Ultrasound-mediated destabilization and drug release from liposomes comprising dioleoylphosphatidylethanolamine

Novel sonosensitive doxorubicin-containing liposomes comprising dioleoylphosphatidylethanolamine (DOPE) as the main lipid constituent were developed and characterized in terms of ultrasound-mediated drug release in vitro. The liposome formulation showed high sonosensitivity; where approximately 95% doxorubicin was released from liposomes after 6 min of 40 kHz US exposure in buffered sucrose solution. This represented a 30% increase in release extent in absolute terms compared to liposomes comprising the saturated lipid analogue distearoylphosphatidylethanolamine (DSPE), and a 9-fold improvement in release extent when compared to standard pegylated liposomal doxorubicin, respectively. Ultrasound release experiments in the presence of serum showed a significantly reduction in sonosensitivity of DSPE-based liposomes, whilst the release properties of DOPE-based liposomes were essentially maintained. Dynamic light scattering measurements and cryo-transmission electron microscopy of DOPE-based liposomes after ultrasound treatment indicated liposome disruption and formation of various lipid structures, corroborating the high release extent. The results point to the potential of DOPE-based liposomes as a new class of drug carriers for ultrasound-mediated drug delivery.     

Self-Assembled Methoxy Poly(Ethylene Glycol)-Cholesterol Micelles for Hydrophobic Drug Delivery

To promote the application of methoxy poly(ethylene glycol)-cholesterol (mPEG—Chol), mPEG–Chol was used to prepare core-shell micelles encapsulating poorly water-soluble docetaxel (DTX-PM) by modified cosolvent evaporation method. Approaches to enhance DTX entrapment efficiency (EE) and minimize particle size were investigated in detail, including organic and aqueous phase composition, organic/aqueous phase ratio, and polymer concentration. In optimal formulation, micelles had higher EE (97.6%) and drug loading (4.76%) with the diameter of 13.76 ± 0.68 nm and polydispersity index of 0.213 ± 0.006. Transmission electron microscopy (TEM) showed that the micelles were spherical, and differential scanning calorimetry (DSC) analysis proved that DTX was successfully entrapped into mPEG–Chol micelles. The in vitro cytotoxicity experiments displayed that blank micelles had no effect on the growth of SKOV-3, BXPC-3, A549, and HepG-2 cells, demonstrating that mPEG–Chol was one of the biocompatible biomaterials. The half inhibition concentration of DTX-PM on SKOV-3, BXPC-3, A549, and HepG-2 cells were 10.08, 7.6, 28.37, and 125.75 ng/mL, respectively. DTX-PM had the similar antitumor activity to free DTX, indicating that mPEG–Chol was a promising micellar vector for hydrophobic drug delivery. In addition, this work provided a new and facile approach to prepare drug-loaded micelles with controllable performances. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1054–1062, 2013     

Physicochemical properties and antioxidant activity of gamma-oryzanol-loaded liposome formulations for topical use

The objective of this study is to prepare the γ-oryzanol-loaded liposomes and investigate their physicochemical properties and antioxidant activity intended for cosmetic applications. Liposomes, Composing phosphatidylCholine (PC) and Cholesterol (Chol), CHAPS or sodium taurocholate (NaTC) were prepared by sonication method. γ-oryzanol-loaded liposomes were prepared by using 3, 5 and 10% γ-oryzanol as an initial concentration. The formulation factors in a particular type and composition of lipid and initial drug loading on the physicochemical properties (i.e., particle size, zeta potential, entrapment efficiency, drug release) and antioxidant activity were studied. The particle sizes of bare liposomes were in nanometer range. The γ-oryzanol-loaded liposomes in formulations of PC/CHAPS and PC/NaTC liposomes were smaller than PC/Chol liposomes. The incorporation efficiency of 10% γ-oryzanol-loaded PC/Chol liposomes was less than γ-oryzanol-loaded PC/CHAPS liposomes and PC/NaTC liposomes allowing higher in vitro release rate due to higher free γ-oryzanol in buffer solution. The antioxidant activity of γ-oryzanol-loaded liposomes was not different from pure γ-oryzanol. Both γ-oryzanol-loaded PC/CHAPS liposomes and PC/NaTC liposomes were showed to enhance the antioxidant activity in NHF cells. γ-oryzanol-loaded PC/Chol liposomes demonstrated the lowest cytotoxicity in NHF cells. It was conceivably concluded that liposomes prepared in this study are suitable for γ-oryzanol incorporation without loss of antioxidant activity.     

