Evaluation of Physicochemical Properties, Pharmacokinetics, Biodistribution, Toxicity, and Contrast-Enhanced Cancer MRI of a Cancer-Targeting Contrast Agent, MT218.

OBJECTIVES: Preclinical assessments were performed according to the US Food and Drug Administration guidelines to determine the physicochemical properties, pharmacokinetics, clearance, safety, and tumor-specific magnetic resonance (MR) imaging of MT218, a peptidic gadolinium-based MR imaging agent targeting to extradomain B fibronectin for MR molecular imaging of aggressive tumors. MATERIALS AND METHODS: Relaxivity, chelation stability, binding affinity, safety-related target profiling, and effects on CYP450 enzymes and transporters were evaluated in vitro. Magnetic resonance imaging was performed with rats bearing prostate cancer xenografts, immunocompetent mice bearing murine pancreatic cancer allografts, and mice bearing lung cancer xenografts at different doses of MT218. Pharmacological effects on cardiovascular, respiratory, and central nervous systems were determined in rats and conscious beagle dogs. Pharmacokinetics were tested in rats and dogs. Biodistribution and excretion were studied in rats. Single and repeated dosing toxicity was evaluated in rats and dogs. In vitro and in vivo genotoxicity, in vitro hemolysis, and anaphylactic reactivity were also performed. RESULTS: At 1.4 T, the r1 and r2 relaxivities of MT218 were 5.43 and 7.40 mM -1 s -1 in pure water, 6.58 and 8.87 mM -1 s -1 in phosphate-buffered saline, and 6.54 and 8.70 mM -1 s -1 in aqueous solution of human serum albumin, respectively. The binding affinity of MT218 to extradomain B fragment is 3.45 µM. MT218 exhibited no dissociation of the Gd(III) chelates under physiological conditions. The peptide degradation half-life ( t1/2 ) of MT218 was 1.63, 5.85, and 2.63 hours in rat, dog, and human plasma, respectively. It had little effect on CYP450 enzymes and transporters. MT218 produced up to 7-fold increase of contrast-to-noise ratios in the extradomain B fibronectin-rich tumors with a dose of 0.04 mmol/kg for at least 30 minutes. MT218 had little pharmacological effect on central nervous, cardiovascular, or respiratory systems. MT218 had a mean plasma elimination half-life ( t1/2 ) of 0.31 and 0.89 hours in rats and dogs at 0.1 mmol/kg, respectively. No detectable Gd deposition was observed in the brain at 6 hours postinjection of MT218 at 0.1 mmol/kg in rats. MT218 was not mutagenic and had no mortality or morbidity in the rats or dogs up to 1.39 and 0.70 mmol/kg/d, respectively. The no observed adverse effect level of MT218 in Sprague-Dawley rats was 1.39 mmol/kg for single dosing and 0.46 mmol/kg/d for repeated dosing. The no observed adverse effect level in dogs was 0.07 mmol/kg/d. MT218 exhibited no genotoxicity, hemolysis, and anaphylactic reactivity. CONCLUSION: The preclinical assessments showed that the targeted contrast agent MT218 has high r1 and r2 relaxivities, satisfactory physicochemical properties, pharmacokinetic, and safety profiles and produces effective tumor enhancement in multiple cancer types in rats and mice at reduced doses.

Manganese(II) EOB-Pyclen Diacetate for Liver-Specific MRI.

MRI is increasingly utilized for the diagnosis of liver disease and focal liver lesions. Although liver-targeted gadolinium-based contrast agents (GBCAs) have high efficacy, there continue to be safety concerns regarding release of toxic Gd(III) ions. Herein, Mn(EOB-PC2A) is synthesized as a nongadolinium alternative for liver-specific MRI. Mn(EOB-PC2A) has an r1 relaxivity of 2.8 mM-1 s-1 in Dulbecco's phosphate-buffered saline (DPBS) and 5.9 mM-1 s-1 in saline containing human serum albumin at 1.5 T. It has a strong uptake in hepatocytes with minimal toxicity and demonstrated robust liver-specific enhancement at a dose of 60 µmol/kg. Mn(EOB-PC2A) is a promising liver-specific contrast agent for liver MRI.

