MR Molecular Imaging of Prostate Cancer with a Peptide-Targeted Contrast Agent in a Mouse Orthotopic Prostate Cancer Model

Purpose  

To study the effectiveness of a peptide targeted nanoglobular Gd-DOTA complexes for MR molecular imaging of prostate cancer in a mouse orthotopic PC-3 prostate cancer model.     

Folate and TAT Peptide Co-Modified Liposomes Exhibit Receptor-Dependent Highly Efficient Intracellular Transport of Payload In Vitro and In Vivo

Purpose

Using different chain lengths of PEG as linkers to develop a novel folate (FA) and TAT peptide co-modified doxorubicin (DOX)-loaded liposome (FA/TAT-LP-DOX) and evaluate its potential for tumor targeted intracellular drug delivery.

Methods

FA/TAT-LP-DOX was prepared by pH gradient method and post-insertion method and the optimal ligand density was screened by MTT assay. In vitro evaluation was systematically performed through cytotoxicity assay, cellular uptake studies, subcellular localization and cellular uptake mechanism in folate receptor (FR) over-expressing KB tumor cells. In vivo tumor targeted delivery of FA/TAT-LP-DOX was also studied by in vivo fluorescence imaging in a murine KB xenograft model.

Results

The particle size and zeta potential determination indicated that FA and TAT were successfully inserted into the liposome and cationic TAT peptide was completely shielded. With the optimal ligand density (5% of FA and 2.5% TAT), the FA/TAT-LP-DOX exhibited improved cytotoxity and cellular uptake efficiency compared with its single-ligand counterparts (FA-LP-DOX and PEG/TAT-LP-DOX). Competitive inhibition and uptake mechanism experiments revealed that FA and TAT peptide played a synergistic effect in facilitating intracellular transport of the liposome, and association between FA and FA receptors activated this transport process. In vivo imaging further demonstrated the superiority of FA/TAT-LP in tumor targeting and accumulation.

Conclusions

Folate and TAT peptide co-modified liposome using different chain lengths of PEG as linkers may provide a useful strategy for specific and efficient intracellular drug delivery.     

89Zr-Labeled Paramagnetic Octreotide-Liposomes for PET-MR Imaging of Cancer

Purpose

Dual-modality PET/MR platforms add a new dimension to patient diagnosis with high resolution, functional, and anatomical imaging. The full potential of this emerging hybrid modality could be realized by using a corresponding dual-modality probe. Here, we report pegylated liposome (LP) formulations, housing a MR T₁ contrast agent (Gd) and the positron-emitting ⁸⁹Zr (half-life: 3.27 days), for simultaneous PET and MR tumor imaging capabilities.

Methods

⁸⁹Zr oxophilicity was unexpectedly found advantageous for direct radiolabeling of preformed paramagnetic LPs. LPs were conjugated with octreotide to selectively target neuroendocrine tumors via human somatostatin receptor subtype 2 (SSTr2). ⁸⁹Zr-Gd-LPs and octreotide-conjugated homolog were physically, chemically and biologically characterized.

Results

⁸⁹Zr-LPs showed reasonable stability over serum proteins and chelator challenges for proof-of-concept in vitro and in vivo investigations. Nuclear and paramagnetic tracking quantified superior SSTr2-recognition of octreotide-LP compared to controls.

Conclusions

This study demonstrated SSTr2-targeting specificity along with direct chelator-free ⁸⁹Zr-labeling of LPs and dual PET/MR imaging properties.     

Efficient siRNA Delivery Using a Polyamidoamine Dendrimer with a Modified Pentaerythritol Core

Purpose

Delivery of siRNA into cells remains a critical challenge. Our lab has shown a novel polyamidoamine (PAMAM) dendrimer with modified pentaerythritol derivative core (PD dendrimer) to exhibit high plasmid DNA transfection efficiency and low cytotoxicity. Here, we evaluate PD dendrimer as a siRNA carrier.

Methods

Agarose gel electrophoresis and AFM were used to confirm formation of generation 5 (G5)-PD dendrimer/siRNA nanoparticles (NPs). G5 PD dendrimer/anti-luciferase siRNA NPs were used to transfect SK Hep-1 cells with stable luciferase expression. Effects of various endocytic pathway inhibitors on uptake of G5 PD dendrimer/siRNA NPs in SK Hep-1 cells were also investigated.