Novel folated and non-folated pullulan bioconjugates for anticancer drug delivery

Two new anticancer polymer therapeutics were designed for tumour cell targeting. The bioconjugates were synthesised by pullulan derivatisation with either doxorubicin or doxorubicin and folic acid. Pullulan was activated by periodate oxidation and functionalised by reductive conjugation with cysteamine and 1.9 kDa PEG(NH₂)₂. The cysteamine thiol groups were conjugated to doxorubicin through a pH-sensitive hydrazone spacer while the pending PEG-NH₂ functions of one derivatised pullulan batch were conjugated to folic acid to obtain one of the two polymer therapeutics. The reaction intermediates and the final products were characterised by mass spectrometry, UV–vis analysis and reverse phase and gel permeation chromatography. The folic acid-free derivative [(NH₂ PEG)-Pull-(Cyst-Dox)] contained 6.3% (w/w) doxorubicin while the folic acid-doxorubicin-coupled derivative [(FA-PEG)-Pull-(Cyst-Dox)] contained 6% (w/w) doxorubicin and 4.3% (w/w) folic acid. Photon correlation spectroscopy showed that (NH₂ PEG)-Pull-(Cyst-Dox) and (FA-PEG)-Pull-(Cyst-Dox) assembled into particles of about 150 and 100 nm diameter, respectively. The two bioconjugates displayed similar drug release profiles either at pH 7.4 buffer or in plasma, where less than 20% of doxorubicin was released within three days. At pH 5.5, both conjugates underwent complete drug release in about 40 h. In vitro studies carried out with KB tumour cells over-expressing folic acid receptor showed that both free doxorubicin and (FA-PEG)-Pull-(Cyst-Dox) were rapidly taken up by the cells, while the internalisation of the non-folated derivative was significantly slower. Cell viability studies did not show relevant difference between the two bioconjugates. After 72 h of incubation with folic acid receptor non-expressing MCF7 cells, the IC₅₀ values of doxorubicin, (NH₂PEG)-Pull-(Cyst-Dox) and (FA-PEG)-Pull-(Cyst-Dox) were 0.3 μM, 1.2 μM and 3.1 μM, respectively. After incubation with KB cells over-expressing folic acid receptor, the IC₅₀ values were 0.4 μM, 1.8 μM and 1.1 μM, respectively. Pharmacokinetic studies showed that 4 h after intravenous administration of the conjugates to Balb/c mice about 40% of the administered drug equivalent dose was present in the bloodstream while in the case of unconjugated doxorubicin, 80% of the drug was cleared within 30 min.These findings suggest that the novel doxorubicin–pullulan bioconjugates possess suitable properties for passive tumour targeting. On the other hand, folic acid conjugation has been found to have limited effect on selective cell up-take.     

Stability of liposomal formulations in physiological conditions for oral drug delivery

The stability of liposomal formulations is a key issue in drug delivery. Liposomes made of egg phosphatidylcholine (EPC), cholesterol (Chol), sphingomyelin (SM), and gangliosides (GM1 and GM type III) were incubated in different media to determine their stability. Mixtures containing GM1 or GM type III were found to be the most stable, and both showed similar stability trends in plasma at 37°C. EPC/Chol was the most susceptible to lysis in plasma. In acid media (pH 2), the highest stability corresponded to EPC/Chol, whereas in bile and pancreatin, liposomes with GM1 and GM type III were more stable than those containing SM. This study suggests that among the formulations used as oral drug carriers, those containing GM1 and GM type III have higher possibilities of surviving through the gastrointestinal tract.     

Stability of Liposomal Formulations in Physiological Conditions for Oral Drug Delivery

The stability of liposomal formulations is a key issue in drug delivery. Liposomes made of egg phosphatidylcholine (EPC), cholesterol (Chol), sphingomyelin (SM), and gangliosides (GM1 and GM type III) were incubated in different media to determine their stability. Mixtures containing GM1 or GM type III were found to be the most stable, and both showed similar stability trends in plasma at 37°C. EPC/Chol was the most susceptible to lysis in plasma. In acid media (pH 2), the highest stability corresponded to EPC/Chol, whereas in bile and pancreatin, liposomes with GM1 and GM type III were more stable than those containing SM. This study suggests that among the formulations used as oral drug carriers, those containing GM1 and GM type III have higher possibilities of surviving through the gastrointestinal tract.     

