Direct measurement of the extravasation of polyethyleneglycol-coated liposomes into solid tumor tissue by in vivo fluorescence microscopy

The extravasation of liposomes of different sizes into solid tumors after i.v. injection was visualized by in vivo fluorescence microscopy in mouse neuroblastoma C-1300-bearing mice. Liposomes composed of distearoylphosphatidylcholine/cholesterol (1/1 molar ratio) and 6 mol% distearoylphosphatidylethanolamine derivative of polyethyleneglycol (PEG) were prepared. The PEG-coated liposomes were fluorescently labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) as a liposome marker or with doxorubicin (DXR) as an aqueous-phase marker. Liposomes with an average diameter of 100–200 nm showed the greatest tumor accumulation. With time after injection of DiI-labeled liposomes, the tumor interstitial fluorescence intensity increased. Most fluorescent spots were located outside and around the vessel wall, indicating extravasation of intact liposomes. The perivascular distribution was heterogeneous. We also obtained the same fluorescence localization pattern with DXR released from extravasated liposomes after injection of DXR-encapsulated liposomes. No fluorescence from extravasated liposomes was detected in normal s.c. tissue; the fluorescent spots were observed only in the vessel wall. Our results indicate that small-size long-circulating liposomes are able to traverse the endothelium of blood vessels in tumors and extravasate into interstitial spaces. Moreover, encapsulated drug was released from extravasated liposomes in the tumor.     

Traumatic brain injury opens blood–brain barrier to stealth liposomes via an enhanced permeability and retention (EPR)-like effect

The opening of the tight junctions in the blood–brain barrier (BBB) following traumatic brain injury (TBI) is hypothesized to be sufficient to enable accumulation of large drug carriers, such as stealth liposomes, in a similar manner to the extravasation seen in tumor tissue via the enhanced permeability and retention (EPR) effect. The controlled cortical impact model of TBI was used to evaluate liposome accumulation in mice. Dual-radiolabeled PEGylated liposomes were administered either immediately after induction of TBI or at increasing times post-TBI to mimic the likely clinical scenario. The accumulation of radiolabel in the brain tissue ipsilateral and contralateral to the site of trauma, as well as in other organs, was evaluated. Selective influx of liposomes occurred at 0–8?h after injury, while the barrier closed between 8 and 24?hr after injury, consistent with reports on albumin infiltration. Significantly enhanced accumulation of liposomes occurred in mice subjected to TBI compared to anaesthetized controls, and accumulation was greater in the injured versus the contralateral side of the brain. Thus, stealth liposomes show potential to enhance drug delivery to the site of brain injury with a wide range of encapsulated therapeutic candidates.     

Targeted thermosensitive liposomes: an attractive novel approach for increased drug delivery to solid tumors

Introduction: Currently available chemotherapy is hampered by a lack in tumor specificity and resulting toxicity. Small and long-circulating liposomes can preferentially deliver chemotherapeutic drugs to tumors upon extravasation from tumor vasculature. Although clinically used liposomal formulations demonstrated significant reduction in toxicity, enhancement of therapeutic activity has not fully met expectations.

Areas covered: Low drug bioavailability from liposomal formulations and limited tumor accumulation remain major challenges to further improve therapeutic activity of liposomal chemotherapy. The aim of this review is to highlight strategies addressing these challenges. A first strategy uses hyperthermia and thermosensitive liposomes to improve tumor accumulation and trigger liposomal drug bioavailability. Image-guidance can aid online monitoring of heat and drug delivery and further personalize the treatment. A second strategy involves tumor-specific targeting to enhance drug delivery specificity and drug internalization. In addition, we review the potential of combinations of the two in one targeted thermosensitive-triggered drug delivery system.

Expert opinion: Heat-triggered drug delivery using thermosensitive liposomes as well as the use of tumor vasculature or tumor cell-targeted liposomes are both promising strategies to improve liposomal chemotherapy. Preclinical evidence has been encouraging and both strategies are currently undergoing clinical evaluation. A combination of both strategies rendering targeted thermosensitive liposomes (TTSL) may appear as a new and attractive approach promoting tumor drug delivery.     

