higher cellular retention of drug than treatment with drug in solution. Cytotoxicity studies demonstrated that doxorubicin in nanoparticles resulted in significantly higher and more sustained cytotoxicity than drug in solution. Conclusions AOT-alginate nanoparticles significantly enhance the cellular delivery of basic, water-soluble drugs. This translates into enhanced therapeutic efficacy for drugs like doxorubicin that have intracellular site of action. Based on these results, AOT-alginate nanoparticles appear to be suitable carriers for enhanced and sustained cellular delivery of basic, water-soluble drugs.