Efficacy of Targeted ECO/miR-200c Nanoparticles for Modulating Tumor Microenvironment and Treating Triple Negative Breast Cancer as Non-invasively Monitored by MR Molecular Imaging.

PURPOSE: To investigate the effectiveness of targeted ECO/miR-200c in modulating tumor microenvironment and treating triple negative breast cancer (TNBC) using non-invasive magnetic resonance molecular imaging (MRMI) of extradomain B fibronectin (EDB-FN) with a targeted MRI contrast agent. METHODS: MDA-MB-231 and Hs578T TNBC cells were transfected with RGD-PEG-ECO/miR-200c. Invasive and migratory potential was evaluated using transwell, scratch wound, and spheroid formation assays. Athymic nude mice bearing orthotopic MDA-MB-231 and Hs578T xenografts were treated with weekly i.v. injection of RGD-PEG-ECO/miR-200c nanoparticles at 1.0 mg/kg/week RNA for 6 weeks. MRMI of EDB-FN was performed using a targeted contrast agent MT218 [ZD2-N3-Gd(DO3A)] on a 3 T MRS 3000 scanner. T1-weighted images were acquired following intravenous injection of MT218 at dose of 0.1 mmol/kg using a fast spin echo axial sequence with respiratory gating. RESULTS: Systemic administration of RGD-PEG-ECO/miR-200c nanoparticles in mice bearing orthotopic TNBC xenografts significantly suppressed tumor progression without toxic side-effects. MRMI with MT218 revealed that the treatment significantly suppressed tumor proliferation as compared to the control. MRMI also showed that the miR-200c treatment altered tumor microenvironment by reducing EDB-FN expression, as evidenced by decreased contrast enhancement in both MDA-MB-231 and Hs578T tumors. The reduction of EDB-FN was confirmed by immunohistochemistry. CONCLUSIONS: Targeted delivery of miR-200c with RGD-PEG-ECO/miR-200c nanoparticles effectively modulates tumor microenvironment and suppresses TNBC proliferation in animal models. MRMI of tumor EDB-FN expression is effective to non-invasively monitor tumor response and therapeutic efficacy of RGD-PEG-ECO/miR-200c nanoparticles in TNBC.

Environment-Responsive Lipid/siRNA Nanoparticles for Cancer Therapy.

RNA interference (RNAi) is a promising technology to regulate oncogenes for treating cancer. The primary limitation of siRNA for clinical application is the safe and efficacious delivery of therapeutic siRNA into target cells. Lipid-based delivery systems are developed to protect siRNA during the delivery process and to facilitate intracellular uptake. There is a significant progress in lipid nanoparticle systems that utilize cationic and protonatable amino lipid systems to deliver siRNA to tumors. Among these lipids, environment-responsive lipids are a class of novel lipid delivery systems that are capable of responding to the environment changes during the delivery process and demonstrate great promise for clinical translation for siRNA therapeutics. Protonatable or ionizable amino lipids and switchable lipids as well as pH-sensitive multifunctional amino lipids are the presentative environment-responsive lipids for siRNA delivery. These lipids are able to respond to environmental changes during the delivery process to facilitate efficient cytosolic siRNA delivery. Environment-responsive lipid/siRNA nanoparticles (ERLNP) are developed with the lipids and are tested for efficient delivery of therapeutic siRNA into the cytoplasm of cancer cells to silence target genes for cancer treatment in preclinical development. This review summarizes the recent developments in environment-response lipids and nanoparticles for siRNA delivery in cancer therapy.

Magnetic Resonance Molecular Imaging of Extradomain B Fibronectin Improves Imaging of Pancreatic Cancer Tumor Xenografts.

