Xueming Wu

Targeted systemic delivery of a therapeutic siRNA with a multifunctional carrier controls tumor proliferation in mice

In this study, novel peptide-targeted delivery systems were developed for systemic and targeted delivery of therapeutic siRNA based on a multifunctional carrier, (1-aminoethyl)iminobis[N-(oleicylcysteinylhistinyl-1-aminoethyl)propionamide] (EHCO), which showed pH-sensitive amphiphilic cell membrane disruption. EHCO formed stable nanoparticles with siRNA. Targeted siRNA delivery systems were readily formed by surface modification of the nanoparticles. PEGylation of the siRNA/EHCO nanoparticles significantly reduced nonspecific cell uptake. The incorporation of a bombesin peptide or RGD peptide via a PEG spacer resulted in receptor-mediated cellular uptake and high gene silencing efficiency in U87 cells. Fluorescence confocal microscopic studies demonstrated that EHCO/siRNA nanoparticles and PEG modified EHCO/siRNA nanoparticles were able to facilitate endosomal escape of the siRNA delivery systems. Systemic administration of a therapeutic anti-HIF-1alpha siRNA with the peptide-targeted delivery systems resulted in significant tumor growth inhibition than a nontargeted delivery system or free siRNA via intravenous injection in nude mice bearing human glioma U87 xenografts. The results indicate a great promise of the multifunctional carrier EHCO for systemic and targeted delivery of therapeutic siRNA to treat human diseases with RNAi.

Peptide-targeted Nanoglobular Gd-DOTA monoamide conjugates for magnetic resonance cancer molecular imaging.

Effective imaging of a cancer molecular biomarker is critical for accurate cancer diagnosis and prognosis. CLT1 peptide was observed to specifically bind to the fibrin-fibronectin complexes presented in tumor extracellular matrix. In this study, we synthesized and evaluated CLT1 peptide-targeted nanoglobular Gd-DOTA monoamide conjugates for magnetic resonance (MR) imaging of the fibrin-fibronectin complexes in tumor. The targeted nanoglobular contrast agents were prepared by conjugating peptide CLT1 to G2 and G3 nanoglobule (lysine dendrimers with a cubic silsesquioxane core) Gd-DOTA monoamide conjugates via click chemistry. The T(1) relaxivities of peptide-targeted G2 and G3 nanoglobules were 7.92 and 8.20 mM(-1) s(-1) at 3T, respectively. Approximately 2 peptides and 25 Gd-DOTA chelates were conjugated onto the surface of 32 amine groups of G2 nanoglobule, and 3 peptides and 43 Gd-DOTA chelates onto the surface of 64 amine groups of G3 nanoglobule. The peptide-targeted nanoglobular contrast agents showed greater contrast enhancement than the corresponding nontargeted agents in tumor at a dose of 0.03 mmol-Gd/kg in female athymic mice bearing MDA-MB-231 human breast carcinoma xenografts. The targeted MRI contrast agents have a potential for specific cancer molecular imaging with MRI.

Peptide targeted tripod macrocyclic Gd(III) chelates for cancer molecular MRI.

Rational design and develop of targeted contrast agents binding to cancer-related proteins will achieve more accurate cancer diagnosis and prognosis by magnetic resonance (MR) imaging. CREKA is a tumor-homing pentapeptide (Cys-Arg-Glu-Lys-Ala) specifically homes to fibrin-fibronectin complexes abundantly expressed in tumor microenvironment. In this study, we developed and evaluated a CREKA peptide targeted multiplexed Gd-MR probe (CREKA-Tris-Gd(DOTA)3) for MR imaging of breast tumors. CREKA and azide bearing Gd(III) was attached to a maleimide-functional trialkyne scaffold via thiol-maleimide and azide-alkyne click chemistry, respectively. CREKA-Tris-Gd(DOTA)3 has a well-defined structure with a molecular weight of 2914 Da. The T1 relaxivity of CREKA-Tris-Gd(DOTA)3 is 8.06 mM(-1) s(-1) per Gd (24.18 mM(-1) s(-1) per molecule) at room temperature and 3 T. Fluorescence imaging showed high binding specificity of CREKA to a 4T1 breast tumor model in mice while it was not found for the scrambled CREKA (CERAK). The CREKA peptide-targeted contrast agent resulted in greater contrast enhancement than the corresponding CERAK agent and the commercialized contrast agent ProHance(®) in tumor at a dose of 0.1 mmol Gd/kg in female athymic mice bearing 4T1 breast carcinoma xenograft. This small molecular contrast agent was easily excreted from body after imaging indicated low toxicity. The targeted MRI contrast agent has a potential for specific cancer molecular imaging with MRI.

