A Liposomal Drug Platform Overrides Peptide Ligand Targeting to a Cancer Biomarker,Irrespective of Ligand Affinity or Density

One method for improving cancer treatment is the use of nanoparticle drugs functionalized with targeting ligands that recognize receptors expressed selectively by tumor cells. In theory such targeting ligands should specifically deliver the nanoparticle drug to the tumor, increasing drug concentration in the tumor and delivering the drug to its site of action within the tumor tissue. However, the leaky vasculature of tumors combined with a poor lymphatic system allows the passive accumulation, and subsequent retention, of nanosized materials in tumors. Furthermore, a large nanoparticle size may impede tumor penetration. As such, the role of active targeting in nanoparticle delivery is controversial, and it is difficult to predict how a targeted nanoparticle drug will behave in vivo. Here we report in vivo studies for α_vβ₆-specific H2009.1 peptide targeted liposomal doxorubicin, which increased liposomal delivery and toxicity to lung cancer cells in vitro. We systematically varied ligand affinity, ligand density, ligand stability, liposome dosage, and tumor models to assess the role of active targeting of liposomes to α_vβ₆. In direct contrast to the in vitro results, we demonstrate no difference in in vivo targeting or efficacy for H2009.1 tetrameric peptide liposomal doxorubicin, compared to control peptide and no peptide liposomes. Examining liposome accumulation and distribution within the tumor demonstrates that the liposome, and not the H2009.1 peptide, drives tumor accumulation, and that both targeted H2009.1 and untargeted liposomes remain in perivascular regions, with little tumor penetration. Thus H2009.1 targeted liposomes fail to improve drug efficacy because the liposome drug platform prevents the H2009.1 peptide from both actively targeting the tumor and binding to tumor cells throughout the tumor tissue. Therefore, using a high affinity and high specificity ligand targeting an over-expressed tumor biomarker does not guarantee enhanced efficacy of a liposomal drug. These results highlight the complexity of in vivo targeting.     

A pretargeted nanoparticle system for tumor cell labeling

Nanoparticle-based cancer diagnostics and therapeutics can be significantly enhanced by selective tissue localization, but the strategy can be complicated by the requirement of a targeting ligand conjugated on nanoparticles, that is specific to only one or a limited few types of neoplastic cells, necessitating the development of multiple nanoparticle systems for different diseases. Here, we present a new nanoparticle system that capitalizes on a targeting pretreatment strategy, where a circulating fusion protein (FP) selectively prelabels the targeted cellular epitope, and a biotinylated iron oxide nanoparticle serves as a secondary label that binds to the FP on the target cell. This approach enables a single nanoparticle formulation to be used with any one of existing fusion proteins to bind a variety of target cells. We demonstrated this approach with two fusion proteins against two model cancer cell lines: lymphoma (Ramos) and leukemia (Jurkat), which showed 72.2% and 91.1% positive labeling, respectively. Notably, TEM analysis showed that a large nanoparticle population was endocytosed via attachment to the non-internalizing CD20 epitope.     

Specific association of thiamine-coated gadolinium nanoparticles with human breast cancer cells expressing thiamine transporters

Thiamine (vitamin B(1)) was investigated as a tumor-specific ligand for gadolinium nanoparticles. Solid nanoparticles containing gadolinium hexanedione (1.5 mg/mL) were engineered from oil-in-water microemulsion templates and coated with thiamine ligands. Thiamine ligands were synthesized by conjugating thiamine to either distearoylphosphatidylethanolamine (DSPE) or fluorescein via a poly(ethylene glycol) (PEG) spacer (Mw 3350). The efficiency of thiamine ligand attachment to nanoparticles was evaluated using gel permeation chromatography (GPC). Cell association studies were carried using a methotrexate-resistant breast cancer cell line, MTX(R)ZR75, transfected with thiamine transporter genes (THTR1 and THTR2). Thiamine-coated nanoparticle association with THTR1 and THTR2 cells was significantly greater than that with control breast cancer cells (MTX(R)ZR75 transfected with the empty expression vector pREP4) (p < 0.01; t-test). The nanoparticle cell association was significantly dependent on the extent of thiamine ligand coating on nanoparticles, expression of thiamine transporters in cells, temperature of incubation, and the concentration of competitive inhibitor (free thiamine). Further studies are warranted to assess the potential of the engineered thiamine-coated gadolinium (Gd) nanoparticles in neutron capture therapy of tumors.     

