In situ T-cell transfection by anti-CD3-conjugated lipid nanoparticles leads to T-cell activation, migration, and phenotypic shift.

Ex vivo programming of T cells can be efficacious but is complex and expensive; therefore, the development of methods to transfect T cells in situ is important. We developed and optimized anti-CD3-targeted lipid nanoparticles (aCD3-LNPs) to deliver tightly packed, reporter gene mRNA specifically to T cells. In vitro, targeted LNPs efficiently delivered mCherry mRNA to Jurkat T cells, and T-cell activation and depletion were associated with aCD3 antibody coating on the surface of LNPs. aCD3-LNPs, but not non-targeted LNPs, accumulated within the spleen following systemic injection, with mCherry and Fluc signals visible within 30 min after injection. At 24 h after aCD3-LNP injection, 2-4% of all splenic T cells and 2-7% of all circulating T cells expressed mCherry, and this was dependent on aCD3 coating density. Targeting and transfection were accompanied by systemic CD25+, OX40+, and CD69+ T-cell activation with temporary CD3e ligand loss and depletion of splenic and circulating subsets. Migration of splenic CD8a+ T cells from the white-pulp to red-pulp, and differentiation from naïve to memory and effector phenotypes, followed upon aCD3-LNP delivery. Additionally, aCD3-LNP injection stimulated the secretion of myeloid-derived chemokines and T-helper cytokines into plasma. Lastly, we administered aCD3-LNPs to tumor bearing mice and found that transfected T cells localized within tumors and tumor-draining lymph nodes following immunotherapy treatment. In summary, we show that CD3-targeted transfection is feasible, yet associated with complex immunological consequences that must be further studied for potential therapeutic applications.

Development of thermosensitive resiquimod-loaded liposomes for enhanced cancer immunotherapy.

Resiquimod (R848) is a toll-like receptor 7 and 8 (TLR7/8) agonist with potent antitumor and immunostimulatory activity. However, systemic delivery of R848 is poorly tolerated because of its poor solubility in water and systemic immune activation. In order to address these limitations, we developed an intravenously-injectable formulation with R848 using thermosensitive liposomes (TSLs) as a delivery vehicle. R848 was remotely loaded into TSLs composed of DPPC: DSPC: DSPE-PEG2K (85:10:5, mol%) with 100 mM FeSO4 as the trapping agent inside. The final R848 to lipid ratio of the optimized R848-loaded TSLs (R848-TSLs) was 0.09 (w/w), 10-fold higher than the previously-reported values. R848-TSLs released 80% of R848 within 5 min at 42 °C. These TSLs were then combined with αPD-1, an immune checkpoint inhibitor, and ultrasound-mediated hyperthermia in a neu deletion (NDL) mouse mammary carcinoma model (Her2+, ER/PR negative). Combined with αPD-1, local injection of R848-TSLs showed superior efficacy with complete NDL tumor regression in both treated and abscopal sites achieved in 8 of 11 tumor bearing mice over 100 days. Immunohistochemistry confirmed enhanced CD8+ T cell infiltration and accumulation by R848-TSLs. Systemic delivery of R848-TSLs, combined with local hyperthermia and αPD-1, inhibited tumor growth and extended median survival from 28 days (non-treatment control) to 94 days. Upon re-challenge with reinjection of tumor cells, none of the previously cured mice developed tumors, as compared with 100% of age-matched control mice. The dose of R848 (10 µg for intra-tumoral injection or 6 mg/kg for intravenous injection delivered up to 4 times) was well-tolerated without weight loss or organ hypertrophy. In summary, we developed R848-TSLs that can be administered locally or systematically, resulting in tumor regression and enhanced survival when combined with αPD-1 in mouse models of breast cancer.

Systemic Immunotherapy with Micellar Resiquimod-Polymer Conjugates Triggers a Robust Antitumor Response in a Breast Cancer Model.

