Correlation of ablation volume with renal function loss after cryoablation in solitary functioning kidneys.

PURPOSE: The purpose of this study was to retrospectively analyze the degree of renal function deterioration after renal cryoablation in patients with a solitary functioning kidney based on ablation volume. MATERIALS AND METHODS: Over a 15-year period, 81 percutaneous cryoablations were performed in solitary functioning kidneys. After exclusion of patients with baseline end-stage renal disease(ESRD) and insufficient follow up, analysis was performed on 65 procedures in 52 patients (40 male, mean age 63.5 years). The post-cryoablation renal function was based on the lowest serum creatinine within 6 months post-procedure. Renal function change was defined as percentage glomerular filtration rate(GFR) change. Volumetric analysis was performed on the target lesion, renal parenchyma, and ablation zone. RESULTS: The median tumor diameter was 2.0cm (range 0.8-4.7cm). The median baseline GFR decreased from 56.4 mL/min/1.73m2 (range 17.5-89.7) to 46.9 (range 16.5-89.7) at median 95 days (p<0.001), equating to an -7.9% median renal function change (range -45.0 to +30.7). All patients had stage 2 or worse chronic kidney disease and baseline function did not correlate with renal function change. The median volume of ablated parenchyma was 19.7mL (range 2.4-87.3mL), equating to 8.1% (range 0.7-37.2%) of total parenchyma. The volume of parenchymal volume ablated correlated significantly with renal function loss, while age, hypertension, and diabetes mellitus did not. No patients developed ESRD within 1 year after cryoablation. CONCLUSION: Cryoablation in solitary functioning kidneys resulted in a modest reduction in renal function, even in patients with chronic kidney disease and ablations up to 20% of renal parenchymal volume.

Evaluating Radioactive Analogs of Doxorubicin to Quantify ChemoFilter Binding and Whole-Body Positron Emission Tomography/Magnetic Resonance Imaging for Drug Biodistribution.

PURPOSE: To evaluate radiolabeled doxorubicin (Dox) analogs as tracers of baseline Dox biodistribution in vivo during hepatic intra-arterial chemotherapy and to assess the efficacy of ChemoFilter devices to bind Dox in vitro. MATERIALS AND METHODS: In an in vitro static experiment, [fluorine-18]N-succinimidyl 4-fluorobenzoate ([18F]SFB) and [fluorine-18]fluorobenzoyl-doxorubicin ([18F]FB-Dox) were added to a beaker containing a filter material (Dowex cation exchange resin, single-stranded DNA (ssDNA) resin, or sulfonated polymer coated mesh). In an in vitro flow model, [18F]FB-Dox was added into a Dox solution in phosphate-buffered saline, and the solution flowed via a syringe column containing the filter materials. In an in vitro flow experiment, using micro-positron emission tomography (PET), images were taken as [18F]SFB and [18F]FB-Dox moved through a phantom. For in vivo biodistribution testing, a catheter was placed into the common hepatic artery of a swine, and [18F]FB-Dox was infused over 30 seconds. A 10-minute dynamic image and three 20-minute static images were acquired using 3T PET/MR imaging. RESULTS: In the in vitro static experiment, [18F]FB-Dox demonstrated 76.7%, 88.0%, and 52.4% binding to the Dowex resin, ssDNA resin, and coated mesh, respectively. In the in vitro flow model, the first-pass binding of [18F]FB-Dox to the Dowex resin, ssDNA resin, and coated mesh was 76.7%, 74.2%, and 76.2%, respectively, and the total bound fraction was 80.9%, 84.6%, and 79.9%, respectively. In the in vitro flow experiment using micro-PET, the phantom demonstrated a greater amount of [18F]FB-Dox bound to both filter cartridges than of the control [18F]SFB. In in vivo biodistribution testing, the first 10 minutes depicted [18F]FB-Dox moving through the right upper quadrant of the abdomen. A region-of-interest analysis showed that the relative amount increased by 2.97 times in the gallbladder and 1.08 times in the kidney. The amount decreased by 0.74 times in the brain and 0.57 times in the heart. CONCLUSIONS: [18F]FB-Dox can be used to assess Dox binding to ChemoFilters as well as in vivo biodistribution. This sets the stage for the evaluation of ChemoFilter effectiveness in reducing systemic toxicity from intra-arterial chemotherapy.