Determination of Fundamental Morphological Parameters of Supported Nanoparticle Ensembles

A new model to extract important morphological parameters of noble metal nanoparticle ensembles with a broad size and shape distribution is presented. The technique is based on a rigorous simulation of the inhomogeneously broadened extinction profiles of nanoparticle ensembles. As input data, only experimentally accessible parameters, such as the amount of deposited material, the nanoparticle number density, and the relative size distribution of the nanoparticles, are used. The model can be applied to oblate nanoparticles, which exhibit a strong correlation between their shape and size, e.g., to supported nanoparticles generated, for example, by deposition of atoms and subsequent nucleation or by gas phase deposition. Both methods are standard preparation techniques to generate well-defined nanoparticle ensembles under ultra high vacuum conditions. We apply our model to gold and silver nanoparticles on sapphire and TiO₂ supports and obtain a perfect agreement between the calculated and experimental data. More importantly, we could extract the functional dependence between the axial ratio and the radius of the nanoparticles within the ensemble and, therewith, the most probable axial ratio in the ensemble. In addition, the extinction spectrum of a nanoparticle ensemble irradiated with nanosecond pulsed laser light during growth has been successfully modeled. This demonstrates, that the model is able to describe shape changes of resonantly heated nanoparticles within the ensemble. By using the coverage as a free parameter, we could calculate from the extinction spectrum the average particle radius as well as the amount of desorbed atoms after irradiation with laser light. In summary, the model allows a fast, easy, but extensive morphological characterization of nanoparticle ensembles that exhibit a broad size and shape distribution.     

New magnetic nanoparticles for biotechnology

Paramagnetic carriers, which are linked to antibodies enable highly specific biological cell separations. With the colloidal synthesis of superparamagnetic Co and FeCo nanocrystals with superior magnetic moments the question about their potential to replace magnetite as the magnetically responsive component of magnetic beads is addressed. Starting from a magnetic analysis of the corresponding magnetophoretic mobility of Co and FeCo based alloys their synthesis and resulting microstructural and magnetic properties as function of the underlying particle size distribution are discussed in detail. The stability of the oleic acid ligand of Co nanocrystals has been investigated. The oxidation kinetics were quantified using magnetic measurements. As a result, this ligand system provides sufficient protection against oxidation. Furthermore, the kinetics of the synthesis of Fe(50)Co(50) nanoparticles has been monitored employing Fourier transform infra red (FT-IR) spectroscopy and is modeled using a consecutive decomposition and growth model. This model predicts the experimentally realized FeCo nanoparticle composition as a function of the particle size fairly well. High-resolution transmission electron microscopy (HRTEM) was performed to uncover the resulting microstructure and composition on a nanometer scale.     

Molecular dynamics simulation study of a pulmonary surfactant film interacting with a carbonaceous nanoparticle

This article reports an all-atom molecular dynamics simulation to study a model pulmonary surfactant film interacting with a carbonaceous nanoparticle. The pulmonary surfactant is modeled as a dipalmitoylphosphatidylcholine monolayer with a peptide consisting of the first 25 residues from surfactant protein B. The nanoparticle model with a chemical formula C₁₈₈H₅₃ was generated using a computational code for combustion conditions. The nanoparticle has a carbon cage structure reminiscent of the buckyballs with open ends. A series of molecular-scale structural and dynamical properties of the surfactant film in the absence and presence of nanoparticle are analyzed, including radial distribution functions, mean-square displacements of lipids and nanoparticle, chain tilt angle, and the surfactant protein B peptide helix tilt angle. The results show that the nanoparticle affects the structure and packing of the lipids and peptide in the film, and it appears that the nanoparticle and peptide repel each other. The ability of the nanoparticle to translocate the surfactant film is one of the most important predictions of this study. The potential of mean force for dragging the particle through the film provides such information. The reported potential of mean force suggests that the nanoparticle can easily penetrate the monolayer but further translocation to the water phase is energetically prohibitive. The implication is that nanoparticles can interact with the lung surfactant, as supported by recent experimental data by Bakshi et al.     

