Hepatocyte-targeting gene delivery using a lipoplex composed of galactose-modified aromatic lipid synthesized with click chemistry

Highly efficient drug carriers targeting hepatocyte is needed for treatment for liver diseases such as liver cirrhosis and virus infections. Galactose or N-acetylgalactosamine is known to be recognized and incorporated into the cells through asialoglycoprotein receptor (ASGPR) that is exclusively expressed on hepatocyte and hepatoma. In this study, we synthesized a galactose-modified lipid with aromatic ring with click chemistry. To make a complex with DNA, termed ‘lipoplex’, we prepared a binary micelle composed of cationic lipid; dioleoyltrimethylammoniumpropane (DOTAP) and galactose-modified lipid (D/Gal). We prepared lipoplex from plasmid DNA (pDNA) and D/Gal and examined the cell specificity and transfection efficiency. The lipoplex was able to interact with ASGPR immobilized on gold substrate in the quartz-crystal microbalance (QCM) sensor cell. The lipoplex induced high gene expression to HepG2 cells, a human hepatocellular carcinoma cell line, but not to A549 cells, a human alveolar adenocarcinoma cell line. The treatment with asialofetuin, which is a ligand for ASGPR and would work as a competitive inhibitor, before addition of the lipoplexes decreased the expression to HepG2 cells. These results indicate that D/Gal lipoplex was incorporated into HepG2 cells preferentially through ASGPR and the uptake was caused by galactose specific receptor. This delivery system to hepatocytes may overcome the problems for gene therapy and be used for treatment of hepatitis and hepatic cirrhosis.     

肝细胞靶向pH敏脂质体的制备及性质分析

为了制备具有肝细胞特异靶向性和pH敏感性的脂质体,设计并合成了四种带有半乳糖残基的导向分子,与具有pH敏感性的DC-chol/DOPE混合制备脂质体,通过质粒转染实验、受体竞争抑制实验和红细胞溶血等实验选出最佳转染活性的十八醇-半乳糖甙(18-gal)脂质体,并证明其具有肝细胞特异受体介导的靶向性和pH敏感性,且细胞毒性较小,可以作为一种潜在的肝细胞靶向转运系统得到进一步发展.     

Receptor-mediated uptake of asialoganglioside liposomes: sub-cellular distribution of the liposomal marker in isolated liver cell types

The use of asialo GM1-containing small unilamellar liposome preparations in vivo caused a 2.8-fold increase in the uptake by the liver as compared with the control (neutral) preparations (without asialo GM1). The uptake of negatively charged dicetylphosphate and dipalmitoyl phosphatidic acid-containing small unilamellar liposomes was found to be 1.6-and 1.8-fold respectively higher than that of the neutral preparations. In studies with isolated liver cell types, inhibition of the galactosylated liposome uptake by asialofetuin indicated a possible involvement of hepatic galactose receptors in the recognition of asialo GM1 liposomes by the hepatic parenchymal cells, which in turn were found to be mainly responsible for the enhanced incorporation of these liposomes in the liver. Sub-cellular distribution studies with isolated liver cell types indicated lysosomal localization of the liposomes both in parenchymal and nonparenchymal cells, and it has been proposed that the asialo GM1 liposomes are cointernalized with asialofetuin through a common lysosomal route of ligand internalization.     

Sustained Liver Targeting and Improved Antiproliferative Effect of Doxorubicin Liposomes Modified with Galactosylated Lipid and PEG-Lipid

