Synthesis and characterization of novel protein nanodots as drug delivery carriers with an enhanced biological efficacy of melatonin in breast cancer cells

Melatonin is a potent antioxidant, chemotherapeutic and chemo preventive agent against breast cancer. However, its short half-life is one of the major limitations in its application as a therapeutic drug. To overcome this issue, the green-emitting protein nanodot (PND) was synthesized by a one-step hydrothermal method for loading melatonin. The synthesized pH-7 and pH-2 PND showed a quantum yield of 22.1% and 14.0%, respectively. The physicochemical characterization of both PNDs showed similar morphological and functional activities. Furthermore, the biological efficacy of melatonin-loaded PND (MPND) was evaluated in a breast cancer cell line (MDA-MB-231) for live-cell imaging and enhanced nano-drug delivery efficacy. Interestingly, the permeability of neutral pH PND in both cell cytoplasm and nucleus nullifies the limitations of real-time live-cell imaging, and ensures nuclear drug delivery efficacy. Neutral pH PND showed better cell viability and cytotoxicity as a fluorescence bioimaging probe compared to acidic PND. The bioavailability and cell cytotoxicity effect of MPND on MDA-MB-231 breast cancer cells were studied through confocal and migration assay. Results showed that MPND causes enhanced bioavailability, better cellular uptake, and inhibition of the migration of breast cancer cells as compared to the drug alone. Besides, the synthesized MPND showed no sign of fluorescence quenching even at a high concentration of melatonin, making it an ideal nanocarrier for bioimaging and drug delivery.

A novel nanodot-using protein has been synthesized for the live cell imaging and drug delivery of melatonin in breast cancer cells. Its unique properties hold potential for various biomedical applications in the field of bioimaging and drug delivery.     

Safety and efficacy of various combinations of injectable anesthetics in BALB/c mice.

Four combinations of drugs--ketamine-xylazine, ketamine-xylazine-acepromazine (KXA), ketamine-xylazine-buprenorphine, and ketamine-xylazine-carprofen--were compared for their ability to produce anesthesia in BALB/c mice. Induction time, anesthetic duration, blood pressure, pulse rate, and time to recovery were recorded. The anesthesia induced by each anesthetic combination was assessed by using reflex responses to standardized stimuli. The KXA combination produced stable physiologic parameters and was associated with the longest duration of anesthesia (40 +/- 8 min); immobility was produced in all other groups (38 +/- 5 min), but a surgical plane of anesthesia could not be confirmed. All anesthetic protocols produced significant hypotension. No deaths occurred. We recommend KXA as a safe and reliable anesthetic for mice requiring a surgical plane of anesthesia.     

一种新型分子探针——荷瘤裸鼠体内实验显像

目的通过荷瘤裸鼠体内实验研究验证hTERT反义分子探针(99mTc-hTERT ASON)作为新型分子显像剂的可行性,探讨其在整体动物水平上的应用价值。方法以双功能螯合剂法制备放射性核素99mTc标记的反义分子探针(99mTc-hTERT ASON)。建立荷MCF-7乳腺癌动物模型,进行体内生物分布与显像实验。所有结果通过统计学软件SPSS12.0进行分析。结果99mTc-hTERT ASON标记率达到76%±5%(n=5),放射性化学纯度达到96%以上,比活度为1850kBq/μg。99mTc-hTERT ASON主要分布在肾脏与肝脏组织;99mTc-hTERT ASON的肿瘤摄取率与肿瘤/非肿瘤比值(T/NT)均高于对照组(P<0.05);6h时99mTc-hTERT ASON的肿瘤/血液比值(T/B)与肿瘤/肌肉比值(T/M)分别为2.02与8.85。在注射99mTc-hTERT ASON后4～8h,荷瘤裸鼠肿瘤部位出现明显放射性浓聚。结论99mTc-hTERT ASON能特异性地聚集于肿瘤组织中,且T/NT比值较高,有望应用于肿瘤的基因显像研究。     