On the design of composite protein-quantum dot biomaterials via self-assembly

Incorporation of nanoparticles during the hierarchical self-assembly of protein-based materials can impart function to the resulting composite materials. Herein we demonstrate that the structure and nanoparticle distribution of composite fibers are sensitive to the method of nanoparticle addition and the physicochemical properties of both the nanoparticle and the protein. Our model system consists of a recombinant enhanced green fluorescent protein-Ultrabithorax (EGFP-Ubx) fusion protein and luminescent CdSe-ZnS core-shell quantum dots (QDs), allowing us to optically assess the distribution of both the protein and nanoparticle components within the composite material. Although QDs favorably interact with EGFP-Ubx monomers, the relatively rough surface morphology of composite fibers suggests EGFP-Ubx-QD conjugates impact self-assembly. Indeed, QDs templated onto EGFP-Ubx film post-self-assembly can be subsequently drawn into smooth composite fibers. Additionally, the QD surface charge impacts QD distribution within the composite material, indicating that surface charge plays an important role in self-assembly. QDs with either positively or negatively charged coatings significantly enhance fiber extensibility. Conversely, QDs coated with hydrophobic moieties and suspended in toluene produce composite fibers with a heterogeneous distribution of QDs and severely altered fiber morphology, indicating that toluene severely disrupts Ubx self-assembly. Understanding factors that impact the protein-nanoparticle interaction enables manipulation of the structure and mechanical properties of composite materials. Since proteins interact with nanoparticle surface coatings, these results should be applicable to other types of nanoparticles with similar chemical groups on the surface.     

Covalent immobilization of cholesterol oxidase on self-assembled gold nanoparticles for highly sensitive amperometric detection of cholesterol in real samples

A novel scheme for the fabrication of gold nanoparticle modified cholesterol oxidase based bioelectrode is presented and its application potential for cholesterol biosensor is investigated. The fabrication procedure is based on the deposition of gold nanoparticles on the 1,6-hexanedithiol modified gold electrode, functionalization of the surface of deposited gold nanoparticles with carboxyl groups using 11-mercaptoundecanoic acid and then covalent immobilization of cholesterol oxidase on the surface of gold nanoparticle film using the N-ethyl-N'-(3-dimethylaminopropyl carbodimide) and N-hydroxysuccinimide ligand chemistry. The assembly process of the bioelectrode is investigated using atomic force microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. The gold nanoparticle film on the electrode surface provided an environment for the enhanced electrocatalytic activities and thus resulted in enhanced analytical response. The resulting bioelectrode is further applied to the amperometric detection of cholesterol and exhibited a linear response to cholesterol in the range of 0.04-0.22 mM with a detection limit of 34.6 μM, apparent Michaelis-Menten constant (K(m)(app)) of 0.062 mM and a high sensitivity of 9.02 μA mM(-1). The fabricated bioelectrode is successfully used for the selective determination of cholesterol in human serum samples.     

Correlated Optical Spectroscopy and Electron Microscopy Studies of the Slow Ostwald-Ripening Growth of Silver Nanoparticles under Controlled Reducing Conditions

Metallic nanoparticles display distinct localized surface plasmon resonance (LSPR) properties that depend on their size, shape, and composition and that can be monitored to characterize their growth. Utilizing LSPR properties, we report the first investigation of ambient temperature formation of trioctylamine (TOA)-stabilized spherical silver nanoparticles (AgNPs) of ～3.0-nm diameter by mild reduction of AgClO₄ with the weak reducing agent heptamethyltrisiloxane in organic solvent. The appropriate choice of experimental conditions caused slow reduction, which allowed the study of the nanoparticle growth process by time-resolved UV–visible spectroscopy and transmission electron microscopy (TEM). The linear nanoparticle growth kinetics from 50 min to end of the reaction derived from LSPR changes, the absence of a bimodal size distribution during the initial stage of the reduction process from TEM analysis, and the single crystallinity of the resulting AgNPs suggested a diffusion-controlled Ostwald-ripening growth process. It was also found that in addition to its stabilizing ability, TOA acted as a catalyst and facilitated Ag⁺ reduction. Furthermore, a modest increase in reaction temperature caused a substantial enhancement in the AgNP formation rate, and low concentration of stabilizing ligand yielded an increase in size and dispersity.     