In this study, a cleavable PEG-lipid (methoxypolyethyleneglycol 2000-cholesteryl hemisuccinate, PEG₂₀₀₀-CHEMS) linked via ester bond and galactosylated lipid ((5-cholesten-3β-yl) 4-oxo-4-[2-(lactobionyl amido) ethylamido] butanoate, CHS-ED-LA) were used to modify doxorubicin (DOX) liposome. DOX was encapsulated into conventional liposomes (CL), galactosylated liposomes (modified with CHS-ED-LA, GalL), pegylated liposomes (modified with PEG₂₀₀₀-CHEMS, PEG-CL), and pegylated galactosylated liposomes (modified with CHS-ED-LA and PEG₂₀₀₀-CHEMS, PEG-GalL) using an ammonium sulfate gradient loading method and then intravenously injected to normal mice. Both PEG-GalL DOX and GalL DOX gave relatively high overall drug targeting efficiencies to liver ((T _e)_liver) and were mainly taken up by hepatocyte. However, PEG-GalL DOX showed unique “sustained targeting” characterized by slowed transfer of DOX to liver and reduced peak concentrations in the liver. The biodistribution and antitumor efficacy of various DOX preparations were studied in hepatocarcinoma 22 (H22) tumor-bearing mice. The inhibitory rate of PEG-GalL DOX to H22 tumors was up to 94%, significantly higher than that of PEG-CL DOX, GalL DOX, CL DOX, and free DOX, although the tumor distribution of DOX revealed no difference between PEG-GalL DOX and PEG-CL DOX. Meanwhile, the gradual increase in the liver DOX concentration due to the sustained uptake of PEG-GalL DOX formulations resulted in lower damage to liver. In conclusion, the present investigation indicated that double modification of liposomes with PEG₂₀₀₀-CHEMS, and CHS-ED-LA represents a potentially advantageous strategy in the therapy of liver cancers or other liver diseases.     

Dual-targeting for brain-specific liposomes drug delivery system: Synthesis and preliminary evaluation

The treatment of glioma has become a great challenge because of the existence of brain barrier (BB). In order to develop an efficient brain targeting drug delivery system to greatly improve the brain permeability of anti-cancer drugs, a novel brain-targeted glucose-vitamin C (Glu-Vc) derivative was designed and synthesized as liposome ligand for preparing liposome to effectively deliver paclitaxel (PTX). The liposome was prepared and its particle size, zeta potential, encapsulation efficiency, release profile, stability, hemolysis and cytotoxicity were also characterized. What’s more, the cellular uptake of CFPE-labeled Glu-Vc-Lip on GLUT₁- and SVCT₂-overexpressed C6 cells was 4.79-, 1.95-, 4.00- and 1.53-fold higher than that of Lip, Glu-Lip, Vc-Lip and Glu?+?Vc-Lip. Also, the Glu-Vc modified liposomes showed superior targeting ability in vivo evaluation compared with naked paclitaxel, non-coated, singly-modified and co-modified by physical blending liposomes. The relative uptake efficiency was enhanced by 7.53 fold to that of naked paclitaxel, while the concentration efficiency was up to 7.89 times. What’s more, the Glu-Vc modified liposomes also displayed the maximum accumulation of DiD-loaded liposomes at tumor sites with the strongest fluorescence in the brain in vivo imaging. Our results suggest that chemical modification of liposomes with warheads of glucose and vitamin C represents a promising and efficient strategy for the development of brain-specific liposomes drug delivery system by utilizing the endogenous transportation mechanism of the warheads.     

Liposomes modified with double-branched biotin: A novel and effective way to promote breast cancer targeting

Although active targeting liposomes with cancer-specific ligands can bind and internalize into cancer cells, only a few high-efficiency liposomes have been developed so far because traditional single branched ligand modified liposomes generally failed to deliver adequate therapeutic payload. In this paper, we broke the traditional design concept and synthesized the double branched biotin modified cholesterol (Bio₂-Chol) for the first time. On this basis, different biotin density modified liposomes ((Bio-Chol)Lip, (Bio-Chol)₂Lip and (Bio₂-Chol)Lip) were successfully prepared and used as active targeting drug delivery systems for the treatment of breast cancer. The in vitro and in vivo breast cancer-targeting ability of these liposomes were systemically studied using paclitaxel (PTX) as the model drug. And the uptake mechanism of (Bio₂-Chol)Lip was investigated. The results showed that (Bio₂-Chol)Lip had the best breast cancer-targeting ability compared with naked paclitaxel, unmodified Lip, (Bio-Chol)Lip and (Bio-Chol)₂Lip. In particular, the relative uptake efficiency (RE) and concentration efficiency (CE) of (Bio₂-Chol)Lip were respectively enhanced by 5.61- and 5.06-fold compared to that of naked paclitaxel. Both distribution data and pharmacokinetic parameters suggested that the double branched biotin modified liposome ((Bio₂-Chol)Lip) is a very promising drug delivery carrier for breast cancer.     

