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Previously, we showed in the human umbilical vein endothelial cells (HUVECs) model that a liposome formulation of melphalan lipophilic prodrug (MlphDG) decorated with selectin ligand tetrasaccharide Sialyl Lewis X (SiaLeX) undergoes specific uptake by activated cells and in an in vivo tumor model causes a severe antivascular effect. Here, we cultured HUVECs in a microfluidic chip and then applied the liposome formulations to study their interactions with the cells in situ under hydrodynamic conditions close to capillary blood flow using confocal fluorescent microscopy. The incorporation of 5 to 10% SiaLeX conjugate in the bilayer of MlphDG liposomes increased their consumption exclusively by activated endotheliocytes. The increase of serum concentration from 20 to 100% in the flow resulted in lower liposome uptake by the cells. To elucidate the possible roles of plasma proteins in the liposome–cell interactions, liposome protein coronas were isolated and analyzed by shotgun proteomics and immunoblotting of selected proteins. Proteomic analysis showed that a gradual increase in SiaLeX content correlated with the overall enrichment of the liposome-associated proteins with several apolipoproteins, including the most positively charged one, ApoC1, and serum amyloid A4, associated with inflammation, on the one hand, and a decrease in the content of bound immunoglobulins, on the other. The article discusses the potential interference of the proteins in the binding of liposomes to selectins of endothelial cells.

To assess the stability and efficiency of liposomes carrying a phospholipase A2-sensitive phospholipid-allocolchicinoid conjugate (aC-PC) in the bilayer, egg phosphatidylcholine and 1-palmitoyl-2-oleoylphosphatidylglycerol-based formulations were tested in plasma protein binding, tubulin polymerization inhibition, and cytotoxicity assays. Liposomes L-aC-PC10 containing 10 mol. % aC-PC in the bilayer bound less plasma proteins and were more stable in 50% plasma within 4 h incubation, according to calcein release and FRET-based assays. Liposomes with 25 mol. % of the prodrug (L-aC-PC25) were characterized by higher storage stability judged by their hydrodynamic radius evolution yet enhanced deposition of blood plasma opsonins on their surface according to SDS-PAGE and immunoblotting. Notably, inhibition of tubulin polymerization was found to require that the prodrug should be hydrolyzed to the parent allocolchicinoid. The L-aC-PC10 and L-aC-PC25 formulations demonstrated similar tubulin polymerization inhibition and cytotoxic activities. The L-aC-PC10 formulation should be beneficial for applications requiring liposome accumulation at tumor or inflammation sites.

Jing Xing,*,1,2 Xiquan Zhang,*,3Zhe Wang,4 Huanqing Zhang,3 Peng Chen,1,2Gaoxin Zhou,5 Chunlong Sun,6 Ning Gu,1,2Min Ji1,21School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, People’s Republic of China; 2School of Biological Science and Medical Engineering and Collaborative Innovation Center of Suzhou Nano Science and Technology, Southeast University, Suzhou 215123, People’s Republic of China; 3Nanjing Institute of Pharmaceutical Research and Development, Chia-Tai Tianqing Pharmaceutical Group Co. Ltd, Nanjing 210023, People’s Republic of China; 4Emergency Department, The Second Affiliated Hospital of Nanjing Medical University, Nanjing 210029, People’s Republic of China; 5School of Biomedical Engineering, Shenzhen University, Shenzhen 518071, People’s Republic of China; 6College of Biological and Environmental Engineering, Binzhou University, Binzhou 256603, People’s Republic of China*These authors contributed equally to this workBackground: SN38 (7-ethyl-10-hydroxy camptothecin), as a potent metabolite of irinotecan, is highly efficacious in cancer treatment. However, the clinical utility of SN38 has been greatly limited due to its undesirable properties, such as poor solubility and low stability.Materials and methods: In order to overcome these weaknesses, moeixitecan, a lipophilic SN38 prodrug containing a SN-38, a trolox, a succinic acid linker, and a hexadecanol chain, was loaded into liposomal nanoparticles by ethanol injection method.Results: Experiments showed that the moeixitecan-loaded liposomal nanoparticles (MLP) with a diameter of 105.10±1.49 nm have a satisfactory drug loading rate (90.54±0.41%), high solubility and stability, and showed sustained release of SN38. Notably, MLP exhibited better antitumor activity against human colon adenocarcinoma cells than irinotecan, a FDA-approved drug for the treatment of advanced colorectal cancer. Furthermore, xenograft model results showed that MLP outperformed irinotecan in terms of pharmacokinetics, in vivo therapeutic efficacy and safety. Finally, we used molecular dynamic simulations to explore the association between the structure of MLP and the physical and functional properties of MLP, moeixitecan molecules in MLP folded themselves inside the hydrocarbon chain of the lipid bilayer, which led an increased acyl chain order of the lipid bilayer, and therefore enhanced the lactone ring stability protecting it from hydrolysis.Conclusion: Our MLP constructing strategy by liposome engineering technology may serve a promising universal approach for the effective and safe delivery of lipophilic prodrug.Keywords: SN38, lipophilic prodrug, liposomes, molecular dynamic simulations, cancer therapy

