Geoffrey von Maltzahn

Biodegradable luminescent porous silicon nanoparticles for in vivo applications

Nanomaterials that can circulate in the body hold great potential to diagnose and treat disease. For such applications, it is important that the nanomaterials be harmlessly eliminated from the body in a reasonable period of time after they carry out their diagnostic or therapeutic function. Despite efforts to improve their targeting efficiency, significant quantities of systemically administered nanomaterials are cleared by the mononuclear phagocytic system before finding their targets, increasing the likelihood of unintended acute or chronic toxicity. However, there has been little effort to engineer the self-destruction of errant nanoparticles into non-toxic, systemically eliminated products. Here, we present luminescent porous silicon nanoparticles (LPSiNPs) that can carry a drug payload and of which the intrinsic near-infrared photoluminescence enables monitoring of both accumulation and degradation in vivo. Furthermore, in contrast to most optically active nanomaterials (carbon nanotubes, gold nanoparticles and quantum dots), LPSiNPs self-destruct in a mouse model into renally cleared components in a relatively short period of time with no evidence of toxicity. As a preliminary in vivo application, we demonstrate tumour imaging using dextran-coated LPSiNPs (D-LPSiNPs). These results demonstrate a new type of multifunctional nanostructure with a low-toxicity degradation pathway for in vivo applications.

Computationally Guided Photothermal Tumor Therapy Using Long-Circulating Gold Nanorod Antennas

Plasmonic nanomaterials have the opportunity to considerably improve the specificity of cancer ablation by i.v. homing to tumors and acting as antennas for accepting externally applied energy. Here, we describe an integrated approach to improved plasmonic therapy composed of multimodal nanomaterial optimization and computational irradiation protocol development. We synthesized polyethylene glycol (PEG)-protected gold nanorods (NR) that exhibit superior spectral bandwidth, photothermal heat generation per gram of gold, and circulation half-life in vivo (t(1/2), approximately 17 hours) compared with the prototypical tunable plasmonic particles, gold nanoshells, as well as approximately 2-fold higher X-ray absorption than a clinical iodine contrast agent. After intratumoral or i.v. administration, we fuse PEG-NR biodistribution data derived via noninvasive X-ray computed tomography or ex vivo spectrometry, respectively, with four-dimensional computational heat transport modeling to predict photothermal heating during irradiation. In computationally driven pilot therapeutic studies, we show that a single i.v. injection of PEG-NRs enabled destruction of all irradiated human xenograft tumors in mice. These studies highlight the potential of integrating computational therapy design with nanotherapeutic development for ultraselective tumor ablation.

Micellar Hybrid Nanoparticles for Simultaneous Magnetofluorescent Imaging and Drug Delivery

Multifunctional nanoparticles have the potential to integrate therapeutic and diagnostic functions into a single nanodevice.[1–9] To date, several types of hybrid nanosystems containing multiple different types of nanoparticles have been developed that allow multi-modal imaging. For example, formulations containing quantum dots (QD) and magnetic iron oxide nanoparticles (MN) provide a means to perform simultaneous fluorescent optical imaging and magnetic resonance imaging (MRI).[10–15] While these nanocomposites have been used for in vitro magnetic cell separation and in vitro cell targeting, there are limited in vivo studies, particularly for cancer imaging and therapy, due to poor stability or short systemic circulation times generally observed for these more complicated nanostructures.[16, 17] Herein, we introduce long-circulating, micellar hybrid nanoparticles (MHN) that contain MN, QD, and the anti-cancer drug doxorubicin (DOX) within a single polyethylene glycol (PEG)-phospholipid micelle and provide the first examples of simultaneous targeted drug delivery and dual-mode NIR-fluorescent and MR imaging of diseased tissue in vitro and in vivo.