The survival of pancreatic cancer patients can be greatly improved if their disease is detected at an early, potentially curable stage. Magnetic resonance molecular imaging (MRMI) of oncoproteins is a promising strategy for accurate, early detection of the disease. Here, we test the hypothesis that MRMI of extradomain-B fibronectin (EDB-FN), an abundant oncoprotein in the tumor extracellular matrix, can overcome the stromal barriers of pancreatic cancer to facilitate effective molecular imaging and detection of small tumors. Specimens of normal, premalignant, and malignant human pancreatic tissues were stained with a peptide-fluorophore conjugate (ZD2-Cy5.5) to assess EDB-FN binding and expression. MRMI with ZD2-N3-Gd(HP-DO3A) (MT218) specific to EDB-FN and MRI with Gd(HP-DO3A) were performed in three murine models bearing human pancreatic cancer xenografts, including a Capan-1 flank model, a BxPC3-GFP-Luc and a PANC-1-GFP-Luc intrapancreatic xenograft model. Tumor enhancement of the contrast agents was analyzed and compared. Staining of human tissue samples with ZD2-Cy5.5 revealed high EDB-FN expression in pancreatic tumors, moderate expression in premalignant tissue, and little expression in normal tissue. MRMI with MT218 generated robust intratumoral contrast, clearly detected and delineated small tumors (smallest average size: 6.1 mm2), and out-performed conventional contrast enhanced MRI with Gd(HP-DO3A). Quantitative analysis of signal enhancement revealed that MT218 produced 2.7, 2.1, and 1.6 times greater contrast-to-noise ratio (CNR) than the clinical agent in the Capan-1 flank, BxPC3-GFP-Luc and PANC-1-GFP-Luc intrapancreatic models, respectively (p < 0.05). MRMI of the ECM oncoprotein EDB-FN with MT218 is able to generate superior contrast enhancement in small pancreatic tumors and provide accurate tumor delineation in animal models. Early, accurate detection and delineation of pancreatic cancer with high-resolution MRMI has the potential to guide timely treatment and significantly improve the long-term survival of pancreatic cancer patients.

Improving Radiation Response in Glioblastoma Using ECO/siRNA Nanoparticles Targeting DNA Damage Repair.

Radiation therapy is a mainstay in the standard of care for glioblastoma (GBM), thus inhibiting the DNA damage response (DDR) is a major strategy to improve radiation response and therapeutic outcomes. Small interfering RNA (siRNA) therapy holds immeasurable potential for the treatment of GBM, however delivery of the siRNA payload remains the largest obstacle for clinical implementation. Here we demonstrate the effectiveness of the novel nanomaterial, ECO (1-aminoethylimino[bis(N-oleoylcysteinylaminoethyl) propionamide]), to deliver siRNA targeting DDR proteins ataxia telangiectasia mutated and DNA-dependent protein kinase (DNApk-cs) for the radiosensitzation of GBM in vitro and in vivo. ECO nanoparticles (NPs) were shown to efficiently deliver siRNA and silence target protein expression in glioma (U251) and glioma stem cell lines (NSC11, GBMJ1). Importantly, ECO NPs displayed no cytotoxicity and minimal silencing of genes in normal astrocytes. Treatment with ECO/siRNA NPs and radiation resulted in the prolonged presence of γH2AX foci, indicators of DNA damage, and increased radiosensitivity in all tumor cell lines. In vivo, intratumoral injection of ECO/siDNApk-cs NPs with radiation resulted in a significant increase in survival compared with injection of NPs alone. These data suggest the ECO nanomaterial can effectively deliver siRNA to more selectively target and radiosensitize tumor cells to improve therapeutic outcomes in GBM.

Noninvasive assessment and therapeutic monitoring of drug-resistant colorectal cancer by MR molecular imaging of extradomain-B fibronectin.