Resiquimod is an immunopotent toll-like receptor 7/8 agonist with antitumor activity. Despite being potent against skin cancers, it is poorly tolerated systemically due to toxicity. Integrating resiquimod into nanoparticles presents an avenue to circumvent the toxicity problem. Herein, the preparation of degradable nanoparticles with covalently bound resiquimod and their systemic application in cancer immunotherapy is reported. Dispersion in water of amphiphilic constructs integrating resiquimod covalently bound via degradable amide or ester linkages yields immune-activating nanoparticles. The degradable agonist-nanoparticle bonds allow the release of resiquimod from the carrier nanoparticles. In vitro assays with antigen presenting cells demonstrate that the nanoparticles retain the immunostimulatory activity of resiquimod. Systemic administration of the nanoparticles and checkpoint blockade (aPD-1) to a breast cancer mouse model with multiple established tumors triggers antitumor activity evidenced by suppressed tumor growth and enhanced CD8+ T-cell infiltration. Nanoparticles with ester links, which hydrolyze more readily, yield a stronger immune response with 75% of tumors eliminated when combined with aPD-1. The reduced tumor growth and the presence of activated CD8+ T-cells across multiple tumors suggest the potential for treating metastatic cancer.

Liposomal Cu-64 labeling method using bifunctional chelators: poly(ethylene glycol) spacer and chelator effects.

Two bifunctional Cu-64 chelators (BFCs), (6-(6-(3-(2-pyridyldithio)propionamido)hexanamido)benzyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA-PDP) and 4-(2-(2-pyridyldithioethyl)ethanamido)-11-carboxymethyl-1,4,8,11-tetraazabicyclo(6.6.2)hexadecane (CB-TE2A-PDEA), were synthesized and conjugated to long-circulating liposomes (LCLs) via attachment to a maleimide lipid. An in vitro stability assay of (64)Cu-TETA, (64)Cu-TETA-PEG2k, and (64)Cu-CB-TE2A-PEG2k liposomes showed that more than 86% of the radioactivity remains associated with the liposomal fraction after 48 h of incubation with mouse serum. The in vivo time activity curves (TAC) for the three liposomal formulations showed that approximately 50% of the radioactivity cleared from the blood pool in 16-18 h. As expected, the in vivo biodistribution and TAC data obtained at 48 h demonstrate that the clearance of radioactivity from the liver slows with the incorporation of a poly(ethylene glycol)-2k (PEG2k) brush. Our data suggest that (64)Cu-TETA and (64)Cu-CB-TE2A are similarly stable in the blood pool and accumulation of radioactivity in the liver and spleen is not related to the stability of Cu-64 chelator complex; however, clearance of Cu-64 from the liver and spleen are faster when injected as (64)Cu-TETA-chelated liposomes rather than (64)Cu-CB-TE2A-chelated liposomes.

Lipid-shelled vehicles: engineering for ultrasound molecular imaging and drug delivery.