Results

Agarose gel electrophoresis indicated that G5 PD dendrimer and siRNA formed NPs at weight ratios >0.5:1. G5 PD dendrimer showed effective luciferase gene silencing when weight ratio was 3.0:1 and above. Treatment with endocytosis inhibitors showed that clathrin-mediated endocytosis was the main endocytic pathway by which G5-PD dendrimer/siRNA NPs enter the cell.

Conclusions

These results show that the novel G5 PD dendrimer has high siRNA delivery activity and is promising as a delivery agent for its therapeutic application.     

Increased Active Tumor Targeting by An αvβ3-Targeting and Cell-Penetrating Bifunctional Peptide-Mediated Dendrimer-Based Conjugate

Purpose

A bifunctional RGDTAT peptide-modified PEG-PAMAM dendrimer conjugate RGDTAT-PEG-PAMAM (RTPP) was established for the targeted treatment of αvβ3-overexpressing tumor cells.

Methods

The RGDTAT peptide was synthesized and attached to PAMAM using PEG to construct the RTPP conjugate. The methotrexate (MTX) encapsulated RTPPM complex was prepared and characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM) and in vitro release. The targeting ability was then studied in cells and tumor-bearing nude mice using fluorescence microscopy, confocal fluorescence microscopy, flow cytometry, and in vivo imaging. The cytotoxicity and pharmacokinetics of the RTPPM complex was also evaluated in cells and rats.

Results

The successful synthesis of the RTPP conjugate was confirmed by ¹H-NMR. DLS and TEM measurements revealed that the size was 37 nm and the complex had a spherical shape. RTPP and RTPPM were taken up by αvβ3-overexpressing cells more efficiently than by αvβ3-lowexpressing cells. The RTPP conjugate localized to the cell nucleus and accumulated in the tumor more efficiently than did the conjugates without RGDTAT. The pharmacokinetic study of the RTPPM complex showed sustained drug release.

Conclusions

The bifunctional peptide-mediated dendrimer-based RTPP conjugate can serve as a promising nanocarrier for targeted drug delivery to improve anti-tumor activity.

     

Asymmetric Peptide Dendrimers are Effective Linkers for Antibody-Mediated Delivery of Diverse Payloads to B Cells in Vitro and in Vivo

Purpose

Safe, targeted delivery of therapeutics remains a focus of drug/gene delivery, the aim being to achieve optimal efficacy while minimising off-target delivery. Dendrimers have a vast array of potential applications and have great potential as gene and drug delivery tools. We previously reported the development of peptide dendrimers that effectively complexed DNA and that have distinct advantages over conventional spherical dendrimers. Here, to expand the application of peptide-based low generation dendrimers we tested their capacity to be transformed into linkers for antibody-based targeting of diverse payloads.

Methods

Peptide-based low-generation asymmetric dendrimers were generated and conjugated to partially-reduced antibodies specific for B cell surface antigens or an irrelevant antigen. Preservation of antigen binding by the antibodies and targeting of the conjugated dendrimers carrying a small molecule (biotin) or plasmid DNA payloads was tested.

Results

Peptide-based low generation dendrimers were efficiently and site-specifically conjugated to antibodies with retention of antigen-binding capacity. Altering the branching termini of dendrimers facilitated delivery of diverse payloads in vitro and in vivo.

Conclusions

We propose that safe, non-toxic peptide dendrimers, which are readily synthesised and modifiable for a variety of applications, form the basis of a new family of biocompatible “linkers” with substantial potential for targeted delivery applications.     

Fluorescence Imaging of the Lymph Node Uptake of Proteins in Mice after Subcutaneous Injection: Molecular Weight Dependence

Purpose

To use noninvasive fluorescence imaging to investigate the influence of molecular weight (MW) of proteins on the rate of loss from a subcutaneous (SC) injection site and subsequent uptake by the draining lymph nodes in mice.