Liposome accumulation in irradiated tumors display important tumor and dose dependent differences

Radiation therapy may affect several important parameters in the tumor microenvironment and thereby influence the accumulation of liposomes by the enhanced permeability and retention (EPR)-effect. Here we investigate the effect of single dose radiation therapy on liposome tumor accumulation by PET/CT imaging using radiolabeled liposomes. Head and neck cancer xenografts (FaDu) and syngenic colorectal (CT26) cancer models were investigated. Radiotherapy displayed opposite effects in the two models. FaDu tumors displayed increased mean accumulation of liposomes for radiation doses up to 10 Gy, whereas CT26 tumors displayed a tendency for decreased accumulation. Tumor hypoxia was found negatively correlated to microregional distribution of liposomes. However, liposome distribution in relation to hypoxia was improved at lower radiation doses. The study reveals that the heterogeneity in liposome tumor accumulation between tumors and different radiation protocols are important factors that need to be taken into consideration to achieve optimal effect of liposome based radio-sensitizer therapy.     

Enhancement of anticancer activity in antineovascular therapy is based on the intratumoral distribution of the active targeting carrier for anticancer drugs

We previously observed the enhanced anticancer efficacy of anticancer drugs encapsulated in Ala-Pro-Arg-Pro-Gly-polyethyleneglycol-modified liposome (APRPG-PEG-Lip) in tumor-bearing mice, since APRPG peptide was used as an active targeting tool to angiogenic endothelium. This modality, antineovascular therapy (ANET), aims to eradicate tumor cells indirectly through damaging angiogenic vessels. In the present study, we examined the in vivo trafficking of APRPG-PEG-Lip labeled with [2-(18)F]2-fluoro-2-deoxy-D-glucose ([2-(18)F]FDG) by use of positron emission tomography (PET), and observed that the trafficking of this liposome was quite similar to that of non-targeted long-circulating liposome (PEG-Lip). Then, histochemical analysis of intratumoral distribution of both liposomes was performed by use of fluorescence-labeled liposomes. In contrast to in vivo trafficking, intratumoral distribution of both types of liposomes was quite different: APRPG-PEG-Lip was colocalized with angiogenic endothelial cells that were immunohistochemically stained for CD31, although PEG-Lip was localized around the angiogenic vessels. These results strongly suggest that intratumoral distribution of drug carrier is much more important for therapeutic efficacy than the total accumulation of the anticancer drug in the tumor, and that active delivery of anticancer drugs to angiogenic vessels is useful for cancer treatment.     

Liposomal drug delivery systems: an update review

The discovery of liposome or lipid vesicle emerged from self forming enclosed lipid bi-layer upon hydration; liposome drug delivery systems have played a significant role in formulation of potent drug to improve therapeutics. Recently the liposome formulations are targeted to reduce toxicity and increase accumulation at the target site. There are several new methods of liposome preparation based on lipid drug interaction and liposome disposition mechanism including the inhibition of rapid clearance of liposome by controlling particle size, charge and surface hydration. Most clinical applications of liposomal drug delivery are targeting to tissue with or without expression of target recognition molecules on lipid membrane. The liposomes are characterized with respect to physical, chemical and biological parameters. The sizing of liposome is also critical parameter which helps characterize the liposome which is usually performed by sequential extrusion at relatively low pressure through polycarbonate membrane (PCM). This mode of drug delivery lends more safety and efficacy to administration of several classes of drugs like antiviral, antifungal, antimicrobial, vaccines, anti-tubercular drugs and gene therapeutics. Present applications of the liposomes are in the immunology, dermatology, vaccine adjuvant, eye disorders, brain targeting, infective disease and in tumour therapy. The new developments in this field are the specific binding properties of a drug-carrying liposome to a target cell such as a tumor cell and specific molecules in the body (antibodies, proteins, peptides etc.); stealth liposomes which are especially being used as carriers for hydrophilic (water soluble) anticancer drugs like doxorubicin, mitoxantrone; and bisphosphonate-liposome mediated depletion of macrophages. This review would be a help to the researchers working in the area of liposomal drug delivery.     