Previously, a liposomal formulation of a chemotherapeutic agent melphalan (Mlph) incorporated in a fluid lipid bilayer of natural phospholipids in the form of dioleoylglyceride ester (MlphDG) was developed and the antitumor effect was confirmed in mouse models. The formulation composed of egg phosphatidylcholine (ePC), soybean phosphatidylinositol (PI), and MlphDG (8:1:1, by mol) showed stability in human serum for at least 4–5 h. On the contrary, replacing PI with pegylation of the liposomes, promoted fast dissociation of the components from the bilayer. In this work, interactions of MlphDG-liposomes with the most abundant plasma protein—albumin—in function of the presence of PI in the formulation were explored using Fourier transform infrared spectroscopy. The release of MlphDG from the liposomes was studied by asymmetrical flow field-flow fractionation (AF4) using micelles formed by a polyethylene glycol conjugate with phosphatidylethanolamine to mimic the physiological lipid sink like lipoproteins. Our results show that PI actually protects the membrane of MlphDG-liposomes from the protein penetration, presumably due to pairing between the positively charged MlphDG and negatively charged PI, which compensates for the heterogeneity of the lipid bilayer. The AF4 technique also evidences high stability of the formulation as a drug carrier.

The lipophilic prodrug of hydrophobic drugs with well-designed molecular structures can form stable pure prodrug nanoparticles (NPs), but rapid NPs aggregation in plasma greatly restricted their direct use for intravenous chemotherapy. To address this, DSPE-mPEG2000 and Cremophor EL are two of the most widely used lipophilic PEG derivatives to enhance their colloidal stability in plasma. However, their drug delivery performances have never been comparatively studied. Here, a redox-responsive lipophilic prodrug of SN38 was chosen as the model drug for such comparative investigations. We found that Cremophor EL/NPs having a small diameter (∼15 nm) and poor kinetic stability displayed an enhanced cell internalization, higher cytotoxicity and prolonged circulation time as compared with DSPE-mPEG2000/NPs. Most importantly, these superiorities further resulted in a much more potent antitumor activity in CT26 colorectal cancer xenograft, but the increased loss of body weight was also noted. These results suggested that Cremophor EL could be more advantageous than DSPE-mPEG2000 in terms of the improvement of antitumor activity, but the enhanced toxicity warranted further attention in the future study.