转铁蛋白修饰脂质体的制备及其肝癌靶向性研究

目的：肿瘤的靶向治疗是当前研究的热点,肝肿瘤细胞表面有大量的转铁蛋白受体表达,而正常组织较少,因此本研究制备转铁蛋白（TF）修饰的脂质体（TFLPs）,并对其肝肿瘤靶向性进行研究。方法：采用薄膜分散法制备普通脂质体,考察其形态,粒径,电位。通过体外血清稳定性模拟脂质体进入体内后的稳定性。通过HepG2肿瘤细胞对TFLPs的摄取实验考查脂质体与肝癌细胞的亲和力。构建荷瘤裸鼠模型,考查TFLPs在荷瘤裸鼠体内的分布。结果：所制备的TFLPs平均粒径为108．8±9．5nm,Zeta电位为．1．80±0．73mV。学期稳定性试验结果显示,TFLPs在24h内具有良好的血清稳定性。体外细胞摄取实验表明,HepG2细胞对TFLPs的摄取效率是普通长循环脂质体（LPs）的3．4倍。荷瘤裸鼠肝组织和肿瘤组织切片结果显示,TFLPs比LPs具有更好的肿瘤靶向性。结论：该脂质体制备方法简单,与LPs相比,经转铁蛋白修饰可显著提高肿瘤细胞对脂质体的摄取,TFLPs是一种潜在高效的肝癌靶向给药系统。     

Construction and characterization of an anti-asialoglycoprotein receptor single-chain variable-fragment-targeted melittin

Antibody-therapeutic agent conjugation to be delivered specifically to tumor cells is required for many target-based therapeutic strategies. In the present study, a recombinant immunotoxin was constructed by which melittin was fused to an anti-asialoglycoprotein receptor (ASGPR) single-chain variable fragment antibody (C1), and targeting ability and cytolytic efficacy of the fusion protein were studied. Our results suggested that the recombinant 29.4 kDa protein C1M was expressed in Escherichia coli as a soluble style. Binding of C1M to the surface of hepatocellular carcinoma (HCC) cells was confirmed by both immunohistochemistry and flow cytometry assays. C1M kept the hemolytic activity of melittin and exhibited cytolytic capacity to HepG2 cells at a concentration of 1.5 μg/mL, under which erythrocytes would not be lysed. The effects were greatly inhibited by coadministration with asialoorosomucoid, a natural ligand for ASGPR. These results suggested that C1M conferred targeting and ASGPR-specific cytotoxicity to HCC cells. This work makes it possible to further investigate its antihepatoma efficacy in vivo.     

Galactose-decorated pH-responsive nanogels for hepatoma-targeted delivery of oridonin

Nanogels based on the polymers of galactosylated chitosan-graft-poly (N-isopropylacrylamide) (Gal-CS-g-PNIPAm) were used as carriers of oridonin (ORI) for tumor targeting. Three ORI-loaded nanogels with various degrees of galactose substitution were prepared, and their characteristics were evaluated. The release behavior of ORI from these nanogels was pH-dependent, and the release could be accelerated under mildly acidic conditions. The cytotoxicity of ORI-loaded nanogels was pH-sensitive. ORI-loaded nanogels exhibited a higher antitumor activity than drug-loaded nanogels without galactosylation, and the anticancer activity increased in relation to increases in the number of galactose moieties of the nanogels in HepG2 cells. In contrast, the cytotoxicity of ORI-loaded nanogels against MCF-7 cells decreased compared with that of drug-loaded nanogels without galactosylation. Results demonstrated that these nanogels could enhance the uptake of ORI into HepG2 cells via asialoglycoprotein receptor-mediated endocytosis. These galactose-decorated pH-responsive nanogels were well-suited for targeted drug delivery to liver cancer cells.     