Ultrasound pressure waves can map the location of lipid-stabilized gas micro-bubbles after their intravenous administration in the body, facilitating an estimate of vascular density and microvascular flow rate. Microbubbles are currently approved by the Food and Drug Administration as ultrasound contrast agents for visualizing opacification of the left ventricle in echocardiography. However, the interaction of ultrasound waves with intravenously-injected lipid-shelled particles, including both liposomes and microbubbles, is a far richer field. Particles can be designed for molecular imaging and loaded with drugs or genes; the mechanical and thermal properties of ultrasound can then effect localized drug release. In this Account, we provide an overview of the engineering of lipid-shelled microbubbles (typical diameter 1000-10 000 nm) and liposomes (typical diameter 65-120 nm) for ultrasound-based applications in molecular imaging and drug delivery. The chemistries of the shell and core can be optimized to enhance stability, circulation persistence, drug loading and release, targeting to and fusion with the cell membrane, and therapeutic biological effects. To assess the biodistribution and pharmacokinetics of these particles, we incorporated positron emission tomography (PET) radioisotopes on the shell. The radionuclide (18)F (half-life approximately 2 h) was covalently coupled to a dipalmitoyl lipid, followed by integration of the labeled lipid into the shell, facilitating short-term analysis of particle pharmacokinetics and metabolism of the lipid molecule. Alternately, labeling a formed particle with (64)Cu (half-life 12.7 h), after prior covalent incorporation of a copper-chelating moiety onto the lipid shell, permits pharmacokinetic study of particles over several days. Stability and persistence in circulation of both liposomes and microbubbles are enhanced by long acyl chains and a poly(ethylene glycol) coating. Vascular targeting has been demonstrated with both nano- and microdiameter particles. Targeting affinity of the microbubble can be modulated by burying the ligand within a polymer brush layer; the application of ultrasound then reveals the ligand, enabling specific targeting of only the insonified region. Microbubbles and liposomes require different strategies for both drug loading and release. Microbubble loading is inhibited by the gas core and enhanced by layer-by-layer construction or conjugation of drug-entrapped particles to the surface. Liposome loading is typically internal and is enhanced by drug-specific loading techniques. Drug release from a microbubble results from the oscillation of the gas core diameter produced by the sound wave, whereas that from a liposome is enhanced by heat produced from the local absorption of acoustic energy within the tissue microenvironment. Biological effects induced by ultrasound, such as changes in cell membrane and vascular permeability, can enhance drug delivery. In particular, as microbubbles oscillate near a vessel wall, shock waves or liquid jets enhance drug transport. Mild heating induced by ultrasound, either before or after injection of the drug, facilitates the transport of liposomes from blood vessels to the tissue interstitium, thus increasing drug accumulation in the target region. Lipid-shelled vehicles offer many opportunities for chemists and engineers; ultrasound-based applications beyond the few currently in common use will undoubtedly soon multiply as molecular construction techniques are further refined.

An imaging-driven model for liposomal stability and circulation.

Simultaneous labeling of the drug compartment and shell of delivery vehicles with optical and positron emission tomography (PET) probes is developed and employed to inform a hybrid physiologically based pharmacokinetic model. Based on time-dependent estimates of the concentration of these tracers within the blood pool, reticuloendothelial system (RES) and tumor interstitium, we compare the stability and circulation of long-circulating and temperature-sensitive liposomes. We find that rates of transport to the RES for long-circulating and temperature-sensitive particles are 0.046 and 0.19 h(-1), respectively. Without the application of exogenous heat, the rates of release from the long-circulating and temperature-sensitive particles circulating within the blood pool are 0.003 and 0.2 h(-1), respectively. Prolonged lifetime in circulation and slow drug release from liposomes result in a significantly greater drug area under the curve for the long-circulating particles. Future studies will couple these intrinsic parameters with exogenous heat-based release. Finally, we develop a transport constant for the transport of liposomes from the blood pool to the tumor interstitium, which is on the order of 0.01 h(-1) for the Met-1 tumor system.

Copper-doxorubicin as a nanoparticle cargo retains efficacy with minimal toxicity.

Repeated administration of chemotherapeutics is typically required for the effective treatment of highly aggressive tumors and often results in systemic toxicity. We have created a copper-doxorubicin complex within the core of liposomes and applied the resulting particle in multidose therapy. Copper and doxorubicin concentrations in the blood pool were similar at 24 h (∼40% of the injected dose), indicating stable circulation of the complex. Highly quenched doxorubicin fluorescence remained in the blood pool over tens of hours, with fluorescence increasing only with the combination of liposome disruption and copper trans-chelation. At 48 h after injection, doxorubicin fluorescence within the heart and skin was one-fifth and one-half, respectively, of fluorescence observed with ammonium sulfate-loaded doxorubicin liposomes. After 28 days of twice per week doxorubicin administration of 6 mg/kg, systemic toxicity (cardiac hypertrophy and weight and hair loss) was not detected with the copper-doxorubicin liposomes but was substantial with ammonium sulfate-loaded doxorubicin liposomes. We then incorporated two strategies designed to enhance efficacy, mTOR inhibition (rapamycin) to slow proliferation and therapeutic ultrasound to enhance accumulation and local diffusion. Tumor accumulation was ∼10% ID/g and was enhanced approximately 2-fold with the addition of therapeutic ultrasound. After the 28-day course of therapy, syngeneic tumors regressed to a premalignant phenotype of ∼(1 mm)(3) or could not be detected.

