Combined radiofrequency ablation and adjuvant liposomal chemotherapy: effect of chemotherapeutic agent, nanoparticle size, and circulation time.

PURPOSE: To evaluate the effects of liposomal chemotherapeutic agent, nanoparticle size, and liposome circulation time on tissue coagulation and intratumoral drug uptake when radiofrequency (RF) ablation is combined with adjuvant intravenous liposomal chemotherapy in an animal breast tumor model. MATERIALS AND METHODS: Ninety-one R3230 mammary adenocarcinoma nodules were implanted in 48 Fischer rats. First, standardized RF ablation was combined with intravenous liposomal doxorubicin, cisplatin, or 5-fluorouracil (35 tumors each). Second, three different-sized doxorubicin-containing nanoparticle preparations were combined with standardized RF ablation. Last, two doxorubicin-containing liposome preparations with different blood elimination half-lives were combined with RF ablation. Coagulation diameter and interstitial doxorubicin concentration were measured 48 hours after treatment and compared with use of statistical analysis. RESULTS: All combinations of RF with liposomal chemotherapy caused significantly greater tumor necrosis than RF alone (P<.05). Significantly increased necrosis was observed with intravenous liposomal RF/doxorubicin and RF/cisplatin compared with intravenous liposomal RF/5-fluorouracil (P<.01). Greater coagulation was observed with RF combined with 100-nm nanoparticles compared with 20-nm or 250-nm nanoparticles (P=.01 and P=.04, respectively). Additionally, greater intratumoral doxorubicin uptake was observed in the group treated with 20-nm nanoparticles compared with those treated with other sizes of nanoparticles (P<.05). RF plus liposomal doxorubicin produced greater coagulation and intratumoral doxorubicin uptake than RF plus 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (P<.05). CONCLUSION: When combined with RF ablation, modification of adjuvant intravenous liposomal chemotherapy, including nanoparticle size, circulation time, and chemotherapeutic agent, can influence intratumoral drug accumulation and tissue coagulation.

Combination radiofrequency ablation with intratumoral liposomal doxorubicin: effect on drug accumulation and coagulation in multiple tissues and tumor types in animals.

PURPOSE: To determine whether use of radiofrequency (RF) ablation combined with intravenously (IV) administered liposomal doxorubicin, as compared with use of RF ablation or doxorubicin alone, facilitates increased tissue coagulation and interstitial drug accumulation in animal models. MATERIALS AND METHODS: The institutional animal care and use committee approved this study. In experiment 1, multiple canine sarcomas were implanted in seven mildly immunosuppressed dogs and grown to a mean diameter of 4.8 cm. Tumors were assigned to three treatment groups: internally cooled RF ablation (12 minutes, 2000-mA pulsed technique) followed by IV liposomal doxorubicin (10 mg per animal) (n = 6), RF ablation alone (n = 6), and liposomal doxorubicin alone (n = 4). In experiment 2, the livers and kidneys of 10 rabbits and the thigh muscles of 10 rats were randomly assigned to one of two treatment groups: conventional RF ablation (90 degrees C +/- 2, 5 minutes) followed by IV liposomal doxorubicin (5 mg per rabbit, 1 mg per rat) or RF ablation alone (n = 5, each). Coagulation diameter and interstitial doxorubicin concentration (tissues were homogenized in acid alcohol, with doxorubicin extracted for 24 hours at 5 degrees C and quantified with fluorimetry) were measured 48 hours after treatment and compared. Multivariate analysis of variance and subsequent pairwise t tests (alpha = .05, two-tailed test) were performed. RESULTS: Data are means +/- standard errors of the mean. A larger diameter of tumor destruction was observed in canine sarcomas treated with RF ablation-liposomal doxorubicin (3.7 cm +/- 0.6) compared with that in tumors treated with RF ablation (2.3 cm +/- 0.1) or liposomal doxorubicin (0.0 cm +/- 0.0) alone (P < .01). A new finding was a completely necrotic red zone (1.6 cm +/- 0.7) surrounding the central RF ablation-induced white coagulation zone. Greater but nonuniform drug uptake was observed particularly in this red zone (77.0 ng/g +/- 18.2) compared with uptake in the central zone (15.1 ng/g +/- 3.2), peripheral area of untreated tumor (38.9 ng/g +/- 8.0), and tumors treated with liposomal doxorubicin alone (43.9 ng/g +/- 6.7 for all regions) (P < .01 for all individual comparisons). In experiment 2, use of combined therapy led to increased coagulation in all tissues (liver: 17.6 mm +/- 3.1, P = .03; kidney: 11.0 mm +/- 3.1, P = .03; muscle: 13.1 mm +/- 1.3, P < .01) compared with use of RF ablation alone (liver, 13.4 mm +/- 1.5; kidney, 7.9 mm +/- 0.7; muscle, 8.6 mm +/- 0.5). Combined therapy, as compared with liposomal doxorubicin therapy alone, was also associated with increased doxorubicin accumulation in liver, kidney, and muscle (1.56 microg/g +/- 0.34, 4.36 microg/g +/- 1.78, and 3.63 microg/g +/- 1.43, respectively, vs 1.00 microg/g +/- 0.18, 1.23 microg/g +/- 0.32, and 0.87 microg/g +/- 0.53, respectively) (P < or = .01 for all individual comparisons). CONCLUSION: Use of RF ablation combined with liposomal doxorubicin facilitates increased tissue coagulation and interstitial doxorubicin accumulation in multiple tissues and tumor types and may be useful for treatment of large tumors and achieving an ablative margin within the untreated tissue surrounding RF ablation-treated tumors.