Radiosynthesis and biological evaluation of an fluorine-18 labeled galactose derivative [18F]FPGal for imaging the hepatic asialoglycoprotein receptor

The asialoglycoprotein receptor (ASGPR) is abundantly expressed on the surface of hepatocytes where it recognizes and endocytoses glycoproteins with galactosyl and N-acetylgalactosamine groups. Given its hepatic distribution, the asialoglycoprotein receptor can be targeted by positron imaging agents to study liver function using PET imaging. In this study, the positron imaging agent [¹⁸F]FPGal was designed to specifically target hepatic asialoglycoprotein receptor and its effectiveness was assessed in in vitro and in vivo models. The radiosynthesis of [¹⁸F]FPGal required 50 min with total radiochemical yields of [¹⁸F]FPGal from [¹⁸F]fluoride as 10% (corrected radiochemical yield). The K_d of [¹⁸F]FPGal to ASGPR in HepG2 cells was 1.99 ± 0.05 mM. Uptake values of 0.55% were observed within 30 min of incubation with HepG2 cells, which could be blocked by 200 mM d(+)-galactose (<0.1%). In vivo biodistribution analysis showed that the liver accumulation of [¹⁸F]FPGal at 30 min was 4.47 ± 0.96% ID/g in normal mice compared to 1.33 ± 0.07% ID/g in hepatic fibrotic mice (P < 0.01). Reduced uptake in the hepatic fibrosis mouse models was confirmed through PET/CT images at 30 min. Compared to normal mice, the standard uptake value (SUV) in the hepatic fibrosis mice was significantly lower when assessed through dynamic data collection for 1 h. Therefore, [¹⁸F]FPGal is a feasible PET probe that provide insight into ASGPR related liver disease.     

Hydrophobized triphenyl phosphonium derivatives for the preparation of mitochondriotropic liposomes: choice of hydrophobic anchor influences cytotoxicity but not mitochondriotropic effect

Context: Nanocarrier-based strategies to achieve delivery of bioactives specifically to the mitochondria are being increasingly explored due to the importance of mitochondria in critical cellular processes.

Objective: To test the ability of liposomes modified with newly synthesized triphenylphosphonium (TPP)–phospholipid conjugates and to test their use in overcoming the cytotoxicity of stearyl triphenylphosphonium (STPP)-modified liposomes when used for delivery of therapeutic molecules to the mitochondria.

Methods: TPP–phospholipid conjugates with the dioleoyl, dimyristoyl or dipalmitoyl lipid moieties were synthesized and liposomes were prepared with these conjugates in a 1?mol% ratio. The subcellular distribution of the liposomes was tested by confocal microscopy. Furthermore, the liposomes were tested for their effect on cell viability using a MTS assay, on cell membrane integrity using a lactate dehydrogenase assay and on mitochondrial membrane integrity using a modified JC-1 assay.

Results: The liposomes modified with the new TPP–phospholipid conjugates exhibited similar mitochondriotropism as STPP-liposomes but they were more biocompatible as compared to the STPP liposomes. While the STPP-liposomes had a destabilizing effect on cell and mitochondrial membranes, the liposomes modified with the TPP–phospholipid conjugates did not demonstrate any such effect on biomembranes.

Conclusions: Using phospholipid anchors in the synthesis of TPP–lipid conjugates can provide liposomes that exhibit the same mitochondrial targeting ability as STPP but with much higher biocompatibility.     

Asialoglycoprotein receptor and liposome synergistically mediate the gene transfer into primary rat hepatocytes

Gene transfer into primary rat hepatocytes was performed by employing cationic liposome as DNA carrier and the specific ligand of hepatic asialoglycoprotein receptor (ASGPR), asialofetuin, as liver-targeting ligand. The resuits showed that asialofetuin, when added to the gene transfer complexes, could significantly increase the hepatocyte transfeetion efficiency, and alleviate the cellular toxicity of Lipofectin. Several synthetic ligands of ASGPR (galactosyl albumin) could also increase the transfection efficiency of hepatocyte like asialofetuin. It was proved that ASGPR and cationic liposome could synergistically mediate the gene transfer into primary rat hepatoeytes. This novel gene delivery system provided a safer, more simple and efficient gene transfer method for primary hepatocytes, and showed prospecting application in hepatic gene therapy.     

