Elucidating the anomalous membrane permeability of Ag(I), Cu(II), Zn(II) and Au(III) towards new nanoreactor strategies for synthesizing metal nanoparticles.

The main structural element defining the cell is the lipid membrane, which is an integral part of regulating the fluxes of ion and nutrition molecules in and out of the cell. Surprisingly, copper ions were found to have anomalous membrane permeability. This led us to consider a broader spectrum of cations and further a new approach for using liposomes as nanoreactors for synthesis of metal and metal alloy nanoparticles. In the present study, the high membrane permeability of Cu2+ and its neighbouring transition elements in the periodic table was investigated. The permeability of Ni2+, Cu2+, Zn2+, Ag+, Au3+, Mg2+, Ca2+ and Lu3+ was assessed, and we report that Zn2+, Cu2+, Ag+ and Au3+ surprisingly are able to cross lipid bilayers. This knowledge is highly relevant for understanding trafficking of cations in biological systems, as well as for design of novel nanoparticle and nanoreactor systems. An example of its use is presented as a platform for synthesizing single highly uniform gold nanoparticles inside liposomal nanoreactors. We envision that this approach could provide a new nanoreactor methodology for forming highly structurally constrained uniform metal and metal alloy nanoparticles, as well as new methods for in vivo tracking of liposomes.

Multimodal soft tissue markers for bridging high-resolution diagnostic imaging with therapeutic intervention.

Diagnostic imaging often outperforms the surgeon's ability to identify small structures during therapeutic procedures. Smart soft tissue markers that translate the sensitivity of diagnostic imaging into optimal therapeutic intervention are therefore highly warranted. This paper presents a unique adaptable liquid soft tissue marker system based on functionalized carbohydrates (Carbo-gel). The liquid state of these markers allows for high-precision placement under image guidance using thin needles. Based on step-by-step modifications, the image features and mechanical properties of markers can be optimized to bridge diagnostic imaging and specific therapeutic interventions. The performance of Carbo-gel is demonstrated for markers that (i) have radiographic, magnetic resonance, and ultrasound visibility; (ii) are palpable and visible; and (iii) are localizable by near-infrared fluorescence and radio guidance. The study demonstrates encouraging proof of concept for the liquid marker system as a well-tolerated multimodal imaging marker that can improve image-guided radiotherapy and surgical interventions, including robotic surgery.

Remote loading of liposomes with a 124I-radioiodinated compound and their in vivo evaluation by PET/CT in a murine tumor model.

Long circulating liposomes entrapping iodinated and radioiodinated compounds offer a highly versatile theranostic platform. Here we report a new methodology for efficient and high-yield loading of such compounds into liposomes, enabling CT/SPECT/PET imaging and 131I-radiotherapy. Methods: The CT contrast agent diatrizoate was synthetically functionalized with a primary amine, which enabled its remote loading into PEGylated liposomes by either an ammonium sulfate- or a citrate-based pH transmembrane gradient. Further, the amino-diatrizoate was radiolabeled with either 124I (t1/2 = 4.18 days) for PET or 125I (t1/2 = 59.5 days) for SPECT, through an aromatic Finkelstein reaction. Results: Quantitative loading efficiencies (>99%) were achieved at optimized conditions. The 124I-labeled compound was remote-loaded into liposomes, with an overall radiolabeling efficiency of 77 ± 1%, and imaged in vivo in a CT26 murine colon cancer tumor model by PET/CT. A prolonged blood circulation half-life of 19.5 h was observed for the radiolabeled liposomes, whereas injections of the free compound were rapidly cleared. Lower accumulation was observed in the spleen, liver, kidney and tumor than what is usually seen for long-circulating liposomes. Conclusion: The lower accumulation was interpreted as release of the tracer from the liposomes within these organs after accumulation. These results may guide the design of systems for controlled release of remote loadable drugs from liposomes.

Mouse Positron Emission Tomography Study of the Biodistribution of Gold Nanoparticles with Different Surface Coatings Using Embedded Copper-64.