Intratumoral delivery of brachytherapy and immunotherapy by a thermally triggered polypeptide depot.

Biomaterial-based approaches for a combination of radiotherapy and immunotherapy can improve outcomes in metastatic cancer through local delivery of both therapeutic modalities to the primary tumor to control local tumor growth and distant metastases. This study describes an injectable depot for sustained intratumoral (i.t.) delivery of an iodine-131 (131I) radionuclide and a CpG oligodeoxynucleotide immunostimulant, driven by the thermally sensitive phase transition behavior of elastin-like polypeptides (ELPs). We synthesized and characterized an ELP with an oligolysine tail (ELP-K12) that forms an electrostatic complex with CpG for delivery from an ELP depot and evaluated the ability of the complex to enhance local and systemic tumor control as a monotherapy and in combination with 131I-ELP brachytherapy. I.t delivery of CpG from an ELP-K12 depot dramatically prolongs i.t. retention to more than 21 days as compared to soluble CpG that is only retained within the tumor for <24 h. ELP-K12 also enhances CpG delivery by increasing cellular uptake of CpG to generate greater toll-like receptor 9 (TLR9) activation than CpG alone. I.t. treatment with an ELP-K12/CpG depot slows primary tumor growth and reduces lung metastases in a poorly immunogenic 4 T1 syngeneic breast cancer model whereas i.t treatment of CpG alone has no significant effect on primary tumor growth or metastases. Notably, a combination of 131I-ELP brachytherapy and ELP-K12/CpG delivered i.t. inhibited 4 T1 tumor growth and strongly decreased the development of lung metastases, leading to a synergistic improvement in mouse survival. These preclinical results demonstrate that injectable ELP depots may provide a useful approach for the delivery of combination radio- and immuno-therapy to treat metastatic disease.

Tumor Subtype Determines Therapeutic Response to Chimeric Polypeptide Nanoparticle-based Chemotherapy in Pten-deleted Mouse Models of Sarcoma.

PURPOSE: Nanoparticle-encapsulated drug formulations can improve responses to conventional chemotherapy by increasing drug retention within the tumor and by promoting a more effective antitumor immune response than free drug. New drug delivery modalities are needed in sarcomas because they are often chemoresistant cancers, but the rarity of sarcomas and the complexity of diverse subtypes makes it challenging to investigate novel drug formulations. EXPERIMENTAL DESIGN: New drug formulations can be tested in animal models of sarcomas where the therapeutic response of different formulations can be compared using mice with identical tumor-initiating mutations. Here, using Cre/loxP and CRISPR/Cas9 techniques, we generated two distinct mouse models of Pten-deleted soft-tissue sarcoma: malignant peripheral nerve sheath tumor (MPNST) and undifferentiated pleomorphic sarcoma (UPS). We used these models to test the efficacy of chimeric polypeptide doxorubicin (CP-Dox), a nanoscale micelle formulation, in comparison with free doxorubicin. RESULTS: The CP-Dox formulation was superior to free doxorubicin in MPNST models. However, in UPS tumors, CP-Dox did not improve survival in comparison with free doxorubicin. While CP-Dox treatment resulted in elevated intratumoral doxorubicin concentrations in MPNSTs, this increase was absent in UPS tumors. In addition, elevation of CD8+ T cells was observed exclusively in CP-Dox-treated MPNSTs, although these cells were not required for full efficacy of the CP nanoparticle-based chemotherapy. CONCLUSIONS: These results have important implications for treating sarcomas with nanoparticle-encapsulated chemotherapy by highlighting the tumor subtype-dependent nature of therapeutic response.

Nanoparticle formulation improves doxorubicin efficacy by enhancing host antitumor immunity.