Effect of gold nanoparticle on stability of the DNA molecule: A study of molecular dynamics simulation

An understanding of the mechanism of DNA interactions with gold nanoparticles is useful in today medicine applications. We have performed a molecular dynamics simulation on a B-DNA duplex (CCTCAGGCCTCC) in the vicinity of a gold nanoparticle with a truncated octahedron structure composed of 201 gold atoms (diameter ～1.8 nm) to investigate gold nanoparticle (GNP) effects on the stability of DNA. During simulation, the nanoparticle is closed to DNA and phosphate groups direct the particles into the major grooves of the DNA molecule. Because of peeling and untwisting states that are occur at end of DNA, the nucleotide base lies flat on the surface of GNP. The configuration entropy is estimated using the covariance matrix of atom-positional fluctuations for different bases. The results show that when a gold nanoparticle has interaction with DNA, entropy increases. The results of conformational energy and the hydrogen bond numbers for DNA indicated that DNA becomes unstable in the vicinity of a gold nanoparticle. The radial distribution function was calculated for water hydrogen–phosphate oxygen pairs. Almost for all nucleotide, the presence of a nanoparticle around DNA caused water molecules to be released from the DNA duplex and cations were close to the DNA.     

Tumor-specific hybrid polyhydroxybutyrate nanoparticle: surface modification of nanoparticle by enzymatically synthesized functional block copolymer

A new approach to functionalize the surface of hydrophobic nanocarrier through enzymatic polymerization was demonstrated. The effective coupling between the hydrophobic surface of PHB nanoparticle and PHB chain grown from the enzyme fused with a specific ligand provided a simple way of functionalizing nanoparticles with active protein layers in aqueous environment. PHB nanoparticles loaded with model drug molecule, Nile red, were prepared through oil-in-water emulsion solvent evaporation method and the surface of nanoparticles were functionalized with tumor-specific ligand, RGD4C, fused with PHA synthase that drove the coupling reaction. The functionalized PHB nanoparticles showed a specific affinity to MDA-MB 231 breast cancer cells indicating that the tumor-specific ligand, RGD4C, was effectively displayed on the surface of PHB nanoparticles through enzymatic modification and confers targeting capability on the drug carrier.     

转铁蛋白导向脂质体核磁造影剂纳米粒子（TfNIR-LipNBD-Magnevist）——一种肿瘤靶向磁共振造影剂

造影剂辅助的核磁共振成像是目前肿瘤诊断的最吁方法之一。但是由于核磁共振成像内在的低灵敏性以及造影剂的非特异性,导致肿瘤早期诊断较为困难。文章将一种新的肿瘤靶向核磁造影剂纳米粒子应用于早期肿瘤的影像诊断。这种新的肿瘤靶向核磁造影剂纳米粒子由配体转铁蛋白（Tf）、纳米水平的正电脂质体（Lip）载体和临床常用的造影剂Magnevist（Tf^NIR-Lip^NBD-Magnevist）三部分构成。另外转铁蛋白和脂质体粒子上,亦标记了荧光物质用于确定转铁蛋白一脂质体一造影剂纳米粒子的靶向性,以及肿瘤的光学影像诊断。在体外实验中,利用激光共聚焦显微镜和光学影像证明了靶向纳米粒子介导的细胞内吞和特异性结合。在裸鼠肿瘤模型中,造影剂纳米粒子Tf^NIR-Lip^NBD-Magnevist经尾静脉注入后,显著增强了肿瘤内信号与周围组织的对比度。由造影剂纳米粒子介导的肿瘤内信号显著强于单独Magnevist辅助的肿瘤内信号。同时,利用光学影像方法,在肿瘤内检测到特异的荧光信号。其结果进一步支持了转铁蛋白一脂质体一造影利（Tf^NIR-Lip^NBD-Magnevist）纳米粒子的靶向性和肿瘤影像诊断的有效性。     

Engineering tumor-targeted gadolinium hexanedione nanoparticles for potential application in neutron capture therapy