Tumor-specific delivery of gemcitabine with activatable liposomes.

Gemcitabine delivery to pancreatic ductal adenocarcinoma is limited by poor pharmacokinetics, dense fibrosis and hypo-vascularization. Activatable liposomes, with drug release resulting from local heating, enhance serum stability and circulation, and the released drug retains the ability to diffuse within the tumor. A limitation of liposomal gemcitabine has been the low loading efficiency. To address this limitation, we used the superior solubilizing potential of copper (II) gluconate to form a complex with gemcitabine at copper:gemcitabine (1:4). Thermosensitive liposomes composed of DPPC:DSPC:DSPE-PEG2k (80:15:5, mole%) then reached 12 wt% loading, 4-fold greater than previously reported values. Cryo transmission electron microscopy confirmed the presence of a liquid crystalline gemcitabine­copper mixture. The optimized gemcitabine liposomes released 60% and 80% of the gemcitabine within 1 and 5 min, respectively, at 42 °C. Liposomal encapsulation resulted in a circulation half-life of ~2 h in vivo (compared to reported circulation of 16 min for free gemcitabine in mice), and free drug was not detected within the plasma. The resulting gemcitabine liposomes were efficacious against both murine breast cancer and pancreatic cancer in vitro. Three repeated treatments of activatable gemcitabine liposomes plus ultrasound hyperthermia regressed or eliminated tumors in the neu deletion model of murine breast cancer with limited toxicity, enhancing survival when compared to treatment with gemcitabine alone. With 5% of the free gemcitabine dose (5 rather than 100 mg/kg), tumor growth was suppressed to the same degree as gemcitabine. Additionally, in a more aggressive tumor model of murine pancreatic cancer, liposomal gemcitabine combined with local hyperthermia induced cell death and regions of apoptosis and necrosis.

Combining activatable nanodelivery with immunotherapy in a murine breast cancer model.

A successful chemotherapy-immunotherapy solid-tumor protocol should accomplish the following goals: debulk large tumors, release tumor antigen for cross-presentation and cross-priming, release cancer-suppressive cytokines and enhance anti-tumor immune cell populations. Thermally-activated drug delivery particles have the potential to synergize with immunotherapeutics to accomplish these goals; activation can release chemotherapy within bulky solid tumors and can enhance response when combined with immunotherapy. We set out to determine whether a single protocol, combining locally-activated chemotherapy and agonist immunotherapy, could accomplish these goals and yield a potentially translational therapy. For effective delivery of free doxorubicin to tumors with minimal toxicity, we stabilized doxorubicin with copper in temperature-sensitive liposomes that rapidly release free drug in the vasculature of cancer lesions upon exposure to ultrasound-mediated hyperthermia. We found that in vitro exposure of tumor cells to hyperthermia and doxorubicin resulted in immunogenic cell death and the local release of type I interferons across murine cancer cell lines. Following intravenous injection, local activation of the liposomes within a single tumor released doxorubicin and enhanced cross-presentation of a model antigen at distant tumor sites. While a variety of protocols achieved a complete response in >50% of treated mice, the complete response rate was greatest (90%) when 1 week of immunotherapy priming preceded a single activatable chemotherapeutic administration. While repeated chemotherapeutic delivery reduced local viable tumor, the complete response rate and a subset of tumor immune cells were also reduced. Taken together, the results suggest that activatable chemotherapy can enhance adjuvant immunotherapy; however, in a murine model the systemic adaptive immune response was greatest with a single administration of chemotherapy.

A novel method to label preformed liposomes with 64Cu for positron emission tomography (PET) imaging.