Strategies for improving the intratumoral distribution of liposomal drugs in cancer therapy

ABSTRACT

Introduction: A major limitation of current liposomal cancer therapies is the inability of liposome therapeutics to penetrate throughout the entire tumor mass. This inhomogeneous distribution of liposome therapeutics within the tumor has been linked to treatment failure and drug resistance. Both liposome particle transport properties and tumor microenvironment characteristics contribute to this challenge in cancer therapy. This limitation is relevant to both intravenously and intratumorally administered liposome therapeutics.

Areas covered: Strategies to improve the intratumoral distribution of liposome therapeutics are described. Combination therapies of intravenous liposome therapeutics with pharmacologic agents modulating abnormal tumor vasculature, interstitial fluid pressure, extracellular matrix components, and tumor associated macrophages are discussed. Combination therapies using external stimuli (hyperthermia, radiofrequency ablation, magnetic field, radiation, and ultrasound) with intravenous liposome therapeutics are discussed. Intratumoral convection-enhanced delivery (CED) of liposomal therapeutics is reviewed.

Expert opinion: Optimization of the combination therapies and drug delivery protocols are necessary. Further research should be conducted in appropriate cancer types with consideration of physiochemical features of liposomes and their timing sequence. More investigation of the role of tumor associated macrophages in intratumoral distribution is warranted. Intratumoral infusion of liposomes using CED is a promising approach to improve their distribution within the tumor mass.     

The size of liposomes: a factor which affects their targeting efficiency to tumors and therapeutic activity of liposomal antitumor drugs

The size of liposomes has been shown to be an important factor in the efficient delivery of an antitumor agent to a tumor. In this paper, the effects of the size of liposomes on the pharmacokinetics of liposomes and liposome-encapsulated drugs are discussed with reference to: (1) the circulation amount and residence time of liposomes in the blood, (2) the accumulation of liposomes in the tumor, and (3) in vivo drug release from liposomes. In addition, the effect of size on therapeutic activity (antitumor efficacy and toxicity) of a liposomal anticancer preparation is discussed. Finally we discuss the importance of liposome size in the design of a more effective liposomal antitumor preparation.     

三磷酸腺苷脂质体的研究进展

本文综述了近年来三磷酸腺苷(ATP)脂质体的研究进展.脂质体作为新载体可以提高ATP在体内的稳定性,并且能靶向于缺血的心肌和缺血的大脑,用于器官移植中能提高冷冻贮存器官的能级状态,提高移植的成活率,故制备稳定的ATP脂质体具有重要的意义.本文还综述了ATP脂质体的制备方法,探讨如何进一步提高脂质体的包裹率和靶向性,以用于临床.     

Heat-Induced Drug Release Rate and Maximal Targeting Index of Thermosensitive Liposome in Tumor-Bearing Mice

To evaluate the rate of drug release at the tumor and maximal drug targeting after administration of thermosensitive liposomes with hy-perthermia, a theoretical and experimental method was derived assessing the fraction of drug released from liposomes in a single pass through the heated tumor, F, and the drug targeting index when drug release occurs completely in response to heat (F = 1), DTI_max. The F and DTImax were evaluated for four types of liposomes (LUV-1 and LUV-2, thermosensitive large unilamellar liposomes; LUV-3, a nonthermosensitive large unilamellar liposome; and SUV-1, a thermosensitive small unilamellar liposome) using reported data on blood liposome levels and tumor drug levels after the liposomes were administered to tumor bearing mice. DTI_max values for LUV-1 and SUV-1 were approximately 6, while the value for LUV-2 with a relatively large systemic clearance was only 2.3. The F values for LUV-1, LUV-2, and SUV-1 with hyperthermia were 0.71, 1.17, and 0.34, respectively, whereas the values for these liposomes without hyperthermia and for LUV-3 with or without hyperthermia were nearly zero. These results confirm earlier findings that LUV-1 and LUV-2 release CDDP almost completely at the heated tumor and that the large DTI value obtained in LUV-1 (DTI = 4.6) was due to its high heat sensitivity and its small systemic clearance.     