联合包载Combretastatin A-4和阿霉素的长循环脂质体的体外体内初步研究

目的制备一种具有程序释药功能的联合包载抗肿瘤药物阿霉素(doxorubicin,DOX)和新生血管抑制剂(combretastatin A-4,CA-4)的新型长循环脂质体并进行体内药动学评价。方法以卵磷脂、胆固醇和聚乙二醇磷脂衍生物(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-polyethylene gly-col,DSPE-PEG)为载体材料,采用薄膜分散法和硫酸铵梯度法分步包载CA-4和DOX制备得到脂质体。通过单因素考察,筛选最佳处方和制备工艺,并评价其体外释放情况。采用正常大鼠考察该制剂在静脉注射后的药动学行为。结果 m(卵磷脂)∶m(胆固醇)∶m(DSPE-PEG)为85∶10∶5、m(药)∶m(脂材)为1∶20、水化脂材浓度为50 mmol.L-1时,CA-4的包封率最高(>85%),DOX的包封率也在95%以上,平均粒径小于80 nm。体外释放结果表明联合包载DOX和CA-4的脂质体在pH7.4磷酸盐介质中可以快速释放CA-4,而DOX释放相对较慢。体内药动学实验结果表明,脂质体包载可明显增加CA-4的体内循环时间,但CA-4的联用并未对DOX体内药动学行为产生明显干扰。结论联合包载CA-4和DOX的脂质体可有效地实现程序释药,并延长药物体内循环时间,将可能成为肿瘤治疗的新策略。     

Surface functionalization of doxorubicin-loaded liposomes with octa-arginine for enhanced anticancer activity

Doxorubicin-loaded PEGylated liposomes (commercially available as DOXIL® or Lipodox®) were surface functionalized with a cell-penetrating peptide, octa-arginine (R8). For this purpose, R8-peptide was conjugated to the polyethylene glycol–dioleoyl phosphatidylethanolamine (PEG–DOPE) amphiphilic co-polymer. The resultant R8–PEG–PE conjugate was introduced into the lipid bilayer of liposomes at 2 mol% of total lipid amount via spontaneous micelle-transfer technique. The liposomal modification did not alter the particle size distribution, as measured by Particle Size Analyzer and transmission electron microscopy (TEM). However, surface-associated cationic peptide increased zeta potential of the modified liposomes. R8-functionalized liposomes (R8-Dox-L) markedly increased the intracellular and intratumoral delivery of doxorubicin as measured by flow cytometry and visualizing by confocal laser scanning microscopy (CLSM) compared to unmodified Doxorubicin-loaded PEGylated liposomes (Dox-L). R8-Dox-L delivered loaded Doxorubicin to the nucleus, being released from the endosomes at higher efficiency compared to unmodified liposomes, which had marked entrapment in the endosomes at tested time point of 1 h. The significantly higher accumulation of loaded drug to its site of action for R8-Dox-L resulted in improved cytotoxic activity in vitro (cell viability of 58.5 ± 7% for R8-Dox-L compared to 90.6 ± 2% for Dox-L at Dox dose of 50 μg/mL for 4 h followed by 24 h incubation) and enhanced suppression of tumor growth (348 ± 53 mm³ for R8-Dox-L, compared to 504 ± 54 mm³ for Dox-L treatment) in vivo compared to Dox-L. R8-modification has the potential for broadening the therapeutic window of pegylated liposomal doxorubicin treatment, which could lead to lower non-specific toxicity.     

Folate-decorated maleilated pullulan–doxorubicin conjugate for active tumor-targeted drug delivery

A novel folate-decorated maleilated pullulan–doxorubicin conjugate (abbreviated as FA–MP–DOX) for active tumor targeting was set up. The structure of this conjugate was confirmed by ¹H NMR analysis. Furthermore, the conjugation efficiency, drug release property and stability of the conjugate were determined. The cellular uptake and cytotoxicity were assessed by using ovarian carcinoma A2780 cells as in vitro cell model. In vitro DOX release from FA–MP–DOX conjugate occurred at a faster rate at acidic pH compared to neutral pH (7.4). After 30 h of incubation at pH 2.5, 5.0 and 7.4 the released free DOX was about 68.71%, 50.08% and 26%, respectively. Based on the IC₅₀ values, the conjugate was found more effective with ovarian carcinoma A2780 cells than the parent drug after 48 h culture. These results suggested that FA–MP–DOX conjugate could be a promising doxorubicin carrier for its targeted and intracellular delivery.     