Previously, we showed that incorporation of methotrexate (MTX) in the form of a lipophilic prodrug (MTXDG) in 100-nm lipid bilayer liposomes of egg phosphatidylcholine can allow one to reduce toxicity and improve the antitumor efficiency of MTX in a mouse model of T-cell leukemic lymphoma. However, in our hemocompatibility tests in vitro, MTX liposomes caused complement (C) activation, obviously due to binding on the liposome surface and fragmentation of the C3 complement factor. In this work, we studied the interactions of MTX liposomes carrying stabilizing molecules phosphatidylinositol (PI), ganglioside GM1, or a lipid conjugate of N-carboxymethylated oligoglycine (CMG) in the bilayer with subpopulations of human blood leukocytes. Liposomes labeled with BODIPY-phosphatidylcholine were incubated with whole blood (30 min and 1 h, 37C), blood cells were lysed with a hypotonic buffer, and the fluorescence of the liposomes bound but not internalized by the leukocytes was quenched by crystal violet. Cell suspensions were analyzed by flow cytometry. Incorporation of MTXDG dramatically enhanced the phagocytosis of liposomes of any composition by monocytes. Neutrophils consumed much less of the liposomes. Lymphocytes did not accumulate liposomes. The introduction of PI into MTX liposomes practically did not affect the specific consumption of liposomes by monocytes, while CMG was likely to increase the consumption rate regardless of the presence of MTXDG. The GM1 ganglioside presumably shielded MTX liposomes from phagocytosis by one of the monocyte populations and increased the efficiency of monocyte uptake by another population, probably one expressing C3b-binding receptors (C3b was detected on liposomes after incubation with blood plasma). MTX liposomes were shown to have different effects on TNF- production by activated leukocytes, depending on the structure of the stabilizing molecule.

Chan Wu,1,* Yang Zhang,1,2,* Daoqiu Yang,3,* Jinfeng Zhang,4 Juanjuan Ma,1 Dan Cheng,1 Jianming Chen,1,5 Li Deng1 1Department of Pharmaceutics, School of Pharmacy, Second Military Medical University, Shanghai 200433, People’s Republic of China; 2Department of Pharmacy, Shanghai Tenth People’s Hospital, School of Medicine, Tongji University, Shanghai 200072, People’s Republic of China; 3Department of Dermatology, 107th Hospital of PLA, Yantai 264000, People’s Republic of China; 4Department of Traditional Chinese Medicine, Shanghai Hospital of Chinese Integrative Medicine, Shanghai, People’s Republic of China; 5Tasly Diyi Pharmaceutical Group Co., Ltd, Jiangsu 223002, People’s Republic of China *These authors contributed equally to this work Background: Many novel drug delivery systems have been extensively studied to exploit the full therapeutic potential of SN38, which is one of the most potent antitumor analogs of camptothecins (CPTs), whose clinical application is seriously hindered by poor water solubility, low plasmatic stability, and severe toxicity, but results are always unsatisfactory.Methods: In this study, combining the advantages of prodrug and nanotechnology, a lipophilic prodrug of SN38, SN38-PA, was developed by conjugating palmitic acid to SN38 via ester bond at C10 position, and then the lipophilic prodrug was encapsulated into a long-circulating liposomal carrier by film dispersion method.Results: The SN38-PA liposomes were characterized as follows: an average particle size of 80.13 nm, an average zeta potential of -33.53mv, and the entrapment efficiency of 99%. Compared with CPT-11, SN38-PA liposome was more stable in close lactone form, more efficient in conversion rate to SN38, and more potent in cytotoxicity against tumor cells. Pharmacokinetic study showed that SN38-PA liposome had significantly enhanced plasma half-life (t1/2) value of SN38 and increased area under the curve (AUC) of SN38, which was 7.5-fold higher than that of CPT-11. Biodistribution study showed that SN38-PA liposome had more active metabolite SN38 in each tissue. Finally, the pharmacodynamic study showed that SN38-PA liposome had higher antitumor effect with the antitumor inhibition rate of 1.61 times than that of CPT-11.Conclusion: These encouraging data merit further investigation on this novel SN38-PA liposome. Keywords: SN38, lipophilic prodrug, long-circulating liposome, pharmacokinetics, biodistribution, pharmacodynamics, CPT-11