Strategies that enhance the host antitumor immune response promise to revolutionize cancer therapy. Optimally mobilizing the immune system will likely require a multi-pronged approach to overcome the resistance developed by tumors to therapy. Recently, it has become recognized that doxorubicin can contribute to re-establishing host antitumor immunity through the generation of immunogenic cell death. However, the potential for delivery strategies to further enhance the immunological effects of doxorubicin has not been adequately examined. We report herein that Chimeric Polypeptide Doxorubicin (CP-Dox), a nanoparticle formulation of doxorubicin, enhances antitumor immunity. Compared to free doxorubicin, a single intravenous (IV) administration of CP-Dox at the maximum tolerated dose increases the infiltration of leukocytes into the tumor, slowing tumor growth and preventing metastasis in poorly immunogenic 4T1 mammary carcinoma. We demonstrate that the full efficacy of CP-Dox is dependent on CD8+ T cells and IFN-γ. CP-dox treatment also repolarized intratumoral myeloid cells towards an antitumor phenotype. These findings demonstrate that a nanoparticle drug is distinct from the free drug in its ability to productively stimulate antitumor immunity. Our study strongly argues for the use of antitumor immunotherapies combined with nanoparticle-packaged chemotherapy.

One-week glucose control via zero-order release kinetics from an injectable depot of glucagon-like peptide-1 fused to a thermosensitive biopolymer.

Stimulation of the glucagon-like peptide-1 (GLP1) receptor is a useful treatment strategy for type 2 diabetes because of pleiotropic effects, including the regulation of islet hormones and the induction of satiety. However, the native ligand for the GLP1 receptor has a short half-live owing to enzymatic inactivation and rapid clearance. Here, we show that a subcutaneous depot formed after a single injection of GLP1 recombinantly fused to a thermosensitive elastin-like polypeptide results in zero-order release kinetics and circulation times of up to 10 days in mice and 17 days in monkeys. The optimized pharmacokinetics leads to 10 days of glycemic control in three different mouse models of diabetes, as well as to the reduction of glycosylated hemoglobin levels and weight gain in ob/ob mice treated once weekly for 8 weeks. Our results suggest that the optimized GLP1 formulation could enhance therapeutic outcomes by eliminating peak-and-valley pharmacokinetics and improving overall safety and tolerability. The design principles that we established should be broadly applicable for improving the pharmacological performance of other peptide and protein therapeutics.

A quantitative study of the intracellular fate of pH-responsive doxorubicin-polypeptide nanoparticles.

Nanoscale carriers with an acid-labile linker between the carrier and drug are commonly used for drug delivery. However, their efficacy is potentially limited by inefficient linker cleavage, and lysosomal entrapment of drugs. To address these critical issues, we developed a new imaging method that spatially overlays the location of a nanoparticle and the released drug from the nanoparticle, on a map of the local intracellular pH that delineates individual endosomes and lysosomes, and the therapeutic intracellular target of the drug-the nucleus. We used this method to quantitatively map the intracellular fate of micelles of a recombinant polypeptide conjugated with doxorubicin via an acid-labile hydrazone linker as a function of local pH and time within live cells. We found that hydrolysis of the acid-labile linker is incomplete because the pH range of 4-7 in the endosomes and lysosomes does not provide complete cleavage of the drug from the nanoparticle, but that once cleaved, the drug escapes the acidic endo-lysosomal compartment into the cytosol and traffics to its therapeutic destination-the nucleus. This study also demonstrated that unlike free drug, which enters the cytosol directly through the cell membrane and then traffics into the nucleus, the nanoparticle-loaded drug almost exclusively traffics into endosomes and lysosomes upon intracellular uptake, and only reaches the nucleus after acid-triggered drug release in the endo-lysosomes. This methodology provides a better and more quantitative understanding of the intracellular behavior of drug-loaded nanoparticles, and provides insights for the design of the next-generation of nanoscale drug delivery systems.

Injectable polypeptide micelles that form radiation crosslinked hydrogels in situ for intratumoral radiotherapy.