Positively Charged Surfactant-like Peptides Self-assemble into Nanostructures

A new type of surfactant peptide designed to mimic the properties of cationic lipid systems is described. These cationic surfactant peptides are approximately 2 nm in length with a cationic, hydrophilic head consisting of one to two residues of lysine or histidine followed by a hydrophobic tail of six alanine, valine, or leucine residues. In water, these surfactant peptides form ordered structures with dynamic behaviors. At pH below the pI values of the peptides, dynamic light scattering showed two distinct structural populations with average diameters of 50 nm (>95%) and 100-200 nm (<5%). Transmission electron microscopy visualization of quick-frozen samples revealed these populations to likely represent nanotubes and nanovesicles, respectively, showing great interplay between them. Above the pI, these structures are absent, having further self-assembled into large membranous sheets. These cationic surfactant peptides are distinct from other anionic surfactant peptides and have different applications, possibly being useful as carriers for encapsulation and delivery of a number of small water-insoluble molecules and large biological molecular systems, including negatively charged nucleic acids.

Cooperative nanomaterial system to sensitize, target, and treat tumors

A significant barrier to the clinical translation of systemically administered therapeutic nanoparticles is their tendency to be removed from circulation by the mononuclear phagocyte system. The addition of a targeting ligand that selectively interacts with cancer cells can improve the therapeutic efficacy of nanomaterials, although these systems have met with only limited success. Here, we present a cooperative nanosystem consisting of two discrete nanomaterials. The first component is gold nanorod (NR) “activators” that populate the porous tumor vessels and act as photothermal antennas to specify tumor heating via remote near-infrared laser irradiation. We find that local tumor heating accelerates the recruitment of the second component: a targeted nanoparticle consisting of either magnetic nanoworms (NW) or doxorubicin-loaded liposomes (LP). The targeting species employed in this work is a cyclic nine-amino acid peptide LyP-1 (Cys-Gly-Asn-Lys-Arg-Thr-Arg-Gly-Cys) that binds to the stress-related protein, p32, which we find to be upregulated on the surface of tumor-associated cells upon thermal treatment. Mice containing xenografted MDA-MB-435 tumors that are treated with the combined NR/LyP-1LP therapeutic system display significant reductions in tumor volume compared with individual nanoparticles or untargeted cooperative system.

Systematic Surface Engineering of Magnetic Nanoworms for in vivo Tumor Targeting

In the design of nanoparticles that can target disease tissue in vivo, parameters such as targeting ligand density, type of target receptor, and nanoparticle shape can play an important role in determining the extent of accumulation. Herein, a systematic study of these parameters for the targeting of mouse xenograft tumors is performed using superparamagnetic iron oxide as a model nanoparticle system. The type of targeting peptide (recognizing cell surface versus extracellular matrix), the surface coverage of the peptide, its attachment chemistry, and the shape of the nanomaterial [elongated (nanoworm, NW) versus spherical (nanosphere, NS)] are varied. Nanoparticle circulation times and in vivo tumor-targeting efficiencies are quantified in two xenograft models of human tumors (MDA-MB-435 human carcinoma and HT1080 human fibrosarcoma). It is found that the in vivo tumor-targeting ability of the NW is superior to that of the NS, that the smaller, neutral CREKA targeting group is more effective than the larger, positively charged F3 molecule, that a maximum in tumor-targeting efficiency and blood half-life is observed with approximately 60 CREKA peptides per NW for either the HT1080 or the MDA-MB-435 tumor types, and that incorporation of a 5-kDa polyethylene glycol linker improves targeting to both tumor types relative to a short linker. It is concluded that the blood half-life of a targeting molecule-nanomaterial ensemble is a key consideration when selecting the appropriate ligand and nanoparticle chemistry for tumor targeting.

Protease-Triggered Unveiling of Bioactive Nanoparticles

Nanomaterials modified with biological recognition motifs acquire a myriad of functions that can be exploited for the diagnosis and treatment of cancer. Nevertheless, while bioactive domains can be used to target nanoparticles to cell receptors, shuttle them across cell membranes, and activate cell signaling, such modifications typically include cationic or hydrophobic regions that lead to rapid reticuloendothelial system (RES) clearance of particles from the blood, ultimately reducing particle accumulation in tumors. [1,2] Further functionalization with hydrophilic polymers like poly(ethylene glycol) (PEG) can improve blood half-lives and tumor accumulation, but often at the expense of efficient ligandmediated nanoparticle binding. [3‐5] To address this tradeoff between improved biodistribution and optimal functionality on nanoparticles, we present a general strategy for reversibly veiling bioactive domains on nanoparticles using sterically protective polymers. We demonstrate that these materials effectively accumulate via the hyperpermeable vasculature of tumors and can be activated by cancer-secreted proteases to unveil hidden functional domains.