Synthesis of a novel glycoconjugated chitosan and preparation of its derived nanoparticles for targeting HepG2 cells

In the study, a novel chitosan (CS) derivative conjugated with multiple galactose residues in an antennary fashion (Gal-m-CS) was synthesized. A galactosylated CS (Gal-CS) was also prepared by directly coupling lactobionic acid on CS. Using an iontropic gelation method, CS and the synthesized Gal-CS and Gal-m-CS were used to prepare nanoparticles (CS, Gal-CS, and Gal-m-CS NPs) for targeting hepatoma cells. TEM examinations showed that the morphology of all three types of NPs was spherical in shape. No aggregation or precipitation of NPs in an aqueous environment was observed during storage for all studied groups, as a result of the electrostatic repulsion between the positively charged NPs. Little fluorescence was observed in HepG2 cells after incubation with the FITC-labeled CS NPs. The intensity of fluorescence observed in HepG2 cells incubated with the Gal-m-CS NPs was stronger than that incubated with the Gal-CS NPs. These results indicated that the prepared Gal-m-CS NPs had the highest specific interaction with HepG2 cells among all studied groups, via the ligand-receptor-mediated recognition.     

Liver uptake and hepato-biliary transfer of galactosylated proteins in rats are determined by the extent of galactosylation

The effect of molecular mass and surface density of galactose residues on hepatic uptake and subsequent biliary excretion of galactosylated proteins was investigated in rats. Several proteins with different molecular weights (15-70 kDa) and different numbers of galactose units were synthesized and radiolabeled with 111In. Galactosylated proteins were administered i.v. to anaesthetized rats and samples of plasma and bile were collected for 3 h. Liver was harvested at the end of the experiments and the radioactivity of all samples was measured. Galactosylated proteins accumulated primarily in the liver and 2-10% of the administered dose appeared in the bile, mainly in undegraded form. The hepatic uptake clearance (Cl liver) and biliary excretion rate constant (kbile) of galactosylated proteins were calculated. No direct effect of molecular weight was observed, however, on increasing the galactose density, Cl liver increased from about 4 to 400 ml/h whereas kbile gradually decreased from about 0.057 to 0.007 (h-1). In conclusion, both hepatic uptake and biliary excretion of galactosylated proteins were found to be affected by the extent of galactosylation.     

Biochimica et biophysica acta,vol. 646 (1981)

Endocytosis of asialo-glycoproteins in hepatocytes is mediated by a lectin-like receptor with specificity for d-galactose. Early events of receptor-ligand interactions have been studied by ultrastructural analysis. Hepatocytes were isolated from the rat liver by collagenase perfusion and incubated with a galactosylated electron dense marker (gold-Gal-BSA, galactosylated bovine serum albumin adsorbed onto colloidal gold particles). Initial binding of gold-Gal-BSA particles occurs to receptors diffusely distributed at hepatic microvilli of the former space of Dissé. No lectin activity was found in membrane areas that had formed in situ the region of hepatic cell contact or bile canaliculi. Microaggregation of receptor-ligand complexes is seen as an early consequence of particle binding. Microaggregates contain 2–5 particles and are located outside coated pits. After prolonged incubation larger clusters are formed, these are found associated with coated membrane areas. It is concluded that at least three steps precede the uptake of galactosylated proteins by hepatocytes. These are: (i) binding of ligand at diffusely distributed binding sites; (ii) local microaggregation of receptor-ligand complexes; (iii) formation of larger clusters and association with coated pits.     

Asialoglycoprotein receptor and liposome synergistically mediate the gene transfer into primary rat hepatocytes

Gene transfer into primary rat hepatocytes was performed by employing cationic liposome as DNA carrier and the specific ligand of hepatic asialoglycopmtein receptor (ASGPR), asialofetuin, as liver-targeting ligand. The results showed that asialofetuin, when added to the gene transfer complexes, could significantly increase the hepatocyte transfection efficiency, and alleviate the cellular toxicity of Lipofectin. Several synthetic ligands of ASGPR (galactosyl albumin) could also increase the transfection efficiency of hepatocyte like asialofetuin. It was proved that ASGPR and cationic liposome could synergistically mediate the gene transfer into primary rat hepatocytes. This novel gene delivery system provided a safer, more simple and efficient gene transfer method for primary hepatocytes, and showed prospecting application in hepatic gene therapy.     