Microemulsions (oil-in-water) have been employed as templates to engineer nanoparticles containing high concentrations of gadolinium for potential application in neutron capture therapy of tumors. Gadolinium hexanedione (GdH), synthesized by complexation of Gd(3+) with 2,4-hexanedione, was used as the nanoparticle matrix alone or in combination with either emulsifying wax or PEG-400 monostearate. Solid nanoparticles (<125 nm size) were obtained by simple cooling of the microemulsions prepared at 60 degrees C to room temperature in one vessel. The feasibility of tumor targeting via folate receptors was studied. A folate ligand was synthesized by chemically linking folic acid to distearoylphosphatidylethanolamine (DSPE) via a poly(ethylene glycol) (PEG; MW 3350) spacer. To obtain folate-coated nanoparticles, the folate ligand (0.75% w/w to 15% w/w) was added to either the microemulsion templates at 60 degrees C or nanoparticle suspensions at 25 degrees C. Efficiencies of folate ligand attachment/adsorption to nanoparticle formulations were monitored by gel permeation chromatography. Cell uptake studies were carried out in KB cells (human nasopharyngeal epidermal carcinoma cell line), known to overexpress folate receptors. The uptake of folate-coated nanoparticles was about 10-fold higher than uncoated nanoparticles after 30 min at 37 degrees C. The uptake of folate-coated nanoparticles at 4 degrees C was 20-fold lower than the uptake at 37 degrees C and comparable to the uptake of uncoated nanoparticles at 37 degrees C. Folate-mediated endocytosis was further verified by the inhibition of folate-coated nanoparticles uptake by free folic acid. It was observed that folate-coated nanoparticles uptake decreased to approximately 2% of its initial value with the coincubation of 0.001 mM of free folic acid. The results suggested that these tumor-targeted nanoparticles containing high concentrations of Gd may have potential for neutron capture therapy.     

Analysis of distribution of ligands adsorbed on DNA fragments

The effect of finite fragment length on the distribution pattern of bound protein along the DNA fragment is considered. If the size of the binding site for a ligand on DNA is comparable with the length of the DNA fragment fluctuations in the amount of ligand bound to the fragment create some difficulties for evaluating the distribution pattern of ligand on DNA. A mathematical approach is developed which enables one to calculate the distribution pattern of ligand on DNA provided that the number of bound ligand on the DNA fragment is known. Expression are also obtained to treat the effects of fluctuations in the number of ligand molecules bound to the DNA fragment on the distribution pattern of ligand. A new procedure is proposed which may be useful for locating the preferable binding sites for ligand on DNA on the basis of footprinting experiments.     

Dynamics of Receptor-Mediated Nanoparticle Internalization into Endothelial Cells

Nanoparticles offer a promising medical tool for targeted drug delivery, for example to treat inflamed endothelial cells during the development of atherosclerosis. To inform the design of such therapeutic strategies, we develop a computational model of nanoparticle internalization into endothelial cells, where internalization is driven by receptor-ligand binding and limited by the deformation of the cell membrane and cytoplasm. We specifically consider the case of nanoparticles targeted against ICAM-1 receptors, of relevance for treating atherosclerosis. The model computes the kinetics of the internalization process, the dynamics of binding, and the distribution of stresses exerted between the nanoparticle and the cell membrane. The model predicts the existence of an optimal nanoparticle size for fastest internalization, consistent with experimental observations, as well as the role of bond characteristics, local cell mechanical properties, and external forces in the nanoparticle internalization process.     

Computational Modeling of Tumor Response to Drug Release from Vasculature-Bound Nanoparticles

Systemically injected nanoparticle (NPs) targeting tumor vasculature offer a venue for anti-angiogenic therapies as well as cancer detection and imaging. Clinical application has been limited, however, due to the challenge of elucidating the complex interplay of nanotechnology, drug, and tumor parameters. A critical factor representing the likelihood of endothelial adhesion is the NP vascular affinity, a function of vascular receptor expression and NP size and surface-bound ligand density. We propose a theoretical framework to simulate the tumor response to vasculature-bound drug-loaded NPs and examine the interplay between NP distribution and accumulation as a function of NP vascular affinity, size, and drug loading and release characteristics. The results show that uniform spatial distribution coupled with high vascular affinity is achievable for smaller NPs but not for larger sizes. Consequently, small (100 nm) NPs with high vascular affinity are predicted to be more effective than larger (1000 nm) NPs with similar affinity, even though small NPs have lower drug loading and local drug release compared to the larger NPs. Medium vascular affinity coupled with medium or larger sized NPs is also effective due to a more uniform distribution with higher drug loading and release. Low vascular affinity hampered treatment efficacy regardless of NP size, with larger NPs additionally impeded by heterogeneous distribution and drug release. The results further show that increased drug diffusivity mainly benefits heterogeneously distributed NPs, and would negatively affect efficacy otherwise due to increased wash-out. This model system enables evaluation of efficacy for vascular-targeted drug-loaded NPs as a function of critical NP, drug, and tumor parameters.     