Radiolabeling of liposomes with 64Cu (t(1/2)=12.7 h) is attractive for molecular imaging and monitoring drug delivery. A simple chelation procedure, performed at a low temperature and under mild conditions, is required to radiolabel preloaded liposomes without lipid hydrolysis or the release of the encapsulated contents. Here, we report a 64Cu postlabeling method for liposomes. A 64Cu-specific chelator, 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (BAT), was conjugated with an artificial lipid to form a BAT-PEG-lipid. After incorporation of 0.5% (mol/mol) BAT-PEG-lipid during liposome formulation, liposomes were successfully labeled with 64Cu in 0.1 M NH4OAc pH 5 buffer at 35 degrees C for 30-40 min with an incorporation yield as high as 95%. After 48 h of incubation of 64Cu-liposomes in 50/50 serum/PBS solution, more than 88% of the 64Cu label was still associated with liposomes. After injection of liposomal 64Cu in a mouse model, 44+/-6.9, 21+/-2.7, 15+/-2.5, and 7.4+/-1.1 (n=4) % of the injected dose per cubic centimeter remained within the blood pool at 30 min, 18, 28, and 48 h, respectively. The biodistribution at 48 h after injection verified that 7.0+/-0.47 (n=4) and 1.4+/-0.58 (n=3) % of the injected dose per gram of liposomal 64Cu and free 64Cu remained in the blood pool, respectively. Our results suggest that this fast and easy 64Cu labeling of liposomes could be exploited in tracking liposomes in vivo for medical imaging and targeted delivery.

Transmitted ultrasound pressure variation in micro blood vessel phantoms.

Silica, cellulose and polymethylmethacrylate tubes with inner diameters of ten to a few hundred microns are commonly used as blood vessel phantoms in in vitro studies of microbubble or nanodroplet behavior during insonation. However, a detailed investigation of the ultrasonic fields within these micro-tubes has not yet been performed. This work provides a theoretical analysis of the ultrasonic fields within micro-tubes. Numerical results show that for the same tube material, the interaction between the micro-tube and megaHertz-frequency ultrasound may vary drastically with incident frequency, tube diameter and wall thickness. For 10 MHz ultrasonic insonation of a polymethylmethacrylate (PMMA) tube with an inner diameter of 195 microm and an outer diameter of 260 microm, the peak pressure within the tube can be up to 300% of incident pressure amplitude. However, using 1 MHz ultrasound and a silica tube with an inner diameter of 12 microm and an outer diameter of 50 microm, the peak pressure within the tube is only 12% of the incident pressure amplitude and correspondingly, the spatial-average-time-average intensity within the tube is only 1% of the incident intensity.

Cholesterol transport from liposomal delivery vehicles.

Rapid internalization of drugs from delivery vehicles via non-endocytotic pathways is an important goal. The transport of imaging probes attached to cholesterol and introduced via a liposomal formulation is considered here, in order to evaluate the intracellular distribution and kinetics of small molecular cargo that might be attached to cholesterol or phospholipids. The internalization efficiencies of two fluorescent cholesterol analogues, one carrying a fluorophore on the head of the cholesterol molecule 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoate (BODIPY)-cholesteryl ester (CE) (BODIPY-CE) and the other on the tail (25-[N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)-methyl]amino]-27-norcholesterol (NBD-cholesterol)), were compared with those of other phospholipid molecules (NBD-phosphatidylcholine (PC) and NBD-phosphatidylethanolamine (PE)) using a liposomal formulation (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 85.5 M%; 1,2 distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2k), 9.5 M%; fluorescent analogue, 5 M%). The rate and transfer efficiency were NBD-cholesterol>BODIPY-CE>NBD-PC>NBD-PE. NBD-cholesterol, delivered by liposomes with an average diameter of 100 nm, localized in the perinuclear region and lipid storage droplets, with transfer observed in as little as 5 min. NBD-cholesterol transport was approximately constant with time, suggesting a unidirectional mode of entry. In the absence of PEG within the liposome, the transfer rate decreased. Filipin, a caveolae-blocking agent, caused 70% inhibition of cholesterol internalization in treated cells, suggesting that cholesterol internalization follows a caveolae-mediated pathway.