Methods

Bevacizumab (149?kDa), bovine serum albumin (BSA, 66?kDa), ovalbumin (44.3?kDa) or VEGF-C156S (23?kDa), labeled with the near infrared dye IRDye 680, were injected SC into the front footpad of SKH-1 mice. Whole body non-invasive fluorescence imaging was performed to quantitate the fluorescence signal at the injection site and in axillary lymph nodes.

Results

The half-life values, describing the times for 50% loss of proteins from the injection site, were 6.81?h for bevacizumab, 2.85?h for BSA, 1.57?h for ovalbumin and 0.31?h for VEGF-C156S. The corresponding axillary lymph node exposure, represented as the area of the % dose versus time curve, was 6.27, 5.13, 4.06 and 1.54% dose ? h, respectively.

Conclusions

Our results indicate that the rate of loss of proteins from a SC injection site is inversely related to MW of proteins, while lymph node exposure is proportionally related to the MW of proteins in a mouse model.     

EphA2 Targeting Pegylated Nanocarrier Drug Delivery System for Treatment of Lung Cancer

Purpose

Evaluation of tumor targeting pegylated EphA2 peptide coated nanoparticles (ENDDs) of a novel anticancer agent DIM-C-pPhC₆H₅ (DIM-P) and Docetaxel (DOC) and investigate its antitumor activity and potential for treatment of lung cancer.

Methods

Nanoparticles were prepared with DIM-P and DOC (NDDs) using Nano-DeBEE. ENDDs were prepared by conjugating NDDs with 6His-PEG2K-EphA2 peptide and characterized for physicochemical properties, binding assay, cytotoxicity, cellular uptake studies, drug release and pharmacokinetic parameters. Anti-tumor activity of ENDDs was evaluated using a metastatic H1650 and orthotopic A549 tumor models in nude mice and tumor tissue were analyzed by RT-PCR and immunohistochemistry.

Results

Particle size and entrapment efficiency of ENDDs were 197?±?21 nm and 95?±?2%. ENDDs showed 32.5?±?3.5% more cellular uptake than NDDs in tumor cells. ENDDs showed 23?±?3% and 26?±?4% more tumor reduction compared to NDDs in metastatic and orthotopic tumor models, respectively. In-vivo imaging studies using the Care stream MX FX Pro system showed (p?Conclusions The results emanating from these studies demonstrate anti-cancer potential of DIM-P and the role of ENDDs as effective tumor targeting drug delivery systems for lung cancer treatment.     

Tumor-Targeted Responsive Nanoparticle-Based Systems for Magnetic Resonance Imaging and Therapy

Purpose

Design and synthesis of a tumor responsive nanoparticle-based system for imaging and treatment of various cancers.

Methods

Manganese oxide nanoparticles (Mn₃O₄ NPs) were synthesized and modified with LHRH targeting peptide or anti-melanoma antibodies (cancer targeting moieties) and a MMP2 cleavable peptide (a possible chemotactic factor). Nanostructured lipid carriers (NLCs) were used to entrap the BRAF inhibitor, vemurafenib, and enhance cytotoxicity of the drug. Size distribution, stability, drug entrapment, cytotoxicity and genotoxicity of synthesized nanoparticles were studied in vitro. Enhancement of MRI signal by nanoparticles and their body distribution were examined in vivo on mouse models of melanoma, ovarian and lung cancers.

Results

Uniform, stable cancer-targeted nanoparticles (PEGylated water-soluble Mn₃O₄ NPs and NLCs) were synthesized. No signs of cyto-,genotoxicity and DNA damage were detected for nanoparticles that do not contain an anticancer drug. Entrapment of vemurafenib into nanoparticles significantly enhanced drug toxicity in cancer cells with targeted V600E mutation. The developed nanoparticles containing LHRH and MMP2 peptides showed preferential accumulation in primary and metastatic tumors increasing the MRI signal in mice with melanoma, lung and ovarian cancers.

Conclusions

The proposed nanoparticle-based systems provide the foundation for building an integrated MRI diagnostic and therapeutic approach for various types of cancer.     