Tumor-targeted liposomes: doxorubicin-loaded long-circulating liposomes modified with anti-cancer antibody.

Commercially available doxorubicin-loaded long-circulating liposomes (Doxil, Alza Pharmaceuticals) were modified with the monoclonal nucleosome (NS)-specific 2C5 antibody (mAb 2C5) that recognizes a broad variety of tumors via the tumor cell surface-bound NSs. For incorporation into liposomes, mAb 2C5 was modified with poly(ethylene glycol)-phosphatidyl ethanolamine conjugate (PEG-PE) with the free PEG terminus activated with the p-nitrophenylcarbonyl group (pNP-PEG-PE). Derivatives of mAb 2C5 containing a variable number of PEG-PE residues (10-32) per protein molecule were prepared with a reasonably good preservation of the antibody specific activity even at the highest degree of modification. PEG-PE-modified antibody quantitatively incorporated into the liposomal membrane of doxorubicin-loaded liposomes with a loss of not more than 20% of the encapsulated doxorubicin. 2C5-targeted Doxil liposomes acquired the ability to recognize NSs and specifically bind to various tumor cells. Doxorubicin-loaded long-circulating liposomes modified with the mAb 2C5 kill various tumor cells in vitro with the efficiency higher than non-targeted doxorubicin-loaded liposomes.

PEGylated dextran as long-circulating pharmaceutical carrier.

Dextran-polyethylene glycol (PEG) conjugates were synthesized by activating dextran hydroxy groups with carbonyldiimidazole, introducing amino groups by attaching ethylenediamine, and reacting amino groups with a succinimidyl-activated derivative of PEG. Conjugates with an average of 12 and 21 PEG (5 kDa) residues per single dextran (73 kDa) molecule were prepared. These conjugates have circulation half-lives of 5.3 h and 7.0 h, respectively, compared to 4.0 h for non-PEGylated dextran. The modification of dextran with PEG inhibits the uptake of polymer by the major organ of the reticuloendothelial system, the liver. Dextran-PEG conjugates may represent a convenient platform for long-circulating pharmaceutical preparations.

Micelles from lipid derivatives of water-soluble polymers as delivery systems for poorly soluble drugs.

Polymeric micelles have a whole set of unique characteristics, which make them very promising drug carriers, in particular, for poorly soluble drugs. Our review article focuses on micelles prepared from conjugates of water-soluble polymers, such as polyethylene glycol (PEG) or polyvinyl pyrrolidone (PVP), with phospholipids or long-chain fatty acids. The preparation of micelles from certain polymer-lipid conjugates and the loading of these micelles with various poorly soluble anticancer agents are discussed. The data on the characterization of micellar preparations in terms of their morphology, stability, longevity in circulation, and ability to spontaneously accumulate in experimental tumors via the enhanced permeability and retention (EPR) effect are presented. The review also considers the preparation of targeted immunomicelles with specific antibodies attached to their surface. Available in vivo results on the efficiency of anticancer drugs incorporated into plain micelles and immunomicelles in animal models are also discussed.