Plasmonic Resonance Excited Extinction Spectra of Cross-Shaped Ag Nanoparticles

Plasmonic properties of cross-shaped Ag nanoparticles are investigated theoretically using finite-difference time-domain algorithm. Electric field (E-field) distribution of a single cross-shaped Ag nanoparticle with different shape parameters and patterned nanoparticles with different periods were presented. Both red shift and blue shift of the extinction spectra were observed. The simulation results demonstrated that the strong E-field intensity is located at sharp corner of the nanoparticles. And E-field intensity of the nanoparticle array is much stronger than that of a single Ag nanoparticle. Enhancement of the large localized E-field originating from the nanoparticles was analyzed. Corresponding influence of “hot spots” effect on enhancing Raman scattering was discussed as well.     

Protein structural changes induced by glutathione-coated CdS quantum dots as revealed by Trp phosphorescence

We evaluated the potential of tryptophan (Trp) phosphorescence spectroscopy for investigating conformational states of proteins involved in interaction with nanoparticles. Characterization of protein–nanoparticle interaction is crucial in assessing biological hazards related to use of nanoparticles. We synthesized glutathione-coated CdS quantum dots (GSH-CdS), which exhibited an absorption peak at 366 nm, indicative of 2.4 nm core size. Chemical analysis of purified GSH-CdS suggested an average molecular formula of GSH₁₈S₅₆Cd₆₀. Investigations were conducted on model proteins varying in terms of isoelectric point, degree of burial of the Trp probe, and quaternary structure. GSH-CdS fluorescence measurements showed improvement in nanoparticle quantum yield induced by protein interaction. Trp phosphorescence was used to examine the possible perturbations in the protein native fold induced by GSH-CdS. Phosphorescence lifetime measurements highlighted significant conformational changes in some proteins. Despite their small size, GSH-CdS appeared to interact with more than one protein molecule. Rough determination of the affinity of GSH-CdS for proteins was derived from the change in phosphorescence lifetime at increasing nanoparticle concentrations. The estimated affinities were comparable to those observed for specific protein–ligand interactions and suggest that protein–nanoparticle interaction may have a biological impact.     

Transferrin-containing, cyclodextrin polymer-based particles for tumor-targeted gene delivery

Transferrin is a well-studied ligand for tumor targeting due to upregulation of transferrin receptors in numerous cancer cell types. Here, we report the development of a transferrin-modified, cyclodextrin polymer-based gene delivery system. The delivery system is comprised of a nanoparticle (formed by condensation of a cyclodextrin polycation with nucleic acid) that is surface-modified to display poly(ethylene glycol) (PEG) for increasing stability in biological fluids and transferrin for targeting of cancer cells that express transferrin receptor. A transferrin-PEG-adamantane conjugate is synthesized for nanoparticle modification. The transferrin conjugate retains high receptor binding and self-assembles with the nanoparticles by adamantane (host) and particle surface cyclodextrin (guest) inclusion complex formation. At low transferrin modification, the particles remain stable in physiologic salt concentrations and transfect K562 leukemia cells with increased efficiency over untargeted particles. The increase in transfection is eliminated when transfections are conducted in the presence of excess free transferrin. The transferrin-modified nanoparticles are appropriate for use in the systemic delivery of nucleic acid therapeutics for metastatic cancer applications.     

The Effect of Ag Nanoparticles on Surface-Enhanced Luminescence from Au Nanovoid Arrays

Studies comparing the effect of adding two different nanoparticle compositions on the plasmonic properties of Au nanovoid arrays were undertaken. Surface-enhanced resonance luminescence and surface-enhanced resonance Raman studies comparing dispersed Ag nanoparticles and Ag nanoparticle aggregates on gold nanovoid arrays were undertaken. These studies showed that using Ag nanoparticle aggregates increased both luminescence and Raman efficiency relative to when dispersed nanoparticles were used; in addition, these studies also showed that adding dispersed Ag nanoparticles supported a more reproducible enhancement in luminescence and Raman across the substrate compared to using Ag nanoparticle aggregates. Finite element analysis simulations indicated that surface plasmon polariton distribution in the sample was affected by the presence of the Ag nanoparticles on the Au nanovoid array.