Sustained Analgesia Achieved Through Esterase-Activated Morphine Prodrugs Complexed with PAMAM Dendrimer

Purpose

Design and evaluate the in vitro and in vivo efficacy of two extended release morphine formulations developed for IV administration by complexing esterase activated morphine prodrugs to surface-modified, generation 5 (G5) poly(amidoamine) (PAMAM) dendrimer.

Methods

Prodrugs were synthesized, complexed with PAMAM dendrimer, characterized via ultra performance liquid chromatography (UPLC), nuclear magnatic resonance (NMR), and tested in vitro using rat plasma vs. saline control and in an in vivo rat and guinea pig pain model (modified Randall and Selitto test).

Results

We demonstrated that complexation with dendrimer allowed the solubilization of the prodrugs for in vivo applications without the need for salt, and that the structural design of the morphine prodrugs allowed the controlled release of morphine which extended the action of morphine-induced analgesia in an animal pain model from 2 h (control) to 6 h (Morphine Prodrug A).

Conclusion

The concept of complexing/solubilizing appropriately designed esterase-sensitive prodrugs with dendrimer to enhance the sustained release of these drugs may be a useful pharmacokinetic strategy for a range of therapeutics.     

Up-regulating Blood Brain Barrier Permeability of Nanoparticles via Multivalent Effect

Purpose

To investigate the multivalent effect for up-regulating the intracerebral delivery of nanoparticles via receptor-mediated transcytosis.

Methods

Nanoparticles labeled with near-infrared (NIR) fluorophore and different numbers of angiopep-2 peptides that specifically target low-density lipoprotein receptor-related protein (LRP) on the brain capillary endothelial cells were developed. Bio-distribution studies quantified the intracerebral uptakes of these nanoparticles at 2 and 24 h after intravenous injection. In vivo NIR fluorescence imaging, ex vivo autoradiographic imaging and 3D reconstructed NIR fluorescence imaging revealed the nanoparticle distribution pattern in brain. Fluorescence microscopic imaging identified the nanoparticle locations at the cellular level.

Results

The multimetirc association between the angiopep-2 peptides labeled on the nanoparticle and the LRP receptors on the brain capillary endothelial cells significantly increased the intracerebral uptake of the nanoparticles. Nanoparticle Den-Angio4 labeled four angiopep-2 peptides achieved the highest BBB traverse efficacy. After penetrating the BBB, Den-Angio4 distributed heterogeneously and mainly located at hippocampus, striatum and cerebellum in the brains.

Conclusions

The multivalent effect significantly enhances the BBB permeability of nanoparticles. Den-Angio4 as a nanoparticle prototype provides a two order targeted strategy for diagnosis or treatment of central nerver system diseases by first traversing the BBB via receptor-mediated endocytosis and secondly targeting the leisions with high receptor expression level.     

Biocompatible Glycol Chitosan-Coated Gold Nanoparticles for Tumor-Targeting CT Imaging

Purpose

The application of gold nanoparticles (AuNPs) in biomedical field was limited due to the low stability in the biological condition. Herein, to enhance stability and tumor targeting ability of AuNPs, their surface was modified with biocompatible glycol chitosan (GC) and the in vivo biodistribution of GC coated AuNPs (GC-AuNPs) were studied through computed tomography (CT).

Methods

Polymer-coated gold nanoparticles were produced using GC as a reducing agent and a stabilizer. Their feasibility in biomedical application was explored through CT in tumor-bearing mice.

Results

Stability of gold nanoparticles increased in the physiological condition due to the GC coating layer on the surface. Tomographic images of tumor were successfully obtained in the tumor-xenografted animal model when the GC-AuNPs were used as a CT contrast agent. The tumor targeting property of the gold nanoparticles was due to the properties of GC because GC-AuNPs were accumulated in the tumor, while most of heparin-coated nanoparticles were found in the liver and spleen.

Conclusions

The polymer properties on the surface played an important role in the behavior of gold nanoparticles in the biological condition and the enhanced stability and tumor targeting property of nanoparticles were inherited from GC on the surface.     

Opening Up the Optical Imaging Window Using Nano-Luciferin

Purpose

The objective of this study was to formulate nanoparticles of D-luciferin (Nano-Luc), DiR (Nano-DiR) and dual functional nanoparticles with DiR and luciferin (Nano-LucDiR) for in-vivo imaging as well as tracking of the nanoparticles in tumors.

