Multistage nanovectors: from concept to novel imaging contrast agents and therapeutics

Abstract

Over the last few decades a great variety of nanotechnology based platforms have been synthesized and fabricated to improve the delivery of active compounds to a disease site. Nanoparticles currently used in the clinic, and the majority of nanotherapeutics/nanodiagnostics under investigation, accommodate single- or multiple- functionalities on the same entity. Because many heterogeneous biological barriers can prevent therapeutic and imaging agents from reaching their intended targets in sufficient concentrations, there is an emerging requirement to develop a multimodular nanoassembly, in which different components with individual specific functions act in a synergistic manner. The multistage nanovectors (MSVs) were introduced in 2008 as the first system of this type. It comprises several nanocomponents or "stages", each of which is designed to negotiate one or more biological barriers. Stage 1 mesoporous silicon particles (S1MPs) were rationally designed and fabricated in a nonspherical geometry to enable superior blood margination and to increase cell surface adhesion. The main task of S1MPs is to efficiently transport nanoparticles that are loaded into their porous structure and to protect them during transport from the administration site to the disease lesion. Semiconductor fabrication techniques including photolithography and electrochemical etching allow for the exquisite control and precise reproducibility of S1MP physical characteristics such as geometry and porosity. Furthermore, S1MPs can be chemically modified with negatively/positively charged groups, PEG and other polymers, fluorescent probes, contrast agents, and biologically active targeting moieties including antibodies, peptides, aptamers, and phage. The payload nanoparticles, termed stage 2 nanoparticles (S2NPs), can be any currently available nanoparticles such as liposomes, micelles, inorganic/metallic nanoparticles, dendrimers, and carbon structures, within the approximate size range of 5-100 nm in diameter. Depending upon the physicochemical features of the S1MP (geometry, porosity, and surface modifications), a variety of S2NPs or nanoparticle "cocktails" can be loaded and efficiently delivered to the disease site. As demonstrated in the studies reviewed here, once the S2NPs are loaded into the S1MPs, a variety of novel properties emerge, which enable the design of new and improved imaging contrast agents and therapeutics. For example, the loading of the MRI Gd-based contrast agents onto hemispherical and discoidal S1MPs significantly increased the longitudal relaxivity (r1) to values of up to 50 times larger than those of clinically available gadolinium-based agents (~4 mM(-1) s(-1)/Gd(3+) ion). Furthermore, administration of a single dose of MSVs loaded with neutral nanoliposomes containing small interfering RNA (siRNA) targeted against the EphA2 oncoprotein enabled sustained EphA2 gene silencing for at least 21 days. As a result, the tumor burden was reduced in an orthotopic mouse model of ovarian cancer. We envision that the versatility of the MSV platform and its emerging properties will enable the creation of personalized solutions with broad clinical implications within and beyond the realm of cancer theranostics.

Figure 1

Schematic summary of possible MSV mechanisms of action. Central compartment: hemispherical or disc-shaped nanoporous silicon S1MP are engineered to exhibit an enhanced ability to marginate within blood vessels and adhere to disease-associated endothelium. Once positioned at the disease site, the S1MP can: (top right) release the drug/siRNA loaded S2NP to achieve the desired therapeutic effect, prior to complete biodegradation of the carrier particle; release an imaging agent (top left) or external energy activated S2NP (e.g. gold nanoparticles, nanoshells- bottom right). Another possible mechanism of action is cell based delivery of the MSV into the disease loci followed by triggered release of the S1MP/S2NP from the cells (bottom left).

Figure 2

Fabrication of S1MP. A1: Patterned SiN layer and trenches etched into silicon. A2: Electrochemically etched S1MPs with release layer. A3: S1MP array on wafer after removal of SiN. A4: Cross-section of hemispherical S1MP. B1: Photoresist pattern on LTO capped porous silicon film with release layer. B2: Particle array on wafer after RIE. B3: Discoidal S1MP array on wafer after LTO removal. B4: Released discoidal S1MPs. C1: Silver nanopattern etched into silicon forming porous silicon nanowires. C2: Nanowire barcode under white light. C3: SEM image of nanowire barcode. C4: 3-channel confocal microscopy images of nanowire barcode with green Q-dot loaded in small pore segment and red Q-dot in bigger pore segment.

Figure 3

Loading and release of S2NPs to/from S1MPs. (A) Loading of negatively and positively charged SPIONs into discoidal oxidized (-) S1MP and the association of gold nanoparticles-phage displaying targeting peptide assemblies on top of hemispherical S1MP; (B) co-loading and (E) co-release of quantum dots (QD) and PEGylated single-wall carbon nanotubes (SWNT) to/from a hemispherical particle with differential porosities; (D) schematic presentation of the payload release from S1MP in the process of degradation; (F) tuning degradation kinetics of the hemispherical 3.2 μm S1MP through covalent conjugation of PEGs possessing various molecular weights; loading (C) and release (G) of siRNA containing liposome (DOPC) into the hemispherical 1.6 μm S1MP Compiled from Refs. (12, 18, 23, 28) with permissions from the publishers.

Figure 4

Intracellular delivery of MSV. (A) SE micrograph of early stage S1MP phagocytosis in an endothelial cell. (B) TE micrographs show a secreted SPION-loaded vesicle (left) and an internalized MSV with dual cell localization (right; endosome and cytosol) (28). (C) Live confocal images of endothelial mitosis in the presence of internalized S1MP, showing how S1MP in the parent cell are split between the two daughter cells.

Figure 5

MSVs as advanced imaging agents. (A) The longitudinal relaxivity, r₁, of the MSV based new MRI nanoconstructs measured by a benchtop relaxometer as compared with the corresponding Gd-based CAs (26); (B) Axial spin- (top) and gradient- (bottom) echo magnetic resonance images of NMR tubes containing PBS (blank), S1MP and MSV loaded with SPIONs (MDS_lo and MDS_hi) (28). (C) In vivo NIR imaging following intravenous administration of 3.2 μm hemispherical S1MPs tagged with Dylight 750 (31).

Figure 6

Systemic delivery of siRNA-DOPC using S1MP results in long-lasting in vivo gene silencing. Mice with SKOV3ip1 orthotopic ovarian tumors were injected with S1MP-EphA2-siRNA-DOPC or left nontreated. (A) Western blot to measuring EphA2 expression levels at predetermined time-points. (B) densitometric analysis of normalized EphA2 expression by β-actin; (C) immunohistochemical analysis of EphA2 expression in the SKOV1ip3 tumor; (D) therapeutic efficacy of sustained EphA2-siRNA-DOPC delivery by S1MP in SKOV3ip1 bearing mice. Treatment groups (n = 10): (a) saline, (b) S1MP, (c) nonsilencing control siRNA-DOPC, (d) S1MP-nonsilencing control-siRNA-DOPC, (e) EphA2-siRNA-DOPC, (f) S1MP-EphA2-siRNA-DOPC (MSV). SiRNA-DOPC was intravenously injected biweekly at a dose of 5 μg siRNA. S1MP-EphA2-siRNA-DOPC was injected as a single administration in 3 weeks at a dose of 15 μg siRNA. Reproduced from (23) with permission from AACR.