Antineoplastic resistance represents a multifaceted challenge for cancer therapy and diagnostics. Extensive molecular heterogeneity, even within neoplasms of the same type, can elicit distinct outcomes of administering therapeutic pressures, frequently leading to the development of drug-resistant populations. Improved success of oncotherapies merits the exploration of precise molecular imaging technologies that can detect not only anatomical but also molecular changes in tumors and their microenvironment, early on in the treatment regimen. To this end, we developed magnetic resonance molecular imaging (MRMI) strategies to target the extracellular matrix oncoprotein, extradomain-B fibronectin (EDB-FN), for non-invasive assessment and therapeutic monitoring of drug-resistant colorectal cancer (CRC). Methods: Two drug-resistant CRC lines generated from parent DLD-1 and RKO cells by long-term treatment with 5'-FU and 5'-FU plus CB-839 respectively, were characterized for functional and gene expression changes using 3D culture, transwell invasion, qRT-PCR, and western blot assays. Contrast-enhanced MRMI of EDB-FN was performed in athymic nu/nu mice bearing subcutaneous tumor xenografts with 40 µmol/kg dose of macrocyclic ZD2-targeted contrast agent MT218 [ZD2-N3-Gd (HP-DO3A)] on a 3T MRS 3000 scanner. Immunohistochemistry was conducted on patient specimens and xenografts using anti-EDB-FN antibody G4. Results: Analyses of TCGA and GTEx databases revealed poor prognosis of colon cancer patients with higher levels of EDB-FN. Similarly, immunohistochemical staining of patient specimens showed increased EDB-FN expression in primary colon adenocarcinoma and hepatic metastases, but none in normal adjacent tissues. Drug-resistant DLD1-DR and RKO-DR cells were also found to demonstrate enhanced invasive potential and significantly elevated EDB-FN expression over their parent counterparts. MRMI of EDB-FN with 40 µmol/kg dose of MT218 (60% lower than the clinical dose) resulted in robust signal enhancement in the drug-resistant CRC xenografts with 84-120% increase in their contrast-to-noise ratios (CNRs) over the non-resistant counterparts. The feasibility of non-invasive therapeutic monitoring using MRMI of EDB-FN was also evaluated in drug-resistant DLD1-DR tumors treated with a pan-AKT inhibitor MK2206-HCl. The treated drug-resistant tumors failed to respond to therapy, which was accurately detected by MRMI with MT218, demonstrating higher signal enhancement and increased CNRs in the 4-week follow-up scans over the pre-treatment scans. Conclusions: EDB-FN is a promising molecular marker for assessing drug resistance. MRMI of EDB-FN with MT218 at a significantly reduced dose can facilitate effective non-invasive assessment and treatment response monitoring of drug-resistant CRC, highlighting its translational potential for active surveillance and management of CRC and other malignancies.

Preclinical Assessment of the Effectiveness of Magnetic Resonance Molecular Imaging of Extradomain-B Fibronectin for Detection and Characterization of Oral Cancer.

PURPOSE: Oral squamous cell carcinoma (OSCC) has not seen a substantial improvement in patient survival despite therapeutic advances, making accurate detection and characterization of the disease a clinical priority. Here, we aim to demonstrate the effectiveness of magnetic resonance imaging (MRI) with the targeted MRI contrast agent MT218 specific to extradomain-B fibronectin (EDB-FN) in the tumor microenvironment for detection and characterization of aggressive OSCC tumors. PROCEDURES: EDB-FN expression was evaluated in human normal tongue and OSCC specimens with immunohistochemistry. Invasiveness of human CAL27, HSC3, and SCC4 OSCC cells was analyzed with spheroid formation and transwell assays. EDB-FN expression in the cells was analyzed with semiquantitative real-time PCR, western blotting, and a peptide binding study with confocal microscopy. Contrast-enhanced MRI with MT218 was performed on subcutaneous OSCC mouse models at a dose of 0.04 mmol/kg, using gadoteridol (0.1 mmol/kg) as a control. RESULTS: Strong EDB-FN expression was observed in human untreated primary and metastatic OSCC, reduced expression in treated OSCC, and little expression in normal tongue tissue. SCC4 and HSC3 cell lines demonstrated high invasive potential with high and moderate-EDB-FN expression, respectively, while CAL27 showed little invasive potential and low-EDB-FN expression. In T1-weighted MRI, MT218 produced differential contrast enhancement in the subcutaneous tumor models in correlation with EDB-FN expression in the cancer cells. Enhancement in the high-EDB-FN tumors was greater with MT218 at 0.04 mmol/kg than gadoteridol at 0.1 mmol/kg. CONCLUSIONS: The results suggest EDB-FN has strong potential as an imageable biomarker for aggressive OSCC. MRMI results demonstrate the effectiveness of MT218 and the potential for differential diagnostic imaging of oral cancer for improving the management of the disease.