Preparation, characterization, and biodistribution study of technetium-99m-labeled leuprolide acetate-loaded liposomes in ehrlich ascites tumor-bearing mice

The purpose of this study was to prepare conventional and sterically stabilized liposomes containing leuprolide acetate in an attempt to prolong the biological half life of the drug, to reduce the uptake by reticuloendothelial system (RES), and to reduce the injection frequency of intravenously administered peptide drugs. The conventional and sterically stabilized liposomes containing leuprolide acetate were prepared by reverse phase evaporation method and characterized for entrapment efficiency and particle size. Radiolabeling of leuprolide acetate and its liposomes was performed by direct labeling with reduced technetium-99m. Its biodistribution and imaging characteristics were studied in ehrlich ascites tumor (EAT)-bearing mice after labeling with technetium-99m. The systemic pharmacokinetic studies were performed in rabbits. A high uptake by tumor was observed by sterically stabilized liposome containing leuprolide acetate compared with free drug and conventional liposomes. The liver/tumor uptake ratio of free drug, conventional (LL), and sterically stabilized liposomes (SLL5000 and SLL2000) was found to be 20, 7.99, 1.63, and 1.23, respectively, which showed the increased accumulation of sterically stabilized liposomes in tumor compared with the free drug and conventional liposomes at 24 hours postinjection. Liver uptake of sterically stabilized liposomes was still 7-fold less than the conventional liposomes. The marked accumulation of liposomes in the tumor-bearing mice was also documented by gamma scintigraphic studies. The findings demonstrate the distribution of these liposomes within solid tumor and prove that the sterically stabilized liposomes experience increased tumor uptake and prolonged circulation half life. Hence these findings will be relevant for the optimal design of long circulating liposomes for the peptide drugs and for targeting of liposomes toward tumor.     

Translocation of liposomes into cancer cells by cell-penetrating peptides penetratin and tat: a kinetic and efficacy study

Unlike conventional liposomes, sterically stabilized liposomes, with their smaller volume of distribution and reduced clearance, preferentially convey encapsulated drugs into tumor sites. Despite these improvements, intracellular delivery is hampered by the stable drug retention of the liposomes, which diminishes the efficacy of the liposomal drug. To facilitate uptake of liposomal drugs into cells, two cell-penetrating peptides, penetratin (PEN) and TAT, derived from the HIV-1 TAT protein, were studied. In contrast to control peptides, both TAT and PEN enhanced the translocation efficiency of liposomes in proportion to the number of peptides attached to the liposomal surface. A peptide number of as few as five could enhance the intracellular delivery of liposomes. The kinetics of uptake was peptide- and cell-type dependent. Intracellular accumulation of TAT-liposomes increased with incubation time, but PEN-liposomes peaked at 1 h and then declined gradually. After treatment with 1 microg/ml doxorubicin equivalents of liposome for 2 h, TAT increased the doxorubicin uptake of A431 cells by 12-fold. However, the improvement of uptake of liposomal doxorubicin was not reflected by cytotoxicity in vitro or tumor control in vivo. Our results demonstrated that merely adding CPP to a liposome encapsulating anticancer drug was inadequate in improving its antitumor activity. An additional approach to enhance the intracellular release of the encapsulated drug is obviously necessary.     

Passive targeting of doxorubicin with polymer coated liposomes in tumor bearing rats.

The purpose of this study was to reveal the effectiveness of the polymer coated liposomes as a carrier of the anticancer drug doxorubicin in intravenous administration. The size controlled doxorubicin-loaded liposomes (egg phosphatidylcholine : cholesterol = 1:1 in molar ratio) were coated with hydrophilic polymers (polyvinyl alcohol; PVA and hydroxypropylmethylcellulose; HPMC) having a hydrophobic moiety in the molecules (PVA-R, HPMC-R). The existence of a thick polymer layer on the surface of the polymer coated liposomes was confirmed by measuring the change in particle size and the amount of polymer on the liposomal surface. The polymer coating effects on the tumor accumulation of the drug encapsulated in the liposomes were evaluated in Walker rat carcinoma 256 cell bearing rats. The doxorubicin-loaded liposomes coated with PVA-R and HPMC-R showed higher drug accumulation into the tumor site by prolonging the systemic circulation in tumor-bearing rats. The targeting efficiency of the polymer coated liposomes calculated with the total and tumorous clearance of the drug was ca. 5 times larger than that of non-coated liposomes. We ascertained that polymers having a hydrophobic moiety in the molecule such as PVA-R and HPMC-R are suitable materials for modifying the surfaces of the doxorubicin-loaded liposome to improve its targeting properties.     