Recombinant human tumor necrosis factor-α covalently conjugated to long-circulating liposomes

Recombinant tumor necrosis factor-α (rHuTNF) was covalently conjugated to a phospholipid, N-glutaryl phosphatidylethanolamine (NGPE). The resultant rHuTNF-NGPE conjugates were incorporated into liposomes composed of phosphatidylcholine (PC) and cholesterol (Chol) with or without polyethyleneglycol conjugated to phosphatidylethanolamine (PEG3000-PE). Efficient incorporation (35–50%) of rHuTNF-NGPE conjugates into liposomes was obtained for both PC/Chol and PC/Chol/PEG3000-PE liposomes. An in vitro cytotoxicity assay showed that rHuTNF-NGPE conjugates incorporated into liposomes exhibit a reduced biological activity as compared to the free rHuTNF. Biodistribution studies using ¹²⁵I-labeled rHuTNF showed a significant increase in the circulation time of rHuTNF by incorporation into PC/Chol/PEG3000-PE liposomes, but not conventional PC/Chol liposomes. However, studies using a radioactive lipid as a liposome marker showed that incorporation of rHuTNF-NGPE conjugates resulted in increased clearance from the blood and accumulation in the spleen and liver of both liposomal formulations. The liposome clearance from the blood depends on the protein/lipid ratio of liposomes. The higher the protein/lipid ratio, the higher the liposome clearance from the blood and accumulation in the spleen and liver, suggesting that accumulation of rHuTNF-bound liposomes in the spleen and liver involves interactions with TNF-receptors in these organs.     

Optimization and characterization of a sphingomyelin/cholesterol liposome formulation of vinorelbine with promising antitumor activity

Vinorelbine (VRL) is a particularly lipophilic member of the vinca alkaloids which, as a class of drugs, exhibit improved cytotoxicity and therapeutic activity through increased duration of exposure. Here, we describe and optimize a sphingomyelin/cholesterol (SM/Chol) liposome formulation of VRL to maximize in vivo drug retention, plasma circulation time, and therapeutic activity. VRL was efficiently encapsulated (>90%) into 100 nm liposomes using an ionophore-mediated loading method. VRL retention in SM/Chol liposomes after intravenous injection in mice was dependent on drug-to-lipid ratio (D/L), with higher D/L ratios exhibiting increased drug retention (0.3 > 0.2 > 0.1, wt/wt) and improved pharmacokinetics. Cryo-electron microscopic examination of a high D/L ratio formulation indicated that the intravesicular regions of these liposomes were electron dense compared with empty liposomes. The optimized, high D/L ratio SM/Chol VRL formulation showed promising activity against subcutaneous B16 melanoma tumors compared with VRL or SM/Chol formulations of vincristine or vinblastine. Finally, the stability of the formulation was excellent (<5% drug leakage, >99% intact VRL, no changes in liposome size after 1 year at 2-8 degrees C). The optimized drug retention properties of the SM/Chol formulation of VRL, combined with its promising antitumor activity and pharmaceutical stability, make this formulation an excellent candidate for future clinical development.     

Effect of liposome size on peritoneal retention and organ distribution after intraperitoneal injection in mice