Composition,Antigenic Properties and Hepatocyte Surface Expression of the Woodchuck Asialoglycoprotein Receptor

Abstract

We have purified woodchuck hepatic asialoglycoprotein receptor (ASGPR) by ligand affinity chromatography and have identified it as a heterooligomeric complex comprised of two subunits with molecular masses of 40 and 47 kD, designated as woodchuck hepatic lectin 1 and 2 (WHL1 and WHL2), respectively. With the help of antisera generated against the soluble, bioactive woodchuck and rabbit ASGPRs and anti-subunit monospecific antibodies, distinct antigenic specificity of each of the ASGPR polypeptide subunits and interspecies immunologic cross-reactivity of the receptor polypeptides displaying comparable molecular masses were documented. In contrast to the purified woodchuck receptor, WHL2 antigenic reactivity was not identifiable in woodchuck hepatocyte plasma membranes unless the intact membranes were exposed to an asialylated ligand or a soluble membrane fraction was incubated with anti-receptor antibody. These findings imply that both WHL1 and WHL2 are expressed on the hepatocyte surface and contribute to ligand binding, since antibody specific to either subunit blocks ligand attachment. Our results also indicate that ligand binding modifies antigenic properties of the membrane expressed ASGPR.     

In vitro delivery of curcumin with cholesterol-based cationic liposomes

A new cholesterol-based cationic lipid was synthesized; liposomes prepared on its basis were evaluated as drug delivery vehicles for curcumin. Free and liposome-encapsulated curcumin cytotoxicity against HeLa, A549, HepG2, K562 and 1301 cell lines was assessed. Liposomal curcumin with ED₅₀ values ranging from 2.5–10 μM exhibited 2–8 times higher cytotoxicity than free curcumin. The synthetic cholesterol-based cationic lipid also enhanced cellular uptake of curcumin into tested cells. Cationic liposome alone showed low cytotoxicity at high doses with ED₅₀ values of 90–210 μM.     

Galactosylated N-2-hydroxypropyl methacrylamide-b-N-3-guanidinopropyl methacrylamide block copolymers as hepatocyte-targeting gene carriers

As alternatives of viral and cationic lipid gene carriers, cationic polymer-based vectors may provide flexible chemistry for the attachment of targeting moieties. In this report, galactosylated N-2-hydroxypropyl methacrylamide-b-N-3-guanidinopropyl methacrylamide block copolymers (galactosylated HPMA-b-GPMA block copolymers, or abbreviated as GHG) were prepared in order to develop hepatocyte targeting gene transfection carriers. The block copolymers were synthesized by aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization of N-2-hydroxypropyl methacrylamide (HPMA) and N-3-aminopropyl methacrylamide (APMA), followed by galactosylation and guanidinylation. The molecular weight of GHG copolymers determined by static light scattering method was in the range from 48?600 to 76?240 g/mol. In addition, the galactose content in the GPMA block in the copolymers was determined to be 6.5-8.0 mol % according to the sulfuric acid method. The GHG copolymers complexed completely with plasmid DNA (pDNA) to show positive zeta-potential values with diameter 100-250 nm from charge ratio of 4, which demonstrated the excellent DNA condensing ability of guanidino groups. Furthermore, the MTT assay data of GHG/pDNA complexes on HepG2 cells and HeLa cells indicated that GHG copolymers had significantly lower cytotoxicity than PEI. In addition, the copolymers with GPMA component from 30.23% showed higher transfection efficiency than PEI at charge ratio of 12 in HepG2 cells. The result revealed that the conjugation of galactose groups in the copolymers brought asialoglycoprotein-receptor (ASGP-R) mediated transfection. The employing of HPMA component decreased the aggregation of protein in transfection presence of serum. The GHG copolymers combined the advantages of galactose moieties, guanidino groups, and HPMA component might show potential in safe hepatocyte targeting gene therapy.