Anna A Alekseeva,1 Ekaterina V Moiseeva,1 Natalia R Onishchenko,1 Ivan A Boldyrev,1 Alexander S Singin,2,† Andrey P Budko,2 Zoya S Shprakh,2 Julian G Molotkovsky,1 Elena L Vodovozova1 1M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, 2N.N. Blokhin Russian Cancer Research Center, the Ministry of Health of the Russian Federation, Moscow, Russian Federation †Alexander S Singin passed away on October 4, 2011 Abstract: In a previous study, a formulation of methotrexate (MTX) incorporated in the lipid bilayer of 100-nm liposomes in the form of diglyceride ester (MTX-DG, lipophilic prodrug) was developed. In this study, first, the interactions of MTX-DG liposomes with various human and mouse tumor cell lines were studied using fluorescence techniques. The liposomes composed of egg phosphatidylcholine (PC)/yeast phosphatidylinositol/MTX-DG, 8:1:1 by mol, were labeled with fluorescent analogs of PC and MTX-DG. Carcinoma cells accumulated 5 times more MTX-DG liposomes than the empty liposomes. Studies on inhibitors of liposome uptake and processing by cells demonstrated that the formulation used multiple mechanisms to deliver the prodrug inside the cell. According to the data from the present study, undamaged liposomes fuse with the cell membrane only 1.5–2 hours after binding to the cell surface, and then, the components of liposomal bilayer enter the cell separately. The study on the time course of plasma concentration in mice showed that the area under the curve of MTX generated upon intravenous injection of MTX-DG liposomes exceeded that of intact MTX 2.5-fold. These data suggested the advantage of using liposomal formulation to treat systemic manifestation of hematological malignancies. Indeed, the administration of MTX-DG liposomes to recipient mice bearing T-cell leukemic lymphoma using a dose-sparing regimen resulted in lower toxicity and retarded lymphoma growth rate as compared with MTX. Keywords: liposomes, methotrexate, lipophilic prodrug, endocytosis, hematological malignancies, leukemia/lymphoma

Anna A Alekseeva,1 Ekaterina V Moiseeva,1 Natalia R Onishchenko,1 Ivan A Boldyrev,1 Alexander S Singin,2,† Andrey P Budko,2 Zoya S Shprakh,2 Julian G Molotkovsky,1 Elena L Vodovozova1 1M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, 2N.N. Blokhin Russian Cancer Research Center, the Ministry of Health of the Russian Federation, Moscow, Russian Federation †Alexander S Singin passed away on October 4, 2011 Abstract: In a previous study, a formulation of methotrexate (MTX) incorporated in the lipid bilayer of 100-nm liposomes in the form of diglyceride ester (MTX-DG, lipophilic prodrug) was developed. In this study, first, the interactions of MTX-DG liposomes with various human and mouse tumor cell lines were studied using fluorescence techniques. The liposomes composed of egg phosphatidylcholine (PC)/yeast phosphatidylinositol/MTX-DG, 8:1:1 by mol, were labeled with fluorescent analogs of PC and MTX-DG. Carcinoma cells accumulated 5 times more MTX-DG liposomes than the empty liposomes. Studies on inhibitors of liposome uptake and processing by cells demonstrated that the formulation used multiple mechanisms to deliver the prodrug inside the cell. According to the data from the present study, undamaged liposomes fuse with the cell membrane only 1.5–2 hours after binding to the cell surface, and then, the components of liposomal bilayer enter the cell separately. The study on the time course of plasma concentration in mice showed that the area under the curve of MTX generated upon intravenous injection of MTX-DG liposomes exceeded that of intact MTX 2.5-fold. These data suggested the advantage of using liposomal formulation to treat systemic manifestation of hematological malignancies. Indeed, the administration of MTX-DG liposomes to recipient mice bearing T-cell leukemic lymphoma using a dose-sparing regimen resulted in lower toxicity and retarded lymphoma growth rate as compared with MTX. Keywords: liposomes, methotrexate, lipophilic prodrug, endocytosis, hematological malignancies, leukemia/lymphoma