Peritoneal carcinomatosis is a serious concern when treating digestive or ovarian tumors. Treatment with systemic chemotherapy suffers from poor penetration of cytotoxic agents into the peritoneal cavity and is not quite effective. Local delivery of drugs, especially as controlled-release delivery systems like liposomes, could provide sustained and higher drug levels and reduce systemic toxicity.In order to investigate the effect of liposome size on peritoneal retention, liposomes composed of distearoylphosphatidylcholine and cholesterol (DSPC/CHOL, molar ratio 2:1) were prepared at four sizes of 100, 400, 1000 and 3000 nm. Subsequently, these liposomes were labeled with ^99mTc complex of hexamethylpropyleneamineoxime (^99mTc-HMPAO) and injected into mouse peritoneum. Then, mice were sacrificed at eight different time points and the percentage of injected radiolabel in the peritoneal cavity and the organ distribution in terms of percentage injected dose/gram tissue (%ID/g) were obtained.Results showed that the free label (^99mTc-HMPAO) was cleared very rapidly from the cavity so that after 5 min and 7 h only 6.89 ± 2.51% and 0.91 ± 0.51% of the injected dose was recovered, respectively. However, for the liposomal formulations, this recovery value ranged from 8.47 ± 1.62% to 29.99 ± 12.06% at 7 h. Peritoneal retention of the vesicles was increased with their size, and the highest retention rate was obtained with 1000 nm liposomes with an AUC value 15.51 times that of ^99mTc-HMPAO. In blood, as expected, 100 nm liposomes showed much higher levels because of their greater stability. Their greater blood concentration also caused increased levels in the heart and kidneys, although their organ to blood AUC ratio was the lowest.Overall, among the different sized neutral liposomes investigated, the 1000 nm vesicles seemed to be the most optimal, achieving a greater peritoneal level and retention.     

Dendronized nanoconjugates of lysine and folate for treatment of cancer

Poly-l-lysine (PLL) dendrimers are currently being investigated as antiangiogenic agent for therapy of cancer. In this study, we report folate conjugated poly-l-lysine dendrimers (FPLL) as an efficient carrier for model anticancer drug, doxorubicin hydrochloride (Dox); for pH sensitive drug release, selective targeting to cancer cells, anticancer activity and antiangiogenic activity. This nanoconjugate of Dox showed initial rapid in vitro release followed by gradual slow release, and the drug release was found to be pH sensitive with greater release at acidic pH. In the CAM assay and tubule formation assay with HUVEC, Dox-FPLL formulation showed the significant antiangiogenic activity confirming that activity of PLL was not compromised by the presence of Dox and folic acid. The ex vivo investigations with human breast cancer cell lines MCF-7 showed enhanced cytotoxicity of Dox-FPLL with significantly enhanced intracellular uptake (p < 0.001). The in vivo therapeutic potential of nanoconjugate was determined in MCF-7 breast cancer xenograft model in tumor-bearing mice. Dox-FPLL increased the concentration of Dox in tumor by 121.5-fold after 24 h in comparison with free Dox formulation. The folate conjugated dendrimeric Dox showed superior anti-tumor activity in tumor xenograft model with significantly prolonged survival determined by Kaplan Meier survival analysis (p < 0.001).     

PEGylated estradiol benzoate liposomes as a potential local vascular delivery system for treatment of restenosis

This study was directed towards the preparation and optimization of PEGylated (PEG, poly(ethylene glycol)) estradiol benzoate (ESB)-loaded liposomes to be used for the treatment of restenosis by local vascular delivery. Various liposomal formulations were prepared by thin film hydration method followed by sonication. Response surface methodology was applied to study the influence of three different independent variables, on the response of entrapment efficiency (%EE). Liposomes were characterized in terms of size, zeta potential, %EE and release profile. Incorporation of ESB was higher in egg phosphatidylcholine (EPC) liposomes, whereas the drug was displaced from liposomes, as the cholesterol (Chol) content of liposome increased. The optimum formulation composed of EPC/dioleyloxy trimethylammonium propane/distearoylphosphatidylethanolamine-PEG2000 with a molar proportion of 8.5:1:0.5 had the highest EE. In?vivo studies in the balloon-injured rat carotid arteries revealed the potential of ESB-loaded liposomes as efficient local and controlled drug delivery systems to reduce restenosis.     

Novel long-circulating liposomes containing peptide library-lipid conjugates: synthesis and in vivo behavior