Synthesis and evaluation of nanoglobular macrocyclic Mn(II) chelate conjugates as non-gadolinium(III) MRI contrast agents

Because of the recent observation of the toxic side effects of Gd(III) based MRI contrast agents in patients with impaired renal function, there is strong interest on developing alternative contrast agents for MRI. In this study, macrocyclic Mn(II) chelates were conjugated to nanoglobular carriers, lysine dendrimers with a silsesquioxane core, to synthesize non-Gd(III) based MRI contrast agents. A generation 3 nanoglobular conjugate of Mn(II)-1,4,7-triaazacyclononane-1,4,7-triacetate-GA amide (G3-NOTA-Mn) was also synthesized and evaluated. The per ion T(1) and T(2) relaxivities of G2, G3, G4 nanoglobular Mn(II)-DOTA monoamide conjugates decreased with increasing generation of the carriers. The T(1) relaxivities of G2, G3, and G4 nanoglobular Mn(II)-DOTA conjugates were 3.3, 2.8, and 2.4 mM(-1) s(-1) per Mn(II) chelate at 3 T, respectively. The T(1) relaxivity of G3-NOTA-Mn was 3.80 mM(-1) s(-1) per Mn(II) chelate at 3 T. The nanoglobular macrocyclic Mn(II) chelate conjugates showed good in vivo stability and were readily excreted via renal filtration. The conjugates resulted in much less nonspecific liver enhancement than MnCl(2) and were effective for contrast-enhanced tumor imaging in nude mice bearing MDA-MB-231 breast tumor xenografts at a dose of 0.03 mmol Mn/kg. The nanoglobular macrocyclic Mn(II) chelate conjugates are promising nongadolinium based MRI contrast agents.

An Effective Targeted Nanoglobular Manganese(II) Chelate Conjugate for Magnetic Resonance Molecular Imaging of Tumor Extracellular Matrix

Stable manganese(II) chelates are of great interest for the design and development of safe and effective non-gadolinium(III)-based targeted MRI contrast agents for MR cancer molecular imaging. In this study, a CLT1 peptide targeted G3 nanoglobular Mn(II)-DOTA monoamide conjugate was designed and synthesized as a targeted MRI contrast agent for molecular imaging of the fibrin-fibronectin complexes or oncofetal fibronectin in tumor stroma. The targeted contrast agent comprised 2 peptides and 42 Mn(II)-DOTA chelates on the surface of the G3 nanoglobule. The T(1) and T(2) relaxivities of the targeted agent at room temperature were 3.13 and 8.74 mM(-1) s(-1) per Mn(II) chelate at 3 T (tesla), respectively. It had a well-defined nanosize (5.2 nm) and could be readily excreted via renal filtration. The targeted nanoglobular contrast agent specifically bound to tumor tissue, resulting in significant tumor contrast enhancement with minimal nonspecific enhancement in the liver of tumor bearing mice as compared to a nontargeted control at a dose as low as 0.03 mmol-Mn/kg. The targeted G3 nanoglobular Mn(II)-DOTA conjugate is promising as a targeted non-gadolinium(III)-based MRI contrast agent for MR cancer molecular imaging.

Synthesis and evaluation of a polydisulfide with Gd–DOTA monoamide side chains as a biodegradable macromolecular contrast agent for MR blood pool imaging

Macromolecular Gd(III)-based contrast agents are effective for contrast-enhanced blood pool and cancer MRI in preclinical studies. However, their clinical applications are impeded by potential safety concerns associated with slow excretion and prolonged retention of these agents in the body. To minimize the safety concerns of macromolecular Gd contrast agents, we have developed biodegradable macromolecular Gd contrast agents based on polydisulfide Gd(III) complexes. In this study, we designed and synthesized a new generation of the polydisulfide Gd(III) complexes containing a macrocyclic Gd(III) chelate, Gd-DOTA monoamide, to improve the in vivo kinetic inertness of the Gd(III) chelates. (N6-Lysyl)lysine-(Gd-DOTA) monoamide and 3-(2-carboxyethyldisulfanyl)propanoic acid copolymers (GODC) were synthesized by copolymerization of (N6-lysyl)lysine DOTA monoamide and dithiobis(succinimidylpropionate), followed by complexation with Gd(OAc)3. The GODC had an apparent molecular weight of 26.4 kDa and T1 relaxivity of 8.25 mM(-1) s(-1) per Gd at 1.5 T. The polymer chains of GODC were readily cleaved by L-cysteine and the chelates had high kinetic stability against transmetallation in the presence of an endogenous metal ion Zn(2+). In vivo MRI study showed that GODC produced strong and prolonged contrast enhancement in the vasculature and tumor periphery of mice with breast tumor xenografts. GODC is a promising biodegradable macromolecular MRI contrast agent with high kinetic stability for MR blood pool imaging.