Cooperative Nanoparticles for Tumor Detection and Photothermally Triggered Drug Delivery

The ability of one structural type to perform multiple medical diagnostic or therapeutic functions is often cited as an advantageous characteristic of nanomaterials that cannot be achieved with organic small molecules.[1–3] Although there are now many examples of nanosystems that integrate multiple functions into a single structure, the designs can reduce the efficacy of the individual functions due to space and surface-chemistry limitations in the tiny platforms. For example, magnetic nanoparticles and drug molecules can be co-encapsulated in liposomes to simultaneously perform multiple functions, such as magnetic resonance imaging, magnetic drug delivery and hyperthermia,[4] but the loading capacity and the stability are typically compromised relative to a single-component liposome. There has been some effort to develop intrinsically multifunctional nanomaterials such as magnetic nanocapsules and luminescent porous silicon nanoparticles to overcome such problems,[5,6] although these more complicated structures may lose versatility in terms of the types of payloads they can carry. Access to the payloads can also be limited, reducing the ability to control their release. Separating functions into two or more nanoparticle formulations is one means to simplify the problem. If two separate nanomaterials can be engineered to synergistically cooperate in their diagnostic or therapeutic functions, it is possible that the overall dosage can be reduced, minimizing side effects and providing a safer transition to the clinic.

Functional delivery of siRNA in mice using dendriworms.

Small interfering RNAs (siRNAs) mediate cleavage of specific, complementary mRNA sequences and thus regulate gene expression. Not surprisingly, their use for treatment of diseases that are rooted in aberrant gene expression, such as cancer, has become a paradigm that has gained wide interest. Here, we report the development of dendrimer-conjugated magnetofluorescent nanoworms that we call “dendriworms” as a modular platform for siRNA delivery in vivo. This platform maximizes endosomal escape to robustly produce protein target knockdown in vivo, and is tolerated well in mouse brain. We demonstrate that siRNA-carrying dendriworms can be readily internalized by cells and enable endosomal escape across a wide range of loading doses, whereas dendrimers or nanoworms alone are inefficient. Further, we show that dendriworms carrying siRNA against the epidermal growth factor receptor (EGFR) reduce protein levels of EGFR in human glioblastoma cells by 70−80%, 2.5-fold more efficiently than commercial cationic lipids. Dendriworms were well-tolerated after 7-days of convection-enhanced delivery to the mouse brain and in an EGFR-driven transgenic model of glioblastoma, anti- EGFR dendriworms led to specific and significant suppression of EGFR expression. Collectively, these data establish dendriworms as a multimodal platform that enables fluorescent tracking of siRNA delivery in vivo, cellular entry, endosomal escape, and knockdown of target proteins.

Ligand-Clustered “Patchy” Nanoparticles for Modulated Cellular Uptake and In Vivo Tumor Targeting†

Despite the evident success of using a multivalent approach to increase efficacy of targeted delivery, a clear understanding of how multiple ligands behave collectively to influence the uptake of nanoparticle cell-targeting agents has not been reached. Although when present in large quantity, multivalent ligands can increase binding avidities to cells, it is also conceivable, that the manner in which these ligands are presented to the cell may have a significant effect on uptake. Here we examine this parameter using a linear dendritic polymer construct that enabled us to pattern the surfaces of nanoparticles with variable sized ligand clusters in different spatial arrangements. We demonstrate for the first time the clear impact of folate presentation on intracellular uptake both in vitro and in vivo. The findings presented here suggest that the nature of ligand presentation on a nanoparticle surface may play an important role in drug targeting; the results suggest potential impact for other targeting moieties and provide a framework for further refinement of future multivalent targeting strategies.