Long-circulating liposomes radiolabeled with [18F]fluorodipalmitin ([18F]FDP).

Synthesis of a radiolabeled diglyceride, 3-[(18)F]fluoro-1,2-dipalmitoylglycerol [[(18)F]fluorodipalmitin ([(18)F]FDP)], and its potential as a reagent for radiolabeling long-circulating liposomes were investigated. The incorporation of (18)F into the lipid molecule was accomplished by nucleophilic substitution of the p-toluenesulfonyl moiety with a decay-corrected yield of 43+/-10% (n=12). Radiolabeled, long-circulating polyethylene-glycol-coated liposomes were prepared using a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] ammonium salt (61:30:9) and [(18)F]FDP with a decay-corrected yield of 70+/-8% (n=4). PET imaging and biodistribution studies were performed with free [(18)F]FDP and liposome-incorporated [(18)F]FDP. Freely injected [(18)F]FDP had the highest uptake in the liver, spleen and lungs. Liposomal [(18)F]FDP remained in blood circulation at near-constant levels for at least 90 min, with a peak concentration near 2.5%ID/cc. Since [(18)F]FDP was incorporated into the phospholipid bilayer, it could potentially be used for radiolabeling a variety of lipid-based drug carriers.

Dynamic contrast enhanced MRI detects changes in vascular transport rate constants following treatment with thermally-sensitive liposomal doxorubicin.

Temperature-sensitive liposomal formulations of chemotherapeutics, such as doxorubicin, can achieve locally high drug concentrations within a tumor and tumor vasculature while maintaining low systemic toxicity. Further, doxorubicin delivery by temperature-sensitive liposomes can reliably cure local cancer in mouse models. Histological sections of treated tumors have detected red blood cell extravasation within tumors treated with temperature-sensitive doxorubicin and ultrasound hyperthermia. We hypothesize that the local release of drug into the tumor vasculature and resulting high drug concentration can alter vascular transport rate constants along with having direct tumoricidal effects. Dynamic contrast enhanced MRI (DCE-MRI) coupled with a pharmacokinetic model can detect and quantify changes in such vascular transport rate constants. Here, we set out to determine whether changes in rate constants resulting from intravascular drug release were detectable by MRI. We found that the accumulation of gadoteridol was enhanced in tumors treated with temperature-sensitive liposomal doxorubicin and ultrasound hyperthermia. While the initial uptake rate of the small molecule tracer was slower (k1=0.0478±0.011s-1 versus 0.116±0.047s-1) in treated compared to untreated tumors, the tracer was retained after treatment due to a larger reduction in the rate of clearance (k2=0.291±0.030s-1 versus 0.747±0.24s-1). While DCE-MRI assesses a combination of blood flow and permeability, ultrasound imaging of microvascular flow rate is sensitive only to changes in vascular flow rate; based on this technique, blood flow was not significantly altered 30min after treatment. In summary, DCE-MRI provides a means to detect changes that are associated with treatment by thermally-activated particles and such changes can be exploited to enhance local delivery.

Intracellular trafficking of a pH-responsive drug metal complex.