Methods

Nano-Luc and Nano-LucDiR were prepared using different lipids, and subsequently characterized for loading and entrapment efficiency, physical properties, release profile, toxicity and stability. We utilized Response Surface Methodology (RSM) to optimize the nanoparticles using design of experiment (DOE Vr.8.0). Nano-Luc was evaluated against free luciferin to establish its pharmacokinetic parameters in mice. In-vivo imaging of tumors and tracking of nanoparticles was carried out with an IVIS® Spectrum-CT (Caliper) using xenograft, orthotopic and metastatic tumor models in BALB/c nude mice with different cell lines and different routes of nanoparticle administration (subcutaneous, intraperitoneal and intravenous).

Results

Particle size of both Nano-Luc and Nano-LucDiR were found to be <200 nm. Nano-Luc formulation showed a slow and controlled release upto 72 h (90%) in vitro. The optimized Nano-Luc had loading efficiency of 5.0 mg/ml with 99% encapsulation efficiency. Nano-Luc and Nano-LucDiR formulations had good shelf stability. Nano-Luc and Nano-LucDiR enhanced plasma half-life of luciferin compared to free luciferin thus providing longer circulation of luciferin in plasma enabling imaging of tumors for more than 24 h. Nano-LucDiR allowed simultaneous bioluminescent and fluorescent imaging to be conducted, with three-dimensional reconstruct of tumors without losing either signal during the acquisition time.

Conclusion

Nano-Luc and Nano-LucDiR allowed prolonged reproducible in-vivo imaging of tumors, especially during multimodality 3D imaging.     

Biodistribution Studies of Nanoparticles Using Fluorescence Imaging: A Qualitative or Quantitative Method?

Purpose

The biodistribution of Lipid/Calcium/Phosphate (LCP) nanoparticles (NPs) in tumor-bearing mice was investigated using fluorescence imaging. A quantitative validation of this method was done by ³H and ¹¹¹In labeling of the nanoparticles.

Methods

The biodistribution of LCP NPs containing oligonucleotides was investigated using three different probes: Texas-Red labeled oligonucleotides, ³H-labeled oligonucleotides, and ¹¹¹In-labled calcium phosphate.

Results

A discrepancy was found between the radioactivity and the fluorescence signals. Signals from ³H and ¹¹¹In exhibited very similar distribution patterns, suggesting that liver and spleen were the major accumulation sites. However, fluorescence imaging indicated that tumor accumulation was predominant. We further confirmed that the fluorescence signals in both liver and spleen were greatly attenuated compared with those in the tumor due to the intrinsic tissue absorption and scattering. Near-infrared (NIR) dye Cy5.5 also suffered from the same problem, in that the quantitative data from whole organs was dramatically affected by absorption and scattering properties of the tissue.

Conclusions

Careful attention must be paid to the quantification and interpretation of fluorescence imaging measurements when comparing different tissues.     

CEACAM5 is correlated with Angio/Lymphangiogenesis of Prostatic Lesions

Objective

The aim of this study was to examine the expression patterns of CEACAM5 in prostatic non-neoplastic and neoplastic lesions and further investigate its relationship with tumor microvessel density(MVD) and lymphatic vessel density(LVD).

Methods

CEACAM5 expression was detected using immunohistochemical staining in a serial sections of the benign prostatic hyperplasia (BPH), prostate intraepithelial neoplasia (PIN) and prostate carcinoma (PCa) lesions. MVD and LVD were quantified in CEACAM5 positive areas by dual-labelling with CD34 and D2-40 respectively.

Results

Both PIN and PCa had significantly higher expression for CEACAM5 than BPH which has no positive expression for CEACAM5(P<0.05). In PIN and PCa, CEACAM5 staining showed different expression patterns in terms the of most of membranous staining for PIN, less membranous staining and more cytoplasmic staining for PCa. MVD results showed that PCa and PIN had more angiogenesis than BPH tissue. The value of MVD in PCa tissue was correlated with tumor Gleason grading (P<0.05). LVD results showed that neoplastic lesions had more lymphangiogenesis than non-neoplastic lesion.