Tumor localization and antitumor efficacy of novel sapphyrin compounds

Sapphyrins are pentapyrrolic metal-free expanded porphyrins with potential medical use as anticancer agents. The novel sapphyrin derivative, PCI-2050, functionalized with 2-[2-(2-methoxyethoxy)ethoxy]ethoxy groups to enhance solubility and a modified bipyrrole moiety was found to be more potent in inducing apoptosis than the previously described sapphyrin PCI-2000. Because some sapphyrins may localize to tumors, we took advantage of the intrinsic fluorescence of these compounds to develop a flow cytometry-based assay to track sapphyrin biodistribution in tumor-bearing mice. Ex vivo analysis of sapphyrin-injected animals revealed that PCI-2050 preferentially localized to tumor, whereas PCI-2000 distributed into normal tissues rather than tumor. PCI-2050 uptake in xenograft tumor cells and resultant tumor cell cytotoxicity was dose dependent. To investigate structure-activity relationships, we focused on PCI-2050 and three derivatives that differ by their alkyl substituents on the bipyrrole moiety: PCI-2051, PCI-2052, and PCI-2053. Treatment of Ramos cells in culture or treatment of Ramos xenograft-bearing animals with each of the sapphyrins followed by ex vivo growth of tumor cells revealed the same pattern of cytotoxicity: PCI-2050 > PCI-2052 > PCI-2051 > PCI-2053. Thus, subtle changes in the alkyl substituents on the bipyrrole moiety result in significant changes in antitumor activity. PCI-2050 displayed significant antitumor efficacy in both Ramos and RKO xenograft models without hematologic, hepatic, or renal abnormalities as assessed by complete blood counts and serum chemistries. On the basis of these findings, it is concluded that the sapphyrin PCI-2050 warrants further evaluation as a potential anticancer agent due to its intrinsic proapoptotic activity and tumor localization ability.     

强磁场联合化疗对S_(180)荷瘤小鼠的影响

目的　观察磁疗结合化疗药物表阿霉素对小鼠S180 肿瘤模型的治疗效果。方法　 6 0只S180 荷瘤小鼠随机分 4组 :磁疗 化疗组 ,单纯化疗组 ,单纯磁疗组和空白对照组 ,每组 15只。于接种后 2 4h分别进行各项处理 ,10d后处死动物分离瘤块 ,称湿重并作病理检查。结果　磁疗 化疗组肿瘤湿重 (0 .32± 0 .13) g ,显著低于各组 ,单纯磁疗组 (0 .95± 0 .42 )g、单纯化疗组 (0 .49± 0 .2 2 ) g亦显著低于空白对照组 (1.5 5± 0 .6 7) g。病理检查可见磁疗 化疗组肿瘤坏死最明显。 结论　磁疗结合化疗组具有显著的抑瘤效果 ,磁疗可作为肿瘤治疗的辅助手段。     

Synthesis and antitumor activity of novel chalcone derivatives

A novel series of chalcone derivatives containing pyrimidinyl group were synthesized and evaluated for their cytotoxic activities in vitro against various human cancer cell lines. Most of the prepared compounds showed potential cytotoxicity against several human cancer cell lines. The compound 5g displayed more potent cytotoxic activities against human cancer cell lines in comparison with Curcumin.     

Enhancement of the fraction of the active form of an antitumor drug topotecan via an injectable hydrogel

Poly(D,L-lactic acid-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(D,L-lactic acid-co-glycolic acid) (PLGA-PEG-PLGA) hydrogels were tried as implants to encapsulate antitumor drug topotecan (TPT), a derivative of camptothecin (CPT). Despite of water solubility of TPT, the in vitro release of this low-molecular-weight drug from hydrogels sustained for 5 days with only a mild initial burst. The antitumor efficacy of the released TPT was further validated in S180-bearing mice. Surprisingly, the fraction of the active lactone form of TPT was increased to above 50% in the hydrogel matrix, while the fraction was just about 10% in phosphate buffer saline under physiological pH at 37 °C. This significant effect was interpreted not by the local acidic pH within the hydrogel, but by the increase of pK_a of the carboxylate group of the open-ring form due to the hydrophobic interaction between the amphiphilic polymer and TPT. Theoretical analysis via a pK_a-related mechanism was also performed, which was consistent with our experimental measurements. Hence, this study has revealed that an appropriate biomaterial could, via drug-material interactions, enhance the drug efficacy by increasing the active fraction of some drugs which exhibit a reversible conversion between the active and inactive structures.     

Synthesis and characterization of novel poly(ethylene glycol)-lipid conjugates suitable for use in drug delivery.