Rapid uptake of intravenously injected liposomes by the mononuclear phagocyte system has limited their use as drug delivery vehicles. Recently, various long-circulating liposomes have been prepared by incorporating glycolipids or other amphiphilic molecules into the lipid bilayer of conventional liposomes. The purpose of the present study was to design a new class of biodegradable membrane modifiers that would increase the half-life of liposomes in vivo. Using solid-phase peptide synthesis, synthesized were 30-residue random libraries consisting of a random sequence of glycine, beta-alanine and gamma-aminobutyric acid. The libraries were coupled to stearic acid (SA) or phosphatidylethanolamine (PE). The resulting amphiphilic conjugates were mixed with egg phosphatidylcholine (PC) and cholesterol (Chol) in a 6:47:47 ratio, and unilamellar liposomes were prepared. For comparison, plain PC/Chol (50:50) liposomes, as well as liposomes containing polyethylene glycol (PEG)-SA/PC/Chol (6:47:47) and PEG-PE/PC/Chol (6:47:47) were also prepared. Calcein was entrapped in the liposomes, which were given intravenously to rats at a dose of 9.2 mumol lipid/kg, and the amount of intact liposomes present in serum was followed with time. While the conventional liposomes had a short elimination half-life (28 min), the liposomes modified with library-PE had a much longer half-life (170 min), while library-SA provided no improvement of the liposome pharmacokinetics. PEG-PE greatly improved the half-life of the liposomes (400 min) while PEG-SA only provided a marginal improvement. All liposome preparations were cleared in a biphasic fashion. In conclusion, a novel biodegradable lipopeptide conjugate was designed that endows liposomes with a prolonged circulation time in vivo. The pharmacokinetic profile of these modified liposomes was drastically improved over that of conventional liposomes. Since the library is prepared by solid-phase synthesis, length and/or composition could easily be modified in order to modulate the clearance profile of the liposomes. Tailoring of the pharmacokinetic profile of the liposomes depending on their intended application may allow for a greater flexibility of use than PEG-PE.     

Principles of rational design of thermally targeted liposomes for local drug delivery

Drug release from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes occurs close to the main transition temperature T_m = 41 °C. The exact release temperature can be adjusted by additional lipids, which shift T_m. A major issue is drug leakage at 37 °C. We here describe a novel approach with improved drug retention yet rapid release. To obtain spherical, smooth liposomes we included: i) 2 mol% cholesterol, to soften bilayers (Lemmich et al 1997), ii) lipids, which due to their spontaneous curvature stabilize the negative and positive curvatures of the inner and outer leaflets of unilamellar liposomes. In addition to differential scanning calorimetry (DSC) and fluorescence spectroscopy, the lipid mixtures were analyzed by a Langmuir balance for their elastic properties and lipid packing, aiming at high elasticity modulus C_S^− 1. Maxima in C_S^− 1 coincided with minima in the free energy of lateral mixing. These liposomes have reduced drug leakage, yet retain rapid release.From the Clinical EditorThis paper reports the development of optimized DPPC liposomes for drug delivery, with reduced drug leakage but maintained rapid release.     

Antitumor activity of doxorubicin encapsulated in hexadecylphosphocholine (HePC) liposomes against human xenografts on Scid mice

Doxorubicin was encapsulated into liposomes composed of hexadecylphosphocholine:egg yolk phosphatidylcholine:stearylamine (HePC.EPC:SA) 10:10.0.1 (molar ratio) (1) and EPC:SA 10:0.1 (molar ratio) (2). Liposomal formulations 1 and 2, as well as free doxorubicin and free HePC, were tested in vitro against HCT116 human colon cancer cell lines and peripheral blood mononuclear cells (PBMCs) obtained from healthy donors, using the sulphorodamine B assay. The activity of doxorubicin was retained or slightly improved when entrapped into liposomes 1 and 2, while liposomal formulation 1 incorporating doxorubicin was found to be less toxic against normal cells. The liposomes were tested in vivo against human colon cancer xenografts in scid mice. The antitumor activities of liposomes 1 and 2 were statistically similar to that of free doxorubicin, but their toxicity was significantly lower. Based on these results, the combination of HePC and doxorubicin in one liposomal formulation may be justified for further evaluation.     

Intracellular trafficking of nuclear localization signal conjugated nanoparticles for cancer therapy