Background: SN38 (7-ethyl-10-hydroxy camptothecin), as a potent metabolite of irinotecan, is highly efficacious in cancer treatment. However, the clinical utility of SN38 has been greatly limited due to its undesirable properties, such as poor solubility and low stability. Materials and methods: In order to overcome these weaknesses, moeixitecan, a lipophilic SN38 prodrug containing a SN-38, a trolox, a succinic acid linker, and a hexadecanol chain, was loaded into liposomal nanoparticles by ethanol injection method. Results: Experiments showed that the moeixitecan-loaded liposomal nanoparticles (MLP) with a diameter of 105.10±1.49 nm have a satisfactory drug loading rate (90.54±0.41%), high solubility and stability, and showed sustained release of SN38. Notably, MLP exhibited better antitumor activity against human colon adenocarcinoma cells than irinotecan, a FDA-approved drug for the treatment of advanced colorectal cancer. Furthermore, xenograft model results showed that MLP outperformed irinotecan in terms of pharmacokinetics, in vivo therapeutic efficacy and safety. Finally, we used molecular dynamic simulations to explore the association between the structure of MLP and the physical and functional properties of MLP, moeixitecan molecules in MLP folded themselves inside the hydrocarbon chain of the lipid bilayer, which led an increased acyl chain order of the lipid bilayer, and therefore enhanced the lactone ring stability protecting it from hydrolysis. Conclusion: Our MLP constructing strategy by liposome engineering technology may serve a promising universal approach for the effective and safe delivery of lipophilic prodrug.

Chan Wu,1,* Yang Zhang,1,2,* Daoqiu Yang,3,* Jinfeng Zhang,4 Juanjuan Ma,1 Dan Cheng,1 Jianming Chen,1,5 Li Deng1 1Department of Pharmaceutics, School of Pharmacy, Second Military Medical University, Shanghai 200433, People’s Republic of China; 2Department of Pharmacy, Shanghai Tenth People’s Hospital, School of Medicine, Tongji University, Shanghai 200072, People’s Republic of China; 3Department of Dermatology, 107th Hospital of PLA, Yantai 264000, People’s Republic of China; 4Department of Traditional Chinese Medicine, Shanghai Hospital of Chinese Integrative Medicine, Shanghai, People’s Republic of China; 5Tasly Diyi Pharmaceutical Group Co., Ltd, Jiangsu 223002, People’s Republic of China *These authors contributed equally to this work Background: Many novel drug delivery systems have been extensively studied to exploit the full therapeutic potential of SN38, which is one of the most potent antitumor analogs of camptothecins (CPTs), whose clinical application is seriously hindered by poor water solubility, low plasmatic stability, and severe toxicity, but results are always unsatisfactory.Methods: In this study, combining the advantages of prodrug and nanotechnology, a lipophilic prodrug of SN38, SN38-PA, was developed by conjugating palmitic acid to SN38 via ester bond at C10 position, and then the lipophilic prodrug was encapsulated into a long-circulating liposomal carrier by film dispersion method.Results: The SN38-PA liposomes were characterized as follows: an average particle size of 80.13 nm, an average zeta potential of -33.53mv, and the entrapment efficiency of 99%. Compared with CPT-11, SN38-PA liposome was more stable in close lactone form, more efficient in conversion rate to SN38, and more potent in cytotoxicity against tumor cells. Pharmacokinetic study showed that SN38-PA liposome had significantly enhanced plasma half-life (t1/2) value of SN38 and increased area under the curve (AUC) of SN38, which was 7.5-fold higher than that of CPT-11. Biodistribution study showed that SN38-PA liposome had more active metabolite SN38 in each tissue. Finally, the pharmacodynamic study showed that SN38-PA liposome had higher antitumor effect with the antitumor inhibition rate of 1.61 times than that of CPT-11.Conclusion: These encouraging data merit further investigation on this novel SN38-PA liposome. Keywords: SN38, lipophilic prodrug, long-circulating liposome, pharmacokinetics, biodistribution, pharmacodynamics, CPT-11