Liposomal formulations have been used to encapsulate and deliver a wide variety of therapeutic and diagnostic agents. Their circulation can be prolonged by the addition of neutral, hydrophilic polymers such as poly(ethylene glycol) (PEG) to the outer surface. An extended circulation lifetime allows them to take advantage of the enhanced permeability and retention effect (EPR), resulting in increased delivery to target sites. Incorporation of PEG also prevents aggregation and aids in the formation of uniform, small mono-disperse particles. This is often accomplished with the use of PEG-lipid conjugates, PEG molecules with a hydrophobic domain to anchor them into the liposomal bilayer upon formulation. Here we present data showing that some commonly used PEG-lipids are chemically unstable due to the presence of carboxylic ester bonds. This instability limits their utility in aqueous environments common to many liposomal preparations. To address this problem, we designed and synthesized three alternative PEG-lipids. Using SPLP (PEG-stabilized liposomal vesicles encapsulating plasmid DNA) as a model system, we investigated the properties of the novel PEG-lipids. An accelerated stability study was conducted at 37 degrees C for 42 days to confirm chemical stability and an in vivo model was used to assess the pharmacokinetics, toxicity and activity of the SPLP. We show that the novel PEG-lipids are more stable in liposomal formulation, less toxic upon systemic administration, and accordingly, are suitable replacements for the PEG-lipids described previously.     

Synthesis of novel naphthalene-heterocycle hybrids with potent antitumor,anti-inflammatory and antituberculosis activities

Multitarget-directed drugs (hybrid drugs) constitute an efficient avenue for the treatment of multifactorial diseases. In this work, novel naphthalene hybrids with different heterocyclic scaffolds such as nicotinonitrile, pyran, pyranopyrazole, pyrazole, pyrazolopyridine, and azepine were efficiently synthesized via tandem reactions of 3-formyl-4H-benzo[h]chromen-4-one 1 with different nucleophilic reagents. Analysis of these hybrids using PASS online software indicated different predicted biological activities such as anticancer, antimicrobial, antiviral, antiprotozoal, anti-inflammatory, etc. By focusing on antitumor, anti-inflammatory, and antituberculosis activities, many compounds revealed remarkable activities. While 3c, 3e, and 3h were more potent than doxorubicin in the case of HepG-2 cell lines, 3a–e, 3i, 6, 8, 10, 11, and 12b were more potent in the case of MCF-7. Moreover, compounds 3c, 3h, 8, 10, 3d, and 12b manifested superior activity and COX-2 selectivity to the reference anti-inflammatory Celecoxib. Regarding antituberculosis activity, 3c, 3d, and 3i were found to be the most promising with MIC less than 1 μg mL⁻¹. The molecular docking studies showed strong polar and hydrophobic interactions with the novel naphthalene-heterocycle hybrids that were compatible with experimental evaluations to a great extent.

Novel naphthalene-heterocycle hybrids were synthesized via tandem reactions of 3-formylchromone with different nucleophilic reagents. Various hybrids revealed potent antitumor and anti-inflammatory as well as promising antituberculosis activities.     

Proapoptotic and antitumor activities of the HMG-CoA reductase inhibitor, lovastatin, against Dalton's lymphoma ascites tumor in mice