Effective MR Molecular Imaging of Triple Negative Breast Cancer With an EDB-Fibronectin-Specific Contrast Agent at Reduced Doses.

MR molecular imaging (MRMI) of abundant oncogenic biomarkers in tumor microenvironment has the potential to provide precision cancer imaging in high resolution. Extradomain-B fibronectin (EDB-FN) is an oncogenic extracellular matrix protein, highly expressed in aggressive triple negative breast cancer. A targeted macrocyclic gadolinium-based contrast agent (GBCA) ZD2-N3-Gd(HP-DO3A) (MT218), specific to EDB-FN, was developed for MRMI of aggressive breast cancer. The effectiveness of different doses of MT218 for MRMI was tested in MDA-MB-231 and Hs578T human triple negative breast cancer models. At clinical dose of 0.1 and subclinical dose of 0.04 mmol Gd/kg, MT218 rapidly bound to the extracellular matrix EDB-FN and produced robust tumor contrast enhancement in both the tumor models, as early as 1-30 min post-injection. Substantial tumor enhancement was also observed in both the models with MT218 at doses as low as 0.02 mmol Gd/kg, which was significantly better than the clinical agent Gd(HP-DO3A) at 0.1 mmol Gd/kg. Little non-specific enhancement was observed in the normal tissues including liver, spleen, and brain for MT218 at all the tested doses, with renal clearance at 30 min. These results demonstrate that MRMI with reduced doses of MT218 is safe and effective for sensitive and specific imaging of aggressive breast cancers.

Formulation of Biocompatible Targeted ECO/siRNA Nanoparticles with Long-Term Stability for Clinical Translation of RNAi.

Nanoparticle based siRNA formulations often suffer from aggregation and loss of function during storage. We in this study report a frozen targeted RGD-polyethylene glycol (PEG)-ECO/siß3 nanoparticle formulation with a prolonged shelf life and preserved nanoparticle functionality. The targeted RGD-PEG-ECO/siß3 nanoparticles are formed by step-wised self-assembly of RGD-PEG-maleimide, ECO, and siRNA. The nanoparticles have a diameter of 224.5 ± 9.41 nm and a zeta potential to 45.96 ± 3.67 mV in water and a size of 234.34 ± 3.01 nm and a near neutral zeta potential in saline solution. The addition of sucrose does not affect their size and zeta potential and substantially preserves the integrity and biological activities of frozen and lyophilized formulations of the targeted nanoparticles. The frozen formulation with as low as 5% sucrose retains nanoparticle integrity (90% siRNA encapsulation), size distribution (polydispersity index [PDI] ≤20%), and functionality (at least 75% silencing efficiency) at -80°C for at least 1 year. The frozen RGD-PEG-ECO/siß3 nanoparticle formulation exhibits excellent biocompatibility, with no adverse effects on hemocompatibility and minimal immunogenicity. As RNAi holds the promise in treating the previously untreatable diseases, the frozen nanoparticle formulation with the low sucrose concentration has the potential to be a delivery platform for clinical translation of RNAi therapeutics.

Systemic Delivery of Tumor-Targeting siRNA Nanoparticles against an Oncogenic LncRNA Facilitates Effective Triple-Negative Breast Cancer Therapy.