Preparation and in vitro synergistic anticancer effect of vitamin K3 and 1,8-diazabicyclo[5,4,0]undec-7-ene in poly(ethylene glycol)-diacyllipid micelles.

Polymeric micelles consisting of poly(ethylene glycol)-distearoyl phosphoethanolamine conjugates (PEG-DSPE) loaded with Vitamin K3 (VK3) to 0.2 mg of drug/mg of carrier and with 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) to 0.06 mg of drug/mg of carrier were prepared. These micelles were stable for as long as 6 months during storage at 4 degrees C and did not change their size or release the incorporated drugs. Co-encapsulation of VK3 and DBU into PEG-DSPE micelles resulted in synergistic anticancer effects against both murine and human cancer cells in vitro. The synergism may be explained by the fact that the presence of DBU promotes the escape of drug-loaded micelles from the endosomes of cancer cells directly into the cytoplasm as demonstrated by fluorescent microscopy.

Improving microcirculation is more effective than substitution of red blood cells to correct metabolic disorder in experimental hemorrhagic shock.

Microcirculatory perfusion deficits and impaired tissue oxygenation in nonvital organs frequently occur after hemorrhage and they contribute to potentially lethal complications. The aim of this study was to test the influence of colloid osmotic pressure, viscosity, and red blood cell (RBC) content of the resuscitative fluid on metabolic disorder, perfusion, and oxygenation in peripheral tissues. Awake hamsters were subjected to hemorrhage of 50% and were resuscitated with 25% of blood volume with solutions containing 6% pegylated bovine albumin only (PEG-BSA 0) and 6% PEG-BSA mixed with autologous RBCs to reach 4 g/dL (PEG-BSA 4) and 8 g/dL (PEG-BSA 8) of hemoglobin. PEG-BSA had a viscosity of 4.2 cP and a COP of 116 mmHg. Microhemodynamics and tissue pO2 were assessed in the hamster chamber window preparation with intravital microscopy. Arterial base excess tended to be lower than baseline for PEG-BSA 0 and PEG-BSA 4 (ns), whereas base deficit remained significantly decreased for PEG-BSA 8 (P<0.05 vs. baseline). Oxygen extraction was 91% +/- 2% of the oxygen delivery for PEG-BSA 0 compared with 85% +/- 2% for PEG-BSA 8 (P<0.05). Functional capillary density was 61%, 47%, and 45% for PEG-BSA 0 (P<0.05 vs. other groups), PEG-BSA 4 and PEG-BSA 8, respectively. We conclude that arterial base excess and oxygen extraction ratio in the tissue was better restored if a higher fraction of PEG-BSA and less RBCs were infused. This was attributed to a more homogeneous distribution of oxygen, as reflected by functional capillary density. Our results suggest that the transfusion trigger in hemorrhagic shock may be shifted toward lower hemoglobin concentrations if highly viscous and oncotic solutions are used.

Increased accumulation of PEG-PE micelles in the area of experimental myocardial infarction in rabbits.

Micelles prepared from polyethyleneglycol/phosphatidyl-ethanolamine conjugates (PEG-PE) with a size of 7-20 nm and zeta-potential of approximately -18 mV were administered i.v. to rabbits with experimental myocardial infarctions. Micelles demonstrated a prolonged circulation in the blood (half-life of 2 h) and accumulated in the infarction zone with efficiency more than 8-fold higher as compared to a non-damaged part of the heart muscle. Obtained results suggest that the enhanced permeability and retention (EPR) effect is the primary mechanism of accumulation of microparticles in the infarct areas, and that drug carriers such as PEG-PE micelles can be used for the delivery of therapeutic or diagnostic agents to an area of myocardial infarction.

Micelles from polyethylene glycol/phosphatidylethanolamine conjugates for tumor drug delivery.

Micelles prepared from polyethylene glycols of various lengths conjugated with phosphatidylethanolamine (PEG-PE) were loaded with various poorly soluble anticancer agents. PEG-PE micelles selectively accumulated in Lewis lung carcinoma (LLC) tumors implanted in mice. Modification of the micelles with tumor specific antibodies further enhanced the efficiency of tumor accumulation.

Immunomicelles: targeted pharmaceutical carriers for poorly soluble drugs.

