Enhancing the Biodistribution and Physicochemical Properties of Gold Nanoparticles by Modifying their Surface Characteristics

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The Influence of Surface Protein Adsorption on Gold Nanoparticle Intratumoral Distribution and Retention

Nanomedicines’ inability to penetrate throughout the entire volume of a tumor due to heterogeneous distribution within the tumor mass remains a crucial limiting factor for a vast range of theranostic applications, including image-guided radiation therapy. Despite many studies conducted on the topic having shown the efficacy and biocompatibility of colloidal gold nanoparticles (GNPs), the biological effects of GNPs in the tumor microenvironment, including the particle–protein interaction and the consequent impact on cellular pathways and contrast enhancement remain unclear. In this regard, further investigations on how GNP surface passivation affects X-ray attenuation as well as in vivo biodistribution will clarify several aspects still under discussion in the scientific community, which so far have limited the clinical translation of their cancer-related applications. We aim to evaluate the influence of protein surface adsorption on the GNP biodistribution in Lewis lung carcinoma (LLC) tumor-bearing mice using high-resolution computed tomography (CT) pre-clinical imaging. We hypothesize that, by controlling the adsorption of proteins on the GNP surface, we can influence the intratumoral distribution and retention of the particles. GNPs approximately 34 nm in diameter were synthesized with a surface plasmon peak at ~530 nm, surface passivated with bovine serum albumin (BSA) to reduce opsonization and improve colloidal stability, and characterized with standard methods. Modulation of BSA adsorption on the GNPs was observed by tuning the pH of the immobilization medium from acidic to alkaline, which we quantified using Langmuir isotherms. CT phantom imaging was used to determine X-ray attenuation as a function of GNP concentration and surface functionalization. The in vitro study for evaluating the uptake of GNPs by LLC cells highlighted a difference in the internalization depending on the surface functionalization. In both cases, macropinocytosis was the trafficking mechanism, but while endosomes with citrate-GNPs can be found in different stages of maturation, cells treated with BSA-GNPs presented larger vesicles up to 1 μm in diameter. The in vivo study was performed by injecting intratumorally, concentrating GNPs into LLC solid tumors grown on the right flank of 6-week-old female C57BL/6 mice. Ten days post-injection, follow-up assessments with CT imaging showed the distribution and retention of the particles in the tumor. CT attenuation quantification based on bioimaging analysis for each time point was conducted. In vivo results showed significant heterogeneity in the intratumoral biodistribution of GNPs dependent on surface passivation. BSA-GNPs perfused predominately along the tumor periphery with few depositions throughout the entire tumor volume. This response can be explained by the abnormal and heterogeneous vascular structure of the LLC tumor, suggesting perfusion rather than permeability as the limiting factor for tumor accumulation of the GNPs. Despite the perivascular cluster accumulation, the BSA-GNP distribution diverged from that obtained after unpassivated, citrate-GNP intratumoral injections. In conclusion, our investigations have shown that surface passivation of GNPs is able to influence the mechanism of cellular uptake in vitro and their in vivo intratumoral diffusion, highlighting the spatial heterogeneity of the solid tumor

Improvements in Gold Nanorod Biocompatibility with Sodium Dodecyl Sulfate Stabilization

Due to their well-defined plasmonic properties, gold nanorods (GNRs) can be fabricated with optimal light absorption in the near-infrared region of the electromagnetic spectrum, which make them suitable for cancer-related theranostic applications. However, their controversial safety profile, as a result of surfactant stabilization during synthesis, limits their clinical translation. We report a facile method to improve GNR biocompatibility through the presence of sodium dodecyl sulfate (SDS). GNRs (120 × 40 nm) were synthesized through a seed-mediated approach, using cetyltrimethylammonium bromide (CTAB) as a cationic surfactant to direct the growth of nanorods and stabilize the particles. Post-synthesis, SDS was used as an exchange ligand to modify the net surface charge of the particles from positive to negative while maintaining rod stability in an aqueous environment. GNR cytotoxic effects, as well as the mechanisms of their cellular uptake, were examined in two different cancer cell lines, Lewis lung carcinoma (LLC) and HeLa cells. We not only found a significant dose-dependent effect of GNR treatment on cell viability but also a time-dependent effect of GNR surfactant charge on cytotoxicity over the two cell lines. Our results promote a better understanding of how we can mediate the undesired consequences of GNR synthesis byproducts when exposed to a living organism, which so far has limited GNR use in cancer theranostics

Hyaluronate-Thiol Passivation Enhances Gold Nanoparticle Peritumoral Distribution When Administered Intratumorally in Lung Cancer

Biofouling is the unwanted adsorption of cells, proteins, or intracellular and extracellular biomolecules that can spontaneously occur on the surface of metal nanocomplexes. It represents a major issue in bioinorganic chemistry because it leads to the creation of a protein corona, which can destabilize a colloidal solution and result in undesired macrophage-driven clearance, consequently causing failed delivery of a targeted drug cargo. Hyaluronic acid (HA) is a bioactive, natural mucopolysaccharide with excellent antifouling properties, arising from its hydrophilic and polyanionic characteristics in physiological environments which prevent opsonization. In this study, hyaluronate-thiol (HA-SH) (MW 10 kDa) was used to surface-passivate gold nanoparticles (GNPs) synthesized using a citrate reduction method. HA functionalized GNP complexes (HA-GNPs) were characterized using absorption spectroscopy, scanning electron microscopy, zeta potential, and dynamic light scattering. GNP cellular uptake and potential dose-dependent cytotoxic effects due to treatment were evaluated in vitro in HeLa cells using inductively coupled plasma—optical emission spectrometry (ICP-OES) and trypan blue and MTT assays. Further, we quantified the in vivo biodistribution of intratumorally injected HA functionalized GNPs in Lewis Lung carcinoma (LLC) solid tumors grown on the flank of C57BL/6 mice and compared localization and retention with nascent particles. Our results reveal that HA-GNPs show overall greater peritumoral distribution (** p < 0.005, 3 days post-intratumoral injection) than citrate-GNPs with reduced biodistribution in off-target organs. This property represents an advantageous step forward in localized delivery of metal nano-complexes to the infiltrative region of a tumor, which may improve the application of nanomedicine in the diagnosis and treatment of cancer