By taking advantage of the ability of 64Cu to bind nonspecifically to gold surfaces, we have developed a methodology to embed this radionuclide inside gold nanoparticles (AuNPs). 64Cu enables the in vivo imaging of AuNPs by positron emission tomography (PET). AuNPs have a multitude of uses within health technology and are useful tools for general nanoparticle research. 64Cu-AuNPs were prepared by incubating AuNP seeds with 64Cu2+, followed by the entrapment of the radionuclide by grafting on a second layer of gold. This resulted in radiolabeling efficiencies of 53 ± 6%. The radiolabel showed excellent stability when incubated with EDTA for 2 days (95% radioactivity retention) and showed no loss of 64Cu when incubated with 50% mouse serum for 2 days. The methodology was chelator-free, removing traditional concerns over chelator instability and altered AuNP properties due to surface modification. Radiolabeled 64Cu-AuNP cores were prepared in biomedically relevant sizes of 20-30 nm and used to investigate the in vivo stability of three different AuNP coatings by PET imaging in a murine xenograft tumor model. We found the longest plasma half-life (T1/2 about 9 h) and tumor accumulation (3.9%ID/g) to result from a polyethylene glycol coating, while faster elimination from the bloodstream was observed with both a Tween 20-stabilized coating and a zwitterionic coating based on a mixture of sulfonic acids and quaternary amines. In the in vivo model, the 64Cu was observed to closely follow the AuNPs for each coating, again attributed to the excellent stability of the radiolabel.

Affinity Induced Surface Functionalization of Liposomes Using Cu-Free Click Chemistry.

Functionalization of nanoparticles is a key element for improving specificity of drug delivery systems toward diseased tissue or cells. In the current study we report a highly efficient and chemoselective method for post-functionalization of liposomes with biomacromolecules, which equally well can be used for functionalization of other nanoparticles or solid surfaces. The method exploits a synergistic effect of having both affinity and covalent anchoring tags on the surface of the liposome. This was achieved by synthesizing a peptide linker system that uses Cu-free strain-promoted click chemistry in combination with histidine affinity tags. The investigation of post-functionalization of PEGylated liposomes was performed with a cyclic RGDfE peptide. By exploring both affinity and covalent tags a 98 ± 2.0% coupling efficiency was achieved, even a diluted system showed a coupling efficiency of 87 ± 0.2%. The reaction kinetics and overall yield were quantified by HPLC. The results presented here open new possibilities for constructing complex nanostructures and functionalized surfaces.

Investigation of enzyme-sensitive lipid nanoparticles for delivery of siRNA to blood-brain barrier and glioma cells.

Clinical applications of siRNA for treating disorders in the central nervous system require development of systemic stable, safe, and effective delivery vehicles that are able to cross the impermeable blood-brain barrier (BBB). Engineering nanocarriers with low cellular interaction during systemic circulation, but with high uptake in targeted cells, is a great challenge and is further complicated by the BBB. As a first step in obtaining such a delivery system, this study aims at designing a lipid nanoparticle (LNP) able to efficiently encapsulate siRNA by a combination of titratable cationic lipids. The targeted delivery is obtained through the design of a two-stage system where the first step is conjugation of angiopep to the surface of the LNP for targeting the low-density lipoprotein receptor-related protein-1 expressed on the BBB. Second, the positively charged LNPs are masked with a negatively charged PEGylated (poly(ethylene glycol)) cleavable lipopeptide, which contains a recognition sequence for matrix metalloproteinases (MMPs), a class of enzymes often expressed in the tumor microenvironment and inflammatory BBB conditions. Proteolytic cleavage induces PEG release, including the release of four glutamic acid residues, providing a charge switch that triggers a shift of the LNP charge from weakly negative to positive, thus favoring cellular endocytosis and release of siRNA for high silencing efficiency. This work describes the development of this two-stage nanocarrier-system and evaluates the performance in brain endothelial and glioblastoma cells with respect to uptake and gene silencing efficiency. The ability of activation by MMP-triggered dePEGylation and charge shift is demonstrated to substantially increase the uptake and the silencing efficiency of the LNPs.

Synthesis of Cross-Linked Polymeric Micelle pH Nanosensors: An Investigation of Design Flexibility.