Noninvasive evaluation of antiangiogenic effect in a mouse tumor model by DCE-MRI with Gd-DTPA cystamine copolymers.

The efficacy of polydisulfide-based biodegradable macromolecular Gd(III) complexes, Gd-DTPA cystamine copolymers (GDCC), for assessing tumor microvascular characteristics and monitoring antiangiogenesis therapy was investigated in a mouse model using dynamic contrast-enhanced MRI (DCE-MRI). The mice bearing human colon tumor xenografts were intraperitoneally injected with an antiangiogenesis agent Avastin three times in a week at a dose of 200 mug/mouse. DCE-MRI with GDCC of 40 kDa (GDCC-40) was performed before and at 36 h after the first treatment with Avastin and at the end of treatment (7 days). Gd(DTPA-BMA) was used as a low molecular weight control. The tumor vascular parameters, endothelial transfer coefficient K(trans) and factional plasma volume f(PV), were calculated from the DCE-MRI data with a two-compartment model. The K(trans) and f(PV) in tumor periphery estimated by DCE-MRI with GDCC-40 before and after the antiangiogenesis treatment correlated well to tumor growth before and after the treatment in the tumor model. In contrast, the parameters estimated by Gd(DTPA-BMA) did not show significant correlation to the therapeutic efficacy. This study demonstrates that DCE-MRI with the biodegradable macromolecular MRI contrast agent can provide effective assessment of the antiangiogenic efficacy of Avastin in the animal tumor model based on measured vascular parameters in tumor periphery.

Synthesis and evaluation of a peptide targeted small molecular Gd- DOTA monoamide conjugate for MR molecular imaging of prostate cancer

Tumor extracellular matrix has an abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. Innovative design and development of safe and effective targeted contrast agents to these biomarkers would allow effective MR cancer molecular imaging with high spatial resolution. In this study, we synthesized a low molecular weight CLT1 peptide targeted Gd(III) chelate CLT1-dL-(Gd-DOTA)(4) specific to clotted plasma proteins in tumor stroma for cancer MR molecular imaging. CLT1-dL-(Gd-DOTA)(4) was synthesized by conjugating four Gd-DOTA monoamide chelates to a CLT1 peptide via generation 1 lysine dendrimer. The T(1) relaxivity of CLT1-dL-(Gd-DOTA)(4) was 40.4 mM(-1) s(-1) per molecule (10.1 mM(-1) s(-1) per Gd) at 37 °C and 1.5 T. Fluorescence imaging showed high binding specificity of CLT1 to orthotopic PC3 prostate tumor in mice. The contrast agent resulted in improved tumor contrast enhancement in male athymic nude mice bearing orthotopic PC3 prostate tumor xenograft at a dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for high-resolution MR molecular imaging of prostate tumor.

Synthesis and evaluation of a nanoglobular dendrimer 5-aminosalicylic acid conjugate with a hydrolyzable Schiff base spacer for treating retinal degeneration

Biocompatible dendrimers with well-defined nanosizes are increasingly being used as carriers for drug delivery. 5-Aminosalicylic acid (5-ASA) is an FDA-approved therapeutic agent recently found effective in treating retinal degeneration of animal models. Here, a water-soluble dendrimer conjugate of 5-ASA (AGFB-ASA) was designed to treat such retinal degeneration. The drug was conjugated to a generation 2 (G2) lysine dendrimer with a silsesquioxane core (nanoglobule) by using a hydrolyzable Schiff base spacer. Incubation of nanoglobular G2 dendrimer conjugates containing a 4-formylbenzoate (FB) Schiff base spacer in pH 7.4 phosphate buffers at 37 °C gradually released 5-ASA. Drug release from the dendrimer conjugate was significantly slower than from the low molecular weight free Schiff base of 5-ASA (FB-ASA). 5-ASA release from the dendrimer conjugate was dependent on steric hindrance around the spacer. After intraperitoneal injection, the nanoglobular 5-ASA conjugate provided more effective 7-day protection against light-induced retinal degeneration at a reduced dose than free 5-ASA in Abca4(-/-)Rdh8(-/-) mice. The dendrimer 5-ASA conjugate with a degradable spacer could be a good candidate for controlled delivery of 5-ASA to the eye for treatment of retinal degeneration.

Polydisulfide manganese(II) complexes as non-gadolinium biodegradable macromolecular MRI contrast agents

To develop safe and effective manganese(II) ‐based biodegradable macromolecular MRI contrast agents.