To prepare immunomicelles, new targeted carriers for poorly soluble pharmaceuticals, a procedure has been developed to chemically attach mAbs to reactive groups incorporated into the corona of polymeric micelles made of polyethylene glycol-phosphatidylethanolamine conjugates. Micelle-attached antibodies retained their ability to specifically interact with their antigens. Immunomicelles with attached antitumor mAb 2C5 effectively recognized and bound various cancer cells in vitro and showed an increased accumulation in experimental tumors in mice when compared with nontargeted micelles. Intravenous administration of tumor-specific 2C5 immunomicelles loaded with a sparingly soluble anticancer agent, taxol, into experimental mice bearing Lewis lung carcinoma resulted in an increased accumulation of taxol in the tumor compared with free taxol or taxol in nontargeted micelles and in enhanced tumor growth inhibition. This family of pharmaceutical carriers can be used for the solubilization and enhanced delivery of poorly soluble drugs to various pathological sites in the body.

Peptide and protein drug delivery to and into tumors: challenges and solutions.

The potential of peptide and protein anticancer agents has yet to be realized owing to the many unresolved problems concerning their delivery to the site of a tumor and into tumor cells. However, our understanding of the mechanisms underlying the biological fate and biodistribution of protein and peptide drugs has advanced to the stage where methods that use or influence these mechanisms are now available. There are different approaches that can improve the stability, longevity and targeting of peptides and proteins in the body, such as their modification with various soluble polymers, incorporation into microparticular drug carriers, enhanced permeability and retention effect-based tumor targeting and the use of targeting moieties. Furthermore, new approaches to intracellular drug delivery, including the use of transduction proteins and peptides, are being developed. These advances promise the delivery of a new generation of anticancer drugs.

Polyethylene glycol-diacyllipid micelles demonstrate increased acculumation in subcutaneous tumors in mice.

PURPOSE: The purpose of this work is to study the potential of micelles prepared from amphiphilic polyethelene glycol/phosphatidylethanolamine (PEG-PE) conjugates as a particulate drug delivery system capable of accumulation in tumors via the enhanced permeability and retention (EPR) effect. METHODS: Micelles were prepared from PEGs of different molecular lengths conjugated with PE. The micelles were characterized by fluorescence-based critical micellization concentration (CMC) measure ments, dynamic light scattering, and HPLC. Blood clearance an tumor accumulation of 111In-labeled micelles were studied in mic with subcutaneously established Lewis lung carcinoma (LLC) and EL4 T lymphoma (EL4) tumors. RESULTS: Various versions of PEG-PE conjugates with PEG blocks ranging from 750 to 5000 Da formed very stable low CMC micelles at all concentrations down to 10(-5) M. The size of the micelles varie between 7 and 35 nm depending on the length of the PEG block. Micelles remained intact after prolonged incubation with the blood serum. Upon intravenous administration into mice, the micelles demonstrated circulation longevity, and they efficiently and selectively accumulated in both subcutaneous Lewis lung carcinoma and EL4 T lymphoma tumors. CONCLUSIONS: PEG-PE conjugates form very stable, long-circulating micelles. These micelles efficiently accumulate in tumors in vivo an may potentially be used as a tumor-specific delivery system for poorly soluble anticancer drugs.

Radio-frequency ablation increases intratumoral liposomal doxorubicin accumulation in a rat breast tumor model.