A monoclonal antibody against the rat colon carcinoma CC531 was covalently coupled to liposomes containing a dipalmitoylated derivative of the anticancer drug FUdR as a prodrug in their bilayers. We investigated the in vitro interaction of these liposomes with CC531 target cells and the mechanism by which they deliver the active drug FUdR intracellularly to the cells by monitoring the fate of the liposomal bilayer markers cholesterol-[C-14]oleate and [H-3]cholesteryloleylether as well as the H-3-labeled prodrug and colloidal gold as an encapsulated liposome marker. After binding of the immunoliposomes to the cell surface, only limited amounts were internalized as demonstrated by a low level of hydrolysis of liposomal cholesterol ester and by morphological studies employing colloidal gold-labeled immunoliposomes. By contrast, already within 24 h immunoliposome-incorporated FUdR-dP was hydrolyzed virtually completely to the parent drug FUdR intracellularly. This process was inhibited by a variety of endocytosis inhibitors, indicating that the prodrug enters and is processed by the cells by a mechanism involving an endocytic process, resulting in intracellular FUdR concentrations up to 3000-fold higher than those in the medium. Immunoliposomes containing poly(ethyleneglycol) (PEG) chains on their surface, with the antibody coupled either directly to the bilayer or at the distal end of the PEG chains were able to deliver the prodrug into the tumor cells at the same rate as immunoliposomes without PEG. Based on these observations, we tentatively conclude that during the interaction of the immunoliposomes with the tumor cells the lipophilic prodrug FUdR-dP is selectively transferred to the cell surface and subsequently internalized by constitutive endocytic or pinocytic invaginations of the plasma membrane, thus ultimately delivering the prodrug to a lysosomal compartment where hydrolysis and release of parent drug takes place. This concept allows for an efficient delivery of a liposome-associated drug without the need for the liposome as such to be internalized by the cells. (C) 1999 Elsevier Science B.V. All rights reserved.

Interactions between paclitaxel and its squalenoyl prodrug with dimyristoylphosphatidylcholine (DMPC) monolayer at the air/water interface were studied. Paclitaxel is an antineoplastic drug, largely used as anti-cancer agents. Because its low aqueous solubility, Cremophor EL is used as excipient for its formulation. However, it has been shown that Cremophor causes serious side effects. Several attempts have been made to develop a safer formulation such as the synthesis of lipophilic prodrug. In particular we have synthesized a paclitaxel prodrug obtained by conjugation with 1,1,2-trisnorsqualenoic acid to improve the physico-chemical properties of this antineoplastic drug. The miscibility of these compounds with DMPC monolayer were studied analyzing thermodynamic properties as well as excess Gibbs free energies, compressibility modulus and mixed monolayer isotherms. The results allowed to evaluate the spatial organization of the compounds and suggested that the prodrug can efficiently be incorporated in the DMPC monolayer.

A previous study has shown that lipophilic prodrugs can be delivered efficiently to normal lung endothelium by incorporation into liposomes covalently conjugated to monoclonal antibody (mAb) 34A against the lung endothelial anticoagulant protein thrombomodulin. In the present study, the potential use of these lung-targeted immunoliposomes (34A-liposomes) for delivery of a lipophilic prodrug, 3',5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine (dpFUdR), to the tumor-bearing lung was examined using BALB/c mice bearing experimental lung metastasis induced by i.v. injection of EMT-6 mouse mammary tumor cells. Immunohistochemical examination of the tumor-bearing lung showed specificity of mAb 34A to lung endothelium. Tumor cells appeared to localize just outside of the normal blood vessels and were within a small diffusion distance from the mAb 34A-binding sites. In-111-labeled 34A-liposomes containing monosialoganglioside (G(M1)) were prepared that included [H-3]-dpFUdR at 3.0 mol% in the lipid mixture. In vitro cell binding studies further demonstrated that 34A-liposomes bound specifically to normal mouse lung cells that expressed thrombomodulin but not to EMT-6 cells. Biodistribution study showed efficient and immunospecific accumulation of [H-3]-dpFUdR incorporated into 34A-liposomes in the lung at a level parallel with that of In-111-labeled 34A-liposomes, indicating that the drug is delivered to the target organ in intact liposomes. Liposomal dpFUdR appeared to be metabolized in the lung to the parent drug FUdR at a rate slower than in the liver and spleen, Furthermore, treatment of lung-metastasis-bearing mice with dpFUdR incorporated into 34A-liposomes on days 1 and 3 after tumor cell injection resulted in a significant increase in the median survival time of treated mice as compared with control mice (%T/C value, 165%). dpFUdR either dispersed in emulsion or incorporated into antibody-free liposomes was ineffective in prolonging the survival of mice. These results indicate the potential effectiveness of organ-specific immunoliposomes containing a lipophilic prodrug for the targeted therapy of metastatic tumors.