The design flexibility that polymeric micelles offer in the fabrication of optical nanosensors for ratiometric pH measurements is investigated. pH nanosensors based on polymeric micelles are synthesized either by a mixed-micellization approach or by a postmicelle modification strategy. In the mixed-micellization approach, self-assembly of functionalized unimers followed by shell cross-linking by copper-catalyzed azide-alkyne cycloaddition (CuAAC) results in stabilized cRGD-functionalized micelle pH nanosensors. In the postmicelle modification strategy, simultaneous cross-linking and fluorophore conjugation at the micelle shell using CuAAC results in a stabilized micelle pH nanosensor. Compared to the postmicelle modification strategy, the mixed-micellization approach increases the control of the overall composition of the nanosensors. Both approaches provide stable nanosensors with similar pKa profiles and thereby nanosensors with similar pH sensitivity.

Injectable Colloidal Gold for Use in Intrafractional 2D Image-Guided Radiation Therapy.

In the western world, approximately 50% of all cancer patients receive radiotherapy alone or in combination with surgery or chemotherapy. Image-guided radiotherapy (IGRT) has in recent years been introduced to enhance precision of the delivery of radiation dose to tumor tissue. Fiducial markers are often inserted inside the tumor to improve IGRT precision and to enable monitoring of the tumor position during radiation therapy. In the present article, a liquid fiducial tissue marker is presented, which can be injected into tumor tissue using thin and flexible needles. The liquid fiducial has high radio-opacity, which allows for marker-based image guidance in 2D and 3D X-ray imaging during radiation therapy. This is achieved by surface-engineering gold nanoparticles to be highly compatible with a carbohydrate-based gelation matrix. The new fiducial marker is investigated in mice where they are highly biocompatible and stable after implantation. To investigate the clinical potential, a study is conducted in a canine cancer patient with spontaneous developed solid tumor in which the marker is successfully injected and used to align and image-guide radiation treatment of the canine patient. It is concluded that the new fiducial marker has highly interesting properties that warrant investigations in cancer patients.

Injectable colloidal gold in a sucrose acetate isobutyrate gelating matrix with potential use in radiation therapy.

External beam radiation therapy relies on the ability to deliver high radiation doses to tumor cells with minimal exposure to surrounding healthy tissue. Advanced irradiation techniques, including image-guided radiation therapy (IGRT), rely on the ability to locate tumors to optimize the therapeutic benefit of these techniques. Today, radiopaque fiducial tissue markers are placed in or around tumors, for example, in prostate cancer patients to enhance the precision of daily and/or real-time IGRT. A liquid injectable fiducial marker (nanogel) is developed based on PEGylated gold nanoparticles and sucrose acetate isobutyrate (SAIB) with improved properties compared to current solid fiducial markers. The developed nanogel is investigated in vitro and subsequently evaluated in vivo in immunocompetent NMRI mice. The nanogel shows high CT-contrast and excellent stability in vivo over a period of 12 weeks. The nanogel is found to be biocompatible and well tolerated. No induction of the inflammatory cytokines INF-γ, IL-6, or TNF-α is observed throughout the study period. The developed nanogel seems to be a safe injectable fiducial marker ideally suited for IGRT that may further enhance the effect of radiation.

Quantitative evaluation of bioorthogonal chemistries for surface functionalization of nanoparticles.

We present here a highly efficient and chemoselective liposome functionalization method based on oxime bond formation between a hydroxylamine and an aldehyde-modified lipid component. We have conducted a systematic and quantitative comparison of this new approach with other state-of-the-art conjugation reactions in the field. Targeted liposomes that recognize overexpressed receptors or antigens on diseased cells have great potential in therapeutic and diagnostic applications. However, chemical modifications of nanoparticle surfaces by postfunctionalization approaches are less effective than in solution and often not high-yielding. In addition, the conjugation efficiency is often challenging to characterize and therefore not addressed in many reports. We present here an investigation of PEGylated liposomes functionalized with a neuroendocrine tumor targeting peptide (TATE), synthesized with a variety of functionalities that have been used for surface conjugation of nanoparticles. The reaction kinetics and overall yield were quantified by HPLC. Reactions were conducted in solution as well as by postfunctionalization of liposomes in order to study the effects of steric hindrance and possible affinity between the peptide and the liposome surface. These studies demonstrate the importance of choosing the correct chemistry in order to obtain a quantitative surface functionalization of liposomes.