Long noncoding RNAs (lncRNAs), by virtue of their versatility and multilevel gene regulation, have emerged as attractive pharmacological targets for treating heterogeneous and complex malignancies like triple-negative breast cancer (TNBC). Despite multiple studies on lncRNA functions in tumor pathology, systemic targeting of these "undruggable" macromolecules with conventional approaches remains a challenge. Here, we demonstrate effective TNBC therapy by nanoparticle-mediated RNAi of the oncogenic lncRNA DANCR, which is significantly overexpressed in TNBC. Tumor-targeting RGD-PEG-ECO/siDANCR nanoparticles were formulated via self-assembly of multifunctional amino lipid ECO, cyclic RGD peptide-PEG, and siDANCR for systemic delivery. MDA-MB-231 and BT549 cells treated with the therapeutic RGD-PEG-ECO/siDANCR nanoparticles exhibited 80-90% knockdown in the expression of DANCR for up to 7 days, indicating efficient intracellular siRNA delivery and sustained target silencing. The RGD-PEG-ECO/siDANCR nanoparticles mediated excellent in vitro therapeutic efficacy, reflected by significant reduction in the invasion, migration, survival, tumor spheroid formation, and proliferation of the TNBC cell lines. At the molecular level, functional ablation of DANCR dynamically impacted the oncogenic nexus by downregulating PRC2-mediated H3K27-trimethylation and Wnt/EMT signaling, and altering the phosphorylation profiles of several kinases in the TNBC cells. Furthermore, systemic administration of the RGD-PEG-ECO/siDANCR nanoparticles at a dose of 1 mg/kg siRNA in nude mice bearing TNBC xenografts resulted in robust suppression of TNBC progression with no overt toxic side-effects, underscoring the efficacy and safety of the nanoparticle therapy. These results demonstrate that nanoparticle-mediated modulation of onco-lncRNAs and their molecular targets is a promising approach for developing curative therapies for TNBC and other cancers.

Synthesis and Evaluation of pH-Sensitive Multifunctional Lipids for Efficient Delivery of CRISPR/Cas9 in Gene Editing.

CRISPR/Cas9 system is a promising approach for gene editing in gene therapy. Effective gene editing requires safe and efficient delivery of CRISPR/Cas9 system in target cells. Several new multifunctional pH-sensitive amino lipids were designed and synthesized with modification of the amino head groups for intracellular delivery of CRISPR/Cas9 system. These multifunctional pH-sensitive amino lipids exhibited structurally dependent formulation of stable nanoparticles with the DNA plasmids of CRISPR/Cas9 system with the sizes ranging from 100 to 200 nm. The amino lipid plasmid DNA nanoparticles showed pH-sensitive hemolysis with minimal hemolytic activity at pH 7.4 and increased hemolysis at acidic pH (pH = 5.5, 6.5). The nanoparticles exhibited low cytotoxicity at an N/P ratio of 10. Expression of both Cas9 and sgRNA of the CRISPR/Cas9 system was in the range from 4.4% to 33%, dependent on the lipid structure in NIH3T3-GFP cells. The amino lipids that formed stable nanoparticles with high expression of both Cas9 and sgRNA mediated high gene editing efficiency. ECO and iECO mediated more efficient gene editing than other tested lipids. ECO mediated up to 50% GFP suppression based on observations with confocal microscopy and nearly 80% reduction of GFP mRNA based on RT-PCR measurement in NIH3T3-GFP cells. The multifunctional pH-sensitive amino lipids have the potential for efficient intracellular delivery of CRISPR/Cas9 for effective gene editing.

Optimization of ZD2 Peptide Targeted Gd(HP-DO3A) for Detection and Risk-Stratification of Prostate Cancer with MRI.