We previously developed a pH-responsive copper-doxorubicin (CuDox) cargo in lysolipid-based temperature-sensitive liposomes (LTSLs). The CuDox complex is released from the particle by elevated temperature; however, full release of doxorubicin from CuDox requires a reduced pH, such as that expected in lysosomes. The primary goal of this study is to evaluate the cellular uptake and intracellular trafficking of the drug-metal complex in comparison with intact liposomes and free drug. We found that the CuDox complex was efficiently internalized by mammary carcinoma cells after release from LTSLs. Intracellular doxorubicin and copper were 6-fold and 5-fold greater, respectively, after a 0.5h incubation with the released CuDox complex, as compared to incubation with intact liposomes containing the complex. Total cellular doxorubicin fluorescence was similar following CuDox and free doxorubicin incubation. Imaging and mass spectrometry assays indicated that the CuDox complex was initially internalized intact but breaks down over time within cells, with intracellular copper decreasing more rapidly than intracellular doxorubicin. Doxorubicin fluorescence was reduced when complexed with copper, and nuclear fluorescence was reduced when cells were incubated with the CuDox complex as compared with free doxorubicin. Therapeutic efficacy, which typically results from intercalation of doxorubicin with DNA, was equivalent for the CuDox complex and free doxorubicin and was superior to that of liposomal doxorubicin formulations. Taken together, the results suggest that quenched CuDox reaches the nucleus and remains efficacious. In order to design protocols for the use of these temperature-sensitive particles in cancer treatment, the timing of hyperthermia relative to drug administration must be examined. When cells were heated to 42°C prior to the addition of free doxorubicin, nuclear drug accumulation increased by 1.8-fold in cancer cells after 5h, and cytotoxicity increased 1.4-fold in both cancer and endothelial cells. Endothelial cytotoxicity was similarly augmented with mild hyperthermia applied prior to treatment with released CuDox. In summary, we find that the drug-metal complex formed in temperature-sensitive particles can be internalized by cancer and endothelial cells resulting in therapeutic efficacy that is similar to free doxorubicin, and this efficacy can be enhanced by elevated temperature.

Ultrasound ablation enhances drug accumulation and survival in mammary carcinoma models.

Magnetic resonance-guided focused ultrasound (MRgFUS) facilitates noninvasive image-guided conformal thermal therapy of cancer. Yet in many scenarios, the sensitive tissues surrounding the tumor constrain the margins of ablation; therefore, augmentation of MRgFUS with chemotherapy may be required to destroy remaining tumor. Here, we used 64Cu-PET-CT, MRI, autoradiography, and fluorescence imaging to track the kinetics of long-circulating liposomes in immunocompetent mammary carcinoma-bearing FVB/n and BALB/c mice. We observed a 5-fold and 50-fold enhancement of liposome and drug concentration, respectively, within MRgFUS thermal ablation-treated tumors along with dense accumulation within the surrounding tissue rim. Ultrasound-enhanced drug accumulation was rapid and durable and greatly increased total tumor drug exposure over time. In addition, we found that the small molecule gadoteridol accumulates around and within ablated tissue. We further demonstrated that dilated vasculature, loss of vascular integrity resulting in extravasation of blood cells, stromal inflammation, and loss of cell-cell adhesion and tissue architecture all contribute to the enhanced accumulation of the liposomes and small molecule probe. The locally enhanced liposome accumulation was preserved even after a multiweek protocol of doxorubicin-loaded liposomes and partial ablation. Finally, by supplementing ablation with concurrent liposomal drug therapy, a complete and durable response was obtained using protocols for which a sub-mm rim of tumor remained after ablation.

The effect of size on the acoustic response of polymer-shelled contrast agents.

In this technical note, we study three polymer-shelled microbubble contrast agents manufactured by POINT Biomedical Corporation that have identical shell composition and mean volumetric diameters of 0.74 microm, 0.91 microm and 1.33 microm. We investigate the effect of agent size on the amplitude, frequency and probability of acoustic echoes received in response to five-cycle, 2.25-MHz pulses of varying pressure. We find that the amplitude and frequency response from the three agents is not significantly different. However, significant differences among the agents do exist in the probability of response to acoustic interrogation: at a pressure of 1.06 MPa, an echo from the 1.33 microm agent is 5 times as likely as an echo from the 0.91 microm agent and 18 times as likely as an echo from the 0.74 microm agent. We hypothesize that there exists an effective pressure-dependent threshold diameter above which single polymer-shelled agents respond to acoustic interrogation.

Influence of lipid shell physicochemical properties on ultrasound-induced microbubble destruction.