Intratumoral radiation therapy - 'brachytherapy' - is a highly effective treatment for solid tumors, particularly prostate cancer. Current titanium seed implants, however, are permanent and are limited in clinical application to indolent malignancies of low- to intermediate-risk. Attempts to develop polymeric alternatives, however, have been plagued by poor retention and off-target toxicity due to degradation. Herein, we report on a new approach whereby thermally sensitive micelles composed of an elastin-like polypeptide (ELP) are labeled with the radionuclide (131)I to form an in situ hydrogel that is stabilized by two independent mechanisms: first, body heat triggers the radioactive ELP micelles to rapidly phase transition into an insoluble, viscous coacervate in under 2 min; second, the high energy ß-emissions of (131)I further stabilize the depot by introducing crosslinks within the ELP depot over 24h. These injectable brachytherapy hydrogels were used to treat two aggressive orthotopic tumor models in athymic nude mice: a human PC-3 M-luc-C6 prostate tumor and a human BxPc3-luc2 pancreatic tumor model. The ELP depots retained greater than 52% and 70% of their radioactivity through 60 days in the prostate and pancreatic tumors with no appreciable radioactive accumulation (≤ 0.1% ID) in off-target tissues after 72h. The (131)I-ELP depots achieved >95% tumor regression in the prostate tumors (n=8); with a median survival of more than 60 days compared to 12 days for control mice. For the pancreatic tumors, ELP brachytherapy (n=6) induced significant growth inhibition (p=0.001, ANOVA) and enhanced median survival to 27 days over controls.

Doxorubicin-conjugated polypeptide nanoparticles inhibit metastasis in two murine models of carcinoma.

Drug delivery vehicles are often assessed for their ability to control primary tumor growth, but the outcome of cancer treatment depends on controlling or inhibiting metastasis. Therefore, we studied the efficacy of our genetically encoded polypeptide nanoparticle for doxorubicin delivery (CP-Dox) in the syngeneic metastatic murine models 4T1 and Lewis lung carcinoma. We found that our nanoparticle formulation increased the half-life, maximum tolerated dose, and tumor accumulation of doxorubicin. When drug treatment was combined with primary tumor resection, greater than 60% of the mice were cured in both the 4T1 and Lewis lung carcinoma models compared to 20% treated with free drug. Mechanistic studies suggest that metastasis inhibition and survival increase were achieved by preventing the dissemination of viable tumor cells from the primary tumor.

The margination propensity of ellipsoidal micro/nanoparticles to the endothelium in human blood flow.

Particle shape is becoming increasingly recognized as an important parameter for the development of vascular-targeted carriers (VTCs) for disease treatment and diagnosis. However, limited research exists that investigates how particle shape coupled with hemodynamics affects VTC margination (localization and adhesion). In this study, we investigate the effects of particle shape parameters (volume, aspect ratio, axis length) on the margination efficacy of targeted spheres and prolate ellipsoids (rods) to an inflamed endothelial wall from human blood flow in an in vitro model of human vasculature. Overall, particles with 2 µm equivalent spherical diameters (ESD) display higher margination than particles with either 1 µm or 500 nm ESDs. Interestingly, rod-shaped microparticles (1 µm or 2 µm ESD) with high aspect ratios display significantly improved margination compared to spheres of equal volume, particularly under high shear rates and disturbed flow profiles. Nanorods (500 nm ESD), even with high aspect ratio, do not display enhanced margination compared to that of equivalent spheres, which suggests that nanorods, like nanospheres, display minimal margination due to their inability to effectively localize to the vessel wall in the presence of RBCs.

Fabrication of biodegradable spheroidal microparticles for drug delivery applications.

Particle shape, in addition to size, is becoming increasingly recognized as important in the design of drug carriers for in vivo use. However, few methods exist for fabricating non-spherical particles from biodegradable polymers. This work describes for the first time the fabrication of biodegradable spheroidal microparticles using the simple oil-in-water emulsion solvent evaporation technique (O/W ESE). Unloaded and paclitaxel-loaded spheroids were fabricated from poly(lactic-co-glycolic acid) (PLGA), and the shape and size of fabricated spheroids were manipulated by controlling fabrication process parameters including stir speed, aqueous and oil phase viscosity, aqueous phase pH, and the polymer molecular weight and end group. The presented data show that high aqueous phase viscosity, basic aqueous phase pH and hydrophilic polymer side chains and end groups are all conditions that favor the formation of spheroidal particles. The described technique is advantageous over methods currently described in the literature in its simplicity in setup, high particle yield and adaptability to a wide range of biodegradable polymers and therapeutics.