BACKGROUND: Diet rich in fat have a clear effect on the tumor incidence in humans. Increased level of lipid peroxidation were found in colon, liver, breast and kidney carcinogenesis. Although the beneficial effects of statins for cardiovascular diseases are well established, their importance in the area of cancer therapeutics has recently gained recognition. Many studies of lovastatin in in vitro systems and experimental animals have been reported as an effective antitumor agent. However, phase I/II clinical trials in cancer patients demonstrated a minor to non-significant responses. Hence more studies in different tumor models using doses corresponding to that used to reduce lipid in human are required to support the antitumor activity. METHODS: The antitumor activity was evaluated using Daltons' Lymphoma Ascites (DLA) cell line-induced ascites tumor model in mice. Proapoptotic activity was evaluated in DLA cell line induced ascites animals after the treatment of lovastatin. Apoptosis was analyzed morphologically by staining with Giemsa and biochemically by observing the laddering of DNA in agarose gel electrophoresis. In vitro cytotoxic activity of lovastatin was studied by trypan blue dye exclusion method. Lipid peroxidation inhibiting activity was demonstrated in Fe2+-ascorbate induced rat whole liver homogenate. RESULTS: Lovastatin dose dependently inhibited the ascites tumor growth at 4 and 16 mg/kg body wt (p.o). The percentage increase in life span (%ILS) in the 16 mg/kg treated group was 61.8% (P<0.01). Single dose of lovastatin (16 mg/kg body wt, p.o) was also effective to accelerate the apoptosis in the ascites tumor bearing mice that was evident from the multiple fragmentation of DNA in gel electrophoresis. Further the morphological analysis of DLA cells aspirated from the lovastatin treated animals showed a significant (P<0.01) increase of apoptotic cells (15.5+/-3%) than the control animals (6.5+/-1%). Concentration of lovastatin required for the 50% of the cytotoxicity was 37+/-5 microg/ml. Lovastatin at its low concentrations were effective to inhibit lipid peroxidation. CONCLUSIONS: The antitumor activity of lovastatin against the ascites tumor is due to its proapoptotic and cytotoxic activities. Lovastatin at low concentrations inhibited Fe2+ induced lipid peroxidation in in vitro system. The proapoptotic and lipid peroxidation inhibiting activities of the lipid lowering drug lovastatin may further suggest its possible therapeutic use as a cancer chemopreventive agent.     

链球菌制剂SM对荷瘤小鼠红细胞免疫功能的影响的研究

目的　探讨链球菌制剂SM对荷瘤小鼠红细胞免疫功能的影响。方法　将昆明种小鼠制成荷瘤模型 ,随机分为肿瘤治疗组和未治疗组 ,同时设健康对照 ,分别给予链球菌制剂SM或PBS治疗 ,从小鼠眼房静脉窦取血 ,采用红细胞酵母菌混合花环法 ,检测S180荷瘤小鼠红细胞C3b受体花环率 (RBC -C3bRR)、红细胞免疫复合物花环率(RBC -ICR)和直向肿瘤红细胞花环率 (RBC -TR)。结果　S180荷瘤小鼠红细胞RBC -C3bRR、RBC -ICR和RBC -TR分别为 5 .5 0± 1.6 8% ,19.38± 4 .6 5 %和 8.78± 3.82 %。经链球菌制剂SM治疗后 ,则分别为 10 .2 5± 2 .0 % ,12 .6 3± 3.79%和 14 .80± 5 .74 %。两者比较具有显著性差异。结论　链球菌制剂SM能明显提高S180荷瘤小鼠红细胞免疫功能。     

Synthesis, characterization, and tumor selectivity of a polyphosphazene-platinum(II) conjugate.

A new amphiphilic poly(organophosphazene) was synthesized by stepwise nucleophilic substitutions with a hydrophilic methoxy poly(ethylene glycol) with an average molecular weight of 350 (MPEG350) and a hydrophobic glycyl-L-glutamate as side groups, and then an antitumor (dach)platinum(II) (dach: trans-(+/-)-1,2-diaminocyclohexane) moiety was conjugated to the polymer using the dipeptide as a spacer. This polymeric platinum conjugate was found to be accumulated in the tumor tissue to a remarkably greater extent than in the normal tissue (tumor/tissue ratio >4), probably due to the excellent EPR effect and the long circulating properties of the polymer conjugate (t1/2beta=6.2 h and AUC=4020 nmol h/ml) compared with carboplatin (t1/2beta=0.42 h and AUC=120 nmol h/ml). The polymer conjugate also exhibited high in vitro cytotoxicity comparable to cisplatin against several human tumor cells tested.     

SBHL对S180荷瘤小鼠肿瘤生长及免疫功能的影响

目的探讨SBHL对肿瘤的作用效果及其抗肿瘤免疫效应机制。方法选用S180荷瘤小鼠,采用腹腔注射SBHL[103、0、50 mg/(kg.d)],连续注射10 d,观察肿瘤生长及SBHL对小鼠免疫功能的影响。结果 SBHL能显著抑制荷瘤小鼠S180肉瘤的生长。SBHL对荷瘤小鼠受抑的细胞免疫功能具有明显正向调节作用,能提高T淋巴细胞增殖及IL-2产生能力,增强NK和LAK细胞活性。结论 SBHL能明显抑制肿瘤的生长,SBHL的免疫增强效应可能是其发挥抗肿瘤作用的重要途径。     

Synthesis,characterization, and in vitro anti-cholinesterase screening of novel indole amines