Conclusion

CEACAM5 had different expression patterns in prostatic non-neoplastic and neoplastic lesions, and these various expression patterns may be correlated with tumor progression through promoting tumorous angiogenesis or lymphangiogenesis.     

Evaluation of a 99mTc-Labeled AnnexinA5 Variant for Non-invasive SPECT Imaging of Cell Death in Liver, Spleen and Prostate

Purpose

We investigate radio-labeling and pharmacokinetics of a new AnnexinA5 variant (HYNIC-cys-AnxA5) and then assess its utility for the non-invasive detection of cell death in liver, spleen and prostate.

Methods

AnnexinA5 binds to phosphatidylserine expressed on the surface of apoptotic and necrotic cells. Contrary to other AnnexinA5 variants, the new cys-AnxA5 allows for site-specific conjugation of a hydrazinonicotinamide-maleimide moiety and subsequent radio-labeling with ^99mTc at a position not involved in the AnxA5-phosphatidylserine interaction. Distribution of ^99mTc-HYNIC-cys-AnxA5 was studied in rats, both invasively and via SPECT/CT. Cycloheximide was used to induce cell death in liver and spleen, whereas apoptosis in the prostate was induced by castration.

Results

HYNIC-cys-AnxA5 was efficiently and reproducibly labeled with ^99mTc. Blood clearance of radioactivity after iv-injection was adequately described by a two-compartment model, the renal cortex representing the main site of accumulation. Cycloheximide treatment resulted in increased accumulation of intravenous-injected ^99mTc-HYNIC-cys-AnxA5 in liver and spleen over controls, which correlated well with TUNEL staining for cell death in corresponding tissue sections. However, the increase in TUNEL-positive prostate epithelial cells observed following castration was not paralleled by greater ^99mTc-HYNIC-cys-AnxA5 accumulation.

Conclusion

^99mTc-HYNIC-cys-AnxA5 appears a suitable tracer for assessment of cell death in liver and spleen, but not prostate.     

Design and Modular Construction of a Polymeric Nanoparticle for Targeted Atherosclerosis Positron Emission Tomography Imaging: A Story of 25% <Superscript>64</Superscript>Cu-CANF-Comb

Purpose

To assess the physicochemical properties, pharmacokinetic profiles, and in vivo positron emission tomography (PET) imaging of natriuretic peptide clearance receptors (NPRC) expressed on atherosclerotic plaque of a series of targeted, polymeric nanoparticles.

Methods

To control their structure, non-targeted and targeted polymeric (comb) nanoparticles, conjugated with various amounts of c-atrial natriuretic peptide (CANF, 0, 5, 10 and 25%), were synthesized by controlled and modular chemistry. In vivo pharmacokinetic evaluation of these nanoparticles was performed in wildtype (WT) C57BL/6 mice after ⁶⁴Cu radiolabeling. PET imaging was performed on an apolipoprotein E–deficient (ApoE^?/?) mouse atherosclerosis model to assess the NPRC targeting efficiency. For comparison, an in vivo blood metabolism study was carried out in WT mice.

Results

All three ⁶⁴Cu-CANF-comb nanoparticles showed improved biodistribution profiles, including significantly reduced accumulation in both liver and spleen, compared to the non-targeted ⁶⁴Cu-comb. Of the three nanoparticles, the 25% ⁶⁴Cu-CANF-comb demonstrated the best NPRC targeting specificity and sensitivity in ApoE^?/? mice. Metabolism studies showed that the radiolabeled CANF-comb was stable in blood up to 9 days. Histopathological analyses confirmed the up-regulation of NPRC along the progression of atherosclerosis.

Conclusion

The 25% ⁶⁴Cu-CANF-comb demonstrated its potential as a PET imaging agent to detect atherosclerosis progression and status.

     

<Emphasis Type="Italic">αvβ</Emphasis>3 Integrin Receptor Targeting and Near-Infrared Imaging of Solid Tumors Using Surface-Modified Nanoliposomes

Purpose

Abundance of receptors on tumor vasculature presents a prominent target for theranostic applications. The alphavbeta3 integrin receptors expressed on vascular endothelial cells during angiogenesis were therefore considered targets for imaging. Non-invasive visualization of tumor growth and/or delivery systems can appreciate tumor localization and disposition kinetics of carriers, respectively. Herein, we report near-infrared fluorescence imaging (NIRFI) of solid tumors using targeted fluorescence nanoliposomes in vivo.