Effects of Surface Protein Adsorption on the Distribution and Retention of Intratumorally Administered Gold Nanoparticles

The heterogeneous distribution of delivery or treatment modalities within the tumor mass is a crucial limiting factor for a vast range of theranostic applications. Understanding the interactions between a nanomaterial and the tumor microenvironment will help to overcome challenges associated with tumor heterogeneity, as well as the clinical translation of nanotheranostic materials. This study aims to evaluate the influence of protein surface adsorption on gold nanoparticle (GNP) biodistribution using high-resolution computed tomography (CT) preclinical imaging in C57BL/6 mice harboring Lewis lung carcinoma (LLC) tumors. LLC provides a valuable model for study due to its highly heterogenous nature, which makes drug delivery to the tumor challenging. By controlling the adsorption of proteins on the GNP surface, we hypothesize that we can influence the intratumoral distribution pattern and particle retention. We performed an in vitro study to evaluate the uptake of GNPs by LLC cells and an in vivo study to assess and quantify the GNP biodistribution by injecting concentrated GNPs citrate-stabilized or passivated with bovine serum albumin (BSA) intratumorally into LLC solid tumors. Quantitative CT and inductively coupled plasma optical emission spectrometry (ICP-OES) results both confirm the presence of particles in the tumor 9 days post-injection (n = 8 mice/group). A significant difference is highlighted between citrate-GNP and BSA-GNP groups (** p < 0.005, Tukey’s multiple comparisons test), confirming that the protein corona of GNPs modifies intratumoral distribution and retention of the particles. In conclusion, our investigations show that the surface passivation of GNPs influences the mechanism of cellular uptake and intratumoral distribution in vivo, highlighting the spatial heterogeneity of the solid tumor

Intratumoral Gold Nanoparticle-Enhanced CT Imaging: An in Vivo Investigation of Biodistribution and Retention

This study aims to evaluate the in vivo distribution of Gold Nanoparticles (GNPs) at different time points after intratumoral (IT) injection, exploiting their properties as contrast agents for Computed Tomography (CT). GNPs approximately 40 nm in diameter were synthesized with a surface plasmon peak at ~530 nm, capped with Bovine Serum Albumin (BSA) to improve colloidal stability, and characterized with standard methods. CT phantom imaging was performed to quantify X-ray attenuation as a function of GNP concentration and surface functionalization and to determine the appropriate particle dose for in vivo studies. Concentrated GNPs were intratumorally (IT) injected into Lewis Lung Carcinoma (LLC) solid tumors grown on the right flank of 6-week old female C57BL/6 mice. Ten days post-injection, follow up CT imaging was performed to assess the distribution and retention of the particles in the tumor. Using the CT attenuation quantification, images for each timepoint were segmented, and 3D volumes rendered, to conduct biodistribution analyses. The successful retention and permanence of the GNPs into the solid tumor after ten days suggests the significance of GNPs as a potential theranostic agent

Quantitative high-resolution 7T MRI to assess longitudinal changes in articular cartilage after anterior cruciate ligament injury in a rabbit model of post-traumatic osteoarthritis

Objective To demonstrate an ultra-high field (UHF) 7 T delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) protocol for quantitative post-traumatic osteoarthritis (PTOA) detection and monitoring in a rabbit anterior cruciate ligament transection (ACLT) model. Design ACL transections were performed unilaterally in 5 rabbits (33-weeks-old, 3.5 ± 0.5 kg) to induce PTOA. MRI exams were performed at 7 T prior to and 2, 4, 7 and 10-weeks after ACLT using a modified dGEMRIC protocol. Voxel-based T1 and T2 maps were created over manually drawn femoral cartilage ROIs from the center of the tibial plateau to the posterior meniscus. Femoral, tibial, and patellar epiphyses were harvested 10-weeks post-surgery and processed for μCT imaging and histology. Results Quantitative analysis revealed a 35% and 39% decrease in dGEMRIC index in the medial ACLT knee compartment 7- and 10-weeks post-surgery, respectively (p = 0.009 and p = 0.006) when compared to baseline. There was no significant change in the lateral ACLT compartment or in either compartment of the control knees. Visual inspection of histology confirmed PTOA in the ACLT knees. Osteophytes were found only in ACLT knees (osteophyte volume in femur: 94.53 ± 44.08 mm3, tibia: 29.35 ± 13.79 mm3, and patella: 3.84 ± 0.92 mm3) and were significantly larger in the medial compartments of the femur than lateral (p = 0.0312). Conclusion The dGEMRIC technique quantitatively applied at 7 T UHF-MRI demonstrates site-specific cartilage degeneration in a large animal PTOA model. This should encourage further investigation, with potential applications in drug and therapeutic animal trials as well as human studies