A previous study has shown that lipophilic prodrugs can be delivered efficiently to normal lung endothelium by incorporation into liposomes covalently conjugated to monoclonal antibody (mAb) 34A against the lung endothelial anticoagulant protein thrombomodulin. In the present study, the potential use of these lung-targeted immunoliposomes (34A-liposomes) for delivery of a lipophilic prodrug, 3',5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine (dpFUdR), to the tumor-bearing lung was examined using BALB/c mice bearing experimental lung metastasis induced by i.v. injection of EMT-6 mouse mammary tumor cells. Immunohistochemical examination of the tumor-bearing lung showed specificity of mAb 34A to lung endothelium. Tumor cells appeared to localize just outside of the normal blood vessels and were within a small diffusion distance from the mAb 34A-binding sites. In-111-labeled 34A-liposomes containing monosialoganglioside (G(M1)) were prepared that included [H-3]-dpFUdR at 3.0 mol% in the lipid mixture. In vitro cell binding studies further demonstrated that 34A-liposomes bound specifically to normal mouse lung cells that expressed thrombomodulin but not to EMT-6 cells. Biodistribution study showed efficient and immunospecific accumulation of [H-3]-dpFUdR incorporated into 34A-liposomes in the lung at a level parallel with that of In-111-labeled 34A-liposomes, indicating that the drug is delivered to the target organ in intact liposomes. Liposomal dpFUdR appeared to be metabolized in the lung to the parent drug FUdR at a rate slower than in the liver and spleen, Furthermore, treatment of lung-metastasis-bearing mice with dpFUdR incorporated into 34A-liposomes on days 1 and 3 after tumor cell injection resulted in a significant increase in the median survival time of treated mice as compared with control mice (%T/C value, 165%). dpFUdR either dispersed in emulsion or incorporated into antibody-free liposomes was ineffective in prolonging the survival of mice. These results indicate the potential effectiveness of organ-specific immunoliposomes containing a lipophilic prodrug for the targeted therapy of metastatic tumors.

A monoclonal antibody against the rat colon carcinoma CC531 was covalently coupled to liposomes containing a dipalmitoylated derivative of the anticancer drug FUdR as a prodrug in their bilayers. We investigated the in vitro interaction of these liposomes with CC531 target cells and the mechanism by which they deliver the active drug FUdR intracellularly to the cells by monitoring the fate of the liposomal bilayer markers cholesterol-[C-14]oleate and [H-3]cholesteryloleylether as well as the H-3-labeled prodrug and colloidal gold as an encapsulated liposome marker. After binding of the immunoliposomes to the cell surface, only limited amounts were internalized as demonstrated by a low level of hydrolysis of liposomal cholesterol ester and by morphological studies employing colloidal gold-labeled immunoliposomes. By contrast, already within 24 h immunoliposome-incorporated FUdR-dP was hydrolyzed virtually completely to the parent drug FUdR intracellularly. This process was inhibited by a variety of endocytosis inhibitors, indicating that the prodrug enters and is processed by the cells by a mechanism involving an endocytic process, resulting in intracellular FUdR concentrations up to 3000-fold higher than those in the medium. Immunoliposomes containing poly(ethyleneglycol) (PEG) chains on their surface, with the antibody coupled either directly to the bilayer or at the distal end of the PEG chains were able to deliver the prodrug into the tumor cells at the same rate as immunoliposomes without PEG. Based on these observations, we tentatively conclude that during the interaction of the immunoliposomes with the tumor cells the lipophilic prodrug FUdR-dP is selectively transferred to the cell surface and subsequently internalized by constitutive endocytic or pinocytic invaginations of the plasma membrane, thus ultimately delivering the prodrug to a lysosomal compartment where hydrolysis and release of parent drug takes place. This concept allows for an efficient delivery of a liposome-associated drug without the need for the liposome as such to be internalized by the cells. (C) 1999 Elsevier Science B.V. All rights reserved.