Methods

Fluorescence nanoliposomes surface modified with cRGD-peptide were injected into CD1 athymic (nu/nu) mice bearing C6 glioblastoma xenografts (300 mm³). At different time points, mice were subjected to NIRFI for visualization of tumor xenografts and nanocarrier tracing in vivo.

Results

NIRFI showed tumor localization of 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl indotricarbocyanine iodide (DiR₁₈)-incorporated-targeted liposomes with maximum tumor-to-tissue occurring at 24-h post-liposome administration. Interaction of integrin receptors with targeted liposomes had contributed to an intense NIRF signal. Molecular studies showed an elevated expression of alphavbeta3 integrin receptors in tumor xenografts.

Conclusion

From the studies, it can be concluded that non-invasive localization of tumors and tracing of liposome carriers had been achieved using receptor targeting and NIRFI approaches.

     

Mitochondrial Delivery of Doxorubicin by Triphenylphosphonium-Functionalized Hyperbranched Nanocarriers Results in Rapid and Severe Cytotoxicity

Purpose

To develop a novel hyperbranched polymer-based nanocarrier for efficient drug delivery to cell mitochondria. Also to study for the first time the cytotoxic effect of doxorubicin via mitochondria-specific delivery system.

Methods

We introduced alkyltriphenylphosphonium groups (TPP) to a poly(ethylene imine) hyperbranched polymer (PEI). We harnessed the hydrophobic assembly of these alkylTPP functionalized PEI molecules into ~100 nm diameter nanoparticles (PEI-TPP) and further encapsulated the chemotherapy agent doxorubicin (DOX), to produce the mitotropic nanoparticles PEI-TPP-DOX.

Results

By administering PEI-TPP-DOX to human prostate carcinoma cells DU145, we found that: (i) PEI-TPP-DOX specifically localized at cell mitochondria as revealed by the inherent DOX fluorescence; (ii) in contrast to the slow apoptotic cell death incurred by DOX over the period of days at micromolar concentrations, PEI-TPP-DOX triggered rapid and severe cytotoxicity within few hours of incubation and at submicromolar incubation concentrations. This cytotoxicity was mainly found to be of a necrotic nature, not precluding autophagy related death pathways to a smaller extent.

Conclusions

We have elaborated a versatile mitotropic nanocarrier; furthermore, using this platform, we have developed a mitochondrial-doxorubicin formulation with exceptional cytocidal properties, even in nanomolar concentrations.

In Vivo Fluorescence Imaging of IgG1 Aggregates After Subcutaneous and Intravenous Injection in Mice

Purpose

To monitor the biodistribution of IgG1 aggregates upon subcutaneous (SC) and intravenous (IV) administration in mice and measure their propensity to stimulate an early immune response.

Methods

A human mAb (IgG1) was fluorescently labeled, aggregated by agitation stress and injected in SKH1 mice through SC and IV routes. The biodistribution of monomeric and aggregated formulations was monitored over 47 days by fluorescence imaging and the early immune response was measured by quantifying the level of relevant cytokines in serum using a Bio-plex assay.

Results

The aggregates remained at the SC injection site for a longer time than monomers but after entry into the systemic circulation disappeared faster than monomers. Upon IV administration, both monomers and aggregates spread rapidly throughout the circulation, and a strong accumulation in the liver was observed for both species. Subsequent removal from the circulation was faster for aggregates than monomers. No accumulation in lymph nodes was observed after SC or IV administration. Administration of monomers and aggregates induced similar cytokine levels, but SC injection resulted in higher cytokine levels than IV administration.

Conclusion

These results show differences in biodistribution and residence time between IgG1 aggregates and monomers. The long residence time of aggregates at the SC injection site, in conjunction with elevated cytokine levels, may contribute to an enhanced immunogenicity risk of SC injected aggregates compared to that of monomers.