PURPOSE: To determine whether intratumoral accumulation of liposomal doxorubicin or free unencapsulated doxorubicin is increased when combined with radio-frequency (RF) ablation. MATERIALS AND METHODS: Two 1.2-1.5-cm R3230 mammary adenocarcinomas were grown within the mammary fat pads of 19 female Fischer rats. One tumor of each pair was treated with RF ablation (tip temperature, 70 degrees C +/- 2 [SD]; 120 mA +/- 75) for 5 minutes, whereas the other tumor was a control. Intravenous liposomal doxorubicin (1 mg in 500 micro L, n = 6) or intravenous free unencapsulated doxorubicin (n = 7) was administered immediately following RF ablation. Doxorubicin was extracted in acid alcohol from tumors 24 hours following RF ablation, and fluorescent spectrophotometry was used to quantify extracted doxorubicin. Comparisons of intratumoral doxorubicin accumulation in tumors treated with RF ablation and in untreated tumors were analyzed with parametric (paired Student t test) and nonparametric (Wilcoxon rank sum test) statistics. Findings at autoradiography with densitometry (six additional tumors) demonstrated the spatial distribution of the intratumoral accumulation of liposomal doxorubicin. RESULTS: When RF ablation preceded administration of liposomal doxorubicin, mean intratumoral doxorubicin concentration was 5.6 micro g/g +/- 2.1 (range, 1.9-7.7 micro g/g), whereas 1.0 micro g/g +/- 0.4 (range, 0.5-1.5 micro g/g) was present in control tumors not treated with RF ablation (P <.05). Thus, there was a mean 7.1-fold +/- 4.9 increase in intratumoral doxorubicin accumulation following RF ablation (range, 2.1-14.5-fold) compared with the amount without RF pretreatment (P <.05). Increased intratumoral accumulation was not seen in animals receiving free doxorubicin with (mean, 0.4 micro g/g +/- 0.1) or without (mean, 0.8 micro g/g +/- 0.4) RF pretreatment (P =.07). Autoradiographic findings demonstrated accumulation of liposomal doxorubicin in a peripheral rim of tumor adjacent to the zone of coagulation. CONCLUSION: RF ablation augments the delivery of systemic antineoplastic agents such as liposomal doxorubicin.

Liposome clearance in mice: the effect of a separate and combined presence of surface charge and polymer coating.

The purpose of our work was to compare the biodistribution of liposomes with different surface properties. Phosphatidylcholine (PC)/cholesterol (Chol) liposomes were prepared containing 6% mol of a charged lipid (stearylamine, SA; phosphatidic acid, PA; or phosphatidyl serine, PS) and/or polyethylene glycol (PEG)-PE of different MW (750 and 5000). zeta-Potentials and liposome clearance in mice were investigated. In vitro, the attachment of PEG in a similar fashion neutralizes the effect of any charged component. In vivo, the chemical nature of a charged lipid becomes important. Both short PEG750 and longer PEG5000 inhibit the clearance of positively charged SA-liposomes, while only longer PEG5000 inhibits the clearance of negatively charged PA-liposomes and none of the PEGs inhibit the clearance of negatively charged PS-liposomes. The opsonins with different molecular size may be involved in the clearance of liposomes containing different charged lipids.

Percutaneous tumor ablation: increased necrosis with combined radio-frequency ablation and intravenous liposomal doxorubicin in a rat breast tumor model.

PURPOSE: To determine whether a combination of intravenous liposomal doxorubicin and radio-frequency (RF) ablation increases tumor destruction compared with RF alone in an animal tumor model. MATERIALS AND METHODS: R3230 mammary adenocarcinoma 1.4-1.8-cm- diameter nodules were implanted subcutaneously in 132 female Fischer rats. Initially, tumors were treated with (a) conventional, monopolar RF (mean, 250 mA +/- 25 [SD] at 70 degrees C +/- 1 for 5 minutes) ablation alone, (b) RF ablation followed by intravenous administration of 1 mg of liposomal doxorubicin, (c) RF ablation followed by intravenous administration of 1 mg of empty liposomes, (d) RF ablation and direct intratumoral administration of liposomal doxorubicin, or (e) no treatment. Subsequently, the dose (0.06-2.00 mg) of liposomal doxorubicin, the timing of administration (3 days before to 3 days after RF ablation), and the time of pathologic examination (0-72 hours after treatment) were varied. RESULTS: Mean coagulation diameter for treated tumors follows: 6.7 mm +/- 0.6, RF ablation alone; 11.1 mm +/- 1.5, RF ablation and intravenous administration of empty liposomes (P <.05, compared with RF ablation alone); and 8.4 mm +/- 1.1, RF ablation with intratumoral administration of liposomal doxorubicin (P <.05, compared with RF ablation alone). Maximal increased mean coagulation diameter (13.1 mm +/- 1.5) was observed with a combination of liposomal doxorubicin and RF ablation (P <.001, for all comparisons). The increased coagulation for combination therapy developed over 48 hours after therapy. Coagulation diameter did not vary with the doxorubicin concentration range and was not dependent on the timing of administration of liposomal doxorubicin from 3 days before to 24 hours after RF ablation. CONCLUSION: Intravenous administration of liposomal doxorubicin can improve RF ablation, since it increases coagulation diameter in solid tumors compared with RF ablation alone or a combination of RF ablation with administration of empty liposomes.