Doxorubicin (DOX) is an anticancer drug with an intracellular site of action in the nucleus. For high antitumour activity, it should be effectively internalized into the cancer cells and accumulate in the nucleus. In this study, we have prepared a nuclear localization signal conjugated doxorubicin loaded Poly (d,l-lactide-co-glycolide) nanoparticles (NPs), to deliver doxorubicin to the nucleus efficiently. Physico-chemical characterization of these NPs showed that the drug is molecularly dispersed in spherical and smooth surfaced nanoparticles. NPs (～226 nm in diameter, 46% encapsulation efficiency) under in vitro conditions exhibited sustained release of the encapsulated drug (63% release in 60 days). Cell cytotoxicity results showed that NLS conjugated NPs exhibited comparatively lower IC₅₀ value (2.3 μM/ml) than drug in solution (17.6 μM/ml) and unconjugated NPs (7.9 μM/ml) in breast cancer cell line MCF-7 as studied by MTT assay. Cellular uptake studies by confocal laser scanning microscopy (CLSM) and fluorescence spectrophotometer showed that greater amount of drug is targeted to the nucleus with NLS conjugated NPs as compared to drug in solution or unconjugated NPs. Flow cytometry experiments results showed that NLS conjugated NPs are showing greater cell cycle (G2/M phase) blocking and apoptosis than native DOX and unconjugated NPs. In conclusion, these results suggested that NLS conjugated doxorubicin loaded NPs could be potentially useful as novel drug delivery system for breast cancer therapy.     

Ketorolac entrapped in polymeric micelles: preparation, characterisation and ocular anti-inflammatory studies

Two types of ligand anchored multilamellar liposomes (MLVs) containing amphotericin B (Amp B) were prepared. The MLVs consisting of soya phosphatidylcholine (PC) and cholesterol (Chol) were coated with O-palmitoyl mannan (OPM). Similarly, the MLVs with the same Amp B content consisting of soya PC, Chol and phosphatidylethanolamine (PE) were prepared and covalently anchored with p-aminophenyl-mannopyranoside (PAM). The surface modified MLVs and their plain counterparts were characterised for size, shape, lamellarity, entrapment efficiency and ligand density. The stability in serum and in vivo bio-distribution in albino rats were also determined. It was observed that extent of accumulation of liposomal Amp B in macrophage rich organs, particularly liver, spleen and lungs was significantly high when compared against the free drug. The rates and extent of accumulation were found to increase further on ligand anchoring. In either of the cases, the macrophagic uptake of ligand anchored liposomes was inhibited significantly on pre-injection of hydrolysed mannan, being suggestive of receptor mediated uptake of ligand anchored liposomes. Comparison of biodistruibution pattern of ligand anchored MLVs revealed that PAM linked liposomes exhibited a higher hepato-splenic accumulation where as drug accumulation in lungs was highest in the case of OPM coated liposomes. It was thus observed that mannopyranoside is a specific ligand for targeting bioactives to the macrophages of liver and spleen while OPM could preferentially negotiate the targeting of bioactives to the alveolar macrophages.     

Novel galactosylated liposomes for hepatocyte-selective targeting of lipophilic drugs

Novel galactosylated neutral liposomes containing cholesten-5-yloxy-N-(4-((1-imino-2-beta-D-thiogalactosylethyl)amino)butyl)formamide (Gal-C4-Chol) as a "homing" device were developed for hepatocyte-selective drug targeting. Distearoylphosphatidylcholine (DSPC)/cholesterol (Chol) (60:40) and DSPC/Chol/Gal-C4-Chol (60:35:5) liposomes were prepared and labeled with [3H]cholesteryl hexadecyl ether (CHE). [3H]Prostaglandin E1 (PGE1) and [14C]probucol were incorporated in liposomes as model lipophilic drugs. After intravenous injection of the liposomes, mice were sacrificed at suitable time periods, and the lung, liver, kidney, spleen, and heart were excised. DSPC/Chol/Gal-C4-Chol liposomes rapidly disappeared from the blood, and 85% of the dose had accumulated in the liver within 10 min compared with hepatic accumulation of DSPC/Chol liposomes of 12%. The liver was perfused with collagenase, and liver parenchymal cells (PC) and liver nonparenchymal cells (NPC) were separated by centrifugal differentiation to determine the cellular distribution. The PC/NPC ratios for DSPC/Chol/Gal-C4-Chol and DSPC/Chol liposomes were 15.1 and 1.1, respectively. The hepatic uptake of DSPC/Chol/Gal-C4-Chol liposomes, but not that of DSPC/Chol liposomes, was significantly inhibited by the predosing of galactosylated bovine serum albumin. [14C]Probucol and [3H]PGE1 incorporated in DSPC/Chol/Gal-C4-Chol liposomes was also efficiently delivered to the liver. In conclusion, newly developed galactosylated liposomes have been proven to be a useful carrier for hepatocyte-selective targeting that will have many practical applications.