抗肿瘤药物脂质体粒径对肿瘤靶向性的影响

脂质体抗癌药物的粒径在它将药物运载到肿瘤组织的过程中是一个重要的因素。在本文中，脂质体粒径在药代动力学中所发挥的作用从以下几方面进行了研究：（1）脂质体在血液中的分布和滞留时间；（2）脂质体在肿瘤组织中的积聚；（3）脂质体从肿瘤毛细血管中渗漏和它们在肿瘤组织中的滞留。最后，讨论了脂质体粒径在设计抗肿瘤脂质体药物时的重要性。     

Trends and developments in liposome drug delivery systems

Since the discovery of liposomes or lipid vesicles derived from self-forming enclosed lipid bilayers upon hydration, liposome drug delivery systems have played a significant role in formulation of potent drugs to improve therapeutics. Currently, most of these liposome formulations are designed to reduce toxicity and to some extent increase accumulation at the target site(s) in a number of clinical applications. The current pharmaceutical preparations of liposome-based therapeutics stem from our understanding of lipid-drug interactions and liposome disposition mechanisms including the inhibition of rapid clearance of liposomes by controlling size, charge, and surface hydration. The insight gained from clinical use of liposome drug delivery systems can now be integrated to design liposomes targeted to tissues and cells with or without expression of target recognition molecules on liposome membranes. Enhanced safety and heightened efficacy have been achieved for a wide range of drug classes, including antitumor agents, antivirals, antifungals, antimicrobials, vaccines, and gene therapeutics. Additional refinements of biomembrane sensors and liposome delivery systems that are effective in the presence of other membrane-bound proteins in vivo may permit selective delivery of therapeutic compounds to selected intracellular target areas.     

Effect of surface ligand density on cytotoxicity and pharmacokinetic profile of docetaxel loaded liposomes

Various biotin-modified liposomes incorporated with docetaxel (DTX) were prepared to study the effect of surface biotin density on the pharmacokinetic profile of the liposome. Four types of liposomes such as PEG modified liposome (PDL), 0.5% (mol) biotin modified liposome (0.5BDL), 1% (mol) biotin modified liposome (1BDL) and 2% (mol) biotin modified liposome (2BDL) were prepared using thin film dispersion method. The prepared liposomes were characterized by measuring encapsulation efficiency (EE), particle size, Zeta-potential, physical stability and drug release profiles in vitro. MTT assay was performed to elevate the cytotoxicity of liposomes on MCF-7 cells. In vivo evaluation was further performed to investigate the effect of biotin surface density on the pharmacokinetic profiles. All the prepared liposomes exhibited high encapsulation efficiency, small particle size, narrow particle distribution and sustained release profiles in vitro. In MTT assay, 0.5BDL showed largest tumor cell toxicity, compared with DTX solution. All liposomes containing DTX showed prolonged blood circulation in vivo, and 0.5BDL showed the longest circulation time among the biotin modified liposome. Surface modification of liposome had a negative impact on the circulation of liposomes in the blood, which needs to be considered when designing the ligand mediated targeting delivery systems. A proper amount of biotin liposome with 0.5% molar ratio is expected to produce the best anti-tumor effect.     

MRI evaluation of the antitumor activity of paramagnetic liposomes loaded with prednisolone phosphate