The aim of this work is to optimize a peptide targeted macrocyclic MRI contrast agent for detection and risk-stratification of aggressive prostate cancer. The optimized agent was prepared using click chemistry in the presence of CuSO4 and ascorbate at room temperature. The T1 and T2 relaxivities of ZD2-N3-Gd(HP-DO3A) are 5.44 and 7.10 mM-1 s-1 at 1.4 T, and 5.53 and 7.81 mM-1 s-1 at 7 T, respectively, higher than the previously reported ZD2-Gd(HP-DO3A). The specific tumor enhancement of the agent was investigated in male nude mice bearing aggressive PC3 human prostate cancer xenografts and slow-growing LNCaP tumor xenografts. Contrast enhanced MR images were acquired using a 2D spin-echo sequence and a 3D FLASH sequence with a 7 T small animal scanner. ZD2-N3-Gd(HP-DO3A) produced robust contrast enhancement in aggressive PC3 tumors and little enhancement in slow-growing LNCaP tumors. It produced 400% and 100% CNR increases in the T1-weighted 2D spin-echo MR images and 3D FLASH images of PC3 tumors, respectively, for at least 30 min at a dose of 0.1 mmol/kg. In contrast, less than 20% CNR increase was observed in the LNCaP tumors with both sequences. The optimized targeted contrast agent has higher relaxivities and are effective to detect aggressive PC3 tumors and differentiate the aggressive cancer from the slow-growing LNCaP prostate cancer in contrast enhanced MRI. ZD2-N3-Gd(HP-DO3A) has the promise for accurate detection and risk-stratification of aggressive prostate cancer.

Influence of Molecular Structure on the In Vivo Performance of Flexible Rod Polyrotaxanes.

Polyrotaxanes, a family of rod-shaped nanomaterials comprised of noncovalent polymer/macrocycle assemblies, are being used in a growing number of materials and biomedical applications. Their physiochemical properties can vary widely as a function of composition, potentially leading to different in vivo performance outcomes. We sought to characterize the pharmacokinetic profiles, toxicities, and protein corona compositions of 2-hydroxypropyl-ß-cyclodextrin polyrotaxanes as a function of variations in macrocycle threading efficiency, molecular weight, and triblock copolymer core structure. We show that polyrotaxane fate in vivo is governed by the structure and dynamics of their rodlike morphologies, such that highly threaded polyrotaxanes are long circulating and deposit in the liver, whereas lung deposition and rapid clearance is observed for species bearing lower 2-hydroxypropyl-ß-cyclodextrin threading percentages. Architecture differences also promote recruitment of different serum protein classes and proportions; however, physiochemical differences have little or no influence on their toxicity. These findings provide important structural insights for guiding the development of polyrotaxanes as scaffolds for biomedical applications.

A neutral polydisulfide containing Gd(III) DOTA monoamide as a redox-sensitive biodegradable macromolecular MRI contrast agent.

This work aims to develop safe and effective gadolinium (III)-based biodegradable macromolecular MRI contrast agents for blood pool and cancer imaging. A neutral polydisulfide containing macrocyclic Gd-DOTA monoamide (GOLS) was synthesized and characterized. In addition to studying the in vitro degradation of GOLS, its kinetic stability was also investigated in an in vivo model. The efficacy of GOLS for contrast-enhanced MRI was examined with female BALB/c mice bearing 4T1 breast cancer xenografts. The pharmacokinetics, biodistribution, and metabolism of GOLS were also determined in mice. GOLS has an apparent molecular weight of 23.0 kDa with T1 relaxivities of 7.20 mM(-1) s(-1) per Gd at 1.5 T, and 6.62 mM(-1) s(-1) at 7.0 T. GOLS had high kinetic inertness against transmetallation with Zn(2+) ions, and its polymer backbone was readily cleaved by L-cysteine. The agent showed improved efficacy for blood pool and tumor MR imaging. The structural effect on biodistribution and in vivo chelation stability was assessed by comparing GOLS with Gd(HP-DO3A), a negatively charged polydisulfide containing Gd-DOTA monoamide GODC, and a polydisulfide containing Gd-DTPA-bisamide (GDCC). GOLS showed high in vivo chelation stability and minimal tissue deposition of gadolinium. The biodegradable macromolecular contrast agent GOLS is a promising polymeric contrast agent for clinical MR cardiovascular imaging and cancer imaging.