We present the first study of the effects of monolayer shell physicochemical properties on the destruction of lipid-coated microbubbles during insonification with single, one-cycle pulses at 2.25 MHz and low-duty cycles. Shell cohesiveness was changed by varying phospholipid and emulsifier composition, and shell microstructure was controlled by postproduction processing. Individual microbubbles with initial resting diameters between 1 and 10 microm were isolated and recorded during pulsing with bright-field and fluorescence video microscopy. Microbubble destruction occurred through two modes: acoustic dissolution at 400 and 600 kPa and fragmentation at 800 kPa peak negative pressure. Lipid composition significantly impacted the acoustic dissolution rate, fragmentation propensity, and mechanism of excess lipid shedding. Less cohesive shells resulted in micron-scale or smaller particles of excess lipid material that shed either spontaneously or on the next pulse. Conversely, more cohesive shells resulted in the buildup of shell-associated lipid strands and globular aggregates of several microns in size; the latter showed a significant increase in total shell surface area and lability. Lipid-coated microbubbles were observed to reach a stable size over many pulses at intermediate acoustic pressures. Observations of shell microstructure between pulses allowed interpretation of the state of the shell during oscillation. We briefly discuss the implications of these results for therapeutic and diagnostic applications involving lipid-coated microbubbles as ultrasound contrast agents and drug/gene delivery vehicles.

The pharmacokinetics of Zr-89 labeled liposomes over extended periods in a murine tumor model.

(89)Zr (t1/2=78.4h), a positron-emitting metal, has been exploited for PET studies of antibodies because of its relatively long decay time and facile labeling procedures. Here, we used (89)Zr to evaluate the pharmacokinetics of long-circulating liposomes over 168h (1week). We first developed a liposomal-labeling method using p-isothiocyanatobenzyl-desferrioxamine (df-Bz-NCS) and df-PEG1k-DSPE. Df-Bz-NCS was conjugated to 1mol% amino- and amino-PEG2k-DSPE, where the 1mol% df-PEG1k-DSPE was incorporated when the liposomes were formulated. Incubation of (89)Zr with df, df-PEG1k, and df-PEG2k liposomes for one hour resulted in greater than 68% decay-corrected yield. The loss of the (89)Zr label from liposomes after incubation in 50% human serum for 48h ranged from ~1 to 3% across the three formulations. Tail vein administration of the three liposomal formulations in NDL tumor-bearing mice showed that the (89)Zr label at the end of the PEG2k brush was retained in the tumor, liver, spleen and whole body for a longer time interval than (89)Zr labels located under the PEG2k brush. The blood clearance rate of all three liposomal formulations was similar. Overall, the results indicate that the location of the (89)Zr label altered the clearance rate of intracellularly-trapped radioactivity and that df-PEG1k-DSPE provides a stable chelation site for liposomal or lipid-based particle studies over extended periods of time.

Self-assembled 20-nm (64)Cu-micelles enhance accumulation in rat glioblastoma.

There is an urgent need to develop nanocarriers for the treatment of glioblastoma multiforme (GBM). Using co-registered positron emission tomography (PET) and magnetic resonance (MR) images, here we performed systematic studies to investigate how a nanocarrier's size affects the pharmacokinetics and biodistribution in rodents with a GBM xenograft. In particular, highly stable, long-circulating three-helix micelles (3HM), based on a coiled-coil protein tertiary structure, were evaluated as an alternative to larger nanocarriers. While the circulation half-life of the 3HM was similar to 110-nm PEGylated liposomes (t1/2=15.5 and 16.5h, respectively), the 20-nm micelles greatly enhanced accumulation within a U87MG xenograft in nu/nu rats after intravenous injection. After accounting for tumor blood volume, the extravasated nanoparticles were quantified from the PET images, yielding ~0.77%ID/cm(3) for the micelles and 0.45%ID/cm(3) for the liposomes. For GBM lesions with a volume greater than 100mm(3), 3HM accumulation was enhanced both within the detectable tumor and in the surrounding brain parenchyma. Further, the nanoparticle accumulation was shown to extend to the margins of the GBM xenograft. In summary, 3HM provides an attractive nanovehicle for carrying treatment to GBM.