The design of long circulating liposomes co-loaded with the glucocorticoid prednisolone phosphate (PLP) and the amphiphilic paramagnetic contrast agent Gd-DOTAMA(C(18))(2) allowed the MRI-guided in vivo visualization of the delivery and biodistribution of PLP, as well as the monitoring of drug efficacy. The performance of this theranostic probe was investigated in a mouse model bearing a melanoma B16 syngeneic tumor. The release kinetics of the drug were evaluated in vitro where it displayed a peculiar behavior characterized by a fast process (completed in few hours) involving only a small portion (<5%) of the drug. Interestingly, the incorporation of the amphiphilic imaging reporter in the liposomal bilayer slightly increased the amount of the fast-release portion (<10%), thus suggesting that it could be attributed to a drug fraction embedded in the liposomal bilayer. In fact, the release of a hydrophilic imaging probe encapsulated in the inner core of the same long circulating liposomes formulated for carrying the drug, displayed different, single-step, kinetics. The in vivo monitoring of the antitumor activity of the nanomedicine revealed that the incorporation of the MRI probe into the liposome bilayer did not significantly affect the drug efficacy. The in vivo experiments also indicated a relevant and fast liposome uptake from macrophage-rich organs like spleen and liver, which reduced the tumor accumulation of the liposomes. The accumulation of the amphipatic MRI label caused the occurrence of a long-term residual T(1) contrast still detectable 1week after injection.     

In Vitro andIn Vivo Studies of Different Liposomes Containing Topotecan

Liposome as a carrier of topotecan (TPT), a promising anticancer drug, has been reported in attempt to improve the stability and antitumor activity of TPT. However, the biodistribution pattern of TPT liposome in vivo and PEG-modified liposome containing TPT have not been studied systemically. In this paper, the in vitro stability and in vivo biodistribution behavior of several liposomes containing TPT with different lipid compositions and PEG-modification were studied. Compared with the 'fluid' liposome (S-Lip) composed of soybean phosphatidylcholine (SPC), the 'solid' liposome (H-Lip) composed of hydrogenated soybean phosphatidylcholine HSPC decreased the leaking efficiency of TPT from liposome and enhanced the stability of liposome in fetal bovine serum (FBS) or human blood plasma (HBP). The results of biodistribution studies in S180 tumor-bearing mice showed that liposomal encapsulation increased the concentrations of total TPT and the ratio of lactone form in plasma. Compared with free TPT, S-Lip and H-Lip resulted in 5- and 19-fold increase in the area under the curve (AUC(0-->infinity)), respectively. PEG-modified H-Lip (H-PEG) showed 3.7-fold increase in AUC(0-->infinity) compared with H-Lip, but there was no significant increase in t(1/2) and AUC(0-->infinity) for PEG-modified S-Lip (S-PEG) compared with S-Lip. Moreover, the liposomal encapsulation changed the biodistribution behavior, and H-Lip and H-PEG dramatically increased the accumulation of TPT in tumor, and the relative tumor uptake ratios were 3.4 and 4.3 compared with free drug, respectively. There was also a marked increase in the distribution of TPT in lung when the drug was encapsulated into H-Lip and H-PEG. Moreover, H-PEG decreased the accumulation of TPT in bone marrow compared with unmodified H-Lip. All these results indicated that the membrane fluidity of liposome has an important effect on in vitro stability and in vivo biodistribution pattern of liposomes containing TPT, and PEG-modified 'solid' liposome may be an efficient carrier of TPT.     

Liposomes equipped with cell penetrating peptide BR2 enhances chemotherapeutic effects of cantharidin against hepatocellular carcinoma

A main hurdle for the success of tumor-specific liposomes is their inability to penetrate tumors efficiently. In this study, we incorporated a cell-penetrating peptide BR2 onto the surface of a liposome loaded with the anticancer drug cantharidin (CTD) to create a system targeting hepatocellular carcinoma (HCC) cells more efficiently and effectively. The in vitro cytotoxicity assay comparing the loaded liposomes’ effects on hepatocellular cancer HepG2 and the control Miha cells showed that CTD-loaded liposomes had a stronger anticancer effect after BR2 modification. The cellular uptake results of HepG2 and Miha cells further confirmed the superior ability of BR2-modified liposomes to penetrate cancer cells. The colocalization study revealed that BR2-modified liposomes could enter tumor cells and subsequently release drugs. A higher efficiency of delivery by BR2 liposomes as compared to unmodified liposomes was evident by evaluation of the HepG2 tumor spheroids penetration and inhibition. The biodistribution studies and anticancer efficacy results in vivo showed the significant accumulation of BR2-modified liposomes into tumor sites and an enhanced tumor inhibition. In conclusion, BR2-modified liposomes improve the anticancer potency of drugs for HCC.