Positron emission tomography evaluation of somatostatin receptor targeted 64Cu-TATE-liposomes in a human neuroendocrine carcinoma mouse model.

Targeted therapeutic and diagnostic nanocarriers functionalized with antibodies, peptides or other targeting ligands that recognize over-expressed receptors or antigens on tumor cells have potential in the diagnosis and therapy of cancer. Somatostatin receptors (SSTRs) are over-expressed in a variety of cancers, particularly neuroendocrine tumors (NETs) and can be targeted with somatostatin peptide analogs such as octreotate (TATE). In the present study we investigate liposomes that target SSTR in a NET xenograft mouse model (NCI-H727) by use of TATE. TATE was covalently attached to the distal end of DSPE-PEG(2000) on PEGylated liposomes with an encapsulated positron emitter (64)Cu that can be utilized for positron emission tomography (PET) imaging. The biodistribution and pharmacokinetics of the (64)Cu-loaded PEGylated liposomes with and without TATE was investigated and their ability to image NETs was evaluated using PET. Additionally, the liposome accumulation and imaging capability was compared with free radiolabelled TATE peptide administered as (64)Cu-DOTA-TATE. The presence of TATE on the liposomes resulted in a significantly faster initial blood clearance in comparison to control-liposomes without TATE. PEGylated liposomes with or without TATE accumulated at significantly higher quantities in NETs (5.1±0.3 and 5.8±0.2 %ID/g, respectively) than the free peptide (64)Cu-DOTA-TATE (1.4±0.3 %ID/g) 24 h post-injection. Importantly, (64)Cu-loaded PEGylated liposomes with TATE showed significantly higher tumor-to-muscle (T/M) ratio (12.7±1.0) than the control-liposomes without TATE (8.9±0.9) and the (64)Cu-DOTA-TATE free peptide (7.2±0.3). The higher T/M ratio of the PEGylated liposomes with TATE suggests some advantage of active targeting of NETs, although no absolute benefit in tumor accumulation over the non-targeted liposomes was observed. Collectively, these data showed that (64)Cu-loaded PEGylated liposomes with TATE conjugated to the surface could be promising new imaging agents for visualizing tumor tissue and especially NETs using PET.

Engineering liposomes and nanoparticles for biological targeting.

Our ability to engineer nanomaterials for biological and medical applications is continuously increasing, and nanomaterial designs are becoming more and more complex. One very good example of this is the drug delivery field where nanoparticle systems can be used to deliver drugs specifically to diseased tissue. In the early days, the design of the nanoparticles was relatively simple, but today we can surface functionalize and manipulate material properties to target diseased tissue and build highly complex drug release mechanisms into our designs. One of the most promising strategies in drug delivery is to use ligands that target overexpressed or selectively expressed receptors on the surface of diseased cells. To utilize this approach, it is necessary to control the chemistry involved in surface functionalization of nanoparticles and construct highly specific functionalities that can be used as attachment points for a diverse range of targeting ligands such as antibodies, peptides, carbohydrates and vitamins. In this review we provide an overview and a critical evaluation of the many strategies that have been developed for surface functionalization of nanoparticles and furthermore provide an overview of how these methods have been used in drug delivery systems.

Solid-phase synthesis of PEGylated lipopeptides using click chemistry.

A versatile methodology for efficient synthesis of PEGylated lipopeptides via CuAAC "Click" conjugation between alkyne-bearing solid-supported lipopeptides and azido-functionalized PEGs is described. This new and very robust method offers a unique platform for synthesizing PEGylated lipopeptides with a high level of complexity. These molecules, obtained in a single purification step, are ideally suited for functionalization of solid-supported lipid bilayers and liposomal drug delivery systems and are particularly valuable in enzyme activation strategies.