Stealth filaments: Polymer chain length and conformation affect the in vivo fate of PEGylated potato virus X.

Nanoparticles hold great promise for delivering medical cargos to cancerous tissues to enhance contrast and sensitivity of imaging agents or to increase specificity and efficacy of therapeutics. A growing body of data suggests that nanoparticle shape, in combination with surface chemistry, affects their in vivo fates, with elongated filaments showing enhanced tumor targeting and tissue penetration, while promoting immune evasion. The synthesis of high aspect ratio filamentous materials at the nanoscale remains challenging using synthetic routes; therefore we turned toward nature's materials, developing and studying the filamentous structures formed by the plant virus potato virus X (PVX). We recently demonstrated that PVX shows enhanced tumor homing in various preclinical models. Like other nanoparticle systems, the proteinaceous platform is cleared from circulation and tissues by the mononuclear phagocyte system (MPS). To increase bioavailability we set out to develop PEGylated stealth filaments and evaluate the effects of PEG chain length and conformation on pharmacokinetics, biodistribution, as well as potential immune and inflammatory responses. We demonstrate that PEGylation effectively reduces immune recognition while increasing pharmacokinetic profiles. Stealth filaments show reduced interaction with cells of the MPS; the protein:polymer hybrids are cleared from the body tissues within hours to days indicating biodegradability and biocompatibility. Tissue compatibility is indicated with no apparent inflammatory signaling in vivo. Tailoring PEG chain length and conformation (brush vs. mushroom) allows tuning of the pharmacokinetics, yielding long-circulating stealth filaments for applications in nanomedicine.

Multifunctional PEG retinylamine conjugate provides prolonged protection against retinal degeneration in mice.

A polyethylene glycol (PEG) retinylamine (Ret-NH2) conjugate PEG-GFL-NH-Ret with a glycine-phenylalanine-leucine (GFL) spacer was synthesized for controlled oral delivery of Ret-NH2 to treat retinal degenerative diseases, including Stargardt disease (STGD) and age-related macular degeneration (AMD). The peptide spacer was introduced for sustained release of the drug by digestive enzymes in the gastrointestinal tract. The pharmacokinetics experiments showed that the PEG conjugate could control the sustained drug release after oral administration and had much lower nonspecific liver drug accumulation than the free drug in wild-type female C57BL mice. In the mean time, the conjugate maintained the same concentration of Ret-NH2 in the eye as the free drug. Also, PEG-GFL-NH-Ret at a Ret-NH2 equivalent dose of 25 mg/kg produced complete protection of Abca4(-/-)Rdh8(-/-) mouse retinas against light-induced retinal degeneration for 3 days after oral administration, as revealed by OCT retina imaging, whereas free Ret-NH2 did not provide any protection under identical conditions. The polymer conjugate PEG-GFL-NH-Ret has great potential for controlled delivery of Ret-NH2 to the eye for effective protection against retinal degenerative diseases.

Biodistribution and clearance of a filamentous plant virus in healthy and tumor-bearing mice.

AIM: Nanoparticles based on plant viruses are emerging biomaterials for medical applications such as drug delivery and imaging. Their regular structures can undergo genetic and chemical modifications to carry large payloads of cargos, as well as targeting ligands. Of several such platforms under development, only few have been characterized in vivo. We recently introduced the filamentous plant virus, potato virus X (PVX), as a new platform. PVX presents with a unique nanoarchitecture and is difficult to synthesize chemically. METHODS: Here, we present a detailed analysis of PVX biodistribution and clearance in healthy mice and mouse tumor xenograft models using a combination of ex vivo whole-organ imaging, quantitative fluorescence assays and immunofluorescence microscopy. RESULTS & CONCLUSION: While up to 30% of the PVX signal was from the colon, mammary and brain tumor tissues, remaining particles were cleared by the reticuloendothelial system organs (the spleen and liver), followed by slower processing and clearance through the kidneys and bile.