A monoclonal antibody against the rat colon carcinoma CC531 was covalently coupled to liposomes containing a dipalmitoylated derivative of the anticancer drug FUdR as a prodrug in their bilayers. We investigated the in vitro interaction of these liposomes with CC531 target cells and the mechanism by which they deliver the active drug FUdR intracellularly to the cells by monitoring the fate of the liposomal bilayer markers cholesterol-[14C]oleate and [3H]cholesteryloleylether as well as the 3H-labeled prodrug and colloidal gold as an encapsulated liposome marker. After binding of the immunoliposomes to the cell surface, only limited amounts were internalized as demonstrated by a low level of hydrolysis of liposomal cholesterol ester and by morphological studies employing colloidal gold-labeled immunoliposomes. By contrast, already within 24 h immunoliposome-incorporated FUdR-dP was hydrolyzed virtually completely to the parent drug FUdR intracellularly. This process was inhibited by a variety of endocytosis inhibitors, indicating that the prodrug enters and is processed by the cells by a mechanism involving an endocytic process, resulting in intracellular FUdR concentrations up to 3000-fold higher than those in the medium. Immunoliposomes containing poly(ethyleneglycol) (PEG) chains on their surface, with the antibody coupled either directly to the bilayer or at the distal end of the PEG chains were able to deliver the prodrug into the tumor cells at the same rate as immunoliposomes without PEG. Based on these observations, we tentatively conclude that during the interaction of the immunoliposomes with the tumor cells the lipophilic prodrug FUdR-dP is selectively transferred to the cell surface and subsequently internalized by constitutive endocytic or pinocytic invaginations of the plasma membrane, thus ultimately delivering the prodrug to a lysosomal compartment where hydrolysis and release of parent drug takes place. This concept allows for an efficient delivery of a liposome-associated drug without the need for the liposome as such to be internalized by the cells.

A previous study has shown that lipophilic prodrugs can be delivered efficiently to normal lung endothelium by incorporation into liposomes covalently conjugated to monoclonal antibody (mAb) 34A against the lung endothelial anticoagulant protein thrombomodulin. In the present study, the potential use of these lung-targeted immunoliposomes (34A-liposomes) for delivery of a lipophilic prodrug, 3',5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine (dpFUdR), to the tumor-bearing lung was examined using BALB/c mice bearing experimental lung metastasis induced by i.v. injection of EMT-6 mouse mammary tumor cells. Immunohistochemical examination of the tumor-bearing lung showed specificity of mAb 34A to lung endothelium. Tumor cells appeared to localize just outside of the normal blood vessels and were within a small diffusion distance from the mAb 34A-binding sites. In-111-labeled 34A-liposomes containing monosialoganglioside (G(M1)) were prepared that included [H-3]-dpFUdR at 3.0 mol% in the lipid mixture. In vitro cell binding studies further demonstrated that 34A-liposomes bound specifically to normal mouse lung cells that expressed thrombomodulin but not to EMT-6 cells. Biodistribution study showed efficient and immunospecific accumulation of [H-3]-dpFUdR incorporated into 34A-liposomes in the lung at a level parallel with that of In-111-labeled 34A-liposomes, indicating that the drug is delivered to the target organ in intact liposomes. Liposomal dpFUdR appeared to be metabolized in the lung to the parent drug FUdR at a rate slower than in the liver and spleen, Furthermore, treatment of lung-metastasis-bearing mice with dpFUdR incorporated into 34A-liposomes on days 1 and 3 after tumor cell injection resulted in a significant increase in the median survival time of treated mice as compared with control mice (%T/C value, 165%). dpFUdR either dispersed in emulsion or incorporated into antibody-free liposomes was ineffective in prolonging the survival of mice. These results indicate the potential effectiveness of organ-specific immunoliposomes containing a lipophilic prodrug for the targeted therapy of metastatic tumors.