The present study involved the targeted synthesis and characterization of novel indole amines with anti-acetylcholinesterase profiling. A series of proposed indole amines was virtually screened against human acetylcholinesterase. A few indole amines (23, 24, and 25) showing strong enzyme binding in the in silico studies were synthesized in the laboratory and characterized using spectroscopic (IR, UV, NMR, single crystal XRD) and spectrometric (EIMS, HR-EIMS) methods. The indole amine 23 was crystallized from EtOH and analyzed with single crystal XRD. These ligands interacted with the PAS site in the enzyme, and their binding may disrupt the activity. The in vitro acetylcholinesterase inhibition studies revealed that the IC₅₀ values for indole amines 25 and 24 (4.28 and 4.66 μM, respectively) were comparable to that of galantamine (4.15 μM) and may be studied further as cost-effective acetylcholinesterase inhibitors.

The present study involved the targeted synthesis and characterization of novel indole amines with anti-acetylcholinesterase profiling.     

Development and characterization of a novel peroral peptide drug delivery system.

Novel drug delivery systems were developed for peroral administration of peptide and protein drugs for site specific mechanical fixation at the gut wall and with specific release patterns. These so-called shuttle systems were designed by using superporous hydrogels (SPH) and SPH composite (SPHC) as the conveyor of a core which contained the model compound N-alpha-benzoyl-L-arginine ethylester (BAEE). Two different types of shuttle systems were evaluated: (a) core inside the shuttle system, and (b) core attached to the surface of shuttle system. Each of these systems was made of two parts: (1) the conveyor system made of SPHC which is used for keeping the dosage form at specific site(s) of the GI tract by mechanical interaction of the dosage form with the intestinal membranes, and (2) the core containing the active ingredient and incorporated in the conveyor system. The effect of formulation composition of the core on the release profile of BAEE was investigated by changing the type and amount of excipients in the formulations. In addition, the effect of various enteric-coat layers on the release profile and dissolving of the dosage form was investigated. The systems were also characterized for trypsin inactivation and Ca(2+) binding. The release profile of BAEE from the core formulation consisting of PEG 6000 microparticles or small tablets showed the desired burst release. When these core formulations were incorporated into the conveyor system made of SPH and SPHC, a suitable time-controlled release profile was obtained. Changing the type, concentration and thickness of the enteric-coat layer influenced the starting time of BAEE release from the dosage form, which indicates the necessary lag time for dissolving of the dosage form at any desired specific site of drug absorption in the intestine. Both SPH and SPHC were found to partly inhibit the activity of trypsin, due to two mechanisms: Ca(2+) binding and entrapment of the enzyme in these polymers. In conclusion, the presently developed delivery systems demonstrate suitable in vitro characteristics with an appropriate time-controlled release profile, making these systems very promising for effective peroral delivery of peptide and protein drugs.     

Ethosomes - novel vesicular carriers for enhanced delivery: characterization and skin penetration properties.

This work describes a novel carrier for enhanced skin delivery, the ethosomal system, which is composed of phospholipid, ethanol and water. Ethosomal systems were much more efficient at delivering a fluorescent probe to the skin in terms of quantity and depth, than either liposomes or hydroalcoholic solution. The ethosomal system dramatically enhanced the skin permeation of minoxidil in vitro compared with either ethanolic or hydroethanolic solution or phospholipid ethanolic micellar solution of minoxidil. In addition, the transdermal delivery of testosterone from an ethosomal patch was greater both in vitro and in vivo than from commercially available patches. Skin permeation of ethosomal components, ethanol and phospholipid, was demonstrated in diffusion-cell experiments. Ethosomal systems composed of soy phosphatidylcholine 2%, ethanol 30% and water were shown by electron microscopy to contain multilamellar vesicles. 31P-NMR studies confirmed the bilayer configuration of the lipids. Calorimetry and fluorescence measurements suggested that the vesicular bilayers are flexible, having a relatively low T(m) and fluorescence anisotropy compared with liposomes obtained in the absence of ethanol. Dynamic light scattering measurements indicated that ethanol imparted a negative charge to the vesicles. The average vesicle size, as measured by dynamic light scattering, was modulated by altering the ethosome composition. Experiments using fluorescent probes and ultracentrifugation showed that the ethosomes had a high entrapment capacity for molecules of various lyophilicities.